| ObjectiveAccording to statistics from the world health organization(WHO),there were more than 18 million new cases of cancer in 2018,with more than 9.55 million deaths from cancer,and cervical cancer ranked eighth among new tumors of all genders and ages.In the treatment of tumor,there are five commonly used treatment methods: surgery,chemotherapy,radiotherapy,immunotherapy and tumor hyperthermia.However,due to high toxicity,high cost,long period,high recurrence rate and safety,the survival rate of tumor patients is still not up to the ideal level.The cervical cancer vaccine is less effective for non-HPV infections and for developing cervical cancer.The aim of this study was to explore the potential mechanism of the combination of the more effective small molecule compound metformin and the HIV inhibitor nelfinavir on cervical cancer.Autophagy is an important direction in the treatment of cancer,and the production of reactive oxygen species is closely related to the occurrence of cancer.This paper will further reveal the mechanism of combined action of metformin and nelfinavir on cervical cancer and its relationship with autophagy,and explore whether the combination of metformin and nelfinavir can enhance the autophagy of cervical cancer cells through the mitochondrial SITR3/mROS pathway,so as to achieve the anti-cervical cancer effect.Methods1.HeLa and SiHa cells without treatment,treated with metformin(10mmol/L),nelfinavir(4?mol/L)and combined drug(metformin 10mmol/L + nelfinavir 4?mol/L)were detected by transmission electron microscopy,and the accumulation of autophagy vesicles was observed.2.Western Blot was used to detect the expression of autophagy related proteins,including: LC3II/I,Beclin-1,Atg7,Atg3,and SIRT3 related to the production of mitochondrial reactive oxygen species(mROS)after processing with metformin and(or)nelfinavir.3.Assess the cytotoxicity of metformin and(or)nelfinavir to natural killer(NK)cells by lactate dehydrogenase(LDH)cytotoxicity assay;western blot was used to detect the expression of NK cytotoxic related protein MICA.4.The xenograft model of cervical cancer in nude mice was used to evaluate the anti-tumor effect of the combination of drugs: the xenograft model of nude mice was constructed,and the body weight and tumor size of nude mice were weighed every three days after administration,so as to observe the effect of combined(single)use of drugs on the growth and tumor size of nude mice with transplanted tumor.The expression of autophagy-related proteins,proteins associated with mROS production and NK cytotoxicity were verified by western blot.Results1.Transmission electron microscopy results showed that compared with metformin or nelfinavir alone,metformin combined with nelfinavir treatment resulted in significant accumulation of autophagosomes in HeLa and SiHa cell lines,suggesting that the combination of drugs may be associated with autophagy.2.Western blot(in vivo and in vitro samples)results showed that compared with metformin or nelfinavir alone,the expressions of HeLa and SiHa autophagy marker proteins LC3II/I was increased and the expressions of Beclin-1,Atg3 and Atg7 were decreased after the treatment with metformin combined with nelfinavir.Increased expression of SIRT3 associated with mROS production suggests that autophagy produced by combination therapy may be related to SIRT3/mROS.3.LDH cytotoxicity test showed that the NK cell-mediated toxicity was enhanced after the treatment of metformin combined with nelfinavir.Western blot assay showed that the expression of MICA,a protein associated with enhanced NK cytotoxicity increased,suggesting that the combination of the two drugs enhanced the killing effect of NK cells on cervical cancer cells HeLa and SiHa.4.The results of xenotransplantation of cervical cancer in nude mice showed that metformin combined with nelfinavir was more effective in inhibiting tumor growth than metformin or nelfinavir alone,and the expression of autophagy related proteins LC3II/I,Beclin-1,SIRT3 related to mROS production,and MICA related to enhanced NK cytotoxicity were also changed.ConclusionMetformin and nelfinavir act synergistic on cervical cancer,causing changes in autophagy related proteins: the expression of LC3II/I is up-regulated,and the expression of Beclin-1,Atg3,and Atg7 is down-regulated.At the same time,up-regulation of mitochondrial SIRT3 causes the increase of mROS and induces autophagy in cervical cancer.In addition,MICA on the cell surface can be up-regulated to enhance the lysis of NK cells,thus promoting the apoptosis of cervical cancer cells and inhibiting the growth of cervical cancer. |
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