Archives

  • 2022-08
  • 2022-07
  • 2022-06
  • 2022-05
  • 2022-04
  • 2021-03
  • 2020-08
  • 2020-07
  • 2020-03
  • 2019-11
  • 2019-10
  • 2019-09
  • 2019-08
  • 2019-07
  • SCH 58261 br So far only indirect

    2022-07-06


    So far, only indirect evidences on the beneficial or detrimental effect of autophagic degradation in experimental cancer cachexia are available. The present work is aimed at overcoming such limita-tions by directly modulating (blocking or activating) autophagy in order to measure the resulting effects on muscle mass and quality.
    Results
    Beclin-1 deficiency does not prevent muscle wasting in tumor-bearing mice
    To understand whether muscle wasting in cancer cachexia could be prevented by blocking stress-induced (beclin-1-regulated) autophagy, beclin-1 was knocked down in the tibialis anterior (TA) muscle via electroporation of a specific shRNA, leading to a 50% reduction of the protein levels (Fig. 1a). As expected, in C26-bearing mice TA mass was reduced (39%) when electroporated with scramble sequence, while it was moderately pro-tected against loss (24%) in condition of beclin-1 deficiency. Such a difference could not be appreci-ated in non-electroporated muscles such as the gastrocnemius (Fig. 1b).
    However, beclin-1 knockdown did not result in improved TA fiber cross-sectional area (CSA; Fig. 1c) and induced p62 accumulation in the muscle of both control and tumor-bearing (TB) animals, in the latter going beyond the already increased SCH 58261 (Fig.1d). Morphological data were con-firmed biochemically (Fig. 1e), showing that beclin-1 loss-of-function prompted an abnormal p62 increase and beclin-1-independent LC3B accumulation, likely due to a reduced autophagy flux, as previously suggested [13]. Similar results were obtained in a different experimental setting, by blocking autopha-gy through electroporation of a vector harboring a non-phosphorylatable BCL2 AAA knock-in mutant (see Ref. [14] for details). In this experiment, TA mass was increased by BCL2 AAA expression in control mice (151 ± 12 versus 178 ± 20 mg % i.b.w., p = 0.018), whereas no effect was observed in TB mice (112 ± 27 versus 107 ± 31 mg % i.b.w.). Fiber CSA analysis showed no differences upon BCL2
    AAA expression. On the whole, although cancer-induced muscle wasting is associated with in-creased autophagy, tissue-specific inhibition seems unable to counteract the depletion.
    Muscle wasting is improved by formoterol while maintaining autophagy
    Changing perspective by focusing on anti-wasting treatments, autophagy was measured in TB mice treated with formoterol, a selective β2-agonist that effectively counteracted cachexia in both TB mice and cancer patients [15,16]. Consistently, in mice
    Fig. 1. Muscle specific autophagy inhibition does not spare cancer-induced wasting. (a) Beclin-1 knockdown in the tibialis anterior (TA) muscle of control (C, n = 6) and C26-bearing male mice (C26, n = 7). (b) TA and gastrocnemius (GSN) mass and (c) TA fiber cross-sectional area (CSA) in the above-mentioned groups. (d) Immunofluorescence images of SCH 58261 TA muscle sections stained for p62 (red) and nuclei (blue). (e) LC3B and p62 Western blotting analyses in TA homogenates. *p b 0.05 versus SCR; **p b 0.01 versus SCR.
    2676 Autophagy and mitochondria in cancer cachexia
    Fig. 1 (legend on previous page)
    Autophagy and mitochondria in cancer cachexia 2677
    bearing the C26 tumor, formoterol counteracted the loss of body weight, muscle mass and strength, without protecting from adipose wasting (Fig. 2a). In agreement with previous observations [5], Beclin-1 and LC3B-II levels increased in C26 TB mice (Fig. 2b), while treatment with formoterol only moderately reduced LC3B-II levels (Fig. 2b), not allowing to draw conclusions on formoterol activity on the autophagy flux. An autophagy flux study was then performed in TB mice at early cachexia stages, treating systemically for 2 days with colchi-cine, in order to avoid premature animal death (see Ref. [5]). The results showed that the protection against muscle wasting exerted in the C26 hosts by formoterol was not associated with reduced autophagy flux (Fig. 2c).