| BackgroundGastric cancer (GC) is the fourth most common cancer and the second leading cause of cancer deaths worldwide. The formation, progression, invasion and metastasis of tumor is a complex, multistep process. Cyclooxygenase-2 (COX-2) is upregulated and plays an important role in carcinogenesis of gastric cancer. Deregulation of COX-2 expression leads to an increased abundance of PGs, specifically PGE2, which can potentially affect most of the events in cancer development, including proliferation, resistance to apoptosis, angiogenesis, immune suppression and invasion. Because of its important role in tumor formation, progression, invasion and metastasis, COX-2 is considered as a promising target for cancer therapy.Nonsteroidal anti-inflammatory drugs (NSAIDs), some of the most commonly used drugs around the world, could inhibit both isoenzymes (COX-1 and COX-2). A large body of evidence from epidemiological studies and clinical trials indicated that NSAIDs are effective in preventing adenoma recurrence and reducing the incidence of gastric cancer to some extent. However, the prolonged use of non-selective NSAIDs is associated with side effects such as abdominal pain, peptic ulcer, gastrointestinal bleeding and/or perforation of gastroduodenal ulcers. The cardiovascular side effects of COXIBs limit their use in healthy individuals. Therefore, immense effort has been devoted to developing molecules that could inhibit COX-2 with fewer side effects.Artesunate, a derivative of artemisinin isolated from Artemisia annua L., has been approved by the Chinese government for the treatment of malaria, especially against cerebral malaria. Studies demonstrated that it possesses a number of biological activities, including hepatoprotective, antiviral, anti-inflammatory, antioxidative, anti-allergic, antidiabetic, and antibacterial effects. Artesunate has been shown to exhibit anti-proliferative effects and apoptosis-inducing activities in various types of tumor cell lines in vitro and in vivo. However, whether artesunate has any function on COX-2 in gastric cancer has not yet been reported. It was found that the levels of EP1, one of the PGE2 receptor (EP), increased in gastric cancer. We postulate that COX-2 and metabolic product, PGE2, and EP1 may be the important mechanism involved in the effect of artesunate in gastric cancer. Here, in the present study, we investigated whether artesunate inhibited gastric cancer cell growth, invasion and metastasis by reducing COX-2 expression, PGE2 synthesis and EP1 expression.Part 1 Artesunate inhibits cell proliferation and induces apoptosis on human gasric cancer cells through inhibition of COX-2 expression, PGE2 synthesis and EP1 expressionPurpose To observe the effect of artesunate on cell proliferation and apoptosis, and investigate whether artesunate inhibits cell proliferation and induces apoptosis by targeting COX-2, PGE2 and EP1 in gastric cancer cells.Methods The gastric cancer cells (HGC-27, BGC-823 and MGC-803) served as the experimental subjects. After treatment by artesunate at different concentrations respectively at different time, the cell survival was determined by the MTT method. HGC-27 cells, as the most sensitive of three types of cancer cells, were treated with PGE2 or ONO-DI-004 and with and without artesunate for different time and the cell survival was determined. HGC-27 cells were cultured in vitro. After treatment by artesunate at different concentrations for 48 h, cell apoptosis was detected by flow cytometry. Western blot were used for COX-2, EP1, Bax and Bcl-2 proteins analysis. Spectrophotometer was used to detect the cell Caspase-3 and Caspase-9 activities. The expression of PGE2 in the culture medium was detected by ELISA. The mitochondrial membrane potential was evaluated by flow cytometry analysis after Rho123 probe staining. MTT method and flow cytometry were uesed to detected the growth and apoptosis of colon cancer cell line treated with celecoxib. Chemically synthesized siRNA targeting COX-2 (COX-2 siRNA) was transfected into HGC-27 cells, and the expressions of COX-2 mRNA and protein were analyzed by RT-PCR and Western blot, then MTT method and flow cytometry were uesed to detected the growth and apoptosis of gastric cancer cell line.Results After treatment with artesunate, the proliferation of these cell lines was significantly inhibited, especially in the HGC-27 cells, which showed a dose-and time-dependent manner. Flow cytometry assays demonstrated that artesunate significantly induced apoptosis in HGC-27 cells. Artesunate reduces COX-2 expression, prostaglandin E2 (PGE2) synthesis and EP1 expression in HGC-27 cells. Treatment with PGE2 or ONO-DI-004 promoted the cell proliferation and artesunate could inhibit PGE2-or ONO-DI-004-induced proliferation of HGC-27 cells. Celecoxib inhibited cell proliferation and induced apoptosis in HGC-27 cells. After trasfection of COX-2 siRNA, the expressions of COX-2 mRNA and protein decreased, and we could observe inhibition of cell proliferation and induction of apoptosis. Treatmentwith increasing doses of artesunate led to increased expression of Bax and decreased expressions of anti-apoptotic Bcl-2, and the stimulation of caspase-3 and caspase-9 activity, as well as decreased mitochondrial membrane potential.Conclusions Artesunate could inhibit cell proliferation and induce apoptosis in a in gastric cancer cells, which was associated with a ruduction in the levels of COX-2, PGE2 and EP1.Part 2 Artesunate inhibits human gastric cancer cell invasiveness by inhibition of epithelial-to-mesenchymal transition via reduction of COX-2 expression, PGE2 synthesis and EP1 expressionPurpose To explore the effect of artesunate on the invasive potential of human gastric cancer cells and investigate whether artesunate inhibits cell invasiveness by inhibition of epithelial-to-mesenchymal transition via reduction of COX-2 expression, PGE2 synthesis and EP1 expression.Methods After treatment by artesunate at different concentrations for 24 h, the invasion and migration of HGC-27 cells were tested by transwell assay and wound healing assay. The expression of E-cadherin was analyzed by immunofluorescence staining. And the proteins related to PI3K/Akt, NF-?B, and ERK/MAPK signaling pathway and EMT were analyzed by Western blot. HGC-27 cells were treated with PAM, PGE2, or ONO-DI-004 and with and without artesunate, and transwell assay and Western blot were carried out. The transwell assay and Western blot were carried out after treatment with celecoxib and PDTC, and trasfection of COX-2 siRNA.Results After treatment with artesunate, the invasion and migration of HGC-27 cells were inhibited. Artesunate reduced COX-2 expression, PGE2 synthesis and EP1 expression and inhibited the proteins related to PI3K/Akt, NF-?B, and ERK/MAPK signaling pathway and EMT. Treatment with PMA, PGE2, or ONO-DI-004 promoted the cell invasion and artesunate could inhibit PMA-, PGE2-, or ONO-DI-004-induced invasion of HGC-27 cells. Celecoxib inhibited cell invasion. After trasfection of COX-2 siRNA, the invasion and the proteins related to PI3K/Akt and ERK/MAPK signaling pathway and EMT of HGC-27 cells was inhibited.Conclusions Artesunate inhibits human gastric cancer cell invasiveness, and the underlying mechanism may be its reversal of epithelial-to-mesenchymal transition by inhibition of I3K/Akt and ERK/MAPK signaling pathway via reduction of COX-2 expression, PGE2 synthesis and EP1 expression. |
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