Oxiditive Stress biomarkers levels in blood sample of Iraqi Breast cancer patients

 

Hadeel Saeed Hadi1*, Shaymaa Abdulzahra Abbas2

1Apharmacist, Wasit Health Department, Ministry of Health and Environment, Wasit, Iraq.

2Department of Clinical Laboratory Sciences, Collage of Pharmacy, University of Baghdad, Baghdad, Iraq.

*Corresponding Author E-mail: hadeel.Sadi1200m@copharm.uobaghdad.edu.iq

 

ABSTRACT:

Background: Breast cancer is a malignant tumor arising from epithelial cells of glandular lacteferons ducts or terminal ductilobular unit (TDLM) of the breast which is affected by the oxidative stress and the defensive mechanisms against it. Objective: to reveal the correlation between the levels of superoxide dismutase (CuZn SOD), glutathione peroxidase (GSH-Px), glutathione reductase (GR), reduced glutathione (GSH) and sulfhydryl groups (SH) with breast cancer with/without metast. Methods: A case control study was performed on 30 female patients with metastasized breast cancer, 30 female patients with non- metastasized breast cancer and 30 female patients with benign breast tumor. All patients were recruited from Al-Karama teaching hospital at Wasit city in Iraq, for the period from October 2021 to February 2022. Results: The levels of GSH-PX, GR, GSH, and sulfhydral groups were significantly reduced in breast cancer patients in comparison with healthy controls whereas SOD levels were upregulated in breast cancer patients in comparison with healthy controls subjected to the current study. Conclusions: It is concluded that  the activities of superoxide dismutase (CuZn SOD), glutathione peroxidase (GSH-Px), glutathione reductase (GR), as well as concentrations of reduced glutathione (GSH) and sulfhydryl groups (SH) have a significant impact on the pathogenisis and progression of breast cancer.

 

KEYWORDS: Breast Tumor, Superoxide Dismutase (CuZn SOD), Glutathione Peroxidase (GSH-Px), Glutathione Reductase (GR), Reduced Glutathione (GSH), Sulfhydryl Groups (SH).

 

 


INTRODUCTION: 

Breast cancer is a malignant tumor arising from epithelial cells of glandular lacteferons ducts or terminal ductilobular unit (TDLM) of the breast. Breast carcinoma is classified as either non-invasive (carcinoma insitu) or invasive, depending on whether or not the tumor has started to grow outside the basement membrane1,2. Invasive carcinomas are cancers in which the cancer cells diffuse to surrounding connective tissues and metastasize to distant organs of the body3,4. Around two-thirds of breast carcinomas arise from epithelial cells of the ducts, called ductal carcinoma, and around one third from lobules, called lobular carcinoma5.

 

Other less common histological groups are identified as inflammatory, medullary, apocrine, mucinous and tubular carcinomas6-8.

 

It is considered as one of the most common cancers in womenglobally, accounting for about 570,000 deaths in 20159. More than 1,500,000 women (25% of all women with cancer) are diagnosed with breast cancer annually throughout the world10,11. Breast cancer is a metastatic cancer and can commonly transfer to distant organs such as the bone, liver, lung and brain, which mainly accounts for its incurability12. Early diagnosis of the disease can lead to a good prognosis and a high survival rate13,14.

 

Redox and ROS are also of particular importance in tumor cell signaling pathways associated with cancer progression through their roles in tumorigenesis, tumor cell migration, and tumor cell survival. Compared to healthy non-malignant cells, tumor cells have aberrant ROS homeostatic characteristics. Most cancerous cells have a higher ROS set point than associated non-cancerous cells. In many types of tumor cells these higher levels support their growth, proliferation, metastasis and survival in various microenvironments or conditions. Low to medium levels of ROS can lead to genetic instability and tumor suppression15.

 

To protect themselves, body maintains complex systems of multiple types of antioxidants, such as glutathione, vitamin C and vitamin E as well as enzymes such as catalase(CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione- S-transferase (GST). These components or enzymes are involved in multiple biochemical reactions to prevent the harmful oxidative damage. Certainly, the genetic polymorphisms of these enzymes and their different expression levels are correlated to the individual’s susceptibility to DNA damage and cancer risk16.

 

The aim of this study was to reveal the relationship between some oxidative stress biomarkers in the blood of breast cancer patients with/without metastasis. These include the activities of superoxide dismutase (CuZn SOD), glutathione peroxidase (GSH-Px), glutathione reductase (GR), as well as concentrations of reduced glutathione (GSH) and sulfhydryl groups (SH).

 

MATERIAL AND METHODS:

A case control study was done on 30 female patients with metastasized breast cancer, 30 female patients with non- metastasized breast cancer and 30 female patients with benign breast tumor. All patients and controls were recruited from Al-karama teaching hospital at Wasit city in Iraq, for the period from October 2021 to February 2022. Ages of all studied groups were ranged from 18-60 years old. The control group comprised of 30 age and BMI apparently healthy females.

 

The practical part of the study was conducted at the department of laboratory sciences, College of Pharmacy, University of Baghdad, Baghdad, Iraq.

 

In the current research women were eligible for this study if they had a suspicious breast lesion (newly diagnosed) which was recorded by clinical breast examination and/or imaging technology. Patients were subjected to physical breast examination, mammography and approved by histopathologist.

 

This work was performed according to the Helsinki 11 declaration confirmed by The Ethics Committee of the College of Pharmacy in University of Baghdad. All participants were informed about the purpose and the expected benefits of this study before agreement of participation was documented.

 

Inclusion Criteria:

The patients included in the current study if they meet the following criteria:

·       Adult female (18-60) years old.

·       Patients with histopathological diagnosis of breast cancer.

·       Willing to participate.

 

Exclusion Criteria:

·       Male patients were excluded.

·       History of chronic renal failure.

·       History of hepatic diseases .

·       History of autoimmune diseases.

·       History or newly diagnosed with diabetes mellitus or hypertention

·       History of myocardial infraction

 

Specimens Collection and Preparation:

A fasting five milliliters of venous blood sample was withdrawn from each patient and healthy adult (controls). The sample was transferred into a gel containing tube, left at room temperature for at least 30 minutes for clotting, then centrifuged at 1000rpm for 10 minutes to obtain serum (which kept frozen until analysis), The  separated serum was used for measuring the following variables: Human Reduced Glutathione (GSH), Human Superoxide Dismutase (Cu-Zn) (SOD), Human Glutathiome Reductase (GR), Human Glutathione Peroxidase (GSH-PX) and Sulfhydryl group according to the manufacturer instructions.

 

Statistical analysis:

The results obtained in this study were expressed as median±SD, and statistical comparisons were conducted with the use of Kruskal-Wallis test for a comparison among more than 2 non-normally distributed groups considered p<0.05 as statistically significant. Correlations among all studied parameters were tested by the Pearson correlation test, and all statistical analyses used in this study were carried out by using the IBM SPSS Statistics for Windows, Version 20.0 (Armonk, NY: IBM Corp). The normality of distribution was checked using Shapiro- Wilk and Kolmogorov- Smirnov tests.

 

RESULTS:

Somedemographic characteristics of the studied groups were summarized in table 1. Table 1 showed non-significant differences in age and body mass index (BMI) among all studied groups.


 

Table (1): Demographic characteristics of the patients with benign, metastasized and non-metastasized malignant tumor in comparison with controls.

 

Control

Benign breast tumor

Metastasized Malignant breast tumor

Non-Metastasized Malignant breast tumor

n

30

30

30

30

Age (year)

40.32±4.57

40.73±5.02

41.83±4.17

40.83±3.97

P-value with control

 

0.12

0.54

0.47

BMI (Kg/cm3)

27.43±4.84

27.73±5.02

27.93±5.14

27.85±4.64

P-value with control

 

0.61

0.47

0.54

 


Results illustrated in table 2 revealed that the levels of all antioxidant parameters subjected to the current study were significantly varied from each other in that the difference in the level of GSH in 4 groups has highly significant statistical difference (p-value is <0.001) with a highest median was reported in control group and lowest median was reported inmetastasized breast cancer group (figure 1). Moreover, the difference in the level of SOD in the studied groups has highly significant statistical difference (p-value <0.001) with highest median was reported in breast cancer free form metastasis group and lowest median was reported in benign group (figure 2). Additionally, highly significant differences in the levels of GR were obtained among all studied groups (p-value <0.001) in which the benign group showed the highest median while the lowest levels were reported in breast cancer free from metastasis group (figure 3). Furthermore, GSH-PX showed highly significant levels among the studied groups (p-value <0.001) in that controls demonstrated the highest level and the metastasized breast cancer group showed the lowest levels (figure 4). Finally, among the 4 studied group, Sulfhydryl group showed highly significant differences (p-value <0.001) with highest median was reported in control group and lowest median was reported in breast cancer free from   metastasis   group (figure 5).

 

Table (2) Descriptive Data the oxidative stress biomarkers in the studied 4 groups subjects.

 

Control

Benign

Met.B.C

B.C without Met

p-value

GSH

Median

14.597

4.477

2.999

3.526

<0.001

SD

4.535

1.090

1.008

0.793

SOD

Median

189.000

150.450

549.896

596.250

<0.001

SD

17.042

2.696

23.14

30.491

GR

Median

119.076

128.182

100.277

50.007

<0.001

SD

43.728

31.728

21.774

17.192

GSH-PX

Median

93.107

37.435

34.253

39.181

<0.001

SD

32.199

26.449

7.485

8.216

SH group

Median

7.500

4.980

3.440

3.025

<0.001

SD

1.229

0.842

0.732

1.093

 

Figure (1) Assessment of GSH in studied groups

 

 

Figure (2) Assessment of SOD in studied groups

 

 

Figure (3) Assessment of GR in studied groups

 

Figure (4) Assessment of GSH-PX in studied groups

 

Figure (5) Assessment of Sulfhydryl group in studied groups

 

Results demonstrated in table 3 revealed that there were highly significant positive correlations between the levels of GSH and the levels of GR, GSH-PX and sulfhydryl group and also positive significant correlation between GR levels and the levels of GSH-PX and sulfhydryl group. On the other hand, significant negative correlations were observed between the levels of SOD and the levels of GSH, GSH-PX and sulfhydryl group

 

Table (3): Correlation between oxidative stress biomarkers

Variables

SOD

GR

GSH-PX

Sulfhydryl group

GSH

r

-0.377**

0.404**

0.627**

0.703**

p

0.000

0.000

0.000

0.000

SOD

r

 

-0.514**

-0.169

-0.525**

p

 

0.000

0.065

0.000

GR

r

 

 

0.263**

0.484*

p

 

 

0.004

0.000

GSH-PX

r

 

 

 

0.543**

p

 

 

 

0.000

 

DISCUSSION:

Breast cancer has become the most common cancer and the leading cause of cancer-related deaths in females worldwide, according to a status report on the global cancer burden provided by Globocan 2018. Recent studies have shown that reactive oxygen species (ROS) are involved in the molecular mechanisms of breast cancer occurrence and development17.

 

Current work aimed to assess the levels of several oxidative stress markers to elucidate their possible role in the cancer development. The markers subjected to the present research include the activities of superoxide dismutase (CuZn SOD), glutathione peroxidase (GSH-Px), glutathione reductase (GR), as well as concentrations of reduced glutathione (GSH) and sulfhydryl groups (SH).

 

Results obtained in the current study revealed that GSH levels were significantly reduced in breast tumor patients to reach the lowest levels in metastasized breast cancer patients in a comparison with controls that showed the highest GSH levels among all the studied groups. Benign breast cancer patients showed a higher GSH levels than that of non-metastasized BC which in turn showed a higher GSH levels than metastasized patients as demonstrated in table (1). Previous studies demonstrated thatmolecular changes in the GSH antioxidant system and disturbances in GSH homeostasis have been implicated in tumor initiation, progression, and treatment response14 which is consistent with the current work which indicate that breast cancer severity accompaied with a disturbance and a significant reduction in GSH levels that indicate a higher oxidative stress status.

 

In parallel with the significant reduction in the levels of GSH, GSH-PX and sulfhydral groups showed a nearly similar pattern of reduction that reached to the lowest GSH-PX activities and a lowest sulfhydral groups level in metastasized breast cancer patients in a comparison with the highest activities and levels in the healthy subjects. Several studies implicated the association of dysfunctional GSH-PX and GR and cancer risk16 which is in consistent with the results postulated in the current work in which the levels of GSH-PX and GR were significantly reduced in breast cancer in comparison with either healthy subjects or benign tumer patients. Functional sulfhydryl groups (‑SH) in thiols act as substrates for antioxidant enzymes and as a free radical scavenger. As a result of the elevation in the ROS levels as a consequence of the disturbance in the defensive mechanism which is represented by the antioxidant enzymes activities reduction, sulfhydral group also showed a significant reduction in breast tumor patients in comparison with controlswhich is compatible with the previous study which demonstrated that the thiol levels in platelets of breast cancer patients, free was significantly lower in both benign and malign groups than in control group18.

The above discussed results were confirmed by the highly significant positive correlation among GSH, GSH-PX, GR and sulfhydral groups which indicate that all these parameters were related directly and strongly in al subjected indivduals as tabulated in table (3).

 

In contrary to GSH and related oxiditive stress parameters that discused above, SOD activities showed a different pattern which obviated in the significant elevation in cancer patient in comparison with controls and also benign breast tumor patients which is disageed with privious study which demonstrated that the activities of SOD were significantly reduced in cancer patient as a result of the increase in ROS activities19 whereas a more recent study demonstrated that the expression of SOD enzyme upregulated leading to a significant increase in the activities of SOD in a response to an increase in the oxidative stress status20. These findings also accompaied by the negative significant correlation between SOD and GSH, GR and sulfhydral groups that means an inversily relationship were obtain between SOD and these oxidative stress markes.

 

It was concluded that the acitivities of GSH-PX and GR and the levels of GSH, and sulfhydral groups were significantly reduced in cancer patients in comparison with controls whereas SOD activities upregulated in cancer patients in comparison with healthy controls subjected to the current study that indicate the impact of these markeres on the pathogenisis and progression of breast cancer.

 

REFERENCES:

1.      Arjun Patidar, S.C.Shivhare, Umesh Ateneriya, Sonu Choudhary. A Comprehensive Review on Breast Cancer. Asian J. Nur. Edu. & Research. 2012; 2(1):28-32.

2.      Saad AH,Rabeea IS, Salih HN. Adherence and Beliefs to Adjuvant Hormonal Therapy in Patients with Breast Cancer: A Cross-Sectional Study(Conference Paper). Iraqi J Pharm Sci. 2021;30(Suppl.):31-39.doi: 10.31351/vol30issSuppl.pp31-39

3.      Dange VN, Shid SJ, Magdum CS, Mohite SK. A Review on Breast cancer: An Overview. Asian J. Pharm. Res. 2017; 7(1): 49-51. doi: 10.5958/2231-5691.2017.00008.9

4.      Yadav AR, Mohite SK. Cancer- A Silent Killer: An Overview. Asian J. Pharm. Res. 2020; 10(3):213-216. doi: 10.5958/2231-5691.2020.00036.2

5.      Jayashree V, Velraj M. Breast Cancer and various Prognostic Biomarkers for the diagnosis of the disease: A Review. Research J. Pharm. and Tech. 2017; 10(9): 3211-3216. doi: 10.5958/0974-360X.2017.00570.4

6.      Sudhakar GK, Pai V, Pai A. An overview on current Strategies in Breast Cancer Therapy. Research J. Pharmacology and Pharmacodynamics. 2013; 5(6): 353-355.

7.      Tiwari A, Singh M, Sahu B. Risk Factors for Breast Cancer. International Journal of Nursing Education and Research. 2022; 10(3):276-2. doi: 10.52711/2454-2660.2022.00065.

8.      Sinn HP, Kreipe H. A Brief Overview of the WHO Classification of Breast Tumors, 4th Edition, Focusing on Issues and Updates from the 3rd Edition. Breast Care. 2013;8:149–154.

9.      Alrubaye YSJ, Mohammed MB, Abdulamir HA. Exosome and Breast Cancer. Research Journal of Pharmacy and Technology. 2022; 15(3):1393-7. doi: 10.52711/0974-360X.2022.00232.

10.   Hariramani N, Jayanthi S. A Systematic Review of Intrinsic Factors and its Influence in Breast Cancer. Research J. Pharm. and Tech. 2018; 11(8): 3543-3546. doi: 10.5958/0974-360X.2018.00654.6.

11.   Stewart BW, and Wild CP. World Cancer Report 2014. Geneva, Switzerland: WHO Press; 2014.

12.   Assaf A, Al-Saleh J. Evaluation the Serum vascular endothelial growth factor C (VEGF-C) value in breast cancer patients. Research J. Pharm. and Tech. 2020; 13(9):4277-4279. doi: 10.5958/0974-360X.2020.00755.6

13.   Karale PA, Karale MA, Utikar MC. Advanced Molecular Targeted Therapy in Breast Cancer. Res. J. Pharmacology and Pharmacodynamics. 2018; 10(1): 29-37. doi: 10.5958/2321-5836.2018.00006.X.

14.   DeSantis CE, Fedewa SA, Goding Sauer A, Kramer JL, Smith RA, Jemal A. Breast cancer statistics, 2015: Convergence of incidence rates between black and white women. CA Cancer J Clin. 2016;66(1):31-42. doi: 10.3322/caac.21320.

15.   Kennedy L, Sandhu JK, Harper ME, Cuperlovic-Culf M. Role of Glutathione in Cancer: From Mechanisms to Therapies. Biomolecules. 2020;10(10):1429. doi: 10.3390/biom10101429.

16.   Khan MA, Tania M, Zhang D, Chen, H.  Antioxidant enzymes and cancer. Chinese Journal of Cancer Research. 2010;22(2): 87–92. doi:10.1007/s11670-010-0087-7.

17.   Zhang ML, Wu HT, Chen WJ, Xu Y, Ye QQ, Shen JX, Liu J. Involvement of glutathione peroxidases in the occurrence and development of breast cancers. J Transl Med. 2020;18(1):247. doi: 10.1186/s12967-020-02420-x..

18.   Eryilmaz MA, Kozanhan B, Solak I, Çetinkaya and D, Neselioglu S, Erel &. Thiol-disulfide homeostasis in breast cancer patients. J Can Res Ther. 2019;15:1062-6.

19.   Sahu A, Varma M, Kachhawa K. A Prognostic Study of MDA, SOD and Catalase in Breast Cancer Patients. International J. Sci. Res. 2015;4 (5):157-159.

20.   Lunetti P, Di Giacomo M, Vergara D, De Domenico S, Maffia M, Zara V, Capobianco L, Ferramosca A. Metabolic reprogramming in breast cancer results in distinct mitochondrial bioenergetics between luminal and basal subtypes. FEBS J. 2019;286(4):688-709. doi: 10.1111/febs.14756.

 

 

 

 

 

Received on 12.08.2022            Modified on 07.09.2022

Accepted on 01.10.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(5):2364-2368.

DOI: 10.52711/0974-360X.2023.00389