Rania Azar

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

BackgroundAdult 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-κB) 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-κB 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-κB 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.ResultsA 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-κB. This culminated in the inhibition of the activity of MMP-9 and their expression both at the transcriptional and translational levels.ConclusionsThe 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-κB pathway, and hence has the potential to be integrated in the treatment of this disease as a natural potent anticancer agent.

Epigallocatechin-3-gallate Inhibits Tax-dependent Activation of Nuclear Factor Kappa B and of Matrix Metalloproteinase 9 in Human T-cell Lymphotropic Virus-1 Positive Leukemia Cells

Epigallocatechin-3-gallate (EGCG) is the most abundant polyphenol molecule from green tea and is known to exhibit antioxidative as well as tumor suppressing activity. In order to examine EGCG tumor invasion and suppressing activity against adult T-cell leukemia (ATL), two HTLV-1 positive leukemia cells (HuT-102 and C91- PL) were treated with non-cytotoxic concentrations of EGCG for 2 and 4 days. Proliferation was significantly inhibited by 100 μM at 4 days, with low cell lysis or cytotoxicity. HTLV-1 oncoprotein (Tax) expression in HuT- 102 and C91-PL cells was inhibited by 25 μM and 125 μM respectively. The same concentrations of EGCG inhibited NF-kB nuclearization and stimulation of matrix metalloproteinase-9 (MMP-9) expression in both cell lines. These results indicate that EGCG can inhibit proliferation and reduce the invasive potential of HTLV-1- positive leukemia cells. It apparently exerted its effects by suppressing Tax expression, manifested by inhibiting the activation of NF-kB pathway and induction of MMP-9 transcription in HTLV-1 positive cells.

Berberis libanotica extract targets NF-κB/COX-2, PI3K/Akt and mitochondrial/caspase signalling to induce human erythroleukemia cell apoptosis

The aim of this study was to describe and understand the relationship between cyclooxygenase-2 (COX-2) expression and apoptosis rate in erythroleukemia cells after apoptosis induction by Berberis libanotica (Bl) extract. To achieve this goal we used erythroleukemia cell lines expressing COX‑2 (HEL cell line) or not (K562 cell line). Moreover, we made use of COX‑2 cDNA to overexpress COX‑2 in K562 cells. In light of the reported chemopreventive and chemosensitive effects of natural products on various tumor cells and animal models, we postulated that our Bl extract may mediate their effects through apoptosis induction with suppression of cell survival pathways. Our study is the first report on the specific examination of intrinsic apoptosis and Akt/NF-κB/COX‑2 pathways in human erythroleukemia cells upon Bl extract exposure. Even if Bl extract induced apoptosis of three human erythroleukemia cell lines, a dominant effect of Bl extract treatment on K562 cells was observed resulting in activation of the late markers of apoptosis with caspase-3 activation, PARP cleavage and DNA fragmentation. Whereas, we showed that Bl extract reduced significantly expression of COX‑2 by a dose-dependent manner in HEL and K562 (COX‑2+) cells. Furthermore, in regard to our results, it is clear that the simultaneous inhibition of Akt and NF-κB signalling can significantly contribute to the anticancer effects of Bl extract in human erythroleukemia cells. We observed that the Bl extract is clearly more active than the berberine alone on the induction of DNA fragmentation in human erythro-leukemia cells.

Downregulation of sphingosine kinase-1 induces protective tumor immunity by promoting M1 macrophage response in melanoma

The infiltration of melanoma tumors by macrophages is often correlated with poor prognosis. However, the molecular signals that regulate the dialogue between malignant cells and the inflammatory microenvironment remain poorly understood. We previously reported an increased expression of sphingosine kinase-1 (SK1), which produces the bioactive lipid sphingosine 1-phosphate (S1P), in melanoma. The present study aimed at defining the role of tumor SK1 in the recruitment and differentiation of macrophages in melanoma. Herein, we show that downregulation of SK1 in melanoma cells causes a reduction in the percentage of CD206highMHCIIlow M2 macrophages in favor of an increased proportion of CD206lowMHCIIhigh M1 macrophages into the tumor. This macrophage differentiation orchestrates T lymphocyte recruitment as well as tumor rejection through the expression of Th1 cytokines and chemokines. In vitro experiments indicated that macrophage migration is triggered by the binding of tumor S1P to S1PR1 receptors present on macrophages whereas macrophage differentiation is stimulated by SK1-induced secretion of TGF-β1. Finally, RNA-seq analysis of human melanoma tumors revealed a positive correlation between SK1 and TGF-β1 expression. Altogether, our findings demonstrate that melanoma SK1 plays a key role in the recruitment and phenotypic shift of the tumor macrophages that promote melanoma growth.

Contribution of HIF-1α in 4E-BP1 Gene Expression

The eukaryotic translation initiation factor 4E (eIF4E) is necessary for the translation of capped mRNAs into proteins. Cap-dependent mRNA translation can be however inhibited by the eIF4E-binding protein 1 (4E-BP1). The hypophosphorylated forms of 4E-BP1 indeed sequester eIF4E and thus block translation initiation and consequent protein synthesis. Different reports indicate that, in addition to hypophosphorylation, 4E-BP1 function can be also regulated at the level of protein expression. This is the case in contact-inhibited cells or in cells exposed to hypoxia. The molecular mechanisms responsible for 4E-BP1 protein accumulation in these conditions remain however unknown. In the present study, we found that 4E-BP1 gene promoter contains a hypoxia-responsive element (HRE) that mediates 4E-BP1 gene upregulation via the hypoxia-inducible factor-1 alpha (HIF-1α) transcription factor. Gene reporter assays then revealed that the presence of such HRE in the promoter of 4E-BP1 gene is involved in 4E-BP1 accumulation in contact-inhibited cells and in cells exposed to hypoxia. We also reveal that the TGF-β–dependent transcription factor SMAD4 cooperates with HIF-1α to fully activate 4E-BP1 gene transcription under hypoxia. These data therefore suggest that HIF-1α contributes to 4E-BP1 gene expression under different conditions. Mol Cancer Res; 11(1); 54–61. ©2012 AACR.

Effects of nutrients on matrix metalloproteinases in human T-lymphotropic virus type 1 positive and negative malignant T-lymphocytes

Experimental and clinical studies have revealed the effectiveness of a specific nutrient synergy (SNS) mixture composed of ascorbic acid (AA), lysine, proline, arginine, epigallocatechin gallate (EGCG) and other micronutrients in targeting crucial physiological mechanisms involved in cancer progression and metastasis. HTLV-1 causes adult T-cell leukemia (ATL). The spread and metastases of ATL as well as other tumors has been associated with matrix metalloproteinases, especially the gelatinases MMP-2 and MMP-9. The objective of this study was to investigate whether SNS, AA and EGCG affects the gelatinolytic activity of MMP-2 and its transcriptional and translational levels in HTLV-1-positive and -negative malignant T-cells. The results indicated that SNS and EGCG caused a dose-dependent decline in the activity, transcription and translation of MMP-2 after treatment with SNS and EGCG, while AA was only able to inhibit the activity at maximum doses tested and to some extent, the protein expression levels of MMP-2, without affecting their transcriptional levels. The highest activity was noted in the case of SNS which is likely to be due to a synergistic effect of the different constituents in the formulation. These results point towards the potential integration of SNS in the anti-invasive treatment of ATL and related diseases.

Inhibition of Proliferation and Induction of Apoptosis by Thymoquinone via Modulation of TGF Family, p53, p21 and Bcl-2α in Leukemic Cells

INTRODUCTION Adult T-cell leukemia (ATL) is an aggressive form of malignancy caused by human T- cell lymphotropic virus 1 (HTLV-1). Currently, there is no effective treatment for ATL. Thymoquinone has been reported to have anti-cancer properties. OBJECTIVE The aim of this study is to investigatthe effects of TQ on proliferation, apoptosis induction and the underlying mechanism of action in both HTLV-1 positive (C91-PL and HuT-102) and HTLV-1 negative (CEM and Jurkat) malignant T-lymphocytes. MATERIALS AND METHODS Cells were incubated with different thymoquinone concentrations for 24h. Cell cytotoxicity was assayed using the CytoTox 96® Non-Radioactive Cytotoxicity Assay Kit. Cell proliferation was determined using CellTiter 96® Non-Radioactive Cell Proliferation. Cell cycle analysis was performed by staining with propidium iodide. Apoptosis was assessed using cell death ELISA kit. The effect of TQ on p53, p21, Bcl-2 protein expression was determined using Western blot analysis while TGF mRNA expression was determined by RT-PCR. RESULTS At non-cytotoxic concentrations of TQ, it resulted in the inhibition of proliferation in a dose dependent manner. Flow cytometric analysis revealed a shift in the cell cycle distribution to the PreG1 phase which is a marker of apoptosis. Also TQ increase DNA fragmentation. TQ mediated its anti-proliferative effect and apoptosis induction by an up-regulation of TGFβ1, p53 and p21 and a down-regulation of TGF-α and Bcl-2α. CONCLUSION Thymoquinone presents antiproliferative and proapoptotic effects in ATL cells. For this reason, further research is required to investigate its possible application in the treatment of ATL.

Effects of Ascorbic Acid on Tax, NF-κB and MMP-9 in Human T-cell Lymphotropic Virus Type 1 Positive Malignant T-Lymphocytes

BACKGROUND HTLV1 is a retrovirus that infects CD4-positive cells and leads to Adult T-cell leukemia by constitutive activation of nuclear factor kappa B. Ascorbic acid (AA) is an essential nutrient that possess anti-proliferative and pro-apoptotic activity against a number of malignant cell lines. This study delineates the effect of AA on Tax protein expression as well as NF-κB and MMP9 activity in two HTLV1-positive leukemia cells (HuT-102 and C91-PL). METHODS The cytotoxic and antiproliferative effect of AA were studied by LDH release and MTT tests, respectively. The proteins expression level was assessed by western blotting. RT-PCR was used to study mRNAs level. Finally, ELISA/EMSA and Zymography were used to evaluate NF-κB and MMP-9 activities, respectively. RESULTS Cell lines were treated with non-cytotoxic concentrations of AA for 48h and 96h, which resulted in a significant inhibition of proliferation at a concentration of 50µg/ml at 96h in both cell lines. The same concentration inhibited Tax protein expression as well as the NF-κB nuclearization and DNA binding activity. The inhibitory effect of AA on MMP9 protein expression and activity started at 100µg/ml and 50µg/ml in HuT-102 and C91-PL cells respectively, with no effect at the transcriptional levels of MMP-9 in either one of the two cell lines. CONCLUSION These results indicated that while AA exerted its anti-proliferative effect on the NF- κB activation pathway by suppressing Tax expression, its effects on MMP9 seemed to be independent of this mechanism and follow a different approach.

Effects of specific nutrients on tax-dependent activation of NF-κB and MMP-9 in human T-cell lymphotropic virus -1 positive malignant T-lymphocytes

Effects of specific nutrients on tax-dependent activation of NFB and MMP-9 in human T-cell lymphotropic virus -1 positive malignant T-lymphocytes Steve Harakeh, Mona Diab-Assaf, Rania Azar, Esam Azhar, Ghazi A. Damanhouri, Adeel Chaudary, Haitham Yacoub, Mourad Assidi, Muhammad M. Abu-Elmagd, Mohammed H. Alqahtani, Adel M. Abuzenadah , Taha Kumosani, Aleksandra Niedzwiecki, Mathias Rath, Elie Barbour