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Abstract 3792: Upregulation of TNF-α by ethanol extract of Moringa oleifera leaves in benzene-induced leukemic Wister rat: a possible mechanism of anticancer property

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     RRJoOH (2014) 16-21 © STM Journals 2014. All Rights Reserved Page 16   Research & Reviews: Journal of Oncology and Haematology ISSN: 2319-3387 (online) Volume 3, Issue 3 www.stmjournals.com Up-Regulation of TNF- α by Ethanol E xtract of Moringa Oleifera in Benzene-Induced Leukemic Wister Rat: A Possible Mechanism of Anticancer Property E.O. Akanni  1  , A.L. Adedeji  2  * , R.A. Akanni  3  , J.K. Oloke  4    1 Medical Laboratory Science Department, College of Health Sciences, Ladoke Akintola University of Technology, P.M.B. 4400, Osogbo, Nigeria 2 Biochemistry Department, College of Health Sciences, Ladoke Akintola University of Technology, P.M.B. 4000, Ogbomoso, Nigeria 3 Medical Microbiology & Parasitology Department, College of Health Sciences, Ladoke Akintola University of Technology, P.M.B. 4400, Osogbo, Nigeria 4 Pure & Applied Biology Department, Ladoke Akintola University of Technology, P.M.B. 4000, Ogbomoso, Nigeria Abstract  Ethanol extract of Monringa oleifera (EMO) has been shown to possess anti-cancer  properties in animal models. The mechanisms of action of its anticancer properties have not been fully demonstrated. We induced leukemia in two groups of seven rats each by intravenous injection of benzene chromasolv solution (0.2 mL) 48 hourly for 4 weeks.  EMO (0.2 ml of 100 mg/mL) was administered orally by gavage, after leukemia induction, daily for 4 weeks to one of the groups while the other group was not treated. The third group served as the normal control. At the end of the treatment, the rats were immobilized by cervical dislocation and blood samples were collected by cardiac  puncture. Plasma concentrations of Beta- 2 Microglobulin (β  2  M), Perforins (PF),  Interleukin-2 (IL2), Interferon-gamma (IFN- γ) and Tumour Necrosis Factor  -alpha (TNF- α) we re determined by standard commercial ELISA. Results showed that leukemia was induced within 4 weeks as significantly elevated leukocyte count and plasma β  2  M concentrations over the baseline were noted (p < 0.05). Plasma IL2 and TNF- α were also  significantly elevated while IFN- γ decreased significantly in EMO treated rats compared with untreated groups. The plasma concentration of PF remained statistically unchanged. Since IL2 and PF are associated with cytotoxicity of CD8 T lymphocytes, reduced expression of PF in the face of increasing plasma concentration of IL-2 suggests that  EMO may not exert its anticancer properties via direct cytotoxicity. We thus concluded that increased expression TNF- α would transl  ate to enhanced apoptosis and may be a  possible mechanism of action. Keywords:  Moringa oleifera, anticancer mechanism, cytokines * Author for Correspondence    E-mail: aladedeji@lautech.edu.ng INTRODUCTION Benzene has been demonstrated to induce cancer especially of the myeloid cell by different proposed mechanisms and phenolic metabolites such as phenol, hydroquinone, catechol, and 1,2,4-benzenetriol have been implicated for its carcinogenicity [1, 2]. Tumor-specific T-cells are normally activated to inhibit tumor growth. T-cells in response to tumors secrete cytokine. Also cells expressing CD4 secrete TNF- α  which specifically attacks malignant cells [3]. Extensive preclinical studies documented a direct cytostatic and cytotoxic effect of TNF against subcutaneous human xenograft and lymph node metastasis in immunodeficient mice, as well as a variety of immunomodulatory effects on various immune effector cells, including macrophages and T-cells. Medicinal plants provide an inexhaustible source of anticancer drugs in terms of both   Anticancer mechanism of Moringa oleifera Akanni et al.  RRJoOH (2014) 16-21 © STM Journals 2014. All Rights Reserved Page 17   variety and mechanism of action. Accumulating reports have suggested that many naturally-occurring substances exhibit cancer chemotherapeutic effects [4]. Moringa oleifera has potent antiproliferative activity and apoptosis inducing capacity on tumor cell line. It also increases the cytotoxicity of chemotherapy on pancreatic cancer cells [5, 6]. Furthermore, a recent study has also confirmed these findings [7]. The mechanisms of anticancer properties of Moringa oleifera are still emerging. Cytokines are secreted by hematopoietic and non-hematopoietic cell types and are critical for both innate and adaptive immune responses. The expression of cytokines by cells may be altered in immunological, inflammatory, infectious and neoplastic disease states. Since cytokines in turn exert their effects by influencing gene activation, growth, differentiation, functional cell surface molecule expression and cellular effector function that can facilitate tumor destruction [8], we sought to provide a  possible mechanism of Moringa oleifera anticancer properties by studying its modulatory effect on selected cytokines in  benzene-induced leukemic animal model. MATERIALS AND METHODS The Plant Extract The fresh leaves  Moringa oleifera  collected from Osogbo was identified at the Department of Botany, Obafemi Awolowo University, Ile-Ife, Nigeria. 100 g of air dried leaves were taken. The dried leaves were then grinded into fine powder using an electric blender. The fine  powder was then weighed and then soaked with measured quantity of ethanol (for every 10 ml of ethanol, 1 g of  Moringa oleifera  powder). The mixture was stirrer and allowed to soak for 48 h. Thereafter it was filtered using and the filtrate collected and concentrated to dryness at temperature 45 o C. The eventual residue was carefully scrapped, weighed and stored at − 20 o C until further analyses were carried out. Experimental Rats Twenty-one adult Wister rats weighing  between 150 and 200 g were obtained from the animal house of the college of Health Sciences, Ladoke Akintola University of Technology, Osogbo. The rats were randomly arranged in separate wooden cages in seven  per group. They were allowed to acclimatize for a period of seven days before the commencement of the experiment. The animals were maintained at room temperature 28 o C (±2) with 12 h light/dark cycle and also allowed unrestricted access to water and rat chow. We induced leukemia in two groups of seven rats each by intravenous injection of  benzene chromasolv (Sigma-Aldrich Cat No 270709) solution (0.2 mL) 48 hourly for 4 weeks. EMO (0.2 ml of 100 mg/ml) was administered orally by gavage, after leukemia induction, daily for 4 weeks to one of the groups while the other group was not treated. The third group served as the normal control. At the end of the treatment, the rats were immobilized by cervical dislocation and blood samples were collected by cardiac puncture. Plasma were separated by centrifugation and stored at − 20 o C until analyzed. Selected cytokines were measured using standard commercial enzyme-linked immunosorbent assay kits. Statistical Analyses Data were presented as mean ± standard deviation (SD). Statistical analysis was  performed using GraphPad, p-values of < 0.05 were considered significant. RESULTS AND DISCUSSION Medicinal plants provide an inexhaustible source of anticancer drugs in terms of both variety and mechanism of action. Accumulating reports have suggested that many naturally-occurring substances exhibit cancer chemotherapeutic effects [4].  Moringa oleifera  has potent antiproliferative activity and apoptosis inducing capacity on tumor cell line. It also increases the cytotoxicity of chemotherapy on pancreatic cancer cells [5, 6]. Furthermore, a recent study has also confirmed this claim [7]. The mechanisms of anticancer properties of  Moringa oleifera is still emerging. Since cytokines in turn exert their effects by influencing gene activation, growth, differentiation, functional cell surface molecule expression and cellular effector function that can facilitate tumor destruction [8], we sought to provide a possible mechanism of  Moringa oleifera anticancer  properties by studying its modulatory effect on selected cytokines in animal model bearing  benzene-induced leukemia.   Research & Reviews: Journal of Oncology and Haematology Volume 3, Issue 3  ISSN: 2319-3387 (online)  RRJoOH (2014) 16-21 © STM Journals 2014. All Rights Reserved Page 18   Our results presented in Table 1 showed that leukemia was induced within 4 weeks as significantly elevated leukocyte count [5.84 ± 0.92 versus  12.03 ± 0.49] was observed from  baseline and post-treatment parameters respectively. We had earlier confirmed induction of leukemia by benzene injection [9]. The blood film appearance was consistent with acute myeloid leukemia after intravenous injection of benzene chromasolv solution (0.2 mL) 48 hourly for 4 weeks. Plasma β 2 M concentration was also elevated over the control were as [69.1 ± 5.5 versus 74.5 ± 5.0 µg/mL], (p < 0.05). Plasma β 2 M concentration, a marker of leukemia, also suggested a degree of cancer induction in our study. Different mechanisms of leukemia induction by benzene have been proposed [10]. One of such mechanisms has been shown to be decreased immunosurveillance [11, 12]. Whatever the case, significantly reduction in leukocyte count was observed in EMO treated group (Table 1). We discovered that plasma IL-2 [261 ± 30 versus 315 ± 62 ng/L] and TNF- α [ 187 ± 18 Vs 220 ± 18 ng/L] were also significantly elevated (p < 0.05) in EMO treated rats compared with untreated groups while IFN- γ [67 ± 7.4 versus  55 ± 9.4 ng/ml] decreased significantly in EMO treated rats compared with untreated groups (p < 0.05) (Figure 1). Cytokines are released in response to a diverse range of cellular stresses that profoundly affect several stages of cancer formation, growth of tumours in vivo, progression and playing a significant role in immunosurveillance against malignant cells [13]. The results of present study show that Moringa Oleifera leaf ethanol extract significantly increase plasma concentration of TNF- α and IL -2 and significantly reduce IFN- γ concentration in  benzene-induced leukemic Wister rats. This translated to increased expression of TNF- α and IL-2 and of course reduced expression of IFN- γ.  In acute leukaemia, cytokines could be  produced both by leukemic and normal cells [14, 15]. Cytokines have been reported to be important regulators of blast proliferation, but the responses to cytokines have been variable [16]. Therefore the roles of cytokines in the  pathogenesis of acute leukaemia are not very clear. However, Plasma concentrations of cytokines have been shown to correlation cellular expression [17]. TNF is a pleiotropic cytokine, which modulates the responsiveness of immune system by stimulation the expression of a number of other regulatory cytokines that are harmful to cancer cells [18]. Kim et al.  had earlier reported that exposure of HL-60 cells to a Korean herbal drug formula resulted into an increase in TNF- α secretion. Similarly, we discovered that our extract stimulated the expression of IL-2 [19]. TNF and other cytokines were also reported to be markedly upregulated in peripheral mononuclear blood cells following ex-vivo  administration of aqueous extract of Carica papaya leaves extract [20]. Admittedly, IL-2 is capable of inducing the synthesis of various cytokines such as TNF- α , IFN- γ  [21]. Although, IL-2 with TNF synergistically activates and increases NK cytolytic function [22], IL-2 has no direct cytolytic or cytostatic effects on tumour cells. The ability of Natural Killer and IL-2-activated killer cells to induce cytotoxicity in a variety of tumour cell targets was found to be impaired in the absence of  perforin [23].  NK-mediated anti-tumor response is likely initiated by the production of IFN- γ  by activated Type I NKT cells, leading to the recruitment of NK and CD8 +  T-cells which directly lead to tumor cell lysis.  NKT-mediated cytotoxic activity has also been demonstrated to occur through several mechanisms including perforin/granzyme, Fas ligand, and TNF-related apoptosis-inducing ligand [24  –  26]. TNF-induced apoptosis is mediated primarily through the activation of Type I receptors, the death domain of which further recruits many different signaling  proteins, which together are considered part of an apoptotic cascade. Furthermore, reactive oxygen intermediates, ceramide,  phospholipases, and serine proteases are also implicated in TNF-induced apoptosis [27]. Other mechanisms of TNF-induced apoptosis are still emerging. Although these processes were not captured in the present study, the significant elevation in the plasma   Anticancer mechanism of Moringa oleifera Akanni et al.  RRJoOH (2014) 16-21 © STM Journals 2014. All Rights Reserved Page 19   concentration of both TNF and IL-2 without concurrent increase in plasma concentration of  perforin (Figure 1) may rule out direct cytotoxicity by CD8 T lymphocytes on leukemic cells. This led us to summarize that an enhanced apoptosis as a possible mechanism of action ethanol extract of Moringa oleifera in our benzene-induced leukemic animal model. Table 1:  Haematological Parameters of Rats Bearing Benzene-Induced Leukemia Treated with  Ethanol Extract of Moringa Oleifera (EMO). Treatment Group WBC/µl RBCx10 9 /µl HB (g/dl) PCV (%) Leukamia + EMO 6.1±1.4 a  7.5±0.2 a  13.1±0.4 a  44.7±1.4 a  Leukemia untreated 12.0±0.5  b  3.8±0.4  b  7.5±0.6  b  25.60±1.2  b   Normal control 5.8 ±0.9 a  8.2±0.5 a  13.4±0.8 a  48.9 ±4.0 c   Column with different superscripts are significant different (p < 0.05). Fig. 1:   Bar Chart Showing the Effect of Ethanol Extract of Moringa Oleifera Leaves on Plasma Concentrations of Selected Cytokines in Benzene-Induced Leukemic Rats. 20 mg/200 µl EMO Were  Administered Orally by Gavage, to Benzene-Induced Leukemia Rats Daily for 4 Weeks. Bar Caring  Different Letter on Each Parameter Are Statistically Different (p < 0.05). TNF- α = Tumour Necrosis  Factor-alpha (ng/l), IL2 = Interleukin-2 (ng/ml), IFN- γ  = Interferon-Gamma (ng/ml) and  PF = Perforins (ng/ml). CONCLUSION We conclude that enhanced expression of TNF- α is a possible mechanism of action Moringa oleifera extract in rats bearing  benzene-induced leukemia. ACKNOWLEDGEMENT We appreciate the financial support of TETFUND Nigeria. The support of LAUTECH Animal House staff, our Research Assistants and Students are also appreciated.  REFERENCES 1.   Smith MT. The Mechanism of Benzene-induced Leukemia: A Hypothesis and Speculations on the Causes of Leukemia.   Environ Health Perspect.  2006; 104(Suppl 6): 1219  –  25p. 2.   Vaughan AT, Betti CJ, Villalobos MJ, et  al.  Surviving apoptosis: A possible mechanism of benzene-induced leukemia.  Chem Biol Interact  . 2005; 153  –  4: 179  –  85p.    Research & Reviews: Journal of Oncology and Haematology Volume 3, Issue 3  ISSN: 2319-3387 (online)  RRJoOH (2014) 16-21 © STM Journals 2014. 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