3.1 Screening of compounds to inhibit growth of gastric cancer AGS cells
Gastric cancer is a worldwide common disease which is in desperate need of new
therapeutic approaches. Autophagy has been reported to play a vital role in the tumorigenesis and development of gastric cancer [16]. It is not only a survival mechanism, but also participating in the process of cell death [17, 18] Targeting autophagy has been proposed to be a promising adjuvant therapy for gastric cancers.
CCK-8 assay was firstly applied to assess the growth inhibitory effects of 10 µM compound from the Topscience Preclinical Compound Library (Catalog No.L3410, 450 compounds) against gastric cancer AGS cells. We found that 32 compounds inhibited the proliferation of AGS cells, with compound 4-(-2-[7-amino-2-{2-furyl}{1,2,4}triazolo{2,3-a}{1,3,5}triazin-5-yl-amino]ethyl)phenol (ZM-241385) whose chemical structure was shown in Fig. 1A be the most potent and fully killing all the AGS cells at 48h. To further confirm the inhibitory effect of ZM-241385, increasing concentrations (0, 0.1, 1, 5 10 µM) of ZM-241385 were used in the CCK-8 assay at different times (from 0h to 48h). As shown in Fig. 1B, ZM-241385 inhibited the viability of AGS cells in a dose-dependent and time-dependent manner. 1 µM, 5 µM and 10 µM ZM-241385 significantly inhibited cell viability after treatment for 12h, and cell viability was inhibited by 42% with 5 µM ZM-241385 treatment for 24h. Therefore, 5 µM ZM-241385 treatment for 24h was used in subsequent experiments.
To explore whether autophagy in AGS cells was affected by ZM-241385, western blot was applied the test the expression level of autophagy related proteins in AGS cells, including LC3B and Beclin-1. LC3 is an autophagosomal biomarker and has three isoforms, including LC3A, LC3B and LC3C, which are distributed in different tissues. LC3B is the most widely investigated and detected member in cancers [19]. It has been shown that LC3B is highly expressed in the cytoplasm of gastric cancer cells of 58% of gastric cancer patients, while it is not shown in noncancerous gastric epithelial cells [20]. Not surprisingly, in this study, we found that LC3B was activated in AGS cells (Fig. 1C), which is consistent with other studies [20, 21]. Beclin-1 is an essential factor to initiate autophagy and thus participates in various physiological and pathological processes, such as tumour formation [22]. Studies have shown that Beclin-1 is highly expressed in 50.9% of gastric cancer tissues, while is not or weakly expressed in nonmalignant gastric tissues [23]. Similarly, as shown in Fig. 1C, Beclin-1 was also highly expressed in AGS cells. These data suggest that autophagy was induced in AGS cell. In addition, compound ZM-241385 further stimulated LC3B formation and Beclin-1 activation in a dose dependent manner (Fig. 1C). These results indicate that ZM-241385 activated autophagy in AGS cells.
3.2 Compound ZM-241385 induces autophagy in AGS cells via activating AMPK
Autophagy is governed by multiple signaling pathways, including AMPK, mTOR, and ERK1/2, et al [24]. AMPK and ERK1/2 are crucial pathways positively regulate autophagy, while Akt/mTOR is the main pathway that negatively regulates autophagy [24]. To uncover the molecular mechanism of ZM-241385-induced autophagy in AGS cells, western blot was performed to assess the effect of ZM-241385 on the phosphorylation levels of the pathway members mentioned above. As shown in Fig. 2A, the phosphorylation of AKT, mTOR and p-ULK1(Ser757) was decreased by ZM-241385. Besides, the AMPK pathway was activated, shown as an elevated expression of p-AMPK and p-ULK1(Ser556) (Fig. 2B). The phosphorylation level of ERK was not changed (Fig. 2C). These results suggest that ZM-241385 induced autophagy in AGS cells by activating AMPK pathway and attenuating mTOR pathway.
Subsequently, siRNA against AMPK was applied to determine the role of AMPK pathway in ZM-241385 induced autophagy. It was shown that ZM-241385 induced p-AMPK(Thr172) was successfully suppressed by siRNA against AMPK (Fig. 2D). Furthermore, the activated LC3B and ULK1(Ser556) by compound ZM-241385 were attenuated (Fig. 2D). These data suggest that AMPK is essential for ZM-241385 to induce autophagy in GAS cells.
The above data suggest that AMPK/ULK1 pathway plays a vital role in ZM-241385 induced autophagy in AGS cells.
3.2 Compound ZM-241385 induces apoptosis in gastric cancer AGS cells
Numerous antitumor agents exert their effects through the induction of cell apoptosis. To investigate whether the inhibitory effect of ZM-241385 on cell proliferation is due to the induction of cell apoptosis, western blot analysis was used to investigate the apoptotic effects of ZM-241385 on AGS. cells. We found that the expression of apoptosis related proteins, including cleaved-caspase 3, cleaved-caspase 8, cleaved-caspase 9 and cleaved-PARP was significantly up-regulated by ZM-241385 (Fig. 3A&B). Besides, ZM-241385 increased Bax expression and decreased Bcl-2 expression (Fig. 3C). These data indicate that ZM-241385 induced apoptosis in AGS cells in a concentration dependent manner.
Caspase-specific inhibitors were than used to further confirm the function of activated caspase in ZM-241385-induced apoptosis. It was shown that all the specific inhibitors, including Z-DEVE-FMK (caspase-3), Z-IETD-FMK (caspase-8), or Z-LEHO-FMK (caspase-9), attenuated ZM-241385-induced cell proliferation (Fig. 3D). These data indicated that ZM-241385 induced AGS cell apoptosis to inhibit cell proliferation, which is dependent on the activation of caspase-3, caspase-8, and caspase-9.
3.4 Inhibition of autophagy enhances ZM-241385 induced cell death
It is well known that autophagy prevents cancer development. Conversely, once cancer is established, increased autophagic flux often enables tumour cell survival and growth. Many anticancer agents can activate autophagy, which might either induce cell death, or play a cryoprotection role [10, 11, 12, 13, 14, 15]. Targeting autophagy has been an effective approach to enhance cell apoptosis, thus, to enhance the therapeutic effect of anticancer agents [9].
To investigate the relationship between autophagy and apoptosis in AGS cells induced by ZM-241385, 10 µM chloroquine (CQ), which inhibits autophagosome degradation thus blocks the formation of autolysosomes, was used to block autophagy. As shown in Fig. 4A, compared with ZM-241385-treated cells, the cell viability was decreased after the addition of chloroquine, indicating that inhibition of autophagy improved the response of AGS cells to ZM-241385. In addition, western blot results demonstrated that chloroquine further increased the expression of apoptosis protein induced by ZM-241385, including cleaved-caspase 3, cleaved-caspase 9 and cleaved-PARP (Fig. 4B), which further demonstrated that ZM-241385 induced apoptosis in AGS cells was enhanced by chloroquine. Collectively, these data indicate that autophagy is beneficial for AGS cell survival and blocking autophagy can enhance the apoptotic inhibitory effects of ZM-241385 on AGS.
Autophagy has been shown to be a survival mechanism among several tumour types, including ovarian cancer, gastric cardnoma, breast cancer, et al [25, 26, 27]. The relationship between autophagy and cancer cell survival has not been fully uncovered. It is partly because that the enhanced autophagy may provide nutrients for cancer cell survival [27] or protect cells from undergoing programmed cell death [28]. Therefore, it is logical to improve cancer cell response to anticancer agents by inhibiting autophagy.
As shown in this study, autophagy is beneficial to maintain the survival of GAS cells, which potentiates AGS cells resistance to ZM-241385 or other anticancer cells. Therefore, applying autophagy inhibitors to destroy the protective effect can enhance cell apoptosis induced by ZM-241385 or other anticancer drugs.
In conclusion, we have screened a compound, ZM-241385, which suppressed AGS cell growth and induced cell apoptosis. Besides, ZM-241385 activated autophagy through an activation of AMPK signal pathway. In addition, ZM-241385 induced autophagy plays a cytoprotective role in ZM-241385 induced apoptosis in AGS cells. These results demonstrate that compound ZM-241385 combined with autophagy inhibitors may act as a new potential anticancer strategy for human gastric cancer.