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. Author manuscript; available in PMC: 2022 Feb 1.
Published in final edited form as: Anticancer Drugs. 2021 Feb 1;32(2):189–202. doi: 10.1097/CAD.0000000000000997

3,3'-diindolylmethane exerts anti-proliferation and apoptosis induction by TRAF2-p38 axis in gastric cancer

Yang Ye a,1, Fen Ye a,b,1, Xue Li a,1, Qi Yang c, Jianwei Zhou d, Wenrong Xu e, Michael Aschner f, Rongzhu Lu a,g,**, Shuhan Miao h,*
PMCID: PMC7790923  NIHMSID: NIHMS1623338  PMID: 33315588

Abstract

3,3'-diindolylmethane (DIM), an active phytochemical derivative extracted from cruciferous vegetables, possesses anticancer effects. However, the underlying anti-cancer mechanism of DIM in gastric cancer remains unknown. Tumor Necrosis Factor (TNF) receptor associated factor 2 (TRAF2), one of signal transduction proteins, plays critical role in proliferation and apoptosis of human gastric cancer cells, but there still are lack of practical pharmacological modulators for potential clinical application. Here, we further explored the role of TRAF2 in inhibiting cell proliferation and inducing apoptosis by DIM in human gastric cancer BGC-823 and SGC-7901 cells. After treating BGC-823 and SGC-7901 cells with DIM for 24 h, cell proliferation, apoptosis and TRAF2-related protein were measured. Our findings showed that DIM inhibited the expressions of TRAF2, activated p-p38 and its downstream protein p-p53, which were paralleled with DIM-triggered cells proliferation inhibition and apoptosis induction. These effects of DIM were reversed by TRAF2 overexpression or p38 MAPK specific inhibitor (SB203580). Taken together, our data suggest that regulating TRAF2/p38 MAPK signaling pathway is essential for inhibiting gastric cancer proliferation and inducing apoptosis by DIM. These findings broaden the understanding of the pharmacological mechanism of DIM’s action as a new modulator of TRAF2, and provide a new therapeutic target for human gastric cancer.

Keywords: TRAF2; 3,3'-diindolylmethane; gastric cancer; apoptosis; cell proliferation; phytochemicals

1. Introduction

Gastric cancer is one of the most common malignancies and remains the second leading cause of cancer-related mortality worldwide. Despite much more progresses in diagnosis and treatment, gastric cancer is still clinically detected at an advanced stage, thus resulting in higher mortality rates [1, 2]. Therefore, it is essential to develop novel targets to improve chemopreventive and chemotherapeutic efficacy.

Tumor necrosis factor (TNF)-α may cause cancer by disrupting apoptosis and cell survival pathways [3]. However, TNF receptor associated factor 2 (TRAF2), one of transduction proteins, can facilitate TNF binding to specific cytokine receptors [4–6]. TRAF2 deletion exaggerates cell death induced by TNF related apoptosis inducing ligands (TRAIL) and Fas through extrinsic apoptotic pathways [7]. Studies have shown that TRAF2 is closely related to tumors [8, 9], clinically high expression of TRAF2 promotes tumor metastasis and is associated with prognosis of gastric cancer [10, 11]. In addition, TRAF2 can also interact with p38 [12, 13], a member of serine/threonine mitogen-activated protein kinase (MAPK) family which is critical in regulation of cell growth and death in tumor cells [14]. Once phosphorylated, p38 subsequently interacts with various substrates and regulates differentiation, cell cycle, apoptosis and autophagy [15]. Notably, phosphorylation of p38 MAPK delays the progression of colon cancer, chondrosarcoma, gastric cancer, prostate cancer and other cancers [16–19]. Studies have shown that the regulation of TRAF2 and p38 MAPK signaling pathways can play a common role in cells, such as apoptosis [20], suggesting that TRAF2 is related to the p38 MAPK signaling pathway to some extent, p38 MAPK has been reported to interact with TRAF2 and also regulates the growth and death of tumor cells [21], accordingly, TRAF2/p38 MAPK-dependent pathway may be a novel target for preventive and chemotherapy of cancer. However, the mechanism of TRAF2 and p38 MAPK signaling pathways in gastric cancer is still unclear, and the specific regulatory relationship between them needs to be further explored.

3,3'-diindolylmethane (DIM), derived from cruciferous vegetables [22], has been shown to have anticancer effects in vitro and in vivo [23]. DIM inhibited TNF-α and TGF-β induced metastasis and invasion in breast cancer [24], DIM has been reported to inhibit colon cancer growth by activating caspase-8 or down-regulating survivin [25], mediated inhibition of angiogenesis and invasion in prostate cancer by regulate MMP-9 and uPA of NF-κB [26], meanwhile, DIM could up-regulate Ca2+ and activate the p38 MAPK signaling pathway, thereby inhibiting the proliferation and promoting the apoptosis of liver cancer in vitro [27]. Relevant studies have also demonstrated that DIM can inhibit gastric cancer by activating Hippo, Akt/FOXM signaling pathways [28, 29], however, the exact underlying mechanisms for DIM against gastric cancer development and progression can be further expanded. This study investigated the role of TRAF2/p38 pathway in DIM-induced proliferation inhibition and apoptosis induction in human gastric cancer BGC-823 and SGC-7901 cells. We found that DIM as a novel modulator for TRAF2 in gastric cancer cells, activates p-p38 and subsequently inhibits cell proliferation and promotes apoptosis.

2. Materials and methods

2.1. Patients and tissue specimens

A total of 15 formalin-fixed, paraffin-embedded human gastric tissue samples were collected. Samples selected from each patient included cancerous tissues and corresponding adjacent non-cancerous tissues. None of the patients received any treatment (chemotherapy, radiation therapy, and/or immunotherapy) before surgical resection. All tissue blocks were obtained from the Department of Pathology at the Affiliated People’s Hospital of Jiangsu University during January to September, 2019. Signed informed consents were obtained from all subjects and all aspects of the study were approved the Ethics Committee of Jiangsu University (No. 2012258).

2.2. Chemicals

3,3'-diindolylmethane (DIM), p38 MAPK specific inhibitor SB203580, 3-(4, 5-dimethylthiazole-yl)-2, 5-diphenyltetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were purchased from Sigma (St. Louis, MO, USA). Phosphate-buffered saline (PBS) was purchased from Gibco BRL Life Technologies (Grand Island, NY, USA). Polyoxyethylene (Tween 20) sorbitan monolaurate was obtained from Calbiochem (Billerica, MA, USA). In Situ Cell Death Detection Kit was purchased from Roche (Roche, Penzberg, Germany). Primary antibodies against cyclin-dependent kinase 4 (CDK4) (12790T), Cyclin D1 (2978T), p21 (2947T), cleaved caspase-3 (9664T), cleaved PARP (5625T), Bcl-2 (3498T), Bax (2772S), p-p53 (2521T), p-p38 (4511S), p38 (8690T), and TRAF2 (4724T) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Primary antibodies against GAPDH and secondary antibodies (anti-rabbit IgG and anti-mouse IgG) were obtained from Sigma (St. Louis, MO, USA).

2.3. Cell culture

The human gastric cancer cell lines BGC-823, SGC-7901 and human gastric mucosal epithelial cell GES-1 were purchased from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). Cells were cultured in RPMI-1640 medium (Gibco Life Technologies, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Tianhang Biological Technology Co, Ltd, Hangzhou, Zhejiang, China), 100 U/ml penicillin and 100 µg/ml streptomycin (Gibco Life Technologies, Grand Island, NY, USA). All cells were maintained in a 100% humidified atmosphere of 5% CO2 at 37°C.

2.4. Tissue immunohistochemistry (IHC)

Waxed tissue blocks of samples of gastric cancer patients were fixed in 10% formalin, sectioning and embedding. After deparaffinization and rehydration, boiling samples with 10 mmol/L citrate buffer (pH 6.0) for 10 min to retrieve antigen; next washing the slides with PBS. Sections were blocked with 2% bovine serum albumin in PBS for 30 min and then incubated with 1:100 rabbit anti-human TRAF2 antibodies overnight at 4°C. After washing with PBS, sections were treated with streptavidin peroxidase-conjugated secondary antibody (Maixin Biotechnology Co., Ltd. Fuzhou, China). Tissue sections were counterstained with hematoxylin and 3,3'-diaminobenzidine, and observed and recorded under an optical microscope.

2.5. Assessment of cell proliferation (MTT)

For MTT assay, BGC-823/SGC-7901 were seeded in 96-well plates at 3000 cells/well in RPMI-1640 media supplemented with 10% FBS, each condition was set up for six parallel samples, with the cells incubated for 24 h. DIM (0, 10, 20, 40, 60, 80, 100 and 120 μM) treatments were lasted for 12, 24 or 48 h. Next, 10μl 5% MTT (10% of the total volume) was added for additional 3–4 h at 37°C. The reaction was terminated by adding 150 µl DMSO. The absorbance of the reaction mixture was measured at 490 nm. Assays were performed according to the manufacturer’s protocol.

2.6. Cell apoptosis analysis (TUNEL)

Apoptotic cells were determined by terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP)-biotin nick end labeling (TUNEL, Roche Diagnostics, Switzerland). Briefly, 3 ×104 cells were seeded into 24-well plates and incubated for 24 h. The cells were then treated with different concentrations of DIM (0, 20, 40, 60 and 80 µM) for 24 h. After washing with PBS, the cells were fixed in 4% paraformaldehyde for 20 min at room temperature. 3% H2O2 was used to quench endogenous peroxidase for 15 min at room temperature. Next, the cells were processed for 5 min in 0.3% Triton X-100 dissolved in PBS to increase cell membrane permeability. Cell DNA fragments were labeled with peroxidase, which were then reacted with the peroxidase substrate. The apoptosis rate was calculated under common fluorescence microscope.

2.7. Western blotting assay

Cells lysates were prepared in RIPA buffer (Pierce, Rockford, IL. USA), 20 µg of protein of cellular were subjected to 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto PVDF membranes. The membranes were blocked for 2 h with 5% nonfat dry milk/BSA in TBST (Tris-base buffer solution containing of 0.1% Tween 20) at room temperature and incubated with the corresponding primary antibody (all in 1:1000 dilutions in TBST/BondTM Primary Antibody Diluent) overnight at 4°C. Next, the membranes were washed three times with TBST and incubated with secondary antibody for 1 h. Target protein bands were detected with an enhanced chemiluminescence system (Dingguo Bio) according to the manufacturer’s instructions. Bands were then scanned and quantified by Lane software (Beijing Saizhi Pioneering Technology Co. LTD, Beijing, China). GAPDH content was used as an internal loading control. All measurements were conducted in triplicate and repeated independently for at least three times.

2.8. Specific inhibitor treatment

BGC-823 and SGC-7901 cells were pretreated with or without 10 μM p38MAPK specific inhibitor SB203580 for 3 h, followed by treatment with or without 80 μM DIM for another 24 h.

2.9. Total RNA extraction and quantitative real-time PCR (qRT-PCR)

Total RNA was extracted from BGC-823 and SGC-7901 cells using the TRIzol reagent (Invitrogen). Five hundred nanograms of total RNA were reverse-transcribed to cDNA using an RT reagent kit (TaKaRa), which was then amplified with SYBR green dye on a CFX96 Real-Time PCR Detection System (Bio-Rad). The relative quantification of mRNA levels was calculated using the standard 2-DDCt relative quantification method. The primers for human TRAF2 and β-actin were designed and synthesized by Shanghai Generay Biotech Co., Ltd (Shanghai, China), and the sequences were as follows:

TRAF2:

Forward primer: 5’-AGACAAGATTGAAGCCCTGAG-3’.

Reverse primer: 5’-CCGTACCTGCTGGTGTAGAA-3’.

β-actin:

Forward primer: 5’-CTACCTCATGAAGATCCTCACCGA-3’.

Reverse primer: 5’-TTCTCCTTAATGTCACGCACGATT-3’.

2.10. Transfection with overexpression plasmids (TRAF2)

TRAF2 overexpression vectors were designed and synthesized by the Guangzhou GeneCopoeia Company (Guangzhou, Guangdong, China). The BGC-823 and SGC-7901 cells were plated in antibiotic-free medium at 30–40% confluence, approximately 24 h prior to transfection. Following the manufacturer’s instructions, the TRAF2 overexpression plasmids were transfected into BGC-823 and SGC-7901 cells using the Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA). Further treatments were conducted 48 h after transfection. The cells were then treated with or without 80 μM DIM for another 24 h.

2.11. Statistical analysis

Results were expressed as mean ± SD, and data were obtained from at least 3 separately performed experiments. Differences between groups were determined by one-way ANOVA followed by the Student-Newman–Keuls test with the InStat statistical program (GraphPad Software, San Diego, CA, USA). All differences were considered statistically significant at a value of p < 0.05.

3. Results

3.1. DIM inhibits proliferation in BGC-823 and SGC-7901 human gastric cancer cells.

The structural formula and typical vegetable sources of DIM were shown in Fig. 1A and Fig. 1B. DIM significantly inhibited the proliferation of BGC-823 and SGC-7901 cells in a time- and concentration-dependent manner. To investigate the specific effects of DIM on gastric cancer cells, we carried out analogous treatments in non-cancerous human gastric mucosal epithelial GES-1 cells (control cells). After treatment for 24 h, DIM significantly inhibited the proliferation of BGC-823 and SGC-7901 cells at doses of ≥60 μM in a concentration-dependent manner (Fig. 1C), and the proliferation inhibition of GES-1 cells only at concentrations ≥100 μM (Fig. 1D). Compared with GES-1, the decrease of cell proliferation in BGC-823 and SGC-7901 was more pronounced at the same concentrations of DIM, for example, the inhibition of cell viability of BGC-823 and SGC-7901 by DIM increased by 10.7%, 29.0% compared with GES-1 under the treatment of 80 μM DIM for 24h (Fig. 1E).

Fig. 1. Effect of DIM on cell proliferation in BGC-823, SGC-7901 and GES-1 cells.

Fig. 1.

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A: The structural formula of DIM. B: The typical vegetable sources of DIM. C: BGC-823 and SGC-7901 cells were treated with indicated concentrations of DIM for 12, 24, 48 h, cell viability was determined by MTT assay. D: Non-cancerous human gastric mucosal epithelial cell GES-1 cells were treated with the indicated concentrations of DIM for 12, 24 or 48 h, and cell viability was determined with the MTT assay. E: Degree of proliferative inhibition in BGC-823, SGC-7901 cells and GES-1 cells upon 24 h DIM treatment. F: Protein expression was measured after treatment of DIM for 24 h of CDK4, Cyclin D1, and p21 in the BGC-823 and SGC-7901 cells, GAPDH was used as a loading control. G: Each band density of p21, CDK4, and Cyclin D1 was quantified and normalized to the expression of GAPDH; each value represents the mean ± SD of triplicates (*p < 0.05).

Expression of proliferation and cell cycle-related protein markers in BGC-823 and SGC-7901 cells were analyzed by means of western blotting. The protein level of anti-proliferative p21, one of cyclin-dependent kinase inhibitors, was significantly increased in a concentration-dependent manner after 24 h treatment with DIM. However, two basic regulators of cellular G1-S transition (Cyclin D1 and CDK4) were decreased (Fig. 1F–G).

3.2. DIM induces apoptosis in BGC-823 and SGC-7901 human gastric cancer cells.

TUNEL analysis confirmed that the number of apoptotic cells significantly increased in BGC-823 and SGC-7901 cells treated with DIM. DIM treatment (20, 40, 60 or 80 μM) increased the apoptotic rates to 3.5%, 14%, 34% and 83%, respectively (only 1.5% in the control). A similar trend was observed in SGC-7901 cells treated with DIM, increased the apoptotic cells to 3%, 6%, 32% and 80% respectively (0.5% in the control) (Fig. 2A–B). These results suggested that DIM enhanced apoptosis in gastric cancer cells in a concentration-dependent manner.

Fig. 2. Effect of DIM on apoptosis in BGC-823 and SGC-7901 cells.

Fig. 2.

Fig. 2.

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A: The BGC-823 and SGC-7901 cells were treated with various concentrations of DIM for 24 h and subsequently stained with TUNEL, observed with a common fluorescence microscope. Scale bars, 100 µm. B: The ratio of apoptotic cells treated with DIM in BGC-823 and SGC-7901 cells, respectively (*p < 0.05). C: Protein expression of cleaved PARP, cleaved caspase-3, Bax, and Bcl-2 in the BGC-823 and SGC-7901 cells upon DIM treatment, GAPDH was used as a loading control. D: Each band density was quantified and normalized to the expression of GAPDH. E: The Bax/Bcl-2 ratio of various DIM concentrations was calculated in BGC-823 and SGC-7901 cells. Each value represents the mean ± SD of triplicates (*p < 0.05).

To further address the pro-apoptotic mechanism of DIM in BGC-823 and SGC-7901 cells, the expression of Bcl-2, Bax, cleaved PARP and cleaved caspase-3 were evaluated by western blotting. Cleaved PARP and cleaved caspase-3 were increased in BGC-823 and SGC-7901 cells, but the expression of the anti-apoptotic protein Bcl-2 was decreased. Furthermore, levels of the pro-apoptotic protein Bax were significantly increased in BGC-823 and SGC-7901 cells treated with DIM at 20, 40, 60 or 80 μM compared to the control group (Fig. 2C–D). Additional analysis illustrated that DIM induced a significant increase in the Bax/Bcl-2 ratio (Fig. 2E).

3.3. Inhibition of gastric cancer cell proliferation by DIM is dependent on the activation of the p38 MAPK pathway.

The expression of p-p38, and phosphorylated p53 (p-p53) were up-regulated after 24 h treatment with DIM both in BGC-823 and SGC-7901 cells (Fig. 3A–B), while the expression of total p38 did not change. However, when p38 MAPK specific inhibitor SB203580 was added prior to DIM treatment, proliferation inhibition of BGC-823 and SGC-7901 cells by DIM was attenuated (Fig. 3C). Furthermore, effects of DIM on the expression of p-p53, proliferation-related proteins (p21, CDK4, and Cyclin D1), and apoptosis-related proteins (cleaved PARP, cleaved caspase-3, Bax, and Bcl-2) were also partially reversed (Fig. 3D–E). Treatment with SB203580 alone (100 nM, 1 μM, 10 μM, and 20 μM) did not affect cell proliferation (data not shown). These results suggested that p38 MAPK pathway was involved in DIM-induced inhibition of gastric cancer.

Fig. 3. Effect of DIM and/or p38 MAPK specific inhibitor SB203580 on cells proliferation and apoptosis related protein expression.

Fig. 3.

Fig. 3.

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A: The expression of p-p38 and p-p53 in BGC-823, SGC-7901 cells after treatment with DIM for 24h, respectively. GAPDH was used as a loading control. B: Each band was quantified and normalized with density of GAPDH, each value represents the mean ± SD of triplicates (* p < 0.05). C: BGC-823 and SGC-7901 cells were treated with DIM and with or without SB203580, respectively, cell proliferation was determined by MTT assay (* p < 0.05). D: Protein expression of p-p53, p21, Cyclin D1, CDK4, cleaved PARP, cleaved caspase-3, Bax, and Bcl-2 was measured after treatment of SB203580 and DIM in the BGC-823 and SGC-7901 cells, GAPDH was used as a loading control. E: Lane 1, control; Lane 2, DIM alone; Lane 3, DIM with SB203580. Each band was quantified and normalized with expression of GAPDH, each value represents the mean ± SD of triplicates (* p < 0.05).

3.4. TRAF2 is highly expressed in human gastric cancers.

Among the 15 samples, the expression of TRAF2 in 12 patients’ cancer tissues was significantly elevated compared to the adjacent gastric tissues (Fig. 4A). TRAF2 expression was also significantly higher both in BGC-823 and SGC-7901 cells compared to non-cancerous human gastric mucosal epithelial cell (GES-1), 25% and 88% higher than that in GES-1 cells, respectively (Fig. 4B).

Fig. 4. Expression of TRAF2 in human cancer tissues and BGC-823, SGC-7901 cells, and its effect on proliferation and apoptosis of gastric cancer cells after TRAF2 knockdown.

Fig. 4.

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A: Representative photomicrographs of immunohistochemical staining of TRAF2 in paraffin sections of human gastric cancer and adjacent tissues. (a): cancerous tissue, (b): adjacent non-cancerous tissue. Scale bars, 100 µm. B: The expressions of TRAF2 protein level in non-cancerous human gastric mucosal epithelial GES-1 cell, gastric cancer BGC-823 and SGC-7901 cells. Each value represents the mean ± SD of triplicates (* p < 0.05).

3.5. DIM regulates proliferation and apoptosis through the TRAF2/p38 MAPK pathway.

DIM significantly down-regulated the protein expression of TRAF2 in BGC-823 and SGC-7901 cells in a concentration-dependent manner (Fig. 5A). In addition, we analyzed the expression of the TRAF2 gene with qRT-PCR. As shown in Figure 5B, DIM treatment did not affect TRAF2 mRNA levels. We may implicate that DIM had a major effect on TRAF2 expression at the protein level, with no significant effect at the transcriptional level. To further clarify the role of TRAF2, we determined the inhibitory effect of DIM on proliferation in gastric cancer cells was significantly ameliorated by TRAF2 overexpression (Fig. 5C).

Fig. 5. Effect of DIM on TRAF2 expression and combined effect of DIM and TRAF2 overexpression plasmids on cell proliferation and apoptosis-related proteins expression.

Fig. 5.

Fig. 5.

Fig. 5.

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A: Protein expression of TRAF2 after treatment with DIM for 24 h of TRAF2 in the BGC-823 and SGC-7901 cells. GAPDH was used as a loading control. Quantification from three independent measures of each band was shown. B: BGC-823 and SGC-7901 cells were treated with DIM (0, 20, 40, 60, or 80 μM, 24 h), TRAF2 mRNA was evaluated. The density of mRNA was normalized to β-actin and plotted relative to the control. C: BGC-823 and SGC-7901 cells were treated with or without TRAF2 overexpression plasmid, cell proliferation was determined by MTT assay. D: Protein expression of p-p38, p-p53 after treatment with TRAF2 overexpressed plasmids and DIM in the BGC-823 and SGC-7901 cells, GAPDH was used as a loading control. E: Protein expression of proliferation and apoptosis-related proteins after treatment with TRAF2 overexpression plasmids and DIM in the BGC-823 and SGC-7901 cells, GAPDH was used as a loading control. F: Lane 1, control; Lane 2, DIM; Lane 3, DIM with TRAF2 overexpression plasmids; Lane 4, DIM with negative control. Each band was quantified and normalized with expression of GAPDH, each value represents the mean ± SD of triplicates (*p < 0.05).

TRAF2 overexpression also significantly blocked the up-regulation of p-p38 and p-p53 by DIM (Fig. 5D). In addition, TRAF2 overexpression reversed the DIM-induced inhibition of CDK4, Cyclin D1 and Bcl-2. However, DIM-induced expression of p21, cleaved PARP, cleaved caspase-3 and Bax were markedly attenuated by TRAF2 overexpression (Fig. 5E–F).

4. Discussion

In this study, it was demonstrated for the first time that DIM inhibited the expression of TRAF2 and activated the p38 MAPK signaling pathway, resulting in proliferation inhibition and apoptosis induction. These effects were reversed by TRAF2 overexpression and p38 MAPK inhibition. These findings suggest TRAF2 may be a novel target for chemotherapy of gastric cancer.

Our research group carried out an in-depth study on the anticancer effect of DIM [30], we recently reported that DIM can inhibit gastric cancer both in vivo and in vitro by regulating autophagy [31], and that DIM can activate p-p38 by releasing calcium ions to induce cell apoptosis and inhibit liver cancer proliferation [27]. Inhibition of cell proliferation and promoting apoptosis is one of the main methods to study tumor inhibition [32, 33], our research direction is mainly based on this. Given that the p38 MAPK pathway is involved in the inhibition of various cancers by exerting its anti-proliferation, pro-apoptotic and autophagic effects [34], we considered whether the p38 MAPK signaling pathway was involved in DIM inhibition of gastric cancer. Our experimental results showed that the DIM suppression of gastric cancer is associated with the activation of the p38 MAPK signaling pathway, and the proliferation inhibition and apoptosis induction by this pathway mediated gastric cancer cells.

TRAF2 has been reported to regulate TNF and NF-κB signaling to inhibit apoptosis of cancer cells [35], high expression of TRAF2 can inhibit TRAIL-induced apoptosis of tumor cells [36], meanwhile, our previous studies have also confirmed that DIM can enhance TRAIL and induce apoptosis of gastric cancer cells [7], so can DIM directly regulate the expression of TRAF2 in gastric cancer cells to trigger apoptosis? Relevant literature showed that high expression of TRAF2 can promote the development of tumor [10], our results indicate that TRAF2 is significantly overexpressed in gastric cancer tissues and cells (Fig. 4A–B), and DIM can down-regulate the protein expression level of TRAF2 in a concentration-dependent manner, we began to explore the possible regulatory mechanisms of TRAF2 in DIM inhibition of gastric cancer cells. Reports indicate that berberine [37, 38], 2α- hydroxyursulic acid [34], biotin A (BA) and ginsenoside Rh in combination [40] can all play an anticancer effect through down-regulating TRAF2 and then activating p38 MAPK signaling pathways. We speculated that phosphorylation of p38 MAPK representing a downstream effector of TRAF2 inhibition in DIM’s mechanism of inhibiting gastric cancer cells. Indeed, when we applied SB203580 or TRAF2 overexpression plasmids, the expression of downstream proteins of p-p38 in DIM-treated cells was reversed, proliferation inhibition and apoptosis promotion by DIM was attenuated (Fig. 3C, 5C). Notably, when SB203580 and DIM were combined to treat gastric cancer cells, TRAF2 expression was not significantly changed compared with the DIM group (data did not show), while p-p38 levels were significantly lower with the TRAF2 overexpressed plasmid and DIM than with DIM alone, the above results are consistent with our hypothesis. Therefore, we may imply that TRAF2 was the upstream regulatory protein of p38 MAPK pathway in the inhibition of gastric cancer by DIM and that the TRAF2/p38 MAPK pathway plays critical role in DIM inhibited gastric cancer.

Activated p53 induces apoptosis and autophagy in human tumors [41, 42], give full play to its anti-cancer ability [43], p53 activation ensue via the p38 MAPK pathway [44]. In addition, we found DIM enhanced p-p38 and p-p53 (Fig. 3A), both overexpression of TRAF2 and inhibition of p38MAPK by SB203580 attenuated the enhancement of p-p53 (Fig. 3D–E, 5D). Thus we may verify our hypothesis that DIM inhibits TRAF2 to activate p-p38, subsequently activate p-p53 to regulate proliferation and apoptosis in gastric cancer (Fig. 6). These findings deepen the understanding of the pharmacological mechanism of DIM action as a novel modulator of TRAF2, and provide a new putative therapeutic target for gastric cancer.

Fig. 6. Potential mechanisms of action of DIM in regulating cell proliferation and apoptosis in BGC-823 and SGC-7901 human gastric cancer cells through down-regulating TRAF2.

Fig. 6.

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After treated with DIM, TRAF2 was downregulated, p-p38 and p-p53 was activated. On the one hand, activated p-p53 inhibits the proliferation of gastric cancer cells by up-regulating p21 to inhibit CDK4 and CyclinD1. On the other hand, p-p53 promotes the apoptosis of gastric cancer cells by activating Bax and inhibiting Bcl-2 expression, increased the expression of cleaved caspase-3, and cleaved PARP. Ultimately realizes the inhibition of gastric cancer cells.

However, further studies are needed to better understand the underlying mechanisms of DIM-modulating TRAF2 and to identify the direct binding sites of DIM with TRAF2. Moreover, animal experiments are essential to confirm the functional and efficacious concentrations of DIM as TRAF2 modulators for chemoprevention or chemotherapy of gastric cancer.

5. Conclusion

For the first time, we offer evidence that the TRAF2 can be used as the target of DIM in inhibiting gastric cancer. Our results showed that TRAF2 is highly expressed in gastric cancer tissues and cells compared with normal tissues and cells. DIM inhibited the proliferation and promoted apoptosis of gastric cancer cells, accompanied by suppressing TRAF2 expression and the activation of p38 MAPK. In conclusion, DIM may represent a novel candidate regulator for TRAF2, and targets TRAF2/p38 MAPK signal pathway to increase apoptosis and inhibit proliferation in gastric cancers.

Acknowledgements

The present study was supported by the National Natural Science Foundation of China (81502800), Jiangsu University youth talent cultivation program 2016, Young Science and Technology Talent Support Project of Jiangsu Association of Science and Technology 2018 and the Research Foundation for Advanced Talents of Jiangsu University (14JDG044). Partial support (MA) was provided by grants from the National Institute of Environmental Health sciences R01 ES10563 and R01 ES07331.

Abbreviations:

CDK4

cyclin-dependent kinases-4

DIM

3,3'-diindolylmethane

MAPK

mitogen-activated protein kinase

NF-κB

nuclear factor-κB

PARP

poly(ADP-ribose) polymerase

ROS

reactive oxygen species

TGF

transforming growth factor

TNF

tumor necrosis factor

TRAF2

TNF receptor associated factor 2

TRAIL

TNF related apoptosis inducing ligands

Footnotes

Conflict of interest

Nothing to declare.

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