**1. Introduction**

174 Apoptosis and Medicine

[92] Dreiseitel A, Oosterhuis B, Vukman KV, Schreier P, Oehme A, Locher S, Hajak G, Sand PG (2009) Berry anthocyanins and anthocyanidins exhibit distinct affinities for the

efflux transporters bcrp and mdr1. Br. J. Pharmacol. 158:1942-1950.

The number of scientific papers devoted to the study of caspases has lately being constantly growing. Caspases are a family of cysteine-dependent aspartate specific proteases. Caspases play an essential role in the apoptosis, necrosis and inflammation processes. Apoptosis is asynchronous programmed cell death, which helps maintain the physiological balance and genetic stability of the organism through self-destruction of genetically modified defect cells. Apoptosis may happen also in normal cells, e.g., during embryogenesis. During apoptosis, activated endogenous nucleases cleave DNA into fragments, but cell membranes and the intracellular matter remain intact, nor is there tissue damage or leukocytic infiltration. In contrast to apoptosis, necrosis is a pathological form of cell death caused by acute damage, rupture of the membrane, release of the cytoplasm content, and the inflammatory process induced thereby [1, 2].

As of now, at lease 14 caspases have been described from mammals: 8 caspases are involved in apoptosis, 5 activate anti-inflammatory cytokines, and one acts in keratinocyte differentiation. This division is, however, rather arbitrary – we know from a number of papers that some apoptotic caspases may, depending on the conditions, participate in other cell life processes, such as proliferation [3], differentiation [4, 5], modification of susceptibility to leukocyte lysis [2, 6].

During apoptosis, different caspases perform different functions. Depending on the phase at which those proteins enter the apoptotic cascade one distinguishes initiator (apical) and effector (executioner) caspases. E.g., caspase-2, -8, -9, -10, and -12 are initiator ones; caspase-3, -6, -7 – effector ones. All caspases are originally inactive, but activated when needed by cleavage of a small fragment by initiator caspases. Initiator caspase activation is more sophisticated – by special protein complexes: apoptosomes, PIDDosomes, DISC (*death-*

© 2012 Volkova and Poltorak, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Volkova and Poltorak, licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*inducing signalling complexes*) [7, 8]. Under certain conditions effector caspases may act as initiator ones to accelerate apoptotic reactions.

Cellular Caspases: New Targets for the Action of Pharmacological Agents 177

**Figure 1.** Derivatives of quinoline and pyridine

and low side effects is therefore an important task.

In current clinical practices, quinoline derivatives are used as drugs with high pharmacological potential – photoprotective, anti-inflammatory, immunomodulating, antioxidant, antiproliferative, antiaggregatory, hypolipidemic, and hypoglycemic. This group of chemical compounds and their intracellular metabolites can be highly competitive as structural analogs of key molecules of the cell (nitrogen bases, nucleosides, nucleotides, various co-enzymes, etc.), and take effect by activating or inhibiting a certain cell process. The compounds with such biological effects most frequently used in the oncology practice

Derivatives of N-bearing heterocycles are very effective in treatment of tumors of various histogenesis and location, including leukemia, but many of them have limited applicability because of high toxicity. Finding the compounds that feature both high anti-tumor activity

are 5-fluorouracil, cytarabine, methotrexate, vinblastine, dactinomycin, and others.

Research into the phenomenon of programmed cell death started in the late 1960s. One of the pioneers in the sphere was John Kerr, who studied the death of hepatocytes in rats with acute liver failure [9]. In 1972, a team of British scientists headed by Kerr first coined the term apoptosis to denote programmed cell death. The authors described two phases of the process (formation of apoptotic bodies, and their phagocytosis by other cells), and stressed that apoptosis is an active and controlled process. Yet, nothing was known at the time about the factors and mechanisms of this type of cell death. Studies of the structure of caspases began only in 1994 [10, 11]. In 1996, it was found that cytochrome c together with ATP promote activation of caspase-3 [12]. Caspases are now investigated in various model systems both *in vitro* and *in vivo*. The most popular systems in use are tumor cell lines of various histogenesis. Most studies employ hemopoietic tumor cells (lines *К562*, *HL-60*, *Raji*, *HEL*, etc.), cells derived from solid tumors (lines *HeLa*, *RPTC*, *НСТ 116*, etc.), as well as stem cells. Through this approach one can not just study the structure, activation mechanisms and basic functions of caspases in normal cells, but also identify the biological role of these enzymes in emerging and progressing pathological processes, first of all, oncologic and immuno-inflammatory ones. Besides, the chemical reagents used in those model systems usually activate or inhibit cell processes, including apoptosis. These facts open up immense opportunities in the analysis of the activity of the substances considered for applicability as components of new targeted drugs. The clue to the effectiveness of the chemical reagents for the treated cells will be the changes in the susceptibility of those cells to cytotoxic lysis of blood leukocytes, namely natural killer cells and cytotoxic Т-lymphocytes. Since proteases play an important part in cytotoxic lysis, caspases in this case can also be viewed as candidate molecules whose activity can be modulated to accelerate or damp apoptosis, as well as modify the susceptibility of pathological cells to leukocytolysis.

Hence, the primary objective of the chapter is to demonstrate by means of the experimental data the crucial role of caspases in the induction and progress of tumor cell apoptosis, as well as in the modification of tumor cell susceptibility to the cytotoxic lysis of natural killer cells upon treatment with chemical compounds, part of which have been newly synthesized. The model system used in our study was the human erythromyeloid leukemic cell line *К562*  (Russian Cell Culture Collection, Institute of Cytology, Russian Academy of Sciences, St. Petersburg, Russia), as well as *К562/2-DQO* and *К562/4-NQO* subline cells. To induce apoptosis and modulate cell susceptibility to the lytic action of blood leukocytes we used a group of N-bearing heterocyclic reagents – derivatives of quinoline and pyridine: 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)quinoline-1-oxide – *4-NSQO*, and 4-(4' dimethylaminostyryl)pyridine-1-oxide – *4-DPyO* (Figure 1).

Cellular Caspases: New Targets for the Action of Pharmacological Agents 177

**Figure 1.** Derivatives of quinoline and pyridine

176 Apoptosis and Medicine

initiator ones to accelerate apoptotic reactions.

*inducing signalling complexes*) [7, 8]. Under certain conditions effector caspases may act as

Research into the phenomenon of programmed cell death started in the late 1960s. One of the pioneers in the sphere was John Kerr, who studied the death of hepatocytes in rats with acute liver failure [9]. In 1972, a team of British scientists headed by Kerr first coined the term apoptosis to denote programmed cell death. The authors described two phases of the process (formation of apoptotic bodies, and their phagocytosis by other cells), and stressed that apoptosis is an active and controlled process. Yet, nothing was known at the time about the factors and mechanisms of this type of cell death. Studies of the structure of caspases began only in 1994 [10, 11]. In 1996, it was found that cytochrome c together with ATP promote activation of caspase-3 [12]. Caspases are now investigated in various model systems both *in vitro* and *in vivo*. The most popular systems in use are tumor cell lines of various histogenesis. Most studies employ hemopoietic tumor cells (lines *К562*, *HL-60*, *Raji*, *HEL*, etc.), cells derived from solid tumors (lines *HeLa*, *RPTC*, *НСТ 116*, etc.), as well as stem cells. Through this approach one can not just study the structure, activation mechanisms and basic functions of caspases in normal cells, but also identify the biological role of these enzymes in emerging and progressing pathological processes, first of all, oncologic and immuno-inflammatory ones. Besides, the chemical reagents used in those model systems usually activate or inhibit cell processes, including apoptosis. These facts open up immense opportunities in the analysis of the activity of the substances considered for applicability as components of new targeted drugs. The clue to the effectiveness of the chemical reagents for the treated cells will be the changes in the susceptibility of those cells to cytotoxic lysis of blood leukocytes, namely natural killer cells and cytotoxic Т-lymphocytes. Since proteases play an important part in cytotoxic lysis, caspases in this case can also be viewed as candidate molecules whose activity can be modulated to accelerate or damp apoptosis, as

well as modify the susceptibility of pathological cells to leukocytolysis.

dimethylaminostyryl)pyridine-1-oxide – *4-DPyO* (Figure 1).

Hence, the primary objective of the chapter is to demonstrate by means of the experimental data the crucial role of caspases in the induction and progress of tumor cell apoptosis, as well as in the modification of tumor cell susceptibility to the cytotoxic lysis of natural killer cells upon treatment with chemical compounds, part of which have been newly synthesized. The model system used in our study was the human erythromyeloid leukemic cell line *К562*  (Russian Cell Culture Collection, Institute of Cytology, Russian Academy of Sciences, St. Petersburg, Russia), as well as *К562/2-DQO* and *К562/4-NQO* subline cells. To induce apoptosis and modulate cell susceptibility to the lytic action of blood leukocytes we used a group of N-bearing heterocyclic reagents – derivatives of quinoline and pyridine: 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)quinoline-1-oxide – *4-NSQO*, and 4-(4'


In current clinical practices, quinoline derivatives are used as drugs with high pharmacological potential – photoprotective, anti-inflammatory, immunomodulating, antioxidant, antiproliferative, antiaggregatory, hypolipidemic, and hypoglycemic. This group of chemical compounds and their intracellular metabolites can be highly competitive as structural analogs of key molecules of the cell (nitrogen bases, nucleosides, nucleotides, various co-enzymes, etc.), and take effect by activating or inhibiting a certain cell process. The compounds with such biological effects most frequently used in the oncology practice are 5-fluorouracil, cytarabine, methotrexate, vinblastine, dactinomycin, and others.

Derivatives of N-bearing heterocycles are very effective in treatment of tumors of various histogenesis and location, including leukemia, but many of them have limited applicability because of high toxicity. Finding the compounds that feature both high anti-tumor activity and low side effects is therefore an important task.
