Objective： To investigate whether the change of drug resistance degree could be evaluated by apoptotic rate in ovarian cancer cell lines. Methods： Human epithelia ovarian cancer cell line A2780 and its platinum （DD...Objective： To investigate whether the change of drug resistance degree could be evaluated by apoptotic rate in ovarian cancer cell lines. Methods： Human epithelia ovarian cancer cell line A2780 and its platinum （DDP） resistance cell line AD4 were used. They were divided into 4 groups respectively （A2780-DDP group, A2780-DDP＋VRM group, AD4-DDP group and AD4-DDP＋VRM group）. 3-（4, 5- dimethylthiazol-2-yl）-2, 5-diphenyl-2H-tetrazolium bromide （MTT） was used to measure the multiple of drug resistance. The expression of drug-resistance genes （mdrl, TopoⅡα and GSTπ） was detected by using reverse transcription polymerase chain reaction （RT-PCR）. Semi-quantity assay was proceed by rate the of multidrug resistance genes to G3 PDH gene. Apoptosis was measured by DNA gel electrophoresis and flow cytometry respectively. The advantages and disadvantage of evaluating drug-resistance with these three methods were analyzed. Results： The 50% inhibition concentration （IC50） of A2780 and AD4 was 19.2 μg/mL and 66 μg/mL respectively, and the resistance fold of the AD4 was 3.4. Some drug-resistance genes could be detected by RT-PCR in A2780 and AD4 cell lines. The expression of mdrl was only （0.09±0.03）×10^-2 ： 1 and （0.10±0.02） × 10^-2：1 respectively （rate to G3 PDH gene） with the difference being not significant between them. The expression of TopoⅡα in the A2780 cells was （2.60±0.12）×10^-2：1 and （0.11±0.03）× 10^-2：1 in the AD4 cells respectively with the difference between them being significant. On the contrary, the expression of GSTπ in A2780 cells was lower than in AD4 cells, and the ratio was （0.11±0.03）×10^-2：1 and （3.13±0.14）×10^-2：1 respectively with tile difference being significant between them. There was no significant difference among the genes expression after the drugs were given for 6 h, 12 h and 24 h. couldn＇t reflect the change of drug-resistance timely. DNA gel electrophoresis used to detect apoptosis was only a qualitative analysis. Different drug resistance degrees may be detected by flow cytometry as early as few hours after drugs were given, which realized the earlier and quantities detection of drug resistance. Conclusion： Detection of apoptosis with flow cytometry may not be affected by the variety of drug-resistance genes, suggested this was a general, quantitative and objective method to reflect drug resistance.展开更多
Characterized by insidious onset,fast progress,wide epidemic with poor therapeutic effect and shortsurvival period,primary hepatocarcinoma is one ofthe malignant tumors that threatens the human health.Rich experiences...Characterized by insidious onset,fast progress,wide epidemic with poor therapeutic effect and shortsurvival period,primary hepatocarcinoma is one ofthe malignant tumors that threatens the human health.Rich experiences and gratifying achievements havebeen obtained by Chinese medical workers.展开更多
Recent studies have suggested an involvement of processing pathways for the initiation of cellular responses induced by topoisomerase-targeting drugs. Here, we showed that cellular exposure to camptothecin （CPT） ind...Recent studies have suggested an involvement of processing pathways for the initiation of cellular responses induced by topoisomerase-targeting drugs. Here, we showed that cellular exposure to camptothecin （CPT） induced formation of topoisomerase I cleavable complex （TOPlcc）, degradation of TOP1 and activation of DNA damage responses （DDR）. Transcription and proteasome-dependent proteolysis, but not replication, were involved in CPTo indneed TOPl degradation, while none of above three processing activities affected TOPlcc formation. Replication- and transcription-initiated proeessing （RIP and TIP） of TOPlee were identified as two independent pathways, which contribute distinctly to various CPT-activated DDR. Specifically, in cycling cells, RIP-processed TOPlec triggered the CPT-induced RPA pbosphorylation. At higher CPT dosages, the TIP pathway is required for other DDR activation, including ATM, p53 and Chkl/2 phosphorylation. The TIP pathway was further demonstrated to be S-phase independent by using three nonreplicating cell models. Furthermore, the effect of proteasome inhibitors mimicked that of transcription inhibition on the CPT-induced activation of DDR, suggesting the involvement of proteasome in the TIP pathway. Interestingly, the TIP pathway was important for TOPlcc-activated, but not ionization radiationactivated ATM, p53 and Chk2 phosphorylation. We have also found that pharmacological interferences of TIP and RIP pathways distinctively modulated the CPT-induced cell killing with treatments at low and high dosages, respec- tively. Together, our results support that both RIP and TIP pathways of TOPlcc are required for the activation of CPT-induced DDR and cytotoxicity.展开更多
基金This work was supported by a grant from National Natural Sciences Foundation of China (No. 30070786).
文摘Objective： To investigate whether the change of drug resistance degree could be evaluated by apoptotic rate in ovarian cancer cell lines. Methods： Human epithelia ovarian cancer cell line A2780 and its platinum （DDP） resistance cell line AD4 were used. They were divided into 4 groups respectively （A2780-DDP group, A2780-DDP＋VRM group, AD4-DDP group and AD4-DDP＋VRM group）. 3-（4, 5- dimethylthiazol-2-yl）-2, 5-diphenyl-2H-tetrazolium bromide （MTT） was used to measure the multiple of drug resistance. The expression of drug-resistance genes （mdrl, TopoⅡα and GSTπ） was detected by using reverse transcription polymerase chain reaction （RT-PCR）. Semi-quantity assay was proceed by rate the of multidrug resistance genes to G3 PDH gene. Apoptosis was measured by DNA gel electrophoresis and flow cytometry respectively. The advantages and disadvantage of evaluating drug-resistance with these three methods were analyzed. Results： The 50% inhibition concentration （IC50） of A2780 and AD4 was 19.2 μg/mL and 66 μg/mL respectively, and the resistance fold of the AD4 was 3.4. Some drug-resistance genes could be detected by RT-PCR in A2780 and AD4 cell lines. The expression of mdrl was only （0.09±0.03）×10^-2 ： 1 and （0.10±0.02） × 10^-2：1 respectively （rate to G3 PDH gene） with the difference being not significant between them. The expression of TopoⅡα in the A2780 cells was （2.60±0.12）×10^-2：1 and （0.11±0.03）× 10^-2：1 in the AD4 cells respectively with the difference between them being significant. On the contrary, the expression of GSTπ in A2780 cells was lower than in AD4 cells, and the ratio was （0.11±0.03）×10^-2：1 and （3.13±0.14）×10^-2：1 respectively with tile difference being significant between them. There was no significant difference among the genes expression after the drugs were given for 6 h, 12 h and 24 h. couldn＇t reflect the change of drug-resistance timely. DNA gel electrophoresis used to detect apoptosis was only a qualitative analysis. Different drug resistance degrees may be detected by flow cytometry as early as few hours after drugs were given, which realized the earlier and quantities detection of drug resistance. Conclusion： Detection of apoptosis with flow cytometry may not be affected by the variety of drug-resistance genes, suggested this was a general, quantitative and objective method to reflect drug resistance.
文摘Characterized by insidious onset,fast progress,wide epidemic with poor therapeutic effect and shortsurvival period,primary hepatocarcinoma is one ofthe malignant tumors that threatens the human health.Rich experiences and gratifying achievements havebeen obtained by Chinese medical workers.
文摘Recent studies have suggested an involvement of processing pathways for the initiation of cellular responses induced by topoisomerase-targeting drugs. Here, we showed that cellular exposure to camptothecin （CPT） induced formation of topoisomerase I cleavable complex （TOPlcc）, degradation of TOP1 and activation of DNA damage responses （DDR）. Transcription and proteasome-dependent proteolysis, but not replication, were involved in CPTo indneed TOPl degradation, while none of above three processing activities affected TOPlcc formation. Replication- and transcription-initiated proeessing （RIP and TIP） of TOPlee were identified as two independent pathways, which contribute distinctly to various CPT-activated DDR. Specifically, in cycling cells, RIP-processed TOPlec triggered the CPT-induced RPA pbosphorylation. At higher CPT dosages, the TIP pathway is required for other DDR activation, including ATM, p53 and Chkl/2 phosphorylation. The TIP pathway was further demonstrated to be S-phase independent by using three nonreplicating cell models. Furthermore, the effect of proteasome inhibitors mimicked that of transcription inhibition on the CPT-induced activation of DDR, suggesting the involvement of proteasome in the TIP pathway. Interestingly, the TIP pathway was important for TOPlcc-activated, but not ionization radiationactivated ATM, p53 and Chk2 phosphorylation. We have also found that pharmacological interferences of TIP and RIP pathways distinctively modulated the CPT-induced cell killing with treatments at low and high dosages, respec- tively. Together, our results support that both RIP and TIP pathways of TOPlcc are required for the activation of CPT-induced DDR and cytotoxicity.
