Supplementation with Fish Oil and Selenium Protects Lipolytic and Thermogenic Depletion of Adipose in Cachectic Mice Treated with an EGFR Inhibitor

Abstract

Lung cancer and cachexia are the leading causes of cancer-related deaths worldwide. Cachexia is manifested by weight loss and white adipose tissue (WAT) atrophy. Limited nutritional supplements are conducive to lung cancer patients, whereas the underlying mechanisms are poorly understood. In this study, we used a murine cancer cachexia model to investigate the effects of a nutritional formula (NuF) rich in fish oil and selenium yeast as an adjuvant to enhance the drug efficacy of an EGFR inhibitor (Tarceva). In contrast to the healthy control, tumor-bearing mice exhibited severe cachexia symptoms, including tissue wasting, hypoalbuminemia, and a lower food efficiency ratio. Experimentally, Tarceva reduced pEGFR and HIF-1α expression. NuF decreased the expression of pEGFR and HIF-2α, suggesting that Tarceva and NuF act differently in prohibiting tumor growth and subsequent metastasis. NuF blocked LLC tumor-induced PTHrP and expression of thermogenic factor UCP1 and lipolytic enzymes (ATGL and HSL) in WAT. NuF attenuated tumor progression, inhibited PTHrP-induced adipose tissue browning, and maintained adipose tissue integrity by modulating heat shock protein (HSP) 72. Added together, Tarceva in synergy with NuF favorably improves cancer cachexia as well as drug efficacy.

Keywords: cachexia; fish oil; selenium yeast; target therapy; white adipose tissue browning.

Figure 1

Anticancer and antimetastatic effect of the combination of Tarveva and NuF in tumor-bearing mice. (A). The study involved a treatment regimen combining Tarceva with NuF in mice with tumors. On day 7, Lewis lung carcinoma (LLC) cells (3 × 10⁵) were injected subcutaneously into the right dorsal side of C57BL/6 mice. Tumor volume was measured using the following formula: 1/2 (x^2y), where x represents tumor width and y represents tumor length. Tumor-bearing mice were randomly assigned to four groups as follows: the control group with no treatment (T), the TT group (Tarceva at 2 mg/kg/day), the TN group (NuF at 1 g/mouse/day), and the TTN group (Tarceva at 2 mg/kg/day combined with NuF at 1 g/mouse/day). After 28 days, mice were sacrificed, and tumors, gastrocnemius muscles, white adipose tissue, brown adipose tissue (BAT), and lungs were collected for further analysis. (B). MTS assay for determining the inhibition of LLC cell growth by Tarveva. (C). Tumor weight. Results are based on three independent replicates. (D). Tumor weight distribution. (E). The average number of lung metastatic nodules. Representative photos of the lungs; arrows point to the metastatic nodules. (F). The image on the left displays the expression level of EGFR and its phosphorylated form in tumors from each group. Meanwhile, the image on the right illustrates the quantified ratio of phosphorylated EGFR to total EGFR (pEGFR/total EGFR) following treatment with Tarceva and NuF. ^# p < 0.05, ^## p < 0.01, ^### p < 0.001, and ^#### p < 0.0001 compared to the T group. Data are expressed as means ± SD. N = 5–6 samples per group. Each group consisted of 5 to 6 mice.

Figure 2

Anticachexic effect of the combination of Tarveva and NuF in tumor-bearing mice. (A) Albumin level. (B) Gastrocnemius muscle (Gastroc), epididymal fat (WAT) and interscapular brown adipose tissue (BAT) weight. (C) Image of BAT. (D) H&E staining image of WAT. Scale bar = 200 µm. ^# p < 0.05 as compared to the T group. Different letters in the groups represent significant differences. Data are expressed as means ± SD. N = 5–6 samples per group.

Figure 3

Co-administration of Tarveva and NuF inhibited adipocyte dysfunction factor in tumors. (A) Representative Western blots of IL-6, PTHrP, and β-actin in tumors from LLC tumor-bearing mice. (B) Relative mRNA expression levels of Il6 and PTHrP were measured via RT-qPCR. Values are means of fluorescence signals expressed as a percentage of no-treatment tumor mice (T), and normalization to the Gapdh mRNA. (C) Western blot analysis for the expression of HIF-1α and β-actin in tumors. The graph represents the relative densitometric intensity of each band normalized to β-actin. (D) Western blot analysis for the expression of HIF-2α and β-actin in tumors. ^# p < 0.05 and ^## p < 0.01 as compared to the T group. * p < 0.05 and ** p < 0.01 as compared between two groups. Data are expressed as means ± SD. N = 5–6 samples per group.

Figure 4

NuF suppresses the expression of thermogenic and lipolytic factors in white adipose tissue (WAT). (A). Representative Western blots of pHSL, total HSL, UCP-1, and β-actin in WAT from LLC tumor-bearing mice. (B). Relative mRNA expression levels of Il6, Ucp1, Argl, and HSL were measured via RT-qPCR. Values are means of fluorescence signals expressed as a percentage of health control mice (NT group), and normalization to the Gapdh mRNA. (C). A representative Western blot of HSP25, HSP72, and β-actin expression in WAT. The graph represents the relative densitometric analysis of each band normalized to β-actin. (D). Diagram showing the hypothesized underlying mechanism for NuF inhibition of tumor progression and adipose tissue atrophy. UCP1, uncoupling protein 1. ATGL, adipocyte triglyceride lipase. HSL, hormone-sensitive lipase. HSP, Heat shock protein. ^# p < 0.05, ^## p < 0.01, and ^### p < 0.001 as compared to the T group. *** p < 0.001 as compared between the two groups. Data are expressed as means ± SD. N = 5–6 samples per group.