| Cisplatin (DDP) ,as an anti-cancer drug, used commonly in clinic, displays a broad spectrum of tumour cell lines ,one of effective chemotherapeutic agents in union-treating osteosarcoma. But due to the development of resistance to DDP and it's noxious side-effect during treatment, the use of DDP is limited in clinic, the development of resistance is related tightly with the increasing level of excision repair cross complementation-1(ERCC-1) in tumor's cell, the key to treat tumor is how to decrease the level of ERCC-1. Ubiquitin-proteasome pathway is one of major way to subdivide protein selectively in organism, participates in many important physiological and biochemical process in cell, proteasome inhibitor can induce multi-species tumor cell apoptosis in human being by blocking ubiquitination pathway, is deemed to develop an encouraging anti-cancer agent. Some literatures indicated that proteasome inhibitor can prevent cisplatin-DNA adduct repair and potentiate cisplatin-induced apoptosis by decreasing ERCC-1 mRNA expression, but in bone and soft connective tissue tumor, no correlative literature was reported.In this experiment we choose proteasome inhibitor(Z-LLL-CHO) and DDP, observe the depressant effect of proteasome inhibitor and DDP on human osteosarcoma MG-63 cells continually, study if proteasome inhibitor can increase osteosarcoma cells' sensibility to DDP and co-induct MG-63 cell apoptosis, offer reference to bolting new anti-cancer agent.Methods(1) Cell survival rate was tested by MTT. Divide MG-63 cells into four groups: the first group, disposal by a series of different concentration DDP;the second group, based on the first group, added 0.5umol/L proteasome inhibitor;the third group, based on the first group, added 1.0umol/L proteasome inhibitor;the fourth group was control group. Inoculation cells 48, 72 hours in 96 orifice, then added MTT and dimethyl sulfoxide, measured absorbance under 570nm and calculated cell survival rate.(2) Cell apoptotic morphology was observed by electron microscopy. Divide MG-63 cells into three groups: the first group, disposal by a series of different concentration DDP;the second group, based on the first group, added 1.0umol/L proteasome inhibitor;the third group was control group. Inoculation cells 12, 24, 48 hours, then prepare sections, cell morphology was observed by electron microscopy.(3) Cell apoptotic rate was analyzed by flow cytometry(FCM). Divide MG-63 cells into three groups: the first group, disposal by DDP(10ug/ml);the second group, based on the first group, added 1.0umol/L proteasome inhibitor;the third group was control group. Inoculation cells 12 , 24 , 48 hours, then disposal cells and detected by FCM.(4) The transcription level of excixion repair cross complementation-1 (ERCC-1) was tested by reverse transcription polymerase reaction (RT-PCR).Theproup of cells was same as FCM, Inoculation cells 24 hours, then extracted total RNA, after processing RT-PCR, agarose gel electrophoresis was used to detect the transcription level of ERCC-1.Results(1) The toxic effect of DDP and proteasome inhibitor to osteosarcoma cells was disclosed by MTT. Compared to cells treated with cisplatin alone, cells treated with cisplatin and proteasome inhibitor showed lower survival rate, Compared to cells treated with 0.5umol/L proteasome inhibitor, ceHs treated with 1.0umol/L proteasome inhibitor showed lower survival rate. Cells treated with 50ug/ml cisplatin for 48 hours, survival rate was (64.66±0.05) %, treated with 50ug/ml cisplatin and 0.5umol/L proteasome inhibitor for 48 hours, survival rate was (56.38 ± 0.03) %, 72 hours was (29.44 ± 0.04) %, but cells treated with 50ug/ml cisplatin and 1.0umol/L proteasome inhibitor for 48 hours, survival rate was (2.95 ± 0.01) %. The data showed that all treated groups existed significant difference compared to contrast group by Dunnet-t analysis (p<0.05).(2) The morphology of osteosarcoma cell apoptosis induced by DDP and proteasome inhibitor was observed .The morphology of normal cell was rule, kytoplasm and nucleus were integrity, located in the cellular center and chromatin was uniformity. Osteosarcoma cell manifested apoptosis after treated with cisplatin for 24 hours: cell shrank, the membrane structure kept integrity, nucleus showed crescent-shaped shape, located at the plasma edge, there were a few vacuoles in the plasma. Osteosarcoma cell manifested distinctive apoptotic morphology after treated with cisplatin and proteasome inhibitor: cell volume reduced and apoptotic body extro-prominent at cell membrane was formed, nucleus showed crescent-shaped shape, there were many vacuoles in the plasma.(3) The apoptotic rate of osteosarcoma cell apoptosis induced by DDP andproteasome inhibitor was detected. The apoptotic rate of osteosarcoma cell begun manifest change after treated with cisplatin and proteasome inhibitor for 24 hours. Treated with cisplatin alone ,the apoptotic rate was (14.37±2.37) %, treated with cisplatin and proteasome inhibitor the apoptotic rate was (50.93 ±4.84) %,compared to the former, the apoptotic rate was raised significantly (P<0.05) .(4) The effect of transcription level of ERCC-1 of osteosarcoma cell treated with DDP and proteasome inhibitor. Treated with cisplatin alone,the transcription level of ERCC-1 was raised significantly, from 0.45 ± 0.06 to 0.53 ± 0.10 (P<0.05 ), treated with cisplatin and proteasome inhibitor, the transcription level of ERCC-1 depressed significantly, from 0.53 ± 0.10 to 0.33 ± 0.01 (P<0.05) .ConclusionsProteasome inhibitor could increase the killing effect of cisplatin on osteasarcoma cells and promote cisplatin-induced osteosarcoma apoptosis. These effects may be associated with the decreased transcription of excision repair cross complementation-1.
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