Babaodan overcomes cisplatin resistance in cholangiocarcinoma via inhibiting YAP1

Abstract

Context: Cholangiocarcinoma with highly heterogeneous, aggressive, and multidrug resistance has a poor prognosis. Although babaodan (BBD) combined with cisplatin improved non-small cell lung cancer efficacy, its impact on overcoming resistance in cholangiocarcinoma remains unexplored.

Objective: This study explored the role and mechanism of BBD on cisplatin resistance in cholangiocarcinoma cells (CCAs).

Materials and methods: Cisplatin-resistant CCAs were exposed to varying concentrations of cisplatin (25-400 μg/mL) or BBD (0.25-1.00 mg/mL) for 48 h. IC₅₀ values, inhibition ratios, apoptosis levels, DNA damage, glutathione (GSH) levels, oxidized forms of GSH, total GSH content, and glutaminase relative activity were evaluated using the cell counting kit 8, flow cytometry, comet assay, and relevant assay kits.

Results: BBD-reduced the cisplatin IC₅₀ in CCAs from 118.8 to 61.83 μg/mL, leading to increased inhibition rate, apoptosis, and DNA damage, and decreased expression of B-cell lymphoma-2, p-Yes-associated protein 1/Yes-associated protein 1, solute carrier family 1 member 5, activating transcription factor 4, and ERCC excision repair 1 in a dose-dependent manner with maximum reductions of 78.97%, 51.98%, 54.03%, 56.59%, and 63.22%, respectively; bcl2-associated X and gamma histone levels were increased by 0.43-115.77% and 22.15-53.39%. The impact of YAP1 knockdown on cisplatin-resistant CCAs resembled BBD. GSH, oxidized GSH species, total GSH content, and glutaminase activity in cisplatin-resistant CCAs with BBD treatment also decreased, while YAP1 overexpression countered BBD's effects.

Discussion and conclusion: This study provides a scientific basis for BBD clinical application and provides a new direction for BBD biological mechanism research.

Keywords: Apoptosis; DNA damage; glutathione; yes-associated protein 1.

Figure 1.

Babaodan (BBD) antagonized cisplatin resistance in cholangiocarcinoma cells (CCAs) by advancing the inhibition ratio, apoptosis, and inhibiting glutathione (GSH) synthesis. (a) The inhibition ratio curve and the IC₅₀ of cisplatin in CCAs were measured using the cell counting kit 8 (n = 5). (b) according to the inhibition of cisplatin and cisplatin with BBD, 1 mg/mL of BBD was chosen for the follow-up study (n = 5). (c) the inhibition ratio curve and the IC₅₀ of cisplatin with 1 mg/mL BBD in CCAs were also measured using the cell counting kit 8 (n = 5), and the IC₅₀ was significantly reduced compared to cisplatin treatment alone. (d) The apoptosis of CCAs was detected by flow cytometry (n = 3). BBD increased apoptosis levels in a dose-dependent manner. (e) TTGSH, (f) relative activity of GLS, (g) GSH, and (h) GSH/GSSG levels in CCAs with cisplatin treatment were inhibited by BBD treatment in a dose-dependent manner. (mean ± standard deviation) *p < 0.05, **p < 0.01, vs. control group; ^#p < 0.05, ^##p < 0.01, vs. CDDP group.

Figure 2.

Babaodan (BBD) promoted the effect of cisplatin on DNA damage in cholangiocarcinoma cells (CCAs). (a) characteristic photos showed the DNA damage (×400, scale bar = 50 μm). The comet assay measured (b) the tail moment and (c) the percentage of tail DNA. The combination of cisplatin and BBD increased them compared to cisplatin alone (n = 3). (mean ± standard deviation) *p < 0.05, **p < 0.01, vs. control group; ^#p < 0.05, ^##p < 0.01, vs. CDDP group.

Figure 3.

Apoptosis, glutathione (GSH) synthesis, and DNA damage-related protein expression in cholangiocarcinoma cells (CCAs) with co-treatment of babaodan (BBD) and cisplatin. Western blot was used to measure protein levels (n = 3). BBD decreased (a) the bcl-2 level and (b) increased the bax level in a dose-dependent manner. Change in (c) cle-caspase-3/caspase-3 levels with BBD treatment was not statistically significant. In CCAs involving cisplatin, the expression levels of (d) p-YAP1 relative to total YAP1, (e) SLC1A5, and (f) ATF4, which are involved in GSH synthesis, were observed to decrease in a dose-dependent manner following BBD treatment. (g) the γH2Ax level was increased by 1 mg/mL of BBD treatment, and (h) the ERCC1 level was inhibited by treatment with 0.5 and 1 mg/mL of BBD treatment. Representative protein bands are shown in the (i), (j), and (k). (mean ± Standard deviation) *p < 0.05, **p < 0.01, vs. control group; ^#p < 0.05, ^##p < 0.01, vs. CDDP group.

Figure 4.

Babaodan (BBD) antagonized cisplatin resistance in cholangiocarcinoma cells (CCAs) by inhibiting YAP1. (a, b) The YAP1 mRNA of CCAs with siYAP1 and YAP1 cDNA transfection was measured using quantitative real-time PCR (n = 3). ^##p < 0.01 vs. siNC group; ^▲▲p < 0.01 vs. NC group. (c) The inhibition ratio was detected using the cell counting kit 8 (n = 5). (d) apoptosis was measured by flow cytometry (n = 3) and increased in CCAs with YAP1 knockdown. Additionally, YAP1 overexpression antagonized the inhibition effect of BBD on CCA proliferation with cisplatin treatment. (e) Representative images of flow cytometry are shown. The levels of (f) TTGSH, (g) relative activity of GLS, (h) GSH, and (i) GSH/GSSG in CCAs with YAP1 knockdown, and cisplatin/BBD co-treatment were decreased and were reversed by YAP1 overexpression (n = 5). (mean ± standard deviation) ⁺p < 0.05, ⁺⁺p < 0.01 vs. CDDP group; ^#p < 0.05, ^##p < 0.01, vs. CDDP + BBD group.

Figure 5.

YAP1 Overexpression antagonized the promotional effect of babaodan (BBD) on DNA damage in cholangiocarcinoma cells with cisplatin. (a) The characteristic photos showed the DNA damage (×400, scale bar = 50 μm). The comet assay was used to measure (b) tail moment and (c) percentage of tail DNA, and YAP1 overexpression inhibited (b) tail moment and (c) percentage of tail DNA in cholangiocarcinoma cells with co-treatment of cisplatin and BBD (n = 3). (mean ± standard deviation) ⁺p < 0.05, ⁺⁺p < 0.01, vs. CDDP group; ^#p < 0.05, ^##p < 0.01, vs. CDDP + BBD group.

Figure 6.

The extent of apoptosis, glutathione (GSH) synthesis, and the expression of DNA damage-related proteins were assessed in cholangiocarcinoma cells (CCAs) subjected to YAP1 knockdown or YAP1 overexpression. Western blot was used to measure protein levels (n = 3). With cisplatin incubation, YAP1 knockdown and 1 mg/mL babaodan (BBD) treatment decreased the (a) Bcl-2 level and (b) increased bax level, while the change in (c) cle-caspase-3/caspase-3 levels with BBD treatment was not statistically significant. In the CCAs dealing with cisplatin, the expression levels of (d) p-YAP1/YAP1, (e) ATF4, and (f) SLC1A5 were decreased by YAP1 knockdown and BBD treatment. Additionally, the (g) γH2Ax level was increased and (h) the ERCC1 level was inhibited by YAP1 knockdown and BBD treatment. YAP1 overexpression antagonized the effect of BBD on these proteins. Representative protein bands are shown in (i), (j), and (k). (mean ± standard deviation) ⁺p < 0.05, ⁺⁺p < 0.01, vs. CDDP group; ^#p < 0.05, ^##p < 0.01, vs. CDDP + BBD group.