Calpain inhibitors inhibit mitochondrial calpain activity to ameliorate apoptosis of cocultured myoblast.

Cancer cachexia is a fatal syndrome associated with muscle regeneration disability. Tumor factors induce the apoptosis of myoblasts to impair the regeneration of skeletal muscle. Cancer cachectic myoblast apoptosis is associated with mitochondria injury. It has been reported that activated mitochondrial calpain caused mitochondria injury in mouse cardiomyocytes and pulmonary smooth muscle. We wondered if mitochondrial calpains exist in skeletal myoblast and their potential role in myoblast apoptosis of cancer cachexia. We used a transwell to build a novel myoblast-carcinoma cell coculture model to simulate the cancer cachexia environment in vitro. Calpain inhibitors, calpastatin (CAST) and calpeptin (CAPT), were used during coculture. We found for the first time that two calpains (calpain-1 and calpain-2) and CAST were present in the mitochondria of myoblast. The activation of mitochondrial calpain decreased mitochondrial complex I activity, promoted mitochondrial permeability transition pore opening, and impaired mitochondrial membrane potential in myoblast during coculture, which induced myoblasts apoptosis. CAST and CAPT protected myoblasts from apoptosis by inhibiting mitochondrial calpain activity, which may attenuate or even reverse cancer cachectic muscle atrophy by improving muscle regeneration ability. Our study provides a new perspective for understanding the mechanism of cancer cachexia, and will further contribute to treat cancer cachexia by focusing on the mitochondrial calpain activity.

Acylated and unacylated ghrelin relieve cancer cachexia in mice through multiple mechanisms.

Cancer cachexia is a wasting syndrome resulting from decreased protein synthesis and increased protein degradation. Calpain-dependent cleavage of myofilament is the initial step of myofilament degradation and plays a critical role in muscle atrophy. Ghrelin is a multifunctional hormone known to improve protein synthesis and inhibit protein degradation. However, its mechanism of action is not fully understood. Here we investigated whether acylated ghrelin (AG) and unacylated ghrelin (UnAG) could protect against cancer cachexia in mice bearing CT26 colorectal adenocarcinoma. We found for the first time that both AG and UnAG could inhibit calpain activity in skeletal muscle of cancer cachectic mice. AG and UnAG also improved tumor-free body weight, grip strength, muscle mass, epididymal fat mass, and nutritional state in tumor-bearing (TB) mice. Moreover, AG and UnAG reduced serum tumor necrosis factor-± concentration, increased Akt activity, and downregulated atrogin-1 expression in TB mice. Our results may contribute to the development of an AG/UnAG-based therapy for cancer cachexia.

Rosiglitazone and imidapril alone or in combination alleviate muscle and adipose depletion in a murine cancer cachexia model.

Rosiglitazone (RGZ) and imidapril improve cancer cachexia via different mechanisms. Therefore, we hypothesized that combination therapy of RGZ+imidapril would further attenuate cancer cachexia in vivo. After injection with colon-26 adenocarcinoma for 9 days, BALB/c mice were randomly divided into the following four treatment groups for 7 days (n = 8 per group): (1) placebo, (2) RGZ, (3) imidapril, and (4) RGZ+imidapril. Eight healthy control animals were also assessed. Body weight, tumor volume, gastrocnemius muscle and epididymal adipose mass, serum metabolic markers and cytokines, and the expression of nuclear factor-κB and two E3 ubiquitin ligases, atrogin-1 and MuRF-1, were measured. From days 14 to 16, all treatments significantly reduced tumor volume (P < 0.05). From days 10 to 16, improvements in the tumor-free body weight were observed in the RGZ and RGZ+imidapril groups. In addition, significant improvements in both gastrocnemius muscle and epididymal adipose mass were observed in all treatment groups (all, P < 0.05). Furthermore, all treatments significantly increased tumor necrosis factor alpha levels as compared to those observed in the healthy control animals (P < 0.001). Insulin levels significantly increased in the placebo group as compared to those in the healthy control group (P < 0.05), which were reduced in all the treatment groups (P < 0.05). Finally, whereas all treatments significantly reduced atrogin-1 levels as compared to the placebo group (all, P < 0.05), significant reductions in MuRF-1 levels were only observed in the RGZ and RGZ+imidapril groups (both, P < 0.05). Thus, all three treatments reduce tumor growth and alleviate cancer cachexia; however, synergistic effects of RGZ+imidapril combination therapy were not observed.

SPERT gene silencing inhibits the growth of human colon cancer xenograft tumor in nude mice via p38MAPK/HSP27 signaling pathway.

Colorectal cancer is one of the most common gastrointestinal malignancies and is also a disease of genetic heterogeneity. Our previous studies have shown that SPERT (sprermatid-associated protein) gene may be an underlying oncogene that is associated with the progression of the disease in colorectal cancer patients, and SPERT gene silencing can inhibit the proliferation of colorectal tumor cells and promote cell apoptosis. Here, we use the stably transfected human colorectal cancer cell line RKO to construct an animal xenograft model and study the effect of SPERT gene silencing on animal xenografts. The results showed that SPERT gene silencing can inhibit tumor growth in animals. In addition, through signaling pathway analysis, we found that the p38MAPK/HSP27 signaling pathway may be the molecular mechanism by which SPERT gene silencing inhibits the growth of xenograft tumors in nude mice. Combined with previous data, SPERT gene silencing has the same inhibitory effect on tumor growth in vitro and in vivo. These data suggest that SPERT gene may be a potential target for the treatment of colorectal cancer in clinic.

Inhibition of mitochondrial and cytosolic calpain attenuates atrophy in myotubes co-cultured with colon carcinoma cells.

Cancer cachexia is a life-threatening syndrome characterized by muscle atrophy. Cancer cachectic muscle atrophy (CCMA) is associated with mitochondrial injury. Mitochondrial calpains have been reported to induce mitochondrial injury in mouse cardiomyocytes and pulmonary smooth muscle. In the present study, the presence of calpain in the mitochondria of skeletal muscle and its potential role in CCMA were investigated. Transwell plates were used to develop a myotube-carcinoma cell co-culture model to simulate the cancer cachexia environment in vitro. The calpain inhibitors, calpastatin (CAST) and calpeptin (CAPT), were used to inhibit calpain activity in myotubes during co-culture. Calpain-1, calpain-2 and CAST were found to be present in mouse myotube mitochondria. Co-culture activated calpain in both cytoplasm and mitochondria, which caused myotube atrophy. CAST and CAPT treatment prevented calpain activation in both cytoplasm and mitochondria, which inhibited myotube atrophy during co-culture. Additionally, CAST and CAPT treatment increased mitochondrial complex I activity, decreased mitochondrial permeability transition pore opening and improved mitochondrial membrane potential in myotubes during co-culture. In addition, CAST and CAPT treatment increased AKT/mTOR activity, inhibited FoxO3a activity and decreased atrogin-1 content in myotubes during co-culture. The present findings provide new insights to understand the mechanism of CCMA and further help the development of focused approaches to treat CCMA by manipulating the mitochondrial and cytosolic calpain activity.

shRNA­induced knockdown of the SPERT gene inhibits proliferation and promotes apoptosis of human colorectal cancer RKO cells.

Colorectal cancer is the third most common type of cancer and the fourth leading cause of cancer­related deaths worldwide. Although several genes have been identified to contribute to the pathogenesis of colorectal cancer, there are still many genes with unidentified functions in colorectal cancer. This study aimed to investigate the effect of shRNA­induced knockdown of the SPERT gene on the proliferation and apoptosis of human colorectal cancer RKO cells. SPERT was screened based on the TCGA dataset, and SPERT expression, cell growth, proliferation and apoptosis were detected in shSPERT­ and shCtrl­transfected RKO cells. In addition, the SPERT­related biological pathways were detected using a PathScan® Signaling Antibody Array Kit. We detected lower SPERT expression in shSPERT­transfected RKO cells than in shCtrl­transfected cells at both the translational and transcriptional levels (P<0.05), and an MTT assay revealed a clear­cut decrease in the proliferation of shSPERT­transfected RKO cells relative to shCtrl­transfected RKO cells (P<0.01). A Caspase­Glo® 3/7 assay detected an increase in the caspase­3/7 activity and the number of apoptotic cells in the shSPERT­transfected RKO cells than in the shCtrl­transfected cells (P<0.01), and flow cytometry detected a higher apoptotic rate in the shSPERT­transfected RKO cells than in the shCtrl­transfected cells (20.65±0.26 vs. 5.93±0.06%, respectively, P<0.01). Elevated levels of phosphorylated p44/42 MAPK (ERK1/2), Akt, Bad, HSP27, p38 MARK and Chk2, and elevated PARP and caspase­3 expression levels were detected in shSPERT­transfected RKO cells compared with the shCtrl­transfected cells (P<0.05). The results of the current study demonstrated that knockdown of SPERT suppresses colorectal cancer cell growth and promotes apoptosis. SPERT may serve as an oncogene and may be a potential target for the treatment of colorectal cancer.

Acylated and unacylated ghrelin inhibit apoptosis in myoblasts cocultured with colon carcinoma cells.

Cancer cachexia is a life­threatening syndrome associated with myofiber damage. Tumor factors impair muscle regeneration by promoting myoblast apoptosis. Ghrelin is a multifunctional hormone with an anti­apoptotic effect, but its mechanism of action is not fully understood. In the present study, we investigated whether the coculturing of C2C12 myoblasts with CT26 colon carcinoma cells may induce myoblast apoptosis, and whether acylated ghrelin (AG) and unacylated ghrelin (UnAG) may exert anti­apoptotic effects. We found that the coculture induced myoblast apoptosis and increased tumor necrosis factor (TNF)­α concentrations in the culture medium. Moreover, the coculture increased c­Jun N­terminal kinase (JNK) activity, suppressed Akt activity, increased the mitochondrial Bax/Bcl­2 ratio, impaired mitochondrial membrane potential (Δψm), increased the cytosolic cytochrome c levels, and activated the caspase­3/poly (ADP­ribose) polymerase (PARP) cascade in myoblasts. We also found that either AG or UnAG inhibited these changes. The present study describes a novel in vitro model that can be employed to investigate cancer­induced myoblast apoptosis, and our findings suggest a possible use for AG and UnAG in treating cancer cachexia.

Calpain inhibitors ameliorate muscle wasting in a cachectic mouse model bearing CT26 colorectal adenocarcinoma.

Cancer-related cachexia involves increased protein breakdown through various proteolytic pathways, including the ubiquitin-proteasome pathway (UPP). We hypothesized that a calcium- and calpain-dependent pathway might play a crucial role during the proteolytic procedure, and that pathway interventions would ameliorate cancer cachexia in vivo. After being inoculated with CT26 adenocarcinoma cell culture subcutaneously, BALB/c mice developed cachexia in 12 days. They were then administered with different types of calpain inhibitors individually or in combination for 7 consecutive days. Eighteen healthy mice were also assessed as a control group. Changes in body weight, gastrocnemius muscle mass, tumor volume, food intake, survival time, and serum nutritional markers were monitored. Also measured were the levels of calpains, E3 ubiquitin ligases, and apoptosis-associated markers in gastrocnemius muscle. Our study showed that the intraperitoneal administration of calpain inhibitors significantly improved tumor-free body weight and gastrocnemius muscle mass in all treatment groups. Treatment with calpain inhibitors also ameliorated cachexia-associated negative effects in metabolic profiles and increased survival time in most of the tumor-bearing mice compared with the cachexia controls. Furthermore, calpain inhibitors reduced the calpain activity and the expression of MuRF-1 and atrogin-1 in all treatment groups, while increasing the level of cleaved caspase-3 and BAX and lowering the level of BCL-2 in some groups. These results justify further evaluation of calpain inhibitors both alone and in combination with other candidate agents as a potential new therapeutic strategy for treating cancer cachexia.

Acylated and unacylated ghrelin inhibit atrophy in myotubes co-cultured with colon carcinoma cells.

Cancer cachexia is a result of increased protein degradation and decreased protein synthesis. The multifunctional circulating hormone ghrelin promotes synthesis and inhibits degradation of muscle protein, but its mechanism of action is not fully understood. Here, we investigated whether co-culturing C2C12 myotubes with CT26 colon carcinoma cells induces myotube atrophy, and whether acylated ghrelin (AG) and unacylated ghrelin (UnAG) had anti-atrophic effects. We found that co-culture induced myotube atrophy and increased tumor necrosis factor-alpha (TNF-α) and myostatin concentrations in the culture medium. Moreover, co-culture down-regulated myogenin and MyoD expression, inhibited the Akt signaling, up-regulated ubiquitin E3 ligase expression, and activated the calpain system and autophagy in myotubes. Both AG and UnAG inhibited these changes. Our study describes a novel in vitro model that can be employed to investigate cancer cachexia, and our findings suggest a possible use for AG and UnAG in treating cancer cachexia.

Treatment of cancer cachexia in mice by combination of dsRNA-dependent protein kinase inhibitor and medroxyprogesterone acetate.

Inhibitor of dsRNA-dependent protein kinase (PKRI) and medroxyprogesterone acetate (MPA) improve cancer cachexia via different mechanisms. We aimed to compare these two drugs, alone or in combination, in cancer cachexia in mice. Forty male BABL/c mice aged 6-8 weeks were randomly divided into PKRI, MPA, PKRI+MPA, placebo, and healthy control groups. The first 4 groups were injected with colon-26 adenocarcinoma and fed for 12 days and then treated with PKRI and MPA alone or in combination for 7 days. Body weight, tumor volume, wet weight of gastrocnemius muscle, serum levels of nutritional markers and cytokines were measured. The tumor growth (volume and weight) of mice treated with PKRI, MPA alone or PKRI+MPA was slower than that of placebo group. Wet weight of gastrocnemius muscle was significantly higher in PKRI and PKRI+MPA-treated than in placebo animals (P<0.01). All tumor-bearing mice had a significantly lower level of blood glucose, higher level of serum triglyceride and lower level of serum albumin compared with healthy control (P<0.001). However, PKRI, MPA and PKRI+MPA groups had a significant higher level of blood glucose and lower level of serum triglyceride compared with placebo group (P<0.001). All tumor bearing mice had a significant higher level of serum TNF-α, IL-1 and IL-6 compared with healthy control (P<0.001). Serum level of TNF-α and IL-6 was significantly lower in PKRI and PKRI+MPA-treated than in placebo animals (P<0.01). PKRI alone and combination therapy with PKRI and MPA reduce tumor growth and may alleviate cachexia.

Combined treatment with GH, insulin, and indomethacin alleviates cancer cachexia in a mouse model.

Cancer-induced cachexia involves weight loss, catabolic activity, and inflammation. We have evaluated the effects of various treatments (GH, insulin (INS), indomethacin (IND), and all possible combinations) on cancer cachexia in a mouse model. BALB/c mice that were implanted with colon-26 adenocarcinoma developed cachexia in 9 days. Body weight, tumor volume, tumor-free weight, inflammatory cytokines (tumor necrosis factor-α (TNF-α), interleukin 6 (IL6)), and nutritional markers (glucose (Glu), albumin, triglycerides (TGs)) of treatment and control groups were monitored. In the cachexia group, there was a significant decrease in tumor-free bodyweight by day 11. Treatment with GH+INS+IND significantly alleviated tumor-free bodyweight reduction and cachexia-induced changes in nutritional markers and cytokines, and prolonged survival time. GH+INS+IND treatment was more effective than other treatment combinations in elevating Glu and TGs, reducing TNF-α and IL6 levels, and prolonging survival time. In conclusion, GH+INS+IND alleviated cachexia symptoms in a murine model of cancer cachexia.