Introduction
Esophageal cancer (EC), ranked as the sixth leading cause of cancer-related mortality worldwide, is one of the most highly malignant and aggressive cancers (1,2). Esophageal squamous cell carcinoma (ESCC) is the predominant histological subtype, accounting for more than 90% of EC cases in China (3). Although screening technology and multimodality therapies have remarkably improved during the past decade, the prognosis of EC remains dismal and the 5-year overall survival rate is still below 15% (4). Accumulative studies have tried to demonstrate the molecular and biological mechanisms that lead to EC. A series of risk factors for EC have been established, such as epidermal growth factor receptor (EGFR), Her-2, p53 and heat shock proteins (HSPs), which have been found to be associated with the progression of EC (5–8). However, the mechanisms of oncogenesis of EC have not been completely clarified. Therefore, the characterization of molecular markers involved in the pathophysiological process of ESCC is essential.
The HSPs family, as molecular chaperones, are biochemical regulators, which function in mediating cell growth, apoptosis, migration and protein homeostasis (9). HSPs are induced in response not only to cellular stress, but also to other environmental, physical and chemical stresses (10). HSPs are classified into 6 major family members according to molecular weight: HSP100, HSP90, HSP70, HSP60, HSP40 and small HSPs (11). HSP90 is one of the most abundant HSPs and more than 200 types of HSP90 client proteins have been found. A series of previous studies have demonstrated that HSP90 is activated and upmodulated in a wide variety of human tumors, such as head and neck squamous cell cancer (HNSCC) (12), colon carcinoma (13) and other adenocarcinomas (14). During the progression of cancers, HSP90 has a key role in the regulation of cell cycle growth, signaling, migration and transcription factors, which may lead to tumorigenesis (15–17). However, there is a paucity of data on the relationship between activation of HSP90 and the malignancy of EC.
Hydrogen sulfide (H2S), as a specific toxic gas, has been qualified as the third gasotransmitter following nitric oxide (NO) and carbon monoxide (CO) (18–20). Endogenous H2S is synthesized from L-cysteine by two key enzymes: cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE) which are mainly expressed in the enteric neurons and smooth muscle of the stomach and colon (21,22). Recently, numerous scientific investigations have proven the extensive physiological and pathophysiological properties of H2S on progress of cancer. Previous findings have demonstrated that H2S promotes cancer cell growth, proliferation, migration and invasion (23–29), owing to its vascular relaxant and angiogenesis effects. H2S enhances the supply of nutrients and blood to the tumor cells and tissues (29). Our latest research also showed that exogenous H2S promoted cancer cell proliferation/anti-apoptosis/angiogenesis/migration effects via amplifying the activation of NF-κB (30) and p38 MAPK/ERK1/2-COX-2 pathways (31). However, research focused on the effect of exogenous H2S on esophageal EC109 cells and its potential mechanisms is lacking. Hence, we investigated whether exogenous H2S contributes to cancer progress and explored these potential effects via activation of HSP90 pathways in esophageal EC109 cells.
Materials and methods
Materials
NaHS, a donor of H2S, was obtained from Sigma Chemicals Co. (St. Louis, MO, USA), stored at 2–4°C and protected from sunlight. GA (a specific inhibitor of HSP90 pathway) was also purchased from Sigma Chemicals Co. The Cell Counting Kit-8 (CCK-8) was supplied by Dojindo Laboratory (Kumamoto, Japan). Fetal bovine serum (FBS) and RPMI-1640 medium were obtained from Gibco-BRL (Grand Island, NY, USA). Anti-MMP2, anti-HSP90, anti-cleaved caspase-3, anti-bcl-2 antibody and anti-bax antibodies were supplied by Cell Signaling Technology (Boston, MA, USA). Horseradish peroxidase (HRP)-conjugated secondary antibody and BCA protein assay kit were obtained from KangChen Bio-tech, Inc. (Shanghai, China). Enhanced chemiluminescence (ECL) solution was purchased from KeyGen Biotech (Nanjing, China). Enzyme-linked immunosorbent assay (ELISA) was supplied by ExCell Bio Co. (Shanghai, China).
Cell culture and treatments
The human esophageal carcinoma cells EC109 (EC109 cells) were supplied by Sun Yat-sen University Experimental Animal Center (Guangzhou, Guangdong, China). The EC109 cells were grown in RPMI-1640 medium supplemented with 10% FBS under an atmosphere of 5% CO2 and at 37°C with 95% air. The EC109 cells were treated with 500 µmol/l NaHS for 24 h or co-treated with 500 µmol/l NaHS and 20 µmol/l GA for 24 h.
Western blot analysis
After the indicated treatments, the cells were harvested and lysed with cell lysis solution at 4°C for 30 min. The total proteins were quantified using the BCA protein assay kit. Loading buffer was added to cytosolic extracts, and then after boiling for 6 min, the same amounts of supernatant from each sample were fractionated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The total proteins were then transferred into polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% fat-free milk for 60 min in fresh blocking buffer [0.1% Tween-20 in Tris-buffered saline (TBS-T)] at room temperature, and incubated with either anti-MMP2 (1:1,000 dilution), anti-HSP90 (1:1,000 dilution), anti-bax (1:1,000 dilution), anti-bcl-2 (1:1,000 dilution) and anti-cleaved caspase-3 antibodies (1:1,000 dilution) in freshly prepared TBS-T with 3% fat-free milk overnight with gentle agitation at 4°C. Membranes were washed for 5 min with TBS-T for 3 times and incubated with HRP-conjugated goat anti-rabbit secondary antibody at a concentration of 1:3,000 dilution (Kangchen Biotech, Shanghai, China), in TBS-T with 3% fat-free milk for 1.5 h at room temperature. Then, membranes were washed 3 times with TBS-T for 5 min. The immunoreactive signals were visualized via the ECL. In order to quantify the protein expression, the X-ray film was scanned and analyzed with ImageJ 1.47i software. The experiment was carried out 3 times.
Measurement of cell viability
The EC109 cells were seeded in 96-well plates at some concentration of 1×104/ml and incubated at 37°C. The CCK-8 assay was employed to assess the cell viability of EC109 cells. After the indicated treatments, 10 µl CCK-8 solution at a 1/10 dilution was added to each well and then the plate was incubated for 1.5 h in the incubator. Absorbance at 450 nm was assayed using a microplate reader (Molecular Devices, Sunnyvale, CA, USA). The means of the optical density (OD) of 3-wells in the indicated groups were used to calculate the percentage of cell viability according to the formula below: Cell viability (%) = (OD treatment group/OD control group) × 100%. The experiment was carried out 5 times.
ELISA for detection of VEGF in culture supernatant
EC109 cells were cultured in 96-well plates. After the different indicated treatments, the level of VEGF in the culture media was tested by ELISA according to the manufacturer's instructions. The experiment was performed at least 5 times.
Transwell migration assay
The EC109 cells were harvested and washed twice with phosphate-buffered saline (PBS). After washing, 1×105 cells were resuspended in 200 µl Dulbecco's modified Eagle's medium (DMEM), and added to the upper chamber of the Transwell membrane (Transwell permeable support with a 5.0-µm polycarbonate membrane, 6.5-mm insert and 24-well plate; Corning Costar, Tewksbury, MA, USA), and 600 µl of 10% FBS-DMEM was added to each bottom chamber. Four groups in the upper chamber were included in the assay: i) control; ii) NaHS, NaHS (500 µmol/l); iii) NaHS + GA (a specific inhibitor of HSP90 pathway), NaHS (500 µmol/l) + GA (20 µmol/l); iv) GA, GA (20 µmol/l). After 24 h at 37°C, cells that migrated to the lower chambers were counted. Triplicate experiments were performed with each group, and the means and standard deviations were calculated.
Statistical analysis
All data are presented as the mean ± SEM. Differences between groups were analyzed by one-way analysis of variance (ANOVA) using SPSS 13.0 (SPSS, Inc., Chicago, IL, USA) software, and followed by LSD post hoc comparison test. Statistical significance was considered at P<0.05.
Results
NaHS promotes cell proliferation in EC109 esophageal cells
In order to test the effect of exogenous H2S on human EC cell proliferation, the dose-response study with varying doses (200, 400, 600, 800 and 1,000 µmol/l) of NaHS (a donor of H2S) for 24 h was performed to calculate the effective doses of NaHS. As shown in Fig. 1A, the doses of NaHS from 200 to 1,000 µmol/l markedly promoted cell proliferation, leading to an increase in cell viability and reaching a peak at 400 µmol/l. Therefore, 400 µmol/l NaHS was used in the subsequent time-response study with different treatment times (12, 24, 36, 48 and 60 h). As shown in Fig. 1B, treatment of EC109 cells with 400 µmol/l NaHS for the indicated times all markedly promoted cell proliferation, reaching the maximal proliferative effect at 24 h. Based on the aforementioned results, EC109 esophageal cells were treated with 400 µmol/l NaHS for 24 h in all subsequent experiments.
NaHS upregulates the expression levels of HSP90 in EC109 esophageal cells
We observed the effects of NaHS on the expression levels of HSP90. As shown in Fig. 2A and B, exposure of EC109 cells for the indicated time (12 and 24 h) to 400 µmol/l NaHS markedly enhanced the expression of HSP90, reaching a peak at 24 h.
GA (a specific inhibitor of HSP90 pathway) reduces the cell proliferation in EC109 esophageal cells
EC109 cells were treated with different doses of GA (10, 20, 40, 60 and 80 µmol/l) for 24 h. As shown in Fig. 3A, the doses of GA from 10 to 80 µmol/l markedly reduced cell proliferation, dropping to a bottom at 80 µmol/l.
GA alleviates NaHS-induced cell proliferation in EC109 cells
As shown in Fig. 3B, exposure of EC109 cells to 400 µmol/l NaHS for 24 h induced cell proliferation, leading to an increase in cell viability. However, the increased cell viability was repressed by co-treatment with different doses of GA (a specific inhibitor of HSP90 pathway) for 24 h. As shown in Fig. 3A, at the dose of 10 µmol/l, the cell viability did not change. On the contrary, the dose of GA from 20 to 80 µmol/l significantly suppressed the cell proliferation, leading to a decrease in cell viability and reaching the minimum at 20 µmol/l. According to the aforementioned results, EC109 cells were co-treated with 400 µmol/l NaHS and 20 µmol/l GA for 24 h in all following experiments.
NaHS alleviates the expression level of cleaved caspase-3 and bax, and upregulates the expression levels of bcl-2 in EC109 esophageal cells
In order to observe the effects of NaHS on the expression levels of cleaved caspase-3, bax and bcl-2 in EC109 cells, EC109 cells were exposed to 400 µmol/l NaHS for different times (1, 3, 6, 9, 12 and 24 h). As shown in Fig. 4A, NaHS significantly enhanced the expression levels of bcl-2 reaching a peak at 12 h, whereas the expression level of caspase-3 and bax was markedly decreased.
GA inhibits NaHS-induced increased expression levels of bcl-2 and upregulates NaHS-induced decreased caspase-3 expression in EC109 esophageal cells
As shown in Fig. 4E, EC109 cells were exposed to 400 µmol/l NaHS for 24 h. The expression levels of bcl-2 were significantly increased; on the contrary, the expression level of caspase-3 and bax were markedly decreased. Notably, co-treatment of EC109 cells with 400 µmol/l NaHS and 20 µmol/l GA for 24 h considerably depressed NaHS-induced increased expression levels of bcl-2; however, expression of caspase-3 and bax was considerably downregulated. Treatment of cells with 20 µmol/l GA for 24 h did not alter the basal expression levels of caspase-3, bax or bcl-2.
NaHS upregulates the expression level of MMP-2
In order to observe the effects of NaHS on the expression levels of MMP-2 in EC109 cells, EC109 cells were exposed to 400 µmol/l NaHS for different times (1, 3, 6, 9, 12 and 24 h). As shown in Fig. 5A, NaHS significantly enhanced the expression levels of MMP-2, which peaked at 24 h. Notably, co-treatment of EC109 cells with 400 µmol/l NaHS and 20 µmol/l GA for 24 h considerably depressed NaHS-induced increased expression levels of MMP-2. Treatment of cells with 20 µmol/l GA for 24 h did not alter the basal expression levels of MMP-2.
The migration rate and Transwell migration assay
As shown in Fig. 5E, NaHS strengthened the migration rate in EC109 esophageal cells while GA depressed NaHS-induced increased migration rate. Treatment of cells with 20 µmol/l GA for 24 h did not alter the migration rate compared with control group.
GA suppresses NaHS-induced upregulated production of VEGF in EC109 esophageal cells
As shown in Fig. 6, the level of VEGF was markedly increased in NaHS-induced EC109 cells compared with the control group (P<0.01). However, the increased level of VEGF was significantly suppressed by co-treatment with GA and NaHS.
H<sub>2</sub>S demonstrates proliferation, anti-apoptosis, angiogenesis and migration effects on EC109 esophageal cells via amplifying the activation of HSP90 pathway
We found that NaHS upregulated HSP90 activity resulting in an elevated rate of H2S production level, which in turn modulated protein expressions of caspase-3, bax, bcl-2, MMP-2 and VEGF. The downregulated caspase-3 and bax directly induced anti-apoptosis, led to decreased apoptosis and increased cell viability of EC109 cells. MMP-2 contributes to cancer cell invasion and migration. The increased production of VEGF stimulates angiogenesis, promoting the supply of nutrients and blood to the tumor. Conversely, the above properties of H2S were significantly inhibited by the co-condition of 400 µmol/l NaHS and 20 µmol/l GA for 24 h.
Discussion
In the present study, we demonstrated a novel finding of H2S on esophageal EC109 cells and provided evidence to reveal its potential mechanisms. These findings support our hypothesis that preconditioning with exogenous H2S mediates proliferation, anti-apoptotic, angiopoiesis and migration effects in esophageal cancer. The molecular mechanisms of H2S are not yet fully understood. It is known that H2S is produced in the body mainly by two crucial enzymes, CBS and CSE, which are mainly found in the central nervous system (CNS) (32). A recent study emphasized that H2S played an important role in various physiological and pathological processes of the nervous system as a neuromodulator and neuroprotectant (33). Furthermore, H2S could exert protection to nerve cancer cells, such as PC12 cells (34). However, the performance of H2S on the cancer cells are comparatively complicated and extremely controversial. On the one hand, H2S has shown its anticancer ability based on anti-inflammatory effect, anti-apoptosis and activation of some signal pathways (35,36). On the other hand, H2S can exert totally opposite properties via amplifying the activation of NF-κB pathway (30) and p38 MAPK/ERK1/2-COX-2 pathways (31) in other cancer cells. In order to confirm our hypothesis, EC109 cells were treated with NaHS (a donor of H2S) and some typical pathway-related biomarkers were detected. Unexpectedly, we found two interesting results. Firstly, the optimal concentration of NaHS that induced maximal effect of proliferation was 400 µmol/l, which was in the range of physiological doses of H2S (0.2–1 mmol/l). This indicated that H2S may participate in the esophageal cancer growth. Secondly, previous studies have suggested that HSPs can prevent pro-apoptotic signaling and apoptosis (10). Therefore, it is necessary to assess apoptotic factors and apoptosis in EC109 cells. Treatment of cells with 400 µmol/l NaHS for 24 h markedly diminished cell apoptosis by upregulating the expression of bcl-2 and decreasing the expression of caspase-3 and bax, which are pro-apoptotic Bcl-2 family proteins. Moreover, the aforementioned NaHS-induced effects were inhibited by GA. HSPs can inhibit the activity of pro-apoptotic Bcl-2 proteins to prevent permeabilization of the outer mitochondrial membrane and release of apoptogenic factors (10). The disruption of apoptosome formation represents another mechanism by which HSPs can prevent caspase activation and induction of apoptosis. The aforementioned results were consistent with previous studies (15–17). These data demonstrated that H2S exerted its cell proliferation and anti-apoptosis effects in EC109 cells via activating the HSP90 pathway, and H2S may be involved in esophageal cancer growth under physiological conditions. Given that the previous study showed that H2S-protected PC12 cells from formaldehyde induced apoptosis (34), our findings imply that H2S exerts a cytoprotective effect for EC109 cells.
A large number of experiments have shown that H2S can contribute to VEGF production (37–41). The present study also found that H2S significantly increased the production of VEGF in EC109 cells, and the effect was similarly suppressed by the specific HSP90 pathway inhibitor. VEGF is one of the most potent and pivotal angiogenic factors, and is crucial for the persistent proliferation and metastasis of tumor cells (42). Therefore, we hypothesized that H2S promotes the supply of blood and nutrients to the tumor via angiogenesis effect. Further studies are needed to explore our hypothesis in vivo. It is well known that tumor invasion and metastasis require increased expressions of MMPs. Among the MMPs, MMP-2 and MMP-9 have been thought to be key enzymes in this process since they degrade type IV collagen, which is one of the important components of extracellular matrix (43). Growing evidence reveals that the upregulated expression of MMPs, particularly the gelatinase (MMP-2 and MMP-9), is closely associated with metastasis potential in several types of carcinomas (44–47). The present study demonstrated that HSP90 activation strongly increases the expression of MMP-2 protein in EC109 cells, which indicated that H2S was involved in EC109 cell invasion and migration.
To investigate the complicated mechanism for NaHS induced pro-proliferative effect, anti-apoptosis, angiogenesis and migration in EC109 cells, we studied the HSP90 pathway, which has been previously demonstrated, linked to cancer progression by regulation of cell proliferation, signaling and apoptosis (15–17). It has been reported that HSP90 can be activated by various stimuli both in normal and in cancer cells (10). Herein, we found that NaHS activated HSP90 pathway in EC109 cells. Notably, GA, an inhibitor of HSP90, blocked NaHS-induced HSP90 pathway activation by decreasing expression levels of bcl-2, MMP-2 and VEGF, and increasing caspase-3 and bax expression. These results suggest that HSP90 activation is necessary in NaHS-induced EC109 cell progression.
In conclusion, H2S-induced cell proliferation, anti-apoptosis, angiogenesis and migration in EC109 esophageal cells. These effects may be mediated by the activation of HSP90 pathway, leading to overexpression levels of MMP-2, bcl-2 and VEGF, downregulation of caspase-3 and bax, increased cell viability, and decreased number of apoptotic cells. In esophageal cancer, the findings provide novel insight into a unified concept and identify H2S as an endogenous tumor-promoting factor and anticancer drug target. The deeper mechanism of H2S in EC109 esophageal cells is still unclear and needs to be further investigated.