Journal: Molecular pharmaceutics

Article Title: Enhancing the Efficacy of Melanocortin 1 Receptor-Targeted Radiotherapy by Pharmacologically Upregulating the Receptor in Metastatic Melanoma

doi: 10.1021/acs.molpharmaceut.9b00512

Figure Lengend Snippet: MC1R expression is upregulated in human melanoma cells upon treatment with BRAFi and HDACi. (A) qRT-PCR analysis of MC1R mRNA expression in BRAFV600E A2058 cells after 24 h of incubation with BRAFi dabrafenib (Dabr) and HDACi 4-phenylbutyrate (PBA) and in BRAFWT MEWO cells after 24 h treatment with HDACi PBA and vorinostat (Vor) (n = 3). Data are presented as normalized mean MC1R mRNA ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 vs controls; (B) flow cytometry histograms and protein expression of MC1R in A2058 cells after 24 h treatment with BRAFi: Vem (5 μM) and Dabr (2 μM); HDACi: PBA (2 mM) and Vor (2 μM); and in MEWO cells after incubation with PBA (2 mM) and Vor (2 μM). Experiments were conducted in triplicate. Data are expressed as relative expression of MC1R vs isotype control (mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001 vs controls); (C) MC1R binding with [125I]NDP-α-MSH in A2058 and MEWO cells following incubation with Dabr (1−10 μM), Vor (0.5−10 μM), and PBA (0.5−10 mM) for 12−24 h (n = 4). Data are expressed as MC1R-ligand binding vs dimethyl sulfoxide (DMSO)-treated cells (mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs controls); all experiments were performed in duplicate (n = 2).

Article Snippet: Human melanoma cell lines used for this study included BRAF-mutant (BRAF V600E ) A2058 and BRAF wild-type (BRAF WT ) MEWO cells obtained from American Type Culture Collection, and they were used within 15 passages for all experiments.

Techniques: Expressing, Quantitative RT-PCR, Incubation, Flow Cytometry, Control, Binding Assay, Ligand Binding Assay

Journal: Molecular pharmaceutics

Article Title: Enhancing the Efficacy of Melanocortin 1 Receptor-Targeted Radiotherapy by Pharmacologically Upregulating the Receptor in Metastatic Melanoma

doi: 10.1021/acs.molpharmaceut.9b00512

Figure Lengend Snippet: Upregulation of MC1R in melanoma cells is mediated by the transcription factor MITF. Immunoblotting analysis of MC1R and MITF expressions in (A) BRAFV600E cells (A2058) following 24 h incubation with dabrafenib (Dabr) (1−10 μM) and vorinostat (Vor) (1−10 μM) and in (B) BRAFWT cells (MEWO) following 24 h exposure to HDACi vorinostat (Vor) (1−10 μM) and 4-phenylbutyrate (PBA) (1−10 mM); immunoblotting analysis of MC1R in (C) A2058 and (D) MEWO cells in response to 24 h incubation with Dabr (1 μM), Vor (1 μM), or PBA (1 mM), with normal MITF expression (negative scrambled Dsi-RNA) and attenuated MITF expression (MITF Dsi-RNA). All experiments were conducted in duplicate (n = 2).

Article Snippet: Human melanoma cell lines used for this study included BRAF-mutant (BRAF V600E ) A2058 and BRAF wild-type (BRAF WT ) MEWO cells obtained from American Type Culture Collection, and they were used within 15 passages for all experiments.

Techniques: Western Blot, Incubation, Expressing

Journal: Molecular pharmaceutics

Article Title: Enhancing the Efficacy of Melanocortin 1 Receptor-Targeted Radiotherapy by Pharmacologically Upregulating the Receptor in Metastatic Melanoma

doi: 10.1021/acs.molpharmaceut.9b00512

Figure Lengend Snippet: Combination of BRAF, HDAC inhibitors, and MC1R-targeted 212Pb α-particle therapy significantly impairs BRAFV600E melanoma A2058 tumor growth and improves survival. (A) Representative IHC staining of MC1R in A2058 melanoma-bearing animals that were treated with HDACi PBA (90 mg kg−1, i.p., q.d.), BRAFi vemubrafenib (10 mg kg−1, p.o., b.i.d.) (n = 2); (B) 2 h postinjection SPECT/CT imaging of A2058 melanoma in athymic nu/nu mice treated with vemurafenib (Vem 10 mg kg−1, p.o.) and 4-phenylbutyrate (PBA 90 mg kg−1, i.p.) using [203Pb]DOTA-MC1L as the imaging tracer. Organs of interest are indicated as T (tumor), K (kidney), and B (bladder); (C) average tumor volume for each group of animals after treatments were initiated; data are expressed as mean ± SD; statistical analysis: *p < 0.05, **p < 0.01; (D) overall fractional survival in each treatment cohort over 100 days (n = 9−10 per group): vemurafenib (Vem) (10 mg kg−1, p.o., b.i.d.), 4-phenylbutyrate (PBA) (90 mg kg−1, i.p. q.d.), and 212Pb α-particle therapy (212Pb) (single dose of 5.2 MBq [212Pb]DOTA-MC1L). Statistical analysis: ****p <0.0001 212Pb versus control; ****p < 0.0001 212Pb versus Vem; **p < 0.01 212Pb/Vem vs 212Pb; **p < 0.01 212Pb/Vem/PBA vs 212Pb/Vem.

Article Snippet: Human melanoma cell lines used for this study included BRAF-mutant (BRAF V600E ) A2058 and BRAF wild-type (BRAF WT ) MEWO cells obtained from American Type Culture Collection, and they were used within 15 passages for all experiments.

Techniques: Immunohistochemistry, Single Photon Emission Computed Tomography, Imaging, Control

Journal: Frontiers in Oncology

Article Title: Clinicopathological correlations in 38 cases of gastroenteropancreatic high-grade neuroendocrine neoplasms

doi: 10.3389/fonc.2024.1399079

Figure Lengend Snippet: Illustrates the distribution of BRAF V600E mutation in GEP HG-NENs. A statistically significant disparity (**p=0.0086) was observed between NET G3 and SCNEC (A) . The distribution of KRAS mutation in GEP HG-NENs is also shown in the figure (B) .

Article Snippet: In our study, the BRAF V600E mutation was identified in 32.4% of the HG-NEN cases.

Techniques: Mutagenesis

Journal: Journal of Clinical Medicine

Article Title: Frequency of BRAF Mutations in Dysplastic Nevi, Lentigo Maligna, and Melanoma In Situ

doi: 10.3390/jcm13164799

Figure Lengend Snippet: The frequency of BRAF mutations varies across diagnoses.

Article Snippet: Three reaction mixtures were prepared for each sample: (i) mix with Mutation Detection Assay for V600E mutation (BRAF_476_mu; Assay ID: Hs00000111_mu), (ii) mix with Mutation Detection Assay for V600K mutation (BRAF_473_mu; Assay ID: Hs00000769_mu), (iii) Reference Assay (BRAF_rf; Assay ID: Hs00000172_rf).

Techniques: Mutagenesis

Journal: Journal of Clinical Medicine

Article Title: Frequency of BRAF Mutations in Dysplastic Nevi, Lentigo Maligna, and Melanoma In Situ

doi: 10.3390/jcm13164799

Figure Lengend Snippet: Presence and frequency of BRAF mutation between patients with ND, MIS, and LM.

Article Snippet: Three reaction mixtures were prepared for each sample: (i) mix with Mutation Detection Assay for V600E mutation (BRAF_476_mu; Assay ID: Hs00000111_mu), (ii) mix with Mutation Detection Assay for V600K mutation (BRAF_473_mu; Assay ID: Hs00000769_mu), (iii) Reference Assay (BRAF_rf; Assay ID: Hs00000172_rf).

Techniques: Mutagenesis

Journal: Journal of Clinical Medicine

Article Title: Frequency of BRAF Mutations in Dysplastic Nevi, Lentigo Maligna, and Melanoma In Situ

doi: 10.3390/jcm13164799

Figure Lengend Snippet: Age distribution of patients with and without BRAF mutation.

Article Snippet: Three reaction mixtures were prepared for each sample: (i) mix with Mutation Detection Assay for V600E mutation (BRAF_476_mu; Assay ID: Hs00000111_mu), (ii) mix with Mutation Detection Assay for V600K mutation (BRAF_473_mu; Assay ID: Hs00000769_mu), (iii) Reference Assay (BRAF_rf; Assay ID: Hs00000172_rf).

Techniques: Mutagenesis

Journal: Journal of Clinical Medicine

Article Title: Frequency of BRAF Mutations in Dysplastic Nevi, Lentigo Maligna, and Melanoma In Situ

doi: 10.3390/jcm13164799

Figure Lengend Snippet: Correlation of BRAF mutation and localization.

Article Snippet: Three reaction mixtures were prepared for each sample: (i) mix with Mutation Detection Assay for V600E mutation (BRAF_476_mu; Assay ID: Hs00000111_mu), (ii) mix with Mutation Detection Assay for V600K mutation (BRAF_473_mu; Assay ID: Hs00000769_mu), (iii) Reference Assay (BRAF_rf; Assay ID: Hs00000172_rf).

Techniques: Mutagenesis

Journal: Journal of Clinical Medicine

Article Title: Frequency of BRAF Mutations in Dysplastic Nevi, Lentigo Maligna, and Melanoma In Situ

doi: 10.3390/jcm13164799

Figure Lengend Snippet: Tumor size distribution in patients with and without BRAF mutation. ∗ and ◦ represent outliers.

Article Snippet: Three reaction mixtures were prepared for each sample: (i) mix with Mutation Detection Assay for V600E mutation (BRAF_476_mu; Assay ID: Hs00000111_mu), (ii) mix with Mutation Detection Assay for V600K mutation (BRAF_473_mu; Assay ID: Hs00000769_mu), (iii) Reference Assay (BRAF_rf; Assay ID: Hs00000172_rf).

Techniques: Mutagenesis

Journal: Journal of Clinical Medicine

Article Title: Frequency of BRAF Mutations in Dysplastic Nevi, Lentigo Maligna, and Melanoma In Situ

doi: 10.3390/jcm13164799

Figure Lengend Snippet: Correlation between the presence of the BRAF mutation and a previous melanoma diagnosis.

Article Snippet: Three reaction mixtures were prepared for each sample: (i) mix with Mutation Detection Assay for V600E mutation (BRAF_476_mu; Assay ID: Hs00000111_mu), (ii) mix with Mutation Detection Assay for V600K mutation (BRAF_473_mu; Assay ID: Hs00000769_mu), (iii) Reference Assay (BRAF_rf; Assay ID: Hs00000172_rf).

Techniques: Mutagenesis, Biomarker Discovery

Journal: Frontiers in Immunology

Article Title: Identification and validation of a KRAS-macrophage-associated gene signature as prognostic biomarkers and potential therapeutic targets in melanoma

doi: 10.3389/fimmu.2025.1566432

Figure Lengend Snippet: Functional investigation of CLEC4A’s role in melanoma cell behavior. (A) CLEC4A mRNA expression in melanoma cell lines. (B) Verification of CLEC4A knockdown/overexpression in A375 cells. (C-E) Proliferation assays (EdU, colony formation, CCK-8). (F, G) Migration and invasion assays (wound healing, Transwell). *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: Human melanoma cell lines, including A375 (known to harbor the BRAF V600E mutation), SK-MEL-2, A2058, and MV3, were procured from the American Type Culture Collection (ATCC; Manassas, VA, USA) ( ) and cultured under standardized conditions to ensure consistency and reliability of experimental results.

Techniques: Functional Assay, Expressing, Knockdown, Over Expression, CCK-8 Assay, Migration

Journal: Oncology Letters

Article Title: BRAF V600E -induced KRT19 expression in thyroid cancer promotes lymph node metastasis via EMT

doi: 10.3892/ol.2019.10360

Figure Lengend Snippet: Antibody list.

Article Snippet: The wild type (WT) BRAF WT (pcDNA3.1, cat. no. 40775; Addgene, Inc.) with its empty vector were transfected to the cells followed by Lipofectamine 2000 instruction and mutant BRAF V600E (pBabe, cat. no. 15269; Addgene, Inc.) with its empty vector were transfected into retrovirus packaging phoenix cells (with an VSV.G envelope plasmid; cat. no. 14888; Addgene, Inc.), after 48 h culturing the virus supernatant was harvested and filtered.

Techniques:

Journal: Oncology Letters

Article Title: BRAF V600E -induced KRT19 expression in thyroid cancer promotes lymph node metastasis via EMT

doi: 10.3892/ol.2019.10360

Figure Lengend Snippet: The TCGA_THCA dataset was used for the following analysis. (A) Compared with the BRAF wild type group, the expression of KRT19 was increased in BRAF V600E group, however, not in BRAF wild type group. The (B) wild type BRAF or (C) V600E mutated BRAF overexpression plasmids were transfected into 8505C cells, and may be overexpressed significantly compared with their empty vectors, BRAF WT /BRAF V600E . (D) mRNA and (E) protein expression levels of the BRAF V600E mutation group exhibited higher expression levels of KRT19 in 8505C cells when compared with BRAF wild type group. BRAF, B-Raf Proto-Oncogene, Serine/Threonine Kinase; TCGA, The Cancer Genome Atlas Network; THCA, thyroid cancer; KRT19, keratin 19; WT, wild type.

Article Snippet: The wild type (WT) BRAF WT (pcDNA3.1, cat. no. 40775; Addgene, Inc.) with its empty vector were transfected to the cells followed by Lipofectamine 2000 instruction and mutant BRAF V600E (pBabe, cat. no. 15269; Addgene, Inc.) with its empty vector were transfected into retrovirus packaging phoenix cells (with an VSV.G envelope plasmid; cat. no. 14888; Addgene, Inc.), after 48 h culturing the virus supernatant was harvested and filtered.

Techniques: Expressing, Over Expression, Transfection, Mutagenesis

Journal: Oncology Letters

Article Title: BRAF V600E -induced KRT19 expression in thyroid cancer promotes lymph node metastasis via EMT

doi: 10.3892/ol.2019.10360

Figure Lengend Snippet: The TCGA_THCA dataset was used for the following analysis. (A and D) Heatmap and GSEA analysis in EMT and BRAF V600E tumor vs. wild type BRAF tumor samples from the TCGA_THCA dataset. (B and C) Heatmap of EMT genes in the top 10 samples exhibiting high KRT19 expression vs. 10 samples with the lowest KRT19 expression from the TCGA dataset. (E) Western blot analysis of EMT markers in the 8505C cell line transfected with controls or si-KRT19. Knockdown of KRT19 was associated with increased E-cadherin and reduced N-cadherin levels when compared with the control group. BRAF, B-Raf Proto-Oncogene, Serine/Threonine Kinase; TCGA, The Cancer Genome Atlas Network; THCA, thyroid cancer; GSEA, Gene Set Enrichment Analysis; EMT, epithelial-mesenchymal-transition; KRT19, keratin-19; siRNA, small interfering RNA; FDR, false discovery rate; ES, enrichment score.

Article Snippet: The wild type (WT) BRAF WT (pcDNA3.1, cat. no. 40775; Addgene, Inc.) with its empty vector were transfected to the cells followed by Lipofectamine 2000 instruction and mutant BRAF V600E (pBabe, cat. no. 15269; Addgene, Inc.) with its empty vector were transfected into retrovirus packaging phoenix cells (with an VSV.G envelope plasmid; cat. no. 14888; Addgene, Inc.), after 48 h culturing the virus supernatant was harvested and filtered.

Techniques: Expressing, Western Blot, Transfection, Knockdown, Control, Small Interfering RNA

Journal: Cancer letters

Article Title: Synergistic efficacy of irinotecan and sunitinib combination in preclinical models of anaplastic thyroid cancer

doi: 10.1016/j.canlet.2017.09.032

Figure Lengend Snippet: Antiproliferative effects of sunitinib (SU) and SN-38 in vitro on 8305C (A and C, respectively), and FB3 (B and D, respectively) cell lines. The antiproliferative effects of the drugs were studied after 72 h of exposure. The data are presented as percentage of vehicle-treated cells. The concentrations of drug that reduced cell proliferation by 50% (IC50) vs controls were calculated by a nonlinear regression fit of the mean values of the data obtained in triplicate experiments (i.e. at least 9 wells for each concentration). Columns and bars, mean values ± S.E., respectively. *, P < 0.001 vs. control.

Article Snippet: The human ATC cell line 8305C ( BRAF V600E mutated) – established from undifferentiated thyroid carcinomas of a 67 year-old-female patient – was from DSMZ (Braunschweig, Germany, DSMZ no.: ACC 133) [ 18 ], whereas the human ATC cell line FB3 ( HRas Q61R mutated) was obtained from Prof. Fulvio Basolo of the University of Pisa, Pisa, Italy [ 6 ].

Techniques: In Vitro, Concentration Assay

Journal: Cancer letters

Article Title: Synergistic efficacy of irinotecan and sunitinib combination in preclinical models of anaplastic thyroid cancer

doi: 10.1016/j.canlet.2017.09.032

Figure Lengend Snippet: CI (Combination Index) and DRI (Dose Reduction Index) values for the drug combinations at 25%, 50%, and 75% levels of inhibition of 8305C and FB3 cell proliferation. SN-38, active metabolite of irinotecan; SU, sunitinib.

Article Snippet: The human ATC cell line 8305C ( BRAF V600E mutated) – established from undifferentiated thyroid carcinomas of a 67 year-old-female patient – was from DSMZ (Braunschweig, Germany, DSMZ no.: ACC 133) [ 18 ], whereas the human ATC cell line FB3 ( HRas Q61R mutated) was obtained from Prof. Fulvio Basolo of the University of Pisa, Pisa, Italy [ 6 ].

Techniques: Inhibition

Journal: Cancer letters

Article Title: Synergistic efficacy of irinotecan and sunitinib combination in preclinical models of anaplastic thyroid cancer

doi: 10.1016/j.canlet.2017.09.032

Figure Lengend Snippet: Accumulation of SN-38 (ng · mg−1 protein) in 8305C (A) and FB3 (B) cell lines after exposure to 1 μM SN-38 alone and in combination with sunitinib (SU). Columns and bars indicate the mean percentage values (±S.D.) vs. treated cells with SN-38 alone. ABCG2 gene expression (2−ΔΔCt) and ABCG2 (ng · mg−1 protein) protein levels in 8305C (C and E, respectively) and FB3 (D and F, respectively) cells exposed to sunitinib or with vehicle alone for 72 h. Data are expressed as percentage of vehicle-treated cells. Columns and bars, mean values ± S.D., respectively. *P < 0.05 vs. vehicle-treated controls. The quantitation of gene expression was performed using the ΔΔCt calculation, where Ct is the threshold cycle; the amount of target, normalized to the endogenous control, glyceraldehyde 3-phosphate dehydrogenase, and relative to the calibrator (vehicle treated control cells), is given as 2−ΔΔCt. The quantitation of protein levels was performed by ELISA. The optical density was determined using a Multiskan Spectrum microplate reader set to 450 nm. The results were expressed as nanograms of ABCG2 per milligram of total protein. All experiments were repeated, independently, three times.

Article Snippet: The human ATC cell line 8305C ( BRAF V600E mutated) – established from undifferentiated thyroid carcinomas of a 67 year-old-female patient – was from DSMZ (Braunschweig, Germany, DSMZ no.: ACC 133) [ 18 ], whereas the human ATC cell line FB3 ( HRas Q61R mutated) was obtained from Prof. Fulvio Basolo of the University of Pisa, Pisa, Italy [ 6 ].

Techniques: Expressing, Quantitation Assay, Enzyme-linked Immunosorbent Assay

Journal: Cancer letters

Article Title: Synergistic efficacy of irinotecan and sunitinib combination in preclinical models of anaplastic thyroid cancer

doi: 10.1016/j.canlet.2017.09.032

Figure Lengend Snippet: VEGF (A) and CSF-1 (B) gene expression (2−ΔΔCt) in 8305C and FB3 cells and (C) CSF-1 (ng · mg−1 protein) protein levels in FB3 cells exposed to sunitinib (SU) or with vehicle alone for 72 h. Columns and bars, mean values ± S.D., respectively. *P < 0.05 vs. vehicle-treated controls. Amplifications were normalized to glyceraldehyde 3-phosphate dehydrogenase, and the quantitation of gene expression was performed using the ΔΔCt calculation, where Ct is the threshold cycle; the amount of target, normalized to the endogenous control and relative to the calibrator (vehicle-treated control cells), is given as 2−ΔΔCt. The quantitation of protein levels was performed by ELISA. The optical density was determined using a Multiskan Spectrum microplate reader set to 450 nm. The results were expressed as nanograms of CSF-1 per milligram of total protein. All experiments were repeated, independently, three times.

Article Snippet: The human ATC cell line 8305C ( BRAF V600E mutated) – established from undifferentiated thyroid carcinomas of a 67 year-old-female patient – was from DSMZ (Braunschweig, Germany, DSMZ no.: ACC 133) [ 18 ], whereas the human ATC cell line FB3 ( HRas Q61R mutated) was obtained from Prof. Fulvio Basolo of the University of Pisa, Pisa, Italy [ 6 ].

Techniques: Expressing, Quantitation Assay, Enzyme-linked Immunosorbent Assay

Journal: Cancer letters

Article Title: Synergistic efficacy of irinotecan and sunitinib combination in preclinical models of anaplastic thyroid cancer

doi: 10.1016/j.canlet.2017.09.032

Figure Lengend Snippet: In vivo antitumor effects of the single drugs and three different combination schedules of sunitinib (SU) and irinotecan (CPT-11) on 8305C tumors xenotransplanted in mice (A); Immunohistochemistry quantification of CD31 (B) and Capase-3 positive cells (C) in 8305C tumor xenografts administered with vehicle, SU at 25 mg/kg every 3 days, CPT-11 100 mg/kg every week through i.p. injection, and their combinations. Symbols/columns and bars, mean values ± S.D., respectively.*P < 0.001 vs. vehicle-treated controls. #P < 0.001 vs. sunitinib-treated group.

Article Snippet: The human ATC cell line 8305C ( BRAF V600E mutated) – established from undifferentiated thyroid carcinomas of a 67 year-old-female patient – was from DSMZ (Braunschweig, Germany, DSMZ no.: ACC 133) [ 18 ], whereas the human ATC cell line FB3 ( HRas Q61R mutated) was obtained from Prof. Fulvio Basolo of the University of Pisa, Pisa, Italy [ 6 ].

Techniques: In Vivo, Immunohistochemistry, Injection