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

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 openlabel, 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 RNAto-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.