The alcohol-abuse deterrent disulfiram (DSF) is shown to have a highly selective toxicity against melanoma in culture, inducing a largely apoptotic response, with much lower toxicity against several other cell lines. Melanoma cell lines derived from different stages (radial, vertical, and metastatic phase) were all sensitive to DSF treatment in vitro; melanocytes were only slightly affected. A required role of extracellular Cu is demonstrated for DSF toxicity. Low concentrations of DSF alone decreased the number of viable cells, and the addition of CuCl2 significantly enhanced the DSF-induced cell death to less than 10% of control. Significantly, the intracellular Cu concentration of melanoma cells increased rapidly upon DSF treatment. Both the intracellular Cu uptake and the toxicity induced by DSF were blocked by co-incubation with bathocuproine disulfonic acid (BCPD, 100 μM), a non-membrane-permeable Cu chelator. Chemical studies demonstrated a complicated, extracellular redox reaction between Cu(II) and DSF, which forms the complex Cu(deDTC)2 in high yield, accompanied by oxidative decomposition of small amounts of disulfiram. The Cu complex has somewhat higher activity against melanoma and is suggested to be the active agent in DSF-induced toxicity. The redox conversion of DSF was unique to Cu(II) and not engendered by the other common biological metal ions Fe(II or III), Mn(III), and Zn(II). The implications of this work are significant both in the possible treatment of melanoma as well as in limiting the known side-effects of DSF, which we propose may be diminished by cotreatment to decrease adventitious Cu.

Flavokawain A is the predominant chalcone from kava extract. We have assessed the mechanisms of flavokawain A's action on cell cycle regulation. In a p53 wild-type, low-grade, and papillary bladder cancer cell line (RT4), flavokawain A increased p21/WAF1 and p27/ KIP1, which resulted in a decrease in cyclin-dependent kinase-2 (CDK2) kinase activity and subsequent G 1 arrest. The increase of p21/WAF1 protein corresponded to an increased mRNA level, whereas p27/KIP1 accumulation was associated with the down-regulation of SKP2, which then increased the stability of the p27/KIP1 protein. The accumulation of p21/ WAF1 and p27/KIP1 was independent of cell cycle position and thus not a result of the cell cycle arrest. In contrast, flavokawain A induced a G 2 -M arrest in six p53 mutant-type, highgrade bladder cancer cell lines (T24, UMUC3, TCCSUP, 5637, HT1376, and HT1197). Flavokawain A significantly reduced the expression of CDK1-inhibitory kinases, Myt1 and Wee1, and caused cyclin B1 protein accumulation leading to CDK1 activation in T24 cells. Suppression of p53 expression by small interfering RNA in RT4 cells restored Cdc25C expression and down-regulated p21/WAF1 expression, which allowed Cdc25C and CDK1 activation, which then led to a G 2 -M arrest and an enhanced growth-inhibitory effect by flavokawain A. Consistently, flavokawain A also caused a pronounced CDK1 activation and G 2 -M arrest in p53 knockout but not in p53 wild-type HCT116 cells. This selectivity of flavokawain A for inducing a G 2 -M arrest in p53-defective cells deserves further investigation as a new mechanism for the prevention and treatment of bladder cancer.

Melanoma cells have a poor ability to mediate oxidative stress, which may be attributed to constitutive abnormalities in their melanosomes. We hypothesize that disorganization of the melanosomes will allow chemical targeting of the melanin within. Chemical studies show that under oxidative conditions, synthetic melanins demonstrate increased metal affinity and a susceptibility to redox cycling with oxygen to form reactive oxygen species. The electron paramagnetic resonance (EPR)-active 5,5¢-dimethyl-pyrollidine N-oxide spin adduct was used to show that binding of divalent Zn or Cu to melanin induces a pro-oxidant response under oxygen, generating superoxide and hydroxyl radicals. A similar pro-oxidant behaviour is seen in melanoma cell lines under external peroxide stress. Melanoma cultures grown under 95% O 2 ⁄5% CO 2 atmospheres show markedly reduced viability as compared with normal melanocytes. Cu-and Zn-dithiocarbamate complexes, which induce passive uptake of the metal ions into cells, show significant antimelanoma activity. The antimelanoma effect of metal-and oxygen-induced stress appears additive rather than synergistic; both treatments are shown to be significantly less toxic to melanocytes.

No abstract

Objectives: Although MHC class II molecules can be expressed on dendritic cells (DC), macrophages and B-cells in lymph nodes and have the potential to activate CD4 T cells, it is believed that the dendritic cells are the major antigen presenting cells for T lymphocytes. They are located at portals of antigen entrance and when located in paracortical areas are more efficient antigen processors than macrophages and B-cells whose antigen-presenting role is limited by their location away from the paracortex. Here we focus on DC expression of MHC-class II molecules and the interaction with OPD4 + activated T cell in same nodal quadrants. We also evaluated the overall CD68 + macrophage density in sentinel nodes (SN) and non-sentinel nodes (NSN). Methods: Eighteen sets of metastasis-free SN and NSN from melanoma patients were evaluated for HLA-DR + DC and OPD4 + activated T lymphocytes. Immunohistochemical single staining was performed for OPD4 + activated T-cells and cocktail-triple staining to distinguish HLA-DR + DC from CD20 + B cells and CD68 + macrophages. The densities, area occupied by HLA-DR + /CD68 ) CD20 ) DC and OPD4 + activated T-cells, CD68 + macrophages and coefficient of variation of density of HLA-DR + /CD68 ) CD20 ) DC and OPD4 + activated T-cells by quadrants were measured by a Simple-PCI imaging system. We randomly subdivided the SN and NSN into quarters and evaluated the areas of five maximum density of HLA-DR + /CD68 ) CD20 ) DC in each quarter for both HLA-DR + /CD68 ) CD20 ) DC and OPD4 + T lymphocytes. Results obtained: The area occupied by HLA-DR + /CD68 ) CD20 ) DC was 5.30 ± 6.98% in SN and 11.95 ± 5.79% in NSN (P ¼ 0.017); the density of HLA-DR + /CD68 ) CD20 ) DC was 112.45 ± 100.42 in SN and 231.55 ± 49.22 in NSN (P ¼ 0.002). The coefficient of variation of HLA-DR + / CD68 ) CD20 ) DC density was significant higher among the four quarters in SN compared with in NSN (44.49 ± 19.58, 27.55 ± 14.87, P ¼ 0.012). The density of OPD4 + activated T-cells was significant lower in SN than in NSN (1329.1 ± 680.9, 1546.7 ± 699.1; P ¼ 0.012). The area occupied by OPD4 + activated T-cells was lower in SN but the difference was not significant (15.19 ± 12.66 and 17.94 ± 13.65%; P ¼ 0.832). The coefficient of variation of OPD4 + T-cell density by quadrants (32.5 ± 39.3 in SN, 27.0 ± 24.3 in NSN, P ¼ 0.417) was not significant. Overall the density of D68 + macrophages was 105.12 ± 59.6 in SN and 136.18 ± 82.7 in NSN (P ¼ 0.081); while the area occupied by CD68 + macrophages was significant lower in SN rather than in NSN (0.69 ± 0.46%, 1.09 ± 0.66, P ¼ 0.015). Conclusion: Lymph nodes influenced by melanoma are immune modulated. The antigen presentation by APCs via MHC-class II to CD4 T-cells is inhibited in SN compared with NSN. Higher variations in DC density between the different quadrants of SN suggests that the area of the SN receiving lymph via the tumor-derived afferent lymphatic may be immune-suppressed prior to seeding of metastases.