â… . Effects Of The Combination Of Topoisomerase I Inhibitors And Celecoxib, A Selective Cylooxygenase-2 Inhibitor On Colorectal Cancer â…¡. Studies On Antitumor Activity Of New Angiogenetic Inhibitors And Their Mechanisms

AIM: To investigate the antitumor properties of camptothecins (CPTs) combined with celecoxib, a selective cylooxygenase-2(COX-2) inhibitor, in vitro and in vivo, and the effects of celecoxib on irinotecan (CPT-11)-induced diarrhea in mice.METHODS: Four human colon cancer cell lines, HT-29, HCT-8, HCT-116 and Caco-2 were exposed to increasing concentration(10^-5, 10^-4, 10^-3,10^-2, 10^-1, 1, 10,100μmol/L ) of three Topoâ… inhibitors, CPT-11, CPT and topotecan(TPT), and celecoxib(1 and 5μmol/L) as well as a combination of each topoisomerase I (Topo I )inhibitor, then MTT assay was performed to evaluate the effects of adjunct use of celecoxib on chemosensitivity to CPTs. HT-29 cells were divided into several treatment groups, control group, celecoxib treatment group, CPT treatment group and combined CPT with celecoxib treatment group. Flow cytometry was used to evaluate the apoptotic rates and cell cycle distribution. Expression of COX-2 and apoptosis related proteins, Bcl-2, Caspase-3 and P53, were determined by immunocytochemical method. HT-29 cell line-xenografts model was established. The nude mice bearing tumor were divided into five groups, namely control, CPT-11(25 mg/kg per day) treatment group, celecoxib (60 mg/kg) treatment group and combined celecoxib (30 mg/kg and 60 mg/kg, respectively) with CPT-11 treatment group. After being treated with drugs, the effects of those drugs on tumor growth and the severity of late diarrhea induced by CPT-11 were assessed.RESULTS: Celecoxib significantly decreased the IC₅₀ of the CPTs in the four colon cancer cell lines in vitro, but the degrees of reduction of IC₅₀ depended on the level of COX-2 expression. In HT-29 cells, the apoptotic rates were 51.4 % in co-treated groups (24.4% in CPT treated groups, P＜0.01), and the proportion in the G0/G1 phase were 49.1% in co-treated group (5.5% in CPT treated groups, P＜0.01). The expression of COX-2 and Bcl-2 were down-regulated; the expression of P53 and Caspase-3 were up-regulated alter co-treatment with CPT and celecoxib in HT-29 cells. In vivo, treatment with celecoxib at 60mg/kg in conjunction with CPT-11 (25 mg/kg per day for three consecutive days) significantly reduced tumor growth by 78.77% (P＜0.01 compared with vehicle; P＜0.05 compared with CPT-11), and decreased the score of diarrhea induced by CPT-11(P＜0.01 compared with CPT-11).CONCLUSION: Celecoxib enhances antitumor properties of CPTs in colon cancer in vitro and in vivo, and this enhancement may be associated with an increase in apoptosis and cell cycle arrest. Additionally, celecoxib reduces the severity of diarrhea and body weight loss induced by CPT-11 significantly. New blood vessel formation (angiogenesis) is a fundamental event in the process of tumor growth and metastatic dissemination. Hence, the molecular basis of tumor angiogenesis has been of keen interest in the field of cancer research. The vascular endothelial growth factor (VEGF) pathway is well established as one of the key regulators of this process. The VEGF/VEGF-receptor axis is composed of multiple ligands and receptors with overlapping and distinct ligand-receptor binding specificities, cell-type expression, and function. Activation of the VEGF-receptor pathway triggers a network of signaling processes that promote endothelial cell growth, migration, and survival from pre-existing vasculature. In addition, VEGF mediates vessel permeability, and has been associated with malignant effusions. More recently, an important role for VEGF has emerged in mobilization of endothelial progenitor cells from the bone marrow to distant sites of neovascularization. The well-established role of VEGF in promoting tumor angiogenesis and the pathogenesis of human cancers has led to the rational design and development of agents that selectively target this pathway. Studies with various anti-VEGF/VEGF-receptor therapies have shown that these agents can potently inhibit angiogenesis and tumor growth in preclinical models. Recently, an anti-VEGF antibody (bevacizumab), when used in combination with chemotherapy, was shown to significantly improve survival and response rates in patients with metastatic colorectal cancer and thus, validate VEGF pathway inhibitors as an important new treatment modality in cancer therapy. In order to develop new anti-angiogenic agents, two kinds of drugs were studied in this part and we also tried to elucidate the mechanisms underlying the effects of these compounds. The first candidate, new derivative of phthalazine, was designed as a VEGF-R kinase inhibitor synthesized by associate professor Zhiqiang Feng, which inhibited the signaling transduction of VEGF/VEGF-R; the second one, LGD1069, is a selective agonist of retinoid X receptor (RXR), which down-regulated the secretion of VEGF in tumor cells through the JNK and ERK pathway.The results are as follows:1. Antitumor and antiangiogenesis properties of FVE-3.1.1 Antitumor activity of FVE-3In vitro, FVE-3 was found to inhibit significantly the growth of cancer cells derived from different tissues, including human colorectal adenocarcinoma cells (HT-29, HCT-8), human ovarian cancer cells (A2780), human gastric adenocarcinoma cell (BGC-823), human lung adenocacinoma cells (A549), human uterine cervix cancer cells (HeLa), human breast cancer cells (MCF-7), human melanoma cells (A375), mouse melanoma cells (B16), highly metastatic mouse melanoma cells (B16-BL6, derived from B16), mouse melanoma cell transfected by mekkl gene and pCDNA 3.1 (m1B16 and pB16, respectively). MTT assay showed that its IC₅₀ toward these tumor cells was 2.25～9.17μmol/L. GI₅₀ evaluated by SRB assay was 1.06～4.3μmol/L in three human colorectal adenocarcinoma cell line (HT-29, HCT-8 and HCT-116). Additionally, the colony formation ability in HT-29 and HCT-8 cells was inhibited significantly by FVE-3. (p＜0.05, p＜0.01).In vivo, FVE-3 administered by i.g. prevented, in a dose-dependent manner, the growth of Lewis lung cancer and hepatic cancer H22 in mice. FVE-3 administered at 37.5 and 75mg/kg/day caused a 28.0% and 53.6% inhibition in Lewis lung tumor growth, respectively. The similar results were also shown in hepatic cancer model, the tumor growth inhibited by 45.1% and 50.4%. In addition, two human colorectal cancer models, HCT-116 and HT-29, in nude mice were used to evaluate the antitumor properties of FVE-3 in vivo. In this setting, FVE-3 inhibited tumor growth 39.7% and 42.0% in HCT-116 tumor (p＜0.05 compared with vehicle-treated animals). To further demonstrate the value of this candidate, we tested it in another tumor model (HT-29). Under this condition, FVE-3 given at 100 and 200mg/Kg/day, caused a similar inhibition to HCT-116 tumors (30.4% and 43.5%, respectively).1.2. Antiangiogenesis properties of FVE-3The anti-proliferation effect of FVE-3 on human umbilical vein endothelial cell (ECV/304 and HUVEC) was demonstrated by SRB assay with GI₅₀ as 4.38 and 1.11μmol/L. Three-dimensional in vitro angiogenesis system showed that FVE-3 inhibited tube formation of ECV/304 cells seeded on Matrigel; the multi-cellular capillary-like structure formation rate was suppressed to 93.99%, 44.54% and 35.73% of untreated control by 8μmol/L, 16μmol/L and 32μmol/L FVE-3, respectively. The same model was used to assess the effects of FVE-3 on the tube formation of HUVEC cells. In this setting, the vessel formation rate was 87.66%, 19.12% and 14.52%, respectively. Additionally, the invasion through reconstituted basement membrane of ECV/304 cells induced by fibronectin (FN) was significantly restrained by FVE-3 at differentconcentrations, and the inhibitory rate of 8μmol/L, 16μmol/L and 32μmol/L FVE-3 on migration potential was 9.72%, 21.73% and 72.16%, respectively. To further demonstrate the anti-angiogenic effects of this candidate in vivo, two tumor angiogenic models were used. In hollow fiber assay, the fiber containing A549, MCF-7 and HT-29 cells induced marked angiogenesis; However, FVE-3 administrated i.p. once daily inhibited the induction of angiogenesis. The inhibitory effects of FVE-3 were dose-dependent. Furthermore, Immunohistochemical evaluation of HCT-116 tumors after FVE-3 treatment was used. Compared with control, FVE-3 dramatically reduced the expression of CD31 in a dose-dependent manner. With RT-PCR analysis, FVE-3 treated ECV/304 and HUVEC cells for 24h inhibited VEGF-Rs gene expression. When cells were treated with FVE-3 for 48h, the contents of p-VEGF-R, p-ERK, MEK-1 and COX-2 protein in ECV/304 cells were decreased dramatically. It was shown that the positive area was lower than vehicle-treated animals significantly.Totally, the compound, FVE-3, showed antitumor efficacy attributable to antiangiogenic activity, it is very probable that the efficacy is a result of this mechanism that it inhibits the VEGF-R2 kinase.2. Antitumor and antiangiogenesis properties of LGD1069, a selective RXR agonist.2.1 Antitumor activity of LGD1069 in vitro and in vivoEffects of LGD1069 on the growth of different cancer cell lines were assayed by MTT and SRB assay. MTT test showed that the IC₅₀ of LGD1069 to A2780 and A549 cells were in the range of 29.3～34.6μmol/L. GI₅₀ evaluated by SRB assay was 23.43～26.04μmol/L. The inhibition of LGD1069 on growth of A2780 and A549 cells was in a dose-dependent manner, and the colony formation ability of A2780 and A549 cells was inhibited by LGD1069 (p＜0.05) significantly, too. In vivo, oral LGD1069 at two different doses, 30mg/kg and 60mg/kg, prevented, in a dose-dependent manner, the growth of Lewis lung cancer and cervical cancer U14 of mice. We next investigated, using a xenografted A549 model, whether the anti-tumor effects of LGD1069 on human lung adenocacinoma growth could also be observed. LGD1069 administered at 30 and 60mg/kg/day caused a 53.5% and 64.2% (p＜0.05) inhibition in A549 tumor growth, respectively. 2.2 Mechanisms of action of LGD10692.2.1 Effects of LGD1069 on apoptosis in cancer cellsTo investigate the characteristic of growth inhibition caused by LGD1069, flow cytometric analysis was performed. The cells treated with increasing concentrations of LGD1069 dose-dependently increased the percentage of apoptotic cells (p＜0.01). In addition, the similar results were shown by DNA ladder assay. Western Blotting analysis was performed to observe the expression of apoptosis related proteins, Bcl-2 and caspase-3. Except Caspase-3, other proteins expression was decreased in a dose-dependent manner.2.2.2 Antiangiogenesis properties of LGD1069The anti-proliferation effect of LGD069 on human umbilical vein endothelial cell (ECV/304) was demonstrated by MTT and SRB assay with IC₅₀ of 28.28μmol/L and with GI₅₀ of 17.24μmol/L. Three-dimension in vitro angiogenesis system showed that LGD1069 inhibited tube formation of ECV/304 cells seeded on Matrigel, the multicellular capillary-like structure formation was suppressed to 72.8%, 30.8% and 13.2% of untreated control by 7.5, 15 and 30μmol/L LGD1069, respectively. To further demonstrate the antiangiogenic effects of LGD1069 in vivo, two tumor angiogenic models were used. In hollow fiber assay, the fiber containing A549,MCF-7 and HT-29 cells induced marked angiogenesis; However, LGD1069 administrated i.g. once daily inhibited the induction of angiogenesis. The inhibitory effects of LGD1069 were dose-dependent and 30, 45 and 60 mg/kg of LGD1069 reduced the dense capillary network about 50%, 54% and 55.2% of the control, respectively. Furthermore, Immunohistochemical evaluation of A549 tumors after LGD1069 treatment was used. Compared with control, LGD1069 dramatically reduced the expression of CD31 in a dose-dependent manner. With RT-PCR analysis, RXRs, bFGF and VEGF gene expression in A549 cells was inhibited by LGD1069 treatment. In order to evaluate the effects of LGD1069 on the level of VEGF product, an ELISA assay was used, and it was shown that LGD1069 inhibited the secretion of VEGF in A549 cells significantly. In addition, the contents of p-JNK, p-ERK, and COX-2 protein in A549 cells were decreased dramatically, when cells were treated with LGD1069 for 48h.In addition, effects of LGD1069 on invasion and adhesion of endothelial cells were detected. 7.5μmol/L LGD1069 suppressed 38.7% ECV/304 cells to invade through reconstituted basement membrane, while treatment with 30μmol/L LGD1069 for 24h, invasive potential of ECV/304 cells decreased to 14.2% as compared with control. After treated with LGD1069 for 2h, the adhesive ability of ECV/304 cells with basement membrane components (matrigel) was markedly reduced in a dose-dependent manner. Furthermore, the effects of LGD1069 on the expression of invasion related genes were evaluated by RT-PCR assay. It was shown that LGD1069 decreased the RNA level of uPA and MMP-2 in A549 cells, but increased the expression of TIMP-1 gene.In summary, it was found that LGD1069 inhibited growth of various cancer cells in vitro and in vivo, and the probable mechanisms were first, induction of apoptosis in tumor cells by activating its receptors, and inhibition of tumor-induced angiogenesis. The underlying story may down-regulate the expression of VEGF through JNK and ERK signaling transduction.