RESUMO
BACKGROUND: Tumors of the head and neck present aggressive pathological behavior in patients due to high expression of CDK/CCND1 proteins. P276-00, a novel CDK inhibitor currently being tested in clinic, inhibits growth of several cancers in vitro and in vivo. The pre clinical activity of P276-00 in head and neck cancer and its potential mechanisms of action at molecular level are the focus of the current studies. METHOD: We have investigated the anti-cancer activity of P276-00 in head and neck tumors in vitro and in vivo. Candidate gene expression profiling and cell based proteomic approaches were taken to understand the pathways affected by P276-00 treatment. RESULTS: It was observed that P276-00 is cytotoxic across various HNSCC cell lines with an IC50 ranging from 1.0-1.5 µmoles/L and culminated in significant cell-cycle arrest in G1/S phase followed by apoptosis. P276-00 treatment suppressed cell proliferation through inhibition of CCND1 expression, reduced phosphorylation of retinoblastoma protein and abrogative transcription of E2F1 gene targets. Further, we observed that apoptosis was mediated through P53 activation leading to higher BAX/BCL-2 ratio and cleaved caspase-3 levels. It was also seen that P276-00 treatment reduced expression of tumor micro-environment proteins such as IL-6, secreted EGFR and HSPA8. Finally, P276-00 treatment resulted in significant tumor growth inhibition in xenograft tumor models via lowered proliferative activity of E2F1 and aggravated P53 mediated apoptosis. CONCLUSION: In summary, we have observed that P276-00 inhibits cyclin-D/CDK4/P16/pRB/E2F axis and induces apoptosis by increased P53 phosphorylation in HNSCC cells. These results suggest a novel indication for P276-00 in head and neck cancer with a potential role for IL-6 and HSPA8 as candidate serum biomarkers.
Assuntos
Antineoplásicos/farmacologia , Carcinoma de Células Escamosas/tratamento farmacológico , Flavonas/farmacologia , Regulação Neoplásica da Expressão Gênica , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Animais , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Ciclina D/metabolismo , Ciclina D1/metabolismo , Quinases Ciclina-Dependentes/antagonistas & inibidores , Fator de Transcrição E2F1/metabolismo , Receptores ErbB/metabolismo , Perfilação da Expressão Gênica , Proteínas de Choque Térmico HSC70/metabolismo , Humanos , Concentração Inibidora 50 , Interleucina-6/metabolismo , Camundongos , Camundongos SCID , Transplante de Neoplasias , Fosforilação , Proteína do Retinoblastoma/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Proteína Quinase CDC2/metabolismo , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ciclo-Oxigenase 2/metabolismo , Doxorrubicina/administração & dosagem , Sinergismo Farmacológico , Flavonas/administração & dosagem , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/genética , Camundongos , Camundongos SCID , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína Supressora de Tumor p53/genéticaRESUMO
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.
Assuntos
Quinases Ciclina-Dependentes/antagonistas & inibidores , Desoxicitidina/análogos & derivados , Flavonas/uso terapêutico , Neoplasias Pancreáticas/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Sequência de Bases , Linhagem Celular Tumoral , Primers do DNA , Desoxicitidina/farmacologia , Desoxicitidina/uso terapêutico , Flavonas/farmacologia , Humanos , Neoplasias Pancreáticas/patologia , Inibidores de Proteínas Quinases/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , GencitabinaRESUMO
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.
Assuntos
Inibidores da Angiogênese/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Fosfatidilinositol 3-Quinases/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Piridinas/farmacologia , Pirimidinas/farmacologia , Animais , Processos de Crescimento Celular/efeitos dos fármacos , Hipóxia Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células Endoteliais da Veia Umbilical Humana , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Masculino , Camundongos , Neovascularização Patológica/tratamento farmacológico , Neoplasias da Próstata/irrigação sanguínea , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Transdução de Sinais/efeitos dos fármacos , Topotecan/farmacologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
Assuntos
Apoptose/efeitos dos fármacos , Quinase 9 Dependente de Ciclina/antagonistas & inibidores , Flavonas/farmacologia , Mieloma Múltiplo/tratamento farmacológico , Transcrição Gênica/efeitos dos fármacos , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Western Blotting , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Quinase 9 Dependente de Ciclina/genética , Quinase 9 Dependente de Ciclina/metabolismo , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Relação Dose-Resposta a Droga , Humanos , Camundongos , Camundongos SCID , Microscopia Confocal , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Interferência de RNA , RNA Polimerase II/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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 approximately 30-fold higher than cisplatin. Studies to show tumor sensitivity using clonogenic assay in 22 human xenografts indicated that P276-00 was approximately 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 G(2)-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 G(1). 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 G(1) 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.
Assuntos
Antineoplásicos/uso terapêutico , Quinases Ciclina-Dependentes/antagonistas & inibidores , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos , Flavonas/farmacologia , Fase G2/efeitos dos fármacos , Neoplasias Experimentais/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cisplatino/uso terapêutico , Quinases Ciclina-Dependentes/metabolismo , Citometria de Fluxo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Neoplasias Experimentais/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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 of the flavone's 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 IC(50)s <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 IC(50) 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 Ser(780) 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.