| ObjectiveColorectal cancers (CRCs) ranks third as a cause of cancer -related mortality all over the world, and accounts for 8.5% of all new diagnosed cancer patients. Epidemiological,clinical,animal and laboratory studies have, all provided evidence for prostaglandins ( PGs) in the tumorigenesis and development. Cy-clooxygenase(COX) is the key enzyme in the conversion of eicosanoids to PGs. Two isoforms of COX have been identified,namely COX - 1 and COX -2. COX -1 is the constitutive gene and regulate normal activity of human body, such as stomatch mucosa protection and platelet agglutination;whereas COX - 2 is in-ducible gene. Cytokines, growth factors, oncogenes, stress stimulating and mito-gen have been found to induce COX - 2 up - expression. Studies in recent years have founded that COX - 2 played a key role in tumourigenesis and development. PGE2, a major COX - 2 derived product, has been reported to stimulate the tumor cell growth and inhibit cell apoptosis. There are considerable reasons to believe that production of PG2 could be reduced through selectively inhibiting COX - 2 in order to inhibit the tumor cell proliferation and promote the apoptosis. Therefore,it is important to reduce the production of the PGs in prevention, treatment and prognosis of cancers.Non - steroidal anti - inflammatory drugs ( NSAIDs) are inhibitors of COX. It can be divided into three categories based on the difference of inhibition role: COX -1 selective inhibitors, COX - 2 selective inhibitors, COX non - selective inhibitors. Studies have showed the potential of the NSAIDs for anticancer. Itcan inhibit tumor cell proliferation and induce its apoptosis. Although the precise mechanism for the anticancer effects of NSAIDs are unknown, many studies have showed NSAIDs might play anticancer role by inducing cell cycle arrest and apoptosis and inhibiting the angiogenesis, tumor infiltration and metastasis through different signal conduct routes. Meanwhile, we donot understand the role of bcl - 2 and bax in which processs, and the relation of COX - 2, bcl - 2, and bax for expression on primary CRCs, metastasis, and normal colorectal tissues. Therefore, we conducted the test of COX -2, bcl -2 and bax together for the first time.Our studies aim to investigate inhibitive effect of COX - 2 selective inhibitor -Celecoxib on human colon cancer cell LSI74 -T, and roles on cell cycle and expressions of COX - 2 and apoptosis - related factors, try to firstly clarify the relation of COX -2, bcl -2 and bax. In order to dope out the possibility and significance of this kind of drugs to treat CRCs, we surveyed the expressions of COX - 2, bcl - 2 and bax proteins on primary CRCs, metastatic hepatic lesions , and corresponding normal mucosa. Also, we conduct comparison between the expressions of COX - 2, bcl - 2 and bax proteins, and clinical and prognostic outs.Experimental Methods1. MTT assay: To observe the proliferation of LS174 - T cell under the treatment with Celecoxib.2. FCM Assay for Analysis of DNA content;To observe the change of LS174 - T cell cycle under the treatment of Celecoxib.3. RT - PCR and Western - bloting assay: To observe the expression of COX -2N bcl -2 vbax mRNA and proteins after Celecoxib treatment by semi-quantitative method.4. Collecting clinical and prognostic data of post -operated CRCs patients. Immunohistochemical test: To observe the expression of COX - 2, bcl - 2 and bax proteins on 85 CRCs and 15 samples of normal colorectal mucosa." 5. Western - blot and RT - PCR methods were used to test the expression ofCOX - 2, bcl - 2 and bax mRNA and proteins on these fresh and iced samples of 40 CRCs and 10 normal colorectal mucosa.6. Western - blot, RT - PCR and immunohistochemical tests were used to test the expression of COX - 2, bcl - 2 and bax mRNAs and proteins on these samples of primary CRCs, metastatic hepatic lesions and corresponding normal mucosa.7. Statistical Methods: Statistical analysis was performed using SPSS for windows 13. 0 software. A P - value of less than 0. 05 was considered to indicate statistical significance, and all tests and P values reported are two tailed. The results of human colon cancer cell line LS174 - T treated with Celecoxib was expressed as mean s.. And comparisons of means were carried out by one way ANOVA - Dunnett t test;Explore was used to review data;Crosstabs method was used to compare groups with respect to expressions of three factors and differences in baseline characteristics. The Kaplan Meier product - limit estimator was used to estimate cancer specific survival and general survival. Survival between groups was compared by the Log rank test and Breslow test in general and stratification. The Cox proportional hazards model was used for multivariate analyses to estimate the joint effect of prognostic factors on survival. In the model fitting procedures, backward stepwise selection was used to determine more precise models. The covariable was entered the model if it had a less than 0.05 level of significance, and was removed if it's P value was larger than 0.10.Experimental Results1. The results of MTT assay indicated that Celecoxib induced significant growth inhibition in LSI 74 - T cell line in a good time - dependent and dose -dependent manner. The inhibition rate was increasing along with the concentration and time increasing.2. The results of FCM assay for analysis of DNA content showed that LS174 - T cells were arrested in G0/Gj stages following treatment with Celecoxib. A-long with augmented concentration and prolonged time of Celecoxib, the percentage of LS174 - T cells in Go/G, stages increased significantly, so did cellapoptosis rate.3. RT - PCR and Western - bloting assay semiquantitative analysis showed in human colon cancer cell line LSI74 - T, the COX -2^bcl -2 mRNA and protien expressions decreased under Celecoxib, however, the bax mRNA expression increased gradually accompanied with augmented dose and prolonged time.4. Immunohistochemical assay results showed that in all cases, COX - 2, bcl - 2 and bax expressions were predominantly localized in tumor cells, whereas some stromal cells, endothelial cells, and colonic normal mucosa far from the cancer tissue stained weakly or completely unstained. In cancer cells, COX - 2, bcl - 2 and bax expressions were observed mainly in the cytoplasm and nuclear envelope. COX -2 and bcl -2 expressions rate were statistically significant between CRCs tissue and normal colonic mucosa, nor was bax expression. COX -2 expression rate was high in the colon cancer tissue of female, invading disease , lymph node metastasis, and C and D stages;bcl - 2 expression rate was low in the colon cancer tissue of lymph node metastasis, and C and D stages;bax expression rate was high in the colon cancer tissue of lymph node metastasis, and C and D stages. There was positive correlation between bax and COX - 2 expressions in the colon cancer tissue;there was negative correlation between bcl - 2 and bax expressions;there was negative correlation between bcl - 2 and COX - 2 expressions. The 5 - year survival rate was high in COX - 2 negative or bcl -2 positive patients, and difference was significant statistically.5. Expression rate of COX -2 protein was increasing by degrees in the rae-tastatic CRC tissues from the liver, primary CRC, and corresponding normal mucosa from the same individual;and expression rate of bax protein and mRNA was decreasing by degrees;and bcl - 2 exhibited slightly higher levels in the primary tumors than in their hepatic metastases. Western - blot and RT - PCR test results of fresh and iced samples were close to those immunohistochemical assay results.DiscussionCyclooxygenase -2 (COX -2) is involved in inhibition of apoptosis, pro-moting cell growth, and angiogenesis and as such is a target for drug development. The COX - 2 enzyme is frequently overexpressed in colorectal carcinoma. The aim of this study was to determine the effects of celecoxib on the growth inhibition , induction of apoptosis, and mechanism of apoptosis in colorectal carcinoma cells. Baseline expression of COX - 2 enzyme was determined by Western blot analysis in human colon cancer cell LS174 - T. The obvious tendency in growth inhibition was observed in LSI74 -T cells (high COX -2 expression). Meanwhile, apoptosis were significantly increased in these cells.Another study had recently shown that down - regulation of COX - 2 mRNA and protein expression was observed in the pancreatic cancer cells, which had high COX - 2 expression, treated with celecoxib. Followed by significant down - regulation of nuclear factor - kB , and induction of growth inhibition and apoptosis in pancreatic cells. However, after treating with celecoxib, those pancreatic cells with low COX - 2 expression did not have the overt growth inhibition, induction of growth inhibition and apoptosis, and down - regulation of COX - 2 mRNA and protein expression, which confirmed that celecoxib, COX - 2 selective inhibitor, inhibited the growth of pancreatic cancer cells and induced the apoptosiss through specifically down - regulation of mRNA and protein in these cells.The seminal epidemiological observation that nonsteroidal anti - inflammatory drugs (NSAIDs) prevent colon and possibly other cancers has spurred novel approaches to cancer prevention. The known inhibitory effect of NSAIDs on the eicosanoid pathway prompted studies focusing on cyclooxygenase (COX) and its products. The increased prostaglandin E2 levels and the overexpression of COX -2 in colon and many other cancers provided the rationale for clinical trials with COX -2 inhibitors for cancer prevention or treatment. Theirefficacy in the prevention of sporadic colon and other cancers remains unknown;one COX -2 inhibitor has been withdrawn because of side effects, and there are concerns about whether these effects are class - specific.There is evidence to suggest that COX -2 may not be the only or ideal eicosanoid pathway targetfor cancer prevention. Six sets of observations support this notion: the relatively late induction of COX - 2 during carcinogenesis;the find-ing that NSAIDs may not require inhibition of COX -2for their effect;the modest effect of coxibs in cancer prevention;that .currently available coxibs have multiple non - COX -2 effects that may account for at least some of their efficacy;the possibility that concurrent inhibition of COX -2 in non -neoplastic cells may be harmful;and the possibility that COX -2 inhibition may modulate alternative eicosanoid pathways in a way that promotes carcinogenesis. Given the limitations of COX -2 - specific inhibitors and the biological evidence mentioned a-bove, we agree with the views of Rigas and Kashfi that targets other than COX -2 should be pursued as alternative or complementary approaches to cancer prevention.This study inherited the former experimental considerations, the first part of which clarified the role of Celecoxib to inhibit proliferation of LSI74 -T human colon cancer cell, and to promote cell apoptosis, meanwhile reducing the expression of COX - 2 both in protein and mRNA level, more important for the first time identified the change of bcl - 2 and bax, two significant factors correlating with apoptosis, revealed the apoptotic pathway Celecoxib conducted;the second part of which, deploying the matured techniques of immunohistochemis-try, Western - blot and RT - PCR, analyzed the expression of COX - 2, bcl - 2 and bax on primary CRCs, metastasis, and normal colorectal tissues. Therefore, we conducted the test of COX - 2, bcl - 2 and bax together for the first time, meanwhile, associated which with clinical factors and survival outputs.We clarified the effects of celecoxib on the growth inhibition in human colon cancer cell LS174 - T, partly coming from or promoting induction of apoptosis , in which processes there was a conspicuous modulation of celecoxib for bcl- 2 and bax. What would COX - 2 selective inhibitors, such as celecoxib, do on the colorectal carcinoma in vivo? Now that COX - 2 selective inhibitors suppress growth of colon cancer cells with high COX - 2 expression in vitro, COX -2 expression level of the colorectal carcinoma in vivo would make a forecast for the potential value of COX - 2 selective inhibitors to treat this kind of malignan: cy.According to the results of second part of experiment, there was high COX-2 expressions in most colorectal carcinomas, and which correlated with theprogression of disease and poor prognosis. So we consider that COX - 2 participates the tumorigenesis and development of colon carcinoma, and should be taken as a target of therapy. The expression levels of COX - 2, bax and bcl - 2 in the colorectal carcinoma verified the results of experiments in prophase further, that celecoxib, COX -2 selective inhibitor, induces apoptosis of human colon cancer cell by bcl - 2 pathway. This kind of COX - 2 selective inhibitor could be the alternative for treating female patients with colorectal carcinoma and disease in advanced stage.Conclusions1. Selective COX -2 inhibitor Celecoxib can inhibit proliferation of LS174- T human colon cancer cell and promote cell apoptosis.2. Celecoxib can restrain COX - 2 and bcl - 2 expression, and boost bax expression in LS174 - T cell at both transcription and protein levels.3. There is high expression of COX -2 and bcl -2 in human CRCs tissue. There is high expression of COX — 2 and bax in patients of invasive disease. In terminal stage, bcl -2 expression is low. There is positive correlation between COX - 2 and bax expressions. There is negative correlation between bax and bcl- 2 expressions, also between COX - 2 and bcl - 2 expressions. The 5 - year survival rate is high in COX - 2 negative or bcl - 2 positive patients. Our results imply that COX - 2 expression may be associated with the invasive and metastat-ic stage of CRC;bcl - 2 may be associated with the forepart stage of the disease.4. Selective COX - 2 inhibitor Celecoxib may turn into a hopeful drug for treating CRCs.
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