Withaferin A and Ovarian Cancer Antagonistically Regulate Skeletal Muscle Mass

Abstract

Cachexia is a complex wasting syndrome that overwhelmingly affects the majority of late-stage cancer patients. Additionally, there are currently no efficacious therapeutic agents to treat the muscle atrophy induced by the cancer. While several preclinical studies have investigated the molecular signals orchestrating cachexia, very little information exists pertaining to ovarian cancer and the associated cachexia. Work from our lab has recently demonstrated that the steroidal lactone Withaferin A (WFA) is capable of attenuating the atrophying effects of ovarian cancer in a preclinical mouse model. However, it remained to be determined whether WFA's effect was in response to its anti-tumorigenic properties, or if it was capable of targeting skeletal muscle directly. The purpose of this study was to uncover whether WFA was capable of regulating muscle mass under tumor-free and tumor-bearing conditions. Treatment with WFA led to an improvement in functional muscle strength and mass under tumor-bearing and naïve conditions. WFA and ovarian cancer were observed to act antagonistically upon critical skeletal muscle regulatory systems, notably myogenic progenitors and proteolytic degradation pathways. Our results demonstrated for the first time that, while WFA has anti-tumorigenic properties, it also exerts hypertrophying effects on skeletal muscle mass, suggesting that it could be an anti-cachectic agent in the settings of ovarian cancer.

Keywords: atrophy; cachexia; catabolism; ovary; satellite cells.

FIGURE 1

Withaferin A increases the grip strength and myofiber size of female NSG mice. Quantification of mean (A) forelimb and (B) total limb grip strength normalized to body weight in tumor-free and tumor-bearing vehicle-treated, WFA 2 mg/kg, and WFA 4 mg/kg groups at week four post-xenografting of A2780 cells. N = 10 in each group. (C) Quantification of tibialis anterior (TA), gastrocnemius (GA), and quadriceps femoris (QF) muscle wet weight normalized by initial body weight. N = 7 in each group. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, value significantly different from corresponding value of tumor-free vehicle-treated group by two-way ANOVA followed by Tukey’s multiple comparison test post hoc analysis. ^@p < 0.05, value significantly different from corresponding value of tumor-bearing vehicle-treated group.

FIGURE 2

Withaferin A rescues skeletal muscle at the structural level. (A) Representative images of H&E-stained transverse TA muscle sections. Scale bar = 50 μm. Inset images magnified from whole image. Quantification of average (B) myofiber cross-sectional area (CSA) and (C) minimal Feret’s diameter in TA muscle. N = 10 in all groups. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, value significantly different from corresponding value of tumor-free vehicle-treated group by two-way ANOVA followed by Tukey’s multiple comparison test post hoc analysis. ^@p < 0.05, value significantly different from corresponding value of tumor-bearing vehicle-treated group.

FIGURE 3

Withaferin A is a potent activator of satellite cells. (A) Representative images of transverse TA muscle sections after immunostaining for Pax7 (red color), MyoD (yellow color), and Laminin (green color) proteins in tumor-free and tumor-bearing groups. Nuclei were identified by counterstaining with DAPI (blue color). Scale bar = 25 μm. Quantification of (B) the total number of Pax7⁺ cells per Laminin⁺ myofiber, (C) the proportion of Pax7⁺/MyoD^– cells, and (D) the proportion of Pax7⁺/MyoD⁺ cells. N = 10 in all groups. Solid white arrow denotes Pax7⁺/MyoD^– cells. Solid yellow arrow denotes Pax7⁺/MyoD⁺ cells. Dashed yellow arrow denotes Pax7^–/MyoD⁺ cells. (E) Relative mRNA levels of Pax7 and Myod1 in GA muscle. N = 10 in all groups. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, value significantly different from corresponding value of tumor-free vehicle-treated group by two-way ANOVA followed by Tukey’s multiple comparison test post hoc analysis. ^@p < 0.05, value significantly different from corresponding value of tumor-bearing vehicle-treated group.

FIGURE 4

Withaferin A and ovarian cancer differentially transcriptionally regulate the UPR. Relative mRNA levels of select markers of the unfolded protein response (UPR) in the GA muscle of tumor-free and tumor-bearing groups. N = 10 in all groups. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, value significantly different from corresponding value of tumor-free vehicle-treated group by two-way ANOVA followed by Tukey’s multiple comparison test post hoc analysis. ^@p < 0.05, value significantly different from corresponding value of tumor-bearing vehicle-treated group.

FIGURE 5

Withaferin A and ovarian cancer’s effect on the UPR at the protein level. (A) Representative immunoblots for components of the PERK and IRE1α arms of the UPR in QF muscle samples in the tumor-free and tumor-bearing vehicle-treated and WFA 4 mg/kg groups. N = 3 in all groups. (B) Densitometric quantification of western blot results. (C) Representative images of spliced (sXBP-1), unspliced (uXBP-1) and total XBP-1 levels in GA muscle samples from tumor-free and tumor-bearing animals. Unrelated gene (β-Actin) was used as a load control for normalization. N = 3 per group. (D) Densitometric quantification of transcript levels of XBP-1 and β-Actin. N = 3 per group. “n.s.” = no significant differences. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, value significantly different from corresponding value of tumor-free vehicle-treated group by two-way ANOVA followed by Tukey’s multiple comparison test post hoc analysis. ^@p < 0.05, value significantly different from corresponding value of tumor-bearing vehicle-treated group.

FIGURE 6

Withaferin A reduces activation of the UPS. (A) Representative immunoblots for poly ubiquitinated proteins and unrelated protein GAPDH in tumor-free and tumor-bearing vehicle-treated or WFA 4 mg/kg QF muscle samples. N = 3 per group. (B) Densitometric quantification of poly ubiquitinated proteins in QF muscle samples. Relative mRNA levels of (C) Traf6 and (D) select muscle-specific E3 ubiquitin ligases in GA muscle samples. N = 10 in all groups. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, value significantly different from corresponding value of tumor-free vehicle-treated group by two-way ANOVA followed by Tukey’s multiple comparison test post hoc analysis. ^@p < 0.05, value significantly different from corresponding value of tumor-bearing vehicle-treated group.

FIGURE 7

Withaferin A downregulates activation of the ALS. (A) Relative mRNA levels of select markers of autophagy in GA muscle samples from tumor-free and tumor-bearing mice. N = 10 in all groups. (B) Western blot analysis of common markers of autophagy in QF muscle samples. N = 3 per group. (C) Densitometric quantification of western blot images. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001, value significantly different from corresponding value of tumor-free vehicle-treated group by two-way ANOVA followed by Tukey’s multiple comparison test post hoc analysis. ^@p < 0.05, value significantly different from corresponding value of tumor-bearing vehicle-treated group.

FIGURE 8

The induction of cachexia by ovarian cancer. Proposed mechanistic model showing ovarian cancer’s effect on satellite cell homeostasis and the UPR to facilitate skeletal muscle atrophy, and the proposed mechanism(s) through which WFA interferes with the induction of cachexia.