Study on mechanism of action of Tongdao Kaijie pill in regulating biological behavior of esophageal cancer cells through Wnt/β-catenin signaling pathway
Objective To explore the effects of Tongdao Kaijie pill on the biological behavior of esophageal cancer(EC)cells and its regulatory role with Wnt/β-catenin signaling pathway.Methods From June 2022 to July 2023,SD rats were randomly grouped into low,medium,and high-dose groups and normal group.The low,medium,and high-dose groups were given 1.05 g/(kg·d),2.1 g/(kg·d),and 4.2 g/(kg·d)Tongdao Kaijie pill by gavage,and se-rum was collected and stored for future use.The human esophageal cancer cell line Eca109 was conventionally cultured and randomly separated into a Control group(10％control group serum treatment),a low concentration traditional Chi-nese medicine group(ZY-L group,10％low dose medicated serum),a medium concentration traditional Chinese medicine group(ZY-M group,10％medium dose medicated serum)High concentration traditional Chinese medicine group(ZY-H group,10％high-dose medicated serum)and high concentration traditional Chinese medicine+W nt activator SB216763 group(ZY-H+SB216763 group,10％high-dose medicated serum+20 μmol/L SB216763).CCK-8 method was applied to detect cell proliferation ability.Scratch experiments were applied to detect cell migration ability.Transwell experiment was applied to detect cell invasion ability.Flow cytometry was applied to detect cell apop-tosis rate.Real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)method was applied to measure the expression levels of Wnt3a and β-catenin mRNA in cells.Western Blot method was applied to detect the expression of Wnt3a,β-catenin,and apoptosis related proteins in cells.BALB/C nude mice were divided into model group,low[1.58 g/(kg·d)],high-dose Chinese medicine group[3.16 g/(kg·d)]and high-dose Chinese medicine+sb216763 group[3.16 g/(kg·d)Chinese medicine+2 mg/kg SB216763],with 12 mice in each group.The nude mouse model of esophageal cancer was established,and the tumor growth of nude mice was observed and recorded,and the tumor volume was compared.Results Compared with the control group,the invasion number of Eca109 cells[(119.49±13.20),(85.66±9.38)vs(148.26±16.07)]in the medium and high concentration traditional Chinese medicine groups was decreased P＜0.05).Compared with the high concentration traditional Chinese medicine group,the invasion number of Eca109 cells[(128.06±13.25)vs(85.66±9.38)]in the high concentration traditional Chi-nese medicine+SB216763 group was increased P＜0.05).Compared with the control group,optical density(OD)450(0.40±0.04,0.22±0.02 vs 0.58±0.06,0.71±0.07,0.53±0.05 vs 0.97±0.10)in medium and high con-centration traditional Chinese medicine groups at 48 h and 72 h was decreased P＜0.05).Compared with the high con-centration traditional Chinese medicine group,the OD of the high concentration traditional Chinese medicine+SB216763 group was increased at 48 h and 72 h(0.43±0.04 vs 0.22±0.02,0.76±0.08 vs 0.53±0.05)P＜0.05).Compared with the control group,the scratch healing rate[(33.65±3.88)％,(14.49±1.83)％vs(57.24±5.36)％]in the medium concentration traditional Chinese medicine group and the high concentration tradi-tional Chinese medicine group were decreased P＜0.05).Compared with the high concentration traditional Chinese medicine group,the scratch healing rate[(37.50±3.40)％vs(14.49±1.83)％]in the high concentration tradi-tional Chinese medicine+SB216763 group was higher P＜0.05).Compared with control group,the concentration of Chinese traditional medicine group,the high concentration of Chinese traditional medicine group apoptosis rate[(19.56±2.13)％,(37.49±4.02)％vs(6.50±0.80)％]were increased(P＜0.05);Compared with the high concentration traditional Chinese medicine group,the apoptosis rate of the high concentration traditional Chinese medicine+SB216763 group[(21.33±2.08)％vs(37.49±4.02)％]was decreased P＜0.05).Compared with the control group,the mRNA(0.64±0.06,0.31±0.03 vs 1.00±0.00,0.70±0.07,0.36±0.04 vs 1.00±0.00)and protein 0.54±0.05,0.26±0.03 vs 0.89±0.09,0.67±0.07,0.34±0.03 vs 0.95±0.10)expressions of Wnt3a,3-catenin and Bcl-2 protein(0.58±0.06,0.28±0.03 vs 0.83±0.08)in medium concentration Chinese medicine group and high concentration Chinese medicine group were decreased,and the protein expression of Bax(0.56±0.06,0.78±0.08 vs 0.27±0.03)was increased P＜0.05).Compared with the high concentration Chinese medicine group,the mRNA(0.57±0.06 vs.0.31±0.03,0.65±0.07 vs 0.36±0.04)and protein(0.50±0.05 vs 0.26±0.03,0.62±0.06 vs 0.34±0.03)expressions of Wnt3a,3-catenin and Bel-2 protein(0.55±0.06 vs 0.28±0.03)in the high concentration Chinese medicine+SB216763 group were increased,and the expression of Bax protein(0.61±0.06 vs 0.78±0.08)was decreased P＜0.05).Compared with model group,the tumor volume[(603.27±85.41)mm2 to(862.46±134.22)mm]in high-dose traditional Chinese medicine group was signifi-cantly decreased(P＜0.05).Compared with the high-dose traditional Chinese medicine group,the tumor volume[(362.44±54.66)mm2 vs(603.27±85.41)mm]in the high-dose traditional Chinese medicine+SB216763 group was significantly increased(P＜0.05).Conclusion Tongdao Kaijie pill may inhibit the malignant biological be-havior of EC cells by down-regulating the Wnt/β-catenin signaling pathway.
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