2025 AMA Research Challenge – Member Premier Access

October 22, 2025

Virtual only, United States

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Abstract Title: Sildenafil and Rapamycin potentiate chemo-immunotherapy-induced breast cancer cell killing while attenuating its cardiotoxicity

Background: Although Doxorubicin (DOX) and Trastuzumab (HER2 inhibitor) demonstrate synergistic effects on repressing breast cancer and enhancing patient survival, cardiotoxicity remains an undesirable consequence. Thus, cardioprotective drugs become necessary. Sildenafil (Sild) (an inhibitor of cGMP-specific phosphodiesterase 5) combined with DOX can increase the chemotherapeutic efficacy against prostate cancer, while attenuating DOX-induced cardiotoxicity. Rapamycin (Rapa) (an inhibitor of mammalian target of rapamycin, mTOR) also protects against myocardial ischemia/reperfusion injury. As mTOR signaling is overactivated in multiple cancers, including breast cancer, we examined the effects of combination therapy with Sild and Rapa on cardiotoxicity and cytotoxicity with DOX±HER2 inhibitor in human cardiomyocytes and HER2-positive breast cancer cells.

Methods: To determine the role of tumor-derived endogenous factors in causing cardiac injury after DOX+HER2 inhibitor treatment, we designed experiments using spheroid cultures of ErbB2/HER2 positive breast cancer cells (SKBR3) and human cardiomyocytes (AC-16). Spheroid cultures of SKBR3 were treated for 24 hours based on the following groups: (1) control (no treatment), (2) DOX (1 µM), (3) AG-825 (HER2 inhibitor, 5 µM), (4) DOX + AG-825, (5) DOX + AG-825 + Sild (10 µM) + Rapa (100 nM), (6) DOX + AG-825 + Sild, (7) DOX + AG-825 + Rapa, and (8) Sild + Rapa. After replacing treatment media with fresh media, SKBR3 cells were further incubated for 48 hours and medium was collected from each group. Then, AC-16 cells were treated with the medium from drug-treated SKBR3 spheroids for 48 hours. SKBR3 and AC-16 cell death were evaluated with Trypan blue staining and imaging. SKBR3 cell viability was assessed using MTS assay. Western Blots were conducted to evaluate protein expression profiles in AC-16 cells.

Results: DOX+AG-825 significantly increased SKBR3 cell death compared to DOX or AG-825 alone. Sild and Rapa combined significantly augmented the cytotoxic effects of DOX+AG825 on SKBR3 cells compared to individual drug treatments by intensifying DOX+AG-825-induced cell death. The media from DOX+AG825-treated SKBR3 spheroids induced AC-16 death, which was mitigated by DOX+AG825+Sild+Rapa-treated SKBR3 spheroid medium. Western Blots revealed DOX±AG-825 increased cleaved PARP levels, which was suppressed with Sild+Rapa co-treatment, indicating protection against DOX+AG-825-induced cardiomyocyte apoptosis. DOX+AG-825+Sild+Rapa reduced phospho-mTOR and phospho-S6, which represents inhibition of mTOR, specifically mTORC1 activity. Phospho-AKT was increased following DOX+AG-825+Sild+Rapa co-treatment, indicating mTORC2 complex activation and protection against DOX+AG-825 toxicities.

Conclusion: Through modulating mTOR-signaling, combination treatment with DOX+AG-825+Sild+Rapa enhanced chemoimmunotherapy-induced breast cancer cell killing while protecting cardiac cells compared to DOX±AG-825 treatment.

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