Dynamic Roles of Signaling Pathways in Maintaining Pluripotency of Mouse and Human Embryonic Stem Cells.

Culturing of mouse and human embryonic stem cells (ESCs) in vitro was a major breakthrough in the field of stem cell biology. These models gained popularity very soon mainly due to their pluripotency. Evidently, the ESCs of mouse and human origin share typical phenotypic responses due to their pluripotent nature, such as self-renewal capacity and potency. The conserved network of core transcription factors regulates these responses. However, significantly different signaling pathways and upstream transcriptional networks regulate expression and activity of these core pluripotency factors in ESCs of both the species. In fact, ample evidence shows that a pathway, which maintains pluripotency in mouse ESCs, promotes differentiation in human ESCs. In this review, we discuss the role of canonical signaling pathways implicated in regulation of pluripotency and differentiation particularly in mouse and human ESCs. We believe that understanding these distinct and at times-opposite mechanisms-is critical for the progress in the field of stem cell biology and regenerative medicine.

Molecular pharmacology of multitarget cyclin-dependent kinase inhibitors in human colorectal carcinoma cells.

BACKGROUND: Colorectal cancer (CRC) is a leading cause of cancer death. Certain signaling pathways are implicated in colorectal carcinogenesis. Cyclin-dependent kinases (CDKs) are commonly hyperactivated in CRC and hence multitarget CDK inhibitors serve as promising therapeutic drugs against CRC. OBJECTIVE: Off-target effects of multitarget CDK inhibitors with differential CDK inhibitory spectrum viz. P276-00 (also known as riviciclib), roscovitine and UCN-01 on CRC cell lines of varied genetic background were delineated. METHOD: Protein expression was analyzed for key signaling proteins by western blotting. ß-catenin localization was assessed using immunofluorescence. HIF-1 transcriptional activity and target gene expression were studied by reporter gene assay and RT-PCR respectively. Anti-migratory and anti-angiogenic potential was evaluated by wound healing assay and endothelial tube formation assay. RESULTS: CDK inhibitors modulated various signaling pathways in CRC and for certain proteins showed a highly cell line-dependent response. Riviciclib and roscovitine inhibited HIF-1 transcriptional activity and HIF-1α accumulation in hypoxic HCT116 cells. Both of these drugs also abrogated migration of HCT116 and in vitro angiogenesis in HUVECs. CONCLUSION: Anticancer activity of multitarget CDK inhibitors can be certainly attributed to their off-target effects and should be analyzed while assessing their therapeutic utility against CRC.

Molecular Milieu of Autophagy in Cervical Cancer and its Therapeutic Implications.

Cervical cancer is a common death-causing cancer among women in developing countries. Majority of the cases are triggered by persistent infections with high-risk Human Papillomavirus (HPV16 and 18). Metastasis, disease relapse, and drug resistance are common among patients in advanced stages of cancer despite the available therapies. Consequently, new prospective targets are needed for this disease. Autophagy is professed to have implications in cervical cancer progression as well as cancer dormancy. This article reviews the role of autophagy in cervical cancer progression and the modulation of the autophagy pathway by HPV. Further, various therapeutic agents that target autophagy in cervical cancer are discussed.

The story of EGFR: from signaling pathways to a potent anticancer target.

EGFR is a member of the ERBB family. It plays a significant role in cellular processes such as growth, survival and differentiation via the activation of various signaling pathways. EGFR deregulation is implicated in various human malignancies, and therefore EGFR has emerged as an attractive anticancer target. EGFR inhibition using strategies such as tyrosine kinase inhibitors and monoclonal antibodies hinders cellular proliferation and promotes apoptosis in cancer cells in vitro and in vivo. EGFR inhibition by tyrosine kinase inhibitors has been shown to be a better treatment option than chemotherapy for advanced-stage EGFR-driven non-small-cell lung cancer, yet de novo and acquired resistance limits the clinical benefit of these therapeutic molecules. This review discusses the cellular signaling pathways activated by EGFR. Further, current therapeutic strategies to target aberrant EGFR signaling in cancer and mechanisms of resistance to them are highlighted.

Promising Anticancer Activity of Multitarget Cyclin Dependent Kinase Inhibitors against Human Colorectal Carcinoma Cells.

BACKGROUND: Colorectal cancer (CRC) is the third leading cause of cancer death worldwide, and its incidence is steadily rising in developing nations. Cell cycle aberrations due to deregulation of cyclin dependent kinases (CDKs) and cyclins are common events during colorectal carcinogenesis. Yet, efficacy of multitarget CDK inhibitors as therapeutic agents has not been much explored against CRC. OBJECTIVE: The anticancer potential of multitarget CDK inhibitor riviciclib (also known as P276-00), was investigated against CRC cell lines of varied genetic background. METHODS: Cytotoxicity of riviciclib - potent CDK1, CDK4 and CDK9-specific inhibitor was evaluated in vitro. Further, its effect on clonogenic potential, cell cycle, apoptosis and transcription was tested using colony forming assay, flow cytometry and western blot analysis, respectively. Also, efficacy of riviciclib in combination with standard chemotherapeutic agents was assessed. Dependency of CRC cells on specific CDKs for their survival was confirmed using siRNA studies. RESULTS: Riviciclib exerted significant cytotoxicity against CRC cells and inhibited their colony forming potential. It induced apoptosis along with inhibition of cell cycle CDKs and cyclins as well as transcriptional CDKs and cyclins. Moreover, dual combination of riviciclib with standard chemotherapeutic drugs exhibited synergism in CRC cells. siRNA studies indicated that CRC cells are dependent on specific CDKs for their survival which are targets of riviciclib. CONCLUSION: This study provides evidence that multitarget CDK inhibitors can serve as promising therapeutic agents against CRC alone or in combination.

A flow cytometric journey into cell cycle analysis.

Cell cycle involves a series of changes that lead to cell growth and division. Cell cycle analysis is crucial to understand cellular responses to changing environmental conditions. Since its inception, flow cytometry has been particularly useful for cell cycle analysis at single cell level due to its speed and precision. Previously, flow cytometric cell cycle analysis relied solely on the measurement of cellular DNA content. Later, methods were developed for multiparametric analysis. This review explains the journey of flow cytometry to understand different molecular and cellular events underlying cell cycle using various protocols. Recent advances in the field that overcome the shortcomings of traditional flow cytometry and expand its scope for cell cycle studies are also discussed.

Flow cytometry: principles, applications and recent advances.

Flow cytometry (FCM) is a sophisticated technique that works on the principle of light scattering and fluorescence emission by the specific fluorescent probe-labeled cells as they pass through a laser beam. It offers several unique advantages as it allows fast, relatively quantitative, multiparametric analysis of cell populations at the single cell level. In addition, it also enables physical sorting of the cells to separate the subpopulations based on different parameters. In this constantly evolving field, innovative technologies such as imaging FCM, mass cytometry and Raman FCM are being developed in order to address limitations of traditional FCM. This review explains the general principles, main applications and recent advances in the field of FCM.

Potentiation of in vitro and in vivo antitumor efficacy of doxorubicin by cyclin-dependent kinase inhibitor P276-00 in human non-small cell lung cancer cells.

BACKGROUND: In 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. METHODS: Synergistic 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. RESULTS: The 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. CONCLUSION: These 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.

P276-00, a cyclin-dependent kinase inhibitor, modulates cell cycle and induces apoptosis in vitro and in vivo in mantle cell lymphoma cell lines.

BACKGROUND: Mantle cell lymphoma (MCL) is a well-defined aggressive lymphoid neoplasm characterized by proliferation of mature B-lymphocytes that have a remarkable tendency to disseminate. This tumor is considered as one of the most aggressive lymphoid neoplasms with poor responses to conventional chemotherapy and relatively short survival. Since cyclin D1 and cell cycle control appears as a natural target, small-molecule inhibitors of cyclin-dependent kinases (Cdks) and cyclins may play important role in the therapy of this disorder. We explored P276-00, a novel selective potent Cdk4-D1, Cdk1-B and Cdk9-T1 inhibitor discovered by us against MCL and elucidated its potential mechanism of action. METHODS: The cytotoxic effect of P276-00 in three human MCL cell lines was evaluated in vitro. The effect of P276-00 on the regulation of cell cycle, apoptosis and transcription was assessed, which are implied in the pathogenesis of MCL. Flow cytometry, western blot, immunoflourescence and siRNA studies were performed. The in vivo efficacy and effect on survival of P276-00 was evaluated in a Jeko-1 xenograft model developed in SCID mice. PK/PD analysis of tumors were performed using LC-MS and western blot analysis. RESULTS: P276-00 showed a potent cytotoxic effect against MCL cell lines. Mechanistic studies confirmed down regulation of cell cycle regulatory proteins with apoptosis. P276-00 causes time and dose dependent increase in the sub G1 population as early as from 24 h. Reverse transcription PCR studies provide evidence that P276-00 treatment down regulated transcription of antiapoptotic protein Mcl-1 which is a potential pathogenic protein for MCL. Most importantly, in vivo studies have revealed significant efficacy as a single agent with increased survival period compared to vehicle treated. Further, preliminary combination studies of P276-00 with doxorubicin and bortezomib showed in vitro synergism. CONCLUSION: Our studies thus provide evidence and rational that P276-00 alone or in combination is a potential therapeutic molecule to improve patients' outcome in mantle cell lymphoma.

Molecular evidence for increased antitumor activity of gemcitabine in combination with a cyclin-dependent kinase inhibitor, P276-00 in pancreatic cancers.

BACKGROUND: P276-00 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. METHODS: Cytotoxic 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. RESULTS: The 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. CONCLUSIONS: 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. Phase IIb clinical trials of P276-00 in combination with gemcitabine in pancreatic cancer patients are ongoing.

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

BACKGROUND: Hypoxia-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. METHODS: To 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. RESULTS: P3155 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. CONCLUSION: We 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.

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.