Effect Of Aliskiren On The Progress Of Cancer Cachexia And Its Mechanism

Objective To observe the effects of ASK, a small-molecule renin inhibitor, on cancer cachexia at different time points through the establishment of cachexia model mice. To investigate its effects on the inflammatory cytokines, oxidative stress and the expression of autophagy-lysosomal system and ubiquitin-proteasome pathway- related factors.Methods 64 healthy male BALB/c mice were randomly divided into four groups: healthy controls group(HC), cancer cachexia group(CA), ASK prevention group(AP)and ASK treatment group(AT).To induce cancer cachexia, C26 cells growing in log phase were detached and resuspended as single cells at 1×107/m L, and 100 μL C26 cell suspension was injected subcutaneously into the left axilla of CA,AP and AT. Following C26 cell injection, all mice were monitored daily for the appearance of skin and hair, mental state, BW, tumor growth, and spontaneous activity. Mice in a preceived ASK(10 mg/kg BW) in PBS intragastrically on day 5 after C26 injection, when tumor nodules were palpable. Mice in AT received ASK(10 mg/kg BW) in PBSintragastrically on day 12 after C26 injection, when cachexia was well developed. Simultaneously, HC and CA were given the same amount of PBS instead. Whole body strength and coordination was assessed on day 20 after C26 cell injection using a Grip Strength Meter and the rotarod performance test. Eight mice from each group were sacrificed, on day 20 after C26 inoculation, with blood immediately collected from the retroorbital venous sinus and tumor tissue as well as the bilateral gastrocnemius muscles isolated and weighed. The gastrocnemius muscle was stained with hematoxylin and eosin(HE).Serum angiotensin(Ang) I and Ang II levels and both serum and muscular tumor necrosis factor-alpha(TNF-α) and interleukin-6(IL-6) levels were determined by ELISA. The activities of superoxide dismutase(SOD) and glutathione peroxidase(GSH-Px) were determined by xanthine oxidase mold method(hydroxyl ammonia) and reduced glutathione assay. The levels of ? OH and malondialdehyde(MDA) were determined by thiobarbituric acid(TBA) method and Fenton reaction. The m RNA and protein expression of Bnip3, LC3, Beclin1, Mu RF1, Atrogin1 and Caspase3 were detected by Real-time PCR and Western bolt. The other eight from each group were continued to be monitored daily and used to assess whole-body functions(as described below) and survival time.Results 1 General conditions and changes in body mass of mice On day 5 after C26 cell inoculation, tumor nodules were palpablethrough the skin. On day 12, mice in the CA and AT groups showed obvious signs of cachexia, including rough skin, disheveled fur, reduced motility, and dramatic weight loss(P<0.05, compared with mice in the HC group). By day 20, mice in the CA group presented the most dramatic weight loss, followed by those in the AT group, and the least weight loss was observed in mice of the AP group(P<0.05). Consistently, the percentage decrease in tumor-free body mass from day 0 to day 20 was most significant in mice in the CA group(32.67%), and this percentage was significantly higher than that in the AT group(21.21%) or the AP group(12.42%)(P<0.05). 2 Tumor tissue and skeletal muscle consumption ASK as a treatment agent or an early prevention strategy led to reductions in both tumor mass and tumor volume(P<0.05, compared with those in the CA group), with the most robust anti-tumor effects observed in the AP group(P<0.05). While, ASK as a treatment agent or an early prevention strategy led to addition in both gastrocnemius mass and crosscut area of gastrocnemius muscle(P<0.05, compared with those in the CA group), with the most robust anti-muscle wasting effects observed in the AP group(P<0.05). 3 Whole-body fancation, locomotor activity and mouse survival time Treatment with ASK on day 12(AT group) improved grip strength and the latency-to-fall during the rotarod test by 21.17% and 16.98%, and prevention using ASK on day 5 improved these two parameters by 39.06%and 34.56%(P<0.05, compared with the AT group). Similar alterations in locomotor activity also were noticed in different groups, and the best locomotor activity among tumor-bearing mice was observed in the AP group(P<0.05, compared with the AT group).Functionally, ASK resulted in significantly improved survival in the AT and AP groups(P<0.05, compared with the CA group), and the best survival was observed in the AP group(P<0.05, compared to the AT group). 4 RAS and inflammatory cytokine levels of mice ASK led to serum up-regulation of Ang I and II to a much lower degree, with less upregulation observed in the AP group than in the AT group(P<0.05).The serum and muscular levels of pro-inflammatory cytokines TNF-α as well as IL-6 in the AT and AP groups were lower than those in CA group, with less upregulation observed in the AP group than in the AT group(P<0.05). 5 Indicators of oxidative stress in mice gastrocnemius Following ASK treatment, the muscular activities of SOD and GSH-Px were increased, whereas the levels of ? OH and MDA were decreased respectively in AP and AT group(P<0.05, compared with the CA group). AP group was more effective compared with the AT group(P<0.05). 6 The expression of autophagy-lysosomal system and ubiquitin proteasome pathway- related factors in mouse gastrocnemius muscle The m RNA and protein level of Bnip3, LC3, Beclin1, Mu RF1 andAtrogin1 in gastrocnemius muscle were dramatically inhibited and the ratio of LC3-Ⅱ/ LC3-Ⅰwere decreased in the AT and AP groups(P<0.05, compared with the CA group), with more robust inhibition achieved in the AP group(P<0.05, compared with the AT group).Conclusions ASK can attenuate the loss of body mass, inhibit the growth of tumor, improve whole-body functions, inhibit muscle wasting, enhanced locomotor activity and prolong mouse survival. The prevention effect of ASK is superior to that of treatment. ASK exhibited potent anti-cachexia activities. These activities may be achieved through inhibiting RAS activation, decreasing systematic inflammation, downregulating oxidative stress, and which in turn to inhibit autophagy-lysosomal system and ubiquitin-proteasome pathway.