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ACTIVITY OF APOPTOSIS MARKERS IN HPV-ASSOCIATED CERVICAL PATHOLOGIES
Artemova O.
Medical Institute of FSBEI HE "Penza State University " Assistant, Department of Obstetrics and Gynecology, Medical Institute of FSBEI HE "Penza State University"
Abstract
Current development of medicine demonstrates the presence of pathological processes at the molecular level. In particular, the phenomenon of apoptosis, being programmed cell death, is being actively studied. In order to determine the role of apoptosis in the etiology and pathogenesis of such HPV-associated cervical pathologies as leukoplakia and cervical intraepithelial neoplasia I, cervical status in women of fertile age was investigated. The study has revealed that an apoptosis-resistant phenotype develops in HPV-positive patients with cervical intraep-ithelial neoplasia I.
Keywords: ectocervical dysplasia, leukoplakia of the cervix, caspase-1, caspase-3, apoptosis-resistant phe-notype.
At present, the problem of cervical pathology is still relevant. Despite numerous studies, diagnostic criteria and treatment options, there are cervical pathologies in the general pathology of gynecological morbidity (1). Currently, the most urgent problem in the field of obstetrics and gynecology is the combination of changes in the cervix with the activity of human papillomavirus (HPV). HPV types (4, 5) were detected in 94% of identified cases of cervical cancer. Due to special attention of the World Health Organization (WHO) to widespread atypical cervical processes, the screening programs (1, 2, 4, 13) have been introduced, aimed at the earliest detection of precancerous diseases of the cervix (2, 4, 5, 7). During formation of atypical changes in the cervix, a virus DNA can penetrate the genome of an infected cell (7, 8, 9), stimulating proliferative processes therein (10, 11, 21). A modern stage of medical science development is characterized by investigating the mechanisms of pathological processes at the molecular level (14). In particular, the phenomenon of apoptosis is being actively studied as
programmed cell death (6, 9, 11, 13, 14). Thus, the development of pathological changes in the cervix under the influence of human papillomavirus is accompanied by a violation of the induction of apoptosis mechanisms (6, 14, 18, 19), where the main role is played by enzymes functionally connected with differentiation clusters (15, 17). One of these enzymes are cell caspases (6, 14), which are the main links in the apoptosis process, and are responsible both for the intracellular formation of the apoptotic signal and for the final stage of this process (7, 8, 14, 20). There are studies devoted to cervical cancer processes, but it is not determined at what stage the formation of apoptotic changes and possible variants of this process (10, 12, 13) begin. An effective secondary prevention of atypical processes in the cervix is an adequate prediction of cervical HPV-associated pathologies by evaluating caspase-1 and caspase-3 markers.
Purpose of the study is to determine the degree of the apoptotic program violation by evaluating caspase-1 and caspase-3 expression in cervical mucus
in HPV-positive patients of fertile age with cervical intraepithelial neoplasia I (CIN I), and leukoplakia of the cervix.
Materials and Research methods
Sixty-five women of fertile age from 18 to 45 years old took part in the research. Group I consisted of 35 patients with HPV-associated leukoplakia of the cervix. Group II consisted of 15 women with HPV-associated CIN I. The control group consisted of 15 healthy women.
In working with patients, the ethical principles presented by the WMA Helsinki Declaration "Ethical Principles of Scientific and Medical Research with Human Participation" (as amended in 2008), "Rules of Clinical Practice in the Russian Federation" (Order of the Ministry of Health and Social Development of the Russian Federation No. 266 dated June 19, 2003), and
National Standard of the Russian Federation "Good Clinical Practice" (2005) were observed.
The inclusion criteria in research studies were histologically confirmed leukoplakia of the cervix or cervical intraepithelial neoplasia I; presence of human papillomavirus; absence of therapy with immunomodulatory drugs during the last seven months; absence of exacerbation of chronic inflammatory processes in the small pelvis; absence of sexually transmitted infections.
The criteria for exclusion from the research were an age interval from 18 to 45 years old; positive pregnancy test; severe somatic pathology; taking drugs affecting the studied parameters. Women with HPV-associated status were divided into groups by age (Table 1).
Table 1.
Age distribution of the examined patients
Age group 18-20 years old 21-30 years old 31-39 years old 40-45 years old
Number of patients in total 9 (22.5%) 15(37.5%) 18 (45%) 8 (20%)
Group I 9 (25.7%) 10 (28.6%) 8 (22.86%) 8(22.8%)
Group II 0 5 (33.3%) 10 (66.7%) 0
A comparative analysis of age categories and the presence of cervical atypical processes have revealed a predominance of the age interval of 21-39 years old, which indicates long-term virus persistence in the epithelium until clinical changes appear. An analysis of social data has not revealed direct dependence on the place of work, marital status, and educational level among HPV-positive patients. Age data, body mass index, features of reproductive function, sexual debut, number of sexual partners, and obstetric history (special emphasis is given to the number and course of pregnancies, and the number of abortions) were taken into account when working with anamnestic indicators of the patients. In analyzing clinical and anamnestic data, there were no significant differences in the groups with HPV-positive patients and the control group, regarding body mass index. When assessing the data of the gynecological history, there were no significant differences in the duration of menstruation and the number of pregnancies.
An assessment of patients' clinical condition was carried out on the basis of the examination data, according to the Order of the Ministry of Healthcare of the Russian Federation No. 572H dated November 01, 2012 "On approval of the Procedure for health care in "Obstetrics and Gynecology (except for assisted reproductive technology)". Smears for purity degree and bacterial culture were taken, and cervical oncocytology and colposcopy were performed. An examination for sexually transmitted infection (STIs) was carried out by the polymerase chain reaction (PCR) method when conducting a comprehensive examination.
Cytological technique is one of the leading methods for the diagnosis of precancerous and malignant processes in the cervix. The material was taken from the three zones: the ectocervix, the junction zone, and the lower third of the endocervix. Then, Papanicolaou (Pap) stain of cervical smear was carried out, and a 95% solution of ethyl alcohol being the solvent for better color differentiation.
During the research, all patients underwent PCR diagnosis of papillomavirus infection. For this purpose, AmpliSens® HPV HCR-genotype-FRT test system (Russia) was used. It is a set of reagents for identifying and genotyping high carcinogenic risk (HCR) of HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59 types in real-time. The method is based on the simultaneous amplification (multiplex PCR) of DNA sections of E1-E2 genes of three HPV groups, and DNA section of p-globin gene used as an endogenous internal control. PCR analysis for DNA presence of 14 HPV types is performed in a single test tube. All amplifiable fragments have similar length.
The next stage was the procedure of cervical col-poscopy. To conduct this examination of patients, the OLYMPUS OCS-500 colposcope with 15-30x magnification was used. Shape, size, relief, and color of the cervix and external orifice were determined during the colposcopy. The border of the squamous and cylindrical epithelium, as well as their structure and presence of additional formations were evaluated. After that, the cervix was treated with 3% acetic acid solution and 3% Lugol's aqueous solution (Schiller test).
The documentation of colposcopy results was carried out graphically using the clock-face method.
6
Fig. 1. Graphic visualization of lesion localization by clock position
During the colposcopy, the classification of the In- Committee at the World Congress (July 2011, Rio de ternational Federation for Cervical Pathology and Col- Janeiro), was used. To unify the obtained results, a poscopy (IFCPC), revised by IFCPC Nomenclature modified Reid colposcopic index was calculated.
Table 2.
Colposcopy prognosis of the histological conclusion by the Reid index
Number of points Histology
0-2 probably, CIN I
3-4 CIN I or CIN II
5-8 probably, CIN II-III
To confirm leukoplakia of the cervix or cervical intraepithelial neoplasia I, the patients underwent target biopsy of the cervix. Bioptates of the affected cervical areas were studied taken by the method of radio wave surgery using the Fotek apparatus, which allowed minimally affecting the substrate with physical energy to obtain quality results. It was important to obtain a sufficiently large bioptate (about 5 mm in width) and to take the connective tissue of the cervix (up to 6 mm in depth) along with squamous epithelium. The biopsy area was documented, and the material was marked and sent for histological examination. In the laboratory, it was fixed in paraffin or frozen in a thermostat. Then the paraffin block or frozen piece was cut, and slices were placed on glass, stained and examined under a microscope. Upon completion of the histological examination, the obtained samples were evaluated according to the WHO classification of 2014.
To determine the level of cysteine proteases (caspase-1 and caspase-3), an intake of cell scraping was carried out from the transformation zone and from the cervical canal. The obtained material from the cervical canal was placed in phosphate-buffered saline using urogenital disposable sterile probe (type F-1 Cytobrush), and delivered to the laboratory for further processing.
To determine caspase-1 and caspase-3 level, a reagent kit by Cloud-Clone Corp. (CCC) was used intended for quantitative determination of CASP3 by the ELISA (Enzyme Linked Immunosorbent Assay) sandwich method in tissue homogenates, cell lysates, cell culture supernatants, and other human biological fluids.
Statistical processing of the obtained results was carried out by the analysis of variance and correlation analysis using Microsoft Excel 7.0 (Windows Office 2007) with determination of the Fisher criterion (F-
criterion) to assess nonparametric group indicators of small samples, and Student criterion (t) for independent groups.
Results and Discussion.
The first stage was a cytological examination of the patients. The overwhelming majority of women had normal cytological smears in both groups. In analyzing the data of cytological examination, 37 patients (74%) had normal results, 12% (6 patients) had low-grade squamous intraepithelial lesion (LSIL), and leukoplakia of the cervix was found in 14% (7 patients). The next stage was a colposcopic examination. Changes in patients with leukoplakia corresponded to abnormal colposcopy, moreover, obvious signs of leukoplakia were observed only in 19 (38%) patients, and mild manifestations in the form of fine punctation or mosaics, which may correspond to CIN I, were detected in 16 (32%) patients. Pronounced changes in the epithelium corresponding to high-grade squamous intraepithelial lesion (HSIL), not typical for CIN I were observed in three cases in patients of Group 2, which amounted to 7.5%. When analyzing data of colposcopic examination with grading of colposcopic signs (clinical colposcopic index and the Reid index (CRI), it was determined that in 79.8% of patients with leukoplakia of the cervix, evaluation by CRI scale was 1-2 points; in 81.3% of patients with CIN I it was 2 points; and in 18.1% of patients it was 3 points. Thus, colposcopy, referring to subjective research methods, having a sensitivity of 80% and a specificity of 60% (1, 2, 19) does not always allow us to assess the degree of damage to the epithelium, but helps to identify areas for target multifocal biopsy.
In HPV-typing using the polymerase chain reaction method, it was found that in the studied groups there was a combination of highly oncogenic HPV types, and HPV type 16 being the most common one.
Thus, the analysis of colposcopic, cytological and histological results allows concluding that the histological method gives the largest information on the state of the cervical epithelium in HPV-positive patients with leukoplakia of the cervix and CIN I.
To determine the severity degree of the cervical changes and to evaluate the risk of developing atypical processes, an assessment of apoptotic process in the cervical mucus obtained during the cytological intake of the material was carried out. Determination of the level of caspase-1 (Table 3) and caspase-3 (Table 4) was another stage of the research.
Table 3.
The level of caspase-1 in research groups
Group name Caspase-1
Control group 0.060 ±0.01*
Leukoplakia 0.059 ± 0.01*
CIN I 0.061 ±0.02*
In the group of patients with leukoplakia, HPV type 16 was found in 27 (78%) women, and in 13 (89%) patients with CIN I. HPV type 18 was detected in 24 (69%) women of Group 1, and in 12 (81%) patients of Group 2. HPV type 33 was registered in seven (19%) patients with leukoplakia, and in five (34%) patients with CIN I.
To establish a final diagnosis, a multifocal cervical biopsy with a histological examination was performed. The results of this histological examination served as the basis for the formation of research groups.
In determining the level of caspase-1 in the control and research groups, the obtained results were within sensitivity limit of the method, which indicates the absence of an inflammatory reaction in the cervical epithelium induced by HPV, since caspase-1 plays a key role in cellular immunity (6, 11, 12, 14) as a mediator of the inflammatory response. Activating this caspase-1 via formation of an inflammation complex, it initiates an anti-inflammatory response by cleavage and, thus, an activation of two inflammatory cytokines, interleukin 1ß and interleukin 18, as well as pyroptosis, being the programmed lytic cell death pathway (11, 14, 21). Then, an escape of cytokines from the cell additionally induces an inflammatory response in neighboring cells (14).
In clinical conditions, an assessment of violation degree of the apoptotic program in HPV-associated cervical pathology by determining caspase-3 expression
was carried out. Studying effector caspase-3, changes were obtained characterizing the progression degree of HPV presence in cervical epithelial cells. Due to the lack of normal values for caspase under study, the results in the immunological control group were taken as normal indicators. In research groups, the activity of effector caspase-3 was higher relative to the control level, and increased as cervical pathology progressed against the background of the virus. These results confirm the formation of apoptosis resistant process in HPV-affected cells (9, 11, 12, 21) due to activation of caspase genes in changed areas of the cervical zone. It supplements the previous studies confirming the data on atypical progression with CIN III in cervical cancer, and an increase in caspase-3 level (13, 15).
Table 4.
The level of caspase-3 in research groups
Group name Caspase-3
Control group 0.188 ±0.02*
Leukoplakia 0.718 ± 0.05*
CIN I 2.393 ±0.03*
An increase in severity degree of a pathological process in the cervix was accompanied with determination of caspase-3 expression (Table 4). This is explained by the fact that there are inactive monomeric precursors in the caspase cells, requiring cleavage of proenzyme and subsequent dimerization for activation. The implementation of these reactions is possible with successive mutual activation of caspases. The starting moment for such transformations is DNA damage, which in turn launches a caspase cascade. It can follow either external or internal pathway, but no matter what pathway the cascade is launched, caspase-3 is its effector caspase. The mechanisms for HPV-associated cervical pathologies formation, such as leukoplakia or CIN I, can be considered as expression of effector caspase-3 level. The studied cysteine protease is able to increase with introduction of HPV DNA into the host cell. Determination results of caspase-3 have shown different
values in the expression level of apoptosis marker in the cervical epithelium as HPV-associated pathology progresses.
Determining the level of caspase-3 in case of leu-koplakia and CIN I proves the validity of an in-depth dispensary observation and eventual active treatment of these patients. The obtained data on change of the apop-totic program and, as a consequence, local immunity are a pathogenetic justification for complex therapy using antiviral drugs with an immunomodulatory effect.
Conclusion
A key role in the process of pyroptosis is played by cytokines, which, when released, induce an inflammatory process. One of the final stages to implement inflammatory reactions is caspase-1. The indicators were not statistically different when determining its level in control and research groups, which points out the absence of inflammatory cervical
process in HPV-positive patients with leukoplakia of the cervix and CIN I.
For a long time, reparative processes have been limited exclusively by epithelium of the cervical zone under the influence of human papillomavirus. Introducing HPV DNA into the host cell, a cascade of apoptotic reactions is launched, where the final stage is activation of effector caspase-3. The determination results of caspase-3 have shown different values in the expression level of apoptosis marker in the cervical epithelium as HPV-associated pathology progresses. Therefore, an assessment of cervical zone by means of determining the level of apoptotic caspase-3 in HPV-positive patients are prognostic and diagnostic criteria for assessing the severity of the identified pathology.
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