SKP2 Is Associated With Paclitaxel Resistance In Prostate Cancer Cells

Objectives:Prostate cancer is the most common tumor diagnosed in men in the United States. In recent years, the incidence of prostate cancer in men is rising in china. Patients with hormone-refractory prostate cancer are ofen treated with paclitaxel, but most of them eventually develop drug resistance. Therefore, it is urgent need to find hormone refractory drug-resistant prostate cancer new therapeutic targets in the clinical treatment of prostate cancer. In the present study, we investigated the effects of Skp2 and its inhibitors on cell viability and proliferation in paclitaxel resistant PCa cells. So as to provide a new theoretical basis for the clinical treatment of prostate cancer.Methods:1. Detect the index of drug resistance of androgen resistance paclitaxel-resistant cells PC-3-TxR and DU145-TxR constructed in our lab by using MTS method.2. In addition to Skp2, detect the expression of Fbxw8 and other Cullin-RING E3 ligase components CUL1-7 in Pca paclitaxel-resistant cells and parental cells by using Real time quantitative RT-PCR and Western blot.3. The expression of Skp2 in PC3-TxR and DU145-TxR by lentivirus-mediated Skp2 shRNA interference. Detect the efficiency of Skp2 interference by using Real time quantitative RT-PCR and Western blot.4. To determine the effects of Skp2 interference and Skp2 inhibitor combined chemotherapeutics paclitaxel on PCa cells proliferation by using MTS assay.5. The Western blotting was applied in substrates of Skp2 such as p21, p27 and FOXO1 in paclitaxel resistant Pca cells and their parental cells.6. Detect the changes of EMT markers expression of E-cadherin and Vimentin in the paclitaxel-resistance Pca cells the level of mRNA and protein by using Real time quantitative RT-PCR and Western blot. Further detect the expression of E-cadherin and Vimentin in the level of mRNA and protein in paclitaxel resistant PCa cells PC3-TxR and DU145-TxR by shRNA interference the expression of Skp2.7. We examined the expression of Skp2-related cell cycle proteins in Skp2 silencing paclitaxel resistant PCa cells. Explore the related posssible signaling pathways and molecular mechanisms of the Skp2 involved in paclitaxel resistant PCa cells.8. Detect the proliferation and cell viability of Skp2 inhibitor C1 and compound 25 by using MTS method for PCa paclitaxel-resistant cells and parental cells.9. Detect the expression of p21 and p27 substrates of Skp2 through Skp2 inhibitor C1 and compound 25 combined with paxlitaxel in paclitaxel resistant PCa cells.Results:The results of MTS showed that 12.5 nM of paclitaxel treatment for 48 or 72 hours significantly inhibited the growth of PC3 cells. While in PC3-TxR cells,200 nM of paclitaxel treatment for 48 or 72 hours significantly inhibited cell growth. The IC50 of 72 hours of parental cells PC3 or DU145 are 18.42 or 10.91 nM. The IC50 of 72 hours of drug resistant cells PC3-TxR or DU145-TxR are respectively 166.47 or 3245.63 nM. The expression of Skp2 was significantly increased in paclitaxel resistant prostate cancer cells in the level of protein and mRNA by using Western blot and Real time quantitative RT-PCR. We established the stable interference of Skp2 cell lines PC3-TxR Skp2shRNA and DU145-TxR Skp2shRNA in paclitaxel resistant prostate cancer cells. Knockdown of Skp2 increases sensitivity to paclitaxel in paclitaxel-resistant prostate cancer cells. The results of Skp2 inhibitor combined with conventional chemotherapeutics paclitaxel showed that comparing with paclitaxel alone group, the paclitaxel plus Skp2 inhibitor group significantly decreased the cell viability. Western blotting showed that E-cadherin was significantly reduced in PC-3-TxR and DU145-TxR. Otherwise, the expression of mesenchymal markers including Vimentin and Skp2 was elevated compared with their parental cells. p27, one of the Skp2 substrates, was significantly decresed in paclitaxel-resistant cells compared with parental cells. Immunofluorescence further observed the expression and localization of EMT markers and Skp2. Knockdown of Skp2 restores the expression of p27 in paclitaxel-resistant prostate cancer cells. Furthermore, paclitaxel or Skp2 inhibitor treatment alone led to the accumulation of p27 in paclitaxel-resistant prostate cancer cells. Importantly, there is a synergetic effect of the accumulation of p27 in the combination treatment of paclitaxel with Skp2 inhibitors.Conclusion:This present study showed that Skp2 is up-regulated in paclitaxel resistant PCa cells.Knockdown of Skp2 increases sensitivity to paclitaxel and restored the expression of p27 in paclitaxel-resistant PCa cells. Skp2 inhibitors combined with paclitaxel also reverse the resistance of paclitaxel-resistant PCa cells to paxlitaxel. The molecular mechanism may be through Skp2-p27 pathway.