Celastrus Orbiculatus Extract Could Inhibit Human Colorectal Carcinoma HT-29Cells Metastasis Via Suppression Of The MTOR Signaling Pathway

Colorectal cancer is one of the most frequent cancers worldwide and is a leading cause of cancer mortality. About50-60%of patients with colorectal cancer will experience metastasis. Metastasis is one of the remarkable characteristics of colorectal cancer and also the primary cause of colorectal cancer mortality. The process of metastasis occurs by a series of steps including vessel formation, cell attachment, migration, invasion, and cell proliferation, and is regulated by extremely complicated mechanisms. Among them, mTOR pathway has drawn a great attention. The mammalian target of rapamycin (mTOR), a highly conserved and ubiquitously expressed serine/threonine (Ser/Thr) kinase, plays a critical role in the regulation of tumor cell invasion, migration and cancer metastasis.mTOR is considered as a downstream target of Akt and involved in the translational initiation of many survival proteins through its activation of p70S6kinase (p70S6K) and4E-BP1. Recent study also reveals that mTOR signaling promotes cell migration and invasion may through increased expression and proteolytic activities of MMP-9. MMP-9is an important member of matrix metallo proteinases which act an important role in the proteolytic destruction of extracellular matrix and basement membranes, and is essential for tumor invasion and metastasis. Therefore, the inhibition of mTOR pathway could be a potential treatment for inhibiting metastasis of colorectal cancer.The Celastraceae plant Celastrus Orbiculatus(Celastrus Orbiculatus Thunb., Nansheteng, Celastrus), which is widely distributed in China, has been used as a folk medicine in China for the treatment of many diseases, including arthritis and other inflammatory diseases. Our previous study found that C. orbiculatus ethyl acetate extract (COE) displays anti-cancer effects in vitro and in vivo through the inhibition of proliferation, angiogenesis, invasion and adhesion ability.In this study, we investigated the anti-metastasis effects of COE in human colorectal carcinoma HT-29cells for the first time. We found that COE inhibited the proliferation, invasion and migration activity of HT-29cells. We further investigate whether COE could inhibit the mTOR signaling pathway with special reference to the process of metastasis. The data showed that COE down-regulated mTOR signaling pathway. Taken together, our observations indicate that COE exerts an inhibitory effect on several essential steps of metastasis, including proliferation, migration and invasion of HT-29cells. The inhibitory effect of COE might be in part related with the down-regulation of mTOR pathway. As evidenced by our results, COE could be recognized to be a potential candidate for the development of a preventive agent for cancer metastasis.The findings will be described in two parts as follows.PartⅠEffects of Celastrus Orbiculatus Extract on proliferation, migration and invasion of human colorectal carcinoma HT-29cellsObjective:To investigate in vitro the effects of Celastrus Orbiculatus Extract (COE) on proliferation, migration and invasion in human colorectal carcinoma HT-29cells.Methods:The human colorectal carcinoma HT-29cells were divided into negative and positive control group and COE groups (20,40,80,160,320μg/mL, respectively). The cytotoxicity effect was measured by using the MTT assay. The invasion ability of HT-29cells was assayed by transwell invasion assay. The migration ability of HT-29cells was assayed by wound-healing repair assay and transwell migration assay.Results:COE suppressed the proliferation of HT-29cells in a does-dependent manner. The concentrations of20,40,80μg/mL of COE significantly inhibited the invasion and migration activity of HT-29cells (P<0.05or P<0.01).Conclusions:COE could significantly inhibited proliferation, invasion and migration activity of HT-29cells. Part ⅡEffect of COE on the expression of mTOR signalling pathwayObjective:To investigate whether Celastrus Orbiculatus Extract (COE) could inhibit the mTOR signaling pathway with special reference to the process of metastasis.Methods:After HT-29cells were incubated with various concentrations (20,40,80and160μg/mL) of COE for24h, total protein was extracted. Then western blot was carried out to examine protein expression of each group. The protein we detected consisted of mTOR, its upstream regulators and downstream effectors such as Akt, p70S6K,4EBP1and MMP-9.Results:COE treatment dose-dependently inhibited the expression of mTOR and phospho-mTOR, phospho-4E-BP1, phospho-p70S6K, p70S6K and MMP-9compared to the control group, whereas there was no significant effect on Akt. Conclusions:The mechanisms of the anti-metastasis activity of COE might be due to the down-regulation of mTOR signaling pathway.