| Colorectal cancer (CRC) is the third common malignancies worldwide, which incidence is approximately7per100,000, with approximately5,000new cases and3,200deaths annually. Therefore, it remains a major cause of morbidity and mortality each year. Currently, surgery is still the curative treatment for CRC and postoperative chemotherapy is considered an effective method of colon cancer treatment. But its side effect reaction caused a severe reduction in quality of life. With a long history of seeking bioactive anticancer compounds, natural products are fertile resources to look for potential antineoplastic drugs due to the rich experiences and medicinal plants of traditional folk medicine, i.e., traditional Chinese medicine (TCM). Sophora alopecuroides L. is a TCM, which is used to treat flammitory and Quinolizidine alkaloids have been identified to be the major components of Sophora alopecuroides L. and accumulated evidence indicates that these alkaloids may potentially exhibit anti-cancer property. More than twenty types of alkaloids have been isolated from the plant, including matrine, oxymatrine, sophocarpine, sophoramine, aloperine and others, the structure of which are shown in. In previous bioassay screening, we found quinolizidine alkaloids show potent antiproliferative effects. We have tested the anti-tumor effects of Quinolizidine alkaloids, which have been reported to display anti-tumor effects in human cancer cells in vitro. Research has shown that the molecular mechanism of Quinolizidine alkaloids is related with physiological cell death. Programmed cell death, or apoptosis, is a fundamental process essential for both development and maintenance of tissue homeostasis. Impairment of normal apoptosis can lead to tumour or autoimmune disease. They may lead to the activation of Bax, which plays a major role in the initiation and execution phases of apoptosis. However, little is known about the mechanisms associated with the effect of aloperine against CRC.In the present study, we examined the anti-tumor effect of aloperine in human colorectal cancer SW620cells, and evaluate its potential mechanism of in vitro. Our research provides some basis for the development of resource-rich S. alopecuroides L.Methods3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazo-lium bromide (MTT) assay. Cells were seeded into96-well plates at the density of5000/well for24h, then treated with quinolizidine alkaloids at24h,48h,72h, respectively. The anti-proliferative effects of alkaloids in SW620human colon cancer cells were assessed using the MTT assay. To examine levels of apoptosis, SW620cells treated with aloperine were detected by hematoxylin-eosin (H.E.), hoechst33258staining, propidium iodide staining (PI), Annexin-V/PI and JC-1by flow cytometry and cell cycle distribution assays. The quantitative real-time PCR assay was used to evaluate the expression of p16, CDK4, cyclin D1, Rb, Akt, Bax and Bcl-2mRNA at the genetic level. To investigate the molecular mechanisms underlying alterations in cell apoptosis, the proteins cell cycle-related p16, phosphor-Rb (Ser780), cyclin D1, CDK4, caspase-3, caspase-9, PARP, Bax, Bcl-2, Akt, p-Akt(Ser473) and p-Akt(Thr308) were determined by western blotting analysis.Result 1. In vitro anti-CRC effects of quinolizidine alkaloids.The effect of quinolizidine alkaloids on cell growth was investigated using MTT assay on several human cell lines. The antiproliferative effects of quinolizidine alkaloids (aloperine, matrine, sophoridine, sophocarpine, cytisine and oxymatrine) were detected by MTT assay. Above mentioned compounds decreased cell viabilities of SW620cells with the IC50values of1.02±0.092,3.587±0.045,3.612±0.169,3.350±0.121,3.356±0.092and3.720±0.268mM at72h, respectively. Compared with other alkolids, aloperine showed potent anti-tumor activity. Aloperine potently inhibited the growth of SW620cells with IC50values of1.767±0.016mM,1.218±0.032mM and1.021±0.092after24h,48h and72h treatment, respectively. Thus, we examined the cytotoxicities of aloperine on Human colorectal cancer cell lines SW620, DLD1, HCT116, HT29and174-t. The IC50by aloperine in SW620cells was significantly lower than in other cell lines at24h,48h and72h, respectively. It is obvious that the SW620cells were more sensitive to aloperine and showed a concentration-dependent (F=92.553, p=0.000) and time-dependent (F=4.418, p<0.05) decrease in cell growth.2. Aloperine induces cell cycle arrest in GO/G1phaseWe tested the influence of cell cycle in SW620treated with aloperine. As shown in Figure2-A, aloperine induced GO/Gl arrest of the cell cycle in SW620cells. And the ratio of GO/G1phase significantly increased from61.73±1.70%in control cells to84.13±1.01%at24h. These results suggested that aloperine significantly inhibited cellular proliferation of SW620cells via the GO/G1phase arrest of the cell cycle (P<0.05). Western blot analysis has shown the expression of complete P16(INK4a) and cyclin D1were activated in the SW620cells exposed to aloperine for different concentrations (P<0.05). Aloperine significantly decreased levels of CDK-4and phosphor-Rb (Ser780), which were significantly inhibited at different concentration (P<0.01). Real time RT-PCR was performed to evaluate the expression of pl6, CDK4, cyclin D1and Rb. It was found that aloperine could decrease CDK4mRNA level, and increase the mRNA expressions of p16and cyclinDl. We observed that the expression of p16in dose of1,1.5and2mM was significantly higher than the control in a concentration fashion (F=61.429, P=0.000)(Figure.2B). CDK4was decreased at the concentration range of1.5to2mM (p<0.05). While, the effects of aloperine on the expression of cyclinDl caused a markedly increase in mRNA expression.3. Aloperine induces apoptosis though mitochondrial pathwayCell death was assayed morphologically by HE staining and fluorescence microscopy of Hoechst33258, PI staining. Compared with untreated cells, drug groups were characterized by shrinkage of cells and nucleus. Typical markers of apoptosis such as significant chromatin condensation, nuclear deformation, and disassembly were also observed in drug-treated cells. The ratios of apoptotic cells were determined by flow cytometry. As shown in Fig.3-B, the population of apoptotic cells increased in a concentration-dependent manner after aloperine treatment for24h. Consistent with the above results, the apoptotic rate increased significantly (p<0.05) from25.40±8.58%to67.78±4.74%as the sample concentrations increased from1to2mM. The results indicated that aloperine could inhibit the growth of SW620cells inducing early and late apoptosis. From the data of Fig.3-C, Energized mitochondria of controls demonstrated a moderate ratio of J-aggregate+/JC1+mitochondria (8.10±1.57%). The ratio increased from22.32±1.92%to55.43±4.81%and in aloperine groups, reflecting mitochondrial hyper-polarization.In order to determine the effect of aloperine on mitochondrial pathway and death receptor pathway, we used western blot analysis to evaluate caspase-3, caspase-9and Bcl-2, Bax and PARP expression. The results show aloperine significantly decreased levels of activated caspase-3and caspase-9. Western blot analysis showed that the expression of PARP in drug groups was significantly lower than those in the control cells.1-2mM aloperine significantly decreased the level of Bcl-2(P<0.05). The levels of Bax were remarkably increased after treatment with lmM and2mM aloperine compared with the control (P<0.05).Real time RT-PCR was performed to evaluate the expression of Bcl-2and Bax. It was found that aloperine could decrease Bcl-2mRNA level, and increase the mRNA expressions of Bax. Bcl-2was markedly decreased at the concentration range of1to2mM (p<0.05). While, the effects of aloperine on the expression of Bax caused a markedly increase in mRNA expression and1mM aloperine was the optimum concentration.4. Aloperine induces cell cycle arrest and apoptosis though Akt pathwayWe examined the phosphor-Akt (Ser473), phosphor-Akt (Thr308) and total Akt by Western blot. As shown in the result, Western blot analysis revealed that phosphor-Akt (Ser473) and phosphor-Akt (Thr308) were inactive in SW620cells exposed to aloperine for different concentration. The results demonstrated that1-2mM aloperine decreased the levels of phosphor-Akt (Ser473) and phosphor-Akt (Thr308) at24h. The expression of Akt exhibited no significant differences between the drug groups and the control group (P>0.05). Real time RT-PCR was performed to evaluate the expression of total Akt. In accordance with the above result, the mRNA level of Akt exhibited no significant differences between the drug groups and the control group (P>0.05).ConclusionIn our study showed that aloperine exerts anti-colorectal cancer effects though G0/G1phase arrest and mitochondrial Dependent apoptosis, which may be associated with Akt signaling pathway. |
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