Apoptotic Gene Expression in Sinecatechins-Treated External Genital and Perianal Warts

Abstract

Dear Editor

External genital and perianal warts (EGW)—the most common viral sexually transmitted disease in the United States—are caused by the human papillomavirus (HPV), a family of DNA viruses with more than 150 genotypes characterized to date (7). In 2006, sinecatechins ointment 15% (trade name Veregen^®) was approved by the Food and Drug Administration for the treatment of EGW. Sinecatechins ointment is comprised of a proprietary blend of eight different catechins, the chief family of flavonoids believed to account for the wide array of health benefits attributed to green tea (12). In decreasing order of concentration, the four major catechins found in green tea are epigallocatechin gallate, epigallocatechin, epicatechin gallate, and epicatechin.

Prior to their application for treating viral disease, catechins had been associated with a wide array of health benefits (10). Although it is believed that the majority of these health benefits are due to the catechins' antioxidative activity, green tea catechins have also displayed poorly understood antiproliferative and antiviral properties (10). In vitro, epigallocatechin gallate can induce apoptotic growth inhibition of four HPV-infected tumor cell lines, and in vivo, green tea catechins were found to be effective in treating cervical HPV lesions, as determined by positive morphological changes of cervical lesions and a decrease of HPV DNA levels following treatment (8,11). However, despite these observations and FDA approval nearly a decade ago, the mechanism of action of sinecatechins-induced growth inhibition of EGW is unknown.

To begin to answer this question, we performed an open-label, single-site study enrolling subjects with a clinical diagnosis of EGW, and used specialized microarrays to determine the expression-level changes specific to apoptosis of EGW before and after sinecatechins treatment. A total of 30 subjects were recruited for the study, 24 male and 6 female, with a mean age of 39.2±10.6 years. Of these, 18 subjects remained enrolled and were available for follow-up for the duration of the study. Veregen^® ointment, 15%, was dispensed to the patient with instructions to apply to the target warts three times daily for 16 weeks.

Three biopsies were taken from each patient—excised at baseline (B1), at the first visit with 50% or more clearance of target warts (B2), and at the first visit with complete clearance of target lesions (B3). Tissue samples were stored in RNAlater (Ambion) solution until processing, and nucleic acids were extracted from the samples using Trizol reagent (Sigma).

HPV types from extracted DNA samples were detected by a nested polymerase chain reaction (PCR) approach as previously described (1), and copy number was determined with a custom-made real time PCR kit (Quantification of HPV6_15979, L1 protein, L1 gene, PrimerDesign Ltd.). Extracted and applied RNA, measured at A260/A280 nm, produced a ratio between 1.8 and 2.0. Subjects were stratified based on their response to treatment, which was quantitatively determined by measuring change in viral copy number between B1 and B3. Subjects were classified as virological responders (VR) if viral copy number decreased by at least 60% from B1, and were classified as virological nonresponders (VNR) if viral copy number remained the same or increased from B1. Of these, seven were found to be VR, and 11 were defined as VNR. In addition to the detection of HPV-6 in all lesions, one VR and three VNR were positive for co-infection with other HPV types (VR: HPV-18 and -35; VNR: HPV-7, -8, and -35).

Next, the Applied Biosystems High Capacity RNA-to-cDNA master mix was utilized for cDNA synthesis, and analysis of apoptotic gene expressions was then performed using TaqMan array 96-well plates. Initially, gene expression level was surveyed individually as an independent variable, and subsequently, group-based response values were generated using DataAssist™ v3.01 Software. Quantification of group-based responses for each gene were determined by calculating a fold change from biopsy 1 to biopsy 2 (B1_2), from biopsy 1 to biopsy 3 (B1_3), and from biopsy 2 to biopsy 3 (B2_3). Gene expression changes were categorized as biologically significant if there was at least a twofold change. To determine statistical significance of these fold changes, the statistical significance of these fold changes was evaluated using a nonparametric two-sided Wilcoxon signed-rank test with a significance level of p<0.05.

Of the 92 genes assessed in the Apoptosis microarray (Supplementary Table S1; Supplementary Data are available online at www.liebertpub.com/vim), no genes in the VNR group were found to have biologically significant changes between any of the time points. The differentially expressed genes in the VR group were downregulated by factors ranging from 0.18 to 0.42, with none upregulated. From B2_3, there were no genes demonstrating statistically or biologically significant changes.

In the VR group, six genes were found to have biologically significant expression changes between B1_2 (Table 1). Two of those genes—BIRC3 (baculoviral IAP repeat containing 3) and BIRC5 (baculoviral IAP repeat containing 5/Survivin)—are anti-apoptotic genes that previously have been shown to be upregulated by the HPV oncoprotein E6 via a NF-κB-related mechanism (2,4). The reduction in levels of mRNA of BIRC3 and BIRC5 that we observed in the VR of this current study is consistent with the observed reduction of viral load in lesions. Furthermore, the significant concomitant reduction in REL, a component of the NF-κB complex, suggests that the effects of E6 on apoptosis are being blocked by treatment with sinecatechins, at least in responders.

Table 1.

Comparison Between Biopsy Groups in Virological Responders

Comparison between biopsy groups in viral responders	Gene	Relative fold change	p-Value
Biopsy 1 to biopsy 2	BIRC3	0.20590	0.0469
	BIRC5	0.36028	0.0469
	LTA	0.17863	0.0313
	LTB	0.17664	0.0156
	REL	0.41592	0.0156
	TNF	0.20424	0.0313
Biopsy 1 to biopsy 3	FASL	0.31459	0.0496
	TNF	0.15287	0.0156

Differential expression of genes was determined using RNA array data, and compared between the biopsies obtained. Statistical analyses were performed and fold changes were calculated from baseline expression. Statistical significance was set at p<0.05.

Another upregulated gene between B1_2 in VR was TNF (tumor necrosis factor). TNF can serve both pro- and anti-apoptotic roles, depending on interactions with and concentrations of other proteins in the TNF pathway (3). Predominantly, however, TNF tends to be anti-apoptotic through its interaction with NF-κB (3), so it is not surprising to see TNF expression reduced in VR.

Similarly, B1_3 comparison also yielded significant changes in TNF as well as FASL (Fas ligand). TRAIL (TNF-related apoptosis-inducing ligand) and FASL can trigger apoptosis when binding with their respective receptors, and HPV oncoprotein E5 has been shown to inhibit FAS and TRAIL-induced apoptosis (5). In the present study, we found mRNA levels of FASL to be reduced in VR between B1_3. Although there have been no studies in the literature examining the levels of FASL mRNA in EGW, FASL upregulation is seen in a majority of cervical squamous cell tumors (6). Upregulation of FASL may be a normal cellular response when the apoptosis-related machinery of infected cells is compromised by viral proteins, and the reduction in FASL levels may constitute evidence of a return-to-normal apoptotic function in VR once the lesions have been resolved.

Inhibition of apoptosis has been demonstrated in the pathogenesis of both high- and low-risk HPV types, and thus facilitation of apoptotic mechanisms is likely a major factor in the therapeutic success of catechins for EGW (9). Overall, our study identified seven genes that were downregulated in VR, although these findings should be confirmed by specific reverse transcriptase (RT)-PCR for each gene. Due to the complex cross-talk in the TNF, NF-κB, apoptosis, and related pathways, as well as the limited knowledge of the precise molecular targets of green tea catechins, it is not possible to put forth a conclusive explanation for the effectiveness of sinecatechins treatment based solely on an apoptotic gene expression study. However, a future study investigating a larger population of genes might provide more detailed insight into the mechanisms behind this promising treatment.

Footnotes

Author Disclosure Statement

Stephen K. Tyring received study grants from Fougera Pharmaceuticals, Inc. No other conflicts of interest reported.

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Supplementary Material

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