通关藤联合XELOX方案促进人结直肠癌HCT116细胞双硫死亡的作用

Effect of Marsdenia tenacissima combined with XELOX solution on disulfide apoptosis in human colorectal cancer HCT116 cells

目的探讨通关藤联合XELOX方案对人结直肠癌HCT116细胞双硫死亡的作用及相关机制。方法体外培养结直肠癌HCT116细胞,分别用0、0.3、0.6、1.2、2.4、4.8μg/ml浓度的卡培他滨,0、10、20、40、80、160μg/ml浓度的奥沙利铂,0、15、30、60、120、240 mg/ml浓度的通关藤,0、4、8、16、32、64μg/ml浓度的葡萄糖抑制剂BAY-876处理HCT116细胞;以及经25μg/ml BAY-876预处理后再经以上各药物、各浓度处理HCT116细胞。将HCT116细胞分为阴性对照组(不做任何处理)、卡培他滨组(4.0μg/ml)、奥沙利铂组(90μg/ml)、通关藤组(140 mg/ml)、XELOX方案组(4.0μg/ml卡培他滨+90μg/ml奥沙利铂)、通关藤联合XELOX方案组(140 mg/ml通关藤+4.0μg/ml卡培他滨+90μg/ml奥沙利铂)。BAY-876处理组为以上各组别中,每组加入葡萄糖抑制剂BAY-87625μg/ml。采用MTT法检测细胞增殖情况,AnnexinⅤ-FITC/PI双染法检测细胞凋亡情况,邻甲苯胺法检测葡萄糖浓度,NADPH比色法检测NADPH水平,胱氨酸摄取荧光法检测胱氨酸荧光强度,半胱氨酸比色法检测半胱氨酸含量。结果经0、0.3、0.6、1.2、2.4、4.8μg/ml浓度卡培他滨,0、10、20、40、80、160μg/ml浓度奥沙利铂,0、4、8、16、32、64μg/ml浓度BAY-876,以及经25μg/ml葡萄糖抑制剂BAY-876预处理后再经上述各药物、各浓度处理HCT116细胞后,细胞存活率差异均有统计学意义(F=644.60,P<0.001;F=417.30,P<0.001;F=1028.00,P<0.001;F=1066.00,P<0.001;F=847.70,P<0.001),且随着各药物浓度增加,HCT116细胞活性均逐渐下降(均P<0.05);经0、15、30、60、120、240 mg/ml浓度通关藤,以及经25μg/ml葡萄糖抑制剂BAY-876预处理后再经上述浓度药物处理HCT116细胞后,细胞存活率差异均具有统计学意义(F=107.50,P<0.001;F=619.70,P<0.001),且随着药物浓度增加,HCT116细胞活性呈现先上升后下降的趋势(均P<0.05)。AnnexinⅤ-FITC/PI双染法结果显示,阴性对照组绿色、红色、黄色荧光强度均较弱,各用药组绿色荧光与红色荧光的表达增强,其中卡培他滨组红色荧光、黄色荧光占比较大;奥沙利铂组及通关藤组绿色荧光占比较小;XELOX方案组的绿色荧光和黄色荧光占比多于卡培他滨组及奥沙利铂组;通关藤联合XELOX方案组绿色荧光、红色荧光占比最大。经葡萄糖抑制剂BAY-876预处理后,各组细胞中绿色、黄色荧光均明显增多,其中,卡培他滨组和奥沙利铂组的绿色荧光、黄色荧光明显增多;XELOX方案组的3种荧光明显多于卡培他滨组和奥沙利铂组;通关藤联合XELOX方案组,3种荧光占比最大。阴性对照组、卡培他滨组、奥沙利铂组、通关藤组、XELOX方案组、通关藤联合XELOX方案组以及经葡萄糖抑制剂BAY-876处理HCT116细胞后以上各组的葡萄糖浓度分别为(19.91±0.13)、(22.82±0.88)、(11.87±0.14)、(17.93±0.14)、(10.53±0.10)、(7.56±0.08)μg/ml,(11.44±0.10)、(11.73±0.72)、(8.98±0.40)、(14.25±0.33)、(6.77±1.50)、(1.56±0.17)μg/ml,差异均有统计学意义(F=762.60,P<0.001;F=118.80,P<0.001);与无处理的各组相比,经葡萄糖抑制剂BAY-876处理HCT116细胞后的细胞内葡萄糖浓度均显著降低(t=86.50,P<0.001;t=16.90,P<0.001;t=11.83,P<0.001;t=17.79,P<0.001;t=4.35,P=0.012;t=54.34,P<0.001)。各组NADPH水平分别为(131.80±2.61)、(93.87±1.00)、(136.50±3.69)、(105.70±0.84)、(146.90±2.94)、(105.00±2.25)nmol/gProt,(92.33±0.23)、(88.63±0.31)、(97.33±2.02)、(81.77±1.33)、(102.80±1.61)、(85.13±0.45)nmol/gProt,差异均有统计学意义(F=225.60,P<0.001;F=125.50,P<0.001);与无处理的各组相比,经葡萄糖抑制剂BAY-876处理HCT116细胞后的细胞内NADPH水平均显著降低(t=26.11,P<0.001;t=8.62,P<0.001;t=16.13,P<0.001;t=26.38,P<0.001;t=22.78,P<0.001;t=14.97,P<0.001)。各组胱氨酸荧光强度分别为607.30±8.76、655.70±6.57、647.10±19.35、737.80±6.34、756.00±8.65、846.60±11.70,929.60±6.88、1049.00±22.35、1021.00±29.49、1094.00±16.17、1137.00±10.08、1230.00±46.57,差异均有统计学意义(F=188.00,P<0.001;F=48.32,P<0.001);与无处理的各组相比,经葡萄糖抑制剂BAY-876处理HCT116细胞后的细胞内胱氨酸荧光强度均显著升高(t=50.09,P<0.001;t=29.26,P<0.001;t=18.34,P<0.001;t=35.53,P<0.001;t=49.66,P<0.001;t=13.83,P<0.001)。各组半胱氨酸含量分别为(457.00±30.69)、(581.20±30.69)、(326.40±5.49)、(374.20±5.54)、(565.30±5.54)、(246.80±30.69)μmol/L,(100.30±16.57)、(472.90±19.10)、(262.70±28.65)、(348.70±9.55)、(533.40±11.03)、(30.23±5.49)μmol/L,差异均有统计学意义(F=110.00,P<0.001;F=423.50,P<0.001);与无处理的各组相比,经葡萄糖抑制剂BAY-876处理HCT116细胞后的细胞内半胱氨酸含量均显著降低(t=17.71,P<0.001;t=5.19,P=0.006;t=3.78,P=0.019;t=4.00,P=0.016;t=4.47,P=0.011;t=12.03,P<0.001)。结论通关藤联合XELOX方案可通过双硫死亡程序促进人结直肠癌HCT116细胞的死亡。

Objective To investigate the effect and related mechanism of Marsdenia tenacissima combined with XELOX solution on disulfide apoptosis in human colorectal cancer HCT116 cells.Methods The human colorectal cancer HCT116 cells were cultured in vitro and treated with different concentrations of capecitabine(0,0.3,0.6,1.2,2.4,4.8μg/ml),oxaliplatin(0,10,20,40,80,160μg/ml),Marsdenia tenacissima(0,15,30,60,120,240 mg/ml),and the glucose inhibitor BAY-876(0,4,8,16,32,64μg/ml),respectively.Furthermore,the HCT116 cells were pre-treated with 25μg/ml of BAY-876,followed by exposure to the specified concentrations of each drug group.HCT116 cells were divided into the following groups:negative control group(no treatment),capecitabine group(4.0μg/ml),oxaliplatin group(90μg/ml),Marsdenia tenacissima group(140 mg/ml),XELOX solution group(4.0μg/ml capecitabine+90μg/ml oxaliplatin),and Marsdenia tenacissima combined with XELOX solution group(140 mg/ml Marsdenia tenacissima+4.0μg/ml capecitabine+90μg/ml oxaliplatin).BAY-876 treatment groups refer to the groups which the glucose inhibitor BAY-87625μg/ml was added to each of the above groups.The cell proliferation was assessed using the MTT assay.Apoptosis was determined through AnnexinⅤ-FITC/PI double staining.The concentration of glucose was quantified using the o-toluidine method.NADPH levels were measured by colorimetry.Cystine uptake fluorescence assay was utilized to quantify the fluorescence intensity of cystine,and cysteine content was determined using cysteine colorimetry.Results After 0,0.3,0.6,1.2,2.4,4.8μg/ml concentration of capecitabine,0,10,20,40,80,160μg/ml concentration of oxaliplatin,0,4,8,16,32,64μg/ml concentration of BAY-876,and after pre-treatment with 25μg/ml glucose inhibitor BAY-876,HCT116 cells were treated with the above drugs and concentrations,there were statistically significant differences in cell survival rate(F=644.60,P<0.001;F=417.30,P<0.001;F=1028.00,P<0.001;F=1066.00,P<0.001;F=847.70,P<0.001),and with the increase of each drug concentration,the activity of HCT116 cells decreased gradually(all P<0.05).After 0,15,30,60,120,240 mg/ml concentration of Marsdenia tenacissima,and after pre-treatment with 25μg/ml glucose inhibitor BAY-876,HCT116 cells were treated with the above drugs,there were statistically significant differences in cell survival rate(F=107.50,P<0.001;F=619.70,P<0.001),and with the increase of drug concentration,the activity of HCT116 cells increased first and then decreased(all P<0.05).AnnexinⅤ-FITC/PI staining showed that the intensity of green,red and yellow fluorescence was weaker in the negative control group.The expression of green fluorescence and red fluorescence was enhanced in each drug group.In capecitabine group,the red fluorescence and yellow fluorescence were larger.The proportion of green fluorescence in oxaliplatin group and Marsdenia tenacissima group was smaller.The proportion of green fluorescence and yellow fluorescence in XELOX solution group was higher than that in capecitabine group and oxaliplatin group.Marsdenia tenacissima combined with XELOX solution group had the highest proportion of green and red fluorescence.After pre-treatment with the glucose inhibitor BAY-876,the green and yellow fluorescence of the cells in each group increased significantly.The green and yellow fluorescence of capecitabine group and oxaliplatin group increased significantly.The fluorescence of XELOX solution group was significantly higher than that of capecitabine group and oxaliplatin group.In Marsdenia tenacissima combined with XELOX solution group,the proportion of fluorescence expressing three colors was the largest.In the negative control group,capecitabine group,oxaliplatin group,Marsdenia tenacissima group,XELOX solution group,Marsdenia tenacissima combined with XELOX solution group,and the groups after BAY-876 pre-treatment,the glucose concentration of HCT116 was(19.91±0.13),(22.82±0.88),(11.87±0.14),(17.93±0.14),(10.53±0.10),(7.56±0.08),(11.44±0.10),(11.73±0.72),(8.98±0.40),(14.25±0.33),(6.77±1.50),and(1.56±0.17)μg/ml,respectively,with statistically significant differences(F=762.60,P<0.001;F=118.80,P<0.001).Compared with the untreated groups,the intracellular glucose concentration of HCT116 cells treated with BAY-876 was significantly decreased(t=86.50,P<0.001;t=16.90,P<0.001;t=11.83,P<0.001;t=17.79,P<0.001;t=4.35,P=0.012;t=54.34,P<0.001).NADPH levels in each group were(131.80±2.61),(93.87±1.00),(136.50±3.69),(105.70±0.84),(146.90±2.94),(105.00±2.25),(92.33±0.23),(88.63±0.31),(97.33±2.02),(81.77±1.33),(102.80±1.61),and(85.13±0.45)nmol/gProt,respectively,with statistically significant differences(F=225.60,P<0.001;F=125.50,P<0.001);Compared with the untreated groups,the intracellular NADPH levels of HCT116 cells treated with BAY-876 was significantly decreased(t=26.11,P<0.001;t=8.62,P<0.001;t=16.13,P<0.001;t=26.38,P<0.001;t=22.78,P<0.001;t=14.97,P<0.001).The fluorescence intensity of cystine in each group was 607.30±8.76,655.70±6.57,647.10±19.35,737.80±6.34,756.00±8.65,846.60±11.70,929.60±6.88,1049.00±22.35,1021.00±29.49,1094.00±16.17,1137.00±10.08,and 1230.00±46.57,respectively,with statistically significant differences(F=188.00,P<0.001;F=48.32,P<0.001).Compared with the untreated groups,the intracellular fluorescence intensity of cystine of HCT116 cells was significantly increased after treatment with BAY-876(t=50.09,P<0.001;t=29.26,P<0.001;t=18.34,P<0.001;t=35.53,P<0.001;t=49.66,P<0.001;t=13.83,P<0.001).The contents of cysteine in each group were(457.00±30.69),(581.20±30.69),(326.40±5.49),(374.20±5.54),(565.30±5.54),(246.80±30.69),(100.30±16.57),(472.90±19.10),(262.70±28.65),(348.70±9.55),(533.40±11.03),(30.23±5.49)μmol/L,respectively,with statistically significant differences(F=110.00,P<0.001;F=423.50,P<0.001).Compared with the untreated groups,the intracellular contents of cysteine of HCT116 cells treated with BAY-876 was significantly decreased(t=17.71,P<0.001;t=5.19,P=0.006;t=3.78,P=0.019;t=4.00,P=0.016;t=4.47,P=0.011;t=12.03,P<0.001).Conclusion Marsdenia tenacissima combined with XELOX solution can promote HCT116 cell death through disulfide apoptosis.