Gaoxin Lei1, Menglong Xu2, Zhipan Xu2, Chenchen Lu2, Shuhua Tan3. 1. State Key Laboratory of Natural Medicines, School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, PR China; Vaccine Engineering Center of China Medical City, Taizhou, 225300, Jiangsu, PR China. 2. State Key Laboratory of Natural Medicines, School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, PR China. 3. State Key Laboratory of Natural Medicines, School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, PR China. Electronic address: tanshuhua163@163.com.
Abstract
OBJECTIVES: To investigate the antitumor activity of a novel agonistic single chain fragment variable (scFv) antibody TR2-3 targeting death receptor 5 (DR5) combined with cisplatin in vitro and in vivo. METHODS: The in vitro cytotoxic effects of TR2-3 and cisplatin, alone or in combination on human cancer cell lines COLO205 and MDA-MB-231 were evaluated using the MTT assay. The apoptosis in cancer cells was evaluated by an Annexin V-PE apoptosis detection kit and flow cytometry. The mRNA and protein levels of DR5 were analyzed by real-time PCR and Western blot, respectively. Additionally, the in vivo antitumor activity of TR2-3 combined with cisplatin was evaluated in a xenograft model. RESULTS: The combination treatment with TR2-3 and cisplatin for 24 h on COLO205 and MDA-MB-231 cells showed significant cytotoxicity effects by MTT assay, compared with the alone treatment. Consistent with cell viability results, the cisplatin enhanced the apoptosis-inducing effects of TR2-3 in the COLO205 cells and MDA-MB-231 cells by flow cytometry. In addition, treatment with cisplatin alone for 24 h resulted in significantly up-regulating the mRNA and protein levels of DR5 in both COLO205 and MDA-MB-231 cell lines by q-PCR and Western blot assay. Moreover, the cytotoxic effects of TR2-3 can be blocked by adding the soluble DR5, and the blocking rate can be greatly reduced by co-treatment with cisplatin. These results indicated that cisplatin sensitized COLO205 and MDA-MB-231 cancer cells to TR2-3-mediated apoptosis by up-regulation of DR5 expression. Furthermore, combination therapy with TR2-3 and cisplatin enhanced tumor growth inhibition compared to treatment with TR2-3 or cisplatin alone in mice bearing COLO205 xenograft tumors. CONCLUSIONS: Our findings suggest that cisplatin enhanced the antitumor activity of TR2-3 in COLO205 and MDA-MB-231 cancer cells through up-regulation of DR5 expression. The TR2-3 combined with cisplatin may be a promising treatment for cancer therapy.
OBJECTIVES: To investigate the antitumor activity of a novel agonistic single chain fragment variable (scFv) antibody TR2-3 targeting death receptor 5 (DR5) combined with cisplatin in vitro and in vivo. METHODS: The in vitro cytotoxic effects of TR2-3 and cisplatin, alone or in combination on humancancer cell lines COLO205 and MDA-MB-231 were evaluated using the MTT assay. The apoptosis in cancer cells was evaluated by an Annexin V-PE apoptosis detection kit and flow cytometry. The mRNA and protein levels of DR5 were analyzed by real-time PCR and Western blot, respectively. Additionally, the in vivo antitumor activity of TR2-3 combined with cisplatin was evaluated in a xenograft model. RESULTS: The combination treatment with TR2-3 and cisplatin for 24 h on COLO205 and MDA-MB-231 cells showed significant cytotoxicity effects by MTT assay, compared with the alone treatment. Consistent with cell viability results, the cisplatin enhanced the apoptosis-inducing effects of TR2-3 in the COLO205 cells and MDA-MB-231 cells by flow cytometry. In addition, treatment with cisplatin alone for 24 h resulted in significantly up-regulating the mRNA and protein levels of DR5 in both COLO205 and MDA-MB-231 cell lines by q-PCR and Western blot assay. Moreover, the cytotoxic effects of TR2-3 can be blocked by adding the soluble DR5, and the blocking rate can be greatly reduced by co-treatment with cisplatin. These results indicated that cisplatin sensitized COLO205 and MDA-MB-231 cancer cells to TR2-3-mediated apoptosis by up-regulation of DR5 expression. Furthermore, combination therapy with TR2-3 and cisplatin enhanced tumor growth inhibition compared to treatment with TR2-3 or cisplatin alone in mice bearing COLO205 xenograft tumors. CONCLUSIONS: Our findings suggest that cisplatin enhanced the antitumor activity of TR2-3 in COLO205 and MDA-MB-231 cancer cells through up-regulation of DR5 expression. The TR2-3 combined with cisplatin may be a promising treatment for cancer therapy.