Xuesong Wu, Timothy W. Wang, George M

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Gallium Maltolate Inhibits Human Cutaneous T-Cell Lymphoma Tumor Development in Mice Xuesong Wu, Timothy W. Wang, George M. Lessmann, Jamal Saleh, Xiping Liu, Christopher R. Chitambar, Sam T. Hwang Journal of Investigative Dermatology Volume 135, Issue 3, Pages 877-884 (March 2015) DOI: 10.1038/jid.2014.476 Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 GaM has cytotoxic effects on cultured CTCL cells. (a) The trypan blue exclusion assay measuring the effect of GAM exposure on the growth rate of Hut78 and HH cells. Cells were incubated with GaM at indicated concentrations, and live cells were counted after trypan blue staining on day 0, 1, 2, and 3. (b) The apoptosis assay for GaM-treated Hut78 cells. Cultured Hut78 cells were exposed to GaM at 100 μM for 1 day. Cells were stained with Annexin V and 7-AAD and analyzed by FACS. (c) GaM produces a dose-dependent decrease in the cellular oxygen consumption rate in HuT78 cells. Cells were incubated with GaM for 8 hours prior to assay. CTCL, cutaneous T-cell lymphoma; GaM, gallium maltolate. Journal of Investigative Dermatology 2015 135, 877-884DOI: (10.1038/jid.2014.476) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Administration of GaM inhibits CTCL tumor growth in mouse models. (a) Schematic GaM treatment regimen. Hut78 cells were implanted SC in NSG mice on day 1. GaM was administered by peritumoral injection at a dosage of 400 μg per mouse/day for 5 consecutive days starting on day 2 (1st wk), day 8 (2nd wk), or day 15 (3rd wk), respectively. Equal volume of PBS (200 μl) was injected instead of GaM for 1st wk as control. (b) Representative pictures of 1st wk GaM- and PBS-treated mice. Red arrows indicate subcutaneous areas where tumor formation was seen in PBS-treated, whereas not in GaM-treated mice. (c) H&E staining for maximum cross-sections of tumors isolated from GaM-treated (2nd wk) and control groups. (d) Sizes and (e) images of flank tumors 4 weeks after implantation. Student’s t-test was performed between each GaM-treated group and PBS control. CTCL, cutaneous T-cell lymphoma; GaM, gallium maltolate; H&E, hematoxylin and eosin. Journal of Investigative Dermatology 2015 135, 877-884DOI: (10.1038/jid.2014.476) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Gallium maltolate (GaM) alters expression levels of inflammatory cytokines in Hut78 cells. (a) Upregulated genes in GaM vs. PBS-treated Hut78 cells by inflammatory cytokines and receptor PCR array (fold change >2.5). (b) Real-time PCR analysis of the expression levels of IL-10, IL-13, CXCL10, and 11 in GAM/PBS-treated Hut78 cells. y-Axis shows relative quantification after correction to glyceraldehyde-3-phosphate dehydrogenase expression. *P<0.05, **P<0.001, and ***P<0.0001 compared with the control group. Journal of Investigative Dermatology 2015 135, 877-884DOI: (10.1038/jid.2014.476) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 GaM treatment activates oxidative stress and p53 pathways in Hut78 cells. (a) Upregulated genes in GaM vs. PBS-treated Hut78 cells by Pathway Finder PCR array (fold change >2.5). (b) Activation of the p38/MAPK pathway was detected by western blot in GaM-treated CTCL cell. Image acquisition and analysis was carried out through Image Lab software. Journal of Investigative Dermatology 2015 135, 877-884DOI: (10.1038/jid.2014.476) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions