New Combination Therapies with Cell Cycle Agents

Abstract

Cancer cells have deregulated cell cycle progression with overexpression of positive regulators and inhibition of negative regulators giving them unlimited replication potential. Therefore, development of agents targeting the deregulated cell cycle has been considered as an ideal strategy for cancer therapy in recent years. Cell cycle based agents have been categorized as CDK inhibitors, Cdc25 inhibitors, checkpoint inhibitors and mitotic inhibitors. These drugs have shown tremendous pre-clinical effectiveness but their efficacy in clinic has been modest along with various side effects. Alternatively, their combination with chemotherapeutic drugs has been studied in different pre-clinical and clinical settings. The initial reports from these combination studies have been encouraging and might be useful in lowering the cancer burden.

Introduction

Cancer is one of the major health problems and causes unbearable morbidity and mortality worldwide [1, 2]. Deregulated cell cycle progression has been considered as the hallmark of cancer progression, and therefore, is a practical target for anti-cancer drug development [3–6]. The present review details various categories of cell cycle agents namely CDK inhibitors, Cdc25 inhibitors, checkpoint inhibitors and mitotic inhibitors; along with their anticancer efficacy and clinical limitations. Chemotherapy has been the frontline treatment against cancer for almost last half century [7], and is also discussed briefly. The main focus of the review is on the combination studies of chemotherapeutic drugs (DNA damaging agents and microtubule poisons) with selective cell cycle modulator-based agents. Various pre-clinical and clinical combination studies with probable mechanism for synergy have also been discussed in detail. The review covers the advancements, the problems, and the lessons learnt in last decade in the direction of developing new cell cycle modulator-based combination therapies for cancer eradication.

Cell Cycle Progression: Normal vs. Cancer Cells

The cell cycle is the mechanism through which cells divide, and is an orderly and tightly regulated phenomenon involving four phases (G1, S, G2 and M) [5, 6]. The gap phases (G1 and G2) separate the DNA synthesis (S phase) and mitosis (M phase) (Figure 1). The progression through these phases is controlled by a number of CDKs which are heterodimeric complexes composed of a catalytic kinase subunit and a regulatory cyclin subunit [5, 6]. Cyclin D-associated kinases CDK4 and CDK6, as well as cyclin E-CDK2 complexes are known to sequentially phosphorylate the retinoblastoma protein (Rb), resulting in the release of E2F1 (Figure 1), which then transcribes proteins needed for G1 to S transition [6, 8, 9]. Similarly, cyclin A-associated kinases CDK2 and CDK1 (also known as CDC2) and cyclin B-CDK1 complexes are required for orderly S-phase progression and the G2M transition, respectively [6, 9] (Figure 1). The activity of CDKs is regulated by both inhibitory and activating phosphorylation at various sites, as well as by different CDK inhibitors such as INK4 family members (p16^Ink4, p15^Ink4, p18^Ink4 and p19^Ink4) and CIP/KIP family members (p21^waf1, p27^kip1 and p57^kip2) [6, 9]. Other than cell cycle regulatory CDKs, newer CDKs/cyclins with housekeeping as well as cell-cycle related roles have been reported and these have been termed as ‘non-cycling CDKs/cyclins’[10]. One of the members of non-cycling CDKs/cyclins family, CDK7/cyclin H (also known as CDK activating kinase) has been reported to regulate CDKs activity [6, 10]. Further, CDK7/cyclin H, CDK8/cyclin C and CDK 9/cyclin T have been shown to regulate the expression of RNA polymerase II promoting the elongation of nascent transcripts [6, 10]. A more in-depth understanding of the non-cycling CDKs/cyclins might help to have a better idea about cell cycle regulation as well as mechanism of action of various CDK inhibitors.

Figure 1.

The basis of cell cycle regulation involving cyclins-CDKs-CDKIs, Cdc25 phosphatases, cellular checkpoint and DNA damage sensors. Abbreviations: ATM: Ataxia telangiectasia mutated; ATR: Ataxia-telangiectasia-mutated and Rad3-related; DNA-PK: DNA-dependent protein kinase

As shown in figure 1, cell remains in quiescent phase (G0) and its entry into the cell cycle is governed by the restriction point, which is a transition point beyond that the cell cycle progression is independent of external stimuli such as exposure to mitogen activation or nutrients [6]. Another checkpoint known as replication checkpoint monitors the progression through S phase and controls the ability of cell to enter mitosis. This checkpoint is known to involve the activations of ATM, ATR or DNAPK kinases with subsequent activation of Chk1 and Chk2, and results in damage repair, cell cycle arrest or apoptosis, depending upon the extent of DNA damage [11–15] (Figure 1). Similarly, during mitosis, there is spindle assembly check point which inhibits the onset of anaphase until all kinetochores are properly attached to spindle microtubules and set under tension during metaphase, thus, prevents the missegregation of chromosomes [16]. Overall, these checkpoints regulate orderly progression of cell cycle and ensure genetic fidelity between daughter cells.

During carcinogenesis, cell cycle is deregulated due to overexpression of positive regulators (CDKs and cyclins) and a loss in function of CDK inhibitors [6, 9]. The Cdc25 overexpression and genetic alterations in Chk2 have also been identified in a wide spectrum of human tumors [17, 18]. Furthermore, in most cancer cells, G1 checkpoint malfunctions either due to inhibitory mutations in most of the regulators (p53, Rb, INK4) or due to activating mutations in oncogenes (cyclin D1, Ras, erbB, EGFR). Overall, all these alterations in the cell cycle regulatory molecules result in an uncontrolled cancer cell growth.

Cell Cycle as Therapeutic Target

Since an aberrant cell cycle progression is considered as the key for cancer cell growth, agents targeting the cell cycle have been considered ideal for cancer treatment [3, 6, 9, 19–21]. These drugs target the abnormal expression of CDKs, Cdc25s or affect the cellular checkpoints resulting in cell cycle arrest followed by induction of apoptosis in cancer cells. Based upon their targets, cell cycle inhibitory agents have been categorized as listed in Table 1.

Table 1.

Different classes of cell cycle agents, their mechanisms of action and clinical status.

Class	Compounds#	Mechanism of action	Stage	References
CDK inhibitors	Pan CDK inhibitors Flavopiridol***, R-547*, Silibinin**, SNS-032**, AG-024322 Selective CDK inhibitors Cdk4/6 specific PD-0332991*, CINK4 CDK2,1 specific AT-7519**, PNU-252808, BMS-387032* AZ703, NU6102, NU6140, CYC202**, SQ-67563, E7070**	Target CDK activation and causes cell cycle arrest resulting in growth inhibition and apoptosis	* In phase I clinical trial ** Phase II clinical trial *** Phase II clinical trial	[3, 6 and references therein, 9, 20, 22, 23, 24, 25, 34]
Cdc25 phosphatase inhibitors	BN82685, BN82002, NSC663284, JUN-1111, Caulibugulone, PM20, TPY-835, IRC-083864, Thiazolidine dione, 5169131, Adociaquinone B, ARQ-501*	Cdc25 inhibitors activate cellular checkpoint, induces cell cycle arrest and apoptosis.	* In phase I clinical trial	[17, 35 and references therein, 36]
Checkpoint inhibitors	UCN01**, Go6976, SB-218078, ICP-1, CEP-3891, CHIR-124, Debromohimenialdesine, TAT-S216A, CEP-6367, PD0166285, Okadaic acid, PF-00394691, PD-321852, Fostriein, 13-hydroxy-15-ozoapatlin, Isogranulatimide, XL844*, 17AAG*	Inhibits cell cycle arrest in cancer cells, which then enter M phase with damaged chromosomes, activates spindle check-point and causes mitotic catastrophe.	*Phase I clinical trial ** Phase II clinical trial	[38 and references therein, 40, 41, 42]
Mitotic inhibitors	Kinesin inhibitors KSP/Eg5 inhibitors Ispinesib (SB-715992)**, SB-743921** MK-0731*, ARRY-520* CENP-E inhibitor GSK923295A Mitotic kinase inhibitor Plk1 inhibitor BI2536**, GSK461364A**, Cyclapolin I DAP-81 Aurora kinase inhibitor VX-680**, PHA-739358**, AZD1152* MLN-8054**, R763*, CYC116* PF-03814735*, AT-9283**	Mitotic inhibitor prevents the formation of bipolar spindles and normal assembly of chromosomes, activating the spindle checkpoint and resulting in M-phase arrest and initiate apoptosis.	* Phase I clinical trials ** Phase II clinical trial *** Phase III clinical trial	[6, 16 and references therein, 44, 45]

^#Compounds without any star (*) are still at pre-clinical stage

CDK Inhibitors

As discussed earlier, CDKs regulate the cell cycle progression, and their activity is increased in cancer cells. Accordingly, pursuits for the drugs that inhibit CDKs have been the intense area of research for last two decades, and numerous CDK inhibitors have been identified (Table 1). These drugs have been classified as pan-CDK inhibitors or selective CDK inhibitors [9]. Flavopiridol and CYC-202 (R-roscovitine) are the earliest known CDK inhibitors and have undergone numerous clinical trials; however, their efficacy had been modest [22–25]. One of the reasons behind their modest clinical success is their non-selective action affecting normal as well as cancer cells. In this regard, it will be pertinent to mention that other than cell cycle progression each of the CDKs has unexpected roles in specialized cell types. For example, the role of CDK2 in germ cells maturation, and the role of CDK4 in the proliferation of pancreatic β cells and endocrine cells have been shown [23]. Therefore, the inhibitors of these CDKs are expected to cause many adverse effects. Further, in clinical trials CDK inhibitors have encountered problems related with their dosing, schedule of administration and their target specificity. Accordingly, the new generation of CDK inhibitors with better potency (e.g. SNS-032, AT-7519, R-547 etc.) are being tested in pre-clinical and clinical settings [23]. Silibinin is another pan-CDK inhibitor, which is widely known for its hepatoprotective and cancer chemopreventive properties [26–28]. It has been shown to modulate cyclin-CDK-CDKI axis resulting in cell cycle arrest in variety of cancer cell lines in vitro and in vivo [29–33]. Silibinin has recently completed phase I clinical trial and now its efficacy is being evaluated in phase II clinical trial in prostate cancer patients [34].

Lately, there has been a lot of debate over the choice of CDK inhibitors. It is being realized that identification of predictive biomarkers for various cancers might be useful in selecting the CDK inhibitor as treatment option. For example, CDK4 inhibitor alone can protect mammary gland cells from Ras- or Her2-, but not Myc-, induced tumorigenesis [23]. Similarly, CDK1 inhibition alone can provide relevant therapeutic effects in Myc-induced lymphomas and hepatoblastomas [23]. These results suggest that identification of these biomarkers and genetic context of CDK inhibitors action might provide significant therapeutic value. Further, CDK inhibitors like flavopiridol and rocovitine have been shown to target CDK9/cyclin T resulting in the reduced efficiency of transcriptional elongation, which might promote apoptosis or inhibit cell proliferation [10]. Therefore, the effect of CDK inhibitors on non-cell cycling CDKs/cyclins might also determine their effect, but still more studies are needed to understand the effect of other CDK inhibitors on these non-cycling CDKs/cyclins.

Cdc25 Phosphatase Inhibitors

The Cdc25 phosphatases (A, B and C) serve as key activators of CDKs by removing the inhibitory phosphorylation, and thereby, play a central role in the checkpoint response to DNA damage [35] (Figure 1). The overexpression of Cdc25A and Cdc25B has been reported in numerous human tumors and is linked with poor clinical prognosis [17]. Therefore, the Cd25 phosphatases have been targeted for anticancer drug development, and represent a promising therapeutic approach for the treatment of cancer. Various Cdc25 phosphatase inhibitors are listed in Table 1; among them, ARQ-501 has been engaged in phase I clinical trials in patients with advanced and chemotherapy unresponsive solid tumors [35]. Another noteworthy Cdc25 inhibitor BN82685 has been reported to be active in vivo by oral administration and to inhibit the growth of the human pancreatic tumor Mia PaCa-2 xenografted in athymic nude mice [36].

Checkpoint Inhibitors

DNA damaging agents are known to activate the cellular checkpoints (Chk1 and Chk2) via DNA damage sensor protein kinases namely ATM, ATR and DNA-PK (Figure 1) [12, 37]. These activated checkpoints kinases phosphorylate Cdc25 phosphatases causing their inactivation whereby downstream CDKs remain inhibited resulting in cell cycle arrest, which provides the cells extra time to repair the damage [6, 12]. Accordingly, the rationale behind the development of checkpoint inhibitors is that their treatment would target the cellular checkpoints and abrogate the cell cycle arrest imposed by DNA damaging agents resulting in an unscheduled entry into mitosis and mitosis-associated death in tumor cells [38, 39]. Since, cancer cells already have a malfunctioning G1 checkpoint, inhibitors specifically targeting G2 checkpoints are of greater interest [38–40]. Various molecules like Chk1, Chk2, PP2A, 14-3-3 and Wee1 have been suggested as the key targets for checkpoint abrogation [38], and numerous checkpoint inhibitors are listed in Table 1. Among all the checkpoint inhibitors, UCN-01 is most clinically advanced, and is in phase I/II clinical trials in cancer patients [38, 41–43].

Mitotic Inhibitors

Mitotic inhibitors include inhibitors of microtubule, mitotic kinesins and mitotic kinases. Microtubule inhibitors are non-specific in action and have been categorized as chemotherapeutic agents, and therefore, only mitotic kinesins and kinases are discussed here, which play an important role during mitosis in centrosome maturation, spindle assembly, chromosome segregation, activation of anaphase-promoting complex (APC/C), cytokinesis and the activation of the spindle checkpoint [16, 44]. Aurora kinase family members (A, B and C) have been regarded as the key mitotic kinases regulating the divergent functions in mitotic control. Aurora-A kinase is mainly involved in centrosome function, mitotic entry, and spindle assembly, whereas Aurora-B participates in chromatin modification, microtubule-kinetochore attachment, spindle checkpoint, and cytokinesis [16, 45]. Aurora-A and -B kinases, despite having high structural homology, differ in their sub-cellular localization as well as in their regulation [45]. It has been reported that abnormal expression of Aurora A or Aurora B in cancer cells results in anomalous spindle formation, compromised spindle checkpoint and failure of cytokinesis resulting in polyploidy or aneuploidy [45]. Therefore, targeting Aurora kinases in cancer cells has been suggested as a sound strategy.

In recent years, the field of the mitotic inhibitors’ discovery and development has exploded, and numerous of them are already in clinical development (Table 1). Among these, ispinesib (KSP/Eg5 inhibitors), BI2536 (Plk1 inhibitor) and VX-680 (Aurora kinase inhibitor) are most effective and clinically advanced agents [16, 46, 47]. These inhibitors have been shown to result in the activation of spindle checkpoint and mitotic arrest followed by induction of apoptosis, though, their exact mechanism of action is still unknown [16].

Efficacy and Limitations of Cell Cycle Inhibitors

The cell cycle based agents have shown excellent pre-clinical effectiveness but their efficacy in the clinic has been modest and far below expectations. Most of the clinically advanced cell cycle agents like flavopiridol, UCN01, VX-680, ispinesib etc. have shown serious toxicities in the clinic [6, 16, 43, 48], which could be due to a lack of specificity. Furthermore, the agents like UCN01 have shown unique pharmacological problems in the clinic related to their binding with high affinity to human alpha1-acid glycoprotein [43]. Overall, identification of the pharmacological doses, schedule of administration and related efficacy of these agents in the clinic have been the key issues yet to be answered. Accordingly, it has been suggested that these agents could play a better role as a partner with chemotherapeutic agents, and therefore, cell cycle agents are being evaluated in various new combination therapies for cancer eradication.

Cancer Chemotherapy

Cancer chemotherapy has been the frontline approach for cancer treatment in last several decades. The use of nitrogen mustard for lymphoma treatment during 1940s was the first step to the realization that cancer could be treated by pharmacological agents [49]. This was followed by the use of folic acid antagonist, purines analogues, and platinum- and taxol-based drugs [7]. The majority of the chemotherapeutic drugs can be divided in to alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase poisons, etc., and have been described in detail earlier [7]. The major limitation that has restricted the usefulness of most of the cancer chemotherapy agents is their non-specificity with broader cytotoxicity against dividing cells. For this reason, more recently, there is a growing interest in developing drugs that target a specific molecular alteration in cancer cells. One successful example is tyrosine kinase inhibitor imatinib (Gleevac) which has been used against CML with abnormal protein kinase BCR-ABL [50]. Despite these advances, the use of chemotherapy has been limited by the associated toxicity and side effects, higher costs, and the development of drug resistance. Overall, the cancer remains a major cause of illness and death, and conventional cytotoxic chemotherapy has been unable to cure most cancers especially those at advanced stage.

Cell Cycle Agents in Combination with Chemotherapeutic Agents

It has been reported that cell cycle mediated drug resistance limits the potential benefits of standard chemotherapeutic drugs in clinic [51], which could be overcome by better understanding the effect of chemotherapeutic agents on cell cycle and by appropriate sequencing and scheduling of the agents in the combination therapy [51] (Figure 2). For example, the treatment with chemotherapeutic drugs mostly a) interferes with DNA synthesis, b) introduces DNA damage, or c) inhibits the function of mitotic spindle [16]; and these effects lead to activation of cellular checkpoint followed by cell cycle arrest, which might partly be responsible for the cell cycle based resistance (Figure 2). In such scenarios, the presence of another appropriate cell cycle based agent might inhibit the cell cycle based resistance along with increasing the potency of chemotherapeutic drug as illustrated in detail in Figure 2. Accordingly, there is an emphasis on using the cell cycle agent in combination with chemotherapy [52]. These combinations with different targets could better challenge the cancer, which has multiple mechanisms of survival. Furthermore, the use of agents in combination might also reduce the chances of development of drug resistance to any one agent. In this regard, different classes of cell cycle agents have been studied in combination with chemotherapeutic drugs in numerous pre-clinical and clinical investigations, as discussed below.

Figure 2.

Proposed model suggesting the ideal combination therapy for cancer treatment. In step I, cancer is treated with only a chemotherapeutic agent, while in step II, a suitable cell cycle agent is also added.

CDK Inhibitors in Combination Studies

Various CDK inhibitors have been studied in combination with chemotherapeutic drugs and many of them are in clinical trials [6, 51]. Flavopiridol is the most studied CDK inhibitor in this regard, and has been combined with taxols, irinotecan, gemcitabine, cisplatin, etc. [6, 51, 53]. A combination of paclitaxel and flavopiridol in phase I study has shown promising results in patients with chemotherapy refractory malignancies such as prostate, lung and esophagus [54]. In another phase I clinical trial in pancreatic, breast and ovarian cancer patients, the combination of docetaxel and flavopiridol has shown encouraging partial responses [6, 55]. The combination of irinotecan and flavopiridol was also shown to have significant partial responses in patients with gastric, esophagus, colorectal, adrenocortical, and hepatocellular cancers [6, 56].

Another pan-CDK inhibitor silibinin has been shown to sensitizes prostate cancer cells to cisplatin-, carboplatin-, doxorubicin- and mitoxantrone-induced cell growth inhibition, cell cycle arrest and/or apoptotic death [57–59]. Silibinin combination with these platinum drugs and doxorubicin has also shown synergistic effect towards cell growth inhibition and apoptotic death in breast cancer cells [60]. The combination of silibinin has been shown to increase the efficacy and reduce the toxicity of doxorubicin in lung cancer cells in xenograft model [61]. Silibinin infusion before cisplatin treatment has also been shown to decrease cisplatin-associated glomerular and tubular kidney toxicity [62]. Another in vitro study in human testicular cancer cell lines has suggested that silibinin does not affect the anti-tumor activity of cisplatin or ifosfamide [62].

With regard to a mechanistic base in selecting combination approaches, several studies have shown that cell death after the exposure of taxanes occurs as cell exits from abnormal mitosis. Because degradation of cyclin B1-CDK1 is required for the exit from mitosis, its inhibition by CDK inhibitors after chemotherapeutic drugs facilitates mitotic exit and hastens cell death. In this regard, it has also been shown that spindle checkpoint activation also induces survival pathway that depends upon CDK1-mediated phosphorylation and stabilization of survivin, which is an apoptotic inhibitor and mitotic regulator [16]. Accordingly, it is rationalized that the inhibition of CDK1 activity would prevent the phosphorylation and accumulation of survivin, thereby effectively removing a survival signal and enhancing apoptosis [48]. Therefore, combining the chemotherapeutic drugs with CDK1 inhibitor could be one of the mechanisms to overcome the increased cancer cell survival eventually leading to an enhanced apoptotic death (Figure 2). In another study, Motwani et al. have shown that DNA damaging agent SN-38 induces cell cycle arrest without cell death in human colon cancer HCT116 cells. The addition of flavopiridol to SN-38-treated HCT166 cells caused cell death in vitro and in vivo [63]. The increased apoptotic death in the presence of flavopiridol was associated with higher activation of caspase-3 and cleavage of p21 and XIAP (an inhibitor of apoptosis). Jung et. al. have also shown that the addition of flavopiridol to gemcitabine-treated human gastrointestinal cancer cells is associated with reduction in the ribonucleotide reductase M2 subunit (RR-M2), a rate limiting enzyme in DNA synthesis, thereby, enhancing the apoptosis and anti-tumor activity of gemcitabine [64].

Overall, these studies suggest that combining CDK inhibitors with chemotherapeutic drugs might reduce the toxicity and increase the efficacy of chemotherapeutic drugs, while also decreasing the chances of drug resistance development.

Cdc25 Inhibitors in Combination Studies

Cdc25 inhibitors have been studied pre-clinically for their efficacy in combination with chemotherapeutic drugs. It has been reported that combining the low concentrations of BN82685 and paclitaxel inhibits proliferation of colon cancer cells, suggesting that combination of Cdc25 inhibitors with microtubule targeting agents may be of therapeutic interest [65].

Checkpoint Inhibitors in Combination Studies

As summarized above, the checkpoint inhibitors in the presence of DNA damaging agents result in inhibition of cell cycle arrest, and cells enter in mitosis phase with DNA damage, which activates the spindle checkpoint resulting in mitotic arrest followed by the activation of apoptotic pathway known as ‘mitotic catastrophe’[16]. In this regard, the combination of UCN-01 has been shown to enhance the antitumor efficacy of nucleoside analogs such as cytarabine, fludarabine and gemcitabine [39]. Furthermore, UCN-01 combination with cisplatin [66], topotecan [67], fluorouracil [68], carboplatin [69] and irinotecan [70] has completed phase I clinical trial in patients with solid tumors. Based upon encouraging results from these combinations, several additional phase I and II clinical trials for leukemia, lung cancer and advanced solid tumors are currently underway. Recently, the in vitro and in vivo studies have shown that XL-844, an orally available and specific inhibitor of Chk1 and Chk2, enhances the anti-tumor activity of gemcitabine in human pancreatic cancer cells [71]. Currently, XL-844 is undergoing phase I clinical trial as a single agent as well as in combination with gemcitabine in adults with advanced malignancies. Other Chk1 inhibitors have also shown encouraging results in pre-clinical studies. For example, Chk1 inhibitor CHIR-124 has been shown to enhance topoisomerase I poison-induced apoptosis in breast cancer cells in cell culture and orthotopic xenograft model [72]. Another Chk1 inhibitor PF-00394691 has also been shown to potentiate the antitumor activity of gemcitabine, irinotecan and cisplatin without increasing the host toxicity in a tumor xenograft model [39].

Mitotic Inhibitors in Combination Studies

It has been shown that the treatment with mitotic inhibitors (microtubule poison) results in activation of spindle checkpoint and mitotic arrest followed by mitotic slippage and induction of apoptosis [16]. However, cancer cells have been reported to have weak spindle checkpoint along with activation of various pro-survival signals in the presence of mitotic inhibitors [16]. In this regard, overexpression of Aurora-A in cancer cells has been demonstrated to result in an abrogation of the spindle checkpoint leading to resistance towards taxol [16]. Therefore, combining taxol-based agents with mitotic kinase inhibitors might decrease the chemoresistance and increase the drug efficacy. Indeed, the inhibition of Aurora A kinase has been shown to enhance the chemosensitivity of pancreatic cancer cells towards taxanes [73]. Similarly, the downregulation of mitotic kinase Plk1 has been shown to increase the sensitivity of breast cancer cells towards paclitaxel [74]. Plk1 inhibitor, ON01910, has been shown to enhance the effect of several chemotherapeutic agents, and its clinical trials with conventional chemotherapeutic drugs are currently underway [75, 76]. A completed phase I clinical trial of ispinesib and docetaxel in patients with advanced solid tumors has shown partial responses with acceptable toxicity profile [77]. These encouraging reports warrant more clinical studies with the combination of mitotic inhibitors and chemotherapeutic drugs.

Critical Element in Combination Studies: Lessons Learnt

The completion of various combination studies has shown that the sequence of drug use is the most critical element determining the success of combination. One agent can impact the cell cycle in such a manner that next agent administered immediately in sequence becomes less effective. For example, in vitro and in vivo studies have shown that when flavopiridol is used at the same time or before the paclitaxel or docetaxel treatment, there is a decrease in the efficacy of paclitaxel or docetaxel [6, 48]. This is due to the fact that flavopiridol induces cell cycle arrest and prevents cells from entering M-phase that is where paclitaxel and docetaxel are most active. Importantly and in support of the thought that the sequence of drug use is most critical element in determining the success of combination therapies, the reverse sequence of paclitaxel or docetaxel followed by flavopiridol is associated with an increased induction of apoptosis [6, 48].

An additional important aspect of these combination strategies is that the cell cycle based agents together with chemotherapeutic agents have also shown toxicity, which indicates that further molecular understanding is required regarding the pharmacologic inhibition of drug targets in clinical settings. For example, increased myelosuppression was seen in the phase I combination trial of UCN-01 with topotecan at doses of topotecan lower than the ones when the drug is used as a single agent, suggesting that combination might have synergistic effect in normal cells as well [39].

Another critical element of combination strategy is that one drug should stay sufficiently long enough in the target tumor tissue to sensitize most of the cells towards the other drug. Therefore, the guidelines are needed for the optimal scheduling, which would provide the levels and exposure time required for optimal biological response by combination therapies.

Conclusion and Future Directions

Cell cycle based agents have shown tremendous promise and potential against cancer; however, they are not fully effective by themselves. Similarly, cancer chemotherapies, which are the mainstream treatments for various human malignancies, are plagued by toxicity and the development of drug resistance, decreasing their overall clinical usefulness. Combining these two different categories of drugs has shown decreased toxicity and chemoresistance along with increased efficacy, suggesting that this could be an ideal approach to lower the cancer burden (Figure 2); however, we still have a long way to go with this strategy, specifically because only in recent year it is being realized that the better understanding of cell cycle regulatory molecules is the pre-requisite for the development of better drugs used either alone or in combination to eradicate different cancers. In this regard, recent studies have also shown various non-traditional roles for cell cycle molecules. For example Cdc25 phosphatases are traditionally considered to play a role in the activation of CDKs; however, now they are also identified to play a role in microtubule dynamics including a correct assembly of the mitotic spindle, etc [65]. Further, the identification of predictive biomarkers in tumors could be useful to select the patients for the treatment with cell cycle based agent alone or in combination. For example, the status of p53 (wild type or mutated) might determine the success of a particular treatment strategy. These and several additional new findings are expected to help us better understand the potential additional roles of cell cycle inhibitors toward both their efficacy and associated side effects. In future, advances in these areas and refinement of dosing and scheduling of drugs will be the key for establishing a standard cell cycle based combination therapies against cancer.