The Study On Leptin And Colon Cancer

Leptin is the protein-related product encoded by obesity gene. It is mainly secreted by adipose tissue and can regulate metabolism of adipose and balance of energy. And it is closely related with pathogenesis and development of various malignant tumors. This research aimed to investigate the effect of leptin on colorectal carcinoma and its possible anticancer mechanism about NSAIDs from four aspects which included the expressions of leptin and its receptor on colorectal carcinoma tissues, the effect of leptin on growth and apoptosis of HT-29 colorectal carcinoma cell lines, the effect of leptin on COX-2 expression of HT-29 colorectal carcinoma cell lines and the effect of NSAIDs on leptin within HT-29 cell lines.Chapter 1 the expression of leptin and its receptor on colorectal carcinoma tissuesAim: To investigate the expression of leptin and its receptor in human colorectal carcinoma tissues at differentiation clinical stages so as to show the role of leptin in the pathogenesis of colorectal carcinoma and the relationship between leptin and clinical pathological stages of colorectal carcinoma.Methods: Seventy-two specimen of colorectal carcinoma had been collected. To study the expression location of leptin and its receptor in colorectal carcinoma tissues using immunohistological methods and RT-PCR and to classify them by calculating positive cell rate and semi-quantitative analysis.Results and discussions: The cells with stained brownish yellow granules in cytoplast were regarded as the positive cells Those stained brownish yellow granules in cellular membrane showed positive expression of leptin receptor. Those stained violet granules in cytoplast showed positive expression of leptin mRNA. The positive expression rates of leptin and its receptor were 87.8% and 88.24% respectively.There were 0% and 66.18% respectively in normal colorectal tissues (P<0.01; P<0.05). The positive expression rates of leptin in tubular adenocarcinoma, papillary adeno--carcinoma, myxoadenocarcinoma, signet ring cell carcinoma and undifferentiated carcinoma were 88.24%, 86.67%, 85.71%, 78.57% and 75% respectively.And the positive expression rates of leptin's receptor were 94.12%, 86.67%, 78.57%, 78.57% and 75%. There was no significant difference in the positve expression rate of leptin as well as its receptor in colorectal carcinima and the pathological types (P>0.05). Duringthe TNM stages,the positive expression rates of leptin in A,B stage and C, D stage were 85.2% and 88.4% respectively.And those of its receptor were 89.4% and 90.7%. There was no siginificant difference in expression of leptin and its receptor between two groups. The postive expression rates of leptin and its receptor without lymphoid metastasis were 86.7% and 87.3%; while those with lymphoid metastasis were 89.4% and 90.7%.There was no significant difference in positive expression rates of leptin and its receptor between two groups.Those data showed that leptin could be regarded as a new marker for early diagnosis of the colorectal carcinoma.To interdict leptin as well as its receptor might be an auxiliary method for treating colorectal carcinoma.Conclusions: There is a high level expression for leptin and its receptor in colorectal carcinoma tissues. There is no obvious correlation between the expression of them and pathological differential grade. Leptin might be a new marker for early diagnosis of colorectal carcinoma.Chapter 2 Effect of leptin on growth and apoptosis of HT-29 colorectal carcinoma cell linesAims: To investigate the effect of leptin on growth and apoptosis of HT-29 colorectal carcinoma cell lines.Methods: HT-29 cell lines were cultured along walls and then were given leptin at different concentration levels (0.5ng/ml, 50ng/ml, lOOng/ml, 200ng/ml). Meanwhile, epithelial growth factor was taken as the positive control. To study the effect of leptin on growth and proliferation of HT-29 colorectal carcinoma cell lines through measuring growth curve and the MTT (2.5-diphenyltetrazolium bromide) method. Cell cycle and apoptotic rate were measured by using the flow cytometry.Results and discussions: According to growth curve, the cell amounts from HT-29 cell lines increased in proportion to leptin concentration varying from 5ng/ml to 200ng/ml which showed leptin accelerated proliferation of HT-29 cell lines dose-independently. However, the cell proliferation amounts of 200ng/ml leptin group were still less than that of the positive control group (EGF). The results from MTT method was similar to those from cell counting method. Leptin at different concentration levels was added to HT-29 cell lines and then promote growth of HT-29 cell lines time and dose-dependently. So the proliferation rate varies from 5.1% to 67.9%. Leptin also could affect cell cycle of HT-29 cell lines obviously by using the flow cytometry. The cell ratio in Go/Gi period decreased gradually and that in S, G2/M period increased gradually compared with blank control group as the concentration of leptin multipled. But leptin had no effect on apoptosis of cells. Cell accounting andMTT method showed that leptin could accelerate growth and proliferation of HT-29 colorectal carcinoma cell lines. Results from the flow cytometry suggested that leptin could promote proliferation through affecting colorectal carcinoma cell cycles and made no influence in apoptosis of cells.All above evidences suggested that leptin,a multifunctional cytokine, was closely related with the pathogenesis of colorectal carcinoma.Conclusions: Leptin could promote growth and proliferation of HT-29 cell lines which was affected by its concentrations and experimental time.Chapter 3 Effect of leptin on COX-2 expression of HT-29 colorectal carcinoma cell linesAims: To investigate the effect of leptin on COX-2 expression of HT-29 colorectal carcinoma cell lines and further study possible the effect and mechanism of promoting growth of cell lines for leptin.Methods: HT-29 cell lines were cultured along walls similar to Chapter 2.Three groups consisted of the blank control group, the positve control croup and the leptin group at different concentration levels. Leptin was added into each well at different concentration level (5ng/ml, 50ng/ml, lOOng/ml, 200ng/ml) in the experimental group. Nutrient solution was added into each well in the blank control group. EGF at the concentration of 1 OOng/ml was added into each well in the positive control group. Total RNA were extracted according to operation manual of Trizol. RNA molecular weight was appraised quantitatively using the method of Shambrook (RNA was denaturalized and then separated by agar gel electrophoresis, and the size of RNA was measured after they were stained by othidium bromide and were observed under ultraviolet light. The effect of leptin on C0X-2mRNA expression of HT-29 cells was measured by using RT-PCR. Suspending solution of HT-29 cells was inoculated at the concentration of 5xlO4/each well in the 24-well culture plate. The culture solution was changed after each 24 hours. The cells in three groups was cultured after 24 hours and then the upper layer was collected so as to let Prostaglandin E2 to be measured.Results and discussions: The expression levels of C0X-2mRNA in the blank positive group and 5ng/ml, 50ng/ml, 1 OOng/ml, 200ng/ml leptin group were 0.673 + 0.072, 0.678 + 0.07, 0.756+0.068, 0.956 + 0.039, 1.137 + 0.045 respectively. Statistical analysis showed that the difference between the blank control group and the leptin group (the concentration of leptin was more than 50ng/ml) in the expression level of C0X-2mRNA was significant (PO.01). Nevertheless, the expression level of COX-2mRNA was dose-dependently correlated with leptin concentration.And theexpression level of C0X-2mRNA in the positive control group was 1.485 + 0.068 which was different significantlu from that in the leptin group at different levels (PO.01). PGE2 contents in the upper layer of culture solution of HT-29 cells in the blank positive group and 5ng/ml, 50ng/ml, lOOng/ml, 200ng/ml leptin group were 86.5 + 8.4, 89.4 + 8.8, 107.5±7.5, 125.1+6.8, 146.3 + 7.2 respectively. PGE2 contents of HT-29 cells in leptin group were increased obviously in proportion to leptin (more than 5ng/ml) which was obviously less than that in the positive group (182.3 + 6.8) (PO.01). According to some researches, there was the abnormal COX-2 expression in various malignant tumors which showed that COX-2 was closely related with human malignant tumors. The high level expression of COX-2 in colorectal carcinoma tissues was mainly represented in COX-2 protein and mRNA.The COX-2 protein and mRNA were higher than those in paracancerous normal tissues.Therefore, the abnomal expression of COX-2 played an important role in early diagnosis of colorectal carcinoma, metastasis judgement, prevention of precancerous lesion as well as treatment and prognosis estimation. Something like arachidonic acid could promote growth and proliferation of colorectal carcinoma cells by producing prostaglandin through catalysis of COX-2. PGE2 was closely related with colorectal carcinoma. So the PGE2 contents in colorectal carcinoma could indirectly reflect catalysis level of COX-2. This research had confirmed the fact that leptin could promote secretion of COX-2 in HT-29 cells at the level of mRNA and increase PGE2 concentration dose-dependently. It suggested that leptin, as a multifunctional cytokine, could accelerate growth and proloferation of HT-29 cells by enhancing catalysis of COX-2 in tumor cells.Conclusions: Leptin could enhance COX-2 at the level of mRNA and then promote catalysis of COX-2 as as to improve the production of PGE2. But it is less obvious than EGF.Chapter 4 Effect of NSAIDs on leptin of HT-29 cell linesAims: To study the effect of NSAIDs on HT-29 cell lines after the intervention of leptin and to investigate the possible anticancer mechanism of NSAIDs.Methods: HT-29 cell lines were cultured along walls similar to Chapter 2.Five groups consisted of the blank control group, the solvent group, the leptin group (200ng/ml), NS-398 (10|iM) +leptin (200ng/ml) group, Aspirin (2mM) +leptin (200ng/ml) group. The proliferation level of cells was observed in growth curve and then studied using the MTT method.The first experimental group, the second experimental group and the control group were established.In the first experimentalgroup, leptin at the different concentration (5/ngml, 50ng/ml, 1 OOng/ml, 200ng/ml) was added and aspirin (2mM) was then added. While in the second experimental group, leptin at the different concentration (5/ngml, 50ng/ml, 1 OOng/ml, 200ng/ml )was added and NS-398 (lOuM) was then added. In the control group no NSAIDs but leptin like those two experimental group were added. Cell cycle, proliferation levels and apoptotic rate were detected by using the flow cytometry. The distribution percentage of DNA histogram in each period was calculated using DNA cell cycle distribution software. Cell cycle includes Go/Gi period, S period, G2/M period. The cell percentage in each period the cell amounts in this period/the total amounts of cellsxlOO%. The proliferation index the cell amounts in S+G2/M period/the total amounts of cells xlOO%. The effect of NSAIDs on PGE2 expression in HT-29 cell lines induced by leptin was measured using ELISA.Results and discussions: According to the growth curve, the cells in the blank group and the solvent group grew normally, and the result in the leptin group (200ng/ml) was similar to that in chapter two. While the cell amounts decreased obviously as time went on after the intervention of Aspirin and NS-398 which was less than those in the blank group and the solvent group. Results of MTT showed that the growth of HT-29 cells was obviously suppressed time-dependently during 72 hours.Results from the flow cytometry showed that Aspirin and NS-398 had the antiblastic effect on HT-29 cells intervened by leptin previously.Compared with the leptin group (200ng/ml), the cell percentage in Go/Gj period increased evidently and that in S period and G2/M period decreased sharply.But compared with the blank control group and the solvent group, it is similar to the above results which was more significant than the leptin group. And the apoptotic rates of the NS-398 and Aspirin groups were higher than those of any other three groups. According to results from ELISA, the PGE2 content of the blank group and the solvent group were similar to each other. However, the PGE2 content was enhanced obviously in the leptin group. And the PGE2 contents in the NS-398 group and in the Aspirin group were both significantly less than that in the leptin group. The anticancer mechanism of NSAIDs is still unclear. Most scholars thought that it could reduce the PGE2 content and promote the leukotrienes level through inhibiting COX-2. Leptin, as a growth factor of epithelial cells and vascular epithelial cells, could strengthen proliferation and differentiation of cells, enhance aggressiveness of cells and regulate immune function of human body, which were the requirements for growth and development of malignant tumors. Therefore, leptin might play an important role in the pathogenesis and development of colorectal carcinoma. In chapter three, leptin could promote COX-2 and PGE2 and then enhance the growth and proliferation of HT-29 cells.But the intervention of Aspirin and NS-398 could suppress the growth of HT-29 cells according to the results from the growth curve, the MTT method and the flow cytometry respectively. It suggested that NSAIDs such as Aspirin and NS-398 could inhibit leptin from stimulating the growth of HT-29 cells. Because NSAIDs might inhibit COX-2 and then reduce PGE2 content. Or it might induce the apoptosis of tumor cells. And NSAIDs might intervene leptin by affecting its signal pathway which still deserved further investigation.Conclusions: NSAIDs such as Aspirin and NS-398 could inhibit leptin from promoting the growth and proliferation of HT-29 colorectal carcinoma cell lines for they might interdict COX-2, induce apoptosis and affect the signal pathway.