Raf/ERK/Nrf2 signaling pathway and MMP-7 expression involvement in the trigonelline-mediated inhibition of hepatocarcinoma cell migration

Abstract

Background: Trigonelline occurs in many dietary food plants and has been found to have anti-carcinogenic activity. Trigonelline is also found in coffee which is one of the most widely consumed beverages. Many epidemiological studies have reported that coffee consumption has an inverse relationship with the risk of cirrhosis or hepatocellular carcinoma. It would be interesting to investigate whether trigonelline is an ideal chemoprevent agent to prevent cancer progression.

Methods: The protein expression was performed by western blotting. The trigonelline content in snow pea (Pisum sativum) was analyzed by high-performance liquid chromatography (HPLC). The migratory activity of human hepatocarcinoma cells (Hep3B) was assessed by using a wound migration assay. The percentage of each phase in the cell cycle was analyzed on a FACScan flow cytometer. Gene expression was detected by real-time reverse transcriptase-polymerase chain reaction techniques. Native gel analysis was performed to analyze the activity of superoxide dismutase (SOD), catalase and glutathione peroxidase.

Results: According to the data of HPLC analysis, P. sativum, which is a popular vegetable, has relatively high content of trigonelline. Our findings suggest that trigonelline is an efficient compound for inhibiting Hep3B cell migration. Trigonelline inhibited the migration of hepatoma cells at concentrations of 75-100 µM without affecting proliferation. Raf/ERK/Nrf2 protein levels and further downstream antioxidative enzymes activity, such as SOD, catalase, and glutathione peroxidase, significantly decreased after treatment with 100 µM of trigonelline for 24 h. The migration inhibition of trigonelline is also related to its ability to regulate the matrix metalloproteinases 7 (MMP-7) gene expression.

Conclusions: In this study, protein kinase Cα (PKCα) and Raf/ERK/Nrf2 signaling pathway and MMP-7 gene expression were involved in the trigonelline-mediated migration inhibition of Hep3B cells. We also demonstrated that trigonelline inhibits Hep3B cell migration through downregulation of nuclear factor E2-related factor 2-dependent antioxidant enzymes activity. This study analyzed the trigonelline content in a popular vegetable, snow pea, as a representative proof to prove that trigonelline is often found in the daily intake of food. Our finding suggested that trigonelline should be a useful chemopreventive agent derived from the daily intake of food to prevent cancer progression.

Keywords: MMP-7; Pisum sativum; Raf/ERK/Nrf2 signaling pathway; chemotherapeutic agent; hepatocarcinoma Hep3B cells; migration; trigonelline.

Fig. 1

The trigonelline content in P. sativum was analyzed by HPLC. (A) Snow pea used in this study was purchased from a traditional market in December in Taiwan Taichung city. (B) Pure trigonelline (5 µg/ml) showed a retention time of 1.728 min. HPLC analysis of the n-hexane (C), methanol (D), n-butanol (E), and water (F) soluble fractions of P. sativum exhibited one peak about at 1.72 min, which was merged with that of trigonelline standard. For qualitative analysis of trigonelline in the fractions of P. sativum, 15 µg/ml trigonelline was added to the four fractions of P. sativum and then analyzed by HPLC (C′, D′, E′, and F′). Results are representative of three independent experiments.

Fig. 2

Evaluation of cytotoxicity after incubation of Hep3B cells with trigonelline. Cells were incubated with vehicle alone or with 50, 75, or 100 µM trigonelline for 24 and 48 h. After incubation, the viable cells were measured by MTT assay. The data are presented as proportional viability (%) by comparing the treated group with the untreated group, the viability of which was assumed to be 100%. All results are expressed as the mean percentage of control ±SD of triplicate determinations from four independent experiments.

Fig. 3

The effects of trigonelline on the protein levels of Nrf2 (pSer40), Nrf2 upstream kinases, and Nrf2-regulated detoxification genes in Hep3B cells. The effects of trigonelline on the protein levels of PKCα, c-Raf (pSer259), ERK (pThr202/Tyr204), p38 (pThr180/Tyr182), Nrf2 (pSer40), catalase, Cu/Zn-SOD, and Mn-SOD were detected by western blot analysis. Cells were incubated with vehicle alone or with 50, 75, or 100 µM trigonelline for 24 and 48 h. Cell lysates were subjected to SDS–PAGE [6% for Nrf2 (pSer40); 7% for PKCα; 8% for c-Raf (pSer259); 10% for catalase; 12% for ERK (pThr202/Tyr204), and p38 (pThr180/Tyr182); 13% for caspase-3, caspase-9, and Mn-SOD; and 15% for Cu/Zn-SOD], and then probed with primary antibodies as described in Materials and Methods section. Results are representative of three independent experiments.

Fig. 4

Effect of trigonelline on the migration of Hep3B cells as revealed by the wound assay. Hep3B cells were wounded as described in the Materials and Methods section. After washing, fresh culture medium, containing vehicle alone or various concentrations of trigonelline, was added. Photographs were taken after 48 h of incubation in the absence (A) or the presence (B–D) of trigonelline. Concentrations of trigonelline: 50 µM (B), 75 µM (C), and 100 µM (D). Results are representative of three independent experiments.

Fig. 5

Effects of trigonelline on the mRNA expression of MMP-2, -7, and -9 in Hep3B cells. Trigonelline-induced gene expression of MMP-2, -7, and -9 was detected by real-time RT-PCR. Hep3B cells were incubated with vehicle alone or with 50, 75, or 100 µM trigonelline for 24 h. RNA samples were prepared from control and trigonelline-treated cells. Results were expressed as fold changes and were normalized to GAPDH. Data are represented as mean±SD. *p<0.05 compared to the control values. Results are representative of three independent experiments.

Fig. 6

Effects of trigonelline on superoxide dismutase (SOD), catalase, and glutathione peroxidase (Gpx) activity in Hep3B cells. Cells were treated with vehicle alone or with 50, 75, or 100 µM trigonelline for 24 and 48 h. Native gel analysis was performed to analyze the activity of SOD, catalase and glutathione peroxidase. Proteins were separated by electrophoresis through a 10% native PAGE gel. Activities of SOD, catalase, and glutathione peroxidase were analyzed as described in Materials and Methods section. Results are representative of three independent experiments.