The effect of cancer-induced cachexia on mitochondrial fusion and fission

Stradovnik, Kaja (author), Kreft, Marko (mentor), Grefte, Sander (co-mentor)

Cancer 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.

• Language: Slovenian
• Keywords: cancer-induced cachexia, mitochondria, gene expression, mitochondrial fusion and fission, RT-qPCR, western blot, cachectic markers, myosin switching
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Place of publishing: Ljubljana
• Publisher: [K. Stradovnik]
• Year: 2021
• PID: 20.500.12556/RUL-132816
• UDC: 601.4:577.21:575.116.4:576.311.347(043.2)
• COBISS.SI-ID: 83912963
• Publication date in RUL: 04.11.2021

Secondary language

• Language: English
• Title: Učinek rakave kaheksije na fuzijo in fisijo mitohondrijev

Abstract

Kaheksija raka je težka bolezen, ki se izrazi v končni fazi raka. Zaradi škodljivih učinkov kaheksije in slabe napovedi preživetja bolnikov je treba kaheksijo prepoznati v zgodnjih fazah in razviti preventivne terapije. Medtem ko učinek kaheksije na mitohondrijsko dinamiko ostaja negotov zaradi nasprotujočih si rezultatov, je za izboljšanje znanja o njegovih kompleksnih molekularnih mehanizmih potrebno razumevanje regulacije njegovih mediatorjev. Zato je bil namen našega magistrskega dela opazovanje učinka dveh rakavih kondicioniranih gojišč na ekspresijo genov mitohondrijske dinamike. Metode: Šestdnevne diferencirane miocevke C2C12 so bile 24 ur izpostavljene mediju za kondicioniranje adenokarcinoma debelega črevesa C26 (C26), mediju pljučnega karcinoma Lewis (LLC), in mediju lipopolisaharida (LPS). Kondicionirane celice so bile uporabljene za določanje izražanja fuzijskih (Mfn1, Opa1) in fisijskih genov (Dnm1l, Fis1, Mff), markerjev kaheksije (Atrogin1 in MuRF1) in genov miozina (Myh1, Myh4, Myh7). Ekspresijo ključnega proteina mitohondrijske fisije, Drp1, smo določili s prenosom western. Kasneje je bil izveden dodatni eksperiment, kjer je bilo 7-dnevno diferenciranje izvedeno sočasno s kondicioniranjem s prej omenjenimi mediji. Rezultati: Po 24-urni inkubaciji je le LLC medij signifikantno povečal gensko izražanje enega izmed kahektičnih markerjev, Atrogin1. Sprememba miozinske genske ekspresije se je odrazila le pri 24-urni inkubaciji z medijem LPS, kjer je bila ekspresija Myh4 znatno znižana. Geni mitohondrijske dinamike niso kazali znatnih sprememb v številčnosti RNA. V svojem dodatnem poskusu smo pri vseh tretmajih opazili vse pričakovane kahektične spremembe izražanja genov, kot sta bila močno povišana ekspresija obeh markerjev kaheksije in opaženo miozinsko gensko preklapljanje, značilno za kaheksijo. Izražanje genov, povezanih z mitohondrijsko fuzijo (Mfn1, Opa1) je bilo močno povišano, medtem ko je bila ekspresija genov mitohondrijske fisije znižana (Dnm1l, Mff), ali nespremenjena (Fis1). Sklepi: Po 24 urah kondicioniranja z mediji C26, LLC in LPS je bilo opaziti le malo genskih sprememb značilnih za kaheksijo. To kaže, da tovrstno kondicioniranje za naš in vitro model ni bilo ustrezno. Naši predlogi, ki razlagajo pomanjkanje značilnega genskega izražanja, so podaljšan čas diferenciacije miocevk C2C12, kratek čas kondicioniranja in nizka serumska koncentracija znotraj medijev. Medtem ko redke opažene spremembe genske ekspresije namigujejo na razliko v začetni indukciji kaheksije, inducirane z medijem LLC in LPS. Presenetljivo, 7-dnevno kondicioniranje nakazuje na nov vidik vpletenosti mitohondrijske dinamike pri kaheksiji, s povišanjem mitohondrijske fuzije in zmanjšanjem mitohondrijske fisije.

• Keywords: rakava kaheksija, mitohondrij, genska ekspresija, mitochondrijska fusija in fisija, kahektični markerji, miozinsko preklapljanje, RT- qPCR, western blot