| IntroductionOvarian cancer is still the greatest contributor for gynecological malignancies. Despite significant initial response rates for advanced ovarian carcinoma using Paclitaxel-Platinum combination chemotherapy, the vast majority of patients relapse and develop a drug-resistant disease with an overall 5-year survival of <50%. Acquired resistance to Paclitaxel is one of the most significant reasons for its failure in chemotherapy. Previous studies have demonstrated that Paclitaxel resistance is attributable to various mechanisms: increased drug efflux that results from up-regulation of membrane transporters (e.g., P-gp); alterations in the expression of β-tubulin isotypes; changes in apoptotic regulatory proteins (e.g. Bcl-2); and the overexpression of TRAG-3/CSAG2. However, limited achievements have been obtained in the development of means to combat resistance according to the possible mechanisms. Thus, the molecular mechanism of Paclitaxel resistance has yet to be precisely defined and further research is needed.Heat shock protein 27(HSP27, also called HSP25), is one of the small heat shock proteins family. HSP27 plays an important role in regulating wide range of homeostatic and pathogenic activities such as the correct folding of proteins, cell proliferation and survival et al. HSP27's expression could be inhibited by Homo sapiens scaffold attachment factor B (SAFB), which could depress the transcription of HSP27 by binding to the HSP27 promoter. Investigations show that HSP27 is over-expression in the chemoresistant tumors. HSP27 is a dependent prognostic factor for ovarian tumors and it is also suggested that HSP27's over-expression relates theresponse of ovarian cancer cells to chemotherapeutic drugs. But there are contradictory and limited data about the relationship between HSP27 and ovarian cancer's drug resistance, so the specific relation between them still needs further study. HSP27 could enhance chemoresistance by inhibiting apoptosis and stabilizing the cytoskeleton system, and Paclitaxel's fanction depends on the microtubule cytoskeleton. Our Previous study found that HSP27 was up-regulated in Paclitaxel-resistant cell line SKOV3-TR30 compared with its maternal cell line SK0V3, suggesting the role of HSP27 playing in ovarian cancer SK0V3's Paclitaxel resistance. Therefore, in this study we aimed to further explore the possible role of HSP27 playing in Paclitaxel resistance by investigating the effects of Paclitaxel on HSP27's expression and cyto-location.ObjectivesTo investigate the relationship between HSP27 and Paclitaxel-resistance, and to explore the possible role of HSP27 playing in the SK0V3's Paclitaxel-resistance.MethodsThe epithelial ovarian cancer cell line SK0V3 and its Paclitaxel-resistant sub-line SKOV3-TR30 were selected. Based on cell culture, Western Blot and Immunofluorescence were performed to analyze the expression level and intracellular localization of HSP27 in the two cell lines after exposure to Paclitaxel. SAFB-siRNA and HSP27-siRNA (Shanghai Genchem limited Company) were used to change the expression level of HSP27. Cell growth rate and early apoptosis level were detected by MTT assay and Annexin V/PI staining combined with Flow Cytometry, respectively.Results1. After exposed to 50nM, 150nM, 300nM, 500nM, 1000nM Paclitaxel, HSP27 in the SK0V3 was inhibited depending on Paclitaxel's concentrations. There was significant difference between any two groups except for 50nM and 150nMgroups. After exposed to Paclitaxel for 24hr, 48hr, 72hr, HSP27 in the SK0V3 was inhibited depending on time. There was significant difference between any two groups except for 24hr and 48hr groups. Paclitaxel induced the nucleus-translocation of HSP27 in SKOV3. Paclitaxel had no significant influence on the expression and distribution of HSP27 in SKOV3-TR30.2. UP-regulation of HSP27 after SAFB-siRNA increased Paclitaxel sensitivity of SK0V3. Down-regulation of HSP27 after HSP27-siRNA increased Paclitaxel resistance of SKOV3-TR30.3. The early apoptotic level of down-regulation of HSP27 in SKOV3-TR30 was significantly higher after exposed to 500nM Paclitaxel than before.Conclusions1. The expression of HSP27 enhanced the Paclitaxel-resistance of ovarian cancer.2. Inhibiting apoptosis induced by Paclitaxel was the possible mechanism of HSP27 involving in the SKOV3's Paclitaxel-resistance. |
