Kalpana Joshi

In vitro antitumor properties of a novel cyclin-dependent kinase inhibitor, P276-00

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 IC50 below 250 nmol/L. In this report, we have described the properties of one of the best compound, P276-00 of the flavones series. P276-00 shows 40-fold selectivity toward Cdk4-D1, compared with Cdk2-E. The specificity toward 14 other related and unrelated kinases was also determined. P276-00 was found to be more selective with IC50s <100 nmol/L for Cdk4-D1, Cdk1-B, and Cdk9-T1, as compared with other Cdks, and less selective for non-Cdk kinases. It showed potent antiproliferative effects against various human cancer cell lines, with an IC50 ranging from 300 to 800 nmol/L and was further compared for its antiproliferative activity against cancer and normal fibroblast cell lines. P276-00 was found to be highly selective for cancer cells as compared with normal fibroblast cells. To delineate its mechanism of action, the effect of P276-00 on cell cycle proteins was studied in human breast cancer cell line (MCF-7) and human non–small cell lung carcinoma (H-460). A significant down-regulation of cyclin D1 and Cdk4 and a decrease in Cdk4-specific pRb Ser780 phosphorylation was observed. P276-00 produced potent inhibition of Cdk4-D1 activity that was found to be competitive with ATP and not with retinoblastoma protein. The compound also induced apoptosis in human promyelocytic leukemia (HL-60) cells, as evidenced by the induction of caspase-3 and DNA ladder studies. These data suggest that P276-00 has the potential to be developed as an anti-Cdk chemotherapeutic agent. [Mol Cancer Ther 2007;6(3):918–25]

P276-00, a novel cyclin-dependent inhibitor induces G1-G2 arrest, shows antitumor activity on cisplatin-resistant cells and significant in vivo efficacy in tumor models

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 ∼30-fold higher than cisplatin. Studies to show tumor sensitivity using clonogenic assay in 22 human xenografts indicated that P276-00 was ∼26-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. Asynchronous population of human prostate carcinoma (PC-3) and human promyelocytic leukemia (HL-60) cells when exposed to P276-00 showed arrest of slow-growing PC-3 cells in G2-M with no significant apoptosis observed up to 72 h. Unlike PC-3, significant apoptosis was seen in fast-growing HL-60 cells at 6 h. However, synchronized human non–small cell lung carcinoma (H-460) and human normal lung fibroblast (WI-38) cells showed arrest of cells in G1. H-460 cells undergo apoptosis, which increases with longer exposure to the compound and also after exposure to P276-00 for 48 h followed by recovery. In contrast, the normal cells (WI-38) remain arrested in G1 with no significant apoptosis up to 72 h of exposure and also after 48 h of P276-00 treatment followed by recovery, confirming our previous results that P276-00 was less effective against normal cells compared with cancer cells. After promising in vitro results, P276-00 was checked for in vivo efficacy in murine tumor and human xenograft models. Growth inhibition of murine colon cancer (CA-51) was significant when P276-00 was administered i.p. at 50 mg/kg daily for 20 treatments. However, in murine lung carcinoma model (Lewis lung), an increased dose of 60 mg/kg (30 mg/kg twice daily) administered every alternate day i.p. for seven treatments showed significant inhibition in the growth. Further studies were undertaken to establish the efficacy profile of P276-00 in human tumor xenograft models. In the two xenograft models studied, P276-00 showed potent in vivo antitumor potential. Compound P276-00 at a dose of 35 mg/kg administered daily via the i.p. route for 10 days showed significant (P < 0.05) inhibition in the growth of human colon carcinoma HCT-116 xenograft. Furthermore, P276-00 at a dose of 50 mg/kg once daily and 30 mg/kg twice daily administered via i.p. route for 20 treatments significantly (P < 0.05) inhibited growth of human non–small cell lung carcinoma H-460 xenograft. Thus, the in vitro cellular potency, together with in vivo antitumor activity, confirms the potential of P276-00, a cyclin-dependent kinase inhibitor as an anticancer molecule. [Mol Cancer Ther 2007;6(3):926–34]

Cyclin-dependent kinase inhibitor, P276-00 induces apoptosis in multiple myeloma cells by inhibition of Cdk9-T1 and RNA polymerase II-dependent transcription

P276-00 is a novel cyclin-dependent kinase inhibitor especially potent for Cdk9-T1, Cdk4-D1 and Cdk1-B. Multiple myeloma (MM) is a B-cell malignancy characterized by the accumulation of malignant plasma cells. Treatment of MM cell lines with P276-00 resulted in apoptosis that correlated with transcription inhibition and a significant decline in Mcl-1 protein levels with the appearance of cleaved PARP in these cells. In vivo studies of P276-00 confirmed antitumor activity in RPMI-8226 xenograft. These results suggest that P276-00 causes multiple myeloma cell death by disrupting the balance between cell survival and apoptosis through inhibition of transcription and downregulation of Mcl-1.

A novel inhibitor of hypoxia-inducible factor-1α P3155 also modulates PI3K pathway and inhibits growth of prostate cancer cells.

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.

Development of novel inhibitors targeting HIF-1α towards anticancer drug discovery

Hypoxia-inducible factor-1α (HIF-1α) is a critical regulatory protein of cellular response to hypoxia, and regulates the transcription of many genes involved in key aspects of cancer biology, including immortalization, maintenance of stem cell pools, cellular dedifferentiation, vascularization, and invasion/metastasis. HIF-1α has been implicated in the regulation of genes involved in angiogenesis, for example, VEGF and is associated with tumor progression. In the last decade, over expression of HIF-1α has been demonstrated in many common human cancers and emerging as a validated target for anticancer drug discovery. Here we report the discovery of newly designed and synthesized pyridylpyrimidine based potent and selective inhibitors of HIF-1α. P2630 has been found as potent antiproliferative, antiangiogenic and orally efficacious compound in PC-3 xenograft mice model.

Design and synthesis of novel furoquinoline based inhibitors of multiple targets in the PI3K/Akt-mTOR pathway.

We herein report the design and synthesis of furoquinoline based novel molecules (16-36) and their in vitro multiple targeted inhibitory potency against PI3K/Akt phosphorylation and mTOR using cell based and cell-free kinase assay. In particular, compound 23 in addition to PI3K-mTOR inhibitory potency, it has shown potent inhibition of hypoxia-induced accumulation of HIF-1alpha protein in U251-HRE cell line. The inhibitory activities of compound 23 were confirmed by Western blot analysis, using human non-small cell lung carcinoma H-460 cell line and glioblastoma U251 cell lines.

Abstract A67: Molecular evidence of increased antitumor activity of gemcitabine in combination with a Cdk inhibitor, P276 in pancreatic cancer

Purpose: Gemcitabine is an inhibitor of ribonucleotide reductase and DNA synthesis and is a standard of care for the treatment of pancreatic cancer. The present study investigates whether the cdk inhibitor P276 would be synergistic with gemcitabine in pancreatic cancers. Experimental design: Various pancreatic cancer cell lines were treated with gemcitabine and P276 and the cytotoxicity was assessed using the combination index. The effect of gemcitabine and P276 on cell cycle and apoptosis was analyzed by flow cytometry. Several genes that are known to inhibit apoptosis and contribute to chemoresistance were analyzed using western blot analysis and RT‐PCR. Finally, in vivo efficacy of the combination was studied in Panc‐1 xenograft model in SCID mice. Results: The combination of gemcitabine followed by P276 was found to be synergistic in various pancreatic cancer cell lines Panc‐1, Capan‐1, BxPc‐3, AsPc‐1 and MiaPaca‐2 as assessed by the combination index using the Compusyn software. Mechanistic studies indicated enhancement of apoptosis in Panc‐1 cells as seen by flow cytometry and increase in the antiapoptotic Bcl‐2 protein by western blot analysis. Several genes that are known to inhibit apoptosis and contribute to chemoresistance such as p8, cox‐2 and survivin were assessed to investigate the basis for the observed chemosensitizing effects of P276. P276 potentiated the gemcitabine induced cytotoxicity by downregulation of p8, cox‐2 and survivin. In contrast, cox‐2 was found to be significantly up regulated when the cells were exposed to gemcitabine alone, while there was no effect on p8 or survivin as compared to control, suggesting the potential mechanism of acquired chemoresistance to gemcitabine. BNIP3 the tumor suppressor is also downregulated in most pancreatic cancer cell lines and is associated with chemoresistance to gemcitabine. Interestingly BNIP3 was significantly up regulated in the combination and P276 alone but not by gemcitabine alone. In addition to in vitro results, we show here that gemcitabine in combination with P276 is much more effective as an antitumor agent compared with either agent alone in our Panc‐1 xenograft tumor model in SCID mice. Conclusion: These data demonstrate that the gemcitabine and a Cdk inhibitor, P276 combination displays synergistic cytotoxic activity, induces apoptosis and sensitizes the pancreatic cells to gemcitabine by modulating the pro‐ and anti apoptotic proteins. In vivo evidence of the synergistic combination is also demonstrated in Panc‐1 xenograft model. The 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. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A67.