HDAC1 was involved in placental breast cancer resistance protein regulation in vitro: A preliminary study

1Department of Pediatric Cardiology, West China Second University Hospital, Sichuan University, Chengdu, China 2The Cardiac development and early intervention unit, West China Institute of Women and Children’s Health, West China Second University Hospital, Sichuan University, Chengdu, China 3Key Laboratory of Birth Defects and Related Diseases of Women and Children (Sichuan University), Ministry of Education Chengdu, Sichuan, China 4West China Medical School of Sichuan University, Chengdu, China


| INTRODUC TI ON
Placental breast cancer resistance protein (BCRP, encoded by ABCG2 gene), predominantly located on apical brush-border membrane of the syncytiotrophoblast, plays a critical role in controlling transplacental transfer rates of various drugs. 1 Investigations on placental BCRP regulation are most likely to offer great promise for progress in individualized and safe pharmacotherapy during pregnancy. Currently, data regarding epigenetic regulation of placental BCRP are lacking. As core members of class Ⅰ histone deacetylases (HDACs), HDAC1, 2 and 3 are abundantly expressed in trophoblast cells and involved in an extremely broad spectrum of gene regulation in the placenta. [2][3][4] Recent work in tumour cells, [5][6][7] along with our previous findings in the placenta, 8 implied that HDAC1/2/3 might play a significant role in placental BCRP regulation. Importantly, given the discovery of dietary bioactive compounds that can affect HDAC1/2/3 expression and activity, 9 it will be possible to clinically manipulate placental BCRP safely and effectively by means of epigenetics during pregnancy. Therefore, we carried out this preliminary study to investigate the effect of HDAC inhibition on placental BCRP expression and to further determine the role of HDAC1/2/3 in the placental BCRP regulation in vitro, which might shed some light on the pathway of placental BCRP regulation from the perspective of epigenetics

| MATERIAL S AND ME THODS
After being authenticated by specific biomarkers (CK7, hPL and hCG positive; HLA-G negative) to confirm its cell identity, 10

| D ISCUSS I ON
To our knowledge, our study was the first one to elucidate that HDAC1 was most likely to be involved in the positive regulation of placental BCRP expression and functionality in vitro. This finding contrasted with the best-documented and most common biological function of HDAC1, acting as a transcriptional repressor by histone modifications to regulate downstream genes. 5 However, it was also previously documented that HDAC1 could modify non-histone proteins and function as a positive regulator of target gene expression either directly or as a coactivator in multiprotein complexes. 11,12 On account of cell-specific regulation and individual roles in respective gene regulation of HDACs, the regulatory pathway of HDAC1 on placental BCRP still needs to be further clearly elucidated, which could provide more novel therapeutic targets for controlling drug delivery across the placenta.
F I G U R E 1 Trichostatin regulation of HDAC1/HDAC2/HDAC3/BCRP mRNA and protein expressions after 24 (A and B) and 48 hours (C and D) of incubation in BeWo. Differences among different groups were assessed using one-way ANOVA followed by Tukey's honestly significant difference multiple range test. N = 3 for each group. Data are presented as Means ± SEM. *P ＜ 0.05, **P ＜ 0.01, ***P ＜ 0.001 Notably, although HDAC1 was found to be involved in BCRP regulation through specific siRNA methodology, some divergent results were found using the methodology of TSA inhibition. For instance, down-regulation of ABCG2 mRNA occurred, whereas none of the altered HDAC1/2/3 mRNA was observed after 0.5 and 1.0 μmol/L TSA exposure at 24 hours ( Figure 1A), implying that TSA seemed to inhibit BCRP expression not only through HDAC1mediated pathway but also likely through certain undefined-regulator dependent manners (eg, other HDAC isoforms or non-HDAC regulation factors), owing to its broader influence on regulatory genes in comparison with siRNA methodology. Additionally, the inconsistent tendency of BCRP mRNA and protein ( Figure 1A/B) seems to be somewhat dependent on half-life, stability or abundance of the protein, 13 which may confer the delayed reduction of BCRP protein level.
Some limitations of the present study must be considered. Firstly, as non-syncitialized BeWo cells primarily consist of cytotrophoblast that most appropriately model early gestation, extrapolation of these data from this preliminary study to the whole gestational stage is difficult and more experimental models (ie, syncytializing and/or syncytialized trophoblast cells, primary placental cells, animal models, etc) will definitely be needed to validate our results.
Additionally, apart from suppression of placental BCRP, HDAC1 inhibition could result in potential effect on other placental and non-placental targets, 14,15 which might have major phenotypic implications. Therefore, more studies should be further undertaken to verify the safety of HDAC1 inhibition, particularly for foetal development. Moreover, given some inconsistent or divergent results between TSA and siRNA methodologies, future studies are therefore required to clarify the effect of TSA on placental BCRP expression F I G U R E 2 Effect of HDAC1/HDAC2/HDAC3 silencing on breast cancer resistance protein (BCRP) expression and functional activity in BeWo cells. After 48 hours of transfection, the mRNA (A) and protein levels (B) were analysed using qRT-PCR and Western blot respectively. Representative fluorescent images of BCRP (green staining) were obtained using immunofluorescence microscopy (C). Quantitative fold changes in BCRP expression were analysed using Image J version 1.44 (D). BCRP functionality was measured by the intracellular retention of Hoechst 33342 (5 μg/mL) in the presence or absence of the BCRP-specific inhibitor Ko143 (1 μmol/L). Intracellular fluorescence was analysed by the fluorescence plate reader (E). Data are expressed as Means ± SEM. *P ＜ 0.05, **P ＜ 0.01, ***P ＜ 0.001; asterisk represents differences among siRNA-transfected groups; line represents differences between efflux in absence of Ko143 and in presence of Ko143 or function, aiming to further testify the current data and more importantly, to uncover some regulators besides HDAC1/2/3 involved in this process.
Despite these limitations, we took the first step and made some preliminary exploration of placental BCRP regulation from the perspective of epigenetics in vitro, which was currently lacking in the literature. Obviously, these data obtained from current pilot experiments cannot fully reflect the physiological and biochemical changes in placenta; However, our present findings might expand the limited knowledge with respect to epigenetic regulation of placental BCRP and provide some clues for further studies.

CO N FLI C T O F I NTE R E S T
None of authors have declared any conflict of interests.