HMGB1-mediated autophagy confers resistance to gemcitabine in hormone-independent prostate cancer cells.

As a main treatment of prostate cancer, castration therapy has been widely applied in the clinic. However, the therapeutic strategy for hormone-independent prostate cancer (HIPC) was not satisfied. Gemcitabine is an important chemotherapeutic agent that has been approved for the treatment of numerous human solid tumors, including HIPC, whereas the gemcitabine resistance has become a serious problem in clinical chemotherapy. In the present study, the mechanisms of resistance to gemcitabine were investigated in HIPC cell lines. The results demonstrated that the autophagy markers were induced significantly in HIPC cells subsequent to gemcitabine treatment. Meanwhile, administration of gemcitabine to HIPC cells increased the expression of high mobility group box1 (HMGB1). Furthermore, the gemcitabine-induced autophagy response was attenuated in stable HIPC cells harboring HMGB1 shRNA. Notably, the HIPC cells stably transfected with HMGB1 shRNA or treated with autophagy inhibitors were more sensitive to gemcitabine compared with the control group. These data suggested that inhibition of HMGB1 increased the sensitivity to gemcitabine by decreasing autophagy response in HIPC cells. Overall, the present findings demonstrate a new mechanism for the resistance to gemcitabine in HIPC cell lines.

[Prokaryotic expression, purification and identification of NY-ESO-1/GST fusion protein in E.coli].

AIM: To construct an expression plasmid for NY-ESO-1 gene and identify the expression of recombinant protein NY-ESO-1/GST in E.coli. METHODS: NY-ESO-1 segment was amplified from the testis cDNA library by RT-PCR and cloned into the prokaryotic expression vector pGEX4T-1 downstream tagged by GST to construct the expression plasmid pGEX-4T1-NY-ESO-1. The recombinant vector was transformed to BL21 (DE3) and NY-ESO-1/GST fusion protein was induced expression by IPTG. The protein was purified by urea elution and identified by SDS-PAGE and Western blotting. RESULTS: The NY-ESO-1 segment was successfully amplified and its sequence was identical with that published in GenBank. The BL21 (DE3) pLysS containing the pGEX-4T1-NY-ESO-1 expressed a M(r); 44 000 fusion protein under the induction of IPTG. The purity of the protein was 90%. Western blotting proved that NY-ESO-1/GST had a specific reaction with anti-GST mAb. CONCLUSION: The prokaryotic expression vector of NY-ESO-1 has been constructed and the fusion protein NY-ESO-1/GST of high purity is successfully expressed.