Herbal melanin modulates tumor necrosis factor alpha (TNF-[alpha]), interleukin 6 (IL-6) and vascular endothelial growth factor (VEGF) production.Abstract
Recent studies have indicated that cytokines can enhance immunogenicity immunogenicity /im·mu·no·ge·nic·i·ty/ (-je-nis´it-e) the property enabling a substance to provoke an immune response, or the degree to which a substance possesses this property. and promote tumor regression. However, the means for modulating cytokine production are not yet fully investigated. In this study we report the effects of a herbal melanin melanin (mĕl`ənĭn), water-insoluble polymer of various compounds derived from the amino acid tyrosine. It is one of two pigments found in human skin and hair and adds brown to skin color; the other pigment is carotene, which contributes , extracted from Nigella sativa L., on the production of three cytokines [tumor necrosis factor tumor necrosis factor
n. Abbr. TNF
A protein that is produced in the presence of an endotoxin, especially by monocytes and macrophages, is able to attack and destroy tumor cells, and exacerbates chronic inflammatory diseases. alpha (TNF-[alpha]), interleukin 6 (IL-6) and vascular endothelial growth factor Vascular endothelial growth factor (VEGF) is an important signaling protein involved in both vasculogenesis (the de novo formation of the embryonic circulatory system) and angiogenesis (the growth of blood vessels from pre-existing vasculature). (VEGF VEGF vascular endothelial growth factor. )], by human monocytes, total peripheral blood mononuclear cells (PBMC PBMC Peripheral Blood Mononuclear Cell ) and THP-1 cell line. Cells were treated with variable concentrations of melanin and the expression of TNF-[alpha], IL-6 and VEGF mRNA in cell lysates and secretion of proteins in the supernatants were detected by RT-PCR and ELISA ELISA (e-li´sah) Enzyme-Linked Immuno-Sorbent Assay; any enzyme immunoassay using an enzyme-labeled immunoreactant and an immunosorbent.
n. . Melanin induced TNF-[alpha], IL-6 and VEGF mRNA expression by the monocytes, PBMC and THP-1 cell line. On the protein level, melanin significantly induced TNF-[alpha] and IL-6 protein production and inhibited VEGF production by monocytes and PBMC. In the THP-1 cell line melanin induced production of all three cytokine proteins. These observations raise the prospects of using N. sativa L. melanin for treatment of diseases associated with imbalanced cytokine production and for enhancing cancer and other immunotherapies.
[c] 2005 Elsevier GmbH. All rights reserved.
Keywords: Nigella sativa; Melanin; Cytokines; Immunomodulation
Nigella sativa L. (family: Ranunculaceae) is a herbaceous plant growing in the Mediterranean countries and Western Asia. The plant has been considered for millennia as one of the greatest healing herbs and has long been used for strengthening the immune system and for protection against various diseases (Khan, 1999). Voluminous research has been carried out on the medicinal properties of the seeds as antioxidants (Atta and Imaizumi, 1998), antimicrobial (Morsi, 2000), anti-inflammatory (Al-Ghamdi, 2001) and anticancer agents (Awad, 2003; Farah and Begum, 2003; Salomi et al., 1992). Various extractions, previously obtained from N. sativa seeds, included volatile and stable oils, whole and fractionated proteins, carbohydrates, crude fibers and minerals (Khan, 1999). More recently, melanin has been shown to occur abundantly in the seed coats of N. sativa L. (Hassib, 1998). Melanin is a pigment that causes darkness in animal and plant tissues. Although there has been little understanding of melanin morphology, structure and even size, there has been some agreement regarding the molecular units that comprise the polymerized structure. Typical units include dihydroxyindole (DHI DHI
see dairy herd improvement. ), dihydroxyindole-carboxylic acid (DHICA), and 5,6-indolequinone (IQ) (Stark et al., 2003; Tian et al., 2003). Melanins are divided into three groups: allomelanins in the plant kingdom and eumelanins and phaeomelanins in the animal kingdom. All melanins show a large content of stable free radicals, that are easily detectable by electron spin resonance electron spin resonance (ESR)
or electron paramagnetic resonance (EPR)
Technique of spectroscopic analysis (see spectroscopy) used to identify paramagnetic substances (see (ESR ESR - Eric S. Raymond ) and all show pronounced radical scavenging ability and metal chelation Chelation
The process by which a molecule encircles and binds to a metal and removes it from tissue.
Mentioned in: Heavy Metal Poisoning
chelation properties, both of these properties have been related to their general protective and antioxidant behavior (Pathak, 1995). Most of the plant melanins have not yet been given adequate attention in terms of their potential therapeutic uses. This study has been prompted by recent findings that melanin, from a number of botanical sources, has been found to be an immunologically active modulator of cytokines (Avramidis et al., 1998; Mohagheghpour et al., 2000; Pugh et al., 2005; Pasco et al., 2005). None of these studies have mentioned N. sativa L. as a source of melanin.
Cytokines are relatively low-molecular-weight proteins produced by many cell types (Feghali and Wright, 1997). They are pharmacologically active, exhibiting both beneficial and pathologic effects on the target cells. Imbalanced expression of cytokines has been implicated in the progression of many diseases (Arend and Gabay, 2004). Tumor necrosis factor alpha (TNF-[alpha]) is a major immune response-modifying cytokines produced primarily by cells of monocytic lineage. TNF-[alpha] has been shown to enhance antitumor responses and to promote tumor regression (Peron et al., 1999). Interleukin 6 (IL-6) is a cytokine produced by a number of normal and transformed cells (Bartold and Haynes, 1991; Van Meir et al., 1990). IL-6 promotes or inhibits the growth of tumor cells depending upon the cell type (Lu et al., 1995). Vascular endothelial growth factor (VEGF) is a potent multifunctional angiogenic cytokine (Brown et al., 1997). VEGF overproduction o·ver·pro·duce
tr.v. o·ver·pro·duced, o·ver·pro·duc·ing, o·ver·pro·duc·es
To produce in excess of need or demand.
o has been implicated in hematologic hematological, hematologic
pertaining to or emanating from blood cells.
total and differential white cell counts, hematocrit estimation, erythrocyte count. malignancies (Di Raimondo et al., 2000).
In the present study we report on possible immunogenic im·mu·no·gen·ic
Producing an immune response.
producing immunity; evoking an immune response. activity of a melanin extract from N. sativa, as evidenced by its effects on the expression of the three cytokines: TNF-[alpha], IL-6 and VEGF by monocytes, mixed PBMC cultures and THP-1 cell line THP-1. Our results indicate that melanin extracted from N. sativa significantly modulates the expression of TNF-[alpha], IL-6 and VEGF by the human monocytes, PBMC and THP-1 cell line. This finding suggests that N. sativa melanins may have an immunoregulatory activity that could contribute to future therapeutic interventions relating to diseases associated with imbalanced cytokine production and cancer.
Materials and methods
Preparation and characterization of N. sativa L. herbal melanin
Melanin has been extracted from the seed coats of the well-known herb N. sativa via alkali solublization and acid aggregation; purified by washing in excess amounts of highly distilled water and vacuum drying. The melanin nature of the extract has been verified via the standard analytical techniques: ESR, infra red (IR), ultraviolet-visible (UV-VIS), Nuclear Magnetic Resonance nuclear magnetic resonance: see magnetic resonance.
nuclear magnetic resonance (NMR)
Selective absorption of very high-frequency radio waves by certain atomic nuclei subjected to a strong stationary magnetic field. (NMR NMR: see magnetic resonance. ), XRD, Fluorescence, Solubility studies, amino acid composition and elemental analysis. The elemental analysis of carbon, hydrogen and nitrogen gave the (wt/wt percent) ratios of: 50.29, 5.77 and 4.4, respectively. The amino acid composition of the extract is as given in Table 1. These results are similar to the relative amino acid compositions, obtained for other melanins, published by Lisa Zeise (Zeise, 1995). The characterization of the extracted N. sativa melanin, using UV-VIS spectra for various concentrations, is as shown in Fig. 1a. The spectra show the well-known monotonically increasing absorption of melanin for photons of decreasing wavelength. The IR spectrum of NS melanin powder (1% in K Br pellets) (data not shown) agrees very well with the results reported in earlier studies of melanins from other sources (Bilinska, 1996). These spectra are characterized by strong absorption near the wave numbers of 1200, 1660, 2800-3100, 3400[cm.sup.-1]. All melanin are rich in natural stable free radicals that are easily detectable by ESR. ESR of the NS melanin, using an x-band spectrometer, has shown a single symmetrical line of narrow width (7-8G) at a g-value of 2.004 without a resolvable hyperfine interaction, as shown in Fig. 1b. The spin concentration for this resonance has been calculated to be around [10.sup.17] spins per gram at room temperature. The number of spins showed a little variation with variation in the extraction method. However, the line intensity increased, in accordance with the source intensity, on exposure of the NS melanin to UV (250 nm) radiations in situ. Both of the XRD and the NMR has shown very dense, overlapping broad spectral lines that are not amenable to detailed analysis. This is typical of melanins because of the interference of the free radicals with the NMR spectrum and the broad diffractions by the highly heterogeneous amorphous melanins in XRD.
[FIGURE 1 OMITTED]
The dark black to brown powders which we extracted from N. sativa (and would refer to, henceforth as herbal melanin (HM)), satisfy, in accordance with the above-mentioned studies, the standard criteria for melanin pigments as suggested by Zeise (Zeise, 1995).
The HM dried powders were dissolved in 0.1 M NaOH solution at a concentration of 1 g/l. The pH of the melanin solution was subsequently adjusted to pH 7.0, using concentrated HCl and filtered through 0.4 [micro]m filters. Melanin stock solutions for experimental use were prepared in distilled water at concentrations of 0.1-1 g/l.
Cell culture conditions
Human monocytic THP-1 cells were obtained from American Type Culture Collection (ATCC ATCC American Type Culture Collection, see there , Rockville, MD, USA). The cells were maintained in RPMI RPMI Rapid Prototyping & Manufacturing Institute
RPMI Roswell Park Memorial Institute
RPMI Royal Park Memorial Institute (culture medium) 1640 (ATCC, Rockville, MD, USA), supplemented with 10% fetal bovine serum Fetal bovine serum ( or foetal bovine serum) is serum taken from the fetuses of cows. Fetal Bovine Serum (or FBS) is the most widely used serum in the culturing of cells. In some papers the expression foetal calf serum is used. and 1% penicillin-streptomycin (Gibco BRL BRL
In currencies, this is the abbreviation for the Brazilian Real.
The currency market, also known as the Foreign Exchange market, is the largest financial market in the world, with a daily average volume of over US $1 trillion. ) in a humidified atmosphere of air with C[O.sub.2] 5% at 37[degrees]C. The medium was replaced 24 h prior to experiments by serum-free RPMI 1640 medium to avoid the effects of serum on gene expression.
Blood cells isolation
In order to isolate human peripheral blood mononuclear cells (PBMC), blood was collected from healthy human volunteers (age ranging 22-45). Informed consent was obtained before a 30 ml volume of peripheral blood was collected by venipuncture venipuncture /veni·punc·ture/ (ven?i-pungk´chur) surgical puncture of a vein.
ve·ni·punc·ture or ve·ne·punc·ture
n. in sterilized ethylenediamine tetra-acetic acid (EDTA EDTA: see chelating agents. ) tubes. PBMCs were separated on Ficoll-Paque density gradients (Pharmacia Biotech, Uppsala, Sweden) and cells from the interface were washed twice with calcium and magnesium free phosphate buffer saline (PBS PBS
in full Public Broadcasting Service
Private, nonprofit U.S. corporation of public television stations. PBS provides its member stations, which are supported by public funds and private contributions rather than by commercials, with educational, cultural, ) and seeded at a density of 1 x [10.sup.6] cells/ml per well in 24-well microtiter plates in RPMI-1640 medium. For isolation of monocytes, PBMC were plated for 2 h and the non-adherent cells were removed with three changes of warm PBS. Pure monocytes were then positively selected by means of anti-CD 14-coated microbeads (MiniMACS separation column; Milteny Biotec, Auburn, CA) following manufacturer's instructions. Flow cytometry analysis, using CD-14 and CD-45 antigen expression, showed that more than 90% of the cells were monocytes.
Induction and analysis of cytokine mRNA levels
Monocytes and PBMC (1 x [10.sup.6] cells/ml) were treated with HM melanin solutions at concentrations of 50 and 100 [micro]g/ml. The expression of TNF-[alpha], IL-6 and VEGF mRNA was tested 3 h later. THP-1 cells were similarly treated with HM and mRNA expression was tested after 3 h for TNF-[alpha] and after 24 h for IL-6 and VEGF. Escherichia coli lipopolysaccharide lipopolysaccharide /lipo·poly·sac·cha·ride/ (-pol?e-sak´ah-rid)
1. a molecule in which lipids and polysaccharides are linked.
2. (LPS LPS - Sets with restricted universal quantifiers.
["Logic Programming with Sets", G. Kuper, J Computer Sys Sci 41:44-64 (1990)]. ) was used as a positive control stimulus for cytokines induction at a concentration of 10 [micro]g/ml (E. coli LPS 026:B5 Sigma). Control cells from monocytes and PBMC taken from the same subject and control THP-1 cells were used as controls.
Total cellular RNA RNA: see nucleic acid.
in full ribonucleic acid
One of the two main types of nucleic acid (the other being DNA), which functions in cellular protein synthesis in all living cells and replaces DNA as the carrier of genetic was extracted from monocytes, PBMC and THP-1 cells using TRIzol reagent (Invitrogen) according to manufacturer's instructions. Two micrograms of total RNA were reverse transcribed into single stranded cDNA in a reaction mixture (30 [micro]l) containing 1 x reaction buffer (50 mM Tris-HCl pH 8.3, 75 mM KCl and 3 mM Mg[Cl.sub.2]), 0.5 mM deoxynucleoside triphosphate triphosphate /tri·phos·phate/ (tri-fos´fat) a salt containing three phosphate radicals.
A salt or ester containing three phosphate groups. mixture, 1 U of RNasin per [micro]l, 1.5 mM. oligo(dT) primer, and 10 U of Moloney murine leukemia virus The murine leukemia virus belongs to the gammaretroviral genus of the Retroviridae family of viruses, their hosts are vertebrates. It is a Type VI: positive sense ssRNA viruses that replicates through a DNA intermediate, reverse transcriptase. reverse transcriptase (Clontech Laboratories, Inc., Palo Alto, Calif.) per [micro]l at 42[degrees]C for 1 h. Reverse transcription was inactivated at 95[degrees]C for 5 min and the products were kept on ice until needed for the PCR PCR polymerase chain reaction.
polymerase chain reaction
Polymerase chain reaction (PCR) .
Amplification of cytokine cDNA was carried out using PCR primers obtained from Aragene Company (Aragene, Riyadh, Saudi Arabia). The primers used for TNF-[alpha] sense and antisense, respectively, were CTT-CTG-CCT-GCT-GCA-CTT-TGG-A and TCC-CAA-AGT-AGA-CCT-GCC-CAG-A. Conditions for RT-PCR were 30 cycles of 94[degrees]C for 30 s, 62[degrees]C for 1 min, 72[degrees]C for 2 min. Primers used for IL-6 were ATG-AAC-TCC-TTC-TCC-ACA-AGC-GC and GAA-GAG-CCC-TCA-GGC-TGG-ACT-G. Conditions for RT-PCR were 30 cycles of 94[degrees]C for 30 s, 65[degrees]C for 2 min, and 72[degrees]C for 2 min. The primers and RT-PCR conditions used for VEGF and VEGF isoforms (VEGF 121, VEGF165 and VEGF189) were described previously (El-Obeid et al., 2004). The house keeping gene, [beta]-actin, was used as an internal control for RT-PCR assay. The primers used for [beta]-actin were ATC-TGG-CAC-CAC-ACC-TTC-TAC-AAT-GAG-CTG-CG and CGT-CAT-ACT-CCT-GCT-TGC-TGA-TCC-ACA-TCT-GC. Conditions for RT-PCR were 40 cycles of 94[degrees]C for 45 s, 61[degrees]C for 45 s and 72[degrees]C for 2 min. The products were separated on a 2% agarose gel using electrophoresis and visualized by ethidium bromide staining.
Induction and analysis of cytokine protein levels
Protein secretion was tested in order to find out whether the changes in the cytokine mRNA levels following HM treatment were paralleled by changes in the cytokines produced. Cells from monocytes, PBMC and THP-1 were seeded at 1 x [10.sup.6] cells/ml in 12-well dishes. Cells were treated with HM (10, 50 and 100 [micro]g/ml) or LPS (10 mg/ml) for 24 h before collecting the supernatants. The levels of cytokine proteins produced in the supernatant were assayed by ELISA (R & D Sys., Minneapolis, MN, USA) following manufacturer's instruction. Supernatants were used as collected or at diluted concentrations. RPMI-1640 media were used as negative controls. Additional control was obtained by incubating 100 [micro]g HM in RPMI-1640 media for 24 h at 37[degrees]C. Duplicate readings for each standard, control and sample were recorded. The average absorbance absorbance /ab·sor·bance/ (-sor´bans)
1. in analytical chemistry, a measure of the light that a solution does not transmit compared to a pure solution. Symbol .
2. for each duplicate set of standards, controls, and samples was calculated using a standard curve. Concentrations were determined by extrapolation on the standard curve. It was verified that the addition of different concentrations of HM, in supernatants collected from HM treated cells, did not interfere with the measurements by determining the concentrations of the cytokines from control standard curves. These were constructed by plotting optical density values obtained from cytokine standards diluted in medium incubated with 100 [micro]g/ml HM for 24 h at 37[degrees]C. Repeated assays showed that HM did not affect the outcome of the cytokine assay (data not shown).
Cellular toxicity assay
The toxicity of HM on THP-1 was determined by the 3-(4,5dimethylthiazoly1-2)-2, 5-diphenyltetrazolium bromide (MTT) cell proliferation assay according to manufacturer's instruction (MTT# 30-101K. ATCC Rockville, MD, USA). Briefly, THP-1 cells were plated in triplicate at [10.sup.3] cells/ml and, subsequent to 6 h incubation at 37[degrees]C in 5% C[O.sub.2] incubator, HM was added at the concentration of 10, 50 and 100 [micro]g/ml. The medium was also used as a control to provide the blank for absorbance readings. Four hours before the 24-h time point, 10 ml of the MTT Reagent were added for 2 h followed by 100 ml of the Detergent Reagent. After 2 h incubation in the dark, the plates were read for absorption at 570 nm ([A.sub.570]) in an automatic ELISA plate reader. The results were quantified as optical density (O.D.) at 570 nm of cultures exposed to HM or non-exposed media.
All values were expressed as means [+ or -] standard error. Statistical differences were estimated by Mann-Whitney Test. Values of p<0.05 were considered significant.
Cytokine mRNA expression in human monocytes, PBMC and THP-1
RT-PCR showed low expression of 517 bp TNF-[alpha] transcript (Fig. 2, lane a) and 627 bp IL-6 transcript (Fig. 2, lane b) for control monocytes, which became more pronounced on addition of LPS (10 [micro]g/ml). Treatment with HM at 50 and 100 [micro]g/ml clearly induced both TNF-[alpha] and IL-6 mRNA expression as compared with control cells. Similarly, control PBMC showed low expression of TNF-[alpha] and IL-6 mRNA (Fig. 3, lanes a, b). Addition of HM and LPS up-regulated the expression of both cytokines.
Both control and LPS-treated monocytes (Fig. 2, lanes c, d) and PBMC (Fig. 3, lanes c, d) expressed the 334-bp band of the VEGF transcripts and VEGF isoforms VEGF121(435 bp) and VEGF165 (567 bp) transcripts, coding for the smallest secretable VEGF isoforms (Robinson and Stringer, 2001), while the larger cytoplasmic associated variant VEGF 189 (639 bp) was not expressed. Addition of HM at 50 [micro]g/ml slightly induced mRNA expression of the detected VEGF isoforms in monocytes (Fig. 2, lanes c, d) and PBMC (Fig. 3, lanes c, d). The house keeping gene, [beta]-actin, indicated comparable levels of the internal mRNA in the control and treated monocytes (Fig. 2, lane e) and PBMC (Fig. 3, lane e).
[FIGURE 2 OMITTED]
[FIGURE 3 OMITTED]
Control THP-1 cells expressed low levels of TNF-[alpha], IL-6, VEGF and VEGF 121, 165, 189 isoforms mRNA but not IL-6 mRNA. Addition of LPS and HM slightly induced the expression of the three cytokines (data not shown).
Cytokines secretion in human monocytes and PBMC
As shown in Fig. 4a control monocytes produced low levels of TNF-[alpha] protein. Addition of LPS (10 [micro]g/ml) strongly induced the production of TNF-[alpha] protein, as was previously reported (Bruggen et al., 1999). Our results clearly demonstrate that HM at 10, 50 and 100 [micro]g/ml induced TNF-[alpha] production in a dose-dependent manner. Similarly, control monocytes, that expressed low levels of IL-6, produced high levels of IL-6 on treatment with LPS or HM (Fig. 4b). Significant increase in TNF-[alpha] and IL-6 production was shown on using HM at concentration of 100 [micro]g/ml (p = 0.014 and p = 0.025, respectively). Both TNF-[alpha] and IL-6 results for protein expression agreed with our RT-PCR results.
Control monocytes constitutively secreted a mean of 67.81 pg/ml of VEGF (Fig. 4c). LPS induced the expression of VEGF to 89.14 pg/ml. In contrast to TNF-[alpha] and IL-6 results, the addition of HM inhibited the production of VEGF protein to very low or to undetectable levels giving a mean of 22.9 pg/ml (0.00-45.81 pg/ml). However, no inhibitory effect of HM on VEGF transcripts was detected by our RT-PCR.
PBMC spontaneously secreted a mean of 396.07 pg/ml (20.00-772.15 pg/ml) of TNF-[alpha] and 253.15 pg/ml (200.00-306.30) of IL-6 that were induced by LPS treatment (data not shown). On treating the cells with HM at 10, 50 and 100 [micro]g/ml the basal secretions of both of TNF-[alpha] and IL-6 were strongly induced to levels that could not be read by the machine. The supernatants had to be diluted down to 1:100 for TNF-[alpha] and 1:200 for IL-6 in order to carry out the assay. At the concentration of 100 [micro]g/ml, HM increased TNF-[alpha] production to about 45-fold over that of the control and increased IL-6 production to about 318-fold over that of the controls.
Detectable levels of VEGF have been secreted by the control and LPS-treated PBMC. Treatment with HM suppressed VEGF production by PBMC from 83.49 pg/ml (51.95-115.02 pg/ml) that secreted by control cells to 20.50 pg/ml (3.88-37.12 pg/ml) secreted by HM-treated cells.
[FIGURE 4 OMITTED]
Cytokines secretion in THP-1 cells
A very low level of TNF-[alpha] has been detected in the culture supernatant of control THP-1 having a mean of 21.35 pg/ml (Fig. 5a). This result is supported by our RT-PCR results, indicating that THP-1 cells have constitutively produced low levels of TNF-[alpha]. Treatment of the cells with LPS significantly increased TNF-[alpha] production (p = 0.020). Addition of HM at 50 and 100 [micro]g/ml increased TNF-[alpha] production to 276.80 and 249.42 pg/ml, respectively.
Fig. 5b shows no expression of IL-6 protein by control THP-1 cells. LPS significantly induced IL-production (p = 0.037). HM at 50 and 100 [micro]g/ml induced IL-6 production to 16.85 and 7.42 pg/ml, respectively.
As shown by Fig. 5c, control THP-1 cells constitutively secreted a mean of 346.40 pg/ml of VEGF protein that have been induced by LPS treatment to 911.70 pg/ml and by HM (50 and 100 [micro]g/ml) to 912.37 and 769.97 pg/ml, respectively.
Results of cellular toxicity assay
In order to check if the concentrations of HM used in the study caused cell toxicity or affected the viability of the cells we performed MTT cell proliferation assay. THP-1 cells were cultured in the presence and absence of 10, 50 and 100 [micro]g/ml of HM. Fig. 6 showed that HM is not toxic to the cells even at the highest employed concentrations (p = 0.166) as determined by four separate experiments.
In this study, we tested the effect of melanin extracted from N. sativa L. on the production of TNF-[alpha], IL-6 and VEGF in human monocytes, PBMC and THP-1 cell line on the transcriptional and translational levels. Melanin significantly induced the production of TNF-[alpha] and IL-6 and inhibited the production of VEGF by monocytes and PBMC. Treatment of THP-1 cell line with melanin induced the production of TNF-[alpha], IL-6 and VEGF.
[FIGURE 5 OMITTED]
Previous studies on the effects of N. sativa on the immune system have shown that total extracts of N. sativa or its fractionated proteins could modulate cytokines production (Haq et al., 1999; Swamy and Tan, 2000). However, in these studies the actual effective component(s) in N. sativa underlying the results obtained remained to be identified. On the other hand, recent studies have shown that both synthetic melanin (Mohagheghpour et al., 2000) and plant melanin (Avramidis et al., 1998) can also modulate cytokines production.
Our data are, however, the first to specify that melanin extracted from N. sativa has a direct modulatory effect on cytokine production by monocytes, PBMC and THP-1 cells. RT-PCR results showed that HM up-regulated the expression of TNF-[alpha] and IL-6 mRNA in monocytes and PBMC in a dose-dependent manner. These findings were corroborated through ELISA determination of TNF-[alpha] and IL-6 protein expression under the same experimental conditions. The levels of both cytokines were significantly elevated following treatment with HM. VEGF expression was also found to change after melanin treatment. In contrast to its action on TNF-[alpha] and IL-6, HM inhibited the secretion of basal VEGF by monocytes and BPMC.
Because of potential variability in cytokine production by monocytes and PBMC from different individuals (Jacob et al., 1990; Webb and Chaplin, 1990), we used the human monocytic cell line, THP-1, that exhibits high inducibility for TNF-[alpha] gene transcription similar to that of freshly isolated monocytes (Yao et al., 1997). HM induced mRNA expression and increased the spontaneous production of TNF-[alpha], IL-6 and VEGF by the THP-1 cells, thus providing further evidence of its modulatory effect on cytokines. Recently, Pasco and coworkers (Pugh et al., 2005) tested the effect of Echinacea echinacea (ĕk'ənā`shēə), popular herbal remedy, or botanical, believed to benefit the immune system. It is used especially to alleviate common colds and the flu, but several controlled studies using it as a cold medicine have melanin on THP-1 cells and reported that it increased the production of IL-1[beta] in a dose-dependent manner. They also showed that melanin has a direct effect on mice mucosal immune function that results in an increased production ex vivo of interferon-[gamma], IgA and IL-6.
[FIGURE 6 OMITTED]
The genes of TNF-[alpha], IL-6 and VEGF in human monocytes and PBMC are generally under strict inductional control. Nonetheless, a variety of stimuli, such as endotoxins and other cytokines, are known to modulate their expression (Bruggen et al., 1999; Sakuta et al., 2001; Tosato and Jones, 1990). The exact mechanism(s) by which melanin molecules affect genes transcription and cytokines production are not yet determined, though their roles as antioxidants, free radical scavengers plus their possession of a rich content of stable free radicals are well documented (Bustamante et al., 1993; Enochs et al., 1993; Zeise, 1995; Pathak, 1995; Riley, 1997; Sichel et al., 1991). Melanin molecules regulate gene expression of several cytokines by modulating the genes and transcription factors that are involved in the immune responses (Marui et al., 1993; Weber et al., 1994; Munoz et al., 1996). Nathens et al. (2001) have shown that antioxidant pyrrolidine pyrrolidine /pyr·rol·i·dine/ (pi-rol´i-din) a simple base, (CH2)4NH, obtained from tobacco or prepared from pyrrole.
n. dithiocarbamate increases LPS-stimulated TNF-[alpha] release in murine macrophages. They suggested stimulatory and species-dependent role of antioxidant on gene expression (Nathens et al., 2001). The increased secretion of TNF-[alpha] and IL-6 by monocytes, PBMC and THP-1 cells, after HM treatment, might suggest a similar possibility of a modulatory effect of melanin as an antioxidant and free radical scavenger free radical scavenger Free radical inactivator Any compound that reacts with free radicals in a biological system, ↓ free radical-induced damage, and protects against the indirect effects of free radicals produced by ionizing radiation, etc Examples on cytokines expression.
The use of cytokines and cytokine genes such as TNF-[alpha] and IL-6 to promote tumor regression/rejection has been established in various mouse tumor models (Colombo et al., 1992; Fearon et al., 1990). Recently, Xiao et al. have shown that IL-6 could inhibit the growth and development of colon cancer (Xiao et al., 2000). Thus, HM might be applied, within novel approaches, for boosting of anticancer immunotherapy.
VEGF is an important angiogenic cytokine with critical roles in tumor angiogenesis (Dvorak, 2002). Different strategies have been developed to inhibit VEGF and the kinase activity of VEGFR VEGFR Vascular Endothelial Growth Factor Receptor (Melnyk et al., 1999; Chen et al., 1997; Ogawa et al., 2002). Our results indicate that HM significantly inhibits VEGF production by monocytes and PBMC. This result might have a profound effect on therapeutic strategies aimed at inhibiting VEGF overproduction and related diseases (Bell et al., 1999; Dvorak, 2002; Kim et al., 1993).
Modulation of cytokines production by a herbal extract from a well-known herbal spice is interesting in view of its safety as a harmless non-toxic natural product in common use. The results presented here should also merit further detailed studies of correlation of the properties of melanin with the well-known folk medicinal attributes of N. sativa L.
We are thankful to Prof. M.H. Faris for reading and commenting on the manuscript, Dr. Gamal Mohamed for assistance with the statistical analysis and Mr. Ibrahim Awad Mohammed for preparation of the drawings.
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Bustamante, J., Bredeston, L., Malanga Noun 1. malanga - tropical American aroid having edible tubers that are cooked and eaten like yams or potatoes
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A. El-Obeid (a), S. Al-Harbi (a), N. Al-Jomah (a), A. Hassib (b,*)
(a) Biological Repository Center, Department of Molecular Pathology, King Faisal Specialist Hospital History
King Faisal Specialist Hospital was established on 450,000 square meters of land which was donated by King Faisal who laid the cornerstone of this hospital in 1970 (the current total land of the hospital is 920,000 square meters). & Research Center, Riyadh, Saudi Arabia
(b) Faculty of Science, King Saud University King Saud University (جامعة الملك سعود) is Saudi Arabia's oldest and premier university, located in the capital Riyadh. , Riyadh, Saudi Arabia
Received 25 November 2004; accepted 17 March 2005
Abbreviations: TNF-[alpha], tumor necrosis factor alpha; IL-6, interleukin 6; VEGF, vascular endothelial growth factor; PBS, phosphate buffer saline; PBMC, peripheral blood mononuclear cells; MTT, 3-(4, 5 dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide; HM, herbal melanin
*Corresponding author. Tel.: +966 1 4676437; fax: +966 1 4608185.
E-mail address: firstname.lastname@example.org (A. Hassib).
Table 1. Relative amino acid composition (wt/wt %, SD: 0.032) in Nigella sativa L. melanin Amino acid % Ratio wt/wt Amino acid % Ratio wt/wt Aspartic 4.01 Isoleucine 1.68 Threonine 1.91 Leucine 3.05 Serine 1.85 Tyrosine 1.83 Glutamic acid 7.97 Phenylalanin 1.92 Glycine 2.36 Histidine 1.14 Alanine 2.01 Lysine 1.64 Valine 2.40 Arginine 3.58 Methionine 0.52