Ellagic acid induces esophageal squamous cell carcinoma cell apoptosis by modulating SHP‐1/STAT3 signaling

Abstract

Ellagic acid (EA) has been reported to have antiproliferative and antioxidant properties, but its function in esophageal squamous cell carcinoma (ESCC) has not been investigated yet. In the current study, EA was found have a significant anti‐tumor activity in ESCC. In specific, EA inhibited ESCC cell survival in both of a concentration‐ and time‐dependent manner. And our results showed that EA promoted ESCC cell apoptosis, including inducing the cleavages of PARP, and inhibiting the expression of anti‐apoptotic proteins. In mechanistic, EA markedly suppressed STAT3‐driven luciferase activity, and inhibited both of the endogenous and cytokines‐induced STAT3 activation in ESCC cells. Further investigations indicated that EA could significantly upregulate SHP‐1 expression, a negative modulator of STAT3 signaling. In contrast, knockdown of SHP‐1 could attenuate the effects of EA on inhibiting ESCC cell survival. Moreover, we found that EA could inhibit RNF6 expression, an E3 of SHP‐1, and overexpressing RNF6 could also significantly attenuate the effects of EA on inhibiting ESCC cell survival, which further revealed that EA could inhibit STAT3 signaling by modulating RNF6/SHP‐1 axis. Our present study indicated that EA could be as a novel STAT3 inhibitor for the treatment of ESCC.

1. INTRODUCTION

Esophageal squamous cell carcinoma (ESCC) is a common pathological type of esophageal cancer, which accounts for about 90% of the total cases of esophageal cancer. ¹ And China is the country with the highest incidence and mortality of ESCC in the world, accounting for about 50% of the world's new cases every year. ² The typical symptom of ESCC is the progressive dysphagia. ESCC patients cannot swallow dry food, then semi liquid food, and finally water and saliva, which forces the patients to pay close attention to the treatment. ³ In general, early ESCC patients can be treated by surgery, while late ESCC patients can only take radiotherapy, chemotherapy and other comprehensive treatment, and their prognosis is relatively poor. ⁴ Therefore, it is urgent to find effective drugs for the treatment of ESCC.

STAT3 is an important transcription factor which is involved in the tumorigenesis of many tumors, including ESCC. STAT3 is overexpressed and overactivated in ESCC, which can promote the cell proliferation, migration and invasion of ESCC cells, and targeting STAT3 signaling can be an effective strategy for the development of anti‐ESCC drugs. ⁵ Several cytokines and growth factors can trigger STAT3 activation through tyrosine phosphorylation, such as interleukin‐6 (IL‐6), epidermal growth factor (EGF), platelet‐derived growth factor (PDGF), fibroblast growth factor (FGF), and so forth. ⁶ In contrast, the nonreceptor protein tyrosine phosphatase (PTP) named SHP‐1 can downregulate the phosphorylation of STAT3, which is proved to be a negative modulator of STAT3 signaling. ⁷ It has been reported that regorafenib exerted its anti‐tumor effects by suppressing STAT3 signaling through enhancing SHP‐1 activity in colorectal cancer. ⁸ SHP‐1 was also reported to be ubiquitinated and degraded into 26S proteasome, and the ubiquitin ligase RNF6 could activate STAT3 signaling by modulating SHP‐1 ubiquitination in gastric cancer and colorectal cancer.9, 10

In the current study, ellagic acid (EA) was found to exert its anti‐tumor activity by suppressing STAT3 in ESCC cells. In mechanism, EA could inhibit the expression of RNF6 and then stabilize SHP‐1 in ESCC cells. Our present study indicated that EA could be as a potential anti‐tumor drug candidate for the treatment of ESCC.

2. MATERIALS AND METHODS

2.1. Cells, cell culture, and chemicals

ESCC cell lines Eca‐109 and TE‐1 were purchased from the Cell Bank of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). The human normal esophageal epithelial cell line Het‐1A was obtained from ATCC. EC9706, KYSE70, and KYSE450 were kindly provided by Dr Jiali Tao. ¹¹ All cells were cultured in RPMI 1640 medium with 10% FBS and 1% penicillin/streptomycin. EA was purchased from Selleck Chemicals, Houston.

2.2. Cell survival analysis

ESCC cells were treated with increasing concentrations of EA for indicated time, and then cell viability was detected by MTT assay through using the MTT Cell Proliferation and Cytotoxicity Assay Kit (Beyotime, Beijing, China) according to the manufacturer's protocol.

2.3. Cell apoptosis analysis

ESCC cell lines were treated with indicated EA for 24 hours, followed by Annexin V and propidium iodide (PI) staining (Beyotime, Beijing, China). Then, cell apoptosis was analyzed on a flow cytometer.

2.4. Western blot

To detect the proteins' expression, western blot was carried out as described previously. ⁹ Primary antibodies against PARP, Bcl‐2, Mcl‐1, β‐actin, p‐STAT3 (Tyr705), STAT3, and SHP‐1 were obtained from CST, Danvers, Massachusetts. Anti‐Myc tag and RNF6 antibodies were obtained from Sigma‐Aldrich, Inc.

2.5. Luciferase assay

EC9706 cells were transfected with empty vector (EV) or pSTAT3‐Luc plasmids along with renilla plasmids by using Lipofectamine 2000 (Invitrogen) for 24 hours, and then transfected cells were treated with increasing concentrations of EA for 24 hours, followed by luciferase assay by using Dual‐Luciferase Reporter Assay System (Promega, Madison) according to the manufacturer's protocol.

2.6. Cycloheximide (CHX) chase assay

CHX chase assay was carried out as described previously. ¹² In brief, EC9706 cells were treated with 10 μM EA or 50 μg/mL CHX for indicated time, followed by western blot. And at the end, optical density of western blot was analyzed.

2.7. Plasmids, siRNAs, and gene transfection

The human gene RNF6 was amplified by PCR, and subcloned into pcDNA3.1 vector as described previously. ¹² The siRNAs for SHP‐1 were purchased from Ribobio Biotechnology Co., Ltd (Guangzhou, China). The plasmids and siRNAs were transfected into ESCC cells by using Lipofectamine2000 (Invitrogen) according to the manufacturer's protocol.

2.8. Statistical analysis

All the data analyzed in this study were presented as mean ± SD, and the experiments were repeated three times. Student's t test was used to detect the difference between two groups, and ANOVA test was used to detect the differences between multi‐groups. P value less than .05 was considered to have significant difference in this study.

3. RESULTS

3.1. Ellagic acid inhibits cell survival and induces cell apoptosis in ESCC cells

To evaluate the anti‐tumor activity of EA in ESCC, five ESCC cell lines were treated with increasing concentrations of EA. As shown in Figure 1A,B, EA obviously inhibited the cell survival of five ESCC cell lines in a dose‐dependent manner. But EA had little effects on the normal esophageal epithelial cells (Figure 1C). Then, EC9706 and KYSE450 cells were incubated with EA for different time, and it showed that EA markedly suppressed ESCC cell survival in a time‐dependent manner (Figure 1D,E).

FIGURE 1.

Ellagic acid inhibits ESCC cell survival. A, The chemical structure of ellagic acid (EA). B & C, five ESCC cell lines and the normal esophageal epithelial cell line Het‐1A were treated with indicated EA for 24 hours, followed by MTT assay. D & E, EC9706 and KYSE450 cells were treated with indicated EA for 24, 48, or 72 hours, followed by MTT assay. *P < .05, **P < .01

Above results indicated that EA inhibited ESCC cell survival, and it is known that cell apoptosis is closely related to cell survival. Thus, cell apoptosis was then analyzed. As shown in Figure 2A, the flow cytometry showed that EA induced a significant increase of Annexin V⁺ cells of ESCC. Additionally, EA markedly induced the cleavages of PARP (Figure 2B), and also downregulated anti‐apoptotic proteins' expression, including Bcl‐2 and Mcl‐1 (Figure 2C). These results indicated that EA induced ESCC cell apoptosis.

FIGURE 2.

Ellagic acid induces ESCC cell apoptosis. A, EC9706 and KYSE450 were treated with indicated EA for 24 hours, followed by cell apoptosis analysis. B & C, ESCC cells were treated with indicated EA for 24 hours, followed by western blot

3.2. Ellagic acid inhibits STAT3 signaling in ESCC cells

Next, we used luciferase assay to detect the effects of EA on related signaling pathways, and we found EA could significantly suppress STAT3‐driven luciferase activity (Figure 3A), which suggested EA may inhibit STAT3 signaling in ESCC cells. To further confirm it, ESCC cells were incubated with EA, and endogenous phosphorylated STAT3 were examined. As shown in Figure 3B, EA obviously inhibited STAT3 phosphorylation in ESCC cells. To further investigate the specificity of EA in inhibiting STAT3 signaling, ESCC cells were starved and incubated with IL‐6 and EA. As shown in Figure 3C, IL‐6 induced STAT3 activation, but EA markedly attenuated IL‐6‐triggered STAT3 activation in ESCC cells. And IL‐6‐induced p‐STAT3 could partly rescue EA‐induced ESCC cell death (Figure 3D). In addition, we also found that EA‐induced cell death was correlated with endogenous STAT3 activity in different esophageal cell lines (Figures 3E and 1B,C).

FIGURE 3.

Ellagic acid inhibits STAT3 signaling in ESCC cells. A, EC9706 cells transfected with empty vector (EV) or pSTAT3‐Luc plasmids along with renilla plasmids were treated with indicated EA for 24 hours, followed by luciferase assay. B, ESCC cells were treated with indicated EA for 24 hours, followed by western blot. C, Following starvation overnight, EC9706 cells were incubated with indicated EA for 12 hours, then stimulated with IL‐6 for 20 minutes, followed by western blot. D, EC9706 cells were incubated with indicated EA or along with 30 ng/mL IL‐6 for 24 hours, followed by MTT assay. E, Indicated cells were prepared for western blot. *P < .05, **P < .01

3.3. Ellagic acid upregulates SHP‐1 expression in ESCC cells

To further investigate how EA suppressed STAT3 signaling in ESCC cells, the expression of SHP‐1, a negative modulator of STAT3, was evaluated. As shown in Figure 4A, EA markedly upregulated SHP‐1 expression in a dose‐dependent manner. In addition, when ESCC cells were incubated with EA, the half‐life of SHP‐1 was apparently prolonged (Figure 4B,C). Moreover, knockdown of SHP‐1 significantly abolished EA‐induced ESCC cell death (Figure 4D,E). These results indicated that EA inhibited STAT3 signaling by upregulating SHP‐1 expression in ESCC cells.

FIGURE 4.

Ellagic acid upregulates SHP‐1 expression in ESCC cells. A, ESCC cells were treated with indicated EA for 24 hours, followed by western blot. B & C, EC9706 cells were treated with 10 μM EA or 50 μg/mL cycloheximide (CHX) for indicated time, followed by B, western blot, and C, the optical density was analyzed. D & E, EC9706 cells were transfected with siNC, siSHP‐1#1, or siSHP‐1#2 for 24 hours, and then cells were treated with indicated EA for 24 hours, followed by D, western blot and E, MTT. *P < .05

3.4. Ellagic acid inhibits RNF6 expression in ESCC cells

The above finding that EA stabilized SHP‐1 protein prompted us to evaluate RNF6 expression, a ubiquitin ligase of SHP‐1. As shown in Figure 5A, we found that EA markedly inhibited RNF6 expression in ESCC cells. And when ESCC cells were overexpressed with RNF6, the cells were more resistant to EA than the control (Figure 5B,C). Moreover, we found overexpressing RNF6 could partly revert EA‐increased SHP‐1 expression in ESCC cells (Figure 5D,E). Above results suggested EA upregulated SHP‐1 expression by suppressing RNF6 expression in ESCC cells.

FIGURE 5.

Ellagic acid inhibits RNF6 expression in ESCC cells. A, ESCC cells were treated with indicated EA for 24 hours, followed by western blot. B & C, EC9706 cells were transfected with EV or Myc‐RNF6 for 24 hours, and then cells were treated with indicated EA for 24 hours, followed by B, MTT and C, western blot. D & E, EC9706 cells were transfected with EV or Myc‐RNF6 for 24 hours, and then cells were treated with indicated EA for 24 hours, followed by D, western blot, and E, optical density was measured. *P < .05, **P < .01

4. DISCUSSION

Our present study demonstrated the anti‐tumor properties of EA as well as its mechanism of action in ESCC cells. It has been reported that EA has antiproliferative and antioxidant properties, which is found in many vegetables, fruits and nuts, such as pomegranates, strawberries, raspberries, and so forth. ¹³ And many experimental evidences showed that EA displayed significant antitumor, antiangiogenic, and antimetastatic activities. ¹⁴ The anticarcinogenic effect of EA has been shown in some types of tumors, including prostate cancer, lung cancer, osteosarcoma, breast cancer, and so forth.15, 16, 17, 18 Consistent with previous studies, EA also showed good antitumor activity in ESCC. We found that EA inhibited ESCC cell survival by inducing cell apoptosis.

Our further results showed that EA markedly inhibited STAT3 activation in ESCC cells, which was consistent with previous studies reported in HeLa and PC3 cells.19, 20 In the two papers, it was only found that EA downregulated STAT3 phosphorylation in HeLa and PC3 cells, and did not study the specific mechanism of EA, which was very superficial. In our present study, we firstly found that EA could significantly inhibit the luciferase activity driven by STAT3. Then, we detected the endogenous levels of phosphorylated STAT3, and found that EA could significantly inhibit STAT3 activation in ESCC cells. Additionally, we also found that EA could markedly suppress IL‐6‐triggered STAT3 activation in ESCC cells. These results further confirmed that EA could be as a novel inhibitor of STAT3 signaling. Moreover, SHP‐1 was a negative modulator of STAT3 signaling, and SHP‐1 could be degraded into 26S proteasome triggered by the ubiquitin ligase RNF6. ¹⁰ Then, we detected the effect of EA on SHP‐1 and RNF6 expression. And our results showed that EA could markedly upregulate SHP‐1 expression by suppressing RNF6 expression in ESCC cells. And overexpression of RNF6 could apparently attenuate EA‐induced ESCC cell death, which further suggested EA inhibited STAT3 signaling by modulating RNF6/SHP‐1 axis in ESCC cells. However, this article has not specifically evaluated the anti‐ESCC activity of EA in vivo, as well as some pharmaceutical experiments, which will be studied in our future work.

5. CONCLUSION

Collectively, EA displayed anti‐tumor activity by suppressing STAT3 signaling in ESCC. EA could be as a potential drug candidate for the treatment of ESCC.

CONFLICT OF INTEREST

All authors declare no conflict of interest.

Xu Y‐Y, Wang W‐W, Huang J, Zhu W‐G. Ellagic acid induces esophageal squamous cell carcinoma cell apoptosis by modulating SHP‐1/STAT3 signaling. Kaohsiung J Med Sci. 2020;36:699–704. 10.1002/kjm2.12224