| BackgroundColorectal cancer, a malignant tumor of the digestive system, is the second most common cause of cancer-related mortalities. Invasion and metastasis are major causes of treatment failure in patients with colorectal cancer. The formation, progression, invasion and metastasis of tumor is a complex, multistep process. While perturbation of the WNT-signalling pathway is believed to account for the initiation of colorectal tumours, the aberrant expression of cyclooxygenase-2 (COX-2) that occurs in the majority of colorectal tumours is thought to play a crucial role during colorectal cancer development. Deregulation of COX-2 expression leads to an increased abundance of prostaglandin E2 (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 colorectal 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.The evidence that treatment of familial adenomatous polyposis (FAP) patients with celecoxib significantly reduced the polyp burden in a randomized controlled trial led to the FDA approving celecoxib (brand name Celebrex) for use in patients with FAP at 400 mg twice a day. Therefore, immense effort has been devoted to developing molecules that could inhibit COX-2 with fewer side effects.Paeonol (2-hydroxy-4-methoxyacetophenone), a major active extract from the root bark of Paeonia suffruticosa Andrews, possesses a number of biological activities, including anti-inflammatory, anti-oxidant, antiangiogenic, anti-allergic and anti-oxidation. Paeonol 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 paeonol has any function on COX-2 in colorectal cancer has not yet been reported. We postulate that COX-2 and metabolic product, PGE2, may be the important mechanism involved in the effect of paeonol in colorectal cancer. Here, in the present study, we investigated whether paeonol inhibited colorectal cancer cell growth, invasion and metastasis by reducing COX-2 expression and PGE2 synthesis.Part 1 Paeonol inhibits cell proliferation and induces apoptosis on human colorectal cancer cells through inhibition of COX-2 expression and PGE2 synthesisPurpose To observe the effect of paeonol on cell proliferation and apoptosis, and investigate whether its antitumor effect is associated with reduction in COX-2 expression and a decrease in the levels of PGE2 in colorectal cancer cells.Methods The colorectal cancer cells (LoVo, HCT-116 and SW620) served as the experimental subjects. After treatment by paeonol at different concentrations respectively at different time, the cell survival was determined by the MTT method. LoVo cells, as the most sensitive of three types of cancer cells, were treated with PGE2 and with and without paeonol for different time and the cell survival was determined. LoVo cells were cultured in vitro. After treatment by paeonol at different concentrations for 48 h, cell apoptosis was detected by flow cytometry. Western blot were used for COX-2, Bax, Bcl-2, Caspase-3 and Caspase-9 proteins analysis.The concentration of intracellular calcium was investigated by laser confocal sanning microscope with calcium-flourecent probes-Fluo-3/AM. 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 LoVo 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 colon cancer cell line.Results After treatment with paeonol, the proliferation of these cell lines was significantly inhibited, especially in the LoVo cells, which showed a dose-and time-dependent manner. Flow cytometry assays demonstrated that paeonol blocked the cell cycle at the G1 to S transition and significantly induced apoptosis in LoVo cells. Paeonol reduces cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) synthesis in LoVo cells. Treatment with PGE2 promoted the cell proliferation and paeonol could inhibit PGE2-induced proliferation of LoVo cells. Celecoxib inhibited cell proliferation and induced apoptosis in LoVo 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 paeonol led to intracellular calcium accumulationoccurred, and increased expression of Bax and decreased expressions of anti-apoptotic Bcl-2, and the stimulation of caspase-3 and caspase-9 activity, and decreased mitochondrial membrane potential.Conclusions Paeonol could inhibit cell proliferation and induce apoptosis in a in colorectal cancer cells, which was associated with a ruduction in the levels of COX-2 and PGE2.Part 2 Paeonol inhibits human colorectal cancer cell invasiveness by inhibition of epithelial-to-mesenchymal transition via reduction of COX-2 expression and PGE2 synthesisPurpose To explore the effect of paeonol on the invasive potential of human colorectal cancer cells and investigate whether paeonol inhibits cell invasiveness by inhibition of epithelial-to-mesenchymal transition via reduction of COX-2 expression and PGE2 synthesis.Methods After treatment by paeonol at different concentrations for 24 h, the invasion and migration of LoVo 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-κB, and ERK/MAPK signaling pathway and EMT were analyzed by Western blot. LoVo cells were treated with PAM and with and without paeonol, 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 paeonol, the invasion and migration of LoVo cells were inhibited. Paeonol reduced COX-2 expression and PGE2 synthesis and inhibited the proteins related to PI3K/Akt, NF-κB, and ERK/MAPK signaling pathway and EMT. Treatment with PMA promoted the cell invasion and paeonol could inhibit PMA-induced invasion of LoVo 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 LoVo cells was inhibited.Conclusions Paeonol inhibits human colorectal 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 and PGE2 synthesis. |
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