BIX‐01294 enhanced chemotherapy effect in gastric cancer by inducing GSDME‐mediated pyroptosis

Abstract Adjuvant chemotherapy in combination with surgery is expected to be a curative strategy for gastric cancer. However, drug resistance remains an obstacle in effective chemotherapy. Therefore, understanding the potential mechanisms of chemotherapy induced gastric cancer cell death is of great importance. We demonstrated that BIX‐01294 (BIX) at low concentration could induce autophagic flux by converting LC3B‐I to LC3B‐II and directly activate autophagy associated cell death in gastric cancer cell lines at high concentration. BIX at low concentration could help obtain sensitivity of gastric cancer cells to chemotherapy with significantly reduced cell viability. Interestingly, BIX combined Cis (BIX + Cis) treated SGC‐7901 cells display pyroptosis related cell death with large bubbles blown around the membrane, significantly decreased cell viability, elevated lactate dehydrogenase release and increased percentage of propidium iodide and Annexin‐V double positive cells. Furthermore, the cleavage of gasdermin E (GSDME) and caspase‐3 but not GSDMD was detected by immunoblotting and the knockout of GSDME switched pyroptosis into apoptosis in the BIX + Cis combined treated group. Furthermore, the deficiency of Beclin‐1 to inhibit BIX induced autophagic flux completely blocked BIX + Cis combined treated induced cell pyroptosis related cell death. Additionally, BIX + Cis in vivo treatment could inhibit tumor growth, which could be reversed by the deficiency of Beclin‐1 and be delayed by the deficiency of GSDME. In conclusion, our data was the first to reveal that BIX enhanced the anticancer chemotherapy effect by induced GSDME‐mediated pyroptosis through the activation of autophagic flux in gastric cancer cells.

cancer are markedly elevated and gastric cancer constitutes the second leading cause of cancer-related death (Bray et al., 2018;Chen et al., 2016). Currently, adequate surgical resection is still expected to be the most curative option for gastric cancer, while chemotherapy or neoadjuvant therapy is usually applied in combination with surgery for locally advanced cancer to prevent recurrence and metastasis after surgery (Van Cutsem, Sagaert, Topal, Haustermans, & Prenen, 2016), it is broadly accepted that chemotherapy induces gastric cell death, and drug resistance and tumor growth are influenced by drug-induced cancer cell death. Therefore, understanding the mechanism of chemotherapyinduced gastric cancer cell death is eagerly awaited. Autophagy, a conserved cellular self-digestive process for maintaining homeostasis, degrades proteins and organelles through lysosomal degradation (Levy, Towers, & Thorburn, 2017). However, recent studies have shown that excessive activated autophagy moved toward autophagic cell death (Maiuri, Zalckvar, Kimchi, & Kroemer, 2007;Pei et al., 2016). Recently, increasing evidence suggested that activation of autophagy can enhance the sensitivity of anticancer therapeutics (Levy et al., 2017;Pei et al., 2016;Zhang, Yin, & Sui, 2018). Moreover, rapamycin, an autophagy inducer, has been approved by the U.S. Food and Drug Administration for cancer treatment (Gentzler, Altman, & Platanias, 2012). Therefore, the autophagic pathway might be a promising therapeutic target for cancer chemotherapy. BIX-01294 (BIX; a diazepin-quinazolin-amine derivative) was discovered to be a selective inhibitor for euchromatic histone-lysine N-methyltransferase 2 (EHMT2) histone methyltransferase (HMTase) initially (Chang et al., 2009). Recently, BIX has been found to be an effective inducer of autophagy and even to induce cell death at a high dosage (Kim et al., 2013). Moreover, BIX has also been demonstrated to increase glioma cell radiosensitivity (Gursoy-Yuzugullu et al., 2017). However, whether BIX can enhance the sensitivity of gastric cancers to chemotherapy and the potential mechanisms remain unknown.
Pyroptosis, characterized by cell swelling and pore formation, is a form of lytic cell death (Kovacs & Miao, 2017). The Gasdermin superfamily constitutes of conserved proteins, including gasdermin A, B, C, D, E, and DFNB59, most of which have now been shown to have pore-forming activity and then induce pyroptosis (Ding et al., 2016).
Gasdermin D (GSDMD) can be cleaved by activated caspase-1 or caspase-11/4/5 to release its N-terminal pore-forming domain (PFD; Kayagaki et al., 2015). The PFD oligomerizes to pore the membrane and then drive swelling and membrane rupture (Kayagaki et al., 2015;Liu et al., 2016). GSDME/DFNA5, a nonsyndromic hearing impairment gene, resembles the membrane pore formation effects of GSDMD in being cleaved by active caspase-3 and induce characteristics of pyroptosis (Wang et al., 2017). Meanwhile, GSDME has been demonstrated to be highly expressed in gastric tumors and be cleaved by activated caspase-3 in a previous study . However, the role of GSDME in chemotherapy for gastric cancers remains largely unclear.
In this study, we, first, confirmed that BIX could enhance the sensitivity of gastric cancer to chemotherapy. Moreover, we found that BIX combined Cis (BIX + Cis) could induce GSDME-mediaed pyroptosis in gastric cancer. Finally, we demonstrated BIX-induced autophagic flux play a vital role in enhancing the sensitivity of gastric cancer cells to chemotherapy. Logan, UT), 100 U/ml penicillin and 100 mg/ml streptomycin (Gibco) and 2 mM L-glutamine (Gibco). All cells were kept at 37°C in a 5% CO 2 humidified incubator. SGC-7901, MKN-45, and AGS were pretreated with or without BIX (B9311; Sigma-Aldrich) for 2 hr, and then treated with cis-platinum (Cis; 1134357; Sigma-Aldrich) at the indicated concentrations and different time points.

| In vivo nude mouse model
The male BALB/c nude mice (6-8 weeks) were purchased from the Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). (1 mg/kg/2 day) for another 15 consecutive days. After the experiment, all mice were euthanized by the carbon dioxide method.
Tumor volume was recorded as: V (mm 3 ) = length (mm) × width 2 (mm 2 )/2. All animal studies were approved by the Institutional Care and Use Committee, Experimental Animal Centre of Jinzhou Medical University (Jinzhou, China).

| Microscopy images
Cells treated with BIX or BIX combined Cis, 1 × 10 5 SGC-7901 cells were seeded into in 24-well plates and treated as indicated. Static bright-field images were photographed by using an Olympus IX71 microscope.

| Flow cytometry analysis
After 12 hr treatment of Cis, SGC-7901 cells were digested with trypsin digestion solutions without ethylenediaminetetraacetic acid (T1350; Solarbio), harvested and incubated with APC Annexin-V (BD Biosciences) for 15 min at room temperature protected from light following the manufacturer's instructions. Washing cells with PBS was followed by propidium iodide (PI; 421301; Biolegend) incubation for 5 min. Two hundred microliter 1× binding buffer (BD Biosciences) was added to each sample and data were acquired by flow cytometer (BD Biosciences, CA).

| Statistical analysis
Statistical analysis and graphs were performed using GraphPad Prism 7.0 software (GraphPad Software, San Diego, CA). One-way analysis of variance followed by Bonferroni's multiple comparisons post hoc test was used to compare the difference of three or more groups and Student's t test was used to compare the difference of two groups.
All data was represented as the mean ± SEM of at least three independent experiments. In all statistical tests, a p < .05 was considered statistically significant. Therefore, we measured the LDH release, which would leak into extracellular space from the cytosol after the loss of membrane integrity (Evavold et al., 2018). As expected, the LDH release was significantly increased in the BIX + Cis combined treated group but not by Cis or BIX alone (Figure 2c). Furthermore, the loss of membrane integrity also resulted in intracellular nucleic acids stained by the membrane impermeable dye PI (Evavold et al., 2018). Flow cytometry in Figure 2d showed that the percentage of Annexin V and Collectively, these data suggest that BIX pretreatment would promote Cis induced gastric cancer cells pyroptosis.

| BIX combined cis induced gastric cancer cells pyroptosis dependent on the cleavage of GSDME
GSDMD has been demonstrated to play a critical role in inducing pyroptosis by eliciting pore formation on the cell membrane (Tamura & Shiroishi, 2015). We first detected the full length and N-terminal of GSDMD by immunoblotting to further explore the potential mechanism of BIX combined Cis induced gastric cancer cells pyroptosis. However, no cleavage of GSDMD (GSDMD-N) after the treatment of BIX + Cis could be detected (Figure 3a). We then detected the cleavage of another member of the gasdermin family, gasdermin E (GSDME), which was reported to exert pyroptosis depending on the cleavage of caspase-3 (Cleaved CASP3). Both N-terminal of GSDME and Cleaved CASP3 were obviously detected by immunoblotting in the BIX-Cis combined treated group ( Figure 3a). Using the CRISPR-Cas9 technology to KO the GSDME gene in SGC-7901 cells (Figure 3b), we found that cell swelling and bubble blowing was diminished in the BIX-Cis combined treated group, indicating that the cleavage of GSDME was necessary for BIX + Cis-induced pyroptosis in SGC-7901 cells (Figure 3c). Moreover, LDH release was completely blocked and cell viability was significantly increased in the BIX + Cis combined treated GSDME deficiency group compared with the WT group (Figure 3d). However, the GSDME deficiency could not completely block cell death after BIX + Cis treatment (Figure 3d). In the GSDME-deficient group with BIX + Cis combined treatment, the percentage of Annexin V and PI double-positive late apoptotic cells was significantly lower, but the percentage of Annexin V single-positive early apoptotic cells was significantly higher than that in WT group (Figure 3e). These results indicated that BIX switched Cis-induced gastric cancer cells apoptosis to pyroptosis depending on the cleavage of GSDME.

| BIX enhanced anticancer effect by autophagic flux
As demonstrated above, BIX can strongly induce autophagy related cell death, therefore, we proposed that BIX induced autophagic flux play a vital role on BIX + Cis triggered pyroptosis. To validate our hypothesis, we used the CRISPR-Cas9 technology to knock out the  Figures 4f and 4h), however, the deficiency of Beclin-1 but not GSDME completely blocked the effect of BIX + Cis effects in vivo (Figures 4g and 4i).

| DISCUSSION
BIX, a pluripotency inducer via EHMT2 inhibition, was used as a replacement of Oct3/4 in generating induced pluripotent stem cells from mouse fetal neural precursor cells in the absence of Sox2 (Shi et al., 2008). Recently, it is reported that BIX increases the transcription of Beclin-1 to activate autophagy and even induces DENG ET AL.

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| 1895 autophagy-associated cell death (Park et al., 2016). However, emerging evidence suggests that induction of autophagy can not only limit tumorigenesis in the long-term, but also promote autophagic cell death and enhance the sensitivity of anticancer therapeutics in various types of cancer (Sun et al., 2017). The precise molecule mechanism of how BIX enhances anticancer therapeutics has not been elucidated yet. In Kim et al.'s (2013) study, they have found that BIX induced breast cancer autophagy-associated cell death via EHMT2/G9a dysfunction and reactived oxygen species production (Kim et al., 2013). However, compared with 1 μM for inducing strong autophagic flux, 10 μM BIX may be too high to be applied in the clinic.
In our study, we found that BIX can also induce autophagy of gastric cancer cells in a dose-and time-dependent manner. Meanwhile, the ability of the BIX induced gastric cancer cell line, SGC-7901, MKN-45, and AGS death was increased with the dosage, which was similar to the effect of BIX on other cancers (Huang, Zou, Lin, Ma, & Huang, 2017;Zhu et al., 2018). Interestingly, we found that a low dose of BIX (1 μM) can help obtain the sensitivity of gastric cancer to a low dose of chemotherapy, which was further confirmed in vivo experiments. Overall, our results suggested that BIX might be a new promising targeted therapeutic agent in the clinic.

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| 1897 pyroptosis to apoptosis, which confirmed that BIX combined Cis induced pyroptosis in gastric cancer dependent on the cleavage of GSDME and caspase-3. However, the deficiency of Beclin-1 in SGC-7901 completely blocked BIX combined Cis induced pyroptosis as well as apoptosis, which suggested that BIX enhances the sensitivity of anticancer therapeutics in gastric cancer through the activation of autophagic flux. The in vivo results indicated that the KO of GSDME or Beclin-1 could both inhibit the effect of BIX + Cis treatment in vivo. However, the effect of Beclin-1 KO on BIX + Cis anticancer efficiency was stronger than that of GSDME KO, which was similar to what we found in vitro. These findings indicated that BIX-Cis induced GSDME-midiated pyroptosis through the activation of autophagic flux.
DFNA5 (GSDME) has been reported to induce high methylation in the putative gene promoter and is downregulated in many tumor cell lines, including breast, gastric, and colon cancer cell lines (Wang et al., 2017). But the human breast cancer line MCF-7 and mouse mammary carcinoma cell line EMT6 have been found to express high levels of GSDME (Wang et al., 2017). And we found that SGC-7901 also expresses a high level of GSDME, which is consistent with a recent study . Due to the heterogeneity of tumor cells, the high expression of GSDME may hint that GSDME probably exerts an alternative function in tumor cells. It has been reported that the higher expression of GSDME caused more extensive pyroptosis (Wang et al., 2017). Although Gsdme −/− mice were often more healthy than wild-type mice after the treatment of cisplatin (20 mg/kg), we did not observe obviously abnormality after the treatment of cisplatin at low concentrations (1 mg/kg/ 2 day). This may be because of the very high level of GSDME in SGC-7901, which makes it more sensitized to BIX-Cis treatment. BIX may change the state of GSDME methylation dependent inhibiting G9a, which sensitizes the tumor to chemotherapy. Therefore, the combination of BIX and Cis maybe a good treatment for the gastric cancer expressed very high level of GSDME or very low level of GSDME.
In conclusion, we were the first to report that BIX can induce autophagic flux and enhance the anticancer effect in gastric cancer.
Moreover, our study also demonstrated that BIX combined Cis can induce GSDME-mediated pyroptosis in gastric cancer cell lines.
Furthermore, BIX combined Cis induced cell death dependent on the activation of autophagic flux.