The Resistant Mechanisms Of Ovarian And Cervical Cancer Cells To DCA And The Corresponding Sensitizing Strategies

BackgroundOvarian and cervical cancers are two most common female tumor worldwide and serious threats to women’s health.The mortality of ovarian cancer and the morbidity of cervical cancer rank top among several types of gynecological cancers.At present,platinum and taxol-based chemotherapies are still standard paradigms in addition to surgery,but their side effects are severe and the chemoresistance has also emerged.Therefore,it is urgent to explore novel strategies as alternatives of traditional chemotherapy.There are growing evidences that the unique metabolism is a new essential target of most solid tumors.Targeting key metabolic pathways can significantly kill numerous cancer cells.Among various metabolic drugs,dichloroacetate(DCA)has shown charming prospect because of its positive function in cancer therapy.DCA,a mitochondria-targeting small molecule,has been recently considered as a promising nontoxic antitumor drug that facilitates apoptosis in cancer cells.It acts as an inhibitor of pyruvate dehydrogenase kinase(PDK)and subsequently increases the activity of pyruvate dehydrogenase(PDH),which accelerates the flux of carbohydrates into mitochondria and thereby enhances aerobic oxidation of glucose.However,we found that the level of Mcl-1 and COX2 were induced with the treatment of DCA in ovarian and cervical cancer cells respectively which result in the insensitivity of ovarian and cervical cancer cells to DCA.While the mechanism remains unknown which needs to be further studied.In the present study,we investigated the resistant mechanisms of ovarian and cervical cancer cells to DCA and the corresponding sensitizing strategies in vitro and in vivo.The work was displayed in two parts as below.Part 1:DCA and metformin synergistically suppress the growth of ovarian cancer cellsObjectiveTo investigate whether DCA and metformin could synergistically suppress the growth of ovarian cancer cells and the resistant mechanism of ovarian cancer cells to DCA in vitro and in vivo.Methods1.The ovarian cancer cells were treated with DCA and metformin,then CCK8 assay was used to detect the cell viability,Western blot and caspase 3 activity assay were used to examine whether DCA and metformin could induce apoptosis.2.Western blot was used to explore the expression of Bcl-2,Mcl-1 and Bcl-xL in SKOV3 and OVCAR3 with the treatment of DCA and metformin.After silencing Mcl-1 with siRNA,the proliferation and apoptosis of cells were detected with treatment of DCA.Then cells were transfected with pCMV-Mcl-1 and the effects of proliferation and apoptosis were measured with the treatment of DCA and metformin.3.Real-time PCR was used to examine the transcription level of Mcl-1 in present of DCA.After treated with translational inhibitor(CHX),then the level of Mcl-1 was treated with DCA treatment.Western blot was used to screen the molecular which were related to the stability of Mcl-1 protein.And then detected the level of Mcl-1 and the cytotoxic of DCA after inhibited p-ERK using U0126.Examine the level of p-ERK with metformin treatment and explore the effect of Mcl-1 level in present of MG132.4.Ovarian cancer cells were transfected with or without GFP-LC3,after that Western blot was used to identify the effect of autophagy with the treatment of DCA and metformin.5.After treated with DCA and metformin,the glucose accumulation and lactate were detected.The XF Cell Mito Stress Test Kit was used to calculate the mitochondrial respiration rate with the treatment of DCA and metformin.6.Western blot was used to explore the influence of DCA and metformin on the level of PDH.The concentrations of lactate were examined with interference of PDH.After transfected with PDK,the killing effect of DCA and metformin was detected.7.The repression effect of DCA and metformin was evaluated using ovarian xenograft tumor models.Results1.DCA and metformin synergistically suppress the growth of kinds of cancer cells especially ovarian cancer cells.Moreover,the apoptotic bodies,level of cleaved-PARP and activity of caspase3 were synergistically induced after treated with DC A and metformin compared to themselves alone.2.DC A alone significantly increases the level of Mcl-1 protein in ovarian cancer cells.Silence of Mcl-1 by siRNA enhances the DCA-mediated inhibition of the cell viability and augmented DCA-induced apoptosis.Moreover,ectopic expression of Mcl-1 dramatically alleviates the sensitizing effect of Met to DCA on cell viability and apoptosis.DCA upregulates Mcl-1 via enhancing the phosphorylation of ERK and GSK-3β to decrease the degradation of Mcl-1 protein and metformin suppresses Mcl-1 translation through inhibiting p-mTOR.3.DCA also promotes the level of autophagy.Inhibition of autophagy by chloroquine(CQ)or silence of ATG7 dramatically enhanced the DCA-induced apoptosis and cytotoxicity.In addition,metformin diminishes DCA-induced protective autophagy.4.Metformin increases the lactate production and glucose consumption,which is remarkably alleviated by DCA.DCA dramatically attenuates metformin decreased ratio of OCR/ECAR.DCA inhibits the level of p-PDH.Silence of PDH with siRNA significantly attenuates the metformin-induced lactate production and Ectopic expression of PDK attenuates apoptosis induced by the cotreatment with DCA and metformin.ConclusionIn the present study,we demonstrated that cotreatment with DCA and metformin can repress the growth of ovarian cancer cells via increasing apoptosis.Metformin attenuates DCA-induced Mcl-1 and protective autophagy,while DCA alleviates metformin-induced excessive lactate accumulation and glucose consumption via inhibit the activity of PDK and subsequently increase the activity of PDH,which promotes the flux of carbohydrates into mitochondria and thereby enhances aerobic oxidation of glucose.The reciprocal benefits of the two agents contribute an intense apoptosis to kill ovarian cancer cells more effectively.We for the first time revealed that DCA and metformin synergistically suppress the growth of ovarian cancer cells.These results may pave a way for developing novel strategies for the treatment of ovarian cancer based on the combined use of DCA and metformin.Part 2 Inhibition of COX2 enhances the chemosensitivity of dichloroacetate in cervical cancer cellsObjectiveTo investigate the effect of DCA to cervical cancer cells in vivo and in vitro and the mechanism of DCA induced COX2 and to explore the corresponding sensitizing strategies.Methods1.Real time cell electronic sensing(RT-CES)analysis was used to measure the cytotoxicity effect of DCA in cervical cancer cells after treated with the different concentrations of DCA.Western blot and qPCR were used to detect the level of COX2 and the inflammatory factors.2.After inhibition of COX2 using celecoxib or siRNA,RT-CES,CCK8,flow cytometry and Western blot were used to examine the influence of DCA in cervical cancer cells.3.Reporter assay was used to identify whether DCA increased COX2 via transcription level.Act D was used to determine the role of DCA in RNA stability.4.Screen the potential RNA binding protein which may be associated with COX2 and then overexpress QKI,the levels of COX2 were detected by Western blot and qPCR,the influence of DCA was examined by flow cytometry and CCK8.5.Cervical cancer xenografts in nude mice were used to confirm the sensitivity of DCA to cervical cancer after silencing of COX2.Results1.RT-CES and CCK8 assay confirms that DCA can kill cervical cancer cells only in a high concentration.Simultaneously,DCA increases the level of COX2 and inflammatory factors.2.Inhibition COX2 using celecoxib or siRNA remarkably promotes the apoptosis of cervical cancer cells and subsequently induce the killing effect of DCA.3.DCA increases COX2 via enhancing its mRNA stability by suppressing the RNA binding protein.4.Inhibition COX2 using celecoxib can enhance the chemosensitivity of cervical cancer cells to DCA in nude mice model.ConclusionDCA could suppress the growth of cervical cancer cells only at a high concentration,indicating that DCA is relatively ineffective in cervical cancer cells.It is because of the elevation of COX2 and inflammatory factors which result in the resistant of DCA.Inhibition of COX2 with celecoxib or siCOX2 increased the sensitivity of cervical cancer cells to DCA.DCA induces COX2 via decrease the degradation of COX2 mRNA by downregulating QKI.These results indicate that COX2 is a novel resistance factor of DCA,and combination of celecoxib with DCA may be beneficial to the treatment of cervical cancer.