| Mitomycin C(MMC)is a natural antibiotic that is now used as chemotherapeutic drug for cytotoxic tumors.It is used in the clinical treatment of a variety of malignant tumorsand has achieved good therapeutic effects.However,the drug resistance of cancer cells to MMC affects its clinical application and treatment efficiency,which has become a difficult problem that must be overcome when MMC is used to treat cancer.Pleiotrophin(PTN)is a heparin-bound soluble protein,which forms a family of heparin-bound cytokines together with Midkine(MK).PTN is highly expressed during embryonic development,and is overexpressed in a variety of malignant tumors,including breast cancer,prostate cancer,cervical cancer,etc.,and has certain anti-apoptotic activity on some cancer cells.Studies have shown that some anti-apoptotic proteins related to MMC resistance have been found,but no PTN protein has been found.In this paper,the PTN protein is obtained from Escherichia coli containing the foreign protein expression vector,and then purified,and at the same time its activity is determined by acting on the cell.The PTN protein was applied to MCF-7,MDA-MB-231,HeLa,and SiHa cells stimulated by different concentrations of MMC.The experimental results showed that the IC50 value of the cells in the MMC-stimulated administration group was significantly higher than that of the control group,which proved that PTN has a certain anti-apoptotic activity in the process of MMC stimulation of the cells,and it reduced the sensitivity of the cells to MMC;When MMC stimulated MCF-7,MDA-MB-231,HeLa,and SiHa cells,a gradient concentration of PTN was added at the same time,the flow cytometry results proved that PTN protein has a concentration-dependent anti-apoptotic effect on cells.In order to verify the effect of PTN on the sensitivity of MMC,we transfected MCF-7,MDA-MB-231,HeLa,SiHa cells with PTN interference plasmids to silence the expression of PTN gene and protein in the cells,by detecting the flow cytometry rate of MMC-treated cells,obtained the change of cell sensitivity to MMC.After transfecting sh-NC,sh-PTN-369,and sh-PTN-521 into MCF-7,MDA-MB-231,HeLa,and SiHa cells respectively for 24 hours,the interference efficiency of the two interfering plasmids at the m RNA gene level and the protein level was compared,and agarose gel electrophoresis and western blot experiments were performed.The results showed that the interference efficiency of sh-PTN-521 was more ideal.The IC50 values of MCF-7,MDA-MB-231,HeLa and SiHa cells silenced by MMC-stimulated PTN were significantly lower than those of control cells,which enhanced the sensitivity of cells to MMC.Further flow cytometry results showed that there was no significant difference in apoptosis between the sh-NC cells treated by MMC and control cells.In the cells transfected with sh-PTN-521 plasmid,the apoptosis rate induced by MMC increased to a certain extent compared with the previous two groups,which proved that the silencing of PTN enhanced the sensitivity of cells to MMC.The results of western blot experiments proved that the expression of C-PARP(Cleaved-Poly ADP ribose polymerase)protein in the cells of the MMC administration group with PTN gene silenced was significantly higher than that of the control group,which further proved that PTN could reduce the sensitivity of MMC.In summary,PTN affect the sensitivity of these tumor cells to MMC in different cell lines of breast cancer and cervical cancer.Moreover,the proposed activity of PTN lays the foundation for subsequent research on the mechanism of MMC drug resistance. |
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