Background: Response rates to gemcitabine (Gem), the most commonly used chemotherapy in pancreatic cancer (PC), remain quite low, which is partly due to de novo chemo-resistance, in addition to over-expression of ribonucleotide reductase M2 subunit (RR-M2), a rate-limiting enzyme in DNA synthesis that is regulated by E2F-1. Moreover, E2F-1 is a key transcriptional factor necessary for transition to S phase, cellular proliferation, viability, and has recently been correlated with poor differentiation and survival in PC. Thus, E2F-1 represents a unique therapeutic target in PC. We have previously reported E2F-1 targeted effects in prostate cancer using thymoquinone (TQ), an herbal agent that spares normal cells. Aim: The aim of our study was to determine the effects of TQ either alone or in combination with Gem in PC, both in vitro and in vivo. Methods and Results: We observed that TQ inhibited proliferation of PC cell lines (AsPC-1, BxPC3, MIAPaCa-2, and PANC-1) but not that of immortalized human pancreatic ductal epithelial (HPDE) cells. We showed that TQ synergistically potentiated pro-apoptotic effects of Gem in PC cell lines. Using PANC-1 (Gem-resistant) cells as our model system, we showed that TQ down-regulated E2F-1 and increased p21 (Cip1) and p27 (Kip1) expression. We demonstrated that TQ inhibited expression of anti-apoptotic proteins including IAP-1, survivin, and Bcl-2 and proliferative proteins including Cyclin D1. We also showed that TQ inhibited expression of proteins involved in invasion and metastasis including COX-2, VEGF, and MMP-9. Further, TQ significantly potentiated Gem effects in reduction of E2F-1 and anti-apoptotic proteins along with increase of p21 and p27. In a PANC-1 heterotopic xenograft model, TQ given daily orally (50 mg/kg) in combination with Gem (25 mg/kg intraperitoneally twice weekly) for 4 weeks significantly reduced tumor volume as compared to control vehicle, TQ alone, or Gem alone groups. We showed that in vivo suppression of tumor growth was associated with potent reduction in E2F-1, c-myc, and Cyclin D1 with concomitant increase in p21 and p27 expression in tumor explants. Furthermore, we demonstrated that TQ + Gem significantly reduced proliferation index (Ki-67+ cells) and increased apoptosis (in situ TUNEL assay) in tumor explants. Conclusion: Collectively, we showed that TQ potently potentiated cytotoxic effects of gemcitabine in PC cells both in vitro and in vivo. These effects could be in part due to downregulation of E2F-1, necessary for proliferation, survival, and chemo-resistance of PC.

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 2759.

100th AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO

American Association for Cancer Research
2009