Cancer-induced muscle wasting, which commonly occurs in cancer cachexia, is characterized by impaired quality of life and poor patient survival. To identify an appropriate treatment, research on the mechanism underlying muscle wasting is essential. Thus far, studies on muscle wasting using cancer cachectic models have generally focused on early cancer cachexia (ECC), before severe body weight loss occurs. In the present study, we established models of ECC and late cancer cachexia (LCC) and compared different stages of cancer cachexia using two cancer cachectic mouse models induced by colon-26 (C26) adenocarcinoma or Lewis lung carcinoma (LLC). In each model, tumor-bearing (TB) and control (CN) mice were injected with cancer cells and PBS, respectively. The TB and CN mice, which were euthanized on the 24th day or the 36th day after injection, were defined as the ECC and ECC-CN mice or the LCC and LCC-CN mice. In addition, the tissues were harvested and analyzed. We found that both the ECC and LCC mice developed cancer cachexia. The amounts of muscle loss differed between the ECC and LCC mice. Moreover, the expression of some molecules was altered in the muscles from the LCC mice but not in those from the ECC mice compared with their CN mice. In conclusion, the molecules with altered expression in the muscles from the ECC and LCC mice were not exactly the same. These findings may provide some clues for therapy which could prevent the muscle wasting in cancer cachexia from progression to the late stage.
Cachexia has two well-known features: weight loss (mainly due to loss of skeletal muscle and body fat) and inflammation. This syndrome is prevalent in cancer patients, and muscle wasting is the most prominent symptom of cancer cachexia. It is well known that muscle wasting in cancer cachexia is directly related to the poor quality of life of cancer patients and even impacts their survival (
To date, for both practical and ethical reasons, studies on muscle wasting have mainly depended on the use of murine models. Among the many available models, the colon-26 adenocarcinoma (C26) and Lewis lung carcinoma (LLC) models are the most commonly used (
Many studies have shown that an intricate regulatory network is involved in muscle wasting (
Myostatin, which functions specifically as a negative regulator of skeletal muscle growth, is present at a higher level in serum of cancer cachectic mice than in those of normal healthy mice (
The activity of FoxO3a is inhibited by an important transcriptional coactivator, peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1α), which is stimulated by signals that maintain energy and nutrient homeostasis and involved in important metabolic pathways in muscular tissue (
CCAAT/enhancer binding protein beta (C/EBPβ) is an important transcription factor involved in cellular metabolism and inflammation (
Histone deacetylases (HDACs) are the most well known for their roles in the regulation of muscle development and differentiation (
Additionally, the roles of microRNAs in skeletal muscle damage and regeneration induced by atrophy have emerged (
Although a lot of information has been reported about muscle wasting in cancer cachexia, few studies have been focused on whether muscle wasting in early cancer cachexia (ECC) differs from that in late cancer cachexia (LCC). It has been established that the development of tumors can be divided into different phases (
Colon-26 adenocarcinoma cells (C26 cells) (Medical Science Experimentation Center of Sun Yat-Sen University, China) and Lewis lung carcinoma cells (LLC cells) (Shanghai Branch of Chinese Academy of Science, China) were cultured in Dulbeccos modified Eagles medium (DMEM) plus 10% fetal bovine serum (FBS) with 1% penicillin/streptomycin at 37°C and 5% CO2. Before injection of C26 cells into CD2F1 mice (C26 model) or injection of LLC cells into C57BL/6 mice (LLC model), cells were counted and resuspended at 5×107 cells/ml in sterilized PBS. The right flanks of the mice were shaved, and they were administered a subcutaneous (s.c.) injection of either 5×106 C26 cells or LLC cells suspended in 100 µl sterilized PBS (tumor-bearing mice, TB mice) or 100 µl sterilized PBS without cells (control mice, CN mice). Eight-week-old male CD2F1 or C57BL/6 mice were allocated randomly into one of four experimental groups: i) tumor-bearing mice in early cachexia (ECC mice); ii) tumor-bearing mice in late cachexia (LCC mice); iii) ECC-matched control mice (ECC-CN mice); and iv) LCC-matched control mice (LCC-CN mice). The animals were monitored daily and were euthanized separately at 24 days (ECC and ECC-CN mice) and 36 days (LCC and LCC-CN mice) following injection (
To determine the myofiber cross-sectional area (CSA), hematoxylin and eosin (H&E) staining was performed on a middle cross-section of the tibialis anterior. Images were acquired using a digital camera and were quantified using ImageJ software (NIH, Bethesda, MD, USA). Within each section, five view fields with 100 myofibers per field were measured (
To visualize the outlines of myofibers, 10 µm sections were obtained from the middle of the tibialis anterior. The sections were then incubated with Alexa Fluor 350-conjugated wheat germ agglutinin (Invitrogen, Carlsbad, CA, USA) for 2 h and subsequently washed in PBS. Images were acquired using a digital camera (
RNA was extracted from quadriceps muscles using TRIzol reagent (Invitrogen) according to the manufacturers instructions. The concentration and purity of the RNA solution were determined by Epoch microplate spectrophotometer (BioTek Instruments, Inc., Winooski, VT, USA). RNA (1 µg) was used for reverse transcription. Reverse transcription of mRNA was performed using a RevertAid First-Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc., Rockford, IL, USA) in a total reaction volume of 10 µl. Dilution (1:10) of the RT product was used as template for the quantitative real-time PCR (qPCR). qPCR was performed with the 2X SYBR-Green Mix (Thermo Fisher Scientific) using a LightCycler® 480 (Roche Diagnostics, Mannheim, Germany) in a total reaction volume of 10 µl with the primers from Sangon Biotech, Co., Ltd. (Shanghai, China). The amplification procedure was 95°C pre-denaturation for 10 min followed by 95°C for 15 sec, 60°C for 10 sec and 72°C for 30 sec for a total of 40 cycles. The data were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression and the relative expression was calculated using the formula: 2−ΔCt (ΔCt = Ct gene - Ct GAPDH). The primer sequences were as follows: Myostatin: F-AGTGGATCTAAATGAGGG CAGT and R-GTTTCCAGGCGCAGCTTAC; PGC1α: F-AA CCACACCCACAGGATCAGA and R-TCTTCGCTTTAT TGCTCCATGA; FoxO3a: F-GCAAGCCGTGTACTGTGGA and R-CGGGAGCGCGATGTTATCC; MuRF1: F-AGCAT CAAGATCCGTCTGACA and R-CCAGAGCCGTCCACA ACAAT; Atrogin1: F-ACACATCCTTATGCACACTGG and R-TCTCCATCCGATACACCCACA; GAPDH: F-GGTGAA GGTCGGAGTCAACGG and R-GAGGTCAATGAAGGGG TCATTG.
The quadriceps muscles were homogenized, and total protein was extracted using RIPA protein lysis buffer (P1003; Beyotime Institute of Biotechnology, Nantong, China) with freshly added protease inhibitor cocktail and phenylmethylsulphonyl fluoride (PMSF). The protein concentration of the samples was measured using BCA method. A total of 80 µg of protein was subjected to a 10% SDS-PAGE gel to separate the proteins by gel electrophoresis, and they were then transferred onto polyvinylidene fluoride (PVDF) (0.45 µm; Millipore, Boston, MA, USA) membranes. The membranes were blocked for 1 h at 37°C in 5% (w/v) non-fat dried skim milk (blocking buffer) and incubated with primary antibodies in blocking buffer overnight at 4°C. The primary antibodies were as follows: anti-atrogin1 antibody (#AP2041), purchased from ECM Biosciences, Versailles, KY, USA; anti-PGC1α antibody (ab54481), purchased from Abcam, Cambridge, MA, USA; anti-Phospho-FoxO3a (#9466) and anti-FoxO3a (#2497) antibodies obtained from Cell Signaling Technology, Danvers, MA, USA; anti-C/EBPβ (sc-7962), anti-HDAC1 (sc-7872), anti-HDAC2 (sc-7899), and anti-HDAC3 (sc-11417) antibodies acquired from Santa Cruz Biotechnology, Santa Cruz, CA, USA. The membranes were washed and incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (Invitrogen) in blocking buffer for 2 h at room temperature. Finally, the membranes were washed before detection. Quantitative analyses of protein expression were performed using ImageJ software (
All values were represented as the mean ± standard error (SEM) unless stated otherwise. Differences between group means were determined using the Students t-test with Graphpad Prism 5 unless otherwise specified. A two-sided P-value of <0.05 was considered to indicate statistically significant result.
For the C26 model mice, at 24 days following C26 tumor implantation, the body weights of the ECC-CN and ECC mice were both increased (
The tumor-free masses of the ECC mice were reduced by ~18 and 13% compared with those of ECC-CN mice for the C26 and LLC models, respectively. A similar finding was observed for the LCC mice, but with higher rates of reduction (~28 and 29% compared with the C26 and LLC model LCC-CN mice, respectively). The tumor-free masses of the LCC mice were obviously less than those of the ECC mice for both models (
For the above reasons, we defined the TB mice sacrificed on the 24th day as ECC mice (their tumor-free body masses were decreased by <20%) and the TB mice sacrificed on the 36th day as LCC mice (their tumor-free body masses were decreased by >20%).
As previously reported, C26 cachexia results in skeletal muscle, epididymal adipose and heart mass losses (
C26 cancer cachexia has been reported to result in a large increase in the mass of the spleen (
Representative images of H&E-stained tibialis anterior middle cross sections from the mice in each group are shown in
To determine the mRNA levels of some molecules involved in muscle wasting, five prominent molecules were selected for analysis in each group. The mRNA levels of these molecules did not obviously change in the ECC mice of both models, except for that of atrogin1, which was increased in the ECC mice compared with the ECC-CN mice for the C26 model, but not the LLC model (
To explore the underlying mechanism of the increased severity of cancer cachexia in the LCC mice compared with the ECC mice, the protein levels of some crucial molecules involved in muscle wasting, such as atrogin1, FoxO3a, PGC1α, C/EBPβ and class I HDACs, were determined. The protein level of atrogin1 was increased in the TB mice compared with their matched CN mice for both models (
Furthermore, the protein levels of three class I HDACs were determined, and those of HDAC1 and HDAC3 were found to be slightly increased in the LCC mice compared with the LCC-CN mice for both models, while only HDAC2 was increased in the LLC model LCC mice (
Cancer cachexia has been widely studied. A previous report demonstrated that lipid metabolism in adipose tissue differs between C26 model ECC and LCC mice. ECC was defined by the author as occurring no more than 12 days following C26 tumor implantation, when the white adipose tissue mass in cachectic mice is moderately reduced (34–42%) and weight loss is <10% of the initial body weight (
Prior to this study, many research groups focused on muscle wasting only in ECC. Thus, we questioned whether the molecules regulating muscle wasting in LCC are similar to those in ECC. The aim of the present study was to determine the differences between muscle wasting in ECC and LCC.
The alterations in the tumor-free body masses, the masses of various tissues and the cross-sectional areas (CSAs) of muscle fibers differed between the ECC and LCC mice and their matched CN mice. These results demonstrated that obvious differences existed between ECC and LCC. From this point of view, the definitions of ECC and LCC in the C26 and LLC models were also feasible.
Myostatin plays an important role in many types of muscle atrophy (
Currently, increasing numbers of studies are focusing on the molecules that affect the myostatin-FoxO3a-atrogin1 axis. We found that the molecules involved in muscle wasting were not exactly the same in the ECC and LCC mice of each model. In addition, we focused on the molecules that were altered only in the muscles from the LCC mice. Although the mRNA level of PGC1α was not altered in the TB mice, its protein level was decreased in the LCC mice, but not in the ECC mice, of both models. These results indicated that C/EBPβ, HDAC1 and HDAC3 might play roles in promoting cancer cachexia, especially during the late stage. Correspondingly, PGC1α might play an opposite role. As previously reported, muscles from the TB mice had a higher level of phosphorylated C/EBPβ, along with a modest increase in total C/EBPβ, on day 14 for the LLC model (
Many studies have verified that the levels of microRNAs are altered in muscles from cancer cachectic mice. We used different miRNA target-predicting algorithms (for example, TargetScan and RegRNA) to identify potential miRNAs that could affect the aforementioned genes. We found conserved miR-30c sites in the 3UTRs of atrogin1, FoxO3a and HDAC3 (
By comparing the changes in the expression of crucial molecules involved in muscle wasting in both the ECC and LCC mice, we confirmed that some molecules exhibited varying degrees of change in our models. Although the expression levels of several other molecules did not obviously change in the ECC mice, they were significantly altered in the LCC mice, such as PGC1α, C/EBPβ and HDACs. However, it is still difficult to conclude that these unchanged molecules do not play roles in the ECC mice. For instance, the role of HDACs in muscle wasting has been realized in recent years, and pharmacological interventions with HDAC inhibitors have been shown to increase myofiber size and counter the functional decline of dystrophic muscles (
In conclusion, our results have revealed that the expression levels of several molecules are altered in muscles from LCC mice, but not in those from ECC mice. From our results we deduce that these changes may promote muscle wasting in late cancer cachexia. The data in this study may facilitate the further understanding of the underlying mechanism involved in the development of cancer cachexia. However, our present study on muscle wasting in late cancer cachexia merely sheds light on the underlying mechanism, which remains poorly understood. Thus, further investigation is warranted to delineate the foundation of late cancer cachexia to provide a solid basis for the clinical prediction and prevention of muscle wasting in cancer cachexia.
The present study was supported by the National Natural Science Foundation of China (NSFC; grant no. 81272560), the Open Research Foundation of the State Key Laboratory of Virology of Wuhan University (grant no. 2014KF007), the Hubei Province Scientific and Technical Project (grant no. 2011CDB366), and the Hubei Provincial Health Project (grant no. WJ2015MB020) to H.Y. The study was also supported by the National Natural Science Foundation of China (grant nos. 30872924, 81072095 and 81372760), the Program for New Century Excellent Talents in University from the Department of Education of China (NCET-08-0223), and the National High Technology Research and Development Program of China (863 Program) (2012AA021101) to X.Z.
Body weight curves and tumor mass in each group. Body weights of CD2F1 mice injected subcutaneously with C26 cells or PBS were recorded for (A) 24 days and (B) 36 days. Body weights of C57BL/6 mice injected subcutaneously with LLC cells or PBS were recorded for (C) 24 days and (D) 36 days. (E) The tumor masses from the C26 model ECC and LCC mice. (F) The tumor masses from the LLC model ECC and LCC mice. (A-D) All body weights were normalized to the percentage of the initial body weight. (E and F) The tumor masses of TB mice were normalized to the percentage of the tumor masses of their matched ECC mice. *P<0.05, #P<0.01.
The tumor-free body masses and masses of various tissues in C26 model CN and TB mice. The (A) tumor-free body, (B) quadriceps, (C) tibialis anterior, (D) soleus, (E) gastrocnemius, (F) heart, (G) spleen, and (H) epididymal fat masses from C26 model ECC and LCC mice. (A-H) The masses of tissues from the TB mice were normalized to the percentage of the masses of the tissues from their matched CN mice. *P<0.05, **P<0.01, ***P<0.001).
The tumor-free body masses and masses of various tissues in LLC model CN and TB mice. The (A) tumor free body, (B) quadriceps, (C) tibialis anterior, (D) soleus, (E) gastrocnemius, (F) heart, (G) spleen, and (H) epididymal fat masses from LLC model ECC and LCC mice. (A-H) The masses of tissues from the TB mice were normalized to the percentage of the masses of the tissues from their matched CN mice. *P<0.05, **P<0.01, ***P<0.001.
Middle cross sections of tibialis anterior muscles from mice in each group. (A) Representative images of H&E-stained cross sections of tibialis anterior muscles from mice in each group. Bar represents 100 µm. (B) Representative images of cross sections of tibialis anterior muscles incubated with wheat germ agglutinin to allow for visualization of muscle fiber membranes (blue). Bar represents 50 µm.
Cross-sectional areas of myofibers. The average CSAs of myofibers in tibialis anterior muscles from mice of the (A) C26 model and (B) LLC model. Analysis of the size distributions of myofibers in tibialis anterior muscles from mice of the (C) C26 model and (D) LLC model. (A and B) The average CSA of myofibers in the TB mice were normalized to the percentage of the average CSA of myofibers in their matched CN mice. ***P<0.001.
The mRNA levels of genes involved in muscle wasting. (A) The mRNA levels of genes in muscles from C26 model ECC mice. (B) The mRNA levels of genes in muscles from C26 model LCC mice. (C) The mRNA levels of genes in muscles from LLC model ECC mice. (D) The mRNA levels of genes in muscles from LLC model LCC mice. (A-D) The data for the TB mice were normalized to those for their matched CN mice. *P<0.05, **P<0.01, ***P<0.001.
The protein levels of genes involved in muscle wasting in C26 model mice. The protein levels of atrogin1, p-FoxO3a, FoxO3a, PGC1α, C/EBPβ, HDAC1, HDAC2 and HDAC3 in muscles from C26 model (A) ECC mice and (B) LCC mice. Densitometric analysis of molecules detected by western blot analysis in muscles from C26 model (C) ECC mice and (D) LCC mice. (C and D) The data for the TB mice were normalized to those for their matched CN mice. *P<0.05, **P<0.01, ***P<0.001.
The protein levels of genes involved in muscle wasting in LLC model mice. The protein levels of atrogin1, p-FoxO3a, FoxO3a, PGC1α, C/EBPβ, HDAC1, HDAC2 and HDAC3 in muscles from LLC model (A) ECC mice and (B) LCC mice. Densitometric analysis of molecules detected by western blot analysis in muscles from LLC model (C) ECC mice and (D) LCC mice. (C and D) The data for the TB mice were normalized to those for their matched CN mice. *P<0.05, **P<0.01, ***P<0.001.
The expression of miR-30c in muscles from mice in each group. (A) Prediction of the conserved miR-30c sites in the 3UTRs of atrogin1, FoxO3a, and HDAC3 was performed using TargetScan. Prediction of the conserved miR-30c site in the 5UTR of PGC1α was performed using RegRNA. (B) The expression of miR-30c in muscles from C26 model ECC and LCC mice. (C) The expression of miR-30c in muscles from LLC model ECC and LCC mice. (B and C) The data for the TB mice were normalized to those for their matched CN mice. *P<0.05, **P<0.01.
Changes in tumor-free body mass, muscle mass, organ mass, and fat mass in the C26 model.
24 days | 36 days | |||||
---|---|---|---|---|---|---|
Control (CN) | P-value | C26 tumor bearing (TB) | Control(CN) | P-value | C26 tumor bearing (TB) | |
n | 4 | 5 | 4 | 5 | ||
Tumor-free body mass (g) | 27.74±0.74 | 0.001 |
22.92±0.73 | 27.15±0.62 | 0.001 |
19.60±0.90 |
Quadriceps (mg) | 134.90±7.30 | 0.001 |
114.97±7.69 | 161.93±20.82 | 0.001 |
80.09±13.07 |
Tibialis anterior (mg) | 51.76±3.75 | 0.01 |
44.93±5.03 | 56.24±7.39 | 0.001 |
31.89±6.43 |
Gastrocnemius (mg) | 128.56±10.80 | 0.01 |
111.99±6.67 | 144.61±10.06 | 0.001 |
83.98±10.89 |
Soleus (mg) | 6.73±2.46 | 0.05 |
4.90±0.84 | 6.46±1.80 | 0.01 |
4.47±0.85 |
Heart (mg) | 139.60±6.05 | 0.05 |
120.94±13.33 | 149.65±13.61 | 0.001 |
87.26±3.28 |
Spleen (mg) | 77.83±5.14 | 0.001 |
218.84±45.36 | 90.33±4.14 | 0.001 |
293.70±49.96 |
Epididymal fat (mg) | 559.78±114.77 | 0.001 |
194.54±68.95 | 401.48±60.37 | 0.001 |
33.24±14.99 |
Twenty-four days-CN vs. 24 days-TB
24 days-CN vs. 24 days-TB
24 days-CN vs. 24 days-TB
36 days-CN vs. 36 days-TB
36 days-CN vs. 36 days-TB.
Changes in tumor-free body mass, muscle mass, organ mass, and fat mass in the LLC model.
24 days | 36 days | |||||
---|---|---|---|---|---|---|
Control (CN) | P-value | C26 tumor bearing (TB) | Control (CN) | P-value | C26 tumor bearing (TB) | |
n | 4 | 6 | 4 | 6 | ||
Tumor-free body mass (g) | 22.61±1.16 | 0.05 |
19.87±1.39 | 23.19±2.10 | 0.001 |
16.49±1.05 |
Quadriceps (mg) | 117.04±8.50 | 0.01 |
100.13±10.23 | 116.18±6.16 | 0.001 |
64.49±11.48 |
Tibialis anterior (mg) | 50.88±6.95 | 0.05 |
44.24±5.69 | 50.26±4.91 | 0.001 |
30.27±4.32 |
Gastrocnemius (mg) | 131.11±7.58 | 0.05 |
119.15±12.54 | 134.11±7.04 | 0.001 |
85.55±5.83 |
Soleus (mg) | 7.73±0.89 | 0.001 |
6.29±0.55 | 7.10±0.64 | 0.001 |
5.34±0.67 |
Heart (mg) | 100.23±5.61 | 103.05±8.24 | 142.80±33.31 | 124.58±35.09 | ||
Spleen (mg) | 72.05±7.50 | 0.001 |
198.63±30.27 | 67.90±13.51 | 0.05 |
220.72±85.35 |
Epididymal fat (mg) | 339.38±97.72 | 0.05 |
159.32±100.88 | 465.55±121.66 | 0.001 |
18.16±5.69 |
Twenty-four days-CN vs. 24 days-TB
24 days-CN vs. 24 days-TB
24 days-CN vs. 24 days-TB
36 days-CN vs. 36 days-TB)
36 days-CN vs. 36 days-TB.