EGCG has been shown to have growth inhibitory and pro-apoptotic activity against a number of human cancer cell lines (reviewed in (3
)). These effects have been shown to occur via a number of mechanisms including induction of oxidative stress. In vivo
, EGCG has been shown undergo extensive Phase II metabolism resulting in the formation of methylated, glucuronidated, and sulfated metabolites, as well as cysteine conjugates (10
). Previous studies with glutathione conjugates of MDMA have shown that these metabolites retain the neurotoxic effects of the parent compounds, and have the ability to undergo redox cycling and induce oxidative stress (22
). Here we investigated the in vitro
anticancer, anti-inflammatory, and pro-oxidative effects of analogous metabolites of EGCG: 2′-cysteinyl-EGCG and 2″-cysteinyl-EGCG.
The growth inhibitory effects of both 2′-cysteinyl-EGCG () and 2″-cysteinyl-EGCG () against HT-29 and HCT-116 human colon cancer cells, as well as INT-407A and IEC-6 immortalized intestinal cells, were determined using the MTT assay. Both compounds dose-dependently inhibited the growth of all cell lines tested. 2″-Cysteinyl-EGCG was significantly more potent than 2′-cysteinyl-EGCG resulting in approximately 80% growth inhibition of HCT-116, IEC-6 and INT-407A cells at 40 μM. HT-29 cells were the least sensitive to the growth inhibitory effects of either 2″-cysteinyl-EGCG or 2′-cysteinyl-EGCG (less than 20% growth inhibition at 40 μM).
Figure 2 Growth inhibitory effects of 2′-cysteinyl-EGCG (A) and 2″-cysteinyl-EGCG (B) against HT-29, HCT-116, IEC-6 and INT-407A cells. Cells were treated for 48 h and growth inhibition was determined by the MTT assay. Each point represents n = (more ...)
These results are in contrast to those from studies by us and others on the growth inhibitory activity of other EGCG metabolites. For example, M4 had significantly less growth inhibitory activity than EGCG (5
). These results suggest that an intact A, C, and D-ring system are necessary for growth inhibitory activity. Likewise, the major methylated metabolites of EGCG, 4′-O
-methylEGCG and 4′,4″-di-O
-methylEGCG, have much less growth inhibitory activity than EGCG indicating that the intact trihydroxy ring structures are also critical for growth inhibitory activity (18
Both 2′-cysteinyl-EGCG and 2″-cysteinyl-EGCG demonstrated significant anti-inflammation related activity in the LPS-stimulated RAW264.7 murine macrophage cell line model. LPS stimulation increased arachidonic acid release and NO production by 1.8 and 6.7-fold, respectively compared to control (). Both EGCG-cysteine conjugates (20 μM) reduced the aberrant arachidonic acid release and NO production to unstimulated levels.
Figure 3 Effect of 2′-cysteinyl-EGCG, and 2″-cysteinyl-EGCG on NO production (A) and aberrant arachidonic acid release (B) by LPS-stimulated RAW264.7 murine macrophage cells. Data represent the mean of n = 3. Error bars represent the standard deviation. (more ...)
By contrast, we have previously reported that EGCG had no significant effect on LPS-mediated NO production and arachidonic acid release by RAW264.7 cells (29
). M4 and M6 have differential effects on NO production by LPS-stimulated macrophages. M4, which has an intact gallate ring, was shown to inhibit the production of NO by activated RAW264.7 cells, whereas M6 was not (21
). Overall our new findings with 2′-cysteinyl-EGCG and 2″-cysteinyl-EGCG demonstrate the importance of an intact trihydroxy ring structure for the inhibition of NO production.
Previous studies with EGCG and EGCG-glucuronides have demonstrated that glucuronidation of EGCG on the B- or D-ring has no effect on the ability of the compound to prevention aberrant arachidonic acid release by HT-29 cells, however, glucuronidation of EGCG on the 7-OH position on the A-ring significantly reduced the inhibitory activity of EGCG (30
). These results suggest that the A-ring is more important for this effect than the B- or D-ring.
We and others have reported that EGCG is unstable under cell culture conditions and forms oligomers and H2
. The ability of 2″-cysteinyl-EGCG to produce H2
was compared to that of EGCG (). Incubation of equimolar concentrations of EGCG and 2″-cysteinyl-EGCG (50 μM) produced similar concentrations of H2
after 24 h incubation. The kinetics of H2
formation, however, were accelerated for 2″-cysteinyl-EGCG compared to EGCG. The time to half-maximal H2
level were 15 and 60 min for 2″-cysteinyl-EGCG and EGCG, respectively. These results are similar to those reported by Monks, et al. on the pro-oxidant activity of glutathione-conjugated catechol metabolites of MDMA (22
). Those authors suggest that the neurotoxic effects of MDMA are at least in part due to the enhanced redox cycling activity of these glutathione-conjugated metabolites, and that in the case of MDMA, glutathione conjugation represents a maladaptive metabolic response.
Figure 4 H2O2 generation by EGCG and 2″-cysteinyl-EGCG under cell culture conditions. EGCG or 2″-cysteinyl-EGCG (50 μM) were incubated in serum-free cell culture medium at 37°C under 5% CO2 atmosphere. Each point represents the (more ...)
In summary, the present results demonstrate that, in contrast to other Phase II metabolites of EGCG, 2′-cysteinyl-EGCG and 2″-cysteinyl-EGCG retain the growth inhibitory and anti-inflammatory capacity of EGCG in vitro, and are more pro-oxidative. Whether these effects are observed in vivo, and by extension whether these metabolites contribute to the cancer preventive effects of EGCG, remains to be determined.