Metronomic 5-Fluorouracil Delivery Primes Skeletal Muscle for Myopathy but Does Not Cause Cachexia

Abstract

Skeletal myopathy encompasses both atrophy and dysfunction and is a prominent event in cancer and chemotherapy-induced cachexia. Here, we investigate the effects of a chemotherapeutic agent, 5-fluorouracil (5FU), on skeletal muscle mass and function, and whether small-molecule therapeutic candidate, BGP-15, could be protective against the chemotoxic challenge exerted by 5FU. Additionally, we explore the molecular signature of 5FU treatment. Male Balb/c mice received metronomic tri-weekly intraperitoneal delivery of 5FU (23 mg/kg), with and without BGP-15 (15 mg/kg), 6 times in total over a 15 day treatment period. We demonstrated that neither 5FU, nor 5FU combined with BGP-15, affected body composition indices, skeletal muscle mass or function. Adjuvant BGP-15 treatment did, however, prevent the 5FU-induced phosphorylation of p38 MAPK and p65 NF-B subunit, signalling pathways involved in cell stress and inflammatory signalling, respectively. This as associated with mitoprotection. 5FU reduced the expression of the key cytoskeletal proteins, desmin and dystrophin, which was not prevented by BGP-15. Combined, these data show that metronomic delivery of 5FU does not elicit physiological consequences to skeletal muscle mass and function but is implicit in priming skeletal muscle with a molecular signature for myopathy. BGP-15 has modest protective efficacy against the molecular changes induced by 5FU.

Keywords: 5-fluorouracil; NF-B; cachexia; chemotherapy; desmin; dystrophin; p38; skeletal muscle.

Figure 1

The effect of 5-fluorouracil (5FU) and 5FU with BGP-15 (5FU+BGP) treatment on body composition and muscle size indices. Body composition parameters were measured and presented as pre- and post-treatment data points for (A) body, (B) lean and (C) fat mass. (D) Hindlimb skeletal muscles extensor digitorum longus (EDL), soleus (SOL), and tibialis anterior (TA) were weighed post-treatment and data presented as raw mass and muscle to body mass ratios (# p = 0.07; compared to 5FU). TA cross-sections were H&E-stained and underwent histological fibre size analysis with data presented as (E) percentage relative frequency distribution of the muscle fibre cross-sectional area (CSA) and (F) mean muscle fibre CSA. (G) Representative images of H&E-stained TA cross-sections are displayed. Scale bar = 100 m. n = 7–8 for body composition indices; n = 4–8 for muscle weights; n = 5–7 for histology. Data are mean ± SEM.

Figure 2

The effect of 5-fluorouracil (5FU) and 5FU with BGP-15 (5FU+BGP) treatment on skeletal muscle contractile function. Extensor digitorum longus (EDL) and soleus (SOL) muscles underwent ex vivo assessment of contractile functional properties, with (A) force–frequency relationships and (B) force production characteristics analyzed, including; Peak twitch force (P_t), Absolute tetanic force production (P_o), twitch to tetanus ratio (P_t/P_o), physiological cross-sectional area (pCSA) and Specific force production (sP_o). n = 4–8 for ex vivo contractile function. Data are mean ± SEM.

Figure 3

The effect of 5-fluorouracil (5FU) and 5FU with BGP-15 (5FU+BGP) treatment on expression of cytoskeletal structural proteins. Western blotting experiments were undertaken in tibialis anterior (TA) muscle homogenate, with samples probed for (A,B) cytoskeletal structural proteins including; laminin (LAM), -dystroglycan (-DGC), -sarcoglycan (-SGC), dystrophin (DYS), dystrobrevin (DBVN), syntrophin (SYTN), desmin (DES) and talin (TAL). (C) Phosphorylated (Ser473) and total Akt were probed for as an indicator of mammalian target of rapamycin Complex 2 (mTORC2) activity. (D) Ankrd2 was probed for as a marker of mechano-sensitvit. (E) Representative images for phosphorylated Akt^Ser473, total Akt and Ankrd2 displayed. Protein expression was normalised to total protein derived from Coomassie Brilliant Blue (CBB) staining and presented relative to vehicle (VEH) control group. * = p < 0.05; n = 4 for VEH and n = 6–8 for 5FU and 5FU+BGP groups for Western blotting. Data are mean ± SEM.

Figure 4

The effect of 5-fluorouracil (5FU) and 5FU with BGP-15 (5FU+BGP) treatment on molecular markers of cellular stress. Western blotting experiments were undertaken on tibialis anterior muscle homogenate. Samples were probed for (A) phosphorylated (Thr180/Tyr182) and total p38, (B) phosphorylated (Thr202/Tyr204) and total ERK1/2, (C) phosphorylated (Thr183/Tyr185) and total JNK, (D) phosphorylated (Ser536) and total NF-B subunit protein p65. Data are presented as phosphorylated to total protein ratios and normalised to total protein derived from Coomassie Brilliant Blue (CBB) staining and expressed as a relative percentage of the vehicle (VEH) control grou (E) Western blotting and CBB representative images are displayed. * = p < 0.05; n = 4 for VEH and n = 6–8 for 5FU and 5FU+BGP groups. Data are mean ± SEM. ** = p < 0.01.

Figure 5

The effect of 5-fluorouracil (5FU) and 5FU with BGP-15 (5FU+BGP) treatment on skeletal muscle oxidative capacity and mitochondrial dynamics signalling. Succinate dehydrogenase (SDH) staining was performed on tibialis anterior (TA) cross-sections. (A) SDH representative images displayed, with data presented as (B) overall SDH intensity and (C) SDH intensity separated based on oxidative fibre phenotype, i.e., low oxidative, more oxidative and highly oxidative. TA muscle homogenate was analyzed for (D) citrate synthase (CS) activity, as a marker of mitochondrial density. Further, TA homogenate was utilized in Western blotting experiments with samples probed for (E) HSP-70, (F) PARP-1 and (G) a suite of proteins related to mitochondrial dynamics including; TFAM, PGC-1, PGC-1, OPA1 and DRP1. Protein expression was normalised to total protein derived from Coomassie Brilliant Blue (CBB) staining and presented relative to vehicle (VEH) control group. (H) Western blotting and CBB representative images are displayed. * = p < 0.05; n = 6–8 for SDH histology; n = 4 for VEH and n = 6–8 for 5FU and 5FU+BGP groups for CS activity and Western blotting. Data are mean ± SEM.

Figure 6

The effect of BGP-15 (BGP), 5-fluorouracil (5FU) and 5FU with BGP (5FU+BGP) treatment on HSP-70 expression and cell viabilityC2C12 myotubes were treated with BGP, 5FU and 5FU with BGP before being prepared as lysates for Western blotting experiments. (A) C2C12 lysates were then probed for HSP-70, with protein expression normalised to total protein derived from Coomassie Brilliant Blue (CBB) staining data and data presented as a percentage of the vehicle control group (VEH). (B) Cell viability was analyzed via the resazurin cell viability assay, with data presented as a percentage of VEH. (C) Representative images for the cell viability assay are displayed. Scale bar = 100 m. * = p < 0.05; n = 3–4 for all C2C12 experiments. Data are mean ± SEM.