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Virology
HPV persistence is the most important risk factor of tumor development, as in the case of cervical cancer, and it still represents a serious clinical challenge. EGCG, vitamin B12, folic acid, and HA are natural molecules that demonstrated beneficial role in treating HPV-induced lesions. Therefore, is reasonable to think that their association may have a promising action against HPV persistence.
- EGCG
- folic acid
- vitamin B12
- hyaluronic acid
1. Introduction
Epidemiological studies over the last decade have reported that human papillomavirus (HPV) infections are commonly sexually transmitted worldwide [1]. Recent data indicate that sexually active individuals will acquire an HPV infection at least once during their lives [2].
The pathogenic agents of HPV infection are papillomaviruses, non-enveloped circular double-stranded DNA viruses with a tropism for squamous epithelium and mucosal tissues [3]. Over 100 HPV types have been described, and 40 of them may infect the genital tract [4]. HPV infections may often cause cancer, mainly occurring at the transformation zones—“weak points”—between different types of epithelia [3]. HPVs are indeed the primary etiological agent for several cancers, including most of cervical cancers and anal, penile, vulvar, vaginal, and oropharyngeal tumors [5]. However, although HPV infection concerns both women and men, the high susceptibility to transformation following the infection, makes the weak point of the uterine cervix an ideal tissue to complete the viral lifecycle [6].
According to the oncogenic potential, papillomaviruses are classified into low-risk (LR) and high-risk (HR) HPV types. LR-HPV types, including types 1, 2, 6, and 11, are non-carcinogenic types as they do not induce cancerous lesions. They can lead to benign lesions (warts, condylomas, or recurrent respiratory papillomatosis) or, in rare cases, precancerous lesions [7]. Instead, HR types, including 16, 18, 31, 33, 35,45, 51, 52, 56, 58, 59, 66, and 68, are classified as carcinogenic types according to the IARC (International Agency for Research on Cancer) because they induce 99.7% of cervical cancers [8] and other anogenital cancers [9,10,11].
Often, HPV infections affecting cervical tissue can induce cervical intraepithelial (CIN) lesions of various degrees. These lesions, depending on their severity grade, can spontaneously revert or progress toward tumor development. Pre-malignant changes represent histological abnormalities ranging from atypical squamous cells of undetermined significance (ASCUS) and low-grade cervical dysplasia (LSIL/CIN1) that could progress to moderate dysplasia (CIN2) or severe dysplasia/carcinoma in situ (CIN3/CIS).
Following histologic diagnosis, the treatment of the HPV-induced lesions depends on the degree of severity: while ASCUS/CIN1 need no treatments at the time of diagnosis as most of them regress spontaneously, high-grade lesions (HSIL/CIN2 or CIN3) may undergo conservative surgical procedures, including ablative methods that destroy the affected cervical tissue [12]. Nevertheless, women with HSIL (CIN2/3) still have an increased risk of developing recurrent CIN2 or worse (CIN2+) after a loop electrosurgical excision procedure (LEEP) [13].
More than 90% of HPV infections are asymptomatic, transient, and regress within 6–12 months from the onset, thanks to the viral clearance activity of the immune system [14]. However, in some cases, the infection can persist over a year, thus increasing the probability of developing malignant tumors [14]. Prospective studies revealed that the prevalence of HPV includes a mix of both incident and persistent infections that have accumulated over time due to the lack of clearance [15].
Persistence is a peculiar concept in HPV infection, even though it is not homogeneously measured. Most authors refer to persistence as those infections having two consecutive positive HPV DNA tests with undetermined time intervals [14]; many other authors define persistence using the time to clearance, while others mean persistence as the same virus strain present in the same patient for more than nine months. Moreover, defining HPV persistence is further complicated considering the different methods of HPV laboratory detection, testing intervals, and HPV categorization [16]. However, despite the several definitions, persistence remains a key event in HPV infection, as it can trigger the development of malignant tumors. Persistence is associated with almost all cervical and anogenital cancers but also with head and neck cancers [17]. Several risk factors, such as genetics and lifestyle, can significantly increase the probability of developing persistent infections [18,19]. For instance, the carcinogens in cigarette smoke may increase the viral load and the probability of tumoral transformation in HPV-infected epithelial cells [20]. In addition, smoking or alcohol use are risk factors for persistent oral and genital HPV infections [21]. Given the prevalence of co-infections with multiple HPV types, several authors also indicated such factors as a predictor of persistent infection [22].
In general, the persistence of HPV infection—due to HR types—especially HPV types 16 (HPV16) and 18 (HPV 18)—is responsible for many genital [23] as well as oropharyngeal cancers [24]. If undetected and untreated, these types of lesions slowly progress in an average of 5–14 years due to the increased possibility of viral genome integration.
When HPV infection persists, genomic instability can increase viral genome integration into the host genome [25]. Once the viral genome has integrated into the host genome, this leads to a breakpoint in the E2 genetic sequence, resulting in the de-repression of the E6 and E7 viral oncogenes. The carcinogenic process, started with the E6 and E7 de-repression, includes the accumulation of additional alterations in the host genome that lead to the invasive cancer phenotype. For instance, E6 and E7 proteins impact the function of p53 and pRb proteins, two fundamental tumor suppressors [26,27].
Viral persistence often correlates with the ability to escape the immune response of the host. Indeed, the innate host immune response against HPV infection represents the first line of defense due to the recognition by pattern recognition receptors (PRRs) that activate downstream signaling pathways [28]. However, the HPV virus has developed evasion strategies to bypass immune surveillance, including the modulation of cytokine and chemokine expression, the alteration of antigen presentation process, and the down-modulation of the interferon (IFN) pathway and adhesion molecules [29]. Although integrated viral DNA is a hallmark of many HPV-associated cancers, in some cases, cancer cells may exhibit either integrated HPV DNA, extrachromosomal viral DNA, or a mix of both [30]. For instance, in HPV18-induced cancers, integrated sequences are prevalent, while in the case of HPV16-induced tumors, both genomic integration and the presence of episomes coexist [31].
To date, HPV persistence remains untargeted. The prevention of cervical cancer relies primarily on HPV vaccination (primary prevention) and other strategies (secondary prevention), such as cervical cancer screening programs, with the aim to detect and treat pre-cancerous lesions before they progress to cancer. Unfortunately, these strategies are not equally distributed worldwide [32], thus leaving millions of women without options to receive protection against HPV infections. Moreover, prophylactic HPV vaccines may reduce—but not eliminate—the risk of cervical cancer without altering the course of existing HPV infections.
2. Epigallocatechin Gallate Prevents Human Papillomavirus
EGCG is one of the major bioactive polyphenolic components of green tea—known as catechins—with immunostimulatory, antioxidant, antiproliferative, and pro-apoptotic activity [34]. In the context of HPV infections, extensive research, both in vitro and in vivo, reported the role of EGCG in regulating viral infection and preventing cervical cancer.
Several in vitro studies demonstrated that EGCG has an antiproliferative activity by interfering with the HPV life cycle and suppressing the oncogenes and oncoproteins E6/E7, which are responsible for the viral oncogenic activity and cancer development. In particular, a recent in vitro study reported that EGCG inhibits the growth of premalignant HPV18-positive keratinocytes by stimulating the degradation of E6 and E7 proteins through the ubiquitin-proteasome pathway [35]. The suppression of E6 and E7 proteins correlates with the up-regulation of tumor-suppressor genes as p53, pRb, and p21 [36,37,38], thus resulting in the apoptosis of the cervical cancer-derived cell line, Caski cells (HPV16 positive) and Hela cells (HPV18 positive). Indeed, both E6 and E7 interfere with cell cycle regulation by binding p53 and pRB, thus preventing apoptosis [39]. Interestingly, beyond the modulation of p53 and pRb in Caski cells (HPV16 infected) and Hela cells (HPV18 infected), EGCG may target most of the mechanisms involved in cancer transformation and progression, such as cellular proliferation, microtubule stability, angiogenetic processes, and cellular apoptosis. According to a recent work, a dosage of 100 µM of EGCG inhibits telomerase activity—as an effective method for anticancer protection—in different cellular models of HPV infection: (i) HPV18-immortalized ectocervical cells (HEC-18), (ii) transformed HPV18-immortalized human ectocervical cells (HEC-18T), (iii) HPV18-immortalized endocervical cells (HEN-18), and (iv) serum-adapted HPV18-immortalized human endocervical cells (HEN-18S). These results highlighted the ability of EGCG to reduce the cellular growth rate in human HPV-infected endocervical and ectocervical cells [40]. Furthermore, increasing concentrations of EGCG (10 uM, 25 uM, and 50 uM) for 24, 48, and 72 h inhibited cell proliferation and modulated RNA polymerase III in a dose-dependent manner in HeLa cells (HPV18-infected) [41]. Since microtubule cytoskeletal structures play a key role in proliferation, signalling, and migration in cancer cells [42], microtubules and tubulin—as their monomer—are popular targets for anticancer drugs [43]. In this regard, microtubule depolymerization is another mechanism targeted by the antiproliferative activity of EGCG in HeLa cells (HPV18-infected) [42]. Indeed, the IC50 dose (50 mM) rapidly disrupted microtubule networks, and higher doses (75 and 100 mM) of EGCG drastically distorted microtubule structure, making the cells almost round-shaped. In addition, other studies on HeLa cells revealed that EGCG treatment prevents the spreading of cancer cells by keeping their round shape and reducing in a dose-dependent manner the expression of metalloproteinases (MMP-2 and MMP-9). These latter, by degrading various components of the extracellular matrix (ECM), play a critical role in cancer invasion, migration, metastasis, and tumorigenesis [44,45]. EGCG also suppresses the angiogenetic process through which tumoral cells obtain nutrients and oxygen by down-modulating the expression of vascular endothelial growth factor (VEGF) [46].
In addition, EGCG can prevent cancer progression by inducing apoptosis in cervical cancer cells. In vitro studies in both HeLa and SiHa cells (HPV16 positive) demonstrated that EGCG promoted apoptosis in a time-dependent manner by increasing the expression of pro-apoptotic genes such as p53 and caspase-3 [47,48]. Another mechanism by which EGCG stimulates apoptosis in cancer cells is through mitochondrial perturbation, correlated to an excess in hydrogen peroxide. A dosage of 60 mM EGCG for 12 h in serum-free medium HeLa cells can trigger the permeabilization of the lysosomal membrane. Hence, lysosomal proteases and hydrolytic enzymes are released into the cytosol leading to HeLa cell death [49].
A recent paper highlighted the activity of EGCG to counteract one of the escaping mechanisms of HPV. Indeed, the virus can escape the immune system surveillance by interrupting the type I IFN signalling pathway and establishing a persistent infection. When keratinocytes are transfected with type 2 HPV (HPV2) E7, the mRNA and protein expression of type I IFN signaling pathway components are significantly downregulated. EGCG pre-treatment can reverse it by significantly up-regulating them, so reinforcing innate antiviral immunity against HPV2 [50].
Despite this molecular evidence, different clinical studies demonstrated the efficacy of EGCG, both as a topical and oral formulation, in recovering HPV infections. Tatti and colleagues reported that an EGCG-based formulation is effective for topically treating external genital warts, which are a cutaneous manifestation of a proliferative disorder due to LR subtypes, such as HPV6 and HPV11 [51]. Two clinical trials at phase III organized as multicenter, randomized, double-blind, and controlled, evaluated the effectiveness of treatment with EGCG-based ointment (10% or 15%) on external genital warts showing a reduction in baseline warts area by at least half, compared with 52.2% in the control group (p < 0.05) [51].
Later, a systematic review and meta-analysis [52], including three randomized studies, evaluated the positive effects of an EGCG-based topical formulation in the treatment of warts. These studies screened a total of 660 men and 587 women and confirmed the efficacy and safety of EGCG for the topical treatment of genital warts [53,54,55].
Besides these beneficial effects on external warts, another randomized controlled-clinical study investigated the efficacy of green tea extracts in different forms in patients with HPV and pre-cancerous lesions of various degrees. Ahn and collaborators demonstrated that after 8–12 weeks of oral intake of EGCG (200 mg), cervical lesions regressed, and the clearance of the virus increased, thus preventing the progression toward a more severe grade [56]. A total of 6 among 10 patients receiving EGCG capsules orally exhibited a positive response, in contrast to the untreated control group, in which only 4 of the 39 patients exhibited a positive response. As stated by the authors, about 10% of the response rate in the untreated group may correlate to differences in the immune status of the patients or other unknown factors.
Taken together, these data emphasize the powerful antiviral activity of EGCG in individuals with HPV infection. Overall, EGCG mediates antiproliferative, antioxidant, antimetastatic, and pro-apoptotic activities in HPV-infected cell lines, leading to beneficial effects in HPV-infected patients and representing a promising molecule in the treatment of HPV persistence.
This entry is adapted from the peer-reviewed paper 10.3390/pathogens12030416
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