Journal: Cancer research

Article Title: Inhibition of BCL2 family members increases the efficacy of copper chelation in BRAF V600E -driven melanoma

doi: 10.1158/0008-5472.CAN-19-1784

Figure Lengend Snippet: A, Schematic diagram of high throughput screen of 2123 Selleckchem bioactive compound library at 100nM alone or in combination with the IC20 of TTM (dashed red line) in A375 or WM88. B,C, Normalized Percent Inhibition (NPI) of TTM + Compound versus NPI Compound graph for indicated cells treated with Selleckchem bioactive compound library alone or in combination with IC20 of TTM. Hits (blue circles) are defined as NPI TTM + Compound ≥ 20 (dashed red line). D,E, Observed effect versus expected effect graph for indicated cells of Hits from B and C. Hits (blue circles) are defined as Bliss Index ≥ 1.5 (dashed red line). F,G, Graphical representation of Hits defined as NPI TTM + Compound ≥ 20 and Bliss Index ≥ 1.5 in indicated cells. H, Venn diagram relationship between Compound Hits. I, NPI of 5 overlapping Hit Compounds from H alone or in combination with IC20 of TTM (dashed red line). J,K, Scatter dot plot of %ATP normalized to VEH ± s.e.m. of indicated cells treated with vehicle or indicated concentrations of drugs. L,M,N,O, Scatter dot plot of %ATP normalized to VEH ± s.e.m. of indicated cells treated with VEH or indicated concentrations of drugs. Results were compared using a one-way ANOVA followed by a Tukey’s multi-comparisons test. One asterisk, P<0.05, Two asterisks, P<0.01, Three asterisks, P<0.001, Four asterisks, P<0.0001. n=3.

Article Snippet: 293T/17 (ATCC, catalog #CRL-11268), A375 (ATCC, catalog #CRL-1619), WM88 (Rockland, catalog #WM88-01-0001), WM3311 (Rockland, catalog #WM3311-01-0001), WM3743 (Rockland, catalog #WM3743-01-0001) cells were purchased from the indicated companies and maintained in Dulbecco’s Modified Eagle Media (DMEM, Gibco) supplemented with 10% v/v fetal bovine serum (FBS, GE Lifesciences) and 1% penicillin-streptomycin (P/S, Gibco).

Techniques: High Throughput Screening Assay, Drug discovery, Inhibition

Journal: Cancer research

Article Title: Inhibition of BCL2 family members increases the efficacy of copper chelation in BRAF V600E -driven melanoma

doi: 10.1158/0008-5472.CAN-19-1784

Figure Lengend Snippet: A,C, Relative CellTiter-Glo® cell viability ± s.e.m. of indicated cells stably expressing two independent Dox-inducible shRNAs (#1 and #2) against indicated genes treated with indicated concentrations of TTM without or with Dox n=3. B,D, Scatter dot plot of TTM IC50 ± s.e.m in indicated cells cells stably expressing two independent Dox-inducible shRNAs against indicated genes treated without or with Dox. Results were compared using a two-way ANOVA followed by a Sidak’s multi-comparisons test. Three asterisks, P<0.001, Four asterisks, P<0.0001. n=3. E,G, Relative CellTiter-Glo® cell viability ± s.e.m. of indicated cells treated without or with indicated concentrations of TTM and increasing concentrations of indicated BH3 mimetics. n=3. F,H, Scatter dot plot of BH3 mimetic IC50 ± s.e.m in indicated cells treated without or with indicated concentrations of TTM and increasing concentrations of indicated BH3 mimetics. n=3. Results were compared using a two-way ANOVA followed by a Tukey’s multi-comparisons test. n=3. I,J, Graphical representation of Bliss Index (observed effect versus expected effect) from A375 (E) and WM88 (G) at the indicated drug combinations. Synergistic combinations are indicated by Bliss Index values >1. Two asterisks, P<0.01, Three asterisks, P<0.001, Four asterisks, P<0.0001.

Article Snippet: 293T/17 (ATCC, catalog #CRL-11268), A375 (ATCC, catalog #CRL-1619), WM88 (Rockland, catalog #WM88-01-0001), WM3311 (Rockland, catalog #WM3311-01-0001), WM3743 (Rockland, catalog #WM3743-01-0001) cells were purchased from the indicated companies and maintained in Dulbecco’s Modified Eagle Media (DMEM, Gibco) supplemented with 10% v/v fetal bovine serum (FBS, GE Lifesciences) and 1% penicillin-streptomycin (P/S, Gibco).

Techniques: Stable Transfection, Expressing

Journal: Cellular & Molecular Biology Letters

Article Title: Melanoma cells induce dedifferentiation and metabolic changes in adipocytes present in the tumor niche

doi: 10.1186/s11658-023-00476-3

Figure Lengend Snippet: Characterization of cancer-associated adipocytes (CAAs). Adipocytes were cocultured with melanoma cells (lines: WM1341D, A375, WM9, and Hs294T) present on Transwell inserts, and then the expression of leptin, adiponectin, resistin, and FABP4 was established in obtained CAAs, as well as nondifferentiated 3T3L1 fibroblasts and control adipocytes, using real-time PCR analysis. For quantification, the level of expression was normalized to the expression of EEF2 and presented as the percent of control (untreated adipocytes). The mean of at least three biological repetitions ± standard deviation (SD) is shown. Asterisks indicate statistically significant differences between control adipocytes and 3T3L1/CAAs. The significance levels were set at p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***), or p ≤ 0.0001 (****). FABP4 fatty acid-binding protein 4, EEF2 eukaryotic translation elongation factor 2

Article Snippet: Briefly, 2 days after reaching confluence, cells were treated with a mixture of rosiglitazone (Merck, Darmstadt, Germany), insulin (Sigma Aldrich, Burlington, Massachusetts, USA), dexamethasone (Sigma Aldrich, Burlington, Massachusetts, USA), and 3-isobutyl-1-methylxanthine (IBMX; Merck, Darmstadt, Germany) for 48 h. They were then incubated with insulin for another 48 h. The influence of melanoma cells on adipocytes was assessed using four melanoma cell lines: A375 (CRL-1619) and Hs294T (HTB-140), obtained from the ATCC, and WM1341D (WM1341D-01-0001) and WM9 (WM9-01-0001) cell lines purchased from Rockland Immunochemicals, Inc. (Royersford, Pennsylvania, United States).

Techniques: Expressing, Control, Real-time Polymerase Chain Reaction, Standard Deviation, Binding Assay

Journal: Cellular & Molecular Biology Letters

Article Title: Melanoma cells induce dedifferentiation and metabolic changes in adipocytes present in the tumor niche

doi: 10.1186/s11658-023-00476-3

Figure Lengend Snippet: Features of fibroblasts exhibited by CAAs. A Adipocytes were cocultured with melanoma cells ( lines: WM1341D, A375, WM9 and Hs294T) present on Transwell inserts and then obtained CAAs as well as the non-differentiated 3T3L1 fibroblasts and control adipocytes were seeded on coverslips coated with gelatin-FITC (green). After 16 h of incubation, cells were fixed and stained using phalloidin-Alexa Fluor 568 to visualize F-actin (red). Areas of gelatin degradation are visible as dark holes on a green background. White arrows indicate stress fibers. Scale bar: 25 μm. B Western blot analysis of protein level of vimentin and TGFβ receptor III (TGFβRIII) in cell lysates of 3T3L1 cells; control adipocytes, and adipocytes cultured with melanoma cells growing on Transwell inserts. The signal was normalized to total protein content assessed by Ponceau S staining. The mean of at least three biological repetitions ± SD is shown. Asterisks indicate statistically significant differences between control adipocytes, 3T3L1 fibroblasts and CAAs. The significance levels were set at p ≤ 0.05 (*), p ≤ 0.01 (**), or p ≤ 0.0001 (****).

Article Snippet: Briefly, 2 days after reaching confluence, cells were treated with a mixture of rosiglitazone (Merck, Darmstadt, Germany), insulin (Sigma Aldrich, Burlington, Massachusetts, USA), dexamethasone (Sigma Aldrich, Burlington, Massachusetts, USA), and 3-isobutyl-1-methylxanthine (IBMX; Merck, Darmstadt, Germany) for 48 h. They were then incubated with insulin for another 48 h. The influence of melanoma cells on adipocytes was assessed using four melanoma cell lines: A375 (CRL-1619) and Hs294T (HTB-140), obtained from the ATCC, and WM1341D (WM1341D-01-0001) and WM9 (WM9-01-0001) cell lines purchased from Rockland Immunochemicals, Inc. (Royersford, Pennsylvania, United States).

Techniques: Control, Incubation, Staining, Western Blot, Cell Culture

Journal: Cellular & Molecular Biology Letters

Article Title: Melanoma cells induce dedifferentiation and metabolic changes in adipocytes present in the tumor niche

doi: 10.1186/s11658-023-00476-3

Figure Lengend Snippet: Loss of lipid content in cancer-associated adipocytes. Adipocytes were cocultured with melanoma cells (lines: WM1341D, A375, WM9, and Hs294T) present on Transwell inserts. A After coculture, obtained CAAs, control adipocytes, and 3T3L1 fibroblasts were seeded on coverslips, fixed, and stained with Lipid Spot 488 to visualize lipid droplets. Cell nuclei were stained using Hoechst 33,342 reagent. Scale bar: 25 μm. B To quantify the amount of neutral lipids, cells were fixed and then stained with Oil Red O, and the amount was measured spectrophotometrically. C Expression of PLIN1 (perilipin1) in 3T3L1 cells, control adipocytes, and CAAs established by real-time PCR analysis. For quantification, the samples were normalized against the expression of EEF2. For all experiments, the control constitutes normal adipocytes. In the case of quantitative analyses, the mean of at least three biological repetitions ± SD is shown. Asterisks indicate statistically significant differences between control cells and 3T3L1/CAAs. The significance levels were set at p ≤ 0.01 (**), p ≤ 0.001 (***), or p ≤ 0.0001 (****)

Article Snippet: Briefly, 2 days after reaching confluence, cells were treated with a mixture of rosiglitazone (Merck, Darmstadt, Germany), insulin (Sigma Aldrich, Burlington, Massachusetts, USA), dexamethasone (Sigma Aldrich, Burlington, Massachusetts, USA), and 3-isobutyl-1-methylxanthine (IBMX; Merck, Darmstadt, Germany) for 48 h. They were then incubated with insulin for another 48 h. The influence of melanoma cells on adipocytes was assessed using four melanoma cell lines: A375 (CRL-1619) and Hs294T (HTB-140), obtained from the ATCC, and WM1341D (WM1341D-01-0001) and WM9 (WM9-01-0001) cell lines purchased from Rockland Immunochemicals, Inc. (Royersford, Pennsylvania, United States).

Techniques: Control, Staining, Expressing, Real-time Polymerase Chain Reaction

Journal: Cellular & Molecular Biology Letters

Article Title: Melanoma cells induce dedifferentiation and metabolic changes in adipocytes present in the tumor niche

doi: 10.1186/s11658-023-00476-3

Figure Lengend Snippet: CAA secretome analysis. To identify proteins secreted by control adipocytes and CAAs cocultured with melanoma cells (lines: WM1341D, A375, WM9) present on Transwell inserts, conditioned media were analyzed using cytokines ( A ) and angiogenesis ( B ) arrays. Based on obtained signals, quantitative analysis was performed. Densitometric data were normalized to reference spots and are presented in the form of heatmaps, where darker blue indicates a higher increase in signal. CCL2 (MCP-1) C–C motif chemokine ligand 2, IL6 interleukin 6, CXCL1 C-X-C motif chemokine ligand 1, PTX3 pentraxin 3, IGFBP-2 insulin-like growth factor binding protein 2, IGFBP-3 insulin-like growth factor binding protein 3, VEGF vascular endothelial growth factor, PDGF-AA platelet-derived growth factor AA, TIMP-1 tissue inhibitor of metalloproteinases 1, TSP-1 thrombospondin 1, MMP8 matrix metalloprotease 8

Article Snippet: Briefly, 2 days after reaching confluence, cells were treated with a mixture of rosiglitazone (Merck, Darmstadt, Germany), insulin (Sigma Aldrich, Burlington, Massachusetts, USA), dexamethasone (Sigma Aldrich, Burlington, Massachusetts, USA), and 3-isobutyl-1-methylxanthine (IBMX; Merck, Darmstadt, Germany) for 48 h. They were then incubated with insulin for another 48 h. The influence of melanoma cells on adipocytes was assessed using four melanoma cell lines: A375 (CRL-1619) and Hs294T (HTB-140), obtained from the ATCC, and WM1341D (WM1341D-01-0001) and WM9 (WM9-01-0001) cell lines purchased from Rockland Immunochemicals, Inc. (Royersford, Pennsylvania, United States).

Techniques: Control, Binding Assay, Derivative Assay

Journal: Cellular & Molecular Biology Letters

Article Title: Melanoma cells induce dedifferentiation and metabolic changes in adipocytes present in the tumor niche

doi: 10.1186/s11658-023-00476-3

Figure Lengend Snippet: Influence of melanoma cells on lactate secretion ( A ), ions ( B , C ), and glucose transport ( D ) by 3T3L1 cells, control adipocytes, and CAAs. CAAs were obtained from adipocytes cultured with melanoma cells (lines: WM1341D, A375, WM9, and Hs294T) present on Transwell inserts. A The level of secreted lactate was measured using a chemiluminescent reaction in a cell-conditioned medium devoid of serum, in which cells were grown for 24 h. The expression of NHE1 ( B ), MCT1 ( C ), and GLUT1 ( D ) was estimated through real-time PCR analysis. For quantification, the samples were normalized against the expression of EEF2. The mean of at least three independent repetitions ± SD is shown. Asterisks indicate statistically significant differences between control cells and 3T3L1/CAAs. The significance levels were set at p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***), or p ≤ 0.0001 (****). NHE1 sodium/hydrogen exchanger 1, MCT1 monocarboxylate transporter 1, GLUT1 glucose transporter 1, EEF2 eukaryotic translation elongation factor 2

Article Snippet: Briefly, 2 days after reaching confluence, cells were treated with a mixture of rosiglitazone (Merck, Darmstadt, Germany), insulin (Sigma Aldrich, Burlington, Massachusetts, USA), dexamethasone (Sigma Aldrich, Burlington, Massachusetts, USA), and 3-isobutyl-1-methylxanthine (IBMX; Merck, Darmstadt, Germany) for 48 h. They were then incubated with insulin for another 48 h. The influence of melanoma cells on adipocytes was assessed using four melanoma cell lines: A375 (CRL-1619) and Hs294T (HTB-140), obtained from the ATCC, and WM1341D (WM1341D-01-0001) and WM9 (WM9-01-0001) cell lines purchased from Rockland Immunochemicals, Inc. (Royersford, Pennsylvania, United States).

Techniques: Control, Cell Culture, Expressing, Real-time Polymerase Chain Reaction

Journal: Cellular & Molecular Biology Letters

Article Title: Melanoma cells induce dedifferentiation and metabolic changes in adipocytes present in the tumor niche

doi: 10.1186/s11658-023-00476-3

Figure Lengend Snippet: Effect of coculture with adipocytes on the proliferation of melanoma cells. Melanoma cells (lines: WM1341D, A375, WM9, and Hs294T) were cocultured with adipocytes present on Transwell inserts for 7 days. Next, melanoma cells were seeded on a 96-well plate, and their proliferation rate was calculated as a ratio of the spectrophotometric signal after 48 h to the spectrophotometric signal at T0. Results are expressed as the mean (fold change) ± SD of three independent experiments. Asterisks above the bars express significance vs. control. The significance levels were set at p ≤ 0.05 (*), p ≤ 0.01 (**), and p ≤ 0.001 (***)

Article Snippet: Briefly, 2 days after reaching confluence, cells were treated with a mixture of rosiglitazone (Merck, Darmstadt, Germany), insulin (Sigma Aldrich, Burlington, Massachusetts, USA), dexamethasone (Sigma Aldrich, Burlington, Massachusetts, USA), and 3-isobutyl-1-methylxanthine (IBMX; Merck, Darmstadt, Germany) for 48 h. They were then incubated with insulin for another 48 h. The influence of melanoma cells on adipocytes was assessed using four melanoma cell lines: A375 (CRL-1619) and Hs294T (HTB-140), obtained from the ATCC, and WM1341D (WM1341D-01-0001) and WM9 (WM9-01-0001) cell lines purchased from Rockland Immunochemicals, Inc. (Royersford, Pennsylvania, United States).

Techniques: Control