The effect of cancer-induced cachexia on mitochondrial fusion and fissionStradovnik, Kaja (Avtor) Kreft, Marko (Mentor) Grefte, Sander (Komentor) cancer-induced cachexiamitochondriagene expressionmitochondrial fusion and fissionRT-qPCRwestern blotcachectic markersmyosin switchingCancer cachexia is a devastating disease resulting in end-stage cancer. There is a need to recognize cachexia in early stages and develop prevention therapies. While the effect of cachexia inducing treatments on mitochondrial dynamics remains uncertain due to limited, ambiguous results, understanding the regulation of its mediators is needed to improve the knowledge about its complex molecular mechanisms. Therefore, our study aimed at observing the effect of C26 colon adenocarcinoma (C26) conditioned medium (CM) and Lewis lung carcinoma conditioned medium (LLC) CM on mitochondrial dynamics. Methods: Six days of differentiated C2C12 myotubes were exposed to C26 CM, LLC CM, and lipopolysaccharide (LPS) medium for 24 hours. The harvested cells were used to determine the expression of fusion (Mfn1, Opa1) and fission genes (Dnm1l, Fis1, Mff), cachectic markers (Atrogin1 and MuRF1), and myosin genes (Myh1, Myh4, Myh7). Protein expression of Drp1, a crucial protein in mitochondrial fission, was determined with western blot. An additional experiment was performed subsequently where the conditioning was performed alongside the differentiation process for 7 days. Cells were left overnight for attachment, later to be conditioned with the prior-mentioned CMs. Results: Only LLC treatment was able to significantly upregulate one of the cachectic markers, Atrogin1, after 24 hours of incubation. Myosin gene expression was shown to be altered after LPS treatment, where Myh4 was significantly downregulated. Mitochondrial fusion and fission-related genes do not show significant alteration in RNA abundance. In our additional experiment, all the expected cachexia-related gene expression changes were observed, as both cachectic markers were highly upregulated and adequate myosin expression switching was shown in all treatments compared to our negative control. Conclusions: Upon 24 hours of C26, LLC, and LPS treatment, hardly any changes were observed, indicating that the treatment regime was not adequate and should be adjusted. The possible explanations for the literature discrepancy are the differentiation time of C2C12 myotubes, short conditioning time, and low serum concentration. We observed the difference in initial cachexia inducement between LLC CM and LPS. Interestingly, 7-day conditioning suggests a novel insight into mitochondrial dynamics pathway involvement, where upregulation of mitochondrial fusion and decreased fission were proposed.[K. Stradovnik]20212021-11-04 07:15:02Magistrsko delo/naloga132816UDK: 601.4:577.21:575.116.4:576.311.347(043.2)VisID: 202715COBISS_ID: 83912963sl