Effects Of Celecoxib In Combination With Oxaliplatin On Xenograft Tumor Growth Of Colon Cancer In Nude Mice And Its Mechanisms

Objective:Inflammatory mediators such as cytokines, eicosanoids, and growth fac-tors are thought to play a critical role in the initiation and maintenance of can-cer cell survival and growth. One of these mediators, PGE2, is produced in large amounts by tumors. PGE2 is produced from arachidonic acid by either of two enzymes: COX-1 or COX-2. COX-1 is constitutively expressed in most tissues, whereas COX-2 is induced in inflammatory cells and in human tumors by cytokines and tumor promoters. Both COX isozymes can be inhibited by traditional NSAIDs, such as aspirin and indomethacin. COX-2 has a major influence on cancer biology and applicable to many tumor types, it is involved in many aspects of carcinogenesis. A proposed mechanism for the antitumor effect of COX-2 inhibitors was that they inhibit the growth of newly formed blood vessels, COX-2-derived prostanoids were shown to modulate cytokine synthesis, to influence cell proliferation and apoptosis, and to modulate the nuclear translocation and function of tumor suppressor gene products.Colorectal cancer (CRC) is the second leading death cause of malignancy in the western countries and the incidence is increasing recent years in China. Several studies show that regularly taking aspirin or other conventional NSAIDs provides a 40–50% reduction in relative risk of death by colon cancer, indicating that inhibition of COX in humans has a chemopreventive effect. COX-2 inhibition also demonstrates chemopreventive activity against colon carcinogenesis. Taken together, these data provide strong evidence for the importance of COX-2 enzyme activity in oncogenesis.COX-2 is an enzyme that can be inhibited with available medica-tions—COX-2 inhibitors, but it is naive to expect COX-2 inhibitor alone can have a deadly impact on the cancer process in which multiple aberrations are involved. Therefore, a combined use of COX-2 specific inhibitors and che-motherapy might result in a combination of cytotoxic and antiangiogenic ef-fects or, possibly, overcome multi-drug resistance, and enhance anti-cancer efficacy. Survivin, one of the targets of the Wnt-signaling pathway counteracts cell death and controls mitotic progression. It is undetectable in normal adult tissues and becomes prominently expressed in nearly all human cancers. NSAIDs are able to downregulate survivin by inhibition of the Wntsignaling pathway as demonstrated in vitro. In vivo study was not found.Oxaliplatin in combination with other anti-cancer drugs, such as fluorouracil and leucovorin, has been used as a conventional therapy for spread or metastasized colorectal cancer. We hypothesized that a combined use of celecoxib, a specific COX-2 inhibitor, with oxaliplatin might potentially have synergistic effects for the treatment of colon cancer. In present research, we generated HT-29 human colon cancer xenograft models by injecting HT-29 cells subcutaneously into the back of nude mice, different doses of celecoxib was administered alone or in combination with oxaliplatin, and the effects on tumor growth, cell apoptosis, survivin expression and angiogenesis were evaluated. The results indicated a significant synergistic effect of anti-cancer when celecoxib was used in combination with oxaliplatin.Methods:1 To establish a xenograft animal model,human colon cancer HT-29 cells were injected into the BALB/c nude mice subcutaneously. After 16 days,mice were randomly divided into four groups:control group,the first cele-coxib group(25mg/kg/d),the second celecoxib group(50mg/kg/d),the third celecoxib group ( 75mg/kg/d ) and the fourth celecoxib group(100mg/kg/d). Celecoxib was administered respectively. Tumour volumes were measured every week. Mice were sacrificed 35 days after the treatment. Xenograft cancer tissues were used to measure the expression level of COX-2, survivin and the microvessel density (MVD) by immunohistochemistry, mRNA level of VEGF by RT-PCR and cell apoptosis by TUNEL method. 2 To establish a xenograft animal model,human colon cancer HT-29 cells were injected into the BALB/c nude mice subcutaneously. After 16 days,mice were randomly divided into four groups:control group,oxaliplatin group,celecoxib group,and celecoxib in combination with oxaliplatin group. Medicine was administered respectively. Tumour volumes were measured every week. Mice were sacrificed 35 days after the treatment. Xenograft can-cer tissues were used to measure the expression level of COX-2 and the mi-crovessel density (MVD) by immunohistochemistry,and mRNA level of VEGF by RT-PCR.3 The expression of COX-2, MVD and LVD were studied by immuno-histochemistry in 64 cases of colorectal adenocarcinomas, the relationships among COX-2, MVD, LVD and the clinicopathological characteristics were analyzed. Seven advanced colorectal cancer patients whose tumor, could not be removed,were treated with celecoxib enema twice a day for fifteen days, the change of LVD and MVD were evaluated.Results:1 Different doses of celecoxib suppress xenograft tumor growth of co-lon cancer in nude mice through inhibiting angiogenesis and survivin expres-sionAll the mice(n=50, 100%) developed tumors 5 to 11 days after cell in-oculation. No change in body weight and activity of the mice in early phase. In the late phase, most mice administered with drugs had reduced activity, especially mice in the third and fourth celecoxib group, two mice in the third and two mice in the fourth group died during the experiment. There was no difference in body weight of mice between groups.After the treatment was finished, the tumor volume of each group was as follows: control group, 1326±238mm3;the first celecoxib group, 923±171mm3;the second celecoxib group, 863±177mm3;the third celecoxib group, 729±179mm3;the fourth celecoxib group, 647±205mm3. Different doses of celecoxib prevented the growth of tumors in nude mice. At the end of the experiment, celecoxib inhibited the tumor growth by 34.94%; 39.20%; 53.50%; 59.20% in celecoxib group one, two, three and four respectively. Tumor treated with celecoxib grew slowly. Tumor in the fourth celecoxib group grew more slowly than tumors in the first celecoxib group.In order to explore the mechanisms of the inhibition on tumor growth of celecoxib we performed immunohistochemistry analysis of COX-2 protein and survivin protein in tumor samples. Mice treated with different doses of celecoxib had a statistically significant reduction in tumor levels of COX-2 and survivin protein in comparison with mice in control group (P < 0.05). COX-2 protein level in the third and fourth celecoxib group and survivin pro-tein level in fourth celecoxib group was significantly lower than that in cele-coxib group one(P < 0.05). COX-2 and survivin protein expression was sup-pressed dose dependently.In comparison with control group, VEGF mRNA level was significantly reduced when mice were treated with different doses of celecoxib. There was no difference in VEGF mRNA level among celecoxib-treated groups (P > 0.05). Different doses of celecoxib significantly inhibited xenograft tumor angiogenesis. There was no difference in MVD among celecoxib-treated groups (P > 0.05).Further more, celecoxib induced apoptosis was determined in four groups by TUNEL assay. The cell apoptosis index of control group, the first, second and fourth group are 0.46±0.20,2.72±1.15,2.82±1.07,3.91±1.12 respectively. Mice treated with celecoxib had a statistically significant in-crease in tumor cell apoptosis in comparison with mice in control group (P < 0.05). Cell apoptosis index of the fourth group was higher than that of the first and second celecoxib group. There are no differences among other groups.2 Effects of celecoxib in combination with Oxaliplatin on xenograft tumor growth of colon cancer in nude mice and its mechanismsAll the mice (n=42, 100%) developed tumors 5 to 11 days after cell in-oculation. No change in body weight and activity of the mice in early phase. In the late phase, most mice administered with drugs had reduced activity. There was no difference in body weight of mice between groups. One in the oxaliplatin group got diarrhoea, no mice died until the end of the experiment.The tumor volume of each group at the end of the experiment was as follows: control group, 1326±238mm3;celecoxib group, 923±171mm3;oxaliplatin group, 963±192mm3;combination use group, 604±213mm3. Both celecoxib and oxaliplatin prevented the growth of HT-29 tumors in nude mice. At the end of the experiment, celecoxib and oxaliplatin inhibited the HT-29 tumor growth by 35% and 31% respectively (P < 0.05 compared with control group). Administering both agents at the same time caused a 63% tumor inhibition (P < 0.05 compared with the other three groups, respec-tively).In order to explore the mechanisms of the inhibition on tumor growth of celecoxib and oxaliplatin, we performed immunohistochemistry analysis of COX-2 protein in tumor samples. COX-2 staining was found only in the cyto-plasm. Mice treated with celecoxib had a statistically significant reduction in tumor levels of COX-2 in comparison with mice in control group (P < 0.05). In comparison with control group, COX-2 level in oxaliplatin group is sig-nificantly elevated (P < 0.05), COX-2 level in combination group was signifi-cantly lower than that in control group (P < 0.05).In comparison with control group, VEGF mRNA level was significantly reduced when mice were treated with celecoxib alone, or in combination with oxaliplatin, and elevated when mice were treated with oxaliplatin alone (P < 0.05). There was no difference in VEGF mRNA level between celecoxib group and combination group (P > 0.05). Celecoxib alone, or in combination with oxaliplatin, significantly inhibited xenograft tumor angiogenesis, but not oxaliplatin alone. MVD values in celecoixb group and combination group were significantly lower than that of control group (P < 0.05).3 COX-2 staining was mainly found in the cytoplasm which scattered as brown stain. Strong expression of COX-2 was found in 78.13% of the patients, weak expression in 14 cases (21.87%). COX-2 expression in Dukes A and B colorectal cancer and mucous adenocacinoma were lower than that in Dukes C colorectal cancer and nomal adenocarcinoma. The expression of COX-2 was correlated with tumor stage (p=0.003) and histological type (p=0.004)in colo-rectal carcinoma. MVD and LVD in COX-2 strong expression group were higher than the value in COX-2 weak expression group (p<0.05). Seven pa-tients were treated with celecoxib enema, treatment of celecoxib enema could reduce MVD but not LVD in colorectal carcinoma.Conclusions:1 Celecoxib can suppress tumour growth dose dependently partly by inhibiting suvivin expression. Low dose of celecoxib can decrease xenograft colon cancer angiogenesis which play an important role in the metastatic po-tential of colorectal cancer2 Celecoxib can suppress tumour growth and angiogenesis alone or in combination with Oxaliplatin. Celecoxib can enhance the antitumor effects of oxaliplatin through their synergistic role in inhibiting different targets. Cele-coxib in combination with oxaliplatin may play an important role in attenuat-ing the metastatic potential of colorectal cancer through decreasing angio-genesis.3 It is possible that COX-2 was involved in the tumor formation and development of colorectal adenocarcinoma through promotion of angiogene-sis and lymphangiogenesis. Celecoxib enema can suppress angiogenesis in colorectal adenocarcinoma.