Nitidine Chloride Is Involved In Regulation Of Proliferation And Apoptosis In Colorectal Carcinoma Cells

Background:Colorectal cancer (CRC) is a serious health problem in western countries. In developed countries and developing countries, the incidence of Colorectal cancer ranks the third of all tumors. The incidence of colorectal cancer in the male comes after lung cancer and prostate cancer, with 50-65 years old as popular. The incidence of colorectal in the female comes after breast cancer, ranking the second. Each year there are about one million new cases Worldwide, among those about 550,000 men and 470,000 women. This suggests that colorectal cancer is becoming an increasingly important public health issue.Colorectal cancer accounts for 13% of all tumors. Similar to many other cancers, it exhibits differences in developing regions and developed regions. At the same time, in the past 20 years, due to advances in surgical techniques, and improved use of adjuvant chemotherapy, radiotherapy and improvement of primary and secondary prevention campaigns, survival in colorectal cancer increased and mortality reduced. About 50% of newly diagnosed patients will progress to metastatic cancer, and the average lifetime in about 50-60% of patients will come up to five years. In general, colorectal cancer has been in the late stage when diagnosed, which is due to the rapid transfer. Therefore, high transfer rate through the blood formation is one of the main obstacles to achieve a more effective treatment. The incidence of colorectal cancer is different in different parts of the world. These geographic differences may be due to different regional genetic inheritances and different local eating habits. Colorectal cancer risk factors include personal and family history and environmental and dietary habits. The following people have 2-3 more times of incidence of colorectal cancer than the general population, such as a family history of benign or malignant intestinal adenomas,45 women before the age of 45 years old getting cancers such as ovarian, uterine and breast cancer. It is worthy of noting that at least 15% of colorectal cancers appear familial aggregation. Pathogenesis of colorectal cancer as well as molecules involved in tumor progression is complex, which is due to progress in gene regulation of epithelial cell differentiation and cell mutations accumulation. Based on the origins and manifestations colorectal cancer can be divided into the following three types:(1) dissemination type, refers to the absence susceptibility gene mutation exists but does not show any hereditary.60-80% of colorectal cancer is of this type. (2) Familial aggregation type, no relevant gene was found, accounting for 20-40% of cases. Population studies have shown that the presence of family members related by blood sporadic colon cancer, colorectal cancer risk is two to three times of the general population. (3) genetic type, two kinds of tendencies tumor variation can be distinguished by presence or absence of adenomatous polyps.1) familial adenomatous polyposis (FAP), patients are with multiple polyps. Without preventive surgery at age 40, one or more adenomas may become malignant; 2) hereditary non-polyposis colorectal cancer (HNPCC), It is a malignant tumor with a very high risk of developing to non -gastrointestinal cancer.Recently, natural herbal drugs have been used in the treatment of various diseases including cancer for a long time with less side effects. Thus herbal medicines are becoming a new type of anticancer drugs and gained widely attention. Therefore, the emergence of new natural drugs may provide a new method for cancer treatment. Nitidine chloride is an important component in a Chinese herbal medicine. Nitidine chloride is extracted from Zanthoxylum nitidum and is a natural chemical alkaloid. Studies have shown that nitidine chloride has a variety of biological activities including anti-inflammatory, antimalarial, antibacterial, and anti-cancer activity. Recent studies have shown that nitidine chloride can promote cell apoptosis and inhibit occurrence and progress of human cancers. Nitidine chloride inhibits cycle protein B1 and p53 dependent pathways to prevent cell G2/M phase, which inhibits the growth of a wide variety of cancer cells. Another study showed that nitidine chloride inhibits tumor cell growth in liver cancer through JAK1/STAT3 pathway. In addition, nitidine chloride can inhibit the proliferation of cells in liver cancer and kidney cancer and the metastasis of breast cancer cell via c-Src/FAK pathway.Many evidences show that mitogen-activated protein kinase (MAPK) signaling pathway in colorectal cancer is a major regulator of cell proliferation pathways, playing an important role in colon cancer occurrence and progression. MAPKs contain three major subfamilies:the extracellular-signal-regulated kinases (ERK MAPK, Ras/Rafl/MEK/ERK); the c-Jun N-terminal or stress-activated protein kinases (JNK or SAPK); and MAPK14. Many evidences prove that MAPK pathway involves in invasion and metastasis of human colorectal tumor cells. And inhibitors of MAPK pathway in colorectal cancer are in clinical trials as new anti-drugs.This study investigated the effects of chlorination nitidine onproliferation and apoptosis of colorectal cancer cells. Nitidine chloride was found to significantly inhibit the proliferation of colorectal cancer cells and promote apoptosis of colorectal cancer; The main mechanism of inhibiting cell proliferation is blocking MAPK signaling pathway, and activating Bax, caspase and other apoptosis-related proteins to promote apoptosis. These results suggest that nitidine chloride may become a new clinical chemotherapy drugs, thereby providing value to improve the prevention and treatment of colorectal cancer.Objective:1.To investigate the effects of NC on proliferation and survival in colorectal cancer cells;2. To investigate the effects of NC on apoptosis in colorectal cancer cells;3.To investigate the specific effect of NC on colorectal cancer and its related signaling pathways.Methods:Using human colorectal cancer cell line HCT116, MTT test is used to assess the viability and proliferation of cells. Annexin V-FITC/propidium iodide (PI) and TUNEL assay experiments are used to analyze apoptosis. Western blot experiment is used to analyze protein. The data are expressed as the mean±standard deviation (SD). All of the experiments were repeated at least three times. Comparisons among values for all groups were performed by one-way analysis of variance (ANOVA). Holm’s t-test was used for analysis of differences between different groups. Differences were considered to be significant at P＜0.05.Results:1. NC inhibits the proliferation of colorectal carcinoma cellsTo investigate the role of NC on colorectal carcinoma cells proliferation, MTT assay was measured as described in the materials and methods. NC inhibited the proliferation of colorectal carcinoma cells in a time- and dose-dependent manner. To further assess the result, the effects of NC on colorectal carcinoma cells proliferation was detected by [3H] thymidine uptake. The result demonstrated that NC over a range of concentrations (5,10 and 20 μM) can significantly inhibits colorectal carcinoma cells proliferation. Considering that the significant effect appeared when the NC concentration was 10 and 20 μM, these concentrations (10 and 20 μM) of NC were chosen for the following experiments. These data taken together suggested that NC effectively inhibited colorectal carcinoma cells proliferation.2. NC inhibit ERK phosphorylation in colorectal carcinoma cellsNitidine chloride can significantly inhibit cell proliferation. Then Our studying team explored the mechanism of chloride nitidine Inhibiting cell proliferation. MAPK signaling pathway plays an important role in tumor genesis and development. After treating cells for 24h with NC in 10 and 20μM concentrations, Our studying team detected the expression of MAPK signaling pathway proteins. The results showed that after treated with nitidine chloride, ERK phosphorylation level of cells decreased, indicating that nitidine chloride likely inhibitied cell proliferation by affecting ERK.3. NC induces the apoptosis in colorectal carcinoma cellsTo determine whether NC-induced growth inhibitory effect was due to NC’s effect on adducing colorectal carcinoma cells apoptosis, the cells were treated with NC for 24 hours. As demonstrated in Annexin V-FITC/PI assay, NC effectively increased apoptosis in colorectal carcinoma cells at a dose of 10 and 20 μM. The percentage of cells undergoing apoptotic cell death increased from 6.9±1.3% to 34.8±6.8%,36.9±7.2% after being exposed to 10 and 20 μM NC for 24 hours. TUNEL is used to evaluate the fragmented DNA of cells apoptosis. After NC (0,10 and 20μM) treatment for 24 h, the cells were stained with TUNEL and DAPI and analyzed by fluorescence microscopy.10 and 20 μM NC could effectively increase apoptotic cells. The significant induction of apoptosis after NC treatment demonstrated its anti-cancer effect on colorectal carcinoma cells.4. NC induces colorectal carcinoma cells apoptosis via alteration of Bcl-2 family proteins and caspase activationSince nitidine chloride can induce apoptosis, our studying team ulteriorly discussed the mechanism of nitidine chloride-induced apoptosis. Our studying team first examined the effect of nitidine chloride on apoptosis-related proteins Bcl-2, Caspase-9, Caspase-3 and p53’s expression.The Western blot results indicated that following the treatment with 10 and 20 μM NC, the expression of anti-apoptotic protein Bcl-2 was decreased and the pro-apoptotic protein Bax were increased. Meaningwhile, the expression of cleaved Caspase-9, Caspase-3 and p53 were also upregulated. These results suggest that by inducing the expression of Bax, thereby activating Caspase-9, Caspase-3 and p53, nitidine chloride induces apoptosis in colorectal cancer cells.5. ERK inhibitors cooperate nitidine chloride to promote cell apoptosisTo further explore mechanism of nitidine chloride inhibiting cell growth and promoting apoptosis, our studying team combined nitidine chloride with ERK inhibitor U0126. The result showed the proliferation of cells was significantly reduced, indicating that ERK inhibitors significantly improved the effect of nitidine chloride (NC) induced inhibition of colorectal cancer cell activity. Observing 3H thymidine uptake results also came to similar conclusions, which confirmed that blocking ERK activation can significantly improve the effect of nitidine chloride-induced inhibition of colorectal cancer cell proliferation.6. The mechanism of ERK inhibitors cooperating nitidine chloride to promote cell apoptosisSince ERK inhibitors significantly cooperated nitidine chloride to promote cell apoptosis, our studying team ulteriorly examined the expression of apoptosis-related proteins by combining nitidine chloride with ERK inhibitor U0126 to treat cells. The results showed that inhibiting ERK activity with U0126 increased expression of NC-induced Bax, increased caspase-3 and caspase-9 expression, and decreased expression of Bcl-2, which further proved that NC-induced apoptosis may depend on ERK.Conclusion:1. NC could inhibit the proliferation and survival of HCT116 cells in concentration-dependent and time-dependent manner;2. NC could induce apoptosis of HCT116 cells in concentration-dependent and time-dependent manner;3. NC inhibits the proliferation and survival of colorectal carcinoma cells via inhibiting the phosphorylation of ERK pathway.Innovation:1.We firstly discovered that NC could inhibit the proliferation and survival of HCT116 cells in concentration-dependent and time-dependent manner;2.We firstly discovered NC could induce apoptosis of HCT116 cells in concentration-dependent and time-dependent manner;3.We firstly discovered NC inhibits the proliferation and survival of colorectal carcinoma cells via inhibiting the phosphorylation of ERK pathway.Limitation:1. In this study, the effect of nitidine chloride on colorectal cancer was mainly confined to in vitro experiments, while relevant animal model experiments were not performed.2. Pathogenesis of colorectal cancer is a complex signaling network, the present study only explores the link between nitidine chloride and ERK, MAPK,however, the link between nitidine chloride and other signaling pathways is not explored.