Journal: Cancers

Article Title: Targeting p53 for Melanoma Treatment: Counteracting Tumour Proliferation, Dissemination and Therapeutic Resistance

doi: 10.3390/cancers13071648

Figure Lengend Snippet: SLMP53-2 inhibits melanoma cell growth through induction of cell cycle arrest and apoptosis. ( A ) IC 50 values of SLMP53-2 in A375, G361, MEWO and SK-MEL-5 melanoma cells obtained by colony formation assay for 11 days; data were normalized to DMSO and correspond to mean ± SEM, n = 5 (two replicates each). ( B ) Colony formation assay for A375, G361, MEWO and SK-MEL-5 melanoma cells treated with SLMP53-2 for the indicated concentrations. Images are representative of five independent experiments. ( C ) Effect of SLMP53-2 on growth and morphology of A375 cells for the indicated time points; images are representative of five independent experiments (scale bar = 100 μm, magnification = ×100). ( D ) Apoptosis (Annexin V-positive cells) was evaluated in A375 cells after 24, 48 and 72 h of treatment with 12 μM SLMP53-2. ( E ) Cell cycle analysis in A375 cells was determined after 24, 48 and 72 h of treatment with 12 μM SLMP53-2. In ( D , E ), data are mean ± SEM, n = 5; values are significantly different from DMSO: * p < 0.05, one-way ANOVA followed by Tukey’s test. ( F , G ) Effect of SLMP53-2 on three-day-old A375 spheroids, for up to 8 days of treatment. In G , data are mean ± SEM, n = 5; values are significantly different from DMSO: * p < 0.05, one-way ANOVA followed by Tukey’s test. ( H , I ) Evaluation of spheroid formation after 10 days of treatment with SLMP53-2; treatment was performed at the seeding time of A375 cells. In I , data are mean ± SEM, n = 5; values are significantly different from DMSO: * p < 0.05, one-way ANOVA followed by Tukey’s test. In ( F , H ), images are representative of five independent experiments; scale bar = 100 μm; magnification = 100×.

Article Snippet: A total of 5.0 × 10 5 /flask A375 cells were treated with 12 and 18 μM SLMP53-2 for 4 h; after cell lysis and protein lysate separation, 300 μg of total protein was incubated with 10 μL of mouse monoclonal anti-p53 (DO-1) or mouse immunoglobulin G (IgG, negative control) from Santa Cruz Biotechnology (Frilabo, Porto, Portugal), overnight at 4 °C.

Techniques: Colony Assay, Cell Cycle Assay

Journal: Cancers

Article Title: Targeting p53 for Melanoma Treatment: Counteracting Tumour Proliferation, Dissemination and Therapeutic Resistance

doi: 10.3390/cancers13071648

Figure Lengend Snippet: SLMP53-2 has p53-dependent growth inhibitory effect in melanoma cells with enhancement of p53 transcriptional activity. ( A – C ) Colony formation assay for silenced p53 (sip53) and control (CTRL) A375 cells treated with SLMP53-2, allowed to grow for 11 days. In ( A ), silencing efficacy of p53 by siRNA is shown; immunoblots are representative of five independent experiments and GAPDH was used as loading control; data plotted were normalized to CTRL and correspond to mean ± SEM, n = 5; values are significantly different from CTRL: * p < 0.05, unpaired Student’s t -test. In ( B ), images are representative of five independent experiments. In ( C ), data are normalized to DMSO and correspond to mean ± SEM, n = 5; values of sip53 cells significantly different from CTRL cells: * p < 0.05, two-way ANOVA followed by Sidak’s test. ( D , E ) Protein levels of p53 transcriptional targets in A375 cells treated with SLMP53-2 for 24 h (p53, MDM2, PTEN, Cyclin D1, p21 and KILLER) or 48 h (GADD45, PUMA, BCL-2, BCL-xL and BAX). In ( D ), immunoblots are representative of five independent experiments; GAPDH was used as loading control. In ( E ), quantification of protein expression levels is shown; values with DMSO were set as 1; data are mean ± SEM, n = 5. ( F ) mRNA levels of p53 target genes were determined by RT-qPCR in A375 cells after 24 h treatment with SLMP53-2; fold change is relative to DMSO; data are mean ± SEM, n = 5; values are significantly different from DMSO: * p < 0.05, two-way ANOVA with Dunnett’s multiple comparison test.

Article Snippet: A total of 5.0 × 10 5 /flask A375 cells were treated with 12 and 18 μM SLMP53-2 for 4 h; after cell lysis and protein lysate separation, 300 μg of total protein was incubated with 10 μL of mouse monoclonal anti-p53 (DO-1) or mouse immunoglobulin G (IgG, negative control) from Santa Cruz Biotechnology (Frilabo, Porto, Portugal), overnight at 4 °C.

Techniques: Activity Assay, Colony Assay, Control, Western Blot, Expressing, Quantitative RT-PCR, Comparison

Journal: Cancers

Article Title: Targeting p53 for Melanoma Treatment: Counteracting Tumour Proliferation, Dissemination and Therapeutic Resistance

doi: 10.3390/cancers13071648

Figure Lengend Snippet: SLMP53-2 enhances p53 stabilization by disrupting the p53–MDM2 interaction and interferes with the miRNA network in melanoma cells. ( A ) p53 protein levels in A375 melanoma cells treated for 24 h with 12 µM SLMP53-2 or solvent followed by cycloheximide treatment from 0 to 2 h (CHX; 150 μg/mL). ( B ) Quantification of p53 protein expression levels; immunoblots are representative of five independent experiments; GAPDH was used as loading control. Values for cells nontreated with cycloheximide (0 h) were set as 1; data are mean ± SEM, n = 5. ( C , D ) Coimmunoprecipitation (Co-IP) was performed in A375 cells treated with SLMP53-2 for 4 h. In C, representative immunoblots of five independent experiments are shown—whole-cell lysate (Input). p53 from IP was used as loading control. In D, quantification of protein expression levels relative to DMSO is shown (set as 1). Data shown are mean ± SEM, n = 5. ( E ) Expression levels of miR-145 and miR-23a in A375 cells after 24 h of treatment with SLMP53-2 were determined by RT-qPCR; fold of change is relative to DMSO; data are mean ± SEM, n = 5; values are significantly different from DMSO: * p < 0.05, one-way ANOVA followed by Tukey’s test. ( F , G ) Protein levels of miR-145 target genes, in A375 cells treated with SLMP53-2 for 24 h. In ( F ), immunoblots are representative of five independent experiments; GAPDH was used as loading control. In ( G ), quantification of protein expression levels is shown; values with DMSO were set as 1; data are mean ± SEM, n = 5.

Article Snippet: A total of 5.0 × 10 5 /flask A375 cells were treated with 12 and 18 μM SLMP53-2 for 4 h; after cell lysis and protein lysate separation, 300 μg of total protein was incubated with 10 μL of mouse monoclonal anti-p53 (DO-1) or mouse immunoglobulin G (IgG, negative control) from Santa Cruz Biotechnology (Frilabo, Porto, Portugal), overnight at 4 °C.

Techniques: Solvent, Expressing, Western Blot, Control, Co-Immunoprecipitation Assay, Quantitative RT-PCR

Journal: Cancers

Article Title: Targeting p53 for Melanoma Treatment: Counteracting Tumour Proliferation, Dissemination and Therapeutic Resistance

doi: 10.3390/cancers13071648

Figure Lengend Snippet: SLMP53-2 inhibits melanoma cell migration and invasion. ( A ) A375 and SK-MEL-5 confluent cells were treated with 2 or 4 μM SLMP53-2, respectively; cells were observed at 24 and 32 h (A375) and 30 and 48 h (SK-MEL-5) in the wound-healing assay. Images are representative of five independent experiments; scale bar = 100 μM; magnification = 100×. ( B ) Quantification of wound closure using randomly selected microscopic fields (six fields per sample). Data are mean ± SEM, n = 5; values are significantly different from DMSO: * p < 0.05, two-way ANOVA followed by Sidak’s test. ( C ) Effect of 2 μM SLMP53-2 on migration of A375 and SK-MEL-5 cells after 24 h of treatment. The relative number of migratory cells was determined by analysis of fluorescence signal intensity; values with DMSO were set as 1. Data are mean ± SEM, n = 5 (two replicates each); values are significantly different from DMSO: * p < 0.05, Student’s t -test. ( D ) Effect of 2 μM SLMP53-2 on the invasion of A375 and SK-MEL-5 cells after 24 h of treatment. Cells able to invade through an ECMatrix layer were quantified by fluorescence signal; values with DMSO were set as 1. Data are mean ± SEM, n = 5 (two replicates each); values are significantly different from DMSO: * p < 0.05, Student’s t -test. ( E ) Effect of SLMP53-2 on lactate secretion by A375 and SK-MEL-5 cells after 8 h of treatment. Cell density for each sample was used to normalize relative luminescence units (RLU) signal. Data are mean ± SEM, n = 5 (two replicates each); values are significantly different from DMSO: * p < 0.05; unpaired Student’s t -test.

Article Snippet: A total of 5.0 × 10 5 /flask A375 cells were treated with 12 and 18 μM SLMP53-2 for 4 h; after cell lysis and protein lysate separation, 300 μg of total protein was incubated with 10 μL of mouse monoclonal anti-p53 (DO-1) or mouse immunoglobulin G (IgG, negative control) from Santa Cruz Biotechnology (Frilabo, Porto, Portugal), overnight at 4 °C.

Techniques: Migration, Wound Healing Assay, Fluorescence

Journal: Cancers

Article Title: Targeting p53 for Melanoma Treatment: Counteracting Tumour Proliferation, Dissemination and Therapeutic Resistance

doi: 10.3390/cancers13071648

Figure Lengend Snippet: SLMP53-2 interferes with key molecular players in epithelial-to-mesenchymal transition (EMT) and angiogenesis. ( A – D ) Protein expression levels of crucial regulators of EMT and angiogenesis in A375 ( A , B ) and SK-MEL-5 ( C , D ) melanoma cells after 48 h of treatment with SLMP53-2 (in A375 cells, β-catenin was detected for 8 h and E-cadherin and TWIST for 24 h of treatment). Immunoblots are representative of five independent experiments; GAPDH was used as a loading control. In ( B , D ), quantification of protein expression levels is shown; values with DMSO were set as 1; data are means ± SEM, n = 5.

Article Snippet: A total of 5.0 × 10 5 /flask A375 cells were treated with 12 and 18 μM SLMP53-2 for 4 h; after cell lysis and protein lysate separation, 300 μg of total protein was incubated with 10 μL of mouse monoclonal anti-p53 (DO-1) or mouse immunoglobulin G (IgG, negative control) from Santa Cruz Biotechnology (Frilabo, Porto, Portugal), overnight at 4 °C.

Techniques: Expressing, Western Blot, Control

Journal: Cancers

Article Title: Targeting p53 for Melanoma Treatment: Counteracting Tumour Proliferation, Dissemination and Therapeutic Resistance

doi: 10.3390/cancers13071648

Figure Lengend Snippet: SLMP53-2 sensitizes melanoma cells to clinically used chemotherapeutic agents. ( A – C ) Cells were treated with a concentration range of vemurafenib ( A ), dacarbazine ( B ) and cisplatin ( C ) alone and in combination with 2 μM SLMP53-2, for 48 h, and the growth was analysed by SBR assay. Growth with DMSO was set as 100%. For each combination, the combination index (C.I.) and dose reduction index (D.R.I.) values were obtained. Data are mean ± SEM, n = 5 (two replicates each); values are significantly different from chemotherapeutic drug alone: * p < 0.05; two-way ANOVA followed by Sidak’s test. ( D ) Apoptosis (Annexin V-positive cells) was evaluated in A375 cells after 48 h of treatment with 2 μM SLMP53-2 (SLMP) and 5 µM cisplatin and 0.03 µM vemurafenib. Data are mean ± SEM, n = 5; values are significantly different from drug alone: * p < 0.05, one-way ANOVA followed by Tukey’s test. ( E , F ) Protein expression levels of BCL-2 after 48 h of treatment of SLMP53-2 with cisplatin (cisp) and with vemurafenib (vem). Immunoblots are representative of five independent experiments; GAPDH was used as a loading control. In F , quantification of protein expression levels; values with DMSO were set as 1; data are mean ± SEM, n = 5. ( G ) Cell cycle analysis in A375 cells was determined after 48 h of treatment with 2 μM SLMP53-2 and 2 μM dacarbazine (Dac). Data are mean ± SEM, n = 5; values are significantly different from drug alone: * p < 0.05, one-way ANOVA followed by Tukey’s test. ( H , I ) Protein expression levels of p21 after 48 h treatment of SLMP53-2 with dacarbazine (Dac). Immunoblots are representative of five independent experiments; GAPDH was used as a loading control. In I , quantification of protein expression levels is shown; values with DMSO were set as 1; data are mean ± SEM, n = 5. ( J , K ) Effect of 2 μM SLMP53-2 in combination with 0.027 μM Vemurafenib (Vem) on three-day-old A375 spheroids for up to 8 days of treatment. For the combination, the C.I. value was obtained. Images are representative of five independent experiments; scale bar = 100 μm; magnification = 100×. In ( K ), data are mean ± SEM, n = 5; values are significantly different from DMSO: * p < 0.05, one-way ANOVA followed by Tukey’s test.

Article Snippet: A total of 5.0 × 10 5 /flask A375 cells were treated with 12 and 18 μM SLMP53-2 for 4 h; after cell lysis and protein lysate separation, 300 μg of total protein was incubated with 10 μL of mouse monoclonal anti-p53 (DO-1) or mouse immunoglobulin G (IgG, negative control) from Santa Cruz Biotechnology (Frilabo, Porto, Portugal), overnight at 4 °C.

Techniques: Concentration Assay, Expressing, Western Blot, Control, Cell Cycle Assay

Journal: Cancers

Article Title: Targeting p53 for Melanoma Treatment: Counteracting Tumour Proliferation, Dissemination and Therapeutic Resistance

doi: 10.3390/cancers13071648

Figure Lengend Snippet: Melanoma cells do not develop resistance to SLMP53-2: vemurafenib-resistant melanoma cells show no cross-resistance to SLMP53-2 and are resensitized to vemurafenib by SLMP53-2. ( A ) A375 cells were exposed to six rounds of treatment with 6, 9, 12, 18, 24 and 30 μM of SLMP53-2. IC 50 values were determined at the end of each round by SRB assay after 48 h of treatment. Data were normalized to DMSO and correspond to mean ± SEM, n = 5 (two replicates each); values not significantly different from parental cells: p > 0.05, two-way ANOVA followed by Sidak’s test. ( B ) Representative images of parental, vemurafenib-resistant (Vem-res) A375 cells; scale bar = 100 μm; magnification = 100×. ( C ) Concentration–response curves for vemurafenib in parental and Vem-res A375 cells after 48 h of treatment. Data were normalized to DMSO and correspond to mean ± SEM, n = 6 (two replicates each); values of Vem-res cells significantly different from parental cells: * p < 0.05; two-way ANOVA followed by Sidak’s test. ( D , E ) Protein levels of p-AKT/AKT, p-ERK/ERK, PTEN and MDR-1 in untreated parental and Vem-res A375 cells. In D, immunoblots are representative of five independent experiments; GAPDH was used as loading control. In E, quantification of protein expression levels is shown; values with DMSO were set as 1; data are mean ± SEM, n = 5. ( F ) Concentration–response curves for SLMP53-2 in parental and Vem-res A375 cells after 48 h of treatment. Data were normalized to DMSO and correspond to mean ± SEM, n = 6 (two replicates each); values of Vem-res cells are not significantly different from parental cells: two-way ANOVA followed by Sidak’s test. ( G ) Vem-res A375 cells were treated with a concentration range of vemurafenib alone and in combination with 2 μM of SLMP53-2. Cell growth was evaluated for 48 h of treatment; growth obtained with DMSO was set as 100%. For each combination, the C.I. and D.R.I. values were obtained. Data are mean ± SEM, n = 6 (two replicates each); values are significantly different from vemurafenib alone: * p < 0.05, two-way ANOVA followed by Sidak’s test. ( H ) Representative images of Vem-res A375 cells treated with DMSO, 2 μM SLMP53-2, 1.3 μM vemurafenib (Vem) and the combination (SLMP53-2 + Vem) for 48 h; images are representative of five treatments; scale bar = 100 μm; magnificatio n = 100×. ( I , J ) Protein levels of PTEN, BCL-2, MDR-1 and p-AKT/AKT, in Vem-res cells after 48 h of treatment with 2 µM SLMP53-2. In I, immunoblots are representative of five independent experiments; GAPDH was used as loading control. In J, quantification of protein expression levels is shown; values with DMSO were set as 1; data are mean ± SEM, n = 5.

Article Snippet: A total of 5.0 × 10 5 /flask A375 cells were treated with 12 and 18 μM SLMP53-2 for 4 h; after cell lysis and protein lysate separation, 300 μg of total protein was incubated with 10 μL of mouse monoclonal anti-p53 (DO-1) or mouse immunoglobulin G (IgG, negative control) from Santa Cruz Biotechnology (Frilabo, Porto, Portugal), overnight at 4 °C.

Techniques: Sulforhodamine B Assay, Concentration Assay, Western Blot, Control, Expressing

Journal: Cancers

Article Title: Targeting p53 for Melanoma Treatment: Counteracting Tumour Proliferation, Dissemination and Therapeutic Resistance

doi: 10.3390/cancers13071648

Figure Lengend Snippet: In vivo melanoma antitumour activity of SLMP53-2. C57BL/6-Rag2 −/− IL2rg −/− mice carrying A375 xenografts were treated with 50 mg∙kg −1 SLMP53-2 or vehicle by intraperitoneal injection twice a week for a total of six administrations. ( A ) Tumour volume curves of mice carrying A375 xenografts treated with SLMP53-2 or vehicle. Fold change is relative to the start of treatments; data are mean ± SEM, n = 7; values are significantly different from vehicle: * p < 0.05, two-way ANOVA followed by Sidak’s test. ( B ) Tumour weights measured at the end of the in vivo experiment; data are mean ± SEM, n = 7; values are significantly different from vehicle: * p < 0.05, unpaired Student’s t -test. Representative images of the tumours treated with SLMP53-2 or vehicle at the end of the experiment. ( C ) Body weight of the mice registered during the course of the experiment. Data are mean ± SEM, n = 7; values are not significantly different from vehicle: p > 0.05, two-way ANOVA followed by Sidak’s test. ( D ) Weight of heart, spleen, kidney and livers from animals treated with SLMP53-2 or vehicle. Data are mean ± SEM, n = 7; values are not significantly different from vehicle: p > 0.05, two-way ANOVA followed by Sidak’s test. ( E ) Representative images of p53, Ki-67, BAX, BCL-2, TUNEL, β-catenin, Vimentin, and Slug detection in tumour tissues of A375 xenografts treated with SLMP53-2 or vehicle, collected at the end of treatment (scale bar = 5 μm; magnificatio n = 200×); haematoxylin and eosin (H&E). ( F – H ) Quantification of immunohistochemistry of A375 xenograft tumour tissues treated with SLMP53-2 or vehicle. In F, quantification of the number of Ki-67-positive and -negative cells; values are significantly different from vehicle: * p < 0.05, two-way ANOVA followed by Sidak’s test. In G, quantification of the percentage of positive-staining cells with TUNEL, n = 5; values are significantly different from vehicle: * p < 0.05, unpaired Student’s t -test. In H, quantification of the p53, Vimentin, BAX, BCL-2, β-catenin and Slug staining by evaluation of 3,3′-diaminobenzidine (DAB) intensity is shown, n = 5; values are significantly different from vehicle: * p < 0.05, unpaired Student’s t -test.

Article Snippet: A total of 5.0 × 10 5 /flask A375 cells were treated with 12 and 18 μM SLMP53-2 for 4 h; after cell lysis and protein lysate separation, 300 μg of total protein was incubated with 10 μL of mouse monoclonal anti-p53 (DO-1) or mouse immunoglobulin G (IgG, negative control) from Santa Cruz Biotechnology (Frilabo, Porto, Portugal), overnight at 4 °C.

Techniques: In Vivo, Activity Assay, Injection, TUNEL Assay, Immunohistochemistry, Staining