The Anti-Tumor Effects And Mechanism Of NEO212in Ovarian Cancer

BackgroundOvarian cancer is the most lethal gynecological malignancy,which seriously threatens the health of women.Current treatment for ovarian cancer includes a combination of surgery and chemotherapy.However,ever since the platinum-based chemotherapy was applied to clinic,the 5-year survival rate of ovarian cancer patients has had no obvious improvement in the past 40 years,it is necessary to develop more effective agents to improve the therapeutic effect of ovarian cancer.Temozolomide-perillyl alcohol conjugate(NEO212),a novel temozolomide(TMZ)analog,is developed based on the conjugation of TMZ,a clinically applied alkylating agent,and perillyl alcohol(POH),a natural monoterpene that has been used for the treatment of cancers.This novel compound has recently attracted continuous attention since accumulating data demonstrate that it exhibits stronger anti-cancer potency than either of its individual components in several malignancy such as glioma,triple-negative breast cancer(TNBC),non-small cell lung cancer(NSCLC)and human nasopharyngeal carcinoma(NPC).In this study,we intend to illuminate the potential anti-cancer property of NEO212 in ovarian cancer.Mitochondria as crucial organelles regulate cellular energy generation,calcium and redox homeostasis and apoptosis.To maintain the cellular functions effectively,mitochondria continuously change their structure and morphology through protein machineries controlling fission and fusion process.The fission can create new mitochondria,but it can also promote apoptosis during high levels of cellular stress such as chemical agents.This occurs almost simultaneously with two steps of apoptosis that involve mitochondria: translocation from the cytosol to mitochondria of the pro-apoptotic Bcl-2 family member Bax and Cytochrome C release.The previous studies have demonstrated that NEO212 was capable of inducing ROS accumulation,mitochondrial membrane potential collapse and mitochondrial apoptosis,indicating mitochondria might be one of main targets of chemical agents.Damaged mitochondria can trigger apoptosis,it is crucial for cell survival.An increasing number of researches showed that autophagy can endow cancer cells with the resistant ability to apoptosis,whereas inhibition of autophagy caused apoptotic cell death.Illustrating the relationship between apoptosis and autophagy in cancer cells is vital to find out new strategies for cancer therapy and improve the therapeutic efficiency.Hence,the potence of NEO212 to affect apoptosis and autophagic activity in ovarian cancer cells should be revealed.The function of autophagosome and lysosome can be regulated by some transcriptional factors,such as transcription factor EB(TFEB).TFEB regulates lysosomal biogenesis by stimulating the expression of proteins involved in all steps of lysosome,it is activated by an increased need for lysosomal activity or impaired lysosomal.Interestingly,TFEB is also involved in mitochondrial dysfunction and has been reported to regulate mitochondrial biogenesis via inducing the expression of major related master regulator genes,indicating that mitochondria and lysosomes share strong functional links that could play a fundamental role in physiology and pathology processes.TFEB is regulated by serine phosphorylation mediated by extracellular signal-regulated kinase and serine/threonine kinase AKT.Therefore,we explored the mechanism of TFEB blocking autophagy flow.ObjectiveIn this study,we investigated the potential anti-ovarian cancer activity of NEO212 and the possible mechanisms.Firstly,we illustrated the effects of NEO212 on ovarian cancer in vitro and in vivo.Secondly,we studied that NEO212 induced mitochondrial apoptosis and autophagy.Finally,we investigated NEO212 blocked mitochondrial autophagy flux and its mechanism.Our results provided a new strategy for developing potential therapeutic chemical agent for ovarian cancer.Methods1.The effects of NEO212 on ovarian cancer in vitro and in vivoThree ovarian cancer cell lines A2780,SK-O-V3 and OVCAR-3 were selected.The drugs were TMZ(temozolomide),POH(perillanol),TMZ+POH,NEO212 and DMSO(dimethyl sulfoxide).MTT assay was used to detect the effect of drugs on tumor cell survival rate.The clonogenesis ability of ovarian cancer cells were detected on the plate clonogenesis assay.Flow cytometry was used to the analysis of cell cycle alterations.Western Blot was employed to detect the expression of drug-induced DNA damage related proteins.The nude mice were randomly divided into 5 groups and were injected with DMSO,TMZ,POH,TMZ+POH and NEO212,respectively.The two vertical diameters of xenografts were measured with calipers every other day,the sizes of xenografts were calculated,and the body weights of mice were recorded.The mice were sacrificed after completion of treatment and the xenografts were separated and weighted.The tissue sections were for HE staining and anti-cl-CASP3 immunohistochemical staining.2.NEO212 induces mitochondrial apoptosis and autophagyMitochondria were labeled with fluorescent probe mito-Tracker Green,and the morphological changes of mitochondria were observed under fluorescence microscope.Transmission electron microscopy was used to observe the the morphology of mitochondria and the intracellular autophagosomes.JC-1 staining was used to detect the changes of mitochondrial membrane potential in ovarian cancer cells after drug treatment.Flow cytometry was used to detect mitochondrial apoptosis of ovarian cancer cells.The levels of the apoptosis-related proteins and autophagy related proteins were detected by Western Blot.3.NEO212 blocks mitochondrial autophagy flux and TFEB nuclear translocationThe dual fluorescence autophagy indicator m RFP-GFP-LC3 was used to detect the effect of drugs on autophagy flux.The expression levels of autophagosome and lysosome related proteins,p-ERK and p-AKT were detected by Western Blot.The colocalization of autophagosome and lysosome was observed by anti-LC3 B and antiLAMP1 immunofluorescence staining.The TFEB nuclear translocation of ovarian cancer cells was observed by anti-TFEB immunofluorescence staining.Results1.The effects of NEO212 on ovarian cancer in vitro and in vivoWe first studied the cytotoxicity of NEO212 on ovarian cancer cells in vitro.The results showed that NEO212 had a stronger inhibitory effect on proliferation and clonal formation of ovarian cancer cells compared with DMSO,TMZ,POH and TMZ+POH treatment groups.In addition,NEO212 induced G2/M phase arrest and significantly increased the phosphorylation level of DNA damage-related proteins.The effect of NEO212 was further explored using SK-O-V3 xenograft model in vivo.The results showed that compared with DMSO,TMZ,POH and TMZ+POH treatment groups,NEO212 significantly inhibited the growth of xenograft and induced tumor cell apoptosis more obviously.It is noteworthy that NEO212 did not significantly affect the histology of the major organs in nude mice.2.NEO212 induces mitochondrial apoptosis and autophagyFirstly,we investigated the effect of NEO212 on mitochondrial morphology.Mitochondrial fluorescence probe Mito-Tracker Green(MTG)labeling and electron microscope observation showed that compared with DMSO,TMZ,POH and TMZ+POH treatment groups,NEO212 induced the shorter and less branched fragments of mitochondria.The morphology of mitochondria became round and shorter,and the internal matrix of mitochondria was severely distorted or almost missing.Mitochondrial transmembrane potential(??m)detected by JC-1 probe decreased significantly,indicating that NEO212 impaired mitochondrial function.Flow cytometry showed that the number of apoptotic and dead cells in NEO212 treatment group increased significantly compared with other treatment groups.The important markers of apoptosis,Bax,Cyto C,cl-CASP3 and cl-CASP9,were also significantly increased in NEO212 group.Apoptosis inhibition test showed that Caspase inhibitor could significantly reduce NEO212 induced apoptosis.These results suggest that mitochondrial apoptosis pathway plays a key role in NEO212 induced apoptosis.We then studied the autophagy induced by NEO212 in ovarian cancer cells.The expression of autophagic marker protein LC3 B was significantly increased.However,BECN1 expression had no significant effect,indicating that NEO212 did not directly depend on BECN1 pathway to induce autophagy.By staining endogenous LC3 B,the number of autophagosomes increased significantly.We further studied the relationship between NEO212 induced apoptosis and autophagosome accumulation.The results showed that Earle's equilibrium salt solution induced autophagy and Baf.A1 inhibited autophagy,both of them induced autophagosome accumulation and promoted NEO212 induced apoptosis.3.NEO212 blocks mitochondrial autophagy flux and its mechanismFirst,we studied the effect of NEO212 to block autophagy flux.The dual fluorescence autophagy indicator m RFP-GFP-LC3 showed that m RFP and GFP signals were retained,the quenching of GFP was significantly reduced,the yellow spots increased.EEA1,LAMP1 and LAMP2 were significantly down-regulated,indicating that NEO212 interfered with lysosome maturation and activity.The colocalization of anti-LC3 B and anti-LAMP1 was significantly reduced,indicating that NEO212 blocked the fusion of autophagosome and lysosome.Next,we investigated the inhibition of TFEB nuclear translocation by NEO212.Immunofluorescence staining was performed in Ovarian cancer cells,which showed that TFEB nuclear translocation was significantly inhibited.Flag-TFEB was transfected into A2780 and SK-O-V3 cells,and Flag-TFEB levels in the cytoplasm and nucleus were detected by Western Blot.The results showed that NEO212 induced up-regulation of Flag-TFEB in the cytoplasm and down-regulation in the nucleus.This confirmed that NEO212 blocks TFEB nuclear translocation.Finally,We explored the effect of NEO212 on ERK/AKT phosphorylation.Western Blot analysis showed that the levels of p-ERK and p-Akt increased significantly,which phosphorylated TFEB serine residues,and TFEB remained inactive in the cytoplasm.All the results showed that NEO212 blocks TFEB nuclear translocation.Conclusions:1.NEO212 exerts stronger cytotoxicity than its individual constituents in vitro and inhibits ovarian cancer xenograft proliferation in vivo.2.NEO212 induces mitochondrial apoptosis and promotes mitochondrial dysfunction.NEO212 induces autophagosome accumulation and blocks autophagy flux to promote apoptosis of ovarian cancer cells.3.NEO212 blocks TFEB nuclear translocation through up-regulating ERK/AKT phosphorylation.