Study On The Role Of Cyclooxygenase-2 In Lung Carcinoma: Growth Inhibition Of Lung Carcinoma By Selective Cyclooxygenase-2 Inhibitor

BackgroundLung carcinoma is one of the fatal malignant tumors which threaten human health and life seriously. Even more important, its incidence is rising more rapidly in many countries. Lung carcinoma is the most common cancer in our country recently based on statistic. Although the mechanism in the pathophysiology of lung carcinoma was investigated deeply, the survival time did not prolong significantly. On the average, the overall 5-yr survival rate of lung carcinoma was about 8% accordimg to statistic reports of our courtry in 2002. As a cause of cancer death, it is first in men and third in women. So the poor prognosis for patients with bronchogenic carcinoma requires emphasis on new prevention and treatment strategy.Studies on epidemiology and animal models of colon carcinogenesis have indicated that non-steroidal anti-inflammatory drugs(NSAIDs) have anti-colorectal cancer activity. Recent studies have reported a 40% to 50% lower colorectal caner risk in people who are continuously taking aspirin or other NSAIDs, and the inhibitory effects of NSAIDs on tumorigenesis of the colon were observed in many animal experimental models. Many tissue have the capacity to synthesize prostaglandin(PG) and generally do so when stimulated. If arachidonic acid is acted on by the enzyme cyclooxygenase(COX), then a series of cyclic endoperoxides are formed which are known as PGs. There are different PGs that fall into several groups: mainly thePGEs, PGFs, PGI2(prostacyclin) and thromboxane A2(TXA2). Two types of cyclooxygenase were found in 1991, including COX-1 and COX-2. Multiple lines of evidence suggest that COX-2 expression is up-regulated in several types of human cancers, including colon, lung, breast, stomach and pancreatic cancer. These novel findings suggest that COX-2 may play a role in tumor cells growth.Multiple lines of evidence have demonstrated that the NSAIDs inhibit both cyclooxygenase-1 (COX-1) and COX-2. Recently, it has been reported that selective inhibitors of COX-2 decrease gastrointestinal tumors formation in experimental animals, and these compounds may induce apoptosis and inhibit cell growth in several types of cancer cells. Celecoxib was a novel selective COX-2 inhibitor. In vitro, COX activity assays using isolated COX-1 and COX-2 enzymes from sheep seminal vesicle microsome and placenta, respectively, have confirmed that celecoxib selectively inhibits COX-2. Recently, it has been reported that selective inhibition of COX-2 by celecoxib in mice reduced hematogenous metastasis of human colorectal cancer cells that had high levels of COX-2 expression. In addition, some investigators presume that a certain type of lung carcinoma, such as lung adenocarcinoma has several features in common with colon cancer and may share some pathogenetic mechanisms.With this in mind, we examined whether celecoxib could inhibit the growth of lung carcinoma cells and induce apoptosis in vitro and in vivo, and explored potential mechanisms by which celecoxib might mediate growth inhibition and apoptosis. Based on the results of these studies, it will be important to establish whether selective inhibitors of COX-2 are useful for the treatment of human lung carcinoma.Methods1. We have studied the cytotoxic effect of a specific COX-2 inhibitor, celecoxib, against Lewis lung carcinoma cells grown in cell culture in vitro. The viability of Lewis lung carcinoma cells was measured by MTT assay. In addition, FCM assay were performed to determine the effects of celecoxib on the cell apoptosis and cell cycle distribution.2. Lewis lung carcinoma cell suspension were inoculated subcutaneously to 20 C57BL/6 mice, they were randomized into 2 groups. One day after inoculation of Lewis lung carcinoma cell suspension, treated group began receiving lOOOppm celecoxib in the diet for 24 days. All mice were sacrificed on the 24th day after tumor implantation. The weight of the subcutaneous tumor was detected by electronic balance and the lung metastasis rates of the two groups were detected with anatomical microscope.3. The concentration of VEGF^ MMP-2 expressed in Lewis lung carcinoma cell lines which incubated with 3.1 to 120umol/L celecoxib in vitro and in C57BL/6 mice serum was assayed by enzyme-linked immunoadsordent assay(ELISA).4. The protein expression of COX-2, VEGF, MMP-2, TIMP-2 and CD34 in xenografted tumor tissue and in surgically resected NSCLC tissue samples was measured by immunohistochemical staining.5. Ultramicrostructure of Lewis lung carcinoma cells in two groups' xenografted tumor tissue was observed with transmission electron microscopes.6. The mRNA expression of VEGF, MMP-2 and TIMP-2 in xenografted tumors was measured by demi-quantify Reverse transcriptive polymerase chian reaction.Results1. The viability of Lewis lung carcinoma cells was inhibited by celecoxib in dose and time-dependent manner in vitro. Flow cytometric analysis demonstrated that apoptosis cells of Lewis lung carcinoma were increased in treated group cells (14.93+0.79%) as compared with the control group( P < 0.001).2. Celecoxib inhibited the growth of transplanted Lewis lung carcinoma significantly at dosage of 1000 ppm celecoxib in the diet for 24 days, with the inhibitory rate of 60.1 %. The subcutaneous tumor weight of the treated and control group were found to be (1.37±1.04)g and (3.49 ± 1.75)g (P < 0.001), respectively. The rate of lung metastasis of the treated and control group was 50%(5/10) and 40%(4/10), respectively. There was no significantly difference (P >0.05).3. The expression of VEGF in vitro was inhibited by celecoxib in dose dependent manner. The lowest concentration of VEGF was 41.01+9.05 ng/L when cells acubated with 120nmol/L celecoxib. There was significantly difference as compared with the control group(362.23± 36.00 ng/L) ( P < 0.01). While the expression of MMP-2 both in Lewis lung carcinoma cell lines in vitro and in C57BL/6 mice serum was not inhibited by celecoxib(P >0.05).4. The ultrastructure of xenografted tumor tissue cells in treated group showed typical apoptotic feature under transmission electron microscopes. Typical apoptotic cells and apoptotic bodies could be observed in these treated group cells. The ultrastructure of xenografted tumor tissue cells in control group showed few apoptosis pathologic changes.5. The results of immunohistochemistry showed: there was a positive correlation between expression of COX-2 and VEGF (r=0.696, P < 0.01). The VEGF staining score of tumor tissue in treated group and control group were 2.00 + 0.82 and 2.90+0.88 CP < 0.05) . The average MVD in twogroups were 16.70±7.77 and 23.15 ±4.58 (? <0.05) .The expression scores of COX-2 in the xenografted tumor tissue of treated group was 3.14±0.92, and was higher than that in controls(3.89± 0.85). However, there was no significantly difference (P > 0.05 ) . Meanwhile, the MMP-2 and TIMP-2 protein expression in xenografted tumor tissue determined by immunohistochemical staining was not significantly down-regulated after treatment with celecoxib(P >0.05).6. After 24 days, the results of demi-quantify RT-PCR showed that the mRNA expression of VEGF at xenografted tumor tissue was down-regulated in treated group as compared with that in control group(0.609 + 0.109 vs 1.067 + 0.071) (P < 0.01) . The mRNA expression of MMP-2 and TIMP-2 expression in xenografted tumor tissue determined by RT-PCR was not significantly down-regulated after treatment with celecoxib.7. The overexpression rate of COX-2 in human NSCLC was 66.7%. The overexpression rate of COX-2 in NSCLC was correlated to tumor size, lymph node metastasis, histological type and clinicopathological stage of NSCLC. The overexpression rate of VEGF, MMP-2 and TIMP-2 in 48 sugically removed specimens of NSCLC were 68.8%, 47.9% and 41.7%, respectively. The overexpression rate of COX-2, VEGF and MMP-2 in these patients with lymph node metastasis were 85.0%, 85.0% and 65.0%, respectively. These were significantly higher than those in patients without lymph node metastasis (53.6%,57.1% and 35.7%, respectively) (all P < 0.05) .Conclusions1. celecoxib has inhibitory effects on Lewis lung carcinoma cells both in vitro. Inducing apoptosis and inhibiting proliferation might be the mechanism by which celecoxib exerts its chemopreventive and treatmenteffects.2. Celecoxib has inhibitory effect on Lewis lung carcinoma. The activity of COX-2, which is inhibited by celecoxib, is probably related to the production of angiogenic factors such as VEGF modulated by COX-2 at Lewis lung carcinoma. Antiangiogenesis may be another mechanism by which celecoxib exerts its chemopreventive and treatment effects. But the expression of MMP-2 of Lewis lung carcinoma both in vitro and in vivo could not be inhibited by celecoxib.3. The overexpression of COX-2 was found in NSCLC. COX-2 might play a crucial role in the carcinogenesis and development of NSCLC. COX-2 may stimulate angiogenesis through VEGF upregulation and enhance lymphatic metastasis in patients with NSCLC, which supports the notion that inhibition of COX-2 activity maybe one of the treatment strategy of NSCLC to inhibit its growth and metastasis.