Effect Of MG132 With Cisplatin On Hela Cell Growth, Apotosis And Expression Of GST-Ï€

Objective Observe the effect of proteasome inhibitor MG132combined with cisplatin on Hela cell growth and apotosis, and test theexpression of GST-π's.Methods (1)Invert microscope was used to observe the changes ofcell appearance, and to take photos. HeLa cells were divided into four groups:the first group (control group) was just treated by DMSO for 24h; the secondgroup was treated by 2.5μg/ml cisplatin and DMSO for 24h; the third groupwas treated by 5μmol/l MG132 for 24h; the forth group was treated by5μmol/l MG132 combined with 2.5μg/ml cisplatin for 24h. (Because MG132uses a DMSO compound, the control group and cisplatin group were addedto the DMSO as the MG132 group, and when each group was compared theinfluence of DMSO on the cells could be eliminated.)(2) MTT assay was used to detect the growth inhibiton ratio of HeLacells. The first control group were only treated by 1640 culture medium withcalf serum for 24, 48 and 72 hours respectively; The cisplatin group weretreated by 1.0,2.5,5.0 and 10.0μg/ml cisplatin for 24, 48 and 72 hoursrespectively; the second control group were treated by DMSO; MG132 groupwere treated by 5μmol/l MG132 for 24 and 48 hours respectively; MG132combined with cisplatin group were treated by 1.0,2.5,5.0 and 10.0μg/mlcisplatin, combined with 5μmol/l MG132 for 24 and 48 hours respectively.(3)PI staining flow cytometry was used to confirmed the apoptotic ratesand cell cycles of HeLa cells. HeLa cells were divided into four groups: thefirst group was just treated by DMSO for 48h; the second group was treatedby 2.5μg/ml cisplatin and DMSO for 48h; the third group was treated by5μmol/l MG132 for 48h; the forth group was treated by 5μmol/l MG132 combined with 2.5μg/ml cisplatin for 48h.(4) Strep tavidin-peroxidase conjugated method (SP) ofimmunocytochemistry was used to detect the expression of GST-πin HeLacells. HeLa cells were divided into three groups: the first group was justtreated by DMSO for 24h; the second group was treated by 2.5μg/ml cisplatinand DMSO for 24h; the third group was treated by 5μmol/l MG132combined with 2.5μg/ml cisplatin for 24h.Results (1) Cell changes were observed in an inverted microscope:Cellsin the control group grew well, the cells extended, were transparent, formedirregular angular forms, and were close together. The cisplatin group cellsdecreased in number, bubbles formed in the cytoplasm, their areas decreased,they became round, and the distance between them increased. The MG132combined with cisplatin cells decreased in number even more, and a largenumber of suspended apoptotic cells could be seen with the naked eye.(2) The diffferences of the proliferative inhibition rates among thecisplatin groups with different concentrations were statistically significant(P＜0.05) and the differences of the proliferative inhibition rates among thecisplatin groups with different durations were also statistically significant(P＜0.05). The effect of different concentrations and different durations ofcisplatin on cell growth was interactive(P＜0.05). The diffferences of theproliferative inhibition rates among the cisplatin, MG132, MG132 combinedwith cisplatin groups were also statistically significant(P＜0.05).(3) Flow cytometer analysis of Hela cell apoptosis rates and cell cycles:The MG132 combined with cisplatin group induced apoptosis of Hela cellsmore effectively than the control, cisplatin and MG132 groups, and theirdifferences were statistically significant (P＜0.05). Cell cycles were arrestedat S phase significantly.(4) GST-πexpression in Hela cells detected by strep- -tavidin-peroxidase conjugated method (SP) of immunocytochemistry:Compared with the control and the cisplatin groups, the GST-πexpressionshowed a clear decrease among the MG132 combined with cisplatin groups(P＜0.05).Conclusion 1. Proteasome inhibitor MG132 can enhance the growthrestraint effect of cisplatin on Hela cells and can enhance the induction ofHela cell apoptosis.2. Proteasome inhibitor MG132 combined with cisplatin can decreasethe expression of GST-πin Hela cells. This may be related to thephenomenon of MG132 increasing the sensitivity of Hela cells to cisplatin.