Opportunities in discovery and delivery of anticancer drugs targeting mitochondria and cancer cell metabolism

Advanced Drug Delivery Reviews 61 (2009) 1250–1275

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Advanced Drug Delivery Reviews j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a d d r

Opportunities in discovery and delivery of anticancer drugs targeting mitochondria and cancer cell metabolism☆ Divya Pathania 1, Melissa Millard 1, Nouri Neamati ⁎ Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, School of Pharmacy, Los Angeles, CA 90089, USA

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Article history: Received 14 May 2009 Accepted 18 May 2009 Available online 27 August 2009 Keywords: Warburg effect Aerobic glycolysis Glycolytic inhibitors Targeted drug delivery to cancer Delocalized lipophilic cations Inhibitors of mitochondrial electron transport chain Biosynthetic alterations in cancer cells Mitochondrial redox system Mitochondrial apoptotic machinery Triphenylphosphonium compounds

a b s t r a c t Cancer cells are characterized by self-sufficiency in the absence of growth signals, their ability to evade apoptosis, resistance to anti-growth signals, sustained angiogenesis, uncontrolled proliferation, and invasion and metastasis. Alterations in cellular bioenergetics are an emerging hallmark of cancer. The mitochondrion is the major organelle implicated in the cellular bioenergetic and biosynthetic changes accompanying cancer. These bioenergetic modifications contribute to the invasive, metastatic and adaptive properties typical in most tumors. Moreover, mitochondrial DNA mutations complement the bioenergetic changes in cancer. Several cancer management therapies have been proposed that target tumor cell metabolism and mitochondria. Glycolytic inhibitors serve as a classical example of cancer metabolism targeting agents. Several TCA cycle and OXPHOS inhibitors are being tested for their anticancer potential. Moreover, agents targeting the PDC/PDK (pyruvate dehydrogenase complex/pyruvate dehydrogenase kinase) interaction are being studied for reversal of Warburg effect. Targeting of the apoptotic regulatory machinery of mitochondria is another potential anticancer field in need of exploration. Additionally, oxidative phosphorylation uncouplers, potassium channel modulators, and mitochondrial redox are under investigation for their anticancer potential. To this end there is an increased demand for agents that specifically hit their target. Delocalized lipophilic cations have shown tremendous potential in delivering anticancer agents selectively to tumor cells. This review provides an overview of the potential anticancer agents that act by targeting cancer cell metabolism and mitochondria, and also brings us face to face with the emerging opportunities in cancer therapy. © 2009 Elsevier B.V. All rights reserved.

Abbreviations: AIF, apoptosis inducing factor; AVPI, alanine valine proline isoleucine; BAD, Bcl-2 antagonist of cell death; BAX, Bcl-2 associated protein X; BAK, Bcl-2 homologous antagonist/killer; BH, Bcl-2 homology domain; BIR, baculovirus IAP repeat; 1,3-BPG, 1,3-bisphosphoglycerate; AMF, autocrine motility factor; ANT, adenine nucleotide transporter; ARD1, arrest-defective 1 protein; BCNU, bis-chloronitrosourea; 3-BrP, 3-bromopyruvate; CAD, c-terminal activation domain; CARD, caspase recruitment domain; CBP, CREB binding protein; CCCP, carbonylcyanide-3-chlorophenylhydrazone; COX4/2, cyotochrome oxidase isoform2; CT, computed tomography; DCA, dichloroacetate; 2-DG, 2-deoxyglucose; DHAP, dihydroxy acetone phosphate; DIABLO, Direct Inhibitor of Apoptosis Binding protein with a Low pI; DLC, delocalized lipophilic cation; DNP, 2,4-dinitrophenol; EGFR, epidermal growth factor receptor; ERK, extracellular signal regulated kinase; ETC, electron transport chain; FAD, flavin adenine dinucleotide (oxidized); FADH2, flavin adenine dinucleotide (reduced); 18FBzTPP, 4-(18F-benzyl) triphenylphosphonium; FCCP, p-trifluoromethoxyphenylhydrazone; FDA, Food and Drug Administration; 18F-FDG, 18F-deoxygluxose; FH, fumarate hydratase; FIH, factor inhibiting HIF-1; FNQ, furanonapthoquinone; F-1,6-BP, fructose-1,6-bisphosphate; F-6-P, fructose-6-phosphate; GAPDH, glyceraldehyde-3phosphate dehydrogenase; GPI, glucose phosphate isomerase; Glut, glucose transporter; G-6-P, glucose-6-phosphate; Glyc-3-P, glyceraldehydes-3-phosphate; HBD, hydrogen bonding donor; HBA, hydrogen bonding acceptor; HDAC, histone deacetylase; HIF-1, hypoxia inducible factor-1; HK, hexokinase; HLRCC, hereditary leiomyomatosis/renal cell cancer; PGL, hereditary paraganglioma; Hsp90, heat shock protein 90; 3H-TPP, 3H-tetraphenylphosphonium; IAP, inhibitor of apoptosis protein; IMM, inner mitochondrial membrane; JNK, c-Jun N-terminal kinase; LDH, lactate dehydrogenase; MAPK, mitogen activated protein kinase; MDR, multidrug resistance; MEK, MAPK-ERK kinase; M2-PK, pyruvate kinase M2; MMP, matrix metalloproteinase; MOM, mitochondrial outer membrane; MPP+, 1-methyl-4-phenylpyridinium cation; MPT, membrane permeability transition; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; 99m-Tc-MIBI, 99m-Tc-Sestamibi; MTD, maximum tolerated dose; mTOR, mammalian target of rapamycin; NAD+, nicotinamide adenine dinucleotide (oxidized); NADH, nicotinamide adenine dinucleotide (reduced); NADPH, nicotinamide adenine dinucleotide phosphate (reduced); NCI, National Cancer Institute; NFAT, nuclear factor of activated T cells; NO, nitric oxide; OXPHOS, oxidative phosphorylation; PCD, programmed cell death; PDC, pyruvate dehydrogenase complex; PDT, photodynamic therapy; PDK, pyruvate dehydrogenase kinase; PDP, pyruvate dehydrogenase phosphatase; PEP, phosphoenol pyruvate; PET, positron emission tomography; PHD, prolyl hydroxylase; PI3K, phosphoinositide 3-kinase; PFK, phosphofructokinase; PFKFB, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase; 2-PG, 2phosphoglycerate; 3-PG, 3-phosphoglcerate; PGK, phosphoglycerate kinase; PGM, phosphoglycerate mutase; Pgp, p-glycoprotein; PK, pyruvate kinase; PKCδ, protein kinase C delta; PS, photosensitizer; PSA, prostate specific antigen; PTP, permeability transition pore; pVHL, von Hippel–Lindau protein; ROS, reactive oxygen species; SAR, structure activity relationship; SCO2, synthesis of cytochrome c oxidase2; SDH, succinate dehydrogenase; Smac, Second mitochondria derived activator of caspases; SPECT, single photon emission computed tomography; TIGAR, tp53 induced glycolysis and apoptosis regulator; TCA cycle, tricarboxyclic acid cycle; TNFR, tumor necrosis factor receptor; TPA, triphenylarsonium; TPP, triphenylphosphonium cation; TRAF, TNF receptor associated family of proteins; TKTL1, transketolase like enzyme1; αTOS, α-tocopheryl succinate; TPI, triosephosphate isomerase; Trx/TrxR, thioredoxin/thioredoxin reductase; VDAC, voltage dependent anion channel; XIAP, X-linked inhibitor of apoptosis protein. ☆ This review is part of the Advanced Drug Delivery Reviews theme issue on “Mitochondrial Medicine and Therapeutics, Part II”. ⁎ Corresponding author. Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, School of Pharmacy, PSC304, 1985 Zonal Avenue, Los Angeles, CA 90089, USA. Tel.: +1 323 442 2341; fax: +1 323 442 1390. E-mail address: [email protected] (N. Neamati). 1 First two authors contributed equally. 0169-409X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.addr.2009.05.010

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Contents 1. 2. 3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Rationale for targeting mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Glycolytic inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Hexokinase (HK) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.1. 2-Deoxyglucose (2-DG) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.2. 3-Bromopyruvate (3-BrP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.3. Lonidamine (TH-070) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.4. Methyl jasmonate. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Glucose-6-phosphate isomerase (GPI) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Phosphofructokinase (PFK). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Aldolase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Triosephosphate isomerase (TPI) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6.1. Iodoacetate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6.2. Koningic acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.7. Phosphoglycerate kinase (PGK). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.8. Phosphoglycerate mutase (PGM) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.9. Enolase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.10. Pyruvate kinase (PK) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.11. Lactate dehydrogenase (LDH) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.12. Transketolase like enzyme (TKTL1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Role of HIF-1α in promoting the Warburg effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Mitochondrial bioenergetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. The Citric Acid Cycle is the entry point for aerobic respiration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Enzymes of the Electron Transport Chain (ETC) create proton motive force needed to produce ATP . . . . . . . . . . . 5.3. Mechanisms of respiratory control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4. Targeting OXPHOS enzymes to induce mitochondrial bioenergetic failure . . . . . . . . . . . . . . . . . . . . . . . . 5.4.1. Targeting Complex I, mitochondrial NADH ubiquinone reductase . . . . . . . . . . . . . . . . . . . . . . . 5.4.2. Succinate dehydrogenase links TCA cycle and OXPHOS . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4.3. Cytochrome c reductase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4.4. Cytochrome c oxidase is the final electron acceptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4.5. Targeting the F0F1 ATPase prevents ATP synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. The other side of the coin: biosynthetic drive . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Biosynthesis of nucleotides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Biosynthesis of fatty acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. Biosynthesis of proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.4. Anaplerosis and NADPH production: role of glutaminolysis in biosynthesis . . . . . . . . . . . . . . . . . . . . . . . 7. Critical switch between glycolysis and TCA cycle: pyruvate dehydrogenase complex/pyruvate dehydrogenase kinase (PDC/PDK) . 7.1. Dicholoroacetate (DCA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.2. Radicicol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7.3. Nov3r and AZD7545 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8. Mitochondrial OXPHOS uncouplers and cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9. Mitochondrial DNA mutations in cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10. TCA cycle enzymes as tumor suppressors: succinate dehydrogenase (SDH) and fumarate hydratase (FH) . . . . . . . . . . . . 11. Targeting mitochondrial redox status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.1. Inhibition of mitochondrial thioredoxin reductase disrupts mitochondrial redox balance and induces tumor cell apoptosis. 12. Cancer therapy with mitochondrial potassium channel modulators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13. Targeting mitochondrial apoptotic machinery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.1. Targeting membrane permeability transition (MPT) to induce apoptosis . . . . . . . . . . . . . . . . . . . . . . . . 13.2. Bcl-2 inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13.3. Smac/DIABLO . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14. Small molecule delivery to mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.1. Delocalized lipophilic cations selectively target the mitochondria of tumor cells . . . . . . . . . . . . . . . . . . . . . 14.2. Lipophilic triphenylphosphonium cations rapidly accumulate within mitochondria and have been utilized to target a wide variety of molecules into the mitochondria of tumor cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.3. Lipophilic cations as tumor selective radiotracers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14.4. Lipophilic triphenylphosphonium cations as mitochondrial drug carriers . . . . . . . . . . . . . . . . . . . . . . . . 15. Mitochondrial targeted anti-cancer photodynamic therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16. Conclusion and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction The mitochondrion is the powerhouse of the cell and serves as the major energy source. The mitochondrion consists of two membranes that separate it from the cytosol. The outer and inner mitochondrial membranes divide the mitochondrion into two compartments, the

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intermembrane space (the space between the outer and inner mitochondrial membranes) and the matrix filled inner compartment. The inner mitochondrial membrane (IMM) consists of several folds known as cristae. The VDAC (voltage dependent anion channel) present on the outer membrane regulates the entry of different cellular components into the mitochondria. All the enzymes for the

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TCA cycle (tricarboxylic acid cycle/Krebs cycle/citric acid cycle) reside in the mitochondrial matrix, whereas the respiratory chain and ATP synthase/ATPase are present on the inner membrane [1]. Besides serving as the cell's primary energy source, mitochondria are implicated in the regulation of programmed cell death (PCD, the intrinsic pathway of apoptosis), reactive oxygen species (ROS) generation, and maintenance of calcium homeostasis [2–4]. Mitochondria play an important role in cell survival and cell death, and dysregulation of any form leads to diseases. Mitochondrial dysfunction has been implicated in neurodegenerative and neuromuscular disorders, ischemia-reperfusion injury, diabetes, obesity, inherited mitochondrial diseases and most importantly, cancer [5–7]. A change in cellular bioenergetics is one of the key hallmarks of cancer. The concept of aerobic glycolysis was introduced by Warburg [8]. Since then research has been carried out with the goal of targeting the ‘sweet tooth’ of cancer [9,10]. Aerobic glycolysis provides cancer cells with several growth advantages. The various advantages include growth of cells in adverse microenvironment, generation of substrates for glycosylation reactions, and supply of precursors for biosynthetic reactions [11,12]. Moreover, metabolism independent functions of the glycolytic enzymes provide additional benefits such as resistance to apoptosis, invasiveness, malignancy and transcriptional regulation [13]. In addition to aerobic glycolysis, the malignant phenotype is characterized by additional metabolic changes, de novo lipid and nucleotide biosynthesis, and glutamine dependent anaplerosis (replenishment of TCA cycle intermediates). These metabolic changes are necessary in order to produce sufficient biomass required for rapid cell proliferation. Various TCA cycle intermediates serve as precursors for the biosynthetic reactions. Citrate derived from the TCA cycle acts as a precursor for fatty acid synthesis. Oxaloacteate and α-ketoglutarate provide nonessential amino acids required for protein and nucleotide synthesis. Moreover, cancer cells have higher uptake of glutamine for replenishing the TCA cycle intermediates through glutaminolysis. The entire metabolic profile of cancer cells is regulated by PI3K/Akt/mTOR(PI3K, phosphoinositide 3-kinase; mTOR, mammalian target of rapamycin), HIF-1α (Hypoxia-inducible factor-1α), and c-Myc. Activation of the PI3K pathway increases glucose and nutrient uptake, and increases the expression of enzymes involved in glycolysis and lipid synthesis. Expression of c-Myc is known to regulate glutamine uptake in cells. c-Myc regulates transcription machinery components and plays an important role in protein synthesis. The activity of c-Myc modulates nucleotide, amino acid, fatty acid and polyamine synthesis. The expression of HIF-1α inhibits translation initiation in addition to induction of glycolysis. HIF-1α inhibits the entry of glycolytic end products in the TCA cycle via its effect on pyruvate dehydrogenase kinases (PDKs). The metabolic changes in the cancer cells complement tumor cell requirements for increased biogenesis, increased energy demand and an adaptive response to tumor microenvironment [11]. We have provided a summary of approaches for targeting the metabolism of cancer cells for therapeutic benefit. The focus of this review is on cancer cell metabolism and mitochondrial targeted anticancer approaches. The purpose of this review is to examine the current knowledge of potential targets in this field. We propose that combination therapies can be advantageous for the treatment of cancer. Moreover, targeting several different functions can potentiate the therapeutic benefits. 2. Rationale for targeting mitochondria Otto Warburg was the first to demonstrate that even in ample supply of oxygen cancer cells rely on glycolysis for energy [8]. According to Warburg the reliance of cancer cells on aerobic glycolysis was due to defects in oxidative phosphorylation (OXPHOS) in cells. However, it has now been proved that the defect in OXPHOS is not the

cause of the Warburg effect. Several tumor types have functional OXPHOS and but rely on aerobic glycolysis due to invasive and adaptive benefits [9,14]. Aerobic glycolysis has been observed in almost all cancers. The bioenergetic switch from a higher energy yielding process (TCA cycle) to a lower energy yielding process (glycolysis) is a hallmark of most cancer cells. The increased glycolytic dependency of cancer cells is attributable to mitochondrial defects and malfunctions, nuclear DNA mutations, abnormal expression of metabolic enzymes (fumarate hydratase and succinate dehydrogenase), adaptation to the tumor microenvironment (HIF-1α), and disruption in oncogenic/tumor suppressor signaling (Ras, Akt, p53, pVHL) (Fig. 1). Many forms of cancer exhibit mitochondrial DNA (mtDNA) mutations (Fig. 2). MtDNA mutations can cause inhibition of OXPHOS, increase ROS generation, and aid in tumor adaptation under adverse conditions [15]. Abnormalities in nuclear DNA can also lead to increased aerobic glycolysis in cancer cells. Mutations in nuclear DNA can give rise to abnormal succinate dehydrogenase (SDH) and fumarate hydratase (FH), as well as overexpression of glycolytic, and pentose phosphate pathway enzymes [16]. Transketolase like enzyme, TKTL1 is a pentose phosphate pathway enzyme which is overexpressed in many human tumors. Overexpression leads to increased production of gylceraldehyde-3-phosphate which is utilized for the energy yielding phase of glycolysis [17]. Two TCA cycle enzymes, SDH and FH act as tumor suppressors. SDH catalyzes succinate to fumarate in the TCA cycle. Fumarate is further hydrolyzed to malate by FH. SDH mutations give rise to hereditary paraganglioma (PGL) and phaeochromocytomas. Impaired FH function is implicated in hereditary leiomyomatosis/renal cell cancer (HLRCC) syndromes [18]. The adaptive response to tumor hypoxia leads to stabilization of HIF-1 and/or HIF-2. HIF activation leads to transcriptional activation of the genes for glucose transporters, glycolytic enzymes, and angiogenic factors. HIF-1 may also be elevated under normoxic conditions in tumor cells (Table 1) [19]. Several factors such as pVHL (von Hippel–Lindau protein) inactivation or Ras, Src, or PI3K/Akt activation can lead to stabilization of HIF-1 under normoxia. Oncogenes can also directly activate glycolysis independent of HIF-1. c-Myc binds the promoters of several glycolytic genes (Hexokinase II, Enolase1 and Lactate dehydrogenaseA), glucose transporters, and activates them even under normoxia. Constitutive activation of Akt increases surface expression of the Glut1 glucose transporter. Akt also maintains the mitochondrial association of hexokinase. This association prevents changes in the permeability of the outer mitochondrial membrane and cytochrome c release upon apoptotic stimulation. Akt phosphorylates BAD (Bcl-2 antagonist of cell death) and inhibits the association of pro-apoptotic BAX (Bcl-2 associated protein X) and BAK (Bcl-2 homologous antagonist/killer) [19,20]. Tumor suppressor p53 affects mitochondrial respiration as well. p53 can transactivate SCO2 (synthesis of cytochrome c oxidase2). SCO2 is required for the assembly of the mitochondrial cytochrome c oxidase complex. Moreover p53 stimulates TIGAR (tp53 induced glycolysis and apoptosis regulator), which decreases fructose-2,6bisphosphate and hence suppresses glycolysis [21]. Recent reports have shown the involvement of mitochondrial uncouplers in aerobic glycolysis. It has been suggested that aerobic glycolysis may represent a shift to the oxidative metabolism of nonglucose carbon sources (fatty acid oxidation). Additionally, mitochondrial uncoupling may be responsible for increased resistance to chemotherapy [22].Thus, designing therapeutic strategies for specifically killing cancer cells by exploiting their metabolic alterations seems to be a promising approach. 3. Glycolytic inhibitors Glycolysis refers to the series of enzymatic reactions which convert a molecule of glucose into lactate with the generation of two

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Fig. 1. Opportunities in treating cancer by targeting cancer energy metabolism and mitochondria. Cancer energy metabolism and mitochondria play a crucial role in tumor development. Increased glycolysis is a hallmark of most cancer cells. Various factors contribute to the phenomenon of the Warburg effect seen in tumors. Oncogenic alterations (PI3K/Akt) and HIF-1 stabilization result in increased expression of glucose transporters and glycolytic enzymes. Moreover, glycolysis aids in increasing the cellular anabolic processes by shunting intermediates to the pentose phosphate pathway. Glutaminolysis serves as a mode of replenishing the TCA cycle intermediates that are used up in fatty acid biosynthesis. Glutaminolysis also promotes nucleotide and protein synthesis in the cells. Glutamine uptake is controlled by c-Myc, via regulating the cell surface expression of AST2 and SN2 glutamine transporters [339]. Besides the bioenergetic changes, the apoptotic machinery of the mitochondria also serves as a remarkable target for anti-cancer therapy. Caspases are the key players of apoptosis. Activation of caspases occurs as a consequence of cytochrome c release from the mitochondria and formation of apoptosome. The proapoptotic Bcl-2 proteins inhibit the formation of apoptosome. XIAP (X-linked inhibitor of apoptosis protein) inhibits the activation of caspases. Agents mimicking Smac, DIABLO, the natural inhibitor of XIAP are being studied for their anticancer potential. Furthermore, oncogenic proteins (Akt) promote the association of hexokinase and VDAC (voltage dependent anion channel) exerting an anti-apoptotic effect on cells.

molecules of ATP. Overexpression of glycolytic genes has been seen in numerous cancer cell lines [16]. Glycolytic inhibitors are being designed that target the enzymes involved in the glycolysis pathway.

Some glycolytic enzymes have functions in addition to their enzymatic roles. Hexokinase, glyceraldehydes-3-phosphate dehydrogenase (GAPDH), enolase and LDH are known to function in transcriptional regulation. Hexokinase and GAPDH have also been implicated in the regulation of apoptosis. Glucose-6-phosphate isomerase plays a crucial role in cellular motility and invasion [13]. Therefore inhibiting glycolytic enzymes can provide multifold benefit in cancer therapy. Several glycolytic inhibitors (Fig. 3) are in various stages of preclinical and clinical studies. Table 1 provides a brief overview of glycolytic enzymes implicated in various types of cancer, their inhibitors and the clinical status of these inhibitors. 3.1. Hexokinase (HK)

Fig. 2. Rationale for targeting mitochondria for anticancer therapy. Various factors affect mitochondrial function, and make it an exciting target for anticancer therapy. Mitochondrial DNA mutations result in inhibition of oxidative phosphorylation, increased ROS, and help cancer cells to adapt to adverse conditions. Nuclear DNA mutations can give rise to malfunctional succinate dehydrogenase and fumarate hydratase resulting in PGL (hereditary paraganglioma) and HLRCC (hereditary leiomyomatosis/ renal cell cancer). Moreover, there is an increased expression of glycolytic enzymes as a consequence of nuclear DNA mutations. Additionally, HIF-1 promotes the expression the glycolytic enzymes leading to Warburg effect. Oncogenic and tumor suppressor proteins also function to cause alterations in cellular bioenergetics. Lastly, dysregulation of mitochondrial machinery of apoptosis aids in tumor progression.

Hexokinase catalyzes the first step of glycolysis, phosphorylating hexose (e.g. glucose) to hexose-6-phosphate. There are four tissue specific hexokinase isozymes (I–IV). Hexokinase is physically associated with the outer surface of external membrane of mitochondria through specific binding to VDAC. Akt signaling promotes this association between VDAC and hexokinase. Mitochondrial hexokinase is known to be upregulated in malignant tumor cells. Hexokinase is also known to influence PCD. It can inhibit PCD in cancer cells by affecting the pro- and anti-apoptotic Bcl-2 family proteins. Akt signaling activates mitochondrial hexokinase, which inhibits cytochrome c release and thus apoptosis. Hexokinase inhibition affects cellular metabolism as well as cell survival [23,24]. Several hexokinase inhibitors have been designed for cancer therapy. 3.1.1. 2-Deoxyglucose (2-DG) Hexokinase phosphorylates 2-DG to 2-DG phosphate, so that it cannot participate in the downstream glycolytic steps and in effect

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Table 1 Expression of metabolic enzymes in cancer and their inhibitors.a Target

Status in cancer

Inhibitor

Clinical status of inhibitor

References

Hexokinase

Colorectal cancer, hepatocellular carcinoma, breast cancer

2-Deoxyglucose

Clinical trials terminated

[29,30,37–39, 267–270]

3-Bromopyruvate Lonidamine

Showed efficacy in animal studies Clinical trials terminated in US. Approved in Europe for use. Tested in animals Tested in mitochondrial fractions from cancer cell lines Experimental drugs

[41,267]

Tested in animals

[44,271]

Under development

[271–273]

Antiprotozoal agents

[41,272,274]

Experimental inhibitor Tested in animal models Under development

[41,267,274–278]

Experimental inhibitors

[41,49,272]

Effective against breast cancer cells Experimental inhibitors

[41,267,271,272]

Experimental inhibitors

[51,267,271]

Mannoheptulose Methyl jasmonate Glucose-6-phosphate isomerase

Colorectal cancer

Phospho-fructokinase

HLRCCb uterine tumor

D-fructose-6-phosphate,

b

Aldolase Triosephosphate isomerase

Glyceraldehyde-3-phosphate dehydrogenase Phosphoglycerate kinase

Phosphoglycerate mutase

HLRCC uterine tumor, lung squamous carcinoma Lung squamous carcinoma, pancreatic cancer Colorectal cancer, prostate cancer, pancreatic cancer Colorectal cancer, HLRCCb uterine tumor, pancreatic ductal adenocarcinoma Lung squamous carcinoma

b

Enolase

Colorectal cancer, HLRCC uterine tumor, lung squamous carcinoma

Pyruvate kinase

Colorectal cancer, HLRCCb uterine tumor

Lactate dehydrogenase Pyruvate dehydrogenase kinase

HLRCCb, B cell non-Hodgkin lymphoma Head and neck squamous cancer

Transketolase like enzyme

Uterine, cervix cancer, non small cell lung carcinoma, breast cancer

a b

6-phospho-gluconic acid, N-bromoacetyl aminoethyl phosphate 3-(3-pyridinyl)-1-(4-pyridinyl)-2propen-1-one (3-PO) 3-Fluoro-D-glucose/3-deoxy-D-glucose, 4-deoxy-D-glucose/4-fluoro-D-glucose 2-Carboxyethylphsophonic acid, N-hydroxy-4-phosphono-butanamide, 2-phospho-glyceric acid Iodoacetate Koningic acid 1,3-bisphosphoglyceric acid analogs

Benzene hexacarboxylic acid, 3-phosphoglyceric acid MJE3 Sodium fluoride, phosphonoacetohydoxamic acid, 2-phospho-D-glyceric acid Fluorophosphates, pyridoxal-5'-phosphate, creatine phosphate, L-phospholactate CAP-232/TLN-232 Oxalate, oxamate Dicholoroacetate

Oxythiamine

Completed Phase II clinical trials Experimental inhibitors Phase I trials for metastatic solid tumor; Phase II trials for brain cancer Tested in animals

[41,267,271,279]

[149,280,281] [131,282]

[52,283–285]

Consult reference [16] for a more detailed overview on the expression of glycolytic enzymes in cancer. HLRCC, hereditary leiomyomatosis/renal cell cancer.

competitively inhibits the enzyme. 2-DG decreases the mitochondrial association of hexokinase [25]. Additionally, 2-DG causes abnormal GlcNAcylation of proteins leading to accumulation of misfolded proteins and endoplasmic reticulum stress response [26]. Studies have shown that 2-DG enhances the anticancer activity of drugs like

adriamycin and paclitaxel [27]. 2-DG exhibits synergistic effects with HDAC (histone deacetylase) inhibitors [28]. Phase I clinical trials have been conducted by Threshold Pharmaceuticals to evaluate the safety and efficacy of 2-DG for the treatment of solid tumors. The aim of the trial was to calculate the MTD

Fig. 3. Potential glycolytic inhibitors for anti-cancer therapies. Several glycolytic inhibitors are being evaluated for their anticancer effects.

D. Pathania et al. / Advanced Drug Delivery Reviews 61 (2009) 1250–1275

(maximum tolerated dose) for 2-DG, and to determine the pharmacokinetic parameters of 2-DG and 2-DG/docetaxel combination. The trial was conducted on thirty-four patients having solid tumors that relapsed after chemotherapy. The 2-DG/docetaxel combination showed no pharmacokinetic interactions. Anti-tumor activity was reported for breast cancer, and head and neck tumors. However, further clinical trials with 2-DG have been terminated by Threshold pharmaceuticals [29]. 5-Thioglucose and Mannoheptulose are other examples of glucose analogs that competitively inhibit hexokinase, and also affect the glucose uptake in cells [30,31]. 3.1.2. 3-Bromopyruvate (3-BrP) 3-Bromopyruvate is a synthetic bromo-derivative of pyruvic acid that inhibits hexokinase by alkylation of the sulfhydryl groups of hexokinase [23]. The strong alkylating capacity of 3-BrP brings its selectively towards hexokinase into question [32]. 3-BrP in combination with mTOR inhibitors displays synergistic effects on leukemia and lymphoma cells [33]. 3.1.3. Lonidamine (TH-070) This derivative of indazole-3-carboxylic acid inhibits glycolysis in relatively hypoxic conditions [34]. Lonidamine increases the intracellular content of doxorubicin due to reduced ATP availability [35]. In combination, lonidamine enhances the cytotoxicity of alkylating agents such as cisplatin, melphalan, and BCNU (bis-chloronitrosourea) [36]. Lonidamine has been approved for use in Europe for cancer therapy (450–900 mg daily in three divided doses) [37]. However, phase III clinical trials for lonidamine have been terminated in the U.S. Phase III trials were being carried out by Threshold Pharmaceuticals to investigate the safety and efficacy of lonidamine in treating benign prostatic hyperplasia [38]. 3.1.4. Methyl jasmonate Methyl jasmonate binds to mitochondrial hexokinase and detaches it from VDAC. This causes hexokinase to dissociate from the mitochondria with a concomitant release of cytochrome c. These effects of methyl jasmonate have been validated in mitochondrial fractions isolated from murine melanoma B16, murine colon carcinoma CT26, murine B cell leukemia BCL1, and human T lymphoblastic leukemia Molt-4 cells. It has been proposed that hydrophobic jasmonates interfere with the hydrophobic interactions between hexokinase and VDAC leading to the disruption of the mitochondrial hexokinase-VDAC interaction [39]. 3.2. Glucose-6-phosphate isomerase (GPI) The second enzyme of glycolytic pathway catalyzes the conversion of glucose-6-phosphate into fructose-6-phosphate. GPI promotes proliferation and motility of cancer cells in an autocrine manner, and is also known as autocrine motility factor (AMF). Moreover, it plays a role in invasiveness of tumors by inducing matrix metalloproteinase-3 (MMP-3) [40]. Chemical compounds such as D-fructose-6-phosphate, 6-phosphogluconic acid, and N-bromoacetylethanolamine phosphate are being developed as GPI inhibitors [41].

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sion of these enzymes lead to a higher abundance of fructose-1,6bisphosphate which is shunted into the pentose phosphate pathway for ribose 5 phosphate synthesis [43]. 3PO [3-(3-pyridinyl)-1-(4pyridinyl)-2-propen-1-one] is a small molecule that has shown inhibitory activity against PFKFB3 and thus influences the activity of PFK1 [44]. 3.4. Aldolase Fructose-1,6-bisphosphate is converted into dihydroxy acetone phosphate (DHAP) and glyceraldehyde-3-phosphate by an aldolase catalyzed reaction. There are three isozymes of aldolase, A–C. Small molecules such as 3-deoxy or 3-fluoro-D-glucose and 4-deoxy or 4fluoro-D-glucose are being investigated for their inhibitory potential against this enzyme [41]. 3.5. Triosephosphate isomerase (TPI) This enzyme catalyzes the reversible isomeric conversion of DHAP and glyceraldehyde-3-phosphate. TPI inhibitors are being developed as antiprotozoal drugs. Examples of TPI inhibitors are 2-carboxyethylphosphonic acid, N-hydroxy-4-phosphono-butanamide, 2-phosphoglyceric acid, and ornidazole [41,45]. 3.6. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) GAPDH converts glyceraldehydes-3-phosphate to 1,3-bisphosphoglycerate with a simultaneous reduction of NAD+ to NADH (nicotinamide adenine dinucleotide; oxidized and reduced respectively). There are several GAPDH inhibitors currently under development. 3.6.1. Iodoacetate Iodoacetate reacts with cysteine sulfhydryl groups in the active site of GAPDH [46]. Being a strong alkylating agent, the selectivity of iodoacetate towards GAPDH is debatable. 3.6.2. Koningic acid Koningic acid is a sesquiterpene antibiotic that binds to the sulfhydryl group within the active site of GAPDH [47]. α-Chlorohydrin, ornidazole and 1-chloro-3-hydroxypropanone have also shown anti-GAPDH activity [32]. 3.7. Phosphoglycerate kinase (PGK) PGK catalyzes the first ATP yielding reaction in glycolysis, transferring a phosphate group from 1,3-bisphosphoglycerate onto ADP to generate ATP and 3-phosphoglycerate. Two isozymes of PGK, PGK1 and PGK2 have been identified. PGK has been reported to show disulfide reductase activity. It is secreted by tumor cells and it aids in angiogenic processes. PGK targets the disulfide bonds in plasmin, and initiates the proteolytic release of angiostatin (angiogenesis inhibitor) [48]. Bisphosphonate analogs of 1,3-bisphosphoglyceric acid have been designed and studied as PGK inhibitors [41]. 3.8. Phosphoglycerate mutase (PGM)

3.3. Phosphofructokinase (PFK) The phosphorylation of fructose-6-phosphate to fructose-1,6bisphosphate is catalyzed by PFK1. It is present in three tissue specific isozymes (liver L, muscle M, and platelet P). The activity of PFK1 is an important point of regulation in glycolysis. PFKFB3 (6-phosphofructo2-kinase/fructose-2,6-bisphosphatase 3) is the key enzyme regulating the activity of PFK1. PFKFB3 regulates the synthesis/degradation of fructose-2,6-bisphosphate, the main positive allosteric modulator of PFK1 [42]. PFK1 and PFKFB3 expression has also been implicated in the biosynthetic changes observed in tumor cells. The overexpres-

The internal transfer of phosphate from C-3 to C-2, to generate 2phosphoglycerate from 3-phosphoglycerate is catalyzed by PGM. Benzene hexacarboxylic acid and 3-phosphoglyceric acid are being developed as PGM inhibitors [41]. MJE3 is another small molecule that is under development as a PGM inhibitor [49]. 3.9. Enolase Enolase catalyzes the reversible dehydration of 2-phosphoglycerate to phosphoenol pyruvate. There are several tissue specific

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isozymatic forms of this enzyme. Sodium fluoride, phosphonoacetohydroxamic acid, and 2-phospho-d-glyceric acid are known to inhibit the action of enolase [41]. 3.10. Pyruvate kinase (PK) PK catalyzes the second ATP generating step in glycolysis. PK facilitates the reversible transfer of a phosphate group from phosphoenol pyruvate to ADP, giving ATP and pyruvate as products. There are several isozymes of pyruvate kinase. Tumor cells express the M2PK form of this enzyme. Fluorophosphates, pyridoxal 5′-phosphate, creatine phosphate, oxalate, and L-phospholactate inhibit PK [41]. There are also phosphoenolpyruvate (PEP) analogs with modified phosphate and carboxylate groups being designed to inhibit PK [50]. CAP-232/TLN-232 is a synthetic cyclic heptapeptide which targets PK. A phase II clinical trial in patients with refractory metastatic renal cell carcinoma has been successfully completed for CAP-232. Currently, phase II trials for studying the efficacy of TLN-232 in patients with recurring metastatic melanoma are being conducted [51]. 3.11. Lactate dehydrogenase (LDH) LDH catalyzes the conversion of pyruvate to lactate. A molecule of NADH is oxidized to NAD+ in the course of this reaction. Inhibitors for LDH are being developed as antiprotozoal agents. Oxalate is known to inhibit LDH activity [32]. 3.12. Transketolase like enzyme (TKTL1) TKTL1 is a pentose phosphate pathway enzyme overexpressed in many cancers. Overexpression increases the activity of the pentose phosphate pathway, leading to augmented production of glyceraldehde-3-phosphate. Oxythiamine is a potent inhibitor of TKTL1 [52]. 4. Role of HIF-1α in promoting the Warburg effect HIF-1 is a transcription factor that regulates transcription of several genes involved in cancer metabolism (Table 2). HIF-1 activates gene for glucose metabolism, cell survival/proliferation, angiogenesis, and metastases [53,54]. HIF-1 is a heterodimer of HIF-1α and HIF-1β. The HIF-1β subunit of the heterodimer is constitutively expressed in

cells [55]. HIF-1β can also dimerize with HIF-2α and regulate gene activation [56,57]. Another HIF protein, HIF-3α acts as an inhibitor of HIF-1α [58]. The expression of HIF-1α is regulated via oxygen independent and dependent mechanisms. The protein synthesis of HIF-1α is under the control of the PI3K and ERK-MAPK (ERK, extracellular signal regulated kinase; MAPK, mitogen activated protein kinase) signaling pathways. These pathways are activated via signaling of G-protein coupled receptors, receptor tyrosine kinases, and non-receptor tyrosine kinases [59]. Degradation of HIF-1α is mediated via oxygen dependent mechanisms. Proline hydroxylation of Pro 402 and 564 of HIF-1α by PHD 1-3 (prolyl hydroxylase) targets it to the pVHL tumor suppressor. pVHL acts as recognition marker for E3 ubiquitin protein ligase and thus leads to proteosomal degradation of HIF-1α. PHDs utilize oxygen and α-ketoglutarate, and generate prolyl hydroxylated HIF-1α and succinate. Increased levels of succinate in cells promote HIF-1α stabilization [60]. Acetylation can also regulate the levels of HIF-1α. Lysine-532 of HIF-1α is acetylated by ARD1 (arrest defective1 protein) acetyltransferase. Acetylated HIF-1α interacts with pVHL, which causes its ubiquitination and proteosomal degradation [61]. Furthermore hydroxylation of asparagine-803 by FIH-1 (factor inhibiting HIF-1) inhibits the interaction of HIF-1α with its co-activators p300 and CBP (CREB binding protein) [62]. HIF-1 can also be regulated by gain of function of oncogenes (PI3K etc), and loss of function of tumor suppressors (pVHL). HIF-1α activation has been shown to play an important role in altering the metabolism of cancer cells. It causes increased glycolysis and decreased mitochondrial function in tumors by regulating several genes involved in these processes (Table 2). Several anticancer agents that affect activity or levels of HIF-1α in cells influence HIF-1 without directly targeting it. The agents that target the HIF-1α regulatory systems include inhibitors of PI3K/Akt/mTOR and ERK-MAPK pathways, Hsp90 complexes (Heat shock protein90 complexes), thioredoxin-1, topoisomerase-1, and molecules that cause disruption of microtubules. Recently interest has developed in designing inhibitors that target the interactions of HIF-1 with DNA or its coactivators [63]. Moreover, high throughput screens are being conducted to find small molecule inhibitors of HIF-1α. A list of these compounds is mentioned in Table 3. These agents target HIF-1α and HIF-1β protein–protein interactions, the HIF-1/coactivator interaction or the DNA binding ability of HIF-1 (Table 3).

Table 2 Role of HIF-1 in cancer energy metabolism. Effect of HIF-1 on metabolism

HIF-1 target gene product

Role in metabolism

Ref.

Increase in glycolysis

Glucose transporters Glut1 and Glut3 Hexokinase 2 Phosphoglucose isomerase (autocrine motility factor) Phosphofructokinase1 Aldolase

Glucose uptake in cells Phosphorylation of glucose Conversion of glucose-6-phosphate to fructose-6-phosphate; motility of cancer cells Converts fructose-6-phosphate into fructose-1,6-bisphosphate Catalyses conversion of fructose-1,6-bisphosphate to dihydroxy acetone phosphate (DHAP) and glyceraldehydes-3-phosphate Isomeric conversion of DHAP and glyceraldehydes-3-phosphate Converts glyceraldehydes-3-phosphate into 1,3-bisphosphoglycerate Transfers a phosphate group from 1,3-bisphosphoglycerate onto ADP and generates ATP and 3-phosphoglucerate Converts 3-phophoglycerate into 2-phosphoglcerate Causes dehydration of 2-phosphoglcerate into phosphoenol pyruvate Leads to reversible transfer of phosphate from phosphoenol pyruvate to ADP and produces ATP and pyruvate Reversible conversion of fructose-6-phosphate and fructose-2,6 bisphosphate Conversion of pyruvate to lactate Removal of lactate from the cell Decreases the entry of pyruvate into TCA CYCLE Decrease mitochondrial activity Increased oxygen consumption in hypoxia Increased oxygen consumption in hypoxia

[286–288] [289] [290]

Triosephosphate isomerase Glyceraldehde-3-phosphate dehydrogenase Phosphoglycerate kinase Phosphoglycerate mutase Enolase Pyruvate kinase

Decrease rate of TCA cycle and oxidative phosphorylation

6-phosphofructo-2-kinase/fructose-2,6bisphosphatase 1-4 Lactate dehydrogenase A Monocarboxylate transporter4 Pyruvate dehydrogenase kinase 1 Max interactor 1 Cytochrome oxidase isoform2 (COX4/2) LON protease

[54,288] [54,288] [291] [292] [54,288] [54] [54,288] [293] [294] [54,288,295] [296] [297,298] [299] [300] [300]

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Table 3 Table 3 HIF-1 pathway inhibitors. Mechanism

Inhibitors

Ref.

Inhibit HIF-1 binding to DNA Inhibit HIF-1 mediated transactivation by binding to p300 Antisense that reduces the expression of HIF-1α mRNA and protein Prevents p300 binding to CAD of HIF-1α by promoting FIH binding to CAD Inhibits deubiquitination of HIF-1α and increase its polyubiquitination; inhibits HIF-1α translation Reduces HIF-1α protein synthesis Inhibition of HIF-1α transcriptional activity

Echinomycin/NSC-13502, synthetic polyamides Chetomin RX0047 YC-1 PX-478 103D5R DX-52-1/NSC-607097 NSC-609699,NSC-639174, NSC-606985 NSC-134754,NSC-643735 NSC-50352

[301,302] [303] [304] [305] [306] [307] [308]

Inhibit HIF-1α activity Inhibit PAS-A mediated interaction of HIF-1α and HIF-1β

5. Mitochondrial bioenergetics 5.1. The Citric Acid Cycle is the entry point for aerobic respiration In non-transformed cells, the three-step process of aerobic respiration takes places in the mitochondria beginning with the citric acid cycle. TCA cycle is a series of oxidation and reduction reactions that results in the transfer of hydride ions (2 electrons) from carbon precursors to NAD+ and FAD (flavin adenine dinucleotide, oxidized) resulting in the formation of FADH2 (flavin adenine dinucleotide, reduced), 3 NADH, 2 molecules of carbon dioxide and the regeneration of intermediates to complete the cycle. The NADH and FADH2 produced in TCA cycle stores energy as electron motive force. These electron carriers are shunted to the electron transport chain where they interact with complexes I and II respectively. The unconstrained replicative potential of transformed cells requires the constant production of large amounts of biomaterial in the form of nucleotides and fatty acids and sufficient energy to drive the biosynthetic reaction. Normal metabolic processes simply cannot meet the increased biosynthetic and energetic demands of DNA replication and membrane formation and therefore, co-option of metabolic intermediates is often observed in highly proliferative cancers. Citrate formed by the condensation of acetyl-CoA and oxaloacetate is shunted from TCA cycle into the cytoplasm where it is used in the formation of fatty acids and NADPH (nicotinamide adenine dinucleotide phosphate, reduced) via conversion to lactate. To compensate for the loss of this TCA cycle intermediate, cancer cells consume excess glutamine. Once in the mitochondria, glutamine is converted to the TCA cycle intermediate α-ketoglutarate allowing for the regeneration of oxaloacetate and the continued production of citrate as a precursor for downstream TCA cycle intermediates as well as lipogenesis. Under these circumstances of metabolic re-organization, the citric acid cycle becomes a potent source of citrate for the biosynthetic pathways relating to growth, replication and energy production (Fig. 4) [11].

[309] [310]

and the cytosol. The final step of aerobic respiration utilizes the proton gradient established by the presence of hydrogen ions in the intermembrane space to power the phosphorylation of ADP to ATP by complex IV (Fig. 5). Aerobic respiration is a slower process than glycolysis, but yields higher amounts of ATP. A complete cycle of respiration can generate ~ 30 molecules of ATP from one glucose molecule. 5.3. Mechanisms of respiratory control In normal cells the rate of energy production via oxidative phosphorylation is tightly controlled such that energy production and demand are balanced. OXPHOS is coupled to the consumption of ATP and this is the primary means of respiratory control. Excess ATP and intermediary reaction substrates interact with the matrix exposed portion of Complex IV and allosterically inhibit enzyme function to slow the rate of respiration [64]. Three additional mechanisms of control include allosteric inhibition, tissue specific expression of isozymes and regulation by cell signaling. Allosteric controls have been reported mainly in Complex IV. ATP binds a pocket formed by residues of subunit I, II and IV, the matrix exposed portion of Complex IV, to decrease ADP phosphorylation. In vitro studies have demonstrated that members of the OXPHOS machinery can be phosphorylated [64]. In some cases in vivo studies have validated these results but the effect of these phosphorylations on function remains to be fully understood. Multiple cell signaling pathways have been

5.2. Enzymes of the Electron Transport Chain (ETC) create proton motive force needed to produce ATP The electron motive force stored as NADH and FADH2 is converted to a proton motive force by the enzymes of the electron transport chain in the process of OXPHOS. Complexes I and II are the entry point of electrons into the citric acid cycle. Complex I oxidizes NADH and complex II oxidizes FADH2 to NAD+ and FAD respectively. The electrons liberated by oxidation are transferred to ubiquinol and carried to complex III. Complex III functions to move electrons across the inner membrane, where they are transferred to cytochrome c. Electrons are then carried to Complex IV where they are used to reduce oxygen to water. Each complex harnesses the energy of electron movement to pump hydrogen ions into the intermembrane space. The process of electron transport also generates low levels of ROS, primarily superoxide O− 2 which can readily diffuse into the matrix

Fig. 4. The reliance of cancer cells on TCA intermediates for biosynthetic materials makes mitochondria an attractive target for anti-cancer therapies. In order to meet the increased biosynthetic demands of rapid proliferation, the TCA cycle intermediate, citrate is exported from the mitochondria into the cytosol. Once in the cytosol, it is utilized in the production of energy and lipids. Cancer cells consume excess glutamine to compensate for the loss of citrate in TCA. Glutamine is converted to glutamate in the mitochondria where it enters the TCA in the form of α-ketoglutarate.

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Fig. 5. Schema depicting electron transport and oxidative phosphorylation. Electrons enter the ETC at complexes I and II in the form of NADH and FADH2 respectively. In the process, complexes I, III and IV pump protons across the inner mitochondrial membrane. Accumulation of protons in the intermembrane space creates a charge gradient that is utilized by Complex V (ATP synthase) to drive the synthesis of ATP from ADP and Pi. Specific inhibitors of the OXPHOS machinery are shown in bold.

implicated in the regulation of mitochondrial respiration such as the PI3K/Akt and p38 MAPK pathways as well as Src and EGFR (epidermal growth factor receptor) mediated signaling pathways [65–68]. These same kinases that have been implicated in control of mitochondrial respiration are commonly upregulated in many cancers [69,70]. The relationship between mitochondrial respiratory control and increased expression of pro-oncogenic kinases has not been fully investigated. 5.4. Targeting OXPHOS enzymes to induce mitochondrial bioenergetic failure Several lines of evidence suggest that selective inhibition of tumor cell OXPHOS may be a rational approach for the treatment of cancers (Table 4). The reliance of cancer cells on glycolysis for the production of energy has been attributed to mitochondrial dysfunction and the resultant ROS species. OXPHOS components are the major source of ROS in cells [71]. ROS have been implicated in HIF1α stabilization, DNA damage and disturbances in cellular redox status [72–74]. The instability of dysregulated OXPHOS components may impart a greater sensitivity to antagonists in tumors compared to normal cells and represents one rationale for targeting these metabolic enzymes. Cellular respiration via glycolysis is observed in early embryonic tissue and in stem cells which normally reside in hypoxic niches [75,76]. De-differentiation to a stem-cell like phenotype of self renewal and resistance to apoptosis is observed in hypoxic cancers cells derived from breast, brain and neural origins [77–80]. Hypoxic stabilization allows HIF 1α signaling to maintain the undifferentiated state [81]. Inhibition of respiratory complexes, presumably via inhibition of oxygen sensing mechanisms has been shown to prevent HIF1α stabilization. Increased degradation of HIF1α via inhibition of respiratory chain components could contribute to the restoration of the differentiated phenotype. The metabolic co-option of citrate for the formation of NADPH and fatty acid synthesis compensated by production of α-ketoglutarate via the breakdown of glutamine by mitochondrial glutaminases suggests that this pathway is a mechanism critical to fulfilling the biosynthetic

needs of transformed cells. Evidence in Saccharomyces cerevisiae demonstrated that dysfunction in components of the electron transport system results in decreased expression of components of both the electron transport chain and the citric acid cycle [82]. Although this effect has not been studied extensively in humans it suggests that targeting mitochondrial components of the OXPHOS machinery could be beneficial on several levels, including the interruption of metabolic processes required for cancer cell proliferation (Table 4). 5.4.1. Targeting Complex I, mitochondrial NADH ubiquinone reductase Complex I, NADH ubiquinone reductase, is the largest and most complex enzyme of the electron transport chain and the main entry point of electrons into the electron transport chain. Seven of the fourteen units essential for function are encoded in the mtDNA. The remainder of the forty-six subunits are encoded in nuclear DNA [83]. Targeted inhibition of Complex I has the potential to significantly curtail the flow of electrons through the transport chain to the final electron acceptor oxygen, resulting in bioenergetic depletion. Complex I is embedded in the inner mitochondrial membrane and in the process of electron transfer to ubiquinol, protons are pumped into the intermembrane space while ROS species diffuse into the matrix. Human cell lines derived from patients carrying inherent deficiencies in Complex I activity and number produced higher levels of ROS and exhibit differences in mitochondrial morphology compared to normal matched cells [84]. Prolonged inhibition of complex I in human skin fibroblasts results in increased ROS production accompanied by changes in mitochondrial morphology [85]. Based on these results and that of other studies it can be assumed that pharmacologic inhibition of Complex I would lead to inhibition of OXPHOS, increased ROS production and mitochondrial mediated apoptosis. A wide array of compounds ranging from pesticides and rodent poisons to antibiotics and antihistamines, have been shown to target Complex I [86] but most lack acceptable therapeutic profiles or reasonable specificity to deem them suitable for development as drugs. Initial functional studies of the respiratory complexes were facilitated by the discovery of potent and selective inhibitors. Complex

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Table 4 Drugs targeting components of the mitochondrial respiration machinery. Target

Compound

Complex I

Acetogenins

Activity

Cytotoxic in mammary adenocarcinoma, ovarian and hepatocellular carcinomas, activity not significant in mice, toxicity observed Δ-lac acetogenins No activity currently reported Rhein Induces apoptosis in multiple cancer cell lines. Diphenyliodium Induces apoptosis in prostate cancer cell lines TEMPOL Chemopreventive in mouse models; antiproliferative activity in mammalian cell lines. Potentiates effect of temezolimide in human glioblastoma cell lines MP-6, MP-24 No in vivo or cell based activity currently reported NSC analogs Anti-leukemic activity in cell lines Complex II Malonate Derivatives under investigation as clinical imaging agents α-tocopheryl succinate Induces apoptosis in cancer cell lines inhibits tumor growth in animal models Complex III Stigmatellin Antibacterial agent Benzylisothiocyanate Found in cruciferous plants, chemopreventive in animal models, induces ROS, cell cycle arrest and apoptosis in cancer cell lines Myxothiozol Antibiotic; reversibly blocks cell cycle in Jurkat cells. Antimycin A Antifungal, antibiotic; induces apoptosis in cancer cell lines Complex IV A2E Endogenously produced; induces apoptosis in retinal pigment cells Ditercalinium Antibacterial agent used in commercial preps, anti-tumor intercalating agent F0F1 ATPase AA1 Prevented the growth of human bladder cancer xenografts in nude mice. MKT-077 Completed Phase II clinical trials, withdrawn due to nephrotoxicity. Rhodamine 123 Completed phase I clinical trials for treatment of prostate cancer, showed reasonable efficacy, low toxicity. Oligomycin Shows potent cytotoxicity in NCI60 screen. Apoptolidin Shows potent cytotoxicity in NCI60 screen. Resveratrol Potent antioxidant, displays both chemopreventive and chemotherapeutic properties in cancer cell lines and animal xenograft models

a

Drug-like propertiesa

Ref.

High logP, high number of rotatable bonds High number of rotatable bonds Reasonable drug properties Lacks HBD or HBA No rotatable bonds

[311–313]

Lacks HBD or HBA Reasonable drug properties Reasonable drug properties Reasonable drug properties

[314] [315–317] [318] [319–322]

[323] [106,324] [325] [326] [114,327–329]

Reasonable drug properties Reasonable drug properties

[330] [331,332] [116,333] May have reasonable drug properties [334,335] Reasonable drug properties [336] Reasonable drug properties [123] Reasonable drug properties [124] High logP, high HBA High logP, high HBA Naturally occurring antioxidant

[337] [337] [338]

Drug-like properties refer to adherence to Lipinski's Rule of Five, a rule of thumb measure for predicting druglikeness. HBA-hydrogen bond acceptor, HBD-hydrogen bond donor.

I function was deciphered based on the inhibitory action of rotenone, a non-competitive binder. Rotenone itself exhibits anti-tumor activity in animal models [86]. Complex I antagonists share a number of common properties and thus are classified accordingly. Complex I inhibitors are often grouped (Fig. 6) as class I or II, depending on their ability to bind in a partially competitive or non-competitive manner, respectively. Properties such as kinetics, ubiquinone or semiubiquinone antagonists, and the ability to induce or repress ROS production have also been considered when classifying complex I inhibitors [87–89]. The acetogenins, such as rollinistatin and bullatacin are potent Complex I inhibitors that have shown anti-tumor properties. The terminal α and β unsaturated γ-methylbutyrolactone imparts potency at the target site but shows high reactivity with non-target proteins [90]. Δlac-acetogenins lacking this strong electrophilic moiety are selective but act on different sites within complex I than previously described acetogenins. The sensitivity to Δlac-acetogenins amongst cancer cell lines varies considerably [91]. Rhein and diphenyliodonium act as competitive inhibitors [92]. Rhein acts to inhibit Complex I by blocking electron input. This is in contrast to most known complex I antagonists which function at or near the site of ubiquinone reduction [93]. Diphenyliodonium increases ROS production resulting cell death via overproduction of superoxide ion [94]. Complete inhibition of complex I requires binding at two regions, the hydrophobic site and the hydrophilic site. As their names imply, the hydrophilic is accessible to hydrophilic antagonists while hydrophobic residues protect the hydrophobic site [95]. Oddly, despite the differences in the physical properties of both binding sites there does not seem to be much selectivity amongst the majority of inhibitors, with the exception of the propylpyridium derivatives, MP-6 and MP24 which target the hydrophilic site preferentially [96,97]. Self organizing map analysis of compounds from the NCI (National Cancer Institute) public database of anti-tumor drug screening data revealed a series of five compounds selective for Complex I. The three most potent compounds contained pyrimadinediamine head groups separated by alkyl linkers 10–12 carbons in length and showed antileukemic activity when tested in cell models [98].

The prodrug TEMPOL, a piperidine nitroxide, is reduced by ubiquinol within mitochondria. The resulting hydroxylamine inhibits Complex I and decreases glutathione levels, mitochondrial membrane potential and oxygen consumption and induces apoptosis in cancer cells [99]. Interestingly, addition of triphenylphosphonium as a targeting moiety increased mitochondrial accumulation at the expense of Complex I specificity. MitoTEMPOL has higher mitochondrial membrane affinity and thus interacts preferentially with Co-enzyme Q [100]. Caution must be taken in targeting Complex I inhibitors to the intended site of action (i.e. tumors). Inhibition of complex I as an offtarget affect has been associated with serious drug related toxicities, as illustrated by MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) and flutatminde. MPP+(1-methyl-4-phenylpyridinium cation), the cytotoxic metabolite of MPTP, a contaminant in narcotic street drugs induces parkinsonian type symptoms as a result of ROS-induced dopaminergic neuron cell death. Use of the antiandrogen flutamide for the treatment of prostate cancer, in rare instances has serious toxicity. Inhibition of Complex I by flutamide causes decreases in ATP and glutathione depletion and oxidation leading to hepatitis. Targeted drug delivery to cancer cells is a means by which untoward effects could be avoided. Strategies and methods of rational drug development using moieties such as triphenylphosphonium cations or delocalized lipophilic cations could be employed to combat potentially harmful off-target effects associated with systemic inhibition of complex I by exploiting the higher mitochondrial membrane potentials observed in tumors. 5.4.2. Succinate dehydrogenase links TCA cycle and OXPHOS Succinate dehydrogenase (Complex II) is the only complex of the ETC that is fully encoded in the nuclear DNA. It is not subject to alterations imparted by mutations in mtDNA and is rarely altered in non-germline cancers [101]. Complex II functions in both TCA cycle to catalyze the oxidation of succinate to fumarate, and in OXPHOS by transferring the liberated electrons stored as FADH2 into the electron transport chain. Complex II is located on the IMM. Unlike the other complexes of the ETC, Complex II does not pump protons across the IMM and therefore does not contribute to mitochondrial membrane potential (Fig. 5). Post-translational modifications such as phosphorylation,

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Fig. 6. Structures of Complex I inhibitors.

N-terminal acetylation and methionine oxidation have been observed. The functional consequences of these modifications are still uncertain [102]. Inhibitors of complex II can be classified according to their mechanisms of action. Inhibitors such as antimycin, funiculosin and the hydroxyquinoline-N-oxides block the QD site. Other inhibitors block the transfer of electrons through the QP site. Examples include the 1,2 hydroxy-1-4, benzoquinone derivatives and monoamine oxidase inhibitors. Complex II dysfunction is correlated with pseudohypoxia, enhanced glycolysis, and resistance to apoptosis in patients [103,104]. Inhibition of Complex II with thenoyltrifluoroacetone slows cell cycle progression and increases ROS production and glutathione oxidation [105]. In neuroblastoma cells inhibition of Complex II leads to increased ROS production, p38 MAPK activation and BAX mediated apoptosis [106]. Compounds such as the benzoquinone derivatives, monoamine oxidase inhibitors and Vitamin E analogs contain moieties that resemble ubiquinone and function to inhibit Complex II via competitive binding to the ubiquinone sites. Ether, ester and amide linked redox-silent Vitamin E analogs such as α-tocopheryl succinate (αTOS) (Fig. 7) show potential as anti-cancer agents. αTOS has been shown to inhibit tumor cell growth

and induce apoptosis in animal xenograft models. αTOS competes with ubiquinone in binding to Q sites on Complex II [107]. Displacement of ubiquinone from the distal and proximal Q sites allows the escape of electrons. Reduction of oxygen by these free electrons promotes the formation of ROS that are capable of activating BAX. Once activated BAX dimerizes and initiates the intrinsic pathway of apoptosis. ROS production is required for apoptosis in response to αTOS [108]. Treatment of cells with the TPP conjugated antioxidant MitoQ abrogates ROS production and prevents αTOS-induced cell death [109]. 5.4.3. Cytochrome c reductase Cytochrome c reductase (Complex III) (Fig. 5) is embedded in the mitochondrial inner membrane and functions as a dimer to facilitate the transfer of electrons from reduced ubiquinone to cytochrome c in a process known as the Q cycle. Complex III is a major site of ROS production in the ETC. Under conditions of hypoxia, Complex III increases production of ROS. ROS generation by Complex III in response to decreased oxygen levels acts to stabilize HIF1α leading to induction of the glycolytic pathway and VEGF (vascular endothelial growth factor) mediated angiogenic signaling [72,110]. In this way, Complex III acts as an oxygen sensor. Complex III also acts to mediate MPT

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Fig. 7. Structures of Complex II and III and IV antagonists. Alpha-tocopheryl succinate is a redox-silent vitamin E analog that inhibits complex II. Benzoisothiocyanate and stigmatellin both inhibit Complex III function. Complex IV is inhibited by ditercalinium.

(membrane permeability transition) via its role in the regulation of Ca2+ flux [111]. Myxothiazol (Fig. 7) is a highly potent inhibitor of Complex III belonging to a group of quinol antagonists that includes the strobilurines and oudemansins. The methoxyacrylate group common amongst these inhibitors resembles the structure of ubiquinone such that their binding to the Qo site of Complex III blocks ubiquinol oxidation [112]. The antibiotic antimycin inhibits Complex III function by antagonizing the transfer of electrons at the Q1 center of Complex III. Due to their structural similarities and the ability to mimic ubiquinone binding these compounds may not be selective for Complex III. Stigmatellin (Fig. 7) inhibits Complex III in a different manner. Stigmatellin binds Complex III preventing the transfer of electrons from the Fe2S2 complex to cytochrome c1 in the bc1 complex of Complex III. In this way it acts as a strong oxidant to generate reduced Fe2S2 complex and oxygen radicals [113]. Benzylisothiocyanate (Fig. 7) is a dietary cancer preventive agent that has been shown to inhibit complex III. In MCF7 and MDA-MB-231 cell lines, treatment with benzylisothiocyanate leads to JNK (c-Jun Nterminal kinase) and MAPK p38 mediated activation of BAX and apoptosis. Benzylisothiocyanate showed selectivity for cancer cells as breast epithelial cells were resistant to apoptosis in response to treatment [114]. 5.4.4. Cytochrome c oxidase is the final electron acceptor Electrons are transferred from cytochrome c to cytochrome c oxidase (Complex IV) where they are used in the reduction of oxygen to water. The protons generated in this reaction are pumped across the IMM into the intra-membrane space to further increase the proton motive force generated by the H+ gradient (Fig. 5). Complex IV function is regulated endogenously by multiple mechanisms. Binding of ATP at the matrix side of Complex IV provides allosteric control of enzyme activity thereby regulating the rate of energy production to match ATP demand. Nitric oxide (NO) at low concentrations reversibly inhibits the activity of Complex IV [115]. Multiple Zn2+ and Ca2+ binding sites have been identified in mammalian Complex IV. The functional significance of Ca2+ binding has yet to be determined. Zinc

binding leads to inhibition of proton pumping in particular although solid evidence as to the role of zinc in Complex IV regulation remains elusive. Complex IV is a substrate of multiple Ser/Thr and Tyr kinases involved in cell signaling. Phosphorylation of Complex IV has been shown to have inhibitory or activating effects depending on the type of kinase [65]. N-retinyl-N-retinylidene ethanolamine (A2E) is an endogenously produced lipophilic cation implicated in age related macular degeneration. N-retinyl-N-retinylidene ethanolamine or A2E, is highly selective, inhibiting Complex IV activity by blocking its interaction with cytochrome c in mammalian cell cultures [116]. Ditercalinium (Fig. 7) was designed as a DNA bis-intercalating agent but its anti-tumor properties have been ascribed to its inhibition of Complex IV [117]. 5.4.5. Targeting the F0F1 ATPase prevents ATP synthesis The F0F1 ATPase (Complex V) is located in the mitochondrial inner membrane. The membrane bound F0 subunit transports protons through inner membrane and the F1 unit acts as the ATP synthase. Complex V produces the majority of ATP utilized by the cell. It does so using the proton gradient established by complexes I, III and IV of the ETC to drive the synthesis of ATP from ADP and inorganic phosphate and therefore is a significant target for disrupting energy metabolism (Fig. 5). The beta-F1 subunit of ATPase is significantly reduced in breast and gastric adenocarcinomas, and squamous esophageal and lung carcinomas. A decrease in ATPase activity has also been noted in hepatocellular carcinomas [118–120]. Presumably, the decreased activity of ATP synthase in the presence of a functional ETC would increase mitochondrial membrane potential and could partially account for the selectivity of delocalized lipophilic cations (DLCs) observed in carcinoma cells. Point mutations in the mtDNA encoding subunits of the F0F1 ATPase have been shown to sensitize cancer cells to chemotherapeutic agents such as etoposide [121]. Considering this observation, it is possible that therapeutic agents targeting F0F1 ATPase may synergize with known chemotherapeutic agents in vivo. Numerous compounds have been identified for their ability to inhibit ATP synthesis (Table 4). The Rhodamine dyes, MKT-077 and

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Rhodamine 123 (Fig. 8), have shown the most potential thus far having completed clinical trials for the treatment of solid tumors. Development of the rhodamine dyes prompted the observation that some analogues sequestered into the mitochondria. It was later found that the esterified carboxyl and delocalized positive charge imparted the mitochondria-selective properties seen in some rhodamine analogues. This discovery subsequently led to the identification of a series of mitochondriotropic rhodamines (rhodamine 123) and rhodacyanin dyes (MKT-077). It was noted that the rhodamines exhibited prolonged retention in tumor cells compared to normal cells. Rhodamine DLCs and rhodacyanin dyes were found to have potent antitumor activity in mouse xenograft models and drugs belonging to both classes advanced to and completed Phase I clinical trials [122]. MKT-077 was tested in treatment refractory solid tumors for its ability to inhibit ATP synthesis by selectively targeting tumor mitochondria. Treatment with MK-077 resulted in stable disease in one patient but the number of cycles administered was limited by recurrent nephrotoxicity. Evidence of mitochondrial accumulation was not seen in the early stages of treatment but after prolonged treatment toxicities that correlated with mitochondrial dysfunction were observed. Phase II testing was rejected on the basis of prolonged and recurrent nephrotoxicity seen in nearly all study participants [123]. Rhodamine 123 completed phase I clinical trials for the treatment of prostate cancer and the results were presented in 2005. Rhodamine 123 was well tolerated at 6 infusions over a 28 day treatment schedule reaching a MTD of 96 mg/m2. Drug related toxicities were manifest as hypertension, vomiting and rash all of which subsided 6 h post administration. Preliminary evaluation of Rhodamine 123 efficacy showed increased PSA (prostate specific antigen) doubling time in 80% of patients (8/10 patients). Increased tumor drug levels versus serum levels validates the theoretical basis for the utility of DLCs as anticancer agents and may explain the modest levels of drug related toxicities and increased PSA doubling time observed in patients treated with Rhodamine 123 [124]. Currently there are no reports of Phase II clinical trials of Rhodamine 123 in any type of cancer.

6. The other side of the coin: biosynthetic drive Tumor cells must increase their cellular biomass prior to cell division. The metabolic changes are necessary to provide tumor cells with sufficient substrates for the biosynthetic machinery. Moreover, oncogene products, tumor suppressor gene products and HIF-1 regulate the enzymatic machinery, and availability of substrates for the biosynthesis of cellular macromolecules. These biosynthetic precursors are formed by the metabolism of glucose and glutamine (Fig. 1) [11]. 6.1. Biosynthesis of nucleotides The synthesis of purines and pyrimidines requires ribose-5phosphate, which is derived from the oxidative and non-oxidative branches of the pentose phosphate pathway. Some of the glycolytic intermediates feed the pentose phosphate pathway leading to the synthesis of ribose-5-phosphate. Moreover the non-essential amino acids obtained from glucose and glutamine metabolism are also required for the synthesis of nucleotides [125]. Oncogene and tumor suppressor gene products play an important role in diverting glycolytic metabolites into the branches of the pentose phosphate pathway. Several enzymes of the nucleotide biosynthetic pathway are the target of c-Myc [43]. TIGAR suppresses glycolysis by decreasing the levels of PFK-1 and PGM. TIGAR decreases the expression of PFK1 activator (fructose-2,6-biphosphate) and thus leads to the accumulation of fructose-6-phosphate, which is retained for ribose-5phosphate synthesis by the pentose phosphate pathway [125]. c-Myc and Ras are also known to activate PFK1 [43]. In p53 negative tumors, pyruvate kinase-M2 exists as a dimer (less active form of the enzyme), leading to accumulation of upstream glycolytic intermediates that are subsequently used by the pentose phosphate pathway [125]. Increased expression of HIF-1α regulates the entry of glycolytic intermediates into the pentose phosphate pathway. HIF-1α potentiates the expression of transketolase and pyruvate kinase-M2, which promote the synthesis of ribose-5-phosphate via the pentose phosphate pathway [43].

Fig. 8. Mitochondriotropic delocalized lipophilic cations under investigation for anti-tumor properties.

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6.2. Biosynthesis of fatty acids

7.1. Dicholoroacetate (DCA)

The biosynthesis of fatty acids is required for the formation of lipids which are incorporated into the membranes of cells and organelles. Furthermore, these lipids can also function to modify proteins destined to be associated with membranes. Lipogenic enzymes are overexpressed in tumor cells. The expression of ATP citrate lyase, a lipogenic enzyme favors the Warburg effect by preventing the citrate build up in the cytosol (increased citrate can suppress glycolysis). Oncogenic mutations play an important role in fatty acid synthesis in tumor cells. Activation of the PI3K/Akt pathway promotes the expression of lipogenic enzymes, while suppressing the β-oxidation of fatty acids. Additionally the PI3K/Akt pathway increases the expression of glucose transporters providing substrate for the reactions [125].

DCA is a pyruvate analog that binds the N-terminal region of PDK. Phase I trials for clinical and pharmacological studies of DCA are being carried out in patients with recurrent and/or metastatic solid tumors. Studies of the safety and efficacy of DCA for the treatment of brain cancer are currently in phase II clinical trials [127,130,131].

6.3. Biosynthesis of proteins Both glucose and glutamine metabolism are involved in generating amino acids, tRNAs and ribosomes required for protein synthesis. The ribose-5-phosphate synthesized as a result of shunting of glycolytic intermediates to the pentose phosphate pathway is used up in the synthesis of nucleotides. These nucleotides are the constituents of the protein synthesis machinery of the cells (tRNA, ribosomes). The increased glutaminolysis also adds to the cellular pool of ribose-5phosphate and contributes to protein synthesis. Moreover, metabolism of glucose and glutamine is involved in increasing cellular supply of amino acids for synthesis of proteins [125]. 6.4. Anaplerosis and NADPH production: role of glutaminolysis in biosynthesis The increased demand for biosynthetic precursors depletes the intermediates of glycolysis and the TCA cycle. Citrate produced by TCA cycle is transferred out of the mitochondria to the cytosol, where it is used in fatty acid synthesis. There is a need for replenishment of these metabolic intermediates. Glutamine anaplerosis plays a key role in compensating for the depletion of these metabolites. Glutaminolysis also serves as a source of reducing power, providing NADPH required for nucleotide and fatty acid biosynthesis [125]. The uptake of glutamine by tumor cells is regulated by c-Myc. It also induces the expression and activity of enzymes involved in the biosynthetic processes (Fig. 4) [43]. 7. Critical switch between glycolysis and TCA cycle: pyruvate dehydrogenase complex/pyruvate dehydrogenase kinase (PDC/PDK) The PDC/PDK interaction regulates the entry of pyruvate into the TCA cycle. PDC is a multienzyme complex. It is composed of pyruvate decarboxylase (E1 subunit), dihydrolipoyl acetyltransferase (E2 subunit), and dihydrolipoyldehydrogenase (E3 subunit). PDC catalyzes the oxidative decarboxylation of pyruvate to give rise to acetyl-CoA. PDC activity is controlled by PDKs (1–4) and PDPs (pyruvate dehydrogenase phosphatases 1,2). Phosphorylation and inactivation of PDC by PDK favors glycolysis [126,127]. Recent studies have shown that induction of PDK3 by HIF-1 promotes the metabolic switch and resistance to conventional anticancer drugs like cisplatin and paclitaxel [128]. In vitro assays have shown decreased invasiveness of human head and neck squamous carcinoma (UM-22B) cells upon PDK inhibition. Additionally, a decrease in tumor volume was seen for mouse xenograft models expressing shPDK1. Therefore, inhibition of PDK can serve as an effective anticancer strategy [129]. In addition to its role in cancer, the PDC/PDK interaction has also been implicated in diabetes and some types of heart disease (Table 1) [127]. Some known inhibitors of PDK are listed below and are shown in Fig. 9.

7.2. Radicicol Radicicol binds to the nucleotide (ATP) binding site of PDK3. It is also known to inhibit heat shock protein Hsp90 and TopoVI [130]. 7.3. Nov3r and AZD7545 AZD7545 and other related compounds inhibit PDK2 and PDK1 but do not affect the activity of PDK4. AZD7545 binds to the lipoyl-binding pocket in the N-terminal domain of PDK1 and inhibits the enzyme [127,128,130]. Oximes of diterpenes and triterpenes, analogs of ATP, lactones, and (R)-3,3,3-Trifluro-2-hydroxy-2-ethyl-propionamides also inhibit PDK. However, these are more useful in the treatment of diabetes [132–134]. 8. Mitochondrial OXPHOS uncouplers and cancer Under normal circumstances the process of electron transfer is tightly coupled to the production of ATP. Mitochondrial OXPHOS uncouplers cause the uncoupling of the phosphorylation from the oxidation in the mitochondria (Fig. 2). DNP (2,4-dinitrophenol), FCCP (p-trifluoromethoxyphenylhydrazone) and CCCP (carbonylcyanide3-chlorophenylhydrazone) are classical examples of mitochondrial OXPHOS uncouplers [135]. Studies have revealed that OXPHOS uncouplers can influence tumor cell growth. CCCP, a proton ionophore and a mitochondrial OXPHOS uncoupler showed toxicity in human bladder carcinoma cell line (MGH-U1) and in murine mammary sarcoma cells (EMT-6) [136]. Another study demonstrated that rottlerin synergistically potentiated imatinib induced apoptosis in tumors expressing BCR/ABL. These effects were independent of rottlerin's effect on PKCδ (protein kinase C delta). Furthermore similar results were obtained with FCCP and DNP, two well known mitochondrial uncouplers [137]. Additionally rottlerin and FCCP decreased the HIF-1 transcription activity in PC-3 and DU-145 prostate cancer cells both under hypoxic and normoxic conditions. Moreover the uncoupler decreased the expression of HIF targeted genes [138]. Thus using mitochondrial uncouplers may prove to be a valid strategy for anticancer therapy. Further studies are required to establish this field. 9. Mitochondrial DNA mutations in cancer The circular mitochondrial genome is composed of a circular double stranded DNA molecule ~ 16,000 base pairs in length. MtDNA encodes 12S and 16S rRNA, 22 tRNAs and 13 subunits of the OXPHOS machinery [139]. The remaining OXPHOS proteins as well as ~ 1500

Fig. 9. Pyruvate dehydrogenase kinase inhibitors. Dichloroacetate and radicicol are PDK inhibitors under investigation for their anticancer potential (radicicol is a known Hsp 90 and TOPO VI inhibitor).

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other gene products are encoded by the nuclear genome. MtDNA lacks histones, has limited DNA repair mechanisms and is in proximity to high levels of ROS produced in the course of normal energy metabolism taking place in the mitochondria. These factors contribute to the high rate of mtDNA damage and mutation [140]. Damage and mutations, regardless of their source can result in alterations in gene products that impair mitochondrial function. Mutations that compromise OXPHOS components may initiate cycles of ROS production and subsequent damage to mtDNA culminating in dysregulation of energy metabolism [141]. Mutations in the mtDNA promoter region, aka the D-loop, or in tRNA coding regions may have deleterious effects on gene transcription of OXPHOS components [121]. Mutations in mtDNA have been observed in cancers of the breast, lung, thyroid and prostate. Mutations have also been reported in renal cell carcinoma and hepatocellular carcinoma [142]. Point mutations deletions, insertions, tandem duplications and copy number changes are among the types of alterations that have been identified in human cancers. Both the type and extent of mutations observed have been correlated with increased invasive and metastatic potential, drug resistance and poorer prognosis [143]. MtDNA mutations have also been observed in normal cells in the urine and bodily fluid of cancer patients and thus have been explored as a means of early detection of cancers. Unfortunately, in the studies performed the percentages of patients with detectable mutations in bodily fluid was below optimal for diagnostic testing parameters [144]. The number and uniformity of mtDNA mutations has been observed to change in the course of cancer progression [145]. Depending on the type, mammalian cells generally contain 103–104 copies of mtDNA that randomly distribute to daughter cells upon division. With further cell divisions, mutated DNA may become enriched in certain cells resulting in a phenotype of metabolic dysfunction conducive to the initiation of carcinogenesis. During the process of cancer progression the complement of mitochondria harboring mutations within a given cell progress toward a state of homoplasmy. Studies demonstrate that homoplasmic, mutated mtDNA imparts metastatic potential to cancer cells regardless of the genomic DNA complement. Mitochondria from highly metastatic cancer cells were transferred to cells with low metastatic potential. These cells, when grafted into immune compromised mice, produced tumors that were highly metastatic [146]. 10. TCA cycle enzymes as tumor suppressors: succinate dehydrogenase (SDH) and fumarate hydratase (FH) TCA cycle is a metabolic process which consists of sequential enzymatic reactions linked to OXPHOS. TCA cycle and OXPHOS are the main source of energy in the cell. Recent findings have ascribed remarkable properties to two TCA cycle enzymes, succinate dehydrogenase and fumarate hydratase. Germline mutations in the SDH gene were found to give rise to hereditary paragangliomas and phaeochromocytomas. FH gene germline mutations were observed in hereditary leiomyomas and renal cell cancer [147]. The tumor suppressor activity of these two enzymes is evidenced by emergence of a pseudohypoxic environment in SDH/FH deficient cells. Decreased activity of SDH or FH leads to pseudohypoxia which stabilizes HIF-1α. The PHDs responsible for degradation of HIF-1α use molecular oxygen and α-ketoglutarate as their substrates, to generate succinate and prolyl hydroxylated HIF-1α (degradation of hydroxylated HIF-1α is discussed in the section for HIF-1α inhibitors). Impaired SDH/FH function leads to the accumulation of succinate/fumarate in cells, which causes inhibition of PHDs (details in HIF-1 section) [18]. Loss of SDH function causes increased ROS production. ROS degrade PHDs and thus stabilize HIF-1α. Furthermore, it is proposed that fumarate accumulation leads to activation of some unknown physiological signaling pathways. These pathways may be responsible for development of uterine leiomyoma/renal cancer cell syndromes

[148]. Recently, it has been reported that inhibition of LDH-A can be therapeutically beneficial for hereditary leiomyomas and renal cell cancer treatment. As already stated, FH deficiency leads to stabilization of HIF-1α, which in turn increases the expression of HIF-1α target genes. LDH-A is a HIF-1α targeted gene and is overexpressed in hereditary leiomyomas and renal cell cancer. Studies have revealed that LDH-A inhibition leads to apoptosis of A549 (surrogate FH knockdown cell line) cells via ROS production [149]. 11. Targeting mitochondrial redox status 11.1. Inhibition of mitochondrial thioredoxin reductase disrupts mitochondrial redox balance and induces tumor cell apoptosis The formation of ROS occurs as a by-product of oxidative phosphorylation and increased formation of ROS is a trait often associated with pathological states, including cancer. ROS production results in the modification of proteins via the oxidation of thiol groups of cysteine and methoinine residues. Disulfide linkages formed by ROS may impinge on the functionality, reactivity, interactions and structure of the modified proteins and therefore mechanisms to restore the redox status of the protein and the cell exist within both the cytosol and mitochondria. One mechanism by which the internal environment of cells is maintained in the reduced state occurs through thiol transferase reactions carried out by the thioredoxin/ thioredoxin reductase (Trx/TrxR) system. This system, via disulfide exchange, reverses inter- and intra-protein disulfide linkages that occur in thiols of oxidized cysteine residues [150]. Noteworthy substrates of the Trx/TrxR system include DNA replication enzymes, transcription factors and components of the electron transport chain [151–153]. Recent experimental evidence suggests NADPH-dependent reduction via the Trx/TrxR system may function as a regulatory mechanism thereby influencing the rate of cell division and protein synthesis. Upregulation in the activity of the Trx/TrxR system seen in many tumors is thought to be a result of increased ROS production associated with oncogenic transformation and correlates with resistance to apoptosis and tumor cell proliferation [154,155]. Increased expression of thioredoxin has been observed in numerous cancers, and its increase has been correlated with decreased survival in colorectal cancer and diffuse large B cell lymphoma [154,156]. Elevated levels of Trx are observed in drug resistant cancer cell lines and have been implicated in steroid resistance in the treatment of certain leukemias [157,158]. Disruption of the cellular redox state via inhibition of TrxR, either in the cytosol or the mitochondria has been shown to increase the ratio of oxidized/reduced Trx, increase ROS, modulate cell signaling, sensitize cancer cell lines to chemotherapeutic agents, and induce cell cycle arrest, senescence and apoptosis of cancer cells [159,160]. Maintenance of intracellular redox state is critical to cellular homeostasis therefore tumor selectivity in targeting at the cellular and enzymatic levels are of great interest to the rational design of TrxR targeted compounds. The lipophilic and cationic Au(I) diphosphines (Fig. 8) and the Au(I) N-heterocyclic carbene compounds are goldbased compounds rationally designed to show selectivity for both TrxR and tumor cell mitochondria compared to earlier generation gold (I) and gold (III) based compounds that targeted both gluthione and thioredoxin reductases and were not tumor cell specific [161,162]. 12. Cancer therapy with mitochondrial potassium channel modulators Mitochondrial potassium fluxes are important for controlling the proton motive force in energized mitochondria [163]. It has been reported that the mitochondria-NFAT-Kv (NFAT, nuclear factor of activated T cells) channel axis plays a significant role in the electrical

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remodeling of mitochondria that characterizes human cancers. The downregulation of Kv channels contributes to apoptotic resistance in cancer cells. Normalization of the NFAT-Kv channel by dichloroacetate results in apoptosis [164]. Several agents are being developed that act on mitochondrial potassium channels. The potassium channel openers such as diazoxide, and cromakalim and its derivatives affect the mitochondrial as well as the plasma membrane KATP channels [165]. In vitro assays have revealed the antitumor potential of cromakalim against human neuroblastoma (SK-N-MC) and human astrocytoma (U-373 MG) cells [166]. However certain potassium channel openers such as minoxidil have been shown to stimulate the growth of MCF-7 breast cancer cells, while potassium channel blockers like dequalinium and amioradone inhibit it [167]. Recent studies have shown that glibenclamide, a KATP channel blocker acts as an antitumor agent for the human gastric cell line MGC-803 [168]. These contradictory observations make it difficult to assign a specific characteristic to potassium channel openers/blockers with regard to their effects on the tumor growth. Their lack of specificity for mitochondrial/plasma membrane potassium channels further complicates the scenario. Diazoxide is more specific for mitochondrial channels, but also has cardio protective effects [169]. Additionally it has been reported that pretreatment of cells with diazoxide exerts a protective effect against rotenone induced cell death [170]. Preconditioning with pinacidil and diazoxide exerts protective action against UV induced skin damage [171]. Moreover levosimendan (potassium channel opener) has antiischemic effects [172]. Certain potassium channel openers (NS004 and NS1619) have been shown to inhibit the activity of mitochondria in glioma cells. However these mitochondrial alterations do not lead to any change in the survival of glioma cells [173]. The benzothiazine diazoxide is reported to decrease the division of leukemic cells by causing mitochondrial membrane depolarization [174]. In summary most of the potassium channel modulators lack specificity for the mitochondrial channels, and exert varying effects on the tumor tissue. This necessitates further studies for elucidating the role of mitochondrial potassium channel modulators in cancer therapy. 13. Targeting mitochondrial apoptotic machinery 13.1. Targeting membrane permeability transition (MPT) to induce apoptosis Signaling transduction cascades along the pathway of intrinsic apoptosis descend upon the mitochondria to induce the membrane permeability transition (MPT), the sentinel, irreversible event leading to apoptosis. Membrane permeability transition is a sudden permeation of the mitochondrial inner membrane to solutes of greater than 1500 Da. The sudden permeation is thought to occur upon the opening of a channel called the mitochondrial permeability transition pore (PTP). Membrane permeability transition is characterized by a rapid loss of ΔΨm, accompanied by mitochondrial swelling and outer membrane rupture. Upon rupturing, ATP and the apoptogenic contents of the mitochondrial intermembrane space such as cytochrome c, Smac/DIABLO (second mitochondria derived activator of caspases/Direct Inhibitor of Apoptosis Binding protein with a Low pI), AIF (apoptosis inducing factor) and DNAseG are released into the cytoplasm. Once in the cytoplasm, in the presence of ATP, cytochrome c and AIF associate to form oligomers that recruit caspase 9. Caspase 9 in turn, auto-activates and together with cytochrome c and AIF comprises the apoptosome to execute programmed cell death [175– 177]. Thus, agents capable of modulation and induction of the MPT are of great utility for the treatment of neoplasms. The exact composition of the mitochondrial PTP and the mechanisms governing its response to pro-apoptotic signals are largely unknown although is believed the Bcl-2 family member proteins are intricately involved in the regulation of the PTP [178]. It is generally

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believed that the PTP spans the outer and inner membranes via complexation of mitochondrial VDAC on the outer membrane, adenine nucleotide transporter (ANT) on the inner membrane and cyclophilin D [179]. Cyclophilin D is normally found in the matrix but is believed to associate with ANT upon stimulation of MPT. The composition of the PTP is a topic of ongoing debate. VDAC and ANT co-purify with cyclophilin D by affinity chromatography of mitochondrial extracts [180]. Inhibition of cyclophilin D with cyclosporine A blocks MPT [181]. The ANT ligands bongkrekic acid and atractyloside modulate MPT and induce mitochondrial swelling [182]. In contrast to the evidence obtained by pharmacological inhibition, cell culture based studies on tissues derived from VDAC and ANT deficient mice show that both are dispensable for MPT [183,184]. Despite the conflicting evidence obtained in pharmacological and genetic studies, targeting antagonists to VDAC and ANT have shown potential as anti-cancer therapeutics. Three isoforms of VDAC have been identified thus far. In humans, VDAC1 is ubiquitously expressed, and VDAC2 has been isolated from heart mitochondria [185]. VDACs regulate the movement of metabolites, including ATP, to and from the mitochondria and cytosol [186]. Pro-apoptotic Bcl-2 family members BAX and BAK associate with, and have been shown to cause opening of VDAC [187]. Association of VDAC with Bcl-XL favors a closed conformation that suppresses MPT [188]. In rats VDAC mRNA levels are increased in hepatomas as compared to normal matched tissues. VDAC1 expression in human solid tumor cell lines is increased compared to normal human fibroblasts. Cancer cells harboring mutations in mtDNA express higher levels of VDAC and ANT [189]. The increased levels of VDAC seen in cancer may be explained in part by the Crabtree effect. Under conditions of aerobic glycolysis, hexokinase II is bound to the outer mitochondrial membrane by association with VDAC. ATP produced by ATP synthase in the inner membrane is transported through the channel formed by VDAC and ANT. Hexokinase II, due to its proximity, co-opts the newly formed ATP thereby sequestering it for use in the phosphorylation of glucose to glucose-6-phosphate. The Crabtree effect favors aerobic glycolysis and prevents optimal energy metabolism, i.e. state 3 respiration, for lack of adequate energy supply. In addition, the association of hexokinase II with VDAC limits the amount of calcium that enters the mitochondria further suppressing apoptosis [23]. The VDAC contribution to both the Crabtree effect and MPT suggest that it would be a significant target for anti-cancer therapies. Treatment of isolated mitochondria derived from head and neck carcinomas with cisplatin showed preferential binding to VDAC suggesting it as one potential target for cisplatin cytotoxicity [190]. Furanonapthoquinone (FNQs) (Fig. 10) derivatives have been shown to target VDAC1. FNQs, isolated from the inner bark of tropical trees, exhibit a 10–14 fold higher toxicity in cancer cells versus normal cells. FNQs induce apoptosis via NADH dependent ROS production at the mitochondrial outer membrane causing collapse of the membrane potential, cytochrome c release and caspase 9 activation [191]. Erastin (Fig. 10) binds VDAC 2 and 3 to induce NADH dependent oxidative cell death via the Ras-Raf-MEK pathway. Erastin shows greater activity in cancer cells harboring mutations in HRas, KRas and BRaf [192]. In addition to its proposed role in MPT, ANT is responsible for the exchange of ADP and ATP across the mitochondrial inner membrane [193]. The rate of respiration is tightly linked to the ratio of ATP/ADP within the mitochondria and modulation of ANT activity may provide a means for controlling the rate of ATP synthesis, albeit indirectly. Beutlinic acid, lonidamine and arsenic trioxide have been described as ANT effectors based on their ability to induce MPT, but are not specific for ANT [194]. The ANT2 gene has been shown to be upregulated in a number of hormone dependent cancers. Consistent with previous genetic studies, siRNA did not have deleterious effects on cell functions but was able to potentiate lonidamine induced MPT [195]. The role of ANT in ATP and ADP exchange, its role in MPT and its association with pro-apoptotic molecules such as BID and BAX suggest it would be a good therapeutic target.

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domains. The pro-apoptotic proteins are either “multidomain proteins” (BH1–BH3) or “BH3-only proteins”. All three classes play a crucial role in regulation of apoptosis. Upon apoptotic stimulation, the pro-apoptotic BAX and BAK undergo oligomerization causing mitochondrial membrane permeabilization and the release of cell death promoting factors. The anti-apoptotic Bcl-2 proteins are known to prevent this oligomerization. BH3-only proteins can act as sensitizers (BAD, BIK, and NOXA) or activators (BIM, BID). Activators bind and cause oligomerization of BAX and BAK leading to cytochrome c release. The sensitizers act indirectly by inhibiting the interaction of pro-survival Bcl-2 with activators or BAX/BAK [202,203]. Several strategies have been designed for targeting the Bcl-2 family of proteins. Antisense (G3139), anti-Bcl-2 antibody, anti-Bcl-2 ribozyme, BAK BH3 peptide, and SAHBs (stabilized alpha-helix of Bcl2 domains) of the BH3 domain from Bid have been developed as antiBcl2 therapies. Numerous small molecule inhibitors that bind the BH3 binding site of Bcl-2 have been designed to block the interaction of Bcl-2 with pro-apoptotic proteins and thus promote apoptosis of the cancer cells. ABT-263 and ABT-737 are examples of small molecule inhibitors of the Bcl-2 proteins [203,204]. (For a detailed overview of Bcl-2 inhibitors consult reference [205]). Fig. 10. Structures of MPT modulators.

Cyclophilin D is a peptidyl prolyl-cis trans isomerase involved in protein folding [196]. It normally resides in the mitochondrial matrix. In the event of MPT, cyclophilin D translocates to the inner membrane where it associates with ANT where it is believed to induce a conformational change that augments channel opening [175]. Unlike VDAC and ANT, genetic ablation of cyclophilin D in mice completely prevents MPT suggesting a critical role for cyclophilin D [181,197]. The immunosuppressive drug cyclosporine A inhibits some forms of MPT [198]. Despite this critical role in MPT, overexpression of cyclophilin D is observed in many tumors. Overexpression of cyclophilin D in cell lines imparts resistance to apoptosis [199]. These seemingly opposing roles may be explained by the recent finding that cyclophilin D interacts with Bcl-2 in a pro-survival role [200]. Further investigation of the pro and anti-apoptotic roles of cyclophilin D in apoptosis is needed to fully understand role of cyclophilin D in cell fate determination. The evidence for the composition of the MPT and the relative role each component plays in the execution of apoptosis remains unclear and is a source of on-going debate due to the inherent limitations of current scientific methods of investigation. The inhibition of VDAC and ANT has been shown to modulate MPT. Regardless of whether this modulation is direct or indirect, these promising results warrant continued investigation for the development of anti-cancer therapeutics. 13.2. Bcl-2 inhibitors Apoptosis is the major process that maintains tissue homeostasis. Apoptosis is carried out by an extrinsic pathway (involving death receptors), or an intrinsic pathway which is mediated by mitochondria. Disruption in the physiological balance between cell survival and apoptosis is observed in most cancers. Pro-apoptotic signaling is downregulated in tumors [201]. The Bcl-2 family of proteins is a key player in apoptosis. The Bcl-2 family consists of pro-apoptotic (BAX, BAK) and anti-apoptotic (Bcl-2, Bcl-xl, Bcl-w, Mcl1,Bcl2-A1) members. The communication between the anti and pro-apoptotic Bcl-2 family members governs the destiny of the cell. There is higher expression of anti-apoptotic Bcl-2 family proteins in cancer. The higher occurrence in cancers is responsible for chemo-resistance of some tumors. The Bcl-2 family of proteins is classified into three categories depending on structure and function. All contain the Bcl-2 homology (BH) domain. The anti-apoptotic Bcl-2 proteins consist of four BH

13.3. Smac/DIABLO Apoptotic stimuli lead to permeabilization of the outer membrane of mitochondria with a concomitant release of pro-apoptotic factors into the cytosol. The mitochondrial apoptosis promoting factors include cytochrome c, AIF, Smac/DIABLO and Omi/HtrA2 [206]. The XIAP (X-linked inhibitor of apoptosis) and cIAPs (inhibitor of apoptosis) consist of three tandem BIR (baculovirus IAP repeat) domains and a Cterminal RING domain. The RING domain is characterized by E3 ubiquitin ligase activity. cIAP1 and cIAP2 also contain a CARD domain (CAspase Recruitment Domain). cIAP1/2 have been found to be associated with the TNFR2/TRAF (TNFR, tumor necrosis factor receptor; TRAF, TNF receptor associated family of proteins) signaling complex, whereas the XIAP is implicated in causing inactivation of caspases [207,208]. XIAP interacts with the initiator caspase 9 through its BIR3 domain, and with the effector caspases 3 and 7 through its BIR2 domain [208–210]. Smac/DIABLO is an endogenous inhibitor of IAPs. Smac/DIABLO antagonizes IAPs and positively regulates caspase activity [211,212]. Several types of cancers have upregulated IAPs and hence are able to evade apoptosis [213]. Changes in the expression pattern of Smac/DIABLO have also been observed in numerous tumors. A lower Smac/Diablo expression is reported in renal cell carcinoma and lung cancer [214,215]. However there is de novo expression of Smac in some forms of cervical tumor [216]. Moreover recent findings have shown a higher expression of Smac/DIABLO in recurrent cervical squamous cell carcinoma [217]. Agents that mimic the IAPs–Smac interaction are under development for cancer therapy. Smac mimetics antagonize IAPs and promote apoptosis of tumor cells expressing higher levels of IAPs and lower Smac/DIABLO levels. Furthermore combined treatment with Smac mimetics and other chemotherapeutics is also being studied. For instance, Smac mimetics have shown to potentiate the apoptosis mediated by tamoxifen, paclitaxel, doxorubicin, and irradiation. They also sensitize the TRAIL-resistant tumor cells for apoptosis [218,219]. Smac/DIABLO has also been found to play an important role in promoting pthalocyanine-PDT (photodynamic therapy) induced apoptosis in a BAX dependent manner [220]. Studies have reported that besides activating caspases, Smac mimetics can also stimulate autoubiquitination and degradation of cIAPs, activate NFκB, and lead to TNF-α dependent cell death [221–223]. Several Smac mimics have been designed and are being tested for their potential in cancer therapy. For example, AT-406 is in the late stage of preclinical studies carried out by Ascenta Therapeutics. It has shown promising results in xenograft models of human cancer [224].

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Smac mimetics have been designed based on the interaction of Smac/DIABLO with the IAPs. The N-terminal AVPI (alanine valine proline isoleucine) sequence of Smac is involved in the interaction and serves as the foundation for the designing of Smac mimics [225]. These are either peptidomimetics or small molecules mimicking Smac. Some of the inhibitors possess only one AVPI mimic domain (monovalent), whereas others are divalent. Earlier studies showing the Structure Activity Relationship (SAR) of the modified peptides derived from AVPI tetrapeptide, emphasized the feasibility of designing Smac based XIAP inhibitors [226]. Further modifications of the Smac mimetic peptides led to the improvement in their proteolytic stability and potency. These were synthesized by incorporating capped peptides with unnatural amino acids. Such peptides showed cytotoxic effects in various cell lines and slowed the growth of breast cancer in mouse xenograft models [227]. Structure based designing and synthesis have led to development of potent Smac peptidomimetics showing activity in cell based assays (MDA MB231 and PC-3 cells), and enhancing the apoptosis induced by chemotherapeutic drugs [228]. Several Smac mimetics have been designed by developing peptide isosteres for AVPI tetrapeptide [229]. Small molecule mimics of Smac were developed with the aim of generating better drug entities. Heterocycles were used to replace the amino acid moieties and develop non-peptidic Smac mimetics [230]. Advances in structure based drug design led to the development of conformationally constrained bicyclic Smac mimetics. The nonpeptidic Smac mimetics showed promising results when tested in various cell lines [231,232]. SM-122, SM-130, SM-131 and SM-230 are some examples of Smac mimics that inhibited cell growth in MDAMB-231 breast cancer cells [233,234]. The field of Smac mimics advanced with the development of divalent Smac mimetics. Divalent Smac mimics such as SM-164 showed higher potency in binding, functional and cellular assays as compared to the monovalent mimics. The higher potency of divalent mimics can be attributed to the fact that they target both the BIR2 and BIR3 domains of the XIAP. SM-164 also induces the degradation of cIAPs [235–237]. 14. Small molecule delivery to mitochondria 14.1. Delocalized lipophilic cations selectively target the mitochondria of tumor cells DLCs, due to large hydrophobic surface areas and a delocalized positive charge are readily absorbed through plasma and mitochondrial membranes [238]. The lipophilic nature of DLCs allows for rapid passage through membrane bilayers while the permanent cationic charge, driven by the negative membrane potential, causes selective 100–1000 fold accrual within the negatively charged cytosol and mitochondrial compartments. Studies comparing the uptake of charged molecules such as the anthracyclines and rhodamines show that parameters such as size, lipophilicity and binding properties are a factor in compartmental accumulation, but delocalization of charge is the key element that determines selective mitochondrial accumulation [122]. DLCs are currently in various stages of preclinical and clinical investigation for potential utility in targeting drugs to the mitochondria of tumor cells. DLCs accumulate in cancerous cells to a greater degree than in normal cells due to the more negative plasma and mitochondrial membrane potentials typically observed in tumor cells and offer a unique means by which to deliver drugs preferentially to tumors and spare normal healthy cells in hopes of reducing drug related toxicities. 14.2. Lipophilic triphenylphosphonium cations rapidly accumulate within mitochondria and have been utilized to target a wide variety of molecules into the mitochondria of tumor cells Lipophilic triphenylphosphonium cations (TPP) are the most widely studied and reported on mitochondrial targeting DLC. TPP

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compounds were first utilized as probes for assaying mitochondrial membrane potential as it was observed that the lipophilic nature and cationic charge facilitated the mitochondrial membrane potentialdependent accumulation of TPP within the negatively charged mitochondrial matrix [239]. By exploiting this property, TPP compounds were used for the visualization and evaluation of mitochondrial attributes. For these purposes, the TPP became a targeting motif via which relevant molecules were conjugated to the TPP with the goal of targeting their uptake into the mitochondrial inner membrane. Later, routine screening of synthetic intermediates revealed a significant anti-leukemic activity in isoindolylalkyl phosphonium salts prompting the development of SAR to determine which substituents imparted activity. These SAR studies showed the TPP moiety to be essential for anti-tumor activity. These same SARs showed that variations in the length of the carbon side chains of the isoindolylalkyphopsphonium salts modified the antimitotic properties of the TPP halide moiety. The changes in potency were likely related to changes in hydrophobicity [240]. Mathematical based models predicting the selective uptake of DLCs such as TPPs into tumor cells have been experimentally validated and show the importance of logP values in the range of −2 to 2 [241]. Below this range, absorption of compounds are predicted to be kinetically limited and compounds having higher logP values are predicted to absorb to cytosolic as well as mitochondrial lipids and therefore will lack compartmental selectivity [241]. The rate of mitochondrial TPP uptake in cells can be greatly influenced by hydrophobicity as shown by subsequent studies performed using TPP compounds conjugated to alkyl chains of varying length. Addition of hydrophobic moieties to TPPs results in increased rate of uptake in cellular mitochondria compared to more hydrophilic TPPs, due to more rapid passage through the lipid portion of the plasma membrane bilayer [242]. Once in the cytosol, entry into the mitochondria occurs via adsorption of the TPP containing molecule to the outer surface of the mitochondrial inner membrane, permeation of the hydrophobic region of the bilayer, binding to the inner surface of the inner membrane and finally desorption into the mitochondrial matrix [239]. Increases in TPP uptake through the plasma membrane imparted by alkyl groups or other hydrophobic motifs are tempered by increased adsorption to mitochondrial inner membrane. The increased hydrophobicity may ultimately result in impaired function of therapeutic molecules via sequestration leading to decreased potency, as well as a reduction in drug like properties such as solubility and bioavailability. 14.3. Lipophilic cations as tumor selective radiotracers Due to their selective accumulation in energized mitochondria, numerous DLCs have been investigated as potential contrast agents for the detection, diagnosis and therapeutic management of cancers using PET (positron emission tomography), SPECT (single photon emission computed tomography), CT (computed tomography) and scintigraphy imaging modalities. In addition to accumulation in the mitochondria of tumor cells, DLCs have been demonstrated to rapidly accumulate in other mitochondria enriched tissues such as the heart, liver and kidneys [243–247]. For diagnostic purposes, a radiotracer must be able to detect small lesions with high fidelity. Accumulation in mitochondria-rich, non-cancerous tissue, therefore presents a significant consideration for the development of tumor specific radiotracers. Radiolabelled DLCs such as quaternary ammonium, arsonium and phosphonium complexes were developed to overcome the limitations associated with Thallium 201 based imaging for the differentiation of ischemia and myocardial damage [248]. In the process, biodistribution studies of structural analogs of radioiodinated iodopentenyl-trisubstituted phosphonium, arsonium and ammonium cations provided early insight for the development of tumor specific tracers. Elemental substitutions, i.e. phosphorus in place of arsenic, had little effect on

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heart uptake nor did replacement of the phenyl rings system with a cyclohexyl ring. Ultimately tissue distribution of these radioiodinated compounds was influenced by lipophilicity as alkyl substitution of the ring system produced hydrophilic compounds that favored biliary excretion over cardiac uptake [248]. Additional lipophilic cations such as 3H-tetraphenylphosphonium (3H-TPP) and 4-(18F-benzyl) triphenylphosphonium (18FBzTPP) are described in the literature and have been assessed for their utility in tumor imaging. 3H-TPP administered in mice showed early uptake and rapid clearance from the blood as expected for a radiotracer. The tumor uptake of 3H-TPP was not significantly different from that of 18F-FDG (18F-deoxygluxose), the most common clinically used tumor imaging radiotracer. 3H-TPP did exhibit some advantages over 18 F-FDG. 3H-TPP uptake was not influenced by blood glucose levels, as uptake in cell lines remained unchanged in the presence of increasing concentrations of glucose in culture media. 3H-TPP also showed minimal uptake in brown adipose tissue and at sites of inflammation in tumor and inflammation bearing mice respectively, two common means by which 18F-FDG contributes to false positive readings. The high accumulation in heart and kidney and lower accumulation in brain tissue may limit the diagnostic potential of 3H-TPP to detect small lesions in the brain and abdominal regions [243]. 18 F-FBzTPP uptake was correlated with membrane potential but intracellular accumulation was driven mainly by ΔΨm in accordance with the lipophilic, cationic properties of the TPP substituent. Biodistribution studies in rats and dogs revealed low tumor specificity as high uptake was noted in liver, heart, kidney and spleen. Due to these characteristics 18F-FBzTPP would not be suitable for diagnostic purposes but could be useful for the detection of apoptosis in tumor masses thereby indicating the level of response to treatment, if in fact ΔΨm does decrease in response to treatment [244]. 99 m-Tc-Sestamibi (99m-Tc-MIBI) and 99m-TC-tetrofosmin are FDA (Food and Drug Administration) approved contrast agents for cardiac PET and SPECT imaging and have since been utilized for multiple purposes related to cancer diagnosis and treatment. The biodistribution profile of 99m-Tc-MIBI shows rapid uptake and clearance from the blood with optimal cardiac contrast in the timeframe of 60–90 min [247]. 99m-Tc-MIBI uptake ratios in heart versus lung and heart versus liver were calculated to be 2:1 and 1:1 respectively and the primary target organ was found to be the thyroid [249]. Accumulation of 99m-Tc-MIBI in abdominal organs and the thyroid limit the diagnostic potential on the whole body scale but localized imaging modalities have been successfully applied. Scintigraphy using 99m-TcMIBI has been shown to be as effective as traditional mammography for the detection of small breast lesions and may been even more sensitive than conventional methods for the detection of locoregional recurrence and residual breast cancers [250–252]. Decreased 99 m-Tc-MIBI and 99m-TC-tetrofosmin retention time in tumors has been correlated with increased expression of MDR (multidrug resistance protein) and Pgp (p-glycoprotein) expression and are currently under clinical investigation for their ability to serve as a noninvasive marker for MDR and Pgp expression in breast malignancies to predict response to chemotherapies [253–255]. Over-expression of Bcl-2 inhibited the uptake of 99m-TC-MIBI in breast cancer cell lines [256]. In breast cancer patients, absence of 99m-TC-MIBI uptake correlated highly with overexpression of Bcl-2 in tumors [257]. Inhibition of Bcl-2 to induce MPT and tumor cell apoptosis is currently the subject of intense investigation for the development of therapeutic agents. The development of non-invasive imaging for the detection of Bcl-2 expression in solid tumors would certainly aid drug development and complement patient stratification in the event that Bcl-2 inhibitors demonstrate clinical application. Copper-labeled TPP cations are currently under investigation for their potential as tumor selective PET imaging agents. The short halflife, low β-emission and coordination chemistry of copper are favorable properties that have been exploited for the development of small

molecule radiotracers. First generation triphenylphosphonium and triphenylarsonium (TPA) copper chelates displayed higher selectivity for tumor mitochondria than 99m-Tc-MIBI and 99m-TC-tetrofosmin in mouse xenograft imaging studies but were still retained in liver and heart to a high degree [245]. Further structural refinements of the TPA and TPP chelates showed that moderate lipophilicity provided the best selectivity presumably due to slightly lower uptake kinetics compared to molecules having more positive logP values. The increase in hydrophilicity ensured that uptake was dictated by charge therefore imparting greater tumor selectivity [246]. These experiments were performed in glioma cell lines expressing low levels of PgP and MDR. Further studies will be needed to determine what effect, if any of these modulators will play in uptake and whether copper labeled radiotracers will show additional utility for the prediction of response to treatment. 14.4. Lipophilic triphenylphosphonium cations as mitochondrial drug carriers Triphenylphosphonium conjugated antioxidant compounds, owing to their ability to sequester selectively into tumor mitochondria, have been shown to act as ROS scavengers, induce cell differentiation, inhibit cell proliferation, and suppress tumor growth in animal xenograft models [258–260]. Triphenylphosphonium conjugated antioxidant compounds are currently the focus of intense evaluation for their therapeutic potential in the treatment of numerous disease states defined by mitochondrial dysfunction such as cancer, Parkinson's disease and hepatitis C viral infection. 15. Mitochondrial targeted anti-cancer photodynamic therapy Photodynamic therapy (PDT) harnesses the energy produced by excitation of a photosensitizable molecule to generate ROS via energy transfer. In chemotherapeutic PDT, the drug, a photosensitizer (PS) is administered systemically. A locally applied external light will excite the PS to a higher energy state caused by the absorption of a photon of light. Upon returning to the ground state, the PS will transfer the energy of absorbed light to proximal oxygen molecules producing singlet state oxygen. It is the singlet oxygen that is believed to mediate the primary effects of PDT [261]. The mechanisms of action of PDT are either direct or indirect and vary according to the PS used and the host response. The ROS generated by PDT causes lethal oxidative damage leading to rapid cell death. In addition the photosensitization may also have an antiangiogenic effect, and the resulting loss of oxygen and nutrient supply will indirectly kill the cells. PDT can also stimulate host immune responses that impart anti-tumor activity [262]. Photodynamic therapy using the photosensitizing agent porfimer sodium is FDA approved for the treatment of obstructing esophageal and endobronchial non small cell lung cancer. Many photosensitizing drugs (Fig. 11) are known to target the mitochondria. Depending on the photosensitizer used, the mechanism of action may involve direct or indirect targeting of mitochondria [263]. Cationic photosensitizers vary in mitochondrial affinity based on the extent of their charge and lipophilicity [264]. Further, interactions within the organelle are molecule specific. Cationic photosensitizing dyes directly kill tumor cells as a result of mitochondrial photodamage. Indirect killing of proximal neovascular may also occur as a result of the accumulation of the cation in the mitochondria of proximal endothelial cells [261]. Agents such as porfimer sodium, hemotoporphyrin and the pro-drug ALA, selectively target specific components of the mitochondria [265]. Light interrogation in the presence of these agents causes extensive photodamage. The effects include mitochondrial swelling, morphological perturbations and inhibition of mitochondrial function [266]. Indirect effects may

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Fig. 11. Structures of PDT agents.

occur as the result of redistribution upon photosensitization resulting in mitochondrial depolarization. 16. Conclusion and future directions We provide an overview of the anticancer strategies targeting cancer cell bioenergetics or mitochondria. The use of glycolytic inhibitors is a rational approach for targeting cancer's sweet tooth. Several glycolytic inhibitors have also shown synergy with anticancer drugs. The discovery of methyl jasmonate has provided a new gateway in the cancer bioenergetics targeting. Targeting of glycolysis also decreases the abundance of metabolic intermediates which serve as precursors for macromolecule biosynthesis. Targeting the mitochondrial apoptotic regulatory machinery is also a promising anticancer approach. Recently, interest has developed in exploiting mitochondrial uncouplers and potassium channel modulators as anticancer agents. However more research is required in this field to establish their importance. We suggest using a combination of these agents to target different aspects of cancer bioenergetics and mitochondrial functions to provide synergistic therapies for cancer. Moreover, directing these compounds specifically to their targets would reduce the off target effects and increase the potency. The physiological characteristics of cancer cells, such as higher glucose uptake, and specific mitochondrial features should be exploited for designing cancer cell specific treatments. References [1] B. Alberts, A. Johnson, J. Lewis, M. Raff, K. Roberts, P. Walter, Molecular biology of the cell, Garland Sciencefifth ed., Taylor and Francis Group, New York, 2008. [2] X. Wang, The expanding role of mitochondria in apoptosis, Genes Dev. 15 (2001) 2922–2933. [3] D.F. Babcock, J. Herrington, P.C. Goodwin, Y.B. Park, B. Hille, Mitochondrial participation in the intracellular Ca2+ network, J. Cell Biol. 136 (1997) 833–844. [4] D.D. Gutterman, Mitochondria and reactive oxygen species: an evolution in function, Circ. Res. 97 (2005) 302–304. [5] D.C. Wallace, A mitochondrial paradigm of metabolic and degenerative diseases, aging, and cancer: a dawn for evolutionary medicine, Annu. Rev. Genet. 39 (2005) 359–407. [6] D.C. Chan, Mitochondria: dynamic organelles in disease, aging, and development, Cell 125 (2006) 1241–1252. [7] Kurt Højlund, Martin Mogensen, Kent Sahlin, H. Beck-Nielsen, Mitochondrial dysfunction in type 2 diabetes and obesity, Endocrinol. Metab. Clin. North Am. 37 (2008) 713–731. [8] O. Warburg, On the origin of cancer cells, Science 123 (1956) 309–314. [9] T. Bui, C.B. Thompson, Cancer's sweet tooth, Cancer Cells 9 (2006) 419–420. [10] J.W. Kim, C.V. Dang, Cancer's molecular sweet tooth and the Warburg effect, Cancer Res. 66 (2006) 892–8930. [11] J. Ralph DeBerardinis, Julian J. Lum, Georgia Hatzivassiliou, C. Thompson, The biology of cancer: metabolic reprogramming fuels cell growth and proliferation, Cell Metab. 7 (2008) 11–20.

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