Targeting CAR and Nrf2 improves cyclophosphamide bioactivation while reducing doxorubicin-induced cardiotoxicity in triple-negative breast cancer treatment
Sydney Stern, Dongdong Liang, Linhao Li, Ritika Kurian, Caitlin Lynch, Srilatha Sakamuru, Scott Heyward, Junran Zhang, Kafayat Ajoke Kareem, Young Wook Chun, Ruili Huang, Menghang Xia, Charles C. Hong, Fengtian Xue, Hongbing Wang
Sydney Stern, Dongdong Liang, Linhao Li, Ritika Kurian, Caitlin Lynch, Srilatha Sakamuru, Scott Heyward, Junran Zhang, Kafayat Ajoke Kareem, Young Wook Chun, Ruili Huang, Menghang Xia, Charles C. Hong, Fengtian Xue, Hongbing Wang
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Research Article Oncology Therapeutics

Targeting CAR and Nrf2 improves cyclophosphamide bioactivation while reducing doxorubicin-induced cardiotoxicity in triple-negative breast cancer treatment

Abstract

Cyclophosphamide (CPA) and doxorubicin (DOX) are key components of chemotherapy for triple-negative breast cancer (TNBC), although suboptimal outcomes are commonly associated with drug resistance and/or intolerable side effects. Through an approach combining high-throughput screening and chemical modification, we developed CN06 as a dual activator of the constitutive androstane receptor (CAR) and nuclear factor erythroid 2-related factor 2 (Nrf2). CN06 enhances CAR-induced bioactivation of CPA (a prodrug) by provoking hepatic expression of CYP2B6, while repressing DOX-induced cytotoxicity in cardiomyocytes in vitro via stimulating Nrf2-antioxidant signaling. Utilizing a multicellular coculture model incorporating human primary hepatocytes, TNBC cells, and cardiomyocytes, we show that CN06 increased CPA/DOX-mediated TNBC cell death via CAR-dependent CYP2B6 induction and subsequent conversion of CPA to its active metabolite 4-hydroxy-CPA, while protecting against DOX-induced cardiotoxicity by selectively activating Nrf2-antioxidant signaling in cardiomyocytes but not in TNBC cells. Furthermore, CN06 preserves the viability and function of human iPSC–derived cardiomyocytes by modulating antioxidant defenses, decreasing apoptosis, and enhancing the kinetics of contraction and relaxation. Collectively, our findings identify CAR and Nrf2 as potentially novel combined therapeutic targets whereby CN06 holds the potential to improve the efficacy/toxicity ratio of CPA/DOX-containing chemotherapy.

Authors

Sydney Stern, Dongdong Liang, Linhao Li, Ritika Kurian, Caitlin Lynch, Srilatha Sakamuru, Scott Heyward, Junran Zhang, Kafayat Ajoke Kareem, Young Wook Chun, Ruili Huang, Menghang Xia, Charles C. Hong, Fengtian Xue, Hongbing Wang

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Figure 7

CN06 protects DOX-induced cell death and impaired contractility in hiPSC-CMs.

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CN06 protects DOX-induced cell death and impaired contractility in hiPSC...
(A) Immunostaining of Oct4 and Tra-60α for the pluripotency of hiPSC, and cardiac α-actinin for the pluripotency of hiPSC-CMs. Differentiated hiPSC-CMs were treated with 0.1% DMSO (CT), or CN06 at 1, 5, and 10 μM for 24 hours. (B and C) RT-PCR was used to analyze HO-1 mRNA expression (B), and Western blotting was used to detect the protein expression of HO-1 and Nrf2 (C) (n = 3, data represent mean ± SD, 1-way ANOVA with a Bonferroni post hoc). In separate experiments, hiPSC-CMs were pretreated with vehicle control or CN06 (1, 5, and 10 μM) for 2 hours, followed by a cotreatment with/without DOX (2.5 μM) for another 24 hours. (D) Cell viability was determined using CCK8 reagent. (E) Protein expression of Nrf2, HO-1, and cleaved caspase 3 under the same treatment condition was analyzed using Western blotting. To detect cell contractility, hiPSC-CMs were cultured in the presence of vehicle control or CN06 (10 μM) for 24 hours, followed by a 6-hour cotreatment with DOX (1 μM). (F) Video-based edge detection was used to assess cellular shortening of the hiPSC-CMs during spontaneous contraction (n = 20–35 single cells, data represent mean ± SD, 2-tailed Student’s t test). Analyses of traces were completed using the instrument packaged IonWizard v7.3 data analysis software. (G) The beneficial effects of CN06 as a dual activator of CAR/Nrf2 on CPA/DOX-based treatment of TNBC were depicted in a schematic illustration. All values are normalized to the vehicle control. Statistical significance was determined at **P < 0.01, ***P < 0.001.