Volume 15, Issue 2, Pages 404-410 (February 2007) Inhibitor of Heat-shock Protein 90 Enhances the Antitumor Effect of DNA Vaccine Targeting Clients of Heat-shock Protein  Chi-Chen Lin, Cheng-Fen Tu, Meng-Chi Yen, Ming-Chuan Chen, Wan-Jung Hsieh, Wen-Chang Chang, Wen-Tsang Chang, Ming-Derg Lai  Molecular Therapy  Volume 15, Issue 2, Pages 404-410 (February 2007) DOI: 10.1038/sj.mt.6300014 Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 1 GA downregulates Her-2/neu but upregulates MHC class I and HSP70 expression in MBT-2 tumors overexpressing p185neu. (a) Tumors were treated with escalating amounts of GA for the indicated periods, and tumor lysates were harvested and subjected to Western blot analysis with the indicated antibodies. (b) MBT-2 tumors were treated with various doses of GA, and the surface MHC class I was examined by flow cytometric analysis. Gray histogram plots represent GA treatment. White histogram plots represent the saline controls and the black dotted lines represent the isotype-matched controls. (c) Tumors were treated with GA, and the expression HSP70 was examined by Western blotting, and the fold represents the HSP70/actin ratio. Molecular Therapy 2007 15, 404-410DOI: (10.1038/sj.mt.6300014) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 2 Therapeutic effects of GA and N′-neu DNA vaccine on established MBT-2 tumors. (a) Tumor volume in mice treated with GA was measured every 3 days; bars, ±SD. (b) Lifespan of C3H mice after s.c. challenge with MBT-2 cells; survival data were subjected to Kaplan–Meier analysis. (c) Experimental protocol for combination treatment with GA and N′-neu DNA vaccine. (d) Tumor volume of mice treated with combination of GA and N′-neu DNA vaccine was measured every 3 days; bars, ±SD. (e) Lifespan of C3H mice after s.c. challenge with MBT-2 cells; survival data were subjected to Kaplan–Meier analysis. Numbers in parentheses are the number of mice in treatment groups. *Statistically significant difference compared with the control mice. **Statistically significant difference between the GA and N′-neu DNA vaccine combination and the N′-neu DNA vaccine alone. Molecular Therapy 2007 15, 404-410DOI: (10.1038/sj.mt.6300014) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 3 Humoral and cellular immunity. (a) IgG and IgG subtype of anti-p185neu antibody titers in the serum samples was determined with an ELISA. The data represent the average titer of three mice. (b) Cytotoxicity assay of lymphocytes. Target cells are MBT-2-luciferase cells cultured in vitro. Effector cells were lymphocytes derived from mice treated with the N′-neu DNA vaccine, a combination of GA and the N′-neu DNA vaccine, or saline (control). Cytotoxicity was determined by the luciferase release. Each point represents the average of triplicate wells. (c) Adoptive transfer of splenocytes of immunized mice. MBT-2 tumor-bearing mice were injected intravenously with splenocytes isolated from the mice treated with the N′-neu DNA vaccine or a combination of GA and the N′-neu DNA vaccine as indicated. Tumor bearing control animals received no splenocytes (MBT-2) or received splenocytes from mice that received saline treatments (MBT-2+saline). (d) Cytokine profile, pooled splenocytes from each group of mice were stimulated with p185neu antigen. Supernatants were collected 2 days after stimulation and the concentration of interferon-γ and interleukin-4 were measured with ELISA. (e) Susceptibility of GA-treated tumor cells to cytotoxic lymphocytes. Effector cells were lymphocytes isolated from mice immunized with the N′-neu DNA vaccine or from control mice. Target cells were isolated from established GA-treated or untreated MBT-2-luciferase tumors. Statistically significant differences between treated and control mice are represented with *(P<0.05), **(P<0.01). Molecular Therapy 2007 15, 404-410DOI: (10.1038/sj.mt.6300014) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 4 The effects of CD4+ T-cell depletion or CD8+ T-cell depletion on the therapeutic efficacy of GA and/or the N′-neu-DNA vaccine. (a) Protocol for in vivo depletion of CD4+ or CD8+ T cells. MBT-2 tumor-bearing mice were intraperitoneally injected with 300 μg of anti-CD4 antibody, 500 μg of anti-CD8 antibody, or an isotype-matched control mAb at weekly intervals starting 2 days before the first treatment. (b) Lifespan of C3H mice after s.c. challenge with MBT-2 cells; survival data were subjected to Kaplan–Meier analysis. Molecular Therapy 2007 15, 404-410DOI: (10.1038/sj.mt.6300014) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 5 Therapeutic effects of GA and C′-met DNA vaccines on established B16 tumors in C57BL/6 mice. (a) Therapeutic protocols of GA alone and in combination with the C′-met DNA vaccine. Lifespan of (b) B16 tumor bearing C57BL/6 mice treated with GA alone or (c) a combination of GA and the C′-met DNA vaccine is shown by Kaplan–Meier analysis. Numbers in parentheses are numbers of mice in treatment groups. *Statistically significant differences between treated and control mice (P<0.05). **Statistically significant differences between mice treated with the combination of GA and the C′-met DNA vaccine and mice treated with either GA alone or the C′-met DNA vaccine alone (P<0.05). Molecular Therapy 2007 15, 404-410DOI: (10.1038/sj.mt.6300014) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions