Effect of Marsdenia tenacissima combined with XELOX solution on disulfide apoptosis in human colorectal cancer HCT116 cells
10.3760/cma.j.cn371439-20240422-00092
- VernacularTitle: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
- Author:
Wei WEI
1
;
Zhaoying CAI
;
Yayun QIAN
Author Information
1. 扬州大学附属江都人民医院普外科，扬州 225200
- Keywords:
Colorectal neoplasms;
Capecitabine;
Oxaliplatin;
Marsdenia tenacissima;
Disulfide apoptosis
- From:
Journal of International Oncology
2024;51(9):545-555
- CountryChina
- Language:Chinese
-
Abstract:
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, 4.8 μg/ml), oxaliplatin (0, 10, 20, 40, 80, 160 μg/ml), Marsdenia tenacissima (0, 15, 30, 60, 120, 240 mg/ml), and the glucose inhibitor BAY-876 (0, 4, 8, 16, 32, 64 μg/ml), respectively. Furthermore, the HCT116 cells were pre-treated with 25 μg/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 (4.0 μg/ml), oxaliplatin group (90 μg/ml), Marsdenia tenacissima group (140 mg/ml), XELOX solution group (4.0 μg/ml capecitabine+90 μg/ml oxaliplatin), and Marsdenia tenacissima combined with XELOX solution group (140 mg/ml Marsdenia tenacissima+4.0 μg/ml capecitabine+90 μg/ml oxaliplatin). BAY-876 treatment groups refer to the groups which the glucose inhibitor BAY-876 25 μg/ml was added to each of the above groups. The cell proliferation was assessed using the MTT assay. Apoptosis was determined through Annexin Ⅴ-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, 4.8 μg/ml concentration of capecitabine, 0, 10, 20, 40, 80, 160 μg/ml concentration of oxaliplatin, 0, 4, 8, 16, 32, 64 μg/ml concentration of BAY-876, and after pre-treatment with 25 μg/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=1 028.00, P<0.001; F=1 066.00, 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 25 μg/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). Annexin Ⅴ-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, 1 049.00±22.35, 1 021.00±29.49, 1 094.00±16.17, 1 137.00±10.08, and 1 230.00±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.
