The Effect Of EB1089 On Prostate Cancer DU145 Cell

BackgroundProstate cancer is currently the most frequent malignancy and the second leading cause of cancer-related deaths in men in the United States. In the past few years, detection rate of prostate cancer has been increasing in our country. Now, advanced androgen-independent prostate cancer (AIPC) remains a therapeutic challenge.There is still not an ideal, effective therapy for AIPC. Therefore, it is of clinical significance to study and explore novel therapeutic strategies for AIPC.It is reported that Hedgehog signaling pathway is closely related to prostate cancer. The reported experiments show that human prostate tumors express Hh ligand and Hh-target genes-Gli and a chemical inhibitor of Hedgehog signaling pathway, cyclopamine, inhibits the growth of cultured and xenografted human prostate cancer cell lines and down-regulates cell invasiveness. But cyclopamine have not succeeded in the clinic for its strong side effect and synthesis difficulty. Recent report shows that vitamin D is an inhibitor of Hedgehog signaling pathway and bound to Smo with high affinity in a cyclopamine-sensitive manner, and effectively inhibits Gli activity.Failure of chemotherapy may be caused by multidrug resistance (MDR) mechanisms protecting cancer cells against cytotoxic drugs in prostate cancer. MDR is associated with ATP-binding cassette transporter family protein, including P-glycoprotein(PGP),multidrug resistance-associated protein(MRP) and breast cancer resistance protein(BCRP). It is reported that Hedgehog pathway inhibitor is a potent inhibitor of BCRP and PGP.However, the clinical usefulness of vitamin D is limited by its tendency to cause hypercalcaemia. EB1089, a vitamin D analogue, is more effective than vitamin D to regulate cell growth and differentiation, but with weaker effects on the calcium metabolism. The aim of the paper is to assess whether or not EB1089, as a Hedgehog pathway inhibitor, inhibits cell growth and reverse multidrug resistance by Hedgehog signaling pathway in AIPC DU145 cell.Method and ResultFirst,we surveyed the impact that EB1089 contributed DU145 cell growth, cell cycle and apoptosis by MTT assay, flow cytometry(FCM), microscope and transmission electron microscope, compared with cyclopamine. In result, EB1089(1?10.50?100nmol/L) inhibited DU145 cell proliferation significantly after 24h,48h,72h and showed time and dosage symbiosis. The impact of EB1089 was more effective than that of cyclopamine. After DU145 cell were treated by 1?10?50?100nmol/L EB1089 for 48 hours,G0/G1 cell population of DU145 cell gradually degraded, and apoptosis rate gradually increased. EB1089 could evoke typical apoptosis in DU145 cell, which including cell volume deflating, karyopyknosis, nuclear spallation, nuclear fragmentation apoptotic body formation.Second, we detected expression of Glil?cyclinDl?Bcl-2?Bax mRNA and protein in DU145 cell respectively by Real time PCR and Western blot, after EB1089(1?10?50?100nmol/L) treated DU145 for 48h. As a result, along with effective dosage of EB1089 increasing, there were gradual decrease in the expression of Glil?cyclinDl?Bcl-2 mRNA and protein levels and increase in the expression of Bax levels, after EB1089 treated DU145 cell for 48 hours.Third, we evaluated the effect that combined treatment of EB 1089 and docetaxel contributed DU145 cell growth and EB1089 contributed chemosensitivity of docetaxel-resistant DU145 cell(DU145-DR) by proliferation assays (MTT). We further detected the expression of PGP/MDR1?MRP?BCRP mRNA and protein levels in DU145-DR cell respectively by Real time PCR and Western blot, which before and after was treated by EB1089. The effect manifested as an increasing growth inhibition rate in DU145 cell, under combined treatment compared with single treatment of either EB1089 or docetaxel alone. Docetaxel-induced growth inhibition in DU145-DR was enhanced by EB1089. The reversal index of chemosensitivity of DU145-DR cell was 2.87 and the relative reverse rate of sensitivity is 70.8%. We further demonstrated that EB1089 significantly reduced the expression of PGP/MDR1?BCRP at both the mRNA and protein levels in DU145-DR.ConclusionThis suggests EB1089 can inhibit proliferation, and cause cell-cycle arrest in the G1-phase, induce apoptosis in DU145 due to EB1089 reducing of Gli?cyclinDl?Bcl-2 expression and increasing of Bax expression by Hedgehog signaling pathway. EB1089 can enhance chemosensitivity and reverse drug resistance in DU145-DR cell associated with down-regulation of PGP/MDR?BCRP expression. Combined treatment of EB1089 and docetaxel can synergismly inhibit proliferation in DU145 cell.