*3.3. Effects of FM Supplementation on Macro-Histological Symptoms of Cervical Dysplasia*

The post-trial colposcopic examination revealed normal patterns in 100% of the FM group versus 62% of the placebo group. A sample of macro-histological effects of FM supplementation is shown on Figure 5, where the (a) panel photos were taken in the beginning of the trial, and the panel (b) photos were taken at the cessation of it. Before the administration of FM, ectopic transformed areas, squamous cell metaplasia, and mosaics marked by the iodine-negative areas were evident. After a three-month course of FM, we observed normal tissue basis, the shrinkage of iodine-negative areas, and cervical coagulation. Inflammatory cells in cervical fluid were found in 21% of the FM group versus 40% of the placebo group.

(**a**)

**Figure 5.** *Cont*.

(**b**)

**Figure 5.** Typical colposcopy of CIN I and CIN II lesions before (**a**) and after (**b**) administration of antioxidants. (**a**) Before administration of antioxidants (ectopic transformed areas, squamous cell metaplasia, and mosaics marked by the iodine-negative areas). (**b**) After a three-month course of antioxidants (normal tissue basis, shrinkage of iodine-negative areas, and cervical coagulation). **Left** panels are colposcopic images without iodine staining. **Right** panels are colposcopic images after iodine staining.

#### *3.4. Effects of FM Supplementation on Oxidative and Nitrosative Markers in Cervical Tissue*

The cervical tissue markers of oxidative and nitrosative stress appeared to be selectively modified by the FM treatment of the CIN I-II patients (Table 3). After 3 months, MPO activity was significantly (*p* < 0.01) decreased, as well as nitrate/nitrite and nitrotyrosine levels (*p* < 0.05). Catalase activity showed a trend toward reduction, whilst isoprostane and GSH-peroxidase differences, before and after FM treatment, were not detected at the cervical level.


**Table 3.** Oxidative markers in cervical tissue of CIN I and CIN II patients (n = 50) treated with fermented mangosteen for 3 months. \* *p* < 0.05; \*\* *p* < 0.01.

*3.5. Effects of Fermented Mangosteen Supplementation on Systemic and Topical (Cervical) Ligands of TNF-Alpha Apoptosis*

The results of the three-month-long course of FM supplementation are collected in Figures 6 and 7. The FM treatment remarkably affected circulating ligands of TNF-alphainduced apoptosis in both experimental groups with HPV16- and HPV18-infected females, with or without clinical features of cervical carcinogenesis. In these groups, highly enhanced serum levels of TNF-alpha and IL-2 went down to normal values (Figure 6B,C).

**Figure 6.** Effects of FM supplementation (AO) or placebo on serum content of the apoptosis ligands (**A**—sFAS; **B**—TNF-alpha; **C**—IL-2; **D**—TRAIL) in groups of practically healthy HPV16- and HPV18 infected females (n = 152), and in groups of HPV16- and HPV18-infected females with pre-cancerous cervical lesions (CIN I and CIN II) (n = 98). \* *p* < 0.05 vs. donors; \*\* *p* < 0.05 vs. before treatment.

At the same time, strongly suppressed serum content of TRAIL was statistically significantly restored, although TRAIL levels did not reach the normal values (Figure 6D). The FM supplementation did not induce any significant change in circulating sFAS ligands; we also did not observe any effects on circulating ligands in both placebo groups (Figure 6A).

Regarding the cervical levels of the same ligands, the course of FM, and not of the placebo, led to the statistically significant enhancement of TRAIL, while, again, it did not affect suppressed levels of sFAS (Figure 7A,D). At the cervical level, the background concentrations of TNF-alpha and IL-2 did not differ in HPV-infected females with and without pre-cancerous lesions. They remained within the background range after the cessation of the clinical trial with FM (Figure 7B,C).

**Figure 7.** Effects of fermented mangosteen (FM) supplementation on cervical fluid content of the apoptosis ligands (**A**—sFAS; **B**—TNF-alpha; **C**—IL-2; **D**—TRAIL) in groups of practically healthy HPV16 and HPV18-infected females (n = 152) and in groups of HPV16- and HPV18-infected females with pre-cancerous cervical lesions (CIN I and CIN II) (n = 98). \* *p* < 0.05 vs. donors; \*\* *p* < 0.05 vs. before trial.

#### **4. Discussion**

Clinical and epidemiological studies have revealed that certain pathogens causing persistent infections and chronic inflammation were associated with cancer [1,9,63]. The oncogenic action seems to be induced, maintained, and mediated by chronic inflammation and/or by alterations in host cells caused by the microbial genome [9,64]. Here, we observed (Figure 1) two molecular markers of chronic inflammation, MPO and nitrotyrosine, which were significantly changed in the cervical fluid of the females with cervical cancer (CIN III). As expected, there was a strong positive correlation between NO2 −/NO3 − and nitrotyrosine, the latter being formed under nitration of the amino acid by these nitrative species (Figure 2A). At the same time, we found a quite significant negative correlation between MPO and nitrotyrosine (Figure 2B). Human granulocytes release MPO from intracytoplasmic granules being challenged by microbes or viruses [17]. At the same time, they contain very low levels of inducible nitric oxide synthase [14]. Hence, they do not generate reactive nitrogen species intracellularly [65]. Instead, human granulocytes are likely to use nitrite (NO2 −) from other sources, thus forming nitrating oxidants and, as a consequence, nitrotyrosine in the extracellular environment [13]. Nitrating oxidants are implicated in the host defence and pathogenesis of many diseases [13,15,16]. Nitrative stress contributes to asbestos-induced carcinogenesis [10] through myeloperoxidase (MPO), which plays a crucial role in the asbestos-derived inflammatory response and following asbestos-induced carcinogenesis [11–13]. In the cystic fibrosis airways, MPO acts as a phagocyte-derived NO oxidase that diminishes NO bioavailability and, consequently, its immune protective (anti-bacterial and anti-viral) and anti-inflammatory properties [66]. On these grounds, it

has been suggested that MPO could be a target for therapeutic intervention to attenuate the oxidative burden and preserve essential physiological functions of NO. The inflammatory environment in HPV-infected cervical tissue provides all the factors necessary for the generation of nitrating agents by MPO, granulocyte invasion, and a high local level of NO2 − [13,15,16].

Irreversible tyrosine modifications by inflammatory oxidants such as peroxynitrite include the formation of characteristic markers 3-nitrotyrosine and 3,3- -dityrosine [67,68]. These modifications of critical tyrosine residues in proteins inactivate a variety of enzymes [67] and affect their structures [69]. Of great importance, tyrosine nitration may interfere with signal transduction pathways involving both the kinase-dependent and auto-phosphorylation of tyrosine. Thus, in epidermoid carcinoma cells, tyrosine nitration altered the epidermal growth factor receptor (EGFR) activation through irreversible dimerisation [70]. An alteration of EGFR-mediated signalling pathways in epithelial cells would inevitably affect cell proliferation or differentiation, which may bear implications on carcinogenesis [71–73].

Solid tumours (human gastric, colorectal, cervical, and bronchoalveolar carcinomas, as well as epithelial ovarian cancer) frequently contain inflammatory cells, such as neutrophils, macrophages, and T-lymphocytes [74–77]. Their recruitment is mainly explained by C-X-C chemokines, namely IL-8 (a potent chemoattractant for neutrophils) or C-C chemokines (MCP-1) [63]. The inflammatory cells recruited to tumours become an important additional source of the chemokines [14]; thus, a vicious cycle is formed. It is well-established that activated neutrophils induce prolonged DNA damage in neighbouring cells [78], mainly through the genotoxic effects of ROS and RNS [79,80]. These species are widely recognised as key carcinogenic agents, causing tumour transformation, progression, and metastasis.

As per comprehensive review [81], HPV-encoded intracellular proteins can reshape signalling pathways in a mode that facilitates carcinogenesis through escaping immune surveillance and the impairment of TRAIL-mediated apoptosis. In the present study, we found that circulating levels of two cytokines (TNF-alpha and IL-2) and two ligands (sFAS and TRAIL) contributing to TRAIL-mediated apoptosis were significantly compromised versus normal values in females infected by highly oncogenic HPV forms without any symptoms of cervical pre-cancerous lesions as yet (Figure 3A). The impairment was aggravated in females with pre-cancerous CIN I and CIN II stages, while no differences were seen between healthy non-infected HPV females and those infected by low-risk HPV 10,13. When TNF-alpha, sFAS, and TRAIL proteins were measured in the cervical fluid, there were no differences between groups in the content of sFAS and TNF-alpha; however, local levels of TRAIL were dramatically diminished in all HPV16/18-infected groups of females (with no lesions, CIN I-II, and CIN-III stages) (Figure 3B). At the level of mRNA for sFAS and TRAIL in cervical tissue, the remarkable suppression of both was observed in the group with pre-cancerous states (CIN I-II). In the groups with no dysplasia, infected either by low- or high-risk HPV, mRNA expression was practically at normal levels.

A new approach to target and kill circulating tumour cells has been proposed recently [82]. The method is based on the coating of circulating leukocytes with liposomes loaded by TRAIL. The coated leukocytes could be considered as "un-natural killer cells", because they resemble natural killers activated by IL-2, which overexpresses TRAIL to attack and induce apoptosis in cancer cells. Applying the immunohistochemical approach, Carrero and co-authors [77] showed that in the advanced stage of pre-malignant lesions of the cervix, a progressive stage-dependent CD3/VEGF-positive lymphocyte infiltration correlated with the increased number of superoxide-producing cells, while tissue levels of nitrites and nitrates remained unchanged. However, in this work, only 16% of patients with CIN were infected with HPV; hence, one cannot estimate the impact of oxidative stress on virus-associated carcinogenesis.

Nutritional paradigms and dietary active components in the chemoprevention of carcinogenesis and in the potentiation of anti-cancer therapies have drawn a lot of attention recently (reviewed in [9,20–25]). Resveratrol has been shown to potentiate the apoptotic effects of TRAIL and other death cytokines, as well as chemotherapeutic agents and gamma irradiation [47,83,84]. On these grounds, a novel strategy to enhance the efficacy of TRAILtargeting chemoprevention/therapies has been suggested. However, recent publications raised serious concerns about the feasibility of resveratrol in anti-cancer therapy and possibly in cancer chemoprevention due to its poor pharmacokinetics, low potency, and nephrotoxicity [84]. The publication of our group [85] clearly showed the strong phototoxicity of resveratrol, which is widely promoted for the prevention of UV-induced skin tumours. The anti-proliferative effect of the dietary anti-carcinogenic compound phenyl ethyl isothiocyanate in human cancer stem cells, derived from the human cervical tumourderived HeLa cell line, partially resulted from the up-regulation of death receptors DR4 and DR5 of the TRAIL-mediated apoptotic pathway [86]. The dietary substances cucurbitacins, glycosylated triterpenes [87], luteolin [88], and epigallocatechin gallate [89] have been shown to regulate DNA repair systems and TRAIL-driven signalling in a number of cancer cells.

FM has been suggested as an effective chemopreventive/anti-cancer dietary product on the basis of phytochemical analyses [42], the in vitro and in vivo experiments [40,43–45] showing that its active components, such as polyphenols, terpenes, and xanthons, acted by different mechanisms, i.e., mTOR, deranged cell cycle, autophagy, and p53-dependent apoptosis. Direct antioxidant effects have been shown, as well [44,45]. Clinical data on the anti-cancer or cancer chemopreventive efficacy are completely lacking. In the present placebo-controlled clinical study, we showed, for the first time, that a 3-month-long supplementation with FM in females infected by HPV16/18 of high oncogenic risk, with the first symptoms of cancerous dysplasia (CIN I-II), resulted in the normalisation of macrohistological patterns in cervical tissue (100% of patients) and diminished the invasion of inflammatory neutrophils into the cervix (Figure 5). Along with the observed clinical efficacy, FM supplementation significantly suppressed the MPO presence, nitrative agents NO2 −/NO3 −, and nitrotyrosine in cervical tissue (Table 3). All these species are known for their carcinogenic action (see above). The most impressive findings were on the effects of FM supplementation on the TRAIL protein, circulating as well as localised, in the cervical fluid (Figures 6 and 7). This TNF-alpha-related apoptosis-inducing ligand, an inducer of cancer cell apoptosis through death receptors [84], was initially greatly suppressed in HPV16/18-infected females without symptoms of dysplasia; gradually, its suppression became deeper in HPV16/18-induced cervical dysplasia (CIN I and CIN II) and cervical cancer (CIN III) (Figure 4). Supplementation with FM, and not with the placebo, led to the significant restoration of circulating and local TRAIL levels. Unusually low TRAIL levels in the cervical fluid in the beginning of the study were not accompanied by any significant change in the three other cytokines and ligands (TNF-alpha, IL-2, and sFAS), while the circulating TNF-alpha and IL-2 in serum were highly increased in all HPV16/18 infected females with or without signs of pre-cancer. That, in our opinion, could reflect the generalised chronic inflammatory response to viral infection. Of great importance, FM supplementation completely normalised the circulating levels of both pro-inflammatory cytokines, thus diminishing the burden from chronic inflammation.

On the grounds of the results described here and the literature data, we assumed that FM could exert preventive effects on HPV-induced cervical cancer through several pathways, where one or another of its numerous active ingredients could:


• the presence of numerous active compounds derived from the plant and fermenting microbes in the natural matrix of FM could attenuate their potential individual toxicity, increase bioavailability, and exhibit a synergy between them.

### **5. Conclusions**

Our findings indicate that FM, in combination with anti-viral therapy, could prevent or slow down HPV-associated cervical carcinogenesis, mainly through the suppression of leukocyte recruitment into infected tissue, suppression of general virus-associated chronic inflammation, and restoration of nitric oxide metabolite-initiated TRAIL-dependent apoptosis.

Much more research is needed to elucidate the mechanism(s) of FM's cancer-preventive effects. There are still many critical issues in the wide acceptance of fermented fruits as remedies for health problems, such as the lack of reliable clinical evidence, as well as scarce data on individual active components in fermented fruits and on the molecular mechanisms of their cancer chemopreventive effects. There is also an urgent need to optimise/standardise technologies of fermentation and to develop drugs based on the actives of fermented products.

**Author Contributions:** Conceptualization, Z.K., P.T. and L.K.; methodology, Z.K., I.T., G.I. and J.C.; software, P.T. and W.M.; validation, Z.K., C.D.L. and I.T.; formal analysis, P.T. and C.D.L.; investigation, I.T., G.I. and J.C.; resources, W.M. and L.K.; data curation, P.T. and J.C.; writing—original draft preparation, L.K.; writing—review and editing, W.M. and J.C.; visualization, C.D.L., P.T. and I.T.; supervision, L.K.; project administration, Z.K.; funding acquisition, I.T. and L.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding. The APC was shouldered by Fame Team Internat., Hong Kong, PRC.

**Institutional Review Board Statement:** The study was conducted in accordance with the Declaration of Helsinki and was approved by the Local Ethics Committee (Berbekov's Kabardino-Balkar State Medical University, Nal'chik, Russia), Protocol No. 133-2/2018 of 21 November 2018 for studies involving humans.

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** The data can be shared upon request.

**Acknowledgments:** The authors are grateful to Fame Team Ltd. (Hong Kong, PRC) for covering the publication costs and to Carica Ltd., Manila, the Philippines, for providing fermented mangosteen syrup (BioRex Mangosteen) free of charge.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

