Systemic Study of Selected Anti-Diabetic Agents in Cancer Cachexia

Abstract

Background and objective: Cancer cachexia can be defined as a syndrome involving multiple factors and accompanied with muscle loss, fat loss, reduction in the nutritional intake, alterations in the carbohydrate metabolism and progresses along with systemic inflammation. Cancer cachexia is the cause of 1/4th of deaths in cancer patients. The syndrome has its impact on the quality of life (QOL) of patients, it can be explained by loss of body weight leading to weakness, inability to perform daily chores, prone to fatigue and thus low self-esteem negatively affecting the quality of life. The impact on the cardiovascular system is evident by the wasting of cardiac muscles and the development of cardiovascular complications. Carbohydrate alteration is specifically characterized by increased peripheral glucose and the development of insulin resistance in cancer cachexia. Peripheral glucose levels increase due to activation of hepatic gluconeogenesis, activation of glycolysis and decreased utilization of glucose by the host. The abnormalities in insulin production and utilization result in the development of insulin sensitivity. Thus, regulating peripheral glucose levels and insulin sensitivity can serve as potential strategy for the management of cancer cachexia. In the present study, we hypothesize that regulation of altered carbohydrate mechanism can serve as a pharmacological approach for the management of cancer cachexia. Antidiabetic agents regulate peripheral glucose and insulin levels, as observed in diabetic patients. In both cancer and diabetes, inflammation, elevated peripheral glucose levels and insulin resistance are common factors and they serve as link between diabetes and cancer. Anti-cancer activity of many antidiabetic agents is reported. Thus, repurposing of antidiabetic agents can be an effective pharmacological approach in management of cancer cachexia. So we selected metformin a biguanide, teneligliptin and vildagliptin from DPP4 inhibitors class and empagliflozin and dapagliflozin from SGLT2 inhibitors class as antidiabetic agents to control altered carbohydrate metabolism in cancer cachexia. In light of the above mentioned facuts, the objective of the present study was evaluate the role of metformin, teneligliptin, vildagliptin, empagliflozin and dapagliflozin in management of cancer cachexia and associated cardiovascular complications. Material and methods: The study was carried out using in vitro and in vivo models. For in vitro study, muscle cell line, C2C12 myoblasts was cultured in a 48 well plate, 1*104 cells were seeded and cultured in growth medium at 37oC in 5% CO2. After cells reached 60-70% confluency, differentiation was induced by changing the growth medium with differentiating medium, leading to myotube formation. The formed myotubes were incubated in LLC-1 conditioned media for 24 h and 48 h, respectively. Followed by treatment of myotubes with selected antidiabetic drugs, metformin (1 mM), teneligliptin (1 μM), vildagliptin (1 μM), empagliflozin (1 μM) and dapagliflozin (50 μM) for 24 h. After completion of treatment the myotubes were evaluated for morphometric changes and myotube viability. For in vivo study, two models i.e. B16F1 induced cancer cachexia model and urethane induced cancer cachexia model were used. BALB/c mice of either sex, at 10-12 weeks of age, weighing 25-35 g were taken for both the animal models. For B16F1 induced cancer cachexia model, B16F1 cells were cultured in growth media containing 10% fetal bovine serum and 1% penicillin/streptomycin at 37o C in 5% CO2. A rapid injection of large volume B16F1 cell suspension (1*106 cells) into the mice tail-vein was administered. Hydrodynamic injection involved injection of large volume of 7.5 % body weight within 5 seconds. Following this, the animals were housed for 21 days for tumor induction. BALB/c mice of either sex, at 10-12 weeks of age, weighing 35-40 g were taken for urethane induced cancer cachexia model. Urethane 1 gm/kg was administered to mice by i.p. route for induction of cancer. Urethane was administered every 3rd day for 8 doses for cancer induction. The mice were housed till 60th day for induction of cancer. After induction period was completed in both the animal models, the animals were given treatment with selected antidiabetic agents i.e. metformin (65 mg/kg), teneligliptin (2.6 mg/kg), vildagliptin (6.5 mg/kg), empagliflozin (1.3 mg/kg) and dapagliflozin (0.65 mg/kg) for 28 days. Various parameter were evaluated like tumor markers, body mass markers, inflammatory markers, carbohydrate markers, protein markers, lipid markers and cardiovascular markers. Results: C2C12 myotube evaluation: The relative myotube diameter and myotube viability was significantly decreased after LCM treatment. After LCM treatment the myotubes were treated with selected antidiabetic agents and, metformin, teneligliptin, vildagliptin and dapagliflozin resulted in significant increase in myotube diameter and myotube viability after, but, empagliflozin treatment did not produce significant increase in myotube diameter and myotube viability. Tumor markers: After completion of induction period, tumor markers like organ weight to body weight ratios were increased significantly. Treatment with selected antidiabetic agents significantly decreased the organ weight to tumor weight ratio in both models. Body mass markers: After completion of induction period, body mass markers like body weight change, food intake, water intake, carcass weight and lean mass were reduced significantly and serum ghrelin levels were increased significantly. Treatment with selected antidiabetic agents significantly increased body weight change, food intake, water intake, carcass weight and lean mass and, significantly decreased serum ghrelin levels in both models. Inflammatory markers: After completion of induction period, inflammatory markers like serum TNFα, serum IL6 and serum CRP were increased significantly. Treatment with selected antidiabetic agents significantly decreased TNFα, serum IL6 and serum CRP in both models. Carbohydrate markers: After completion of induction period, carbohydrate markers like serum glucose, serum insulin and serum LDH were increased significantly and serum GLP1 was decreased significantly. Treatment with selected antidiabetic agents significantly decreased serum glucose, serum insulin and serum LDH and significantly increased serum GLP1 in both models. Expression of glycolytic proteins LDH and PDH was increased in diseased group and treatment with selected antidiabetic agents supressed the expression of glycolytic proteins LDH and PDH. Protein markers: After completion of induction period, protein markers like muscle weights and locomotor count were decreased significantly in both models. In B16F1 model, treatment with metformin, vildagliptin and dapagliflozin significantly increased muscle weights and locomotor count, while teneligliptin and empagliflozin failed to improve muscle weights and locomotor count. In urethane model, treatment with selected antidiabetic agents significantly increased muscle weights and locomotor count. Protein expression of MuRF1 was increased in diseased group and treatment with selected antidiabetic agents supressed the expression of MuRF1 in urethane induced cancer cachexia model. Lipid markers: After completion of induction period, lipid markers such as lipid weights and serum lipids were decreased significantly in both models. In B16F1 model, treatment with metformin, teneligliptin, vildagliptin, empagliflozin and dapagliflozin significantly increased brown adipose tissue weight, serum triglyceride, serum HDL and serum VLDL levels. In urethane model, treatment with metformin, teneligliptin, vildagliptin, empagliflozin and dapagliflozin significantly increased sub-cutaneous fat weight, serum triglyceride and serum HDL. Cardiovascular markers: After completion of induction period, cardiovascular markers such as hypertrophic markers, cardiac markers and hemodynamic markers were altered significantly in both models. Treatment with metformin, teneligliptin, vildagliptin, empagliflozin and dapagliflozin significantly improved the hypertrophic markers, cardiac markers and hemodynamic markers in both models. Histopathological analysis: Histopathological analysis in gastrocnemius muscle and heart tissue showed increased muscular dystrophy, irregular cellular architecture, irregular connective tissue and reduced collagen content in disease control group in both models. In mice treated with metformin, teneligliptin, vildagliptin, empagliflozin and dapagliflozin reduced muscular dystrophy, continuous cellular arrangement, continuous connective tissue and increased collagen content was observed as compared to the disease control group in both models. Conclusions: The findings of present study demonstrate that selected antidiabetic agents have a beneficial role in cancer cachexia and associated cardiovascular complications. The selected antidiabetic agents exhibited the anti-cachectic activity by regulating the glucose and insulin metabolism and reducing inflammation.