Dynamic interactions between skeletal muscle and breast cancer cells following chemotherapeutic treatment

Conradie, Daleen (2019-04)

Thesis (MSc)--Stellenbosch University, 2019.

Thesis

ENGLISH ABSTRACT: Background: Breast cancer is the most common cancer found among women of South Africa with the prominent effective form of treatment being chemotherapy. Many cancer patients receiving chemotherapeutic treatment experience skeletal muscle wasting, however, the contribution of muscle wasting to the metastatic properties of breast cancer and response to current treatment strategies has not yet been fully investigated. The aims of this study were to investigate the reciprocal interactions between mouse breast cancer cells (E0771) and mouse myotubes (C2C12) as well as the effects of doxorubicin (DXR) on these interactions. Methods: Conditioned media was collected from two separate cycles. The initial cycle of conditioned media was collected from E0771 breast cancer cells after treatment with/without 1.6 μM of DXR. Myotubes were then treated with/without DXR as well as conditioned media collected during the initial cycle from the E0771 cells. A new series of E0771 cells were then treated with/without DXR as well as with the second cycle of conditioned media collected from the myotubes. Mitochondrial integrity of myotubes was investigated using MitoSOX™ stain analysis while myotube cell viability and integrity was assessed using a Cell Tracker™ stain analysis. Cell viability of E0771 cells was assessed with an MTT assay and the migratory properties (wound closure) using a migration scratch assay. Western blot analyses were used to determined alterations in proliferation, apoptotic, and epithelial-mesenchymal transition (EMT) signaling pathways. Results: Treatment of myotubes with 1.6 μM of DXR significantly induced mitochondrial ROS production (5.580 ± 0.4, p<0.001) when compared to Control but myotube integrity was maintained. Treatment of E0771 cells with 1.6 μM of DXR compared to Control significantly decreased cell viability (60.354% ± 1.237, p<0.001), significantly increased the phosphor/total ERK expression ratio (3.946 ± 0.520, p<0.001), and significantly decreased the cleaved/total PARP expression ratio (0.651 ± 0.027, p<0.001). Additionally, a significant increase in the percentage of wound closure was also observed in the DXR group (16.049% ± 1.11, p<0.01) compared to Control after 24-hours. E0771 cells treated with myotube conditioned media after treatment of DXR (C.DXR), induced a significant decrease in expression of the cleaved/total PARP ratio (0.662 ± 0.097, p<0.01) as well as a significant difference in percentage of wound closure (17.19 ± 0.758, p<0.001) compared to C.Control. Following treatment of the E0771 cells with myotube conditioned media, harvested after the treatment of conditioned media from DXR treated E0771 cells (C.C.DXR), a significant increase in cell viability (121.743% ± 3.442, p<0.05) when compared to C.C.Control. Additionally, comparison of C.C.DXR to C.C.Control observed a significant decrease in expression of total Akt (65.554% ± 17.55, p<0.05), MCM2 (55.167% ± 14.64, p<0.05), and the cleaved/total PARP ratio (0.456 ± 0.111, p<0.001) was observed. Conclusion: Investigation of the dynamic interactions between myotubes and breast cancer cells revealed novel evidence of the influence of the myotube environments on cancer progression. Our study also revealed novel evidence that this myotube environment significantly affected the response of breast cancer cells to the chemotherapeutic treatment of DXR. These findings identified new mechanisms that may promote breast cancer metastasis, which can be utilized to improve chemotherapy in cancer patients.

AFRIKAANSE OPSOMMING: Agtergrond: Borskanker is die mees algemene kanker wat onder die vroue van Suid-Afrika voorkom, met chemoterapie wat tans die mees effektiewe vorm van behandeling is. Baie kankerpasiënte wat chemoterapeutiese behandeling ontvang, ervaar skeletspieratrofie, maar die bydrae van spierverlies tot metastatiese eienskappe van borskanker asook die reaksie op huidige behandeling is nog nie volledig ondersoek nie. Die doel van die studie was om ondersoek in te stel na die wederkerige interaksies tussen muis borskankerselle (E0771) en muis spiervesels (C2C12) asook die effekte van doxorubicin (DXR) op hierdie interaksies. Metodes: Voorgeskrewe media is versamel uit twee afsonderlike siklusse. Die aanvanklike siklus van gekondisioneerde media is uit E0771 borskanker selle versamel na behandeling met/sonder 1.6 μM van DXR. Spiervesels is dan behandel met/sonder DXR sowel as met gekondisioneerde media wat tydens die aanvanklike siklus van die E0771-selle versamel is. ‘n nuwe reeks van E0771-selle is behandel met/sonder DXR en met die tweede siklus gekondisioneerde media wat van die spiervesels versamel is. Mitokondriale integriteit van die spiervesels is ondersoek deur van MitoSOX™ kleuring met fluoresensie mikroskopie te gebruik terwyl die lewensvatbaarheid en integriteit van spiervesels bepaal is met behulp van 'n Cell Tracker ™ kleuring. Die lewensvatbaarheid van E0771-selle is geassesseer met 'n MTT-toets en die migrerende eienskappe (wond sluiting) met behulp van ‘n migrasiekrapassessering. Westerse kladanalise is gebruik om veranderinge in proliferasie-, apoptotiese- en epiteel-mesenkiemale oorgang (EMT) seinweë te bepaal. Resultate Behandeling van spiervesels met 1.6 μM van DXR het 'n aansienlike induksie van mitochondriale ROS-produksie (5.580 ± 0.4, p <0.001) gelewer n vergelyking met die konrole groep (Control), maar die integriteit van spiervesels het behoue gebly. Behandeling van E0771-selle met 1,6 μM DXR in vergelyking met die konrole groep (Control) het die lewensvatbaarheid van die selle aansienlik verminder (60.354% ± 1.237, p <0.001), die fosfo/totale ERK-uitdrukkingsverhouding (3.946 ± 0.520, p <0.001) laat toeneem en die gesplete/totale PARP-uitdrukkingsverhouding (0.651 ± 0,027, p <0,001). Daarbenewens is 'n beduidende verskil in die persentasie wondsluiting ook waargeneem (16.049% ± 1.11, p <0.01) in vergelyking met die konrole groep na 24 uur. E0771 selle behandel met DXR behandelde spiervesel gekondisioneerde media (C.DXR) het 'n beduidende afname in die uitdrukking van die gesplete/totale PARP-verhouding (0.662 ± 0.097, p <0.01) sowel as 'n beduidende verskil in persentasie wondsluiting veroorsaak (17.19 ± 0,758, p <0.001) n vergelyking met die konrole groep (C.Control). Die behandeling van E0771-selle met spiervesel-gekondisioneerde media, na behandeling met gekondisioneerde media van DXR behandeled E0771-selle (C.C.DXR), 'n beduidende toename in die lewensvatbaarheid van die selle (121.743% ± 3.442, p <0.05) in vergelyking met die konrole groep (C.C.Control). Daarbenewens het die vergelyking van C.CDXR tot C.C.Control 'n beduidende afname in die uitdrukking van totale Akt (65.554% ± 17.55, p <0.05), MCM2 (55.167% ± 14.64, p <0.05), en die gesplete/totale PARP verhouding (0.456 ± 0.111, p <0.001) waargeneem. Gevolgtrekking: Ondersoek na die dinamiese interaksies tussen skeletspiervesels en borskankerselle het nuwe bewyse getoon van die invloed van die spiervesel-omgewings op die verloop van kanker. Ons studie het ook nuwe bewyse getoon dat hierdie spierveselomgewing die reaksie van borskankerselle op die chemoterapeutiese behandeling met DXR aansienlik beïnvloed het. Hierdie bevindings het nuwe meganismes geïdentifiseer wat metastase van borskanker beïnvloed en nuwe teikens uitgewys wat moontlik chemoterapie in kankerpasiënte kan verbeter.

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