Journal: Nutrients

Article Title: Induction of p53 Phosphorylation at Serine 20 by Resveratrol Is Required to Activate p53 Target Genes, Restoring Apoptosis in MCF-7 Cells Resistant to Cisplatin

doi: 10.3390/nu10091148

Figure Lengend Snippet: Early phosphorylation of p53 in S20 induced by Resv is necessary for p53-stability in MCF-7 R cells. ( A ) MCF-7 R cells were treated with CDDP (6 μM) with or without Resv (100 μM) and ( B ) MCF-7 cells were treated with Resv (100 μM); both cell cultures were treated for 6 h with specific p53-pS20 site kinase inhibitors: CK1 (60 μM), CHK2 (25 μM) or AMPK (40 μM). Total and phospho-p53 contents are assessed by Western blot using antibodies directed against total p53 (DO-1) or against the specific phosphorylated residue on S20, as indicated. ( C ) MCF-7 and MCF-7 R cells were treated with a DMSO–ethanol vehicle as control or CDDP (6 μM) with resveratrol (100 μM) and cultured in combination with CK1 (60 μM), CHK2 (25 μM) or AMPK (40 μM) inhibitors for 48 h and were double-stained with Annexin V and propidium iodide (PI) followed by flow cytometry analysis to determine apoptotic cells. The viable cells are located in the lower left quadrant (double negative with Annexin V–/PI–). Apoptotic cells (Annexin V+/PI–) appear in the lower right (early apoptosis) and upper right (late apoptosis) quadrant of data plots. Data are presented as a percentage of the cell population. ( D ) The combined results of three independent cytometry analyses depicting the mean levels of total apoptotic cells are shown. Results are presented as the means ± SD. *** p < 0.001 by one-way ANOVA followed by Turkey’s Multiple Comparison test.

Article Snippet: The AMPK inhibitor Compound C (or dorsomorphin), the CK1 inhibitor D4476, the Chk2 inhibitor, anti-rabbit and anti-mouse secondary antibodies, mouse monoclonal anti-phospho-ATM (S1981), rabbit polyclonal anti-ATM, monoclonal anti-p53-HRP (DO-1), and monoclonal anti-BCL-2 were purchased from Santa Cruz Biotechnology (San Diego, CA, USA).

Techniques: Phospho-proteomics, Western Blot, Residue, Control, Cell Culture, Staining, Flow Cytometry, Cytometry, Comparison

Journal: Nutrients

Article Title: Induction of p53 Phosphorylation at Serine 20 by Resveratrol Is Required to Activate p53 Target Genes, Restoring Apoptosis in MCF-7 Cells Resistant to Cisplatin

doi: 10.3390/nu10091148

Figure Lengend Snippet: In the MCF-7 resistant cell variant (MCF-7 R ), Resv attenuates phosphorylation in S15 and S46 of p53 by dephosphorylation and deactivation of ATM. However, it activates kinases CK1, CHK2, and AMPK to induce phosphorylation of p53 in S20 (which is required to activate p53 in order to upregulate BAX and PUMA genes) and modifies the ratio between BCL-2/BAX expression. The BAX protein was increased while BCL-2 protein was decreased, restoring apoptosis and overcoming chemoresistance. On the other hand, the overexpression of BCL-2 in MCF-7 R cells after CDDP treatment maintains the chemoresistance and blocks apoptosis despite the phosphorylation of p53 in S15 and S46 and the upregulation of NOXA and PUMA .

Article Snippet: The AMPK inhibitor Compound C (or dorsomorphin), the CK1 inhibitor D4476, the Chk2 inhibitor, anti-rabbit and anti-mouse secondary antibodies, mouse monoclonal anti-phospho-ATM (S1981), rabbit polyclonal anti-ATM, monoclonal anti-p53-HRP (DO-1), and monoclonal anti-BCL-2 were purchased from Santa Cruz Biotechnology (San Diego, CA, USA).

Techniques: Variant Assay, Phospho-proteomics, De-Phosphorylation Assay, Expressing, Over Expression