Babao dan promotes apoptosis of cisplatin (DDP)-resistant gastric cancer cell line SGC-7901/DDP by inducing autophagy through PI3K/AKT/mTOR pathway modulation

Background: Multidrug resistance (MDR) is a critical reason of cancer chemotherapy failure. Babao dan (BBD) is a classical and famous traditional Chinese patent medicine, which has been reported to has anti-gastric 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. Methods: SGC-7901 and SGC-7901/DDP cells were used in this study, and drug resistance and evaluation of the reversal effect of BBD was determined using MTT assays in SGC7901/DDP cells. Doxorubicin (DOX) and Rhodamin123 (Rho123) staining was performed to assess BBD effects on drug accumulation and eux of drug-resistant gastric cancer cells. Cell apoptosis was directly assessed using DAPI staining. Apoptotic and dead cells were detected by ow cytometry after staining with Annexin V-FITC and propidium iodide (PI). Cyto-ID assays were performed to examine cellular autophagy. Changes in cell protein expression of ABCB1, ABCC1, ABCG2, Bax, Bcl-2, caspase-3, cleaved-caspase-3, LC3, p62, Beclin1 and the PI3K/AKT/mTOR pathway were detected by Western blot. Inhibition of autophagy with 3-MA, chloroquine (CQ) and PI3K antagonist (LY294002) or agonist (740Y-P) , uncovered a role for the potentially downregulated signaling pathway, PI3K/AKT/mTOR. Results: The SGC7901/DDP cell line exhibited multi-drug resistance to DDP, DOX and 5-uorouracil (5-FU) and the drug resistant index (RI) of DDP, DOX and 5-FU were 1.86, 1.50 and 47.70, respectively. BBD reversed the MDR of SGC7901/DDP cells by increasingDOX accumulation, reducing Rh123 eux and down-regulating the expression of ABCB1, ABCC1, ABCG2. Furthermore, BBD induced apoptosis in SGC7901/DDP cells through regulating caspase-3, cleaved-caspase-3, Bax and Bcl-2. Moreover, BBD induced autophagy in DDP-resistant gastric cancer cells via regulating p62, LC3 and Beclin1. Pathway analyses suggested BBD may inhibit PI3K/AKT/mTOR pathway activity and subsequent autophagy induction. Conclusions: BBD may reverse the MDR of gastric cancer cells, and promote autophagic death via inactivation of the PI3K/AKT/mTOR signaling pathway.


Background
Gastric cancer (GC) is the third most common cancer worldwide and is associated with high mortality [1,2] . Gastric cancer incidence and mortality is highest in East Asia, especially in China [3][4][5]. The occurrence of stomach cancer is multi-factorial, being in uenced by environmental bacteria, host responses, genetic and epigenetic molecular changes, Helicobacter pylori infection, among other factors [6,7]. The current primary treatment for gastric cancer is surgical resection; however, some advanced or relapsed GC patients are not candidates for surgical treatment, so chemotherapy serves as a lead alternative therapy, especially for advanced gastric cancer [3]. Although medical advances in gastric cancer have improved, the prognostic and overall survival rates of patients with gastric cancer remain poor due to their limited and complex response to chemotherapy [5]. Multidrug resistance is the main cause of low survival time, which leads to the failure of chemotherapy for tumors and limited e cacy in most patients with gastric cancer. Therefore, how to reverse multidrug resistance remains a major clinical challenge.
Multidrug resistance (MDR) involves different mechanisms that induce cancer cells to develop crossresistance to a variety of structurally and mechanically unrelated chemotherapy agents, thereby limiting the long-term effective use of chemotherapy drugs [8]. One mechanism mediating MDR is through the regulation of cancer cell apoptosis and autophagy by anticancer drugs [9]. Another important mechanism involves increased out ow of drugs from cancer cells by upregulated transport proteins that rely on speci c energy sources such as ABCB1, ABCC1, and ABCG2 [10]. Other mechanisms that may contribute to drug resistance include EMT, DNA damage repair, drug target mutations, and stem cell modi cation [11,12].
Babaodan (BBD) has a long medical history from palace secret recipe. It consists of Moschus, natural Calculus bovis, snake gall, pearl, antelope horn, radix notoginseng and other precious Chinese medicinal materials. Such components act through clearing away wetness-heat, promoting circulation, detoxi cation, and pain relief. Many clinical and experimental studies have demonstrated signi cant antitumor activity by BBD. In addition, these studies highlighted BBD's promising clinical e cacy during posttumor chemotherapy and use as an adjuvant therapy for various cancers, including GC [13][14][15][16].
Preliminary experimental results corresponding to clinical studies suggest BBD could signi cantly inhibit gastric cancer cell proliferation, metastasis, and induce their apoptosis [13,16]. However, the speci c regulatory mechanisms by which BBD reverse MDR have not been well studied. Therefore, the purpose of this study was to further elucidate the role of BBD in reversing gastric cancer cell MDR and its speci c regulatory mechanism through in vitro experiments. Furthermore, our study aimed to provide an experimental basis for the clinical treatment of patients with gastric cancer drug resistance.

Results
BBD reversed the MDR of the SGC-7901/DDP We utilized the Resistance Index (RI) to evaluate the resistance of MDR cells to various anticancer drugs.
The ability of BBD to reverse the resistance of SGC-7901/DDP cells to several chemotherapeutic drugs is shown in Table 2. The reversal effect was evaluated by reversal fold (RF). RF > 1 indicates enhanced drug sensitivity. BBD (0.5 mg/mL) enhanced the sensitivity of SGC-7901/DDP cells to DDP, ADM and 5-FU by 1.55-fold, 4.68-fold, and 3.56-fold, respectively. The results indicated that BBD signi cantly increased the cytotoxicity of anticancer drugs in MDR cells. (Table 2) BBD increased the accumulation of DOX and Rho123 in gastric cancer resistant cells.
The accumulation or e ux of chemotherapeutic drugs from tumor cells into the surrounding tissue in cells are important indicators for assessing MDR reversal. Our ndings showed that 0.25, 0.5 and 1.0 mg/mL BBD increased the accumulation values of DOX to 9.1, 15, and 17.2, over the levels in control cell (5.1), respectively (p < 0.05) ( Figure 1A and 1B) and increases Rho123 to 5.4, 14.7, 21.5, over control cells (2.1), respectively (p < 0.05) ( Figure 1C and 1D). ABC family protein accounts for the accumulation of drugs. Our Western blot results showed that treatment with BBD (0.25, 0.5 or 1.0 mg/mL), signi cantly decreased ABCB1, ABCC1 and ABCG2 expression compared to control (p < 0.05). BBD (0.25, 0.5 or 1.0 mg/mL) reduced the ABCB1, ABCC1 or ABCG2 to (65. 35 Figure 1E and 1F) BBD induced apoptosis of SGC7901/DDP cells Apoptotic inhibition is one of the most important factors that contribute to MDR. As such, apoptotic induction maybe a strategy to overcome the MDR. The pro-apoptotic activity of BBD in SGC7901/DDP cells was tested by assessing DAPI and Annexin-V/PI staining. DAPI staining showed that BBD can induce punctate apoptotic body formation, as shown in Figure 2A and 2B. In cells treated with varying concentrations of BBD (0.25, 0.5, 1.0 mg/mL), Annexin V/PI staining was assessed via ow cytometry. The ndings showed that the percentage of apoptotic cells (7.54 ± 0.84%), (14.49 ± 0.39%) and (14.64 ± 1.36%), respectively. Apoptosis was signi cantly elevated (P 0.05) compared to control (4.54 ± 0.24%), as shown in Figure 2C and 2D. We also found that BBD regulated the expression apoptosis-related proteins ( Figure 2E). The expression levels of Bax and cleaved-caspase-3 were increased by BBD treatment to 156.53% and 124.55 % (P 0.05). Bcl-2 and total caspase-3 levels were reduced to 49.56 % and 19.61% (P 0.05), compared to control cells ( Figure 2F). The above results suggest that BBD overcomes MDR in SGC-

7901/DDP cells via apoptotic induction.
BBD promoted autophagy in SGC-7901/DDP cells To explore whether autophagy contributed to cell death, we assessed BBD treatment effects on autophagy in SGC-7901/DDP cells. The cyto-ID staining results showed that BBD promoted autophagy in SGC-7901/DDP cells ( Figure 3A and 3B). Moreover, we assessed autophagy-related protein expression and found that BBD increases the protein levels of LC3-II and beclin-1, but decreased expression of p62 compared with control cells. (Figure 3E and 3F) We used 3-MA and CQ to further con rm the role of autophagy in SGC-7901/DDP cells. 3-MA (an early inhibitor of autophagy) of 10 mM blocked the formation of LC3-and inhibited apoptosis via regulating Bax and Bcl-2 expression ( Figure 4A and 4B), while CQ (a late inhibitor of autophagy) of 5μM upregulated LC3-and p62 ( Figure 4C and 4D). Based on these results, we concluded that BBD promotes SGC-7901/DDP cell apoptosis though inducing autophagy.
BBD promoted autophagy-induced apoptosis in SGC-7901/DDP cells by inhibiting the PI3K/AKT/mTOR pathway PI3K/AKT/mTOR signaling is a signi cant mechanism for the promotion of cancer biology. We examined whether BBD affects PI3K/AKT/mTOR signaling pathway as a mechanism for its effects on MDR of GC. Our results showed that SGC7901/DDP cells treated with BBD exhibited reduced PI3K, p-PI3K, p-AKT, AKT, and p-mTOR expression ( Figure 5). In addition, PI3K/AKT/mTOR signaling pathway has been shown to promote downregulation of autophagy. To determine whether BBD regulated autophagy-induced apoptosis through the PI3K/AKT/mTOR signaling, we applied LY294002 (an inhibitor of PI3K) and 740Y-P (an activator of PI3K) to the cells. As expected, LY294002 inhibited the PI3K/AKT/mTOR signaling and promoted autophagic apoptosis. Meanwhile, 740Y-P negatively affected autophagic apoptosis. These results con rmed that BBD reversed MDR of GC via PI3K/AKT/mTOR signaling ( Figure 6 and Figure 7).

Discussion
With recent rapid developments in Chinese economy, the continuous improvement of people's living standards, and changes in the environment and diet, the incidence of gastric cancer has increased annually, which is a serious threat to human health. As a result, gastric cancer has become an undeniable social health problem. Over decades of medical research and therapeutic development, several chemotherapeutic regimens based on different anti-cancer drugs have improved the survival of many GC patients. However, chemotherapy in GC patients often fails due to the cancer cells' development of multidrug resistance (MDR) [17]. Therefore, increased emphasis has recently been placed on screening high e ciency and low toxicity drugs that can reverse or diminish tumor resistance.
BBD, a traditional Chinese medicine formula, has the characteristics of a multi-component, multi-target therapy and is widely used in cancer treatment. Preliminary studies have reported that BBD has been widely used to treat patients with various cancers with noted prevention of complications [13,16]. Wang Q et al. found that BBD can inhibit cell growth by inducing autophagy through PI3K/AKT/mTOR signaling and enhancing the anti-tumor effect of cisplatin in non-small cell lung cancer (NSCLC) cells. Still, the regulatory mechanism of BBD in GC multidrug resistance has not been fully clari ed. In the present study, we demonstrate that BBD may reverse MDR by down-regulating PI3K/AKT/mTOR signaling to induce autophagic apoptosis in the human drug-resistant cell line SGC-7901/DDP.
It is well known that ABC transport protein-mediated MDR is one of the important causes of tumor chemotherapy failure [18,19]. Therefore, potential molecular targets or biomarkers must be sought to mitigate MDR. P-gp (MDR1/ABCB1) rely on ATP energy to pump hydrophobic drugs from cells, which reduces intracellular drug concentration and causes resistance in tumor cells [20]. The expression of ABCB1 protein in gastric cancer tissue with chemotherapy-resistant has been reported to be higher than in chemotherapy-sensitive cancer tissue [21]. MRP (ABCC1) can transfer a combination of GSH and related drugs out of cells, causing drug resistance [22]. Chen et al. found that ABCC1 increased the protein and gene expression of ABCC1 in a cancer cell line and induced in vitro gastric cancer cell line (SGC-7901/AS) resistant to arsenic trioxide [22][23][24]. ABCG2 is involved in mediating MDR by reducing drug penetration and intestinal absorption [25]. Accordingly, inhibiting ABCG2 can reduce MDR phenotypes and improve chemotherapy outcomes. Recent literature demonstrates that MDR1, MRP and P-gp protein levels in SGC-7901/DDP and SGC-7901/VCR cells is increased [26]. Therefore, MDR1, ABCC1 and ABCG2 expression were rst measured in SGC-7901/DDP cells. Our ndings indicate that 48h BBD treatment signi cantly lowers ABCB1, ABCC1, and ABCG2 expression in SGC-7901/DDP cells, which is consistent with previous reports.
Apoptosis is an important defense mechanism to eliminate malignant cells and prevent cancer progression. The primary function of many anti-tumor drugs is to induce tumor apoptosis through various apoptosis-related signaling pathways [27]. A modi ed apoptotic responses is a key basic mechanism leading to DDP resistance. One of the important mechanisms for the development of MDR in cancer cells in response to chemotherapy is to inhibit or evade apoptosis [28,29] . There are two main pathways tumor cells are stimulated to undergo apoptosis, the death receptor pathway (exogenous) and the mitochondrial dependent pathway (endogenous). The key regulatory factors of endogenous pathways are Bcl-2 family proteins, including anti-apoptotic factor Bcl-2 and pro-apoptotic factor Bax [30]. Over-expression of Bcl-2 and under-expression of Bax reduces cancer cell sensitivity to chemotherapy drugs avoid apoptosis. Liquiritin (LIQ) has been shown to inhibit drug-induced apoptosis by increasing the expression of Bax and reducing Bcl-2 in cisplatin (DDP)-resistant gastric cancer cells [31]. Of all the caspases, caspase-3 was the one most associated with several apoptotic pathways [32]. Therefore, BBD has been shown to inhibit drug-induced apoptosis in SGC7901/DDP by increasing the expression of Bax and cleaved caspase-3 while reducing Bcl-2 and total caspase-3.
Autophagy is another molecular mechanism that induces cell death. Consequently, autophagy plays a vital role in preventing tumor growth in drug discovery [33,34]. Extensive evidence suggests that increasing autophagy may help reverse MDR [35,36] . For example, Wei F found that LIQ can induce apoptosis and autophagy by the arresting the cell cycle at G0/G1 phase [31], and enhancing the proapoptotic effect of DDP on human gastric cancer SGC7901/DDP cells [31]. Wang Q found that Babaodan inhibited cell growth by inducing autophagy and enhanced the anti-tumor effect of cisplatin in NSCLC cells [37]. Recent studies have con rmed autophagy as a scavenger in the apoptosis-blocked signal pathway, making MDR tumors sensitive to apoptosis [38]. As shown in Figure 3A, uorescence analysis showed that BBD treatment severely disrupted cell morphology. Western blot analysis demonstrated that BBD treatment highly induced autophagy-related signals of LC3 I/II and Beclin 1, but suppressed p62, leading to autophagy in SGC7901/DDP cells ( Figure 3E and 3F) Activation of the PI3K/AKT/mTOR signaling pathway has been shown to have carcinogenic effects in gastric cancer, and its regulatory pathways are closely related to genetic variation, cell proliferation, migration, invasion, cell cycle, apoptosis, autophagy, angiogenesis, multi-drug resistance and cell viability [21,[39][40][41][42][43][44][45][46]. Guo et al. found that Ubenimex inhibited the phosphorylation and activation of the PI3K/AKT/mTOR pathway and down-regulated membrane transporter (such as p-gp and MRP1) expression, resulting in the intracellular accumulation of 5-uorouracil and oxaliplatin [47]. Chen et al. reported that proton pump inhibitors inhibited the V-ATPases/PI3K/AKT/mTOR/HIF-1 alpha signaling pathways and downregulated expression of TSC1/2 in gastric cancer cells and Rheb to reverse multi-drug resistance [48]. In addition, Wang Q found Babaodan induced autophagy and inhibited cell growth through PI3K/AKT/mTOR pathway and enhances anti-tumor effects of cisplatin in NSCLC cells [37]. The PI3K/AKT/mTOR signaling pathway was investigated to further understand the biological function of BBD in GC cells. Surprisingly, we found that several phosphorylated products in this pathway, such as p-PI3K, p-AKT, and p-mTOR were reduced by BBD ( Figures 5). We further explored the role of PI3K/AKT/mTOR pathway in BBD modulation of apoptosis and autophagy. Our results indicate that PI3K inhibition blocked the phoaphorylation and activity of PI3K, AKT, and mTOR, as well as reduced the expression of Bcl-2, p62 and increased Bax, LC31/LC311, and Beclin1. Meanwhile 740Y-P-mediated activation of PI3K had a catalytic effect on p-PI3K , p-AKT, p-mTOR, and increased Bcl-2, and p62 expression and reduced pro-apoptotic and Bax, autophagy-associated LC31/LC311, and Beclin1, as shown in Figures 6 and 7. These results showed that BBD can reverse MDR in gastric cancer cells via inactivating of PI3K/AKT/mTOR signaling to induce apoptosis and autophagy.

Conclusions
In conclusion, the results of the present study indicate that BBD may be a promising candidate for the treatment of MDR in gastric cancer. However, as our study was performed only in vitro, additional studies are needed in animal models or humans to further con rm these therapeutic effects of BBD in gastric cancer. Doxorubicin and Rho123 staining SGC7901/DDP cells were seeded at a density of 4.0×10 5 in 6-well plates and cultured overnight. When the cells reached 50%~60%, different concentrations of BBD (0, 0.25, 0.5, 1.0 mg/mL) were added and incubated for 48 h. Next, the supernatant was discarded and 1 mL PBS was added to wash the cells 3 times. We used 4% paraformaldehyde to x the cells for 10 min, then washed the cells with PBS 3 times. Doxorubicin (5 μM) or Rho123 (5 μM) staining solution was added and incubated with the cells for 15 min, and 1 mL PBS was used to wash 3 times. The samples then were observed and photographed with an inverted uorescence microscope (400×).

DAPI staining
After cell intervention, 4% paraformaldehyde was added to x the cells for 15 min. 10% DAPI staining solution (in PBS) was added to each well and cultured in the darkness for 15 mins. The cells were then washed by PBS 3 times and imaged with a uorescence microscope (400×).

Apoptosis assays
The apoptotic rate was determined by ow cytometry. The AnnexinV-uorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit (Nanjing KeyGene Biotech Co., Ltd. Nanjing, China) were used. SGC-7901/DDP cells in logarithmic growth phase were plated in six-well plates at the density of 4.0×10 5 cells per well and treated with different concentrations of BBD (0, 0.25, 0.5, 1.0 mg/mL) for 48 h, 1×10 6 cells were collected and washed twice with cold PBS. Cells were resuspended in 500 µl 1×binding buffer and then incubated for 15 min at room temperature in the dark following 5 µl Annexin V/FITC and 5 µl PI additions. For each analysis, 10,000 events were recorded.
Cyto-ID autophagy detection SGC-7901/DDP cells were treated with different concentrations of BBD for 48h, the cells were centrifuged, then the 1×10 6 cells were resuspended in 0.5 ml of freshly diluted Cyto-ID green detection reagent (1μL Cyto-ID green detection reagent mixed with RPMI-1640 medium to a nal volumn of 2 mL). After incubation for 30 min at 37℃ in the dark, the cells were determined by ow cytometry.

Western blot analysis
Protein expression of Bax, Bcl-2, caspase-3, cleaved-caspase-3, GAPDH, LC3, p62, Beclin1, PI3K, AKT, p-AKT mTOR and p-mTOR were examined by western blot analysis. After treated with BBD as above. . Cellular proteins of SGC-7901/DDP cells were lysed by radioimmunoassay (RIPA) buffer with inhibitor cocktail (Thermo Fisher Scienti c, USA), and centrifuged at 14,000 rpm for 20 min at 4℃. The nal supernatants were harvested. Protein concentrations were determined by BCA protein assay kit (bovine serum albumin). Total protein (30 µg) was electrophoresed on 10% SDS-PAGE and then transferred to a PVDF membrane. Following incubation with 5% skimmed milk for 1 h at room temperature, Primary antibodies (1:1000) were diluted with TBST solution and incubated with the membranes overnight at 4˚C. Washed 3 times (TBST, 10 min), then incubated with secondary antibodies (HRP-conjugated 1:5000) for 1 h. Finally, we used Image Lab (Bio-Rad Laboratories, Inc., Berkley, California, USA) to detect protein. Three independent experiments were performed in order to get the representative data.

Statistical analysis
All representative data were obtained from three independent experiments. Statistical analyses were performed by IBM SPSS Statistics 21. P values < 0.05 were considered to be statistically signi cant.

Declarations Consent for publication
All authors reviewed and approved the nal manuscript. All authors supported publication of this manuscript.

Availability of data and materials
The data used to support the ndings of this study are original and availability from the corresponding author upon request.

Table1
Experimental results of sensitivity to chemotherapeutic drugs in two groups of cells  Reversal Fold (RF) = alone chemotherapy IC50 / combination chemotherapy IC50