Sulforaphane Induction of p21Cip1 Cyclin-dependent Kinase Inhibitor Expression Requires p53 and Sp1 Transcription Factors and Is p53-dependent

Gautam Adhikary,Gerald M. Wilson,Richard L. Eckert,Wen Xu,Yap Ching Chew

doi:10.1074/jbc.m111.305292

Gautam Adhikary, Gerald M. Wilson + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m111.305292

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Abstract

Sulforaphane (SFN) is an important cancer preventive agent derived from cruciferous vegetables. We show that SFN treatment suppresses normal human keratinocyte proliferation via a mechanism that involves increased expression of p21(Cip1). SFN treatment produces a concentration-dependent increase in p21(Cip1) promoter activity via a mechanism that involves stabilization of the p53 protein leading to increased p53 binding to the p21(Cip1) promoter p53 response elements. The proximal p21(Cip1) promoter GC-rich Sp1 factor binding elements are also required, as the SFN-dependent increase is lost when these sites are mutated. SFN treatment increases Sp1 binding to these elements, and the response is enhanced in the presence of exogenous Sp1 and reduced in the presence of ΔN-Sp3. CpG island methylation alters p21(Cip1) promoter activity some systems; however, expression in SFN-treated keratinocytes does not involve changes in proximal promoter methylation. The promoter is minimally methylated, and the methylation level is not altered by SFN treatment. This study indicates that SFN increases p21(Cip1) promoter transcription via a mechanism that involves SFN-dependent stabilization of p53 and increased p53 and Sp1 binding to their respective response elements in the p21(Cip1) promoter. These results are in marked contrast to the mechanisms observed in skin cancer cell lines and suggest that SFN may protect normal keratinocytes from damage while causing cancer cells to undergo apoptosis.

Highlights

Sulforaphane is an important cancer preventative in skin cancer
To understand the molecular basis for this response, we monitored the impact of SFN on p21Cip1 expression. p21Cip1 was selected as a target because it is a key suppressor of keratinocyte proliferation (40 – 44) and because SFN treatment increases p21Cip1 in other cell types [5, 33, 45]
We first examined the impact of SFN treatment on p21Cip1 promoter methylation, as SFN is known to function as an epigenetic regulator [46, 47] and because the p21Cip1 promoter CpG island can be hypo- or hypermethylated in various cell types [48]

Summary

Background

Sulforaphane is an important cancer preventative in skin cancer. Results: SFN induces p21Cip via a p53-dependent mechanism that involves p53 stabilization. This study indicates that SFN increases p21Cip promoter transcription via a mechanism that involves SFN-dependent stabilization of p53 and increased p53 and Sp1 binding to their respective response elements in the p21Cip promoter These results are in marked contrast to the mechanisms observed in skin cancer cell lines and suggest that SFN may protect normal keratinocytes from damage while causing cancer cells to undergo apoptosis. We further show that Sp1 transcription factors are required and that methylation-related epigenetic regulation (i.e. modulation of methylation of the CpG cluster in the p21Cip proximal promoter) is not involved This is in contrast to our recent findings showing that SFN treatment induces apoptosis in skin cancer cells [34]. These findings suggest that topical SFN treatment on the epidermis will halt proliferation of normal epidermal keratinocytes without inducing apoptosis but will cause resident cancer cells to undergo cell death

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION