Antitumor efficacy of combination of interferon-gamma-inducible protein 10 gene with gemcitabine, a study in murine model

Kai Mei 2 Lian Wang 0 Ling Tian 1 Jingrui Yu 2 Zhixuan Zhang 2 Yuquan Wei 1 0 Department of Pathology, West China College of Stomatology, Sichuan University , Chengdu, 610041 , PR China 1 State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medical School, Sichuan University , Chengdu, 610041 , PR China 2 Department of Oncology, Sichuan Cancer Hospital , Chengdu, 610041 , PR China Background: Interferon--inducible protein 10 (IP-10) is a potent inhibitor of tumor angiogenesis. It has been reported that the antiangiogenic therapy combined with chemotherapy has synergistic effects. Methods: To elucidate the mechanisms of IP-10 gene combined with a chemotherapy agent, we intramuscularly injected pBLAST-IP-10 expression plasmid combined with gemcitabine into tumorbearing mice. Results: The proliferation of endothelial cells was effectively inhibited by IP-10 combined with gemcitabine in vitro. Treatment with pBLAST-IP-10 twice a week for 4 weeks combined with gemcitabine 10 mg/kg (once a week) resulted in sustained high level of IP-10 protein in serum, inhibition of tumor growth and prolongation of the survival of tumor-bearing mice. Compared with administration of IP-10 plasmid or gemcitabine alone, the angiogenesis in tumors were apparently inhibited, and the numbers of apoptotic cells and lymphocytes in tumor increased in the combination therapy group. Conclusion: Our data indicate that the gene therapy of antiangiogenesis by intramuscular delivery of plasmid DNA encoding IP-10 combined with gemcitabine has synergistic effects on tomor by inhibiting the proliferation of endothelail cells, inducing the apoptosis of tumor cells, and recruiting lymphocytes to tumor in murine models. The present findings provided evidence of antitumor effects of genetherapy combined with chemotherapy. - Background Angiogenesis, the formation of new blood vessels, is important not only for normal physiological processes, but also for the development of pathologic conditions such as cancer, rheumatoid and inflammation. Presently, accumulated evidences indicate that the growth and metastasis of solid tumors is dependent on angiogenesis. Therefore, targeting tumor vasculature has been a popular strategy of therapeutics [1,2]. But anti-angiogenesis alone is not sufficient due to the angiogenesis-independent phase of tumor growth. It has been reported that cytotoxic agents can impair neovasculature directly or indirectly [3]. Further, proliferative endothelial cells in new vessels are sensitive to cytotoxic agents [4]. Combination therapy, consisting of low-dose chemotherapy and antiangiogenesis, may produce improved efficacy and reduced toxicity due to the synergistic effect on tumors [4,5]. Interferon--inducible protein 10 (IP-10), a member of the chemokine family, is secreted by activated T cells, fibroblast and endothelial cells [6]. It attracts activated, but not resting, T lymphocytes and NK cells [7-9] via stimulating CXCR3 chemokine receptor [7]. Tumor cell lines stably transfected with the IP-10 gene were rejected by immune-system [10]. Intratumoral injection of IP-10 gene facilitated regression of established mice tumors [11]. IP-10 is also a potent inhibitor of angiogenesis [10,12]. Thus, we hypothesize that IP-10, which is involved in immune (T and NK cells) and antiangiogenic responses, is a good candidate for treatment of malignant tumors. Gemcitabine (GEM), a deoxycytidine analog, is currently used as a therapeutic agent against several solid tumors, such as non-small cell lung cancer (NSCLC), pancreatic cancer and bladder cancer [13]. In local advanced or metastatic NSCLC, gemcitabine has been used as the first-line therapeutic. However, the efficacy of gemcitabine is unsatisfactory with a response rate of only about 36%, and time of tumor progression is 4 to 5 months. [14] Therefore we sought to develop therapeutic agents that would increase the anti-tumor efficacy of gemcitabine. IP-10 may enhance the effects of gemcitabine in combination therapy via angiogenic-independent mechanisms. In 2005, a synergistic effect of their combination on solid tumors was found by our study group [15]. In this study, we try to elucidate the mechanism of combination of IP-10 with gemcitabine. Materials and methods Preparation of IP-10 plasmid pBLAST-IP-10 plasmid (purchased from Invivogen, San Diego, CA, USA) was transformed into E. coli JM109, and cultured at 37C for plasmid isolation by endotoxin-free plasmid maxiprep kit (Roche, Germany). The concentration of pBLAST-IP-10 plasmid was determined by ultraviolet spectrophotometer. Transfection of COS cells with pBLAST-IP-10 COS cells were plated in six-well plates at 2 105 cells/ well in DMEM containing 10% FBS at 37C overnight. Cells at 6080% confluence were transfected with 2 g of plasmid and 6 l lipofectamine (Invitrogen) in serum-free DMEM following the manufacture's instructions. After addition of the lipofectamine-DNA complex, the ce (...truncated)