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Furin targeted drug delivery for treatment of rhabdomyosarcoma in a mouse model

Furin targeted drug delivery for treatment of rhabdomyosarcoma in a mouse model. and shRNA expression was induced by administration of DOX-supplemented food 7 days prior engraftment of tumor cells (Physique ?(Figure5A).5A). Control groups were fed normal food. Tumor growth was monitored over time through caliper measurements and mice were sacrificed once tumors reached a size of 1000 mm3. Rh30 scri tumor growth was comparable with or without DOX supplement and tumors reached a size of 300 mm3 after 25 days. Conversely, early tumor growth was clearly delayed in Rh30 shFAi and shFEi tumors in the presence of DOX and tumors took approximately 50% more time to reach 300 mm3 (Physique ?(Physique5B,5B, shFAi – no DOX: 21 days, DOX: 31 days; shFEi – no DOX: 22 days, DOX: 34 days). After reaching a size of 300 mm3 most Rh30 tumors grew at the same pace independent of the genetic background. To investigate Rupatadine furin silencing efficiency we extracted RNA from tumor tissue at sacrifice and evaluated furin mRNA levels by qRT-PCR. Furin mRNA levels of Rh30 shFAi or shFEi tumors with DOX as compared to respective tumors without DOX were on average 15% and 11%, respectively (Physique ?(Physique5C),5C), confirming Rupatadine effective furin silencing. Despite having low furin levels, once furin silenced Rh30 tumors exceeded a size of approximately 300 mm3, they grew almost as fast as control tumors. However, a clear delay in early tumor growth could be observed, suggesting that furin activity is important in early phases of Rh30 tumor growth and resulted in regression or delayed tumor growth of aRMS Rupatadine tumors. Furin is known to process many growth factors and their receptors, like IGF-1 and 2, IGF1R or transforming growth factor (TGF) [34, 35]. Accordingly, silencing of furin led to significant reduction of cell viability and proliferation rate in all investigated aRMS cell lines. Furin silencing had no effect on MRC5 fibroblast viability, suggesting that aRMS, but not normal cells are dependent on furin activity. A subset of the aRMS cell lines tested was particularly sensitive to furin silencing and underwent BAX/BAK- and caspase-9-dependent apoptosis. This indicates that furin silencing primarily triggers the intrinsic apoptotic pathway. Others have shown that disruption of the IGF1R and/or PI3K/mTOR pathways leads to apoptosis in Rh3 and Rh4 cell lines [12, 33]. In this study we found evidence that activation of the IGF signaling pathway is dependent on furin activity. We therefore hypothesize that incomplete activation of growth factor-dependent pathways is the principal cause of cell death induced upon furin silencing in sensitive cell lines. It remains to be investigated which proapototic IGFBP3 BH3-only proteins are involved, but treatment of Rh3 and Rh4 cells with a combination of the IGF1R inhibitor AEW541 and the dual PI3K/mTOR inhibitor BEZ235 results in BMF-dependent apoptosis [33]. Other proapototic BH3-only proteins linked to growth factor deprivation are BAD, BIM and PUMA. For instance, phosphorylation of BAD through active protein kinase Akt results in binding and sequestration by 14-3-3 scaffold proteins [36, 37]. Thus, lower levels of activated Akt due to decreased IGF signaling upon furin silencing might promote release of BAD and mitochondria pore formation. Further investigations will be required to elucidate the exact mechanism of cell death after silencing of furin. To examine the impact of furin silencing on aRMS tumor growth we engrafted two aRMS cell lines, Rh30 and the sensitive Rh4. In Rh30 xenografts, upon furin silencing, we observed delay of early.