**3. Materials and methods**

Cells of the human erythromyeloid leukemic cell line *К562* were incubated in the following medium: 89% RPMI 1640 (Institute of Poliomyelitis and Viral Encephalitides, Russian Academy of Medical Sciences, Moscow) supplemented with 11% fetal bovine serum (Institute of Epidemiology and Microbiology, Russian Academy of Medical Sciences, Moscow), 2 mM L-glutamine, 40 µg/ml gentamicin sulphate, 50 µM 2 mercaptoethanol (Ferak, Germany) in 95% O2 and 5% С02 at 370С. The cultures were seeded to 1 ml (3 ml) of the medium with the seeding density of 105 (106) cells/well, respectively. Quinoline derivatives in concentrations detailed in the text and figure captions were added to the medium. Cells were counted in Goryaev's chamber. EC50 cells incubated with the reagents were ascertained using the technique [38]. The viability was assessed using the test with 3-(4.5-dimethylthiasol-2-yl)-2.5-diphenyltetrazolium bromide (МТТ) (Sigma, USA).

*Assessment of viability (МТТ-test).* The technique was described in [39]. 100 µL aliquots of cell suspension (105 cells/well) were seeded to a 96-well flat bottom culture plate and treated with 100 µL of the chemical compound solution of the corresponding concentration. All assays were performed in three replicates. The plates were left to incubate for 24, 48, 72, and 96 hours. Three hours before the end of the incubation period the wells were stained with МТТ solution (3-(4.5-dimethylthiasol-2-yl)-2.5-diphenyltetrazolium bromide (Sigma, USA)) to a final concentration of 0.25 mg/ml, and re-incubated in the dark in a humidified atmosphere at 37оС. Then, the supernatants were carefully removed and 200 µL aliquots of DMSO were added to each well. The residue was re-suspended and incubated for 15 min in the dark at room temperature. The optical density was measured at 540 nm with a Labsystems Multiscan Plus reader (LKB, Finland). In this case, staining of the cells incubated in the absence of chemical compounds was regarded as the control. Specific cell death was calculated by formula (1):

$$\text{Induced death}, \%= (1 - (\text{D}\_{\text{exp}} - \text{D}\_{\text{env}} / \text{D}\_{\text{c}} - \text{D}\_{\text{env}})) \times 100 \text{ }\% \tag{1}$$

where Dc is the optical density in control wells (cells without chemical reagent); Dexp – optical density in treated assays (cells with chemical reagent); Denv – optical density of the control environment.

*Tumor cell clones resistant to 2-DQO and 4-NQO* were obtained as described in [38]. Cell resistance to xenobiotics was induced by long-term (over a month) exposure in a culture medium containing *2-DQO* (10-9 M) or *4-NQO* (10-12 M). The concentration of the substances in the culture medium was increased every 14−21 days. The final doses for which resistant cell lines were obtained were 10-5 and 10-8 M, respectively.

*Real-time PCR.* Total RNA from peripheral blood leukocytes and tumor cells was extracted with the "YellowSolve" kit (Clonogen, Russia) following the manufacturer's guidelines. The extracted RNA template was treated with DNase (Sigma, USA). The concentration and purity of the RNA template was determined by spectrophotometry ("SmartSpec Plus", BioRad, USA). RNA nativity was determined by agarose gel electrophoresis. Complementary DNA was synthesized from 1 µg of total RNA using random hexaprimers and MMLV reverse transcriptase following the protocol proposed by the manufacturer (Sileks, Russia). RNA and cDNA samples were stored at -80°С. Gene expression was estimated by real-time PCR. The fluorophore for product detection was the intercalating SYBR Green I dye. Amplification was performed in an "iCycler Thermal Cycler" (BioRad, USA) with "iQ5 Optical System" V2.0 software (BioRad, USA) using real-time PCR assay kits in the presence of SYBR Green I. The PCR reaction mixture was prepared with the component volumes recommended by the manufacturer: we mixed 2.5 µl deoxynucleosidetriphosphates (2.5 mM), 2.5 µl 10-fold PCR buffer with SYBR Green I, 2.5 µl MgCl2 (25 mM), 1 µl aliquots of forward and reverse primers (20 pmol/µl), 0.25 µl Taq-DNA-polymerase (5 U/µl), 2 µl template cDNA, deionized water – up to 25 µl per test tube. The PCR protocol was 15 sec at 95С, 50 sec at 60С (45 cycles). To determine the specificity of primer annealing PCR fragments were melted: for 1 min at 95С, 1 min at 60С, 10 sec at 60С (80 cycles, the temperature raised by 0.5С in each cycle). To exclude the possibility of the template cDNA being contaminated by the genomic DNA PCR was performed for each template under the same conditions with the RNA matrix. Primers for the nucleotide sequences of the investigated genes and the reference gene GAPDH were selected using the Primer Premier software ("Premier Biosoft", USA) or published sources (Table 1). Oligonucleotides were synthesized by the Syntol company (Russia). Gene expression was measured against the amount of GAPDH mRNA using the 2–∆∆Ct method [40]. The resultant reaction products were separated in 8% polyacrilamide gel using the tris-borate buffer. PCR products were stained with 1% ethidium bromide solution and visualized in transmitted UV light using the low-molecular (501–567 bp) pUC19/Msp I fragment length marker (Syntol, Russia).

182 Apoptosis and Medicine

**3. Materials and methods** 

(МТТ) (Sigma, USA).

calculated by formula (1):

control environment.

application of these compounds enable activation of apoptosis in tumor cells by modulating the activity of caspases? Will the susceptibility of the reagent-treated cells lead to leukolysis

Cells of the human erythromyeloid leukemic cell line *К562* were incubated in the following medium: 89% RPMI 1640 (Institute of Poliomyelitis and Viral Encephalitides, Russian Academy of Medical Sciences, Moscow) supplemented with 11% fetal bovine serum (Institute of Epidemiology and Microbiology, Russian Academy of Medical Sciences, Moscow), 2 mM L-glutamine, 40 µg/ml gentamicin sulphate, 50 µM 2 mercaptoethanol (Ferak, Germany) in 95% O2 and 5% С02 at 370С. The cultures were seeded to 1 ml (3 ml) of the medium with the seeding density of 105 (106) cells/well, respectively. Quinoline derivatives in concentrations detailed in the text and figure captions were added to the medium. Cells were counted in Goryaev's chamber. EC50 cells incubated with the reagents were ascertained using the technique [38]. The viability was assessed using the test with 3-(4.5-dimethylthiasol-2-yl)-2.5-diphenyltetrazolium bromide

*Assessment of viability (МТТ-test).* The technique was described in [39]. 100 µL aliquots of cell suspension (105 cells/well) were seeded to a 96-well flat bottom culture plate and treated with 100 µL of the chemical compound solution of the corresponding concentration. All assays were performed in three replicates. The plates were left to incubate for 24, 48, 72, and 96 hours. Three hours before the end of the incubation period the wells were stained with МТТ solution (3-(4.5-dimethylthiasol-2-yl)-2.5-diphenyltetrazolium bromide (Sigma, USA)) to a final concentration of 0.25 mg/ml, and re-incubated in the dark in a humidified atmosphere at 37оС. Then, the supernatants were carefully removed and 200 µL aliquots of DMSO were added to each well. The residue was re-suspended and incubated for 15 min in the dark at room temperature. The optical density was measured at 540 nm with a Labsystems Multiscan Plus reader (LKB, Finland). In this case, staining of the cells incubated in the absence of chemical compounds was regarded as the control. Specific cell death was

where Dc is the optical density in control wells (cells without chemical reagent); Dexp – optical density in treated assays (cells with chemical reagent); Denv – optical density of the

*Tumor cell clones resistant to 2-DQO and 4-NQO* were obtained as described in [38]. Cell resistance to xenobiotics was induced by long-term (over a month) exposure in a culture medium containing *2-DQO* (10-9 M) or *4-NQO* (10-12 M). The concentration of the substances in the culture medium was increased every 14−21 days. The final doses for which resistant

cell lines were obtained were 10-5 and 10-8 M, respectively.

Induced death, % = (1 – (Dexp – Denv / Dc – Denv)) × 100 % (1)

change? It is these and other questions that we shall try to answer below.


**Table 1.** Primers for the nucleotide sequences of the caspase genes under study and the reference gene GAPDH

*The enzyme activity of caspases* was determined by standard technique using specific substrates labeled with fluorescent marker (7-amino-4-trifluoromethylcumarin – AFC) (BioRad, USA), detected by variations in fluorescence or optical density [2]. 50 µl of lytic buffer prepared by mixing 920 µl of bidistilled Н2О, 40 µl of 25-fold reaction buffer and 10 µl of each of the four inhibitors: PMSF (phenylmethylsulfonyl fluoride) (35 mg/ml), pepstatin *А* (1 mg/ml), aprotinin (1 mg/ml), and leupeptin (1 mg/ml), was added to the tumor cells (106 cells). The 25-fold reaction buffer included the following components: 250 мМ HEPES, рН 7.4, 50 mM EDTA, 2.5% CHAPS (3-((3 chloramidopropyl)dimethylammonio)-1-propanesulfonate), 125 mM dithiothreitol. After that, the cells were frozen three times in liquid nitrogen, the cell lysate then centrifuged in a microcentrifuge at 17 000 *G* (40 С) for 30 min, and the supernatant (template) collected. The activity of caspases-3, -6 and -9 was determined in the reaction buffer by mixing the template with the corresponding specific substrate. The substrate for caspase-3 was DEVD (Asp–Glu–Val–Asp), for caspase-6 – VEID (Val–Glu–Ile–Asp), for caspase-9 – LEНD (Leu–Glu–His–Asp). The amount of cleaved AFC was measured by spectrophotometry in FluoroMax ("Horiba-Scientific", Japan) at 395 nm 30, 60, 90, 120, 150, 180 min after the onset of the reaction. Then, the curve of caspase activity depending on the template and substrate incubation time was plotted. Plot ∆S versus ∆t and calculate the slope (∆S/∆t).

$$
\Delta \mathbf{S} = \left[ \mathbf{S}(\mathbf{t}) - \mathbf{B}(\mathbf{t}) \right] - \left[ \mathbf{S}(\mathbf{t}) - \mathbf{B}(\mathbf{t}) \right] \boldsymbol{\uplambda} \qquad \Delta \mathbf{t} = (\mathbf{t} - \mathbf{t}) \tag{2}
$$

Cellular Caspases: New Targets for the Action of Pharmacological Agents 185

Quinoline derivatives were kindly provided by Prof. V.P. Andreev (St. Petersburg State University, Russia). The composition and structure of the resultant compounds were

Reliability of the results was estimated using Student's t-test, and the non-parametric

The first stage of the investigation of the biological activity of quinoline and pyridine derivatives was experiments to determine the effect of varying concentrations of *Q, 2-MeQ, QO, 2-MeQO, 4-NQO, 2-Me-4-NQO, 2-DQO, 4-DQO, 2-NSQO, 4-NSQO*, and *4-DPyO* on the viability of *К562* cells. The resultant data were processed to calculate ЕС50 values of each compound. The results are detailed in Table 2 (the reagents are arranged in the order of decreasing toxicity). It follows from the results that the greatest toxic effect on tumor cells under the stated conditions was demonstrated by *2-Me-4-NQO* and *4-NQO*, and the lowest –

ascertained by elemental analysis, mass, IR-, NMR spectroscopy (1H, 13C, 15N).

Mann−Whitney test.

by *2-NSQO* and *4-NSQO*.

**Reagent ЕС50, µM**

*1. 2-DQO* 2. 208.93 *3. 4-DQO* 4. 221.28 *5. QO* 6. 316.23 *7. 2-NSQO* 8. 570.10 *9. 4-NSQO* 10. 600.50

**Table 2.** Reagent concentrations resulting in 50% death of *К562* cells (ЕС50)

The data presented in Table 2 indicate also that when the methyl radical (*Q → 2-MeQ, QO → 2-MeQO, 4-NQO → 2-Me-4-NQO*) and the nitro group (*QO → 4-NQO, 2-MeQO → 2-Me-4- NQO*) were attached to the quinoline heterocycle, the toxicity of the compounds for *К562* cells increased*.* For example, the ЕС50 of *Q* was 4.47 µM, *2-MeQ* – 1.26 µM; *QO* – 316.23 µM, *2-MeQO* – 23.44 µM; *4-NQO* – 1.05 µM, *2-Me-4-NQO* – 1.04 µM, ЕС50 in the *QO/4-NQO* and *2-MeQO/2-Me-4-NQO* pairs was 316.23 µM, 1.05 µM and 23.44 µM, 1.04 µM, respectively. When *К562* cells were incubated with reagents comprising the N-oxide group (*QO*, 2- *MeQO*), the cell survival rate was, on the contrary, higher than for the cells incubated with *Q*

*2-Me-4-NQO* 1.04 *4-NQO* 1.05 *2-MeQ* 1.26 *Q* 4.47 *2-MeQO* 23.44 *4-DPyO* 33.11

**4. Results** 

S – sample signal at time t, and B – blank signal at time t; ti – time of measurement, t0 – time of initial measurement.

*Cytochrome c reductase activity of microsomes* was measured by spectrophotometry in FluoroMax ("Horiba-Scientific", Japan) at 250 С. The reaction medium contained microsomes (30 µg protein/ml), NADPH or NADH (50 µM), *2-NSQO* or *4-NSQO* (1−100 µM), and cytochrome с (20 µM).

*Determination of the susceptibility of tumor cells to the cytotoxic lysis of human leukocytes.* Lysis was studied in a homologous system. Human peripheral blood leukocytes were isolated from the blood of healthy males by a two-step procedure involving fractionation on a Ficoll-Hypaque gradient, followed by erythrocyte lysis by distilled water. The viability of leukocytes, estimated by the Trypan blue test, was at least 93–95 %.

The test for cytotoxicity of human leukocytes (effector cells) for *К562* cells (target cells) incubated with quinoline derivatives and labeled with 3Н-uridine followed the protocol [44]. Pre-assay incubation of the cultures with the reagents lasted 48 h and 96 h in all variants. The effector cell/target cell ratio was 50:1. The cytotoxic index (CI, %) was calculated by formula (3):

Cytotoxic index = 1 – (cpm) in experimental tests / (cpm) in control tests × 100 %, (3)

where the control was *К562* cell cultures labeled with 3Н-uridine and free of effector-cells.

Quinoline derivatives were kindly provided by Prof. V.P. Andreev (St. Petersburg State University, Russia). The composition and structure of the resultant compounds were ascertained by elemental analysis, mass, IR-, NMR spectroscopy (1H, 13C, 15N).

Reliability of the results was estimated using Student's t-test, and the non-parametric Mann−Whitney test.
