The Chemopreventive Effect Of Curcumin On The Inflammation-induced Esophageal Squamous Cell Carcinoma

ObjectiveEsophageal carcinoma(EC)is the sixth most common cause of death from cancer.North-Central China is one of the highest-risk area,90% of cases are esophageal squamous cell carcinoma(ESCC),although the treatment technology of EC have been improved,the overall 5-year survival rate of EC is still less than 30%.There are many reasons for the occurrence of ESCC,and the mechanism of which is complicated,many studies have found that it is related to the activation of a variety of signaling pathways,but has not yet been clearly understood.In recent years,the studies have shown that chronic infection and inflammation are closely associated with the etiology of cancer.Therefore,researchers continue to seek new ways to understand the molecular mechanisms of esophageal squamous cell carcinoma,thereby improving their clinical efficacy and prognosis,as well as improving drug resistance.At present,the treatment of cancer,including surgery,chemotherapy,radiation therapy and biological therapy have some limitations,with the increasing understanding of the etiology and pathogenesis of tumor,and many scholars found that the tumor could be preventable.At present,the application of chemoprevention in turmors has been widely concerned,it refers to a strategy of using synthetic or natural compounds to prevent the progression of tumors,and it can block the activation of multiple signal pathways in the tumor.Curcumin is a natural polyphenols extracted from turmeric,it not only has antioxidant,anti-inflammatory effect and also can adjust the NF-κB、MAPK signaling pathways and to inhibit the proliferation and induce apoptosis of tumor cells.The JAK-STAT signaling pathway plays an important role in a variety of tumors.STATs are activated by cytokines,growth factors and hormones.STAT3 belongs to the signal transducer and activator of transcription(STAT)family.In normal cells,STAT3 activation is transient and reversible.A large number of researches have found that the persistent constitutive activation of the STAT3 protein is detected in some tumors,which can promote tumor cells proliferation,metastasis and angiogenesis,interference of tumor cells apoptosis and anti-tumor immue response,and also related to the prognosis.Therefore,blocking the function of STAT3 is expected to provide a new important direction for tumor targeted therapy and chemoprevention.This paper is divided into two parts to study the chemopreventive of curcumin on the inflammation-induced esophageal squamous cell carcinoma.We explored the role of STAT3 in ESCC cells through down-regulation of STAT3 by si RNA,and chose the EC9706 and TE13 cells as the experimental object and evaluated that whether curcumin could inhibite proliferation,colonies,induce apoptosis and block cell cycle of ESCC cells through STAT3 signaling pathway.IL-6 is an activator of STAT3.We used IL-6 to act on EC9706 and TE13 cells and co-curcumin to evaluate whether curcumin could block the constitutive activation of STAT3 after IL-6 stimulation.Furthermore,we established patient-drived esophageal squamous cell carcinoma model(PDX model)and evaluated that the curative effect of curcumin on STAT3 in vivo,and to provide theoretical basis for STAT3 as a molecular therapy target and whether curcumin can be a chemotherapeutic drug for inflammation-induced esophageal squamous cell carcinoma in ESCC patient-derived xenograft models.esophageal squamous cell carcinoma by down-regulating the activity of STAT3 PartⅠ: Curcumin inhibited the proliferation and promoted apoptosis ofMethods1.STAT3 si RNA was treated to EC9706 for 48 h,and the cell cycle and apoptosis were detected after downregulation of STAT3.2.CCK-8 cell cytotoxicity experiment: different concentrations of curcumin(0,25,50 and 100μM)on EC9706 and TE13 cells at 24 and 48 h,to determine the cell absorbance and calculate the cell survival rate,and the drug concentration of about 80% was the highest drug concentration in the cell proliferation experiment.3.CCK-8 cell proliferation assay: different concentrations of curcumin(0,4,8,12 and 16μM)on EC9706 and TE13 cells at different time points(0,24,48,72 and 96h),to investigate the inhinitory effect of curcumin on proliferation of ESCC cell lines.4.Soft-agar colony formation assay: different concentrations of curcumin(0,4,8,12 and 16μM)on EC9706 cell to evaluated the clony forming ability.5.Cell cycle and apoptosis experiments: Stained with Annexin V-FITC,Hoechst 33342 and DAPI,we evaluated the effect of different concentrations of curcumin(0,4,8,12 and 16μM)on cell cycle and apoptosis of EC9706 and TE13 cell lines.6.Western blotting was used to detect the expression of STAT3 signaling pathway with the treatment of curcumin on EC9706 and TE13 cell lines.Similarly,the effect of curcumin on STAT3 signaling pathway induced by IL-6 on EC9706 and TE13 cell lines was also detectd by this method.Results1.The results showed that after si RNA-1878 and si RNA-1272 transfected EC9706 cells,and the number of apoptotic cells and S phase cells increased significantly compared with the control group.2.Curcumin(0,25,50 and 100μM)were applied to EC9706 and TE13 cells for 24 and 48 h respectively,the results showed that the cell survival rate of 80% was about 20μM.3.The results of cell proliferation showed that curcumin could significantly inhibit the proliferation of EC9706 and TE13 cells for 0,24,48,72 and 96 h after treated with various concentrations of curcumin(0,4,8,12 and 16μM),concentration-dependently.4.The results of soft-agar clony formation showed that curcumin could suppress the clony numbers of EC9706 cells,concentration-dependently.5.Apoptosis and cycle results showed that curcumin could induce the apoptosis of EC9706 and TE13 cells,and the higher the concentration,the more obvious the induction effect.Curcumin could block the progress of cell cycle and increase the number of S phase cells,concentration-dependently.6.Western blotting showed that curcumin could inhibit the expression of p-STAT3 in EC9706 and TE13 cells,and also inhibited the expression of i NOS and p-NF-κB.IL-6 can enhance the expression of p-STAT3 in EC9706 and TE13 cells,and curcumin also can inhibit the activation of p-STAT3 induced by IL-6.PartⅡ: Curcumin inhibited the growth of ESCC patient derived exnograft(PDX)by down-regulating the activity of STAT3Methods1.The expression of p-STAT3 in esophageal squamous cell carcinoma was detected by Western blotting.2.EG37(high expression p-STAT3)and EG2(low expression of p-STAT3)were selected to construct PDX model.The model was used to evaluate the anti-cancer and chemoprevention of curcumin.3.After the end of treatment,the expression of STAT3 and its regulated-proteins in tumor tissues was detected by Western blotting.4.The expression of STAT3 signaling pathway and the expression of apoptotic protein Caspase-3 were detected by immunohistochemistry.5.The cell differentiation and necrosis were observed by HE staining.6.TUNEL method was to observe the apoptosis of tissue cells after the curcumin treatment.Results1.In the EG37 and EG2 PDX models,curcumin inhibited tumor growth compared to the control group,and the inhibitory effect was more pronounced in thechemoprevention group than the treatment group,and both models the anti-cancer effect of curcumin was more significant in the STAT3 high activation EG37 group.2.Western blotting showed that the expression levels of p-STAT3,i NOS,p-NF-κB and Cox2 were decreased in curcumin-treated and prophylactic groups.3.Immunohistochemistry indicated that the expression levels of p-STAT3 and Cox2 were decreased,the levels of Caspase-3 was upregulated in curcumin-treated and prophylactic groups.4.TUNEL results showed that the apoptosis cells increased significantly in curcumin-treated and prophylactic group.Conclusion1.The results in vitro have shown that curcumin could block the constitutive activation of STAT3 in esophageal squamous cell carcinoma and inhibit the activation of STAT3 induced by IL-6,down-regulate the expression of STAT3-regulated genes p-NF-κB and i NOS,and suppressed the proliferation,clony formation,induction of apoptosis and blockage of cell cycle of ESCC cell lines.2.The results in ESCC patient-derived xenograft(PDX)vivo indicated that that curcumin could inhibit the growth of tumor by inhibiting the constitutive activation of STAT3 and could promote cell apoptosis.3.In vivo and in vitro experiments showed that curcumin may be a chemopreventive agent for inflammation-induced esophageal squamous cell carcinoma.