Human A375 melanoma cells (10 × 10⁶) were implanted s.c. into the right flank of SCID mice. 14 days after cell injection animals were pair-matched (65 mm³ average tumor size) and daily treatment (CA, 120 mg/kg/d in methylcellulose/PBS, n=10) was initiated by oral gavage. Control animals (n=12) received carrier only. (A) Tumor growth curves were obtained by determining average tumor volumes until day 30 after cell injection. Data points are depicted as means ± SEM and statistical comparison between individual data point was performed using the two-sided Student’s t test (*, p < 0.05; **, p < 0.01; ***, p < 0.001). (B) Immunohistochemical staining for PCNA using primary tumor tissue of CA-treated and carrier-treated mice sacrificed on day 34 (n = 2, each group). Paraformaldehyde-fixed, paraffin-embedded 5 μm sections were analyzed using a mouse monoclonal antibody to PCNA followed by biotinylated-streptavidin-HRP/DAB visualization. Hematoxylin counterstaining was also performed. Photographs representative of five high power fields taken per tissue section are shown.

Human A375 melanoma cells were exposed to CA (20 μM, 48 h), and bivariate cell cycle analysis was performed by flow cytometric determination of cellular DNA synthesis (BrdU incorporation) and DNA content (7-AAD staining). The numbers indicate cells in G1, S, and G2/M phase, respectively, in percent of total gated cells (mean ± SD, n=3). The arrow marks a small fraction (approx. 8%) of apoptotic cells in sub-G1.

Melanoma cell invasion through basement membrane extracellular matrix was determined using the CytoSelect^™ Cell Invasion Assay as detailed in Materials and Methods. Invasion of A375 cells (over a 48 h period) left untreated or treated with CA (10 or 20 μM) was assessed by crystal violet staining of cells that passed through the basement membrane clinging to the bottom of the Boyden chamber membrane. (A) CA-Modulation of melanoma cell invasiveness assessed by light microscopy. A representative result of three similar repeats is shown. (B) Colorimetric analysis (absorbance at 560 nm) of CA-modulation of melanoma cell invasiveness (mean ± SD, n=3).

A375 melanoma cells were cotransfected with a plasmid containing a firefly luciferase reporter gene under the control of the NFκB promotor and a plasmid encoding renilla luciferase for normalization of transfection efficiency. (A) Suppression of constitutive NFkB transcriptional activity was examined in cells treated with CA (20 μM) for 12 h. (B) Suppression of TNFα-induced NFkB transcriptional activity was examined in cells that were pretreated with CA, DHCA, COH, and CAC (20 μM each, 3h exposure) followed by TNFα treatment (20ng/mL, 12h exposure) and determination of luciferase activity. Potency of fold-induction is expressed as luciferase activity in relative luminescence units versus untreated control transfectants (mean ± SD, n≥3).

Anti-proliferative activity of CA and CA-derivatives (DHCA, COH, CAC, and MCA; molecular structures as indicated) was assessed using human A375 melanoma cells (mean ± SD, n=3) as described in Materials and Methods. Numbers indicate concentration of test compound in μM. Proliferation was compared with control cells that received mock treatment.

(A) Time course (24, 48, and 72 h) of induction of cell death by exposure to increasing doses of CA (10, 20, and 40 μM) was assessed by flow cytometric analysis of annexinV-FITC/propidium iodide-stained cells. The numbers indicate viable cells (AV⁻, PI⁻, lower left quadrant) in percent of total gated cells (mean ± SD, n=3). (Viability of untreated controls was 92.1 ± 1.0 %; data not shown). (B) CA-induced (10, 20, and 40 μM, 72 h) caspase-3 activation was examined by flow cytometric detection using an Alexa Fluor 488-conjugated monoclonal antibody against cleaved procaspase-3. One representative experiment of three similar repeats is shown.

(A) CA-induced gene expression changes in A375 human melanoma cells. The scatter blot (left panel) depicts differential gene expression as detected by the RT² Human Oxidative Stress Profiler^™ PCR Expression Array technology profiling the expression of 84 oxidative stress-related genes after CA treatment (25 μM, 24 h; three independent experiments). Upper and lower lines represent the cut-off indicating four fold up- or down-regulated expression, respectively. Arrows specify genes with at least 4 fold up-regulated expression versus untreated controls. The table (right panel) summarizes all genes that are at least two-fold upregulated in response to CA treatment. (B) Human A375 melanoma cells were cultured for 24h in the presence of CA (25 μM) or left untreated. Total RNA was prepared and gene expression of HMOX1, CDKN1A, SRXN1, TXNRD1 was then quantitatively examined using real time RT-PCR analysis with normalization for GAPDH expression levels. Relative expression levels in response to mock treatment (control) and exposure to CA were determined in three repeat experiments (n=3, mean ± SD). (C) CA-modulation of cellular heme oxygenase-1 (HO-1) protein levels and (D) CA-modulation of cellular p21 protein levels were examined in total cellular protein extracts by 15% SDS-PAGE followed by immunoblot detection using enhanced chemiluminescence. Detection of α-actin expression served as a loading control. (E) Induction of intracellular oxidative stress in human A375 melanoma cells by treatment with CA. Cells were exposed to CA (10 and 25 μM, 24 h) and intracellular oxidative stress was assessed by 2′,7′-dichloro-dihydrofluorescein diacetate staining followed by flow cytometric analysis. One representative experiment of three similar repeats is shown. (F) Modulation of intracellular glutathione content in A375 melanoma cells exposed to increasing concentrations of CA (10–40 μM, 24 h). Total glutathione content was normalized to protein content. (G) Upregulation of GCLC gene expression assessed by real time RT-PCR analysis with normalization for β-actin expression levels in A375 melanoma cells exposed to increasing concentrations of CA (10–20 μM, 24 h).

A375 cells were pretreated with CA for 3 h and then exposed to TNFα. After 6 h, IL-8 expression was examined in CA- and mock-pretreated cells by flow cytometric analysis using an Alexa 488-conjugated mouse monoclonal antibody directed against human IL-8. (A) Modulation of IL-8 expression by CA (20 μM) pretreatment followed by TNFα stimulation. (B) Dose response relationship of inhibition of TNFα-induced IL-8 expression by CA (10 and 20 μM) pretreatment. One representative experiment out of at least three similar repeats is shown.