Inhibitory Effect And Mechanism Of Alginate Oligosaccharide On Growth And Proliferation Of Prostate Cancer Cells

Prostate cancer is an epithelial malignancy that occurs in the prostate.It has the highest incidence among malignant tumors in adult males in the United States and Europe,and is one of the most important causes of cancer death in men in developed countries.With the gradual improvement of people’s living conditions,the prolonged life expectancy and the great changes in the diet structure,the incidence and mortality of prostate cancer have gradually increased in China.The main treatment methods for prostate cancer are: follow-up without treatment,total prostatectomy,radiotherapy and endocrine therapy.However,these treatments could not inhibit tumor metastasis and are highly toxic to the patient’s normal tissues.Therefore,finding an effective drug for the treatment of prostate cancer is a top priority.In addition,despite significant advances in surgery,chemotherapy,and drug therapy,the incidence is still increasing due to cancer recurrence and metastasis.Therefore,the molecular mechanisms that elucidate the progression of prostate cancer may be helpful in the diagnosis and treatment of research.Alginate oligosaccharide(AOS)is a marine plant oligosaccharide obtained by enzymatic hydrolysis of sodium alginate,which is composed of two structural units:guluronic acid and mannuronic acid.The currently known effects are anti-inflammatory,anti-viral,anti-fungal,anti-tumor effects,in addition to lowering blood lipids,enhancing the body’s antioxidant function and immune regulation.In recent years,a large number of studies have shown that chitosan oligosaccharides could enhance the body’s immunity and inhibit various tumors and cancer cells.However,there are few reports of AOS on anti-tumor.Although AOS has been shown to induce apoptosis in osteosarcoma cells,it has not been reported whether AOS plays a role in the growth and proliferation of prostate cancer cells.Sialic acid is a derivative of nine-carbon monosaccharide and plays an important role in cell recognition,adhesion,and signal transduction.According to the way of sialic acid and sugar residues,it could be divided into three major categories: α2,3-SA,α2,6-SA and α2,8-SA.In particular,changes in the expression of α2,6 and α2,3 linked sialyltransferases are closely related to tumorigenesis.The sialylation modification shields the glycoprotein and glycolipid structure that are associated with it,stabilizes the cell membrane structure,improves thermal stability,and protects cells and macromolecules from attacking by enzymes and the immune system,thereby protecting cells.Clinically,poor prognosis in cancer patients is often associated with abnormal sialylation.Sialic acid is widely distributed in the body,mediating or modulating many physiological and pathological processes such as development,inflammation,pathogen infection,tumor development,etc.,and is closely related to human health and disease.Compared with normal tissues,it was found that the structure of sugar chains on tumor tissues and tumor cells changed significantly.Among them,the significant upregulation of the characteristic sialic acid expression is associated with the occurrence,development,invasion,metastasis and poor prognosis of malignant tumors.The sialyltransferase could be further subdivided into four classes based on substrate specificity and catalytic synthesis to form new glycosidic bond types: ST3 Gal I~VI;ST6Gal I 、 II;ST6GalNAc I~Ⅵ and ST8 Sia I~VI.α2,6 sialylation which is mediated by ST6Gal-1 is especially important in cancer progression.A large number of reports have reported that high expression of ST6Gal-1 is found in colon cancer,liver cancer,breast cancer,prostate cancer and cervical cancer.ST6Gal-1,which was discovered in the previous group,plays an important role in the growth,migration and invasion of prostate cancer DU145 and PC3 cells.In addition,AOS was found to upregulate the expression of α2,6-sialic acid on the surface of prostate cancer cells.Thus,we speculate whether AOS affects prostate cancer cells by affecting the expression of ST6Gal-1 and causing changes in sialic acid.Objective: To investigate the inhibitory effect of AOS on the growth and proliferation of prostate cancer DU145 and PC3 cells;To elucidate the specific molecular mechanism of AOS inhibiting the growth and proliferation of prostate cancer cells by affecting the expression of ST6Gal-1.Method: The effects of AOS treatment on the malignant phenotype of prostate cancer cells(DU145 and PC3)were detected by CCK8,cell colony,scratch test andTranswell;The apoptosis of prostate cancer cells induced by AOS was detected by AV/PI apoptosis assay;The effect of AOS on the cycle of DU145 and PC3 was detected by flow cytometry;The expression of ST6Gal-1 in prostate cancer cells before and after AOS treatment was detected by qPCR and Western-blot;The results that the expression of apoptosis-related proteins and Hippo signaling pathway-related proteins in prostate cancer cells after AOS treatment were detected by Western-blot method.The effect of AOS on the transcriptional activity of ST6Gal-1 promoter and the identification of core functional regions were detected by construction of ST6Gal-1 promoter truncated reporter gene vector and luciferase reporter assay;Bioinformatics predicted the transcription factor that bound to the ST6Gal-1 promoter region;The transcription factor interacting with the core region of the ST6Gal-1 promoter was further validated by Chromatin immunoprecipitation(CHIP);The protein interaction between the transcription factor c-Jun that was interacted with ST6Gal-1 promoter and YAP,the Hippo/YAP signaling pathway effector,was verified by Co-immunoprecipitation(CoIP)and immunofluorescence colocalization;The effect of AOS on the growth and proliferation of prostate cancer cells and its molecular mechanism were analyzed in nude mice.Result: After AOS treatment,the growth and proliferation and the migration rate and invasion ability of prostate cancer DU145 and PC3 cells were decreased;The apoptosis of DU145 and PC3 cells was detected by flow cytometry and the apoptosis rate in the treatment group was higher than the untreated group;The results showed that AOS arrested DU145 and PC3 cells in S phase by flow cytometry;The expression of ST6Gal-1 in AOS-treated prostate cancer cell line was significantly lower than that in the control group.The expression levels of apoptotic protein Bax,cleaved-caspase 9 and cleaved-PARP in the treated group were significantly higher than those in the control group by Western blot,while the expression level of Bcl-2 was decreased compared with the control.The results showed that AOS could activate Hippo signaling pathway by Western blot,and the expressions of related proteins P-YAP,P-MOB1,MOB1,LATS1,MST1,MST2 and SAV1 were increased,while the expression of YAP,an oncoprotein,was decreased compared with the control;The dual luciferase reporter gene assay showed that AOS inhibited the activity of ST6Gal-1 promoter,and the core functional region was a truncated fragment from-308 bp to +1bp in the promoter region;The PROMO database predicts that the transcription factor binding to the ST6Gal-1promoter region is c-Jun which regulates tumor cell proliferation,differentiation,apoptosis and tumorigenesis;The results that c-Jun could bind to the-308bp~+1bp region upstream of ST6Gal-1 gene and AOS could reduce the binding of transcription factor c-Jun to promoter ST6Gal-1 were further confirmed by Chromatin immunoprecipitation(CHIP);co-immunoprecipitation(Co-IP)and immunofluorescence co-localization results showed that the transcription factor c-Jun interacted with the transcriptional coactivator YAP;The results of in vivo experiments showed that the tumor-forming ability of mice in the AOS-treated group decreased and the tumor volume growing in vivo was small and affected the expression of Hippo signaling pathway proteins.Conclusion:1.Alginate oligosaccharide(AOS)could inhibit the growth,proliferation,migration and invasion of prostate cancer cells in a concentration-dependent manner.2.AOS could inhibit the transcription level of ST6Gal-1 gene and inhibit the expression of α2,6-sialic acid in prostate cancer cells.3.AOS could inhibit the regulation of ST6Gal-1 gene promoter by transcription factor c-Jun and transcriptional coactivator YAP,thereby inhibiting the growth and proliferation of prostate cancer cells.