Antitumor Activity And Its Molecular Mechanism Of LCP In Combination With Oxaliplatin In Colon Cancer Cell Lines

BackgroundColon cancer is one of the most common malignant tumors in digestive tract. In recent years, with the improvement of living standards, lifestyle and diet changes, the morbidity rate increased year by year, its incidence ranked NO.3 among all of tumors in our country and its clear upward trend. More note worthy is that the number of colon cancer has surpassed the United States, and the median age patients are earlier than in Europe and America about ten years.Young patients are more common in our country than in Europe.Although the current treatment of colon cancer has made great progress, its five-year survival rate hasn′t improve significantly.Surgery Combinding with chemotherapy is the standard model in treatment of colon cancer. For middle-late stage patients, tumor can't be radical resect or has already metastased systemically, chemotherapy extensively carried out improving the effectiveness of surgery. However, the traditional chemotherapy drugs have many shortcomings: It lacks of selectivity between normal cells and tumor cells. These drug toxisities limit the intensity and frequency of treatment . The current datas show that cytotoxic therapy treatment has reached a plateau, the existing combination of chemotherapy drugs is difficult to significantly improve the survival . With the better understanding the molecular mechanism of tumor, we found many signaling molecules or protein play a key role in cancer recurrence and development. Based on these theories, targeted therapy was developed, which control gene expression and changes of its biological behavior by blocking specific cell signal transduction pathways, or blocking tumor angiogenesis, inhibiting cell growth and proliferation. To use the targeted drug in clinical, it provides a compelling anti-cancer strategy. Compared with traditional chemotherapy, molecular targeted therapy have highly selectivity, low toxicity and high therapeutic index. It′s an urgent desire to find new targets or effective and specific new compounds that acting on these tatgets for doctors.In the performance of a stronger anti-tumor activity, molecular targeted therapy also reduce the toxicity to normal cells. Compared with traditional chemotherapy, molecular targeted therapy has proved highly selective, low toxicity and high therapeutic index,it can be used long-time .This targeted therapy for cancer treatment indicated a new direction.Strong activity in addition to chemotherapy drugs, targeted drugs bring more hope for patients.In addition, the emergence of oral targeted chemotherapy drugs make patients taking treatment at home ,their quality of life can be significantly improved.LCP(Low-Molecular-Weight Citrus Pectin,LCP) is one of the water-soluble polysaccharides which extracts from the peel or the pulp of citrus fruit , by the high PH and after heat treatment, it can be adhere to the phenotype of tumor cell affinity and kill the mutant cells specificity. As the competitive inhibitor of Galectin -3'ligand, it can close the Galectin-3 site of tumor cells surface and prevent the formation and development of tumor cells. Galectin-3 is known as "tumor-associated protein ", it is widely distributed in normal cells and tumor cells, and involved in regulating cell growth and differentiation.It is closely related with almost the whole process in tumor formation, development and transfer.Oxaliplatin is one of the main drug of colon cancer chemotherapy, it is a new platinum derivative. It produces the alkylating composite actting on function of DNA, and contacts between the formation of chain and cross-linking between chains, plays anti-tumor effect by inhibiting DNA synthesis and replication. However, vitro tests in prostate cancer, platinum drugs can make the galectin-3 phosphorylation and transported from the nucleus to the cytoplasm. The high expression of galectin-3 in cytoplasmic phosphorylation can inhibit the expression of Bad so as to stabilize the mitochondrial membrane and inhibit cytochrome C release.This process leads to apoptosis blocked.Thus, the cytoplasmic galectin-3 phosphorylation may be one of the reasons reduce platinum drugs efficiency.Previous studies have confirmed LCP has anti-tumor effect by suppress galectin-3,it influences key proteins and key pathways in apoptosis. With the combination of chemotherapeutic drugs in some experiments, LCP increased the reactivity of chemotherapeutic drugs to induce apoptosis.It also significantly reversed the resistance of tumor cells to chemotherapy.So far,there is no report on LCP combined with oxaliplatin in the literature at home and abroad.Basis:Galectin-3 contains NWGR of the Bcl-2 family, it can inhibit the platinum drug to induced apoptosis.It induces phosphorylation on serine 112 of Bad so as to stabilize the mitochondrial membrane potential , inhibits the activity of caspase-3 and cytochrome C releasing. It inhibits the apoptosis process by blocking endogenous apoptosis pathway.LCP can effective against the galectin-3 and induces apoptosis, thereby increasing the sensitivity of platinum chemotherapy drugs.ObjectiveThe point of penetration of this project is inducing cancer cell apoptosis,we took galectin-3 as the reserch target,in vitro to study: 1) the effect of cell proliferous inhibition and apoptosis induced by LCP combination with Oxaliplatin on human colon carcinoma cell lines in vitro, to explore the anticancer mechanism through the expression of key protein in apoptosis and cell cycle; 2)Discuss whether LCP has sensitivity - increasing effects of platinum chemotherapy drugs in colon cancer and explore its mechanism. Materials and methods1. Cell lines: human colon cancer cell line HCT116, HT-29.2. MTT analysis the proliferation of human colon cancer cells in vitro in different concentrations of Oxaliplatin monotherapy and Oxaliplatin + LCP (concentration of 5mg/ml) after 72h treatment.3. Observed the morphological changes of the above colon cancer cells after different concentrations Oxaliplatin monotherapy and combination therapy by microscope.4. Influence of Oxaliplatin single-agent and combination with LCP on human colon cancer cells apoptosis rate was determined by FACS.5. Change of procaspase-3,8,9,PARP and Bcl-2 were analyzed by Western blot.6. Statistical analysed by statistical software SPSS13.0, means between the two groups were compared by two independent sample t-test,means among different groups were compared by Oneway ANOVA,use LDS when homogeneousness,otherwise count by Dunnett-t.P<0.05 means it has the significant difference.All the measurement data showed by ?χ±s.Results1.Oxaliplatin work on the two kinds of colon cancer cell lines and show the significant inhibit proliferation effect in vitro,this effect is dose-dependent. The Oxaliplatin IC50 values are different between the two cell lines: HCT116: 34.00±0.59umol / L, HT-29: 14.73±0.11 umol / L. LCP work on the two cell lines show the inhibit proliferation effect equally, and also a dose dependency. Because of the high concentration of LCP that can not be dissolved, so MTT experiment can not be completed successfully. In this study, the LCP dissolved at room temperature's concentration is 5mg/ml which is the highest concentration.2.Two groups of colon cancer cells, (Oxaliplatin / Oxaliplatin + LCP) single agent and combination therapy on the inhibition rate of each concentration group are different, with statistical significance (P <0.05).And the medium concentration of Oxaliplatin puls LCP show the best effect of inhibit proliferation in colon cells.3.Observe IC50 dose of Oxaliplatin monotherapy and LCP + Oxaliplatin combination treatment of HCT116 and HT29 cells began to appear morphological changes after 2h and increased with time ,concentration characteristics of apoptosis cells were more obvious, such as chromatin Condensation, membrane blistering, break the nucleus and associated apoptotic bodies appear. The apoptosis of Joint Group is more obvious than single agent group, this result was consistent with the MTT test.4. FACS analysis show that monotherapy and combination therapy can increase the apoptosis proportion in two colon cancer cells,the rate of apoptosis in combination therapy was higher than monotherapy (P <0.01).5.Both monotherapy and combination therapy appeared decrease G0/G1 and S phase cell ratio and increase G2/M phase cell ratio in two colon cancer cells.G2/M phase cell ratio in combination therapy was increase more significantly than monotherapy.(P<0.05)6. Both monotherapy and combination therapy appeared expression down regulation of procaspase-3, 9, PARP protein.Procaspase-3, 9, PARP protein expression in combination therapy was decreased more significantly than monotherapy, procaspase-8 between the two groups showed no significant difference.Compare with the control group,Bcl-2 has no change.Conclusion1. Effect inhibiting the proliferation of colon cancer cells in combination therapy is more visible compared with monotherapy, the moderate concentration of LCP combined Oxaliplatin has the best effect.2. The apoptotic cells ratio induced by drugs increases over time, The effect of Joint Group that work on cell apoptotic ratio action is more significant .3. Both IC50 concentration of single-agent oxaliplatin and the combination could restrain the colon cancer cells arrested in G2/M phase.There is a same cell cycle arrest phase between LCP and Oxaliplatin. It showed superimposition effect combinate them,thus significantly extend cell proliferation cycle.4. Both monotherapy and combination therapy appeared expression down regulation of procaspase-3, 8,9, PARP protein. Compared with monotherapy, the activation function on procaspase-9 of the combination is superior.It suggests that LCP play synergies through apoptosis mitochondrial pathway.