Evaluation Of Vascular Normalization In Human Lung Adenocarcinoma Induced By Epigollatecatech In Gallate And The Efficacy Of Combine Chemotherapy In Its Normalization Time Window

Background:Microvasculature and microenvironment play important roles of proliferation, invasion, metastasis and prognosis in human lung adenocarcinoma, and can be affected by various anti-angiogenic drugs. It has been demonstrated that appropriate anti-angiogenesis treatment tends to normalize previously torturous and malformed vessels, thus normalizing microvasculature and microenvir-onment, which, then translates to delivering oxygen and drugs to tumor cells in a more effective way, and consequently improving the treatment efficacy of both chemotherapy and radiotherapy. The priod of normalization is transient and reversible, so a specific normalization time window can be formed according to the applied drugs, as well as the types and location of the tumor. In this time window, combined chemotherapy can reach a synergistic effect.Currently a large scale of pre-clinical or clinical researches synergistic effect. Epigallocatechin-3-gallate (EGCG), a nature anti-angiogenesis agent refined from green tea, is defined to have multiple effects on angiogenesis factors, such as vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF) and angiopoietins(ANGs). So, we suppose that ECGC can alter human lung adenocarcinoma tumor microvasculature and microenvironment and cause "vessel normalization"phenomenon, and in addition combined chemotherapy exert a synergistic effect in the tumor vessel normalization window caused by EGCG treatment.To demonstrate this assumption, we built a nude mice xenograft tumor model in the cell line of A549human lung adenocarcinoma, examine the structure and function of microvasculature, microenvironment index changes, as well as the efficacy of combined chemotherapy at different time points after treating the model with EGCG.Methods and results:Build a nude mice xenograft tumor model in the cell line of A549human lung adenocarcinoma. Randomly divide the mice into three groups and treat each one with saline, EGCG, bevacizumab, and respectively test the following indexes at the time points of0,2,4,6,9,12day.(1)Vessel structure change index:quantum dots double-lable immunofluorescence assessment of CD31-a-SMA to observe microvasculature, counting CD31-MVD, a-SMA-MVD, and MPI; quantum dots double-lable immunofluorescence assessment of CD31-Collagen IV to observe vessel GBM; Transmission electron microscope to assess endothelial cells, the form and connection of pericytes, the integrity of GBM and the association between tumor cells and vessels.(2)Vessel functional change indexes:quantum dots double-lable immunofluorescence assessment of CD31-lectin to assess vessel perfusion function. Evans blue perfusion to assess vessel permeability.(3)Microenvironment effect change indexes:IFP, PO2. Immunohistochmical label of Pimonidazole to test hypoxia level in the tumor tissue. EGCG treatment of A549cells in mice bearing xenografts in vivo led to a persisting decrease of CD31-MVD, and a gradual decrease of MPI, a transient alevation of vessel perfusion function and permeability and PO2, transient decrease of IFP in tumor tissue. Yet EGCG treated group had a thinner microvasculature GBM which was regularly distributed, but bevacizumab group had simply a tendency of decreased Collage IV expression without significancy (P>0.05). Accordingly, it can be concluded that EGCG caused the vessel normalization of NSCLC xenografts tumor. Additionally, vessel indexes mentioned above had a transient change window in day4to9, yet bevacizumab’s time window was in day2to6. We test cisplatin concentration in tumor tissues when administrated with different combined chemotherapy of EGCG and cisplatin. Full-dose cisplatin (4mg/kg) at dO (pre-window period) in single intraperitoneal administration (Group A), half-dose cisplatin (2mg/kg) at d5(window period) in single intraperitoneal administration (Group B), full-dose cisplatin (4mg/kg) at d5in single intraperitoneal administration(Group C), and use graphite furnace atomic absorption spectrometry to test cisplatin concentration in tumor tissue. Results showed that Group C had a concentration significantly higher than other three groups (1.8μg/gm tissue, p<0.001), and Group B had the assembling concentration as Group A (respectively1.0μg/gm tissue and0.8μg/gm tissue) without significancy (p>0.05). To specify whether combined therapy can affect human lung adenocarcinoma tumor’s sensitivity to chemotherapy and have synergistic effect in the window periods mentioned above, we treated xenograft tumor nude mice with saline (Group A), cisplatin (Group B), EGCG (Group C), EGCG+combining cisplatin on dO (Group D1) and EGCG+combining cisplatin on d5(Group D2). Recorded the time for each tumor to reach the approximately volume of1250mm3then calculate number of tumor growth delay, and calculate tumor suppressing rate to observe tumor growth rate. Results showed that each group had a tumor growth delay of6.3±1.51days,7.5±1.57days,8.3+1.79days,12.1±1.35days and15.4±1.99days. Analysis of tumor growth delay of Group A, B, C, D2suggested that EGCG and cisplatin had synergistic effect as a combined anti-tumor chemotherapy (P<0.01). In addition, this result also revealed that combined treatment groups (D1, D2) had significantly lower xenograft tumor growth rates than other three groups (tumor inhibition rate were respectively5.53%、12.82%、40.18%、 56.88%, p<0.001), and tumor growth rate in D2was significantly lower than D1(P<0.01).Conclusion:EGCG causes vessel normalization in NSCLC xenograft tumor, the window period of with is between Day4to Day9. Combined therapy in this window period can escalate drug concentration in local tumor tissue, and leads to anti-tumor synergistic effect, providing a new strategy for EGCG applying as a complementary chemotherapy drug.