Cellular signaling in sinecatechins-treated external genital and perianal warts: unraveling the mechanism of action of a botanical therapy

Dear Editor,

Derived from green tea leaves, sinecatechins (Veregen^®) ointment, 15% is a topical therapy that is FDA-approved to treat human papillomavirus (HPV)-induced external genital and perianal warts (EGW) in immunocompetent patients aged 18 years and older. In two phase 3 trials enrolling over 1, 000 participants with EGW, a 16-week treatment regimen with sinecatechins ointment resulted in higher rates of complete clearance of all warts — both baseline and newly emerging warts — when compared to vehicle-treated patients (54.9% vs. 35.4%, respectively; p < 0.001) (Tatti et al., 2010). This fnding, taken together with the low recurrence rates (6.8%, p < 0.001), represented a practical improvement over previous clinical trials with other EGW therapies (Tyring, 2012)

Despite its apparent clinical efficacy, the mechanism of action of sinecatechins in the treatment of EGW is largely unknown. Utilizing in vitro biochemical assays, sinecatechins was found to mediate dose-dependent inhibition of several inflammatory proteases, kinases, and oxygenases that have been implicated in the pathogenesis of HPV-induced EGW formation (Tyring, 2012). To this end, other therapeutic mechanisms that have been associated with green tea components include pro-apoptotic, anti-viral, anti-oxidative, anti-angiogenic, and immunostimulatory processes; as a result, it is likely that the mechanism of action specifc to sinecatechins-treated EGW is multifactorial. Recently, we published our fndings from the frst in vivo study on this topic, demonstrating that sinecatechins are capable of down-regulating antiapoptotic genes in the NF-κB pathway (Nguyen et al., 2014). While our data confirmed previous in vitro findings (Tyring, 2012), this report focused solely on apoptotic gene expressions responsive to sinecatechins treatment. In order to evaluate other key cellular mechanisms contributing to pathogenesis of EGW treated with sinecatechins, we sought to perform additional molecular studies on tissues obtained during the clinical study described previously (Nguyen et al., 2014). In this present report, we selected microarrays designed to provide general yet exhaustive assessment of salient genes involved in pro-inflammatory cellular signaling.

Briefly, the open-label, single-site clinical study enrolled 18 immunocompetent subjects with a clinical diagnosis of EGW and employed specialized microarrays to determine the expression level changes of EGW before and after sinecatechins treatment. Veregen^® ointment, 15% was dispensed to patients applied to the target warts 3 times daily for 16 weeks. Three 2-mm punch biopsies were obtained from each patient; the first biopsy was excised at baseline (B1), the second at the frst visit with 50% or more clearance of target warts (B2), and the third at the frst visit with complete clearance of target lesions (B3).

HPV types from extracted DNA samples were detected by a nested PCR approach as previously described (Fuessel-Haws et al., 2004), and copy number was determined with a custom-made real time PCR kit (Quantification of HPV6_15979, L1 protein, L1 gene, PrimerDesign Ltd). Subjects were classified based on their response to treatment, which was quantitatively determined by measuring change in viral copy number between B1 and B3. Subjects were classifed as virological responders (VR) if viral copy number decreased by at least 60% from B1 and were classifed as virological non-responders (VNR) if viral copy number remained the same or increased from B1. Of the 18 participants, seven were found to be VR, and 11 were defned to be VNR.

Following cDNA synthesis, analyses of signal transduction and cell cycle gene expressions were performed on TaqMan array 96-well plates utilizing StepOnePlus^TM Real-Time PCR System (Life Technologies). Quantification of group-based responses for each gene was 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 two-fold change, and statistical significance of these fold changes were evaluated using a non-parametric two-sided Wilcoxon signed-rank test with a signifcance level of p < 0.05.

Of the 192 genes assessed, 17 gene expression changes met criteria for both biological and statistical signifcance; 13 and 4 genes were found to be differentially expressed in VR and VNR, respectively (Table 1). Interestingly, among these differentially expressed genes (DEG), all but two genes (IL2 and CDC2) in VR were down-regulated; in contrast, all four DEG in VNR were upregulated.

Table 1.  Comparison between biopsy groups (B1, B2, B3)in virological responders (VR) and virological non-responders (VNR).

The expression pattern identified in this study confirms our previous findings, providing further evidence that sinecatechins mediates EGW regression through pro-apoptotic expression changes. Notably, while the anti-apoptotic gene CDC2 was found to be down-regulated only in the present study, BIRC1 and BIRC3 — which encode inhibitors of apoptosis proteins and are known targets of HPV E6 — as well as TNF were down-regulated in VR in both studies (Yu et al., 1998; James et al., 2006). BIRC3's consistent down-regulation in response to sinecatechins treatment likely marks the reversal of a viral pathomechanism known to prevent apoptosis. Similarly, TNF can be either pro-or anti-apoptotic but is predominantly the latter, and to this end, the blockage of TNF is a therapeutic strategy in the management of a variety of diseases, including plaque psoriasis and rheumatoid arthritis. This is not the frst time green tea derivatives have been shown to down-regulate TNF; recently, epigallocatechin gallate (EGCG), which is an active constituent in sinecatechins ointment, was shown to effectively down-regulate TNF in vitro (Wang et al., 2014). These fndings provide strong evidence for TNF as a key therapeutic target in the treatment of EGW.

Based on results from this and our previous study, sinecatechins ointment appears to interact with the NF-κB pathway to modulate apoptosis. The NF-κB pathway, however, functions in other biological processes, including inflammation. Another NF-κB associated change includes NFKB1, which encodes the p105 subunit of a host of transcription factors active in the NF-κB pathway. Interestingly, NFKB1 was up-regulated in VNR, which is counterintuitive to the aforementioned down-regulation of NF-κB-pathway molecule BIRC3 in VR. However, taken together with the fnding that the pro-inflammatory IL1A is also upregulated in VNR, the up-regulation of NFKB1 in VNR likely reflects the uncontrolled inflammation in lesions that sinecatechins was unable to suppress. It is unclear whether the inflammation would have occurred independent of sinecatechins application; regardless, inflammatory changes appear to be an important indicator of therapeutic failure. In contrast, several pro-inflammatory genes were down-regulated in VR, including VCAM1, which promotes inflammatory cell migration; CD5, which serves as a marker for T cell proliferation in the inflammatory response; CSF-2, which stimulates differentiation of inflammatory cells such as granulocytes and macrophages; and CXCL9, which encodes the chemoattractant that functions in tandem with interferon-gamma in the recruitment of macrophages (Yusuf-Makagiansar et al., 2002; Saha et al., 2010).

Although the HPV types detected in the VR' lesions are uncommonly associated with malignancy (HPV-6, -18, and -35), dysregulated epithelial cell proliferation is a cardinal pathophysiological feature of EGW. To this end, it is not surprising that several genes implicated in tumor immortalization were down-regulated and up-regulated in VR and VNR, respectively. Cancer-associated genes with decreased expression levels in VR include GREB1 (breast cancer) and TFRC (leukemia and lymphoma) (Aisen, 2004; Liu et al., 2012). Interestingly, TP53, the gene for the well-described tumor suppressor p53, was down-regulated in VR; while the significance of this finding is unclear, this expression change could represent a return-to-normal cellular function in VR as the sinecatechins-treated lesions resolve. The down-regulation of HSPB1, which functions to facilitate cellular stress response, may also underlie a return-to-normal cellular response in VR. In contrast, TERT, which encodes the telomerase reverse transcriptase enzyme, was up-regulated in VNR by approximately 4.85×. Telomerase up-regulation is highly associated with carcinogenesis since upregulation promotes apoptosis bypass and promotes immortalization (Baird, 2010). Finally, CDKN2A was upregulated 2.1× in VNR. CDKN2A encodes p16^INK4A, which inhibits growth signals leading to G1 growth arrest and senescence, and thus, CDKN2A is traditionally known as a tumor suppressor gene. However, it was shown that p16^INK4A is upregulated in HPV-mediated tumors and that this upregulation is necessary for tumor growth in vitro. It is hypothesized that the HPV E7 protein stimulates p16^INK4A, resulting in a molecular state in which the iconoclastic tumor suppressor p16^INK4A actually stimulates cell proliferation (McLaughlin-Drubin et al., 2013). The up-regulation of CDKN2A in our study provides in vivo evidence for this recently described in vitro process, which itself represents a potential therapeutic target.

Taken together with our previous report, current results provide cursory evidence that the sinecatechins' mechanism of action likely entails some degree of modulation of inflammatory and apoptotic processes, in particular, the NF-κB-pathway. It is possible that sinecatechins also upregulate the host immune response, since the only gene to be signifcantly upregulated in VR is the pro-lymphocytic IL2; however, further studies are needed to examine the exact pattern of immune regulation. Moreover, since no DEGs were identifed in VNR in the prior report, the present study provides putative insight into the expression patterns of patients who are not responsive to sinecatechins. Further examination into the mechanism of resistance is warranted, perhaps through the use of gene expression to identify patients who will not benefit from sinecatechins therapy. Overall, these studies provide valuable preliminary in vivo evidence on how sinecatechins may function to treat a highly infectious and debilitating disease.