Journal: Cancers

Article Title: TP53 Mutation-Specific Dysregulation of Store-Operated Calcium Entry and Apoptotic Sensitivity in Triple-Negative Breast Cancer.

doi: 10.3390/cancers17101614

Figure Lengend Snippet: Figure 1. Differential calcium channel gene expression in TNBC TP53 mutant patient samples and cell line versus TP53 wildtype. (A) Differential expression analyses (DEAs) carried out using CCLE breast cancer cell line microarray data and TCGA patient sample RNA-sequencing data. (i) The CCLE volcano plot illustrates the DEGs (Dots) when comparing TNBC TP53 MUT (n = 20) vs. all BC subtype WT (n = 13) with 372 significantly upregulated (lfc ≥2, BH-adj. p-value ≤0.01) and 168 significantly downregulated (lfc ≤−2, BH-adj. p-value ≤0.01) genes. and (ii) the TCGA volcano plot illustrates the DEGs when comparing TNBC TP53 MUT (n = 142) vs. all BC subtypes WT (n = 658) samples with 2282 significantly upregulated and 1389 significantly downregulated genes. (B) (i) Differentially

Article Snippet: In addition, COTI-2, a third-generation thiosemicarbazone p53 reactivator (Selleckchem, Houston, TX, USA; Cat. No. S8580), was prepared at a concentration of 5 mM in DMSO.

Techniques: Gene Expression, Mutagenesis, Quantitative Proteomics, Microarray, RNA Sequencing

Journal: Cancers

Article Title: TP53 Mutation-Specific Dysregulation of Store-Operated Calcium Entry and Apoptotic Sensitivity in Triple-Negative Breast Cancer.

doi: 10.3390/cancers17101614

Figure Lengend Snippet: Figure 2. SOC and tumour biology is altered in TP53 mutant TNBC cell lines compared to WT. (A) Traces of SOC were measured in TNBC cell lines, either TP53 WT CAL-51 (black, n = 7) or mutant

Article Snippet: In addition, COTI-2, a third-generation thiosemicarbazone p53 reactivator (Selleckchem, Houston, TX, USA; Cat. No. S8580), was prepared at a concentration of 5 mM in DMSO.

Techniques: Mutagenesis

Journal: Cancers

Article Title: TP53 Mutation-Specific Dysregulation of Store-Operated Calcium Entry and Apoptotic Sensitivity in Triple-Negative Breast Cancer.

doi: 10.3390/cancers17101614

Figure Lengend Snippet: Figure 3. Impact of CACNA1D manipulation through COTI-2 treatment or siRNA on calcium channel expression and SOC in TP53 mutant TNBC cell lines. (A) Effect of COTI-2 treatment on relative fold change gene expression of calcium channels using qRT-PCR in HDQ-P1 cells (purple) and MDA-MB-157 cells (pink) compared to untreated control. (B) Individual graphs of CANCA1D relative fold change gene expression in COTI-2 treated TP53 mutant cells vs untreated. SOC measured by Fura-2AM ratiometeric in (C) HDQ-P1 or (D) MDA-MB-157 as displayed as a trace over time(s) (i) compared to DMSO control, with individual analysis displaying (ii) average baseline 0 mM calcium, (iii) max TG peak and (iv) SOCE. Mann-Whitney was used for all above comparing two groups. (E) SOC trace for CAL-51 following (Ei) CACNA1D siRNA compared to siNegative or untreated control with individual analysis displaying (ii) average baseline 0 mM calcium, (iii) max TG peak and (iv) SOCE. Kruskal-Wallis with Dunn’s multiple comparisons was used for analysis in (E).

Article Snippet: In addition, COTI-2, a third-generation thiosemicarbazone p53 reactivator (Selleckchem, Houston, TX, USA; Cat. No. S8580), was prepared at a concentration of 5 mM in DMSO.

Techniques: Expressing, Mutagenesis, Gene Expression, Quantitative RT-PCR, Control, MANN-WHITNEY

Journal: Cancers

Article Title: TP53 Mutation-Specific Dysregulation of Store-Operated Calcium Entry and Apoptotic Sensitivity in Triple-Negative Breast Cancer.

doi: 10.3390/cancers17101614

Figure Lengend Snippet: Figure 4. Impact of COTI-2 and TG treatment on Apoptosis. TP53 wildtype CAL-51 and TP53 mutant MDA-MB-157 cells were analyzed for apoptosis following TG and COTI-2 treatment along with DMSO and untreated controls in duplicates. Using the caspase 3/7 dye, apoptosis was recorded every 4 h over a period of 5 days. (A) Shows a trace of apoptotic induction in CAL-51 and MDA-MB-157 in the presence and absence of COTI-2. (B). Responses to (An) at 120 h where graphed and analysed by Mann-Whitney. Apoptosis traces in CAL51 (C) and MDA-MB-157 (D) cells under control (black), DMSO (blue), thapsigargin (TG; green), COTI-2 (red), and combined TG+COTI-2 (purple) treatments are shown. Analysed by Two-way ANOVA Post hoc Tukey. Cell proliferation rates after treatment with TG (green), COTI-2 (red) and TG+COTI-2 (black) measured by acid phosphatase in (E) TP53 WT CAL-51 and (F) T53 mutant MDA-MB-157, with bar graph analysis at 3.125nm. Analysed by One-way ANOVA with post hoc.

Article Snippet: In addition, COTI-2, a third-generation thiosemicarbazone p53 reactivator (Selleckchem, Houston, TX, USA; Cat. No. S8580), was prepared at a concentration of 5 mM in DMSO.

Techniques: Mutagenesis, MANN-WHITNEY, Control

Journal: Cancers

Article Title: TP53 Mutation-Specific Dysregulation of Store-Operated Calcium Entry and Apoptotic Sensitivity in Triple-Negative Breast Cancer.

doi: 10.3390/cancers17101614

Figure Lengend Snippet: Figure 5. Schematic representation of TP53 mutation-dependent regulation of Ca2+ homeostasis and apoptosis via CACNA1D/Cav1.3 modulation in TNBC. Left (TP53 WT): In wild-type TP53 cells, cellular stress (e.g., chemotherapy, hypoxia) activates p53, which upregulates CACNA1D (CaV1.3), leading to increased basal Ca2+ levels. Functional SERCA pumps and IP3Rs ensure proper ER Ca2+

Article Snippet: In addition, COTI-2, a third-generation thiosemicarbazone p53 reactivator (Selleckchem, Houston, TX, USA; Cat. No. S8580), was prepared at a concentration of 5 mM in DMSO.

Techniques: Mutagenesis, Functional Assay

Journal: Investigational New Drugs

Article Title: Degradation of MYC by the mutant p53 reactivator drug, COTI-2 in breast cancer cells

doi: 10.1007/s10637-023-01368-1

Figure Lengend Snippet: Effect of COTI-2 on degradation of MYC. ( a ) MDA-MB-468, ( b ) MDA-MB-453, ( c ) BT549, ( d ) MDA-MB-231, ( e ) SUM159 and ( f ) MCF7. Cells were incubated with different concentrations of COTI-2 for 48 h. Cell lysates were assessed by Western blotting. GAPDH was probed as loading control. Expression fold changes and % of MYC remaining were then calculated and graphed using GraphPad Prism 5. Data plotted are means ± S.E.M (n = 3) and evaluated using the Student’s unpaired, two-tailed t-test

Article Snippet: COTI-2 was purchased from Stratech Scientific Ltd, UK, MYCi 975 (HY-12,960) from MedChemTronica (HY-129,601), Bergkällavägen 37 C, 192 79 Sollentuna, Sweden and both MG-132 and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) form Sigma-Aldrich, Ireland.

Techniques: Incubation, Western Blot, Control, Expressing, Two Tailed Test

Journal: Investigational New Drugs

Article Title: Degradation of MYC by the mutant p53 reactivator drug, COTI-2 in breast cancer cells

doi: 10.1007/s10637-023-01368-1

Figure Lengend Snippet: Effect of COTI-2 on expression of MYC mRNA. ( a ) MDA-MB-231 and ( b ) MDA-MB-468 cells were incubated with DMSO or 32 nM of COTI-2 for 48 h. mRNA expression level were determined by RT-PCR. Data were calculated and graphed using GraphPad Prism 5. Data plotted are means ± S.E.M (n = 3) and evaluated using the Student’s unpaired, two-tailed t-test

Article Snippet: COTI-2 was purchased from Stratech Scientific Ltd, UK, MYCi 975 (HY-12,960) from MedChemTronica (HY-129,601), Bergkällavägen 37 C, 192 79 Sollentuna, Sweden and both MG-132 and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) form Sigma-Aldrich, Ireland.

Techniques: Expressing, Incubation, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test

Journal: Investigational New Drugs

Article Title: Degradation of MYC by the mutant p53 reactivator drug, COTI-2 in breast cancer cells

doi: 10.1007/s10637-023-01368-1

Figure Lengend Snippet: Effect of COTI-2 on the proteasomal degradation of MYC. ( a ) MDA-MB-231 and ( b ) MDA-MB-468 cells were incubated with 20 µM of MG-132 for 3 h, followed by 3 h incubation with DMSO or 2 µM of COTI-2, before harvesting. ( c ) MDA-MB-231 and ( d ) MDA-MB-468 were treated with DMSO or 2 µM of COTI-2 for 3.5 h, followed by incubation with CHX (30 µg/ml). Treated cells were then harvested at the indicated time points. Degradation of MYC was visualized by Western blotting. Data were calculated and graphed using GraphPad Prism 5. Data plotted are means ± S.E.M (n = 3) and evaluated using the Student’s unpaired, two-tailed t-test

Article Snippet: COTI-2 was purchased from Stratech Scientific Ltd, UK, MYCi 975 (HY-12,960) from MedChemTronica (HY-129,601), Bergkällavägen 37 C, 192 79 Sollentuna, Sweden and both MG-132 and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) form Sigma-Aldrich, Ireland.

Techniques: Incubation, Western Blot, Two Tailed Test

Journal: Investigational New Drugs

Article Title: Degradation of MYC by the mutant p53 reactivator drug, COTI-2 in breast cancer cells

doi: 10.1007/s10637-023-01368-1

Figure Lengend Snippet: Effect of COTI-2 on phosphorylation of MYC. ( a ) MDA-MB-468 and ( b ) MDA-MB-231 cells were incubated with 2 µM of COTI-2. Cells were harvested at the indicated time points. Cell lysates were determined by Western blotting using antibodies against pT58 MYC, pS62 MYC, and total MYC. GAPDH was probed as loading control. c , d , e f ) Effect of time of incubation with COTI-2 on relative levels of pT58 versus pS62 in MDA-MB-468 ( c , e ) MDA-MB-468 cells ( d , f ). Data was calculated and graphed using GraphPad Prism 5. Data plotted are means ± S.E.M (n = 3)

Article Snippet: COTI-2 was purchased from Stratech Scientific Ltd, UK, MYCi 975 (HY-12,960) from MedChemTronica (HY-129,601), Bergkällavägen 37 C, 192 79 Sollentuna, Sweden and both MG-132 and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) form Sigma-Aldrich, Ireland.

Techniques: Incubation, Western Blot, Control

Journal: Investigational New Drugs

Article Title: Degradation of MYC by the mutant p53 reactivator drug, COTI-2 in breast cancer cells

doi: 10.1007/s10637-023-01368-1

Figure Lengend Snippet: Effects of combined treatment with COTI-2 and MYCi975 on cell proliferation ( a ) MDA-MB-468, ( b ) MDA-MB-231 and ( c ) BT 549 cells were incubated with various concentrations of COTI-2 and MYCi975 for five days. MTT assays were then carried to detect cell proliferation. Combination index (CI) values were calculated using Compusyn software. CI values < 1 indicate drug-synergy. Data were calculated and graphed using GraphPad Prism 5. Data plotted are means ± S.E.M (n = 3)

Article Snippet: COTI-2 was purchased from Stratech Scientific Ltd, UK, MYCi 975 (HY-12,960) from MedChemTronica (HY-129,601), Bergkällavägen 37 C, 192 79 Sollentuna, Sweden and both MG-132 and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) form Sigma-Aldrich, Ireland.

Techniques: Incubation, Software