Effect of Sinecatechins on HPV-Activated Cell Growth and Induction of Apoptosis.

The medicinal properties of tea leaves have been known for thousands of years.1,2 Tea leaves, produced from the plant Camellia sinensis, contain two unique bioactive compounds—flavonoids and methylxanthines.3 Methylxanthines, including caffeine, theobromine, and theophylline, are a group of alkaloids that are present in small quantities. Flavonols, which are a subtype of flavonoids, and also known as catechins, are the polyphenolic compounds that constitute 25 to 35 percent of green tea and are responsible for most of its beneficial effects.2–4 Epigallocatechin-3-gallate (EGCg) is an important constituent of catechins and considered the predominant therapeutic agent derived from green tea.3–5 Catechins, namely EGCg, have a wide variety of beneficial effects, including assisting in the prevention of chronic diseases ranging from cancer to cardiovascular diseases.3 Spectrum of biological and pharmacological activity of catechins. The spectrum of biological and pharmacological properties of green tea catechins includes antiangiogenic4,6–13 activity, anti-inflammatory and immunostimulatory2,3,5,8,10,11 activity, and antimicrobial potential.14–22 The antineoplastic activity of EGCg includes influencing tumor cell growth by inhibiting proliferation and inducing apoptosis.3,4 EGCg has been shown to inhibit growth and induce apoptosis in head and neck squamous cell carcinoma,6 and liver,8 ovarian,7 prostate,9,10 breast,11 and cervical cell lines.12 Antiproliferative and anticarcinogenic effects of EGCg have also been demonstrated in vitro in lung cancer cells,13 colon tumors, as well as lymphoma and leukemia cells.4 Tumor reduction induced by EGCg has been demonstrated in breast cancer cell lines.11 EGCg was shown to play an important role in inhibiting growth in a human papillomavirus (HPV) type 16-associated human cervical carcinoma cell line, CaSki. EGCg significantly inhibited growth by inducing apoptosis, possibly by negatively influencing cell proliferation at the G1 phase and gene expression pattern in CaSki cells.4 Similar results were seen in human prostate cancer cells, where EGCg was shown to affect mitogenesis and induce apoptosis by causing G0/G1-phase cell cycle arrest.9 Induction of apoptosis and cell cycle arrest at the G1 phase was also seen in ovarian and liver cancer cell lines.7,8 In the androgen-sensitive human prostate carcinoma cells, LNCaP, EGCg was shown to induce apoptosis through stabilization of p53 by phosphorylation on critical serine residues, and by negative regulation of nuclear factor-κB (NF-κB) activity, a family of transcriptional regulators.10 Upregulation of p53 expression was also seen in liver cancer cells,8 and reduction in NF-κB levels and activity by inhibition of the Her-2/neu signaling pathway was seen in breast cancer cells.11 Additionally, EGCg has also been associated with telomerase inhibition, a potentially selective target for treatment of cancer.12 However, despite the various mechanisms investigated, including modulation of signal transduction pathways, regulation of gene expression, induction of apoptosis, inhibition of cell proliferation and transformation, the exact mechanism of action of EGCg in inhibiting neoplastic growth is still unclear. EGCg also influences immunostimulation and anti-inflammatory responses by inducing the release of immune stimulatory agents while suppressing the release of immune inhibitory agents. The anti-inflammatory activity of EGCg may be related to an antioxidant function mediated by nonantioxidant mechanisms.2,3 This includes the ability of EGCg to modulate signal transduction pathways8,10 and inhibit the activity of transcription factors, such as NF-κB10,11 and activator protein 1 (AP-1).2 The antioxidant function of EGCg is characterized by its ability to scavenge reactive oxygen species (ROS),3,5 which also plays a key role in enhancing its anti-inflammatory properties.5 For example, the downstream effect of inhibiting NF-κB activation results in downregulation of interleukin-8 (IL-8), a major neutrophil chemoattractant and inflammatory mediator.2,5 In addition to IL-8, EGCg modulates inflammatory signaling pathways and negatively influences the infiltration of T cells into sites of inflammation.5 Additionally, catechins activate T lymphocytes and induce the release of tumor necrosis factor alpha (TNF-α) and interferon-gamma (IFN-γ). Immunostimulatory activity of catechins includes stimulating macrophages to release cytokines, promoting recruitment of monocytes, dendritic cells, lymphocytes, natural killer cells, and T-helper cells.2 EGCg has demonstrated a broad spectrum of antimicrobial activity against bacteria and fungi, including growth inhibition of Escherichia coli, Staphylococcus aureus, and Trichophyton sp.14,23 Most notable is the ability of catechins, such as EGCg, to protect against viral infections, including rotavirus,14 enterovirus,14,24 adenovirus,16,24 human immunodeficiency virus (HIV),17–19 Epstein-Barr virus (EBV),14 and influenza A and B.20–22 Catechins have exhibited antivirulent activity against HIV through several steps in the HIV life cycle. EGCg not only prevents HIV attachment and cell entry, but also destroys viral particles and virus production by competitively inhibiting HIV-1 deoxyribonucleic acid (DNA) polymerase and reverse transcriptase. Viral transcription is also impacted with a distinct decrease in the expression of messenger ribonucleic acid (mRNA) in the presence of EGCg.17,18 Furthermore, EGCg has been shown to inhibit HIV infection and replication without any observed apoptosis or necrosis.19 EGCg inhibits expression of EBV viral protein by inhibiting transcription of immediate-early genes, thus preventing initiation of EBV lytic cycle.14 In Madin-Darby canine kidney (MDCK) cells, EGCg affected the infectivity of influenza A and B viruses by agglutinating the virus, thus blocking the attachment of the viral particles to the target cell receptors and preventing the virus from absorbing to MDCK cells.20,21 In addition to preventing infection, EGCg was also shown to inhibit replication of the viruses by preventing acidification of endosomes and lysosomes, an essential step in uncoating of the virus and viral replication.21,22 Sinecatechins. Sinecatechins ointment, 15%, is manu-factured from the extract of green tea leaves from Camellia sinensis, consisting of 85- to 95-percent catechins, with EGCg representing the primary catechin at more than 55 percent (Figure 1).25 It is the first botanical agent approved by the United States Food and Drug Administration (FDA) for prescription use and is indicated for use as a topical patient-applied treatment for external genital and perianal warts (EGW).26 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).27 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.27 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.27 Figure 1 Structure of green tea catechins. Although the molecular mechanisms of the antiviral and anticarcinogenic potential of green tea catechins have been elucidated recently in numerous studies, the mechanism of action (MOA) of sinecatechins in the eradication of EGW is unknown. As an anticarcinogenic agent, it has been shown that green tea catechins interfere with different biochemical pathways involved in tumor proliferation, mitogenic signal transduction, cell cycle progression, neoplastic cell transformation, and inflammation.3,4,6–13 As an antiviral agent, catechins have exhibited the ability to inhibit reverse transcriptase and viral transcription, interfere with viral replication, and prevent adherence of viral particles to target cell receptors.14,15,17–22 To study the growth inhibitory potential of sinecatechins in HPV-positive tumor cell lines, four cervical carcinoma cell lines were treated with sinecatechins. The objectives of this study were to investigate growth inhibitory potential of the catechins in human cervical carcinoma cell lines infected with HPV. This includes investigating the impact of catechins on the viability of HPV-16 positive cells (CaSki and SiHa) and HPV-18 positive cells (HeLa and C4-I), assessing the kinetics of the antiproliferative effect of sinecatechins on cervical carcinoma cell lines, and determine whether the effect observed in the viability assays (WST-1) was due to a retardation in cell proliferation or due to reduction of total cell numbers leading to cell death. Furthermore, green tea extracts from different suppliers were evaluated to determine any differences in their growth inhibitory potential and further elucidate the potential MOA of sinecatechins in the eradication of EGW.