Drug efflux pumps in photodynamic therapy

C H A P T E R

8 Drug efflux pumps in photodynamic therapy Michael R. Hamblina,b,c a

Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, MA, United States b Department of Dermatology, Harvard Medical School, Boston, MA, United States cHarvard-MIT Division of Health Sciences and Technology, Cambridge, MA, United States

Abstract Photodynamic therapy involves the combination of photosensitizers (PS) with harmless visible light, that then produce reactive oxygen species to kill cancer cells or pathogenic microbial cells. There is evidence that many different PS are substrates of multidrug efflux pumps (MEPs). It was shown that phenothiazinium dyes such as methylene blue were pumped out of both Gram-positive and Gram-negative bacteria by MEPs, and that antimicrobial photodynamic inactivation could be potentiated by addition of a MEP inhibitor. With regard to cancer cells, some PS related to porphyrins and chlorophyll have been found to be substrates of the ATP binding cassette MEP, called ABCG2. ABCG2 has been found to be over-expressed in cancer stem cells, which accounts for their relative resistance to PDT. However several approaches have been found to overcome the protective effect of PS efflux mediated by ABCG2 in these cancer cells, such as the use of specific MEP inhibitors such as imatinib and fumitremorgin C (FTC). This chapter summarizes the existing laboratory and clinical evidence for the role of efflux pumps in PDT.

Abbreviations ABC

ATP-binding cassette

AHR ALA AlPCS2a aPDI BCRP BPD-MA CD243 CR CSCs

aryl hydrocarbon receptor aminolevulinic acid disulfonated aluminum phthalocyanine antimicrobial photodynamic inactivation breast cancer resistance protein benzoporphyrin derivative mono acid ring A cluster of differentiation 243 complete response cancer stem cells

Drug Efflux Pumps in Cancer Resistance Pathways: From Molecular Recognition and Characterization to Possible Inhibition Strategies in Chemotherapy https://doi.org/10.1016/B978-0-12-816434-1.00008-5

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# 2020 Elsevier Inc. All rights reserved.

252 DMMB EGFR EPI EVs HIF-1alpha HO-1 HPD HPIX HPPH Keap1 MATE MB MDR1 MEPs MFS MPPa MRSA mTHPC Npe6 Nrf2 P-gp PCI PDT Ppa PPAR gamma PPIX PS RND ROS SMR SNP SP TAP TBO TKI TM TOOKAD TPCS2a TPPS2a

8. Drug efflux pumps in photodynamic therapy

dimethylmethylene blue epidermal growth factor receptor efflux pump inhibitor extracellular vesicles hypoxia-inducible factor 1alpha heme-oxygenase hematoporphyrin derivative hematoporphyrin IX 2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide a Kelch-like ECH-associated protein 1 multidrug and toxic-compound extrusion methylene blue multidrug resistance protein 1 multidrug efflux pumps major facilitator superfamily pyropheophorbide a methyl ester methicillin resistant Staphylococcus aureus meta-tetrahydroxyphenyl chlorin monoaspartyl chlorin(e6) nuclear factor erythroid-derived 2 P-glycoprotein photochemical internalization photodynamic therapy pyropheophorbide a peroxisome proliferator-activated receptor gamma protoporphyrin IX photosensitizer resistance nodulation division reactive oxygen species small multidrug resistance single nucleotide polymorphism side population transporter associated with antigen processing toluidine blue O tyrosine kinase inhibitor transmembrane padeliporfin disulfonated meso-tetraphenylchlorin adjacent disulfonated meso-tetraphenylporphine adjacent

Conflict of interest MRH declares the following potential conflicts of interest. Scientific Advisory Boards: Transdermal Cap Inc., Cleveland, OH; BeWell Global Inc., Wan Chai, Hong Kong; Hologenix Inc. Santa Monica, CA; LumiThera Inc., Poulsbo, WA; Vielight, Toronto, Canada; Bright Photomedicine, Sao Paulo, Brazil; Quantum Dynamics LLC, Cambridge, MA; Global Photon Inc., Bee Cave, TX; Medical Coherence, Boston MA; NeuroThera, Newark DE; JOOVV Inc., Minneapolis-St. Paul MN; AIRx Medical, Pleasanton CA; FIR Industries, Inc. Ramsey, NJ; UVLRx Therapeutics, Oldsmar, FL; Ultralux UV Inc., Lansing MI; Illumiheal & Petthera, Shoreline, WA; MB Lasertherapy, Houston, TX; ARRC LED, San Clemente, CA; Varuna Biomedical Corp. Incline Village, NV; Niraxx Light Therapeutics, Inc., Boston, MA. Consulting; Lexington Int, Boca Raton, FL; USHIO Corp, Japan; Merck KGaA, Darmstadt, Germany; Philips Electronics Nederland B.V. Eindhoven, Netherlands; Johnson & Johnson Inc., Philadelphia, PA; SanofiAventis Deutschland GmbH, Frankfurt am Main, Germany. Stockholdings: Global Photon Inc., Bee Cave, TX; Mitonix, Newark, DE.

Introduction to photodynamic therapy

253

Introduction to photodynamic therapy Photodynamic therapy (PDT) is a selective anticancer treatment method and is used for a variety of cancers (on the body surface or endoscopically accessible), predominantly for recurrent cancers that no longer respond to conventional anticancer therapies such as chemotherapy or radiation therapy [1]. PDT is a treatment modality requiring three separate individual components: a photosensitizer (PS), low intensity light irradiation, and ambient oxygen [2]. A photosensitizer is a molecule that localizes in a specific target cell and becomes activated when exposed to a light source. When exposed to a specific wavelength of light the molecule is boosted to an electronic excited singlet state. Normally this is a very-short lived species (ns) but in some cases the excited singlet state can undergo an intersystem crossing (electronic spin flip) to form the much longer-lived excited triplet state (μs). Since oxygen is a triplet in its ground state it is allowed to interact with the PS triplet state by the selection rules. An energy transfer reaction can then take place producing two singlet species (ground state singlet PS and excited state singlet oxygen, 1O2) from two triplet species. The pathway that produces 1O2 is called the Type 2 photochemical pathway. There is another pathway that can take place involving the long-lived PS triplet state. This is called the Type 1 photochemical pathway, and involves an electron transfer (or a hydrogen atom transfer) to form free radicals [3]. If oxygen is involved in this reaction, the reactive oxygen species (ROS), superoxide, hydrogen peroxide and hydroxyl radicals (HO% are produced. Both 1O2 and HO% are highly reactive species and can readily oxidize unsaturated lipids, nucleic acid bases, and certain amino-acids in proteins. This oxidative attack produces oxidative stress and results in cell death by apoptosis and/or necrosis [4]. These pathways are schematically illustrated in the Jablonski diagram in Fig. 1. PDT for cancer is usually carried out by injecting the PS

FIG. 1 Jablonski diagram showing the photophysical and photochemical mechanisms of PDT.

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8. Drug efflux pumps in photodynamic therapy

intravenously, followed after some time (the drug-light interval) by delivery of red or NIR light to the tumor area [5]. PDT has shown to be an effective treatment modality in cancers of the lung, skin, breast, head and neck, digestive tract, pancreas, liver, bladder, ovary, prostate and brain [1]. Moreover in recent years PDT has also been tested as an alternative approach to treat infectious disease, where the pathogens are accessible to topically applied PS and light [6]. The chemical structures of the PS used in PDT can vary considerably, but generally involve unsaturated, extensively-conjugated heterocycles such as tetrapyrroles (porphyrins, chlorins, pheophorbides, phthalocyanines), synthetic dyes (such as methylene blue and rose bengal) and some naturally occurring dyes (such as hypericin and curcumin) [7] see Fig. 2. The PS molecules should be able to localize inside a cancer cell or inside a pathogenic microbial cell

FIG. 2 Individual examples from different classes of basic PS structures used for PDT. Porphyrins (protoporphyrin IX); chlorins (chlorin (e6)); bacteriochlorins (TOOKAD soluble); phthalocyanines (PC 4), phenothiazinium salts (methylene blue); halogenated xanthenes (rose bengal); perylenequinones (hypericin); natural products (curcumin).

Microbial efflux pumps in photodynamic inactivation

255

in order to be able to kill it efficiently upon illumination, because the ROS that are produced are short-lived and cannot diffuse very far. Since intracellular localization is required for efficient PDT, the question then arises whether the PS are substrates of multidrug efflux pumps that have been shown to be responsible for the development of resistance to chemotherapy in cancer cells [8], and to antibiotics in the case of microbial cells [9].

Microbial efflux pumps in photodynamic inactivation Multidrug efflux pumps (MEPs) are now broadly recognized as major components of microbial resistance to many classes of antibiotics [10]. Some MEPs selectively expel specific antibiotics, while others, referred to as multidrug resistance pumps, can mediate efflux of a variety of structurally diverse compounds with differing modes of action. Fig. 3 shows the five major families of MEPs that occur in bacteria and fungi. Phenothiazinium-based PS such as toluidine blue O (TBO), methylene blue (MB), and azure dyes have been employed in antimicrobial photodynamic inactivation (aPDI) research for nearly 80 years [11–13]. Phenothiazinium salts are amphipathic planar molecules that possess one intrinsic quaternary nitrogen atom and have phototoxic efficiency against a broad

FIG. 3 Different types of microbial efflux pumps. MFS is “major facilitator superfamily”; SMR is “small multidrug resistance,” MATE is “multidrug and toxic-compound extrusion,” ABC is “ATP-binding cassette” and RND is “resistance nodulation division.” The first three are commonly found in Gram-positive bacteria, ABC in fungi, and RND in Gram-negative bacteria.

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8. Drug efflux pumps in photodynamic therapy

range of microorganisms, such as Escherichia coli, Staphylococcus aureus, streptococci, Listeria monocytogenes, and Vibrio vulnificus [14–18]. MB has been used as an intrinsic antimicrobial compound in conventional (non-light mediated) antimicrobial therapy research for over a 100 years [11]. It has been reported that photodynamic activity of MB occurs mostly, via the Type I mechanism [19]. Several studies have reported its in vitro activity and in animal models of infection, and MB has received regulatory approval to mediate photodynamic therapy of dental infectious diseases, such as periodontitis and caries [20–22], however, it seems that MB is not the optimum member of the class of phenothiazinium derivatives for antimicrobial photo-inactivation. Wainwright et al. compared five different phenothiazinium dyes as antibacterial PS against methicillinsensitive S. aureus (MRSA). They found that dimethyl methylene blue and new methylene blue were the most active compounds but they had dark toxicity [23]. In 2006, Tegos and Hamblin showed for the first time that phenothiazinium salts were substrates of microbial MEPs [24]. The MEPs in Gram-positive bacterial species are primarily classified as the major facilitator-type (characterized by NorA in S. aureus), while Gramnegative species tend to have three-component MEPs, known as resistance nodulation division (RND) and characterized by MexAB-OprM in Pseudomonas aeruginosa [10]. In 2008, Tegos et al. [25] reported that four different inhibitors of the NorA pump (the diphenyl urea INF271, reserpine, 5-methoxyhydnocarpin, and the polyacylated neohesperidoside) significantly potentiated aPDI of S. aureus mediated by MB, TBO, and 1,9-dimethylmethylene blue (DMMB) and an inhibitor of Gram-negative RND pumps (phenylalanine-arginine beta-naphthylamide) also potentiated light-mediated killing of P. aeruginosa by TBO. The discovery that these efflux pump inhibitors (EPI) had a dramatic effect in potentiating the killing effect of aPDI with phenothiazinium dyes suggested that EPI may have some clinical application in this field. Kishen et al. [26] evaluated the ability of MB and rose bengal (RB) to inactivate biofilms of Enterococcus faecalis. The role of a specific microbial EPI, verapamil hydrochloride in the MB-mediated aPDI of E. faecalis biofilms was also investigated. The results showed that E. faecalis biofilms exhibited significantly higher resistance to aPDI when compared with E. faecalis in suspension. aPDI with cationic MB produced superior inactivation of E. faecalis strains in a biofilm along with significant destruction of biofilm structure when compared with anionic RB. The ability to inactivate biofilm bacteria was further enhanced when the EPI was used with MB. These experiments demonstrated the advantage of MB combined with an EPI to inactivate biofilm bacteria and disrupt biofilm structure. In another study, Prates et al. [27] investigated whether the major fungal MEPs affected the efficiency of MB-mediated aPDI in C. albicans and tested specific inhibitors of these efflux systems to potentiate aPDI. C. albicans wild-type and mutants that over-expressed two classes of MEPs [ATP-binding cassette (ABC) and major facilitator superfamily (MFS)] were tested for aPDI using MB as the PS with and without addition of MEP inhibitors. The uptake and cytoplasmic localization of photosensitizer were ascertained using laser confocal microscopy. Their results showed that ABC MEP over-expression reduced MB accumulation and aPDI killing more than MFS MEP over-expression. Furthermore, by combining MB-aPDI with the ABC inhibitor verapamil, fungal killing and MB uptake were potentiated, while by combining MB-aPDI with the MFS inhibitor INF271, fungal killing and MB uptake were inhibited.

Photosensitizers and P-glycoprotein in cancer cells

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Photosensitizers and P-glycoprotein in cancer cells Early studies The first studies that questioned whether efflux pumps played a role in the effectiveness of PDT for cancer date from as long ago as 1989. Cowled and Forbes [28] had studied several vasoactive drugs in combination with hematoporphyrin derivative (HPD) (either both injected together into tumor-bearing mice 24 h before light delivery) or else the HPD was injected 24 h and vasoactive drugs were injected 2 h before light. Noradrenaline, propranolol, hydralazine and phenoxybenzamine inhibited PDT destruction of tumors if injected 2 h before irradiation. This inhibition was associated with reduced uptake of HPD into tumors. There was no inhibition if irradiation was delayed until 24 h after administration of the vasoactive drug, presumably because HPD uptake continued after the drugs had ceased to affect the vasculature. Verapamil enhanced photodynamic destruction of tumors when administered concurrently with HPD (24 h before light) and the enhancement was associated with increased uptake of HPD into tumors. Verapamil neither increased uptake of HPD nor enhanced PDT killing of cells in vitro. When verapamil was administered after irradiation, regrowth of tumors was inhibited. Other calcium channel blocking agents (diltiazem and nifedipine) had no effect on uptake of HPD or inhibition of regrowth of tumors after PDT. Gossner et al. [29] carried out a similar study (injecting verapamil concurrently with HPD 24 h before light delivery in two different mouse tumor models. They concluded “verapamil does not increase PDT damage concurrently administered with low doses of DHE (HPD) in our in vivo models.”

P-glycoprotein P-glycoprotein 1 (permeability glycoprotein, abbreviated as P-gp) also known as multidrug resistance protein 1 (MDR1) or ATP-binding cassette sub-family B member 1 (ABCB1) or cluster of differentiation 243 (CD243) is a cell membrane protein that pumps many foreign substances out of cells. It is an ATP-dependent efflux pump with broad substrate specificity. It exists in animals, fungi and bacteria and likely evolved as a defense mechanism against harmful substances. P-gp is widely distributed throughout the body. It is particularly expressed in the intestinal epithelium where it pumps xenobiotics that may have been inadvertently ingested (such as toxins or drugs) back into the intestinal lumen, in liver cells where it pumps them into the bile via the bile duct, in the cells of the proximal tubule of the kidney where it pumps them into the urine (in the proximal tubule), and in the capillary endothelial cells composing the bloodbrain barrier and blood-testis barrier, where it pumps them back into the capillaries thus preserving sensitive organs from damage. P-gp is a glycoprotein that in humans is encoded by the ABCB1 gene. P-gp is a well-characterized ABC-transporter (which transports a wide variety of substrates across extracellular and intracellular membranes) of the MDR/TAP subfamily. The normal excretion of xenobiotics back into the gut lumen by P-gp reduces the efficacy of some orally

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administered pharmaceutical drugs, which have been found to be P-gp substrates. Moreover, some cancer cells also over-express large amounts of P-gp, rendering these cancer cells multidrug resistant. Many drugs inhibit P-gp, typically incidentally rather than as their main mechanism of action, and some foods do as well. These substances are often designated P-gp inhibitors. Table 1 lists some of the studies asking whether PS were indeed substrates of P-glycoprotein. As can be seen from Table 1, most PS (especially those derived from tetrapyrrole structures such as porphyrins and chlorins) were found not to be substrates of P-gp [30, 33–35]. The exception was PS compounds derived from rhodamine dyes that were indeed found to be substrates [32, 36]. As far as can be told, there were no other published studies that specifically mentioned other multidrug efflux pumps besides ABCG2 described below.

TABLE 1 Photosensitizer structures investigated as substrates of P-glycoprotein Efflux pump

Result

Author date

PS is not substrate

Dellinger [30] et al. (1992)

PS is substrate

Kessel et al. [31] (1994)

PS is substrate and damages protein

Kessel et al. [32] (1995)

Comparison

PS is not substrate

Datta et al. [33] (1997)

ALA-PPIX

Verapamil

PS is not substrate

Li et al. (2001)

MCF-7 and MCF-7/ DXR breast cancer

Chlorin (e6)

SDZ-PSC 833

Inhibitor Merlin [35] potentiates et al. (2003) PDT independent of P-gp

P-gp

Colo26 and MDCKIIMDR1

Thiorhodamines and selenorhodamines

Verapamil

These PS are substrates

P-gp

MCF7 and MCF7/DXR Foscan, mTHPC

Cell line

Photosensitizer

P-gp

ADM-RFLC and FLC Friend leukemia

Photofrin

P-gp

P388/ADR and P388 leukemia

Copper benzochlorin iminium salt (CDS1)

P-gp

P388/ADR and P388 leukemia

Tetrabromorhodamine 123

P-gP

RT112, T24, EJ138, ALA-PPIX SCaBER, MGH-U1 and MGH-U1(R) bladder cancer

P-gp

NB4/MDR and NOMO-1/ADR leukemia

P-gp

Methods/ inhibitor

Uptake by radiolabel/ DMDP

SDZ-PSC-833 PS is not a and substrate cyclosporin A

Hill et al. (2014)

Reference

[34]

[36]

Teiten et al. [37] (2001)

Role of ABCG2 as a multidrug efflux pump

259

Role of ABCG2 as a multidrug efflux pump ABC transporters and role of ABCG2 As mentioned above, the ATP-binding cassette (ABC) transporters function as a defensesystem against toxic molecules absorbed from the environment, using energy from ATPhydrolysis to pump xenobiotics out of cells, thus reducing the likelihood of organisms being poisoned [38]. ABC transporters comprise a protein superfamily, which is ubiquitous and highly conserved across various life-forms in nature. They are characterized by a consensus ATP-binding region of approximately 90–110 amino acids, including the Walker A and B motifs, between which lies the dodecapeptide linker region (Walker C region). The transporters also usually contain transmembrane (TM) domains, which generally consist of six transmembrane helices that confer substrate specificity. There are 48 known ABC transporters expressed in humans. Although the emergence of ABC transporters was a major advantage in the evolution of advanced life-forms (including humans) by providing a survival advantage, they can also function as an obstacle to drug treatment (especially cancer chemotherapy) or can be a causative factor for various diseases. These efflux pumps are often quite indiscriminate, and therefore can have a wide range of substrates, including many therapeutic drugs used to treat various diseases. In addition, there are at least 13 genetic diseases, including adrenoleukodystrophy, Tangier disease and Dubin-Johnson syndrome, which have been attributed to defects in ABC transporters [38]. ABC genes are divided into seven distinct subfamilies (ABC1, MDR/TAP, MRP, ALD, OABP, GCN20, and White) [39]. ABCG2 (ATP-binding cassette sub-family G member 2) is a protein that in humans is encoded by the ABCG2 gene. The ABCG2 gene is located on chromosome 4q22, spans more than 66 kilobases, and consists of 16 exons [40]. The membrane-associated protein encoded by this gene is classified in the large superfamily of ATP-binding cassette (ABC) transporters. ABCG2 is a member of the White subfamily. ABCG2 has sometimes been referred to as the “Breast Cancer Resistance Protein” BCRP, and functions as a xenobiotic transporter playing a role in multi-drug resistance to chemotherapeutic drugs including mitoxantrone and camptothecin analogs. Significant expression of this protein has been observed in the placenta [41], and it has been shown to have a role in protecting the fetus from xenobiotics in the maternal circulation [42]. ABCG2 has also been shown to play protective roles in blocking absorption of potentially toxic foreign molecules at the apical membrane of the intestines, and at the blood-testis barrier, the blood-brain barrier, and the cell membranes of hematopoietic progenitor cells and other stem cells [43]. ABCG2 occurs in the apical membranes of the liver and kidney, where it enhances excretion of xenobiotics [44]. In the lactating mammary gland, it plays a role in allowing the secretion of important vitamins such as riboflavin and biotin into breast milk [45].

ABCG2 in cancer stem cells ABCG2 is widely expressed in normal stem cells, and it is currently thought that ABCG2 plays an important role in stem cell proliferation and the maintenance of the stem cell

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phenotype. ABCG2 has also been found to be involved with the “side population” phenotype in cancer stem cells (CSCs). Side population is a term derived from flow cytometry, denoting a sub-population of cells that is distinct from the main population, on the basis of the markers employed. Cells in a side population have distinguishing biological characteristics depending on the markers used in identifying them. Side populations (SP) have been identified in cancer cell suspensions and are considered to be the cells that are best at effluxing chemotherapy drugs, accounting for their resistance to chemotherapy. Moreover, ABCG2 expression in tumors may contribute to their formation and progression [46]. Taking into account that the SP phenotype is mainly mediated by ABCG2 and the conserved expression of ABCG2 in stem cells, it has been suggested that ABCG2 may serve as a biomarker of CSCs. Since ABCG2 functions as a high capacity efflux transporter with a wide range of substrates including various chemotherapy drugs, it has been shown to participate in the multidrug resistance of tumors, which is responsible for the eventual failure of many (if not most) usual chemotherapy regimens [47]. CSCs have also been held responsible for the emergence of multi-drug chemotherapy resistance and eventual cancer recurrence. The correlation between the occurrence of the SP and chemoresistance has suggested a close link between ABCG2 and CSCs. Elevated expression of ABCG2 has been observed in a number of putative CSCs from retinoblastoma [48], lung [49], liver [50] and pancreatic cancer [51]. In addition, ABCG2 and CD133 (the widely employed CSC marker) are co-expressed in melanoma [52] and pancreatic carcinoma cell lines [53]. ABCG2 + populations show evidence for self-renewal, generating both ABCG2 + and ABCG2
daughter cells during culture, and have a higher proliferative activity. Moreover, other markers of progenitor cells including cytokeratin 19 and α-fetoprotein are over-expressed in ABCG2 + subpopulations [54].

ABCG2 and photosensitizer efflux The role of ABCG2 in mediating the efflux of dietary tetrapyrrole derivatives was discovered by accident in 2003. ABCG2(
/
) mice were constructed by Jonker et al. in order to study whether anti-cancer drugs such as topotecan could be administered orally in the absence of ABCG2 transporters [55]. The oral availability of topotecan was increased sixfold in ABCG2(
/
) mice, indicating that intestinal ABCG2 did indeed limit the absorption of topotecan from the gut. The ABCG2(
/
) mice did not display any gross signs of phenotypical abnormality, until a few animals suddenly developed severe necrotic ear lesions. Only mice housed in cages on the top shelf of the vivarium racks (closest to the room lighting) developed these lesions, suggesting they were caused by some form of phototoxicity. Further studies showed that all of the ABCG2(
/
) mice were liable to develop ear lesions when exposed to standard fluorescent light, but only when fed with mouse chow containing alfalfa leaf concentrate. This ingredient in mouse chow is known to contain porphyrin and/or chlorin-type intermediates [56]. Phototoxic ear lesions developed 1 week after feeding with this chow, and in some cases lesions also appeared on the tail, snout, and eyes. Phototoxicity was never observed in wild-type mice. Erythrocyte levels of the heme precursor, protoporphyrin IX (which is structurally related to pheophorbide a) were increased 10-fold. Transplantation with wild-type mouse bone-marrow cured the protoporphyria, and reduced the

Role of ABCG2 as a multidrug efflux pump

261

phototoxicity suffered by the mice. It was also pointed out there are various reports that cattle that had been fed with alfalfa have suffered outbreaks of photosensitivity. Given the foregoing information, it was not at all surprising when a number of PS that are used for PDT of cancer, turned out to be substrates of ABCG2 [57]. Table 2 summarizes a set of studies looking at PS with different structures and whether or not they are substrates of ABCG2. Robey et al. reported that pheophorbide a (PPa), pyropheophorbide a methyl ester (MPPa), chlorin (e6), protoporphyrin IX (PPIX), hematoporphyrin IX (HPIX), were all substrates of ABCG2, while meso-tetra(3-hydroxyphenyl)porphyrin, and meso-tetra(3hydroxyphenyl)chlorin (mTHPC, Foscan) were not substrates [59]. Robey at al [68] proposed that PPa could be used as a specific fluorescent probe to measure ABCG2 expression levels in various cancer cell lines and might have potential applications in clinical biopsy samples. FTC-inhibitable PPa efflux was found to correlate with cell surface levels of ABCG2 expression as measured by the anti-ABCG2 antibody 5D3. They found that 100 μM of the cyclindependent kinase inhibitor, UCN-01 or 1 μM of the P-glycoprotein inhibitor, tariquidar could inhibit ABCG2-mediated PPa transport. They showed that this assay could be used to predict susceptibility of the cancer cells to topoisomerase inhibitors topotecan and SN-38, and that ABCG2-transfected HEK-293 cells treated with 1 μM tariquidar, and ABCG2-transfected cells were six- to sevenfold resistant to UCN-01. Liu et al. [62] described energy-dependent efflux of 2-(1-hexyloxethyl)-2-devinyl pyropheophorbide-a (HPPH, Photochlor), endogenous protoporphyrin IX synthesized from 5-aminolevulinic acid, and also benzoporphyrin derivative monoacid ring A (BPD-MA, Verteporfin) in ABCG2 + cell lines, but the first-generation photosensitizer porfimer sodium (Photofrin) and a novel derivative of HPPH conjugated to galactose were only minimally transported. Tracy et al. [69] confirmed that HPPH was a substrate of ABCG2, and suggested that the higher expression of ABCG2 in normal cells such as fibroblasts, could allow selective killing of cancer cells. Morgan et al. [58] tested a series of conjugates of PS derived from chlorophyll-a (pyropheophorbides and purpurinimides) with different groups attached at different positions on the tetrapyrrole macrocycle to examine whether a change in affinity for the ABCG2 pump, would affect the PDT effectiveness of the conjugate. Carbohydrate groups conjugated at positions 8, 12, 13 and 17 (but not at position 3) abrogated the affinity of the PS for ABCG2 regardless of structure or linking moiety. At position 3, affinity was retained with the addition of iodobenzene, alkyl chains and monosaccharides, but not with disaccharides. This observation suggests that structural characteristics at position 3 may play a role in the requirements for binding to ABCG2. The substrate PS, HPPH (2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a) did not lead to killing of the SP fraction of tumor cells, but the non-substrate PS HPPH-Gal (a galactose conjugate of HPPH) was able to eradicate these SP cells by PDT. Asashima et al. [70] reported that ABCG2 was expressed in retinal capillary endothelial cells, which form the inner blood-retinal barrier, and might function to protect the eye from phototoxic xenobiotics. They showed that the ABCG2 inhibitor Ko143 could increase the accumulation of the PS, PPa and PPIX in TR-iBRB cells (conditionally immortalized rat retinal capillary endothelial cells). Jung et al. [71] produced a doxorubicin-resistant A2780 cell line (A2780DR) by incubating A2780 with stepwise increasing concentrations of doxorubicin. A2780DR cells showed lower

TABLE 2

Photosensitizer structures investigated as substrates of ABCG2

Photosensitizer

Was an ABCG2 substrate?

Structure

Protoporphyrin IX, PPIX

NH

HO Hematoporphyrin IX HPIX

HO

OH

HO

O

Laboratory studies

O

HO

NH N

Conflicting reports Not ABCG2 substrate [59] ABCG2 substrate [60, 61]

O O N

N

HO

O

OH

HN

NH

HN HO

O

See above

N

OH

HO

ABCG2 substrate

HN

NH

Hematoporphyrin derivative (HPD)

ALA or MAL [58] (precursors) used in dermatology, skin cancer, premalignant conditions

O

HO N

Reference

In vitro and in vivo (4T1). Correlate PDT resistance with ABCG2 expression Inhibition with imatinib mesylate

HN

O

Clinical application

ABCG2 substrate

N

N

Methods

N HO

O

Many cancer types, GI, lung, brain, bile duct

[59]

Pheophorbide a PPa

NH N

HO

N

O

NH

HO

[59]

ABCG2 substrate

See above

Laboratory studies

[59]

ABCG2 substrate

Colo 26, RIF-1, BCC-1, and HEK-293 428R cells, inhibitio with imatinib mesylate, and RIF1 tumors in vivo

Head and neck cancer

[58, 62]

N HN

O O

2-[1-Hexyloxyethyl]-2-devinyl pyropheophorbide-a HPPH

O

NH N

O

Laboratory studies

O

Pyropheophorbide a

N

See above

HN

O

O

ABCG2 substrate

N HN

O OH Continued

TABLE 2 Photosensitizer structures investigated as substrates of ABCG2—Cont’d Photosensitizer

Structure

2-[1-Hexyloxyethyl]-2-devinyl pyropheophorbide-a-galactose conjugate HPPH-Gal

Was an ABCG2 substrate?

Methods

Clinical application

Not ABCG2 substrate

4T1 cells in vitro Laboratory and in vivo studies

[58]

ABCG2 substrate

In vitro HEK293 cells expressing ABCG2 Inhibition with Fumitremorgin C

Laboratory studies (affects livestock)

[63]

ABCG2 substrate

H1650 and H1650 MX50 ABCG2+ overexpressing cells. HEK-293 cells

Various cancer types (Russia and eastern Europe)

[59]

Reference

O

NH N HO

N HN

O

OH O OH

O

H N

OH Phytoporphyrin (phylloerythrin)

NH N

HO

N HN

O O

Chlorin (e6) Ce6

NH N

O O

OH

N HN OH

OH

O

Monoaspartyl chlorin (e6), NPe6

Benzoporphyrin derivative monoacid ring A BPD

O O

In vitro and Lung cancer, [60, 61] in vivo early stomach A431 and A431/ cancer (Japan) ABCG2 cells Inhibition with Fumitremorgin C

ABCG2 substrate

See above

Wet age related macular degeneration

[59]

Not ABCG2 substrate

See above

Many cancer types including head and neck

[59]

O

NH N

O meso-Tetrahydroxyphenylchlorin mTHPC

O

Not ABCG2 substrate

OH

N HN

O

O

Continued

TABLE 2

Photosensitizer structures investigated as substrates of ABCG2—Cont’d

Photosensitizer

Was an ABCG2 substrate?

Structure

Methods

Clinical application

ABCG2 substrate

HT29 cells, inhibition with proadifen (SKF525A)

Iminoacridine C-1371

ABCG2 substrate

A549 and A549/ In vitro, K1.5 and extracellular HEK293 vesicles (ABCG2 expressing)

[65, 66]

PPa-DR2

Not ABCG2 substrate

PC3, LNCaP, A549, VcaP cells. PS recognized androgen receptor and released NO on illumination

[67]

Hypericin

OH

O

OH

OH

O

OH

HO HO

Clinical trials for fluorescence cystoscopy for detection of bladder cancer

Reference

In vitro prostate cancer

[64]

Role of ABCG2 as a multidrug efflux pump

267

accumulation of PPa and were more resistant to PDT cytotoxicity than A2780. Microarray and Western blot analysis showed that ABCG2 was the only MEP whose expression was upregulated in A2780DR. A2780DR cells also showed upregulation of c-MET. Pharmacological inhibitors of PI3K (LY924) and c-MET (SU112) reversed A2780DR resistance to both doxorubicin and PPa-PDT by reducing levels of ABCG2. Selbo et al. [72] asked whether the amphiphilic PS that are used for photochemical internalization (PCI) were substrates of ABCG2. PCI is a process used to deliver macromolecular drugs into the cytosol and nucleus of cancer cells by allowing escape from endosomes. PS such as disulfonated meso-tetraphenylporphine (TPPS2a), disulfonated meso-tetraphenylchlorin (TPCS2a) and disulfonated aluminum phthalocyanine (AlPcS2a) are administered together with the macromolecular drug and subsequent light delivery leads to photodisruption of the endosomal membrane. Since PCI can be used to destroy cancer stem cells they were pleased to report that none of these PS was a substrate of ABCG2 [73]. Usuda et al. reported [60] contradictory results to those of Liu et al. [62] when they compared Photofrin to another PS used in Japan, called monoaspartyl chlorin (e6) (Npe6). Using A431 human squamous carcinoma cells and their ABCG2 over-expressing counterparts they found that the A431/ABCG2 cells were more resistant to Photofrin-PDT than A431 cells in vitro, and FTC reversed the resistance. PDT using Npe6 did not show this resistance. They examined biopsies of 81 early lung cancer lesions treated with PDT [60, 61]. Fifty seven lesions were from patients treated with Photofrin-PDT and 24 were from the NPe6-PDT patients. All of the 81 cancer lesions were ABCG2-positive (45 strongly positive and 36 positive). Photofrin-PDT gave 73.6% complete response (CR) rates with lower responses seen in ABCG2 strongly positive tumors, while Npe6-PDT gave 91.6% CR, and no difference was seen between ABCG2 expression levels. Ishikawa et al. [74] have reported that PDT itself can induce expression of ABCG2 in cancer cells. The mechanisms for this induction was proposed to be singlet oxygen mediated oxidation of the critical cysteine residues (Cys273 and Cys288) within Keap1 (Kelch-like ECHassociated protein 1) which is a substrate adapter protein for the E3 ubiquitin ligase complex formed by CUL3 and RBX1 [75]. Under normal conditions the Keap1/CUL3/RBX1 complex suppresses the activity (using ubiquitin-mediated degradation) of the transcription factor, Nrf2 (nuclear factor erythroid-derived 2). However when Keap1 is disrupted, Nrf2 is free to enter the nucleus where it can activate transcription of numerous genes related to antioxidant functions. One of these Nrf2 responsive genes, is heme-oxygenase (HO-1), whose function is to catalyze the oxidation of heme to biologically active products including carbon monoxide (CO), biliverdin, and ferrous iron. HO-1 participates in the maintenance of cellular homeostasis and plays an important protective role in the tissues by reducing oxidative stress and attenuating inflammation. Hagiya et al. [76] showed that siRNA-mediated knockdown of Nrf2 or Keap1 abrogated the induction of both ABCG2 and HO-1 by PDT. However, the time course for the induction of ABCG2 was different from that of HO-1. They proposed that the induction mechanism may therefore be indirect by the process of “trans-activation” or cross-talk [77]. Choi et al. [78] confirmed that PPa-PDT was affected by ABCG2 expression and investigated the effect of Nrf2 knockdown using a lentivirus containing a short hairpin RNA plasmid. Stable MDA-MB-231 cells with Nrf2 knockdown showed enhanced accumulation of PPa and greater PDT killing while ABCG2 expression was reduced. Similar results were obtained with MCF-7 breast cancer, HCT116 colon carcinoma, A498 renal carcinoma, and A172 glioblastoma cells.

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Francois and co-workers investigated [79] the induction of hypoxia signaling in human BeWo choriocarcinoma cells using either cobalt chloride or culture in 3% oxygen for 24–48 h. They found increased HIF-1alpha protein signaling along with decreased mRNA and protein expression of ABCG2 by 30–75%. There was impaired efflux of the substrate Hoechst 33342. A number of transcription factors known to regulate ABCG2, including AHR, NRF2 and PPARgamma, were also coordinately down-regulated by 3% oxygen in BeWo cells. By contrast ABCB6 was upregulated by hypoxia [80]. It is also known that the efficiency of PDT is reduced by induction of hypoxic signaling [81]. This is due not only to the lack of oxygen in hypoxic regions of tumors that restricts the amount of singlet oxygen that can be formed [82], but also to the induction of pro-survival pathways by hypoxia [83]. These observations suggest that in some cases the opposite effects of hypoxia may “cancel each other out.” In other words hypoxia may reduce the effects of PDT in general, but increase the effects if the PS happens to be a ABCG2 substrate. There are several known single nucleotide polymorphisms (SNPs) in the ABCG2 gene. These SNPs are thought to affect both the response of patients to chemotherapy (such as irinotecan) and also affect the risk of various diseases [84–86]. Tamura et al. [87] studied how some of these SNPs affect the ability of ABCG2 to pump out photoactive porphyrins such as hematoporphyrin. They constructed variants of the ABCG2 gene and expressed them in insect cells (Spodoptera frugiperda Sf9 cells). Some variants (Q126stop, F208S, S248P, E334stop, and S441N) were defective in hematoporphyrin transport while the F489L variant showed impaired transport activity. Flp-In-293 cells expressing the F208S, S248P, S441N, and F489L variants were sensitive to light when cells were treated with pheophorbide a. These authors suggested that the presence of these SNPs in the ABCG2 gene sequence could affect how much patients receiving PDT suffered from the most common sideeffect of the therapy, namely skin phototoxicity. At present it is unknown why some patients suffer severely from this side-effect (despite avoiding light exposure) while others do not.

Strategies to overcome ABCG2 efflux in PDT Since it was discovered that many PS are substrates of ABCG2, and that this efflux pump mechanism was particularly important in the case of cancer stem cells, several workers have investigated novel strategies to overcome the ABCG2-mediated efflux of PS [88]. These are summarized in Table 3. Because ABCG2 has an ATP-binding pocket that bears some similarities to the ATP-binding pocket of protein tyrosine kinases, it is not surprising that many of the compounds that have been tested as inhibitors of ABCG2 in PDT belong to the class of protein tyrosine kinase inhibitors (TKI) [92]. One of the first reports was from Liu et al. [62] who tested whether the TKI called imatinib mesylate (Gleevec) affected HPPH accumulation by cancer cells in vitro. They also tested its effect on in vitro and in vivo PDT efficacy. Imatinib mesylate increased accumulation of HPPH, PpIX, and BPD-MA from 1.3- to 6-fold in ABCG2 + cells, but not in ABCG2
cells, and enhanced PDT efficacy both in vitro and in vivo. Pan et al. [89] used a different ABCG2 inhibitor, FTC to test potentiation of PDT mediated by MPPa in four human glioma cell lines (U87, A172, SHG-44, and U251). The intracellular MPPa and ROS in A172 receiving MPPaPDT significantly increased after using the ABCG2 inhibitor. Both cell viability and apoptosis in A172 cells undergoing MPPa-PDT were significantly improved with FTC.

269

Strategies to overcome ABCG2 efflux in PDT

TABLE 3

Inhibitors of ABCG2 that have been used to potentiate PDT

Inhibitor

Structure

Imatinib mesylate (Gleevec)

N

N H N

N

H N N

O

Fumitremorgin C

N

O N N

O N H

Model

Reference

HPPH, PpIX, and BPD-MA in RIF1ABCG2+ cells and tumors in vivo

[62]

glioma cell lines (U87, A172, SHG-44, U251) PDT with MPPa

[89]

conditionally immortalized rat retinal capillary endothelial cells TR-iBRB with PPa and PPIX

[70]

U87MG glioma cells in vitro and as tumors in nude mice with ALA-induced PPIX

[90]

PC3, LNCaP, A549, VcaP cells, and PPa

[67]

Flp-In-293 cells and HPIX

[91]

O

Ko-143 O

O H N

N

O HN

O O

N

O

Gefitinib (Iressa) N

N

O HN

O

Cl F

Reserpine O

N

N H H O

H O

H

O

O O

O

O O

Purvalanol A

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8. Drug efflux pumps in photodynamic therapy

Ishikawa et al. [90] reported that the widely-used EGFR inhibitor, gefitinib (Iressa) could inhibit ABCG2. They suggested it could be used in an application whereby ALA-induced PPIX is employed in malignant gliomas. The idea is that ALA is administered to the patient, and then at subsequent brain surgery, intraoperative fluorescence diagnosis can be used to reveal additional infiltrations of malignant tissue within the tumor margin in the brain allowing more complete surgical resection [93]. Kim et al. [94] compared HT29 (ABCG2-high) and SW480 (ABCG2-low) colorectal cancer cells and also over-expressed ABCG2 in SW480 cells. Pyropheophorbide-a (PPPa) showed higher uptake and phototoxicity to SW480 compared to HT29 and SW480/ABCG2 cells. Pretreatment with an ABCG2 inhibitor, Ko143 [(3S,6S,12aS)-1,2,3,4,6,7,12,12aoctahydro-9-methoxy-6-(2-methylpropyl)-1,4-dioxopyrazino[10 ,20 :1,6]pyrido[3,4-b] indole-3-propanoic acid 1,1-dimethylethyl ester] [95], significantly enhanced the PDT effects in HT29 cells and SW480/ABCG2 cells. In vivo studies in a mouse xenograft tumor model, showed that PPPa-PDT of HT29 tumors could be potentiated by administration of Ko143. Barron et al. [96] also used Ko143 as an ABCG2 inhibitor in different cell lines, and reported that its effects were even more pronounced when PPIX was induced by MAL than they were when ALA was used. An et al. [91] tested five potential ABCG2 inhibitors that had previously been shown to be cyclin-dependent kinase inhibitors. They used Flp-In-293 cells and plasma membrane vesicles prepared from Sf9 insect cells and examined hematoporphyrin efflux. Out of the five compounds tested (purvalanol A, WHIP180, bohemine, roscovitine, and olomoucine) purvalanol A was the most active with WHIP180 showing lesser activity. Rapozzi et al. [67] studied PDT of prostate cancer cells (LNCaP, VCaP, and PC3) using a conjugate of PPa with a nonsteroidal anti-androgen compound called DR2 connected by a small pegylated linker. The DR2 component was designed to not only recognize the androgen receptor, but also to release nitric oxide upon photoactivation. They also reported that the conjugate was not a substrate of ABCG2 (as opposed to PPa alone), and that the ABCG2 inhibitor, reserpine could increase the cellular accumulation of Ppa but not that of the conjugate. Goler-Baron and Assaraf [65] reported a novel mechanism in which ABCG2-rich extracellular vesicles (EVs) formed in between neighboring breast cancer cells and sequestered various chemotherapeutic agents thereby keeping them away from killing the cancer cells. PS such as iminoacridines were also found to be substrates of ABCG2 and co-localized with the chemotherapeutic drug topotecan inside EVs. Light delivery potentiated the cytotoxicity in cancer cells but not in normal cells. Another approach that has been used to circumvent ABCG2-mediated efflux of PS is to deliver the PS encapsulated in a polymeric drug delivery vehicle [97]. Wang et al. [98] investigated biodegradable amine functionalized polyacrylamide (AFPAA) nanoparticles as a drug carrier for HPPH. They compared three different loading methods (encapsulation, conjugation, and post-loading). These nanoparticles formed a stable aqueous dispersion.

Conclusions and future directions The question arises just how important in the future of PDT as a medical therapy is the recognition of some PS by efflux pumps both in cancer cells and in microbial cells. The evolutionary origin of this recognition appears to be a protective mechanism against

References

271

photosensitizing tetrapyrrole molecules derived from porphyrins and chlorophylls occurring in the natural diet. The most clinically relevant PS are, PPIX induced by administration of ALA or MAL, and pheophorbide-type molecules such as HPPH (Photochlor). ALA-PDT is widely used in dermatology including non-melanoma skin cancer [99], and in photodiagnosis for bladder cancer [100], and malignant glioma [101]. There are several clinical trials of PDT using HPPH for head and neck cancer [102], oral dysplasia and early squamous cell carcinoma [103], early stage non-small cell lung cancer [104], and pre-cancerous lesions associated with Barrett’s esophagus [105]. Photofrin is still the most widely-employed PS throughout the world, but as discussed above it does not appear to be a good substrate for ABCG2 [59], although there are conflicting reports [60, 61]. Another widely used, powerful clinical PS is Foscan (mTHPC) [106], but this compound was found not to be an ABCG2 substrate [59]. Likewise Npe6 which is clinically used in Japan was found to be not a substrate [60, 61]. ® One PS that should be investigated is TOOKAD soluble that has completed clinical trials of PDT for prostate cancer [107]. Since the molecule is derived from a natural tetrapyrrole structure (bacteriochlorin), it is possible that it could be an ABCG2 substrate. Recently, another bacteriochlorin molecule (called redaporfin or LUZ11) has entered clinical trials for advanced head and neck cancer [108]. Since this molecule is a synthetic tetrapyrrole [109] it would perhaps be unlikely to be an ABCG2 substrate. The future of the clinical use of ABCG2 inhibitors to potentiate PDT is likewise uncertain. In fact the use of ABCG2 inhibitors in cancer chemotherapy in general has only progressed to a very few clinical trials [110]. Going forward, there has been a large effort in synthetic chemistry to prepare new, even more powerful anti-cancer PS with improved pharmacokinetics and biodistribution, and it should be possible to ensure that these new structures are not substrates of ABCG2 (or indeed any other efflux pump). It could be argued that it is the field of antimicrobial PDT for infections that the opportunity to use efflux pump inhibitors is most appealing. This is because the most widely used antimicrobial PS in the clinics (namely methylene blue) is an efflux pump substrate [24], and its activity (PDT microbial killing) can be improved by several logs by combination with an efflux pump inhibitor [25].

Acknowledgment MRH was supported by US NIH grants R01AI050875 and R21AI121700.

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