Research On Mechanisms Underlying The Anti-cancer Activities Of Triazolyl-napthyl Derivative Of ?-amyrin In Nasopharyngeal Carcinoma

PART 1 The pro-apoptotic Effects of Triazolyl-napthyl Derivative of ?-amyrin on Nasopharyngeal Carcinoma Cell Line HK-1Objective:In this study,the triazolyl-napthyl derivative of ?-amyrin(TNB)was designed and synthesized by click chemical method.The apoptotic effects of TNB on nasopharyngeal carcinoma cells were evaluated,and possible molecular mechanisms of TNB inhibiting HK-1 proliferation and inducing apoptosis in human nasopharyngeal carcinoma cells.Methods:The primary product was obtained by adding the cesium carbonate(600mg)and the acetylene propyl bromide(675mg)to four hydrogen furan solution containing ?-(1500mg).The crude product was further purified by column chromatography,and a pure compound(?-ethyl derivatives compound 2300mg)was obtained.The reaction mixture is stirred at room temperature 3 hours later to the tert-butanol-Water(tert-butanol)of the compound(2300mg):Water = 2:1,lOmL)add ascorbic acid sodium(3.5mg)and Cupric sulfate(1.5mg)in the solution until the thin layer chromatography(TLC)Monitoring of the reaction is completed.Adding Naphthalene azide(twice times the amount of ?-)to the mixture,diluted with water and extracted with ethyl acetate after the complete reaction of the OC.The organic phase was subjected to drying,filtration,concentration after the calculation of yield.TNB(0,3,6,12,24,50,100 ?m)were used to treat HK-1 nasopharyngeal carcinoma cells for 24 and 48 hours respectively,and MTT assay was used to determine the inhibitory effect of TNB on cells in vitro.The CCK-8 method was used to support the TNB inhibition of HK-1 cell viability in MTT assay.Subsequently,the HK-1 cells were treated with 0,12,48 and 100 ?m concentrations of TNB after 48 hours,and Hoechst 33258 staining was used to observe the cell morphology of TNB in human nasopharyngeal carcinoma cells which were disturbed by HK-1 under fluorescence microscope.To extract the intracellular DNA,The DNA fragmentation was observed under UV-gel imaging system after the separation and color of ethyl bromide.The effect of TNB on the distribution of HK-1 cell cycle was analyzed by using flow cytometry.And the Annexin V-FITC,The HK-1 cells of nasopharyngeal carcinoma cells were treated with different concentrations of TNB(0,12,48,100 ?m)after 48 hours of apoptosis by iodide(PI)double staining.Detection of ROS in HK-1 cells after TNB treated by DCF fluorescence intensity RT-QPCR and Western blot methods were used to detect the effects of different concentrations(0 ?m,48 u m,100 ?m)TNB on mRNA and protein expression in HK-1 cells,including Cyclin Cyclin,D1 and P21,Apoptosis-related protein Caspase3,Caspase 7,Caspase 8,Caspase 9,Bax,anti-apoptotic molecule Mcl-1,PARP and AIF and cytochrome c.Finally,the expression of ?literal,AKT and Xiap was detected by real PCR and Western-blot method to evaluate whether TNB could regulate apoptosis by affecting PI3K/AKT/XIAP signaling pathway.Results:By using Cu(?)-catalyzed 1,3-coupling cascade reaction,the water-soluble gene could be introduced into ?-alcohol ester,which greatly improved its water solubility.The 3-bit hydroxyl group of ?-was converted to acetylene propyl ether,then the three-key of acetylene was transformed into triazole ring by clicking Chemical Reaction,the yield of coarse product was 38%.The cytotoxic effect of TNB on nasopharyngeal carcinoma cell HK-1 was obvious,and the inhibitory effect was time-dependent and concentration dependent.Hoechst 33258 staining was used to observe the morphological changes of HK-1 in nasopharyngeal carcinoma cells treated by TNB.Under the microscope,the number of cells was significantly reduced after treated with TNB.Cell morphology has changed significantly.Cells showed nuclear fragmentation and nuclear contraction,and cell was rounded,and chromatin condensation to the periphery of the nucleus membrane appeared.Thses apoptotic phenomena become more and more obvious with the increase of TNB concentration.The gel electrophoresis results showed that DNA fragments were not found in untreated cells,but the DNA cleavage bands were observed after 48 hours of TNB treated with concentrations of 12 ?m,48 ?m,and 100?m,as TNB concentrations changed.When the concentration increased to 48 and 100 u m,the DNA cleavage bands appeared clearly,indicating that the cells had apoptosis and were in the late stage of apoptosis.DNA cleavage is another important sign of cell apoptosis,which further confirms that the TNB mixture is the death of nasopharyngeal carcinoma cells by means of cell apoptosis.TNB induces the production of Ros in HK-1 cells,and the concentration and time dependence of TNB were increased after 24h and 48h of different concentrations of TNB were HK-1 cells.In the TNB low concentration(3uM),the difference of Ros was obviously induced by the relative blank control group,the higher the concentration time,the more significant the effect was.So,TNB can induce the increase of ROS level in cells,Ros,a biologically active oxygen molecule produced by intracellular oxygen metabolism or exogenous oxidant,plays an important role in the initiation and regulation of cell apoptosis,so it is clear that Ros participates in and promotes the apoptosis of TNB-induced HK-1 cells.The results of flow cytometry showed that TNB resulted in cell stagnation in the HK-1 cell line of nasopharyngeal carcinoma cells and increased the value of apoptosis by dose-dependent method.In the same time period,the fragments of $literal cells were dose-dependent.The proportion of cells in the S and $literal periods was largely unaffected.Annexin V-FITC and Iodide(PI)double staining showed that the proportion of apoptotic cells was 5.6%in HK-I of untreated nasopharyngeal cancer cells,and the ratio of TNB cells in 12?m HK-1 Group was 29.3%;48?m The proportion of apoptotic cells in TNB treated HK-1 cells was 37.1%,the concentration was 100?m TNB treated HK-1 cells and the proportion was 45.2%.Through the above data,we can see that the number of apoptotic cells increases with increasing concentration.The PCR results showed that the expression of cyclin D1 mRNA was significantly lower than that in the control group,while the expression of P21 and P53 mRNA was significantly higher than that in the control group.Western blotting test results showed that the expression level of cyclin D1 protein was significantly decreased after TNB treatment,but the expression level of P21 and P53 protein increased obviously,and the expression level of Bax protein in apoptosis was obviously increased,However,the expression of Mcl-1 protein in anti apoptotic molecule decreased significantly.In addition,RT-PCR showed that the mRNA expression of CASPASE3,Caspase 7 and Caspase 9 increased significantly after 48 hours of treatment with TNB(0?m,48 ?m,100?m),and with the increase of TNB concentration,The expression of caspase protein increased significantly.When the concentration of TNB was 100?m,the mRNA expression level of caspase-3,caspase-7,and Caspase-9 was almost 4 times times that of the control group,and only the expression of Caspase8 was not affected by TNB,and even the expression was decreased slightly after 48 ?m treatment.However,no significant changes were expressed in the expression of TNB concentration.Compared with the normal control group,the expression of CASPASE3 and Parp,AIF and cytochrome c in the cells was significantly increased after different concentrations of TNB intervention,while the expression of intact Caspase-3 and parp cells decreased significantly.With the increase of TNB concentration,this change is more obvious.The results showed that TNB could promote apoptosis of mitochondria apoptotic signaling pathway activation.At the transcriptional level,TNB can promote mRNA expression in caspase-3,caspase-7 and caspase-9,and in the level of protein expression TNB can be a concentration-dependent promoter of Cleavage-parp,CLEAVAGE-CASPASE3,The expression of AIF and CYTC increased.Real-Time PCR results showed that the relative mRNA expression of PI3K,AKT and XIAP was downregulated when treated the cells with TNB.It is indicated that TNB can reduce the expression of PI3K/AKT/XIAP signaling pathway.Western-blot results showed that the expression of HK-1 signaling pathway protein was significantly downregulated after TNB treatment.Conclusion:The results showed that the TNB products synthesized by clicking Chemical improved the stability and solubility of the five-ring triterpene saponins,which increased the bioavailability of the drugs.We found that TNB inhibited the proliferation of HK-1 nasopharyngeal carcinoma cells by inducing apoptosis and cell stagnation in the G1 stage and by lowering the PI3K/AKT/XIAP signaling pathway.PART 2 In vivo study verifying the superiority of TNB combined radiation-chemotherapy treatments for nasopharyngeal carcinomaObjective:The advantages of TNB combined radiotherapy-chemotherapy were evaluated by comparison of TNB method with radiotherapy and chemotherapy in nude mice.Methods:The nude mice were divided into blank group,simple chemotherapy group(cis-3mg/kg),radiotherapy group,simple TNB group(50mg/kg),chemotherapy+TNB group,radiotherapy+TNB group and chemotherapy+TNB group.Subcutaneous transplantation of HK-1 tumor cells,the effects of different treatment methods on the growth of tumor in nude mice were recorded by the corresponding treatment in nude mice,and the effect of different treatments on the tumors in vivo was detected.Immunohistochemical detection of Bax and Ki67 expression in transplanted tumor tissues of nude mice;in situ end labeling(TUNEL)To detect the apoptosis of tumor cells.All data comes from individual trials.The experimental data were analyzed by means of average plus minus standard difference ?x±s.Using SPSS18.0 software for statistical processing.Results:In vivo experiments,after the transplanted tumor body,compared with the blank control group,there was no significant change in nude mice body weight,and there was no difference between the experimental groups.The results showed that after 4 weeks of growth and treatment,the average tumor weight was 1 in the blank control group.After radiotherapy and chemotherapy and TNB treatment,the experimental groups showed the inhibition of tumor body,and compared with the blank control group has statistical significance.In particular,the results showed that TNB therapy had the effect of radiotherapy and chemotherapy,and TNB combined with radiotherapy and chemotherapy had the best effect on tumor inhibition,which was better than that of a single treatment group or other combined treatment group(p<0.05).The expression of Bax in the experimental treatment group was significantly higher than that in the blank group.The expression of Ki67 was significantly lowered,the expression of Bax in TNB combined with radiotherapy and TNB group was higher than that in individual treatment group,while the expression of Bax in TNB combined chemotherapy group was the highest and the expression of Ki67 was the lowest,which was significantly different from other groups(p<0.05).The results of Tunel test showed that the degree of apoptosis in TNB combined with chemotherapy group was higher than that in other groups,which was statistically different from other groups(p<0.05).The apoptosis rate of TNB combined with chemotherapy and TNB radiotherapy group was higher than that in simple group,and the difference was statistically significant(p<0.05).The results indicated that all the experimental treatment groups could induce tumor cell apoptosis in different degrees,but the TNB combined with radiotherapy and chemotherapy group had the highest apoptosis rate,the treatment effect was the best,the TNB had sensitization effect on radiotherapy and chemotherapy,and TNB combined with radiotherapy could better inhibit the proliferation of tumor cells.Conclusion:In vivo experiments showed that TNB could promote the expression of Bax protein and lowered Ki67 expression,which inhibited the growth of transplanted tumor in HK-1 nude mice.At the same time,TNB can also improve the sensitivity of nasopharyngeal carcinoma transplanted tumor to radiotherapy.Compared with the individual TNB treatment group,TNB combined with radiotherapy was the best for tumor suppressor in nude mice.This may be related to changes in cell cycle distribution,inducing apoptosis,inhibiting cell proliferation and other related mechanisms.