Skeletal muscle atrophy is attenuated in tumor-bearing mice under chemotherapy by treatment with fish oil and selenium

Abstract

Chemotherapy can cause cachexia, which is manifested by weight loss, inflammation and muscle atrophy. However, the mechanisms of tumor and chemotherapy on skeletal muscle proteolysis, remained unclear. In this report, we demonstrated that tumor-induced myostatin in turn induced TNF-α, thus activating calcium-dependent and proteasomal protein degradation. Chemotherapy activated myostatin-mediated proteolysis and muscle atrophy by elevating IL-6. In tumor-bearing mice under chemotherapy, supplementation with fish oil and selenium prevented a rise in IL-6, TNF-α and myostatin and muscle atrophy. The findings presented here allow us to better understand the molecular basis of cancer cachexia and potentiate nutrition supplementation in future cancer chemotherapy.

Keywords: cachexia; chemotherapy; fish oil; muscle atrophy; selenium.

Figure 1. Final body weight gain for line-1, docetaxel or combined treatment mice

After sacrifice we scaled mice body weight into two groups according to the body weight loss percentage. The body weight loss >10% or <10% is designated as severe cachexia or moderate cachexia. (A and C) On day 35 and 42 after tumor implantation, we harvest tumor and subtract tumor weight from final body weight to calculate the percentage of body weight loss for line-1 (A), docetaxel (B), or combined chemotherapy (C) treatment mice, respectively. Data show mean ± SD, n = 5–10 mice/group, each value is an average of three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 denote levels of significant differences between groups.

Figure 2. Immunological profiling for mice bearing tumor, mice bearing tumor with chemotherapeutic treatment, and non tumor-bearing mice (Con)

(A) Total CD3⁺ T, CD3⁺CD4⁺ T helper 2, CD3⁺CD8⁺ T helper 1, CD49b⁺ NK, CD19⁺ B, Gr-1⁺CD11b⁺ MDSC and CD4⁺CD25⁺Fox-p3⁺ Tregs in the spleen of line-1 tumor-bearing (T) and tumor with docetaxel-treated (TD) mice. The number of splenocyte was normalized with the normal mice (Con) regarded as 1. (B) NK-associated cytotoxicity in the spleen of mice. (C) and (D), Gr-1⁺CD11b⁺ MDSC, CD4⁺CD25⁺Fox-p3⁺ Tregs and CD49b⁺ NK cells in the spleen of tumor-bearing and tumor with cisplatin-treated (Cis) mice. (E and F) After LLC (1×10⁵) tumor implantation, mice were treated with cisplatin (5 mg/kg, every four days) or 5-FU (40 mg/kg, once a week). In the end of experiments, mice were sacrificed and examined for tumor weight (E) and body weight gain (F). Data are shown as mean ± SD. n = 5–9 mice/group and each value is an average of three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 denote levels of significant differences between groups.

Figure 3. mRNA and protein levels for genes encoding cachexic factors towards proteolytic signaling molecules after line-1 tumor inoculation (protocol #1)

(A) Western blot analysis for expressions of IL-6, TNF-α, myostatin and β-actin in gastrocnemius muscles from line-1 tumor-bearing mice. The graph represents relative densitometric intensity of each band normalized to β-actin. (B) mRNA levels (left) and protein levels (right) for genes of cachexic factors and proteolysis relative signaling molecules in gastrocnemius muscle. Values are means of fluorescence signals expressed as a percentage of healthy control mice, and normalization to the GAPDH mRNA amount. (C) Immunohistochemistry of gastrocnemius muscle from tumor-bearing mice, where protein expressions are shown for MAFbx (top), MuRF-1(middle) and NF-κB (bottom). Data are shown as mean ± SD. n = 5–8 mice/group and each value is an average of three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 denote levels of significant differences between groups.

Figure 4. mRNA and protein levels for genes encoding cachexic factors and proteolytic relative signaling molecules in docetaxel-induced muscle atrophy in vitro and in vivo (protocol #2)

(A) Western blot analysis for expressions of IL-6, TNF-α, myostatin and β-actin in gastrocnemius muscle. The graph represents relative densitometric intensity of each band normalized to β-actin. (B) mRNA levels (left) and protein levels (right) for genes encoding cachexic factors and proteolysis relative signaling molecules in gastrocnemius muscle from docetaxel injected mice. (C) Cells at 0.5 × 10⁴ were plated per well, and incubated for 24 hr. The cells were treated with various concentrations of docetaxel for 48 hr and the cell viability was measured by the MTS assay (left). P19 cells were treated with a range of concentrations of docetaxel for 0, 24, 48, 72 or 96 hr, and the cell viability was measured by the MTS assay (right). Data are relative to control. (D) The time course effect of docetaxel (80 μg/ml) on mRNA and protein expression related to protein degradation in P19 muscle cell line over 24 hr. Values are means of fluorescence signals expressed as a percentage of healthy control mice or normal P19 cells, which are normalized to the GAPDH mRNA amount. Data are shown as mean ± SD. n = 5–10 mice/group and each value is an average of three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 denote levels of significant differences between groups.

Figure 5. Effects of daily oral administration of background diet or combined nutritional components (addition of fish oil (fo) or selenium yeast (se)) on skeletal muscle atrophy in tumor-bearing mice with chemotherapy (protocol #3)

(A) differences in soleus and gastrocnemius weights at day 42 after intervention with nutritional supplements in tumor-bearing mice after chemotherapy. (B) Effects of serum from mice on P19 cell growth. Serum (10% of assay volume) was added to the P19 cell line, and the cell viability during 24 hours incubation was determined by the MTS assay. (C) Concentrations of plasma phospholipids [16] fatty acids in mice. (D) Western blots analysis for expressions of IL-6, TNF-α and myostatin in gastrocnemius muscles of mice (left). Protein levels for ubiquitin ligases, FoxO-1, cathepsin L and calpain in gastrocnemius muscle during cancer and chemotherapy (right). (E) The model illustration for fish oil plus selenium attenuated muscle atrophy after chemotherapy. Under chemotherapy, tumor-bearing mice exhibited a significant increase in the expression of myostatin that activates FoxO-1, and leads to up-regulation of proteasome ubiquitin ligases MuRF-1 and MAFbX. Con, normal control mice; TD, tumor-bearing mice receiving docetaxel; TD-base, tumor-bearing mice receiving docetaxel and background diet; TD-fo+se, tumor bearing mice receiving background diet with additional fish oil and selenium yeast. Data are shown as mean ± SD. n = 5–9 mice/group and each value is an average of three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 denote levels of significant differences between groups.