Chemotherapy-induced muscle wasting: association with NF-κB and cancer cachexia

Abstract

A compounding feature of greater than 50% of all cancers is the high incidence of the cachexia syndrome, a complex metabolic disorder characterized by extreme weight loss due mainly to the gross depletion of skeletal muscle tissue. Although studies into the cause of cancer cachexia has spanned over multiple decades, little is known about the effects of various cancer treatments themselves on cachexia. For example, chemotherapy agents induce side effects such as nausea and anorexia, but these symptoms do not fully account for the changes seen with cancer cachexia. In this study we examine the effects of chemotherapeutic compounds, specifically, cisplatin in the colon-26 adenocarcinoma model of cancer cachexia. We find that although cisplatin is able to reduce tumor burden as expected, muscle wasting in mice nevertheless persists. Strikingly, cisplatin alone was seen to regulate muscle atrophy, which was independent of the commonly implicated ubiquitin proteasome system. Finally, we show that cisplatin is able to induce NF-κB activity in both mouse muscles and myotube cultures, suggesting that an additional side effect of cancer treatment is the regulation of muscle wasting that may be mediated through activation of the NF-κB signaling pathway.

Keywords: Cancer; NF-κB; cachexia; chemotherapy; skeletal muscle; wasting.

Fig 1.

Cisplatin induces weight loss and muscle atrophy in mice. Four sets of treatment group were used with CD2F1 mice. In group 1, mice were injected with saline (control); in group 2, mice were injected with saline and then treated with cisplatin at days 7, 10, and 14 post-injection (cisplatin); in group 3, mice were injected with C-26 tumor cells (C-26); in group 4, mice were injected with C-26 cells then treated with cisplatin at days 7, 10, and 14 post injection (C-26/cisplatin). (A) Following treatments, body weights (n=4, each group) were recorded at indicated days. (B) Gastrocnemius muscle weight (n=6 each group) were recorded in grams on day 17 post injection. (C) C-26 tumors were removed from untreated and cisplatin treated mice and weighed. (D) H&E stained cryosections of gastrocnemius muscles were analyzed (n=4 per group). (E) Fiber diameter was measured from H&E muscle sections from(D).

Fig 2.

Expression of muscle E3 ubiquitin ligase is dependent on C-26 tumors. (A) Muscles were harvested 17 days post-injection in each of the 4 treatment groups (n=2). Total RNA was prepared and Northerns were performed probing for the muscle ubiqutin ligase gene, MuRF1. To confirm equal loading, blots were stripped and reprobed with GAPDH. (B) MuRF1 Northern was performed with RNA prepared from muscles harvested at various times either from C-26 tumor bearing mice alone (days 15 and 22), or from mice where C-26 tumors were surgically removed (post-op, days 1, 4, or 7). Control designates basal level of MuRF1, while Sham is level of MuRF1 following surgery in mice without tumors. GAPDH was also determined to control for loading control. (C) At indicated days and conditions, gastrocnemius muscles were harvested and fiber diameters were calculated from H&E sections.

Fig 3.

Cisplatin is an inducer of NF-κB in muscle cells. (A) CD2F1 mice were treated with cisplatin alone as described above in legend for Figure 1. At day 17 post-injection, muscle sections were prepared and immunostained for activated p65 (phospho-p65). (B) C2C12 myotube cultures were either left alone (control) or treated with cisplatin for indicated times. Nuclear extracts were prepared and EMSA was performed. Intensity of binding complexes were quantitated by phosphoimaging and recorded underneath each lane. Fold designates the amount of binding complex over that of untreated myotubes, which was set to a value of 1.0. Separate Supershift EMSA were preformed with p50 and p65 antibodies. SS refers to supershift complexes.

Fig 4.

Chemotherapy-induced muscle wasting is not specific to cisplatin. CD2F1 mice were treated with either saline (PBS) or different chemotherapeutic compounds, similar to the regimen described in legends for Figure 1. Following treatment, gastrocnemius muscle sections were prepared and stained for H&E (A) and calculated for fiber diameters (B).