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Hydralazine Sensitizes to the Antifibrotic Effect of 5-Aza-2′-deoxycytidine in Hepatic Stellate Cells

Background: Hepatic stellate cell (HSC) activation is essential for the development of liver fibrosis. Epigenetic machinery, such as DNA methylation, is largely involved in the regulation of gene expression during HSC activation. Although the pharmacological DNA demethylation of HSC using 5-aza-2′-d...

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Autores principales: Asada, Kiyoshi, Kaji, Kosuke, Sato, Shinya, Seki, Kenichiro, Shimozato, Naotaka, Kawaratani, Hideto, Takaya, Hiroaki, Sawada, Yasuhiko, Nakanishi, Keisuke, Furukawa, Masanori, Kitade, Mitsuteru, Moriya, Kei, Namisaki, Tadashi, Noguchi, Ryuichi, Akahane, Takemi, Yoshiji, Hitoshi
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7345531/
https://www.ncbi.nlm.nih.gov/pubmed/32503264
http://dx.doi.org/10.3390/biology9060117
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author Asada, Kiyoshi
Kaji, Kosuke
Sato, Shinya
Seki, Kenichiro
Shimozato, Naotaka
Kawaratani, Hideto
Takaya, Hiroaki
Sawada, Yasuhiko
Nakanishi, Keisuke
Furukawa, Masanori
Kitade, Mitsuteru
Moriya, Kei
Namisaki, Tadashi
Noguchi, Ryuichi
Akahane, Takemi
Yoshiji, Hitoshi
author_facet Asada, Kiyoshi
Kaji, Kosuke
Sato, Shinya
Seki, Kenichiro
Shimozato, Naotaka
Kawaratani, Hideto
Takaya, Hiroaki
Sawada, Yasuhiko
Nakanishi, Keisuke
Furukawa, Masanori
Kitade, Mitsuteru
Moriya, Kei
Namisaki, Tadashi
Noguchi, Ryuichi
Akahane, Takemi
Yoshiji, Hitoshi
author_sort Asada, Kiyoshi
collection PubMed
description Background: Hepatic stellate cell (HSC) activation is essential for the development of liver fibrosis. Epigenetic machinery, such as DNA methylation, is largely involved in the regulation of gene expression during HSC activation. Although the pharmacological DNA demethylation of HSC using 5-aza-2′-deoxycytidine (5-aza-dC) yielded an antifibrotic effect, this drug has been reported to induce excessive cytotoxicity at a high dose. Hydralazine (HDZ), an antihypertensive agent, also exhibits non-nucleoside demethylating activity. However, the effect of HDZ on HSC activation remains unclear. In this study, we performed a combined treatment with 5-aza-dC and HDZ to obtain an enhanced antifibrotic effect with lower cytotoxicity. Methods: HSC-T6 cells were used as a rat HSC cell line in this study. The cells were cultivated together with 1 µM 5-Aza-dC and/or 10 µg/mL of HDZ, which were refreshed every 24 h until the 96 h treatment ended. Cell proliferation was measured using the WST-1 assay. The mRNA expression levels of peptidylprolyl isomerase A (Ppia), an internal control gene, collagen type I alpha 1 (Cola1), RAS protein activator like 1 (Rasal1), and phosphatase and tensin homolog deleted from chromosome 10 (Pten) were analyzed using quantitative reverse transcription polymerase chain reaction. Results: The percentage cell viability with 5-aza-dC, HDZ, and combined treatment vs. the vehicle-only control was 101.4 ± 2.5, 95.2 ± 5.7, and 79.2 ± 0.7 (p < 0.01 for 5-aza-dC and p < 0.01 for HDZ), respectively, in the 48 h treatment, and 52.4 ± 5.6, 65.9 ± 3.4, and 29.9 ± 1.3 (p < 0.01 for 5-aza-dC and p < 0.01 for HDZ), respectively, in the 96 h treatment. 5-Aza-dC and the combined treatment markedly decreased Cola1 mRNA levels. Accordingly, the expression levels of Rasal1 and Pten, which are antifibrotic genes, were increased by treatment after the 5-aza-dC and combined treatments. Moreover, single treatment with HDZ did not affect the expression levels of Cola1, Rasal1, or Pten. These results suggest that HDZ sensitizes to the antifibrotic effect of 5-aza-dC in HSC-T6 cells. The molecular mechanism underlying the sensitization to the antifibrotic effect of 5-aza-dC by HDZ remains to be elucidated. The expression levels of rat equilibrative nucleoside transporter genes (rEnt1, rEnt2, and rEnt3) were not affected by HDZ in this study. Conclusions: Further confirmation using primary HSCs and in vivo animal models is desirable, but combined treatment with 5-aza-dC and HDZ may be an effective therapy for liver fibrosis without severe adverse effects.
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spelling pubmed-73455312020-07-09 Hydralazine Sensitizes to the Antifibrotic Effect of 5-Aza-2′-deoxycytidine in Hepatic Stellate Cells Asada, Kiyoshi Kaji, Kosuke Sato, Shinya Seki, Kenichiro Shimozato, Naotaka Kawaratani, Hideto Takaya, Hiroaki Sawada, Yasuhiko Nakanishi, Keisuke Furukawa, Masanori Kitade, Mitsuteru Moriya, Kei Namisaki, Tadashi Noguchi, Ryuichi Akahane, Takemi Yoshiji, Hitoshi Biology (Basel) Article Background: Hepatic stellate cell (HSC) activation is essential for the development of liver fibrosis. Epigenetic machinery, such as DNA methylation, is largely involved in the regulation of gene expression during HSC activation. Although the pharmacological DNA demethylation of HSC using 5-aza-2′-deoxycytidine (5-aza-dC) yielded an antifibrotic effect, this drug has been reported to induce excessive cytotoxicity at a high dose. Hydralazine (HDZ), an antihypertensive agent, also exhibits non-nucleoside demethylating activity. However, the effect of HDZ on HSC activation remains unclear. In this study, we performed a combined treatment with 5-aza-dC and HDZ to obtain an enhanced antifibrotic effect with lower cytotoxicity. Methods: HSC-T6 cells were used as a rat HSC cell line in this study. The cells were cultivated together with 1 µM 5-Aza-dC and/or 10 µg/mL of HDZ, which were refreshed every 24 h until the 96 h treatment ended. Cell proliferation was measured using the WST-1 assay. The mRNA expression levels of peptidylprolyl isomerase A (Ppia), an internal control gene, collagen type I alpha 1 (Cola1), RAS protein activator like 1 (Rasal1), and phosphatase and tensin homolog deleted from chromosome 10 (Pten) were analyzed using quantitative reverse transcription polymerase chain reaction. Results: The percentage cell viability with 5-aza-dC, HDZ, and combined treatment vs. the vehicle-only control was 101.4 ± 2.5, 95.2 ± 5.7, and 79.2 ± 0.7 (p < 0.01 for 5-aza-dC and p < 0.01 for HDZ), respectively, in the 48 h treatment, and 52.4 ± 5.6, 65.9 ± 3.4, and 29.9 ± 1.3 (p < 0.01 for 5-aza-dC and p < 0.01 for HDZ), respectively, in the 96 h treatment. 5-Aza-dC and the combined treatment markedly decreased Cola1 mRNA levels. Accordingly, the expression levels of Rasal1 and Pten, which are antifibrotic genes, were increased by treatment after the 5-aza-dC and combined treatments. Moreover, single treatment with HDZ did not affect the expression levels of Cola1, Rasal1, or Pten. These results suggest that HDZ sensitizes to the antifibrotic effect of 5-aza-dC in HSC-T6 cells. The molecular mechanism underlying the sensitization to the antifibrotic effect of 5-aza-dC by HDZ remains to be elucidated. The expression levels of rat equilibrative nucleoside transporter genes (rEnt1, rEnt2, and rEnt3) were not affected by HDZ in this study. Conclusions: Further confirmation using primary HSCs and in vivo animal models is desirable, but combined treatment with 5-aza-dC and HDZ may be an effective therapy for liver fibrosis without severe adverse effects. MDPI 2020-06-03 /pmc/articles/PMC7345531/ /pubmed/32503264 http://dx.doi.org/10.3390/biology9060117 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Asada, Kiyoshi
Kaji, Kosuke
Sato, Shinya
Seki, Kenichiro
Shimozato, Naotaka
Kawaratani, Hideto
Takaya, Hiroaki
Sawada, Yasuhiko
Nakanishi, Keisuke
Furukawa, Masanori
Kitade, Mitsuteru
Moriya, Kei
Namisaki, Tadashi
Noguchi, Ryuichi
Akahane, Takemi
Yoshiji, Hitoshi
Hydralazine Sensitizes to the Antifibrotic Effect of 5-Aza-2′-deoxycytidine in Hepatic Stellate Cells
title Hydralazine Sensitizes to the Antifibrotic Effect of 5-Aza-2′-deoxycytidine in Hepatic Stellate Cells
title_full Hydralazine Sensitizes to the Antifibrotic Effect of 5-Aza-2′-deoxycytidine in Hepatic Stellate Cells
title_fullStr Hydralazine Sensitizes to the Antifibrotic Effect of 5-Aza-2′-deoxycytidine in Hepatic Stellate Cells
title_full_unstemmed Hydralazine Sensitizes to the Antifibrotic Effect of 5-Aza-2′-deoxycytidine in Hepatic Stellate Cells
title_short Hydralazine Sensitizes to the Antifibrotic Effect of 5-Aza-2′-deoxycytidine in Hepatic Stellate Cells
title_sort hydralazine sensitizes to the antifibrotic effect of 5-aza-2′-deoxycytidine in hepatic stellate cells
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7345531/
https://www.ncbi.nlm.nih.gov/pubmed/32503264
http://dx.doi.org/10.3390/biology9060117
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