Chemicals
Reagents and chemicals were purchased from sigma Aldrich: FMLP, cytochalasin, HEBES, 3,3′,5,5′- tetramethylbenzidine, N-Methoxy-Suc-(Ala)2-Pro-Val-p-Nitroanilide (10− 4) prepared in 1-Methyl-2-Pyrrolidinone, Curcumin, Doxorubicin, and XTT kit, and Penicillin/Streptomycin 5000Units.
Cell lines and culture conditions
EMT6 (ATCC® CRL-2755™) and NIT1 (ATCC® CRL-2055™) cell lines ATCC were purchased and delivered by BDcome, Algeria. EMT6 was cultured in the DMEM/F12culture medium while NIT1 was cultured in RPMI 1640 medium. The culture media for both lines were supplemented with 15% fetal bovine serum (FBS) and 1% Penicillin/streptomycin 5000 Units of Antibiotics. Cells were incubated at 37°C in 95% Air and 5% CO2. Hanks’ balanced salt solutionHBSS2 and HBSS1 were used for neutrophil isolation and its activity study as described in the methods section.
Neutrophil isolation
Neutrophils were isolated in a procedure similar to that documented by Bouriche and her collaborators [27] from freshly venous blood of healthy adult volunteers who declared that they were non-smokers and hadn't taken any anti-inflammatory drugs. All manipulations were performed in sterile conditions. In brief, 10 ml of fresh blood was drawn into a polypropylene conical tube containing 2 ml of lithium heparin as an anticoagulant. Then, sedimentation of erythrocytes was enhanced by the addition of 1ml of dextran 10%. After sedimentation for 1hr at room temperature, leukocyte-rich plasma was transferred to a sterile 15-mL conical tube and layered on a histopaque 1077 gradient. The gradient was then centrifuged at 1000 rpm for 25 min at 4°C (Rotina R 35, Germany). The supernatant containing mononuclear cells was discarded while the pellet containing granulocytes and red cells was recuperated. The remaining erythrocytes were lysed by suspending the pellet in 1 ml of fresh cold distilled water followed by adequate mixing of suspension for the 30s. Promptly, the tonicity of suspension was restored by adding 5 ml of cold HBSS1 at pH 7.4. The cellular suspension was then centrifuged at 1000 rpm for 10 min at 4°C. This step of hypotonic hemolysis was repeated three times. After the last centrifugation, the supernatant was removed whereas the cells were resuspended in 2 ml of Ca2+ and Mg2+-free HBSS and kept in an ice bath. Cells isolated through this technique were always viable with a rate greater than 95%, as established by the trypan blue exclusion test, and higher than 95% of isolated cells were neutrophils.
Cell counting
The concentration of isolated neutrophils was determined using a Thoma counting chamber (Thoma cell). For this goal, 20 µl of neutrophils suspension was mixed with 380 µl of Turkish solution. The coverslip was set over the Thoma counting chamber. Then, the neutrophil’s suspension was well homogenized using a pipette, transferred to a cell Thoma cell and the neutrophil cells were counted in the 16 squares under the light microscope (Nikon, EclipseTs2). The estimation of neutrophil concentration was done as follows:
[C] = N x 20 x 104
[C]: Cell concentration (cell/ml).
N: Number of cells counted in the 16 squares.
20: Dilution factor.
104: Correction factor used to obtain the number of cells in a volume equal to 1ml.
Cytotoxic effect of curcumin on neutrophils
The effect of curcumin on the viability of neutrophils was evaluated using the trypan blue exclusion test, a cell viability assay. The latter was performed according to the method of Lucisano-Valmet al. [28] with some modifications. Briefly, aliquots of 250µl of neutrophils in HBSS (3 x 106 cells/ml) were incubated for 15 min at 37°C with different concentrations of curcumin dissolved in DMSO (1%, control solvent) and HBSS (negative control). After that, an equal volume of 0.4% trypan blue solution was added to each aliquot. A volume of cell suspension from each aliquot was loaded into a Thoma cell, and stained (non-viable) and unstained (viable) cells were counted under a light microscope (Nikon, EclipseTs2). After staining, counting started during the 3 min after the cells began to stain. Cell viability is considered normal when 90 to 95% of cells are colourless. The viability (%) was calculated using the formula:
$$Viability \left(\%\right)=\left(\frac{Number of nonestained cells}{total cells}\right)x100$$
MPO activity assay
The inhibitory effect of curcumin on MPO activity was measured according to the method of Wanikiat et al. [29] which use the 3,3′,5,5′- tetramethylbenzidine as a substrate. The oxidation of 3,3′,5,5′- tetramethylbenzidine by MPO in the presence of H2O2 lead to the formation of a soluble chromophore. Briefly, the MPO was produced from neutrophils by incubating PMNs (5x 106 cell/ml) in HBSS2 for 15min at 37◦C in the presence of 37.5 µl of FMLP/cytochalasin B (10− 6 M/10− 5 M). The suspension was then centrifuged at 3000 rpm for 10min at 4◦C and the resulting supernatant was further assayed. The assay was carried out on a 96-well microplate. 20 µl of supernatant was pre-incubated at 37◦C for 10 min with 30 µl of different dilutions of curcumin solution. Further, 25 µl of tetramethyl benzidine (1.6 mM), then 100 µl of H2O2 (0.003% prepared in phosphate buffer pH 5.4 containing 0.05% of hexadecyltrimethylammonium bromide) were added to each well of the microplate. The reaction was terminated with 50µl of H2SO4(2N) at 5 min. The absorption was measured at a wavelength of 450 nm with a microtiter plate reader. Enzymatic activity was expressed as a percentage compared with the non-treated control set to 100%.
$$Activity \left(\%\right)=\left(\frac{Absorbance sample}{Absorbance control}\right)x100$$
Elastase activity assay
The inhibitory effect of curcumin on elastase was evaluated as described before [30] with slight modifications. Neutrophil suspension 5.5 million/ml was equilibrated in HBSS2 for 15 min at 37◦C in the presence of FMLP/cytochalasin B (10− 6 M/10− 5 M) as stimulators of degranulation and elastase release. The supernatant containing elastane was then separated by centrifugation at 3000 rpm for 15 min at 4°C. Further, on a 96-well microplate, 75µl of this supernatant was pre-incubated with various concentrations of curcumin (50µl) for 10min at 37 ◦C. 75 µl of a specific substrate (le N-Methoxy-Suc-(Ala)2-Pro-Val-p-Nitroanilide10− 4) was added and the incubation was continued for 40 min at 37◦C. Elastase activity was determined by measuring absorbance at 405 nm of released p-nitroaniline against wells containing non-treated supernatant (without curcumin) which represents 100% of elastase activity.
$$Residual Activity \left(\%\right)=\left(\frac{Absorbance sample}{Absorbance control}\right)x100$$
Cytotoxic effect of curcumin on EMT6 breast cancer and NIT-1cell line
The curcumin’s cytotoxicity toward EMT6 and NIT-1 β cell lines was evaluated using an XTT assay [31]. Curcumin concentrations ranged from 1 to 30 ug/ml and from 3 to 60 ug/ml for EMT6 and NIT-1 β respectively. Doxorubicin was used as a positive control according to the protocol described by Rezgui and his collaborators [32]. Briefly, The EMT6 and NIT1 cell lines were cultured in a supplemented DMEM culture medium (5% fetal bovine serum + 1% Penicillin streptomycin antibiotic) and RPMI1640 (10% FBS and 1% Penicillin streptomycin antibiotic) respectively. Cell lines were deposited in a 96-well plate (104 Cell/190µl/well) and incubated at 37°C and 5% CO2 for 24 hours.
After 2 hours of incubation, the cell lines were treated with different concentrations of curcumin, and the plate was incubated under the same conditions for 72 hours. After 72 h, the culture medium was removed, the wells were washed with PBS, and 200 µl of the fresh medium was added. The XTT1 and XTT2 reagents from the XTT Kit were mixed and 50 ul of this mixture was added to each well. After 4h of incubation at 37°C and 5% CO2, absorbance was measured at 450–500 nm using an automatic plate reader.
Statistical analysis
All experiments were performed in triplicate and the results are expressed as mean ± standard deviation of the mean (SEM) in bar graphs or line graphs. Statistical differences between the means were determined by the one-way ANOVA followed by Tukey’s post hoc test. The differences are considered significant if the p-value is less than 0.05 (p < 0.05). Statistical analysis was carried out using GraphPad Prism 7 Software.