Inhibitory Effects Of Aloperine On Colorectal Carcinoma

Background:Colorectal Carcinoma(CRC) is currently one of the most common malignancy tumors in the world. There were approximately 945,000 new cases of CRC in 2000 in the world, whose morbidity was the third in all diseases of malignant tumor. The morbility of CRC increases sharply in China now. How to effectively prevent and treat CRC has been a key point in the medical field. However, most anti-tumor chemotherapy showed lots of toxic and side effects. With the arrival of global green revolution, more and more concern is laid upon our traditional Chinese medicine. Sophora Alopecuraides, L. is a kind of perennial herb which belongs to Legum inosae.This herb is very abundant in northern-west of China. Its alkaloid content is very rich. More than twenty kinds of alkaloids have been isolated from it, We have poved the effect of thetotal alkaloids of Sophora Alopecuraides, L. on the ulcerative colitis. Because the ulcerative colitis is well correlated with CRC, ALO was found that it could significantly inhibit the growth of the cells. However, little is known about the effect of ALO against CRC.Nowadays, it is of particular significance to use molecular biological methods to study the pharmacologic effects of traditional Chinese medicines and the underlyingpharmacological mechanism, to find new anti-tumar drugs, which has also been a trend in exploiting the Chinese medicine. So it is promising and valuable to study the effects of ALO on CRC and the molecular mechanism in it further.Objective:1. To evaluate whether ALO can inhibit the proliferation of colorectal carcinoma cells and induce apoptosis in vitro.2. To investigate the inhibitory effects of ALO I on the growth of subcutaneous implantation tumor in nude mice. To observe the toxicity of ALO and effects on the immunological function of mice in vivo.3. To study the molecular mechanisms of ALO inducing apoptosis in SW480 cells. The effects of ALO on the expression of Bcl-2, Jak-STATs family were performed to search the potential signal transduction pathway.Methods:1. The effect of ALO on the cell line SW480The effect of proliferative inhibition in SW480, the cell growth curve and the half-inhibition concentration (IC50) were detected by MTT assay. The morphology changes of ALO-treated SW480 cells stained by 33258 were observed through inverted microscope and fluorescence microscopy. The apoptosis of SW480 cells induced by ALO was evaluated by DNA fragmentation analysis, Hochest33258 staining assay, The level of STAT3,STAT5,STAT6 and Bcl-2 protein in SW480 cells were investigated using immunocytochemistry technique.2. The toxicity and the effect on immunological function of ALO on Kunming mice The Kunming mice were administed SRI at different dosages through intraperitoneal injection. The toxic reactions were observed and recorded. The median lethal dose(LD50) was tested.40 Kunming mice were randomly divided into 5 groups: ALO group of high dose, ALO group of low dose, CTX group and the control group. The weight and index of thymus gland and spleen of different mice groups were measured and the pathological change was observed.3. The effect of ALO on tumor inhibition in vivoSW480 cells, which express the common green fluorescence protein stablely, were used to establish the colorectal carcinoma whole-body image optical model of nude mice. The experimental groups were administered ALO and CTX through intraperitoneal injection respectivly, and the control group was treated with N.S. solution. After 4 weeks, the mice were sacrificed. The weight of xenograft tumors were measured to calculate the tumor growth inhibitory rate (IR). The tumorvolumes were measured and the kinetics of tumor formation after subcutaneous injection of SW480 were observed. Sections from nude mice xenograft tumor were subjected to HE staining to reveal the changes of histomorphologism. Transmission electron microscopy was used to test the ultramicrostructure alternations of the xenograft tumor.4. The possible molecular mechanism of ALO induced apoptosis in SW480 cells Total RNA was extracted from SW480 cells treated with ALO. The upstrait and downstrait primers of apoptosis related genes STAT-3, STAT5, Bcl-2 and Bax house-keeping gene P-actin were designed with software primer5.0. The real-time fluorescence quantitative PCR was used to detect mRNA expression of the genes in SW480 and ALO-treated SW480 cells. The relative quantity of gene expression was analyzed by the 2-ΔΔCt method. The cell cycle of SW480 cells treated with ALO were examined by Annexin V-FITC/PI double marked assay with flow cytometry. The rate of apoptosis cells was detected also. Western blotting was used to detect the effect of ALO on the level of Jak-STATs family. Results:1. The effect of ALO on the cell line SW4801.1 Cell growth was detected by MTT assay. After treated with ALO, the growth of cells was inhibited in different degrees. The effect of ALO was significant. The inhibition rate of ALO treated the cells for 48h was analysised by probit regression analysis, and the median lethal dose of SRI was 3.08045 mg/ml. The growth curve of ALO group was significantly lower than that of the control group.With the treating time prolonging, the inhibition rates increased. The results indicatedthat exposure to ALO caused significant growth inhibiton of SW480 in a time-anddose-dependent manner.1.2 The morphology observation revealed that the dimension of S W480 cells gotsmaller, big vacuoles appeared in the cytoplasm and so-called signet-ring cellsformed. The morphological characteristics of apoptosis was apparent, evidenced bycondensation of nucleus, bubble of cytoplasm and fragment of nucleus. Hoechst 33258 staining assay and that after being treated with ALO for 24h and 48h, SW480 cells exhibited typical morphological changes of apoptotic cells under fluorescent microscope. Early apoptotic cells appeared cell rounding, detachment from the substrate, cell shrinkage and membrane blebbing. The late apoptotic cells appeared condensation of chromatin, breakage of nuclear, formation of apoptotic bodies, and so on. Aggregation and margination of apoptotic nuclear chromatin could be observed by transmission electronmicroscope. Some parts of the nuclear membrane disappeared. Some cells were vacuolated. The microvilli disappeared with membrane integratedly.DNA ladder pattern of nucleosome fragmentation was observed with exposure to ALO for 48h and 72h. The expression level of STAT3,STAT5, Bcl-2,Baxprotein was down-regulated while that of protein was up-regulated in the cells treated with ALO compared to the control group.2. The toxicity and the immunological function of ALO on Kunming miceThe acute-term toxicity test was performed to calculate LD50 of intraperitoneally-injected ALO on Kunming mice which was 515.19337mg/kg, limit of reliability is from 59.01544mg/kg to 74.41568mg/kg. ALO had no significant effect on the weight and index of thymus and spleen, while CTX could reduce the index of thymus and spleen and cause atrophia significantly.3. The effect of ALO on tumor inhibition in vivoThe experiment in SW480 tumor-bearing nude mice documented thathigh dosage of ALO had a significant effect on tumor inhibition. The inhibitory rates of ALO and CTX were 62.4±4.3% and 69.0±2.3% respectively. The relative tumour volume was lower than the control group significantly also.The ultrastructure alternations of tumor cells in nude mice xenograft tumor treated with ALO exhibited apoptotic changes.4. The possible molecular mechanism of ALO induced apoptosis in SW480 cellsThe mRNA expression of Bax was increased and STAT3,STAT5,Bcl-2 was decreased in SW480 cells when treated with ALO for 24h and 48h, The alternations of the cell cycle in SW480 cells were examined with flow cytometry assay. Incubation of cells with ALO for 24h and 48h increased the percentage of G0/G1 phase, decreased the percentage of S and G2/M stage also. Annexin V assay showed that after being treated with ALO for 24h and 48h, the apoptosis of cells was significant. The result of Western blotting showed that the apoptosis-ralated protein Jak-STATs were activated in the SW480 exposed to ALO for different time.Treating the cells with SRI significantly increased the translocation of Jak-STATs from the cytosol to the nucleus, which may be lead by the phosphorylation of Jak-STATs which was activated by the phosphorylation of IL-6- Jak-STATs.Conclusion:1. ALO can inhibite the growth of SW480 cells in a time and dose-dependent manner and induces apoptosis in vitro. The median lethal dose of SRI was 3.078045 mg/ml. The morphological characteristics of apoptosis and nucleosome fragmentation can be observed with apoptosis-related genes and protein changing.2. ALO has a significant effect on tumor inhibition in vivo. At the same time, it has no influence on the immune system. ALO has some toxic effect on mice and its LD50 of intraperitoneal injection on Kunming mice is 515.19337mg/kg.3. ALO can increase the level of pro-apoptotic factor Bcl-2 in SW480 cells, it may have influence on the cell cycle to induce apoptosis also.4. ALO can activate apoptosis-associated Caspase-9, Caspase-3, caspase-7 and PARP in the SW480 cells exposed to it for some time.5. Treating the cells with ALO significantly increase the translocation of Jak-STATs from the cytosol to the nucleus, which may be lead by the phosphorylation of Jak-STATs which may be activated by the phosphorylation of Jak-STATs6. We postulate one of the signal transduction pathways of apoptosis ALO-induced is as below:ALO SW480 phosphorylation of STAT dissociation of IL-6-Jak-STATs Bcl-2 family activation apoptosis,which needs more evidences from experiments.