**2. Factors of persistence**

#### **2.1. Vaginal microbiota, HPV and co-infections**

More than ever, the association of a disrupted microbiota and the increasing incidence of chronic human diseases have been addressed [4]. Locally, the microbiota affects the functions and regulates the immunity of epithelial barrier. Therefore, the vaginal microbiome plays an essential role not only in health and dysbiosis, but also in modulation of immune response and, possible, in the carcinogenic process. Additionally, the persistence of risk factors, namely, HPV and other co-infections, may be associated to the disruption of these barriers [5].

required to understand the molecular mechanisms involved in the complex role that bacterial

Cervical cancer is one of the most preventable cancers. However, its progression and above all, the progress towards prevention is often frustrating. Moreover, and despite the continuously growing body of knowledge, the role of factors that affect the human papillomavirus

Indeed, the oncogenic HPVs are a necessary cause of cervical cancer; however, they are not a sufficient cause, being other cofactors implicated in the increase of risk. We have also to consider external factors to the host, such as smoking habits, nutritional and behavioural factors (number of partners and their characteristics, age at onset of sexual activity), hormonal therapies-sexual steroids (oral contraceptives and post-menopausal substitution therapy), herpes simplex infections, *Chlamydia trachomatis* or other sexually transmitted infectious diseases and also nonspecific inflammatory diseases. Genetic and immunological factors and other endogenous co-factors may induce initiation and progression associated with genotoxicity, mutagenicity and irreversible cell

Dysbiosis results from the disruption of equilibrium of the microbiome. Given that the vaginal microbiome composition has been shown to play a role in the HPV infection and the rate of HPV clearance, the vaginal microbiome structure may be associated with the development

Nevertheless, recent and concise data show that composition of the early-life microbiota is critical in the development of the immune system, and how deviations from homeostasis can

Our group has been presenting data that reflects mainly the influence of genetic, epigenetic and environmental including the vaginal microbiota-derived factors in the natural history of

In this scenario, the microbiota and its genome (microbiome) fulfils part of the natural history of cervical cancer. In the last years, it has been characterized HPV-genotypes profile, and bacterial vaginosis (BV) leading to its association with the prevalence of HSIL and progression to

Despite the risk factors status knowledge, we may consider the need of a more proactive behaviour, namely, a strategy for improving the local fora with topic therapy. In this chapter, we will focus in the role of genetic susceptibility associated to the development of cervical cancer. Furthermore, we will discuss opportunities for interventions that modify the microbiome for therapeutic purpose.

More than ever, the association of a disrupted microbiota and the increasing incidence of chronic human diseases have been addressed [4]. Locally, the microbiota affects the functions

HPV associated lesions leading to cervical cancer as a multifactorial disease process.

communities can play in the development of cancer.

16 Cervical Cancer - Screening, Treatment and Prevention - Universal Protocols for Ultimate Control

(HPV) persistence are not yet fully understood.

of cervical cancer secondary to a persistent HPV infection.

invasive cervical cancer (ICC) in adult women.

**2.1. Vaginal microbiota, HPV and co-infections**

proliferation [1, 2].

induce disease later in life [3].

**2. Factors of persistence**

The carcinogenic process in cervical cancer results in systemic and persistent damages, with important changes in immune checkpoints of the involved microenvironment [6]. From the key-players involved in this process, the microbiota influences, locally, physiological functions from the maintenance of barrier homeostasis to the regulation of metabolism, hematopoiesis, inflammation, immunity and other functions systematically [4, 7]. This barrier is supported by immune cells, for example, B cells, which produces IgA that helps to neutralize pathogenic bacteria (**Figure 1**) [8]. When this barrier locally fails it is created a favourable environment for carcinogenesis, the dysregulation of the integrity of vaginal epithelial cells will lead to more susceptibility for infections, causing low-grade chronic inflammation that leads to disease.

**Figure 1.** The presence of certain types of lactobacillus lowers pH and induces H2 O2 , which may contribute to the formation of HOCL by myeloperoxidase. CSTs IV are associated to bacterial vaginosis and the other microorganisms, to pre-cancerous lesions and cervical cancer. The types of groups of bacillus may be preponderant for maintaining the vaginal balance. Four species are most important for the balance of the vagina ecosystem: *L. gasseri* (II) *L. crispatus* (I), *L. jensenii* (V) and *L. iners* (III). The inter-individual genetic polymorphic variations should be integrated in a complex model, since a compromise vaginal microflora and inefficient genetic profile may contribute to the development of cervical cancer; CSTs, community state types; CSTs I, *Lactobacillus crispatus*; CSTs II, *Lactobacillus gasseri*; CSTs III, *Lactobacillus iners*; CSTs IV, *Lactobacillus, Sneathia amnii* and *Fusobacterium*; CSTs V, *Lactobacillus jensenii*.

The collection of microorganisms (or microbiota) populates complex ecosystems where genome is called microbiome and the implications on women health, from conception to the next generation, has been recently discussed [9]. A healthy vaginal microbiome is apparently dominated mainly by Community State Types (CST): *Lactobacillus crispatus* (*CSTs I*)*, Lactobacillus gasseri (CSTs II), Lactobacillus iners (CSTs III)* and *Lactobacillus jensenii* (*CSTs V*), which, regulate for instance, the balance of reactive oxygen species (ROS) (**Figure 1**) [7]. Although vaginal dysbiosis presents biological plausibly by influencing host's innate immune response, susceptibility to infection, and the development of cervical disease, the underlying cause is not yet well understood. Nevertheless, greater diversity in the vaginal microbiota was associated in women with HPV-positive with cervical intra-epithelial neoplasia (CIN) [10].

**2.2. Lactobacilli enhance reactive oxygen species and the role of genetic** 

O2

to allow biologically significant HOCL synthesis in the presence of H<sup>2</sup>

eration, differentiation and migration [15]. Lactobacilli's H<sup>2</sup>

peroxidase may result in toxic concentrations of H2

vagina inducing a steady removal of excess of H2

Bauer proposed that peroxidase, which converts H2

The ROS, comprise a group of oxygen derivatives from distinct oxidation status of O2

cascades that regulates gene expression and fundamental cellular processes such as prolif-

The presence of peroxidases guarantees the generating of hypochlorous acid (HOCL) in the


be responsible for the prevention of vaginosis and also might exert an antitumour effect

presence of peroxidases (such as myeloperoxidase and eosinophil peroxidase) and halides [18]. This points to a role of HOCL as superior antimicrobial compound. The vaginal fluid of the majority of healthy women contains sufficiently high concentration of peroxidase

ating a microbicial vaginal milieu by maintaining a balanced, non-toxic [18]. The papers of Bauer had highlighted the role of lactobacilli in the vaginal flora of healthy premenopausal women pointing to the beneficial effects for the predominance of microorganisms. Lactobacilli adhere to epithelial cells and thus cause sterile prevention of cell infections with undesirable microorganisms. Lactobacilli cause low pH through production of lactate and also release bactericidal compounds (**Figure 1**) [7]. Others ROS are the highly reactive and toxic by-products of oxygen metabolism, which can damage bacterial nucleic acids, proteins

not likely to be beneficial for the vaginal epithelium, because it causes nonselective lesions in nontransformed as well as transformed cells. The combination of lactobacillus and peroxidase is more favourable. Moreover, the lactobacilli in this system can be completely mimicked

This idyllic scenario is considered for normal cells or untransformed cells, which have a wide antioxidant regulatory defence system that serves to prevent the oxidative stress and the development of neoplasms [20]. Nevertheless, the papillomavirus infected cells (in particular by oncogenic types HPV 16, 33, 31) are resistant to this pathway of apoptosis induction. In transformed cells caused by damages induced by HPV, cells lose control of senescence and

Recent work of Kruger and Baeur 2017, confirms that the lactobacillus-derived H<sup>2</sup>

tumour prevention is fully explained by bacterial generation of H2

.−), and hydroxyl free-radical (OH.−) and as well as non-radical

O2

(**Figure 1**). Klebanoff et al. proposed that hydrogen perox-

and generation of HOCL [17].


[14]. The latter has a role as a second messenger molecule in signalling

Microenvironment in Vagina as a Key-Player on Cervical Cancer: Interaction of Polymorphic…

O2

O2

, product of lactobacilli and peroxidase, in the vagina of healthy women might

O2

generated by glucose oxidase, indicating that its contribution for potential

O2 [17]. , such

19

O2 in


O2

per se is

production in the absence of

and cause damage of the mucosa [16].

http://dx.doi.org/10.5772/intechopen.73108

O2

into HOCL, is responsible for cre-

**susceptibility in the vaginal microbiome**

as, superoxide radical anion (O2

a two-electron reduction of O2

[18]. The antimicrobial effect of H<sup>2</sup>

O2

forms, namely H2

H2 O2

ide H2 O2

tobacilli [18].

and cell membranes [19].

O2

p53 activity is abrogated [21].

in vitro by H2

There is a strong relationship between infection with HPV, pre-neoplastic lesions and cervical cancer. Moreover, the prevalence of high-risk HPV genital infection (HSIL) and cervical cancer in adult women has been documented. Therefore, the determination of this specific environment correlated with demographic, behavioural and clinical parameters will contribute to a better knowledge of key-players that triggers the carcinogenic pathways in cervical cancer. Other different risk factors including early age at first intercourse, multiple sex partners and low socioeconomic status also have significant role in disease initiation [11].

The HPV infects only epithelial cells, firstly, throughout the basal layer of the epithelium, probably via microabrasions in the epithelial surface, then the viral DNA is released from the capsid and transported into the nucleus as free genetic material or extrachromosomal episomes (**Figure 1**) [12]. Environmental factors might locally influence the initiation of this invasion, among other co-factors, HPV allows potential tumour cells to escape from lactobacilli-mediated control and interfering with intracellular induction of apoptosis [7].

Recently, changes from pro-inflammatory to anti-inflammatory signals - the cytokine milieu - may affect whether or not an infection is cleared [13] and hypothetically an environment favourable or not for tumour growth, might follow a change in regulation of the expression of pro-inflammatory cytokines. After infection takes place, the microbiome changes and its diversity increases. HPV proteins E2, E6 and E7 enhance IL-10 expression secondary to macrophages type 2 presence. These latter are also enhanced in its activity by TGFβ-1 in cytokines expression and its phagocytic efficacy, which is in turn stimulated by the microbiota present. The increase of diversity in the microbiota, through its toxins (FadA from *Fusobacterium* spp.) will promote a metastasis phenotype similar to what happens in cervical cancer [13].

Moreover, polymorphic genetic variants used as surrogate markers might explain the interindividual variations and the differential immune response causing the persistence and the progression of HPV effects. We had studied some polymorphisms that are associated with genetic susceptibility to cervical infection and increase for risk of acquiring and transmitting HPV infection. These polymorphisms are involved in several pathways, throughout the production of metabolites or other carcinogenic substances, by increasing the susceptibility of the inflamed epithelium or by changing the immune system equilibrium (**Figure 1**).

#### **2.2. Lactobacilli enhance reactive oxygen species and the role of genetic susceptibility in the vaginal microbiome**

The collection of microorganisms (or microbiota) populates complex ecosystems where genome is called microbiome and the implications on women health, from conception to the next generation, has been recently discussed [9]. A healthy vaginal microbiome is apparently dominated mainly by Community State Types (CST): *Lactobacillus crispatus* (*CSTs I*)*, Lactobacillus gasseri (CSTs II), Lactobacillus iners (CSTs III)* and *Lactobacillus jensenii* (*CSTs V*), which, regulate for instance, the balance of reactive oxygen species (ROS) (**Figure 1**) [7]. Although vaginal dysbiosis presents biological plausibly by influencing host's innate immune response, susceptibility to infection, and the development of cervical disease, the underlying cause is not yet well understood. Nevertheless, greater diversity in the vaginal microbiota was associated in women with HPV-positive with cervical intra-epithelial neo-

18 Cervical Cancer - Screening, Treatment and Prevention - Universal Protocols for Ultimate Control

There is a strong relationship between infection with HPV, pre-neoplastic lesions and cervical cancer. Moreover, the prevalence of high-risk HPV genital infection (HSIL) and cervical cancer in adult women has been documented. Therefore, the determination of this specific environment correlated with demographic, behavioural and clinical parameters will contribute to a better knowledge of key-players that triggers the carcinogenic pathways in cervical cancer. Other different risk factors including early age at first intercourse, multiple sex partners and low socioeconomic status also have significant role in disease

The HPV infects only epithelial cells, firstly, throughout the basal layer of the epithelium, probably via microabrasions in the epithelial surface, then the viral DNA is released from the capsid and transported into the nucleus as free genetic material or extrachromosomal episomes (**Figure 1**) [12]. Environmental factors might locally influence the initiation of this invasion, among other co-factors, HPV allows potential tumour cells to escape from lactoba-

Recently, changes from pro-inflammatory to anti-inflammatory signals - the cytokine milieu - may affect whether or not an infection is cleared [13] and hypothetically an environment favourable or not for tumour growth, might follow a change in regulation of the expression of pro-inflammatory cytokines. After infection takes place, the microbiome changes and its diversity increases. HPV proteins E2, E6 and E7 enhance IL-10 expression secondary to macrophages type 2 presence. These latter are also enhanced in its activity by TGFβ-1 in cytokines expression and its phagocytic efficacy, which is in turn stimulated by the microbiota present. The increase of diversity in the microbiota, through its toxins (FadA from *Fusobacterium* spp.)

cilli-mediated control and interfering with intracellular induction of apoptosis [7].

will promote a metastasis phenotype similar to what happens in cervical cancer [13].

inflamed epithelium or by changing the immune system equilibrium (**Figure 1**).

Moreover, polymorphic genetic variants used as surrogate markers might explain the interindividual variations and the differential immune response causing the persistence and the progression of HPV effects. We had studied some polymorphisms that are associated with genetic susceptibility to cervical infection and increase for risk of acquiring and transmitting HPV infection. These polymorphisms are involved in several pathways, throughout the production of metabolites or other carcinogenic substances, by increasing the susceptibility of the

plasia (CIN) [10].

initiation [11].

The ROS, comprise a group of oxygen derivatives from distinct oxidation status of O2 , such as, superoxide radical anion (O2 .−), and hydroxyl free-radical (OH.−) and as well as non-radical forms, namely H2 O2 [14]. The latter has a role as a second messenger molecule in signalling cascades that regulates gene expression and fundamental cellular processes such as proliferation, differentiation and migration [15]. Lactobacilli's H<sup>2</sup> O2 production in the absence of peroxidase may result in toxic concentrations of H2 O2 and cause damage of the mucosa [16]. The presence of peroxidases guarantees the generating of hypochlorous acid (HOCL) in the vagina inducing a steady removal of excess of H2 O2 and generation of HOCL [17].

H2 O2 -producing lactobacilli strains, use a NADH oxidase that directly generates H2 O2 in a two-electron reduction of O2 (**Figure 1**). Klebanoff et al. proposed that hydrogen peroxide H2 O2 , product of lactobacilli and peroxidase, in the vagina of healthy women might be responsible for the prevention of vaginosis and also might exert an antitumour effect [18]. The antimicrobial effect of H<sup>2</sup> O2 -generating lactobacilli is efficiently enhanced in the presence of peroxidases (such as myeloperoxidase and eosinophil peroxidase) and halides [18]. This points to a role of HOCL as superior antimicrobial compound. The vaginal fluid of the majority of healthy women contains sufficiently high concentration of peroxidase to allow biologically significant HOCL synthesis in the presence of H<sup>2</sup> O2 -generating lactobacilli [18].

Bauer proposed that peroxidase, which converts H2 O2 into HOCL, is responsible for creating a microbicial vaginal milieu by maintaining a balanced, non-toxic [18]. The papers of Bauer had highlighted the role of lactobacilli in the vaginal flora of healthy premenopausal women pointing to the beneficial effects for the predominance of microorganisms. Lactobacilli adhere to epithelial cells and thus cause sterile prevention of cell infections with undesirable microorganisms. Lactobacilli cause low pH through production of lactate and also release bactericidal compounds (**Figure 1**) [7]. Others ROS are the highly reactive and toxic by-products of oxygen metabolism, which can damage bacterial nucleic acids, proteins and cell membranes [19].

Recent work of Kruger and Baeur 2017, confirms that the lactobacillus-derived H<sup>2</sup> O2 per se is not likely to be beneficial for the vaginal epithelium, because it causes nonselective lesions in nontransformed as well as transformed cells. The combination of lactobacillus and peroxidase is more favourable. Moreover, the lactobacilli in this system can be completely mimicked in vitro by H2 O2 generated by glucose oxidase, indicating that its contribution for potential tumour prevention is fully explained by bacterial generation of H2 O2 [17].

This idyllic scenario is considered for normal cells or untransformed cells, which have a wide antioxidant regulatory defence system that serves to prevent the oxidative stress and the development of neoplasms [20]. Nevertheless, the papillomavirus infected cells (in particular by oncogenic types HPV 16, 33, 31) are resistant to this pathway of apoptosis induction. In transformed cells caused by damages induced by HPV, cells lose control of senescence and p53 activity is abrogated [21].

The combination of the host genome and microbiome increases genetic variation and phenotypic plasticity, enabling the holobiont to increase its overall fitness [22]. Genome-wide association studies identified cervical cancer susceptibility variants across different populations [23]. Therefore, the input of these and other polymorphic variants, may reflect the interindividuality of response in women with cervical cancer. In this chapter, we will focus on some these polymorphisms involved in the modulation of ROS production.

*2.2.2. Catalase*

into H2

*2.2.3. Myeloperoxidase*

of apoptosis [36].

the H2 O2

The toxic concentrations of H2

H2 O2

cells, spontaneous and enzymatic dismutated O2

activity), will deficiently protects cells from ROS.

fungi, viruses, spermatozoa, or tumour cells [37].

O2

a predominant role in controlling H2

The NOX and catalase (CAT) proteins work in sequence in a metabolic pathway. Transformed

to allow optimal velocity of the ROS interactions [28]. The CAT is a heme enzyme that plays

effects of oxidative stress. In healthy women, this protector effect rises from the conversion of

by a membrane associated catalase and causing control system failure that ultimately results in cell apoptosis failure [26, 29]. Notwithstanding, women with a genetic variant of *CAT* asso-

In humans, the *CAT* gene is located on chromosome 11p13 and its rs1001179 polymorphism (C-262 T) is located on the promoter region and influences transcription and consequent expression of this enzyme and hence the oxidative status of cells and its microenvironment [30]. In a case–control study, we observed a greater risk for developing ICC associated with the homozygote genotype TT of *CAT* polymorphism C-262 T polymorphism of the CAT gene (OR = 3.03, 95% CI 1.46–6.29, P = 0.003) [31]. Similarly to other cancers types, the T variant of this polymorphism is associated to a decreased enzyme activity, generating high levels of ROS [32–34]. The interaction of CC genotype of *p22phox* polymorphism and the TT genotype

Moreover, recent reports have suggested a connection between oestrogen exposure, CAT activity and polymorphism in breast cancer [35]. These findings suggest that CAT genotype modifies the effect of hormone replacement therapy (HRT) use on breast cancer risk and that HRT may affect risk by affecting oxidative stress. This scenario, also might be important in cervical cancer, namely, women with *CAT* TT genotype (associated to a decreased catalase

The oxidative stress conditions are generated by the release of ROS at the infection site by host immune cells such as neutrophils and monocytes. Additionally, resistance of oncogenic papilloma virus-expressing cells to apoptosis induction by the HOCL/hydroxyl anion pathways is likely, as papilloma virus-containing cells are also resistant to intercellular induction

lysosomal enzyme expressed in polymorphonuclear neutrophils, has the potential to kill HPV transformed cells, as a component of an intercellular induced-apoptosis pathway. The MPO was also being pointed as a key-player on controlling of vaginal microenvironment, namely,

overgrowth of potentially pathogenic organisms; in fact, it can be toxic to other bacteria,

Supposedly, there are no resident neutrophils and macrophages on vaginal microenvironment, nevertheless the persistence of risk co-factors and ROS may lead to the recruitment of


could be converted by myeloperoxidase (MPO). MPO, a

O2

ciated with a decrease activity will not contribute to this control system (**Figure 1**).

of *CAT* leads to a higher risk for ICC (OR = 3.95, 95% CI 1.07–14.52, P = 0.032) [31].

− into H2

O and O2, but in cervical cancer transformed cells, the ROS signalling is inhibited

O2

Microenvironment in Vagina as a Key-Player on Cervical Cancer: Interaction of Polymorphic…

and O2· protecting in this way cells from deleterious

by SOD occurs at right density

http://dx.doi.org/10.5772/intechopen.73108

21

#### *2.2.1. NAD(P)H oxidase (NOX)*

The production of O2 .− through NAD(P)H oxidase (NOX) by transformed cells or cervical cancer cells, can be specifically targeted with production of OH− that induces apoptosis of these cells. The spontaneous dismutation of superoxide anions produce H2 O2 , at low pH, causes mutagenic effects that initiate malignant transformation (**Figure 1**). The high local concentrations of ROS through expression of SOD and catalase, it has also the potential to prevent elimination of transformed cells trough ROS/Reactive Nitrogen Species (RNS)-dependent intercellular apoptosis-inducing signalling [14].

The changes in the gut influences the vaginal microbiome, for instance the expression of NOX is modulated by, for example, the presence of *Helicobacter pylori*, which induces an indirect prooxidative mechanism through recruitment of neutrophils and by assembling of their NOX2 components to the cell membrane [1, 24].

NOX are membrane-associated oligomeric proteins that produce O2 · for host defence and other functions. Generation of extracellular O<sup>2</sup> · through NOX is associated with oncogene activation and seems to be required for the control of cell proliferation and maintenance of the transformed state [25]. This protein consists of among other peptides by a regulatory 22-kDa α- subunit (p22phox) and a 91-kDa catalytic β-subunit (gp91phox). The p22phox protein is the NOX element responsible for the regulation of electron transfer to gp91phox [26]. The *p22phox* (*CYBA* gene) polymorphism with rs4673 (C-242 T) causes a functional non-conservative substitution from histidine-72 to a tyrosine residue that decreases its activity [27] (**Figure 1**). Our previous work unravels the association between *CYBA* polymorphism in women with ICC, having been observed a heterosis phenomenon with a protective profile in ICC [14]. This U type curve reflects, on one hand, the homozygote genotype CC leaded to increased ROS production, mainly H2 O2 resulting from dismutation of O2·, which in turn results in excessive cell growth; on the other hand, the homozygote genotype TT lowers ROS production, mainly decreasing O2· mediated apoptosis cell capacity, resulting in a higher risk for the development of tumours in both cases [14]. Updated data from our cohort, we found that the TT genotype of *ph22pho*x polymorphism was a tendency for increased risk in ICC (**Figure 1**) (OR = 3.57, 95% [0.85–13.48], P = 0.057), being age and smoking habits dependent factors.

Women with cervical cancer will have a lower induction of O2· and, consequently, compromising the dismutation by SOD3 of this apoptotic factor into H2 O2 . The continuous modification of vaginal microbiota throughout depletion of lactobacillus or infections with HPV, contribute to increase of pH, influencing the concentrations of ROS in vaginal milieu. Moreover, women with TT genotype of *p22phox* polymorphisms will have a worse response to these important modifications (**Figure 1**).

#### *2.2.2. Catalase*

The combination of the host genome and microbiome increases genetic variation and phenotypic plasticity, enabling the holobiont to increase its overall fitness [22]. Genome-wide association studies identified cervical cancer susceptibility variants across different populations [23]. Therefore, the input of these and other polymorphic variants, may reflect the interindividuality of response in women with cervical cancer. In this chapter, we will focus on some

mutagenic effects that initiate malignant transformation (**Figure 1**). The high local concentrations of ROS through expression of SOD and catalase, it has also the potential to prevent elimination of transformed cells trough ROS/Reactive Nitrogen Species (RNS)-dependent

The changes in the gut influences the vaginal microbiome, for instance the expression of NOX is modulated by, for example, the presence of *Helicobacter pylori*, which induces an indirect prooxidative mechanism through recruitment of neutrophils and by assembling of their

activation and seems to be required for the control of cell proliferation and maintenance of the transformed state [25]. This protein consists of among other peptides by a regulatory 22-kDa α- subunit (p22phox) and a 91-kDa catalytic β-subunit (gp91phox). The p22phox protein is the NOX element responsible for the regulation of electron transfer to gp91phox [26]. The *p22phox* (*CYBA* gene) polymorphism with rs4673 (C-242 T) causes a functional non-conservative substitution from histidine-72 to a tyrosine residue that decreases its activity [27] (**Figure 1**). Our previous work unravels the association between *CYBA* polymorphism in women with ICC, having been observed a heterosis phenomenon with a protective profile in ICC [14]. This U type curve reflects, on one hand, the homozygote genotype CC leaded to increased ROS

sive cell growth; on the other hand, the homozygote genotype TT lowers ROS production, mainly decreasing O2· mediated apoptosis cell capacity, resulting in a higher risk for the development of tumours in both cases [14]. Updated data from our cohort, we found that the TT genotype of *ph22pho*x polymorphism was a tendency for increased risk in ICC (**Figure 1**) (OR = 3.57, 95% [0.85–13.48], P = 0.057), being age and smoking habits dependent factors.

Women with cervical cancer will have a lower induction of O2· and, consequently, compromis-

of vaginal microbiota throughout depletion of lactobacillus or infections with HPV, contribute to increase of pH, influencing the concentrations of ROS in vaginal milieu. Moreover, women with TT genotype of *p22phox* polymorphisms will have a worse response to these important

.− through NAD(P)H oxidase (NOX) by transformed cells or cervical can-

resulting from dismutation of O2·, which in turn results in exces-

O2

that induces apoptosis of these

, at low pH, causes

· for host defence and

. The continuous modification

O2

· through NOX is associated with oncogene

these polymorphisms involved in the modulation of ROS production.

20 Cervical Cancer - Screening, Treatment and Prevention - Universal Protocols for Ultimate Control

cells. The spontaneous dismutation of superoxide anions produce H2

NOX are membrane-associated oligomeric proteins that produce O2

cer cells, can be specifically targeted with production of OH−

intercellular apoptosis-inducing signalling [14].

NOX2 components to the cell membrane [1, 24].

other functions. Generation of extracellular O<sup>2</sup>

O2

ing the dismutation by SOD3 of this apoptotic factor into H2

*2.2.1. NAD(P)H oxidase (NOX)*

The production of O2

production, mainly H2

modifications (**Figure 1**).

The NOX and catalase (CAT) proteins work in sequence in a metabolic pathway. Transformed cells, spontaneous and enzymatic dismutated O2 − into H2 O2 by SOD occurs at right density to allow optimal velocity of the ROS interactions [28]. The CAT is a heme enzyme that plays a predominant role in controlling H2 O2 and O2· protecting in this way cells from deleterious effects of oxidative stress. In healthy women, this protector effect rises from the conversion of H2 O2 into H2 O and O2, but in cervical cancer transformed cells, the ROS signalling is inhibited by a membrane associated catalase and causing control system failure that ultimately results in cell apoptosis failure [26, 29]. Notwithstanding, women with a genetic variant of *CAT* associated with a decrease activity will not contribute to this control system (**Figure 1**).

In humans, the *CAT* gene is located on chromosome 11p13 and its rs1001179 polymorphism (C-262 T) is located on the promoter region and influences transcription and consequent expression of this enzyme and hence the oxidative status of cells and its microenvironment [30]. In a case–control study, we observed a greater risk for developing ICC associated with the homozygote genotype TT of *CAT* polymorphism C-262 T polymorphism of the CAT gene (OR = 3.03, 95% CI 1.46–6.29, P = 0.003) [31]. Similarly to other cancers types, the T variant of this polymorphism is associated to a decreased enzyme activity, generating high levels of ROS [32–34]. The interaction of CC genotype of *p22phox* polymorphism and the TT genotype of *CAT* leads to a higher risk for ICC (OR = 3.95, 95% CI 1.07–14.52, P = 0.032) [31].

Moreover, recent reports have suggested a connection between oestrogen exposure, CAT activity and polymorphism in breast cancer [35]. These findings suggest that CAT genotype modifies the effect of hormone replacement therapy (HRT) use on breast cancer risk and that HRT may affect risk by affecting oxidative stress. This scenario, also might be important in cervical cancer, namely, women with *CAT* TT genotype (associated to a decreased catalase activity), will deficiently protects cells from ROS.

#### *2.2.3. Myeloperoxidase*

The oxidative stress conditions are generated by the release of ROS at the infection site by host immune cells such as neutrophils and monocytes. Additionally, resistance of oncogenic papilloma virus-expressing cells to apoptosis induction by the HOCL/hydroxyl anion pathways is likely, as papilloma virus-containing cells are also resistant to intercellular induction of apoptosis [36].

The toxic concentrations of H2 O2 could be converted by myeloperoxidase (MPO). MPO, a lysosomal enzyme expressed in polymorphonuclear neutrophils, has the potential to kill HPV transformed cells, as a component of an intercellular induced-apoptosis pathway. The MPO was also being pointed as a key-player on controlling of vaginal microenvironment, namely, the H2 O2 -generating *Lactobacillus acidophilus*. In healthy women, this production inhibits the overgrowth of potentially pathogenic organisms; in fact, it can be toxic to other bacteria, fungi, viruses, spermatozoa, or tumour cells [37].

Supposedly, there are no resident neutrophils and macrophages on vaginal microenvironment, nevertheless the persistence of risk co-factors and ROS may lead to the recruitment of inflammatory cells. The presence of MPO on vaginal milieu is activated. Probably, the persistence of death cells recalls of neutrophils to vagina, being MPO important for the control of excess production of HOCl. In the vaginal microenvironment, the MPO catalyzes the reaction between H2 O2 and either thiocyanate ions or a halide, such as iodide, bromide or chloride ions, yielding HOCl, which participates in the oxidative burst during the innate host defence [38]. MPO may act in synergy with other proteins. Therefore, an imbalance between oxidants/ antioxidants could mean a higher chance for mutations and oncogenesis leading to diseases, including cancer—since MPO produces ROS secondary derivatives can be involved in the neoplastic transformation of cells through this pathway.

The NOS3 gene is located in the 7q35–36 region of chromosome 7 and the genetic polymorphism with a great clinical relevance is the 27 bp-VNTR 4b/a intron 4 [40]. In addition, NO produced by endothelial and epithelial cells, also modulates the regulation of vascular endothelial growth factor and is possibly associated to increase in processes of invasiveness and metastasis [41]. Preliminary results from our group, although only with a trend, identify that the A variant of NOS3 polymorphism, which is associated with higher activity of this enzyme, predisposes to

Microenvironment in Vagina as a Key-Player on Cervical Cancer: Interaction of Polymorphic…

http://dx.doi.org/10.5772/intechopen.73108

There is a clear association between the excessive and cumulative exposure to oestrogens and the development of cancer in hormone-sensitive tissues, such as the cervix. Therefore, we found that CYP1A1 and Catechol-o-methyltransferase (COMT) work in a metabolic sequence and their interaction could lead to an alternative pathway of oestrogen metabolism with production of 16-OH-estrone that is more proliferative and less apoptotic [42, 43]. The role of oestrogen and the association of *CSTs* favourable for balance vaginal milieu, was previously debated [44]. Aryl hydroxylase (AhR) the transcription factor of CYP1A1 is also associated with immunosuppression after activation of IL-22 pathway, and the maintenance of intraepithelial in innate

Recently, a very interesting work unlighted the mechanism, where the Lactobacillus-derived H2

urenine levels during Lactobacillus supplementation diminished the treatment benefits.

suppress host kynurenine metabolism, by inhibiting the expression of the metabolizing enzyme, indoleamine 2,3-dioxygenase (IDO1), in the intestine [46]. Moreover, maintaining elevated kyn-

Finally, as suggested by other authors, it may be possible to expand the use of probiotics in the treatment of gynecologic cancers. The study of the role of probiotic bacteria for the prevention of colon and cervical cancer has led to the conclusion the tumour preventive effects of probiotic bacteria might be due to their control of the microbial flora, establishment of beneficial metabolic effects and stimulation of the immune system [37, 47]. Therefore, we can act in

Genetic analysis based on single nucleotide polymorphisms identified genetic variants associated with tumour rejection in mice, which could potentially affect ROS production and NK cell activity. That results also supports that B cells play a detrimental role in antitumour immunity and suggest that targeting B cells could enhance the antitumour response and improve the

The conventional photon radiotherapy for cervical cancer irradiates parts of the healthy tissue. This treatment perturbs the vaginal microbiome and disrupt the epithelial barrier function, permitting translocation of pathogenic bacteria and causing an inflammatory response [48]. The role of probiotic bacteria for the prevention of colon cancer has led to conclusion of the

O2

23

ICC OR = 7.50, 95%CI [0.88–63.9], P = 0.066).

the prevention, specifically, the relapses.

efficacy of therapeutic cancer vaccines [8].

*2.2.5. Catechoestrogens and cytochrome P450 (CYP1A1)*

lymphocytes leading to the mucosal protection from inflammation [45].

**3. Treatment and prevention: microbiota-derived factors**

H2 O2 , and a halide form a powerful antimicrobial system in phagocytes and tissue fluids, which certain microorganisms can serve as the source of H2 O2 for this system. The equilibrium of the production of H2 O2 by *Lactobacilli* in the vagina appears to be a nonspecific host defence mechanism, which can be potentiated by myeloperoxidase that produces HOCl (**Figure 1**).

The production of superoxide through NADPH oxidase from cervical cancer cells, can be specifically targeted with production of OH− radicals that induces selective apoptosis of these cells [16].

The polymorphism in the *MPO* gene induce a transition G463A (rs2333227), in the promotor region of the gene, where the wild-type G allele promotes the binding of transcription factors leading to a higher transcriptional activity than the A allele [39].

We found that women with the GG genotype had lower risk for cervical cancer than the women who displayed the heterozygous genotype GA (OR = 0.546, 95% CI = 0.315–0.939, P = 0.028, OR = 2.210, 95% CI [1.257–3.886], p = 0.008, respectively). The genotype that leads to a higher concentration of ROS (GG) presents itself as a protection factor in comparison to the homozygous genotype (AA) [39]. Moreover, recently, we observed that the A carriers of MPO polymorphism were about 5-fold of increased risk for cervical cancer (OR = 5.41, 95% CI [2.15–13.64], P < 0.0001) (**Figure 1**), being dependent of age (OR = 3.38, 95% CI [0.85–13.48], P = 0.085) and independent of smoking habits (OR = 3.85, 95% CI [1.33–11.11], P = 0.013). The interaction of HOCl and superoxide of transformed cells will generate apoptosis-inducing hydroxyl radicals.

We suggest that there is an association between the H2 O2 -producing strains found in the vaginal microbial flora and high activity of MPO leading to a clearance of the HPV-infected cells, the relation may also lead to the apoptosis of the transformed cells, producing O2. and OH. , acting as a protective factor for a cervical cancer [16, 17, 26].

#### *2.2.4. Reactive nitrogen species*

Nitric oxide is generated by nitric oxide synthase (NOS) and presents 3 isoforms: neuronal (NOS1), endothelial (NOS3) and inducible (NOS2). High local concentrations of ROS through expression of SOD and catalase might be associated to prevention of elimination of transformed cells trough ROS/RNS-dependent intercellular apoptosis-inducing signalling [14, 16]. Conversely the excess of NO inhibits the apoptosis induction associated to H2 O2 and reversely NO-mediated apoptosis induction was inhibited by excess of H2 O2 [29].

The NOS3 gene is located in the 7q35–36 region of chromosome 7 and the genetic polymorphism with a great clinical relevance is the 27 bp-VNTR 4b/a intron 4 [40]. In addition, NO produced by endothelial and epithelial cells, also modulates the regulation of vascular endothelial growth factor and is possibly associated to increase in processes of invasiveness and metastasis [41]. Preliminary results from our group, although only with a trend, identify that the A variant of NOS3 polymorphism, which is associated with higher activity of this enzyme, predisposes to ICC OR = 7.50, 95%CI [0.88–63.9], P = 0.066).

#### *2.2.5. Catechoestrogens and cytochrome P450 (CYP1A1)*

inflammatory cells. The presence of MPO on vaginal milieu is activated. Probably, the persistence of death cells recalls of neutrophils to vagina, being MPO important for the control of excess production of HOCl. In the vaginal microenvironment, the MPO catalyzes the reaction

22 Cervical Cancer - Screening, Treatment and Prevention - Universal Protocols for Ultimate Control

ions, yielding HOCl, which participates in the oxidative burst during the innate host defence [38]. MPO may act in synergy with other proteins. Therefore, an imbalance between oxidants/ antioxidants could mean a higher chance for mutations and oncogenesis leading to diseases, including cancer—since MPO produces ROS secondary derivatives can be involved in the

, and a halide form a powerful antimicrobial system in phagocytes and tissue fluids,

mechanism, which can be potentiated by myeloperoxidase that produces HOCl (**Figure 1**).

The production of superoxide through NADPH oxidase from cervical cancer cells, can be spe-

The polymorphism in the *MPO* gene induce a transition G463A (rs2333227), in the promotor region of the gene, where the wild-type G allele promotes the binding of transcription factors

We found that women with the GG genotype had lower risk for cervical cancer than the women who displayed the heterozygous genotype GA (OR = 0.546, 95% CI = 0.315–0.939, P = 0.028, OR = 2.210, 95% CI [1.257–3.886], p = 0.008, respectively). The genotype that leads to a higher concentration of ROS (GG) presents itself as a protection factor in comparison to the homozygous genotype (AA) [39]. Moreover, recently, we observed that the A carriers of MPO polymorphism were about 5-fold of increased risk for cervical cancer (OR = 5.41, 95% CI [2.15–13.64], P < 0.0001) (**Figure 1**), being dependent of age (OR = 3.38, 95% CI [0.85–13.48], P = 0.085) and independent of smoking habits (OR = 3.85, 95% CI [1.33–11.11], P = 0.013). The interaction of HOCl and superoxide of transformed cells will generate apoptosis-inducing

nal microbial flora and high activity of MPO leading to a clearance of the HPV-infected cells,

Nitric oxide is generated by nitric oxide synthase (NOS) and presents 3 isoforms: neuronal (NOS1), endothelial (NOS3) and inducible (NOS2). High local concentrations of ROS through expression of SOD and catalase might be associated to prevention of elimination of transformed cells trough ROS/RNS-dependent intercellular apoptosis-inducing signalling [14, 16].

the relation may also lead to the apoptosis of the transformed cells, producing O2.

Conversely the excess of NO inhibits the apoptosis induction associated to H2

NO-mediated apoptosis induction was inhibited by excess of H2

O2

and either thiocyanate ions or a halide, such as iodide, bromide or chloride

O2

by *Lactobacilli* in the vagina appears to be a nonspecific host defence

radicals that induces selective apoptosis of these cells

for this system. The equilibrium


O2

O2 [29]. and OH.

and reversely

,

between H2

H2 O2

[16].

O2

of the production of H2

hydroxyl radicals.

*2.2.4. Reactive nitrogen species*

neoplastic transformation of cells through this pathway.

which certain microorganisms can serve as the source of H2

leading to a higher transcriptional activity than the A allele [39].

We suggest that there is an association between the H2

acting as a protective factor for a cervical cancer [16, 17, 26].

O2

cifically targeted with production of OH−

There is a clear association between the excessive and cumulative exposure to oestrogens and the development of cancer in hormone-sensitive tissues, such as the cervix. Therefore, we found that CYP1A1 and Catechol-o-methyltransferase (COMT) work in a metabolic sequence and their interaction could lead to an alternative pathway of oestrogen metabolism with production of 16-OH-estrone that is more proliferative and less apoptotic [42, 43]. The role of oestrogen and the association of *CSTs* favourable for balance vaginal milieu, was previously debated [44].

Aryl hydroxylase (AhR) the transcription factor of CYP1A1 is also associated with immunosuppression after activation of IL-22 pathway, and the maintenance of intraepithelial in innate lymphocytes leading to the mucosal protection from inflammation [45].

Recently, a very interesting work unlighted the mechanism, where the Lactobacillus-derived H2 O2 suppress host kynurenine metabolism, by inhibiting the expression of the metabolizing enzyme, indoleamine 2,3-dioxygenase (IDO1), in the intestine [46]. Moreover, maintaining elevated kynurenine levels during Lactobacillus supplementation diminished the treatment benefits.
