The Roles And Mechanisms Of Dichloroacetate And Metformin Alone And Their Combination In The Killings Of Cervical Cancer Cells

Background and Objective:In recent years,the incidence of cervical cancer has been high,the therapeutics effect of cervical cancer has been not quite satisfactory,especially for advanced and recurrent patients;those patients mainly have been given to chemotherapy and radiotherapy.The side effects of chemotherapy based on cisplatin have been serious,and the efficacy has been poor,therefore the new treatment method is needed.Studies have proved that tumor is a metabolic disorder,the treatment of tumor is focusing on regulating the metabolism of antitumor.Similarly,the study on treatment for cervical cancer by regulating metabolism has attracted much attention.Among several antitumor drugs by regulating the metabolism,dichloroacetate(DCA)and metformin(Met)have shown good antitumor prospects.Studies have shown that DCA has a killing effect for a variety of tumor cells,but no damage to normal cells.The mechanism of DCA inhibits pyruvate dehydrogenase kinase,so that pyruvate dehydrogenase activity is increased,which promote intermediates of glucose metabolism into mitochondrial going on tricarboxylic acid cycle,enhancing glucose oxidation and inhibiting glycolysis and production of lactic acid.The effect of DCA could reverse dysfunction of mitochondrion of tumor cells,activate mitochondrial apoptosis,and kill tumor cells,reduce the accumulation of lactic acid and destroy the microenvironment of tumor cells,which repressed tumor cell to survive.Metformin(Met)has been used as a first-line therapies for type 2 diabetes.Epidemiology studies have shown that metformin could decrease morbitidy of cancer on patients with diabetes.The antitumor effect of metformin is getting attention.Many studies have shown that metformin could kill many kinds of tumor cells including cervical cancer cells.Metformin combination with other chemotherapy drugs could improve the effect of chemotherapy,which could significantly reduce the dose of the chemotherapy drugs and side effects,all of which brought new hope for cancer patients.Metformin played antitumor effect on cancer cells,induced the accumulation of lactic acid,which weakened the antitumor effect of metformin.If we can overcome the side effect,which might have helped enhance antitumor function of metformin.Studies found that cancer cells had the capacity to commandeer a variety of cellular processes to aid in their survival?growth and resistance to therapy.Overexpression of anti apoptotic gene could suppress apoptosis of cancer cells.B-cell lymphoma / leukemia-2(Bcl-2)family is a key protein family that regulates mitochondrial apoptosis pathway.Myeloid cell leukemia-1(Mcl-1)is one of the important antiapoptotic members of the Bcl-2 family,which plays a pivotal role in occurring and developing of tumor.Studies have demonstrated that Mcl-1 was highly expressed in a variety of human tumors,including cervical cancer;Downregulating Mcl-1 could significantly increase the sensitivity of tumor tissue to chemotherapeutic drugs,and improve the effect of tumor therapy.Study found that dichloroacetate could suppress the proliferation of colorectal cancer cells by down-regurating the level of Mcl-1.Recently,Mcl-1 has become an important target for tumor therapy.This study selected cervical cancer He La cells as the model,and investigated the roles and mechanisms of dichloroacetate and metformin alone and their combination in the killings of cervical cancer cells,which might provide basis for clinical treatment of cervical cancer.Main contents:Part one The role and mechanism of dichloroacetate in the killings of cervical cancer cells1.Detecting the proliferation and apoptosis by DCA-induced;2.Detecting the expression of Mcl-1 by DCA-induced;3.Detecting the effect of DCA on the production of lactic acid.Part two The role of metformin in the killings of cervical cancer cells1.Detecting the proliferation and apoptosis by Met-induced;2.Detecting the effect of Met on the production of lactic acid.Part three The role and mechanism of dichloroacetate combination with metformin in the killings of cervical cancer cells1.Detecting the proliferation and apoptosis by dichloroacetate combination with metformin-induced;2.Detecting the expression of Mcl-1 by dichloroacetate combination with metformin-induced;3.Detecting the levels of lactic acid by dichloroacetate combination with metformin-induced.Main methods:1.In vitro culture and passage of He La cells;2.CCK-8 detects the proliferation;3.Flow cytometry detects the apoptosis;4.Western blot and q PCR are used to detect the levels of protein and m RNA;5.Detection of the levels of lactic acid by L-Lactate Assay Kit I;Results:Part one1.Compared with the control group,the survival rate of He La cells was decreased(P<0.01),and the apoptosis rate was increased by DCA-induced(P<0.01);2.DCA decreased the expression level of Mcl-1 protein in He La cells;3.MG132 could suppress the down-regulated of Mcl-1 protein in He La cells by DCAinduced;CHX could not suppress the down-regulated of Mcl-1 protein in cervical cancer He La cells by DCA-induced;4.Compared with the control group,DCA could reduce the level of lactic acid in He La cells(P<0.05).Part two1.Compared with the control group,the survival rate of He La cells was decreased(P<0.05),and the apoptosis rate was increased by Met-induced(P<0.05);2.Compared with the control group,Met could enhance the level of lactic acid in He La cells(P<0.05).Part three1.Compared with DCA or Met group alone,the survival rate of He La cells was significantly decreased(P<0.01),and the apoptosis rate was significantly increased(P<0.01)by the combination of DCA and Met-induced;2.Compared with DCA or Met group alone,the expression levels of Mcl-1 protein in He La cells were significantly decreased by the combination of DCA and Met-induced;3.MG132 could suppress the down-regulated of Mcl-1 protein in He La cells by the combination of DCA and Met-induced;CHX could not suppress the down-regulated of Mcl-1 protein in He La cells by the combination of DCA and Met-induced;4.Compared with the control group,The combination of DCA and Met which was no significant difference on the level of lactic acid in He La cells(P > 0.05);Compared with Met group,The combination of DCA and Met could reduce the level of lactic acid in He La cells(P<0.05).Conclusions:1.DCA could inhibit the proliferation and promote the apoptosis of He La cells,which may be resulted from the downregulation of Mcl-1 protein by DCA-induced proteasomal degradation of Mcl-1 and inhibit the accumulation of lactic acid;2.Met could inhibit the proliferation and promote the apoptosis of He La cells;Meanwhile,Met could enhance the level of lactic acid in He La cells;3.Compared with DCA or Met group alone,the combination of DCA and Met could significantly inhibit the proliferation,promote the apoptosis of He La cells,which may be resulted from the downregulation of Mcl-1 protein by the combination of DCA and Met-induced proteasomal degradation of Mcl-1 and inhibit the accumulation of lactic acid by Met-induced.