N-Acetylcysteine enhances the lung cancer inhibitory effect of epigallocatechin-3-gallate and forms a new adduct

Abstract

The major tea polyphenol, (-)-epigallocatechin-3-gallate (EGCG), inhibits carcinogenesis in many in vivo models. Many potential mechanisms of action have been proposed based on cell line studies, including pro-oxidant activity. In the present study, we studied the effect of N-acetylcysteine (NAC) on the inhibitory effects of EGCG on lung cancer cell growth. We found that NAC (0 -2 mM) dosedependently enhanced the growth inhibitory activity of EGCG against murine and human lung cancer cells. The combination of NAC and EGCG caused an 8.8-fold increase in apoptosis in CL13 mouse and H1299 human lung cancer cells compared to treatment with either agent alone. Addition of 2 mM NAC increased the stability of EGCG in the presence of CL13 cells (t 1/2 = 8.5 h vs. 22.7 h). Intracellular levels of EGCG were increased 5.5-fold by the addition of 2 mM NAC. HPLC and LC-MS analyses of cell culture medium from CL13 cells treated with EGCG and NAC for 24 h revealed that EGCG-2′-NAC was time-dependently formed. This adduct was not formed in the absence of NAC. The present results show that under cell culture conditions, EGCG and NAC interact to form a previously unreported adduct, EGCG-2′-NAC, which may contribute to enhancement of EGCGmediated cell killing.

Covalent Binding of Tea Catechins to Protein Thiols: The Relationship between Stability and Electrophilic Reactivity

“…Previous reports indicated that oxidized EGCg reacts rapidly with a thiol group of cysteine residues in GSH or NAC to form thiol adducts, 19,25) but no EGCg-adducts were detected by HPLC analysis under this experimental condition with a 10-fold molar excess of thiol compounds (data not shown). GSH and NAC have been found to have an antioxidant role by direct scavenging of reactive oxygen species such as hydrogen peroxide.…”

“…17) Recently, it was found that oxidation of polyphenols with a catechol structure in the B-ring leads to the formation of a polyphenol quinone, which reacts electrophilically with thiol groups in GSH or protein thiols to form covalent thiol-flavonoid adducts. 18,19) More recently, we found that 3,4-dihydroxyphenyl acetic acid and EGCg form covalent adducts with protein thiols (-actin and glyceraldehyde-3-phosphate dehydrogenase: GAPDH respectively) via autoxidation (Fig. 2.).…”

The Effect of (−)-Epigallo-catechin-(3)-gallate on Amyloidogenic Proteins Suggests a Common Mechanism

“…A number of possible non-covalent and covalent binding modes for EGCG to misfolded polypeptides have been postulated: non-site specific interaction with exposed hydrophobic surfaces through the hydrophobic effect (Ehrnhoefer et al 2008), site-specific hydrogen bonding (H-bonding) (Maiti et al 2006), aromatic π-π-stacking (Scheraga et al 1962), site-specific covalent binding, such as formation of disulfidebridges via cysteines (Lambert et al 2008) and formation of Schiff bases via primary amines (Ishii et al 2011). EGCG binding to amyloidogenic polypeptides has been analyzed using various biophysical and biochemical methods.…”

Natural Compounds as Therapeutic Agents for Amyloidogenic Diseases

“…A number of possible non-covalent and covalent binding modes for EGCG to misfolded polypeptides have been postulated: non-site specific interaction with exposed hydrophobic surfaces through the hydrophobic effect , site-specific hydrogen bonding (H-bonding) (Maiti et al 2006), aromatic --stacking (Scheraga et al 1962), site-specific covalent binding, such as formation of disulfidebridges via cysteines (Lambert et al 2008) and formation of Schiff bases via primary amines (Ishii et al 2011). EGCG binding to amyloidogenic polypeptides has been analyzed using various biophysical and biochemical methods.…”