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Inhibition of oxidative stress in HeLa cells by chemopreventive agents
R S Bhimani1, W Troll, D Grunberger
1Department of Environmental Medicine, New York University Medical Center, New York 10016-6451.
Abstract:
12-O-Tetradecanoylphorbol-13-acetate (TPA)-mediated oxidative stress in HeLa cells and its inhibition were studied by fluorometric measurement of H2O2 and by 3H-postlabeling of the oxidized bases 8-hydroxyl-2'-deoxyguanosine (8-OHdG) and 5-hydroxymethyl-2'-deoxyuridine (HMdU). TPA treatment (10 fmol/cell) caused approximately 7-fold increase in H2O2 levels (0.1 nmol TPA/ml), and 5-10-fold increase in 8-OHdG and HMdU (10 nmol TPA/ml). Naturally occurring compounds [caffeic acid phenethyl ester (CAPE), (-).epigallocatechin gallate (EGCG), penta-O-galloyl-beta-D-glucose (PGG) and sarcophytol A (Sarp A)] and the anticancer drug tamoxifen (TAM) were tested as potential chemopreventive agents. These agents dose-dependently inhibited TPA-induced H2O2, 8-OHdG and HMdU. The doses required for a 50% decrease in H2O2 were approximately 2.5 microM for TAM; 5 microM for CAPE, EGCG and PGG; and 75 microM for Sarp A. TAM and PGG (10 microM), EGCG (25 microM), and CAPE (50 microM) abolished TPA-mediated H2O2 production, even below the normal cellular levels. TAM (2.5-20 microM) decreased TPA-mediated HMdU and 8-OHdG formation 2-29 times. Maximum inhibition occurred at 20 microM TAM, which caused an approximately 95% decline in HMdU and 8-OHdG. CAPE was effective at 0.5-50 microM. CAPE (25 microM) decreased 8-OHdG 95%, and HMdU 58%, while Sarp A (250 microM) reduced 8-OHdG by 93% and HMdU by 78%. EGCG (1-25 microM) and PGG (1-10 microM) inhibited of 8-OHdG and HMdU dose-dependently. However, higher doses (50 and 100 microM) decreased the efficacy of that inhibition. Of those agents tested, TAM appears to be the most and Sarp A the least effective. Our results point to these 5 compounds as being potential chemopreventive agents, which at very low doses decrease the tumor promoter-mediated oxidative processes.
Insights
This study shows that tamoxifen (TAM) and natural compounds like CAPE, EGCG, PGG, and Sarp A can inhibit oxidative stress and DNA damage caused by TPA in HeLa cells. TAM was the most effective inhibitor of TPA-induced oxidative stress and DNA damage.
Area of Science:
- Biochemistry
- Cell Biology
- Toxicology
Background:
- 12-O-Tetradecanoylphorbol-13-acetate (TPA) is a known inducer of oxidative stress in cells.
- Oxidative stress can lead to DNA damage, specifically the formation of 8-hydroxyl-2'-deoxyguanosine (8-OHdG) and 5-hydroxymethyl-2'-deoxyuridine (HMdU).
- Identifying agents that can inhibit TPA-mediated oxidative stress is crucial for chemoprevention.
Purpose of the Study:
- To investigate the inhibitory effects of naturally occurring compounds and tamoxifen on TPA-induced oxidative stress in HeLa cells.
- To quantify the reduction in hydrogen peroxide (H2O2) and oxidized DNA bases (8-OHdG, HMdU) by these agents.
- To determine the relative efficacy of different chemopreventive agents against TPA-mediated cellular damage.
Main Methods:
- HeLa cells were treated with TPA to induce oxidative stress.
- Hydrogen peroxide (H2O2) levels were measured using fluorometric assays.
- DNA damage was assessed by quantifying 8-OHdG and HMdU using 3H-postlabeling.
- The inhibitory effects of caffeic acid phenethyl ester (CAPE), epigallocatechin gallate (EGCG), penta-O-galloyl-beta-D-glucose (PGG), sarcophytol A (Sarp A), and tamoxifen (TAM) were evaluated dose-dependently.
Main Results:
- TPA treatment significantly increased H2O2, 8-OHdG, and HMdU levels in HeLa cells.
- All tested agents (TAM, CAPE, EGCG, PGG, Sarp A) dose-dependently inhibited TPA-induced H2O2, 8-OHdG, and HMdU.
- Tamoxifen (TAM) demonstrated the most potent inhibition, reducing TPA-mediated oxidative stress and DNA damage by up to 95% at 20 microM.
- CAPE, EGCG, and PGG also showed significant inhibitory effects, with varying optimal doses.
- Sarcophytol A (Sarp A) was the least effective among the tested compounds.
Conclusions:
- Naturally occurring compounds (CAPE, EGCG, PGG, Sarp A) and tamoxifen (TAM) are effective chemopreventive agents against TPA-induced oxidative stress.
- These agents significantly reduce TPA-mediated increases in H2O2 and oxidized DNA bases.
- Tamoxifen shows the highest efficacy in inhibiting TPA-induced oxidative damage, suggesting its potential in chemoprevention strategies.
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