Parthenolide: from plant shoots to cancer roots

Accepted Manuscript Title: Parthenolide: from plant shoots to cancer roots Author: Akram Ghantous Ansam Sinjab Zdenko Herceg Nadine Darwiche PII: DOI: Reference:

S1359-6446(13)00132-3 http://dx.doi.org/doi:10.1016/j.drudis.2013.05.005 DRUDIS 1179

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Please cite this article as: Ghantous, A., Sinjab, A., Herceg, Z., Darwiche, N., Parthenolide: from plant shoots to cancer roots, Drug Discovery Today (2013), http://dx.doi.org/10.1016/j.drudis.2013.05.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Parthenolide: from plant shoots to cancer roots Akram Ghantous1, Ansam Sinjab 2, Zdenko Herceg 1 and Nadine Darwiche 3 1

International Agency for Research on Cancer, Lyon, France German Cancer Research Center (DKFZ), Heidelberg, Germany 3 Biochemistry and Molecular Genetics Department, American University of Beirut, Beirut, Lebanon Corresponding author: Darwiche, N. ([email protected]). Keywords: parthenolide; sesquiterpene lactones; cancer; stem cells; epigenetics; structure activity. Teaser: Parthenolide, a lead anticancer drug in clinical trials, possesses key chemical and biological properties to selectively target tumor and cancer stem cell-specific signaling pathways, as well as epigenetic mechanisms.

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Parthenolide (PTL), a sesquiterpene lactone (SL) originally purified from the shoots of feverfew ( Tanacetum parthenium ), has shown potent anticancer and anti-inflammatory activities. It is currently being tested in cancer clinical trials. Structure--- activity relationship (SAR) studies of parthenolide revealed key chemical properties required for biological activities and epigenetic mechanisms, and led to the derivatization of an orally bioavailable analog, dimethylamino-parthenolide (DMAPT). Parthenolide is the first small molecule found to be selective against cancer stem cells (CSC), which it achieves by targeting specific signaling pathways and killing cancer at its roots. In this review, we highlight the exciting journey of parthenolide, from plant shoots to cancer roots. History of PTL research and drug development

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Originating in the Balkans and cultivated for centuries in Europe, feverfew (Tanacetum parthenium) was used by the ancient Greeks and early Europeans for a variety of ornamental and medicinal purposes (Figure 1). The common plant name is derived from the Latin term ‘febrifugia’, which means to ‘drive out fevers’ [1,2]. Owing to its anti-inflammatory properties, feverfew has been used for centuries to relieve arthritis pain, in addition to being an anticoagulant, a digestive aid, an insect repellent, an inducer of uterine contractions during childbirth and of menstruation, and as treatment for depression, vertigo, kidney stones, infant colic and skin wounds. Nonetheless, it was not until the chronic relieving effects of feverfew leaves were reported in a British health magazine in 1978 that the plant gained in popularity as a phytomedicine [3]. The main active component of this top-selling phytopharmaceutical was identified as the SL PTL, which is mainly found in the plant shoots, or aerial parts, mainly flowers and leaves. PTL amounts in the roots are minute or undetectable [4,5]. PTL concentrations should constitute at least 0.1–0.2% of the dry plant material for the plant to be pharmacologically active [6]. To our knowledge, PTL has not been totally synthesized to date (SciFinder database[LM1]). However, commercially available PTL for research purposes has been extracted with more than 97% purity from Chrysanthemum parthenium leaves (Enzo Life Sciences and Cayman Chemical[LM2]). In 1973, PTL was shown for the first time to have antitumor properties (Figure 1) [7]. In addition, it showed potent anti-inflammatory effects. These biological properties of PTL can be attributed to its strong inhibition of nuclear factor kappa B (NF-кB), as first reported in 1997 (Figure 1) [8], by targeting multiple steps along the NFкB signaling pathway [9–12]. In fact, PTL is often sold as a pharmacological NF-кB inhibitor. Following the patenting of PTL use for cancer inhibition in 2005 (Figure 1) [13], much research has been dedicated to further deciphering the molecular mechanisms of its anticancer properties. PTL was recently shown to target epigenetic factors (Figure 1) [14–16]. In fact, because epigenetic modifications are crucial in tumor promotion, the use of PTL is being rationalized for epigenetic-based chemoprevention [16,17]. The promise that PTL holds in therapy is limited by factors that include off-target effects, particularly at high doses, and increased hydrophobicity, which limits the oral bioavailability and solubility of the drug in blood plasma. New strategies involving low and pharmacological doses of PTL, either alone or in combination with other drugs, have shown potent antitumor potential in vitro and in vivo [17–20]. In addition, the derivatization of a more hydrophilic form of PTL, DMAPT (LC-1), has helped circumvent the far-fetched therapeutic potential of this drug (Figure 2). The improved pharmacokinetic properties of DMAPT result in increased oral bioavailability, high plasma concentrations following oral administration and acceptable toxicology profiles in animal studies [21]. Furthermore, the ability of DMAPT to eradicate selectively acute myeloid leukemia (AML) stem cells led to the initiation of an ongoing phase I clinical trial for its use in hematologic malignancies in the UK (Figure 1, personal communication, Craig Jordan, University of Rochester School of Medicine, New York). In this review, we highlight the phases in PTL drug development in cancer, focusing on SAR studies, administration methods, pharmacology, antitumor activities in animal models, epigenetic mechanisms, and how CSCs (hence cancer roots) are targeted by PTL whereas normal stem cells are spared*. We only briefly describe drug combination treatments and general biological activities of PTL in cancer and inflammation because these were recently reviewed elsewhere [18,19]. PTL in SAR studies

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PTL [4α,5β-epoxy-germacra-1-(10),11-(13)-dien-12,6α-olide] belongs to the SL family of plant secondary metabolites; thus, it has a 15-carbon (15-C) structure comprising three isoprene (5-C) units and a lactone group (cyclic ester) (Figure 2) [22]. It is lipophilic and relatively stable in cell culture assays; its pre-incubation in media containing 0.5% serum for 1 and 3 days decreases its cytotoxic activity by approximately 25% and 75%, respectively, compared with freshly added PTL [23]. SLs have a broad range of biological activities consistently attributed to their often conserved α,! -unsaturated carbonyl structures, such as the α-methylene-γ-lactone or α,β-unsubstituted cyclopentenone (Figure 2). These moieties react by a Michael-type addition with biological nucleophiles, especially cysteine sulfhydryl groups, which are common in proteins [22]. During the 1970s, SLs were disqualified by the National Cancer Institute (USA) for use in therapy because their highly reactive groups were thought to interact with almost any exposed thiol moiety, leading to undesirable cytotoxicity [22,24]. Since 1991, interest in SLs has revived, particularly with the advent of novel methods for SAR analyses, which showed that some SL structures, including stereochemistry and conformational changes, probably restrain nonspecific attack onto thiol groups [22]. Typically, PTL has the αmethylene-γ-lactone but induces specific signaling pathways, enabling it to target tumor cells, including CSCs, while sparing normal stem cells. In the following sections, we highlight the important structural features required for the specificity of PTL towards crucial signaling pathways in cancer and for the increased bioavailability of the equally potent analog, DMAPT.

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NF-!B signaling pathway PTL is a well-known NF-кB inhibitor at noncytotoxic pharmacological concentrations (1–10 M), perhaps owing to its ability to target several components of the NF-кB signaling pathway. Unlike most NF-кB inhibitors, which often have antioxidant properties, the structure of PTL does not confer radical-scavenging activity [8]. Quantitative SAR of NF-κB DNA-binding affinities across 103 SLs, including PTL, correlated NF-κB inhibitory potential with the number of alkylating centers, such as the methylene lactone and conjugated keto or aldehyde functions, but not with lipophilicity [25]. The presence of an α-methylene-γ-lactone was the most important requirement for NF-κB inhibition, and electron affinity and shape index were also significant descriptors [25]. Another study, using PTL derivatives, found an effect of lipophilicity and polarity on NF-кB inhibitory potential [26]. More-polar compounds, bearing hydroxyl groups, are stronger inhibitors of NF-кB-driven transcription, possibly owing to hydrogen bonding with amino acid residues adjacent to the target cysteine in NF-кB [22,26]. The most consistently reported mechanism by which parthenolide inhibits the NF-кB pathway is by directly binding to NF-кB subunits. The exomethylene group of PTL inhibits DNA binding of the p65/NF-κB subunit by alkylating p65 cysteine-38, which is crucial for hydrogen bonding with the sugar–phosphate backbone of DNA [9]. SLs lacking the exomethylene group do not inhibit NF-кB even at 100 M concentrations [8]. The requirement for cysteine-38 is supported by evidence from p65 point mutants in which the replacement of cysteine-38 by serine abrogates the inhibition of NF-кB by PTL [9]. Moreover, pre-incubation of PTL with excess free cysteines abolishes its NF-кB inhibitory potential [8]. However, not all cysteines are targeted by PTL. For instance, p65 cysteine-120 protects against the inhibitory potential of PTL because its substitution with alanine renders p65 more sensitive to PTL [9]. This substitution destabilizes the p65 structure, causing it to be dislodged from DNA at lower PTL concentrations [9]. Unlike p65, the NF-кB p50 subunit is not prevented from DNA binding by PTL concentrations as high as 40 M [9]. Another target in the NF-кB pathway that PTL can inhibit is the IкB kinase (IKK) complex, which phosphorylates the NF-кB inhibitors IкBα and IкBβ, leading to their proteasomal degradation. A PTL affinity reagent was shown to bind to and inhibit IKKβ directly [11]. This occurred through alkylation of cysteine-179 in the activation loop of IKKβ, leading to the stabilization of the downstream IкBα and IкBβ. Mutation of cysteine-179 abolished sensitivity towards PTL [11]. Pull-down assays with biotinylated PTL, confirmed that IKKβ and p65 interact with this drug [27]. However, NF-кB DNA binding can be completely inhibited by PTL with no effect on IKK [9]. An effect on IκB might be observed at higher PTL concentrations, masking the effect on p65, which is preferentially induced at lower concentrations [9,28]. Redox balance Oxidative stress in mammalian cells is counteracted by antioxidant functions, including the glutathione and thioredoxin systems. Biotinylated PTL covalently interacts with thioredoxin, particularly at the critical redox motif of the enzyme, glutamate-cysteine ligase, glutathione peroxidase and glutathiones, reducing their intracellular pools [24,27]. Interestingly, PTL interacts with exofacial protein thiols and attenuates exofacial thioredoxin-I levels [29]. Pretreatment of lymphoma cells with glutathione reduces the interaction between PTL and exofacial thiols and inhibits PTL-mediated activation of JNK/NF-κB pathway [29]. Interaction with thiols most probably occurs via the exomethylene group of PTL. Tubulin carboxypeptidase Microtubules, which are polymers of tubulin, are major components of mitotic spindles during cell division. Tubulins can be tyrosinated by tyrosine ligase (TTL) and detyrosinated by tubulin carboxypeptidase (TCP). TTL or TCP inhibitors can target proliferative cells, such as in tumors. PTL, but not dihydroparthenolide, inhibits TCP, indicating that the unsaturated α,β-lactone is crucial for TCP inhibition [30]. Similarly, the epoxide group (Figure 2) is important because PTL partially loses TCP inhibitory activity when its epoxide is replaced by an alcohol.

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Moreover, costunolide, which is analogous to PTL bearing the α-methylene-γ-lactone but no epoxide group, does not inhibit TCP. TCP inhibition by PTL is not an indirect result of NF-кB inhibition because other NF-кB inhibitors do not target TCP [30].

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DNA methyltransferase 1 PTL induces global DNA hypomethylation in vitro and in vivo by specifically inhibiting DNA methyltransferase 1 (DNMT1) activity without affecting other DNMTs [15]. The underlying mechanism involves the α-methylene lactone of PTL, which alkylates the thiolate of cysteine-1226 in the catalytic domain of DNMT1. Currently, the commonly used DNA methylation inhibitors are the nucleoside analogs decitabine and 5-azacytidine, which show unfavorable toxicity by trapping chunks of DNMTs. The specificity of PTL towards DNMT1 makes it a useful molecular and therapeutic tool, with less toxicity than nucleoside analogs [22].

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Hydrophilic PTL analog PTL has shown high potency against leukemia, particularly AML, in which it was first shown to target CSCs. PTL at 10 M kills 84% of primary AML cells, exhibiting a 50% lethal concentration (LC50) of 1.4 M [31,32]. However, the low bioavailability of PTL is a major limitation for its use in the clinic. SAR studies using leukemic cells attempted to identify PTL derivatives with more water solubility while retaining potency [33]. Reduction of the αmethylene, epoxidation of the endocyclic alkene, or oxidation of the allylic methyl groups reduces activity [32,34,35]. However, conjugate addition of aromatics, particularly a tyramine moiety [35], or aliphatic amines [32] to the α-methylene yields compounds with similar potency and better hydrophilicity. Among those products is the aliphatic acyclic amine, DMAPT (Figure 2), which kills 93% of primary AML cells, exhibiting a LC50 of 1.7 M [32]. Among aliphatic acyclic derivatives, secondary amines, such as DMAPT bearing a dimethyl amino group, are more potent than are primary amines against AML cells. In secondary acyclic amines, analogs with at least one Nmethyl group have higher anti-leukemic activity than those lacking it, and increasing the length of the N-linked chain correlates with decreased potency. The most potent secondary acyclic amines have higher anti-leukemic activities than the most active cyclic amines, in which the optimum ring size is five or six [32]. Recently, hydrophilic PTL derivatives were developed to target CSCs specifically, using multiple myeloma as a model [36]. The fluorinated analog, 13-(3-trifluoromethylphenyl)-PTL, displays greater cytotoxicity toward multiple myeloma CSCs than toward normal stem cells; whereas 13-(4-chlorophenyl)-PTL shows the opposite trend. Fluorinated compounds are attractive clinical agents with potential use as metabolic and imaging probes, such as in positron emission tomography (PET) [36]. Antitumor mechanisms of PTL in vitro

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At the molecular level, PTL modulates signaling pathways that endow it with selective toxicity towards several tumor cell types in vitro (Table S1 in the supplementary material online). In leukemia and/or lymphoma and solid tumor cells, PTL orchestrates a series of cellular responses leading to tumor-specific cell death. The underlying molecular interactions are linked mainly to the ability of PTL to inhibit NF-κB, AP-1, mitogen-activated protein kinase (MAPK), and/or Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling and induce c-Jun N-terminal kinases (JNK) and redox stress, ultimately resulting in gene expression changes, essentially downregulating anti-apoptotic and upregulating pro-apoptotic genes (Table S1 in the supplementary material online) [18,19]. Recently, the chemotherapeutic properties of PTL were attributed to its impact on epigenetic mechanisms (Table S1 in the supplementary material online), which are frequently altered in cancer. Cancer cells express elevated histone deacetylase 1 (HDAC1) activity and are more sensitive to the actions of HDAC inhibitors than are normal cells. PTL is the first example of a small molecule that specifically depletes HDAC1 proteins without affecting other class I/II HDACs in several types of tumor cell [14,16,37]. In fact, PTL causes proteasomal-mediated degradation of HDAC1 and modulates histone structure specifically at the p21 promoter, leading to increased transcription of this gene and p21-mediated cell death [14,16]. Another epigenetic role for PTL in cancer is its ability to alter DNA methylation (Table S1 in the supplementary material online) [15]. Many tumors express elevated levels of the maintenance DNMT1 and de novo DNMT3b, both of which contribute to tumor development by silencing the expression of tumor suppressor genes. PTL induces global DNA hypomethylation in vitro and in vivo by specifically inhibiting DNMT1 in myeloid leukemias and skin cancer [15] (A. Ghantous et al., unpublished). Furthermore, PTL decreases promoter methylation and, thus, reactivation, of the tumor suppressor high in normal-1 (HIN-1) gene [15]. The discovery of novel epigenetic regulators such as PTL, with abilities to inhibit specific DNMTs and HDACs, is essential to the implementation of epigenetic therapies with less toxicity than pan-DNMT or pan-HDAC inhibitors. Antitumor mechanisms and pharmacology of PTL and DMAPT in vivo

Various tumor models and drug administration routes have been tested for PTL and DMAPT in vivo (Table 1). PTL is relatively ineffective when given orally, compared with other drug administration routes (Table 1). Moreover, although PTL can significantly inhibit tumor growth in some in vivo models, it is unable to achieve this effect proportionally to its administered dose because of its high lipophilicity and low solubility in body fluids (Table 1) [38]. This is the main reason that led to the synthesis of the more water-soluble and orally bioavailable analog, DMAPT. When administered intravenously, PTL peak plasma levels only reach 0.1 M after a dose of 4 mg/kg. DMAPT plasma levels are at least 130 times higher (13 M), when administered at just a 2.5-fold higher

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concentration (10 mg/kg) [39]. In rats, DMAPT exhibits 70% oral bioavailability, with the major metabolite being a product of mono N-demethylation [34,39]. Aminoparthenolides can undergo retro-Michael additions to regenerate the parent PTL. However, negligible PTL amounts are detected in rat plasma, and DMAPT shows <3% degradation to PTL after 24 h in cell culture media [34,39]. Low PTL plasma levels might result from precipitation upon injection and entrapment in vascular beds [39] or from the instability of PTL at an acidic pH, where it might be transformed into other metabolites in the gastric environment [26].! Importantly, even when administered orally in the form of DMAPT, or when injected in the form of PTL, both drugs, as single agents, do not eradicate tumor volumes (Table 1). It should be noted that many studies have used low suboptimal concentrations of PTL or DMAPT when used in combination with other drugs (Table 1). One possibility for the potential inefficacy of PTL or DMAPT in vivo is that their plasma protein-binding levels exceed 75% [39]. Therefore, some studies have used drug injections into or near the tumor or ex vivo pretreatment of tumor cells before xenograft implantation, successfully increasing drug potency (Table 1). Notably, PTL and DMAPT might not target all the subpopulations in a tumor, but seem to target preferentially the CSCs, which often constitute a small fraction of the tumor volume. This is emphasized by the fact that both drugs significantly and consistently inhibit tumor metastasis and engraftment, for which CSCs are crucial. This is a highly desirable property of drugs for use in anticancer therapy, but requires combination with other drugs that target the tumor. PTL and DMAPT selectively target cancer stem cells

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The cell population within a tumor is heterogeneous, with one subpopulation termed CSCs or tumor-initiating cells. ‘CSC’ is an operational term to define functionally tumor cells with the potential to self-renew, invade and engraft into new tissues [40]. In general, CSCs have slower proliferation rates compared with more differentiated cancer cells, and are often rare but believed to constitute the ‘root’ or ‘seed’ of the tumor [40,41]. They might be the main reason behind inefficient cancer eradication, standard chemotherapy resistance and tumor relapse [42]. Using acute and chronic myelogenous leukemia stem cell models, PTL was identified in 2005 as the first small molecule that selectively kills CSCs while sparing normal stem cells [31]. This finding has been reproduced in several leukemia and/or lymphoma models and solid tumors (Table 2). PTL is able to selectively target primaryand cell line-derived human CSCs by inhibiting their proliferation, sphere formation and tumor transplantation in murine and canine models (Table 2). Table 2 summarizes the potential mechanisms by which PTL selectively targets CSCs, the most consistent of which is the inhibition of NF-кB (Table 2). One reason for this selectivity might be the higher NF-кB-dependent survival in CSCs relative to normal stem cells [40,43]. However, not all pharmacological NF-кB inhibitors selectively target CSCs, possibly because they target different steps of the NF-кB pathway and/or might be lacking specificity. Using breast CSCs as a model, several NF-кB inhibitors were tested, including antioxidants, NF-кB phosphorylation inhibitors and NF-кB degradation inhibitors [44]. Of these, the antioxidants and the NF-кB phosphorylation inhibitors, including PTL, were shown to inhibit CSC sphere formation preferentially [44]. However, not all inhibitors in these two categories were equally potent in eradicating CSCs, emphasizing the role of other signaling pathways utilized by these molecules to intersect or synergize with their NF-кB inhibitory potential. The selectivity of PTL or DMAPT to CSCs might be because of their ‘double-edged sword’ [45] potential to activate simultaneously p53, by increasing its DNA binding [38] and protein levels with concomitant phosphorylation on serine 15 [21,31], and inhibiting NF-κB (Table 2). Moreover, in prostate CSCs, PTL was shown to inhibit MAPK, JAK/STAT, phosphatidylinositol-3-kinase (PI3K) and NF-кB signaling (Table 2) [38]. These constitute four of the seven major pathways for CSC survival and self-renewal [46]. In fact, compounds targeting these pathways might synergize in eradicating tumors, evidenced by the synergy between PTL and PI3K inhibitors [47] and between small molecule inhibitors of NF-кB and JAK/STAT pathways [48]. Another repeatedly reported mechanism by which PTL selectively kills CSCs is overturning their redox balance, in leukemia and/or lymphoma and solid tumors (Table 2) [24]. Thioredoxin and glutathione systems counteract cellular oxidative stress to maintain redox homeostasis, and PTL targets both systems [22]. Redox signaling is crucial for stem cell biology, wherein oxidation is usually associated with differentiation and reduction with survival and self-renewal [24,49]. Many chemotherapeutic agents induce oxidative stress yet do not differentiate or kill CSCs. However, PTL is distinguished by the virtue of preventing the targeted cell from recovering from an oxidative insult. For example, few hours of PTL treatment causes irreversible depletion, compared with modest and reversible decreases, of glutathione levels in leukemic versus normal hematopoietic stem cells, respectively [24]. The irreversible oxidative stress in PTL-treated CSCs often leads to unrepairable DNA damage, evidenced by increased H2A histone family, member X (H2AX) phosphorylation (Table 2) [21]. Interestingly, PTL does not inhibit the oxidant enzyme, myeloperoxidase, but rather triggers myeloperoxidase-dependent apoptosis in AML stem cells [50]. A new mechanism for targeting CSCs was recently reported for PTL, which was shown to preferentially target ABCB5+ melanoma CSCs (Table 2) [51]. The ABCB5 transporter is a notorious chemoresistance factor in melanoma and is considered a marker of melanoma stem cells [52]. In comparison with the first-line anti-melanoma drug, dacarbazine, which only kills up to 70% of melanoma CSCs, PTL completely abrogates melanospheres at lower doses [51]. Given that PTL is the first small molecule identified to kill CSCs selectively, its gene expression signature was in silico screening template in the Gene Expression Omnibus database used as an

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(http://www.ncbi.nlm.nih.gov/geo/), aiming to identify drugs with similar CSC-selective potential [53]. Two new agents, celastrol and 4-hydroxy-2-nonenal, were identified against AML CSCs [53]. Using similar approaches, small molecules that selectively kill CSCs have been recently identified, namely salinomycin, metformin, lapatinib and mitochondrially targeted vitamin E succinate (MitoVES) [42]. Such molecules can kill CSC fractions in tumors while often sparing the tumor bulk. Conversely, conventional chemotherapeutic drugs, including paclitaxel, often act primarily on replicating bulk tumor cells while sparing the more quiescent CSCs [44]. Therefore, combination treatments with CSC-specific and bulk-specific chemotherapeutic drugs are indispensable for complete eradication of the tumor from its roots up. Concluding remarks

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Here, we have reviewed the chemical and biological mechanisms of PTL in various in vitro and in vivo cancer models and have discussed PTL pharmacology, antitumor potential and administration methods in animal models. This sets up a framework of potential benefits and pitfalls for this promising drug for better use in a clinical setting. One advantage of PTL is its ability to target, at pharmacological (noncytotoxic) concentrations, specific signaling pathways or molecules in cancer, typically NF-кB, redox thiols, HDAC1 and DNMT1. This property emphasizes that the α-methylene-γ-lactone of PTL does not interact with any cellular cysteine, as was previously speculated, and that PTL can be used as an epidrug that exhibits specific epigenetic activities [17]. Many other drugs, particularly known NF-кB inhibitors, also solicit specific cellular responses, but PTL is a prototype because of its ability to target multiple cascades crucial for CSC survival, thus completely eradicating this resistant tumor subpopulation. Permanent epigenetic silencing in CSCs can replace reversible gene repression, locking these cells into a perpetual state of self-renewal and causing tumor relapse [54]. Whether the ability of PTL to target CSCs correlates with its epidrug potential remains an interesting topic for investigation. A pitfall of PTL is its low bioavailability, but efforts to circumvent this limitation have shown promise. The structure of the drug has been modified to obtain equally potent and hydrophilic derivatives, such as DMAPT. Moreover, the permeation of PTL into body tissues could be enhanced by conjugation to nanoparticles or polymers. For instance, the encapsulation of PTL into stealthy liposomes was tested in mouse xenografts [55]. However, PTL or its bioavailable analogs do not completely eradicate tumors. Therefore, they should be used in combination with other drugs or radiotherapy, especially given the fact that PTL sensitizes cancer cells to such therapies (Table 1) [56,57]. Importantly, other drugs have also been used in combination treatments to reduce tumor bulk efficiently, but with little success in complete eradication of the tumor, killing the metastatic seed and preventing clinical relapse. Chemical genomic approaches, based on PTL structure, have identified molecules that maximize eradication of heterogeneous tumor populations, establishing a promising roadmap towards the ultimate clinical goal of killing cancer from its roots up. *[LM3] Review literature search criteria: the information presented in this article was collected by searching PubMed, Medline and EMBASE for

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articles published between 1950 and February 2013, including electronic publications available ahead of print. The search term ‘parthenolide’ yielded approximately 550 articles, from which several were selected based on major findings in cancer, particularly pertaining to CSCs, in vivo studies, SAR and major antitumor mechanisms in vitro. Full articles were acquired, and references were checked for additional publications. Articles discussing SL SAR in cancer and inflammation were also searched for and reported, when relevant to parthenolide.

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Acknowledgments We thank Rose-Mary Boustany for her critical review of the manuscript. This work was supported by the International Agency for Research on Cancer (IARC/WHO) post-doctoral fellowship and the Lebanese National Council for Scientific Research (LNSCR; Lebanon) to A.G. and by the LNCSR and the University Research Board of the American University of Beirut grants to N.D. We apologize to those whose work could not be cited because of space limitations.

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Crooks, P.A. et al. (2012) Use of parthenolide derivatives as antileukemic and cytotoxic agents. University of Kentucky [LM4][LM5] Peese, K. (2010) New agents for the treatment of leukemia: discovery of DMAPT (LC-1). Drug Discov. Today 15, 253–324 Nasim, S. and Crooks, P.A. (2008) Antileukemic activity of aminoparthenolide analogs. Bioorg. Med. Chem. Lett. 18, 3870–3873 Gunn, E.J. et al. (2011) The natural products parthenolide and andrographolide exhibit anti-cancer stem cell activity in multiple myeloma. Leuk. Lymphoma 52, 1085–1097 Salisbury, C.M. and Cravatt, B.F. (2008) Optimization of activity-based probes for proteomic profiling of histone deacetylase complexes. J. Am. Chem. Soc. 130, 2184–2194 Kawasaki, B.T. et al. (2009) Effects of the sesquiterpene lactone parthenolide on prostate tumor-initiating cells: an integrated molecular profiling approach. Prostate 69, 827–837 Cheng, D. et al. (2005) Analytical method development and pharmacokinetics studies with parthenolide (NSC 157035) and a water-soluble analog (NSC 734325). In AACR Meeting Abstracts (Vol. 1), pp. 988, AACR Valent, P. et al. (2012) Cancer stem cell definitions and terminology: the devil is in the details. Nat. Rev. Cancer 12, 767–775 Guzman, M.L. and Jordan, C.T. (2005) Feverfew: weeding out the root of leukaemia. Expert Opin. Biol. Ther. 5, 1147–1152 Zobalova, R. et al. (2011) Drugs that kill cancer stem-like cells. In Cancer Stem Cells Theories and Practice (Shostak, S., ed.), pp. 361–378, InTech Zhou, J. and Zhang, Y. (2008) Cancer stem cells: models, mechanisms and implications for improved treatment. Cell Cycle 7, 1360–1370 Zhou, J. et al. (2008) NF-kappaB pathway inhibitors preferentially inhibit breast cancer stem-like cells. Breast Cancer Res. Treat. 111, 419– 427 Dey, A. et al. (2008) Double-edged swords as cancer therapeutics: simultaneously targeting p53 and NF-kappaB pathways. Nat. Rev. Drug Discov. 7, 1031–1040 Dreesen, O. and Brivanlou, A.H. (2007) Signaling pathways in cancer and embryonic stem cells. Stem Cell Rev. 3, 7–17 Hassane, D.C. et al. (2010) Chemical genomic screening reveals synergism between parthenolide and inhibitors of the PI-3 kinase and mTOR pathways. Blood 116, 5983–5990 Ivanenkov, Y.A. et al. (2011) Small molecule inhibitors of NF-kB and JAK/STAT signal transduction pathways as promising antiinflammatory therapeutics. Mini Rev. Med. Chem. 11, 55–78 Shi, X. et al. (2012) Reactive oxygen species in cancer stem cells. Antioxid. Redox Signal. 16, 1215–1228 Kim, Y.R. et al. (2010) Myeloperoxidase expression as a potential determinant of parthenolide-induced apoptosis in leukemia bulk and leukemia stem cells. J. Pharmacol. Exp. Ther. 335, 389–400 Czyz, M. et al. (2013) Parthenolide reduces the frequency of ABCB5-positive cells and clonogenic capacity of melanoma cells from anchorage independent melanospheres. Cancer Biol. Ther. 14, 135–145 Schatton, T. et al. (2008) Identification of cells initiating human melanomas. Nature 451, 345–349 Hassane, D.C. et al. (2008) Discovery of agents that eradicate leukemia stem cells using an in silico screen of public gene expression data. Blood 111, 5654–5662 Widschwendter, M. et al. (2007) Epigenetic stem cell signature in cancer. Nat. Genet. 39, 157–158 Liu, Y. et al. (2008) A potential target associated with both cancer and cancer stem cells: a combination therapy for eradication of breast cancer using vinorelbine stealthy liposomes plus parthenolide stealthy liposomes. J. Control Release 129, 18–25 Won, Y.K. et al. (2004) Chemopreventive activity of parthenolide against UVB-induced skin cancer and its mechanisms. Carcinogenesis 25, 1449–1458 Zuch, D. et al. (2012) Targeting radioresistant osteosarcoma cells with parthenolide. J. Cell. Biochem. 113, 1282–1291 Wang, C. et al. (2012) Combined effects of FLT3 and NF-kappaB selective inhibitors on acute myeloid leukemia in vivo. J. Biochem. Mol. Toxicol. 26, 35–43 Spagnuolo, P.A. et al. (2013) Inhibition of intracellular dipeptidyl peptidases 8 and 9 enhances parthenolide’s anti-leukemic activity. Leukemia http://dx.doi.org/10.1038/leu.2013.9 Diamanti, P. et al. (2013) Parthenolide eliminates leukemia-initiating cell populations and improves survival in xenografts of childhood acute lymphoblastic leukemia. Blood 121, 1384–1393 Shanmugam, R. et al. (2011) A water soluble parthenolide analog suppresses in vivo tumor growth of two tobacco-associated cancers, lung and bladder cancer, by targeting NF-kappaB and generating reactive oxygen species. Int. J. Cancer 128, 2481–2494 Kishida, Y. et al. (2007) Parthenolide, a natural inhibitor of Nuclear Factor-kappaB, inhibits lung colonization of murine osteosarcoma cells. Clin. Cancer Res. 13, 59–67

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33 34 35 36

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17 Schneider-Stock, R. et al. (2012) Epigenetic mechanisms of plant-derived anticancer drugs. Front. Biosci. 17, 129–173 18 Mathema, V.B. et al. (2012) Parthenolide, a sesquiterpene lactone, expresses multiple anti-cancer and anti-inflammatory activities. Inflammation 35, 560–565 19 Kreuger, M.R. et al. (2012) Sesquiterpene lactones as drugs with multiple targets in cancer treatment: focus on parthenolide. Anticancer Drugs 23, 883–896 20 Pajak, B. et al. (2008) Molecular basis of parthenolide-dependent proapoptotic activity in cancer cells. Folia Histochem. Cytobiol. 46, 129– 135 21 Guzman, M.L. et al. (2007) An orally bioavailable parthenolide analog selectively eradicates acute myelogenous leukemia stem and progenitor cells. Blood 110, 4427–4435 22 Ghantous, A. et al. (2010) What made sesquiterpene lactones reach cancer clinical trials? Drug Discov. Today 15, 668–678 23 Lesiak, K. et al. (2010) Parthenolide, a sesquiterpene lactone from the medical herb feverfew, shows anticancer activity against human melanoma cells in vitro. Melanoma Res. 20, 21–34 24 Pei, S. and Jordan, C.T. (2012) How close are we to targeting the leukemia stem cell? Best Pract. Res. Clin. Haematol. 25, 415–418 25 Siedle, B. et al. (2004) Quantitative structure-activity relationship of sesquiterpene lactones as inhibitors of the transcription factor NFkappaB. J. Med. Chem. 47, 6042–6054 26 Dell’Agli, M. et al. (2009) Inhibition of NF-kB and metalloproteinase-9 expression and secretion by parthenolide derivatives. Bioorg. Med. Chem. Lett. 19, 1858–1860 27 Nasim, S. et al. (2011) Melampomagnolide B: a new antileukemic sesquiterpene. Bioorg. Med. Chem. 19, 1515–1519 28 Merfort, I. (2011) Perspectives on sesquiterpene lactones in inflammation and cancer. Curr. Drug Targets 12, 1560–1573 29 Skalska, J. et al. (2009) Modulation of cell surface protein free thiols: a potential novel mechanism of action of the sesquiterpene lactone parthenolide. PLoS ONE 4, e8115 30 Fonrose, X. et al. (2007) Parthenolide inhibits tubulin carboxypeptidase activity. Cancer Res. 67, 3371–3378 31 Guzman, M.L. et al. (2005) The sesquiterpene lactone parthenolide induces apoptosis of human acute myelogenous leukemia stem and progenitor cells. Blood 105, 4163–4169 32 Neelakantan, S. et al. (2009) Aminoparthenolides as novel anti-leukemic agents: Discovery of the NF-kappaB inhibitor, DMAPT (LC-1). Bioorg. Med. Chem. Lett. 19, 4346–4349

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63 Idris, A.I. et al. (2009) Pharmacologic inhibitors of IkappaB kinase suppress growth and migration of mammary carcinosarcoma cells in vitro and prevent osteolytic bone metastasis in vivo. Mol. Cancer Ther. 8, 2339–2347 64 Nakabayashi, H. and Shimizu, K. (2012) Involvement of Akt/NF-kappaB pathway in antitumor effects of parthenolide on glioblastoma cells in vitro and in vivo. BMC Cancer 12, 453 65 Sweeney, C.J. et al. (2005) The sesquiterpene lactone parthenolide in combination with docetaxel reduces metastasis and improves survival in a xenograft model of breast cancer. Mol. Cancer Ther. 4, 1004–1012 66 Kim, S.L. et al. (2012) Parthenolide suppresses tumor growth in a xenograft model of colorectal cancer cells by inducing mitochondrial dysfunction and apoptosis. Int. J. Oncol. http://dx.doi.org/10.3892/ijo.2012.1587 67 Kim, S.L. et al. (2013) Synergistic antitumor effect of 5-fluorouracil in combination with parthenolide in human colorectal cancer. Cancer Lett. 41, 1547–1553 68 Oka, D. et al. (2007) Sesquiterpene lactone parthenolide suppresses tumor growth in a xenograft model of renal cell carcinoma by inhibiting the activation of NF-kappaB. Int. J. Cancer 120, 2576–2581 69 Kim, I.H. et al. (2012) Parthenolide-induced apoptosis of hepatic stellate cells and anti-fibrotic effects in an in vivo rat model. Exp. Mol. Med. 44, 448–456 70 Vegeler, R.C. et al. (2007) Effect of celecoxib and novel agent LC-1 in a hamster model of lung cancer. J. Surg. Res. 143, 169–176 71 Zhang, D. et al. (2009) Nuclear factor-kappaB inhibition by parthenolide potentiates the efficacy of Taxol in non-small cell lung cancer in vitro and in vivo. Mol. Cancer Res. 7, 1139–1149 72 Gao, Z.W. et al. (2010) Paclitaxel efficacy is increased by parthenolide via nuclear factor-kappaB pathways in in vitro and in vivo human non-small cell lung cancer models. Curr. Cancer Drug Targets 10, 705–715 73 Uchibori, R. et al. (2013) NF-kappaB activity regulates mesenchymal stem cell accumulation at tumor sites. Cancer Res. 73, 364–372 74 Yip-Schneider, M.T. et al. (2007) Suppression of pancreatic tumor growth by combination chemotherapy with sulindac and LC-1 is associated with cyclin D1 inhibition in vivo. Mol. Cancer Ther. 6, 1736–1744 75 Yip-Schneider, M.T. et al. (2008) Effect of celecoxib and the novel anti-cancer agent, dimethylamino-parthenolide, in a developmental model of pancreatic cancer. Pancreas 37, e45–e53 76 Wang, W. et al. (2009) A novel combination therapy with arsenic trioxide and parthenolide against pancreatic cancer cells. Pancreas 38, e114–e123 77 Taguchi, T. et al. (2006) Suppressive effects of dehydroepiandrosterone and the nuclear factor-kappaB inhibitor parthenolide on corticotroph tumor cell growth and function in vitro and in vivo. J. Endocrinol. 188, 321–331 78 Shanmugam, R. et al. (2006) Restoring chemotherapy and hormone therapy sensitivity by parthenolide in a xenograft hormone refractory prostate cancer model. Prostate 66, 1498–1511 79 Shanmugam, R. et al. (2010) A water-soluble parthenolide analogue suppresses in vivo prostate cancer growth by targeting NFkappaB and generating reactive oxygen species. Prostate 70, 1074–1086 80 Tanaka, K. et al. (2005) Prevention of the ultraviolet B-mediated skin photoaging by a nuclear factor kappaB inhibitor, parthenolide. J. Pharmacol. Exp. Ther. 315, 624–630 81 Sohma, I. et al. (2011) Parthenolide, an NF-kappaB inhibitor, suppresses tumor growth and enhances response to chemotherapy in gastric cancer. Cancer Genomics Proteomics 8, 39–47 82 Yi, J. et al. (2010) [Effect of parthenolide on leukemia K562 cells and its leukemia stem cells]. Zhongguo Zhong Yao Za Zhi 35, 219–222 83 Birnie, R. et al. (2008) Gene expression profiling of human prostate cancer stem cells reveals a pro-inflammatory phenotype and the importance of extracellular matrix interactions. Genome Biol. 9, R83

Page 7 of 15

cr

ip t AML

7.5 M PTL

Ex vivo pretreatment before tumor cell implantation 5 M; 100 Ex vivo mg/kg DMAPT pretreatment before tumor cell implantation; gavage; IV

4–10 mg/kg PTL

Ex vivo pretreatment before tumor cell implantation IV, IP

IP

10 g/kg PTL

IP

2.5 M PTL

10 M; 40 mg/kg PTL

Tumor methodology

Animal model

Antitumor mechanism

Refs

18 h

Primary human AML cells injected IV

NOD/SCID mice

Decreased engraftment by >80% of leukemic stem cells

[31]

18 h pretreatment ex vivo; 1 h–12 days in vivo with daily doses

24 h

Treatment A: single 10 mg/kg dose (IV); treatment B: five single doses of 4 mg/kg within 7 days (IP). Both treatments started after tumors reached 100–200 mm3 Treatment started 21 days after transplantation, thrice daily for 21 more days

Treatment started when tumor volume reached 100–300 mm3, and repeated every 2 days for a total of 16 days 24 h

Ac

100 mg/kg DMAPT

Treatment durationc

ce pt

10 M PTL

ALL

Drug delivery

M an

Drug doseb

ed

Cancer type

us

Table 1. Antitumor activities of PTL or DMAPT in vivoa

Ex vivo pretreatment before tumor cell implantation Ex vivo pretreatment before tumor cell implantation; IV

20–24 h pretreatment ex vivo; 9 days in vivo with daily doses after level of engrafted human cells !5%

Primary human AML cells injected IV in mice; canine leukemia cells injected IV (1 and 2 mouse transplantation models) Primary human AML cells injected IV

NOD/SCID mice; dogs with Decreased engraftment by >80% of cells CD34+ leukemia pretreated ex vivo; induced activation of Nrf2 stress response and !H2AX and inhibited NF-!B in mice given 100 mg/kg orally for 1 h; decreased proportion of CD34+ cells in dogs treated IV or orally for 3–12 days

[21]

NOD/SCID mice

Eradicated engraftment of myeloperoxidase-high AML cells

[50]

MV4-11 cells injected SC

Athymic nu/nu mice

Treatment A decreased global hypomethylation by 30%; treatment B decreased tumor growth by 37% and DNMT1 expression

[15]

Primary human AML cells injected IV (1 and 2 mouse transplantation models) THP-1 cells injected SC

NOD/SCID mice

No significant effect on tumor burden, as assessed by proportion of human CD45+ cells in mouse bone marrow, except in combination with PI3K/mTOR inhibitors

[47]

CD34+ primary human AML cells injected in femur

Athymic BALB/c nude mice Decreased tumor growth by 27%, increased apoptosis, decreased Bcl-2 and cyclin D1 expression, and inhibited NF-!B NOD/SCID mice No significant effect on tumor cell engraftment, except in combination with dipeptidyl peptidase inhibitor

Primary B-ALL and T- NSG mice ALL and sorted B-ALL (CD34+/CD19+, CD34+/CD19–, or CD34–) or T-ALL (CD34+/CD7–) cells injected IV (1 and 2 mouse

Decreased engraftment by >80% of cells pretreated ex vivo in most cases; increased survival in all animals; inhibited NF-!B

[58]

[59]

[60]

Page 8 of 15

ip t Treatment started 1–7 days post-tumor implantation, twice daily up to 60 days Once daily for 17 (IV model) or 25 (SC model) days

1 mg/kg PTL

IP

Once daily for 10 days

Brain

10 mg/kg PTL

IP

Once daily for 22 days

Breast

40 mg/kg PTL

Gavage

10 mg/kg PTL

IV

In ‘metastasis survival model’, tumors removed after 6 weeks’ implantation and treatment started 1 day later and continued once daily for 45 more days. In ‘orthotopic model’, tumors were left in place, and treatment started 2 weeks after implantation and continued once daily for 28 more days One dose on 5th and another on 8th day after tumor cell implantation, then mice sacrificed on day 18

4 mg/kg PTL

IP

ed

Kidney

Liver

3 g/mouse next to the tumor; 10 mg/kg by gavage PTL 4 mg/kg PTL

ce pt

2.5 mg/kg PTL

Treatment started 5 days post-tumor implantation, thrice weekly, for 23 more days IP Treatment started 5 days post-tumor implantation, thrice weekly, for 23 more days Peritumor injection, Once daily by gavage or thrice weekly gavage next to the tumor for 6 weeks

Ac

Colon

IP

cr

IP, IV

Bone

Gavage

us

100 mg/kg DMAPT 1 mg/kg PTL

W256 cells injected into the cardiac

W256 rats

U87MG cells injected intracerebrally

Athymic BALB/c nu/nu mice

M an

Bladder

transplantation models) UMUC-3 cells injected Athymic nude mice SC LM8 cells injected SC C3H mice or IV

Thrice weekly 1 h before thioacetamide injections, for 2 weeks

TMD231 cells injected Athymic nude mice into mammary fat (female) pad

Decreased tumor growth by 60%; increased p21 and decreased TRAF-2 expression In both models, PTL decreased pulmonary metastasis only when injected on same day as tumor cells and not later, concomitant with decreased NF-!B and VEGF levels Decreased size of osteolytic lesions by up to 75%; reduced tumor size in metaphysis of proximal tibia, osteoclast numbers, eroded surface and number of mononucleated TRAcP+ cells within bone marrow cavity; prevented trabecular bone loss and increase in trabecular separation at proximal tibia Decreased tumor volume by 24%; reduced microvessel density, VEGF and MMP-9 expression In metastasis model: increased survival from 34% to 64%; inhibited NF-!B and decreased metastatic index and lung metastasis deposits. In orthotopic model: decreased tumor growth by 25% and reduced serum levels of IFN! and TNF!

[61] [62]

[63]

[64]

[65]

MCF-7 cells injected SC (1 and 2 mouse transplantation models) HT-29 cells injected SC

BALB/c nude mice (female) PTL, alone or in liposome, decreased tumor volume by 65%

[55]

Athymic nude mice

[66]

SW620 cells injected SC

Athymic nude mice

Decreased tumor volume by 44%; decreased angiogenesis; increased apoptosis Decreased tumor volume by 19%

OUR-10 cells injected SC

Athymic nude mice

Peritumor injection or gavage decreased tumor volume by up to 65%; decreased NF!B, Bcl-x(L), IL-8, VEGF, Cox-2 and MMP-9 expression

[68]

Thioacetamide injected IP

Sprague–Dawley rats

Ameliorated liver fibrosis and restored liver enzyme (AST and ALT) and albumin levels; decreased !-smooth muscle actin and TGF!1 expression; reduced portal inflam mation and intralobular degeneration; induced apoptosis in hepatic stellate cells

[69]

[67]

Page 9 of 15

ip t 5 mg/kg (PTL)

IP

5 mg/kg

IP

Treatment started 2 weeks post-tumor implantation, thrice weekly, for 4 more weeks Thrice weekly for 4 weeks

100 mg/kg DMAPT

Gavage

Treatment started 1–7 days post-tumor implantation, twice daily up to 60 days

Mesenchym e

5 M PTL

Ex vivo pretreatment before tumor cell implantation

6h

Pancreas

Up to 40 Oral gastric lavage mg/kg DMAPT

Once daily for 37 (BxPC-3) or 43 (MiaPaCa-2) days

Up to 40 Orogastric lavage mg/kg DMAPT

Once daily for 32 weeks

Decreased tumor diameter by up to 35%

[70]

No significant inhibition of tumor growth, except in combination with Taxol; inhibited NF-!B Improved mean survival from 95 to 133 days; decreased phosphorylated I!B and cytoplasmic and nuclear expression of p65/NF-!B Decreased tumor growth by 63%; reduced metastatic lung burden; increased p21 and decreased TRAF-2 expression PTL-treated MSCs did not accumulate at tumor sites

[71]

No significant inhibition of tumor growth, except in combination with sulindac; inhibited NF-!B Decreased tumor growth by 63%; inhibited NF-!B

[74]

Athymic nude mice

Decreased tumor growth by 20%

[76]

Treatment started 7 days post-tumor AtT20 cells injected implantation, once daily for 14 more days SC

BALB/C nu/nu mice

[77]

Treatment started 14 days post-tumor CWR22Rv1 cells implantation, once daily for 36 more days injected SC

Athymic nude mice (male)

No significant inhibition of tumor growth, except in combination with dehydroepiandrosterone No significant effect on tumor growth, except in combination with docetaxel; inhibited bFGF- and VEGF-induced angiogenesis; decreased TRAF1 expression Decreased tumor incidence by 50% at day 85 and increased tumor latency period from 61 to 77.5 days Decreased tumor growth by 75%; reduced TRAF-2 expression and increased phosphocJUN Delayed onset of tumor incidence from 13 to 18 weeks; decreased tumor multiplicity by 30%; reduced number of large and increased number of small tumors; decreased UVB-induced epidermal thickness

Prostate

40 mg/kg PTL

Gavage

100 mg/kg DMAPT Skin

1mg/mouse PTL

Ac

40 mg/kg PTL

ce pt

Injection into tumor Treatment started 24h post-tumor implantation, twice weekly for 22 days

Pituitary

5 g/mouse into tumor PTL 8.7 mg/kg PTL

SC

cr

5 weekly doses for 32 weeks

N-nitroso-bis(2Syrian golden hamsters oxopropyl)amine injected SC A549 cells injected SC Athymic nude mice

us

Orogastric lavage

Intratracheal injection Athymic nude mice of H460 cells

M an

40 mg/kg DMAPT

ed

Lung

A549 cells injected SC Athymic nude mice or, for lung metastasis model, IV Luciferase-expressing Balb/c nu/nu mice MSCs injected into ventricle cavity of colon SW480 mice xenografts BxPC-3 or MiaPaCa-2 Athymic nude mice cells injected SC

N-nitrosobis(2oxopropyl)amine injections PANC-1 cells injected SC

Syrian golden hamsters

Gavage

Thrice weekly for 106 days

CD44+ DU145 cells injected SC

NOD/SCID mice (male)

Gavage

CWR22Rv1 or PC-3 Treatment started 7 days post-tumor implantation, once daily for 88 more days cells injected SC

Food pellets

Treatment started, once daily, 1 week before UVB, for 24 more weeks

Testosterone-depleted (female) athymic nude mice SKH-1 mice

UVB

[72]

[61]

[73]

[75]

[78]

[38]

[79]

[56]

Page 10 of 15

ip t cr BALBc nu/nu mice

Decreased proportion of peritoneal nodules by approximatley 30% with no effect on survival, except in combination with paclitaxel

Once daily for 12 days

UVB

DBA/2 mice

IP

JB6P+ cells promoted NMRI-Nu mice ex vivo then injected SC

4 mg/kg PTL

IP

Every other day over a 10-day period and then either stopped upon tumor implantation or continued for 10 additional days when tumors became palpable Treatment started 24 h post-tumor implantation, once daily for 21 more days

us

IP

MKN-45-P cells injected IP

M an

Stomach

Decreased epidermal and melanocyte hyperproliferation by up to 45% Decreased tumor volumes by up to 63%; increased p21 and decreased p65 and cyclin D1 expression

0.25 mg/kg PTL 0.25 mg/kg PTL

a

[80] [16]

[81]

Ac

ce pt

ed

Abbreviations: B-ALL, B-precursor ALL; Bcl-2, B-cell lymphoma 2; Bcl-x(L), B cell leukemia-x long; IL-8, interleukin-8; IP, intraperitoneal; IV, intravenous; MMP-9, matrix metalloproteinase-9; NOD/SCID, nonobese diabetic/severe com bined im m unode! ciency; SC, subcutaneous; T-ALL, T-precursor-ALL; TNF, tumor necrosis factor; TRAF, TNF receptor-associated factor; UVB, ultraviolet B; VEGF, vascular endothelial growth factor. b For comparison purposes, drug doses were converted to a common scale, when appropriate. c Treatment started simultaneously with tumor implantation or initiation, unless indicated otherwise.

Page 11 of 15

Table 2. Cancer stem cells selectively targeted by PTL or DMAPT a

CML Multiple myeloma Solid tumors Bone Breast

Melanoma

[21,31,50,53,59]

Primary B-ALL (CD34+CD10–, CD34+/CD19+, CD34+/CD19–, CD34–, CD133+/CD19+, or CD133+/CD19–) and T-ALL (CD34+CD7–, CD34+CD7+, or CD34–) cells, NSG xenografts of B-ALL or T-ALL Primary blast crisis CML cells (CD34+ or CD34+CD38–); K562 cell line (CD34+CD38–) RPMI-8226 and U266 cell lines (CD20+ and/or CD138–) SaOS2 and LM7 cell lines (CD133! ) MCF-7 and MDA-MB-231 sphere and side population cells (CD44+CD24–); NCR-nu/nu MCF7 xenografts Primary melanosphere-derived cells (CD133+, CD90+, and/or CD49f+) Primary bone marrow-derived mesenchymal stem cells, MSCs (CD73+, CD90+, CD105+, CD11b–, CD34–, and/or CD45–); Balb/c nu/nu MSC xenografts DU145, PC3, VCAP, and LAPC4 cell lines (CD44+); primary prostate cancer cells (CD44+ or CD133+/! 2! 1hi); NOD/SCID DU145 xenografts

ip t

NF-!B inhibition; p53 activation; !H2AX increase; oxidative stress; myeloperoxidase-dependent apoptosis; tumor cell differentiation and apoptosis; transcriptional array response to oxidative stress, unfolded proteins, and NF-!B inhibition Decreased proliferation; apoptosis; NF-!B inhibition

te

d

Prostate

Primary AML cells (CD34+, CD34+CD38–, or CD34+CD38–CD123+); primary AML cells (CD34+CD38–) with high versus low myeloperoxidase expression; NOD/SCID AML xenografts; dogs with CD34+ acute leukemias

[21,60]

Oxidative stress; apoptosis

[21,31,82]

NF-!B inhibition; apoptosis

[36]

Oxidative stress NF-!B inhibition

[57] [44,55]

Targeted cells carrying ABCB5 transporter

[51]

Inhibition of I!B phosphorylation, NF-!B activity and TNF-!–induced VCAM-1 expression

[73]

M

Mesenchyme

Refs

cr

ALL: B-ALL, T-ALL, and Childhood ALL

Antitumor mechanism

us

Leukemia/ Lymphoma AML

Tumor modelb

an

Cancer stem cell

Inhibition of src nonreceptor tyrosine kinase (phosphorylation), MAPK/ELK-1 and PI3K/NF-!B pathways, expression of ELK-1-associated genes, and STAT3 activity; alteration of the JAK/STAT pathway and focal adhesion signaling; increased p53 DNA binding; modulation of DNA binding of transcription factors involved in prostate cancer development

[38,83]

Ac ce p

a Abbreviations: B-ALL, B-precursor ALL; CML, chronic myeloid leukemia; ELK-1, Ets-like transcription factor; STAT-1/3, signal transducer and activator of transcription1/3; T-ALL, T-precursor-ALL; VCAM-1, vascular cell adhesion molecule 1 b Primary cells and cell lines are of human origin. Xenografts were done in mouse animal models.

Figure 1. Timeline of PTL research: milestones are marked in gray, discoveries with major clinical implications are marked in orange, and antitumor effects reported in cancer stem cells are marked in blue. Abbreviations: DMAPT; dimethylamino-parthenolide; NF-B, nuclear factor kappa B; PTL, parthenolide. Figure 2. Structures of (a) parthenolide and (b) dimethylamino-parthenolide (DMAPT).

Page 12 of 15

2004

• Standardization of

ce pt

active components in commercial feverfew preparation • Feverfew is a topselling American phytopharmaceutical

cr

2005

First phase I clinical trial with PTL to evaluate its pharmacokinetic properties and toxicity

2007

PTL inhibits several types of cancer stem cell

2008

• Synthesis of DMAPT: orally bioavailable PTL analog • First report on the epigenetic effect of PTL

2009

2010

Initiation of first clinical trial using DMAPT in patients with leukemia

Ac

Identification of PTL as a tumor inhibitory agent from Magnolia grandiflora

1997

ed

1973 1985 1990

PTL induces apoptosis in hematopoietic cancer stem cells

M an

PTL is purified and shown to inhibit NF-κB

us

First clinical study to assess the effectiveness of feverfew in migraine prevention

Feverfew described for migraines

1597

Use of PTL to inhibit cancer is patented

i

Figure 1

Page 13 of 15

M an

us

cr

i

Figure 2

(a) Parthenolide 1 2

10

9

ed

8 14 5

13

6

4 O

O

15

12

1 10

2

8 14 7

5

3

O 15

13

6

4

11

O

9

O

12

11

N

O

Ac

epoxide

α-methylene

ce pt

3

7

(b) DMAPT

Page 14 of 15

*Highlights (for review)

Parthenolide: from plant shoots to cancer roots Akram Ghantous1, Ansam Sinjab2, Zdenko Herceg1 and Nadine Darwiche3 1

ip t

International Agency for Research on Cancer, Lyon, France German Cancer Research Center (DKFZ), Heidelberg, Germany 3 Biochemistry and Molecular Genetics Department, American University of Beirut, Beirut, Lebanon Corresponding author: Darwiche, N. ([email protected]). 2

cr

us

an

M d te

  

This article highlights phases in parthenolide (PTL) lead anticancer drug development PTL structure–activity relationship studies reveal properties required for potency and bioavailability PTL is selective to biological and/or epigenetic targets, tumors and CSCs PTL targets multiple pathways of tumor survival, seeding and self-renewal PTL pharmacology and use in animal models have clinical implications.

Ac ce p

 

Page 15 of 15