SIRT3 Increases Sensitivity Of Metformin Through Interfering Mitochondrial Function In Ovarian Cells

Clinical studies have shown that the classic drug for the treatment of diabetes-metformin has the effect of inhibiting the development of tumors,and laboratory studies have shown that metformin has an inhibitory effect on many tumor cells.Although the effects of metformin on tumor cells include many molecules,signaling pathways and organelles,mitochondria and energy metabolism are the most widely and deeply targeted.Based on the characteristics of metformin cheap,high efficiency,and safety,the development of metformin anti-tumor drugs not only means that this classic drug is expected to realize the new use of old drugs,but also can provide new strategies and ideas for cancer treatment.Mitochondria and related signaling pathways play multiple roles such as energy balance,redox balance,and apoptosis regulation,and are crucial in the formation and development of tumors.Cancer cells rely on energy metabolism to exert a variety of biological functions.Studies have shown that tumor cells can induce apoptosis when the ATP content in tumor cells is reduced.However,tumor cells have complex metabolic pathways and rich compensatory mechanisms.The effect of metformin on mitochondria is mainly manifested in inhibition of mitochondrial complex 1,disruption of mitochondrial respiratory chain,and energy stress.Studying the effect of metformin on energy metabolism plays a role in anti-tumor activity to better understand the function of mitochondria in tumor biology and provide new clues for investigating anti-tumor strategies.The mitochondrial deacetylase(SIRT3)is regulated by the energy state of the cells.It has been reported in the literature that protein activation of SIRT3 is under stress and energy limitation in adipocytes,cardiomyocytes,and skeletal muscles.SIRT3 maintains normal cellular functions through acetylation of the mitochondrial respiratory chain and redox-related proteins.Although the above studies are based on the premise that normal cells have normal metabolic functions,it is suggested that SIRT3 can activate and exert its effect under stress conditions.However,in tumor cells,it has been reported that SIRT3 promotes apoptosis through multiple pathways such as oxidative stress,increased Bax/Bcl-2 and Bad/Bcl-x L ratio.Our previous work observed that the use of Bcl-2 inhibitor S1 induced apoptosis,OCR oxygen consumption rate in ovarian cancer cells decreased while SIRT3 protein expression increased.Therefore,to explore the regulatory role of SIRT3 in apoptosis can provide new clues to clarify the role of metformin.AMPK also acts as an energy regulator.Metformin was found to promote AMPK activation in myocytes,cardiomyocytes,and tumor cells.AMPK activation restores the energy balance in cells by inhibiting the biosynthetic pathway and promoting catabolism.In normal muscle cells,activation of AMPK was found to promote glucose uptake by the glucose transporter,suggesting that AMPK may play a partial compensatory role in stress.Increased expression of SIRT3 was observed in muscle tissue of mice exercised with CR diet,accompanied by activation of AMPK protein,whereas AMPK activation was reduced in sirt3-/-mice tissues,suggesting a possible link between AMPK and SIRT3.Therefore,to explore the relationship between AMPK and SIRT3 in tumor cells can be used to elucidate the cell-responsive metabolic mechanism in the treatment of tumors targeting mitochondria,which will help to further understand the compensatory mechanism of tumor cell energy metabolism.In this study,we selected human ovarian cancer cell lines as the research object,metformin as a tool drug,and used mitochondrial function as an entry point to analyze energy metabolism in tumors from the perspective of the role of deacetylase SIRT3 in apoptosis.Method:(1)To observe the effect of metformin on the apoptosis sensitivity of ovarian cancer cells SKOV3,A2780 and HO8910.MTT,real-time cell growth curve,and calculation of cell doubling time were used.Hochest33342 staining,Annexin V-PI staining,Western blot were used to detect apoptosis-related proteins of Bcl-2 family,and Cyto C release(2)To observe the effects of metformin on energy metabolism and mitochondrial function,mitochondrial complex I,and mitochondrial respiratory chain in ovarian cancer cells.O xygen consumption rate,lactate production rate and ATP content were detected;DCFH-DA and JC-1 staining flow cytometry were used to detect the effect of metformin on ROS level and mitochondrial membrane potential of ovarian cancer cells.Western blot was used to detect the mitochondrial respiratory chain protein expression,and the kit was used to detect mitochondrial complex I activity.The oxygen consumption rate(OCR)of ovarian cancer cel s was detected.(3)To observe the effect of metformin on SIRT3,and detect the role of SIRT3 in apoptosis.Western blot was used to detect SIRT3 protein expression and mitochondrial protein acetylation level.The kit was used to detect SIRT3 deacetylase activity.Over-expression of SIRT3 and sh SIRT3 plasmids transfected cells,MTT assay the survival rate of metformin,Hochest33342 staining,Annexin V-PI staining,Western blot detection of Bcl-2 family apoptosis-related protein,C yto C release,kit detection of Caspase3/7 activity,the kit detection of ATP content;DCFH-DA,JC-1 staining flow cytometry ROS levels,mitochondrial membrane potential.were used.(4)Explore the relationship between SIRT3 and AMPK and the role of AMPK.Using metformin,sh SIRT3,overexpression of SIRT3 plasmid transfected cells,Western blot was used to detect the expression of AMPK and phosphorylated AMPK.Using the AMPK inhibitor Compound C as a tool,MTT was used to measure cell survival rate,real-time PCR was used to detect GLUT4 gene expression levels,the kit was used to detect glucose consumption,and the kit was used to measure ATP levels.(5)To explore ways to increase the sensitivity of metformin by restricting glycolysis using 2-DG and low-glucose culture medium.The cell survival rate was measured by MTT and the kit was tested for ATP levels.Result:(1)Metformin decreased the survival rate of SKOV3,A2780,HO8910,inhibited the cell growth curve,and increased the doubling time.Metformin led to increased staining of ovarian cancer cells Hochest33342,increased positive rate of Annexin V-PI staining,increased expression of Bcl-2 family pro-apoptotic proteins,decreased expression of anti-apoptotic proteins,and increased release of Cyto C.(2)Metformin promoted glucose uptake and lactic acid production.Metformin increased ovarian cancer cell ROS levels,decreased membrane potential,decreased oxygen consumption,and decreased ATP content.Metformin reduces OCR levels in ovarian cancer cells.The combination of metformin and rotenone does not further promote OCR inhibition by metformin.(3)Metformin promoted SIRT3 protein expression,increased SIRT3 deacetylase activity,and decreased mitochondrial acetylation levels in ovarian cancer cells.sh SIRT3 impaired the inhibition of survival and apoptotic rate of metformin;overexpression of SIRT3 enhanced the inhibition of survival,apoptotic rate of metformin,increased expression of mitochondria-associated pro-apoptotic protein,Cyto C release,and Caspase 3/7 activity.Overexpression of SIRT3 further reduced Metformin-induced reductions in ATP,ROS,and mitochondrial membrane potential.(4)Metformin promoted the expression of AMPK protein and phosphorylation levels,overexpression of SIRT3 promoted AMPK activation,and sh SIRT3 inhibited AMPK activation caused by metformin.Compound C enhanced survival inhibition and ATP production inhibition caused by metformin,and Compound C impaired the increase of GLUT4 gene expression and glucose uptake caused by metformin.(5)2-DG and low-sugar cultures can increase the inhibition of survival of metformin in ovarian cancer,and further decrease the ATP level of ovarian cancer.Conclusion:(1)Metformin has antitumor effects on SKOV3,A2780,and HO8910,and metformin promotes apoptosis of ovarian cancer cel s in the mitochondrial pathway.(2)Although metformin promotes the glycolytic pathway,metformin can inhibit mitochondrial function,mitochondrial membrane potential,mitochondrial respiratory chain,and mitochondrial complex 1 and cause energy stress.(3)metformin can induce SIRT3 protein activation,SIRT3 activation can promote metformin-induced tumor suppression,mitochondrial dysfunction,apoptosis.(4)Metformin and overexpression of SIRT3 can promote the activation of AMPK.AMPK plays a compensatory role by promoting the glycolysis pathway.The inhibition of AMPK/glycolytic pathway can increase the energy stress caused by metformin and increase the anti-tumor effect.(5)Inhibition of glycolytic pathways can enhance the antitumor effect of metformin.In summary,metformin promotes ovarian cancer cell apoptosis and induces SIRT3 activation through its effects on mitochondrial function and energy stress.In the anti-tumor course of metformin,SIRT3 caused energy stress by disrupting mitochondrial function.There is a Energy Stress-SIRT3 positive feedback loop.The inhibition of the glycolysis pathway may be one of the mechanisms that enhance the antitumor effect of metformin-targeted mitochondrial energy metabolism.