Synergistic effects of a combination of dietary factors sulforaphane and (-) epigallocatechin-3-gallate in HT-29 AP-1 human colon carcinoma cells.
OBJECTIVE
The objective of this study was to investigate combinations of two chemopreventive dietary factors: EGCG 20 microM (or 100 microM) and SFN (25 microM) 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 microM SFN and 20 microM EGCG; and 175-fold with 25 microM SFN and 100 microM 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 muM SFN and 20 muM EGCG; and 15-fold with 25 microM SFN and 100 microM 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 microM and 100 microM EGCG, but not 25 microM 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 microM for the low-dose combination which was scavenged to about 1 microM in the presence of SOD.


CONCLUSIONS
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.
Nair
Sujit
S
Graduate Program in Pharmaceutical Science, Department of Pharmaceutics, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, New Jersey 08854, USA.
Hebbar
Vidya
V
Shen
Guoxiang
G
Gopalakrishnan
Avantika
A
Khor
Tin Oo
TO
Yu
Siwang
S
Xu
Changjiang
C
Kong
Ah-Ng
AN
D002392:Catechin / Q000008:administration & dosage / Q000031:analogs & derivatives*; D002470:Cell Survival / Q000187:drug effects; D016922:Cellular Senescence / Q000187:drug effects; D003110:Colonic Neoplasms / Q000188:drug therapy* / Q000473:pathology; D004357:Drug Synergism; D019073:HT29 Cells; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D017879:Isothiocyanates; D017382:Reactive Oxygen Species; D013454:Sulfoxides; D013482:Superoxide Dismutase / Q000494:pharmacology; D013861:Thiocyanates / Q000008:administration & dosage*; D018808:Transcription Factor AP-1 / Q000235:genetics; D015533:Transcriptional Activation
D006877:Hydroxamic Acids; D017879:Isothiocyanates; D017382:Reactive Oxygen Species; D013454:Sulfoxides; D013861:Thiocyanates; D018808:Transcription Factor AP-1; C012589:trichostatin A; D002392:Catechin; C045651:epigallocatechin gallate; D013482:Superoxide Dismutase; C016766:sulforaphane