| Objective: The purpose of this study is to investigate the cardiotoxicity of anticancer drugs in preclinical trial using human embryonic stem cell-derived cardiomyocytes co-cultured with cancer cells,including human hepatoma cells?HepG2?and human breast cancer cells?MCF7?.In addition,by comparing with the existing methods of evaluating cardiotoxicity with cardiomyocytes,it is possible to evaluate whether this method is different from the existing methods and provide data for the preclinical safety of early anticancer drugs.Methods: Three drugs with myocardial toxicity were used,and they are Doxorubicin,Brucine and 5-Fluorouracil.Three cell lines were selected,and they are Human embryonic stem cell-derived cardiomyocytes?hESC-CM?,HepG2 and MCF7.1.The Microplate Reader was used to detect the viability of cancer cells,and the concentrations of co-culture were determined.In order to determine the toxicity of different concentrations of drugs on co-culture cells.2.we used Flow Cytometry to detect the apoptotic rate of cardiomyocytes and cancer cells at 4h,24 h and 48 h after co-culture.3.We used real-time cell analysis system to detect the follows:?1?Before giving anti-cancer drugs,we detect the changes of Beat Rate and Amplitude of monoculture and co-culture hESC-CM cells at 1h,4h,12 h,24h,36 h and 48 h.?2?After giving anti-cancer drugs,we detect the changes of Beat Rate and Amplitude of monoculture hESC-CM cells at 1h,4h,12 h,24h,36 h and 48 h.?3?HepG2 and MCF7 cells were co-cultured with hESC-CM by a real-time cell analysis system and their special cell culture plates.After giving anti-cancer drugs to co-culture cells,we used the real-time cell analysis system with special cell culture plate?insert?to monitor the changes of cardiomyocytes' Beating Rate and Amplitude by analyzing the impedance measurement at 1h,4h,12 h,24h,36 h and 48 h.3.We used IN Cell Analyzer high content cell imaging technology to detect the follows:?1?Before giving anti-cancer drugs,we detect monoculture and co-culture hESC-CM cells' mitochondrial membrane potential at 4h,24 h,and 48 h.?2?After giving anti-cancer drugs,we detect monoculture hESC-CM cells' mitochondrial membrane potential at 4h,24 h,and 48 h.?3?After giving anti-cancer drugs to co-culture cells,we used IN Cell Analyzer high content cell imaging technology to detect cardiomyocytes mitochondrial membrane potential at 4h,24 h,and 48 h.Results:?1?After using the Microplate Reader to detect the viability of cancer cells,we set the drug concentrations of co-culture.The drug concentrations of Doxorubicin were 0.03?mol·L-1,0.1?mol·L-1,0.3?mol·L-1,1.0?mol·L-1,3?mol·L-1,10?mol·L-1,and the drug concentrations of Brucine were 6.25?mol·L-1,12.5?mol·L-1,25?mol·L-1,50?mol·L-1,100?mol·L-1,200?mol·L-1 and the drug concentrations of 5-Fluorouracil were 25?mol·L-1,50?mol·L-1,100?mol·L-1,200?mol·L-1,400?mol·L-1,800?mol·L-1.?2?Flow cytometry was used to determine the apoptotic rate of co-cultured cells.It was found that the apoptotic rate of hESC-CM,HepG2 and MCF7 increased gradually with increasing Doxorubicin concentration.The killing cardiomyocytes effect of Brucine is weaker than its effect on cancer cells,but there are statistical differences.And the cancer cells' apoptotic rate on 5-fluorouracil is higher than that of cardiomyocytes.?3?Using the real-time cell analysis system and its special cell culture plate to monitor cardiomyocytes,the co-culture cardiomyocytes showed a decrease in beating rate and an increase in amplitude compared with monocultured cardiomyocytes.?4?For the system of hESC-CM co-cultured with HepG2,different concentrations of Doxorubicin have significant effect on the cardiomyocytes' beating function.And at 12 h,24h,36 h and 48 h,anti-cancer drugs cause hESC-CM beating disorder.High concentrations of Doxorubicin reduced cardiomyocytes' mitochondrial membrane potential.After giving the system?hESC-CM co-cultured with HepG2?Brucine,100?mol·L-1 and 200?mol·L-1 of drug significantly decreased beating rate and amplitude of cardiomyocytes at 1 h and 4 h,but they gradually recovered after 12 h.100?mol·L-1 and 200?mol·L-1 of Brucine decreased cardiomyocytes' mitochondrial membrane potential at 4 h and 24 h.At 1h,4h and 12 h,the effect of 5-Fluorouracil on cardiomyocytes were not statistically different.At 24 h,36h and 48 h,the beating rate and amplitude of cardiomyocytes were significantly decreased.And at 24 h and 48 h,800?mol·L-1 of 5-Fluorouracil reduce cardiomyocytes' mitochondrial membrane potential.?5?For the system of hESC-CM co-cultured with MCF7,different concentrations of Doxorubicin have significant effects on the cardiomyocytes' beating function.And at 12 h,24h,36 h and 48 h,anti-cancer drugs caused hESC-CM beating disorder.High concentrations of Doxorubicin reduced cardiomyocytes' mitochondrial membrane potential.After giving the system?hESC-CM co-cultured with MCF7?Brucine,100?mol·L-1 and 200?mol·L-1 of drug significantly decrease beating rate and amplitude of cardiomyocytes at 1 h and 4 h,but they gradually recovered after 12 h.50?mol·L-1,100?mol·L-1 and 200?mol·L-1 of Brucine decrease cardiomyocytes' mitochondrial membrane potential at 4 h and 24 h.At 1h,the effect of 5-Fluorouracil on cardiomyocytes were not statistically different.At 4h and 12 h,800?mol·L-1 of 5-Fluorouracil reduced cardiomyocytes' beating rate.At 24 h,36h and 48 h,high concentration of 5-Fluorouracil decreased the beating rate and amplitude of cardiomyocytes.And at 24 h and 48 h,High concentration of 5-Fluorouracil reduced cardiomyocytes' mitochondrial membrane potential.?6?After giving drugs,monoculture cardiomyocytes exhibit different changes compared to co-cultured cardiomyocytes,especially with regard to beating functions.This may be due to the combined effect of cancer cells and drugs given the above results.Conclusion: The co-cultured method can initially simulate the in vivo environment and be used to monitor the effect of anticancer drugs on cardiomyocytes while killing cancer cells.Monoculture cardiomyocytes exhibit different changes compared to co-cultured cardiomyocytes,especially with regard to beating functions.After giving the co-culture system anticancer drugs,the method can accurately reflect drug toxicity on cardiomyocytes and cancer cells. |
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