Peptide-2 from mouse myostatin precursor protein alleviates muscle wasting in cancer-associated cachexia

Abstract

Cancer cachexia, characterized by continuous muscle wasting, is a key determinant of cancer-related death; however, there are few medical treatments to combat it. Myostatin (MSTN)/growth differentiation factor 8 (GDF-8), which is a member of the transforming growth factor-β family, is secreted in an inactivated form noncovalently bound to the prodomain, negatively regulating the skeletal muscle mass. Therefore, inhibition of MSTN signaling is expected to serve as a therapeutic target for intractable muscle wasting diseases. Here, we evaluated the inhibitory effect of peptide-2, an inhibitory core of mouse MSTN prodomain, on MSTN signaling. Peptide-2 selectively suppressed the MSTN signal, although it had no effect on the activin signal. In contrast, peptide-2 slightly inhibited the GDF-11 signaling pathway, which is strongly related to the MSTN signaling pathway. Furthermore, we found that the i.m. injection of peptide-2 to tumor-implanted C57BL/6 mice alleviated muscle wasting in cancer cachexia. Although peptide-2 was unable to improve the loss of heart weight and fat mass when cancer cachexia model mice were injected with it, peptide-2 increased the gastrocnemius muscle weight and muscle cross-sectional area resulted in the enhanced grip strength in cancer cachexia mice. Consequently, the model mice treated with peptide-2 could survive longer than those that did not undergo this treatment. Our results suggest that peptide-2 might be a novel therapeutic candidate to suppress muscle wasting in cancer cachexia.

Keywords: Lewis lung carcinoma; cancer cachexia; mice model; muscle wasting; myostatin.

FIGURE 1

Peptide‐2 is a selective inhibitor of myostatin (MSTN). A, Peptide‐2 significantly inhibited the activity of (SBE)₄‐luc following MSTN stimulation in HepG2 cells. HepG2 cells were cotransfected with a (SBE)₄‐luc reporter construct and pCH110 as an internal marker and stimulated with transforming growth factor‐β (TGF‐β; 5 ng/mL), activin A (10 ng/mL), growth differentiation factor‐11 (GDF‐11; 10 ng/mL), or MSTN (10 ng/mL) for 8 h. Luciferase values were normalized for transfection efficiency. These ligands were preincubated with peptide‐2 (30 nmol/L) or SB‐431542 (10 µmol/L) and were added to the cell. Inhibition efficiency was shown as % of control. All values represent mean ± SD (n = 3). B, Smad2 and Smad3 nuclear localization induced by MSTN were inhibited by peptide‐2. Immunofluorescence staining for Smad2 and Smad3 (Green) in C2C12 cells. Peptide‐2 (30 nmol/L) or SB‐431542 (10 µmol/L) was preincubated with MSTN for 1 h, and added to C2C12 cells for 1 h before staining. Nuclei were counterstained with DAPI (blue). A BZ‐9000 fluorescence microscope was used to visualize the fluorescence. C, Peptide‐2 and the activin type II receptor‐B dominant‐negative receptor (ActRIIB‐Fc) inhibit MSTN‐induced Smad2 phosphorylation. SB‐431542, peptide‐2 (30 nmol/L), ActRIIB‐Fc (0.146 nmol/L), or control‐Fc were preincubated with MSTN for 1 h and added to HepG2 cells for 1 h. These cells were lysed and subjected to western blot analysis using anti‐phospho‐Smad2 (upper panel), anti‐Smad2 Ab (middle panel), and β‐actin (lower panel). Each expression was normalized using the intensity of the band corresponding to β‐actin. Inducibility was calculated relative to the value for cells in the absence of TGF‐β. D, Peptide‐2 significantly inhibited the activity of (CAGA)₁₂‐luc upon MSTN stimulation in HepG2 cells. SB‐431542 (10 µmol/L), peptide‐2 (30 nmol/L), ActRIIB‐FC (0.146 nmol/L), or control‐Fc (0.146 nmol/L) were preincubated with MSTN (10 ng/mL) for 1 h, and added to HepG2 cells for 1 h. HepG2 cells were cotransfected with (SBE)₄‐luc reporter construct and pCH110 as an internal marker and stimulated with MSTN for 8 h. All values represent mean ± SD (n = 3). **P < .01; *P < .05

FIGURE 2

Peptide‐2 inhibited the effect of myostatin (MSTN) and promoted C2C12 myoblast differentiation. A, Expression of myotube differentiation genes. C2C12 cells were cultured with 15% FCS or 2% horse serum (HS) containing medium for 2 d. Cells were stimulated with 10 ng/mL MSTN with or without SB‐431541 (SB) (10 µmol/L) or peptide‐2 (30 nmol/L). Total RNA from these cells was subjected to RT‐PCR for myogenin (upper panel), MylpF (middle panel), and β‐actin mRNAs (bottom panel). B, C2C12 cells cultured in growth medium (15% FCS) reached 90% confluence, and the culture media were switched to differentiation medium (2% HS) for 2 d. These cells were stained with TRITC‐phalloidin (red) and DAPI (blue) to counterstain cell nuclei. The BZ‐9000 fluorescence microscope was used visualize the fluorescence. C, Diameters of C2C12 myotubes was analyzed. Thirty myotubes per experiment were counted over 2 independent experiments, and a representative plot is displayed. Significance was compared to serum‐free control. ***P < .001. Data presented are the mean ± SD

FIGURE 3

Peptide‐2 prolongs survival in cancer cachexia model mice. A, Schematic diagram of the experimental schedule. Peptide‐2 or saline was injected i.m. to the mouse gastrocnemius muscles of both legs a total of 3 times, at 4, 11, 18, and 25 d after cell injection. B, Survival rate of Lewis lung carcinoma (LLC)‐bearing mice with peptide‐2 or saline treatment was analyzed (χ² test, P = .03, power = 0.991). C, Changes in body weights in control and LLC tumor‐bearing mice. Both groups of mice received peptide‐2 or saline at indicated days. Body mass following LLC inoculation was monitored every week at 1, 8, 15, and 22 d after inoculation (n = 6). Representative data are shown. Raw data are shown in Table S1A. D, Tumor volume estimated from width of tumors in LLC‐bearing mice with peptide‐2 or saline treatment. Tumor size was serially measured from above the skin. Tumor volumes were calculated using the formula: length × width ×width × 0.5. Data are expressed as mean ± SD (n = 6). E, Peptide‐2 (Pep‐2) treatment did not alter LLC tumor growth (P = .20) (n = 6). Representative data are shown. F, Body weight of mice excluding tumor weight at day 22. LLC tumor‐bearing mice had significant weight loss compared to controls. **P < .01; *P < .05. Raw data of statistical results are shown in Table S1B

FIGURE 4

Peptide‐2 attenuates muscle wasting induced by cancer cachexia. Peptide‐2 (Pep‐2) or saline was injected i.m. in both legs of control or Lewis lung carcinoma (LLC)‐transplanted mice. A, Photographs of legs of control and cancer cachexia mice. B, Gastrocnemius muscle weight/body weight (n = 6). C, Protein concentration of gastrocnemius muscle (n = 6). D, E, Heart weight/body weight (n = 3‐4) (D) and fat weight/body weight (n = 6) (E) at 22 d after LLC implantation. The weight of each tissue was normalized to the body weight at day 1. ***P < .001; *P < .05. All measurements and statistical analyses are shown in Table [Link], [Link], [Link], [Link]

FIGURE 5

Peptide‐2 (Pep‐2) improves muscle fiber area and strength in cancer cachexia model mice. A, Representative immunofluorescence analysis of dystrophin‐stained gastrocnemius sections from indicated groups. Cryosections of gastrocnemius muscles from healthy control and cancer cachexia model mice treated with saline or Pep‐2 were stained with antidystrophin Ab (green) and DAPI (blue) to counterstain cell nuclei. Sections were imaged using a fluorescence microscope. B, Images were analyzed using a BZ‐H3A software, which automatically identifies individual areas surrounded by the muscle fiber membrane. Box and whisker plots showed the distribution of muscle fiber cross‐sectional area from 3 identical images for each group. The horizontal line in the box indicates the median values, the bottom and top edges indicate the 25th and 75th percentile, respectively. The whiskers extend to the most extreme bottom and top, indicating the minimum and maximum, respectively. In cancer cachexia model mice, the mean area of muscle fibers decreased significantly (***P < .001) indicating muscle atrophy, but treatment with Pep‐2 did not improve the mean value. C, Frequency distribution plots of the area of gastrocnemius muscle fiber. Plots are expressed as a percentage of the total number of fibers. Peptide‐2 increased the area of large muscle fibers in healthy mice and increased the proportion of thick muscle fibers in cachectic mice. D, Grip strength analysis of control and cachexic mice treated with Pep‐2 or saline. Each mouse was tested 5 times and the maximum force demonstrated by each animal was recorded (n = 4). Specific P values are shown in the figure. *P < .05. E, Intramuscular injection of Pep‐2 inhibited Smad2 phosphorylation in gastrocnemius muscle. The gastrocnemius muscle of cancer cachexia model mice treated with saline or Pep‐2 were analyzed. Smad2 phosphorylation was analyzed at 22 d after tumor cell inoculation. These muscles were lysed and subjected to western blot analysis using anti‐phospho‐Smad2 (upper panel), anti‐Smad2 Ab (middle panel), and β‐actin (lower panel) (n = 2)