Mitochondrial ROS accumulation inhibiting JAK2/STAT3 pathway is a critical modulator of CYT997-induced autophagy and apoptosis in gastric cancer

Human GC cell lines SGC-7901, MKN45, AGS, and BGC-823 were purchased from the Cell Bank of the Shanghai Institute for Biological Science (Shanghai, China). All cells were cultured in RPMI-1640 (Hyclone, Thermo Fisher, USA) medium with 10% fetal bovine serum (FBS) (Hyclone). The cells were maintained at 37 °C in a humidified incubator with 5% CO₂.

The fresh GC tumor tissue from GC patient was acquired and washed three times with PBS containing 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA), then, dissociated as small as possible with scissors, digested with collagenase IV (Sigma), 90 min at 37 °C, stopped digestion and centrifugated with 1000 rpm, 3 min, finally, resuspended and cultured with DMEM/F12 (Hyclone) medium containing 10% FBS and 1% penicillin/streptomycin.

CYT997 (MF: C24H30N6O2, MW: 434.53, purity: 99.46%), IL-6 and Mitoquinone (MitoQ) were bought from MCE (Shanghai, China). 3-methyladenine (3-MA) and N-acetylcysteine (NAC) were obtained from Sigma-Aldrich. GAPDH, Cyclin B1, p21, PARP, cleaved PARP, caspase 3, cleaved caspase 3, LC3B, Beclin-1, phosphorylated JAK2 (p-JAK2), JAK2, phosphorylated STAT3^(Tyr705)(p-STAT3), STAT3, Bcl-2, Survivin, Cyclin D1 and PCNA antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA).

Cell proliferation was performed with a CCK-8 kit (Dojindo, Tokyo, Japan). Cells (1× 10⁴) were seeded in 96-well plates. After 6 h, they were treated with different concentrations of CYT997. The OD value was measured at a wavelength of 450 nm. For colony formation assays, 500 cells were plated in each well of a 6-well plate, cultured in RPMI-1640 medium with 10% FBS. CYT997 was added in each well for about 14 days until the cells become visible colonies. Colonies were fixed with 4% paraformaldehyde and stained with crystal violet for 15 min at room temperature. Each experiment was performed in triplicate.

Cells were seeded in six-well plates, pretreated with serum starvation for 12 h, then treated with CYT997 for 12 h. For cell cycle analysis, the cells were collected, fixed in 75% ethanol at 4 °C overnight and stained with PI/RNase staining buffer for 15 min. For apoptosis analysis, cells were collected, washed twice with cold PBS and resuspended in binding buffer containing Annexin V-FITC and PI (Invitrogen Life Technologies, Carlsbad, CA, USA). The cell cycle and apoptosis analyses were performed on the Accuri C6 (BD Biosciences, Mountain View, CA, USA) and the data were analyzed by ModFit LT software (FACSCalibur).

Samples including cells and tissues were lysed in ice-cold RIPA with phenylmethylsulfonyl fluoride and protease inhibitor (Thermo Scientific, Fremont, CA, USA) for 30 min. Lysates were collected, centrifuged and the supernatant was collected. The concentration of protein was measured by a Pierce BCA protein assay kit (Thermo Scientific, Fremont, CA, USA). Equal amounts of protein were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membranes were blocked in 5% non-fat milk at room temperature for 1 h and then incubated with specific primary antibodies at 4 °C overnight, washed 3 times by TBST, and then incubated with secondary antibodies for 1 h. Signals were detected by enhanced chemiluminescence kit (Millipore, Billerica, MA, USA).

Intracellular ROS production was measured by using the Reactive Oxygen Species Assay Kit (Beyotime, Shanghai, China). Cells were seeded in six-well plates overnight and exposed to CYT997 for 12 h.Then, cells were collected, incubated with 10 μM DCFH-DA for 30 min in the dark and washed 3 times. The level of ROS was determined by fluorescence microscopy (Leica, Wetzlar, Germany) and flow cytometry (BD Biosciences; San Jose, CA, USA). Through using MitoSOX Red dye (Invitrogen), mitochondrial superoxide level was detected.

JC-1 Assay Kit (Beyotime, Jiangsu, China) was used to measure the mitochondrial membrane potential according to the manufacturer’s instructions. Briefly, cells after treatment with CYT997 (50 nM) for 12 h, stained with JC-1 for 20 min at 37 °C and then analyzed by a confocal laser scanning microscope and flow cytometry.

Cells were transiently transfected with GFP-LC3 plasmid using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. After 24 h, cells were treated with 50 nM CYT997 for 24 h.Then cells were washed with PBS, incubated with 50 nM of LysoTracker Red DND-99 (Invitrogen) in the dark for 30 min, washed with PBS again, fixed with 4% paraformaldehyde for 15 min and incubated with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min. Images were obtained by using a confocal laser scanning microscope (Leica, Germany).

The coding genes of human STAT3 and JAK2 were cloned into the mammalian expression vector pcDNA3.1. SGC-7901 cells were transiently transfected with the pcDNA3.1-STAT3 plasmid and pcDNA3.1-JAK2 plasmid through using Lipofectamine 3000 according to the manufacturer’s instruction (Invitrogen, Gaithersburg, MD, USA).

The GC tumor tissue from a GC patient was acquired and kept in culture medium on ice for engraftment within 2 h of resection. Then, the tissues were washed with PBS three times. A piece of about 5mm³ tissue was cut and implanted subcutaneously into the flank region of six-week-old BALB/c nude mice by using a trocar. Female athymic BALB/c nude mice (6–8 weeks old) were purchased from Shanghai Experimental Animal Center, Chinese Academy of Science. Until the tumors reached ~ 50 mm³, the mice were randomly assigned to two groups: (1) control group (n = 8), injected intraperitoneally with normal saline (NS). (2) treated group (n = 8): injected intraperitoneally with CYT997 (15 mg/kg) every day. The experiments were approved by the animal research committee in Shanghai Jiao Tong University.

For immunohistochemical staining, slides were deparaffinized, rehydrated, incubated in 3% H₂O₂ to block endogenous peroxidase activity. After antigen retrieval processed by boiling in sodium citrate for 30 min, slides were blocked by using 10% goat serum for 15 min, followed by incubation with specific primary antibodies at 4 °C overnight. Primary tumor samples were immunostained with p-STAT3 (1:50), PCNA (1:3000), cleaved caspase 3 (1:50) and LC3B (1:500). Then slides were washed 3 times, incubated with the second antibody at room temperature for 30 min, washed 3 times and incubated with diaminobenizidine (DAB) for 3 min. Finally, the nuclei were counterstained with Mayer’s hematoxylin.

To explore the effects of CYT997 (Fig.1a) on the proliferation of GC cells, human GC cells SGC-7901, MKN45, AGS, and BGC-823 were treated with CYT997 for 24 h and 48 h. Cell viability was detected by CCK8 assay. CYT997 inhibited proliferation of GC cells in a dose- and time-dependent manner (Fig. 1b). The IC50 values of GC cells SGC-7901, MKN45, AGS, and BGC-823 were 70.35, 77.92, 62.74 and 67.34 nM, respectively, at 24 h and 46.81, 55.80, 39.55 and 45.09 nM, respectively, at 48 h. We also chose osteosarcoma cell 143B as a control reference cell line.143B cells were treated with CYT997 for 24 h and 48 h. The IC50 values were 88.7 nM and 64.8 nM (Additional file 1: Fig. S1). Colony formation assay also showed that the colony formation ability of GC cells was inhibited in the CYT997-treated cells (Fig. 1c and d). To further investigate the mechanism of CYT997-induced suppression of GC cell proliferation, the cell cycle distributions of SGC-7901 and MKN45 cells were determined by flow cytometry. Our results showed that CYT997 induced G2/M arrest in SGC-7901 and MKN45 cells (Fig. 2a). Furthermore, cell cycle-related proteins Cyclin B1 and p21 were upregulated in CYT997 treated GC cells (Fig. 2b; Additional file 1: Fig. S2a).

Apoptosis is also widely believed to be the major antiproliferative mechanism of anticancer drugs in many tumor cell types. Therefore, we also investigated the effect of CYT997 on cell apoptosis in GC cells. Our results showed that CYT997 induced apoptosis in SGC-7901 and MKN45 cells in a dose-dependent manner (Fig. 2c). Furthermore, apoptosis-related protein cleaved caspase 3 and cleaved PARP were significantly increased in GC cells treated with CYT997 (Fig. 2d; Additional file 1: Fig. S2b). Therefore, these results suggested that CYT997 inhibited cell proliferation and induced apoptosis in the GC cells.

Autophagy is a catabolic process in which autophagosomes is formed, and it has double-sided effect in cancer proliferation and death [21, 22]. LC3 and Beclin-1 are key molecular regulator of autophagy and can be used as autophagy marker [23, 24]. To investigate whether CYT997 could trigger autophagy in GC cells, Lyso-Tracker Red, a membrane acidotropic dye probe was used to mark cellular acidic organelles (AVOs), such as lysosomes and autolysosomes. We found that SGC-7901 and MKN45 cells treated with CYT997 showed more AVOs in cytoplasm than control cells (Fig. 3a). Then, GFP-LC3 puncta transfection way was applied to analyze the formation of autophagosomes. The results showed that SGC-7901 and MKN45 cells treated with CYT997 exhibited significantly higher numbers of GFP-LC3 puncta than control cells (Fig. 3a; Additional file 1: Fig. S3a-3b). Additionally, we found that expression of Beclin-1 were upregulated in GC cells treated with CYT997 in a dose-dependent manner (Fig. 3b; Additional file 1: Fig. S3c). To further explore the role of autophagy in CYT997-treated GC cells, SGC-7901 and MKN45 cells were pretreated with autophagy inhibitor 3-MA prior to CYT997 treatment. The outcomes displayed that pretreatment with 3-MA promoted the effect of CYT997 on cell apoptosis (Fig. 3c). Furthermore, the expression of LC3B and Beclin-1 were decreased, while cleaved caspase 3 and cleaved PARP were increased in GC cells (Fig. 3d; Additional file 1: Fig. S3d). Collectively, autophagy may have additional cytoprotective effects by protecting cells against effects of CYT997.

Reactive oxygen species (ROS) are critical regulators of apoptosis [25]. Many chemotherapeutic agents induce tumor cells apoptosis by initially inducing cells to accumulate ROS [26, 27]. To clarify whether CYT997-induced apoptosis was due to ROS production, ROS assay was applied to detect ROS level in GC cells. Our results showed that SGC-7901 and MKN45 cells treated with CYT997 exhibited a distinct enhancement in the DCFH-DA fluorescent signal than control (Fig. 4a and b). These results indicated that CYT997 could increase ROS production in GC cell. To explore the source of ROS, MitoSOX Red dye was used to detect mitochondrial ROS by confocal microscope and flow cytometry. We found that SGC-7901 and MKN45 cells treated with CYT997 showed an obvious improvement in the MitoSOX Red fluorescent signal than control (Fig. 4c). These results indicated that CYT997 increased the production of mitochondrial ROS in GC cells. To further prove mitochondrial ROS, JC-1 assay was used to detect the mitochondrial membrane potential by confocal microscope and flow cytometry. Our results showed that red fluorescent signal was significantly attenuated, while green fluorescent signal was notably enhanced in cells treated with CYT997. The JC-1 red/green fluorescence intensity ratio was distinctly reduced in GC cells treated with CYT997 (Fig. 4d and e). Mitochondrial dysfunction and imbalance often lead to mitochondrial membrane potential change and trigger mitochondrial ROS release [28, 29]. The results suggested that GC cells mitochondrial membrane potential was decreased after CYT997 treatment and mitochondrial function was damage, which increased mitochondrial ROS production. Next, ROS scavengers NAC and MitoQ were used to investigate the effects of antioxidants on CYT997-induced cell apoptosis. The results showed that NAC and MitoQ obviously attenuated the CYT997-induced proliferation inhibition (Fig. 4f and 5a). NAC and MitoQ also distinctly inhibited CYT997-induced cell cycle G2/M arrest and apoptosis in GC cells (Fig. 4g, h, 5b, c). Furthermore, the expression of Cyclin B1, p21, cleaved caspase 3, cleaved PARP, LC3B, Beclin-1 was downregulated in cells pretreated with NAC or MitoQ than only treated with CYT997 (Fig. 4i, 5d; Additional file 1: Fig. S4 and S5a). Therefore, these data indicated that CYT997 could induce apoptosis in GC cells through enhancing mitochondrial ROS.

ROS modulate various cell signaling pathways including STAT3. STAT3, as a transcription factor, plays an important role in tumor cell proliferation and progression [30]. Therefore, we wondered if CYT997 could affect the JAK/STAT3 pathway in GC cells. Our results showed that p-STAT3 was inhibited in CYT997 treated cells. Moreover, p-JAK2 was also inhibited by CYT997, while the JAK2 did not change. Additionally, the major downstream targets of STAT3 were inhibited by CYT997, including Bcl-2, Survivin and Cyclin D1 (Fig. 6a; Additional file 1: Fig. S6a). Cytokine IL-6, a major mediator of inflammation and activator of STAT3, serves to block apoptosis and promote tumor survival. We found that activation of STAT3 induced by IL-6 was reversed by CYT997 (Fig. 6b; Additional file 1: Fig. S6b). Furthermore, IL-6-induced nuclear accumulation of STAT3 was reversed by CYT997 using western blotting and immunofluorescence assay (Fig. 6c and d; Additional file 1: Fig. S6c).

To further certify the molecular mechanism of CYT997, STAT3 and JAK2 plasmid were transfected into SGC-7901 cells respectively. We found that p-STAT3 and its downstream protein were activated by STAT3 overexpression. Overexpression of STAT3 weakened the effects of CYT997 on the expression of p-STAT3, Cyclin D1, Bcl-2 and Survivin in SGC-7901 cells (Fig. 6e; Additional file 1: Fig. S7a). CYT997-induced cell proliferation inhibition, cell cycle arrest and apoptosis were attenuated by STAT3 overexpression in SGC-7901 cells (Fig.6f-h). The expression of Cyclin B1, p21, cleaved caspase 3 and cleaved PARP was partly reversed by STAT3 overexpression (Fig. 6i; Additional file 1: Fig. S7b). The similar results were further proved by JAK2 overexpression (Additional file 1: Fig. S8). Therefore, all results indicated that CYT997 inhibited GC cell proliferation by regulating of JAK2/STAT3 pathway.

Next, we explored whether CYT997 affected the JAK2/STAT3 pathway through ROS generation in GC cells. CYT997-induced inhibition of p-JAK2, p-STAT3, Bcl-2, Survivin and Cyclin D1 was reversed by NAC in SCG-7901 cells (Fig. 6j; Additional file 1: Fig. S7c). In addition, the similar results were found using MitoQ (Additional file 1: Fig. S5b). Collectively, these results suggested that CYT997 inhibited JAK2/STAT3 pathway through ROS generation in GC cells.

To investigate the effect of CYT997 in tumor growth in vivo, mice bearing gastric cancer PDX xenografts were used. When tumors reached a volume of 50 mm³, mice were intraperitoneally injected with normal saline (NS) and CYT997 (15 mg/kg) respectively. Tumor size was measured every other day and tumors were excised after 10 days post injection. We found that CYT997 significantly decreased tumor volume and tumor weight compared with control group (Fig. 7a-c). Furthermore, we also found that the expressions of p-JAK2 and p-STAT3 were decreased after CYT997 treatment. And CYT997 increased the expression of cleaved caspase 3 and LC3B (Fig. 7d-e; Additional file 1: Fig. S9). In addition, we found that there were no obvious changes in body weight and organ-related toxicities were scarce in mice (Fig. 7f and g). Collectively, our results suggest that CYT997 inhibits tumor growth and cell proliferation in vivo.

Primary GC cells were extracted from a GC patient’s tumor tissue and observed by light microscope after CYT997 treatment for 24 h (Additional file 1: Fig. S10a). CYT997 significantly induced cell apoptosis. We found that expression of cleaved caspase 3 and LC3B was upregulated, whereas expression of p-JAK2 and p-STAT3 was downregulated in CYT997-treated primary GC cells (Additional file 1: Fig. S10b-10c). Furthermore, ROS scavenger NAC also distinctly weakened CYT997-induced cell apoptosis (Additional file 1: Fig. S10d). CYT997-induced inhibition of p-JAK2, p-STAT3 was reversed by NAC in primary GC cells (Additional file 1: Fig. S10e). Therefore, these data suggested that CYT997 promoted GC cell apoptosis by inhibiting JAK2/STAT3 pathway.