Study of the Mechanisms of Jadomycin Cytotoxicity in Multidrug Resistant Triple Negative Breast Cancer Cells

Abstract

BackgroundWe previously demonstrated that jadomycins, natural products biosynthesized by the soil bacteria Streptomyces venezuelae, are cytotoxic to drug‐sensitive and multidrug‐resistant breast cancer cells in vitro. Jadomycin cytotoxicity involves the generation of reactive oxygen species (ROS) but the downstream mechanisms contributing to cell death are not completely understood.ObjectiveTo determine if jadomycin anticancer mechanisms involve ROS‐mediated DNA damage and apoptosis in triple negative breast cancer cells in vitro.MethodsMDA‐MB‐231 control (231‐CON) and paclitaxel‐resistant (231‐TXL) breast cancer cells were treated with various concentrations of jadomycin B, F and S or the positive control drug mitoxantrone in the presence and absence of the ROS inducer auranofin or ROS inhibitor N‐acetylcysteine (NAC) for 24 to 36 hours. Western blotting for phosphorylated histone H2AX, a marker of DNA double strand breaks, was used to assess DNA damage. Annexin‐V‐FLUOS and propidium iodide flow cytometric analysis was used to measure apoptosis. The effect of jadomycin treatments on gene expression of topoisomerases IIα and IIβ (Topo IIα and IIβ, respectively) was measured using quantitative PCR. To study the direct inhibition of Topo IIα, Topo IIα decatenation of catenated circular kDNA was measured in the presence and absence of jadomycins.ResultsAll jadomycin treatments and mitoxantrone significantly increased histone H2AX phosphorylation (2.3 – 5.3‐fold) and early apoptosis (1.6 – 3.0‐fold) in 231‐CON and ‐TXL cells versus the vehicle control. NAC and auranofin did not alter jadomycin‐induced DNA damage or early apoptosis. However, auranofin did increase jadomycin‐induced necrosis/late apoptosis (2.7 – 4.5‐fold). Jadomycins caused a significant reduction of Topo IIα and IIβ mRNA expression (4.2 – 8.1‐fold decrease) and directly inhibited Topo IIα in the kDNA decatenation assays.ConclusionsJadomycins induce DNA double strand breaks and apoptosis in drug‐sensitive and multidrug‐resistant MDA‐MB‐231 breast cancer cells. These effects occur independently of ROS generation and may involve gene regulatory and functional inhibition of Topo IIα and IIβ.Support or Funding InformationThe study was funded by the Canadian Breast Cancer Foundation ‐ Atlantic Chapter, the Beatrice Hunter Cancer Research Institute, The Nova Scotia Health Research Foundation and the Dalhousie Pharmacy Endowment. S Hall was a recipient of an Izaak Walton Killam Predoctoral Scholarship. J MacLeod was a recipient of a Beatrice Hunter Cancer Research Institute Cancer Research Training Program Scholarship.