Journal: eBioMedicine

Article Title: Beta-blockers disrupt mitochondrial bioenergetics and increase radiotherapy efficacy independently of beta-adrenergic receptors in medulloblastoma

doi: 10.1016/j.ebiom.2022.104149

Figure Lengend Snippet: IC 50 values of propranolol, carvedilol and nebivolol after 72 h of treatment of non-Wnt human MB cells (DAOY, UW228-2, HD-MB03, ONS-76, D283 Med, D341 Med), mice MB cells (murine SHH-MB) and PDX-isolated cells (G3-PDX7 and SHH-PDX12), determined by GraphPad Prism software. Values are the average of at least three independent experiments ± SD.

Article Snippet: Adherent MB cells were seeded in XF24 V7-PS plates (10,000 cells/well for DAOY; 12,000 cells/well for ONS-76 and UW228-2; 30,000 cells/well for HD-MB03; Agilent, ref. 102340-100) for 24 h. Cells were then exposed to β-blockers and/or IR for 6 or 24 h. Oxygen consumption rate (OCR) was measured using XF cell Mito Stress test (Agilent, ref. 103015-100).

Techniques: Software

Journal: eBioMedicine

Article Title: Beta-blockers disrupt mitochondrial bioenergetics and increase radiotherapy efficacy independently of beta-adrenergic receptors in medulloblastoma

doi: 10.1016/j.ebiom.2022.104149

Figure Lengend Snippet: Increase of IR efficacy by β-blockers in MB cells . HD-MB03 (a) and ONS-76 (b) cell survival analysis by the Alamar Blue assay after 72 h of treatment with IC 20 of propranolol (propra), carvedilol (carve) or nebivolol (nebi) alone and combined to radiotherapy (IR) 2, 5 and 10 Gy. Values are the average of three independent experiments ± standard error of mean (SEM). Quantification of HD-MB03 (c) and ONS-76 (d) cell colonies, using the ImageJ™ software, respectively after 10 or 7 days of treatment with increase concentrations of propranolol (propra) or radiotherapy (IR) 1·8 Gy alone and their combination. Values are the average of three independent experiments ± SEM. * p < 0·05; ** p < 0·005; *** p < 0·001.

Article Snippet: Adherent MB cells were seeded in XF24 V7-PS plates (10,000 cells/well for DAOY; 12,000 cells/well for ONS-76 and UW228-2; 30,000 cells/well for HD-MB03; Agilent, ref. 102340-100) for 24 h. Cells were then exposed to β-blockers and/or IR for 6 or 24 h. Oxygen consumption rate (OCR) was measured using XF cell Mito Stress test (Agilent, ref. 103015-100).

Techniques: Alamar Blue Assay, Software

Journal: eBioMedicine

Article Title: Beta-blockers disrupt mitochondrial bioenergetics and increase radiotherapy efficacy independently of beta-adrenergic receptors in medulloblastoma

doi: 10.1016/j.ebiom.2022.104149

Figure Lengend Snippet: β-blockers enhance IR activity in 3D spheroids of MB cell lines and MB PDX cells. For five consecutive days, the 3D tumour micromasses were exposed to daily low doses (IC 10 ) of β-blockers or radiotherapy (IR) 1·8 Gy alone and their combination. (a) Representative images of DsRed-expressing HD-MB03 spheroids, acquired over time with the JuLi™ Stage system. Treatment efficacy was expressed as a percentage of spheroid growth inhibition vs control spheroids (Ctl), determined by quantifying the DsRed signal with the PHERAstar microplate reader. Scale bars: 500 µm. HD-MB03 spheroid growth measured by acquisition of the DsRed signal over 21 days after treatment with IC 10 of propranolol (propra) (b) , carvedilol (carve) (c) and nebivolol (nebi) (d) alone or combined with radiotherapy (IR) 1·8 Gy. Values are the average of n = 15 samples per condition, from at least four independent experiments ± standard deviation (SD). (e) Spheroid viability assessment by using the CellTiter-Glo® assay in G3-PDX7 cells after 72 h of treatment with IC 10 of propranolol (propra), carvedilol (carve) and nebivolol (nebi) alone or combined with radiotherapy (IR) 1·8 Gy. Values are the average of n = 10 samples per condition, from three independent experiments ± SD. * p < 0·05; ** p < 0·005; *** p < 0·001.

Article Snippet: Adherent MB cells were seeded in XF24 V7-PS plates (10,000 cells/well for DAOY; 12,000 cells/well for ONS-76 and UW228-2; 30,000 cells/well for HD-MB03; Agilent, ref. 102340-100) for 24 h. Cells were then exposed to β-blockers and/or IR for 6 or 24 h. Oxygen consumption rate (OCR) was measured using XF cell Mito Stress test (Agilent, ref. 103015-100).

Techniques: Activity Assay, Expressing, Inhibition, Standard Deviation, Glo Assay

Journal: eBioMedicine

Article Title: Beta-blockers disrupt mitochondrial bioenergetics and increase radiotherapy efficacy independently of beta-adrenergic receptors in medulloblastoma

doi: 10.1016/j.ebiom.2022.104149

Figure Lengend Snippet: Benefits of daily low concentrations of β-blockers combined to IR in ex-vivo MB organotypic model. For five consecutive days, the organotypic cerebellar co-cultures were exposed to daily low doses (IC 10 ) of propranolol alone or combined to radiotherapy (IR) 1·8 Gy. (a) Representative pictures, acquired with the JuLi™ Stage live imaging system, of DsRed-expressing HD-MB03 tumour micromasses grafted in slices of healthy cerebellum. Scale bars: 1 mm. Results were expressed as percentage of growth inhibition in treated vs control organotypic models (Ctl). (b) HD-MB03 tumour growth was measured over 14 days by acquisition of the DsRed signal with the PHERAstar microplate reader (well-scanning mode). Values are the average of n = 15 samples per condition, from five independent experiments ± standard deviation (SD). (c) Representative pictures of HES, KI67 and γH2AX immunostaining in organotypic models, control (Ctl) and treated with IC 10 of propranolol (propra), radiotherapy (IR) 1·8 Gy or their combination. Scale bars: 100 µm. * p < 0·05; ** p < 0·005; *** p < 0·001.

Article Snippet: Adherent MB cells were seeded in XF24 V7-PS plates (10,000 cells/well for DAOY; 12,000 cells/well for ONS-76 and UW228-2; 30,000 cells/well for HD-MB03; Agilent, ref. 102340-100) for 24 h. Cells were then exposed to β-blockers and/or IR for 6 or 24 h. Oxygen consumption rate (OCR) was measured using XF cell Mito Stress test (Agilent, ref. 103015-100).

Techniques: Ex Vivo, Imaging, Expressing, Inhibition, Standard Deviation, Immunostaining

Journal: eBioMedicine

Article Title: Beta-blockers disrupt mitochondrial bioenergetics and increase radiotherapy efficacy independently of beta-adrenergic receptors in medulloblastoma

doi: 10.1016/j.ebiom.2022.104149

Figure Lengend Snippet: β-Adrenergic receptors (β-AR) are not involved in the response of MB cells to β-blockers. Boxplot showing expression of ADRB1 (a) , ADRB2 (b) and ADRB3 (c) by subgroup in MB patient cohort (n = 240). Kaplan-Meier curves reporting patient overall survival stratifying patients by > median and < media expression for ADRB1 (d) , ADRB2 (e) and ADRB3 (f) expression levels. (g) Relative gene expression of ADRB1, ADRB2 and ADRB3 quantified by qRT-PCR using GAPDH as housekeeping gene (n = 8 per condition, from four independent experiments). Calculation has been done by the 2^-∆Ct method. Values are the average of independent experiments ± standard error of mean (SEM). (h) Cell viability analysis by using the Alamar Blue assay after 72h of treatment with radiotherapy (IR) 1·8, 5 and 10 Gy and increase concentrations of propranolol in HD-MB03 cells transfected with siRNA control (Ctl) or siRNA ADRB1&2 . Values are the average of three independent experiments ± SEM, with a biological triplicate in each experiment. * p < 0·05; ** p < 0·005; *** p < 0·001.

Article Snippet: Adherent MB cells were seeded in XF24 V7-PS plates (10,000 cells/well for DAOY; 12,000 cells/well for ONS-76 and UW228-2; 30,000 cells/well for HD-MB03; Agilent, ref. 102340-100) for 24 h. Cells were then exposed to β-blockers and/or IR for 6 or 24 h. Oxygen consumption rate (OCR) was measured using XF cell Mito Stress test (Agilent, ref. 103015-100).

Techniques: Expressing, Quantitative RT-PCR, Alamar Blue Assay, Transfection

Journal: eBioMedicine

Article Title: Beta-blockers disrupt mitochondrial bioenergetics and increase radiotherapy efficacy independently of beta-adrenergic receptors in medulloblastoma

doi: 10.1016/j.ebiom.2022.104149

Figure Lengend Snippet: MB cells resistant to β-blockers are not sensitive to the treatment-mediated alteration of energy metabolism. Mitochondrial oxygen consumption rate (OCR) (a) and ATP production (b) in β-blocker sensitive cells (ONS-76 WT) and in three β-blocker resistant cells (ONS-76 RP, ONS-76 RC, ONS-76 RN), measured using the Seahorse XFe24® analyser. Glycolytic extracellular acidification rate (ECAR) (c) and glycolytic reserve (d) in β-blocker sensitive cells (ONS-76 WT) and in β-blocker resistant cells (ONS-76 RP, ONS-76 RC, ONS-76 RN), measured using the Seahorse XFe24® analyser. ATP production (e) and glycolytic reserve (f) measured with the Seahorse XFe24® analyser in sensitive and carvedilol resistant cells (ONS-76 RC) exposed to IC 20 of propranolol (propra), carvedilol (carve) or nebivolol (nebi) for 24 h. ATP production (g) and glycolytic reserve (h) measured by using the Seahorse XFe24® analyser in HD-MB03 cells exposed for six hours to radiotherapy (IR) 1·8 Gy or IC 10 -IC 20 of propranolol (propra) alone and their combination. All the values are the average of four independent experiments ± standard error of mean (SEM), with a biological triplicate in each experiment. Data were normalised to cell number. * p < 0·05; ** p < 0·005; *** p < 0·001.

Article Snippet: Adherent MB cells were seeded in XF24 V7-PS plates (10,000 cells/well for DAOY; 12,000 cells/well for ONS-76 and UW228-2; 30,000 cells/well for HD-MB03; Agilent, ref. 102340-100) for 24 h. Cells were then exposed to β-blockers and/or IR for 6 or 24 h. Oxygen consumption rate (OCR) was measured using XF cell Mito Stress test (Agilent, ref. 103015-100).

Techniques:

Journal: eBioMedicine

Article Title: Beta-blockers disrupt mitochondrial bioenergetics and increase radiotherapy efficacy independently of beta-adrenergic receptors in medulloblastoma

doi: 10.1016/j.ebiom.2022.104149

Figure Lengend Snippet: Low concentrations of β-blockers enhanced IR-mediated MB cell oxidative stress and DNA damage. (a) Superoxide ions production measured with WST-1 in HD-MB03 cells, six hours after treatment with radiotherapy (IR) 1·8 Gy or IC 5 -IC 10 of propranolol (propra) alone and their combination, and with anti-oxydants (mito-TEMPO (MT) 2·5 µM or Troxerutin (Trox) 1 mM). Data were normalised to cell number. Values are the average of five independent experiments ± standard error of mean (SEM), with a biological quadruplicate in each experiment. (b) Superoxide ions production measured with WST-1 in β-blocker sensitive (ONS-76 WT) and carvedilol resistant ONS-76 cell lines (ONS-76 RC), six hours after treatment with radiotherapy (IR) 1·8 Gy or IC 20 of propranolol (propra) or carvedilol (carve) alone and their combination. Data were normalised to cell number. Values are the average of four independent experiments ± SEM, with a biological quadruplicate in each experiment. Protein expression level of COX-2 (c) and γ-H2AX (d) in HD-MB03 cells exposed to radiotherapy (IR) 1·8 Gy or IC 5 -IC 10 of propranolol (propra) alone and their combination, and with anti-oxydants (mito-TEMPO (MT) 2·5 µM or Troxerutin (Trox) 1 mM). Western blots were quantified using ImageJ™ software; data were normalised to β-Actin. Values are the average of at least five independent experiments ± SEM. * p < 0·05; ** p < 0·005; *** p < 0·001.

Article Snippet: Adherent MB cells were seeded in XF24 V7-PS plates (10,000 cells/well for DAOY; 12,000 cells/well for ONS-76 and UW228-2; 30,000 cells/well for HD-MB03; Agilent, ref. 102340-100) for 24 h. Cells were then exposed to β-blockers and/or IR for 6 or 24 h. Oxygen consumption rate (OCR) was measured using XF cell Mito Stress test (Agilent, ref. 103015-100).

Techniques: Expressing, Western Blot, Software