Hyperthermia Increasing The Sensitivity Of Ovarian Cancer To Chemotherapy Drugs And It's Mechanism

Purpose:To investigate the effect of hyperthermia on chemotherapy sensitivity of ovarian cancer and it's mechanism.Methods:In this study,thermo-sensitive cells A2780 and HO8910 and thermally insensitive cells SKOV3 were used as study carriers to investigate the function of thermotherapy.In the first part of this study,Cell Counting kit-8(CCK8)cytotoxicity test was used to detect the changes of oxaliplatin on IC50 of three types of ovarian cancer cells under thermotherapy.The effects of thermotherapy on the sensitivity to chemotherapy were observe by clonal formation experiment in three ovarian cancer cells.Finally,EDU cells proliferation experiment and cell apoptosis assay were used to observe the effects of thermal therapy on the proliferation and apoptosis of three types of ovarian cancer cells.The influence of thermotherapy on tumor growth and chemotherapy sensitivity in vivo was studied by establishing the model of abdominal transplantation tumor.The thermally sensitive cells A2780 were implanted into the enterocoelia of the immunodeficient nude mice,and the tumor reached a certain volume for thermotherapy and chemistry.Finally,the weight of all tumors in the abdominal cavity of every mouse was compared after treatment.In the second part of this study,In quest of the possible mechanism of the impact of thermal therapy on the sensitivity of ovarian cancer cells to chemotherapy.Then used cellular immunofluorescence assay and comet assay to discover the effects of hyperthermia on chemotherapy-induced DNA damage and repair.Then,western blot assay was designed to explore the effect of hyperthermia on DNA damage repair gap-associated proteins,as well as the effect of thermotherapy on expression levels of proteins which correlation with DNA damage pathway in ovarian cancer cell lines and human ovarian cancer primary cells induced by chemotherapy drugs.Finally,this research used western blot assay to study the effect of PARP1 inhibitor olaparib and overexpressed PARP1 and XRCC1 cells on the expression of above proteins related to DNA damage pathway under the effect of thermo-chemistry and to investigate the possible mechanism.Results:the sensitization effect of thermotherapy was different in different ovarian cancer cell lines.For example,in A2780 and HO8910 cells,Hyperthermia could increase the sensitivity to chemotherapy,but in SKOV3 cells,heat flow meter did not increase the sensitivity to chemotherapy significantly.In heat-sensitive cells,thermotherapy reduced the IC50 of oxaliplatin in ovarian cancer cells.Compared with the chemotherapeutic drug group alone,the clone formation rate of A2780 and HO8910 cells significantly decreased after thermochemistry,suggesting that the sensitivity of tumor cells to chemotherapy increased.On the other hand,the results of EDU experiment and apoptosis experiment showed that compared with the chemotherapy group,the thermo-chemotherapy suppression the expansion and enhance the apoptosis level more remarkable of heat sensitive cells,indicating that hyperthermia plays an important role in the regulation of cell proliferation and apoptosis.The phenomenon that thermotherapy increases the sensitivity of chemotherapy has also been confirmed in vivo experiments.Further studies have shown that thermotherapy can promoted the s-phase cycle arrest by chemotherapeutic drug in Heat sensitive ovarian cancer cells.thermotherapy can make the DNA damage of heat-sensitive cells become more serious in a short time after hot stimulation.Through the comet assay,this study found the DNA tail-pulling was longer and the tail-distance was larger.The number of DNA-damage-related proteins detected by immunofluorescence experiment showed more green ?-h2ax foci.To verify that a separate tepid stimulation effect on DNA damage repair,this study tested the warm stimulation under different processing experiment of base excision repair(BER)and single fracture repair proteins and enzymes involved(HR)scaffold protein--PARP1 and XRCC1,found in 42?stimulation after 20 min,two kinds of protein expression decreased,along with the time,protein expression decreased,but after 60 min,increase heat stimulation time does not reduce the two kinds of protein expression.At the same time,thermo-chemotherapy can increase the expression levels of DNA damage related proteins-phosphorylating ATM and its downstream proteins NBS1 and CHK2 T68.The above changes in protein expression were also verified in primary cells of human ovarian cancer in this research.in order to verify whether the promotion of DNA damage by thermo-chemotherapy is related to the inhibition of DNA damage repair by hyperthermia.This study used PARP1 inhibitor Olaparib combined chemotherapy with hot not sensitive cells,found that DNA damage related proteins have high expression,in order to further validate the link,this study also uses PARP1 and XRCC1 high expression plasmid into heat sensitive A2780 cells of thermochemistry,DNA damage-related protein expression was found to be lower than in normal cells.It is suggested that thermophilic stimulation combined with chemotherapy can promote DNA damage was due to thermophilic stimulation can inhibit DNA damage repair.Conclusions:1.This study confirmed that hyperthermia enhanced chemotherapy sensitivity of ovarian cancer cells.2.Hyperthermia increased cell S stage block in thermally sensitive cells,increased chemotherapeutic drug-induced DNA damage,and decreased the expression of DNA damage repair proteins.3.Research on human primary cells confirms the above theory.4.The mechanism is that in thermally sensitive cells,hyperthermia reduces the expression of DNA damage repair related proteins PARP1 and XRCC1,thereby promoting the upstream ATM phosphorylation induced by chemotherapy drugs and inducing the high expression of downstream NBS1 and CHK2T68,thereby enhancing DNA damage and ultimately leading to increased chemotherapy sensitivity of tumor cells.