INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

WASTING OF SKELETAL MUSCLE mass is a hallmark of cancer cachexia, where the depletion of skeletal muscle mass may be as high as 75% (20). This wasting results in weakness and higher mortality compared with patients without wasting (13, 23). Chemotherapy, which is widely used in the treatment of cancer, unfortunately has a number of nonspecific cytotoxic effects to the host. There is recent evidence to suggest that chemotherapy itself may directly contribute to cancer cachexia, further confounding the treatment of this disease and subsequent recovery by the patient (14). The precise interaction between cancer cachexia and chemotherapy on skeletal muscle mass and protein metabolism has not been addressed. The cytotoxic effects of chemotherapy may cause further skeletal muscle wasting and impede the longer-term ability of skeletal muscle to recover; these possibilities need to be addressed. Furthermore there are no studies that have investigated the processes that might mediate recovery after chemotherapy.

Knowledge of the impact of cancer cachexia and chemotherapy on skeletal muscle protein metabolism is critical to developing treatments to promote recovery of skeletal muscle mass after cancer treatment. In doing so, the potential exists to reduce morbidity, to improve the ability to withstand any subsequent cancer treatments, and to return the individual to an improved physical state. Clearly more research is required on the mechanisms by which chemotherapeutic agents acutely affect skeletal muscle protein turnover and the longer-term ability of skeletal muscle to generate an effective anabolic response to affect restoration of skeletal muscle mass.

The objective of the present research was to measure protein mass and the in vivo rates of protein synthesis and degradation in skeletal muscle during cancer cachexia and chemotherapy and the subsequent recovery from cancer cachexia. We achieved our objective using colon 26 adenocarcinoma (C26) in mice, which results in cachexia characteristic to the human condition, with a relatively small tumor burden (8, 25). To study the recovery of skeletal muscle mass from cancer cachexia, we used cystemustine, a recently developed nitrosourea that cures C26 with 100% efficacy (4, 16).

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Animals, housing, and diet. All animal studies were completed in accordance with the guidelines of the Canadian Council of Animal Care. Male BALB/c ByJ mice (20 g, 4-5 wk old, Jackson Laboratories, Bar Harbor, ME) were housed in individual cages and maintained at 22-23°C on a 12:12-h light-dark cycle commencing at 0800. Mice were given at least 4 days to adjust to their new environment and diet before treatments were imposed. Mice were given free access to water and food (standard rodent chow, UBC Animal Care Centre), with the exception of pair-fed mice.

Tumor model, inoculation, and transplantation. To study the effect of cancer cachexia and chemotherapy on skeletal muscle, an appropriate model must be used to represent the condition observed in human cancer patients. The tumor must be able to induce cachexia and the associated physiological abnormalities while maintaining a reasonably small tumor burden (3). The murine C26 meets these requirements (8, 22, 25). This model results in extensive losses in adipose and skeletal muscle tissue weight, which are not caused by decreased food intake (9, 22, 25, 26).

Chemotherapeutic agent and regimen. To study the recovery of skeletal muscle mass from cancer cachexia, a chemotherapeutic drug that cures the cancer is required. Cystemustine, N-(2-chloroethyl)-N-[2-(methylsulfonyl)ethyl]-N-nitrosourea (C₆H₁₂CIN₃O₄S), a member of the chloroethylnitrosourea family of alkylating antineoplastic agents, has been shown to cure C26 with a 100% efficacy (4, 16). Cystemustine, similar to the other chloroethylnitrosoureas, is a bialkylating agent acting mainly through DNA interstrand cross-links to block tumor cell growth and ultimately induce cellular death (11). Cystemustine also has a degree of relevance to human cancer patients, as phase II clinical trials have been completed (e.g., 30) and current research is being focused on increasing its antineoplastic activity (6).

Experimental treatments. There were four principal treatment groups: 1) healthy mice, 2) healthy mice treated with cystemustine, 3) tumor-bearing mice, and 4) tumor-bearing mice treated with cystemustine. Mice were randomly designated to one of the four treatment groups. Tumors were transplanted into mice in the tumor-bearing groups, whereas mice in the healthy groups were sham injected. About 4 days after the onset of cachexia, chemotherapy was administered to one of the tumor-bearing groups of mice and one healthy group. All other mice were injected with vehicle alone. An additional group of mice (healthy pair-fed mice) was used to control for any possible effects of reduced food intake by tumor-bearing mice resulting from chemotherapy. Healthy mice were initially fed the food intake of tumor-bearing mice. Once the chemotherapeutic agent was administered, healthy pair-fed mice received the food intake of tumor-bearing mice treated with cystemustine. Pair-fed mice received their allotted food between 1000-1100 and 1700-1800. Pair-fed mice were sham injected on the day of tumor transplantation and chemotherapy.

Experimental time points. A group of tumor-bearing and healthy (ad libitum fed) mice were killed on day 2 (2 days before chemotherapy) to determine the acute mechanisms responsible for the initiation of skeletal muscle wasting. Chemotherapy was administered on day 0 (~4 days after the onset of cachexia). The onset of cachexia was determined on the basis of body weight loss. The cachexia causes a sudden and sustained decrease in body weight, starting at about day 4. We defined the onset of cachexia as the first day in which body mass of cachectic mice was significantly lower than that of healthy mice. Healthy mice, tumor-bearing mice, and tumor-bearing mice treated with cystemustine were killed on days 2, 4, and 11 after chemotherapy. Days 2 and 4 were used to determine the acute effects of chemotherapy and the mechanisms initiating recovery, respectively. By day 11, the tumor had fully regressed and therefore served as a good indicator of longer-term recovery.

Protocol used on experimental days. Protein synthesis was measured in vivo using the flooding dose method as described previously (10, 18). Between 1100 and 1400, mice were injected intraperitoneally with 150 µmol phenylalanine/100 g body wt with a specific radioactivity of 0.6 µCi L-2,6-[³H]phenylalanine/µmol (Amersham Canada Limited, Oakville, Canada) in 1.25 ml saline/100 g body wt. After precisely 15 min from time of injection, each mouse was killed by cervical dislocation, and the right and left gastrocnemius muscles were rapidly excised, blotted, weighed, frozen in liquid nitrogen, and stored at 70°C until analyzed.

In vivo rate of protein synthesis and degradation. The flooding dose method is considered to be the method of choice for measuring protein synthesis in vivo in small animals; Davis et al. (5) recently showed that the aminoacyl-tRNA pool, the true precursor pool, equilibrates with the tissue and blood free amino acid pools in skeletal muscle when a flooding dose of phenylalanine is used. To estimate protein synthesis and degradation, muscle samples were analyzed in a manner similar to that reported by McAllister et al. (18) and Garlick et al. (10). The specific radioactivity of free (S_A) tissue and protein-bound (S_B) phenylalanine was measured in each tissue. The fractional rate of protein synthesis (k_syn; %/day) was calculated as: k_syn = (S_B × 100)/(S_A×t) × 100, where t is the time between injection and slaughter (in days). The fractional growth rate (k_growth) for each tissue was determined for each mouse as the average fractional growth rate over 24-48 h immediately proceeding the measurement of protein synthesis (1, 21). The absolute rate of protein synthesis was calculated by multiplying the fractional rate by the phenylalanine mass of that tissue (21).

Statistics. The experiment was completed in two replicates, and all measurements were combined. The total sample size for any one time point and treatment group ranged from 5 to 10 mice. The effects of treatment, day, and the treatment × day interaction were tested using a 2-way ANOVA (23). When a treatment × day interaction occurred, differences between individual means were assessed using t-tests (23). In the present experiment, there was a significant treatment × day interaction present for all parameters tested. Variability was expressed as means ± SE. Differences were considered significant at P < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Characterization of the experimental model. A detailed characterization of the tumor and effect of the tumor and chemotherapy on body weight and food intake is provided in Samuels et al. (22). Briefly, rapid body weight loss (20%) occurred between days 3 and 2. Between days 2 and 11, body weight loss was more gradual; by day 11, it was 30% lower than that of healthy mice. In chemotherapy-treated tumor-bearing mice, body weight was reduced 7% compared with untreated tumor-bearing mice 1 day after treatment. By day 2, body weight of treated mice started to increase, and mice were well into a state of recovery by day 4. By day 11, tumor regression was complete with only traces of tumoral tissue present in a few mice; body weights of cured mice were 5% lower than those of healthy mice. In healthy mice treated with cystemustine, body weight was lower compared with healthy controls on day 2, but was normal by day 3. Cancer cachexia and chemotherapy had a minor effect on food intake.

Skeletal muscle phenylalanine mass and wet weight. At the onset of cachexia (day 2), skeletal muscle phenylalanine mass of tumor-bearing mice was not different from healthy mice (Fig. 1; P > 0.05). However, evidence of wasting was present by day 2, as skeletal muscle phenylalanine mass of tumor-bearing mice was 28% lower than in healthy mice (P < 0.05) and 32% lower (P < 0.05) compared with tumor-bearing mice on day 2. This wasting was not the result of decreased food intake, as pair-fed controls showed no evidence of wasting compared with healthy mice (2.15 ± 0.10 vs. 2.06 ± 0.08 mg wet wt, respectively, on day 2, P > 0.05). Skeletal muscle phenylalanine mass continued to decline in tumor-bearing mice; by the end of the experiment on day 11, it was 43% lower than in healthy mice (P < 0.05) and 42% lower compared with tumor-bearing mice on day 2 (P < 0.05).

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Skeletal muscle phenylalanine mass during cancer cachexia and chemotherapy in mice. A: comparisons among healthy, tumor-bearing, and tumor-bearing mice treated with chemotherapy. B: comparisons between healthy mice and healthy mice treated with chemotherapy. Values for gastrocnemius phenylalanine mass are mean + SE (mg) with n = 5-10 mice per mean. Right and left gastrocnemius muscles were pooled. Values with different letter annotations on the same experimental day are significantly different (P < 0.05). Tumor was colon 26 (C26) adenocarcinoma transplanted on day 18. Chemotherapy was cystemustine (Cyst; C6H12CIN3O4S) administered on day 0 (20 mg/kg).

Mechanisms of skeletal muscle wasting. At the onset of cachexia (day 2), before any losses in protein mass were detected (Fig. 1), the fractional and absolute rates of protein synthesis were lower (38%; P < 0.05) and degradation higher (+125%; P < 0.05) in skeletal muscle of tumor-bearing compared with healthy mice (Figs. 2 and 3). During the protein wasting phase, between days 2 and 11, the fractional (approximately 54%) and absolute (69%) rates of protein synthesis were chronically lower in tumor-bearing than in healthy mice (P < 0.05). The fractional and absolute rates of protein synthesis in tumor-bearing mice were lower on days 2, 4, and 11 compared with day 2 (P < 0.05), but were not different from each other (P > 0.05). The fractional (+67%) and absolute (+22%) rates of protein degradation remained higher on day 2 in tumor-bearing mice compared with healthy mice, although they were lower than they had been on day 2 (P < 0.05). By day 11, the fractional and absolute rates of protein degradation in tumor-bearing mice were not different from healthy mice; as a consequence of the lowered muscle phenylalanine mass in tumor-bearing mice, the absolute rate of protein degradation was lower than it had been on day 4 (P < 0.05). These results are not the product of decreased food intake in tumor-bearing mice; the small reduction in food intake in tumor-bearing mice that occurred before day 1 had no effect on protein synthesis in pair-fed compared with healthy mice (4.7 ± 0.3 vs. 5.0 ± 0.18 %/day, respectively, day 2, P > 0.05). These data clearly demonstrate that skeletal muscle wasting was mediated initially by decreased protein synthesis and increased protein degradation but that chronic wasting was mediated by decreased synthesis.

View larger version (33K): [in this window] [in a new window]   Fig. 2.   In vivo fractional and absolute rates of skeletal muscle protein synthesis during cancer cachexia and chemotherapy in mice. A: comparisons among healthy, tumor-bearing, and tumor-bearing mice treated with chemotherapy. B: comparisons between healthy mice and healthy mice treated with chemotherapy. Values for the fractional rate of protein synthesis (top) are means + SE (%/day). Values for the absolute rate of protein synthesis (bottom) are means + SE (mg phenylalanine incorporated/day). Values represent n = 5-10 mice per mean. Values with different letter annotations on the same experimental day are significantly different (P < 0.05). Tumor was C26 adenocarcinoma. Chemotherapy was cystemustine administered on day 0. See Fig. 1 legend.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   In vivo fractional and absolute rates of skeletal muscle protein degradation during cancer cachexia and chemotherapy in mice. A: comparisons among healthy, tumor-bearing, and tumor-bearing mice treated with chemotherapy. B: comparisons between healthy mice and healthy mice treated with chemotherapy. Values for the fractional rate of protein degradation (top) are means + SE (%/day). Values for the absolute rate of protein degradation (bottom) are means + SE (mg phenylalanine released per day). Values represent n = 5-10 mice per mean. Rates of degradation were determined as the difference between rates of growth and synthesis. Protein degradation could not be estimated in tumor-bearing or healthy mice treated with chemotherapy on day 2 (see MATERIALS AND METHODS). Values with different letter annotations on the same experimental day are significantly different (P < 0.05). Tumor was C26 adenocarcinoma. Chemotherapy was cystemustine administered on day 0. See Fig. 1 legend.

Acute effect of chemotherapy on skeletal muscle protein turnover. Two days after chemotherapy, the fractional and absolute rates of protein synthesis were not lower in treated compared with untreated tumor-bearing mice (Fig. 2). This suggested that chemotherapy had no negative effects on protein synthesis. We considered that the chemotherapy might have a negative effect on protein synthesis on day 1. We therefore measured protein synthesis on day 1 in the second replicate of the experiment. One day after chemotherapy, the fractional rate of protein synthesis in treated tumor-bearing mice was 66% lower than in healthy mice (P < 0.05) (data not shown) and was ~24% lower than in untreated tumor-bearing mice on day 2, although this difference was not statistically significant (data not shown). Protein degradation could not be estimated on day 2 in treated mice because their growth rate was not linear, a criterion for this method.

Mechanism of skeletal muscle recovery in cured mice. Protein synthesis in treated tumor-bearing mice began to increase on day 2 (Fig. 2). The fractional and absolute rates of protein synthesis were ~34% higher on day 2 (P > 0.05) in treated compared with untreated tumor-bearing mice. On day 4, protein synthesis was ~110% higher compared with untreated tumor-bearing mice and ~60% higher compared with treated mice on day 2 (P < 0.05). Protein synthesis approached healthy levels by day 4. The fractional and absolute rates of protein degradation were ~85% lower in treated tumor-bearing mice compared with healthy mice on day 4 (Fig. 3). The profound decrease in protein degradation and the increase in protein synthesis on day 4 corresponds to the day hypertrophy of muscle mass was first detected. These data clearly indicate that recovery of skeletal muscle protein mass was initiated by decreased protein degradation and the restoration of protein synthesis.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

This investigation demonstrated the time course and mechanisms of both skeletal muscle wasting during cancer cachexia and recovery after chemotherapeutic treatment. Of particular interest was the fact that we were able to study recovery of skeletal muscle using a chemotherapeutic agent that effected a cure so that our observations would not be influenced by ongoing or recurrent cancer cachexia. We showed that wasted skeletal muscle has the ability to generate a strong anabolic response after chemotherapy and that this response was primarily driven initially by a substantial decreased protein degradation and later by increased protein synthesis.

Wasting of skeletal muscle from cancer cachexia. Cancer cachexia in C26-bearing mice caused significant wasting of skeletal muscle, and this wasting began shortly after the onset of cachexia, which continued throughout the study. This study measured protein turnover in vivo over a time course to establish the initial and longer-term mechanisms leading to wasting in C26 mice. Protein degradation measured in vivo is determined indirectly as the difference between growth and synthesis and therefore only provides an estimate of protein degradation; these results need to be interpreted with caution. At the very onset of cachexia, protein synthesis was severely reduced and degradation greatly increased in skeletal muscle from tumor-bearing mice. These changes in protein turnover were initiated before any atrophy of skeletal muscle was detected, which just preceded the most catabolic phase of the disease. Protein synthesis in tumor mice remained lower over the experiment but degradation returned to normal. Clearly, initial wasting was mediated by decreased protein synthesis and increased degradation and longer-term wasting solely by decreased synthesis.

Acute effects of chemotherapy on skeletal muscle protein turnover. Skeletal muscle is of interest when studying recovery from cancer cachexia after chemotherapy. It accounts for >45% of whole body protein mass (19), and the profound muscle weakness and wasting associated with cachexia makes skeletal muscle restoration of considerable importance. To understand the process of recovery from cancer cachexia after chemotherapy, the possible negative effects of chemotherapy must also be known and considered. Few studies have given attention to the effect of chemotherapeutic agents in well-controlled experimental studies. Most animal studies have tended to focus on the effects of chemotherapy on healthy animals. The hormonal and cytokine milieu, however, is very different during cancer cachexia (29). Le Bricon et al. (14) demonstrated not only that chemotherapeutic agents have a negative impact on protein metabolism but the effect is more severe in tumor-bearing animals. Thus the effect of chemotherapeutic agents on protein metabolism needs to be addressed in the context of cancer cachexia.

Recovery of skeletal muscle from cancer cachexia after chemotherapy. Complete recovery of skeletal muscle involves restoration of previously existing protein mass. No study to our knowledge has investigated the recovery of skeletal muscle after chemotherapeutic treatment of cancer cachexia. Almost complete recovery of skeletal muscle mass occurred in C26-bearing mice cured with cystemustine. Furthermore, cytotoxicity due to chemotherapy did not appear to seriously impede recovery of skeletal muscle in cured mice. However, this may depend on dose and type of agent used. Le Bricon et al. (14) found that some but not all chemotherapeutic drugs inhibited positive nitrogen balance in tumor-bearing rats.

Perspectives