Enhanced anti-tumor effect of combination therapy with gemcitabine and apigenin in pancreatic cancer

Apigenin is a dietary flavonoid possessing therapeutic potential against cancers. This study was designed to investigate whether combination therapy with gemcitabine and apigenin enhanced anti-tumor efficacy in pancreatic cancer. In vitro, the combination treatment resulted in more growth inhibition and apoptosis through the down-regulation of NF-kappa B activity with suppression of Akt activation in pancreatic cancer cell lines (MiaPaca-2, AsPC-1). In vivo, the combination therapy augmented tumor growth inhibition through the down-regulation of NF-kappa B activity with the suppression of Akt in tumor tissue. The combination of gemcitabine and apigenin enhanced anti-tumor efficacy through Akt and NF-kappa B activity suppression and apoptosis induction.     

Flavopiridol-induced apoptosis during S phase requires E2F-1 and inhibition of cyclin A-dependent kinase activity

Transformed cells are selectively sensitized to apoptosis induced by the cyclin-dependent kinase inhibitor flavopiridol after their recruitment to S phase. During S phase, cyclin A-dependent kinase activity neutralizes E2F-1 allowing orderly S phase progression. Inhibition of cyclin A-dependent kinase by flavopiridol could cause inappropriately persistent E2F-1 activity during S phase traversal and exit. Transformed cells, with high baseline levels of E2F-1 activity, may be particularly sensitive to cyclin A-dependent kinase inhibition, as the residual level of E2F-1 activity that persists may be sufficient to induce apoptosis. Here, we demonstrate that flavopiridol treatment during S phase traversal results in persistent expression of E2F-1. The phosphorylation of E2F-1 is markedly diminished, whereas that of the retinoblastoma protein is minimally affected, so that E2F-1/DP-1 heterodimers remain bound to DNA. In addition, manipulation of E2F-1 levels leads to predictable outcomes when cells are exposed to flavopiridol during S phase. Tumor cells expressing high levels of ectopic E2F-1 are more sensitive to flavopiridol-induced apoptosis during S phase compared with parental counterparts, and high levels of ectopic E2F-1 expression are sufficient to sensitize nontransformed cells to flavopiridol. Furthermore, E2F-1 activity is required for flavopiridol-induced apoptosis during S phase, which is severely compromised in cells homozygous for a nonfunctional E2F-1 allele. Finally, the response to flavopiridol during S phase is blunted in cells expressing a nonphosphorylatable E2F-1 mutant incapable of binding cyclin A, suggesting that the modulation of E2F-1 activity produced by flavopiridol-mediated cyclin-dependent kinase inhibition is critical for the apoptotic response of S phase cells.     

E2F-1 gene therapy induces apoptosis and increases chemosensitivity in human pancreatic carcinoma cells.

Pancreatic cancer is often resistant to conventional chemotherapy. In this study, we examined the role of adenovirus-mediated overexpression of E2F-1 in inducing apoptosis and increasing the sensitivity of pancreatic cancer cells to chemotherapeutic agents. MIA PaCa-2 pancreatic head exocrine adenocarcinoma cells (mutant p53) were treated by mock infection or adenoviruses expressing beta-galactosidase or E2F-1 (Ad-E2F-1) alone or in combination with sublethal concentrations of each chemotherapeutic drug. Cell growth and viability were assessed at selected time points. Apoptosis was evaluated by flow cytometry, characteristic changes in cell morphology and poly (ADP-ribose) polymerase (PARP) cleavage. Western blot analysis was used to examine the expression of E2F-1 and Bcl-2 family member proteins and PARP cleavage. Western blot analysis revealed marked overexpression of E2F-1 at a multiplicity of infection (MOI) of 20 and 70. By 3 days after infection, Ad-E2F-1 treatment at an MOI of 70 resulted in approximately a 20-fold reduction in cell growth and 60% reduction in cell viability as compared to mock-infected cells. Cell cycle analysis, PARP cleavage and changes in cell morphology supported apoptosis as the mechanism of cell death in response to E2F-1. In order to test the efficacy of treatment with a combination of gene therapy and chemotherapy, we utilized concentrations of Ad-E2F-1 which reduced viability to 50% in combination with each chemotherapeutic agent. Cotreatment of the cells with E2F-1 virus and roscovitine (ROS) or etoposide resulted in an additive effect on cell growth inhibition and induction of apoptosis. Interestingly, 5-fluorouracil did not cooperate with Ad-E2F-1 in the mediation of tumor death or inhibition of cell growth. Immunoblotting for Bcl-2 family members revealed no significant changes in the expression levels of Bcl-2, Bcl X(L), Bax or Bak following gene or 'chemogene' therapy with E2F-1. However, a Bax cleavage product was noted which was substantially increased by cotreatment with ROS or etoposide. E2F-1 overexpression initiates apoptosis and suppresses growth in pancreatic MIA PaCa-2 cells in vitro. E2F-1-mediated apoptosis was not associated with significant changes in the expression of Bcl-2 family member proteins in these pancreatic cancer cells. ROS and etoposide, when combined with E2F-1 overexpression, induce apoptosis in an additive manner. This chemogene combination may provide a potentially useful therapeutic strategy for advanced pancreatic cancer.     

Sequence dependent potentiation of gemcitabine by flavopiridol in human breast cancer cells

Summary Purpose. Flavopiridol is a novel cyclin dependent kinase (cdk) inhibitor currently in Phase I and II clinical trials. We investigated the interaction between flavopiridol and gemcitabine in two human breast cancer cell lines.Experimental design. MCF-7 [Estrogen receptor (ER) positive] and MDA-MB-231 cells (ER negative) were treated with sub-cytotoxic concentrations of gemcitabine (G), flavopiridol (F), and flavopiridol followed by gemcitabine (F–G), or gemcitabine followed by flavopiridol (G–F) and assayed for biological activity. Results. Growth inhibition assessed by serial cell counting and MTT assay was highest in the G–F group. Significant increase in apoptosis assessed by flow cytometry, poly-ADP-ribose polymerase (PARP) and Caspase-3 degradation was also highest in the G–F group. Expression of pro-apoptotic Bax was up-regulated and anti-apoptotic Bcl-2 was down-regulated in only the G–F treated cells. Significant up regulation of p21^WAF-1 was demonstrated in the G–F group but not in the reverse regimen treated cells. No effect on protein kinase C (PKC) expression was seen in any of the treated cells. Conclusions. In conclusion, similar to the results in the gastrointestinal cell lines, a sequence dependent potentiation of the effect of gemcitabine by flavopiridol was demonstrated in breast cancer cell lines and it was independent of ER status. This was accompanied by enhanced apoptosis and the up regulation of p21^WAF-1 protein. These results provide rationale for pre-clinical evaluation of this treatment strategy using animal models and in the design of clinical trials of this drug in combination with cytotoxic therapy.     

Enhanced effect of gemcitabine by emodin against pancreatic cancer in vivo via cytochrome C-regulated apoptosis

Gemcitabine is currently the best treatment available for pancreatic cancer, but causes high toxicity. Agents that can enhance the effects of gemcitabine with no or low toxicity are needed for the treatment of pancreatic cancer. Emodin, a natural anthraquinone derivative, is one such agent that has been shown to induce apoptosis in other tumor cells via down-regulation of Bcl-2/Bax and promoting the release of Cytochrome C (CytC), but with very low toxicity. The aim of this study was to evaluate whether emodin can enhance the effect of gemcitabine on pancreatic cancer in?vitro and in?vivo and to investigate the possible mechanisms of the enhancement. In?vitro, emodin inhibited the proliferation of the SW1990 cell line and potentiated the apoptosis induced by gemcitabine, which was demonstrated by activation of caspase-3 in the combination group. In?vivo, tumors from nude mice subcutaneously injected with SW1990 cells and treated with a combination of emodin (40?mg/kg) and gemcitabine (80?mg/kg) showed significant reductions in volume, Ki-67 proliferation index and expression of the Bcl-2/Bax ratio (compared with tumors from mice treated with sodium chloride, emodin alone (40?mg/kg) or gemcitabine alone (125?mg/kg), which induced increasing release of CytC from the mitochondria to the cytoplasm and triggered caspase-3 activation leading to apoptosis. Taken together, our results suggest that emodin improved the anti-tumor effect of gemcitabine, even at a lower dose of gemcitabine which could decrease the toxicity of chemotherapy, on transplanted tumors of the SW1990 cell line through the enhancement of apoptosis induced by gemcitabine, the mechanism of which may be through down-regulation of the Bcl-2/Bax ratio and promoting release of CytC from the mitochondria into the cytoplasm.     

p21对肝癌细胞中survivin转录的影响及调控机制的探讨

目的 研究细胞周期依赖性激酶抑制蛋白p21对人肝癌细胞survivin转录抑制的调控作用,并探讨其相应机制.方法 使用多柔比星处理人肝癌细胞株HepG2,采用脂质体法将质粒pEGFP-C2-p21导入HepG2细胞,用G418筛选转染后的HepG2-p21和HepG2-pEGFP细胞;采用实时定量聚合酶链反应(RQ-PCR)检测p21、p53和survivin的mRNA表达;采用流式细胞仪分析转染后细胞周期的变化;采用逆转录聚合酶链反应(RT-PCR)检测转染后E2F-1和p300的mRNA表达.结果 经多柔比星处理后,HepG2细胞中p21和p53 mRNA的表达先增高,然后逐渐降低;survivin mRNA的表达则相反.转染后,HepG2-p21细胞中p21 mRNA的表达明显增高,分别为HepG2和HepG2-pEGFP细胞的2100.1倍和980.9倍;survivin mRNA的表达却明显降低,分别为HepG2和HepG2-pEGFP细胞的0.5%和0.6%;p53 mRNA的表达差异无统计学意义.流式细胞仪检测显示,HepG2-p21细胞的细胞周期明显阻滞在G0/G1期.HepG2-p21细胞中E2F-1和p300 mRNA的表达水平分别为0.75±0.04和0.18 ±0.06,与HepG2和HepG2-pEGFP细胞比较,含量均明显减低(P＜0.05).结论 p21可能通过抑制survivin的转录调控因子E2F-1和p300 mRNA的表达,从而抑制细胞中survivin的表达,并导致细胞出现G0/G1期阻滞.     

Enhancement of radiation effects by combined docetaxel and flavopiridol treatment in lung cancer cells.

BACKGROUND AND PURPOSE: To evaluate the potential role and mechanism of docetaxel plus flavopiridol in modulating radiosensitivity in vitro and in vivo. PATIENTS AND METHODS: In vitro. H460 human lung carcinoma cells were treated with docetaxel (10 nM for 1 h, at t = 0 h) --> radiation (0-5 Gy, at t = 6 h) --> flavopiridol (120 nM for 24 h, at t = 8 h). Colony forming ability was measured to assess the modulation of sensitivity. Cell cycle redistribution was measured by flow cytometric analysis using propidium iodide. Percent apoptosis was also measured by flow cytometric analysis using 7-amino-actinomycin D staining. In vivo. H460 cell xenografts were used in nude mice. Tumors were grown subcutaneously on the flank, then treated with docetaxel (2.5 mg/kg, at t = 0 h) --> radiation (2 Gy, at t = 6 h) --> flavopiridol (1.25 mg/kg, at t = 8 h) for 5 consecutive days. Tumor growth delay was then measured and compared with the control group. RESULTS: Docetaxel plus flavopiridol enhanced the effect of radiation. The maximum radiopotentiation and apoptosis were observed when the cells were treated with the sequence of docetaxel-->radiation-->flavopiridol both in vitro and in vivo. Flavopiridol and docetaxel induced G1 and G2/M arrest, respectively. CONCLUSIONS: This study shows that docetaxel plus flavopiridol enhances the effects of radiation in vitro and in vivo. Our data suggest that the mechanism of radiopotentiation by combining flavopiridol and docetaxel involves an enhancement of apoptosis and changes of cell cycle by docetaxel and flavopiridol.     

The mdm2 proto-oncogene sensitizes human medullary thyroid carcinoma cells to ionizing radiation

We have analysed the radiation response of a human medullary thyroid carcinoma cell line (MTT), characterized by the absence of a functional p53 protein, and the consequences of MDM2 overexpression in this process. We show that the product of the mdm2 proto-oncogene is able to sensitize MTT cells to ionizing radiation. After radiation treatment, MTT cells display histograms consistent with a G2M arrest. MTT cells expressing MDM2 (MTT-mdm2) are unable to respond to DNA damage with G2M arrest, and display a high percentage of apoptosis. MTT-mdm2 cells show high levels of E2F-1 protein, suggesting that the induction of apoptosis observed upon MDM2 overexpression could be dependent on E2F-1. This observation is further supported with assays showing that E2F-1 binding to specific DNA sequences is enhanced in MTT-mdm2 cells. Likewise, transactivation of reporter constructs exclusively dependent on E2F-1 is also elevated after transfection with MDM2. This effect can be reverted by transient transfection with p19ARF. To link the expression of E2F-1 with the induction of apoptosis, we generated clonal cell lines overexpressing E2F-1. Transfection with E2F-1 results in a low number of outgrowing colonies with reduced proliferation rates, indicating that E2F-1 is deleterious for cell growth. This negative regulation correlates with an increase in the percentage of the cell population with DNA content below 2N, suggesting that E2F-1 promotes apoptosis. Finally, overexpression of E2F-1 sensitizes MTT cells to radiation exposure. We conclude that the effects observed by MDM2 overexpression could be mediated by E2F-1.