| Background and ObjectiveCell apoptosis plays a vital role in the development of tumors.The specific apoptosis inducers can induce the apoptosis of cancer cells and reduce toxic and side effects on the body,making it an important means of cancer treatment.In recent years,many studies have reported that both hedysarum polybotys saccharides(HPS)and selenium can induce the apoptosis and inhibit the proliferation of cancer cells.However,the effect of HPS treatment in oral cancer cells is still unknown.Selenization of HPS forms a novel organic selenium compound,i.e.,selenizated hedysarum polybotys saccharides(SE-HPS),which has the synergistic biological activity of selenium and polysaccharide.However,the effect of SE-HPS treatment on the oral cancer cell remains to be further studied.Therefore,in this study,we aimed to investigate and compare the effect of HPS and SE-HPS on the survival and apoptosis of oral squamous cancer cell.Moreover,the molecular mechanism of SE-HPS on the proliferation and apoptosis of oral squamous cancer cells was further investigated by proteomics to provide the scientific basis for clinical application of traditional chinese medicine.Material and Methods1.Oral squamous cancer cell lines CAL-27,SCC-9,and healthy epithelial cell HOK were cultured in vitro with different concentrations(0,10,25,50,100,200,400μg/mL)of HPS and SE-HPS for different times(0,12,24,36,48 hours).The cell viability was determined by CCK-8 assay and the optimum concentrations of action were screened out.Then CAL-27 or SCC-9 cells were incubated at the best concentration of HPS or SE-HPS for 24 and 48 hours.Their viability was compared by CCK-8 assay.2.CAL-27 and SCC-9 cells were cultured in vitro with different concentrations(0,10,25,50,100,200,400 μg/mL)of HPS and SE-HPS for 48 hours.The apoptotic rates of cancer cells were determined by flow cytometry and the optimum concentrations of action were screened out.Then CAL-27 or SCC-9 cells were incubated at the best concentration of HPS or SE-HPS for 48 hours.Their apoptotic rates were compared by flow cytometry.3.CAL-27 and SCC-9 cells were cultured in vitro with different concentrations(0,25,50 μg/mL)of HPS and SE-HPS for 48 hours.The morphology of apoptotic cells was observed by immunofluorescence staining.4.CAL-27 and SCC-9 cells were cultured in vitro with different concentrations(0,25,50 μg/mL)of HPS and SE-HPS for 48 hours.The RT-qPCR technique was applied to detected the mRNA expression of Bax/Bcl-2,P53,NF-κB,and Fas/Fasl genes.The protein levels of Fas/Fasl were detected through Western blot technique.5.CAL-27 cells were cultured in vitro with different concentrations(0,25,50 μg/mL)of SE-HPS for 48 hours.The protein levels of Caspase 3,8,9 were detected through Western blot technique.After adding the specific inhibitor of Fas/Fasl(KP7-6),the apoptotic rates of CAL-27 cells were detected by flow cytometry and the protein levels of Caspase 3,8,9 were detected through Western blot technique.6.CAL-27 cells were cultured with 25 μg/mL SE-HPS in vitro for 48h.The control group was without SE-HPS treatment.The differential expression of signaling proteins was detected by iTRAQ technology to explore the possible signaling proteins involved in the apoptosis.Bioinformatics method was used to analyze the protein classification,molecular function,biological process,subcellular localization and protein-protein interaction network to determine the key target proteins.RT-qPCR and Western blot were used to validate the expression of some target proteins.Results1.Different concentrations of HPS and SE-HPS significantly inhibited the proliferation of CAL-27 and SCC-9 cells(P<0.05)with a time-dependent manner but not in a concentration-dependent manner.The optimum concentration of CAL-27 cell was 25 μg/mL,and the optimum concentration of SCC-9 cell was 50 μg/mL.The cell viability of SE-HPS group was significantly lower than that of HPS group(P<0.05).2.There was no significant difference in cell activity of HOK cells between HPS and SE-HPS treated group and control group(P>0.05).3.Flow cytometry analysis showed the higher apoptotic rates in CAL-27 and SCC-9 cells cultured with different concentrations of HPS and SE-HPS compared to the control group(P<0.05).The optimum concentration of CAL-27 cell was 25 μg/mL,and the optimum concentration of SCC-9 cell was 50 μg/mL.The apoptosis rates of SE-HPS treated group were higher than that of HPS treated group(P<0.05).4.Fluorescence microscopy showed more apoptotic bodies in the HPS and SE-HPS treated cancer cells compared to the untreated cells.5.HPS and SE-HPS upregulated the mRNA and protein expression of Fas/Fasl in CAL-27 and SCC-9 cells(P<0.05),but the mRNA expression of Bax/Bcl-2,P53 and NF-μB was not affected by HPS and SE-HPS(P>0.05).6.After treated with SE-HPS,the protein levels of Caspase 3,8 and 9 in CAL-27 cells were higher than those of the control group(P<0.05).Blocking Fas/Fasl by KP6-7 downregulated the apoptotic rates and the protein levels of Caspase 3,8 and 9 in CAL-27 cells(P<0.05).7.SE-HPS treatment differentially expressed 131 signaling proteins in CAL-27.Among differentially expressed signaling proteins,66 were upregulated,and 65 were downregulated(P<0.05).CTSB、CTSC、CTSD and other proteins were related to Fas/Fasl apoptosis signal pathway.ASAH1,COX7B,CMC2 and other proteins were related to mitochondrial apoptosis pathway.AQP3,NCK1,BRMS1 and other proteins were associated with tumor proliferation and invasion.The results of RT-PCR and Western blot showed that the gene and protein expression of BRMS1 and DCBLD2 in SE-HPS treatment group were higher than that in control group,and the gene and protein expression of AQP3 and NCK1 were lower than that in control group(P<0.05).Conclusion1.Both HPS and SE-HPS enhanced apoptosis and inhibited the survival of the oral squamous cancer cells.The effect of SE-HPS was better than that of HPS.2.The apoptosis effect of HPS and SE-HPS on oral squamous cancer cells may be possibly via activation of Fas/Fasl pathway.3.SE-HPS treatment could affect the protein expression profile of oral squamous cancer cells CAL-27,suggesting that SE-HPS could inhibit proliferation and induce apoptosis of oral squamous cancer cells by up-regulating or down-regulating the expression of cell growth,apoptosis,energy metabolism,cytoskeleton,ion binding and signal transduction related proteins. |
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