**2. Body**

A major problem in modern cell biology is the search for intracellular targets for pharmacologically active substances, and subsequent development of new generation medicines based on the resultant data. As a rule, molecules involved in basic processes of cell life are viewed as such intracellular targets. The molecules participating in two or more vital processes are of greatest interest. Such are caspases. They are synthesized in the cell as precursors (procaspases). Caspase precursors consist of a prodomain and two subunits: a small one and a large one. Some caspases contain a short linker sequence (around 10 amino acids) between the subunits. Caspases can activate one another: the prodomain is cleaved from the procaspase, and the small and large subunits of the two caspases form an active caspase – heterotetramer (Figure 2), which contains two active centres. Caspases of the initiator (pro-apoptotic and pro-inflammatory) type contain a long prodomain made up of approximately 100 amino acids, whereas the short prodomain of effector caspases contains only 30 amino acid residues. Long prodomains contain various motifs, mainly DED (*death effector domain*) and CARD (*caspase recruitment domain*), but also DID (*death inducing domain*). Each of the procaspases-8 and -10 contains two tandem copies of the DED, whereas CARD was found in caspases-1, -2, -4, -5, -9, -11, -12. Homotypic interactions between DED and CARD recruit procaspases into initiator complexes, activate them and trigger the caspase cascade [13].

Cellular Caspases: New Targets for the Action of Pharmacological Agents 179

Apart from distinctions in the prodomain structure, caspases can be grouped by substrate specificity. E.g., caspases-6, -8 and -9 show preference for cleaving substrates with the valine/leucine−glutamate−threonine/histidine−aspartate (V/L−E−T/H−D) sequence, whereas caspases-3 and -7 selectively cleave the motif made up of the aspartate−glutamate−valine−aspartate (D−E−V−D) sequence. An optimal target for caspase-1 and its related caspases-4 and -5 are the sequences tyrosine−valine−alanine−aspartate

(Y−V−A−D) or tryptophan/leucine−glutamate−histidine−aspartate (W/L−E−H−D) [14].

process.

activation.

*associated DD-protein*) (Figure 3).

initiator caspases then activate effector caspases.

Various forms of post-translational modifications can influence the activity of caspases. Thus, phosphorylation of caspase-9 by the serine/threonine protein kinase Akt inhibits its activity. Such phosphorylation occurs away from the caspase active centre, and presumably hinders the clustering of the caspase subunits into the tetramer [13, 15]. Another variant of post-translational modification of caspases is S-nitrosylation. The NO radical group is tranfered to the cysteine of the caspase active centre and the R−S−NO group is formed [16, 17]. Furthermore, caspases are susceptible to oxidative modifications induced by active forms of oxygen, with disulphides formed in the

Apoptosis can be triggered by various mechanisms that activate caspases. Two major pathways are distinguished: activation involving cell receptors, and mitochondria-mediated

*Caspase activation involving cell receptors***.** The process of apoptosis often begins with ligation of specific extracellular ligands with cell death receptors on the membrane surface. Receptors of the apoptotic signal belong to the TNF (*tumor necrosis factor*) receptors superfamily [18]. The best studied death receptors, whose role in apoptosis has been identified and described, are CD95 (Fas or APO-1), and TNFR1 (p55 or CD120a) (Figure 3). Additional receptors are CARI, DR3 (*death receptor* 3), DR4, DR5, and DR6. All death receptors are transmembrane proteins which share a sequence of 80 amino acids at the cytoplasmic face termed the death domain (DD). It is required for apoptotic signalling [7, 18]. Extracellular regions of death receptors interact with ligand trimers (CD95L, TNF, Apo3L, Apo2L, etc.), and the latter trimerize the death receptors (crosslink 3 molecules of the receptor) [19, 20]. The thus activated receptor interacts with *a* corresponding intracellular adaptor(s). The adaptor for the CD95 (Fas/APO-1) receptor is FADD (*Fasassociated DD-protein*). The adaptor for the TNFR1 and DR3 receptors is TRADD (*TNFR1-*

The adaptor associated with the death receptor interacts with caspases. The "ligand−receptor−adaptor−effector" interaction chain results in the formation of aggregates in which caspases are activated. These aggregates are called apoptosomes, apoptotic chaperones, or death-inducing signalling complexes (DISC). An example of apoptosome is the FasL−Fas−FADD−procaspase-8 complex, in which caspase-8 is activated (Figure 3) [7, 21]. Death receptors can mediate activation of caspases-2; -8, and -10 [22]. The activated

**Figure 2. Proteolytic caspase activation** (adopted from http://www.muldyr.ru/a/a/apoptoz\_- \_fazyi\_apoptoza)

Apart from distinctions in the prodomain structure, caspases can be grouped by substrate specificity. E.g., caspases-6, -8 and -9 show preference for cleaving substrates with the valine/leucine−glutamate−threonine/histidine−aspartate (V/L−E−T/H−D) sequence, whereas caspases-3 and -7 selectively cleave the motif made up of the aspartate−glutamate−valine−aspartate (D−E−V−D) sequence. An optimal target for caspase-1 and its related caspases-4 and -5 are the sequences tyrosine−valine−alanine−aspartate (Y−V−A−D) or tryptophan/leucine−glutamate−histidine−aspartate (W/L−E−H−D) [14].

178 Apoptosis and Medicine

A major problem in modern cell biology is the search for intracellular targets for pharmacologically active substances, and subsequent development of new generation medicines based on the resultant data. As a rule, molecules involved in basic processes of cell life are viewed as such intracellular targets. The molecules participating in two or more vital processes are of greatest interest. Such are caspases. They are synthesized in the cell as precursors (procaspases). Caspase precursors consist of a prodomain and two subunits: a small one and a large one. Some caspases contain a short linker sequence (around 10 amino acids) between the subunits. Caspases can activate one another: the prodomain is cleaved from the procaspase, and the small and large subunits of the two caspases form an active caspase – heterotetramer (Figure 2), which contains two active centres. Caspases of the initiator (pro-apoptotic and pro-inflammatory) type contain a long prodomain made up of approximately 100 amino acids, whereas the short prodomain of effector caspases contains only 30 amino acid residues. Long prodomains contain various motifs, mainly DED (*death effector domain*) and CARD (*caspase recruitment domain*), but also DID (*death inducing domain*). Each of the procaspases-8 and -10 contains two tandem copies of the DED, whereas CARD was found in caspases-1, -2, -4, -5, -9, -11, -12. Homotypic interactions between DED and CARD recruit procaspases into initiator complexes, activate them and trigger the caspase

**Figure 2. Proteolytic caspase activation** (adopted from http://www.muldyr.ru/a/a/apoptoz\_-

**2. Body** 

cascade [13].

\_fazyi\_apoptoza)

Various forms of post-translational modifications can influence the activity of caspases. Thus, phosphorylation of caspase-9 by the serine/threonine protein kinase Akt inhibits its activity. Such phosphorylation occurs away from the caspase active centre, and presumably hinders the clustering of the caspase subunits into the tetramer [13, 15]. Another variant of post-translational modification of caspases is S-nitrosylation. The NO radical group is tranfered to the cysteine of the caspase active centre and the R−S−NO group is formed [16, 17]. Furthermore, caspases are susceptible to oxidative modifications induced by active forms of oxygen, with disulphides formed in the process.

Apoptosis can be triggered by various mechanisms that activate caspases. Two major pathways are distinguished: activation involving cell receptors, and mitochondria-mediated activation.

*Caspase activation involving cell receptors***.** The process of apoptosis often begins with ligation of specific extracellular ligands with cell death receptors on the membrane surface. Receptors of the apoptotic signal belong to the TNF (*tumor necrosis factor*) receptors superfamily [18]. The best studied death receptors, whose role in apoptosis has been identified and described, are CD95 (Fas or APO-1), and TNFR1 (p55 or CD120a) (Figure 3). Additional receptors are CARI, DR3 (*death receptor* 3), DR4, DR5, and DR6. All death receptors are transmembrane proteins which share a sequence of 80 amino acids at the cytoplasmic face termed the death domain (DD). It is required for apoptotic signalling [7, 18]. Extracellular regions of death receptors interact with ligand trimers (CD95L, TNF, Apo3L, Apo2L, etc.), and the latter trimerize the death receptors (crosslink 3 molecules of the receptor) [19, 20]. The thus activated receptor interacts with *a* corresponding intracellular adaptor(s). The adaptor for the CD95 (Fas/APO-1) receptor is FADD (*Fasassociated DD-protein*). The adaptor for the TNFR1 and DR3 receptors is TRADD (*TNFR1 associated DD-protein*) (Figure 3).

The adaptor associated with the death receptor interacts with caspases. The "ligand−receptor−adaptor−effector" interaction chain results in the formation of aggregates in which caspases are activated. These aggregates are called apoptosomes, apoptotic chaperones, or death-inducing signalling complexes (DISC). An example of apoptosome is the FasL−Fas−FADD−procaspase-8 complex, in which caspase-8 is activated (Figure 3) [7, 21]. Death receptors can mediate activation of caspases-2; -8, and -10 [22]. The activated initiator caspases then activate effector caspases.

Cellular Caspases: New Targets for the Action of Pharmacological Agents 181

A presumable alternative pathway for the release of apoptotic proteins from the mitochondrial intermembrane region is formation of a protein channel in the outer mitochondrial membrane. Whichever the pathway, the substances released into the cytoplasm are cytochrome c, procaspases-2, -3, -9, and AIF (*apoptosis inducing factor*) [14]. The release is promoted by Bcl-2 family proteins (Вах-protein). The flavoprotein AIF released from the mitochondrial intermembrane region is an apoptosis effector that would then act

The caspases activated through these pathways directly or indirectly promote the destruction of cell structures. Nuclear lamina proteins are hydrolysed, the cytoskeleton is disrupted, proteins regulating cytoadherence are degraded. Another essential function of effector caspases is inactivation of apoptosis-blocking proteins. To wit, they cleave the inhibitor DFF (*DNA fragmentation factor*), which prevents activation of the apoptotic CAD (*caspase-activated DNase*). Anti-apoptotic proteins of the Bcl-2 family are cleaved. Finally, the action of effector caspases results in dissociation of the regulatory and effector domains of

No matter which apoptotic mechanism the cell chooses, the process can be modulated. At present, the structure and action mechanisms of a great number of apoptosis modulating chemical compounds are known. Most of them influence DNA either directly or indirectly. Nonetheless, if apoptosis can be modulated, then modification of caspase expression is also possible (although there exists caspase-independent apoptosis). The tumor cell apoptosis modulators used in this study are derivatives of two heterocyclic compounds – quinoline and pyridine (Figure 1). This group of reagents, most of which are N-oxide derivatives, holds good promise for *in vitro* study of cell systems, because these compounds comprise several functional activity centres (at the heterocyclic nucleus, functional groups, and radicals). Most published data on the biological activity of quinoline N-oxide derivatives are focused on 4-nitroquinoline-1-oxide (*4-NQO*), whereas information on the activity of other

*4-NQO* is a chemical carcinogen whose biological effects on cells are in many ways similar to the effect of UV light [32]. The action of *4-NQO* is studied using various model systems: prokaryotic cells *in vitro* [33], eukaryotic cells *in vitro* [34], multicellular organisms [35]. The range of the investigated cell life processes and intracellular biomolecules is also diverse. For the neoplastic effect to take place *4-NQO* has to be metabolically activated in the cells to the proximate carcinogen 4-hydroxyaminoquinoline-1-oxide, which, after being acetylated, can covalently bind to DNA, namely adenine and guanine, to form stable monoadducts [36, 37]. Xanthine oxidase can metabolise *4-NQO* to a more reactive superoxide anion (5.5-dimethyl-1-pyrroline-*N*-oxide superoxide radical). Formation of substantial amounts of such structures first of all disturbs the processes of DNA replication and repair, thus inducing apoptosis (or necrosis). If, however, this does not happen, accumulation of multiple mutations in the genome would cause normal cells to transform into tumor cells. Some questions logically arise in this connection: how much similarity is there between the biological effects of the structural analogs of *4-NQO* and those of *4-NQO* itself? Would the

the proteins involved in DNA replication and repair, mRNA splicing [30, 31].

structural analogs of the reagents is, unfortunately, almost totally missing.

independently of caspases [24, 29].

**Figure 3. Receptors of the apoptotic signal** (adopted from http://www.muldyr.ru/a/a/apoptoz\_- \_fazyi\_apoptoza)

*Mitochondria-mediated caspase activation.* The mitochondrial apoptotic signalling pathway is realized through release of apoptogenic proteins from the intermembrane region into the cell cytoplasm. Presumably, there are two major pathways for the release of apoptogenic proteins: through formation of a giant pore followed by rupture of the mitochondrial membrane, or through opening of highly permeable channels on the mitochondrial outer membrane [23]. The apoptosome formation model can be represented as "Cytochrome c−Apaf-1−CARD−procaspase-9". Apaf-1 (*apoptosis protease activating factor-1*) undergoes conformational modifications induced by the reaction involving the loss of ATP energy. Procaspase-9 gets access to the CARD of Apaf-1. The thus activated caspase-9 recruits procaspase-3, which is, in turn, activated to form caspase-3 [24, 25].

In some apoptotic models the cytochrome is released by activation of the РТР (*permeability transition роrе*) [26]. This pore is a compound complex made up of adenine nucleotide transporter (in the inner mitochondrial membrane), voltage-dependent anion channel, otherwise termed porin (in the outer mitochondrial membrane), and cyclophilin D (in the matrix of mitochondria). The 2.6−2.9 nm giant pore is non-specific, and molecules up to 1.5 Da can pass. Pore opening leads to mitochondrial swelling and rupture of the outer membrane. In addition to cytochrome с mitochondria emit other pro-apoptotic factors [23, 25]. Pore opening can be stimulated by inorganic phosphate, caspases, SH-reagents, cell exhaustion by reduced glutathione, formation of active forms of oxygen, uncoupling of oxidative phosphorylation, rise in Ca2+ content in the cytoplasm, effect of ceramide, depletion of the mitochondrial ATP pool, etc. [24, 27, 28].

A presumable alternative pathway for the release of apoptotic proteins from the mitochondrial intermembrane region is formation of a protein channel in the outer mitochondrial membrane. Whichever the pathway, the substances released into the cytoplasm are cytochrome c, procaspases-2, -3, -9, and AIF (*apoptosis inducing factor*) [14]. The release is promoted by Bcl-2 family proteins (Вах-protein). The flavoprotein AIF released from the mitochondrial intermembrane region is an apoptosis effector that would then act independently of caspases [24, 29].

180 Apoptosis and Medicine

\_fazyi\_apoptoza)

**Figure 3. Receptors of the apoptotic signal** (adopted from http://www.muldyr.ru/a/a/apoptoz\_-

recruits procaspase-3, which is, in turn, activated to form caspase-3 [24, 25].

depletion of the mitochondrial ATP pool, etc. [24, 27, 28].

*Mitochondria-mediated caspase activation.* The mitochondrial apoptotic signalling pathway is realized through release of apoptogenic proteins from the intermembrane region into the cell cytoplasm. Presumably, there are two major pathways for the release of apoptogenic proteins: through formation of a giant pore followed by rupture of the mitochondrial membrane, or through opening of highly permeable channels on the mitochondrial outer membrane [23]. The apoptosome formation model can be represented as "Cytochrome c−Apaf-1−CARD−procaspase-9". Apaf-1 (*apoptosis protease activating factor-1*) undergoes conformational modifications induced by the reaction involving the loss of ATP energy. Procaspase-9 gets access to the CARD of Apaf-1. The thus activated caspase-9

In some apoptotic models the cytochrome is released by activation of the РТР (*permeability transition роrе*) [26]. This pore is a compound complex made up of adenine nucleotide transporter (in the inner mitochondrial membrane), voltage-dependent anion channel, otherwise termed porin (in the outer mitochondrial membrane), and cyclophilin D (in the matrix of mitochondria). The 2.6−2.9 nm giant pore is non-specific, and molecules up to 1.5 Da can pass. Pore opening leads to mitochondrial swelling and rupture of the outer membrane. In addition to cytochrome с mitochondria emit other pro-apoptotic factors [23, 25]. Pore opening can be stimulated by inorganic phosphate, caspases, SH-reagents, cell exhaustion by reduced glutathione, formation of active forms of oxygen, uncoupling of oxidative phosphorylation, rise in Ca2+ content in the cytoplasm, effect of ceramide, The caspases activated through these pathways directly or indirectly promote the destruction of cell structures. Nuclear lamina proteins are hydrolysed, the cytoskeleton is disrupted, proteins regulating cytoadherence are degraded. Another essential function of effector caspases is inactivation of apoptosis-blocking proteins. To wit, they cleave the inhibitor DFF (*DNA fragmentation factor*), which prevents activation of the apoptotic CAD (*caspase-activated DNase*). Anti-apoptotic proteins of the Bcl-2 family are cleaved. Finally, the action of effector caspases results in dissociation of the regulatory and effector domains of the proteins involved in DNA replication and repair, mRNA splicing [30, 31].

No matter which apoptotic mechanism the cell chooses, the process can be modulated. At present, the structure and action mechanisms of a great number of apoptosis modulating chemical compounds are known. Most of them influence DNA either directly or indirectly. Nonetheless, if apoptosis can be modulated, then modification of caspase expression is also possible (although there exists caspase-independent apoptosis). The tumor cell apoptosis modulators used in this study are derivatives of two heterocyclic compounds – quinoline and pyridine (Figure 1). This group of reagents, most of which are N-oxide derivatives, holds good promise for *in vitro* study of cell systems, because these compounds comprise several functional activity centres (at the heterocyclic nucleus, functional groups, and radicals). Most published data on the biological activity of quinoline N-oxide derivatives are focused on 4-nitroquinoline-1-oxide (*4-NQO*), whereas information on the activity of other structural analogs of the reagents is, unfortunately, almost totally missing.

*4-NQO* is a chemical carcinogen whose biological effects on cells are in many ways similar to the effect of UV light [32]. The action of *4-NQO* is studied using various model systems: prokaryotic cells *in vitro* [33], eukaryotic cells *in vitro* [34], multicellular organisms [35]. The range of the investigated cell life processes and intracellular biomolecules is also diverse. For the neoplastic effect to take place *4-NQO* has to be metabolically activated in the cells to the proximate carcinogen 4-hydroxyaminoquinoline-1-oxide, which, after being acetylated, can covalently bind to DNA, namely adenine and guanine, to form stable monoadducts [36, 37]. Xanthine oxidase can metabolise *4-NQO* to a more reactive superoxide anion (5.5-dimethyl-1-pyrroline-*N*-oxide superoxide radical). Formation of substantial amounts of such structures first of all disturbs the processes of DNA replication and repair, thus inducing apoptosis (or necrosis). If, however, this does not happen, accumulation of multiple mutations in the genome would cause normal cells to transform into tumor cells. Some questions logically arise in this connection: how much similarity is there between the biological effects of the structural analogs of *4-NQO* and those of *4-NQO* itself? Would the

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

Cellular Caspases: New Targets for the Action of Pharmacological Agents 183

*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,

**Bank № Sequence Source** 

5'-**GAAGGTGAAGGTCGGAGTC**-3' [41] GAPDH R: 5'-**GAAGATGGTGATGGGATTTC**-3'

5'-**ACTGGCTTGTTCAAAGG**-3' [42] CASPASE 6 R: 5'-**CAGCGTGTAAACGGAG**-3'

5'-**ATGGAAGCGAATCAATGGAC**-3' [43] CASPASE 3 R: 5'-**ATCACGCATCAATTCCACAA**-3'

5'-**AACAGGCAAGCAGCAAAGTT**-3' [43] CASPASE 9 R: 5'-**CACGGCAGAAGTTCACATTG**-3'

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

Russia).

GAPDH

**Gene Gene**

GAPDH F: NM\_

CASPASE 6 F: NM\_

CASPASE 3 F: NM\_

CASPASE 9 F: NM\_

002046.3

001226.3

004346.3

001229.2
