Phytosterols regulate expression and function of histone deacetylase enzymes

Five studies investigated the role of PSS on HDAC expression, activity or binding^{(Reference Pradhan, Parbin and Kar39,Reference Pradhan, Parbin and Kausar40,Reference Ishola and Adewole45–Reference Sun, Liu and Feng47)} . Two studies by Pradhan and colleagues in cancer cell lines provided in vitro evidence that PSS promote histone acetylation via HDAC inhibition. SITO inhibited the expression of HDAC1 and HDAC2 in MCF7 and MDA-MB-231 breast cancer cells^{(Reference Pradhan, Parbin and Kar39)} and in PC3 and DU145 prostate cancer cells^{(Reference Pradhan, Parbin and Kausar40)}. In a subsequent HDAC activity assay, HDAC activity was inhibited in a SITO dose-dependent manner that was separate from the effect of the PSS on HDAC expression^{(Reference Pradhan, Parbin and Kausar40)}. A relatively high concentration of 80 μM SITO was used in this in vitro analysis; median serum concentration levels of SITO were previously reported to be 2148 ng/ml (5·2 μM) with a range of 308 ng/ml (0·74 μM) to 19 476 ng/ml (47 μM) in a study of >3000 people^{(Reference Stiles, Kozlitina and Thompson9)}. Ishola and Adewole^{(Reference Sowemimo, Fakoya and Awopetu49)} performed molecular docking experiments and predicted several phytosterols could physically interact with class I and class II HDAC leading to inhibition of activity^{(Reference Ishola and Adewole45)}. Forty-nine compounds isolated from Morinda Lucida, a plant found throughout central and southern Africa and used in traditional medicine for the treatment of cancer^{(Reference Daina, Michielin and Zoete50)}, were compared with the HDACi givinostat for their capacity to bind and inhibit HDAC^{(Reference Ishola and Adewole45)}. Of the forty-nine compounds tested, PSS were among the strongest HDAC-binding factors. Interestingly, CAMP and STIG were predicted to bind in the same HDAC pocket as givinostat and with higher binding affinity. An Absorption–Distribution–Metabolism–Excretion analysis using Swiss online Absorption–Distribution–Metabolism–Excretion web tool indicated that several PSS adhere to the Lipinski rules of five for drug development (Table 3), underlining their suitability for clinical translation^{(Reference Ras, van der Schouw and Trautwein51)}.

Table 3. The oral drug likeness of SITO, CAMP, STIG and ATA by Lipinski’s rule of five^{(Reference Ras, van der Schouw and Trautwein51)}

SITO, sitosterol; CAMP, campesterol; STIG, stigmasterol; ATA, Antcin-A; MLOGP, Moriguchi octanol–water partition coefficient.

Contrary to publications above that found PSS inhibited HDAC expression and function were two publications exploring the effect of PSS in lung diseases that found PSS were associated with increased HDAC expression. In a model of steroid-resistant asthma, HDAC2 protein expression was measured in lung tissue of guinea pigs after the animals were given five daily intraperitoneal injections of 50 mg/kg or 100 mg/kg STIG^{(Reference Hohoayi, Antwi and Amoah46)}. Steroid-resistant asthma animals had fivefold lower expression of HDAC2 compared with healthy controls and the addition of STIG (50 mg/kg or 100 mg/kg) ameliorated this loss of HDAC2 expression. In a study using a mouse coronary obstructive pulmonary disease model and mouse macrophage cell line (Raw264·7), exposure to cigarette smoke extract inhibited HDAC3 expression and induced expression of the histone acetylation coactivators P300 and PCAF. When increasing doses of ERGO (2·5, 5 and 10 mg/kg) were added to the cigarette smoke extract treatment, the cigarette smoke extract mediated loss of HDAC3 was ameliorated^{(Reference Sun, Liu and Feng47)}. Neither the data presented by Hohoayi et al., regarding STIG mediated increases in HDAC2 expression, nor by Sun et al., regarding ERGO induction of HDAC3, included analysis of the effect of the PSS on HDAC in control animals, nor did they directly assess HDAC activity. Given that both disease models are associated with very high free-radical environments, HDAC degradation may be inhibited by PSS via protection from oxidative damage and adduct formation from the high levels of free radical characteristic of both these disease models; indeed, this was proposed by Hohoayi and colleagues as a reason why HDAC2 levels remained high.

The experimental observations showing PSS has HDAC modulatory properties may help explain why PSS are so potent at lowering LDL-cholesterol^{(Reference Ras, Geleijnse and Trautwein12,Reference Musa-Veloso, Poon and Elliot13)} , yet have not been conclusively linked to a reduction in CVD risk^{(Reference Genser, Silbernagel and De Backer10,Reference Zhao, Zhuang and Li52–Reference Yoon and Eom55)} . Whether HDACi are beneficial in terms of CVD prevention is context and end point dependent. In their 2016 review, Yoon and Eom described evidence regarding the use of pharmacological HDACi to reduce risk of several CVD outcomes, such as myocardial infarction and hypertension. However, HDACi is was also linked to worse vascular calcification and indeed, and thrombus was a side effect of significant clinical importance in patients taking vorinostat^{(Reference Soteriou, Kalli and Connell56)}. The molecular mechanisms of action of how PSS inhibit HDAC function in the context of CVD therefore require further elucidation. This would also shed light on the role of PSS in other diseases where HDAC activity is important, such as cancer.

Phytosterols decrease expression of DNA methyltransferase enzymes and reduce global DNA methylation

Two studies investigated whether PSS exposure led to changes in DNA methylation by affecting DNMT expression^{(Reference Pradhan, Parbin and Kar39,Reference Pradhan, Parbin and Kausar40)} . In the first, H₂O₂ was used to induce expression of DNMT1 in MCF7 and MDA-MB-231 breast cancer cells^{(Reference Pradhan, Parbin and Kar39)}. However, in the presence of 40 μM or 60 μM SITO, DNMT1 induction was prevented and both mRNA and protein levels of DNMT were reduced by PSS in both cell lines. Moreover, H₂O₂ downregulated the expression of anti-epithelial to mesenchymal transition factor N-cadherin/CDH1, while lupeol and SITO reversed this pro-metastatic repression of CDH1 at mRNA and protein levels. In prostate cancer cell models, DNMT1 was also reduced following exposure of cell lines (PC-3 and DU-145) to methanolic extract of Paederia foetida leaves, lupeol or SITO, for 24 h or 72 h. The data show SITO inhibited DNMT1 expression and activity, decreased the expression of anti-apoptotic protein Bcl2 and upregulated the expression of pro-apoptotic protein Bax; and, again as for breast cancer cells, CDH1 expression was increased^{(Reference Pradhan, Parbin and Kausar40)}.

Given that DNMT play essential roles in activating or silencing oncogenes and tumour suppressor genes, it is interesting to note that PSS appear to have therapeutic actions in regulating these enzymes, in cell lines at least. Further molecular analyses are required to provide a mechanistic understanding of how PSS are able to regulate activity of DNMT enzymes. Specifically, the use of coarse grained and atomistic computational simulations, which have been applied previously to determine lipid–protein interactions (reviewed in^{(Reference Zhang, Moon and Thorne29)}), molecular docking simulations (such as those in^{(Reference Ishola and Adewole45)} or molecular binding analyses) would determine if physical interactions occur. Further work in pre-clinical models and ultimately dietary interventions in cancer patients will be critical to understanding if PSS-mediated changes to DNA methylation patterns can translate into clinically meaningful effects.

Phytosterols regulate miRNA expression

The link between miRNA and PSS exposure was investigated in six records^{(Reference Jiang, Yang and Yuan37,Reference Xu, Li and Han38,Reference Jiang, Li and Niu41–Reference Kumar, Vani and Hsieh43,Reference Senthil Kumar, Gokila Vani and Hsieh48)} , of which two were screening studies^{(Reference Jiang, Yang and Yuan37,Reference Jiang, Li and Niu41)} . The screening studies evaluated SITO in the context of rat neuronal stem cells^{(Reference Jiang, Yang and Yuan37)}, and human aortic endothelial cells^{(Reference Jiang, Li and Niu41)}. In neuronal stem cells, 40 μM SITO upregulated 30, and downregulated 84, miRNA^{(Reference Jiang, Yang and Yuan37)}. While in the HAEC cells, 4·8 μM SITO upregulated 579 and downregulated 112 miRNA, albeit this was in comparison to an ox-LDL treated group^{(Reference Jiang, Li and Niu41)}. Interestingly, despite being performed in different cell types from different species, and with a ten-fold difference in concentration of SITO utilised, four miRNA were significantly altered by SITO in both studies (miR-17-5p, miR-20a-5p, miR-210-3p and miR-532-3p). Although these were not in the top regulated lists in either study and thus were not validated by PCR. In NSC cells, several SITO-regulated miRNA were found to activate the IGF1 signalling pathway, and treatment was associated with increased cellular proliferation^{(Reference Jiang, Li and Niu41)}. Similarly in HAEC, signalling pathway analysis indicated that SITO treatment enriched for AKT signalling, forkhead box O transcription factors and cell cycle signalling^{(Reference Jiang, Yang and Yuan37)}. Contrary to this, the bronchial epithelial airway cell line model of chronic obstructive pulmonary disease (BEAS-2B) responded to very low concentrations of SITO (60 nM for 24 h) by induction of miR-196-5p and suppression miR-361-5p, leading to inhibition of mTOR signalling and reduced proliferation^{(Reference Shen, Liu and Qu42)}.

In cancer, like in the pulmonary model, PSS did not stimulate proliferative or cell survival signalling, but instead tended to suppress these pathways. In BCa cell lines MCF7 and MDA-MB-231 cells, 32 μM SITO-induced apoptosis via induction of the tumour suppressor miR-10a-5p that in turn limited the activity of PI3K-AKT signalling^{(Reference Xu, Li and Han38)}. Also, in MCF7 and MDA-MB-231 cells, 10 μM ATA upregulated miR-200c^{(Reference Kumar, Vani and Hsieh43)} and miR-708^{(Reference Kumar, Vani and Hsieh44)}. Of note is that miR-708 was significantly downregulated by SITO in HAEC cells^{(Reference Jiang, Li and Niu41)}. ATA inhibited multiple components of the epithelial to mesenchymal transition pathway, a critical step in cancer metastasis. ATA induced miR-200c expression in a p53-dependent manner, which led to repression of transcription factor ZEB1, and molecular effectors of epithelial to mesenchymal transition such as E-cadherin and vimentin^{(Reference Kumar, Vani and Hsieh43)}. ATA suppressed the pro-inflammatory NFκB pathway via miR-708-dependent inhibition of IKKβ ^{(Reference Kumar, Vani and Hsieh44)}.

These data support the hypothesis that PSS regulate important cell fate decisions such as proliferation and differentiation. Importantly, cell fate decisions can be driven in either direction, in cancer PSS are anti-proliferative, anti-metastatic and pro-apoptotic, but in the non-cancer models of NSC and HAEC, PSS-enhanced proliferation and in some cases via upregulation of the same pathways that are downregulated by PSS in cancer. This context dependence indicates the molecular effectors of PSS control over cell fate are yet to be fully elucidated.

Risk of bias and study limitations

The risk of bias score was generated by assessing experimental, statistical and reporting transparency for each included study. Questions regarding experimental protocols were created according to the Minimum Information for Publication of Quantitative Real-Time PCR Experiments guidelines^{(Reference Bustin, Benes and Garson35)} and for reporting guidelines for immunoblotting data^{(Reference Alexander, Roberts and Broughton36)}. See the table in Supplementary (Table S1). Generally, all the studies received a minimal risk of bias rating (Fig. 3). In terms of statistical and experimental transparency, most studies had minimal risk of bias. Other aspects of reporting transparency assessed as low across the studies were: a lack of information on reagent catalogue numbers, reverse transcription and qPCR experimental variables, a lack of size marker reporting in immunoblot images and a lack of reporting across all studies about how PSS were stored and delivered to cell lines (e.g. use of carriers). Apart from these gaps, other criteria were generally sufficiently reported so risk of bias for the included studies was considered low.

Fig. 3. Risk of bias score. The risk of bias assessment for the included studies using a designed multi-point questionnaire. The score presented as green indicated the low risk of bias, red indicated the high risk of bias and yellow indicated the neutral risk of bias.

Perhaps the major limitation of this systematic review was the paucity of studies returned during the search phase. The corollary of which is that we have been unable to adopt previous examples of good practice when reviewing miRNA pathways; namely, requiring a miRNA to be both experimentally independently validated (e.g. QPCR, genetic or other functional confirmation of high-throughput expression data) and reported as differentially regulated in two or more publications to warrant inclusion^{(Reference Katan, Grundy and Jones57)}.

Conclusion

There is international consensus that the high consumption of seeds, nuts, legumes, fruits and vegetables reduces the risk of cardiometabolic disease and cancer. However, further studies characterising the bioactive compounds and mechanisms of action conferring health benefits are required. PSS enriched in plant-based diets lower pathogenic blood lipids^{(Reference Ras, Geleijnse and Trautwein12,Reference Jacobson, Ito and Maki14,Reference Windler, Beil and Berthold58)} , are linked to reduced cancer burden and risk^{(Reference Jiang, Zhao and Xu16,Reference Cioccoloni, Soteriou and Websdale17)} and a range of other diseases. Our systematic review has collated important knowledge regarding how PSS may act as epigenetic modifiers with the following notable observations.

PSS could be amongst the most potent naturally occurring HDACi reported to date^{(Reference Ishola and Adewole45)}. This potency, coupled with PSS’s suitability as orally administered agents as suggested by their high scores in the Lipinski test, indicates that further examination exploring their use as HDACi in clinic is warranted (Table 3)^{(Reference Ras, van der Schouw and Trautwein51)}. Nonetheless, clarity regarding their role in preventing and exacerbating different CVD clinical endpoints is required. As described recently, PSS are very unlikely to exacerbate CVD risk⁽⁶⁰⁾, but gaps remain in understanding the mechanism of action of how they reduce LDL-cholesterol and global histone acetylation analyses may shed light on this phenomenon. PSS inhibit expression and activity of DNMT that methylate CpG islands, leading to re-expression of genes silenced in disease states^{(Reference Pradhan, Parbin and Kar39,Reference Pradhan, Parbin and Kausar40)} . PSS regulate perhaps hundreds of miRNA, but validation of individual miRNA in multiple studies was only possible for five miRNA in total. More screens in more cell and tissue types are required to understand the true scale of miRNA regulation by PSS across tissues.

These epigenetic gene expression regulatory mechanisms impinge on several diseases. In cancer, PSS induce miRNA that lower expression of factors such as ZEB1 that promote the epithelial to mesenchymal transition, a critical step in cancer metastasis^{(Reference Kumar, Vani and Hsieh43)} and suppress miRNA allowing increase in expression of factors that maintain an epithelial phenotype, such as CDH1^{(Reference Kumar, Vani and Hsieh43)}. PSS also inhibit components of inflammatory pathways, including NFκB and mTOR signalling, in models of coronary obstructive pulmonary disease^{(Reference Shen, Liu and Qu42)} indicating a potential dietary route to ameliorating these conditions. Investigations into the epigenetic effects of PSS on many cell types, including those of the immune system, are lacking from the literature thus far. Given the emerging understanding that PSS may ameliorate or even prevent a range of diseases, evaluating the role of PSS in epigenetic reprogramming is of increasing clinical and public health interest.