Cyclin-dependent kinases (Cdk) and their associated pathways represent some of the most attractive targets for the development of anticancer therapeutics. Based on antitumor activity in animal models, a variety of Cdk inhibitors are undergoing clinical evaluation either as a single agent or in combination with other approved drugs. In our anticancer drug discovery program, a novel series of flavones have been synthesized for evaluation against the activity of Cdk4-D1. This enzyme catalyzes the phosphorylation of retinoblastoma protein, thus inhibiting its function. We have identified a series of potent Cdk4-D1 inhibitors with IC 50 below 250 nmol/L. In this report, we have described the properties of one of the best compound,

P276-00, a flavone that inhibits cyclin-dependent kinases, has been identified by us recently as a novel antineoplastic agent. In this study, we have selected a panel of human tumor cell lines and xenografts to allow determination of selectivity and efficacy of P276-00. When tested against a panel of 16 cisplatin-sensitive and cisplatin-resistant cell lines, the antiproliferative potential of P276-00 was found to be f30-fold higher than cisplatin. Studies to show tumor sensitivity using clonogenic assay in 22 human xenografts indicated that P276-00 was f26-fold more potent than cisplatin, and further, it was also found to be active against cisplatin-resistant tumors of central nervous system, melanoma, prostate, and renal cancers. Further, we studied the effects of P276-00 on cell cycle progression by flow cytometry using asynchronous and synchronous population of tumor and normal cells.

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BackgroundP276-00 is a novel cyclin-dependent kinase inhibitor currently in Phase II clinical trials. Gemcitabine is a standard of care for the treatment of pancreatic cancer. The present study investigated the effect of the combination of P276-00 and gemcitabine in five pancreatic cancer cell lines.MethodsCytotoxic activity was evaluated by Propidium Iodide assay. Cell cycle and apoptosis was analyzed by flow cytometry. Genes and proteins known to inhibit apoptosis and contribute to chemoresistance were analysed using western blot analysis and RT-PCR. In vivo efficacy was studied in PANC-1 xenograft model.ResultsThe combination of gemcitabine followed by P276-00 was found to be highly to weakly synergistic in various pancreatic cancer cell lines as assessed by the combination index. Enhancement of apoptosis in PANC-1 cells and decrease in the antiapoptotic protein Bcl-2 and survivin was seen. P276-00 potentiated the gemcitabine-induced cytotoxicity by modulation of proteins involved in chemoresistance to gemcitabine and cell cycle viz. antiapoptotic proteins p8 and cox-2, proapoptotic protein BNIP3 and cell cycle related proteins Cdk4 and cyclin D1. The above results could explain the novel mechanisms of action of the combination therapy. We also show here that gemcitabine in combination with P276-00 is much more effective as an antitumor agent compared with either agent alone in the PANC-1 xenograft tumor model in SCID mice.ConclusionsThe chemosensitzation of pancreatic tumors to gemcitabine would likely be an important and novel strategy for treatment of pancreatic cancer and enable the use of lower and safer concentrations, to pave the way for a more effective treatment in this devastating disease. Phase IIb clinical trials of P276-00 in combination with gemcitabine in pancreatic cancer patients are ongoing.

BackgroundIn the present study, we show that the combination of doxorubicin with the cyclin-dependent kinase inhibitor P276-00 was synergistic at suboptimal doses in the non-small cell lung carcinoma (NSCLC) cell lines and induces extensive apoptosis than either drug alone in H-460 human NSCLC cells.MethodsSynergistic effects of P276-00 and doxorubicin on growth inhibition was studied using the Propidium Iodide (PI) assay. The doses showing the best synergistic effect was determined and these doses were used for further mechanistic studies such as western blotting, cell cycle analysis and RT-PCR. The in vivo efficacy of the combination was evaluated using the H-460 xenograft model.ResultsThe combination of 100 nM doxorubicin followed by 1200 nM P276-00 showed synergistic effect in the p53-positive and p53-mutated cell lines H-460 and H23 respectively as compared to the p53-null cell line H1299. Abrogation of doxorubicin-induced G2/M arrest and induction of apoptosis was observed in the combination treatment. This was associated with induction of tumor suppressor protein p53 and reduction of anti-apoptotic protein Bcl-2. Furthermore, doxorubicin alone greatly induced COX-2, a NF-κB target and Cdk-1, a target of P276-00, which was downregulated by P276-00 in the combination. Doxorubicin when combined with P276-00 in a sequence-specific manner significantly inhibited tumor growth, compared with either doxorubicin or P276-00 alone in H-460 xenograft model.ConclusionThese findings suggest that this combination may increase the therapeutic index over doxorubicin alone and reduce systemic toxicity of doxorubicin most likely via an inhibition of doxorubicin-induced chemoresistance involving NF-κB signaling and inhibition of Cdk-1 which is involved in cell cycle progression.

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BackgroundHypoxia-inducible factor-1 (HIF-1) is a master regulator of the transcriptional response to hypoxia. It is essential for angiogenesis and is associated with tumor progression and overexpression of HIF-1α has been demonstrated in many common human cancers. Therefore, HIF-1α is one of the most compelling anticancer targets.MethodsTo identify HIF-1α inhibitors, luciferase reporter gene assay under hypoxia and normoxia was used. Detailed studies such as western blotting, RT-PCR, immunofluorescence were carried out to elucidate its mechanism of action. Antiangiogenic activity of P3155 was demonstrated by migration assay and tube formation assay. Efficacy study of P3155 was performed on PC-3 xenograft model.ResultsP3155 showed specific HIF-1α inhibition with IC50 of 1.4 μM under hypoxia. It suppressed HIF-1α expression as well as PI3K/Akt pathway and abrogated expression of HIF-1-inducible gene viz. vascular endothelial growth factor (VEGF). P3155 in combination with HIF-1α siRNA showed significant synergistic effect. In addition, it demonstrated significant in vivo efficacy and antiangiogenic potential in prostate cancer cell lines.ConclusionWe have identified a novel HIF-1α inhibitor P3155 that also modulates PI3K/Akt pathway, which may contribute to its significant in vitro and in vivo antitumor activity.

Cell cycle deregulation is one of the first steps that transform normal cells into tumor cells. Cyclin-dependent kinases (Cdks) related alterations such as overexpression and amplification of cell-cycle activators or inactivation of cell cycle inhibitors are among the most common genetic changes in human tumors. The pursuit for drugs that inhibit Cdks has been an intense area of research for more than 15 years. A number of Cdk inhibitors are being evaluated in clinical trials for the treatment of cancer. P1446A-05 has been identified as a Cdk4-D1, Cdk1-B and Cdk9-T inhibitor. P1446A-05 exhibited greater selectivity to Cdk4-D1 and Cdk1-B that are involved in the G1 and G2/M phase of the cell cycle respectively. The IC50 values were 0.09 and 0.025 μM for Cdk4-D1 and Cdk1-B respectively. P1446A-05 also inhibited Cdk9-T, which plays a role in transcription, with an IC50 of 0.022 μM. P1446A-05 was inactive against many non-Cdks with an IC50 of more than 50 μM. In cytotoxicity assay using 30 human cancer cell lines P1446A-05 showed IC50 in the range of 0.5 to 3.9 μM, whereas the cytotoxicity for unstimulated normal human PBMNCs was > 10 μM. P1446A-05 arrested cell cycle progression at G1 and G2 phases and induced apoptosis in human cancer cell lines such as non-small-cell lung cancer (H-460), colorectal carcinoma (HCT-116) and prostate cancer (PC-3) in a dose and time dependent manner. Furthermore, P1446A-05 affected the proteins involved in cell cycle and apoptosis. It down-regulated cyclin D1, inhibited Cdk4 specific phosphorylation (ser780 of retinoblastoma), induced the tumor suppressor protein p53, and reduced the levels of the anti-apoptotic protein Bcl-2. Pharmacokinetic studies revealed that P1446A-05 exhibited an absolute oral bioavailability of 82.48% in mice and 79.07% in rats. It showed dose-dependent increase in 1446A-05 levels in plasma of mice and rats. In in vivo efficacy studies, orally administered P1446A-05 significantly (p<0.001) inhibited the tumor growth in two xenograft models that of colon cancer (Colo-205) and non-small cell lung cancer (H-460) in SCID mice. These data demonstrated that P1446A-05 is an orally active inhibitor of Cdk with a unique target profile that may have therapeutic potential in human malignancies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3054. doi:1538-7445.AM2012-3054