Elsevier

Gene

Volume 553, Issue 2, 15 December 2014, Pages 98-104
Gene

SAHA inhibits the transcription initiation of HPV18 E6/E7 genes in HeLa cervical cancer cells

https://doi.org/10.1016/j.gene.2014.10.007Get rights and content

Highlights

  • The expression of HPV18 E6 and E7 genes were inhibited by SAHA treatment.

  • The recruitment of RNP II to HPV18 promoter was reduced in SAHA treated cells.

  • Histone modifications altered on HPV promoter following SAHA treatment.

  • SAHA blocked the cell division cycle but had little effect on apoptosis.

Abstract

High risk human papillomavirus (HPV) is a well recognized causative agent of cervical cancer. Suberoylanilide hydroxamic acid (SAHA) is a potential anti-cervical cancer drug; however, its effect on the expression of HPV E6 and E7 genes remains unclear. Here, we show that, in SAHA treated HeLa cells, HPV18 E6 and E7 mRNA and protein levels were reduced, HPV18 promoter activity was decreased, and the association of RNP II with HPV18 promoter was diminished, suggesting that SAHA inhibited the transcription initiation of HPV18 E6 and E7 genes. In SAHA-treated HeLa, although the level of lysine 9-acetylated histone H3 in the whole cell extracts increased obviously, its enrichment on HPV18 promoter was significantly reduced which is correlated with the down-regulation of HPV E6 and E7.

Introduction

Cervical cancer is the second common female malignant tumor. Globally, there were more than half million new cases of cervical cancer diagnosed in 2010 (de Sanjose et al., 2010). Carcinogenesis of cervical cancer is highly correlated with human papillomavirus (HPV) infection, which is different from other cancers or malignant tumors. There are more than 100 types of HPVs isolated so far and 13 of them are defined to be high risk HPVs which have a causative relationship with cervical cancer. Among them, HPV 16 and HPV 18 are the two types being most frequently detected in cervical cancer specimens (de Villiers et al., 2004, zur Hausen, 2009).

Genome of HPV is a circular double strand DNA encoding early and late virus genes. The tandemly linked HPV E6 and E7 genes are usually integrated into host cellular genome and are constitutively expressed in HPV-positive cervical cancer cells (zur Hausen, 2009). It has been demonstrated that high risk HPV E6 and E7 proteins contribute to oncogenesis by facilitating cell immortalization, migration, altering cell cycle and apoptosis control, evading host immuno-surveillance, etc. (Ghittoni et al., 2010, Au Yeung et al., 2011, Liu et al., 2009). Introduction of HPV E6 and E7 into normal epithelial cells will immortalize the cells. On the contrary, downregulation of HPV E6 and E7 genes by antisense RNA, siRNA or ectopically expressed HPV E2 which is a repressor of HPV E6/E7 genes resulted in growth arrest and apoptosis of cervical cancer cells (Gu et al., 2011, Hong et al., 2009, Morrison et al., 2011, Sima et al., 2007). Therefore, HPV E6/E7 genes are specific targets for cervical cancer therapy (Shillitoe, 2006, Stern et al., 2012).

In the last 30 years, knowledge about histone post-translational modifications has accumulated quickly and certain chemicals affecting histone modifications are taken as potential anti-cancer drugs. The most successfully developed chemicals are inhibitors of histone deacetylase (HDACi) which are supposed to induce apoptosis and differentiation and inhibit proliferation of cancer cells (Grant and Dai, 2012, Glass and Viale, 2013, Marchion and Munster, 2007). The therapeutic potential of HDACi on HPV-positive cervical cancer was as well investigated in a few of laboratories (Finzer et al., 2001, Finzer et al., 2002, Darvas et al., 2010, Borutinskaite et al., 2012, Shao et al., 2004, Lin et al., 2009, Jin et al., 2010). However, the effects of HDACi on the expression of HPV E6 and E7 oncogenes were less studied (Finzer et al., 2001, Finzer et al., 2002, Darvas et al., 2010, Luczak and Jagodzinski, 2008).

So far, chemists had developed four classes of HDACis, namely small molecule HDACi, depsipeptide HDACi, cyclictetrapeptide HDACi and nonpeptide macrocyclic HDACi (Gryder et al., 2012). Among these HDACis, SAHA (suberoylanilide hydroxamic acid, also named vorinostat, Zolinza™) and FK228 (romidepsin, Istodax™), have been approved by FDA USA for treating refractory C cutaneous T-cell lymphoma (CTCL) in 2006 and 2009, respectively (Gryder et al., 2012, Duvic and Vu, 2007). However, there are still limitations and side effects, for instance, cardiac toxicity and trabecular bone loss, observed during the applications of SAHA and FK228 in cancer patients (Gryder et al., 2012, McGee-Lawrence et al., 2011). Hence, bioinformatics scientists and organic chemists are engaged in designing and synthesizing novel HDACis with side effects (Tambunan and Parikesit, 2012, Spencer et al., 2011). JAHA (Jay Amin hydroxamic acid) was recently synthesized as a SAHA analogue with selective HDACi activity (Spencer et al., 2011). Including JAHA, new compounds with HDACi activity are potential alternatives of SAHA in cancer treatment, which should be further verified in wet laboratory experiments and clinical trials.

In this study, we used HeLa cell which carries HPV18 DNA in cellular genome as a model to investigate whether SAHA, the first HDACi approved by FDA USA for cancer therapy, would affect the transcription of HPV18 E6 and E7 genes, and further, underlying mechanism was explored.

Section snippets

Cell culture and treatments

HeLa cells were cultured in Dulbecco's modified Eagle's medium with l-Glutamine (DMEM; Gibco) supplemented with 10% fetal bovine serum (Sijiqing, Hangzhou China) and 1% Antibiotic/Antimycotic (Solarbio, China). SAHA was purchased from Sigma and dissolved in DMSO.

MTT assay

HeLa cells seeded in 96-well culture plates (1.2 × 104 per well) were treated with SAHA at different concentrations. Twenty-four hours after SAHA treatment, 20 μl of MTT (5 mg/ml, Solarbio, China) was added to each well and incubated for 4 

SAHA inhibits the expression of HPV18 E6/E7 in HeLa cells

To select an optimal dosage and to confirm the HDACi activity of SAHA, HeLa cells were treated with serial diluted SAHA for 24 h as indicated in Fig. 1A. The acetylation level of histone H3 was measured by Western-blot using whole cell extracts. As shown in Fig. 1A, the global level of acetylated histone H3 increased obviously when SAHA concentration reached 2.5 μM, suggesting that SAHA efficiently inhibited HDAC activity in HeLa cells at the concentrations above 2.5 μM. Therefore, 5 μM of SAHA was

Discussion

HDACis were usually utilized to activate epigenetically repressed genes, such as tumor suppressor genes in malignant tumor cells, by boosting the histone acetylation which is well known as a positive marker for gene activation. However, profiling of gene expression in HDACi treated cells reveals that, actually, some genes are upregulated and others are downregulated by HDACi although the underlying mechanism is unclear (Chambers et al., 2003, Joseph et al., 2004). Here, with the promoter

Conflict of interest

These authors claim no actual or potential conflict of interest.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (31301073) and Applied Basic Science and Frontier Technology Program of Tianjin (13JCYBJC38000). Zhejiang University provided starting fund to Y Luo through the National 985 Platform; Y Luo has also been supported in part by the China National 973 project (2014CB542003), China Natural Sciences Foundation project (81372179), Zhejiang Provincial Natural Sciences Foundation project (LY13C070001), and the Fundamental

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