Rhamnazin inhibits pRolifeRation and induces apoptosis of human JuRkat leukemia cells in vitro

antiproliferative and apoptogenic effects of rhamnazin, a dimethoxylated derivative of quercetin, were studied in human acute lymphoblastic leukemia Jurkat cells. The cytotoxicity and apoptogenic activity of rhamnazin in vitro are inferior to that of quercetin. The apoptogenic activity of rhamnazin is realized via mitochondrial pathway and associated with activation of caspase-9 and -3. The additive apoptogenic effect of rhamnazin and suboptimal doses of etoposide, a Dna topoisomerase ii inhibitor, is demonstrated. Therefore, methylation of quercetin modifies its biological effects considerably.

F lavonoids constitute the largest class (over 6,500 compounds) of biologically active plant polyphenols [1].Most of them by far are de rivatives of 2-phenylbenzopyran (flavan) or 2-phe nylbenzopyran-4-one (flavone).Flavonoids display antioxidant properties and may also induce apoptotic cell death, depending on concentration [2].It has been demonstrated that the proapoptotic and cyto static activity of flavonoids could not be explained by their antioxidant or prooxidant properties only.Specific target molecules for flavonoids in tumor cells are currently under spotlight.The data availab le allows us to perceive certain flavonoids as signal transducer analogs, which makes them especially attractive as candidates for specific antitumor phar maceuticals.Importantly, flavonoids are also known to potentiate or modify the effects of cytotoxic and antitumor agents and are thus viewed as suitable for adjuvant therapies.
Quercetin (3,3′,4′,5,7-pentahydroxyflavone) is the most studied of flavonoids.Its well-documented pleiotropic biological activity includes, beyond potent antioxidant properties, an antinflammatory, antiangio genic, cytostatic, and proapoptotic effects (as cited in [3]).Cytotoxic effects of micromolar con centrations of quercetin and its derivatives towards human leukemia cells have been demonstrated [47].Methylated quercetin derivatives with variations in the number and positions of methyl groups are com mon natural substances.Monomethylated deriva tives have been shown to inhibit proliferation and induce apoptosis in cancer cells [8], or to sensitize malignant cells to other cytotoxic agents [9].Yet there is only a limited amount of data concerning efficiency of rhamnazin (3′,7-dimethylquercetin), a quercetin derivative differing only by two methyl groups (Fig. 1), against infection agents [10].An tiproliferative activity of rhamnazin has also been demonstrated recently in vitro in human cancer cell lines [11].To the best of our knowledge, its effects upon leukemia cells have not been studied.Rham nazin interaction with cytotoxic antitumor drugs is also unknown.
Physicalandchemical as well as biological properties of methylated derivatives of quercetin may vary considerably, depending on the number and positions of methyl groups.There are few data relating certain properties of quercetin derivatives, namely their anticancer activity, to structural char acteristics of the particular molecules [12].The aim of the present work was to investigate in vitro bio logical effects of rhamnazin in human acute lympho blastic leukemia Jurkat cells, in particular its ability to induce apoptosis and involvement of caspases in the induction of apoptosis in these cells.The effects of rhamnazin were compared to those of quercetin, which is important for attributing biological effects of flavonoids, e. g. quercetin and its dimethylated de експериментальні роботи doi: http://dx.doi.org/10.15407/ubj87.06.122 rivative, to their structure, and for further targeted selection and synthesis of compounds with particular properties.

materials and methods
Human Tcell acute lymphoblastic leukemia cell line Jurkat was obtained from the National Col lection of Cell Lines of the Institute of Experimen tal Pathology, Oncology and Radiobiology (Kyiv, Ukraine).The cells were grown in RPMI1640 medium with addition of 10% fetal bovine serum (Sigma, USA) and 2 mM of glutamine at 37 °C.The cells were subcultured at density of 0.8×10 6 to 0.9×10 6 per ml.
Rhamnazin (99% pure, Sigma, USA) was dis solved in dimethyl sulfoxide.Quercetin (Merck, USA) was dissolved in ethanol.The stock solutions were stored at 20 °C.Etoposide was purchased from BrystolMyers Squibb SpA (Italy) as commercially available solution at a concentration of 20 mg/ml.
The investigated compounds were added into culture medium in the beginning of the logarithmic cell growth to analyze growth kinetics and cytotoxi city.Final concentrations of ethanol or dimethyl sul foxide in culture media did not exceed 0.5%.Cell viability was determined by trypan blue exclusion.
Cells distribution by phases of cell cycle and percentage of hypodiploid (apoptotic) cells [13] were estimated after incubating them with propidium io dide (50 µg/ml) solution in 0.1% sodium citrate and 0.1% Triton X-100.Cell fluorescence was measured using FACScan flow cytometer (Becton Dickinson, USA).The percentage of cells containing active form of caspase-3 was assessed by flow cytometry using Caspase3, Active Form, mAb Apoptosis Kit: FITC (BD Biosciences Pharmingen, USA) according to the procedure recommended by the manufacturer.The flow cytometry data were analyzed with ModFit LT2.0 software (Verity Software House, USA) and CELLQuest (BD Biosciences Pharmingen, USA).
For Western blot, cells were washed in phos phate buffer solution and lysed on ice in 20 mM TrisHCl (pH 7.4), 1% Triton X100 and 150 mM NaCl with addition of protease inhibitor cocktail (Roche Diagnostics, Germany).The lysate was cen trifuged at 12,000 g, the supernatant was mixed with Laemmli sample buffer and boiled on water bath.Proteins were separated by electrophoresis in 12% PAAG with SDS and transferred to ImmobilonP polyvinylidene difluoride membrane (Millipore, USA).The membranes were blocked for 12 h in 5% nonfat dry milk in phosphate buffered saline with 0.05% Tween 20 at 4 °C, then incubated with an ticaspase9 (5B4 clone recognizing both proform and active form of caspase9) or anticaspase8 (5F7 clone, recognizing only procaspase8) mono clonal antibodies (Immunotech, France).To confirm equal protein loading, each membrane was probed with anti-β-actin MoAb (clone AC-15, Sigma, USA).Antimouse IgG conjugated with horseradish per oxidase (Promega, USA) was used as secondary antibody.The affinity binding bands were identified with enhanced chemiluminescence Amersham ECL Western Blotting Detection kit (GE Healthcare Life Sciences, USA).
The statistical analysis was performed using Student's t-test, the results were considered signifi cant if P < 0.05.

Results and discussion
Jurkat cells were incubated with 15 µM or 150 µM rhamnazin to evaluate its antiproliferative activity.The cells were counted after 24 or 48 h of incubation.According to the results, rhamnazin does not affect Jurkat cell growth kinetics at 15 µM, but inhibits cell growth noticeably if the concentration is increased tenfold (Fig. 2).
As antiproliferative activity is generally reali zed via cell cycle arrest, the next series of experi ments involved investigation of rhamnazin effect on Rhamnazin exhibited low toxicity in Jur kat cells even at the relatively high concentrations (150 µM) with only a minor death fraction as com pared to that induced by quercetin (Fig. 4).These results corroborate the data of N. Li et al. [14], who showed that methylation of flavonols (including quercetin) in 3′ and 4′ position leads to significant decrease or total loss of cytotoxic properties.
In order to evaluate the contribution of apopto sis in overall cell death induced by high rhamnazin concentrations we analyzed in parallel hypodiploid cell fraction and the content of the active form of effector caspase3.As demonstrated in Fig. 5, rham nazin causes apoptotic cell death (Fig. 5), and apo ptosis induction by rhamnazin is accompanied with increased number of cells with the active form of caspase3.The maximum percentage of apoptotic cells and cells containing the active form of cas pase3 was detected after 72 h of incubation with rhamnazin.Delayed cell death and, probably, pro tracted inhibition of cell growth may in part explain cell cycle effects of rhamnazin in comparison to those of quercetin (see Fig. 3).
As at least two main pathways have been iden tified in cells for activation of caspase-3, namely re ceptormediated (via caspase8) and mitochondrial (via caspase9) [15], it was important to determine activation of these initiator caspases in rhamnazin treated cells.To this end, we used immunoblot as say with monoclonal antibodies specific to procas pase8 or active form of caspase9.The active form of caspase9 was detectable in Jurkat cells incubated with rhamnazin or quercetin (Fig. 6, a).On the other hand, the induction of apoptosis in Jurkat cells was not associated with changes in procaspase8 content (Fig. 6, c).There is a good amount of evidence that caspase8 activation is not restricted to receptor

Fig. 3. Distribution of Jurkat cell population incubated for 48 h with rhamnazin or quercetin by cell cycle phases. a -75 μM rhamnazin; B -150 μM rhamnazin; c -40 μM quercetin
Intensity of fluorescence, a. u. mediated apoptosis.Caspase8 may be involved in apoptosis induced by various cytotoxic agents, in cluding etoposide [16], although the significance of this phenomenon for activation of apoptotic effector pathways has not been yet clarified.Our data dis tinctly indicate caspase8 involvement in apoptosis induction by etoposide in Jurkat cells.On the con trary, neither quercetin nor rhamnazin caused sig nificant changes in procaspase-8 content in Jurkat cells, which is in accordance with the results of our previous studies [7].It is worth noting that the pat tern of involvement for various initiator caspases in response to an apoptotic inducer depends on cell type and experimental conditions.For instance, some authors failed to detect caspase8 activation in apoptosis induction by quercetin, which corroborates our results [17].On the other hand, caspase8 activa tion by quercetin has been proven in other studies, i.e. in HL60 cells [18].
Our results indicate the additive total effect by percentage of hypodiploid cells for induction of apoptosis by suboptimal dose of etoposide in cells that had been preincubated with rhamnazin for 72 h (Fig. 7).It is worth to note that the additive effect varied considerably with different modes of expo sure to rhamnazin (data not shown), which requires further study in order to select the optimal conditions for combinations of the investigated compounds.It is well known that flavonoids, quercetin in particular, are not just capable of inducing apoptosis as single agents at higher concentrations, but may also interact with known apoptosis inducers -various classes of cytotoxic chemotherapeutic agents -and potentia te their cytotoxic and proapoptotic effects.For exam ple, quercetin may potentiate cytotoxic effect of doxo rubicin in human hepatocarcinoma SMMC7721 cells and cisplatin in human ovary adenocarcinoma cells [19,20].
Therefore, in experiments with rhamnazin, a quercetin dimethoxylated derivative, we established that methylation of hydroxyl groups in 3′ and 7 po sitions significantly alters the biological activity of quercetin, in particular diminishing its cytotoxic po tential and ability to induce apoptosis in Jurkat cells.However, at higher concentrations (150 µM) rham nazin is capable of inhibiting cell growth, inducing