Novel Endoperoxides Increase Apoptosis only in Cancer Cells by Inducing Oxidative Stress
Authors: Gornick Emily S. , Kim Yechan , Haq Mohammed , Dinsmonaite Ugne , Vavrek Tyler R. , Malik Hamzah , Poch Mark , Rubush David M. , Sarathy Jayashree 
Published: June 2021

Artemisinin (ART), a standard treatment for malaria, is also known to have anti‐cancer properties. Our goal is to develop economical and novel ART analogs that can induce apoptosis in cancer cells at low doses with minimal toxicity. We previously showed that DMR1, a trioxane ART analog, induced apoptosis in T84 human colon cancer cells (Faseb J, 2017:31, 774.24). To explore better analogs, we synthesized HSM2, a novel dioxazinane ART analog with potentially improved pharmacokinetic properties over DMR1, and examined its efficacy in inducing apoptosis in cancer cells. The endoperoxide of ART analogs is the key pharmacophore, and we hypothesize that reactive oxygen species (ROS) generation in cancer cells is responsible for inducing cell death. Therefore, we investigated its role in DMR1/HSM2‐induced cell death.T84 and A549 (lung cancer) cells were grown to confluency in 12‐well plates in fetal bovine serum‐supplemented media. Cells treated (18 hrs) with 0 – 100μM of DMR1 or HSM2 ± 1mM NAC (N‐acteyl cysteine; ROS scavenger) in serum‐free media were stained with FITC‐Annexin V and propidium iodide to measure apoptosis and cell death or CellRox Green and Sytox Red to measure mitochondrial and nuclear ROS and viability, respectively. Samples were assayed by flow cytometry (BD Accuri) and/or imaged using fluorescence microscopy (ZOE‐BioRad). Cytotoxicity was measured by a lactate dehydrogenase (LDH) colorimetric assay.Flow cytometry analysis suggested that DMR1 was more effective in inducing apoptosis in A549 than in T84 cells (% Annexin V+ cells at 18 hr: A549 (n≥3): DMSO: 15 ±1; DMR1: 5μM: 45±4.5, 50μM: 46±4; vs.T84 (n=4) DMSO: 9±1; DMR1: 5 μM: 20±0.4; 10 μM: 15±2; 50 μM: 12±1). Fluorescence imaging revealed that the new ART derivative, HSM2, had a greater apoptotic effect than DMR1 on A549 cells (Image J analysis, in RGB mean pixel intensity 18 hr: DMSO: 1; HSM2: 5 μM: 38±5; 10 μM: 51±14; 50 μM: 29±8, n=3; vs. DMR1: 5 μM: 8±1; 10 μM: 35±2; 50 μM: 21±7, n=3). Similarly, HSM2 had a greater effect on T84 cell death at lower doses than DMR1. In marked contrast, DMR1 and HSM2 (5 – 50μM) did not induce apoptosis in the non‐cancerous BEAS2B cells, a normal bronchial cell line immortalized by viral transformation. At 10 μM, neither DMR1 nor HSM2 caused LDH release in T84 cells (% cytotoxicity, DMSO: 1.3±0.3; DMR1: 1.9±0.2; HSM2; 2.6±0.5; +ve control, 500 μM CDCA: 19.9±0.5; n=3). Flow cytometry measurements demonstrated that DMR1 increased ROS in T84 cells (% CellRox+ cells, DMSO: 7.5±0.2; 10 μM: 29±2; 100 μM: 28±4). Scavenging ROS with NAC (1mM) confirmed its role in ART analog‐induced cell death (Image J Analysis in RGB mean pixel intensity 18 hr: DMSO: 1.4±0.4; NAC: 3.7±0.8; HSM2, 10 μM: 24±6; DMR1, 10 μM: 32±3; HSM2+NAC: 3.2±1, DMR1+NAC: 2.5±1, n=3). Thus, ART analogs are minimally cytotoxic and induce cell death via apoptosis, but not necrosis, in cancer cells. In colon cancer cells, they cause cell death by increasing ROS. We are currently elucidating the mechanisms by which DMR1 and HSM2 cause apoptosis specifically in cancer cells. Synthesizing additional novel anti‐cancer ART analogs with minimal toxicity and understanding their mode of action will help develop potent therapeutic drugs to treat this disease that accounts for ~ 9 million deaths annually.Support or Funding InformationNSF ‐ MRI: DBI‐1427937 to JS and Institutional Funds, Ben. U. to JS, DMR and MPThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.