| OBJECTIVELung cancer is the first cause of cancer death and the most common cancer worldwide, of which more than80%is non-small cell lung cancer (NSCLC). Patients are usually diagnosed in advanced stage and most of them are not sensitive to conventional therapy such as chemotherapy and radiotherapy. Despite of this poor clinical outcome. lung cancer tissue is rich in blood vessels, which provides advantage for the anti-angiogenesis treatment. Tea is the second popular drink after water in the world. It is regarded as an herb in Traditional Chinese Medicine. According to the Syndrome Differentiation Theory, tea has the function of clearing the phlegm and detoxification, which is critical for cancer treatment. And results from our clinical trial as well as lab experiments on Tea Polyphenols (TPs) showed it had anti-cancer and anti-angiogenesis effects. In order to further investigate TP’s effects on the tumor growth and angiogenesis of NSCLC and to explore its possible transduction signaling pathways, we carried out a series of in vitro and in vivo experiments with a focus on NF-κB, AP-1and their related pathways.METHODSFor in vitro experiments, the effects of TP and its main component EGCG on growth inhibition of NSCLC were tested by Methylene Blue Assay. KB and KB-MDR cell lines were used to test TP’s sensitivity and possible reversal effects of multidrug resistance. Luciferase Reporter System was used to study the signaling pathways of TP. For in vivo experiments, Lewis lung cancer xenograft model was established and48implanted mice were randomly assigned to six groups, namely model control group (0.9%NaCl), Thalidomide group (76mg/kg/d), TP low-dose group (112.5mg/kg/d), TP high-dose group (337.5mg/kg/d), TP low-dose combined with Thalidomide group (112.5mg/kg/d+76mg/kg/d), and TP high-dose combined with Thalidomide group (337.5mg/kg/d+76mg/kg/d). All groups received continuous ten-day orally administrated treatment accordingly. Toxicity and anticancer effects were observed and calculated, while different factors (NF-κB, COX-2, Survivin, Akt-2, C-Jun, MMP-2and TIMP-2) related to anti-angiogenesis pathways were tested applying immunohistochemical techniques.RESULTSExperimental results showed that:(1) The IC50of cell growth inhibition for TP in NCI-H1299, NCI-H522, A549and EKVX cell lines were11.8±2.5μg/ml,26.1±5.6μg/ml,61.5±12.7μg/ml and9.4±0.9μg/ml respectively; and for EGCG were20.4±5.2μM,51.3±12.7μM,117.5±28.8μM and20.0±4.7μM respectively.(2) There was no significant difference of TP and EGCG’s IC50between KB and KB-MDR cell lines, and no reversal effects on KB-MDR’s resistance to Taxol were indicated when treated with TP and EGCG.(3) TP inhibited TNF-α induced NF-kB signaling with an IC50of51μg/ml, inhibited TPA induced AP-I signaling with an IC50of18μg/ml, suppressed PGN induced TLR-2signaling with an IC50of83μg/ml, and suppressed ITS induced TLR-4signaling with an IC50of78μg/ml, correspondingly. No significantly changes were showed for Nrf2and Ubiquitin signaling.(4) Compared with model control group. IT high-dose combined vvith Thalidomide group significantly inhibited tumor growth (P<0.05), with a tumor inhibition rate of approximately44%.(5) Comparing to model control group, TP high dose combined with Thalidomide group significantly reduced the expression of NF-kB. COX-2and Survivin (P<0.05), while the expression of Akt-2were not obviously changed in all groups.(6) Comparing to model control group, MMP-2expression was down-regulated in IP groups and their Thalidomide combination groups (P<0.05). whereas TIMP-2expression was up-regulated in IT high close combined with Thalidomide group (P<0.05). there were higher MMP-2/TIMP-2ratios in all IP involved groups, especially for IP high-dose combined with Thalidomide group.CONCLUSIONAccording to our studies, we find out (1) TP and LGCG could inhibit the growth NSCLC cell lines;(2) IP is not resistance to KB-MDR. but cannot reverse its multi-drug resistance either:(3) TP could inhibit NF-kB, AP-1, TLR-2, TLR-4signaling pathways, with a higher potency for NI’-kB and AP-1pathways;(4) TP high dose combined with Thalidomide can inhibit Lewis lung tumor growth;(5) NF-kB signaling pathway is a key pathway of IP’s anti-angiogenesis effects, in which it inhibits NF-kB activation, reduces COX-2expression and Survivin expression in endothclial cells;(6) The anti-angiogenesis effect of TP could also related to down-regulation of MMP-2expression, up-regulation of TIMP-2expression, and restoring of MMP-2/TIMP-2balance. To sum up, Tea polyphenols have been confirmed to have tumor inhibition effect on NSCLC to a certain extent, and it has the potential to be a multi-targeted anti-angiogenesis agent affecting various signaling pathways at different levels. |
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