Angiogenesis Factors Associated with New Breast Cancer Cell Line AMJ13 Cultured in Vitro

Ahmed Majeed Al-Shammari^1*, Worod Jawad Kadhim Allak², Mahfoodha Umran², Nahi Y. Yaseen¹, Ayman Hussien¹

¹Experimental Therapy Department, Iraqi Center for Cancer and Medical Genetic Research, Al-Mustansiriyah University, Baghdad, Iraq.
²Biotechnology Department, Collage of Science, Baghdad University, Baghdad, Iraq.

DOI: 10.4236/abcr.2015.44011                

Abstract

Background: AMJ13 is a new breast cancer cell line that has been established from a 70-year-old Iraqi woman with a histological diagnosis of infiltrating ductal carcinoma. It is the first for an Iraqi population. In breast cancer, angiogenesis provides the tumor tissue, which is rapidly proliferated with oxygen and nutrients, removes wastes and increases the opportunity of cancer cells to invade other organs. Methods: The AMJ13 breast cancer cell line was represented at three different passages and incubated for interval times. Microarray panel of 43 different angiogenesis markers was used to scan the supernatant for the factors. ELISA was used to quantify some of the important angiogenesis factors released in the culture medium and to confirm absence of those who was not detected by the antibody array. RT-PCR was used to confirm the gene expression (mRNA) of studied factors. Results: Microarray analysis showed that TIMP1 and two secreted at highest levels compared to the rest of the factors with low presence of endostatin. Other non-detectable factors by microarray examined by ELISA assay that showed highest expression level of VEGF-A were obtained at earliest passage, while the highest levels of FGF-b were obtained at late passage. The VEGF-D secretion was shown low concentrations at all studied passages. There is no detectable level of EGF protein in different passages and times interval tested. There are no significant differences in secretion of sICAM between different passages and incubation periods. Conclusion is that AMJ13 cell line depends on VEGF-A as main angiogenesis factor to induce micro-vessels supported by low levels of VEGF-D for lymphatic vessels formation. AMJ13 cell line depends on FGF as growth factors as in late passages it was shifted to depend mainly on FGF completely. All of this process may be regulated by TGF-β. TIMP-1 has proangiogentic effect and has feedback talk with TIMP-2. Understanding the angiogenesis process for breast cancer can give us better targets for therapy and more effective treatments.

Keywords

Angiogenesis Factors, Breast Cancer Cell, AMJ13, Tumor Tissue

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	US Department of Agriculture Animal and Plant Health Services. Info Sheet: Bovine Leukosis Virus (BLV) in U.S. Beef Cattle. February 1999.
[2]	Tonini, T., Rossi, F. and Claudio, P.P. (2003) Molecular Basis of Angiogenesis and Cancer. Oncogene, 22, 6549-6556. http://dx.doi.org/10.1038/sj.onc.1206816
[3]	Liekens, S., De Clercq, E. and Neyts, J. (2001) Angiogenesis: Regulators and Clinical Applications. Biochemical Pharmacology, 61, 253-270. http://dx.doi.org/10.1016/S0006-2952(00)00529-3
[4]	Nishida, N., et al. (2006) Angiogenesis in Cancer. Vascular Health and Risk Management, 2, 213-219. http://dx.doi.org/10.2147/vhrm.2006.2.3.213
[5]	Schneider, B.P. and Miller, K.D. (2005) Angiogenesis of Breast Cancer. Journal of Clinical Oncology, 23, 1782-1790. http://dx.doi.org/10.1200/JCO.2005.12.017
[6]	Karamysheva, A.F. (2008) Mechanisms of Angiogenesis. Biochemistry (Moscow), 73, 751-762. http://dx.doi.org/10.1134/S0006297908070031
[7]	Carmeliet, P. (2000) Mechanisms of Angiogenesis and Arteriogenesis. Nature Medicine, 6, 389-395. http://dx.doi.org/10.1038/74651
[8]	Jain, R.K. (2003) Molecular Regulation of Vessel Maturation. Nature Medicine, 9, 685-693. http://dx.doi.org/10.1038/nm0603-685
[9]	Pugh, C.W. and Ratcliffe, P.J. (2003) Regulation of Angiogenesis by Hypoxia: Role of the HIF System. Nature Medicine, 9, 677-684. http://dx.doi.org/10.1038/nm0603-677
[10]	Van Meir, E.G., et al. (1994) Release of an Inhibitor of Angiogenesis upon Induction of Wild Type p53 Expression in Glioblastoma Cells. Nature Genetics, 8, 171-176. http://dx.doi.org/10.1038/ng1094-171
[11]	Wu, H.-C., Huang, C.-T. and Chang, D.-K. (2008) Anti-Angiogenic Therapeutic Drugs for Treatment of Human Cancer. Journal of Cancer Molecules, 4, 37-45.
[12]	Al-Shammari, A.M., Alshami, M., Umran, M., et al. (2015) Establishment and Characterization of a Receptor-Negative, Hormone-Nonresponsive Breast Cancer Cell Line from an Iraqi Patient. Breast Cancer: Targets and Therapy, 7, 223-230. http://dx.doi.org/10.2147/BCTT.S74509
[13]	Würtz, S.O., et al. (2005) Tissue Inhibitor of Metalloproteinases-1 in Breast Cancer. Endocrine-Related Cancer, 12, 215-227. http://dx.doi.org/10.1677/erc.1.00719
[14]	Ikenaka, Y., Yoshiji, H., Kuriyama, S., et al. (2003) Tissue Inhibitor of Metalloproteinases-1 (TIMP-1) Inhibits Tumor Growth and Angiogenesis in the TIMP-1 Transgenic Mouse Model. International Journal of Cancer, 105, 340-346. http://dx.doi.org/10.1002/ijc.11094
[15]	Bourboulia, D., Jensen-Taubman, S., Rittler, M.R., et al. (2011) Endogenous Angiogenesis Inhibitor Blocks Tumor Growth via Direct and Indirect Effects on Tumor Microenvironment. The American Journal of Pathology, 179, 2589-2600. http://dx.doi.org/10.1016/j.ajpath.2011.07.035
[16]	Têtu, B., Brisson, J., Wang, C., et al. (2006) The Influence of MMP-14, TIMP-2 and MMP-2 Expression on Breast Cancer Prognosis. Breast Cancer Research, 8, R28. http://dx.doi.org/10.1186/bcr1503
[17]	Elias, A.P. and Dias, S. (2008) Microenvironment Changes (in pH) Affect VEGF Alternative Splicing. Cancer Microenvironment, 1, 131-139. http://dx.doi.org/10.1007/s12307-008-0013-4
[18]	Marjon, P.L., Bobrovnikova-Marjon, E.V. and Abcouwer, S.F. (2004) Expression of the Pro-Angiogenic Factors Vascular Endothelial Growth Factor and Interleukin-8/CXCL8 by Human Breast Carcinomas Is Responsive to Nutrient Deprivation and Endoplasmic Reticulum Stress. Molecular Cancer, 3, 5670-5674.
[19]	Saponaro, C., Malfettone, A., Ranieri, G., et al. (2013) VEGF, HIF-1alpha Expression and MVD as an Angiogenic Network in Familial Breast Cancer. PLoS ONE, 8, e53070. http://dx.doi.org/10.1371/journal.pone.0053070
[20]	Darrington, E., Zhong, M., Vo, B.-H., et al. (2012) Vascular Endothelial Growth Factor A, Secreted in Response to Transforming Growth Factor-β1 under Hypoxic Conditions, Induces Autocrine Effects on Migration of Prostate Cancer Cells. Asian Journal of Andrology, 14, 745-751. http://dx.doi.org/10.1038/aja.2011.197
[21]	Matei, D., Kelich, S., Cao, L.Y., et al. (2007) PDGF BB Induces VEGF Secretion in Ovarian Cancer. Cancer Biology & Therapy, 6, 1951-1959. http://dx.doi.org/10.4161/cbt.6.12.4976
[22]	Nakamura, Y., Yasuoka, H., Tsujimoto, M., et al. (2003) Prognostic Significance of Vascular Endothelial Growth Factor D in Breast Carcinoma with Long-Term Follow-Up. Clinical Cancer Research, 9, 716-721.
[23]	Stacker, S.A., Caesar, C., Baldwin, M.E., Thornton, G.E., et al. (2001) VEGF-D Promotes the Metastatic Spread of Tumor Cells via the Lymphatics. Nature Medicine, 7, 186-191. http://dx.doi.org/10.1038/84635
[24]	Murphy, L.C. and Dotzlaw, H. (1989) Endogenous Growth Factor Expression in T-47D, Human Breast Cancer Cells, Associated with Reduced Sensitivity to Antiproliferative Effects of Progestins and Antiestrogens. Cancer Research, 49, 599-604.
[25]	O’sullivan, C., Lewis, C.E., Harris, A.L., et al. (1993) Secretion of Epidermal Growth Factor by Macrophages Associated with Breast Carcinoma. The Lancet, 342, 148-149. http://dx.doi.org/10.1016/0140-6736(93)91348-P
[26]	Mori, K., Kurobe, M., Furukawa, S., et al. (1986) Human Breast Cancer Cells Synthesize and Secrete an EGF-Like Immunoreactive Factor in Culture. Biochemical and Biophysical Research Communications, 136, 300-305. http://dx.doi.org/10.1016/0006-291X(86)90909-5
[27]	El-Sayed, L.H.G., Fadali, G., Saad, A., Hafez, E.S. and Shaaban, S. (2010) Expression of MAGE-A Genes and Soluble ICAM-1 in Egyptian Breast Cancer Patients: Possible Prognostic Impact. Journal of the Medical Research Institute, 31, 7-18.
[28]	Thielemann, A., Baszczuk, A., Kopczyński, Z., et al. (2014) The Clinical Usefulness of Assessing the Concentration of Cell Adhesion Molecules sVCAM-1 and sICAM-1 in the Serum of Women with Primary Breast Cancer. Wspólczesna Onkologia, 4, 252-259. http://dx.doi.org/10.5114/wo.2014.43492
[29]	Eggeman, H., et al. (2011) Influence of a Dose-Dense Adjuvant Chemotherapy on sVCAM-1/sICAM-1 Serum Levels in Breast Cancer Patients with 1-3 Positive Lymph Nodes. Anticancer Research, 31, 2617-2622.
[30]	Touvier, M., Fezeu, L., Ahluwalia, N., et al. (2013) Association between Prediagnostic Biomarkers of Inflammation and Endothelial Function and Cancer Risk: A Nested Case-Control Study. American Journal of Epidemiology, 177, 3-13. http://dx.doi.org/10.1093/aje/kws359
[31]	Lai, L., Kadory, S., Cornell, C., et al. (1993) Possible Regulation of Soluble Icam-1 Levels by Interleukin-1 in a Sub-Set of Breast Cysts. International Journal of Cancer, 55, 586-589. http://dx.doi.org/10.1002/ijc.2910550412
[32]	Cross, M.J. and Claesson-Welsh, L. (2001) FGF and VEGF Function in Angiogenesis: Signalling Pathways, Biological Responses and Therapeutic Inhibition. Trends in Pharmacological Sciences, 22, 201-207. http://dx.doi.org/10.1016/S0165-6147(00)01676-X
[33]	Sahni, A., Simpson-Haidaris, P.J., Sahni, S.K., et al. (2008) Fibrinogen Synthesized by Cancer Cells Augments the Proliferative Effect of Fibroblast Growth Factor-2 (FGF-2). Journal of Thrombosis and Haemostasis, 6, 176-183. http://dx.doi.org/10.1111/j.1538-7836.2007.02808.x
[34]	MacCallum, J., Bartlett, J.M.S., Thompson, A.M., et al. (1994) Expression of Transforming Growth Factor Beta mRNA Isoforms in Human Breast Cancer. British Journal of Cancer, 69, 1006-1009. http://dx.doi.org/10.1038/bjc.1994.197
[35]	Buck, M.B. and Knabbe, C. (2006) TGF-Beta Signaling in Breast Cancer. Annals of the New York Academy of Sciences, 1089, 119-126. http://dx.doi.org/10.1196/annals.1386.024
[36]	Pardali, E. and ten Dijke, P. (2008) Transforming Growth Factor-Beta Signaling and Tumor Angiogenesis. Frontiers in Bioscience (Landmark Edition), 14, 4848-4861. http://dx.doi.org/10.2741/3573
[37]	Lebrun, J.-J. (2012) The Dual Role of TGF in Human Cancer: From Tumor Suppression to Cancer Metastasis. ISRN Molecular Biology, 2012, Article ID: 381428.