The Antitumor Effects Of Fatostatin In Endometrial Carcinoma And Its Molecular Mechanism

BackgroundEndometrial carcinoma(EC)is one of the most common gynecological malignant tumors,accounts for 7%of all cancers in women and accounts for 20-30%of gynecological malignant tumors.The average age of onset is 60 years old,and 75%of EC occur in women over 50 years old.In recent years,the incidence rate of EC has been increasing and getting younger in the world.There are two types of EC.Type I is estrogen dependent,and all of them are endometrioid adenocarcinoma.Type II is non-estrogen dependent,the pathogenesis is not related to estrogen and the pathological morphology is rare,such as endometrial serous carcinoma,clear cell carcinoma and adenosquamous carcinoma.Patients with early endometrioid adenocarcinoma have relatively good prognosis,but patients with advanced or relapsed diseaseand patients with II EC have poor prognosis.Surgery and adjuvant chemo-radiotherapy are the first-line therapies used for most patients with EC.However,for patients with advanced or relapsed disease,and for patients who want to retain reproductive function,hormonal regimens,including progestins,luteinizing hormone releasing hormone agonists,anti-estrogens and aromatase inhibitors,are mainly applied.Additionally,progestins have been used as the first option,and the effective rate for progesterone receptor positive EC is up to 80%.But,studies had shown that many patients with ECexhibited no response to the progestins.Thus,new therapeutic targets and drugs are needed.Sterol regulatory element-binding proteins(SREBPs)are critical regulators of lipid homeostasis that function by transcriptionally activating genes that are involved in fatty acid and cholesterol homeostasis.In mammalian cells,three isoforms of SREBP(SREBP-1a,SREBP-lc and SREBP-2),which are coded by two genes(SREBF1 and SREBF2)have been identified.Studies have suggested that SREBP-1 is the main regulator of fatty acid metabolism,while SREBP-2 predominantly regulates cholesterol metabolism.Additionally,several studies have reported that SREBPs function as oncogenes in various malignant tumorsand that SREBPs can promote tumor progression by regulating lipogenesis.In previous studies,our research group demonstrated that the expression level of SREBP-1 was significantly elevated in EC compared with that in healthyendometrium and that the expression levels were positively correlated with cancer progressionand that knockdown of SREBP-1 expression in EC cells suppressed cell proliferation,reduced clonogenic capacity and induced apoptosis.Therefore,these studies revealed that SREBP-1 functions as oncogene in EC and blocking SREBP-regulated metabolic pathways via pharmacological intervention may be a novel therapeutic approach for treating EC.Fatostatin is a chemical inhibitor of the SREBP pathway and inhibits the maturation and nuclear translocation of SREBPs.Kamisuki et al demonstrated that fatostatin increased fatty acid mobilization and oxidation and reduced lipogenesisin obese ob/ob micewhile exhibiting low cytotoxicity.In addition,Li et al revealed that fatostatin demonstrated high antitumor activity against prostate cancer by blocking SREBP-regulated metabolic pathways and androgen receptor signaling in vitro and in vivo.Furthermore,Siqingaowa et al demonstrated that fatostatin decreased pancreatic cancer cell viability and proliferation.Therefore,we hypothesized that fatostatin has antitumor effectsinEC.But so far,there is no relevant research.In our present study,we aim to investigate the antitumor effects of fatostatin in EC and its molecular mechanism and to provide a new theoretical evidence for the treatment of EC.Part One The antitumor effects of fatostatin in endometrial carcinomaObjective:Todetermine the effect of fatostatin on the viability,colony formation ability,invasive and migratory capacities,cell cycle distribution and cell apoptosis of EC cells(Ishikawa and HEC-1A),and to investigate theantitumor effects of fatostatin in EC cells.Methods:In the present study,we determined the effect of fatostatin on the viability of EC cells(Ishikawa and HEC-1A)using MTT assays,and calculated the IC50 values for fatostatin in the Ishikawa and HEC-1A cells by GraphPad Prism 5 software.Then,we determined the effect of fatostatin on the growth rate of each cell line using MTT assays.The effect of fatostatin on EC cell colony formation ability was examined by plate colony formation assays.The effects of fatostatin on the invasive and migratory capacities of EC cells were examined by transwell cell invasion and migration experiments in vitro.Cell cycle and apoptosis analysis were determined using flow cytometry.Results:1.Results of MTT assays showedthat:fatostatin significantly inhibited the viability of both cell lines in a time-and dose-dependent manner;the IC50 values(72-h treatment)for fatostatin in the Ishikawa and HEC-1 A cells were 17.96 and 4.53 pmol/l,respectively;the results of the effect of fatostatin on the growth rates ofboth cell lines with MTT assays showed that compared with the vehicle groups,the OD values of the cells in the fatostatin groups were significantly reduced,having a significantly dose-and time-dependentmanner,P<0.01.2.Results of plate colony formation assays showedthat:compared with the vehicle groups,the colony formation rates of fatostatin groups were significantly decreased and the colony formation abilities were significantly reduced in Ishikawa and HEC-1 A cells,having adose-dependent manner,P<0.01.3.Results of transwell cell invasion experiments in vitroshowedthat:compared with the vehicle groups,the numbers of invading cells of fatostatin groups were significantly decreased and the cell invasion abilities were significantly reduced in Ishikawa and HEC-1 A cells,P<0.01.4.Results of transwell cell migration experiments in vitroshowedthat:compared with the vehicle groups,the numbers of migrating cells of fatostatin groups were significantly decreased and the cell migration abilities were significantly reduced in Ishikawa and HEC-1A cells,P<0.01.5.The main results of cell cycle analysis using flow cytometry in Ishikawa cells showedthat:compared with the vehicle groups,at higher concentrations(20 and 40?M),the percentagesof cells in the G2/M phase of fatostatin groups were significantly increased,P<0.01;the percentagesof cells in the G0-G1 phase and S phase of fatostatin groups were significantly decreased,P<0.05.6.The main results of cell cycle analysis using flow cytometry in HEC-1A cells showedthat:compared with the vehicle groups,at higher concentration(10 ?M),the percentagesof cells in the G2/M phase and S phase of fatostatin groups were significantly increased,P<0.01;the percentagesof cells in the G0-G1 phase of fatostatin groups were significantly decreased,P=0.0002.7.The main results of cell apoptosis analysis using flow cytometry in Ishikawa cells showedthat:compared with the vehicle groups,at higher concentrations(20 and 40?M),the percentagesof apoptotic cells and early apoptotic cells of fatostatin groups were significantly increased,P<0.05;the percentage of lateapoptotic cells was significantly increased only at fatostatin 40?M group,P=0.0447.8.The main results of cell apoptosis analysis using flow cytometry in HEC-1A cells showedthat:compared with the vehicle groups,at higher concentration(10?M),the percentagesof apoptotic cells and early apoptotic cells of fatostatin groups were significantly increased,P<0.01;the percentage of lateapoptotic cellswas only mildly increased,showing no statistical significance,P=0.2658.Conclusion:1.Fatostatin inhibited EC cell viability and the IC50 values(72-h treatment)for fatostatin in the Ishikawa and HEC-1A cells were 17.96 and 4.53 ?mol/l,respectively.2.Fatostatin inhibited the colony formation ability of EC cells.3.Fatostatin inhibited the invasive and migratory capacities of EC cells.4.At higher concentrations,fatostatin promoted the apoptosis of EC cells and arrested the cell cycle in the G2/M phase.5.Fatostatin demonstrated high antitumor activity against EC cells and may be a novel therapeutic strategy for EC treatment.Part Two The molecular mechanism of fatostatin on the antitumor effects of endometrial carcinomaObjective:Todetect the changes of lipid metabolism related protein and the mRNA levels of lipid metabolism related genes in EC cells after treatment with fatostatin,to detect the changes of intracellular free fatty acids and total cholesterol levels in EC cells after treatment with fatostatin,to examine thechanges of apoptosis related protein(caspase-9,caspase-3 and PARP)in EC cells after treatment with fatostatin,and to investigate the molecular mechanism of fatostatin on the antitumor effects of EC cells.Methods:In the present study,the effects of fatostatin on the levels of lipid metabolism related protein and apoptosis related protein(caspase-9,caspase-3 and PARP)in EC cells were determinedby western-blot.The effects of fatostatin on the mRNA levels of lipid metabolism related genes and two chaperones in EC cells were determinedby qRT-PCR.After treatment with fatostatin,the changes of intracellular free fatty acids and total cholesterol in EC cells were assessed using free fatty acid and total cholesterol quantification detection kit,respectively.Results:1.Results of the effects of fatostatin on the expression levels of apoptosis related protein in EC cells by western-blot showedthat:fatostatin significantly decreased the expression levels of apoptosis related protein full-length caspase-9,caspase-3 and PARP and significantly increased the expression levels of cleaved caspase-9,cleaved caspase-3 and cleaved PARP in EC cells,P<0.05.2.Results of the effects of fatostatin on the expression levels of lipid metabolismrelated protein in EC cells by western-blot showedthat:the precursor SREBP-1 and SREBP-2 protein levels were mildly changed,showing no statistical significance,P>0.05;thenuclear SREBP-1 and SREBP-2 protein levels were significantly decreased,P<0.05;the FASN and HMGCR protein levels were also significantly decreased,P<0.05.3.Results of the effects of fatostatin on the mRNA levels of lipid metabolismrelated genes in EC cells by qRT-PCR showedthat:the mRNA expression levels of these genes(ACL,FASN and SCD-1,which are involved in lipogenesis;HMGCS1,HMGCR,MVK,MVDand LDLR,which are involved in cholesterogenesis;and INSIG1 and SCAP,which are two chaperones)were significantly downregulated,P<0.05.4.Results of the changesof intracellular free fatty acidsin EC cells using free fatty,acid quantification detection kitshowedthat:compared with the vehicle groups,the levels of intracellular free fatty acids of fatostatin groups were significantly decreased in Ishikawa and HEC-1A cells,P<0.05.5.Results of the changesof total cholesterol in EC cells using total cholesterol quantification detection kitshowedthat:compared with the vehicle groups,the levels of total cholesterol of fatostatin groups were significantly decreased in Ishikawa and HEC-1A cells,P<0.01.Conclusion:1.Fatostatin significantly reducedthe protein and mRNA levels of lipid metabolismrelated genes and inhibited the SREBP metabolic pathways in EC cells.2.Fatostatin significantly decreased the levels of intracellular free fatty acid and total cholesterol in EC cells,which further confirmed that fatostatin inhibited the SREBP metabolic pathways in EC cells.3.Fatostatin significantly decreased the expression levels of full-length caspase-9,caspase-3 and PARPprotein and significantly increased the expression levels of cleaved caspase-9,cleaved caspase-3 and cleaved PARPprotein in EC cells,which indicated that fatostatin induced caspase-dependent apoptotic death in EC cells.4.Fatostatin inhibited the SREBP metabolic pathways and induced caspase-dependent apoptotic death in EC cells,which may be the potential molecular mechanism of fatostatin on the antitumor effects of EC cells.