| Colorectal cancer (CRC) is a high incidence human malignancy. With an upward trend of incidence, it ranks the third among malignant tumors in the world. In China, the incidence of colon cancer ranks the third as well, behind gastric cancer and esophageal cancer only, and rises every year. Therefore, in-depth study of the pathogenesis and treatment of colon cancer is particularly important.At the moment, typical treatment of colon cancer is combined therapy with surgical treatment. Surgical resection rate can reach60%-70%. After radical resection,50%of patients die of relapse or metastasis.55-80%of recurrent cases occur after2years. The5years survival rate is still hovering around50%-60%and expanding the scope of operation can not help improve the survival rate. Chemotherapy can reduce recurrence, improve clinical efficacy, reduce the incidence of metastasis and save and prolong patient’s survival time.Although surgical treatment has made a great progress, recurrence and metastasis after surgery are major factors of death in colon cancer. Many anti-cancer drugs also bring a lot of side effects. Traditional Chinese medicine plays an increasingly important role because of its relatively insignificant side effects, patient tolerance and good characteristics. The treatment of Traditional Chinese medicine is mainly reflected in the following aspects:(1) Traditional Chinese medicine combined with surgery can reduce tumor recurrence, improve survival quality and strengthen patient immune function: Traditional Chinese medicine can assist therapy after surgical resection of the primary lesion. Traditional Chinese medicine is served by oral and enema before and after colorectal cancer surgery. Tumor growth can be inhibited and immunity be improved. Anti-metastatic and the success rate of surgery can be enhanced.(2) Traditional Chinese medicine combined with chemotherapy to reduce the adverse reactions:Chemotherapy is one of primary treatments in colorectal cancer, but90%of cancer patients with initial chemotherapy have the symptom such as nausea, vomiting, fatigue, etc. Traditional Chinese medicine can reduce intestinal damage for patients with chemotherapy, thus indirectly supporting the cancer treatment. Moreover, it can reduce the incidence of leukopenia caused by chemotherapy and the degree of white blood cells decline. It can significantly reduce the symptoms of nausea, and improve the quality of survival and the clinical symptoms. It can reduce the side reaction of chemotherapy and improve the body’s tolerance to chemotherapy.(3) Enema combined with chemotherapy extends the survival time:the efficacy of Traditional Chinese medicine enema through the rectum is free from the influence of many factors, such as digestive. It can inhibit wound, kill tumors and reduce pains. The enema is easy to operate and conducive to the rehabilitation of patients. Thus such medicine therapy for colorectal cancer becomes more and more important for clinical treatment.Aloperine (ALO) is an alkaloid monomers extracted from the bitter beans (Sophora Alopecuraides, L).Its structure is different from other sophoridine aloperine’s. Aloperine has significant anti-inflammatory effects, anti-viral, analgesic, sedative, etc. The inhibition to Neisseria gonorrhoeae is similar to penicillin. The antimicrobial activity is stronger than matrine. ALO can remove active oxygen free radical and lipid free radicals and can effectively alleviate the inflammation of colitis mice. Studies have shown that ALO has better effect on inhibiting tumor proliferation. The effects on leukemia cell line is better than lung cancer, liver cancer and esophageal cancer entity tumor cell lines, the most sensitive effect happens on the HL60cell lines. ALO can inhibit the proliferation of colon cancer SW480, prompt apoptosis and inhibit activation through JAK/Stat3signaling pathway. Although there have been a few studies on ALO inhibiting the proliferation of tumor cells, its mechanism of action is not fully understood yet.The objective of this study is to further clarify whether ALO, compared with other similar components, has advantages in the inhibitory effect on several kinds of cell lines and ALO inhibits tumor in vivo; understand ALO toxicity, explore its induction of cell cycle arrest and study its molecular mechanism of inducing cell cycle arrest and apoptosis and the signal pathways of inhibition of cell proliferation. Thus it can provide a more in-depth theoretical basis and treatment regimens for the development of ALO. The source of ALO is rich and enjoys low cost. ALO as a new drug will be great help for better utilization of resources, people’s health, ecological environment and economic development. With the combination of traditional Chinese medicine and modern molecular biology techniques, it can promote traditional Chinese medicine to the world.1To compare inhibition effect of gastrointestinal cancers between the total base of sophora alopecuroides (TASA) and eight kinds of alkaloidsThe inhibition of TASA and eight kinds of alkaloids, Sophoridine, Sophocarpine, ALO, matrine, oxysophocarpine, oxymatrine, cytisine and Lehmann were compared for eight kinds of digestive cancer cell lines, including human hepatoma cell lines cells97H, human gastric cancer cell lines BGC823and six different kinds of colon cancer cell lines, SW480, SW620, HCT116, HT29, LS174t and DLD1.The effectiveness of different drugs inhibiting cancer cells was detected by MTT in24,48,72h and the inhibition curves were drawn. IC50s of drug were calculated by SPSS13.0Probit method. The IC50of ALO is less than TASA and other alkaloids’ and is similar with the positive control drug five-fluorouracil (5-Fu)’. The results showed that ALO inhibition rate on the eight kinds of tumor cells was significantly better than other drugs (p<0.001). ALO inhibition rate has significant difference on different cells (F=31.767, P=0.000). ALO has the strongest inhibition on SW480and HT29and the inhibition rates are86.54±22.99%and85.70±16.33%, respectively.2. Aloperine induced apoptosis in colon cancer cell HT29The cells obvious morphological changes were observed under a microscope after ALO affected the cells. Compared with the control group, the cells of ALO group became round. With increasing dose, more and more cells died. The cells became sparse with increased particulate matter in the cytoplasm. The cells were observed under UV light by Hochest33342-PI double staining. The negative control group had few apoptotic cells. Early apoptotic cell with bright blue fluorescence were observed in ALO groups. Negative control group showed a small amount of necrotic cells with red fluorescence. ALO group had more necrotic cells than control group. The apoptosis was detected by Flow cytometry PI/Annexin V-FITC double staining. The apoptosis rate of different concentrations had significant difference (F=28.091, P=0.000)(n=3). No significant difference (F=2.377, P=0.146) among early apoptosis rates. Late apoptosis rates were significantly different (F=38.616, P=0.000).With higher concentration, late apoptotic cells increased significantly. The proliferation inhibition of ALO in HT29cell may be achieved by inducing apoptosis. The cloning experiments showed that ALO at low concentrations can inhibit the proliferation of HT29colon cancer cell lines. The cloning rates were significantly different (F=236.755, p=0.000)(n=3). The cloning rates of0.05mM ALO was64.11±3.87%. With an increase in concentration, cloning rate was reduced. The cloning rate of0.1,0.2mM ALO was41.89±3.91%and19.67±4.25%, respectively.Apoptosis-related proteins were detected by Western blot assay. With increased concentration, Bcl-2protein was significantly lower (F=36.436, p=0.000), whereas Bax protein levels became higher (F=3.472, p=0.071) after the cells were affected by ALO for24h. The results indicated apoptosis. Cleaved PARP protein was significantly higher (F=14.382, p=0.001) and the PARP protein content decreased significantly (F=10.142, p=0.001). It was also a sign of caspase-3activated and cells apoptosis. It indicated that ALO effect on cell proliferation inhibition might be related to apoptosis.3. ALO arrested the cells in G2/M phase and inhibited cell proliferation by Ras/Raf/Erk pathwayALO arrested HT29cells in G2/M phase by flow cytometry. By qPCR assay, ALO concentration has a significant impact on the level of p53and p21genes (F=212.157, p=0.000; F=948.613, p=0.000), and Cyclin B1and Cyclin D1was reduced (F=180.564, p=0.000; F=133.673, p=0.000). The cell cycle related proteins were detected by western blot. With increased concentration and time, Ras protein (F=0.330, p=0.804; F=0.141,p=0.932), Raf1(F=2.395,p=0.144; F=0.288, p=0.833)and Erk1/2(F=1.150, p=0.387; F=0.872, p=0.495) did not vary significantly. Phosphorylated Rafl protein in different time (F=11.609, p=0.003) decreased significantly, and p-Rafl protein in different concentrations decreased without significance (F=3.475, p=0.071).Phosphorylated Erkl protein1/2is significantly lower (F=27.914, p=0.000; F=9.744, p=0.005). The results indicated that ALO probably affected protein inhibit cell cycle by Ras/Raf/Erk pathway to inhibit cell proliferation.4. Aloperine can inhibit HT29colon cancer in vivo.HT29cells were injected in the armpit of healthy BALB/c-nu/nu male nude mice. The xenografts were formed after7-10days. The success rate was100%. There were three groups of ALO according to dose group4,8,16mg/kg by intraperitoneal injection every other day,3times a week.5-Fu, a positive control, was30mg/kg by intraperitoneal injection every other day,3times a week. After the xenograft model were successful, the nude mice were dosed for4weeks. The results indicated that tumor growth can be suppressed by ALO. It had a significant difference in tumor inhibition rate (F=4.216, P=0.018) among groups.5-Fu group was significantly different with ALO low-dose group (p=0.003) and had no significant difference with the ALO medium-high-dose group (p=0.099, p=0.515). ALO low-dose were significantly different with high-dose groups (p=0.015) and had no significant difference with the medium-dose group (p=0.125). The low-dose group was significantly different with high-dose group (p=.299). The inhibition rate of5-Fu group was68.88±1.92%, ALO low-dose group’ was39.93±7.73%, ALO medium-dose group’s was53.85±10.79%and ALO high-dose group’s was63.12±13.77%. Normal control group did not show any abnormal symptoms. During the test, the nude mice weight decreased. The negative control group’s body weight decreased significantly (F=2.172, P=0.040).5-Fu group’s body weight was also significantly reduced (F=3.090, P=0.005). The control group body weight increased significantly (F=2.441, P=0.022). ALO groups’ had no significant difference (F=0.377, P=0.941; F=0.322, P=0.964; F=0.230, P=0.989).All mice livers in the negative group had white spots. HE staining showed that the livers had tumor cell metastasis. The transfer rate was100%. ALO low-dose, medium-dose and5-Fu group was33.33%, higher doses16.67%. The spleen, kidney and heart had no abnormal changes. The livers’AST and ALT had no significant difference (F=1.439, P=0.239; F=0.396, P=0.847). The control group were significantly different with the negative control group (p=0.027). The result showed that the tumors had a certain impact on the liver. ALO had no effect on the mice liver. ALO significantly reduced p-ERKl/2(F=18.621, p=0.001). The results indicated that ALO may inhibit tumor growth by inhibiting the phosphorylation levels of ERK1/2. Immunohistochemistry results also indicated that ALO can inhibit the phosphorylation levels of ERK1/2.5. ALO acute toxicity test by intraperitoneal injectionIn order to investigate the toxicity of ALO, the ALO acute toxicity test was carried out by intraperitoneal injection. The traditional LD50method was used with Kunming mice, male and female. LD50values were calculated based on the mortality of mice with different doses. The LD50is200.54mg/kg weight. It was found that the mortality in females was higher than males. The obvious symptoms of toxicity were convulsions and cyanosis. The mice died within30mins. Toxicity symptoms was lightened1h later and no deaths ever since. The LD50value is far less than sophoridine which LD50is65.19mg/kg weight. The result indicated that the toxicity was less than Sophoridine and its anti-tumor effect was better. Thus ALO as anticancer drug has a better development prospects while acute toxicity results showed ALO had some toxicity.ConclusionIn summary, our study confirms that ALO can inhibit the proliferation of colon cancer cells, induce apoptosis and arrest cell cycle.1The anti-tumor effect of ALO is better than TASA and seven alkaloids in8kinds of digestive cancer cells. ALO had the best inhibition in HT29and SW480 colon cancer cells.2Aloperine inhibited the proliferation in colon cancer cell line HT29and induced apoptosis. Apoptosis rate increases with higher concentration by flow cytometry. With increasing apoptosis, ALO brought more changes in cell morphology. The level of Bcl-2protein reduced and the Bax protein levels increased.3Aloperine arrested HT29cell cycle in G2/M phase. It raised the protein and gene level of p53and p21, lowered the levels of cyclin D1and cyclinB1. ALO inhibited the cell proliferation by inhibiting the RAS/RAF/ERK signaling pathway.4The LD50of ALO by intraperitoneal injection is200.54mg/kg weight and95%confidence interval was189.59-211.84mg/kg weight. The result showed that ALO had some toxicity, mainly neurotoxicity. The mice died within30min and then no death occurred among the surviving mice. It indicated that the toxic effects lasted for a short time.5The success rate of HT29cell xenografts was100%. The inhibition rate of ALO was63.12%which was no significant difference with5-Fu. ALO inhibited the HT29cells metastases. All mice of the negative control group had liver metastases. ALO groups had less liver metastases than the control group.The AST and ALT results showed no significant effect on liver function. Immunohistochemistry and western blot results showed that ALO inhibited the phosphorylation of ERK in tumor protein. ALO may inhibit tumor proliferation by inhibiting p-ERK. |
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