| | Effects of flavonoids on cisplatin-induced apoptosis of HL-60 and L1210 leukemia cellsReceived 7 January 2002; accepted 1 April 2002. Abstract Effects of three flavonoids, quercetin (QU), galangin (GA), and chrysin (ChR) on cisplatin (cis-Pt)-induced apoptosis of human promyelocytic leukemia HL-60 cells and murine leukemia L1210 cells were investigated. The quantitative analysis of apoptotic DNA fragmentation was used to show that preincubation of cells with flavonoids can influence cis-Pt-induced apoptosis in different way. ChR had no effect, QU enhanced, and GA reduced apoptotic DNA fragmentation. It is also shown that combined treatment with QU and cis-Pt showed synergistic effect, however, GA combined with cis-Pt exhibited antagonism on cytotoxicity in L1210 murine leukemia cells. We assume that tested flavonoids affect the important biological activities connected with cancer chemotherapy and chemoprevention as they differently modulated the sensitivity of cells to cis-Pt treatment. QU is presented as pro-apoptotic agent and GA as agent with anti-apoptotic potential.
Abbreviations:
HL-60, human promyelocytic leukemia cell line,
L1210, murine leukemia cell line,
MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide,
QU, quercetin,
GA, galangin,
ChR, chrysin,
PKC, protein kinase C,
H2O2, hydrogen peroxide,
PARP, poly(ADP-ribose) polymerase
1. Introduction  Flavonoids are polyphenolic compounds that occur naturally in foods of plant origin. They have a variety of biological activities, such as anti-allergic, anti-inflammatory, anti-oxidative, free radical scavenging, and anti-mutagenic activities [1]. Many studies have demonstrated that flavonoids are also potent inhibitors of key enzymes taking part in signal transduction. They inhibit several kinases such as PKC, tyrosine kinases, or lipid kinases [2], affect various metabolic pathways such as activation of glycolytic enzymes or protein synthesis [3], promote cell cycle arrest in G0/G1 or G2/M phase, depending on their structure and on the cellular model [4], [5], [6], interact with estrogen type II binding sites to regulate mammary cell growth [7], and induce apoptosis in different cell lines [8], [9]. Some flavonoids are more selective towards cancer cells and may have the potential for reducing side-effects compared with other drugs [10]. Because of extensive intake of flavonoids by humans, it was essential to analyze their potential effect on chemotherapy treatment. Previous reports showed that some flavonoids could potentiate anti-proliferative effects of some chemotherapeutics [11], [12], however, there was no evidence that they also intervent with chemotherapy-induced apoptosis. As the biological activities of chemicals are dependent on the individual structure, we have investigated effects of QU, GA and ChR, three structurally related flavonoids (Fig. 1) on cell viability and cisplatin (cis-Pt)-induced apoptosis of human promyelocytic HL-60 and murine leukemia L1210 cell lines. The aim of the present work was to examine the eligibility of presented agents in cancer chemotherapy or prevention. 2. Materials and methods 2.1. Drugs Platidium (cis-Pt) was obtained from Lachema (Brno, Czech Republic) as a solution for injection. Flavonoids (QU, GA, and ChR) were purchased from Sigma (St. Louis, MO, USA) and dissolved in dimethyl sulphoxide (DMSO, Sigma). The stock solutions of flavonoids (0.1 M) were stored at −20°C. The final concentration of DMSO in the medium was <0.02% and did not affect cell growth [13]. 2.2. Cell culture Human promyelocytic leukemia HL-60 cells were kindly provided by Dr. P. Ujházy (Roswell Park Cancer Institute, Buffalo, USA) and murine leukemia cell line L1210 was obtained from the American Type Culture Collection (Rockville, MD, USA). Cells were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (Grand Island Biological Co., Grand Island, NY, USA), 100U/ml Penicillin G, 100μg/ml streptomycin, and 2mM l-glutamine (Sebac, Germany) in an atmosphere of 5% CO2 in humidified air at 37°C. In all experiments, exponentially growing cells were used. 2.4. Analysis on drug combination The MTT data were analyzed using Calcusyn program to determine the IC50 of each drug alone. The combination index (CI)-isobologram by Chou and Talalay [16] was used to analyze the drug combination. Variable ratios of drug concentrations were used in the studies, and mutually exclusive equations were used to determine the CIs. Each CI was calculated from the mean affected fraction at each drug ratio concentration (triplicate). CI>1, CI=1, and CI<1 indicate antagonism, additive effect, or synergism, respectively. 2.6. Electrophoretic analysis of DNA fragmentation The untreated cells (control) and drug-treated cells (1×106) were harvested, washed in phosphate-buffered saline (PBS) and then lysed in 100μl of solution (10mmol/l TRIS, 10mmol/l EDTA, 0.5% Triton X-100) supplemented with proteinase K (1mg/ml, Serva, Germany). Samples were then incubated at 37°C for 1h and heated at 70°C for 10min. Following lysis, RNA-ase (200μg/ml, Serva) was added and followed by repeated incubation at 37°C for 1h. The samples were electrophoresed at 40V for 3h in 2% (w/v) agarose gels (Sigma) complemented with ethidium bromide (1μg/ml, Sigma) [17]. Separated DNA fragments (DNA ladders) were visualised using UV transilluminator (254nm, Ultra-Lum Electronic UV Transilluminator, USA). The size of DNA fragments was determined by comparing to the DNA molecular weight markers, Superladders-Mid2 200bp ladder (Advanced Biotechologies, UK). 2.7. Quantification of DNA fragmentation The extent of DNA fragmentation of cellular DNA of treated cells was determined by the method of Rauko et al. [18]. Briefly, equal amounts of DNA samples (from 1×106 cells) were electrophoresed and visualized as described above. Photographs of gels were made using digital camera Olympus CAMEDIA C-1400 L (Japan) and elaborated by the software Olympus C-W95. Determination of a relative DNA intensity at the area of DNA ladders (width of area >200 to <1200bp) was performed using the software UTHSCSA Image Tool for Windows (Version 1.28). The values of DNA ladder intensities are presented as the percentage of DNA ladder intensity of cis-Pt treated cells. 3. Results 3.2. The effect of time-different treatment with flavonoids on cisplatin-induced apoptosis of HL-60 cells In studies on the time dependence of treatment with flavonoids (QU and GA), we found that the time of flavonoid addition (20μM) is important for maximal enhancement (QU) or reduction (GA) of cis-Pt-induced apoptotic DNA fragmentation. The significant effects were achieved when flavonoids were added 30min before (QU) or simultaneously (GA) with cis-Pt. After flavonoid pretreatment periods of 40min and longer, no further enhancement or reduction of cis-Pt-induced apoptotic DNA fragmentation occured. Simultaneous treatment (QU) or post-treatment (QU and GA) with flavonoids had little or no effect on cis-Pt-induced apoptosis. Results are shown in Fig. 2c. 3.4. Opposite effects of QU and GA combined with cisplatin on cytotoxicity in L1210 cells To determine the correlation between cytotoxicity and cis-Pt-induced apoptosis modulated by flavonoids, MTT chemosensitivity test and Trypan blue assay were used. Drug combination studies by MTT test demonstrated that QU combined with cis-Pt synergistically enhanced cell death (CI<1), however, GA combined with cis-Pt exhibited antagonism as far as cell death was concerned (CI>1, Table 1). Dose-dependence study of combined application of QU and cis-Pt by MTT test did not reveal any enhancement of synergism when higher concentrations of QU were used. On the contrast, Trypan blue assay proved concentration-dependent enhancement of synergism. CI values obtained by Trypan blue assay and MTT test after the combined treatment with QU and cis-Pt are presented in Table 2. These results correlate with significantly increased apoptotic DNA fragmentation in L1210 cells (see Fig. 3a and b). Therefore, Trypan blue assay is more eligible than MTT test for examination of combined treatment with flavonoids and cis-Pt, because of possible intervention of flavonoids in reduction of MTT. 4. Discussion The anti-neoplastic effects of some agents can influence cells by many different mechanisms, either by preventing initiation and promotion of cancerogenesis or by elimination of abnormal cells by apoptosis. In the process of chemotherapy some agents cooperate with other anti-neoplastic drugs to induce apoptosis and thus may increase/decrease drug efficacy [19], [20]. Therefore, the identification of inhibitors or activators of apoptosis may help in providing more effective strategies for therapeutic intervention. In our laboratory, agents that either prevent induction of damages to DNA by radical scavenging and metal chelating [20], [21] or eliminate the abnormal cells by apoptosis are studied [18], [22]. In the present work, we studied effects of some flavonoids on cis-Pt-induced apoptosis and cytotoxicity in HL-60 and L1210 leukemia cells. GA was presented as the agent with anti-oxidative, free radical scavenging, anti-mutagenic and enzyme modulating activities [19]. Results from in vitro and in vivo studies indicate that GA is capable of suppressing the mutagenicity and clastogenicity of N-methyl-N-nitrosourea [23]. Therefore, it may be useful as a cancer chemopreventive agent against potential long-term health effects from genotoxic environmental compounds [19], [23]. Up to now, only a few studies have been conducted on apoptotis for GA. The ability of GA to block apoptosis was observed with polycyclic aromatic hydrocarbon-induced pre-B-cell apoptosis. This inhibition of apoptosis is specific and was not determined if C2-ceramide and H2O2 were used as inductors of apoptosis [24]. The failure of GA to block H2O2 and C2-ceramide-induced pre-B cell apoptosis indicates that radical scavenging within pre-B cells by GA does not contribute to apoptosis inhibition in this system and signals distal to ceramide generation, such as caspase 8 and caspase 3 activation [25], are not likely to be targeted by this flavonoid. Our results showed that GA acts also as an inhibitor of cis-Pt-induced apoptosis. At present, the reason why the pretreatment of cells with GA results in inhibition of cis-Pt-induced apoptosis is unknown. We assume, this is due to the modulation of enzyme activities involved in chemoprevention. Another anti-neoplastic effect of flavonoids is associated with the elimination of drug-treated cells by apoptosis [8], [9]. As it was presented earlier, QU sensitizes cells to the cytotoxic potential of cis-Pt [26], [27], synergistically enhances the anti-proliferative activity of cis-Pt in vitro by inhibition of PKC [28], increases the anti-tumourous activity of cis-Pt in vivo [29], and are able to induce apoptosis by activating the pre-existing apoptosis machinery, such as induction of caspase-3 activity and degradation of PARP [9]. Therefore, the combination of QU and cis-Pt (also other cytostatic drugs) might be of therapeutic benefit. In our experiments, QU is presented as agent with increased effect on cis-Pt-induced apoptosis and cytotoxicity. Data on agarose gel electrophoresis clearly showed that pretreatment with QU enhanced cis-Pt-induced apoptotic DNA fragmentation in HL-60 and L1210 leukemia cells. Although the exact mechanism of QU intervention to cis-Pt-induced therapy is unclear, we assume that this process related to the application of QU in pretreatment is associated with the regulation of apoptosis. In conclusion, we presented GA as anti-apoptotic agent that had a negative effect on cis-Pt-induced apoptosis. However, QU is presented as pro-apoptotic agent with the perspective potential for cis-Pt-induced apoptosis. Despite of structural similarity of tested flavonoids, we found their modulating effects on cis-Pt efficacy to be contrary. Elucidation of the molecular mechanisms by which flavonoids modulate cis-Pt-induced apoptosis is the topic to be addressed. Further investigations of new flavonoids as agents with cancer-preventive or therapy-increasing effects are required as well. Acknowledgements This investigation was supported by the grants of the Slovak Grant Agency VEGA number 2/7048/2000, 2/1048/21, 1/9152/02, and 2/2094/22. L. 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a Cancer Research Institute, Vlárska 7, 833 91 Bratislava, Slovak Republic b Department of Genetics, Faculty of Sciences, Comenius University, Mlynská dolina, 842 15 Bratislava, Slovak Republic c Institute of Molecular Biology, Dubravská cesta 21, 842 51 Bratislava, Slovak Republic d Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Kuwait University, P.O. Box 24923 Safat, 13110 Kuwait  Corresponding author. Tel.: +421-2-59327-105; fax: +421-2-59327-250.
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