The Effects Of Evodiamine On Proliferation And Migration Of Human Colorectal Cancercells In Vitro And In Vivo And Its Mechanism

Colon carcinoma is a common form of gastrointestinal malignant tumor in gastrointestinal tract, the mortality rate of colon carcinoma occupied the third in the world. Currently, surgery combine chemotherapy is the first choice of the treatment for colon carcinoma. The chemotherapy drugs will cause injury to normal tissue cells in varying degrees while it make effects on anti-tumor cells, and a part of patients abandoned chemotherapy treatment for the pain. Hence, searching for high efficacy and low toxicity anti-cancer drugs is becoming the current focus.Evodiamine(Evo), an alkaloid isolated from the Evodia rutaecarpa Bentham(Rutaceae), has a potential anti-tumor effects on a number of human cancer cells. However, the effects of evodiamine on human colorectal cancer cells and the underlying molecular mechanisms have been poorly determined.Mammalian silent information regulator1(SIRT1) belongs to the sirtuin family of histone deacetylase enzymes, whic is the analogues of silencing information regulator 2(sir2) in human. SIRT1 could make varieties protein’ lysine residues to acetylation relying on NAD +. SIRT1 mainly through deacetylating k26 lysine residues on the H1 histone, k9 lysine residues on the H3 histone and k16 lysine residues on the H4 to modify histone. It is reported that SIRT1 over-expressed in prostate cancer, primary colon cancer and acute myeloid leukemia. Furthermore, other groups showed that SIRT1 over-expressed in Basal cell carcinoma and squamous cell carcinoma. The former study found that evodiamine could inhibit the expression of SIRT1 in human melanoma cells A375 S2.Nuclear factor-κB(NF-κB) is a nuclear transcription factor. In recent years, a large number of studies showed that NF-κB was closely related with the occurrence, proliferation, differentiation, apoptosis, invasion and metastasis of tumor cells. Therefore, it had attracted great attention of many scholars. The study showed that SIRT1 could make NF-κB/P65 subunit acetylation under the assist of NAD+, then it was unable to combine with IκBα. Which induced that the nuclear localization signal couldn’t be exposed and difficult to enter the nuclei, and failed to combine with the specific gene promoter regions(5-’GGGACTTTCC-3’), started the specific genes’ transcription of promoting tumor cell proliferation, invasion and metastasis.Matrix metalloproteinases(MMPs) are implicated in the metastasis of cancer cells. Wen et al. proposed that in MMPs family only MMP-9 gene, its transcription and translation were induce by SIRT1. In addition, it also reported that NF-κB could directly bound to the MMP-9 promoter region and regulated MMP-9 gene in nuclear. Both SIRT1 and NF-κB are related to MMP-9, and MMP-9 as the common downstream proteins of SIRT1 and NF-κB. Hence, we proposed that whether SIRT1 is a key driving force for communal migration?MethodsHuman colorectal cancer cell lines HCT-16 and HT-29 were cultured for 24 h in vitro, then cocultrued with evodiamine at the concentrations of 1.5, 3, 6, 12, 24, 48 and 96 μmol·L-1 for 48 h, respectively. The Survival activity of HCT-16 and HT-29 cells were detected by CCK-8. Migration potential were assessed by Transwell and Scartch assays after cells was treated by evodiamine. Cells treated with Evo(0, 1.5, 3 and 6 μmol·L-1) and Nampt for 48 h, and then analyzed for the expressions of SIRT1, NF-кB and MMP-9 m RNA and protein. Mice were randomized into two groups. Each mouse was inoculated subcutaneously in the right lower quadrant with HCT-116 cells or normal saline in a total volume of 0.2 ml. The tumor-bearing mice were treated by Evo(3mg/kg) once a day. We recorded the weight of mice, the diameter of tumor and so on. After 22 days’ reatment, we collected specimens and counted numbers of metastases to the colon. HE coloration was used to observed the morphologic changes of cells. The expressions of SIRT1, NF-кB and MMP-9 proteins were determined by western blot and immunohistochemical staining.Results1. HCT-116 and HT-29 cells were treated with different concentrations of Evodiamine(1.5, 3, 6, 12, 24, 48 and 96 μmol·L-1) after 48 h, the proliferation of cells was significantly inhibited in a dose-dependent manner. And it could significantly abolish SIRT1-induced cell motility.2. Evo could inhibited the expressions of SIRT1, MMP-9 m RNA and protein,while significantly enhanced the expression of NF-кB m RNA and protein.3. Models of colon cancer were established in animal. Weight of tumor in Evo group(0.63±0.13) g were significantly less than control group(3.00±0.57) g(P<0.01). Tumour volume in control group(1563.48±240.90) mm3 were greater than Evo group(941.98 ± 122.59) mm3(P<0.01). Weight of mice in Evo group(25.60±0.63) g were more heavier than control group(15.54±0.50) g, and the difference was statistically significant(P<0.01). HE staining showed that Evo could promote nuclear condensation and apoptosis in vivo.4. The expressions of NF-кB increase,while the expressions of SIRT1 and MMP9 decreased in Evo group, compared with control group in vivo.ConclusionsEvo-inhibited migration of colorectal cancer cells could mainly through down-regulating the expression of SIRT1, activating the NF-кB signaling path way and then inhibiting the production of MMP-9 in vitro and vivo.