Effects of diallyl trisulfide-induced oxidative stress on proliferation, morphology and cell death in cancer cells

Abstract

Literature indicates that garlic possesses antiproliferative- and antimitotic activity due to containing organosulfur compounds including diallyl trisulfide (DATS). However, the role of reactive oxygen species (ROS) in the antiproliferative- and antimitotic effects exerted by DATS remains elusive. The role of oxidative stress in the effects induced by DATS on cell proliferation, the induction of oxidative stress morphology, cell cycle progression and mitochondrial membrane potential was investigated in this study, as well as the role of some possible sources of oxidative stress as induced by DATS, including dysregulation of antioxidant enzymes including superoxide dismutase (SOD) and catalase were also investigated in breast (MDA-MB-231)- and lung cancer cells (A549). Experiments were conducted in the presence or absence of the ROS scavenger, N-acetyl cystein (NAC) to determine if DATS is dependant on ROS as well as in the presence of sodium azide (SA), N, N-dimethyl thiourea (DMTU), 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (Carboxy-PTIO), D-Mannitol (Mannitol), Tiron and Trolox which scavange scavenge O2•, H2O2, •NO, •OH, O2-, and HO2• respectively, to determine the dependency of the effects of DATS on specific ROS. Exposure to 100 µM DATS for 24 h in the MDA-MB-231 cell line reduced cell growth by 36%; this was abolished in the presence of 2 mM NAC. Co-exposure to SA (6 mM), DMTU (8 mM), Carboxy-PTIO (8 µM), Mannitol (80 mM), Tiron (6 mM) or Trolox (40 µM) with 100 µM DATS for 24 h restored cell growth. Exposure of A549 cells to 300 µM DATS for 24 h reduced cell growth by 42% which in the presence of 2 mM NAC was restored. In addition, co-exposure to SA (10 mM), DMTU (8 mM), Carboxy-PTIO (6 µM), Mannitol (20mM), Tiron (2 mM) or Trolox (20 µM) with 300 µM DATS for 24 h also restored cell growth. Fluorescent microscopy demonstrated an increase in H2O2 production by 67% in the MDA-MB-231 cell line after 24 h. In the A549 cell line, H2O2 production increased by 14%, 16%, and 18% after 48 h of exposure. After 24 h exposure to 100 µM DATS in the MDA-MB-231 cell line cell rounding increased to 46 cells from 5. Cell rounding induced by DATS was significantly inhibited by NAC to 9 and to 8, 5, 16, 12 and to 10 by SA, DMTU, Carboxy-PTIO, Mannitol and Tiron. After exposure of the A549 cells to 300 µM DATS for 24 h cell rounding increased from 4 to 45 which was decreased to 3 by NAC, and to 9, 27, 33, 38, 23 and 23 by SA, DMTU, Carboxy-PTIO, Mannitol, Tiron and Trolox. Cell cycle analysis indicated that exposure to 100 µM DATS in the MDA-MB-231 cell line for 24 h increased the quantity of cells occupying the sub-G1 phase by 17% which was decreased to a 1% increase by NAC. After exposure of the A549 cells to 300 µM DATS for 24 h the percentage of cells in the G2M phase increased by 34% which was restored to 19% by NAC. Exposure of the MDA-MB-231 cells to 100 µM DATS for 24 h resulted in a 1.75-fold increase in mitochondrial membrane potential depolarisation; however, after co-treatment with NAC this decreased to a 1.68-fold change. Exposure of the A549 cells to 300 µM DATS for 24 h resulted in an increase in mitochondrial membrane potential depolarisation to 2.71-fold which was reduced to 1.22-fold after co-treatment with NAC. After 24 h exposure of the MDA-MB-231 cells to 100 µM DATS the percentage of SOD inhibition was recorded as 73% compared to cells propagated in growth medium (63%). After exposure of the A549 cell line to 300 µM DATS for 24 h a significant decrease in SOD inhibition was observed from 85% in cells propagated in growth medium to 79%. After exposure of the MDA-MB-231 cells to 100 µM DATS for 24 h a 40% decrease in catalase enzyme activity was observed relative to cells in growth medium. After exposure of the A549 cells to 300 µM DATS for 24 h catalase activity was decreased by 24%. This study contributes further understanding to the role of oxidative stress in the effects exerted by DATS and other naturally occuring small organosulfur compounds on proliferation, morphology and cell death indution in cancer cell lines. Additionally this study provides insight into the role of specific ROS in the effects of DATS; as well as the modulation of endogenous sources of ROS by DATS, such as SOD, catalase and mitochondrial membrane potential depolarisation.