Serum levels of prostaglandin E2 (PGE2) and interleukin 17 (IL-17) are associated with Angiogenesis and Metastasis in breast cancer patients

 

Maram Bilal^1*, Jumana Al-Saleh², Faihaa Abo Fakher³

¹Department of Biochemistry and Microbiology, Faculty of Pharmacy, Damascus University, Damascus, Syria.

²Assisstant Professor, Department of Biochemistry and Microbiology, Faculty of Pharmacy,

Damascus University, Damascus, Syria.

³Associate Professor, Department of Laboratory Medicine, Faculty of Medicine,

Damascus University, Damascus, Syria.

 

ABSTRACT:

Background: The link between chronic inflammation and tumorogenesis has been noted for a long time. Breast tumor cell-derived prostaglandin E2 (PGE2) induces the production of interleukin 23 (IL-23) that enhances IL-17 secretion. Permanent release of these molecules leads to augmentation of their serum concentrations that may be used as novel clinical markers for diagnosis and prognosis. The aim of this research was to evaluate levels of IL-17 and PGE2 in blood serum of breast cancer patients, and to determine whether these levels correlate with the disease progression Material and Methods: Pre-operative serum samples were collected from 41 patients with breast cancer and 28 patients with benign breast tumors. IL-17 and PGE2 levels were measured using ELISA. Furthermore, correlations between levels of these molecules and clinicopathological parameters were determined. Results: We demonstrated that serum levels of PGE2, but not IL-17, were significantly increased in breast cancer patients as compared with benign tumor patients. Moreover, both IL-17 and PGE2 levels were increased in cancer patients with angiogenesis and lymph node metastasis. A positive correlation was also identified between IL-17 and PGE2 levels. Conclusion: The present study indicates that elevated IL-17 and PGE2 serum concentrations are associated with angiogenesis and metastasis of breast cancer. So, our data suggest that IL-17 and PGE2 could be developed as potential biomarkers of breast cancer diagnosis and prognosis, however more comprehensive results are needed.

 

 

 

INTRODUCTION:

Breast cancer creates a significant public health problem worldwide, and metastasis is considered to be the cause of most deaths in patients¹.

 

Inflammation appears to be a necessity for both metastasis and elimination of tumor cells. Chronic inflammation has been associated with increased tumor incidence and linked to tumorogenesis².

 

Many human cancers exhibit elevated prostaglandin (PG) levels due to upregulation of cyclooxygenase-2 (COX-2), a key enzyme in eicosanoid biosynthesis³. Breast cancer cells secrete PGE2 that plays a role in tumor development and progression, probably through recruitment of leukocytes secreting a variety of cytokines, chemokines and angiogenic factors that promote tumor growth and metastasis⁴.

 

Tumor secreted PGE2 induces IL-23 production in the tumor microenvironment leading to expansion of the newly-defined T helper 17 (Th17) cell subset⁴. IL-17, the hallmark cytokine of Th17, has important roles in protecting the host against extracellular pathogens, but conversely promotes inflammatory pathology in autoimmune disease⁵. In malignancy, IL-17 has a complex role by playing a dual role in the antitumor immunity. On one hand, IL-17 promotes an antitumor cytotoxic T cell response leading to tumor regression. On the other hand, studies have demonstrated that IL-17 plays an intricate role in tumorigenesis, proliferation, metastasis, and angiogenesis⁶. IL-17 has been shown to stimulate vascular endothelial cell migration and regulate production of a variety of proangiogenic factors, such as vascular endothelial growth factor (VEGF) and PGE2 (7). Moreover, evidence is now growing which supports the ability of IL-17 to act as a key diagnostic marker, differentiating between benign and malignant pathology. It could potentially be a significant tool in allowing early detection⁸.

 

Therefore, in the present study we assessed the concentrations of IL-17 and PGE2 in blood serum of breast cancer patients and women with benign breast tumors, and their correlation with clinicopathological parameters of breast cancer.

 

MATERIAL AND METHODS:

The patients were admitted for surgery at Al-Mwasah Teaching Hospital (Damascus), during the period December 2018 – June 2019. Informed written consent was obtained from each patient prior to the specimen collection. 3ml venous blood samples were collected from patients according to the internal review and the Ethics Boards of the Syrian hospitals. Sampling was carried out before any clinical intervention such as chemotherapy, radiotherapy and surgery. Sixty nine female patients recently diagnosed with breast tumors were enrolled in the present study. The age range and the mean age of patients were 22-79 and 45.45±13.01 years respectively. Based on pathologic diagnostic assessment, patients were divided into 2 groups: patients with benign breast tumor (n=28) and patients with breast cancer (n=41). Pathological data [including: tumor grade, lymph node status, angiogenesis] were obtained from medical records of patients, validated by an experienced histopathologist, and summarized in Table 1.

 

Table 1. Histopathologic Information in Breast Cancer Patients

 

Blood specimens were collected in sterile tubes for serum isolation. serum samples were separated from venous blood at room temperature and stored at -30°C until use. Later, serum was assayed by enzyme-linked immunosorbent assay (ELISA) for the presence of IL-17 and PGE2. Concentrations were measured using commercially available ELISA kits (IL-17 and PGE2 ELISA kit, Bender Med Systems, Austria). The procedure was done according to the manufacturers instruction as supplied with kits. The absorbance was read on an ELISA plate reader (Bio Tek, USA) at a wavelength of 450 nm and 405 nm for IL-17 and PGE2, respectively.

 

Statistical analysis:

All statistical analyses were carried out by SPSS software, version 23.0. Student t test was used to assess the statistical significance of difference in median between the 2 study groups. ANOVA test was used for multiple median comparison. For the analysis of correlation, person test was utilized. A P-value of <0.05 was considered to indicate a statistically significant difference.

 

RESULTS:

The concentration of IL-17 in serum samples of patients with breast cancer and patients with benign tumors was determined. Although comparison of 2 given groups indicated an increase of IL-17 in cancer patients, the increase was of no statistical significance (P >0.05; Table 2). Then, the concentration of PGE2 in serum specimens was measured. Comparison of 2 given groups revealed a significant increase of PGE2 in the serum samples of cancer patients (P = 0.001; Table 2). The serum samples from cancer patients revealed a positive statistical correlation between IL-17 and PGE2 levels (P = 0.011, r = 0.392; Figure 1). However, There was no statistical correlation between IL-17 and PGE2 levels in the serum of patients with benign tumors (P >0.05, r = 0.091; Figure 1).

 

Table 2. Comparison of IL-17 and PGE2 Concentration in Serum Samples of patients

 

There was significantly higher concentration of IL-17 in serum of cancer patients with angiogenesis and those with lymph node metastatis (P<0.05, Table 3). There were similar findings regarding PGE2 Levels. We observed significantly higher serum concentration of PGE2 in patients with angiogenic cancer and those with metastatis to lymph nodes (P<0.05, Table 4). However, neither IL-17 nor PGE2 had statistically significant correlation with the tumor grade of cancer patients (Table 3 and 4).

 

Figure 1. Correlation between serum levels of IL-17 and PGE2 in patients with breast cancer and patients with benign breast tumors.

*There was a positive association between the levels of IL-17 and PGE2 in cancer patients. Levels were detected using ELISA and evaluated as pg/ml. IL: Interleukin, PGE2: Prostaglandin E2.

 

Table 3. The difference in median levels of serum IL-17 (pg/ml) in breast cancer patients according to clinicopathological parameters.

^aP-value of student t test as appropriate. ^bP-value of ANOVA test as appropriate.

 

Table 4. The difference in median levels of serum PGE2 (pg/ml) in breast cancer patients according to clinicopathological parameters.

^aP-value of student t test as appropriate. ^bP-value of ANOVA test as appropriate.

 

DISCUSSION:

In the present study, we assessed the abundance of two major inflammatory factors in serum samples from breast cancer patients and patients with benign breast tumors. We used ELISA and measured the levels of IL-17 and PGE2. We also used data from the patients’ records to correlate our findings with clinicopathological results.

 

In the current study, the serum levels of Th17 cells- related cytokine (IL-17) in patients with breast cancer were higher than those in patients with benign tumors, but there was no significant difference between them. In contrast to these results, Rohani Borj et al. demonstrated significant differences in levels of serum IL-17 in breast cancer patients (n=56) as compared with benign tumor patients (n=60)⁹. Lyon et al. also revealed a significant increase in IL-17 serum concentration in breast cancer using Multiplex bead array assay¹⁰. However, most cancer patients in the study of Lyon and associates were at least in their 4th week postsurgery, while Mettler et al. reported that the time since surgery could have affected cytokine values in cancer patients¹¹.

 

In addition to IL-17, We identified significantly elevated concentration of PGE2 in serum of cancer patients compared with benign tumor patients. Our findings support the demonstrated role of COX/PG signaling in malignancy. Elevated COX-2 protein levels have been detected in approximately 40% of invasive breast carcinomas, with individual studies reporting frequencies ranging from 17% to 84%³. Moreover, a work of Mitsuhashi et al. indicated that mammary tumor cells (4T1) secrete many cytokines and lipid molecules, including PGE2¹².

 

Similarly to our results, an increased plasma levels of PGE2 has also been described in colorectal cancer group compared with benign group¹³.

 

Differentiation of Th17 cells from naïve T cells is dependent on signals from TGF-β, IL-6, IL-21 and IL-23 in mice¹⁴. Importantly, IL-23 does not act on naïve T cells, but instead acts on T cells that are already committed to the Th17 lineage. IL-23 enhances the production of IL-17 and stabilizes the Th17 phenotype¹⁵. In humans, it seems that IL-23 enhances IL-17 secretion from Th17 cells⁶. Qian and coworkers demonstrated that breast cancer cell-secreted PGE2 enhanced IL-23 production and tumor-associated Th17 cell development⁴. Importantly, According to our study, the serum levels of PGE2 showed a moderate positive correlation with IL-17 levels in serum of breast cancer patients. These results suggest that PGE2 may play a pivotal role in the regulation of IL-17 secretion in breast cancer. However, we did not find any significant correlation when we examined these two factors in serum of patients with benign tumors.

 

Angiogenesis has received a lot of attention due to its influence on the tumor grade, metastasis, and therefore patients’ prognosis⁸. Numasaki et al. found that Il-17 up-regulates production of a variety of proangiogenic factors, such as vascular endothelial growth factor (VEGF), prostaglandin E1 (PGE1) and PGE2, by fibroblasts as well as tumor cells. IL-17 also promotes angiogenesis through stimulation of vascular endothelial cell migration and cord formation, resulting in tumor progression⁷. A study of Benevides et al. in 2013 suggested that IL-17 may shift the balance between angiogenic and angiostatic factors toward conditions that promote angiogenesis and tumor development of invasive ductal carcinoma (IDC)¹⁶. In agreement with these findings, our results revealed that serum levels of IL-17 in breast cancer patients with angiogenesis had been significantly higher than levels in cancer without angiogenesis.

 

Benevides et al. in 2015 uncovered the mechanism by which the proinflammatory cytokine IL-17 promotes metastatic mammary primary tumor progression; this mechanism is neutrophil dependent. These results support the idea that IL-17 could serve as a biomarker for the prognosis of aggressive breast cancer¹⁷. According to the present data, our results revealed that serum levels of IL-17 in breast cancer patients with metastasis to lymph nodes had been significantly higher than levels in cancer without metastasis.

 

Regarding PGE2, we observed significantly higher serum concentration of PGE2 in patients with metastatis to lymph nodes. Our results were in agreement with evidence from studies in vitro that E-series of prostaglandin (EP) receptors might contribute to metastatic behavior of human cancer cells and murine models of cancer^18-20. An EP receptor expression study by Chang et al. in 2004 indicated that all four subtypes (EP1-4) are expressed in different stages of mammary gland development²¹. Of particular note, It has been shown that breast cancer cells migrate in response to PGE2, and EP4 function was particularly important to migration¹⁸.

 

In addition, we found a significant increase in serum levels of PGE2 in cancer patients with angiogenesis as compared with those without angiogenesis. A previous data strongly suggested that PGE2 secreted by COX2-expressing mammary tumor cells may promot tumor angiogenesis by inducing many proangiogenic factors²¹.

 

Amano and associates indicated that host stromal PGE2-EP3 signaling appears critical for tumor-associated angiogenesis and tumor growth in a mouse tumor implantation model. They also observed that EP3 signaling pathway is relevant to the induction of a potent proangiogenic growth factor, VEGF²².

 

In summary, the present study indicates that preoperative serum PGE2 levels were higher in breast cancer patients as compared to those in benign breast tumor patients. These higher levels could be attributed to tumor-related inflammation and progression. Moreover, PGE2 and IL-17 serum concentrations were associated with breast cancer metastasis and angiogenesis. Thus, these two inflammatory factors may be developed as potential diagnostic and prognostic cancer biomarkers.

 

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Received on 15.12.2019           Modified on 16.02.2020

Accepted on 01.04.2020         © RJPT All right reserved