Cell cycle modulatory and apoptotic effects of plant-derived anticancer drugs in clinical use or...

Review

10.1517/17460441.2.3.361 © 2007 Informa UK Ltd ISSN 1746-0441 361

Cell cycle modulatory and apoptotic effects of plant-derived anticancer drugs in clinical use or developmentNadine Darwiche*, Sara El-Banna & Hala Gali-Muhtasib*†

American University of Beirut, Department of Biology, Beirut, Lebanon*These authors contributed equally to the manuscript

Drugs derived from natural products, particularly from plants, are the leadsof clinically used anticancer agents. Plant-derived anticancer agents in clinicaluse consist of the Vinca alkaloids, vinblastine and vincristine, camptothecinderivatives, paclitaxel, etoposide and teniposide, homoharringtonine andelliptinium. Extensive research has led to the identification of promisingplant-derived anticancer agents in clinical development, namely flavopiridol,combretastatins and roscovitine. These compounds share common antitumoractivities and signaling pathways targeting tumor cell cycle and cell death.This review presents the discovery of plant-derived anticancer agents, theirbiologic activities, with emphasis on cell cycle and apoptotic effects andcombination strategies for treatment optimization.

Keywords: anticancer, apoptosis, cell cycle, plant

Expert Opin. Drug Discov. (2007) 2(3):361-379

1. Introduction

Cancer is the second biggest cause of death after cardiovascular disease. Worldwide,> 20 million people are living with cancer and > 7 million deaths occurred in2005 [201]. By the year 2015, cancer is estimated to kill 9 million people. The mostfrequent cancers are lung, stomach, liver, colorectal and breast.

For thousands of years, natural products derived from plants, microbes or marineorganisms have played an important role in the treatment of cancer [1,2]. In hisrecent review, Newman lists 58 natural and naturally derived anticancer drugs and11 naturally modified drugs [3]. In 2006, > 60% of all the available and presentlyused anticancer drugs were derived from natural sources [4,5]. Over the last halfcentury, most new clinical applications have involved the fighting of cancer by usingplant-derived agents [3].

In more recent history, the use of plants as a source of anticancer agents hasinvolved the isolation of active compounds. The recent advances in drug discoveryand molecular biology techniques have provided the means to standardize theisolation and characterization of plant-derived chemicals, as well as to elucidate theircellular and molecular mechanisms of action.

A promising anticancer drug approach involves the discovery of natural moleculesthat are able to specifically attack the aberrant genetic alterations and deregulatedbiochemical pathways that cause cancer, while sparing normal cells. This new gener-ation of anticancer agents targets neoplastic cells and is more selective and less toxicthan existing drugs used for cancer treatment. In selecting drug targets for cancertherapy, interest has focussed on agents that directly address signal transductionand/or cell cycle and molecular apoptotic regulators [6].

Several anticancer drugs from plant origin have either been introduced to the USmarket or are being at present tested in late-phase clinical trials. Agents in clinical use

1. Introduction

2. Plant-derived anticancer drugs

in clinical use

3. Plant-derived anticancer drugs

in clinical development

4. Conclusion

5. Expert opinion

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Cell cycle modulatory and apoptotic effects of plant-derived anticancer drugs in clinical use or development

362 Expert Opin. Drug Discov. (2007) 2(3)

can be categorized into six major classes of compounds:camptothecins, Vinca alkaloids, taxanes, podophyllotoxins,elliptinium and homoharringtonine.

In 2002, taxanes and camptothecins represented about athird of the global anticancer market [7]. Several derivatives ofall six classes have been synthesized, some of which are in clin-ical use at present. Several promising new agents are in clinicaldevelopment based on selective activity against cancer-relatedmolecular targets. These include three classes of compounds:flavopiridol, combretastatin and roscovitine [5]. Anticancercompounds derived from plants and which are in clinical useor development are summarized in Table 1.

Identifying key cell cycle and apoptotic regulators hasopened new possibilities for cancer therapy. This opportunityof molecular targeting by drugs has seen recent advances inmultiple research disciplines. The combination of structuraland functional genomics and proteomics research has led to adetailed understanding at the cellular and molecular levels ofgenes and proteins that are responsible for cancer progression.This coupled with the emerging field of bioinformatics hasallowed the identification of new therapeutic targets. In addi-tion, the use of combinatorial chemistry with high-throughputscreening for identifying and optimizing drugs complementedwith advanced structural biology has paved the way for a wholenew approach to cancer drug design and discovery.

Although not exhaustive, this review examines the role thatnaturally derived anticancer agents have had in the elucidationof some aspects of the cell cycle and cell death as well as howthese agents have assisted in the treatment of cancers of manytypes as a result of such mechanistic studies. The emphasis ofthis review is on mechanisms of cell cycle regulation and apop-tosis of plant-derived anticancer drugs that are at present usedin the clinic or that are in clinical development.

2. Plant-derived anticancer drugs in clinical use

2.1 Vinca alkaloids: general mechanismsVinca alkaloids (vincristine and vinblastine) were first iso-lated in 1958 from the plant Catharanthus roseus, a plant thatwas traditionally used for the treatment of diabetes [8,9]. Theanticancer effects of Vinca alkaloids were established in1963, and since then they have been used for the treatmentof several cancers [10]. Four types of Vinca alkaloids arewidely used. These include vincristine, vinblastine andvinorelbine, as well as the most recent vinorelbine-deriveddrug, vinflunine. The two Vinca alkaloids, vincristine andvinblastine, have been used for > 30 years for the treatmentof hematologic cancers such as multiple myeloma, acutelymphoblastic leukemia, Hodgkin’s and non-Hodgkin’slymphoma and Wilms’ tumor [5,8,11,12].

Semisynthetic derivatives of Vinca alkaloids that are lesstoxic and more active have been discovered. Vinglycinate wasobtained in 1967 from vincristine, whereas vindesine wasderived from vinblastine and registered in 1980. Vindesine is

at present used for the treatment of melanoma, acutelymphoblastic leukemia and advanced non-small-cell lungcancer (NSCLC) [5,8]. In 1989, the semisynthetic analogvinorelbine was discovered with a wide range of antitumorproperties against advanced breast cancer, NSCLC andgastrointestinal cancers [13-15].

Vinca-alkaloids are microtubule-targeting drugs that bindto α/β-tubulin dimers. They interact with microtubules bydestabilizing them [16]. Recent evidence indicates that bothmicrotubule-stabilizing drugs (taxanes) and micro-tubule-destabilizing drugs (Vinca alkaloids) inhibithypoxia-inducible factor-1α (HIF-1α) accumulation andactivity by disrupting microtubule function [17]. This findingdirectly links β-tubulin drug binding with the inhibition ofthe HIF-1α protein.

2.1.1 Vinca alkaloids: cell cycle and apoptotic mechanismsVinca alkaloids are known to produce a range of effects in cellsdepending on the drug concentration used. High concentra-tions result in extensive depolymerization of the microtubules,whereas lower concentrations induce the depolymerization ofthe microtubule network and the lowest effectiveconcentrations reduce the rate of tubulin addition [18]. Byaltering the function of the mitotic spindle, Vinca alkaloidsprevent cell cycle progression and induce mitotic block andapoptosis [19-22]. They are described to induce G2/M phasearrest before apoptosis, but the molecular mechanisms thatrelate mitotic block and apoptosis are poorly understood. Inresponse to Vinca alkaloids, apoptosis was induced in cancercells through processes that involve protein kinases, as well asthe activation of c-Jun N-terminal kinase (JNK) [23]. Whateverthe concentration of Vinca alkaloids, mitochondria appear tobe the point of convergence for the apoptotic signals [18].

The Bcl-2 family members also play a significant role inVinca alkaloid-induced apoptosis. Treatment of various can-cer cell lines with vincristine induced an increase in theexpression of proapoptotic Bax proteins and a downregulationof the antiapoptotic Bcl-2 proteins [24,25]. Leukemia cellstreated with vincristine showed an increase in the productionof mitochondrial reactive oxygen species (ROS) and the lossof mitochondrial transmembrane potential [26]. The release ofcytochrome c from the mitochondria into the cytosol was trig-gered by vincristine treatment in glioma cells [27]. In addition,several caspases are known to be activated by Vinca alkaloids[26-28] leading to the cleavage of their substrate, poly-ADP-ribose polymerase (PARP), which is involved in DNArepair [28]. Vinorelbine and vinblastine induce Bcl-2inactivation by phosphorylation, which leads to the inductionof apoptosis [23,28].

Recently, mitotic catastrophe has been described as amechanism by which vincristine induces cell death. Thismode of cell death is characterized by the occurrence ofaberrant mitosis, which leads to the formation of tetraploidnon-viable cells with multiple micronuclei that are

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Darwiche, El-Banna & Gali-Muhtasib

Expert Opin. Drug Discov. (2007) 2(3) 363

Table 1. Plant-derived anticancer drugs and analogs in clinical use or development.

Compounds, plant source (origin), year discovered

Analogs (semisynthetic and synthetic)

Cancer types

Vinblastine and vincristine (Vinca alkaloids) Catharanthus roseus (Madagascar) [10]1958 [10] and anticancer properties discovered in 1963 [11]

Vinglycinate – 1967 [194]Vindesine [9] Vinorelbine – 1989 [195] Vinflunine [31]

Vinblastine: advanced Hodgkin's disease and germ cell cancer of the testes, Kaposi’s sarcoma, bladder cancerVincristine: in combinations for acute lymphoblastic leukemia, Hodgkin’s and non-Hodgkin's lymphoma, Wilms' tumor, multiple myeloma, mantle cell lymphoma recurrent or metastatic adrenocortical cancer Vindesine: melanoma, acute lymphoblastic leukemia, advanced non-small-cell lung cancerVinorelbine: breast, non-small-cell lung cancer, gastrointestinal cancersVinflunine: Phase II clinical trials in bladder, non-small-cell lung and breast cancers

PodophyllotoxinPodophyllum peltatum (US) [34]1880 – 1882 [34]

Etoposide (VP-16) 1966Teniposide (VM-26 )1966 [35]Etoposide phosphate [40]

Podophyllotoxin dropped from clinical trials due to severe gastrointestinal side effects Etoposide: lung cancer, germ cell tumors, non-Hodgkin's and Hodgkin's lymphomas, acute leukemia, testicular cancer, prostate cancer, relapsed patients of Wilms' tumor, small-cell lung cancer, retinoblastoma, adenocortical carcinoma Teniposide: lymphomas, bronchial, retinoblastoma, recurrent or metastatic adrenocortical cancer, testicular and ovarian cancersEtoposide phosphate: small-cell lung cancer Phase I clinical trials in ovarian cancer

PaclitaxelTaxus brevifolia (Canada)1971 [196]

Docetaxel 1986 Paclitaxel: prostate, urothelial tract, testicular, bladder, ovarian, breast, non-small-cell lung cancersDocetaxel: bladder, prostate, non-small-cell lung, metastatic breast, lung, ovarian, urothelial, head and neck, gastric and prostate cancers

20(s)-CamptothecinCamptotheca acuminate (China) 1966 [7]

Topotecan – 1991 [89]Irinotecan – 1991 [90]

Camptothecin introduced into clinical trials against gastroinstestinal cancers and urinary bladder tumors until it was dropped in the 1970s due to severe toxicityTopotecan: ovarian and small-cell lung cancersIrinotecan: advanced colorectal, small- and non-small-cell lung, cervical, ovarian, gastric cancers and malignant gliomas

HomoharringtonineCephalotaxusharringtonia var. drupacea (China) [109], 1972 [109]

Undergoing Phase II clinical trials, used for treatment of acute myeloid leukemia, primary and relapsed acute non-lymphocytic leukemia, myeloid leukemia

Ellipticine Ochrosia elliptica (Australia) 1959 [121]

Elliptinium (Bleekeria vitensis)More soluble derivatives:acetate hydroxy and methyl derivatives of elliptinium [197]

Elliptinium: breast cancer Ellipticine and derivatives: osteolytic breast cancer metastases, kidney cancer, brain tumors, acute myeloblastic leukemia

Flavopiridol (semisynthetic from Rohitukine)Amoora rhituka and Dysoxylum binectariferum (India)Anticancer properties discovered in 1992 [139]

Undergoing Phase I and II clinical trials and showing effectivity against refractory renal and gastric cancers

Combretastatin A4Combretum caffrum (South African) [198]1982 [198]

Combretastatin A4 phosphate (prodrug) – 1995 [164]AVE-8062 (synthetic analog) [164] – not yet in clinical trialsOX1-4503 or combretastatin A-1 1987 – not yet in clinical trials [199]

Combretastatin A4 phosphate: Phase I clinical trials against advanced cancer, solid malignanciesPhase II clinical trials ongoing for advanced anaplastic thyroid cancer

Roscovitine (synthetic derivative of olomucine)Raphanus sativus L (China) 1997 [172]

Phase II clinical trials

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morphologically distinguished from apoptotic cells. Inresponse to vincristine, the HL60-derived HCW-2 cell linethat is highly resistant to apoptosis underwent mitoticcatastrophe, which led to caspase-3 activation andoligonucleosomal DNA degradation [29].

2.1.2 Vinflunine: the novel Vinca alkaloidVinflunine (VFL), the novel third-generation fluorinatedVinca alkaloid, was produced by the chemical alteration ofvinorelbine. Promising preclinical activity led to its use inclinical trials for solid tumors [30]. Relative to the other Vincaalkaloids, VFL has some unique properties. It binds to tubulinwith much lower affinity [31]. Such specific tubulin-bindingproperties of VFL result in a considerably different action onmicrotubule dynamics compared with the other Vinca alka-loids [21]. VFL manifests a greater potency in xenograftmodels [19]. Significant in vivo antivascular activity was notedwithin a few hours of administration [32]. In preclinicalmodels, it is much slower to induce resistance thanvinorelbine [33]. Superior antitumor activity in ECV-304tumor cell lines has been described for VFL. Phase II clinicaltrials for bladder, NSCLC and breast cancers showed promis-ing activity and it is now undergoing Phase III clinical trialswith less neurotoxic effects than the other Vinca alkaloids [30].

The mechanism by which VFL induces mitotic block andapoptosis are still unclear. VFL-induced apoptosis was shownto be mediated by caspases-3 and -7 and PARP cleavage [19].Moreover, VFL can induce Bcl-2 phosphorylation and theextent of phosphorylation is dependent on the cell type.VFL-resistant P388 monocytic leukemia cells are character-ized by a relatively high level of expression of the anti-apoptotic protein Bcl-2 [19]. A new mechanism of action ofVFL at low concentrations has been recently described in thehuman neuroblastoma SK-N-SH cell line. Low concentra-tions of VFL that inhibited neuroblastoma cell growth by 50and 70% failed to induce G2/M phase arrest, but led to thesuppression of microtubule dynamics and to a high extent ofapoptosis [18]. In addition, these concentrations induced apostmitotic G1 arrest, which was associated with an increasein p53 and p21 protein levels and nuclear translocation aswell as the upregulation of Bax and its translocation to themitochondria. On the other hand, high concentrations ofVFL blocked neuroblastoma cells at mitosis before apoptosis.

2.2 Podophyllotoxins: general mechanismsPodophyllotoxin was first isolated in 1881 by Podwyssotzki [34]

from podophyllin, the plant extract of Podophyllum peltatum.In 1942, podoplyllotoxin was discovered to have anticanceractivities, but was later discarded due to high toxicity [35].In the mid 1960s the search for semisynthetic analogs withhigher activity and lower toxicity led to the discovery of etopo-side (VP-16) and teniposide (VM-26) [35]. Due to the clinicalefficacy of etoposide, the FDA approved its use as ananticancer agent in 1983. Since then, etoposide has been usedagainst several cancers including lung, germ-cell tumors,

non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, acuteleukemia, testicular, prostate, relapsed patients of Wilms’tumor and small-cell lung cancer (SCLC) [36-38]. Teniposide isused in the treatment of lymphomas, bronchial, testicular andovarian cancers [5,39]. Due to the low solubility of etoposide,analogs such as etoposide phosphate have been discovered [40].This analog is clinically used for the treatment of recurrentovarian cancers and SCLC.

The antimitotic properties of podophyllotoxin contributeto its anticancer effects. Podophyllotoxin is known to bind tomicrotubules, preventing their formation and destabilizingthem. Etoposide’s mechanism of action differs from podo-phyllotoxin. Etoposide acts by inhibiting topoisomerase II(Topo II), an enzyme that induces transient double strandbreaks in the DNA. Etoposide stabilizes a complex of theTopo II enzyme with cleaved DNA, converting this essentialcell division enzyme into a poison and, thus, inducingpermanent breaks in the DNA and triggering cell death [41].

2.2.1 Podophyllotoxins: cell cycle and apoptotic mechanismsA recent study has provided evidence that the activity of theataxia telangiectasia Rad3 related (ATR) checkpoint kinase isrequired for the DNA replication inhibition in the earlyS phase observed in response to etoposide [42]. Followingtreatment, large nuclear foci are formed that contain replica-tion protein A, a single-stranded DNA-binding protein.Etoposide also affects the essential components found in repli-cation factories such as replicative proteins, proliferating cellnuclear antigen (PCNA) and DNA ligase I, proteins that aredispersed by the activation of ATR [42].

Etoposide is also known to induce cell cycle arrest orapoptosis by p53-dependent [43] or -independent pathways[44,45]. When treated with etoposide, the p53 pathway wasactivated in TK6 lymphoblastoid cells, resulting in S phasecell cycle arrest along with the upregulation of Fas andApo-2 receptors [43]. However, etoposide treatment inhuman NSCLC with mutated p53 induced a prolongedG2/M arrest that was accompanied with an upregulation ofthe cyclin-dependent kinase inhibitors (CDKIs), p21 andp16 [44]. In Jurkat T-lymphoma cells, null for Bax and p53,cell death was induced on etoposide treatment [45].Furthermore, c-abl and p73 were phosphorylated causingthe translocation of Bid to the mitochondria, hence,releasing cytochrome c from mitochondira and inducingcaspase activation.

Other mechanisms induced by etoposide include the mod-ulation of p38, MAPK and NF-κB pathways. In response toetoposide treatment, p38 MAPK was found to play a role inthe G2/M phase transition [47]. The addition of the specificp38 inhibitor, SB-203580, in etoposide-treated cellsdecreased the inhibition of Cdc2 and G2/M arrest, henceincreasing apoptosis. Studies on the role of the intrinsic(mitochondrial) and extrinsic (death receptor) pathways inapoptosis induction by etoposide have confirmed the

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involvement of the mitochondrial pathway in leukemic cells[49,50]. Apoptosis induced by etoposide was recently shown toinvolve the transcription factor c-Myc [51].

2.2.2 Podophyllotoxins: enhancing their efficacyDeregulation of intracellular pathways in cancer cells causesresistance to chemotherapeutic drugs. Human astrocytomaADF cells are highly resistant to etoposide treatment. A recentstudy has shown that the sensitivity to apoptosis could berestored in these cells by the combination of etoposide andproteasome inhibitors [52]. Such combination treatmentresulted in the activation of caspases and p53, suggesting thatthis may represent a promising protocol for the treatment ofresistant cell lines. In hepatocellular carcinoma and osteo-sarcoma cells, the transfection of second mitochon-dria-derived activator of caspase was found to sensitize tumorcells to etoposide-induced apoptosis [53].

2.3 Taxanes: general mechanismsTaxanes are a class of alkaloids that possess potent anticanceractivities. The two most common taxanes are paclitaxeland docetaxel, the latter being a semisynthetic analog of pacli-taxel [54]. In the 1960s, paclitaxel was discovered from thePacific Yew tree Taxus brevifolia. This drug is used for thetreatment of ovarian, breast, NSCL, prostate, urological andbladder cancers [55]. Paclitaxel is at present undergoing clinicaltrials for use in combination therapy against SCLC andNSCLC [13,56]. Docetaxel is a second-generation taxane usedfor the treatment of metastatic breast, lung, ovarian, urothe-lial, head and neck, gastric and prostate cancers [39,57]. The useof docetaxel in combination therapy against bladder cancersand advanced lung cancers is under clinical trials [58].

Unlike Vinca alkaloids that interact with themicrotubules by destabilizing them, the primary mechanismof action of taxanes is related to their ability to stabilize themicrotubules by preventing microtubule depolymerisation.Taxanes disrupt microtubule dynamics, hence inhibiting theformation of a normal spindle at metaphase, a process thatis required for subsequent chromosome segregation [59].These effects lead to cell cycle arrest in G2/M phase andeventually to apoptotic cell death. In fact, apoptosis induc-tion has been accepted as the predominant mechanism ofcell death in response to taxane chemotherapy [48]. However,recent studies have indicated that other modes of cell deathmay contribute to the overall therapeutic effects of taxanesand these include drug-induced senescence, mitotic catas-trophe and lytic necrosis [60]. The accumulation of recentdata has indicated that a functional spindle checkpoint isrequired for sensitivity to taxanes and other microtubuleinhibitors in vitro [61].

Although mechanisms of disruption of microtubuledynamics by taxanes are well known, the molecular basis ofapoptotic cell death is not well defined [62]. The cell cycleand cell death mechanisms of the anticancer activities oftaxanes in lung cancer, as well as the signal transduction

pathways involved in their mode of action, have been welldescribed in a recent review [62]. The cell cycle regulatoryand apoptotic mechanisms are described below with anemphasis on recent literature.

2.3.1 Paclitaxel: cell cycle and apoptotic mechanismsCell cycle regulation by paclitaxel varies depending on the celltype and drug concentration. High paclitaxel concentrationspromote mitotic arrest through the activation of the spindlecheckpoint. However, low doses of paclitaxel, which do notalter all of the microtubule network, upregulate p53 proteinsand induce their nuclear accumulation [63]. The nuclearcompartmentalization of p53, in turn, leads to the expressionof a wide network of signals involved in apoptosis inductionthrough the extrinsic death receptor or the intrinsicmitochondrial pathways. These events have been reported inhuman neuroblastoma and ovarian and breast carcinomacells that have undergone treatment with low doses of pacli-taxel [46,64]. Interestingly, in MCF-7 breast cancer cells, shortexposure to low doses of paclitaxel resulted in the activation ofthe p21 promoter via p53, whereas longer times of exposureresulted in an increased coassociation between p21 and thePCNA [65]. In addition, low, but clinically achievable, concen-trations of paclitaxel activate JNK signaling pathways in ovar-ian cancer cells. On the other hand, suprapharmacologicconcentrations of paclitaxel induce lipopolysaccharide-likeeffects in ovarian cancer cells, through the Toll-like receptor 4signaling pathway [48].

Paclitaxel has been recently shown to alter the expressionand phosphorylation of the translation initiation proteins inthe breast cancer cell line MDA-MB-231 [66]. The assembly ofthe multisubunit eukaryotic translation initiation factor (eIF)4E is inhibited by a family of repressor polypeptides, theeIF4E-binding proteins (4E-BPs). The binding of 4E-BPs toeIF4E is regulated by phosphorylation: hyperphosphorylatedisoforms do not interact with eIF4E, whereas hypophosphor-ylated 4E-BP isoforms do. Paclitaxel has been shown toinduce the hyperphosphorylation of 4E-BP1 and, hence,reduce its association with eIF4E [66]. The hyperphosphoryla-tion of 4E-BP1 correlated with G2/M arrest and with anincrease in the phosphorylation of Cdk1 substrates.

Subsequent to microtubule disruption, paclitaxel has beenshown to induce apoptosis. This apoptotic effect has also beendescribed in several cancer cell lines such as breast, human lym-phoblastic leukemia, human osteogenic sarcoma cells, myeloidleukemia HL60 cells and prostate cancer cells [67-71]. In theselatter cells, induction of apoptosis by paclitaxel did require theactivation of cyclin B1-dependent kinase that arrested cells inG2/M and subsequently led to the induction of cell death [67].This drug caused early and late phases of apoptosis in ovariancancer cells and only the early phase was dependent on theactivity of the Ras/Rac/MEK/JNK/AP1 pathway [72]. Paclit-axel-induced apoptosis has been also associated with an increaseof the CDKI p21 [73] concomitantly with a decrease of theantiapoptotic Bcl-2 [65]. Apoptosis induction by paclitaxel in

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MCF-7 breast cancer cells was found to be dependent on JNKactivation and to be mediated by the induction of FOXO3aexpression, a transcription factor downstream of the phosphati-dylinositol-3-kinase/Akt signaling pathway [74]. This wasfollowed by the nuclear localization of FOXO3a, which wasassociated with decreased Akt signaling and increased JNK, p38and extracellular signal-regulated kinase activity. Treatmentwith the JNK inhibitor, SP-600125, abrogated paclit-axel-induced apoptosis, reversed Akt inhibition and eliminatedthe nuclear accumulation of FOXO3a [74]. It is interesting tonote that contradictory results were obtained recently byanother group who showed that JNK activation is not sufficientto cause apoptosis in MCF-7 breast cancer cells [75]. When JNKwas inhibited, MCF-7 cells still accumulated in G2/M phase inresponse to drug treatment [72,75]. These studies confirmed thata signal from the checkpoint acts distal to JNK tocause apoptosis [75].

In other studies, paclitaxel-sensitive and -resistant breast can-cer cells have been found to exhibit different mechanisms of celldeath; cytochrome c was released from the mitochondria inresistant, but not in sensitive cells [68]. About 300-fold higherconcentrations of paclitaxel were required for the induction ofthe cell death response in resistant versus sensitive cells. Thedeath induced by paclitaxel was characterized as anapoptosis-like death and included the activation of caspases-3and -9, but not oligonucleosomal DNA fragmentation [68].

The role of caspases in paclitaxel-induced cell death isevident as low concentrations trigger caspase-8- and10-dependent apoptosis in human lymphoblastic leukemiacell lines and reduce FLICE-like inhibitory protein levels,which are known to prevent apoptosis triggered bydeath-inducing ligands [69]. Paclitaxel has been also shown toinduce caspase-3 activation in HL60 cells and human osteo-genic sarcoma (U2OS) cells [70,71]. The addition of thecaspase-3 inhibitor, z-VAD-fmk, blocked paclitaxel-inducedapoptosis and caspase-3 activation in both cell lines [71].

2.3.2 Docetaxel: cell cycle and apoptotic mechanismsDocetaxel is a semisynthetic analog of paclitaxel that has beenrecently approved for clinical use to treat breast, prostate andSCL cancers [76]. This drug has increased affinity for tubulinand higher anticancer activities when compared with paclit-axel [77]. Docetaxel induces cell death by several mechanisms.A recent study has described a non-apoptotic response todocetaxel treatment in human breast cancer cells of increasingtumor progression (MCF-10A, MCF-7 and MDA-MB-231).The primary mechanism of death induced by docetaxel isdescribed as mitotic catastrophe, which occurs in response toDNA damage. Apoptosis and necrotic cell death are othermechanisms by which docetaxel induces cell death [78]. Theresponse to docetaxel differs according to the type of cancer.In BCap3 human breast cancer cells, it induces mitotic arrestand DNA laddering [79]. In human ovarian cancer cells, itinduces nuclear fragmentation [80]. A recent study suggestedthat docetaxel-induced cell death is caused by JNK-mediated

apoptosis in MCF-7, SK-Br-3 and MDA-MB-231 breastcancer cells [81]. On the other hand, in human oral squamouscell carcinoma, docetaxel-induced apoptosis through amitochondrial-dependent pathway is mediated through theactivation of NF-κB [82].

2.3.3 Taxanes: enhancing their efficacyThe sensitivity of cancer cell lines to paclitaxel could beenhanced by the overexpression of E2F-1, a transcription fac-tor involved in G1/S transition [83]. In U2OS osteosarcomacells, the overexpression of E2F-1 increased cyclin B1 levelsand cdc2 kinase activity and sensitized cells to paclitaxel. Onthe contrary, short hairpin RNA targeting of cyclin B1 inHeLa cells increased their susceptibility to paclitaxel treat-ment [84]. A recent investigation has shown that combiningpaclitaxel with any drug targeting Cdk1, but not Cdk2,would increase the sensitivity of breast cancer cells topaclitaxel treatment [85].

Resistance to paclitaxel and docetaxel develops by over-expression of antiapoptotic genes such as Bcl-XL, c-FLIP andsurvivin [48,86]. Paclitaxel resistance develops when bothextrinsic and intrinsic antiapoptotic genes are overexpressed.However, upregulation of only the intrinsic antiapoptoticgenes induced docetaxel resistance. In MCF-7 cells, over-expression of Bcl-XL decreased their sensitivity to both paclit-axel and docetaxel. However, when c-FLIP levels wereelevated, the cells were resistant to paclitaxel alone [48].

The overexpression of survivin, an inhibitor of apoptosis(IAP) protein, is known to cause resistance in cancer cells tochemotherapeutic drugs. The apoptotic response of MCF-7cells was increased by the combination of retinoic acid withpaclitaxel, a treatment that decreased survivin levels andpromoted aberrant mitotic progression [86].

2.4 Camptothecins: general mechanismsThe pentacyclic alkaloid, 20(S)-camptothecin (CPT) wasfirst isolated in 1966 from the extract of the bark of the Chi-nese tree Camptotheca acuminate [87]. Despite its strong anti-tumor properties against gastrointestinal and urinarybladder tumors, CPT was discarded in the 1970s due tosevere toxicity [5,88]. The search for more soluble analogs hasled to the discovery of topotecan (CPT-11) and irinotecanin 1991 [89,90]. Both compounds were approved by the FDAfor use against ovarian and lung cancers (topotecan) and forSCLC and NSCLC, malignant gliomas and cervical, ovar-ian, gastric and colorectal cancers (irinotecan) [91,92]. Otherless important analogs of CPT include rubitecan, lurtotecan,9-aminocamptothecin, as well as the newly discovereddiflomotecan (BN-80915) and grimatecan (ST-1481) [93].

Camptothecin and its derivatives are selective andreversible inhibitors of DNA Topo I by binding to the inter-face of the Topo I–DNA reaction, known as the cleavagecomplex [94]. This successively traps the catalytic intermedi-ate, causing a break in the DNA strands that triggersapoptosis [95].

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2.4.1 Camptothecins: cell cycle mechanismsSeveral theories have evolved concerning the mechanisms bywhich CPTs induce changes in gene expression levels associatedwith either cell cycle control or apoptosis. Myeloid leukemiacells (HL60) treated with different concentrations of SN-38,the active metabolite of irinotecan, showed a significant loss ofS phase cells and a temporary delay of G1/S phasetransition [96]. Cells in the G1 or G2/M phases entered S phaseonly a few hours after the drug had been removed. In vivo stud-ies with irinotecan in advanced solid tumors of cancer patientscaused an average increase of 137% in S phase cells [97].

Recent studies have determined the role of CPTs in mitoticcatastrophe. The inhibition of the checkpoint kinase 1(Chk 1) by short interfering RNA showed an enhancedCPT-induced S phase arrest, leading to mitotic catastrophe[98]. Double inhibition of both Chk 1 and Chk 2 was not aseffective as Chk 1 inhibition alone. When treated with CPTand SN-38, cancerous cell lines that showed substantial inhi-bition of ATR and Chk 1 were more sensitized to the Topo Ipoisons [99], suggesting a possible role of ATR/Chk 1 pathwayin promoting CPTs antitumor effects. In addition,CPT-induced apoptosis caused increased levels of cyclin B1,along with substantial disorganization of chromosomes andphosphorylation of histone H3 epitopes, providing furtherevidence of mitotic catastrophe that ultimately leads to celldeath [100].

2.4.2 Camptothecins: mechanisms of apoptosisCamptothecins mediate enhanced cell death by upregulatingproapoptotic genes and downregulating antiapoptotic genes.Research has linked the activation of several caspases to thecytotoxicity of CPT. In a NSCLC (H-460), CPT-inducedapoptosis was associated with the activation of caspases-7 and-3 and PARP cleavage prior to the release of cytochrome c andother apoptosis-inducing factors (AIFs) [101]. The role ofPARP 1 in the efficacy of CPT-induced cytotoxicity has beenstudied using PARP 1+/+ and PARP 1-/- mouse embryonicfibroblasts and a PARP 1 inhibitor, AG-14361 [102]. Resultsindicated that the inhibition of PARP 1-dependent base exci-sion repair is a mechanism of CPT-induced antitumoractivity. The enhancement of the TNF apoptotic pathway bytopotecan has been identified as a mechanism by which itinduces antitumor effects [95]. Glioma cells treated with topo-tecan expressed increased sensitivity to the TNF apoptosis-induced ligand (TRAIL)-dependent apoptotic pathway,exhibiting increased expression of the receptor TRAIL R2 andapoptotic cell death.

Another target gene downregulated by topotecan is epider-mal growth factor receptor (EGFR), a direct transcriptionaltarget of the transcription factor c-Jun. Many growthregulatory signaling pathways ultimately interact with EGFR,which is commonly overexpressed in many cancer types. TopoI and c-Jun factor seem to interact in a JNK-dependentmanner to regulate EFGR production [103]. Studiesdemonstrate that the inhibition of c-Jun decreases the

expression of EFGR, leading to a novel mechanism by whichtopotecan may regulate cell proliferation.

2.4.3 Camptothecins: enhancing their efficacyRecent investigations have primarily focussed on novel waysto further enhance the efficacy of the potent cytotoxic CPTdrugs. Studies on the chemoresistant hepatocellularcarcinoma cell lines showed that the inhibition of the Aktpathway by PI3K inhibitors resulted in sensitizing these cellsto SN-38 [104]. In another study, the combined treatment oftopotecan with gemcitabine, a pyrimidine analog used in thetreatment of NSCLC, revealed synergistic effects [105].Irinotecan, followed by 5-fluorouracil treatment also dis-played synergistic effects in HT-29 human colon carcinomacell lines [106,107]. Such synergism occurred due to the activa-tion of caspases-3 and -9, in addition to a 50% reduction ofmitochondrial membrane potential.

2.5 Homoharringtonine: general mechanismsHomoharringtonine has been used in China for decades forthe treatment of ailments such as parasitosis and cancer.Actual clinical studies were not carried out until 1970 when itwas purified and characterized from the native Japanese plantCephalotaxus harringtonia by Powell et al. [108]. Homohar-ringtonine is a cephalotaxus alkaloid that is an ester of cepha-lotaxine. Out of the four esters of cephalotaxine that wereisolated (harringtonine, isoharringtonine, homoharringtonineand deoxy-harringtonine), only harringtonine and homohar-ringtonine showed activity in cancer treatment. Shortly afterhomoharringtonine’s discovery, preclinical trials were startedin China in patients with acute non-lymphocytic leukemiaand were followed by clinical trials in the US. Homohar-ringtonine is used in the treatment of primary acute myeloidleukemia (AML), relapsed acute non-lymphocytic leukemiaand chronic myeloid leukemia (CML).

Homoharringtonine’s anticancer activity is generally due tothe inhibition of protein synthesis [109] and apoptosis induc-tion [110]. It was shown to inhibit translation at the elongationstep, most likely at the first peptide bond. In fact, homohar-ringtonine is able to bind to the peptidyltransferase center in80S ribosome, to hinder peptidylsynthetase activity and tocompete with several peptidylsynthetase inhibitors [111]. Somestudies demonstrated that translation inhibition mostlyoccurs at the G1 and G2 phases of the cell cycle [112] andhomoharringtonine was shown to cause G1 arrest [113].However, this drug inhibits in general cell proliferationwithout any specific cell cycle arrest.

2.5.1 Homoharringtonine: cell cycle and apoptotic mechanismsThe role of homoharringtonine in apoptosis induction is con-firmed in several tumor types (leukemia, lymphoma, neuro-blastoma, carcinoma) and in primary leukemic cells isolatedfrom AML patients [110]. Several experimental approaches,such as DNA flow cytometric analysis, gel electrophoresis and

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electron microscopy, confirmed homoharringtonine-inducedprogramed cell death.

Homoharringtonine induces apoptosis in human T leuke-mic cells (MOLT-3 carrying a wild-type p53), which corre-lates with Bax translocation from cytosol to mitochondria,cytochrome c release and caspase activation, but is independ-ent of ROS generation [114]. Bax upregulation is also observedin homoharringtonine-induced apoptosis in a variety ofhuman myeloid leukemia cell lines [115,116]. Nevertheless,homoharringtonine-induced apoptosis in myeloid leukemiacells was independent of the expression of Bax, but decreasedMcl-1 expression and induced caspase-dependent Bcl-2cleavage [116]. This drug induced apoptosis in HL60 cells isassociated with a reduction in telomerase activity [117].Survivin is recently suggested as a new target for cancer treat-ment and as a new marker for drug sensitivity. The apoptoticeffect of homoharringtonine on malignant hematopoietic cellsis associated with reduced levels of survivin [118].

2.5.2 Homoharringtonine: enhancing their efficacyHomoharringtonine, as a general cell proliferation and pro-tein inhibitor, may be more effective against resistant cancercells if used in combination treatments with nucleoside ana-logs and mitosis inhibitors, although it was originally used 20years ago in China and in the US as a single agent to treatleukemia patients. In fact, synergistic tumor growth inhibi-tory effects have been observed in vitro between homohar-ringtonine, cytarabine (Ara-C), vincristine and daunorubicin.As a result, from the late 1970s many Chinese trials and treat-ment protocols have implemented its combined use in cancertherapy mainly with vincristine, Ara-C and predinosone oronly combined with Ara-C. Although complete remission rateof leukemia patients was enhanced, myelosuppression was theprimary side effect of combined therapy. As the Bcr/Abl tyro-sine kinase inhibitor imatinib became available, treatment ofCML patients drastically improved, but less effectively in theblast phase. A triple combination regimen with interferon-α,Ara-C and homoharringtonine followed by imatinibimproved the overall prognosis of CML patients [119].Recently, a combined homoharringtonine, Ara-C and aclaru-bicin regimen resulted in a high complete remission in youngadult patients with AML [120].

2.6 Ellipticine: general mechanismsEllipticine is an alkaloid that was first isolated in 1959from the leaves of Ochrosia elliptica [121]. It was found tohave strong anticancer properties. However, due to its lim-ited solubility several more soluble derivatives were devel-oped such as elliptinium acetate, 9-hydroxyellipticine,9-hydroxy-N2-methylellipticinium, 9-chloro-N2-methylel-lipticinium, 2-methyl-9-hydroxyellipticinium and9-methoxy-N2-methylellipticinium [122]. Ellipticine and itsmore soluble derivatives are used in the treatment of breastcancer as well as osteolytic breast cancer metastases, kidneycancer, brain tumor and AML.

The mechanism of ellipticine antitumor and cytotoxicactivities is intercalation into DNA and inhibition of Topo IIactivity [123]. Ellipticine intercalates easily into dou-ble-stranded DNA because its size and shape are similar topurine–pyrimidine complementary base pairs [124]. A newmode of ellipticine action was shown to be mediated by itsoxidation with cytochromes P450 and peroxidases and resultsin the formation of covalent DNA adducts through itsactivation to 13-hydroxyellipticine and ellipticine N2-oxidespecies [122]. The oxidation of ellipticine by cytochrome P450enzymes also plays a role in its detoxification.

Several studies have demonstrated the role of ellipticine as aTopo II inhibitor. Ellipticine stimulates Topo II-mediatedDNA breakage by increasing the enzyme’s forward rate ofDNA cleavage and not its DNA religation. It has been shownto bind to Topo II in the presence or absence of DNA [125].However, the formation of a ternary complex betweenTopo II, DNA and drug is crucial for DNA breakage andsubsequent cell death.

2.6.1 Ellipticine: cell cycle and apoptotic mechanismsEllipticine induces cell cycle arrest and apoptosis in severalhuman cancer cell lines. Numerous studies have shown theinvolvement of p53 in ellipticine’s cytotoxic effects. 9-hydrox-yellipticine induces G1 cell cycle arrest and apoptosis inmutant p53 transfected cells, but not in p53-deficientcells [126]. In fact, ellipticine has been found to rescue the tran-scription function of p53 [127]. Ellipticine and 9-hydroxyellip-ticine inhibit p53 phosphorylation, which correlates withtheir cytotoxic effects [126,128].

Ellipticine causes G2/M arrest and apoptosis in MCF-7 andMDA-MB-231 human breast cancer cells. Ellipticine-medi-ated growth inhibition of MCF-7 cells results in elevated pro-tein levels of p53 and the CDKI p21 and p27, triggering ofFas/Fas ligand pathway and disruption of mitochondrial func-tion [129]. Cross-talk between Fas death receptor and mito-chondrial apoptotic pathway amplified the apoptoticsignaling pathway and resulted in the activation of caspases-8and -9. Ellipticine-mediated growth inhibition ofMDA-MB-231 cells increased the expression of Bax anddecreased the expression of Bcl-2, Bcl-XL and X-linked inhib-itor of apoptosis protein (XIAP) [130]. This activation of themitochondrial apoptotic pathway caused cytochrome c releaseand caspase-9 and -3 activation. Ellipticine’s growth suppres-sive effect and apoptosis are abrogated due to thepre-treatment of cells with caspase-9 inhibitors.

Ellipticine’s cytotoxicity was recently attributed to the induc-tion of endoplasmic reticulum stress [131]. 6-propanamine ellipti-cine, a potent ellipticine derivative, causes a conformation changein Bak and cytochrome c release in MDA-MD-231 cells followedby apoptosis induction. Interestingly, this latter compound is ableto induce apoptosis in enucleated cells and to increase the expres-sion of the endoplasmic reticulum chaperones GRP78/BiP andGRP94, strongly implicating its role in induction of endoplasmicreticulum stress.

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3. Plant-derived anticancer drugs in clinical development

3.1 Flavopiridol: general mechanismsFlavipiridol is a semisynthetic flavonoid that was isolated inthe late 1980s from the leaves and stems of the Indian plantAmoora rohituka [132]. When screening for its anticancer activ-ity in 1992, flavipiridol was found to be a potent inhibitor ofcell growth. It is at present undergoing several Phase I and IIclinical trials against advanced solid tumors, endometrial car-cinoma, relapsed and refractory adult acute leukemias [133],relapsed or refractory multiple myeloma, metastatic breastcancer and several other cancers, as summarized in Table 1.

The antitumor activities of flavopiridol are diverse and areattributed to the inhibition of CDKs, transcription andangiogenesis. These compounds have also been shown toinduce apoptosis and differentiation [132,134,135]. The generalanticancer activities of flavopiridol that modulate tumor cellcycle and cell death are discussed in Section 3.1.1.

3.1.1 Flavopiridol: cell cycle mechanisms3.1.1.1 Cyclin-dependent kinase inhibition and cell cycleregulationFlavopiridol can inhibit tyrosine and serine kinases, particu-larly CDKs, due to its direct binding to their ATP-bindingpockets. The ability of flavopiridol to inhibit several kinasescontributes to its effects and the complex mechanism of itsanticancer activities. At submicromolar concentrations,flavopiridol can inhibit Cdk1,-2, -4, -6, -7, and -9 and athigher concentrations it can hinder protein kinase A and C,and the EGFR and receptor-associated protein kinases such asc-Src [136,137]. Flavopiridol inhibits purified CDKs by compet-ing with ATP and inactivating the CDK-activating kinaseCAK, also known as Cdk7, leading to the loss in phosphor-ylation at threonine 160/161, which is necessary for theactivation of most CDKs [138].

In 1992, flavopiridol was first shown to induce G1/S andG2/M cell cycle arrest [139] due to Cdk2 and Cdk1 inhibition[140] and later this drug was demonstrated to be a pan-CDKinhibitor [132]. Further research demonstrated that flavopiri-dol-induced growth arrest is not only due to CDK inhibition,but also due to its suppressive effects on transcription which isnecessary for cell cycle progression. Regulation of the cellcycle requires negative and positive regulators of CDKs. Fla-vopiridol selectively affects the transcription of genes thathave short transcript half-lifes, such as cyclin D1 and Cdk1p21 and p27.3.1.1.2 Inhibition of transcriptionFlavopiridol has pleiotropic effects due to its potent inhibitionof global transcription. As a result, it downregulates numerousproteins, causes cell cycle arrest and apoptosis and also results inside effects. However, flavopiridol is different than the generalinhibitor of transcription actinomycin D and has a uniquemechanism of action that explains its remarkable clinicalpotential [135].

Flavopiridol modulates transcription in several eukaryoticsystems due to its inhibitory effects on Cdk9 and Cdks 7 and8. In fact, it can hinder the activity of the transcriptional fac-tor P-TEFb, a complex of Cdk9 and T-type cyclins [141,142].P-TEFb regulates transcription elongation by phosphorylat-ing the carboxyl-terminal domain of the large subunit ofRNA polymerase II and is, therefore, essential for transcrip-tion [142]. Interestingly, this drug can inhibit P-TEFb and canblock RNA polymerase II transcription at cytotoxic concen-trations, which are easily achieved in human clinical trials.Affected genes may include those involved in the regulation ofcell cycle and apoptosis [143].

3.1.2 Flavopiridol: apoptotic mechanismsFlavopiridol is a powerful anticancer agent because it cantrigger apoptosis in both cycling and non-cyclingtumor cells [132]. It induces cell cycle arrest, sensitizesapoptosis-resistant cancer cells to cell death and synergizeswith other apoptotic agents [144]. The mechanisms by whichflavopiridol induces apoptosis are diverse. Flavopiridol canreduce the expression of antiapoptotic proteins such as Bcl-2,Bcl-XL, Mcl-1, XIAP and survivin and, therefore, enhancesprogramed cell death. As an inhibitor of transcription, it canreduce the levels of the short-lived Mcl-1, sensitizing cells toTRAIL-mediated apoptosis [145] and cell death in aggressivemantle cell lymphoma [146]. Survivin is an IAP memberthat also serves as a mitotic checkpoint. Flavopiridol abrogatesthe phosphorylation of Thr34 on survivin, resulting inan increased degradation of survivin protein levels andenhanced apoptosis [147].

Several studies have demonstrated that flavopiridol-inducedapoptosis is independent of p53 status [136,148-150]. In fact, incells lacking p53, flavopiridol is maximally cytotoxic at the sameconcentrations. Interestingly, flavopiridol can increase p53protein levels due to the inhibition of MDM2 transcription.MDM2 in turn targets p53 for degradation. As p53 proteinlevels depend mainly on translation and protein degradation,but not on transcription, flavopiridol prevents degradation ofp53 by depleting the short-lived MDM2 protein.

Flavopiridol has been shown to activate the mitochondrialcell death pathway associated with cytochrome c release,DNA ladder formation, activation of caspases and PARPand Bid cleavage. This drug can trigger both cas-pase-dependent and -independent cell death pathways dueto the simultaneous release of both cytochrome c and AIFfrom mitochondria [132,151]. For instance, pretreatment ofcells with caspase-3 inhibitors attenuates most of these lattercell death events [152]. This drug can also induce a cas-pase-independent pathway of apoptosis via release of AIF inthe absence of cytochrome c release from mitochondria andindependent of Bcl-2 overexpression [148,153]. It can alsoinhibit activation of the nuclear transcription factor NF-κBby dephosphorylation and inactivation of Akt [154]. Phos-phorylated Akt has been linked to increased cellproliferation, survival and migration. In summary,

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flavopiridol is a potent apoptotic agent that can trigger avariety of cell signaling pathways that are targets forchemotherapy in human cancer.

3.1.3 Flavopiridol: enhancing their efficacyFlavopiridol is an ideal drug to be used in the clinic incombination therapies with other cytotoxic agents because of itspleiotropic antitumor effects [132]. This drug can increase sensi-tivity of S phase cells to different agents by modulating E2F-1transcription factor activity in tumor cells. Certain malignan-cies depend on a crucial oncoprotein for survival, the inhibitionof which can cause apoptosis. Flavopiridol may have preferen-tial cytotoxic effects on cancers overexpressing oncogenes suchas myeloid cell leukemia sequence (Mcl-1), Bcr-Abl and mutantEGFR, c-KIT and signal transducers and activators of tran-scription (STAT)-mediated signaling pathways [146,155,156].In vitro studies have demonstrated a synergy between flavopiri-dol and several other widely used anticancer drugs, such as pacl-itaxel, topotecan, doxorubicin, etoposide, camptothecin andfluorouracil [157,158]. The sequence and timing of drug adminis-tration are crucial factors in determining the success of thosedrug combinatorial programs. For example, flavopiridol is apotent S phase drug and, therefore, can be used successfullyafter any drug that induces tumor cells to enter this phase of thecell cycle, such as aramycin C [137,153].

Flavopiridol can also potentiate the sensitivity of tumor cellsto TNF [144] and TRAIL-mediated signaling pathways [159].Due to its global mechanism of action, flavopiridol prevents atranscriptional response caused by anticancer drugs and enhanceapoptosis caused by paclitaxel, radiation and histone deactety-lase inhibitors. Recently, a new strategy has been suggested incombining flavopiridol and histone deactetylase inhibitors inpromoting mitochondrial injury and cell death in drug-resistantleukemia cells overexpressing Bcl-2 or Bcl-XL [160].

3.2 Combretastatin A4: general mechanismsCombrestatin A4 (CA-4) is a natural stilbenoid that wasisolated from Combretum caffrum, a South African plantknown to be used in traditional medicine for ailments such ashepatitis and malaria [5,161]. In 1989, Petit et al. found thatCA-4 has anticancer properties. However, due to its poorsolubility, a prodrug CA-4 phosphate (CA-4P), has beendeveloped and is at present undergoing Phase I clinical trialsagainst several cancers such as advanced cancers and solidtumors [162]. Several Phase II clinical trials have been initiatedagainst advanced anaplastic thyroid cancer.

The combretastatins are antimitotic cancer drugs thatcause vascular shutdown and tumor necrosis [162,163]. CA-4was first reported as a powerful cell growth and tubulininhibitor [164]. At present, combretastatins are recognized aslow molecular weight vasculature-disrupting agents withpowerful tubulin depolymerizing activities [165]. Unlikeantiangiogenic drugs, which prevent the growth of newblood vessels, combretastatins selectively destroy tumorneovasculature resulting in massive necrosis [166]. These

compounds destabilize tubulin of proliferating, but not ofquiescent endothelial cells [167] and seem to have tubulin iso-type specificity as CA-4 resistance of lung carcinomas hasbeen recently linked to alterations in β-tubulin isotypeexpression [168].

3.2.1 Combretastatin A4: cell cycle and apoptotic mechanismsCells treated with combretastatins at concentrations close toIC50 values arrest after a few hours at the G2/M phase of thecell cycle. CA-4P treatment of proliferating endothelial cellsdamages mitotic spindles, arrests cells at metaphase andleads to the death of mitotic cells with characteristic G2/MDNA content, elevated levels of cyclin B1 and p34cdc2activity [169]. Sustained p34cdc2 activity is responsible formetaphase arrest and pharmacological prevention of entryinto mitosis protected cells from undergoing cell death.CA-4P-mediated programed cell death is associated withprolonged mitotic arrest and is caspase-independent [169,170].However, other studies suggest that endothelial cells mostlyundergo mitotic catastrophe and very few undergo apoptosisfollowing CA-4P treatment [171].

3.2.2 Combretastatin A4: enhancing their efficacyCombretastatins show the greatest efficacy when used incombination with conventional antiproliferative therapies.Although CA-4P selectively target tumor neovasculatureand cause rapid ischemic necrosis, unfortunately rapidtumor regrowth occurs due to few surviving cells. The useof combined CA-4P and antiproliferative therapies, such ascisplatin and radiation, potently improve treatment effi-cacy. In fact, combretastatins target the hypoxic parts ofthe tumor, whereas commonly used chemotherapeuticagents kill the viable rim [163].

3.3 Roscovitine: general mechanismsRoscovitine is a synthetic compound derived from olomucine,a natural compound isolated from the cotyledons of Raphanussativus [172]. Roscovitine, also called CYC202, has undergoneseveral Phase I clinical trials against advanced solid tumors.At present, roscovitine is undergoing Phase II clinical trials inEurope [5]. The purvalanols, which are more potentderivatives, were synthesized and are in preclinicaldevelopment at present [173].

Roscovitine, a purine derivative, acts as a powerful smallmolecule CDKI [174]. It was first described to inhibit theuniversal M-phase promoting factor, Cdk1/cyclin B. Later,roscovitine was cocrystallized with Cdk2 in the ATP-bindingpocket of the kinase [172]. This drug was tested on a wide panelof purified kinases and was found to be highly specific forCDKs with greatest activity against Cdk2/cyclin E, Cdk7/cyc-lin H and Cdk9/cyclin T [175]. Unlike flavopiridol, which hasextensive kinase inhibition activities including CDKs, PKC,PKA and receptor-associated tyrosine kinases, roscovitine isknown as a selective inhibitor of CDKs.

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3.3.1 Roscovitine: cell cycle and apoptotic mechanismsRoscovitine causes cell cycle arrest in G1, S and G2/M phasesand apoptosis in tumor cells by several mechanisms.Short-term treatment of S phase cells with roscovitine hindersDNA synthesis and activates a DNA damage checkpointresponse due to phosphorylation of p53, replication protein Aand histone H2AX [176]. In colon carcinoma cells, roscovitinereduces retinoblastoma protein phosphorylation at multiplesites, inhibits Cdk4, -2 and -1 activities, as well as causing aloss of cyclin D1 [177].

Roscovitine induces apoptosis in tumor cells in vitro and intumor xenografts [178]. It induces cell death by inhibition ofRNA polymerase II-dependent transcription, mostly affectinggene products with short half-lifes such as downregulation ofthe antiapoptotic factor Mcl-1 in multiple myeloma cells [179].Furthermore, roscovitine-induced apoptosis and Mcl-1 rapiddownregulation was shown to be caspase-independent [180].However, in Ewing’s sarcoma family of tumor cells, roscovitinecauses caspase-dependent cell death, upregulates proapoptoticBax and reduces both survivin and XIAP [178]. In breast cancercells, this drug induces apoptosis, increases p53 protein levels,phosphorylation at Ser46 and accumulation in the nucleus, aswell as causing a depolarization of mitochondrial potential andthe release of cytochrome c and AIF [181]. Interestingly, theonset of p53 phosphorylation at Ser46 preceded the upregula-tion of p53 proteins and depolarization of mitochondrialpotential. On the other hand, roscovitine-induced apoptosis inB-cell chronic lymphocytic leukemia is independent ofp53 [182]. In these lymphoma cells, gene expression profilingstudies demonstrated that roscovitine caused reduction of genesinvolved in the regulation of transcription, survival and DNArepair. In NSCLC cells, proteasomes were suggested to play anearly role in roscovitine-induced apoptosis, upstream from thecaspase cascade and mitochondria [183].

3.3.2 Roscovitine: enhancing their efficacyChemotherapy of tumor cells results in a variety of responsesranging from apoptosis to premature senescence. However,DNA repair processes may be also activated and may attenuatethe effects of chemotherapy in tumor cells such as in doxoru-bicin-treated cancer cells. The use of combined roscovitine anddoxorubicin regimen increased the susceptibility of tumor cellsto therapy due to the ability of roscovitine to hinderdoxorubicin-induced DNA repair functions [184]. This prop-erty suggests future directions for the use of combinedroscovitine and chemotherapeutic agents for cancer treatment.

4. Conclusion

Plant-derived anticancer agents have been successfullyexploited against a wide spectrum of human cancers and haveprovided hope for cancers resistant to conventional therapies.

It is evident from this review that plant-derived anticanceragents in clinical use and development share common target-ing mechanisms in tumor cells while sparing the normal ones.

In general, anticancer agents preferentially inhibit the growthof highly proliferative cells and this partly explains their selec-tivity towards tumor cells. For example, the effects of roscovi-tine were observed only in highly proliferating primarykeratinocytes and were reversible on drug withdrawal [185].Many plant-derived anticancer agents may act as small-mole-cule CDKIs (flavopiridol, roscovitine), inhibitors of Topo I(camptothecins) and Topo II (etoposide, podophyllotoxins,ellipticine) and microtubule and tubulin targeting drugs caus-ing depolymerization of microtubules (Vinca alkaloids,podophyllotoxins), depolymerization of tubulin (combreta-statins) or preventing depolymerization of microtubules(taxanes). They may function as angiogenesis inhibitors(flavopiridol) or vascular targeting agents destroying existingtumor neovasculature (combretastatins). They regulate geneexpression of labile cell cycle regulators and antiapoptoticplayers by inhibiting RNA polymerase II-dependent tran-scription (roscovitine, flavopiridol) or by inhibiting transla-tion (homoharringtonine). They trigger numerous signalingpathways leading to cell death, namely mitotic catastrophe(camptothecins, Vinca alkaloids, combretastatins, taxanes),necrosis (taxanes) and/or apoptosis induction (most of thereviewed drugs). Most plant-derived anticancer agents induceintrinsic rather than extrinsic apoptotic pathways. Mostimportantly, these death pathways are p53-dependent orindependent, caspase-dependent or -independent, or may beproteasomal mediated.

These drugs have several antitumor targets, as previouslysummarized, and these pleiotropic mechanisms provide abasis for combining several anticancer agents to targetresidual resistant neoplastic cells. For example, docetaxel isan ideal drug for combination treatments. In fact, docetaxelis endowed with multiple cellular targets working as anantimicrotubule agent, apoptosis inducer, angiogenesisinhibitor, regulator of gene expression and affectingcommonly aberrant signaling pathways in tumor cells suchas the EGFR and Ras. These combination studiesemphasize a multimodality approach to treat cancer and totarget specific patient populations.

A novel approach to cure cancer has been proposed and isbased on combining different therapeutic strategies aiming attargeting cancer-specific signaling pathways and comple-mented by selective drug combinations and tissue selectivetherapy [186]. Ultimately, successful cancer chemotherapy aimsat inducing cell death in cycling cells (cyclotherapy principle)a strategy that selectively kills cancer cells and spares normalones [187]. Understanding anticancer drug mechanisms ofaction provides the basis for combination regimens leading tooptimal therapy.

5. Expert opinion

Plant-derived agents have provided an important source ofnew drug leads and have been the basis of many clinicallyused anticancer medicines. Despite the highly competitive

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pharmaceutical industry, medicines from plant origin havesupplied numerous clinically used anticancer drugs.

The authors, H Gali-Muhtasib and N Darwiche, are mem-bers of an interfaculty initiative (IBSAR: Initiative for Biodi-versity Studies in Arid Regions [202]) that was launched in2002 at the American University of Beirut, Lebanon. Thisinitiative aims at identifying, purifying and characterizing nat-ural compounds from indigenous Middle-Eastern plants withpotential biological activities, including anticancer activities.There are several challenges concerning the discovery ofanticancer drugs from plants which has made such discoveriestraditionally lengthier, very costly and at times disappointing.Some challenges include the availability of plant material andintellectual property rights regulations, the choice of theappropriate high-throughput screening bioassays and the largenumber of marketed active compounds.

The process of drug discovery typically begins with thecollection and identification of the plant by a botanist orplant systematist. In some cases, this collection involvesspecies used traditionally for cancer treatment, whereas inothers it involves species collected randomly for large screen-ing programs. At a second stage, crude extracts from the col-lected plants are subjected to bioassay-guided fractionation inthe hope of isolating and characterizing the active ingredients.To determine the targets of the potential active compounds, awide range of molecular and cellular techniques are used,which aim at determining structure–activity relationships. Inmany instances, such structure–activity relationships areestablished in animal models as well as in cell culture-basedand mechanistic assays.

Once a lead and a promising compound has been identi-fied by in vitro and in vivo assays, then medicinal and com-binatorial chemistry procedures are applied to optimize itsdevelopment. This process of preclinical testing has largelygained from the thousands of new molecular targets identi-fied by the sequencing of the human genome [188]. This istypically followed by a development phase that includespharmacology–toxicology studies (compound absorption,distribution, metabolism, excretion and mode of delivery)

before the drug enters the three phases of clinical trials[189]. Once preclinical and clinical trials are completed withpromising results, these compounds proceed through fur-ther development into FDA-approved medicines. For eachmarketed drug, the length of the process has been esti-mated to take at least 10 years [190] and costs > 800 milliondollars [191].

Other challenges of natural product drug discovery includethe fact that these drugs exist in small quantities in the plantand are insufficient for lead optimization and clinical develop-ment. In addition, plant diversity throughout the world isbeing depleted, which could limit future discoveries ofanticancer plant products.

This could be overcome by establishing collaborations withsynthetic chemists or creating natural product-like librariesthat combine the features of natural products with combina-torial chemistry. The use of synthetic and combinatorialchemistry coupled with molecular modeling andhigh-throughput screening assays have allowed the modifica-tion of existing natural products into more potent and lesstoxic anticancer agents. Numerous examples are mentioned inthis review (Table 1).

All aforementioned challenges in natural drug discoveryhave led to the recent scaling down or elimination of natu-ral product research at the NCI (US) and in many pharma-ceutical companies [192]. Despite these difficulties facinganticancer drug discovery from natural sources, plantproduct research should continue considering that plantsare uniquely endowed with molecular diversity and func-tionality essential for drug discovery [193]. Plant-derivedanticancer compounds are expected to remain essentialcomponents in the search for novel medicines and reaffirmwhat Hippocrates said 25 centuries ago: ‘Let food be thymedicine, and medicine be thy food’.

Acknowledgements

The authors wish to thank A Sawaya for his technical helpwith the references.

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AffiliationNadine Darwiche PhD, Sara El-Banna MSc & Hala Gali-Muhtasib† PhD†Author for correspondenceAmerican University of BeirutDepartment of Biology, Beirut, LebanonTel: +96 11350000; Fax: +961 1 7444461E-mail: [email protected]

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Cell cycle modulatory and apoptotic effects of plant-derived anticancer drugs in clinical use or...

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