Comparative analysis of the phototoxicity induced by BRAF inhibitors and alleviation through antioxidants

Background Small molecules tackling mutated BRAF (BRAFi) are an important mainstay of targeted therapy in a variety of cancers including melanoma. Albeit commonly reported as side effect, the phototoxic potential of many BRAFi is poorly characterized. In this study, we evaluated the phototoxicity of 17 distinct agents and investigated whether BRAFi‐induced phototoxicity can be alleviated by antioxidants. Methods The ultraviolet (UV) light absorbance of 17 BRAFi was determined. Their phototoxic potential was investigated independently with a reactive oxygen species (ROS) and the 3T3 neutral red uptake (NRU) assay in vitro. To test for a possible phototoxicity alleviation by antioxidants, vitamin C, vitamin E phosphate, trolox, and glutathione (GSH) were added to the 3T3 assay of selected inhibitors. Results The highest cumulative absorbance for both UVA and UVB was detected for vemurafenib. The formation of ROS was more pronounced for all compounds after irradiation with UVA than with UVB. In the 3T3 NRU assay, 8 agents were classified as phototoxic, including vemurafenib, dabrafenib, and encorafenib. There was a significant correlation between the formation of singlet oxygen (P = .026) and superoxide anion (P < .001) and the phototoxicity observed in the 3T3 NRU assay. The phototoxicity of vemurafenib was fully rescued in the 3T3 NRU assay after GSH was added at different concentrations. Conclusion Our study confirms that most of the BRAF inhibitors exhibited a considerable phototoxic potential, predominantly after exposure to UVA. GSH may help treat and prevent the phototoxicity induced by vemurafenib

Selective Induction of Cell Death in Melanoma Cell Lines through Targeting of Mcl-1 and A1

Melanoma is an often fatal form of skin cancer which is remarkably resistant against radio- and chemotherapy. Even new strategies that target RAS/RAF signaling and display unprecedented efficacy are characterized by resistance mechanisms. The targeting of survival pathways would be an attractive alternative strategy, if tumor-specific cell death can be achieved. Bcl-2 proteins play a central role in regulating survival of tumor cells. In this study, we systematically investigated the relevance of antiapoptotic Bcl-2 proteins, i.e., Bcl-2, Bcl-xL, Bcl-w, Mcl-1, and A1, in melanoma cell lines and non-malignant cells using RNAi. We found that melanoma cells required the presence of specific antiapoptotic Bcl-2 proteins: Inhibition of Mcl-1 and A1 strongly induced cell death in some melanoma cell lines, whereas non-malignant cells, i.e., primary human fibroblasts or keratinocytes were not affected. This specific sensitivity of melanoma cells was further enhanced by the combined inhibition of Mcl-1 and A1 and resulted in 60% to 80% cell death in all melanoma cell lines tested. This treatment was successfully combined with chemotherapy, which killed a substantial proportion of cells that survived Mcl-1 and A1 inhibition. Together, these results identify antiapoptotic proteins on which specifically melanoma cells rely on and, thus, provide a basis for the development of new Bcl-2 protein-targeting therapies

CIITA Leucine-Rich Repeats Control Nuclear Localization, In Vivo Recruitment to the Major Histocompatibility Complex (MHC) Class II Enhanceosome, and MHC Class II Gene Transactivation

The major histocompatibility complex (MHC) class II transactivator CIITA plays a pivotal role in the control of the cellular immune response through the quantitative regulation of MHC class II expression. We have analyzed a region of CIITA with similarity to leucine-rich repeats (LRRs). CIITA LRR alanine mutations abolish both the transactivation capacity of full-length CIITA and the dominant-negative phenotype of CIITA mutants with N-terminal deletions. We demonstrate direct interaction of CIITA with the MHC class II promoter binding protein RFX5 and could also detect novel interactions with RFXANK, NF-YB, and -YC. However, none of these interactions is influenced by CIITA LRR mutagenesis. On the other hand, chromatin immunoprecipitation shows that in vivo binding of CIITA to the MHC class II promoter is dependent on LRR integrity. LRR mutations lead to an impaired nuclear localization of CIITA, indicating that a major function of the CIITA LRRs is in nucleocytoplasmic translocation. There is, however, evidence that the CIITA LRRs are also involved more directly in MHC class II gene transactivation. CIITA interacts with a novel protein of 33 kDa in a manner sensitive to LRR mutagenesis. CIITA is therefore imported into the nucleus by an LRR-dependent mechanism, where it activates transcription through multiple protein-protein interactions with the MHC class II promoter binding complex

Relationship between alpha-genus human papillomavirus and non-genital seborrheic keratosis: Report of new cases and updated review

Background: Seborrheic keratoses (SK) are the most common acquired benign tumor that affects middle-aged or older adults with great cosmetic concern. Clinical and histopathological similarities of SK and common warts have been addressed by investigating the possible presence of human papillomavirus (HPV) DNA in SK. Previous studies suggested the association between alpha-genus HPV and SK located on genital skin, whereas the causal relationship between alpha-HPV and non-genital SK remains controversial. Aim: This study aimed to clarify the pathogenic involvement of alpha-HPV in the development of non-genital SK. Methods: We analyzed alpha-HPV DNA prevalence and HPV genotypes using a PCR-based microarray on 51 skin samples presenting with histologically confirmed SK without any malignant changes. Correlation between the histological subtype of SK and their HPV DNA-positive reactivity was also evaluated. Results: Of 51 non-genital SK, two (3.9%) skin samples were positive for alpha-HPV DNA;high-risk HPV 31 and low-risk HPV 42 were found. Evaluation of HPV prevalence in different histological types of SK showed that both HPV-positive cases were acanthotic type;14.3% of acanthotic SK lesions were positive, while all of the other types were negative for alpha-HPV. Conclusions: This study demonstrates that alpha-HPV positivity is very rare in common non-genital SK. The rare alpha-HPV-positive SK lesions histologically belonged to the acanthotic type, implying a potential impact of HPV infection on epidermal hyperproliferation. Although a possible association cannot be excluded, our findings suggest that alpha-HPV is not a major causative factor for non-genital SK

Expression of antiapoptotic Bcl-2 proteins in non-malignant skin cells and melanoma cell lines.

<p>(A) Expression of Bcl-2, Bcl-xL, Bcl-w, and Mcl-1 protein in primary human fibroblasts, keratinocytes, and melanocytes was analyzed by immunoblotting. For each cell type, 2 donors are shown. (B) Expression of Bcl-2, Bcl-xL, Bcl-w and Mcl-1 protein in melanocytes isolated from 4 donors. (C) Expression of Bcl-2, Bcl-xL, Bcl-w and Mcl-1 protein in melanoma cell lines of different progression levels. Blot is representative for 2 independent experiments. (D) Levels of A1 mRNA were measured by quantitative RT-PCR. Mean +/− SD of 3 donors is shown for non-malignant cells, mean +/− SD of 3 cell passages is shown for melanoma cell lines. β-actin served as loading control in immunoblots. The same protein sample of melanocytes from donor #2 was loaded on all gels as a reference to allow the comparison of expression levels among the immunoblots shown in A, B, and C. RGP: radial-growth phase (non-invasive); VGP: vertical growth phase (invasive).</p

Chemotherapy adds to cell death induced by Mcl-1 and A1 inhibition.

<p>(A) 1205Lu melanoma cells were treated with the indicated siRNAs together with 1 µM 5-fluorouracil (5-FU) or not (no treatment) and analyzed on day 3 after transfection. Left panel: Cell viability was determined. Viability of control siRNA-treated cells without 5-FU treatment was set to 100%. Right panel: Cell death analysis measured by FACS after staining with Annexin V and propidium iodide. (B) Analysis of cell viability (left panel) or cell death (right panel) of 1205Lu cells on day 6 treated as described in A. (C) Analysis of cell viability (left panel) or cell death (right panel) of primary human fibroblasts on day 6 treated as described in A. If not otherwise indicated, asterisks represent significant increase in cell death compared to control siRNA-treated cells. Mean +/− SD of 3 independent experiments is shown in A, B, and C. AN, Annexin V; PI, propidium iodide.</p

Combined inhibition of Mcl-1 and A1 results in efficient induction of apoptosis in 1205Lu melanoma cells.

<p>(A) 1205Lu melanoma cells were transfected with A1- or Mcl-1-specific siRNAs or control siRNAs as indicated. Cell death was assessed 72 hours after transfection. Mean +/− SD of 3 independent experiments is shown. Asterisks represent significant increase in cell death compared to single inhibition. (B) 1205Lu melanoma cells were transfected with different siRNA sequences targeting A1 or Mcl-1 (A1b, Mcl-1b). Analysis was carried out as described in A. Asterisks represent significant increase in cell death compared to single inhibition. Also, cell death induction by A1b, Mcl-1b, and A1b/Mcl-1b versus control was significant (not indicated in the figure). (C) Caspase activation of 1205Lu cells treated as described in A was assessed by immunoblotting with antibodies detecting the proform as well as the active subunits. Caspase 9 (upper panel) and caspase 3 (lower panel) were analyzed 48 hours after transfection. (D) 1205Lu cells treated with siRNAs as described in A with or without the pan-caspase inhibitor zVAD-FMK (100 µM). Cell death was measured 72 hours after transfection. Mean +/− SD of 3 independent experiments is shown. Asterisks represent significant decrease in cell death of zVAD-FMK-treated cells compared to cells not treated with zVAD-FMK. AN, Annexin V; PI, propidium iodide.</p

Inhibition of Mcl-1 or A1 leads to melanoma-specific cell death.

<p>(A) Primary human fibroblasts and 1205Lu melanoma cells were treated with the indicated siRNAs and expression of Bcl-2, Bcl-xL, Bcl-w, and Mcl-1 was analyzed after 48 hours by immunoblotting. (B) A1 mRNA was measured by quantitative RT-PCR in cells treated with A1-specific- or control siRNA for 48 hours (left and middle panel). Non-targeted Bcl-2 proteins were analyzed at the same time point by immunoblotting (right panel). (C) Cell death of fibroblasts was determined by quantification of Annexin V (AN)- and propidium iodide (PI)-positive cells 72 hours after transfection of the indicated siRNAs. (D) Cell death analysis of 1205Lu melanoma cells treated as described in C. Asterisks indicate a significant increase in cell death versus control siRNA-treated cells. (E) Fluorescence-activated cell sorting (FACS) of apoptotic and dead fibroblasts (upper panel) or 1205Lu melanoma cells (lower panel) treated as described in C. Numbers indicate the portion of cells in each quadrant that defines Annexin V- or propidium iodide-positive or negative cells. Mean +/− SD of at least 3 independent experiments is shown in B, C, and D. α-tubulin served as loading control in immunoblots. Blots are representative for 3 independent experiments.</p

Combined inhibition of Mcl-1 and A1 results in efficient induction of cell death in a panel of melanoma cell lines, but not in keratinocytes.

<p>(A) Primary human keratinocytes (upper left panel), non-invasive melanoma cells (WM3211), invasive (WM793), and metastatic melanoma cells (WM1232, WM239A, WM1158) were transfected with the siRNAs as indicated. Cell death was determined 72 hours after transfection. Mean +/− SD of 3 (keratinocytes, WM1232, WM239A, WM1158) or 5 (WM3211 and WM793) independent experiments is shown. (B) A1 mRNA was measured by quantitative RT-PCR in indicated cells treated with A1-specific- or control siRNA for 48 hours. Mean +/− SD of 3 independent experiments is shown. Asterisks represent significant increase in cell death compared to control siRNA-treated cells. AN, Annexin V; PI, propidium iodide.</p

Representative light microscopy images of 1205Lu melanoma cells (A) or fibroblasts (B) 6 days after transfection of the indicated siRNAs alone (upper panel) or together with 1 µM 5-FU (lower panel).

<p>Scale bar = 0.5 mm.</p