PDTC Relieves Cancer Cachexia By Reducing Muscle Atrophy And Adipose Degradation

Background:Cancer cachexia is a devastating, multifactorial and often irreversible syndrome. It is chiefly used in regard to the loss of skeletal muscle and fat, where the general nutrition is defective. The complex, multifactorial origin of cachexia precludes a uniform pathophysiologic profile. These issues have hindered clinical studies both at a mechanistic level and for targeting therapeutic intervention. There is no uniform standard in the screening model of anti-cachexia compounds, both in vitro and in vivo. In vivo anti-cachexia evaluations is costly and time-consuming, it is delayed the discovery and development of anti-cachexia drugs. Optimization system and representative screening model should be employed in the anti-cachexia activity screening. NF-κB signaling pathway is closely related to cancer cachexia muscle atrophy and fat consumption. Pyrrolidine dithiocarbamate (PDTC) can attenuate the development of cachexia through inhibition of IL-6 synthesis regulated by NF-κB. But the direct effect of PDTC on the fat and skeletal muscle cells and molecular mechanism remains unclear, pending further study.Purpose:We induced muscle atrophy and lipolysis model associated with cancer cachexia by TNF-α and C26 cell culture medium in vitro, and establish C26 tumor model in vivo.Then we use in vitro and in vivo model to evaluate the effect of PDTC, exploring its direct effect on fat and skeletal muscle and possible molecular mechanisms.Methods:(1) The MTT assay, glycerol detection kit, immunofluorescence and Western Blot were used to quantified the effect of mature adipocytes and myotubes which were incubated with TNF-a, building lipolysis and muscle atrophy model.(2) C26 cell culture medium was direct incubated with mature adipocytes.Oil red O staining, triglyceride assay kit, immunofluorescence and Western Blot examine the effect of mature adipocytes and myotubes which were incubated with C26 cell culture medium, building muscle atrophy and lipolysis model.(3) The BALB/c mouse were inoculated with o.1 mL C26 cell suspension (1x107/mL), mice were monitored for body weight, food intake and tumor volume, etc., to build the C26 tumor cachexia model mice.(4) The mature adipocytes and mature myotubes were inoculated with PDTC. Viability was determined using MTT assay or CCK8 cell proliferation assy.(5) Mature 3T3-L1 adipocytes were incubated with PDTC and TNF-α(C26 cell culture medium), lipolysis was quantified by Oil Red O staining, glycerol detection kit and triglyceride assay kit. Western Blot was used to detect the activation of NF-κB, Perilipin and related proteins.(6) Myotube were incubated with PDTC and TNF-a (C26 cell culture medium). Immunofluorescence and Image J were used to detected differentiated myotube diameter which was a index of muscle atrophy. The expression of NF-kB, MyoD, MHC,atrophy associated protein MuRF-1 and Atrogin-1 was dectected by Western Blot.(7) We measured weight, epididymal fat and gastrocnemius muscle tissue, tests blood biochemistry, HE staining and microscopic structural changes of gastrocnemius and epididymal adipose tissue to monitori the impact of PDTC on the cachexia in colon 26 tumor-bearing mice.Result:(1) TNF-a (50 ng/mL) made the release of glycerol reach largest and has no effect on the activity of the fat cells. Phospho-p65 expression was increased with the reducing of perilipin protein. The diameter of myotube was less than the control group when the concentration of TNF-a is 100 ng/mL, and the phospho-p65 was upregulation, MHC was downregulation.(2) With the stimulation of C26 cell culture medium, the triglyceride levels and Perilipin decreased significantly. Myotube diameter was decreased, the phospho-p65 was upregulated, MHC was downregulation.(3) C26 tumor-induced weight loss started on day 12. The tumors grown quickly. The body weight and gastrocnemius weight was decreased.(4) In mature 3T3-L1 adipocytes, PDTC (0-100 uM) had no effect on the viability. In myotube, PDTC(>50 μM) caused dose-dependet decrease in viability.(5) In the lipolysis model, PDTC can reduce glycerol release (TNF-a-induced) when it’s concentration is 5 μM, perilipin expression gradually increased depend on the concentration of PDTC. In the adipocyte which incublated with C26 cell culture medium, PDTC caused a dose-dependent increase in triglyceride. Phospho-p65 and phospho-HSL expression decreased with the increasing concentration of PDTC, Perilipin increased in a dose-dependent manner.(6) In the muscle atrophy model, PDTC alleviate the decrease of tube diameter when it’s concentration are 25 μM and 50 μM. Phospho-p65, MuRF-1 and Atrogin-1 were decreased, MyoD and MHC were increased with increasing concentration of PDTC.(7) In the cancer cachexia model, PDTC relieve the decrease of the body weight, gastrocnemius weight and epididymal fat, improving the levels of blood TG, GLU, ALB,depending on it’s dose.Conclusion:The cancer cachexia model of C26 and the lipolysis model and muscle atrophy which were induced by the TNF-a and C26 cell culture medium were successfully established.This bring a convenient system and platform of the cancer cachexia drug research. PDTC can relieve the symptom of the model by inhibiting the activity of HSL and UPS. It also delayed the decrease of body weight, epididymal fat and gastrocnemius muscle weight of the cancer cachexia mouse, showing a better effect on the cancer cachexia.These results support the fact that PDTC to be a powerful anti-cachexia drug.