Journal: Journal of Biological Chemistry

Article Title: Inhibition of Glycolytic Enzymes Mediated by Pharmacologically Activated p53

doi: 10.1074/jbc.m111.240812

Figure Lengend Snippet: FIGURE 3. p53-dependent ablation of selected metabolic factors upon RITA treatment. A and B, down-regulation of SLC2A1 (GLUT1), HKII (HK2), PFKFB3, SLC2A12 (GLUT12), PDK1, and HIF1A 8 h after treatment with 1 M RITA in p53-positive cancer cell lines as assessed by qRT-PCR (mean S.E. (error bars), n 3). A, down-regulation of selected metabolic genes in colon cancer cell line HCT116 (black bars) and upon inhibition of p53 by small molecule pifithrin- (gray bars). B, down-regulation of selected metabolic genes in wild type p53 human breast cancer cells MCF7 (black bars) and upon inhibition of p53 by small molecule pifithrin- (gray bars). C and D, comparison of changes in mRNA levels of metabolic genes in p53-null cancer cell lines HCT116 TP53/ and Saos2 (mean S.E., n 3). E, p53-dependent down-regulation of selected metabolic factors on protein level as assessed by immunoblotting.

Article Snippet: Small interfering RNA (siRNA) for HIF1 (sc-35561), c-Myc (sc-29226), and HK2 (sc-35621) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and green fluorescent protein (GFP) siRNA, which was used as a control, was purchased from Thermo Scientific Dharmacon.

Techniques: Quantitative RT-PCR, Inhibition, Comparison, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Inhibition of Glycolytic Enzymes Mediated by Pharmacologically Activated p53

doi: 10.1074/jbc.m111.240812

Figure Lengend Snippet: FIGURE 5. Contribution of c-Myc and HIF1 inhibition to the repression of metabolic genes upon p53 reactivation under normoxia and hypoxia, respectively. A, c-Myc protein level is down-regulated upon 1 M but not upon 0.1 M RITA in wild type p53 MCF7 and HCT116 cancer cells, as assessed by Western blot. B, top, the extent of c-Myc depletion by siRNA was assessed by immunoblotting. Bottom, inhibition of c-Myc levels by siRNA led to the down- regulation of the SLC2A1, HKII, and HIF1A mRNA in HCT116 cells, as detected by qRT-PCR (mean S.E. (error bars), n 3). C, RITA down-regulates HIF1 protein levels in a dose- and time-dependent manner under normoxia, as detected by Western blot (short exposure). D, efficient down-regulation of HIF1 by RITA treatment upon its induction by hypoxia mimetic CoCl2 as assessed by immunoblotting (long exposure). E, Western blot analysis revealed the induction of p53 and its target PUMA by 1 and 5 M of RITA in hypoxic conditions. Down-regulation of c-Myc and HK2 correlated with p53 induction. HIF1, which is induced by hypoxia, was down-regulated by RITA as well. F, qRT-PCR analysis revealed transcriptional repression of SLC2A1, HK2, PFKFB3, SLC2A12, and HIF1 in hypoxia upon treatment with 1 M RITA. Shown is the -fold change in hypoxia compared with normoxia (mean S.E., n 3).

Article Snippet: Small interfering RNA (siRNA) for HIF1 (sc-35561), c-Myc (sc-29226), and HK2 (sc-35621) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and green fluorescent protein (GFP) siRNA, which was used as a control, was purchased from Thermo Scientific Dharmacon.

Techniques: Inhibition, Western Blot, Quantitative RT-PCR

Journal: Journal of Biological Chemistry

Article Title: Inhibition of Glycolytic Enzymes Mediated by Pharmacologically Activated p53

doi: 10.1074/jbc.m111.240812

Figure Lengend Snippet: FIGURE6.Sp1cooperateswithp53torepressmetabolicgenes.A,expressionofmetabolicgeneswascomparedbetweencellswithdepletedSp1,untreated or treated with RITA for 4 or 8 h, and control transfected cells, untreated or treated with RITA, using microarray analysis. Data are presented as a heat map. Vertical columns indicate separate arrays, and horizontal rows indicate genes. The rows were standardized by subtracting the mean of the first column. B, Sp1 depletion partially rescued p53-mediated repression of HIF1A, SLC2A12, and HKII as assessed by qRT-PCR. *, statistically significant differences, calculated using Student’s t test. Error bars, S.E. C, Sp1 knockdown rescued MCF7 cells from growth suppression mediated by RITA as assessed by the microscopy analysis of cell morphology.

Article Snippet: Small interfering RNA (siRNA) for HIF1 (sc-35561), c-Myc (sc-29226), and HK2 (sc-35621) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and green fluorescent protein (GFP) siRNA, which was used as a control, was purchased from Thermo Scientific Dharmacon.

Techniques: Control, Transfection, Microarray, Quantitative RT-PCR, Knockdown, Microscopy

Journal: Journal of Biological Chemistry

Article Title: Inhibition of Glycolytic Enzymes Mediated by Pharmacologically Activated p53

doi: 10.1074/jbc.m111.240812

Figure Lengend Snippet: FIGURE 7. Ablation of HK2 contributes to RITA-induced apoptosis. A, HK2 was down-regulated by RITA in a dose-dependent manner, as detected by Western blot analysis in MCF7 and HCT116 cells. B, inhibition of HK2 synergized with 0.1 M RITA. Microscopy analysis shows the extent of cell death induction by 0.1 and 1 M RITA in the presence or absence of HK2 depletion by siRNA. C, down-regulation of HK2 by siRNA synergized with 0.1 M RITA treatment in apoptosis induction in MCF7 cells. Quantification of cell death induction by RITA treatment in the presence or absence of HK2 depletion by siRNA was performed using trypan blue staining. D, combination of 0.5 M RITA with 2-deoxyglucose with 0.5 M RITA potentiated growth inhibition of p53-positive HCT116 and MCF7 cells but not of the HCT116 TP53/ cells. E, model depicting the regulatory pathways governing cancer cell metabolism that are affected upon pharmacological activation of p53 by small molecule RITA. For more details, see “Discussion.”

Article Snippet: Small interfering RNA (siRNA) for HIF1 (sc-35561), c-Myc (sc-29226), and HK2 (sc-35621) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and green fluorescent protein (GFP) siRNA, which was used as a control, was purchased from Thermo Scientific Dharmacon.

Techniques: Western Blot, Inhibition, Microscopy, Staining, Activation Assay