Effects Of Melatonin On TNF-α-mediated Apoptosis Of Cervical Cancer Hela Cells And Its Mechanism

Cervical cancer is the most common malignant tumor of the female reproductive tract.According to the statistics of cancer in 2018,in female malignant tumors worldwide,the annual incidence of cervical cancer is about 570,000,with an incidence rate of 6.6%;the number of deaths is about 310,000,and the mortality rate is 7.5%.Among female tumors,cervical cancer ranks fourth for both incidence and mortality,which seriously threatens women’s health and life.At present,surgery,radiotherapy and chemotherapy have been widely used in the treatment of cervical cancer.However,these traditional treatment schemes have significant effects on early patients,but in advanced cervical cancer and recurrent cervical cancer patients have not achieved satisfactory results.In addition,surgical treatment,radiotherapy and chemotherapy bring many complications to patients,all of which have certain limitations.Therefore,the development of more effective treatment strategies are bound to provide more benefits for the treatment of cervical cancer patients.Recent years immunotherapy have affirmed an exciting development in different human malignancies.Currently,cytokine-based immunotherapy has been shown to inhibit cancer cells invasion and proliferation via multiple mechanisms,thereby affecting the progression of cancer.Previous studies have verified that Tumor Necrosis Factor-α(TNF-α)has an ability to inhibit the survival of cancer cells and has no damage toxicity to normal tissues,suggesting that TNF-α-based immunotherapy may be considered an effective tool with which to intervene against cancer development.However,previous studies have also indicated that the resistance of tumor cells to immunotherapy influences the clinical application and efficacy of treatment.Moreover,increasing evidence suggests that resistance to cytokine immunotherapy contributes to the tumor escape from the apoptptic signal.Therefore,determining methods with which to enhance TNF-α-induced apoptosis is the key to enhance the therapeutic efficacy.Mitochondria,which are oreganelles present in all cells of the human body apart from erythrocytes,play a pivotal role in energy production.It is well known that mitochondria play an important role in the internal pathways of apoptosis in mammalian.Previous studies have indicated that TNF-α induced cancer cells apoptosis via the activation of the caspase-9-dependent apoptotic pathway.Therefore,we hypothesized that rimmunotherapy-resistance may be related to failure to regulate mitochondrial apoptosis.Notably,recent studies found that the mitochondria could sustain itself quantity and quality via mitophagy.Mitophagy refers to the process by which cells selectively degrade excess or damaged mitochondria through autophagy.Mitophagy labels injured mitochondria via LC3-Ⅱ and facilitates the entry of the injured mitochondria to the lysosome,leading to the removal of deficient mitochondria.This protective mechanism helps cancer to block mitochondria-induced apoptosis via timely removing damaged mitochondria.Accordingly;we therefore believe that mitophagy is involved in TNF-α-based immunotherapeutic resistance.Melatonin is an indoleamine hormone synthesized and secreted by the pineal gland of mammals.Melatonin has several beneficial effects on body physiological processes.Nonetheless,in the last few years,the melatonin pro-apoptotic effects in many tumor cells have recently attracted much attention.Besides,many recent studies have highlighted that melatonin holds oncostatic activity through various biological mechanisms such as pro-apoptotic and anti-proliferative actions in breast cancer,colorectal cancer,leiomyosarcoma,renal cell carcinoma and gastrointestinal cancer.So far,various studies have reported that melatonin may be an excellent candidate as anticancer agent or for combined therapy.Indeed,it has been previously described that treatment with the chemotherapeutic drug etoposide in combination with melatonin resulted in increased elimination of leukemia cells derived from a patient with acute myeloid leukemia.It also has been demonstrated that melatonin enhances cisplatin-induced cytotoxicity in different human ovarian cancer cells.Preliminary clinical studies in patients with cancer also demonstrated the benefits of melatonin in association with cancer chemotherapeutic agents.Furthermore,a study has reported that melatonin acts as both a tumor metabolic inhibitor and circadian-regulated kinase inhibitor to promote the sensitivity of breast tumors to doxorubicin(DOX)and drive tumor regression.Notably,several recent studies have validated the inhibitory impact of melatonin on mitophagy.For examples,in acute brain injury,melatonin attenuates traumatic brain inflammation via modulating mitophagy.Moreover,in the human hepatocellular carcinoma cells,melatonin increased the sensitivity of cancer cells to ROS-mediated apoptosis via modification of mitophagy.Few data are available on the effects of therapeutic synergy of TNF-αtogether with melatonin on human cervical cancer cells.Therefore,the aim of this study was to explore the effects of melatonin on TNF-α-induced cervical cancer Hela cells apoptosis by regulating mitochondrial autophagy in vitro and in vivo,and to investigate the possible mechanisms in mitophagy signaling pathway.Part 1 The effect of melatonin on TNF-α-mediated apoptosis of cervical cancer Hela cellsObjectiveThis part aims to investigate the effect of malatonin on TNF-α-mediated apoptosis of cervical cancer Hela cells.Methods1.Hela cells were divided into four groups including control group,TNF-αgroup,melatonin group,melatonin +TNF-α group.2.Methyl thiazolyl tetrazolium(MTT)and lactate dehydrogenase(LDH)assay were used to detect the effects of each group on Hela cells proliferation.3.TdT-mediated terminal deoxynucleotidy transferase-mediated dUTP nick end labeling(TUNEL)was used to detect the effects of each group on Hela cells apoptosis.Enzyme-linked immunosorbent assay(ELISA)was used to detect the activity of caspase-3 and Western blot was used to detect the expression of cleaved caspase-3.4.Flow cytometry(Flow Cytometry,FCM)was used to detect mitochondrial ROS production,mitochondrial permeability transition pore kit was used to detect mitochondrial permeablity transition pore(mPTP)opening rate.The JC-1 assay was used to detect changes in mitochondrial membrane potential(MMP),and immunofluorescence staining was used to detect the expression of Cyt-c in cells.5.Chemiluminescence method was used to detect the production of adenosine triphosphate(ATP),Western blot was used to detect the expressions of mitochondrial respiratory complexes,and ELISA was used to detect mitochondrial lactic acid formation and glucose uptake.Results1.MTT assay showed that TNF-α,melatonin inhibited the proliferation of cervical cancer HeLa cells.Compared with TNF-α alone group,the combined application of melatonin and TNF-α significantly inhibited the proliferation of Hela cells,the difference was statistically significant(P<0.05).2.TUNEL test showed that TNF-α and melatonin alone group induced apoptosis of cervical cancer Hela cells.Compared with TNF-α alone group,the combined application of melatonin and TNF-α significantly induced apoptosis of cervical cancer Hela cells,and the difference was statistically significant(P<0.05).3.ELISA and Western blot showed that TNF-α and melatonin alone group could increase the activity of caspase-3 and the expression of cleaved caspase-3.When compared with TNF-α alone group,melatonin combined with TNF-αsignificantly increased the activity of caspase-3 and the expression of cleaved caspase-3,and the difference was statistically significant(P<0.05).4.TNF-α and melatonin alone group could increase the production of ROS,the mPTP opening rate,and the decrease of MMP.When compared with TNF-α alone group,melatonin combined with TNF-α significantly increased the production of ROS,the mPTP opening rate,and the further decline of MMP.When compared to the TNF-α group,in combination with TNF-α and melatonin effectively promoted the Cyt-c release,the difference was statistically significant(P<0.05).5.Compared with the TNF-α and melatonin alone group,the combined application of melatonin and TNF-α could significantly reduce ATP content,the expressions of mitochondrial respiratory complex,glucose consumption and lactate generation in HeLa cells,the difference was statistically significant(P<0.05).Conclusions1.Melatonin can enhance TNF-α-mediated cervical cancer Hela cells apoptosis.2.Melatonin enhances TNF-α-mediated cervical cancer Hela cells apoptosis via mitochondrial apoptosis pathway.3,Melatonin and TNF-α co-treatment cause mitochondrial energy disorder.Part 2 The mechanism of melatonin enhancing TNF-α-mediated apoptosis of cervical cancer HeLa cellsObjectiveThis part aims to explore the possible mechanism of melatonin enhancing TNF-α-mediated apoptosis of cervical cancer HeLa cells,and to study the possible mitochondrial autophagy signaling pathways.Methods1.Hela cells were divided into four groups including control group,TNF-αgroup,melatonin group,melatonin+TNF-α group,melatonin+TNF-α+FCCP group,melatonin+TNF-α+Brad group.2.Co-staining of mitochondria and lysosome using immunofluorescence was used to detect mitophagy;Western blot was used to detect the expressions of mitochondrial autophagy-related indicators LC3-Ⅱ,Beclinl and P62.3.FCCP,a specific mitophagy activator,was added to cells treated with melatonin and TNF-α to activate mitophagy,and ELISA was used to detect the activities of caspase-3 and caspase-9.4.Byusing siRNA(Small interfering RNA)transfection method to knockdown the gene of Parkin in Hela cells,and then the expressions of Parkin,LC3-Ⅱ and Beclinl in the control group,TNF-α group,TNF-α+ si-Parkin group and TNF-α+melatonin group were detected by Western blot.5.Brad was added to cells treated with melatonin and TNF-α to reactivate the CaMKⅡ pathway,western blot and immunofluorescence staining were used to detect the expressions of p-CaMKⅡ and Parkin.Results1.The mitochondrial and lysosomal fluorescence co-staining results showed that the overlap of mitochondria and lysosome were increased in TNF-α alone group in comparision with the control group;Compared with TNF-α alone group,the overlap of mitochondria and lysosome were decreased in melatonin alone group and melatonin combined with TNF-α group,and the differences were statistically significant(P<0.05).2.Western blot showed that the expressions of LC3-Ⅱ and Beclinl were higher while the expression of P62 was lower in TNF-α alone group than the control group;Compared with TNF-α alone group,the expressions of LC3-Ⅱ and Beclinl were decreased,and the expression of P62 was increased in melatonin alone group and melatonin combined with TNF-α group,and the differences were statistically significant(P<0.05).3.ELISA showed that the activities of caspase-3 and caspase-9 were higer in TNF-α and melatonin alone group than the control group;Compared with TNF-αalone group,the activities of caspase-3 and caspase-9 were increased significantly in melatonin combined with TNF-α group;Compared with the combined application of melatonin and TNF-α group,the activities of caspase-3 and caspase-9 in the melatonin combined with TNF-α and FCCP groups were significantly decreased,and the differences were statistically significant(P<0.05).4.Western blot showed that the expression of Parkin in the TNF-α group was significantly increased compared with the control group;Compared with the TNF-αgroup,the expressions of Parkin in the TNF-α+ si-Parkin group and TNF-α+melatonin group were significantly decreased,and the differences were statistically significant(P<0.05).To understand whether increased Parkin was linked to the TNF-α-mediated mitophagy,after silencing Parkin expression using siRNA,western blot was used to detect the expressions of Beclinl and LC3-Ⅱ.The results showed that the expressions of Beclinl and LC3-Ⅱ in the TNF-α group were significantly increased compared with the control group;but compared with the TNF-α group,the expressions of Beclinl and LC3-Ⅱ in the the TNF-α+ si-Parkin group and TNF-α+melatonin group were significantly decreased,and the differences were statistically significant(P<0.05).5.Western blot and immunofluorescence staining showed that the expressions of p-CaMKⅡ and Parkin were increased in TNF-α and melatonin alone group in comparision with the control group;Compared with TNF-α alone group,the expressions of p-CaMKⅡ and Parkin were decreased in melatonin combined with TNF-α group;Compared with the combined application of melatonin and TNF-αgroup,the expressions of p-CaMKⅡ and Parkin were significantly increased in the melatonin combined with TNF-α and Brad groups,and the differences were statistically significant(P<0.05).ConclusionsMelatonin enhances TNF-α-induced human cervical cancer HeLa cells mitochondrial apoptosis via inactivating the CaMKⅡ/Parkin/mitophagy axis.Part 3 Effects of melatonin on TNF-α-mediated tumor growth and mitophagy of cervical cancer in nude miceObjectiveThe model of the subcutaneous xenografts of human cervical cancer Hela cells was established to explore the effects of melatonin on TNF-α-mediated nude mice xenograft growth and mitophagy.Methods1.Establish the model of human cervical cancer Hela cells subcutaneous xenografts in nude mice.2.Animal groups:Twenty nude mice with cervical cancer Hela cells subcutaneous xenografts were randomly divided into 4 groups,and with 5 nude mice in eaeh group:(1)control group:normal saline;(2)TNF-α group:TNF-α 10μg/kg;(3)melatonin group:melatonin 10mg/kg;(4)TNF-α+ melatonin group:TNF-α10μg/kg+melatonin 10mg/kg.Intraperitoneal inje ction,once a day for twelve days.3.The growth changes of nude mice in each group were observed and recorded.The tumors volume and the changes of weight were measured,and the dynamic growth curve were drawn.At the end of the treatment,nude mice were executed,and the tumors were completely removed,and the size of the tumors was measured and the pictures were taken.4.Hematoxylin Eosin(HE)staining method was used to observe the changes of pathomorpholohy of transplanted tumor cells in in each group transplanted tumors.5.The TUNEL assay was used to detect the apoptosis of transplanted tumors tissue cells.6.Western blot was used to detect the expressions of LC3-Ⅱ and Beclinl.Results1.The model of human cervical cancer Hela cells subcutaneous xenografts in nude mice was successfully established.2.Transplanted tumors volume and weight changes of nude mice in each group.After treatment,TNF-α and melatonin alone group could both decrease the volume of xenografted tumors in comparision with the control group;Compared with the TNF-αalone group,the volumes of the transplanted tumors were decreased more significantly in melatonin combined with TNF-α group,and the differences were statistically significant(P<0.05).After the nude mice were sacrificed,the tumors weight of the nude mice in TNF-α and melatonin alone group were lower than that in control group;Compared with the TNF-α alone group,the tumors weight were decreased more significantly in melatonin combined with TNF-α group,the differences were statistically significant(P<0.05).3.HE staining showed that transplanted tumor tissues of TNF-α group,melatonin group,melatonin combined with TNF-α group could observe obvious apoptosis and necrotic.4.The TUNEL assay showed that the apoptosis index in TNF-α and melatonin alone group were higher than the control group;Compared with TNF-α alone group,the apoptosis index was significantly increased in melatonin combined with TNF-αgroup,and the differences were statistically significant(P<0.05).5.Western blot showed that the expressions of LC3-II and Beclinl were increased in TNF-α alone group compared with the control group;Compared with TNF-α alone group,the expressions of LC3-II and Beclinl were decreased in melatonin alone group and melatonin combined with TNF-α group,the differences were statistically significant(P<0.05).ConclusionsMelatonin enhances TNF-α-mediated apoptosis of the nude mice model of cervical cancer Hela cells via inactivating the mitophagy signaling pathway.