The impact of cruciferous vegetable isothiocyanates on histone acetylation and histone phosphorylation in bladder cancer
Graphical abstract
Introduction
The field of epigenetics, where genomic modifications alter patterns of gene regulation without altering the nucleotide sequence, has emerged as a crucial area for understanding how our genomes function [1], [2], [3]. The ability of epigenetic modulators, both pharmacologic and natural compounds from dietary sources, to impact carcinogenesis is beginning to be elucidated. Unlike genetic mutations, which are difficult to reverse, epigenetic modulation is dynamic and may lead to changes in gene regulation that can be manipulated by drugs or phytochemicals found in foods [4], [5], [6], [7]. Two principal mechanisms have been identified that contribute to epigenetic modulation of transcription, namely histone modification and DNA methylation [8]. DNA methylation in regions with high GC content (CpG-islands) is the most widely studied modification and is associated with gene silencing [9]. Histones are also key players in transcriptional regulation via binding to DNA, a process modulated by acetylation and phosphorylation. The histones are comprised of four core histones (H2A, H2B, H3 and H4) and the linker histone H1. Two H2A–H2B dimers and one H3-H4 tetramer associate to form the histone octamer, which binds to 147 bp of DNA, wrapping 1.65 turns around the histone octamer to form a nucleosome with about 50 bp of DNA between each nucleosome. Histone H1 is called the “linker histone” because rather than forming part of the nucleosome core, it binds the DNA entry/exit points of the nucleosome [9], [10], [11].
Histones are largely globular in structure, except for their N-terminal tails, which are unstructured. It is on these N-terminal tails that histones possess a diverse set of post-translational modifications. These modifications affect chromatin structure and protein-protein interactions and are instrumental in regulating gene transcription. Histone modifications include: acetylation, methylation, phosphorylation, ubiquitination, SUMOlyation, and ADP-ribosylation, among others [9]. A euchromatic gene conformation is usually associated with high levels of acetylation and trimethylated H3K4, H3K36 and H3K79. Conversely, heterochromatin is characterized by low levels of acetylation and high levels of H3K9, H3K27 and H4K20 methylation [9]. Global loss of monoacetylation and trimethylation of histone H4 are common markers detected in human cancer cells [12]. In addition, we have shown a significant increase in phosphorylation of H1 linker histones in the progression from normal bladder epithelial cells to low-grade superficial to high-grade invasive bladder cancer, reflecting bladder carcinogenesis [13], [14], [15], [16].
Histone acetylation status is regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs) [17]. An increase in HAT activity and/or an inhibition of HDAC activity, leads to higher histone acetylation status. HDACs are grouped into four classes, based on their sequence homology to their yeast orthologs: class I (HDAC 1, 2, 3 and 8), class IIa (HDAC 4, 5, 7 and 9), class IIb (HDAC 6 and 10), class III (Sirtuins) and class IV (HDAC 11). Class I, II and IV are referred to as “classical” HDACs and have Zn2 +-dependent catalytic activity, while class III consists of the Sirtuins and require NAD+ as an essential cofactor [8]. Class I HDACs are found exclusively in the nucleus, while class II shuttle between the nucleus and cytoplasm [12]. The term “HDAC inhibitor” (HDACI) is commonly used for compounds that target classical HDACs [18]. HDACIs have been shown to strongly and selectively induce growth arrest, differentiation and apoptosis in many cancer types [8].
Although the effect of HDACIs on the histone code is increasingly appreciated, this may not be the sole mechanism of action responsible for the anti-cancer effect of HDACIs. Emerging evidence points to HDACI modulation of non-histone targets, including tubulin, p53, Ku70, Hsp9 and STAT3, among many others [8]. These targets can be referred to as the acetylome [19]. In addition, HDACIs have been reported to downregulate Akt phosphorylation by interrupting the interaction between protein phosphatase 1 (PP1) with HDAC 1 or 6 [4]. These emerging targets may more aptly explain the effects of HDACIs on cancer.
We have pursued potential mechanisms whereby cruciferous vegetable phytochemicals may impact bladder carcinogenesis based upon evidence from epidemiologic studies [20], [21]. In the past, we showed the positive correlation of H1 phosphorylation and bladder cancer carcinogenesis and progression. We hypothesize that long-term, low-toxicity dietary modulation of the epigenome may inhibit the carcinogenic process, by preventing silencing of tumor suppressor genes, and reducing H1 phosphorylation, among other mechanisms. We and others have shown the ability of broccoli isothiocyanates, namely sulforaphane (SFN) and erucin (ECN), to inhibit bladder cancer growth in vitro and in vivo by inducing cell cycle modulation in the G2/M phase as well as apoptosis [22], [23], [24], [25], [26]. HDACIs have been reported to disrupt the cell cycle in the G2 phase and induce apoptosis which mirrors our findings and led us to suspect epigenetic modulation as a plausible mechanism of action by which these compounds may act. Furthermore, it has been reported that SFN can act as an HDACI [12], [27], [28]. Prior reports by Dashwood and colleagues ([29], [30], [31] and reviewed extensively in [27]and [32]) showed that the isothiocyanate, sulforaphane (SFN), behaved as an HDAC inhibitor in colon cells. This was supported by numerous immunoblot experiments measuring acetylated histones including H3 and H4. Immunoblotting of histone modifications is not always reliable due to cross reactivity and interfering neighboring modifications. This evidence led us to further examine the potential of isothiocyanates to impact histone status thereby influencing their potential as epigenetic modulators of bladder carcinogenesis and progression [33]. When we examined this effect using mass spectrometry we did not observe the same extent of HDAC inhibition in bladder cancer cells, and we feel that it is an important observation. However, quite remarkably, we did see a very significant change in the histone phosphorylation, which is an indicator of altered phosphatase/kinase activity.
Here we show cruciferous vegetable isothiocyanates, namely sulforaphane (SFN) and erucin (ECN) significantly inhibited histone deacetylase (HDAC) activity in vitro in human bladder cancer cells representing superficial to invasive biology, as well as in invasive bladder cancer in vivo xenografts. Individual HDAC activity inhibited by SFN and ECN included HDACs 1, 2, 4 and 6 while HDACs 3, 5, 7–10 and SIRT1–2 activity remained unchanged. Interestingly, global acetylation status of histones H3 or H4 as revealed by LC-MS remained unaltered while the interplay between HDAC inhibition and modest modulation of AcH3 and AcH4 status may be partially explained by decreased histone acetyl transferase activity in isothiocyanate treated cells. In contrast to the small changes in acetylated histone status, a significant decrease in phosphorylation status of all isoforms of histone H1 was observed, concomitant with increased phosphatase PP1β and PP2A activity. Taken together, these findings suggest that cruciferous vegetable isothiocyanates SFN and ECN modulate histone status and act via epigenetic mechanisms in human bladder cancer cells via HDAC inhibition and phosphatase enhancement allowing for reduced levels of histone H1 phosphorylation, a mark we previously correlated with human bladder cancer progression [13]. Therefore, SFN- and ECN-mediated targeting of histone H1 phosphorylation via epigenetic modulation presents a novel direction of research to elucidate epidemiological relationships and perhaps support future studies of food based prevention strategies in high risk cohorts.
Section snippets
Cell culture
RT4, J82 and UMUC3 human bladder cancer cells were purchased from American Type Culture Collection (Manassas, VA) and cultured in Gibco RPMI-1640 medium (Invitrogen Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum, l-glutamine, penicillin and streptomycin. All cells were grown as monolayer cultures at 37 °C in a 95% air/5% CO2 humidified atmosphere.
In vivo mouse xenografts
Mouse xenograft studies were carried out with strict adherence to protocols approved by the Institutional Animal Care
Sulforaphane and erucin cause inhibition of HDAC activity
We have recently shown that broccoli isothiocyanates (ITCs), particularly sulforaphane (SFN) and erucin (ECN) have the ability to significantly inhibit bladder cancer in vitro and in vivo. There is some evidence that SFN can act as an HDACI in prostate and colon cancers [12], [27]. We tested if the ability of broccoli ITCs to inhibit bladder cancer is in association with epigenetic modulation. To do this, we first examined the ability of the broccoli ITCs SFN and ECN to inhibit HDAC activity in
Discussion
The potential of dietary components, such as broccoli isothiocyanates (ITCs), to prevent bladder cancer carcinogenesis and progression through modification of the cancer epigenome, is an attractive hypothesis. We have shown that broccoli ITCs have the ability to significantly inhibit bladder cancer cell growth in vitro in diverse human bladder cancer cell lines (RT4, J82, UMUC3) and in vivo, through an UMUC3 xenograft model [22]. This inhibition was associated with decreases in cell viability,
Conclusions
In conclusion, our findings suggest that cruciferous vegetable isothiocyanates SFN and ECN modulate epigenetic function in human bladder cancer cells via HDAC inhibition and phosphatase enhancement allowing for reduced levels of histone H1 phosphorylation, a potential biomarker we previously correlated with human bladder cancer carcinogenesis and progression [13]. Thus, SFN- and ECN-mediated targeting of histone H1 phosphorylation via epigenetic modulation presents a novel potential
Abbreviations
- AcH3
acetylated histone H3
- AcH4
acetylated histone H4
- ECN
erucin
- HAT
histone acetyl transferase
- HDAC
histone deacetylase
- HDACI
HDAC inhibitor
- IP
immunoprecipitation
- ITCs
isothiocyanates
- LC-MS
liquid chromatography- mass spectrometry
- SFN
sulforaphane
Funding sources
NIH/NCCAM F31AT006486, NIH/NIGMS 5T32GM068412-04, NIH/NCI OSU CCC P30 CA16058 and the Molecular Carcinogenesis and Chemoprevention Program, OSU CCC James Cancer Hospital Shoen Cancer Prevention Research Fund and Bladder Cancer Prevention Fund; OSU Center for Functional Foods and Research Entrepreneurship (CAFFRE). C.R.L. is a recipient of a National Institutes of Health T32 Award in Oncology Training Fellowship at The Ohio State University Comprehensive Cancer Center, T32 CA009338.
Author contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.
Conflict of interest
The authors have no conflict of interest to declare.
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- 1
These authors contributed equally.
- 2
If an author's address is different than the one given in the affiliation line, this information may be included here.
- 3
Present Address: Parker Food Science and Technology Building, 2015 Fyffe Rd., Columbus, OH 43210, USA.
- 4
Present Address: N350 Scott Laboratory 201 W.19th Ave Columbus, OH 43210, USA.