Abstract 18503: Endogenously Released Factors From Doxorubicin-Treated Breast Cancer Spheroids Contribute to Toxicity in Human Cardiomyocytes: Protective Effect of Combination Therapy With PDE5-mTOR Inhibitors

Background: We previously demonstrated that a novel combination therapy with the phosphodiesterase 5 (PDE5) inhibitor (sildenafil, Sild) and mTOR inhibitor (rapamycin, Rapa) potentiates doxorubicin (DOX)-induced breast cancer cell killing, but attenuates cardiotoxicities. However, it remains unclear whether there are any factors released from cancer cells treated with DOX that contribute to the injury in cardiomyocytes, which can be modified by Sild and Rapa in combination. Therefore, we designed experiments using spheroid cultures of breast cancer and human AC-16 cardiomyocytes to determine the role of endogenously released factors following treatment with DOX.

Methods & Results: Spheroid cultures of breast cancer MDA-MB-231 cells were treated with DOX (1 μμ), and/or Sild (10 μμ) and Rapa (100 nμ) for 48 hours. The medium was replaced and spheroids were further incubated for 48 hours in the fresh medium. Thereafter, the AC-16 cardiomyocytes were treated with the medium from drugs-treated spheroids for 48 hours. Our results showed that treatment with DOX and/or Sild and Rapa was highly effective in reducing the size of spheroids as compared to the control ( Fig. A ). Moreover, Western blots showed that p-mTOR/mTOR and p-S6/S6 were induced and p-AKT/AKT was suppressed after DOX treatment in cancer cells, which were normalized following treatment with Sild and Rapa ( Fig. B ). The medium from DOX-treated spheroids caused increase of cardiomyocytes death as assessed by trypan blue exclusion assay, which was attenuated by co-treatment with the medium from Sild and Rapa treated spheroids ( Fig. C ).

Conclusion: These results suggest that cardiotoxicity of chemotherapeutic agents could be partly contributed by the endogenously released unknown factors from tumors, which can be modulated by a novel combination treatment with Sild and Rapa via regulating mTOR-signaling pathways.