peroxidation and bFGF concentration in the HL-60 cell line

Natural and synthetic derivatives of benzo-γ-pyrones (i.e. flavones, chromones, and coumarins) and their synthetic analogues possess a wide range of biological properties in vitro and in vivo. In this paper we investigated the influence of two hydrazone compounds of chromones, 3-{[(2-dimethoxytiophosphoryl)-2-methylhydrazono]-methyl}-chromen-4-one (CH-3) and 2-amino-6-chloro-3-[(2-hydroxyethyl)-hydrazonomethyl]-chromen-4-one (A-12), on lipid peroxidation and bFGF concentration in the HL-60 cells. Both of the studied compounds had a significant influence on bFGF and TBARS in ranges -137.20 ~ 380.26% and -81.66 ~ -28.68%, respectively, in comparison with the control (counted as 0%).


INTRODUCTION
During cancer transformation processes each cell loses its ability to control replications and obtain the values of cancer cells (Ścibior-Bentkowska & Czeczot, 2009).The transformation of a healthy cell into a cancer cell is caused by the activation of proto-oncogenes and/or inactivation of suppressors and mutagenic genes.This process can be triggered by numerous factors including reactive oxygen species (ROS).It has been indicated that chronic, excessive ROS production can lead to oxidative stress, which has been observed in several hematopoietic malignancies including acute and chronic myeloid leukemia (AML and CML, respectively) (Sallmyr, Fan & Rassool, 2008;Hole et al., 2011).However, yet it is not clear whether oxidative stress in leukemia contributes to the suppression or rather to the development, progression, or maintenance of these diseases.
Literature provides us with evidences that tumor-derived ROS may promote cell survival, migration, metastasis, and proliferation (Clerkin et al., 2008;Nishikawa, 2008;Maraldi et al., 2009;Naughton et al., 2009).Zhou et al. suggested that AML relapse is associated with an increase in ROS production (Zhou et al., 2010), therefore indicating the protective role of oxidative stress in leukemic cells.However, excessive ROS production leads to double-stranded DNA breaks and patients with AML and excessive ROS production caused by an internal tandem duplication of fms-like tyrosine kinase 3 (Flt3-ITD) exhibit poorer prognosis (Sallmyr et al., 2008).Therefore, drugs, which decrease the oxidative stress in leukemic cells, may appear beneficial.
Oxidative stress in cancer cells can be associated with its influence on cell membrane lipid peroxidation (LPO) (Kulbacka, 2009).LPO products modify physical properties of cell membranes, causing a decrease in biomembranes fluidity, which leads to cell destruction and organ damage (Gao et al., 2002).LPO can be screened and monitored in cells by measuring the level of thiobarbituric acid reactive substances (TBARS).Previous study recommends the TBARS assay as a good choice for measuring the levels of lipid peroxidation in secondary products, such as MDA, 2,4-decadienols, saturated aldehydes (Esterbauer & Chjeeseman, 1994).It was indicated that MDA is detected in plasma of patients with chronic myeloid leukemia especially during its accelerated phase (Singh et al., 2009).
Cytokines and growth factors are produced in response to injuries and also act locally to modulate cell responses to vascular damage (Sahni et al., 1998).In particular, the basic fibroblast growth factor (bFGF) increases endothelial cell migration and proliferation, but also stimulates angiogenesis in vitro and in vivo, regulates the expression of proteolytic mediators of angiogenesis, including the urokinase receptor plasminogen activator (u-PAR), urokinase plasminogen activator (u-PA), and collagenase.After an injury, bFGF is released from vessel wall cells.
Recent evidence suggests that bone marrow neoangiogenesis plays an important role in the pathogenesis of associated marrow leukemia (Kulimova et al., 2006).Basic fibroblast growth factor can also act as an autocrine cytokine in those diseases.The receptors for VEGF (vascular endothelial growth factor), PDGF (platelet-derived growth factor), and bFGF are members of the split kinase domain family of receptor tyrosine kinases (RTK).Therefore, leukemia cells have receptors that specifically bind bFGF, which in turn stimulates their proliferation (Luizzo & Moscatelli, 1996).
Based on the results of our previous research on the HL-60 cell line (Łazarenkow et al., 2012), we choose two hydrazones of chromone to study their influence on bFGF and TBARS concentrations in human leukemia cells.
Cultivating and exposition of HL-60 cells on studied compounds.HL-60 cells were grown in agreement with our previous studies (Łazarenkow et al., 2012).After cultivating, the cells were counted (6.5•10 6 ) and exposed to A-12 or CH-3 in concentration of 1×IC 50 , (8.1 ± 0.6 and 36.1 ± 2.8 µmol/L, respectively) according to our previous findings (Łazarenkow et al., 2012).After 24 or 48 h of incubation, the cells were gathered, frozen, and keep at the temperature of -80°C for further biochemical analysis.The control cells were treated in the same way but without the examined derivatives.
Determination of lipid peroxidation.After incubation, the cells were counted and 1•10 6 cells were diluted with ice cold 0.01 mol/L phosphate buffer (pH 7.4) containing 0.9% NaCl, sonicated three times (60 s, pause 90 s, amplitude 30%), and centrifuged at 10 000 × g for 15 min.The content of lipid peroxidation products in the supernatant was assayed as thiobarbituric acid reactive substances (TBARS) using the Yagi method (1976) with some modifications.Briefly, 1 mL of solution, containing 0.25% hydrochloric acid, 0.375% thiobarbituric acid (TBA), 15% trichloroacetic acid, and 0.015% butylated hydroxytoluene was added to 100 µL of supernatant.The samples were boiled for 30 min in tightly closed tubes.After cooling to 10°C, 2.5 mL of butanol was added to each tube.After intensive shaking the tubes were centrifuged at 3 800 × g for 10 min.Finally, the upper solvent layer was removed and TBA-reactive substances were measured spectrofluorometrically using a luminescence spectrometer (LS-50, Perkin Elmer, Norwalk, USA) at an excited wavelength of λ = 515 nm and emission of λ = 546 nm.The concentration of TBARS was expressed as equivalents of 1,1,3,3-tetraethoxypropane that was used as the standard.All measurements were represented as means ± S.D. The statistical analysis was performed by ANOVA (analysis of variance) followed by the Shapiro-Wilk test.Statistical significance was set at p ≤ 0.05.
Determination of bFGF concentration in the studied cell line.After incubation, cells were washed with 0.01 mol/L phosphate buffer (pH 7.4), containing 0.9% NaCl, sonicated three times (60 s, pause 90 s, amplitude 30%) and centrifuged (1 200 rpm, 15 min, 4°C).Next, the supernatant was collected and assayed freshly for bFGF using the ELISA kit (Quantikine, R&D System, USA).The assay was performed following the manufacturer's instructions.The results were read using the BIO-TEK El x -800 spectrophotometer apparatus at λ = 562 nm.Final bFGF concentrations were calculated, using standard curve and described in pg/mL.The statistical calculations were prepared using the Stat-Soft Statistica 8.0 PL software.Statistical analysis was performed using t-test and Shapiro-Wilk test.Significance levels were set at p < 0.05 for t-test and p > 0.05 for Shapiro-Wilk test, respectively.

Lipid peroxidation assay
Changes in TBARS levels are presented in Fig. 2. A 24 h incubation of HL-60 cells with CH-3 and A-12 showed a marked reduction in the TBARS concentration (p < 0.05).However, further decline in TBARS level was observed only in case of the CH-3 group (48 hours of incubation, p < 0.001), while in cells treated with A-12 the TBARS level increased.Nevertheless, the TBARS content in the A-12 group after 24h of incubation was still lower than in the control.Our results also showed that CH-3 significantly lowered the TBARS level in HL-60 cells at 24 hours of incubation when compared to the A-12 group (p < 0.05).It may therefore indicate a more potent lipid peroxidationlowering potential of CH-3 in HL-60 cells.
Previous study evidenced that membrane lipid peroxidation may trigger the release of cytochrome c from the mitochondrial inner membrane and phosphatydylserine translocation which are critical events to the elicitation of apoptosis (Ma et al., 1999;Chiou et al., 2003).Decreasing of lipid peroxidation may indicate diminishing ROS generation under the influence of CH-3.Therefore, the lipid-lowering effect of CH-3 on HL-60 cells may be a possible explanation for the delay of HL-60 cell apoptosis observed in our previous report (Łazarenkow et al., 2012).

Investigation of bFGF concentration in the studied cell lines
In our experiment, the level of bFGF was significantly changed after 24 hours of incubation with both studied compounds, especially CH-3 (the difference in action between those compounds is 243%), while A-12 possessed more significant influence on the bFGF level after 48 hours (|x| > 5% in comparison with CH-3).This effect could be associated with inhibition of tyrosine phosphorylation and the mitogenactivated protein kinase pathway (Liekens et al., 1999;Lundin et al., 2003;Karam et al., 2012).
The results regarding A-12 effects on bFGF content after 24 hours of exposition showed similar negative values (-1.752 ± 0.042 pg/mL, Table 1).This indicated an overall lower concentration of bFGF in the HL-60 cell lines treated with A-12.It is possible that A-12 influences bFGF's metabolism or increases its releasing, however, this effect needs to be further analyzed.We also cannot exclude the possible interaction of A-12 with the ELISA kit components, although it is unlikely.All obtained statistical data are in agreement with our assumptions.
Figure 3 sums up the obtained results as it illustrates the percentage relative concentration of bFGF and TBARS in the samples in comparison with the control.Because this figure operates on relative high percentage values, the percentage deviations of these values are not visible on this scale; therefore, to make the Fig. 3 clearly readable, the deviation data was not included.Figure 3 exhibits the negative values of A-12 samples at 24 hours which were converted to [-(Control+|x|)].

CONCLUSIONS
Both of the studied compounds (A-12 and CH-3) had a significant influence on tested biochemical parameters.We observed an antagonism of their influence on the bFGF concentration in HL-60 cells.A-12 proved to be more effective in decreasing TBARS level at 24h of incubation while CH-3 showed a long lasting lipid-lowering effect on HL-60 cells.
Reassuming, the studied hydrazone derivatives of benzo-γ-pyrones prove the purpose of further investigations regarding their influence on proliferative parameters and oxidative stress in leukemic cells.

Figure 1 .
Figure 1.Structure of the studied compounds

Figure 3 .
Figure 3. Percentage comparison of the influence of CH-3 and A-12 on the concentration of bFGF in cells after 24 and 48 hours of incubation.Both compounds were tested in IC 50 concentrations, described in our previous paper (Łazarenkow et al., 2012).

Table 1 . The influence of CH-3 and A-12 on the concentration of bFGF in the HL-60 cell lines after 24 and 48 hours of incubation.
Statistical analysis made with t-test and Shapiro-Wilk (S-W) test.