Skeletal muscle atrophy and dysfunction in breast cancer patients: role for chemotherapy-derived oxidant stress

Abstract

How breast cancer and its treatments affect skeletal muscle is not well defined. To address this question, we assessed skeletal muscle structure and protein expression in 13 women who were diagnosed with breast cancer and receiving adjuvant chemotherapy following tumor resection and 12 nondiseased controls. Breast cancer patients showed reduced single-muscle fiber cross-sectional area and fractional content of subsarcolemmal and intermyofibrillar mitochondria. Drugs commonly used in breast cancer patients (doxorubicin and paclitaxel) caused reductions in myosin expression, mitochondrial loss, and increased reactive oxygen species (ROS) production in C2C12 murine myotube cell cultures, supporting a role for chemotherapeutics in the atrophic and mitochondrial phenotypes. Additionally, concurrent treatment of myotubes with the mitochondrial-targeted antioxidant MitoQ prevented chemotherapy-induced myosin depletion, mitochondrial loss, and ROS production. In patients, reduced mitochondrial content and size and increased expression and oxidation of peroxiredoxin 3, a mitochondrial peroxidase, were associated with reduced muscle fiber cross-sectional area. Our results suggest that chemotherapeutics may adversely affect skeletal muscle in patients and that these effects may be driven through effects of these drugs on mitochondrial content and/or ROS production.

Keywords: cachexia; mitochondria; myotube.

Fig. 1.

Skeletal muscle fiber cross-sectional area (CSA) in all fibers and myosin heavy chain (MHC) I and II fibers (A), myonuclei content in MHC I and II fibers (B), myofilament fractional area (C), and MHC and actin protein content in tissue homogenates (D) in controls and breast cancer patients. Values are means ± SE, with individual data points shown with each bar. For A–C, multiple observations within each individual were averaged to provide a single data point for each volunteer. Representative immunohistochemical images used to measure CSA and myonuclei content for control and cancer patients are shown above A and B, respectively. Scale bars = 100 µm. Representative electron microscopy images used to measure myofilament fractional area (C) for controls and cancer patients are shown above C and D, respectively. Scale bars = 1 µm. AU, arbitrary units. *P ≤ 0.05; †P = 0.07.

Fig. 2.

Skeletal muscle mitochondrial (Mito) content and structure in intermyofibrillar (IMF) and subsarcolemmal (SS) compartments. Representative images for IMF mitochondrial fractional area for controls and cancer patients are shown above A and B, respectively, with average IMF mitochondrial fractional area (A) and average size (B) for controls and breast cancer patients. Representative images for SS mitochondria for controls and cancer patients are shown above C and D, respectively, with average SS fractional area (C) and average number (n) per unit SS area (D). Scale bars = 1 µm for all images. Values are means ± SE, with individual data points shown with each bar. For A–D, multiple observations within each individual were averaged to provide a single data point for each individual. *P < 0.05, **P < 0.01 vs. control.

Fig. 3.

Relationship of mitochondrial oxidant stress to myofiber size and chemotherapy administration. A–C: relationship of myosin heavy chain (MHC) I and II cross-sectional area (CSA) to peroxiredoxin 3 (Prx 3) expression and relationship of MHC II CSA to oxidized Prx 3 in breast cancer patients (■). In A, r value is Pearson’s coefficient; in B and C, r values are Spearman’s rank coefficients. For A and B, n = 9; for C, n = 11. AU, arbitrary units. D and E: effects of 3 days of doxorubicin (Dox, 0.2 µM) or paclitaxel (Taxol, 40 nM) treatment vs. control [Ctrl (DMSO)] on C2C12 myotube Prx 3 expression (D, n = 4/condition) and oxidation, with the latter being shown by Prx 3 dimers [oxidized (ox)] under nonreducing conditions (E). F: quantitation of oxidized Prx 3 (n = 4/condition). Data in D and F are means ± SE, with individual data points shown with each bar. For A–C, multiple observations for CSA within each individual were averaged to provide a single data point for each individual. For D, immunoblots are shown above average data with loading control (GAPDH). Loading controls are not shown for E, as oxidized Prx 3 data are expressed as a fraction of total (oxidized + nonoxidized) Prx 3. *P < 0.05 vs. Ctrl.

Fig. 4.

α-Tubulin and detyrosinated (deTyr) tubulin expression. α-Tubulin and detyrosinated tubulin were assessed following 3 days of treatment of C2C12 myotubes (A) with vehicle [Ctrl (DMSO), n = 6] or paclitaxel (Tax, 40 nM, n = 6) and in controls (n = 11) and breast cancer patients (n = 9) (B). Gel images are provided for both experiments. AU, arbitrary units. Values are means ± SE, with individual data points shown with each bar. *P < 0.05, **P < 0.01 vs. control.

Fig. 5.

Chemotherapeutic effects on myotube myosin content. A and B: effects of 3 days of doxorubicin (Dox, 0.2 µM) or paclitaxel (Taxol, 40 nM) administration vs. vehicle [DMSO (Ctrl)] on myosin content (A) and reactive oxygen species (ROS) production (B), with representative images of myotubes for each experimental condition in A shown above data. Scale bars = 100 μm. C and D: effects of concomitant treatment with the mitochondrial-targeted antioxidant MitoQ (0.25 µM for Dox and 0.125 µM for Taxol) to prevent myosin loss with Dox or Taxol, with representative images of myotubes for each experimental condition shown above data. Scale bars = 100 µm. Q, MitoQ. Values are means ± SE, with individual data points shown with each bar. *P < 0.05, **P < 0.01 vs. Ctrl.

Fig. 6.

A: colocalization of MitoTracker (green) and MitoSOX (red) in a C2C12 murine myotube. Scale bar = 10 μm. B–E: effect of chemotherapeutics [doxorubicin (Dox, 0.25 µM) and paclitaxel (Taxol, 0.125 µM)] and concomitant treatment with the mitochondrial-targeted antioxidant MitoQ (+MitoQ) on mitochondrial content (B and C) and oxidant production (D and E) compared with vehicle (DMSO) control (Ctrl). Values are means ± SE, with individual data points shown with each bar. *P < 0.05, **P < 0.01 vs. Ctrl; #P < 0.01 vs. Taxol.

Fig. 7.

A and B: p38 and ERK1/2 expression and phosphorylation (pp38 and pERK1/2) in tissue homogenates from controls (open bars, n = 11) and breast cancer patients (gray bars, n = 9). Gel images are shown for phosphorylated (phos) and total protein, as well as GAPDH. Ratio, phosphorylated-to-total protein ratio. AU, arbitrary units. Values are means ± SE, with individual data points shown with each bar.