Babao Dan Reverses Multiple-Drug Resistance in Gastric Cancer Cells via Triggering Apoptosis and Autophagy and Inhibiting PI3K/AKT/mTOR Signaling

Abstract

Multidrug resistance (MDR) is a critical reason for cancer chemotherapy failure. Babaodan (BBD) is a famous traditional Chinese patent medicine reported to have antigastric cancer activity. However, the roles and molecular mechanisms of the reversal of MDR of gastric cancer by BBD have not been well described until now. Therefore, the purpose of this study was to elucidate further the role of BBD in reversing the MDR of gastric cancer cells and its specific regulatory mechanism via in vitro experiments. To verify our results, MTT, Doxorubicin (DOX) staining, Rhodamin123 (Rho123) staining, DAPI staining, Annexin V-FITC, propidium iodide (PI), Cyto-ID, and western blot assays were performed. To determine whether BBD triggers apoptosis and autophagy through the PI3K/AKT/mTOR signaling, we also applied 3-methyladenine (3-MA), chloroquine (CQ), and 740Y-P (an activator of PI3K). The results showed that BBD reversed the MDR and induced apoptosis and autophagy of SGC7901/DDP cells. Pathway analyses suggested BBD inhibits PI3K/AKT/mTOR pathway activity and subsequent apoptosis-autophagy induction. Inhibition of autophagy with 3-MA and chloroquine (CQ) was performed to confirm that BBD promoted autophagy. PI3K agonist, 740Y-P, further verified BBD inhibition of PI3K/AKT/mTOR pathway activation. In conclusion, BBD may reverse the MDR of gastric cancer cells, induce apoptosis, and promote autophagy via inactivation of the PI3K/AKT/mTOR signaling pathway.

Figure 1

BBD increased doxorubicin and Rho123 accumulation and downregulated the protein expression of ABCB1, ABCC1, and ABCG2 in gastric cancer-resistant cells. SGC7901/DDP cells were treated with BBD (0.25, 0.5, or 1.0 mg/mL) for 48 h, doxorubicin and Rho123 staining were observed via fluorescence microscope ((a) and (c)), and the average fluorescence intensity of all the cells was calculated in five random fields ((b) and (d)). Expression and quantitative analyses of ABCB1, ABCC1, and ABCG2 protein are shown ((e) and (f)). ^∗P < 0.05 and ^∗∗P < 0.01.

Figure 2

BBD induces apoptosis of SGC7901/DDP cells. SGC7901/DDP cells were treated with BBD (0.25, 0.5, and 1.0 mg/mL) for 48 h, and fluorescence microscopy was used to observe DAPI nuclear staining (a), and the average fluorescence intensity of all nuclei was calculated in five random fields (b). Annexin V/PI staining was used in flow cytometry to assess the proapoptotic effects of BBD (c). Data from apoptotic cell quantification (d). Cleaved caspase-3, caspase-3, Bcl-2, and Bax expression levels were detected by western blot (e). The quantification of each protein is shown (f). ^∗P < 0.05, ^∗∗P < 0.01 versus controls.

Figure 3

BBD promotes autophagy in SGC-7901/DDP cells. Fluorescence microscopy and flow cytometry were performed to detect autophagic activity via Cyto-ID staining of SGC-7901/DDP cells ((a) and (c)). The average fluorescence intensity of cells undergoing autophagy is shown in (b) and (d). LC3I, LC3II, p-62, and beclin 1 expression levels were detected by western blot (e). The quantification of each protein is shown in (f). ^∗P < 0.05 and ^∗∗P < 0.01.

Figure 4

3-MA and CQ were used to further confirm the role of autophagy in SGC-7901/DDP cells. Expression and quantitative analysis of LC3I, LCII, and LC3II/I, and protein expression in SGC701/DDP cells treated with 3-MA (10 mM) ((a) and (b)). Expression and quantitative analysis of LC3I, LC3II, and p-62 protein in SGC701/DDP cells treated with CQ (5 μM) ((c) and (d)). ^∗P < 0.05 and ^∗∗P < 0.01 versus controls.

Figure 5

BBD promotes apoptosis and autophagy via blocking the PI3K/AKT/mTOR pathway in SGC-7901/DDP cells. Expression of PI3K, p-PI3K, AKT, p-AKT, mTOR, and p-mTOR proteins (a). Quantitative analysis of PI3K, p-PI3K, AKT, p-AKT, mTOR, and p-mTOR (b). ^∗∗P < 0.01 versus controls.

Figure 6

740Y-P significantly increased PI3K/AKT/mTOR activity. Expression of PI3K/AKT/mTOR pathway proteins in SGC701/DDP cells treated with BBD or 740Y-P (a). Quantitative analysis of protein expression (b). ^∗P < 0.05, ^∗∗P < 0.01 versus controls.

Figure 7

740Y-P was used to further verify that BBD triggers apoptosis and autophagy by inhibiting PI3K/AKT/mTOR pathway. Bcl-2, Bax, LC3II/I, p-62, and Beclin 1 expression levels were detected by western blot (a). Quantitative analysis of protein expression (b). ^∗P < 0.05, ^∗∗P < 0.01 versus controls.