TOPICS

Sinecatechins Effects on HPV-Induced Enzymes Involved in Inflammatory Mediator Generation

Stephen K. Tyring, MD, PhD, MBA
Departments of Dermatology, Microbiology/Molecular Genetics and Internal Medicine, University of Texas Health Science Center, Houston, Texas

Disclosure: Dr. Tyring has conducted investigator-initiated clinical trials sponsored by PharmaDerm®, a division of Fougera Pharmaceuticals Inc.

Abstract
Background: Based on published studies, the biological properties of green tea catechins are antiviral, antioxidative, anticarcinogenic, antiangiogenic, and immunostimulatory. The United States Food and Drug Administration has approved a topical ointment formulation of sinecatechins, derived from green tea catechins and other tea components, for the treatment of external genital and perianal warts. The exact mechanism of action of sinecatechins in eradication of human papillomavirus-induced external genital and perianal warts is unknown, but may be due to one or more of the mechanisms mentioned. Objective: This study was conducted to investigate the effects of sinecatechins on proteases, inflammatory enzymes, and kinases contributing to human papillomavirus expression and growth. Design: Using commercially available in-vitro biochemical assays, sinecatechins were tested for their activity against matrix metalloproteinase (MMP-1, MMP-2, MMP-7, MMP-9); enzymes involved in oxidative stress (lipoxygenases and cyclooxygenases [COX-1, COX-2]); several growth factors (epidermal growth factor, platelet-derived growth factor, and transforming growth factor-b); and extracellular signal-regulated kinases 1/2. The ability of sinecatechins to inhibit ligand binding of growth factors was also studied. R esults: Sinecatechins showed specific inhibition against a variety of enzymes at concentrations in the micromolar range. With the exception of matrix metalloproteinase-1, all proteases tested were inhibited in a dose-dependent manner. Pronounced inhibition of certain lipoxygenases was observed. Cyclooxygenases were also inhibited, with slight selectivity of greater inhibition against cyclooxygenases-2, the inducible form of cyclooxygenases. Extracellular signal-regulated kinases 1/2 (involved in human papillomavirus tumor cell growth) were also inhibited by sinecatechins at high concentrations, while epidermal growth factor receptor was inhibited at surprisingly low concentrations. In contrast, no inhibition of binding of various growth factors to their corresponding receptors was seen, highlighting the specificity of sinecatechins inhibitory activity. Results demonstrated that sinecatechins specifically inhibit multiple human papillomavirus-induced pathways and molecules, likely contributing to external genital and perianal warts clearance via direct antiviral activity. Conclusion: As expected, sinecatechins inhibited a broad range of enzymes and kinases involved in the generation of inflammatory mediators: proteases, oxygenases, and protein kinases were all inhibited by sinecatechins in the micromolar range.  (J Clin Aesthet Dermatol. 2012;5(1):19–26.)

Numerous published studies have established the health benefits of tea consumption. These health benefits—most specifically those of green tea—have been touted for centuries; however, the bulk of the scientific investigations of green tea have only emerged within the past 30 years or so.[1] Green tea consumption has long been a tradition in China and other Asian countries as a method for preventing and treating a number of diseases. Green tea is mainly produced from the plant Camellia sinensis. Unlike black and oolong teas, green tea is nonfermented; it is processed by drying and steaming the fresh tea leaves, inactivating the polyphenol oxidase, thus preserving the content of the polyphenols.[2] Green tea is a rich source of a wide range of polyphenols, most notably catechins, a group of very active flavonoids, with potent antioxidant activity.1 Catechins represent 60 to 80 percent of the total polyphenols in green tea.[3] The four major catechins in green tea are: epigallocatechin-3-gallate (EGCG), the most abundant catechin in green tea, which represents approximately 59 percent of the total of catechins; epigallocatechin (EGC), which represents approximately 19 percent of the total; epicatechin-3-gallate (ECG), which represents approximately 14 percent; and epicatechin (EC), which represents approximately six percent of the total catechin content.1 These polyphenolic compounds (especially EGCG) are thought to confer green tea’s health benefits.[3]

Green tea catechins are most widely recognized for their antioxidant properties and, in fact, the majority of their health benefits are attributed to this property. This mechanism has led to the evaluation of catechins in numerous diseases associated with reactive oxygen species (ROS), including cancer and cardiovascular and neuro-degenerative diseases.[4] Expression of ROS in cells results in alterations in deoxyribonucleic acid (DNA) structure, enzymatic activity, and host defense mechanism and contributes to cancer development.

Green tea catechins represent a potent group of antioxidants, and, as such, they may play an important role in the prevention and control of cancer development.[5] Flavonols found in green tea exhibit antioxidant activity 20 times greater than vitamin C.[5] Beyond their antioxidative effects, green tea catechins have also demonstrated other effects on cellular and molecular targets linked to cell death and survival. Among the potential health benefits of green tea catechins are their antiviral properties, such as exerting a protective effect against human immunodeficiency virus (HIV) mediated by inhibiting virions to bind to the target cell surface.[2,4] Additionally, effects of green tea against the influenza virus, Herpes simplex virus, and adenovirus have also been demonstrated.[1,5] Other medical benefits of green tea catechins are summarized in Table 1.

Catechins: Biological effects reported in the literature. Numerous mechanisms of action (MOAs) have been attributed to catechins in studies reported in the literature, including their widely known antioxidative activity; additional mechanisms, such as antiviral effects, immunostimulatory activity, and antiproliferative effects, have been reported (Table 2).[4,6–18] Catechins have been reported to scavenge a host of reactive oxygen and nitrogen species, including superoxide and peroxyl radicals, among others. The chemistry explaining this activity is the result of hydrogen atom transfer or single electron transfer reactions involving hydroxyl groups.[6] Green tea catechins may also exert indirect antioxidant activity via inhibitory effects on transcription factors (e.g., nuclear factor-kB [NF-kB], activator protein-1 [AP-1]) and enzymes with activity that increases oxidative stress (e.g., lipoxygenases, cyclo-oxygenases, and inducible nitric oxide).[3,4,6–10] There is evidence from animal models and human studies as well showing that EGCG induces expression of endogenous antioxidant systems (e.g., superoxide dismutases, catalase, and glutathione).[6] Multiple gene products of human papillomavirus (HPV) interfere with cellular pathways, promoting cell growth. Catechins have demonstrated in-vitro inhibitory effects on certain HPV gene products (e.g., retinoblastoma protein [pRb], p53 protein, epidermal growth factor receptor [EGFR] kinase and telomerase) and have also shown antiviral effects in vivo in a number of studies.[7,10–14] EGCG-induced growth inhibition of HPV-transformed cervical cells through early (G1 phase) cell cycle arrest, induction of apoptosis, and regulation of gene expression.[12,14] Green tea catechins in an ointment and oral capsule were found to be effective in treating cervical HPV lesions, as evidenced by positive morphological changes of cervical lesions and a decrease or loss of HPV DNA titers following treatment.[11]

The immunostimulatory effects of catechins have been demonstrated in vitro, specifically inducing a number of proinflammatory cytokines[15] and stimulating macrophages and lymphocytes.[16] Stimulation of peripheral blood mononuclear cells with EGCG in vitro induced the production of interleukin [IL]-1b and tumor necrosis factor-alpha (TNF-a) and also stimulated IL-1a messenger ribonucleic acid (mRNA) expression.[15] The effectiveness of green tea catechins in restoring immune function in the skin has been demonstrated following ultraviolet B (UVB) and UVA-induced immune suppression. Topical application with catechins restored the number of Langerhans cells in the skin of volunteers after their reduction following UV-treatment.[16] Langerhans cells are known to play a key role in the development of cutaneous cell-mediated immune responses.[16] Finally, antiproliferative effects of green tea catechins have been demonstrated in numerous studies.[8–10,17,18] Green tea polyphenols possess cancer chemopreventive effects, anticarcinogenic effects through modulation of signal transduction pathways, and effects on growth factors as well as antiangiogenic effects through inhibition of microvessel formation and effects on matrix metalloproteinase (MMP).[4,17]

Sinecatechins. A patient-applied, topical ointment formulation of catechins (sinecatechins ointment, 15%, Veregen®, PharmaDerm®, a division of Fougera Pharmaceuticals Inc.) was approved by the United States Food and Drug Administration (FDA) for the treatment of external genital and perianal warts (EGW).[19] Sinecatechins ointment, 15%, is the first FDA-approved botanical drug comprising a proprietary blend of eight different catechins and other green tea components. It is a partially purified fraction of the water extract of green tea leaves from Camellia sinensis. Catechins constitute 85 to 95 percent by weight of the total drug substance, with EGCG representing the primary catechin at more than 55 percent (Figure 1).[19] The other seven catechins include EC, EGC, epicatechin gallate, gallocatechin gallate, gallocatechin, catechin gallate, and catechin.
These eight catechins are the most abundant catechins found in green tea extract. Each gram of ointment contains 150mg of sinecatechins in a water-free ointment base. Two Phase 3 clinical trials involving more than 1,000 male and female patients with EGW treated for up to 16 weeks with sinecatechins ointment, 15%, resulted in statistically superior complete clearance rates of all warts (baseline and newly emerging warts) compared with vehicle-treated patients (54.9% vs. 35.4%, respectively, p<0.001).[20] In addition, recurrence of warts was assessed in all patients with complete clearance of all warts; low rates of recurrence (6.8%) were demonstrated with sinecatechins ointment, 15%, when measured at 12 weeks post-treatment.20 As opposed to previous clinical trials of EGW therapies, which only measured the clearance of baseline warts, clearance of all (baseline and new) warts represents a much more relevant therapeutic endpoint.[20]

The mechanism of action (MOA) of sinecatechins in the eradication of EGW is unknown. As described, sinecatechins demonstrate in-vitro antioxidative activity; whether this alone is the MOA in eradication of EGW is unclear. As catechins have demonstrated a host of molecular and cellular mechanisms in published studies (e.g., antioxidative, antiviral, anticarcinogenic, anti-angiogenic, immunostimulatory), it is possible that the efficacy of sinecatechins ointment in eradication of HPV-induced EGW could be attributed to one or more of these mechanisms. Catechins have been shown to have direct inhibitory activities against several enzymes and receptors involved in cancer, angiogenesis, cell cycle regulation, signal transduction, protein degradation, and energy metabolism. An in-vitro study was conducted to investigate the effects of sinecatechins on proteases, enzymes involved in oxidative stress, proteins related to signaling transduction pathways and growth factors, and to further elucidate the potential MOA of sinecatechins in the eradication of EGW.

Methods
The effects of sinecatechins on enzymes involved in the generation of inflammatory mediators (oxygenases involved in prostaglandin and leukotriene generation), proteases involved in tumor invasion, kinases involved in tumor cell signaling, and specific ligand/receptor interactions were tested. Commercially available biochemical assays using purified enzymes and receptors were utilized for all experiments detailed below (MDS Pharma Services, Munich, Germany). For all assays, reference standards were run as an integral part of the assay to validate results obtained. Initially, a single concentration of sinecatechins (10µM) was run for all assays except for the metalloproteinase and proteasome assays, in which a concentration of 1µM was used. Repeated runs of all assays utilizing two sinecatechins concentrations of 50µM and 100µM were then run in triplicate.

Human MMP peptidase assay. Inhibition of human MMP-1, MMP-2, MMP-7, and MMP-9 was measured. Briefly, MMP-1, MMP-2, MMP-7, and MMP-9 proenzymes were each independently activated with APMA (p-aminophenyl-mercuric acetate) for 60 minutes at 37°C. Sinecatechins (1µM, 10µM, and 100µM) were preincubated with 8nM (MMP-1, MMP-2) or 1.9nM (MMP-7, MMP-9) active enzyme in modified (N-morpholino) propane sulfonic acid (MOPS) buffer (pH 7.2) for 60 minutes at 37°C. Each reaction was initiated by addition of 4µM Mca-Pro-Leu-Gly-Leu-Dpa- Ala-Arg-NH2 (Dpa=N-3-2,4-dinitrophenyl)-L-2,3-diamino-propinoyl; Mca=(7-methoxycoumarin-4-yl) acetyl). After incubation for another 120 minutes, the amount of Mca-Pro-Leu-Gly produced was spectrofluorometrically determined. The reference agent in each assay was the tissue inhibitor of MMP-2.

Human 20S proteasome assay. Human erythrocyte 20S proteasome was used. Sinecatechins at 1µM, 10µM, and 100µM concentrations were preincubated with 2.5µg/mL enzyme in modified HEPES buffer (pH 7.5) for 15 minutes at 37°C. The reaction was initiated by addition of 10µM Suc-LLVY-AMC. After another 30 minutes of incubation, the amount of AMC formed was spectrophotometrically determined. The reference agent of this assay was lactacystin.

Human COX and lipoxygenase (LO) assays. In the COX assays, the inhibition of the conversion of arachidonic acid to prostaglandin E2 (PGE2) by recombinant human COX-1 and COX-2 enzymes was analyzed. For the COX-1 assay, 10µM and 50µM of sinecatechins were incubated with human platelets (5×107/mL) in modified HEPES buffer (pH 7.4) for 15 minutes at 37°C. The reaction was initiated by addition of 100µM arachidonic acid for another 15 minutes and terminated by addition of 1N HCl. An aliquot was removed and counted to determine the amount of PGE2 formed. In this assay, the reference agent was indomethacin. For the COX-2 assay, human recombinant COX-2 expressed in insect Sf21 cells was analyzed. Sinecatechins (10µM and 50µM) were preincubated with 0.11U enzyme in modified Tris-HCl buffer (pH 7.7) for 15 minutes at 37°C. The reaction was initiated by the addition of 0.3µM arachidonic acid for another five minutes and terminated by the addition of 1N HCl. An aliquot was removed and counted to determine the amount of PGE2 formed.

The reference agent in this assay was nimesulide. Inhibition of three lipoxygenases (human platelet 5-LO, human platelet 12-LO, and rabbit reticulocyte 15-LO) by 10µM and 50µM of sinecatechins were analyzed. The reference agents in these assays were nordihydroguaiaretic acid (NDGA), baicalein, and PD-146176, respectively.

Human protein tyrosine kinase receptor (EGFR) assay. Protein kinase EGFR from human A431 cells was used. Wells were coated overnight with 10µg/mL poly (Glu:Tyr). Sinecatechins (10µM and 50µM) were preincubated with 10U/mL enzyme in modified HEPES buffer (pH 7.4) for 15 minutes at 25°C. The reaction was initiated by addition of 100µM adenosine triphosphate (ATP) for another 60-minute incubation period and terminated by aspirating the solution. The amount of phosphopoly (Glu:Tyr) produced was determined using a 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) micro well peroxidase substrate system read colorimetrically at 405nm. The reference agent of this assay was tyrphostin 47.

Human protein serine/threonine kinase (extra-cellular signal-regulated kinases [ERK]) assays. Inhibition of human recombinant ERKs ERK1 and mouse recombinant protein kinase ERK2 expressed in Escherichia coli was analyzed in vitro. Sinecatechins (10µM and 50µM) were preincubated with 200ng/mL enzyme in 3-MOPS buffer (pH 7.2) for 15 minutes at 37°C. The reaction was initiated by addition of 50µg/mL myelin basic protein (MBP), 10µM ATP and [g32P] ATP for another 30-minute incubation period and terminated by cooling on ice. An aliquot was removed and counted to determine the amount of [32P] MBP formed. The reference agent of each assay was staurosporine.

Ligand binding assays. Binding of EGF, platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-b), vascular endothelial growth factor (VEGF), and TNF to membrane preparations in the presence of 10µM and 50µM sinecatechins were analyzed in all the assays described below.

Human EGF binding assay. Briefly, 5µg of membrane proteins prepared from human A431 cells were incubated in modified HEPES buffer (pH 7.7) with 0.05nM murine [125I]EGF for 60 minutes at 25°C. Nonspecific binding was determined in parallel incubations by adding 10nM of unlabeled human EGF. Samples were pressed through a 0.2µm filter. Filters were washed then counted to determine the amount of murine [125I]EGF, which had specifically bound. In this setting, specific binding of 95 percent was expected. The reference agent of this assay was human EGF.

Mouse PDGF binding assay. Swiss mouse 3T3 embryo cells were suspended in Dulbecco’s modi?ed Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS). Cells (5×105) were incubated with 20 pM [125I]PDGF-BB for 45 minutes at 25oC. Nonspecific binding was determined in parallel incubations by adding 0.1nM unlabeled PDGF-BB. Cells were centrifuged and specific binding of [125I]PDGF-BB was determined by measuring radioactivity in the pellets. Specific binding of 88 percent was expected. The reference inhibitor was PDGF-BB.

Mouse TGF-b binding assay. Swiss mouse 3T3 embryo cells were suspended in DMEM containing 10% FBS. Cells (5×105/mL) were incubated with 12.5pM [125I] TGF-b1 for 45 minutes at 25oC. Nonspecific binding was determined in parallel incubations by adding 0.1nM of unlabeled TGF-b1. Cells were centrifuged and radioactivity was counted in pellets to determine specifically bound [125I]TGF-b1. Specific binding of 66 percent was expected. The reference inhibitor was TGF-b1.

Human VEGF binding assay. Human umbilical vein endothelial cells were used in modified HEPES buffer (pH 7.4) using standard techniques for whole cell radioligand binding studies. Cells (2×104) were incubated with 0.1nM [125I]VEGF for three hours at 25°C. Nonspecific binding was determined in parallel incubations by adding 3nM of unlabeled VEGF165. Cells were gently washed with buffer and removed from the well with trypsin-EDTA solution and radioactivity was counted to determine specifically bound [125I]VEGF. Specific binding of 85 percent was expected. The reference inhibitor of this assay was VEGF165.

Human TNF, nonselective binding assay. Human U-937 cells were used to prepare membranes in modified Tris-HCl buffer (pH 7.4). A 50µg aliquot was incubated with 28pM [125I]TNF-a for three hours at 4°C. Nonspecific binding was determined in the presence of 40nM of unlabeled TNF-a. Membranes were filtered and washed, and radioactivity of the filters was counted to determine specifically bound [125I] TNF-a. Specific binding of 60 percent was expected. The reference inhibitor of this assay was TNF-a.

Results
Inhibitory effect of sinecatechins on protease activity. MMP and the 20S proteasome are involved in tumor cell proliferation as well as tumor invasion, and were previously described to be inhibited by green tea catechins.[21,22] In a first set of experiments, the effect of sinecatechins on the activity of MMP-2, MMP-7, MMP-9, and the 20S proteasome was analyzed at a concentration of 1µM. Because no inhibition was detectable at this concentration, all protease assays were repeated using a concentration of 1µM, 10µM, and 100µM of sinecatechins. As shown in Figure 2, all proteases, with the exception of MMP-1, were inhibited in a dose-dependent manner, although only at concentrations above 10µM. At 100µM, inhibition was 21 percent (MMP-2), 55 percent (MMP-7), 69 percent (MMP-9), and 59 percent (20S proteasome). In contrast, MMP-1 was not inhibited even at 100µM sinecatechins, suggesting specific inhibitory effect of sinecatechins on metalloproteases.

Inhibitory effect of sinecatechins on enzymes generating inflammatory mediators. The effect of sinecatechins on enzymes involved in the generation of inflammatory mediators was analyzed in different biochemical assays. Among those assays were three lipoxygenases (5-LO, 12-LO, and 15-LO), which are essential enzymes for the production of leukotrienes, the two known cyclooxygenases (COX-1 and COX-2), which are involved in the production of prostaglandins and thromboxanes-cytokines that exert immunosuppressive activity. With the exception of 5-LO, all enzymes were inhibited by sinecatechins (Figure 3). COX-1 and COX-2 were inhibited at 50µM sinecatechins by 27 percent, and 74 percent, respectively. Thus, sinecatechins showed a slight selectivity toward the inhibition of the inducible form of COX (COX-2). A more pronounced inhibition was observed with 12-LO (90% inhibition at 10µM) and 15-LO (100% inhibition, half maximal inhibitory concentration [IC50] 0.67µM). In contrast, 5-LO was not significantly inhibited by sinecatechins at concentrations up to 50µM.

Inhibitory effect of sinecatechins on kinases. Kinases are involved in cell signaling pathways and frequently constitutively active in tumor cells and in HPV-infected cells. Some of these kinases were reported to be influenced in their activity by tea catechins. Therefore, the effect of sinecatechins on the activity of ERK1, ERK2, and EGFR, which are suggested to be involved in tumor cell growth, was investigated. In a pilot experiment, the potential of sinecatechins to modulate kinase activity was analyzed at a concentration of 10µM. Additionally, in the ERK1 and ERK2 assay, sinecatechins were tested at a concentration of 50µM and 100µM. As shown in Figure 4, sinecatechins completely inhibited the human EGFR kinase activity at a concentration of 10µM. ERK1 and ERK2 were also inhibited by sinecatechins, although only at higher concentrations (?80% inhibition at 50µM). Furthermore, a dose-response analysis was performed using the EGF receptor assay. Surprisingly, sinecatechins showed a very high potential to inhibit the activity of the EGFR kinase with an IC50 of 0.029±0.001µM.

Inhibitory effect of sinecatechins on the binding of growth factors. In addition to the potential of sinecatechins to inhibit kinase activity, the effect on ligand binding of different growth factors (EGF, PDGF, TGF-b, and VEGF) as well as TNF was tested. In contrast to the strong inhibitory potential of sinecatechins on the EGFR kinase activity, the interaction between EGF and its ligand was not affected, even at concentrations up to 50µM (Figure 4). Similarly, the interaction of other growth factors with their respective receptors was not influenced. As shown in Figure 4, PDGF, TGF-b, TNF, and VEGF binding to the corresponding receptors was not inhibited by sinecatechins at 50µM concentration.

Discussion
Several publications are available describing the molecular basis of the MOA of green tea catechins.[6–18] Green tea extract has been demonstrated to inhibit enzymes involved in angiogenesis, cell cycle regulation, signal transduction, protein degradation, and energy metabolism. Catechins can directly bind to specific proteins, leading to the inhibition of several enzymes (COX, kinases, and proteases) and to the modulation of transcription factors (e.g., AP-1, NF-kB), which are sensitive to oxidative stress. Green tea extracts and individual catechins have been described as potent inhibitors of metalloproteases MMP-1 (EGCG: IC50=19nM), MMP-7, and the 20S proteasome. Therefore, the aim of the present study was to evaluate the potential of sinecatechins to inhibit the activity of enzymes or to inhibit the binding of receptors to their ligands that have been described in the literature to be affected by catechins. This information could shed light on the MOA of topical sinecatechins ointment in the eradication of EGW. Sinecatechins inhibited several enzymes involved in the generation of intercellular mediators, proteases involved in tumor invasion, and kinases involved in tumor signaling. Sinecatechins inhibitory activity, albeit slightly lower, was found to be similar to the activity of catechins in published literature reports.

No published data are available analyzing the direct inhibition of COX-1 and COX-2 activity by sinecatechins in vitro. Compared with the inhibitory activity of aspirin (IC50 for COX-1 is 4.2µM and for COX-2 is 590µM, historical data from MDS Pharma Services), sinecatechins showed a lower activity toward COX-1, but a higher activity toward COX-2. COX-1 and COX-2 activity was inhibited by 50µM sinecatechins by approximately 30 and 70 percent, respectively. However, it is clearly less active than diclofenac (COX-1: IC50=35nM, COX-2=69nM, historical data from MDS). The direct inhibition of the COX-2 enzyme activity could add onto the NF-kB mediated decrease of cellular COX-2 protein levels by sinecatechins described in several different experimental systems.[3] Indeed, this study demonstrated selective inhibition of the COX-2 enzyme by sinecatechins.
The presence of immune inhibitory cytokines (e.g., COX-2) in the skin can compromise the immune response, favoring progression of HPV. For example, elevated levels of COX-2 have been found in HPV-16 to cause cervical dysplasia, carcinoma in situ, and invasive carcinoma. The immunostimulatory activity shown with sinecatechins suggests that they may contribute to an improved immune response to HPV-induced skin lesions (i.e., EGW). Lipoxygenases have been implicated in a wide range of diseases caused by uncontrolled cell growth and/or regulation, including a variety of inflammatory disorders and cancers. Specifically, 12-LO plays a major role in psoriasis; 5-LO products and 15-LO are involved in other diseases. The 90 to 100-percent inhibition by sinecatechins of 12-LO and 15-LO, respectively, suggests the utility of sinecatechins in LO inhibition. The 0.67µM IC50 found in the 15-LO assay is in the range of EGCG activity described in the literature.[3]

Sinecatechins inhibited several signaling kinases (ERK1/2), which are activated by HPV infection. ERK1/2 inhibitors may be useful therapeutic agents for the treatment of HPV infection as well as cancer and other diseases. The kinases ERK1 and ERK2 were inhibited by sinecatechins, although only at higher concentrations (80% inhibition at 50µM). Published data from cellular assays described in the literature showed comparable activity of EGCG.[10]

The direct inhibition of the ERK1/2 enzyme activity could add to the inhibition of the EGFR activity by sinecatechins described in this study. The combined inhibition of EGFR and ERK1/2 could explain the efficient inhibition of proliferation of several carcinoma cell lines by polyphenols described in the literature.[7,10] The biochemical IC50 value for the inhibition of EGFR kinase is three orders of magnitudes lower (29.2±1.0nM) compared with IC50 determined by cellular assays.[10] In contrast to the results found for the receptor kinase activity or for the other tested growth factors, the interaction of the ligand EGF with its receptor, as well as the interaction of several other growth factors with their respective receptors, were not inhibited by sinecatechins at concentrations up to 50µM.

The molecular activities of sinecatechins demonstrated in this study might well explain its utility in the eradication of EGW. Although the exact MOA of sinecatechins remains unknown, it is unclear if the clearance of EGW may be solely attributed to its known in-vitro antioxidative activity. It is conceivable that the high efficacy rates and low recurrence rates seen following its topical application are likely due to antiviral, immunostimulatory, and antiproliferative activities, in addition to its known antioxidative activity; however, the FDA has not approved the inclusion of these activities in the product label. Most EGW treatments (provider administered and patient applied) rely on destructive methods to eradicate lesions. The sole exception (in addition to sinecatechins) is imiquimod, which does have immunostimulatory activity. These other treatments have not demonstrated the low rates of recurrence or the efficacy against baseline and new warts seen following sinecatechins treatment. Indeed, although no comparative trials with other topical patient-applied EGW therapies (i.e., imiquimod, podofilox) have been conducted to date, published cure rates of other treatments are lower than those seen following sinecatechins and importantly, higher rates of recurrence are reported (13–19% for imiquimod, 40% for cryotherapy, and as high as 91% for podofilox).[20]

Conclusion
As expected, sinecatechins inhibited a broad range of enzymes and kinases involved in the generation of inflammatory mediators. Among these enzymes were proteases, oxygenases, and protein kinases; all inhibited by sinecatechins in the micromolar range. Interestingly, sinecatechins exhibited the strongest inhibitory potential against EGFR activity, with high specificity. This study demonstrates that sinecatechins are potent inhibitors of a variety of enzymes involved in the pathogenesis of HPV infection as well as other viral infections.

The specific inhibition of multiple HPV-induced pathways by sinecatechins reasonably contributes to the clearance of EGW, likely via direct antiviral and immunostimulatory effects. Many of the in-vitro activities of sinecatechins demonstrated in this study result in regulation of cell growth and in the induction of immune mechanisms, both of which may contribute to the clearance of EGW, providing some insight regarding its MOA.

Acknowledgment
The author gratefully acknowledges MediGene AG, Planegg/Martinsried, Germany, and its employees for funding and conducting the study presented in this manuscript. The author also gratefully acknowledges the writing and editorial assistance of Dawn Flynn and Malik Cobb, PA-C.

References
1.    Cabrera C, Artacho R, Giménez R. Beneficial effects of green tea-a review. J Am Coll Nutr. 2006;25:79–99.
2.    Cooper R, Morré DJ, Morré DM. Medicinal benefits of green tea: part I. Review of noncancer health benefits. J Altern Complement Med. 2005;11:521–528.
3.    Higdon JV, Frei B. Tea catechins and polyphenols: health effects, metabolism, and antioxidant functions. Crit Rev Food Sci Nutr. 2003;43:89–143.
4.    Zaveri NT. Green tea and its polyphenolic catechins: medicinal uses in cancer and noncancer applications. Life Sci. 2006;78:2073–2080.
5.    Dufresne CJ, Farnworth ER. A review of the latest research findings on the health promotion properties of green tea.         J Nutr Biochem. 2001;12:404–421.
6.    Lambert JD, Elias RJ. The antioxidant and pro-oxidant activities of green tea polyphenols: a role in cancer prevention. Arch Biochem Biophys. 2010;501:65–72.
7.    Masuda M, Suzui M, Weinstein IB. Effects of epigallocatechin-3-gallate on growth, epidermal growth factor receptor signaling pathways, gene expression, and chemosensitivity in human head and neck squamous cell carcinoma cell lines. Clin Cancer Res. 2001;7:4220–4229.
8.    Bode AM, Dong Z. Signal transduction pathways: targets for green and black tea polyphenols. J Biochem Mol Biol. 2003;36:66–77.
9.    Sachinidis A, Skach RA, Seul C, et al. Inhibition of the platelet-derived growth factor  b-receptor-tyrosine phosphorylation and its downstream intracellular signal transduction pathway in rat and human vascular smooth muscle cells by different catechins. FASEB J. 2002;16:893–895.
10.    Sah JF, Balasubramanian S, Eckert RL, Rorke EA. Epigallocatechin-3-gallate inhibits epidermal growth factor receptor signaling pathway. Evidence for direct inhibition of ERK1/2 and AKT kinases. J Biol Chem. 2004;279: 12755–12762.
11.    Ahn W-S, Yoo J, Huh S-W, et al. Protective effects of green tea extracts (polyphenon E and EGCG) on human cervical lesions. Eur J Cancer Prev. 2003;12:383–390.
12.    Ahn WS, Huh SW, Bae S-M, et al. A major constituent of green tea, EGCG, inhibits the growth of human cervical cancer cell line, CaSki cells, through apoptosis, G1 arrest, and regulation of gene expression. DNA Cell Biol. 2003;22:217–224.
13.    Hastak K, Gupta S, Ahmad N, et al. Role of p53 and NF-kB in epigallocatechin-3-gallate-induced apoptosis of LNCaP cells. Oncogene. 2003; 22:4851–4859.
14.    Yokoyama M, Noguchi M, Nakao Y, et al. The tea polyphenol, (-)-epigallocatechin gallate effects on growth, apoptosis, and telomerase activity in cervical cell lines. Gynecol Oncol. 2004;92:197–204.
15.    Sakagami H, Takeda M, Sugaya K, et al. Stimulation by epigallocatechin gallate of interleukin-1 production by human peripheral blood mononuclear cells. Anticancer Res. 1995;15:971–974.
16.    Elmets CA, Singh D, Tubesing K, et al. Cutaneous photoprotection from ultraviolet injury by green tea polyphenols. J Am Acad Dermatol. 2001;44:425–432.
17.    Ahmad N, Mukhtar H. Green tea polyphenols and cancer: biologic mechanisms and practical implications. Nutr Rev. 1999;57:78–83.
18.    Larsen CA, Dashwood RH, Bisson WH. Tea catechins as inhibitors of receptor tyrosine kinases: mechanistic insights and human relevance. Pharmacol Res. 2010;62:457–464.
19.    Veregen Product Monograph. Melville, NY: PharmaDerm®,      a division of Fougera Pharmaceuticals Inc.; 2010.
20.    Tatti S, Stockfleth E, Beutner KR, et al. Polyphenon E®: a new treatment for external anogenital warts. Br J Dermatol. 2010;162:176–184.
21.    Nam S, Smith DM, Dou QP. Ester bond-containing tea polyphenols potently inhibit proteasome activity in vitro and in vivo. J Biol Chem. 2001;276:13322–13330.
22.    Oneda H, Shiihara M, Inouye K. Inhibitory effects of green tea catechins on the activity of human matrix metalloproteinase 7 (matrilysin). J Biochem (Tokyo). 2003;133:571–576.

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