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Synergistic Effects of a Combination of Dietary Factors Sulforaphane and (−) Epigallocatechin-3-gallate in HT-29 AP-1 Human Colon Carcinoma Cells

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Abstract

Purpose

The objective of this study was to investigate combinations of two chemopreventive dietary factors: EGCG 20 μM (or 100 μM) and SFN (25 μM) in HT-29 AP-1 human colon carcinoma cells.

Methods

After exposure of HT-29 AP-1 cells to SFN and EGCG, individually or in combination, we performed AP-1 luciferase reporter assays, cell viability assays, isobologram analyses, senescence staining, quantitative real-time PCR (qRT-PCR) assays, Western blotting, and assays for HDAC activity and hydrogen peroxide. In some experiments, we exposed cells to superoxide dismutase (SOD) or Trichostatin A (TSA) in addition to the treatment with dietary factors.

Results

The combinations of SFN and EGCG dramatically enhanced transcriptional activation of AP-1 reporter in HT-29 cells (46-fold with 25 μM SFN and 20 μM EGCG; and 175-fold with 25 μM SFN and 100 μM EGCG). Isobologram analysis showed synergistic activation for the combinations with combination index, CI < 1. Interestingly, co-treatment with 20units/ml of SOD, a free radical scavenger, attenuated the synergism elicited by the combinations (2-fold with 25 μM SFN and 20 μM EGCG; and 15-fold with 25 μM SFN and 100 μM EGCG). Cell viability assays showed that the low-dose combination decreased cell viability to 70% whereas the high-dose combination decreased cell viability to 40% at 48 h, with no significant change in cell viability at 24 h as compared to control cells. In addition, 20 μM and 100 μM EGCG, but not 25 μM SFN, showed induction of senescence in the HT-29 AP-1 cells subjected to senescence staining. However, both low- and high-dose combinations of SFN and EGCG attenuated the cellular senescence induced by EGCG alone. There was no significant change in the protein levels of phosphorylated forms of ERK, JNK, p38, and Akt-Ser473 or Akt-Thr308. Besides, qRT-PCR assays corroborated the induction of the luciferase gene seen with the combinations in the reporter assay. Relative expression levels of transcripts of many other genes known to be either under the control of the AP-1 promoter or involved in cell cycle regulation or cellular influx–efflux such as cyclin D1, cMyc, ATF-2, Elk-1, SRF, CREB5, SLCO1B3, MRP1, MRP2 and MRP3 were also quantified by qRT-PCR in the presence and absence of SOD at both 6 and 10 h. In addition, pre-treatment with 100 ng/ml TSA, a potent HDAC inhibitor, potentiated (88-fold) the synergism seen with the low-dose combination on the AP-1 reporter transcriptional activation. Cytoplasmic and nuclear fractions of treated cells were tested for HDAC activity at 2 and 12 h both in the presence and absence of TSA, however, there was no significant change in their HDAC activity. In addition, the H2O2 produced in the cell system was about 2 μM for the low-dose combination which was scavenged to about 1 μM in the presence of SOD.

Conclusion

Taken together, the synergistic activation of AP-1 by the combination of SFN and EGCG that was potentiated by HDAC inhibitor TSA and attenuated by free radical scavenger SOD point to a possible multifactorial control of colon carcinoma that may involve a role for HDACs, inhibition of cellular senescence, and SOD signaling.

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Abbreviations

AP-1:

activator protein

EGCG:

(−) epigallocatechin-3-gallate

HDAC:

histone deacetylase

MAPK:

mitogen-activated protein kinase

qRT-PCR:

quantitative real-time PCR

SFN:

sulforaphane

SOD:

superoxide dismutase

TSA:

Trichostatin A

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Acknowledgements

Sujit Nair is extremely grateful to Ms. Donna Wilson at the Keck Center for Collaborative Neuroscience, Rutgers University, for exhaustive training and extensive discussions that were very helpful in optimizing and validating the quantitative real-time PCR assays. This work was supported in part by RO1-CA073674 and RO1-CA092515 to Ah-Ng Tony Kong from the National Institutes of Health (NIH).

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Nair, S., Hebbar, V., Shen, G. et al. Synergistic Effects of a Combination of Dietary Factors Sulforaphane and (−) Epigallocatechin-3-gallate in HT-29 AP-1 Human Colon Carcinoma Cells. Pharm Res 25, 387–399 (2008). https://doi.org/10.1007/s11095-007-9364-7

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  • DOI: https://doi.org/10.1007/s11095-007-9364-7

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