**4. Results**

184 Apoptosis and Medicine

calculate the slope (∆S/∆t).

of initial measurement.

formula (3):

µM), and cytochrome с (20 µM).

*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

∆S = [S(ti) – B(ti)] – [S(t0) – B(t0)], ∆t = (ti – t0), (2)

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

*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

*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 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

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.

The viability of leukocytes, estimated by the Trypan blue test, was at least 93–95 %.

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 – by *2-NSQO* and *4-NSQO*.


**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*

or *2-MeQ*, respectively (see ЕС50). The toxic effect of the compounds was reduced also by addition of the "*styryl tail*" with the nitro group (*2-NSQO*, *4-NSQO)* to the reagent, whereas substitution of the nitro group with the dimethylamino group (*2-DQO, 4-DQO*) resulted in a 2.72-fold rise in the toxicity (see ЕС50)*.* Translocation of the styryl group within the quinoline ring was also significant: if the group was located closer to the heteroatom (Fig. 1), the compound also became more toxic (EC50 of *2-DQO* was 208.93 µM, that of *4-DQO* – 221.28 µM; EC50 of *2-NSQO* – 570.10 µM, that of *4-NSQO* – 600.50 µM). When the quinoline cycle in the chemical compound was substituted with the pyridine cycle the reagent's toxicity for the cells increased 6.68-fold, e.g., *4-DQO* EC50 was 221.28 µM, whereas *4-DPyO* EC50 was 33.11 µM. This pattern was observed both on the 48 h and on the 96 h of incubation.

Cellular Caspases: New Targets for the Action of Pharmacological Agents 187

Results are presented as the ratio of caspase to GAPDH expression relative to a standard curve for each assay. Drug concentrations: *Q* – 0.3 µМ, *QO* – 10 µМ, *4-NQO* – 0.001 µМ. Incubation time – 48 h. Viability of cultured cells –

**Figure 4.** Caspase-3 (*a*), -6 (*b*) and -9 (*c*) mRNA levels were determined by quantitative real-time RT-

93−95%.

Y axis – relative expression, conventional unit; \* − p<0.05

PCR in *K562* cells treated with *Q*, *QO* and *4-NQO* respectively.

The results above suggest that the toxic effect of the investigated reagents on tumor cells depends on the presence/absence of certain type substituents (electron donors or electron acceptors) in the quinoline cycle, as well as on the direct bond of the named substituents to the quinoline cycle. Thus, attachment of the nitro group (electron acceptor) to the cycle – *QO → 4-NQO, 2-MeQO → 2-Me-4-NQO –* rendered the substance more toxic, whereas inclusion of the substituent in the "*styryl tail*" (*4-NQO → 4-NSQO*) reduced the toxic effect of the reagent 571.90 fold (see ЕС50)*.*

Intracellular activation of aromatic nitrogen-bearing compounds, including *4-NQO* and other quinoline derivatives, is known to involve cytochrome Р-450 and NADPH-dependent cytochrome Р-450 reductase [45], glutathione-S-transferase, and quinone reductase [46]. As the result, there appears a set of metabolites most of which have a higher biological activity, and are potentially capable of interacting with high-molecular cell compounds (proteins, nucleic acids). Since an essential component part of research into the biological activity of chemical reagents *in vitro* is the study of their apoptosis inhibiting action, we shall now report the results on the effect of the investigated group of heterocycles on the expression (at the mRNA level) and enzymatic activity of cellular caspases.

Data on modifications of caspase-3, -6 and -9 mRNA expression in *К562* cells treated with *Q*, *QO* and *4-NQO* for 48 h are presented in Figure 4 (**a−c**). The reagent concentrations applied are detailed in the figure. The results suggest that cell incubation with *4-NQO* caused a rise in mRNA expression in all caspases; treatment with *Q* induced a rise in caspase-3 and -9 mRNA expression; whereas *QO* did not induce caspase expression under the given treatment conditions. A similar pattern was observed in the enzymatic activity (Figure 5 **a−c**). Cell incubation with *4-NQO* promoted the activity of all the caspases, whereas treatment with *Q* activated only caspases-3 and -9 (caspase-6 in this case was induced only on the 96 h). *QО* exhibited an activating effect on caspase-3 also on the 96 h of tumor cell treatment. Note that the activity of caspase-6 in the cells (р<0.05) (Figure 5 **b**) treated with *4- NQO* rose much less than that of caspases-3 and -9 (р<0.01) (Figure 5 **a**, **c**).

Caspase expression in *К562* cells incubated with *2-MeQ, 2-MeQO*, and *2-Me-4-NQO* did not differ from their expression in *Q, QO*, and *4-NQO* treatments, respectively.

Results are presented as the ratio of caspase to GAPDH expression relative to a standard curve for each assay. Drug concentrations: *Q* – 0.3 µМ, *QO* – 10 µМ, *4-NQO* – 0.001 µМ. Incubation time – 48 h. Viability of cultured cells – 93−95%.

Y axis – relative expression, conventional unit; \* − p<0.05

186 Apoptosis and Medicine

the 96 h of incubation.

reagent 571.90 fold (see ЕС50)*.*

or *2-MeQ*, respectively (see ЕС50). The toxic effect of the compounds was reduced also by addition of the "*styryl tail*" with the nitro group (*2-NSQO*, *4-NSQO)* to the reagent, whereas substitution of the nitro group with the dimethylamino group (*2-DQO, 4-DQO*) resulted in a 2.72-fold rise in the toxicity (see ЕС50)*.* Translocation of the styryl group within the quinoline ring was also significant: if the group was located closer to the heteroatom (Fig. 1), the compound also became more toxic (EC50 of *2-DQO* was 208.93 µM, that of *4-DQO* – 221.28 µM; EC50 of *2-NSQO* – 570.10 µM, that of *4-NSQO* – 600.50 µM). When the quinoline cycle in the chemical compound was substituted with the pyridine cycle the reagent's toxicity for the cells increased 6.68-fold, e.g., *4-DQO* EC50 was 221.28 µM, whereas *4-DPyO* EC50 was 33.11 µM. This pattern was observed both on the 48 h and on

The results above suggest that the toxic effect of the investigated reagents on tumor cells depends on the presence/absence of certain type substituents (electron donors or electron acceptors) in the quinoline cycle, as well as on the direct bond of the named substituents to the quinoline cycle. Thus, attachment of the nitro group (electron acceptor) to the cycle – *QO → 4-NQO, 2-MeQO → 2-Me-4-NQO –* rendered the substance more toxic, whereas inclusion of the substituent in the "*styryl tail*" (*4-NQO → 4-NSQO*) reduced the toxic effect of the

Intracellular activation of aromatic nitrogen-bearing compounds, including *4-NQO* and other quinoline derivatives, is known to involve cytochrome Р-450 and NADPH-dependent cytochrome Р-450 reductase [45], glutathione-S-transferase, and quinone reductase [46]. As the result, there appears a set of metabolites most of which have a higher biological activity, and are potentially capable of interacting with high-molecular cell compounds (proteins, nucleic acids). Since an essential component part of research into the biological activity of chemical reagents *in vitro* is the study of their apoptosis inhibiting action, we shall now report the results on the effect of the investigated group of heterocycles on the expression (at

Data on modifications of caspase-3, -6 and -9 mRNA expression in *К562* cells treated with *Q*, *QO* and *4-NQO* for 48 h are presented in Figure 4 (**a−c**). The reagent concentrations applied are detailed in the figure. The results suggest that cell incubation with *4-NQO* caused a rise in mRNA expression in all caspases; treatment with *Q* induced a rise in caspase-3 and -9 mRNA expression; whereas *QO* did not induce caspase expression under the given treatment conditions. A similar pattern was observed in the enzymatic activity (Figure 5 **a−c**). Cell incubation with *4-NQO* promoted the activity of all the caspases, whereas treatment with *Q* activated only caspases-3 and -9 (caspase-6 in this case was induced only on the 96 h). *QО* exhibited an activating effect on caspase-3 also on the 96 h of tumor cell treatment. Note that the activity of caspase-6 in the cells (р<0.05) (Figure 5 **b**) treated with *4-*

Caspase expression in *К562* cells incubated with *2-MeQ, 2-MeQO*, and *2-Me-4-NQO* did not

the mRNA level) and enzymatic activity of cellular caspases.

*NQO* rose much less than that of caspases-3 and -9 (р<0.01) (Figure 5 **a**, **c**).

differ from their expression in *Q, QO*, and *4-NQO* treatments, respectively.

**Figure 4.** Caspase-3 (*a*), -6 (*b*) and -9 (*c*) mRNA levels were determined by quantitative real-time RT-PCR in *K562* cells treated with *Q*, *QO* and *4-NQO* respectively.

Cellular Caspases: New Targets for the Action of Pharmacological Agents 189

Styryl derivatives also produced caspase inhibiting effects on *К562* cells. The results portrayed in Figures 6 and 7 evidence that treatment of tumor cells with *2-DQO, 4-DQO, 2- NSQO*, and *4-NSQO* promoted the activity of caspases-3 and -6, but with the latter two reagents the activity of the stated caspases was higher, and the rise in caspase-9 activity was recorded on the 24 h of application of the compounds to the incubation medium. Expression

at the mRNA level correlated with data on the activity at the enzyme level.

Drug concentrations – 1 µМ. Incubation time – 48 h., Viability of cultured cells – 93−95%. Y axis – ∆S/∆t × 10-4;

**Figure 6.** Caspase-3 and -6 activity in *K562* cells treated with *2-DQO, 4-DQO, 2-NSQO, 4-NSQO*

Drug concentrations – 1 µМ. Incubation time – 24 h, Viability of cultured cells – 93−95%. Y axis – ∆S/∆t × 10-4;

**Figure 7.** Caspase-9 activity in *K562* cells treated with *2-DQO, 4-DQO, 2-NSQO, 4-NSQO* respectively

\* − p<0.05, \*\* p<0.01; caspase-3; caspase-6

respectively.

\* − p<0.05, \*\* p<0.01

Drug concentrations: *Q* – 0.3 µМ, *QO* – 10 µМ, *4-NQO* – 0.001 µМ. Incubation time – 48 h. Viability of cultured cells – 93−95%. Y axis – ∆S/∆t × 10-4; \* − p<0.05

**Figure 5.** Caspase -3 (*a*), -6 (*b*) and -9 (*c*) activity in *K562* cells treated with *Q*, *QO* and *4-NQO* respectively.

Styryl derivatives also produced caspase inhibiting effects on *К562* cells. The results portrayed in Figures 6 and 7 evidence that treatment of tumor cells with *2-DQO, 4-DQO, 2- NSQO*, and *4-NSQO* promoted the activity of caspases-3 and -6, but with the latter two reagents the activity of the stated caspases was higher, and the rise in caspase-9 activity was recorded on the 24 h of application of the compounds to the incubation medium. Expression at the mRNA level correlated with data on the activity at the enzyme level.

Drug concentrations – 1 µМ. Incubation time – 48 h., Viability of cultured cells – 93−95%. Y axis – ∆S/∆t × 10-4; \* − p<0.05, \*\* p<0.01;

caspase-3; caspase-6

188 Apoptosis and Medicine

Drug concentrations: *Q* – 0.3 µМ, *QO* – 10 µМ, *4-NQO* – 0.001 µМ. Incubation time – 48 h.

**Figure 5.** Caspase -3 (*a*), -6 (*b*) and -9 (*c*) activity in *K562* cells treated with *Q*, *QO* and *4-NQO*

Viability of cultured cells – 93−95%. Y axis – ∆S/∆t × 10-4; \* − p<0.05

respectively.

**Figure 6.** Caspase-3 and -6 activity in *K562* cells treated with *2-DQO, 4-DQO, 2-NSQO, 4-NSQO* respectively.

Drug concentrations – 1 µМ. Incubation time – 24 h, Viability of cultured cells – 93−95%. Y axis – ∆S/∆t × 10-4; \* − p<0.05, \*\* p<0.01

**Figure 7.** Caspase-9 activity in *K562* cells treated with *2-DQO, 4-DQO, 2-NSQO, 4-NSQO* respectively

We have already mentioned that the release of cytochrome c from mitochondrial interior to the cell cytoplasm is a crucial stage in the activation of caspase cascades and triggering of apoptosis. In addition to involvement in the reactions of cellular caspase activation, "cytoplasmic" forms of cytochrome c also act as a substrate for microsomal NADPHdependent cytochrome P-450 reductase [47]. This reductase facilitates electron transport to cytochrome P-450, which, in turn, participates in the metabolism of many heterocycles, including those investigated in the present paper. Figure 8 shows the kinetic curve of cytochrome c reduction by microsomes with no and with 10 µM *2-NSQO*. Addition of *2- NSQO* to the medium containing microsome suspension, NADPH, and cytochrome с causes a deceleration of cytochrome c reduction, which progresses with time. Depending on the reagent concentration, the reduction rate decreased at the greatest pace in the first 1−5 min of the reaction. Pre-incubation of microsomes with *2-NSQO* for 5 min, followed by the addition of NADPH and cytochrome c to the reaction medium did not change the protein reduction rate as compared with the variant described above. After the NADPH-cytochrome с reductase activity had been totally inhibited by *2-NSQO*, addition of NADH to the incubation medium partially restored cytochrome c reduction, whereas addition of NADPH and/or cytochrome c did not promote the reaction rate (p>0.05) (Figure 8).

Cellular Caspases: New Targets for the Action of Pharmacological Agents 191

Substitution of *2-NSQO* with its structural analog (*4-NSQO*) yielded a similar pattern, but inhibition of the NADPH-cytochrome с reductase activity proceeded much slower, and addition of NADH to the incubation medium fully restored cytochrome c reduction. Dependence of the NADPH-cytochrome с reductase activity on the concentration of *2- NSQO* and *4-NSQO* is shown in Figure 9. The microsomal protein content being 30 µg/ml, *2- NSQO* in a concentration of 10 µM inhibited the system's enzymatic activity by 50%, and at a concentration of 50 µM and higher the activity dropped by more than 80%. The 10 µM concentration of *4-NSQO* inhibited the enzymatic activity of microsomes by 15%, and the 100 µM concentration – by 50%. The above results suggest that the apoptosis inducing effect of *2-NSQO* and its structural analogs on the cells may be due to the irreversible inhibition of microsomal enzymes with NADPH-cytochrome c reductase activity. Where this effect happens, functioning of the whole electron transport chain in the cells is usually disrupted, there form large amounts of genotoxic products, and the cell eventually dies through

X axis– *2-NSQO* and *4-NSQO* concentrations, µM (logarithmic scale); Y axis – inhibition of the reaction rate (%)

**Figure 9.** Effect of *2-NSQO* (1) and *4-NSQO* (2) on NADPH-cytochrome c reductase activity of the

Another important fact is that when some quinoline derivatives interacted with DNA the greatest hypochromic shift in DNA UV spectra (which evidences the formation of complexes) was observed in treatments with *4-NQO, 4-NSQO, 4-DQO, 4-DPyO*, whereas mixing of *Q* or *2-MeQ* with DNA did not cause changes in the absorption spectra (Table 3). This fact supports the assumption that for such biological effect to appear the latter two

relative to the control. Modifications to the system were made in the first 5 min of the reaction

apoptosis (or necroptosis).

microsomes of *К562* cells.

Cytochrome c and NADPH were added immediately after application of *2-NSQO* to the microsome suspension (2) or after 5 min of pre-treatment of the microsomes with the reagent (3). NADH (50 µM) (4) or NADPH (50 µM) (5) were added to system 2 on the minute of the reading event. Light absorption by the system was measured in phosphate buffer on Labsystems Multiscan Plus reader (LKB, Finland) at 553 nm

**Figure 8.** Cytochrome c reduction by microsomes of *К562* cells with no (1), and with 10 µM *2-NSQO* (2–5).

Substitution of *2-NSQO* with its structural analog (*4-NSQO*) yielded a similar pattern, but inhibition of the NADPH-cytochrome с reductase activity proceeded much slower, and addition of NADH to the incubation medium fully restored cytochrome c reduction. Dependence of the NADPH-cytochrome с reductase activity on the concentration of *2- NSQO* and *4-NSQO* is shown in Figure 9. The microsomal protein content being 30 µg/ml, *2- NSQO* in a concentration of 10 µM inhibited the system's enzymatic activity by 50%, and at a concentration of 50 µM and higher the activity dropped by more than 80%. The 10 µM concentration of *4-NSQO* inhibited the enzymatic activity of microsomes by 15%, and the 100 µM concentration – by 50%. The above results suggest that the apoptosis inducing effect of *2-NSQO* and its structural analogs on the cells may be due to the irreversible inhibition of microsomal enzymes with NADPH-cytochrome c reductase activity. Where this effect happens, functioning of the whole electron transport chain in the cells is usually disrupted, there form large amounts of genotoxic products, and the cell eventually dies through apoptosis (or necroptosis).

190 Apoptosis and Medicine

We have already mentioned that the release of cytochrome c from mitochondrial interior to the cell cytoplasm is a crucial stage in the activation of caspase cascades and triggering of apoptosis. In addition to involvement in the reactions of cellular caspase activation, "cytoplasmic" forms of cytochrome c also act as a substrate for microsomal NADPHdependent cytochrome P-450 reductase [47]. This reductase facilitates electron transport to cytochrome P-450, which, in turn, participates in the metabolism of many heterocycles, including those investigated in the present paper. Figure 8 shows the kinetic curve of cytochrome c reduction by microsomes with no and with 10 µM *2-NSQO*. Addition of *2- NSQO* to the medium containing microsome suspension, NADPH, and cytochrome с causes a deceleration of cytochrome c reduction, which progresses with time. Depending on the reagent concentration, the reduction rate decreased at the greatest pace in the first 1−5 min of the reaction. Pre-incubation of microsomes with *2-NSQO* for 5 min, followed by the addition of NADPH and cytochrome c to the reaction medium did not change the protein reduction rate as compared with the variant described above. After the NADPH-cytochrome с reductase activity had been totally inhibited by *2-NSQO*, addition of NADH to the incubation medium partially restored cytochrome c reduction, whereas addition of NADPH

and/or cytochrome c did not promote the reaction rate (p>0.05) (Figure 8).

Cytochrome c and NADPH were added immediately after application of *2-NSQO* to the microsome suspension (2) or after 5 min of pre-treatment of the microsomes with the reagent (3). NADH (50 µM) (4) or NADPH (50 µM) (5) were added to system 2 on the minute of the reading event. Light absorption by the system was measured in phosphate

**Figure 8.** Cytochrome c reduction by microsomes of *К562* cells with no (1), and with 10 µM *2-NSQO*

buffer on Labsystems Multiscan Plus reader (LKB, Finland) at 553 nm

(2–5).

X axis– *2-NSQO* and *4-NSQO* concentrations, µM (logarithmic scale); Y axis – inhibition of the reaction rate (%) relative to the control. Modifications to the system were made in the first 5 min of the reaction

**Figure 9.** Effect of *2-NSQO* (1) and *4-NSQO* (2) on NADPH-cytochrome c reductase activity of the microsomes of *К562* cells.

Another important fact is that when some quinoline derivatives interacted with DNA the greatest hypochromic shift in DNA UV spectra (which evidences the formation of complexes) was observed in treatments with *4-NQO, 4-NSQO, 4-DQO, 4-DPyO*, whereas mixing of *Q* or *2-MeQ* with DNA did not cause changes in the absorption spectra (Table 3). This fact supports the assumption that for such biological effect to appear the latter two

reagents must first undergo metabolic activation in the cell, and form electrophilic centres within the molecule. Other N-bearing heterocyclic compounds are potentially able to bind to DNA without prior intracellular activation (e.g., through intercalation), so that their biological effects would appear sooner.

Cellular Caspases: New Targets for the Action of Pharmacological Agents 193

Colchicine *4-NQO 2-DQO* 

vinblastine, taxol), and others. *К562/4-NQO* and *К562/2-DQO* cells were tested for susceptibility to the following chemical compounds: DNA intercalating agent ethidium bromide, microtubule polymerization inhibitor colchicine; and quinoline derivatives – *2-*

*К562* 2.5 0.002 1.06 79.7 *К562/4-NQO* 39.8 0.0004 ― 398.2 *К562/2-DQO* 7.9 0.120 79.0 ―

**Table 4. Susceptibility of** *К562/4-NQO* **and** *К562/2-DQO* **cells to cytostatic agents.** Clones resistant to *4-NQO* and *2-DQO* were obtained after [38]. Cell resistance to the xenobiotics was induced by long-term (over 1 month) exposure to 10-12 M *4-NQO* or 10-9 M *2-DQO* in the culture medium. The concentration of the substances was then increased every 14th−21st days. The final concentrations of the reagents for

**ЕС50, µM**

*DQO* and *4-NQO*, respectively. The results are shown in Table 4 and Figure 10.

which resistant cell lines were obtained were 10-8 M for *4-NQO*, and 10-5 M for *2-DQO*.

а – cells were treated with *4-NQO*: 1 – Viability of *К562/2-DQO* cells, 2 – Viability of *К562* cells; b – cells were treated with *2-DQO*: 1 – Viability of *К562/4-NQO* cells, 2 – Viability of *К562* cells; с – cells were treated with colchicines: 1 – Viability of *К562/2-DQO* cells, 2 – Viability of *К562* cells; d – cells were treated with ethidium bromide: 1 – Viability of *К562/2-DQO* cells, 2 – Viability of *К562* cells.

**Figure 10.** Viability of *K562*, *К562/2-DQO* and *К562/4-NQO* cell lines treated with different xenobiotics.

X axis – drug concentration (М). Incubation time – 48 h.

Ethidium bromide

**Cell line** 


**Table 3. Hypochromic effect of DNA upon binding with quinoline and pyridine derivatives.** DNA from chicken erythrocytes was used in the experiment. The spectra were obtained in phosphate buffer on a Labsystems Multiscan Plus reader (LKB, Finland) at 260 nm.

Many chemical compounds, including anticancer drugs, can induce tumor cell apoptosis both *in vivo* and *in vitro* [48−50]. However, a major cause of failure in the application of cytostatic agents in clinical practice is the multiple drug resistance (MDR) phenotype induced in the tumor cells. Multidrug resistance is a condition when tumor cells are unsusceptible to a variety of chemotherapeutic agents differing in chemical structure and the mechanisms through which they affect the cell. MDR is a serious hindrance to success in the treatment of malignant tumors, including leukosis. Latest studies have demonstrated that the molecular mechanisms of MDR are multifarious, and the drug resistance of a cell can depend on various mechanisms triggered at different stages of the toxic impact of the agent on the cell – from restricted accumulation of the drug in the cell to cancellation of the substance-induced cell death programme. Interplay of several protective mechanisms is not uncommon, but one mechanism usually prevails. The best studied mechanisms, whose clinical significance for certain neoplastic forms (such as chronic myeloid leukemia or chronic lymphatic leukemia) has been ascertained, are: activation of transmembrane transport proteins that remove various substances from the cell (namely, Р-glycoprotein – Pgp); activation of the glutathione system enzymes that detoxify the drugs; modifications in the genes and proteins that control apoptosis and cell survival [51].

We have therefore obtained *К562* cell sublines resistant to *2-DQO* and *4-NQO* (*К562/2-DQO, К562/4-NQO*), and characterized their capacity to undergo induced apoptosis under the effect of structurally different chemical reagents as compared with the parental line cells.

The MDR development mechanisms related to inhibition of cytostatic-induced apoptosis have lately been actively investigated. Of greatest interest among the cytostatic agents are DNA-tropic compounds (adriamycin, actinomycin D), antimetabolites (5-fluorouracil, methotrexate), reagents interacting with the mitotic spindle microtubules (vincristine, vinblastine, taxol), and others. *К562/4-NQO* and *К562/2-DQO* cells were tested for susceptibility to the following chemical compounds: DNA intercalating agent ethidium bromide, microtubule polymerization inhibitor colchicine; and quinoline derivatives – *2- DQO* and *4-NQO*, respectively. The results are shown in Table 4 and Figure 10.

192 Apoptosis and Medicine

biological effects would appear sooner.

reagents must first undergo metabolic activation in the cell, and form electrophilic centres within the molecule. Other N-bearing heterocyclic compounds are potentially able to bind to DNA without prior intracellular activation (e.g., through intercalation), so that their

> *4-NQO* 10.71.2 *4-NSQO* 9.71.3 *4-DQO* 9.41.8 *4-DPyO* 7.01.4 *QO* 2.40.6 *Q* 0 *2-MeQ* 0

**Table 3. Hypochromic effect of DNA upon binding with quinoline and pyridine derivatives.** DNA from chicken erythrocytes was used in the experiment. The spectra were obtained in phosphate buffer

Many chemical compounds, including anticancer drugs, can induce tumor cell apoptosis both *in vivo* and *in vitro* [48−50]. However, a major cause of failure in the application of cytostatic agents in clinical practice is the multiple drug resistance (MDR) phenotype induced in the tumor cells. Multidrug resistance is a condition when tumor cells are unsusceptible to a variety of chemotherapeutic agents differing in chemical structure and the mechanisms through which they affect the cell. MDR is a serious hindrance to success in the treatment of malignant tumors, including leukosis. Latest studies have demonstrated that the molecular mechanisms of MDR are multifarious, and the drug resistance of a cell can depend on various mechanisms triggered at different stages of the toxic impact of the agent on the cell – from restricted accumulation of the drug in the cell to cancellation of the substance-induced cell death programme. Interplay of several protective mechanisms is not uncommon, but one mechanism usually prevails. The best studied mechanisms, whose clinical significance for certain neoplastic forms (such as chronic myeloid leukemia or chronic lymphatic leukemia) has been ascertained, are: activation of transmembrane transport proteins that remove various substances from the cell (namely, Р-glycoprotein – Pgp); activation of the glutathione system enzymes that detoxify the drugs; modifications in

We have therefore obtained *К562* cell sublines resistant to *2-DQO* and *4-NQO* (*К562/2-DQO, К562/4-NQO*), and characterized their capacity to undergo induced apoptosis under the effect of structurally different chemical reagents as compared with the parental line cells.

The MDR development mechanisms related to inhibition of cytostatic-induced apoptosis have lately been actively investigated. Of greatest interest among the cytostatic agents are DNA-tropic compounds (adriamycin, actinomycin D), antimetabolites (5-fluorouracil, methotrexate), reagents interacting with the mitotic spindle microtubules (vincristine,

on a Labsystems Multiscan Plus reader (LKB, Finland) at 260 nm.

the genes and proteins that control apoptosis and cell survival [51].

**Reagent Percent of the reagents-related hypochromic** 

**effect of DNA** 


**Table 4. Susceptibility of** *К562/4-NQO* **and** *К562/2-DQO* **cells to cytostatic agents.** Clones resistant to *4-NQO* and *2-DQO* were obtained after [38]. Cell resistance to the xenobiotics was induced by long-term (over 1 month) exposure to 10-12 M *4-NQO* or 10-9 M *2-DQO* in the culture medium. The concentration of the substances was then increased every 14th−21st days. The final concentrations of the reagents for which resistant cell lines were obtained were 10-8 M for *4-NQO*, and 10-5 M for *2-DQO*.

а – cells were treated with *4-NQO*: 1 – Viability of *К562/2-DQO* cells, 2 – Viability of *К562* cells; b – cells were treated with *2-DQO*: 1 – Viability of *К562/4-NQO* cells, 2 – Viability of *К562* cells; с – cells were treated with colchicines: 1 – Viability of *К562/2-DQO* cells, 2 – Viability of *К562* cells; d – cells were treated with ethidium bromide: 1 – Viability of *К562/2-DQO* cells, 2 – Viability of *К562* cells. X axis – drug concentration (М). Incubation time – 48 h.

**Figure 10.** Viability of *K562*, *К562/2-DQO* and *К562/4-NQO* cell lines treated with different xenobiotics.

Measurements of the viability of the cells of the parental line and the derived sublines, incubated for 48 h in the presence of various concentrations of the reagents, revealed substantial differences in the toxic effect of the xenobiotics on the tumor cells. *К562/4-NQO* cells were highly resistant to ethidium bromide and *2-DQO*, but susceptible to colchicine (Table 4, Figure 10). Contrastingly, *К562/2-DQO* cells demonstrated quite high resistance to colchicine and *4-NQO*, but lower resistance to ethidium bromide (Table 4, Figure 10). One should mention that the resistance of *К562/2-DQO* cells to ethidium bromide was 5.03 times lower than that of *К562/4-NQO* cells*.*

Cellular Caspases: New Targets for the Action of Pharmacological Agents 195

The results above prove that the cell sublines we have obtained have become cross-resistant to chemical reagents of the MDR group, and that such resistance may be due, i.a., to

Results are presented as the ratio of caspase to GAPDH expression relative to a standard curve for each assay. Drug concentrations: 0.1 µМ for *К562/2-DQO* cells, 0.001 µМ for *K562* cells, 0.001 µМ for *К562/4-NQO* cells. Incubation time

**Figure 11.** Caspase-3, -6 and -9 mRNA levels were determined by quantitative real-time RT-PCR in

*K562*, *К562/2-DQO* and *К562/4-NQO* cell lines (*a*) treated with colchicine (*b*) respectively.

– 48 h. Viability of cultured cells – 93−95%. Z axis – relative expression, conventional unit.

modifications in caspase expression.

It is now solid knowledge that when subjected to stepped selection for resistance to some cytotoxic agents (plant alkaloids, antibiotics), cells of tumor lines in vitro become crossresistant to quite a number of cytostatics, which differ both in the structure and genesis, and in the effect on different cellular targets [52−54]. The spectra of the drugs to which the cells develop cross-resistance, as well as the mechanisms behind it may vary depending on the selection agent. Grinchuk et al. [55] isolated three adriamycin-resistant clones *С9*, *В2*, and *В3*, through multi-step selection from the *К562* cell population. The cells of the adriamycinresistant clones displayed cross-resistance to colchicine, actinomycin D, and ethidium bromide – agents of the multidrug resistance group. Amplification of the gene *mdr1* was detected in the cells of the resistant clones at DNA hybridization with Southern blot. Karyological analysis of the resistant cells performed at early stages of their incubation with adriamycin showed the genome contained extra genetic material (morphological markers of amplification – double minichromosomes in clones *В2* and *В3*, and uniformly stained regions in the chromosomes of clone *С9* cells). The karyotype modifications are attributed to destabilization of the *К562*/adriamycin cell genome as the cells acquired the MDR phenotype. Thus, distinctions in the resistance to the toxic effect of xenobiotics among cells of different sublines may be related to specific characteristics of the expression of *mdr* genes and then Р glycoprotein, expression of other MDR proteins, genome destabilization, and, not least, changes in the activity of cellular enzymes involved in the first (e.g., cytochrome Р-450) and second (e.g., glutathione-S-transferase) phases of the reagent metabolism in the cells. These and other factors can, under certain conditions, play a decisive role in the modulation of the functional activity of apoptosis inducing agents, including caspases.

Figure 11*a* shows the results on relative expression of caspase-3, -6, and -9 genes in the cells of the *К562* parental line and the derivative sublines (data from RT-PCR analysis). One can see that the mRNA expression of the named caspases in *К562*/*4-NQO* and *К562*/*2-DQO* cells is significantly lower (p<0.05) than in the parental *К562*, and that is a potential explanation of the resistance of the tumor cells to *4-NQO* and *2-DQO*.

In the treatment with colchicine mRNA expression of caspases-3, -6, and -9 was significantly promoted in the parental line *К562* and *К562*/*4-NQO* – p<0.05 (Figure 11 *b*). In *К562*/*2-DQO* cells only caspase-6 expression was activated (p<0.05). Incubation of tumor cells with ethidium bromide promoted the mRNA expression of the three investigated caspases in the parent cells; caspase-3 expression was promoted in *К562*/*2-DQO* cells; no significant changes in caspase mRNA expression was observed in the *К562*/*4-NQO* line. Changes in the enzymatic activity correlated with mRNA expression.

The results above prove that the cell sublines we have obtained have become cross-resistant to chemical reagents of the MDR group, and that such resistance may be due, i.a., to modifications in caspase expression.

194 Apoptosis and Medicine

lower than that of *К562/4-NQO* cells*.*

Measurements of the viability of the cells of the parental line and the derived sublines, incubated for 48 h in the presence of various concentrations of the reagents, revealed substantial differences in the toxic effect of the xenobiotics on the tumor cells. *К562/4-NQO* cells were highly resistant to ethidium bromide and *2-DQO*, but susceptible to colchicine (Table 4, Figure 10). Contrastingly, *К562/2-DQO* cells demonstrated quite high resistance to colchicine and *4-NQO*, but lower resistance to ethidium bromide (Table 4, Figure 10). One should mention that the resistance of *К562/2-DQO* cells to ethidium bromide was 5.03 times

It is now solid knowledge that when subjected to stepped selection for resistance to some cytotoxic agents (plant alkaloids, antibiotics), cells of tumor lines in vitro become crossresistant to quite a number of cytostatics, which differ both in the structure and genesis, and in the effect on different cellular targets [52−54]. The spectra of the drugs to which the cells develop cross-resistance, as well as the mechanisms behind it may vary depending on the selection agent. Grinchuk et al. [55] isolated three adriamycin-resistant clones *С9*, *В2*, and *В3*, through multi-step selection from the *К562* cell population. The cells of the adriamycinresistant clones displayed cross-resistance to colchicine, actinomycin D, and ethidium bromide – agents of the multidrug resistance group. Amplification of the gene *mdr1* was detected in the cells of the resistant clones at DNA hybridization with Southern blot. Karyological analysis of the resistant cells performed at early stages of their incubation with adriamycin showed the genome contained extra genetic material (morphological markers of amplification – double minichromosomes in clones *В2* and *В3*, and uniformly stained regions in the chromosomes of clone *С9* cells). The karyotype modifications are attributed to destabilization of the *К562*/adriamycin cell genome as the cells acquired the MDR phenotype. Thus, distinctions in the resistance to the toxic effect of xenobiotics among cells of different sublines may be related to specific characteristics of the expression of *mdr* genes and then Р glycoprotein, expression of other MDR proteins, genome destabilization, and, not least, changes in the activity of cellular enzymes involved in the first (e.g., cytochrome Р-450) and second (e.g., glutathione-S-transferase) phases of the reagent metabolism in the cells. These and other factors can, under certain conditions, play a decisive role in the modulation of the functional activity of apoptosis inducing agents, including caspases.

Figure 11*a* shows the results on relative expression of caspase-3, -6, and -9 genes in the cells of the *К562* parental line and the derivative sublines (data from RT-PCR analysis). One can see that the mRNA expression of the named caspases in *К562*/*4-NQO* and *К562*/*2-DQO* cells is significantly lower (p<0.05) than in the parental *К562*, and that is a potential explanation

In the treatment with colchicine mRNA expression of caspases-3, -6, and -9 was significantly promoted in the parental line *К562* and *К562*/*4-NQO* – p<0.05 (Figure 11 *b*). In *К562*/*2-DQO* cells only caspase-6 expression was activated (p<0.05). Incubation of tumor cells with ethidium bromide promoted the mRNA expression of the three investigated caspases in the parent cells; caspase-3 expression was promoted in *К562*/*2-DQO* cells; no significant changes in caspase mRNA expression was observed in the *К562*/*4-NQO* line. Changes in the

of the resistance of the tumor cells to *4-NQO* and *2-DQO*.

enzymatic activity correlated with mRNA expression.

Results are presented as the ratio of caspase to GAPDH expression relative to a standard curve for each assay. Drug concentrations: 0.1 µМ for *К562/2-DQO* cells, 0.001 µМ for *K562* cells, 0.001 µМ for *К562/4-NQO* cells. Incubation time – 48 h. Viability of cultured cells – 93−95%. Z axis – relative expression, conventional unit.

**Figure 11.** Caspase-3, -6 and -9 mRNA levels were determined by quantitative real-time RT-PCR in *K562*, *К562/2-DQO* and *К562/4-NQO* cell lines (*a*) treated with colchicine (*b*) respectively.

One of the preconditions for *in vivo* development and progress of tumors is that the tumor cells gain another type of resistance – resistance to factors of anti-tumor immunity. Such factors are, first of all, natural killer (NK) cells and cytotoxic T lymphocytes. The mechanisms of identification of *de novo* tumor cells in the organism by natural killers have not been fully elucidated, but the mechanisms of the lysis of such cells are quite well studied. NK cells and antigen-specific cytotoxic T lymphocytes, as well as these IL-2 activated cells are known to lyse target cells both by perforin-dependent cytolysis, and by inducing Fas-dependent apoptosis [56, 57]. In this case, the target cells can modulate the rate of release of the cytolytic granule content, including perforin and granzymes, or suppress killer activation by reducing the frequency and affinity of the formation of target celleffector cell conjugates, the latter being a quite frequent phenomenon in the system [58]. Perforin is structurally homologous to the *С9* complement component, and has a similar action mechanism (formation of polyperforin pores made up of 12–18 monomers, with the inner diameter of 10–20 nm) [56]. On the other hand, no DNA fragmentation occurs when the complement acts on the cells, wherefore we presume that the action of perforin involves also other factors (namely serine proteases of the granzyme family [58], which can permeate the target cell cytosol via perforin-generated pores in the plasma membrane and trigger apoptotic events, for instance by means of caspase activation.

Cellular Caspases: New Targets for the Action of Pharmacological Agents 197

Drug concentrations: *Q* – 0.3 µМ, *QO* – 10 µМ, *4-NQO* – 0.001 µМ, styryl derivatives – 1 µМ. X-axis: cytotoxic index ratio of effector cells with treated cell lines to control cell lines without treatment, % (cytotoxic index value of control

**Figure 12.** The *К562* cell line sensitivity to cytotoxic lysis by human peripheral blood white cells after

When cells of sublines *К562*/*4-NQO* and *К562*/*2-DQO* were used as the targets, changes in susceptibility to the leukocyte lysis effect were recorded only in the *К562*/*4-NQO−*colchicine

cells is 100 %). Effector cells to target cells ratio is 50:1. Incubation time – 48 h; \* p<0.05.

treatment with quinoline and pyridine derivatives

system (Figure 13).

Some authors have demonstrated in their papers that treatment of tumor cells with chemical reagents modifies their susceptibility to the cytotoxic lysis of homologous effector cells, and the modification may be either for amplification or for reduction of the effect depending on the cell line and the treatment settings. E.g., treatment of *К562* cells with sodium butyrate [59], fagaronine, adriamycin, aclacinomycin А [60] makes the named cells less susceptible to natural killer lysis. Quite a number of lymphoma cell lines (*Raji* and *Daudi* lymphoma cell lines, human lymphoblastoid cell line *NAD-7*, human Т-lymphoblastoid line *Molt-4*) treated with PMA, sodium butyrate, retinoic acid demonstrate higher susceptibility to the cytotoxic activity of natural killers [61−63]. An interesting effect was observed in the case of *CB-1 B* cell lines. Clone *26CB-1* is more resistant to NK cells than clone *13CB-1*. After they had been treated with sodium butyrate, iodinated deoxyuridine, 5-azacytidine, tunicamycin, the susceptibility of clone *26CB-1* cells to cytotoxic lysis increased, whereas the susceptibility of clone *13CB-1* cells remained nearly unmodified [64].

Distinctions in the chemical inducers' structure and mechanisms of action on cells may have different effects on modulation of the expression of positive and negative apoptosis regulators, which quantitative ratios may contribute to establishment of both the initial susceptibility of the cells to natural killer lysis and the post-treatment susceptibility. This may as well be valid for the cell sublines with a more or less differentiated phenotype.

Data in Figure 12 (**a**, **b**) show that the treatment of tumor cells with *4-NQO, 2-NSQO,* and *4- NSQO* for 48 h caused a significant rise (р<0.05) in susceptibility to the lysis of human leukocytes containing NK cells. Caspase expression and cell apoptosis induction were also promoted in this case. In treatments with other quinoline derivatives the cytotoxic index in the cultures did not change. A similar pattern was observed also when incubation lasted 96 h.

One of the preconditions for *in vivo* development and progress of tumors is that the tumor cells gain another type of resistance – resistance to factors of anti-tumor immunity. Such factors are, first of all, natural killer (NK) cells and cytotoxic T lymphocytes. The mechanisms of identification of *de novo* tumor cells in the organism by natural killers have not been fully elucidated, but the mechanisms of the lysis of such cells are quite well studied. NK cells and antigen-specific cytotoxic T lymphocytes, as well as these IL-2 activated cells are known to lyse target cells both by perforin-dependent cytolysis, and by inducing Fas-dependent apoptosis [56, 57]. In this case, the target cells can modulate the rate of release of the cytolytic granule content, including perforin and granzymes, or suppress killer activation by reducing the frequency and affinity of the formation of target celleffector cell conjugates, the latter being a quite frequent phenomenon in the system [58]. Perforin is structurally homologous to the *С9* complement component, and has a similar action mechanism (formation of polyperforin pores made up of 12–18 monomers, with the inner diameter of 10–20 nm) [56]. On the other hand, no DNA fragmentation occurs when the complement acts on the cells, wherefore we presume that the action of perforin involves also other factors (namely serine proteases of the granzyme family [58], which can permeate the target cell cytosol via perforin-generated pores in the plasma membrane and trigger

Some authors have demonstrated in their papers that treatment of tumor cells with chemical reagents modifies their susceptibility to the cytotoxic lysis of homologous effector cells, and the modification may be either for amplification or for reduction of the effect depending on the cell line and the treatment settings. E.g., treatment of *К562* cells with sodium butyrate [59], fagaronine, adriamycin, aclacinomycin А [60] makes the named cells less susceptible to natural killer lysis. Quite a number of lymphoma cell lines (*Raji* and *Daudi* lymphoma cell lines, human lymphoblastoid cell line *NAD-7*, human Т-lymphoblastoid line *Molt-4*) treated with PMA, sodium butyrate, retinoic acid demonstrate higher susceptibility to the cytotoxic activity of natural killers [61−63]. An interesting effect was observed in the case of *CB-1 B* cell lines. Clone *26CB-1* is more resistant to NK cells than clone *13CB-1*. After they had been treated with sodium butyrate, iodinated deoxyuridine, 5-azacytidine, tunicamycin, the susceptibility of clone *26CB-1* cells to cytotoxic lysis increased, whereas the susceptibility of

Distinctions in the chemical inducers' structure and mechanisms of action on cells may have different effects on modulation of the expression of positive and negative apoptosis regulators, which quantitative ratios may contribute to establishment of both the initial susceptibility of the cells to natural killer lysis and the post-treatment susceptibility. This may as well be valid for the cell sublines with a more or less differentiated phenotype.

Data in Figure 12 (**a**, **b**) show that the treatment of tumor cells with *4-NQO, 2-NSQO,* and *4- NSQO* for 48 h caused a significant rise (р<0.05) in susceptibility to the lysis of human leukocytes containing NK cells. Caspase expression and cell apoptosis induction were also promoted in this case. In treatments with other quinoline derivatives the cytotoxic index in the cultures did not change. A similar pattern was observed also when incubation lasted 96 h.

apoptotic events, for instance by means of caspase activation.

clone *13CB-1* cells remained nearly unmodified [64].

Drug concentrations: *Q* – 0.3 µМ, *QO* – 10 µМ, *4-NQO* – 0.001 µМ, styryl derivatives – 1 µМ. X-axis: cytotoxic index ratio of effector cells with treated cell lines to control cell lines without treatment, % (cytotoxic index value of control cells is 100 %). Effector cells to target cells ratio is 50:1. Incubation time – 48 h; \* p<0.05.

**Figure 12.** The *К562* cell line sensitivity to cytotoxic lysis by human peripheral blood white cells after treatment with quinoline and pyridine derivatives

When cells of sublines *К562*/*4-NQO* and *К562*/*2-DQO* were used as the targets, changes in susceptibility to the leukocyte lysis effect were recorded only in the *К562*/*4-NQO−*colchicine system (Figure 13).

Cellular Caspases: New Targets for the Action of Pharmacological Agents 199


virology, cytology, immunology, biochemistry, embryology, and other areas of modern science at the cellular and molecular levels. It is obvious that the problem addressed in this chapter is of high applied value, for the key aim of the effort is to enhance the effectiveness of anticancer therapy. The industry of rational drug design is becoming more and more widespread in practical medicine. The principal concepts in drug design are the target and the drug. The target is a low-molecular biological structure presumably linked to a certain function which disruption would lead to disease, and to which a certain impact should be applied. The most common targets are receptors, enzymes, hormones. The drug is most often a chemical compound (usually a low-molecular one) that specifically interacts with the target, modifying the cell response in one way or another. One of the earliest and most seminal stages of drug design is accurate identification of the target by influencing which one can specifically regulate certain biochemical processes, leaving others unaffected as much as possible. This is not however always feasible: by no means are all diseases caused by dysfunction of just one protein or gene. Preference should therefore be given to the processes' principal biomolecules. In terms of apoptosis, caspases clearly are such biomolecules. Hence, the choice of methods for experimental validation of caspases as potential specific targets for drugs is a topical

Quinoline – *Q*, 2-methylquinoline – *2-MeQ*, quinoline-1-oxide – *QO*, 2-methylquinoline-1 oxide – *2-MeQO*, 4-nitroquinoline-1-oxide – *4-NQO,* 2-methyl-4-nitroquinoline-1-oxide – *2-Me-4-NQO*, 2-(4'-dimethylaminostyryl)quinoline-1-oxide *– 2-DQO,* 4-(4'-dimethylaminostyryl) quinoline-1-oxide – *4-DQO,* 2-(4'-nitrostyryl)quinoline-1-oxide – *2-NSQO,* 4-(4'-nitrostyryl)-

effector domain – DED, caspase recruitment domain – CARD, death inducing domain – DID, р53-induced protein with a death domain – PIDD, death-inducing signalling complex – DISC, DNA fragmentation factor – DFF, caspase-activated DNase – CAD, permeability transition роrе – РТР, apoptosis inducing factor – AIF, tumor necrosis factor receptor – TNFR, death domain – DD, death receptor – DR, Fas-associated DD-protein – FADD, TNFR1-associated

challenge for the nearest future.

quinoline-1-oxide – *4-NSQO*, and 4-(4'

*Department of Biochemistry, School of Medicine, Petrozavodsk State University, Petrozavodsk, Russia* 

*Department of Pathology, School of Medicine,* 

**6. Abbreviations:** 

DD-protein – TRADD.

**Author details** 

Tatyana O. Volkova

Alexander N. Poltorak

*Tufts University, Boston, USA* 

Drug concentrations – 0.1 µМ for *К562/2-DQO* cells, 0.001 µМ for *K562* cells, 0.001 µМ for *К562/4-NQO* cells. X-axis: cytotoxic index ratio of effector cells with treated cell lines to control cell lines without treatment, % (cytotoxic index value of control cells is 100 %). Effector cells to target cells ratio is 50:1. Incubation time – 48 h; \* p<0.05.

**Figure 13.** The *К562* cell line sensitivity to cytotoxic lysis by human peripheral blood white cells after treatment with colchicines.

Thus, treatment of tumor cells with chemical compounds may simultaneously induce cell apoptosis and modulate their susceptibility to the cytotoxic lysis of blood leukocytes. In these treatment conditions the expression and/or activity of intracellular apoptosis regulators, namely caspases, changes, and this change may tell on the effectiveness of the leukocyte lysis effect, which is based on induction of target cell apoptosis. It is essential that the susceptibility of tumor cells to the lysis of leukocytes containing NK cells changed in those variants where the expression and activity of caspases-3, -6, -9 were promoted (cultures treated with *4-NQO, 2-NSQO*, and *4-NSQO*)*.* On the other hand, the equivocal nature of the results proves that the process involves extra intracellular factors. One can therefore assume that similar patterns of the response of tumor cells to treatment with chemical compounds can occur *in vivo* in humans and animals treated with the anticancer drugs stimulating cell apoptosis. This effect can persist when nontoxic or low-toxicity doses are applied, and caspases, being a cell's major apoptosis inducing factor, can be viewed as potential targets for pharmacologically active agents.
