| Objective: Lung cancer is one of the most common malignant tumors in the world. In China, non-small cell lung cancer (non-small cell lung cancer, NSCLC) accounts for more than 80%. Adenocarcinoma accounts for 23.0% ~ 38.5%, and squamous cell carcinoma accounts for 39.4% ~ 53.9%. Because of the atypical incipient symptoms of lung cancer, the lung cancer may have developed into an advanced stage at the moment of clinical diagnosis. Recent studies show that cyclooxygenase-2(COX-2) plays an imporant role in the formation of cancer. COX-2 promotes the hyperplasia of tumor cells, inhibits tumor cell apoptosis, and promotes the invasion and metastasis as well. Looking for inhibition of lymphangiogenesis in cancer chemotherapy drug is a new way to cure cancer.The VEGF-C and VEGFR-3 is the most important signal applied to control the form of lymphangiogenesis. In tumor, the increasment of VEGF-C and VEGFR-3 may promote the increasement of the density of lymphatic vessel and promote its infiltration and local lymph node metastasis. Confirmed previous studies by our group, non-steroidal anti-inflammatory drugs(NSAIDs) inhibit the formation of lung capillaries, recent studies have shown that COX-2 involved in the regulation of the VEGF-A/VEGFR-1, 2 angiogenic pathway. VEGF and its receptors dimerization form a network mechanism. VEGF-A and VEGF-C in tumor cells to form heterodimer, synergistically leading to vascular and lymphatic vessel formation, which can lead to indirectly promote lymphangiogenesis.β-catenin is an important signal transducer, which can decrease cell connection, loose tissue, and loss of the inhibitory functions.β-catenin reflects invasion and metastasis during tumor's development. Abnormal expression ofβ-catenin is significantly associated with non-small cell lung cancer differentiation and lymph node metastasis. Current study showed that indomethicin can stabilizeβ-catenin in colon cancer cells .so that it cannot bind to TCF-4, which blocksβ-catenin-mediated transcription and inhibits the growth and metastasis of tumor cells. 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 Wnt signaling pathway as demonstrated in vitro.As one of the NSAIDs, indomethicin can inhibit the activity of both COX-2 and COX-2. Traditional chemotherapy is not efficient to inhibit lung cancer metastases, so the collaboration of NSAIDs and chemotherapy might provide an efficient way for lung cancer therapy. We detect the Effect of Indomethacin on human lung cancer xenograft in nude mice and the effect of VEGF-C,VEGFR-3,β-catenin and Survivin, to discuss the reason of lung cancer invasion, and to find new methods of curing lung cancer.Methods:1 Xenograft animal model of human lung cancer were established by injecting A549 cells into BALB/c nude mice subcutaneously. Then the mice were randomly divided into 4 groups control group; Indomethacin Group one(1.0mg/kg/d), Indomethacin Group Two (2.5mg/kg/d), Indomethacin Group Three (4.0mg/kg/d). Indomethacin was administered respectively. The mice were sacrificed after administering 42 days.And tumor tissues were used to detect VEGF-C,β-catenin and Microlymphatics density(MLD) by immunohistochemistry and real-time PCR.2 Xenograft animal model of human lung cancer were established by injecting A549 cells into BALB/c nude mice subcutaneously. Then the mice were randomly divided into 4 groups : control group, Indomethacin-treated group, oxaliplatin-treated group, and Indomethacin combined with oxaliplatin-treated group). Medecines were administered respectively. Execute nude mice after administering 42 days, cut and transplant tumor tissue, immunohistochemistry detection of VEGF-C, VEGFR-3,β-catenin and survivin protein expressions, real-time assay tumor VEGF-C and Survivin mRNA expressions. Results:1 Effects of different doses of Indomethacin on tumor lymphangiogenesis andβ-catenin expression in lung cancer xenograft in nude mice.All the mice developed tumors after cell inoculation. In order to explore the mechanisms of the inhibition on tumor growth of Indomethacin we performed immunohistochemistry analysis of VEGF-C, VEGFR-3 andβ-catenin protein in tumor samples. Mice treated with different doses of Indomethacin had a statistically significant reduction in tumor levels of VEGF-C, VEGFR-3 andβ-catenin protein in comparison with mice in control group (P<0.05). Compared with Indomethacin group one, VEGF-C, VEGFR-3 andβ-catenin protein expression levels of Indomethacin group two and group three were significantly lower (P <0.05), and there was no statistically significant difference between group two and three (P> 0.05).According to the real-time fluorescence quantitative analysis of VEGF-C ,VEGFR-3 andβ-catenin mRNA of RT-PCR products, compared with control group, statistical analysis of variance and VEGF-C and VEGFR-3mRNA results showed that the expression of each Indomethacin group was significantly lower (respectively P <0.05). There was no difference inβ-catenin mRNA level among Indomethacin-treated groups (P>0.05).2 Effects of Indomethacin combined with oxaliplatin on tumor lymphangiogenesis and synergistic in lung cancer xenograft in nude mice.We performed immunohistochemistry analysis of VEGF-C, VEGFR-3, andβ-catenin protein in tumor samples. Compared with control group, statistical analysis of variance showed that the expression levels of VEGF-C, VEGFR-3, andβ-catenin protein of Indomethacin-treated group were significantly reduced (respectively, P <0.05). Compared with the control group, the expression levels of VEGF-C protein of oxaliplatin-treated group were evidently increased (P <0.05). Meanwhile the expression levels of survivin protein of oxaliplatin-treated group were significantly reduced (respectively, P <0.05). Compared with the control group, the expression levels of VEGF-C, VEGFR-3, Survivin andβ-catenin protein of Indomethacin combined with oxaliplatin-treated group were significantly reduced (respectively, P <0.05).According to the real-time fluorescence quantitative analysis of VEGF-C and survivin mRNA, compared with the control group, statistical analysis of variance showed that the expression levels of VEGF-C and Survivin mRNA of Indomethacin-treated group and Indomethacin combined with oxaliplatin-treated group were significantly reduced (respectively, P<0.05). Compared with the control group, VEGF-CmRNA of oxaliplatin-treated group were notably increased (P >0.05) . Moreover, the expression levels of Survivin mRNA of oxaliplatin-treated group were significantly reduced (P <0.05).Conclusions:1 Different doses of Indomethacin were able to significantly inhibit the the expression of VEGF-C, VEGFR-3 andβ-catenin of lung cancer xenografts in nude mice. Although the anti-tumor effect of indomethicin is increased as the dose increases, side effects increase as well, so low-dose indomethicin is a good choice for clinical.2 Indomethacin can suppress expression of VEGF-C, VEGFR-3,β-catenin and Survivin when being used alone, further, when being combined with oxaliplatin,it can enhance the effect of oxaliplatin against tumor. Inhibiting VEGF-C/ VEGFR-3 and Wnt pathway maybe its mechanism to induce the tumor metabasis.
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