INTRODUCTION:

Cervical cancer is one of the main causes of death for middle-aged women in the developing world, till now it is completely preventable if precancerous lesions are diagnosed and treated in a timely manner. Different methods are found to control and prevent cervical cancer which involves conventional cytology (Pap smear), liquid-based cytology, human papillomavirus (HPV) screening, and vaccination against HPV (1,2).

Because of the comparatively low incidence of cervical cancer among women worldwide and high prevalence of HPV infection, certain cofactors have been included in the development of cervical cancer (3). These cofactors comprise, but are not limited to, smoking tobacco, high parity (number of children), long-term hormonal contraceptive use, co-infection with the human immunodeficiency virus (HIV): herpes simplex virus-2 (HSV-2): Chlamydia trachomatis (3-5). Sexual behavioral patterns, such as the sexual intercourse at early age and multiple sexual partners (6).

In 2012, Cervical cancer was considered the fourth most common cancer in women, and the seventh overall, with an estimated 528,000 new cases occurs in the less developed regions, where it accounts for almost 12% of all female cancers (7). Iraq has a population of 10.74 million women ages 15 years and older who are at risk of developing cervical cancer. Current estimates indicate that every year 244 women are diagnosed with cervical cancer and 159 die from the disease. Cervical cancer ranks as the 13th most frequent cancer among women in Iraq and the 10th most frequent cancer among women between 15 and 44 years of age. Distribution and Incidence Rate (Per100, 000Population) is 202 case in 2016 (1.41%) and the Incidence rate is 1.08% (8).

The multifunctional enzyme apurinic/apyrimidinic endonuclease 1/redox enhancing factor-1 (APE1/Ref-1) is a critical protein that acts as important regulator of oxidative deoxyribonucleic acid (DNA) damage repair, lead to the maintenance of genome stability. After it was cloned in 1991 as a DNA repair enzyme and as a redox protein later, APE1 has been described as playing a role in many biological contexts. (9–11). APE1 participate in the base excision repair (BER) pathway, and its activity accounts reach over 95 percent of the all AP endonuclease enzyme action in human cells, generally this make it significant to overall BER pathway ability. It regulates the DNA binding affinity of several important transcription factors, including NF-κB; AP-1 and p53 through redox dependent and -independent mechanisms (12). The human APE1 gene is localized on chromosome 14 q11.2-12 and consists of five exons and four introns (13, 14). It has two domain 1- N-terminal domain is important for the redox activity 2-C-terminal is important for DNA repair activity (15). In addition, APE1 has essential roles in regulating cell like apoptosis (cell death programmed), proliferation and differentiation (16). Recent study noted that any changes in APE1 expression patterns are related with carcinogen susceptibility and cancer progression (17). Also APE1 protein can measure in both tissue and serum. As mention previously, DNA damage repair shows essential roles in maintaining genomic stability but increases the progression in cancer growth (18, 19). One study reported that increased APE1 protein levels in non-small cell lung cancer (NSCLC) tissue are associated with a decreased sensitivity to chemotherapy , give influence of carcinoembryonic antigen (CEA) in diagnosis, in addition to that, after treatment, high APE1 level is related with shorter progression-free survival (PFS) therefore this marker may useful in monitoring therapeutic effectiveness (20).

Overexpression or an atypical subcellular distribution pattern of APE1 has been detected in many types of cancer such as breast cancer, NSCLC, head and neck cancer, osteosarcomas, germ cell tumors and ovarian cancer (21–26). These studies recommended that APE1 could be related with lymph node status, proliferation index, survival outcome and resistance to chemotherapy or radiotherapy (17). In NSCLC, previous study reported that APE1 over expression was correlated with induced epithelial-Mesenchymal Transition (EMT) and poor prognosis in vitro and in vivo study (27). While in recent study found the relationship between higher expression of APE1 and stomach cancer positive lymph nodes metastases group than that of negative lymph node group (χ2=5.13, P=0.044) (19).

There is no study about APE1 marker in cervical cancer in Iraq. In the recent study, The prognostic significance of APE1 in cervical cancer tissue was assessed to investigate the pattern of APE1 protein expression and its association with Clinicopathological criteria specially lymphangiogenesis and cancer progression using Immunohistochemistry Technique.

METHOD:

Patients and tissue samples

In this study, 55samples were obtained from un-selected patients age ranged (35-70) years from Baghdad Medical City/teaching laboratories / histopathology department divided to 35 blocks were cervical cancer and 20 samples had no malignant lesions considered as control. 32 samples were squamous cell carcinoma and 3 blocks were adenocarcinoma. The study was approved by Al-Mustansiriyah University/College of pharmacy.

Deparaffin and Immunohistochemistry staining of APE1:

Formalin-fixed paraffin-embedded sections (4 mm) of 55 tissues were deparaffinized in xylene and rehydrated by absolute ethanol. Slides were placed in a target retrieval solution/Dako (pH 9.0) and boiled for 10 min at 98C in microwave. Staining detection kit /My BioSource /UK was used to detect protein expression of APE1according to the manufacturer's instructions method. Slides were then washed with D.W for 10 min, after that TBST buffer (tris-buffered saline +0.1%Tween 20) for 5 min, repeated twice. After quenching of the endogenous peroxidase-blocking agent for 30 minutes in 0.3% H2O2, slides were washed twice with TBST buffer for 5 min. protein-blocking serum was added for 10 min, all tissue sections were incubated with the primary antibody (1:100 dilution, My Bio Source, USA) in hand made a humidified box at 4C for 6 hours. Then sections were treated with secondary antibody (My BioSource/UK) for 30 min. lastly, all sections were visualized using chromogen DAB working solution followed by counter stained with Meyer’s hematoxylin.

Scoring Analysis:

All staining slides were examined by light microscope under 10X, 40X, 100X. Two independently pathologist scored the number and percentage of positive cells expressed APE1present within tumor-cell nuclei manually in 5 HPFs magnification, x40described by Zhang S. et al (2016). Protein expression of this marker was scored for four categories, score 0 (no expression in cells); score 1+( faint/ barely perceptible partial nuclear expression in < 10%of cells); score 2+ (weak to moderate expression of the entire nucleus in > 10% of cells); and score 3+( strong expression of the entire nucleus in > 10% of cells). Score 2+/3+ was consider as high expression, but score 0/1+ as low expression. In addition cytoplasmic expression in the cells were scored according to the intensity. Intensity was scored negative staining, low, moderate and high or strong staining in relation to deep of brown color of this marker.

Statistics:

Statistical significance was determined with by SAS (2012) program which used to detect the effect of difference factors in study parameters. Chi-square test was used to significant compare between percentages, P-value 0.05 consider significant and 0.01 consider highly significant.

RESULTS:

APE1 expression in cervical cancer and normal cervical tissues:

According to the Tab (1) and Fig (1a, b), nuclear staining was of APE1seen in the all of 35 (100 %) cervical cancer samples. Only 29 out of 35 samples (82.85 %) displayed cytoplasmic and nuclear staining together. In addition out of 20 normal cervical tissues,14 (70 % ) samples presented nuclear staining in the luminal epithelium of cervical tissue while There was 6 (30 % ) cases with cytoplasmic and nuclear together as shown in Fig (2a,b) .There is highly significant difference of the APE1 expression in malignant and control cases(0.00027**).

Table 1: Positive Samples nuclear and cytoplasmic - nuclear APEX1 expression in cercal cancer and normal samples

Cytoplasm and nuclear

NO.%

Nuclear NO.%

Total number

Kinds of samples

29(82.85)

6(30)

35(100)

14 (70)

Malignant

Normal

Chi-Square (χ²)

p-value

9.216 **

0.00027 HS

HS: Highly significant at p < 0.01

Figure 1: A: Section of squamous cell carcinoma stained with anti APE1antibody showing nuclear and cytoplasmic localization of APE1in cancer cells. B: stained with anti APE1antibody showing nuclear localization of APE1in cancer cells by using IHC Technique (40X).

Figure 2: A: section of Normal cells stained with anti APE1antibody showing nuclear and cytoplasmic localization of APE1in normal cells. B: stained with anti APE1antibody showing nuclear localization of APE1in normal cells by using IHC Technique(40X).

Nuclear APEX1 expression in cervical cancer and normal samples according to the score.:

The results show the higher positive percentage of malignant cases in nuclear APEX1 expression in score 2 (22.8%, χ²8.29, P-value 0.0037**) which mean high expression of APEX1 than normal cases (0%). While the results in score 1 and 3 showed no significant differences in malignant and normal cases. In normal samples,6 (30%) cases only among 20 cases showed no expression of nuclear APEX1, as illustrated in Fig (3).

Figure 3: Distribution of malignant and normal samples according to the score (percentage, intensity) of APE-1immunohistochemical expression in nuclear localization both in malignant and normal epithelial cells.

Association between APE1 Expression and Clinicopathologic Parameters of cervical tumor.:

The ratio of positive tumor cells to this marker different among samples, Therefore study of IHC data of APE-1 related with some clinicopathologic parameters were done by scoring the number and percentage of positive tumor cells with nuclear staining and categorized to low and high expression as mention above in methods. The APE1 high-level expression cases was 27 (77.1%) while APE1 low-level expression cases was 8 (22.85%) from the total number 35 of malignant samples.

According to the data summarized in Tab (2), it showed demographic criteria association with APE expression patterns. Age, differentiation and histologic type showed highly significant difference (P= 0.0062, 0.0001 and 0.0001 respectively) between the high and low expression of nuclear APEX1expression.

While lymph vascular invasion (positive vs negative group) showed a significant difference (Chi-Square 4.362, p value= 0.0422*) between the high (16/59.2%) and low (4/50%) nuclear expression of APEX1, while lymph node (positive vs negative group) showed no significant association (P-value=0.1476) between the high and low expression of nuclear APEX1.

Table 2. Clinicopathology parameters of cervical cancer patients and their correlation with positive APE-1 high and low expression.

Clinical parameters

low expression=8

No (%)

High expression=27

No (%)

Chi-Square (χ²)

P-value

Variable

No.

Age (year)

> 50

≤ 50

1(12.5)

7(87.5)

9(33.3)

18(66.6)

8.572 **

0.0062

Differentiation

Well + moderately

poorly

8(100)

23(85.1)

4(14.8)

14.037**

0.0001

Histological type

Squamous carcinoma

Adenocarcinoma

8(100)

24(88.9)

3(11.1)

14.794**

0.0001

Lymph vascular invasion

Yes:

No:

4(50)

16(59.2)

11(40.7)

4.362 *

0.0422

Lymph node metastases

Yes:

No:

2(25)

6(75)

8(29.6)

19(70.4)

1.099 NS

0.1476

DISCUSSION:

The distribution of APE1 in many cell has been cited to be varied and complex. Most studies found that APE1 was localized in the nucleus, while in a number of cell types APE1 localized only in cytoplasm or both nuclear and cytoplasmic localization (28).

In the current study, Tab(1), Fig (1a,b) and Fig (2a,b) showed the dominant pattern of APE1 expression was nuclear rather than cytoplasm in both normal and cervical cancer cases in spite of there was mixed expression and high intensity with highly significant difference between malignant and normal groups (0.00027**) according to the localize of expression. While, the level of nuclear APEX1 expression was different in cervical cancer. Present data go in line with pervious studies about many human cell types have variance APE1 localization. These reports showed that breast cancer, APEX1 expression was predominately localized in the nucleus of cancer cell (17), while in other studies, it reported that nuclear and cytoplasm expression were common in breast cancer (30,31). In lung cancer, IHC method was used to found that APE1 nuclear expression was 73.25% in the cancer tissues and dual expression in both nucleus and cytoplasm was 39.53% (20). However level or localization of APE1 expression in human tumor cells were also different as described, also it was clear that these altered in tumor cells and were different compared with control cells (17).

In the present study, Fig (3) reported the percentage of malignant and normal samples according to the score of positive APE-1immunohistochemical expression in nuclear localization both in malignant and normal epithelial cells. data revealed that score 2 was significantly high in malignant samples compared to normal. As mention above the score 2and3 considered high expression which lead to 27/77.1% tissue samples were high APE1expression in cervical cancer tissue versus 8/35 tissue samples were low APE1expression within normal tissue.

A similar result was noted in breast cancers. Moderate to strong nuclear immun-staining (score 2and3) was showed in the 177 (74.1%), while APE1 low-level expression samples were 62 (25.9%) of breast cancer tissues (17). In addition to high percentage (approximately 73%) of score 2 and 3 in non-small cell lung cancer patients in other studies (20).

Nuclear localization of APE1 is thought to indicate that it functions in DNA repair while in cytoplasmic localization has been related with its function in mitochondrial DNA repair or associated with redox regulation of transcription factors (28). In a status of oxidative stress, like cancer, APE1 expression is induced in response to increased radical oxygen species (ROS), and dysregulation of APE1protien allows cancer cells to survive and continue. Also the increased APE1 activity that go along with tumorgenesis, resistance to cytotoxic agents could be enhanced like platinum compounds, alkylating agents and ionizing radiation which used in the treatment of tumors to stimulate DNA damage in abnormal cells (32).Generally high APE1 levels are related to shorter progression-free survival (PFS) (20), apoptosis, proliferation and differentiation (16) and cancer progression (17). In this study we did not find any study about this marker protein expression in cervical cancer using IHC in Iraqi patients.

Association between nuclear APE1 Expression and Clinicopathologic Parameters of cervical cancer was investigated in the current study. Tab (2) demonstrate that high and low expression significantly correlated with age ≤ 50, well+ moderately differention and Squamous carcinoma type with P value= 0.0062**, 0.0001** and 0.0001** respectively. According to the lymphangiogensis study associated with this marker in cancer, lymph vascular invasion (positive vs negative group) in high expression pattern was significant differ from low expression (P value= 0.0422*) while lymph node metastases (positive vs negative group) showed no significant correlation (P-value= 0.1476) between both groups high and low nuclear expression of APE1.

These results not agreement with Woo j. el al. (2014) who noted that clinicopathologic parameters according to high or low APE1 expression levels including: Age of patients, histologic grade, tumor size, lymph vascular invasion and lymph node metastatic did not correlate with APE1 expression (17).

In non-small cell lung cancer, nuclear staining correlated with a low proliferation index, p53-negative and survival benefit. In other studies about breast cancer, nuclear staining associated with decreased angiogenesis and negative lymph nodes (22, 33).

Other study find that APE1 has high expression in gastric cancer samples with lymph node more than in negative lymph node samples (χ2=5.128, P=0.044), with a significant correlation with results of serum (r=0.262 P=0.035) (19).

The small size of samples lead to obtained low number of positive lymph node tissue samples (10/35) or may the lymph node not exist in tissue blocks may be because it is in distal position leading to not appear associated with APE1 expression. Therefore, there is a strong clues to support the use of APEX1 as a prognostic factor in cervical cancer, and analysis of lymphangiogensis including lymph vascular invasion and lymph node metastasis. This need more studies to improve it.

CONCLUSION:

In conclusion, this study identified positive expression of APE1 being a excellent marker to prediction of metastases or study the lymphangiogensis in Iraqi women with cervical cancer. These findings provide clues that tissue positive APE1 possess a potential as diagnostic marker for Iraqi cervical cancer. Additional work should focus on the prediction value on serum APE1 in prognosis of Iraqi patients, using molecular technique to detect polymorphism with big samples size in cervical cancer samples.

ACKNOWLEDGMENT:

The authors would like to thank Al-Mustansiriyah University (www.uomustansiriyah.edu.iq) Baghdad, Iraq for its support in the present work.

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Received on 12.08.2019 Modified on 19.10.2019

Research J. Pharm. and Tech. 2020; 13(7): 3193-3198.

DOI: 10.5958/0974-360X.2020.00565.X