Determination of Autophagy in Human Cervicovaginal Smears by Cytological and İmmunocytochemical Methods

Abstract

Background:

Autophagy is a catabolic process whereby organelles and long-lived proteins are recycled through lysosomes to maintain cellular homeostasis. This process is being widely studied using culture techniques and animal models; however, cervicovaginal smears have not been used to detect autophagy.

Aims:

Our study aims to detect and evaluate autophagy in normal, malignant, infectious, and atypical cells in cervicovaginal smears by using cytological and immunocytochemical methods.

Materials and Methods:

Papanicolaou-stained 200 cervicovaginal smears were examined and 55 of 200 (27.5%) smears containing negative for intraepithelial lesion or malignancy (NILM) with identifiable infections and/or reactive/reparative changes (INF); briefly, NILM-INF (n = 31, 56.4%), atypical (n = 4, 7.3%), and malignant cells (n = 20, 36.3%) were evaluated as a study group. One hundred forty-five of 200 (72.5%) normal smears were accepted as the NILM without any identifiable infections (control group). The autophagy marker protein Microtubule-associated protein 1 light chain 3 A (MAP1LC3A) was used for immunocytochemical examination.

Results:

The staining intensity of the MAP1LC3A protein and autophagy positivity were lower in the malignant cells; however, they were higher in the NILM-INF and atypical cells. A statistically significant correlation between the malignant and normal cells was obtained for the autophagy positivity (P = 0.012). In view of the staining intensity of MAP1LC3A protein by the H-score method, a significant correlation was found between the NILM-INF and the normal cells (P = 0.015).

Conclusions:

Autophagy was detected in various cervicovaginal smears for the first time in this study. Our findings indicate that an autophagy process is essential in infectious cells as well as in the transformation of atypical cells into malignant cells in carcinogenesis.

INTRODUCTION

Autophagy is a highly conserved catabolic process that regulates the recycling of cytoplasmic contents, such as redundant and damaged organelles, misfolded proteins, and phagosomal-escaped microorganisms.[1,2] In this mechanism, cytoplasmic contents are sequestered by an autophagic membrane termed the phagophore, and this membrane elongates and ultimately generates double-membrane vesicles that are called autophagosomes.[1] The outer membrane of the autophagosome fuses with a lysosome to generate an autolysosome.[3] Inside the autolysosome, the inner membrane of the autophagosome and cytoplasmic contents are degraded by acid hydrolases.[4] The resulting degradation products are released back into the cytosol through membrane permeases for reuse.[5] They can be reused as either an energy source to maintain cell viability under unfavorable conditions or as building blocks for new lipids and proteins to protect the cell during various stress conditions.[6,7]

Although autophagy was first identified in mammalian cells more than 50 years ago, the molecular understanding of autophagy began with the identification of autophagy-related (ATG) genes in yeast.[8] To date, genetic analyses in yeast have identified around 40 ATG genes, many of which have orthologs in higher eukaryotes.[9] Among these genes, around 20 ATG genes are referred to as the ‘core’ ATG genes due to the fact that they are required for autophagosome formation in all types of autophagy.[9] One subgroup of these core ATG genes is ATG8/LC3/GABARAP (Microtubule-associated protein 1A/1B-light chain 3/Gammaaminobutyric acid receptor-associated protein) superfamily.[10] Although in yeast there is a single ATG8 protein, in mammals at least eight Atg8 paralogs have been identified.[10] This protein plays a pivotal role in autophagosome formation and autophagosome-lysosome fusion, as well as is used as a general marker of autophagy.[10,11] LC3, approximately 17 kDa soluble protein, is a subset of ATG8 family proteins and is widely investigated and identified as an autophagosome marker in mammalian cells.[12]

Although most of the studies related to autophagy were carried out by using cell culture, immunohistochemistry and animal models,[13-15] there is no previous documentation based on cervicovaginal smears in determining the autophagy for both the normal and malignant exfoliated cells. The purpose of this study was to detect and evaluate the autophagy in infectious, atypical and malignant cells in cervicovaginal smears by using cytologic and immunocytochemical methods.

MATERIALS AND METHODS

Subjects

A total of 200 women aged between 21 and 81 years with various gynecological complaints were examined in the outpatient clinic at Hacettepe University, Department of Obstetrics and Gynecology, Ankara, Turkey. Initially, all women enrolled in this study completed a questionnaire that requested clinical information on age, menstruation date, operation status, and gynecological complaints such as vaginal discharge, itching, and burning. Pregnant women were not included within the scope of this study. Sampling was performed in accordance with the principles of the Helsinki Declaration. All selected women voluntarily signed an informed consent form before enrollment. The study was approved by Hacettepe University, Research Ethics Committee of the Medical Faculty (approval no: LUT 12/69). Both cervicovaginal smears for PAP smear microscopy and the immunocytochemical method were taken from patients at the same time. These smears were obtained from women using a cytobrush before a pelvic examination and were fixed with different techniques for each method. After examining these smears, cases with infectious agents and/or cellular changes or malignant cases were selected as the study group. Cases without any infectious agents, cellular changes, or malignancies were considered as normal and constituted as the control group. We followed the Bethesda classification for the names’ of the cases. Accordingly, “negative for intraepithelial lesion or malignancy (NILM)” was divided into Pap smears with identifiable infections and/or reactive/reparative changes (NILM-INF) refer to “the infectious group,” and NILM without any identifiable infections (NILM) refers to “the control group” in the present study.

PAP smear microscopy

Cervicovaginal smears were fixed onto slides with 96% ethanol without drying in the air and stained by the routine Papanicolaou (PAP) technique. All the stained smears were examined carefully according to the cytological changes and photographed by a camera-attached light microscope (Leica, DM 4000B) [Figure 1].

Figure 1.

(a) The malignant cells are seen in a cervicovaginal smear. The abnormal nuclei of these cells are present with large (arrow) and prominent (double arrows) nucleoli (b) Fungal pseudohyphae (arrow) and blastospores (arrowhead) of fungal infection are seen (c) Atypical intermediate squamous cells with an enlarged nucleus (black arrow) and slight nuclear membrane irregularity (white arrow) in an inflammatory background. (Papanicolaou, ×1000)

Immunocytochemistry

Cervicovaginal smears that were taken for the immunocytochemical technique were fixed onto adhesive slides with 4% paraformaldehyde for ten min and stored at -20°C until immunocytochemical staining. Autophagy was detected by an immunocytochemical technique using the purified rabbit polyclonal antibody MAP1LC3A (AP1805a; Abgent, San Diego, CA) and Novocastra Novolink Polymer Detection System Kit (RE7150-K, Leica Microsystems).

In this staining method, the slides were removed from -20°C storage and placed in citrate buffer (1:10 dilution, pH 7.0) for heat-induced antigen retrieval. They were heated in a microwave oven (Panasonic NN-6450,800W) for five min at a low temperature. After heating, the slides were allowed to cool to room temperature for about 20 min and then washed with phosphate-buffered saline (PBS) twice for five min. Endogenous peroxidase activity was neutralized using two drops of Peroxidase Block (Novocastra Laboratories Ltd, Newcastle on Tyne, UK) for 7 min at room temperature in a moist chamber. After washing with PBS (2 × 5 min), the nonspecific binding was blocked by incubation with two drops of Protein Block for 7 min in a moist chamber under the same conditions as in Peroxidase Block. The diluted (1:100) primary antibody (MAP1LC3A) was added to the slides and incubated overnight at 4°C. After washing with PBS (2 × 5 min), the slides were incubated with two drops of Post Primary Block (Novocastra Laboratories Ltd, Newcastle on Tyne, UK) for 30 min at room temperature to enhance penetration of the subsequent polymer reagent. The slides were washed again with PBS for 2 × 5 min and incubated with two drops of NovoLink polymer (Novocastra Laboratories Ltd, Newcastle on Tyne, UK) for 30 min at room temperature. This reagent recognizes mouse and rabbit immunoglobulins and detects primary antibodies. After this step, the slides were washed with PBS (2 × 5 min) and the color reaction was developed in 3,3’-diaminobenzidine (DAB) solution for 5 min at room temperature and washed with distilled water. Counter-staining of the slides with the hematoxylin was applied for 40 seconds and dehydration was performed through a graded ethanol series, beginning with 70%, 85%, 95%, and 100% ethanol. Dehydrated slides were cleared in xylene, mounted with entellan (Merck), and then viewed under a light microscope (Leica DM 4000B). For the negative control, the same technique of immunocytochemical staining was performed in the absence of the primary antibody. Breast cancer tissue, which expresses MAP1LC3A, served as a positive control.

Evaluation of the immunocytochemical staining

A positive reaction to the MAP1LC3A antibody resulted in a diffuse and brown staining of the cytoplasm of the cells by 3,3’-diaminobenzidine (DAB). The positive and negative staining patterns for this antibody are shown in Figure 2.

Figure 2.

Immunocytochemical staining of epithelial cells using MAP1LC3A antibody. Positive (arrow) and negative staining (arrowhead) of MAP1LC3A protein are shown in the epithelial cells (immunocytochemistry, ×1000)

Firstly, the percentage of cells stained and intensity of staining were determined for the evaluation of immunocytochemical staining. A minimum of 100 cells were counted in at least ten randomly chosen microscopic fields on each slide and the average counts were recorded. As shown in Figure 3, the staining intensity of positive cells was evaluated in three categories as weak (+), intermediate (++), and strong (+++) staining, and the number of cells stained at each intensity was also recorded. Then, the percentages of positive cells were grouped according to the 35^th percentile, as either “low (≤35%)” or “high (>35%)” for autophagy positivity [Table 1].

Figure 3.

The staining intensity of MAP1LC3A protein was evaluated in 3 categories (a) weak (+), (b) intermediate (++), (c) strong (+++) staining intensity of MAP1LC3A protein (immunocytochemistry, ×1000)

Table 1.

Comparison of the study and NILM groups in view of the percentages of positively stained cells for MAP1LC3A protein

Groups	% value (Positivity)		P
	≤35% (Low)	>35% (High)
Study Group (n=55)
Malignant (n=20)	13 (65%)	7 (35%)	*P=0,012
**NILM-INF (n=31)	5 (16.1%)	26 (83.9%)	P=0,083
Atypical Cell (n=4)	1 (25%)	3 (75%)	P=1,00
***NILM Group (n=145)	48 (33.1%)	97 (66.9%)

*P<0.05 were considered statistically significant. **NILM with identifiable infections and/or reactive/reparative changes. ***NILM without any identifiable infections (the control group consisting of normal patients)

Secondly, H-score (Histologic score) method was used to evaluate staining intensity.[16] This method is based on the percentage of cells at different staining intensities and gives a range of 0 to 300. The score was calculated using the following formula:

H-score = 1 × (% of cells stained weakly) + 2 × (% of cells stained intermediately) + 3 × (% of cells stained strongly).[16,17]

According to the distribution of H-score values, the threshold H-score for this study was accepted as 45. The scores were then classified as either low (H-score ≤45) or high (H-score >45) for MAP1LC3A protein expression [Table 2].

Table 2.

Evaluation of the MAP1LC3A protein expression (staining intensity, H-score values) in the study and NILM groups

Groups	H-score		P
	≤45	>45
Study Group (n=55)
Malignant (n=20)	12 (60%)	8 (40%)	P=0,231
**NILM-INF (n=31)	6 (19.4%)	25 (80.6%)	*P=0,015
Atypical Cell (n=4)	0 (0%)	4 (100%)	P=0,138
***NILM (n=145)	63 (43.5%)	82 (56.5%)

*P<0.05 were considered statistically significant, **NILM with identifiable infections and/or reactive/reparative changes. ***NILM without any identifiable infections (the control group consisting of normal patients)

Statistical analyses

Statistical analyses were conducted with Fisher’s exact test and Chi-square test using the SPSS program, version 21. P values < 0.05 were considered statistically significant.

RESULTS

A total of 200 cervicovaginal smears were examined cytologically and 55 of 200 (27.5%) were accepted as the study group. This study group was examined in three categories and evaluated separately. One of these categories was identified as malignant and 20 of the 55 smears (36.3%) were diagnosed as malignant. Thirty-one cervicovaginal smears (56.4%) which had identifiable infections were identified as NILM-INF and 4 cervicovaginal smears (7.3%) were evaluated as atypical gynecological smears. One hundred forty-five of 200 (72.5%) normal cervicovaginal smears were accepted as NILM group (as the control group).

In the cytological examination, malignant, NILM-INF, and atypical gynecological smears were identified. The malignant smears in our study include ovarian adenocarcinoma, endometrial adenocarcinoma, and invasive squamous cell carcinoma cases. Cytologic criteria such as the enlargement of the nucleus and nucleoli, prominent nucleolus, hyperchromatism, and the number of nucleoli were used to detect the malignant cells[18,19] [Figure 1a]. Various infectious smears such as bacterial vaginosis, fungal infections, especially candidiasis, trichomoniasis, human papilloma (HPV) infection, and chlamydial infection were detected. Among them, infectious agents of fungal infection, fungal pseudohyphae and blastospores (budding yeast forms), are shown in Figure 1b.[19,20] Atypical cellular changes were also observed in the slides and these changes generally consist of slightly hyperchromatic nuclei and variations in nuclear size and shape [Figure 1c]. Nuclear enlargement is approximately two-and-a-half or three times that of a normal intermediate squamous cell nucleus.[19] Therefore, a slightly increased nucleus: cytoplasm ratio is seen in the atypical cells and these cells are usually named as ASC-US (Atypical Squamous Cells of Undetermined Significance).[19]

In the immunocytochemical examination, the cytoplasmic positivity for MAP1LC3A was evaluated and autophagy was considered positive in these cells. When the study and NILM groups were compared statistically in view of the number of autophagy-positive cells, autophagy positivity was lower in malignant smears (35%) than in the NILM smears (66.9%). During the statistical examination, a significant correlation was obtained only between the malignant and NILM groups for the autophagy positivity (P = 0.012) [Table 1]. As shown in Table 1, autophagy positivity was higher in the NILM-INF (83.9%) and atypical gynecological smears (75%) than in the NILM group (66.9%).

In our study, “intensity of immunocytochemical staining” was also examined in the autophagy-positive cells of the study and the NILM groups by the H-score method. As seen in Table 2, MAP1LC3A protein expression was observed lower in the malignant smears (40%) than in the NILM smears (56.5%). However, this protein expression was higher in the NILM-INF (80.6%) and atypical gynecological smears (100%) than in the NILM group (56.5%). A statistically significant association was found between the NILM-INF smears and the NILM group (P < 0.05). There were also statistically significant associations between the malignant and NILM-INF groups in terms of both the number of autophagy-positive cells (P = 0.001) and staining intensity (P = 0,003).

DISCUSSION

Autophagy is a catabolic mechanism in which misfolded, damaged, excessive proteins, damaged organelles, and foreign pathogens are delivered to the lysosomes for degradation and recycling.[1,2] This mechanism plays a key role in many aspects of cell physiology, including protein and organelle quality control, prevention of genomic damage, and maintaining homeostasis.[21] Researchers have found that defects in the autophagy pathway or mutations in genes encoding proteins of this pathway cause several diseases including infections, myopathies, neurodegenerative diseases, and cancer in previous studies.[21] Although these studies used animal models, tissue samples, or cell culture methods to investigate autophagy, there is no previous study to observe autophagy in cervicovaginal smears by using immunocytochemical and cytologic methods.[13-15] Therefore, we examined autophagy for the first time in normal, malignant, infectious, and atypical cervicovaginal cells by observing the expression of MAP1LC3A protein, which is a key molecule in autophagy.

Several studies on autophagy-related protein expression in normal, atypical, benign, or malignant cases have been previously reported.[22,23] In one study, the expressions of autophagy proteins, Beclin 1 and MAP1LC3 (LC3), were found low in malignant epithelial ovarian cancer tissues; however, they were high in benign and atypical counterparts. The researchers also found that the expressions of these proteins were lower in advanced-stage ovarian carcinoma. These findings suggested that the decrease in autophagic capacity due to decreasing levels of LC3 and Beclin-1 proteins might be related to tumorigenesis and the development of epithelial ovarian cancer.[22] In another study, Beclin-1 protein expression was investigated in normal and breast carcinoma tissues and cell lines. The expression of this protein was found low in both breast carcinoma tissue samples and human MCF7 breast epithelial carcinoma cell lines compared to normal tissue or cells.[23] The monoallelic deletions of the BECN1 (Beclin 1 autophagy-related gene) were frequently observed in 40-75% of human breast, ovarian, and prostate cancers.[24] They suggested that this deletion was the cause of the decreased expression of Beclin-1 in human breast carcinoma and specific molecular alterations in proteins of autophagy pathways might contribute to tumorigenesis.[23] Recently, Miao et al.[14] have also shown that MAP1LC3A is negatively associated with histological grade and distant metastasis of lung cancer and suggested that LC3A could serve as a potential biomarker in lung cancer progression. In contrast to these studies, Ahn et al.[25] investigated the expression of Beclin-1 in normal, colorectal, and gastric carcinoma tissues and detected that the expression of Beclin-1 was high in these carcinoma tissues, whereas normal cells of these tissues showed no or very weak expression. According to these data, it was suggested that the deletions of one allele of the BECN1 gene might not occur in colorectal and gastric carcinomas and the expression of this protein might play different roles depending on the type of cancer. Similar to this study, Yang et al.[26] observed the higher expression of Beclin-1 in colorectal cancer (CRC) tissues than in normal tissues. They also found that the patients with a high expression of Beclin-1 had a better five-year survival rate compared with the patients who have a low expression of this protein. Therefore, Beclin-1 expression could be used to predict the CRC patients’ prognosis. In our study, we observed that the MAP1LC3A protein expression was lower in malignant smears than in the normal counterparts [Figure 4a and b]. A significant association was also found between the malignant and normal smears in view of the autophagy positivity (P = 0.012) in this study. The decrease in the MAP1LC3A protein expression and lower autophagy in the malignant group of our study were explained by the following: (1) It could be postulated that the cause of this decrease in the protein in question might be the deletions of one allele of the BECN1 gene. Decreased expression of Beclin-1 could lead to defects in the process of autophagy due to its role in autophagosome formation. Thus, the autophagy process could not occur exactly and, in parallel with the expression of the other autophagy proteins such as MAP1LC3A, could decrease. (2) Another reason for the decreased expression of the MAP1LC3A protein and lower autophagy might be the different roles of autophagy in different cancer types. Relatedly, Chakraborty[27] has recently reported that the major actions of autophagy vary in different cancer types, and therefore, this field is still now an active area of research.

Figure 4.

MAP1LC3A protein expression is seen in normal and malignant endocervical cells. (a) Diffuse and strong staining intensity is seen in normal endocervical cells of the NILM group (immunocytochemistry, ×400). (b) Large nucleolus (white double arrows) and very low staining intensity for MAP1LC3A protein in the cytoplasm (arrow) are seen (immunocytochemistry, ×1000)

In the cytologic examination, we also observed various NILM-INF smears as well as malignant smears. There are a few studies on the relationship between autophagy and these infections in the literature. Zhou and Münger examined the autophagy in both normal oral human keratinocyte lines (NOK cells) and the human papillomavirus type 16 (HPV16) E7 protein-expressing human oral keratinocytes (NOK E7 cells).[28] They detected the increased amounts of the autophagosome, which is the hallmark morphological feature of autophagy,[29] by immunofluorescence analysis using microtubule-associated protein 1 light chain 3 (LC3) antibody.[28] Stronger LC3 staining was observed in the NOK E7 cells compared to the NOK cells. The E7 is an oncoprotein that causes the proliferation of cells constantly by inactivating the retinoblastoma tumor suppressor protein, which prevents excessive cell growth by inhibiting cell cycle progression until a cell is ready to divide.[30] Taken together, these data show that the aberrant cell proliferation increased energy requirements in keratinocytes leading to metabolic stress in the cell. Therefore, HPV16 E7 expression causes the increasing energy requirements and it might activate autophagy in keratinocytes.[28] Nicola et al.[31] evaluated the role of autophagy in cellular and host defense against the fungal pathogens Cryptococcus neoformans and Candida albicans. They observed that the autophagosome marker LC3 was present in most macrophage vacuoles containing C. albicans and reported that the clearance of C. albicans by macrophages involves the activation of the autophagy pathway.[31] Consistent with these reports, in our study, the autophagy positivity and staining intensity of MAP1LC3A protein were observed at a higher level in infected cells than in normal cervicovaginal cells. As shown in Table 2, a significant correlation was also found between the NILM-INF and NILM groups in view of the staining intensity (P = 0.015). The cause of the higher autophagy positivity and staining intensity of MAP1LC3A protein in infected cells might be depend on nutrient starvation or energy requirements in the infected cells by different microbial pathogens through diverse mechanisms. Additionally, the autophagy positivity in infected cells might also be through one of the selective types of autophagy called xenophagy, which specifically recognizes and degrades the intracellular bacteria, viruses, and even the ones that escaped from the phagosome in the cell.[32] The surface of these pathogen microorganisms was firstly coated with ubiquitin proteins and these ubiquitinated microorganisms were sequestered into autophagosomes by the interaction between adaptor proteins and LC3 protein, which is in the autophagosome membrane. Therefore, these microorganisms were eliminated rapidly by xenophagy activation. In the literature, although there are some reports about the elimination of intracellular bacteria including Salmonella, Listeria, Shigella Legionella, and Mycobacteria and viruses such as Epstein-Barr virus (EBV) by xenophagy,[33-35] there are no studies on the degradation of some gynecological infectious agents such as Candida spp. and Trichomonas vaginalis. According to these data, it is plausible to suggest that xenophagy might be activated to eliminate the infectious agents in the infected-cervicovaginal cells in the present study, and therefore, MAP1LC3 protein expression was found higher in NILM-INF smears.

Another interesting result in our study was the high intensity of MAP1LC3A protein and autophagy positivity in clue cells (Cc). These cells were the main criteria for the clinical diagnosis of an infection that is called Bacterial vaginosis, which is the most common vaginal infection in women [Figure 5].[36] This result could be derived from the induction of autophagy by anaerobic bacteria, which causes BV or by the xenophagy activation in these cells.

Figure 5.

A diffuse and strong (+++) staining intensity of MAP1LC3A protein is seen in the clue cell (Cc) (immunocytochemistry, ×1000)

Although we found low levels of autophagy positivity and low staining intensity of MAP1LC3A protein in the malignant cells, both of them were observed higher in the atypical cells, such as in ASC-US. Consistent with the other studies, it was postulated that the transformation from an atypical cell into a malignant cell could be prevented by the autophagy process to protect the cells from DNA mutation and carcinogenesis. Because autophagy eliminates damaged molecules and organelles in the cells and this way protects these cells against DNA mutations. However, the accumulation of these mutations could lead to malignant transformation.[37,38] Thus, it might be the cause of observing the high levels of autophagy protein MAP1LC3A in atypical cervicovaginal cells in our study.

In conclusion, we detected the low staining intensity of the MAP1LC3A protein and autophagy positivity in malignant cervicovaginal smears; however, they were higher in the NILM-INF and atypical cells. Our findings indicate that the autophagy process is essential in infectious cells as well as in the transformation of atypical cells into malignant cells in carcinogenesis. This study will be the basis for future studies in this field and further analysis using a larger cohort should help clarify the role of autophagy in malignant, infectious, or atypical cervicovaginal cells.

Financial support and sponsorship

This study was supported by Teaching Staff Training Program (OYP) of Hacettepe University.

Conflicts of interest

There are no conflicts of interest.