Specific nutrient combination effects on tax, NF- κB and MMP-9 in human T-cell lymphotropic virus -1 positive malignant T-lymphocytes

Harakeh et al. BMC Cancer 2015, 15(Suppl 1):S2 http://www.biomedcentral.com/1471-2407/15/S1/-S2 RESEARCH Open Access Specific nutrient combination effects on tax, NF-B and MMP-9 in human T-cell lymphotropic virus -1 positive malignant T-lymphocytes Steve Harakeh1*, Rania Azar2, Esam Azhar1,3, Ghazi A. Damanhouri1, Mourad Assidi4,5, Muhammad Abu-Elmagd4,5,6, Mohammed H. Alqahtani4, Taha Kumosani7, Aleksandra Niedzwiecki8, Mathias Rath8, Ahmed Al-Hejin9, Elie Barbour10, Mona Diab-Assaf2 From 2nd International Genomic Medical Conference (IGMC 2013) Jeddah, Kingdom of Saudi Arabia. 24-27 November 2013 ABSTRACT Background: Adult T-cell Leukemia (ATL) is a disease with no known cure. The disease manifests itself as an aggressive proliferation of CD4+ cells with the human T-cell Lymphotropic virus type 1 (HTLV-1). The leukemogenesis of the virus is mainly attributed to the viral oncoprotein. Tax activates the Nuclear Factor kappa B (NF-B) which stimulates the activity and expression of the matrix metalloproteinase-9 (MMP-9). The objective of this study was to investigate the efficacy of a specific nutrient synergy (SNS) on proliferation, Tax expression, NF-B levels as well as on MMP-9 activity and expression both at the transcriptional and translational levels in two HTLV-1 positive cell lines, HuT-102 and C91-PL at 48h and 96h of incubation. Cytotoxicity of Epigallocatechin-3-gallate (EGCG) was assayed using CytoTox 96 Non-radioactive and proliferation was measured using Cell Titer96TM Nonradioactive Cell Proliferation kit (MTT- based assay). Enzyme linked immunosorbant assay (ELISA) and electrophoretic mobility shift assay (EMSA) were used to assess the effect of SNS on NF-B mobility. Zymography was used to determine the effects of SNS on the activity and secretion of MMP-9. The expression of MMP-9 was done using RT-PCR at the translational level and Immunoblotting at the transcriptional level. Results: A significant inhibition of proliferation was seen in both cell lines starting at a concentration of 200μg/ml and in a dose dependent manner. SNS induced a dose dependent decrease in Tax expression, which was paralleled by a down-regulation of the nuclearization of NF-B. This culminated in the inhibition of the activity of MMP-9 and their expression both at the transcriptional and translational levels. Conclusions: The results of this study indicate that a specific nutrient synergy targeted multiple levels pertinent to the progression of ATL. Its activity was mediated through the NF-B pathway, and hence has the potential to be integrated in the treatment of this disease as a natural potent anticancer agent. Background The human T-cell lymphotropic virus type 1 (HTLV1) is a retrovirus that infects CD4-positive T-cells resulting in the development of adult T-cell leukemia (ATL) in approximately 5% of the cases. ATL manifests as a consequence of the clonal expansion of mature and activated * Correspondence: 1 Special infectious agents unit , King Fahd Medical Research Center, King Abdulaziz University, P.O. Box 80216 Jeddah 21589, Kingdom of Saudi Arabia Full list of author information is available at the end of the article infected CD-4 positive T-cells and is associated with a poor prognosis due to its immunosuppressive and chemotherapy-resistant nature [1]. The causative agent of this disease, HTLV1, is transmitted via breast feeding, transplacentally from mother to child, through sexual contact and blood transfusion [2]; while in vivo transmission occurs through cell-to-cell contact [3]. The HTLV1 oncoprotein, Tax, has been shown to be vital for viral persistence and leukemogenesis due to its pleitoropic effects on cellular proliferation and apoptosis as well as © 2015 Harakeh et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Harakeh et al. BMC Cancer 2015, 15(Suppl 1):S2 http://www.biomedcentral.com/1471-2407/15/S1/-S2 viral replication [4]. As such, targeting Tax has become a novel approach in the treatment of ATL; however, the current options either failed to nullify the issue of relapse [5]. Therefore, it is essential to unravel a nontoxic compound having an inherent ability to inhibit Tax expression and a potential to serve as a therapeutic agent against this aggressive malignancy. The successful documentation of the safety and effectiveness of botanical and dietary natural compounds in cancer prevention has led to the development of a nutrient mixture composed of ascorbic acid (AA), lysine, proline, arginine, epigallocatechingallate (EGCG) and other micronutrients [6]. This natural assortment of nutrients, also known as SNS has exhibited synergistic anticancer properties in a large number of solid cancer cell lines, blocking tumor growth, tumor invasion and MMP expression, both in vitro and in vivo [7]. Not only that, but SNS had an anti-proliferative effect against HTVL-1 positive and negative malignant T-lymphocytes and demonstrated proapoptotic effects with respect to HTLV-1 leukemic cells in specific, via the up-regulation of the pro-apoptotic proteins p53, p21 and Bax, and the down-regulation of the pro-survival Bcl2-a protein [8]. We have also reported enhanced antiproliferative activity of SNS in the presence of polyethylene glycol gold plated nano-particles [9]. SNS was in fact formulated based on the capacity of the individual components to alter key physiological pathways involved in cancer progression and metastasis [10]. In fact, ingredients of SNS were reported to inhibit the destruction of the extracellular matrix (ECM), which is a pre-requisite for cancer cell invasion and metastasis [11]. For example, the biosynthesis of collagen depends on an adequate supply of AA, the amino acids lysine and proline as well as the micronutrients manganese and copper [12]. Therefore, the integration of these nutrients into the formulation would result in strengthening the ECM. Not only that, but lysine is also a natural inhibitor of plasmin-induced proteolysis and, therefore, increases ECM stability by inhibiting the breakdown of collagen fibers [11]. Similarly, N-acetyl cysteine, AA, selenium and EGCG inhibited the invasiveness of tumor cells by blocking the activity of MMPs, which are a unique family of more than 20 proteases responsible for the proteolytic degradation of the ECM, which is essential for the dissemination of cancer cells to secondary sites [12]. The over-expression of MMPs, which is a common occurrence in malignant tumors, is correlated with tumor aggressiveness, stage and prognosis [11,12]. Addition (...truncated)