| Backgrounds and Objectives:Ovarian cancer is a common female malignant tumor,which is also the main cause of death of gynecological malignant tumors in the world.Globally,nearly 240000 women are diagnosed with ovarian cancer every year,and about 150000 patients die of ovarian cancer.Ovarian cancer is the most lethal gynaecological cancer.Ovarian cancer is divided into epithelial cancer,malignant germ cell tumor and sex cord stromal tumor,among which epithelial ovarian cancer is the most common.The origin of ovarian cancer is complex.Due to the deep pelvic cavity of the ovary and unspecific symptom in the early stage,about 70%of the patients are diagnosed in advanced stage,and ovarian cancer cell has spread to the abdominal cavity.In the early stage of ovarian cancer treatment,surgery and chemotherapy are often effective,but most patients's cancer returned after treatment.Drug resistance to chemotherapy is the main cause of high mortality in patients with ovarian cancer.Therefore,it is very important to study the mechanism of ovarian cancer occurrence and development and to find effective potential therapeutic targets,which are beneficial for overcome drug resistance and improve the survival period of patients.CPT1 A,located on the outer membrane of mitochondria,is the key rate limiting enzyme of fatty acid metabolism.It is overexpressed in breast cancer,ovarian cancer,gastric cancer,lung cancer.The overexpression of CPT1A activates fatty acid oxidation metabolism(FAO)via ATP and NADPH production for tumor cells to promote the growth of tumor cells,which becomes an important part of tumor cells with unique metabolic adaptations.More and more studies have shown that CPT1A can be used as a potential molecular target for tumor therapy,but the effect of inhibiting CPT1A alone on tumor cells is limited.Etomoxir can irreversibly inhibit the activity of CPT1A and reduce FAO.In order to improve the anti-tumor effect of etomoxir in the treatment of ovarian cancer,this paper discusses the effect of etomoxir combined with ABT-263 on ovarian cancer cells,and studies the molecular mechanism of the combination of ABT-263 and etomoxir.Methods and Results:1.ABT-263 enhanced etomoxir inhibited the growth and proliferation of ovarian cancer cells(1)The cell death rate of tumor cells after treatmentwith ABT-263 and etomoxir was analyzed.The results showed that the treatment of ABT-263 or etomxir alonehad a limited effect on cell growth,but the combination of ABT-263 and etomxir could significantly induced ovarian cancer cell death.(2)Using western blot to detect the expression of CPT1A in two kinds of immortalized normal ovarian epithelial cell lines and eleven kinds of ovarian cancer cell lines,six kinds of ovarian cancer cell lines with different expression of CPT1A were selected to analysis the cell death rate after the treatment of ABT-263 and etomxir.We found that the combination of ABT-263 and etomxir significantly triggered celldeath in six kinds of cancer cell lines with high expression of CPT1A.(3)Treating CPT1A-knockdown cells with different concentrations of ABT-263,the cell death rate results demonstrated that ABT-263synergistically induced cell death with dose-dependent manners in CPT1A-knockdown cells.(4)The results of CDI confirmed that ABT-263 and etomxir have significant synergistic effect.2.The molecular mechanism of ABT-263 synergistically enhaced etomxir induced apoptosis of ovarian cancer cells(1)Western blot experiments demonstrated that the combination treatment activated Bax and upregulated the expression of cleaved caspase 3,which confirmed that the combination of ABT-263 and etomxir significantly induced cell apoptosis.Meanwhile,knockdown of Bax by sh-RNA significantly resuced cell apoptosis caused by the combination,indicating that ABT-263 and etomxir induced intrinsic apoptotic pathway.(2)Using western blot to detect the expression of the members of Bcl-2 family proteins,the results showed that the combination of ABT-263 and etomxir significantly upregulated the expression of Noxa,knockdown of Noxa significantly reduced the apoptosis of induced by the combination,indicating that Noxa was the upstream molecule of Bax activation and played an important role in the molecular mechanism of two drugs.(3)Screening of Bcl-2 family membersand proteins of signaling pathways in CPT1A-knockdown cells by Western blot demonstrated that knockdown of CPT1A upregulated the expression of Noxa,indicating that CPT1A has a regulatory effect on Noxa.3.CPT1A regulates expression of Noxa at transcriptional level(1)Inhibiting the synthesis of intracellular protein by CHX and detecting the expression of Noxa after the treatment of etomxir,the results suggested that the regulation of CPT1A on Noxa occurred at the pre-translation.(2)Real-time PCR tests showed CPT1A regulates expression of Noxa at transcriptional level.4.Activation of AMPK mediated the upregulation of Noxa expression induced by CPT1A-knockdo wn(1)Screening of the expression of Noxa after the treatment of etomxir in AMPK-al-knockdown and/or AMPK-?2-knockdown cells,we found only when AMPK-al and AMPK-?2 were knockdown at the same time,the upregulation of Noxa caused by etomxir be synergistically reduced,indicating that the activation of AMPK played an important role in the upregulation of Noxa by CPT1A inhibition.(2)Detecting the expression of Noxa in AMPK-al-knockdown and/or AMPK-?2-knockdown cells after the inhibition of ATP production by oligomycin A,the results demonstrated that knockdown of AMPK-al and AMPK-?2 at the same time synergistically resuced the upregulation of Noxa caused by oligomycin A,whichconfirmed the activation of AMPK mediated the upregulation of Noxa induced by CPT 1 A-knockdo wn.Conclusion and significance:In this study,we investigated from molecular and cellular that ABT-263 enhanced etomoxir inhibited the growth and proliferation of ovarian cancer cells,and caused cell death by intrinsic apoptotic pathway,which demonstrated that ABT-263 and etomxir have significant synergistic effect.Molecular mechanism results confirmed that the combination of ABT-263 and etomxir upregulated Noxa,which led to the activation of Bax and the increase of cleaved caspase 3,and induced apoptotic.In addition,CPT1A regulates expression of Noxa at transcriptional level and activation of AMPK mediated the upregulation of Noxa expression induced by CPT1A-knockdown.This study provides experimental basis on CPT1A as a potential treatment target for ovarian cancer,and the combination of ABT-263 and etomoxir provides an effective drug combination in the clinical treatment of ovarian cancer. |
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