| | Cytotoxic and inhibitory effects of 4,4′-dihydroxy chalcone (RVC-588) on proliferation of human leukemic HL-60 cellsReceived 1 August 2001; accepted 19 February 2002.
1. Introduction  Flavonoids compounds are present in normal human diet and represent one of the most important and interesting classes of biologically active compounds. Chalcones (1,3-diphenyl-2-propen-1-ones), considered as the precursor of flavonoids and isoflavonoids are widely distributed in nature from ferns to higher plants, were studied in terms of its multiple biological actions including anti-inflammatory [1], analgesic and antipyretic, anti-mutagenic effects [2], cytotoxic and anti-oxidant activity in vitro and in vivo [3], [4], [5], [6], [7]. Chemically they consist of open-chain flavonoids in which the two aromatic rings are joined by a three-carbon α-, β-unsaturated carbonyl system. Of particular significance, chalcones exhibited varying degrees of inhibition on cell proliferation and demonstrated anticancer properties [8], [9], [10], [11], [12]. Conversion of various acyclic conjugated styryl ketones into the corresponding Mannich bases was often accompanied by increased bioactivity both in vitro and in vivo [13]. Various Mannich bases of chalcones and related compounds displayed significant cytotoxicity towards murine P388 and L1210 leukemia cells as well as a number of other tumor cells [14]. Introduction of hydroxyl groups to chalcone structure has increased the cytotoxic effects [14]. The dihyroxy chalcone, which was found to be the most active tumor reducing agent, was also found to be the most potent inhibitor of lipid peroxidation [15]. It also caused an inhibition on lactate production in Ehrlich ascites tumor cells [16] and has anti-inflammatory effects in anterior ocular inflammation [17]. The synthesis of chalcones for cytotoxic activities appears to be an unexplored field and mechanism of toxicity against various human tumor cell lines is still obscure. Thus, the identification of new chalcone analogues will be important in the continued development of this class of agents as antitumor drugs. In the present study, we have evaluated the cytotoxic and differentiative effect and possible mechanisms underlying its tumor reducing activity of synthetic 4,4′-dihydroxy chalcone (RVC-588), 5,3′-dimethylaminomethyl chalcone (CS-MR1), 4-[2-(p-hydroxybenzoyl) vinyl] phenyl nicotinate (CS-NA1) and 4-hydroxy-3-(dimethyaminomethyl) asetofenone (AF-M1) in HL-60 leukemic tumor cell line. 2. Materials and methods 2.1. Chemicals RVC-588 was synthesized by Dimmock’s method [14]. CS-MR1 was synthesized from RVC-588 by Böhme’s method [14], [18]. CS-NA1 was synthesized as nicotinate ester of RVC-588 in consistent with soft drug approach [14]. AF-MR1 was synthesized from p-hydroxy-asetophenone [14]. CD11b/Mac-1 and CD11c R-phycoerythrin-(R-PE)-conjugated mouse anti-human monoclonal antibodies and CD14 fluorescein isothiocyanate (FITC)-conjugated mouse anti-human monoclonal antibody were purchased from Becton Dickinson Biosciences (San Jose, CA, USA). The cell death detection enzyme-linked immunoabsorbant (ELISA) assay kit, apoptotic DNA ladder kit and DNA molecular weight marker IX were obtained from Roche Biochemicals (Mannheim, Germany). All chemicals and cell cultures mediums were provided from Sigma–Aldrich Chemie GmbH (Deisenhofen, Germany) unless otherwise specified. All chemicals used for synthesis were purchased from Sigma and Merck. All TLC were on Merck Silica Gel 60F254 glass plates which had a layer thickness of 0, 25 mm. 2.3. The melting point of DHC was found to be at 197 °C 4,4′-Dihydroxychalcone was prepared in a high yield (71.43%) by using Claisen–Schmidt reaction. Several spectrophotometric properties of the synthesized drug were investigated. The electronic absorption spectra were obtained by using a Shimadzu UV-160A spectrophotometer (Shimadzu Inc., Kyoto, Japan). The UV spectrum of DHC in methanol was as follows: [λmax ( (1.72), 348 (4.468), 235.5 (4.121), 205 (4.206) nm]. IR spectra were determined by using a Jasco FT-IR-400 spectrophotometer (Jasco International Co., Tokyo, Japan). The IR spectrum of DHC with KBr was as follows: [νmax(cm−1)⇒3292, 1644, 1590, 1511, 1344, 1290, 1221, 1034, 976, 81]. The  NMR spectra were recorded by using Bruker DPX-400 (400MHz) instrument (Brucker Analytik GmbH, Rheinstetten, Germany). The  NMR spectrum of DHC was as follows: [(400MHz, D2O); 10.05 (2H, brs, OH); 8.03 (2H, dd, J=8.70 and 9.60); 7.80 (2H, d, J=8.60); 7.68 ( , Ha, d, J=15.40, E isomer, 7.62 ( , Hb, d, J=15.80, E isomer, 6.88 (2H, d, J=8.70); 6.82 (2H, d, J=8.60) ppm]. The  NMR spectroscopy and IR spectroscopy, when utilized revealed that the olefinic bond in this compound adopted the E-configuration. The spectral and physical data of RVC-588 were consistent with those reported in the literature [19]. The molecular structure of RVC-588, CS-MR1, CS-NA1 and AF-MR1 are shown in Fig. 1. 2.4. Cell cultures and experimental procedures Human myeloid leukemic cell line HL-60 was kindly supplied by Dr. E. Göker (Oncology Department of Ege University School of Medicine, Izmir, Turkey). Cultured cells were grown in RPMI-1640 medium supplemented with 1% non-essential amino acids, 1% l-glutamine, 100U/ml penicillin, 10mg/ml streptomisin and 10% heat inactivated foetal calf serum at 37°C in humidified air containing 5% CO2. The cells were washed and then covered with cell culture medium at the beginning of experiments. For all experiments RVC-588, CS-MR1, CS-NA1 and AF-MR1 were prepared at 1mM stock solution. All compounds were dissolved in 0.05M DMSO. In dose–response study, compounds then added to the cell culture medium directly to obtain different final concentrations (1–20μM). The cells were further incubated at 37°C for 24, 48, 72 or 96h periods in the kinetic study. The control cells were treated with the same amount of vehicle alone. Significant cytotoxic effect was only detected with RVC-588 treatment. Thus, characteristics of RVC-588 induced cytotoxicity was evaluated by further experiments. 2.5. Cell viability and evaluation of RVC-588 induced cytotoxicity The viability of the cells after treatment with increasing concentrations of RVC-588, CS-MR1, CS-NA1 and AF-MR1 were measured using classic trypan blue dye exclusion method. The method is based on the exclusion of the trypan blue by metabolic active living cells. Cells were inoculated onto a 6-well plate at an initial density of 5×105 cells/ml and exposed to varying concentrations of RVC-588, CS-MR1, CS-NA1 and AF-MR1 for 24, 48, 72 and 96h. At the end of the exposure, cell culture medium was removed by vacuum aspiration and the suspended cells were removed from the medium by centrifugation at 1000×g for 10min. A total of 100μl suspensions of the cells were treated with equal amount of a 1:10 (v/v) mixture of 0.4% filtered trypan blue stain in Hanks balanced salt solution (HBSS). Cell counting was performed with a haemocytometer under an inverted phase contrast microscope (Olympus, Tokyo, Japan), and the blue (dead) cells counted by eye under 40× magnification. Viability was expressed as viable cell number and required accurate total cell count information. 2.6. Determination of cell differentiation The morphology was evaluated by light microscopy (100×) by Giemsa staining for RVC-588 treated cells (2μM for 72h). The extent of differentiation by RVC-588 was assessed by cytofluorometry in a fluorescence activated cell sorter scan (FACScan, Becton Dickinson, Mountain View, CA, USA) using monoclonal antibodies to glicoprotein surface markers, including CD11b/Mac-1, CD11c and CD14 (Becton Dickinson Biosciences, San Jose, CA, USA). 2.7. Cell death ELISA assay Cells from control and RVC-588 treatment groups were processed and analyzed for cytotoxic histone-bound DNA fragments using the cell death ELISA kit, essentially as described by the manufacturer. Briefly, cells were plated in 8-well culture at an initial seeding density of 2×104 cells per well and were grown to near confluence. In kinetic studies, to evaluate the mechanism of cellular death by RVC-588 cultures were treated with IC50 dose of 2μM RVC-588 or PBS for 24, 48, 72, or 96h. Following treatment for the requisite period, medium was removed carefully by aspiration and was centrifuged (10min at 1000×g) to collect cells. Then to rupture cells, lysis buffer was added to each well and the plate was incubated with shaking for 30min at room temperature. Lysates were subsequently transferred to Eppendorf microcentrifuge tubes and were centrifuged at 16,000×g for 5min. Triplicate dilutions of cell lysates were placed into wells of a microtiter plate, biotine and anti-DNA conjugated to horseradish peroxidase was added. The plate was then incubated at room temperature for 2h, afterwards, the wells were washed three times to remove unbound antibodies and the nucleosomes were quantitated by determining the amount of peroxidase retained within immunocomplex. The absorbance proportional to the degree of cell viability was determined after the addition of 2,2′-azino-di-[3-ethylbenzthiozolin-sulfonate], as substrate at 15min by an ELISA reader (Bio-Rad, Coda, Hercules, CA, USA) at 595nm. The absorbance of the blank, which contained all reagents but no sample was subtracted from the test results. The apoptosis level was calculated by the formula As/Ao, where As represents the experimental sample absorbance and Ao represents the average absorbance produced in the assay using lysate from control cells. ELISA results were normalized for cell number. 2.8. Apoptotic DNA ladder Suspended cells were pelleted and lysed with lysis solution. After lysis of cultured cells in binding buffer, the lysate was applied to a filter tube with glass fiber fleece and passaged through the glass fiber fleece by centrifugation. Residual impurities were removed by a wash step and subsequently DNA was eluted in elution buffer from the column according to manufacturer’s instructions. Samples were separated by electrophoresis on a 1% agarose/Tris-acetate-EDTA (TAE) gel. After electrophoresis, visualization of DNA band was performed by staining with ethidium bromide and viewing on an ultraviolet transsilluminator (Bio-Rad, Hercules, CA, USA). The gel was photographed under ultraviolet light with polaroid film. 2.9. Statistical analysis Statistical significance was calculated with SPSS 9.0 (SPSS Inc., Chicago, IL, USA). Histograms from flow cytometry or images from ladder analysis were representatives from three independent experiments. Other numerical data were presented as mean from at least three independent experiments and analyzed using one-way ANOVA with Scheffe’s test. A P-value less than 0.05 was considered as statistically significant. 3. Results 4. Discussion Our goals in this study are to demonstrate cytotoxic, differentiative and apoptotic effects of RVC-588 and CS-MR1, CS-NA1 and AF-MR1 on HL-60 cells. RVC-588 showed high growth inhibition potency with IC50 value of 2μM. From a comparison of our results with values reported in the literature, it is interesting RVC-588 showed a growth inhibitory effect in the concentration range (1–2μM) as the most active chalcone analogue previously tested [20]. Nevertheless, the toxicity may diminished by esterification of the compounds to produce soft drugs; as we found no significant effects of CS-MR1, CS-NA1 and AF-MR1 on cell viability in HL-60 cells. In general, hydroxy substituted compounds were found to be the most potent anti-oxidant and cytotoxic in tumor cells [5] and this structure–activity relationship might have a role on high cytotoxicity of RVC-588 in HL-60 cells. Those activities were attributed, in part to alkylating ability of oleofinic groups which are conjugated with a carbonyl function, to guanine bases in DNA [14]. To gain further insight into this aspect we evaluated one of the most active compound, RVC-588, to evaluate their effect on the proliferation, differentiation and apoptosis. The human myeloid HL-60 leukemia cell line is a useful tool for studying the molecular mechanisms involved in the control of the growth and differentiation during myelopoesis. These cells respond to specific chemical stimuli by acquiring either a granulocyte-like phenotype [21] or a monocyte-or-macrophage like phenotype [22]. The acquisition of mature phenotypes can be demonstrated by a variety of differentiation markers [23]. HL-60 cells exposed for 24–96h to medium supplemented with IC50 doses of RVC-588 (2μM) showed no significant increase for CD11b, C11c and CD14 cell surface markers expression for monocytic or granulocytic differentiation. However, statistically significant increases recorded at 72h especially for CD14, is negligible for assessment of differentiation in HL-60 cells. Thus, we found that RVC-588 did not modulate differentiation in HL-60 cells as previously reported [24], [25], [26]. Flow cytometric analysis of B16 cells revealed that chalcones inhibit cell proliferation and induce apoptosis [26] although it is not yet certain that Mannich bases of chalcones play roles in the apoptosis induction processes. We have demonstrated that RVC-588 induces apoptosis insignificantly at low concentrations (2μM) determined by ELISA assay but IC50 dose of RVC-588 caused late apoptotic-cytotoxic smear patterns in time dependent manner. In general, there is a correlation between time-course and apoptosis. Our findings in this study revealed that RVC-588 induces cytotoxicity of HL-60 cells, but has no effect on differentiation. Antitumor activity of RVC-588 is probably due to their unsaturated ketone groups which alkylate the DNA bases [14]. However, chalcones represent a new group of cytotoxic agents and particularly RVC-588 serves as a useful prototypic model molecule. Further studies about the characteristics of chalcone structures and on the mechanism of cell death may yield information that will permit an evaluation of the synthetic chalcone analogues on tumor growth in vivo. Acknowledgements We are thankful to Dr. E. Goker and Msc. N. Selvi for their generous support in the preperation of this manuscript. G. Saydam collected, assembled the data, provided the concept and design and analyzed the data. H.H. Aydin analyzed the data and drafted the manuscript. F. Sahin assembled the data and provided statistical expertise. O. Kucukoglu and E. Erciyas provided study materials. E. Terzioglu helped to collect and assemble the data. F. Buyukkececi provided the funding. S.B. Omay contributed to many of the aspects of this study including the drafting and revision of the article. References [1].
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a Department of Hematology, School of Medicine, Ege University, Bornova, Izmir, Turkey b Department of Biochemistry, School of Medicine, Ege University, Bornova, Izmir, Turkey c Department of Pharmaceutical Chemistry, School of Pharmacy, Ege University, Bornova, Izmir, Turkey d Department of Immunology, School of Medicine, Ege University, Bornova, Izmir, Turkey  Corresponding author. Tel.: +90-232-374-7321; fax: +90-232-374-7321.
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