Apicidin down-regulates human papillomavirus type 16 E6 and E7 transcripts and proteins in SiHa cervical cancer cells

Abstract

Virtually all cervical cancer morbidities are associated with genital skin or mucosa cell infection with human papillomavirus (HPV). The HPV oncogenic proteins E6 and E7 are able to inactivate p53 and Rb proteins, which results in malignant transformation.

Employing quantitative real-time PCR and Western blot analysis, we observed that apicidin histone deacetylase (HDAC) inhibitor significantly reduced HPV16-E6 and -E7 transcripts and protein levels in SiHa cervical cancer cells. Moreover, we found that apicidin lowered HPV16-E6 and -E7 transcript stability and significantly decreased these transcripts’ half-life from approximately 5 h to 2 h and from 6 h to 3 h, respectively. Our results from experiments with protein biosynthesis inhibitor suggest the involvement of an RNase and/or mRNA stabilization protein in HPV16-E6 and -E7 transcript stabilization.

Since the HPV type 16 is associated with most cervical cancer incidence and HDAC inhibitors are being tested in anti-cancer clinical trials, our observations may have clinical significance.

Introduction

Cervical cancer is the most common malignant disease of the female reproductive organs, with an approximate morbidity of 500,000 women per year, of whom 80% live in developing countries [1]. Virtually all cervical cancers are associated with genital skin or mucosa cell infection with human papillomavirus (HPV) [2]. HPVs are small, non-enveloped viruses with an approximately 8-kb circular genome encoding two structural proteins, L1 and L2, and several nonstructural proteins, E1, E2, E4, E5, E6, and E7 [3]. The E1 and E2 proteins are involved in viral replication and transcription. E4 helps release the virus from host cells. The E5 protein potentiates viral gene expression and enhances the malignant transformation properties of E6 and E7, which are major transforming proteins of HPV infected cells [3], [4], [5], [6], [7], [8].

According to DNA sequences of the L1, E6, and E7 genes, more than 100 genotypes of HPV have been detected [9], [10], [11], [12], [13]. Based on the ability of HPV to induce malignant transformation, the HPV types were divided into low-risk and high-risk viruses. The HPV types 16 and 18 belong to the high-risk group, to which belong HPV types that are considered the primary causal agent in morbidity of cervical cancer [14], [15], [16]. HPV type 16 accounts for over 50% of cervical cancer morbidity, whereas HPV type 18 was found in 20% cases of cervical cancers [14].

The HPV16 and 18 oncogenic proteins E6 and E7 are able to cause immortalization of the infected cells [17], [18], [19]. HPV-E6 associates with ubiquitin–protein ligase E6-AP and subsequently interacts with p53, resulting in its degradation in the proteasome [20]. This shortens the half-life of p53 and reduces its concentration in cancer cells compared to normal epithelial cells [21]. Moreover, E6 also increases the telomerase activity above its critical point, thus induce immortality of the cells [22], [23]. The HPV-E7 protein interacts with retinoblastoma protein (Rb) and releases transcription factor E2F, which induces expression of genes involved in cell proliferation [24], [25].

Epigenetic alteration is considered the heritable and reversible changes in gene expression patterns that do not correspond to the DNA sequence [26], [27]. These changes include cytosine methylation in cytosine and guanine dinucleotide (CpG) islands and covalent histone modifications, which modulate the expression of numerous genes [26]. A low degree of methylation of CpG residues in the regulatory sequences of DNA and a high level of histone acetylation correlate with the transcriptional activity of certain genes [26], [27]. Histone acetylation is conducted by acetyltransferases, but their deacetylation is carried out by histone deacetylases (HDACs) [28].

It has been reported that hypermethylation of promoters in patients with cervical carcinoma diminishes or silences the expression of tumor suppressor genes encoding proteins involved in virtually all cancer pathways or cell functions [16]. Moreover, the methylation state of the HPV genome in its regulatory region may also modulate the viral life cycle, infection progress, and viral oncoprotein production in cervical carcinoma [29].

Apicidin is an inhibitor of HDAC and accounts for the in vitro high acetylation level of histones, which correlates with an increase of various genes transcription [30], [31], [32]. However, the eventual effect of HDAC inhibitors on numerous transcript and protein contents in cells should be determined experimentally [33], [34], [35]. We therefore decided to investigate the effect of apicidin on HPV16-E6 and -E7 transcript and protein levels in SiHa cervical cancer cells expressing HPV type 16.