nitrophenols and Skin-Neoplasms

Investigations from multiple laboratories support the existence of melanoma initiating cells (MICs) that potentially contribute to melanoma's drug resistance. ABT-737, a small molecule BCL-2/BCL-XL/BCL-W inhibitor, is promising in cancer treatments, but not very effective against melanoma, with the antiapoptotic protein MCL-1 as the main contributor to resistance. The synthetic retinoid fenretinide N-(4-hydroxyphenyl)retinamide (4-HPR) has shown promise for treating breast cancers. Here, we tested whether the combination of ABT-737 with 4-HPR is effective in killing both the bulk of melanoma cells and MICs. The combination synergistically decreased cell viability and caused cell death in multiple melanoma cells lines (carrying either BRAF or NRAS mutations) but not in normal melanocytes. The combination increased the NOXA expression and caspase-dependent MCL-1 degradation. Knocking down NOXA protected cells from combination-induced apoptosis, implicating the role of NOXA in the drug synergy. The combination treatment also disrupted primary spheres (a functional assay for MICs) and decreased the percentage of aldehyde dehydrogenase (high) cells (a marker of MICs) in melanoma cell lines. Moreover, the combination inhibited the self-renewal capacity of MICs, measured by secondary sphere-forming assays. In vivo, the combination inhibited tumor growth. Thus, this combination is a promising treatment strategy for melanoma, regardless of mutation status of BRAF or NRAS.

Topics: Aldehyde Dehydrogenase; Antineoplastic Agents; Apoptosis; Biomarkers, Tumor; Biphenyl Compounds; Cell Line, Tumor; Cell Survival; Drug Synergism; Drug Therapy, Combination; Fenretinide; Humans; Melanoma; Neoplastic Stem Cells; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Retinoids; Skin Neoplasms; Sulfonamides

Tumour progression and therapy resistance in squamous cell carcinoma of the skin (SCC) is strongly associated with resistance to intrinsic mitochondrial apoptosis. We thus investigated the role of various anti-apoptotic Bcl-2 proteins for apoptosis protection in SCC using the BH3 agonist ABT737 that can overcome multidomain Bcl-2 protein protection. Sensitive SCC cells underwent rapid loss of mitochondrial membrane potential (MMP), subsequent apoptosis concomitant with caspase-3 activation and an early release of mitochondria-derived cytochrome c and smac/DIABLO. In contrast, ABT737 resistance in subsets of SCC cells was not explained by XIAP, important for protection from DR-induced apoptosis in SCC. Of note, ABT737 did not prime SCC cells to DR-induced apoptosis. Interestingly, the ratio of Mcl-1 and Noxa determined sensitivity to ABT737: loss of Mcl-1 rendered resistant cells sensitive to ABT737, whereas loss of Noxa promoted resistance in sensitive cells. In line, suppression of Mcl-1 by the pan-Bcl-2 inhibitor Obatoclax or overexpression of Noxa rendered resistant SCC cells sensitive to BH3 mimetics. Our data indicate that targeting of the Mcl-1/Noxa axis is important to overcome resistance to mitochondrial apoptosis in SCC. Therefore, combination treatment of ABT737 or derivatives with Mcl-1 inhibitors, or inducers of Noxa, may represent a novel option of targeted therapy in metastatic SCC of the skin.

Topics: Antineoplastic Agents; Apoptosis; Biphenyl Compounds; Carcinoma, Squamous Cell; Cell Line, Tumor; Cells, Cultured; Drug Resistance, Neoplasm; Humans; Keratinocytes; Myeloid Cell Leukemia Sequence 1 Protein; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Skin Neoplasms; Sulfonamides

MicroRNAs play important roles in gene regulation, and their expression is frequently dysregulated in cancer cells. In a previous study, we reported that miR-193b represses cell proliferation and regulates cyclin D1 in melanoma cells, suggesting that miR-193b could act as a tumor suppressor. Herein, we demonstrate that miR-193b also down-regulates myeloid cell leukemia sequence 1 (Mcl-1) in melanoma cells. MicroRNA microarray profiling revealed that miR-193b is expressed at a significantly lower level in malignant melanoma than in benign nevi. Consistent with this, Mcl-1 is detected at a higher level in malignant melanoma than in benign nevi. In a survey of melanoma samples, the level of Mcl-1 is inversely correlated with the level of miR-193b. Overexpression of miR-193b in melanoma cells represses Mcl-1 expression. Previous studies showed that Mcl-1 knockdown cells are hypersensitive to ABT-737, a small-molecule inhibitor of Bcl-2, Bcl-X(L), and Bcl-w. Similarly, overexpression of miR-193b restores ABT-737 sensitivity to ABT-737-resistant cells. Furthermore, the effect of miR-193b on the expression of Mcl-1 seems to be mediated by direct interaction between miR-193b and seed and seedless pairing sequences in the 3' untranslated region of Mcl-1 mRNA. Thus, this study provides evidence that miR-193b directly regulates Mcl-1 and that down-regulation of miR-193b in vivo could be an early event in melanoma progression.

Topics: Antimetabolites, Antineoplastic; Apoptosis; Binding Sites; Biphenyl Compounds; Cell Line, Tumor; Cyclin D1; Down-Regulation; Drug Resistance, Neoplasm; Growth Inhibitors; Humans; Melanoma; MicroRNAs; Myeloid Cell Leukemia Sequence 1 Protein; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Skin Neoplasms; Sulfonamides

The mitogen-activated protein kinase (MAPK) pathway is constitutively activated in the majority of melanomas, promoting cell survival, proliferation and migration. In addition, anti-apoptotic Bcl-2 family proteins Mcl-1, Bcl-xL and Bcl-2 are frequently overexpressed, contributing to melanoma's well-documented chemoresistance. Recently, it was reported that the combination of MAPK pathway inhibition by specific MEK inhibitors and Bcl-2 family inhibition by BH3-mimetic ABT-737 synergistically induces apoptotic cell death in melanoma cell lines. Here we provide the first evidence that inhibition of another key MAPK, p38, synergistically induces apoptosis in melanoma cells in combination with ABT-737. We also provide novel mechanistic data demonstrating that inhibition of p38 increases expression of pro-apoptotic Bcl-2 protein PUMA. Furthermore, we demonstrate that PUMA can be cleaved by a caspase-dependent mechanism during apoptosis and identify what appears to be the PUMA cleavage product. Thus, our findings suggest that the combination of ABT-737 and inhibition of p38 is a promising, new treatment strategy that acts through a novel PUMA-dependent mechanism.

Topics: Apoptosis Regulatory Proteins; Biphenyl Compounds; Caspases; Cell Death; Cell Line, Tumor; Cell Survival; Drug Synergism; Enzyme Activation; Gene Knockdown Techniques; Humans; Imidazoles; Melanoma; Myeloid Cell Leukemia Sequence 1 Protein; Nitrophenols; p38 Mitogen-Activated Protein Kinases; Piperazines; Protein Kinase Inhibitors; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Pyridines; Skin Neoplasms; Sulfonamides; Up-Regulation

Even though activating mutations of B-Raf, a kinase atop the MAPK signaling cascade, reportedly sensitize tumor cells to MEK inhibitors, Raf and MEK inhibitors have exhibited limited clinical activity. In this issue of the JCI, Cragg et al. report that MEK inhibition upregulates the proapoptotic Bcl-2 family member Bim but induces little regression of human melanoma xenografts in mice unless the Bcl-2 antagonist ABT-737 is added (see the related article beginning on page 3651). These findings illustrate the potential benefit of simultaneously inhibiting oncogenic kinases and inhibiting Bcl-2 action in solid tumors.

Topics: Animals; Apoptosis Regulatory Proteins; bcl-2 Homologous Antagonist-Killer Protein; Bcl-2-Like Protein 11; Biphenyl Compounds; Humans; Melanoma; Membrane Proteins; Mice; Mitogen-Activated Protein Kinase Kinases; Models, Biological; Nitrophenols; Piperazines; Proto-Oncogene Proteins; Proto-Oncogene Proteins B-raf; Skin Neoplasms; Sulfonamides; Xenograft Model Antitumor Assays

Skin painting experiments showed that two semi-permanent hair colorants applied at low concentrations caused an early appearance of tumours in two strains of mice. Both colorants were mutagenic in S. typhimurium and further investigation revealed that three categories of hair colorant contained mutagenic constitutents.

Topics: Aniline Compounds; Animals; Azo Compounds; Coloring Agents; Cosmetics; Hair Color; Liver; Mice; Mutation; Nitrophenols; Phenylenediamines; Salmonella typhimurium; Skin Neoplasms; Time Factors