INTRODUCTION:

Breast cancer is the second most common cancer in females. In the Syrian Arab Republic; breast cancer accounted for 19% of newly diagnosed tumors and 11.7% of overall cancer deaths^[1].

Breast cancer has several types and is characterized by its ability to spread to other sites within the body. The formation of new lymphatic vessels has become an important topic in scientific research on tumor metastases since the discovery of lymphatic vascular growth factors, the most important of which is VEGF-C^[2].

VEGF-C is a member of vascular endothelial growth factors (VEGF) family. VEGFs are glycoproteins, secreted by many cells, and its receptors are located on different tissues.

They have many vital effects, the most important of which are the permeability of capillaries and are therefore called the vascular permeability factor (VPF). They also promote angiogenesis in response to agitating factors such as cytokines and hypoxia by inducing endothelial migration and proliferation^[3].

VEGF-C is an angiogenic factor in many solid cancers, expressed in most tumors, and associated with metastases and poor prognosis. Excessive expression of VEGF-C has been reported in breast cancer patients, and several studies have examined the relationship between VEGF-C overexpression and poor breast cancer prognosis^[4-9]. Zhang and associates (2016) identified a regulatory mechanism to increase VEGF-C expression in breast cancer cells, contributing to tumor proliferation and increased metastasis^[10].

VEGF-C spreads from tumor cells to surrounding tissues and lymph nodes. The excess amount of VEGF-C spreads into circulation, increasing its serum concentration proportionally to its increased amount in tissues. Zajkowska and colleagues (2018) suggested that the serum VEGF-C value opens a new horizon in the field of breast cancer diagnosis^[11].

The inhibition of VEGF expression has been shown to stop pathological angiogenesis in a wide range of diseases including cancers, leading to the clinical development of VEGF inhibitors for renal cell carcinoma, colon, and rectum tumors.

In this study we determined serum level of VEGF-C in breast cancer patients before and after completing treatment, and compared pre treatment levels with levels in healthy women.

METHODS:

The study included 35 women with breast cancer and 35 healthy women as controls. Five women were excluded from the breast cancer group due to non-completion of the study protocol and loss of communication. We obtained an informed consent from each women at the beginning.

Patients were adjusted in terms of inclusion and exclusion criteria as assessed by oncology team and pathologists.

Inclusion criteria:

Case group: histological diagnosis of breast cancer, in one side.

Control group: normal examination of breasts, normal mammography.

Exclusion criteria:

Women with concurrent or previous tumor story, history of chemo- or radio-therapy, known diseases affecting VEGF-C concentrations such as diabetes, heart diseases, benign ovarian pathologies, endometriosis, polycystic ovarian syndrome.

All patients had surgery at Al-Bairouni Hospital- Damascus University- Damascus, and assays were conducted in the laboratories of the Faculty of Pharmacy at Damascus University.

VEGF-C assay:

The assays were done according to the protocol provided by the manufacturer of VEGF-C ELISA kit (Sun Red Biotechnology Company, Shanghai, China).

We collected a blood sample from each woman, then we prepare the specimen: Coagulation at room temperature 10-20 mins, centrifugation 20-min at the speed of 2000-3000rpm, remove supernatant, centrifuge again if precipitation appeared. We kept the specimens at temperature -08 C till the assay's time.

We added the the specimen to monoclonal antibody enzyme well, pre-coated with VEGF-C monoclonal antibody, then, added VEGF-C antibodies labeled with biotin, and combined with Streptavidin-HRP to form immune complex; then we washed again. Later we added Chromogen Solution A, B, gently mixed, incubated for 10 min at 37 C away from light. At the end the reaction was terminated by addition of stop solution.

The optical densit (OD) was measured within 15 min after adding the stop solution, at 450nm wavelength in an Enzyme Reader. A standard curve was constructed using the standards' concentration provided in the kit, and the OD values of the sample applied on it to calculate the corresponding sample's concentration.

Data analysis:

Data were analyzed using SPSS-17 (SPSS, Inc., Chicago, IL). The data were presented as mean±standard deviation, range. The Kolmogorov – Smirnov test showed that the sample has non-normal distribution, and the variable considered statistically significant when the P-value ≤ 0.01.

RESULTS:

The study involved 65 women (30 women with breast cancer, 35 women with normal breasts). The age characteristics shown in Table 1, without significance.

Table (1): age characteristics

	results
Breast patient	48.33±7.84 (range: 33-63 y)
Controls	48.5±8.05 (range: 35-64 y)

The mean VEGF-C values in cancer's patients was 1122.74±853.97 U/L. the differences were statically significant. Table 2 shows the results.

Table (2) VEGF-C values

		VEGF-C values	p-value
Patients	Pre treatment	1122.74±853.97 (range: 100-3066)	0.007 ^&
	Post treatment	580.16±520.46 (range: 25-2270)	0.007 ^&
controls		334.55±522.6 (range: 15-1721)	0.00 ^*

& Wilcoxon test.

*Mann – Whitney test.

DISCUSSION:

The lymphatic vascular is an important route for the spread of breast cancer. The presence of tumor foci in the lymph nodes is the most important prognostic factor. VEGF-C stimulates the formation of new lymphatic vessels within the tumor, and the spread of tumor cells to lymph nodes. High VEGF-C appears to be associated with lymph node metastases in breast cancer^[12.13].

The study found that the mean values of VEGF-C in women with breast cancer were significantly higher than the mean values in healthy women. The mean values in women with breast cancer before treatment were significantly higher (P<0.01) than the mean values after treatment. The above result is consistent with previous publications^{[8.9.11.12.14]}. A new study found that serological concentrations of VEGF-C have been significantly higher in the tumor group compared to the control^[11]. Wang and colleagues observed in their study that VEGF-C works to develop resistance to chemotherapy in breast cancer patients^[15].

This study is consistent with previous studies on the role of VEGF-C in breast cancer, and identifies significant differences in serological level in patients before and after treatment, supporting the researches to be applied in evaluating treatment response, and tumor recurrence.

CONFLICT OF INTEREST:

The author declares no conflict of interest.

ACKNOWLEDGEMENT:

I would like to thank Dr. Gomanah Al-Saleh for her supporting and advices.

REFERENCES:

1. International Agency for Research on Cancer. GLOBOCAN 2012 (IARC) Section of Cancer Surveillance (8/2/2015) Cancer Incidence and Mortality Worldwide Lyon, France.

2. Ran S, Volk L, Hall K, et al. Lymphangiogenesis and lymphatic metastasis in breast cancer. Pathophysiology 2010;17(4): 229-251.

3. Ferrara N. Vascular endothelial growth factor: Basic science and clinical progress. Endocr Rev. 2004;25(4): 581-611.

4. Zhao YC, Ni XJ, Li Y, et al. Peritumoral lymphangiogenesis induced by vascular endothelial growth factor C and D promotes lymph node metastasis in breast cancer patients. World J Surg Oncol 2012; 10:165.

5. Liu XL, Huang Y, Chen P. Expression of hMAN, VEGF-C and VEGFR-3 in breast cancer tissues and its relation with prognosis. Acad J Sec Mil Med Uni 2013, 10; 1078-1082.

6. Bando H, Weich HA, Horiguchi S, et al. The association between vascular endothelial growth factor-C, its corresponding receptor, VEGFR-3, and prognosis in primary breast cancer: a study with 193 cases. Oncol Rep 2006, 15(3): 653-659.

7. Gisterek I, Matkowski R, Lacko A, et al. Serum vascular endothelial growth factors a, C and d in human breast tumors. Pathol Oncol Res 2010; 16(3):337-344.

8. Gu Y, Qi X, Guo S. Lymphangiogenesis induced by VEGF-C and VEGF-D promotes metastasis and a poor outcome in breast carcinoma: a retrospective study of 61 cases. Clin Exp Metastasis 2008, 25(7):717-725.

9. Lin Y, Liu F, Fan Y, et al. Both high expression of pyruvate kinase M2 and vascular endothelial growth factor C predicts poorer prognosis in human breast cancer. Int J Clin Exp Pathol 2015; 8(7): 8028-8037.

10. Zhang J, Zhang G, Yang S, et al. Macrophage migration inhibitory factor regulating the expression of VEGF-C through MAPK signal pathways in breast cancer MCF-7 cell. World J Surg Oncol, 2016; 14:51.

11. Zajkowska M, Lubowicka E, Fiedorowicz W, et al. Human Plasma Levels of VEGF-A, VEGF-C, VEGF-D, their Soluble Receptor - VEGFR-2 and Applicability of these Parameters as Tumor Markers in the Diagnostics of Breast Cancer. Pathol Oncol Res. 2019;25(4):1477-86

12. Thammineni KL, Thakur GK, Kaur N, et al. Significance of MMP-9 and VEGF-C expression in North Indian women with breast cancer diagnosis. Mol Cell Biochem. 2019;457(1-2):93-103.

13. Liang B, Li Y. Prognostic Significance of VEGF-C Expression in Patients with Breast Cancer: A Meta-Analysis. Iran J Public Health 2014, 43(2):128-135.

14. Zhang Z, Luo G, Tang H, et al. Prognostic Significance of High VEGF-C Expression for Patients with Breast Cancer: An Update Meta Analysis. PLoS ONE 2016, 11(11): e0165725.

15. Wang CA, Harrell JC, Iwanaga R, et al. Vascular endothelial growth factor C promotes breast cancer progression via a novel antioxidant mechanism that involves regulation of superoxide dismutase 3. Breast Cancer Res 2014, 16(5):462.

Received on 14.11.2019 Modified on 11.01.2020

Research J. Pharm. and Tech 2020; 13(9):4277-4279.

DOI: 10.5958/0974-360X.2020.00755.6