Effects Of MAP30from Momordica Charantia On Cell Growth And Apoptosis Of Bladder Cancer Cells

BackgroundBladder cancer is the most common urological malignancy with a higher morbidity and the main therapy methods are exairesis, chemotherapy and radiotherapy at present. These methods are not only costly, but also higher-side effects and non-specificity. Although our traditional Chinese medicine research on bladder tumor starts later, but along with the further research on Chinese herbal medicine, in today the traditional Chinese medicine has made significant progress in prevention and treatment of bladder tumor. The traditional Chinese drugs have the supporting healthy energy to eliminate evils, so it can not only kill the cancer cells, but also adjust the immune balance from the global level. The bitter melons that liked by civilians have many bioactivities such as anti-tumor, depressing blood sugar, anti-virus, anti-bacteria, anti-mutation, immunoregulation and so on. The most attractive bioactive substances isolated from the bitter melons are RIPs. MAP30(Momordica anti-HIV Protein of30KDa) is a kind of RIP I which has potent anti-tumor activity to many kinds of tumor cells, but about the effects of MAP30on cell growth and apoptosis of bladder cancer cells has not yet been reported.ObjectiveIn this study we aim to cloned MAP30gene of the momordica charantia protein, expressed and pured the MAP30protein. And then to explore whether the MAP30could inhibit cell growth and induce apoptosis in the5637human bladder cancer cells in vitro by the detections of MTT and flow cytometry (FCM) analysis.Methods(1) PCR was used to amplify the MAP30gene from bitter melon genome DNA. Then the target gene was inserted into the expression vector pET-28a. The pET-28a (+)-MAP30expression vector was transformed into BL21(DE3). The MAP30protein was expressed in E.coli BL21with IPTG, the protein was identified by SDS-PAGE, purified by Ni-column, and calculated the protein concentration in further.(2) Recombinant MAP30protein were co-cultured with the5637human bladder cancer cells and determined the antiproliferative activities by MTT assay.(3) Recombinant MAP30protein were co-cultured with the5637human bladder cancer cells and the percentage of apoptosis were determined by flow cytometry (FCM) analysis.Results(1) The recombinant plasmid was constructed and the DNA sequence analysis showed that the MAP30gene possesses860bp in length and with100%homology with NCBI. The prokaryotic expression vector pET-28a (+)-MAP30was constructed successfully and the MAP30protein was expressed in E.coli BL21. The relative molecular weight of the protein was30kDa which was identified by SDS-PAGE, and the protein concentration is434μg/ul calculated by BCA assay after purified by Ni-column.(2) After treated with MAP30for24h and48h, the proliferation of5637human bladder cancer cells was significant suppressed in a dose dependent manner which was determined by MTT assay.(3) The FCM assay indicated that After treated with MAP30for24h and48h,200μl/mL and400μl/mL MAP30protein leads to a significant higher percentage of apoptosis of5637human bladder cancer cells than control groups; The percentage of apoptosis after treated with400μl/mL were significant higher than200μl/mL treatment, and the percentage of apoptosis after treated with MAP30for48h was higher than that treated for24h.Conclusion(1) The prokaryotic expression vector pET-28a (+)-MAP30was constructed successfully and the MAP30protein was obtained with a concentration of434μg/μl from E.coli BL21.(2) MAP30protein could inhibit the proliferation of5637human bladder cancer cells in a dose and time dependent manner.(3) MAP30protein could induce5637human bladder cancer cells apoptosis in a dose and time dependent manner.