Journal: Oncotarget

Article Title: miR-638 promotes melanoma metastasis and protects melanoma cells from apoptosis and autophagy

doi:

Figure Lengend Snippet: (A) Heatmap of differentially expressed miRNAs in melanoma patient tissue samples comprising primary melanomas, lymph node metastases and distant cutaneous metastases (ou) (n=24), generated using expression data from TaqMan® low density miRNA arrays. Colors represent normalized Ct values (blue for high Ct values indicates low expression and red for low Ct values indicates high expression of miRNAs). Dendrograms are based on hierarchical clustering with Euclidean distance. (B) miR-638 expression in PM and MM and (C) PM of varying thickness (mm). (D) miR-126* expression in PM and MM and (E) PM of varying thickness (mm). Paired t- test and Pearson's coefficient (Pearson's r) were used to determine significance of correlation between miR-638 expression and tumor thickness. (F, G) Expression levels of miR-638 were analysed in primary melanocytes (n=3) in comparison with (F) PM and MM tissue samples (n=27). (G) miR-638 expression in 9 different melanoma cell lines (HT144, 1F6, Mewo, SK-Mel-147, SK-Mel-19, SK-Mel-28, SK-Mel-29, A375 and BRO). All miRNA expression analyses were performed using TaqMan® qRT-PCR technology. RNU48 expression was used as universal reference control for miRNAs.

Article Snippet: Freshly isolated normal human epidermal melanocyte pellets were purchased from PromoCell, Heidelberg, Germany (C-14043).

Techniques: Generated, Expressing, Comparison, Quantitative RT-PCR, Control

Journal: Oncotarget

Article Title: miR-638 promotes melanoma metastasis and protects melanoma cells from apoptosis and autophagy

doi:

Figure Lengend Snippet: (A) Melanoma cells were transiently transfected with miR-638, antagomir-638 or control-antagomiR and analyzed using whole-genome cDNA microarrays. The Venn diagram at the top displays the overlap between down-regulated and up-regulated genes upon miR-638 and antagomiR-638 overexpression, respectively (n = 3,199). The middle Venn diagram displays the intersection of miRWalk database predicted miR-638 targets and strongly differentially regulated mRNAs (total log 10 fold change (FC) > 2; n = 86). Finally, a subset of targets predicted by at least three different algorithms as high-confidence targets of miR-638 regulation was identified and used in subsequent analyses (n = 30). (B) Heatmap of differentially regulated putative target genes for miR-638. Colors represent fold changes of relative gene expressions of miR-638 or antagomiR-638-transfected cells in comparison with mock transfected control cells (blue color indicates high expression and red color indicates low expression). (C) TP53INP2 mRNA expression analysis for primary melanocytes (P-mel 1 and 2), human fibroblasts (Hu.Fib), and melanoma cell lines, respectively, using TaqMan® gene expression assays. Normalized Ct values were quantitated and compared with the mean TP53INP2 expression values, respectively, across all cell lines. Results are expressed as percentage of relative expression. (D) SK-Mel-147 cells were transfected with a scrambled-control (Co) or miR-638. Fourty eight hours after transfection TP53INP2 mRNA expression was analysed using TaqMan® gene expression assays. GAPDH was used as reference control (mean ± S.E.M). (E) SK-Mel-147 melanoma cells were transfected with scrambled-control (Co) or miR-638. After 48 h of transfection, protein expression for TP53INP2 was analysed using immunoblotting. β-actin was used as loading control. (F) Reporter assay in SK-Mel-147 cells co-transfected with a scrambled miRNA control or miR-638 and with either wild type or single (1X) or double (2X) mutated TP53INP2 3′-UTR cloned in a dual-luciferase constructs (mean ± S.E.M; n= 4). (G) XTT cell proliferation assay using SK-Mel-147 cells transiently overexpressing control plasmid or TP53INP2 cDNA co-transfected with a control miRNA or miR-638 (mean ± S.E.M; n=3). The UV-absorptions were measured at 24 h and 48 h at 492 nm (H) Matrigel invasion assays were performed for SK-Mel-147 cells transiently overexpressing a control plasmid or TP53INP2 cDNA co-transfected with a control miRNA or miR-638. Microscopic pictures were taken at 48 h (mean ± S.E.M, n=3). All biological assays were performed in triplicates and repeated twice individual transfections and assay measurements.

Article Snippet: Freshly isolated normal human epidermal melanocyte pellets were purchased from PromoCell, Heidelberg, Germany (C-14043).

Techniques: Transfection, Control, Over Expression, Comparison, Expressing, Gene Expression, Western Blot, Reporter Assay, Clone Assay, Luciferase, Construct, Proliferation Assay, Plasmid Preparation

Journal: Clinical Cancer Research

Article Title: Anti-EGFR Antibody–Drug Conjugate Carrying an Inhibitor Targeting CDK Restricts Triple-Negative Breast Cancer Growth

doi: 10.1158/1078-0432.CCR-23-3110

Figure Lengend Snippet: Stochastic conjugation of cetuximab to CDK inhibitor and ADC internalization in live breast cancer cells. A, Flow cytometric evaluation of surface EGFR expression (TNBC: MDA-MB-468, HCC1143, HCC1806, MDA-MB-231, HCC1937, SUM149, and CAL51; HER2+: SKBR3; ER+: MCF7, T47D; nontumorigenic epithelial cell model: MCF10A; immune cell model: human B lymphocytes RPMI8866, RPMI8226, and monocytic cell line U937; human primary melanocyte: melanocyte). B, EGFR mRNA expression from the Cancer Cell Line Encyclopedia database showed a positive correlation with surface EGFR measured by flow cytometry in A (Spearman’s rank coefficient, r = 0.723). A high level of correlation was found between EGFR and cyclin E ( r = 0.738), but not with cyclin A or CDK2. Nonsignificant P values are marked as NS. C, Top, Schematic diagram of stochastic ADC conjugation by antibody reduction with TCEP and then conjugation to SNS-032 via MC-Val–Ala-PAB. Middle, HIC analysis confirmed an average DAR of 4.4. Bottom, SEC trace indicates negligible ADC aggregation and minimal free linker–payload (less than 0.8%). D, Surface plasmon resonance analysis demonstrated similar binding affinity ( K D ) for cetuximab (0.73 nmol/L) and ADC (1.28 nmol/L). Isotype IgG1 and isotype ADC showed no measurable binding. E, Monitoring internalization of Fabfluor-pH-labeled cetuximab, ADC, or isotype control (10 nmol/L) by Incucyte live-cell imaging. Phase and red fluorescence time-course images were captured for 24 hours. Images of internalized antibody display in cytosolic, low pH lysosomal vesicle-associated red fluorescence in cells. Scale bar, 0.2 mm. F, Cells were seeded in Matrigel for 5 days, allowing the formation of spheroids. Fabfluor-pH-labeled antibodies or ADC (10 nmol/L) were introduced in the Matrigel and showed rapid internalization in EGFR-high MDA-MB-468 and MDA-MB-231, whereas EGFR-low CAL51 displayed little red fluorescence signals. A low level of internalization was observed for isotype or isotype-ADC controls. Scale bar, 0.5 mm. P values determined by two-tailed unpaired t test of three independent experiments compared with isotype control.

Article Snippet: Human primary epidermal melanocytes were cultured in Melanocyte Growth Medium (Cell Applications Inc.).

Techniques: Conjugation Assay, Expressing, Flow Cytometry, SPR Assay, Binding Assay, Labeling, Control, Live Cell Imaging, Fluorescence, Two Tailed Test

Journal: Advances in Dermatology and Allergology/Postȩpy Dermatologii i Alergologii

Article Title: Effects of tea polyphenols on UVA-induced melanogenesis via inhibition of α-MSH-MC1R signalling pathway

doi: 10.5114/ada.2022.115890

Figure Lengend Snippet: Effects of TPS on the expression of α-MSH. Representative chart trace of α-MSH inhibition by 5, 10, and 15 μg/ml of the TPS on HaCaT cells ( A ) and HEM cells ( B ) with UVA exposure (15 J/cm 2 ) or not by ELISA. Error bars show means ± SEMs. * P < 0.05, ** p < 0.01, *** p < 0.001 versus non-treated cells, # p < 0.05, ## p < 0.01, ### p < 0.001 versus UVA-treated cells

Article Snippet: The following material was purchased from the following manufacturers: tea polyphenols (98% purity), Chinese Academy of Agricultural Sciences, Tea Research Institute; 10% foetal bovine serum (FBS), DMEM and DMSO, Gibco/BRL (Grand Island, NY, USA); Bovine serum albumin (BSA), levodopa (L-DOPA) and nonapeptide-1 acetate salt (N-1A), MedChemExpress (Shanghai, China); and α-MSH ELISA Kit, CUSABIO (Wuhan, China).

Techniques: Expressing, Inhibition, Enzyme-linked Immunosorbent Assay

Journal: Advances in Dermatology and Allergology/Postȩpy Dermatologii i Alergologii

Article Title: Effects of tea polyphenols on UVA-induced melanogenesis via inhibition of α-MSH-MC1R signalling pathway

doi: 10.5114/ada.2022.115890

Figure Lengend Snippet: TPS inhibited melanogenesis through suppressing the α-MSH-MC1R signalling pathway in HaCaT cells and HEM cells. A, B – HEM was pretreated or not with UVA exposure (15 J/cm 2 ) before TPS (10 μg/ml) and N-1A (20 μm) were applied. Melanin contents and the expression of tyrosinase were measured as described in methods. C – HaCaT cells were treated with TPS (10 μg/ml) and/or N-1A (20 μm) in the presence or absence of UVA exposure (15 J/cm 2 ). ELISA was then applied to detect the expression of α-MSH. D – HEM cells were treated with TPS (10 μg/ml) and N-1A (20 μm) in the presence or absence of UVA exposure (15 J/cm 2 ). ELISA was then applied to detect the expression of α-MSH. * P < 0.05, ** p < 0.01, *** p < 0.001 versus non-treated cells, # p < 0.05, ## p < 0.01, ### p < 0.001 versus UVA-treated cells, a p < 0.05, aa p < 0.01, aaa p < 0.001, ns p > 0.05

Article Snippet: The following material was purchased from the following manufacturers: tea polyphenols (98% purity), Chinese Academy of Agricultural Sciences, Tea Research Institute; 10% foetal bovine serum (FBS), DMEM and DMSO, Gibco/BRL (Grand Island, NY, USA); Bovine serum albumin (BSA), levodopa (L-DOPA) and nonapeptide-1 acetate salt (N-1A), MedChemExpress (Shanghai, China); and α-MSH ELISA Kit, CUSABIO (Wuhan, China).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay