A single chemotherapy administration induces muscle atrophy, mitochondrial alterations and apoptosis in breast cancer patients

Figures

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  Figure 1. Standard (neo)adjuvant chemotherapy prescribed in breast cancer patients includes sequential epirubicin‐cyclophosphamide administration (EC) followed by paclitaxel (TAX). This treatment can be dispensed as 3 (100 mg/m2) or 4 (75 mg/m2) EC administrations followed by 9 or 12 TAX (90 mg/m2) administrations. Skeletal muscle biopsies were performed before the first administration of EC (N = 10) or TAX (N = 10) and 4 days later. CT, chemotherapy. W, week.

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  Figure 2. Changes in myofibre cross‐sectional area. Cross‐sectional area of the vastus lateralis muscle fibres from muscle biopsies taken before (pre) and 4 days after (post) the first epirubicin‐cyclophosphamide (EC) or paclitaxel (TAX) administration and representative transversal muscle sections with type I and type IIa muscle fibres staining. Due to limited biopsy materials or poor quality of samples, eight patients were assessed in EC group and nine patients were assessed in TAX group. *P < 0.05; ***P < 0.001.

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  Figure 3. Changes in mitochondrial respiration and reactive oxygen species production. Mitochondrial respiratory capacity quantification (A) and associated mean representative curves for the CI‐linked substrate state, OXPHOS by CI, OXPHOS by CI&CII, OXPHOS by CII and uncoupled CIV on permeabilized fibres from vastus lateralis muscle biopsies taken before (pre) and 4 days after (post) the first epirubicin‐cyclophosphamide (EC) or paclitaxel (TAX) administration. H2O2 production quantification (B) and associated mean representative curves for the CI‐linked substrate state, OXPHOS by CI, OXPHOS by CI&CII, and OXPHOS by CII in permeabilized fibres. Superoxide anion measurement (C) through electron paramagnetic resonance in minced muscle samples. For every index, bars correspond to mean group data, and dots correspond to individual data. Twenty patients were assessed for the EC (N = 10) and TAX (N = 10) groups, except for superoxide anion production, where 1 patient in the TAX group was excluded because of technical issues during the experimentation. *P < 0.05; **P < 0.01. CI, complex I; CII, complex II; CIV, complex IV; OXPHOS, oxidative phosphorylation.

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  Figure 4. Changes in key markers of antioxidant defences and oxidative stress. SOD2 (A), catalase (B) GPx1 (C) and 4‐HNE (D) protein levels from vastus lateralis muscle biopsies taken before (pre) and 4 days after (post) the first epirubicin–cyclophosphamide (EC) or paclitaxel (TAX) administration. Two representative patients in the EC and TAX groups are displayed above each panel with western blot analyses. For every index, bars correspond to mean group data, and dots correspond to individual data. Twenty patients were assessed for the EC (N = 10) and TAX (N = 10) groups, except for 4‐HNE protein levels, where 1 patient was missing for the EC group due to limited biopsy materials. *P < 0.05.

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  Figure 5. Changes in key markers of mitochondrial content and biogenesis. Citrate synthase (A), VDAC (B) and PGC‐1α1 (C) protein levels from vastus lateralis muscle biopsies taken before (pre) and 4 days after (post) the first epirubicin–cyclophosphamide (EC) or paclitaxel (TAX) administration. Two representative patients in the EC and TAX groups are displayed above each panel for western blot analyses. Mitochondrial respiratory capacity per mitochondria (D) for CI‐linked substrate state, OXPHOS by CI, OXPHOS by CI&CII, OXPHOS by CII and uncoupled CIV on permeabilized fibres. The mean of citrate synthase and VDAC protein levels was used for the normalization. For every index, bars correspond to mean group data, and dots correspond to individual data. Twenty patients were assessed for the EC (N = 10) and TAX (N = 10) groups. **P < 0.01; ***P < 0.001. CI, complex I; CII, complex II; CIV, complex IV; OXPHOS, oxidative phosphorylation.

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  Figure 6. Changes in key markers of mitochondrial fusion and fission. MFN2 (A), OPA1 (B), Fis1 (C) and p‐DRP1 (ser 616)/DRP1 (D) protein levels from vastus lateralis muscle biopsies taken before (pre) and 4 days after (post) the first epirubicin–cyclophosphamide (EC) or paclitaxel (TAX) administration. Two representative patients in the EC and TAX groups are displayed above each panel with western blot analyses. For every index, bars correspond to mean group data, and dots correspond to individual data. Twenty patients were assessed for the EC (N = 10) and TAX (N = 10) groups, except for Fis1 protein levels, where one patient was missing for the TAX group due to limited biopsy materials. ***P < 0.001.

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  Figure 7. Changes in key markers of mitophagy. PINK1 (A), Parkin (B) and Mul1 (C) protein levels from vastus lateralis muscle biopsies taken before (pre) and 4 days after (post) the first epirubicin–cyclophosphamide (EC) or paclitaxel (TAX) administration. All values are expressed as the mean ± SD. Twenty patients were assessed for the EC (N = 10) and TAX (N = 10) groups, except for PINK1 and Parkin protein levels, where 1 patient was missing for the TAX group due to limited biopsy materials. **P < 0.01; ***P < 0.001.

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  Figure 8. Changes in the apoptosis process. Bax (A) and Bcl‐2 (B) protein levels from vastus lateralis muscle biopsies taken before (pre) and 4 days after (post) the first epirubicin‐cyclophosphamide (EC) or paclitaxel (TAX) administration. Changes in TUNEL‐positive signal per muscle section (C) with representative transversal muscle sections (D) with DAPI or TUNEL assay for EC and TAX groups. Two representative patients in the EC and TAX groups are displayed above each panel for western blot analyses. For every index, bars correspond to mean group data, and dots correspond to individual data. Twenty patients were assessed for the EC (N = 10) and TAX (N = 10) groups, except for Bax protein levels (TAX, N = 9) and for histological analyses (EC, N = 9; TAX, N = 8) due to limited biopsy materials or poor quality of samples. **P < 0.01; ***P < 0.001.