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

doi:10.3760/cma.j.cn371439-20240422-00092

Abstract

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.

Key words: Colorectal neoplasms, Capecitabine, Oxaliplatin, Marsdenia tenacissima, Disulfide apoptosis

Wei Wei, Cai Zhaoying, Qian Yayun. Effect of Marsdenia tenacissima combined with XELOX solution on disulfide apoptosis in human colorectal cancer HCT116 cells[J]. Journal of International Oncology, 2024, 51(9): 545-555.

Figures/Tables 6

References 31

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药物	细胞活性	F值	P值	药物	细胞活性	F值	P值
卡培他滨（μg/ml）				加入BAY-876后的卡培他滨（μg/ml）
0	100.00±0.00	644.60	<0.001	0	100.00±0.00	1 066.00	<0.001
0.3	86.49±1.08			0.3	87.65±0.64
0.6	77.91±1.08			0.6	76.53±0.91
1.2	75.59±1.64			1.2	73.93±1.33
2.4	64.38±1.96			2.4	64.96±1.50
4.8	47.19±0.58			4.8	46.48±0.70
奥沙利铂（μg/ml）				加入BAY-876后的奥沙利铂（μg/ml）
0	100.00±0.00	417.30	<0.001	0	100.00±0.00	847.70	<0.001
10	75.79±3.20			10	74.03±2.04
20	67.57±2.16			20	65.43±1.12
40	54.36±0.90			40	54.34±0.92
80	51.06±0.89			80	51.38±0.85
160	48.33±0.24			160	47.38±1.07
通关藤（mg/ml）				加入BAY-876后的通关藤（mg/ml）
0	100.00±0.00	107.50	<0.001	0	100.00±0.00	619.70	<0.001
15	108.00±1.98			15	107.50±0.86
30	116.10±4.34			30	121.50±5.18
60	124.10±5.35			60	128.50±1.06
120	75.56±1.59			120	77.90±3.87
240	21.71±1.73			240	20.77±1.23
葡萄糖抑制剂BAY-876（μg/ml）
0	100.00±0.00	1 028.00	<0.001
4	64.99±0.93
8	60.64±1.04
16	58.95±0.90
32	55.62±1.46
64	47.39±1.01

组别	葡萄糖浓度	加入BAY-876后的葡萄糖浓度	t值	P值
阴性对照组	19.91±0.13	11.44±0.10	86.50	<0.001
卡培他滨组	22.82±0.88^a	11.73±0.72	16.90	<0.001
奥沙利铂组	11.87±0.14^ab	8.98±0.40^ab	11.83	<0.001
通关藤组	17.93±0.14^abc	14.25±0.33^abc	17.79	<0.001
XELOX方案组	10.53±0.10^abcd	6.77±1.50^abcd	4.35	0.012
通关藤联合XELOX方案组	7.56±0.08^abcde	1.56±0.17^abcde	54.34	<0.001
F值	762.60	118.80
P值	<0.001	<0.001

组别	NADPH水平	加入葡萄糖抑制剂BAY-876后NADPH水平	t值	P值
阴性对照组	131.80±2.61	92.33±0.23	26.11	<0.001
卡培他滨组	93.87±1.00^a	88.63±0.31^a	8.62	<0.001
奥沙利铂组	136.50±3.69^b	97.33±2.02^ab	16.13	<0.001
通关藤组	105.70±0.84^abc	81.77±1.33^abc	26.38	<0.001
XELOX方案组	146.90±2.94^abcd	102.80±1.61^abcd	22.78	<0.001
通关藤联合XELOX方案组	105.00±2.25^abce	85.13±0.45^abcde	14.97	<0.001
F值	225.60	125.50
P值	<0.001	<0.001

组别	胱氨酸荧光强度	加入葡萄糖抑制剂BAY-876后胱氨酸荧光强度	t值	P值
阴性对照组	607.30±8.76	929.60±6.88	50.09	<0.001
卡培他滨组	655.70±6.57^a	1 049.00±22.35^a	29.26	<0.001
奥沙利铂组	647.10±19.35^a	1 021.00±29.49^a	18.34	<0.001
通关藤组	737.80±6.34^abc	1 094.00±16.17^ac	35.53	<0.001
XELOX方案组	756.00±8.65^abc	1 137.00±10.08^abc	49.66	<0.001
通关藤联合XELOX方案组	846.60±11.70^abcde	1 230.00±46.57^abcde	13.83	<0.001
F值	188.00	48.32
P值	<0.001	<0.001

组别	半胱氨酸含量	加入葡萄糖抑制剂BAY-876后半胱氨酸含量	t值	P值
阴性对照组	457.00±30.69	100.30±16.57	17.71	<0.001
卡培他滨组	581.20±30.69^a	472.90±19.10^a	5.19	0.006
奥沙利铂组	326.40±5.49^ab	262.70±28.65^ab	3.78	0.019
通关藤组	374.20±5.54^ab	348.70±9.55^abc	4.00	0.016
XELOX方案组	565.30±5.54^acd	533.40±11.03^abcd	4.47	0.011
通关藤联合XELOX方案组	246.80±30.69^abcde	30.23±5.49^abcde	12.03	<0.001
F值	110.00	423.50
P值	<0.001	<0.001