Influence ofKinetin on Animals

20.4.2.1 Kinetin Antioxidative Properties

In experiments using animal cells and other organisms it was shown that kinetin influences many processes, regulates proliferation, and has antiaging and antioxidant properties.

Kinetin's antioxidant and scavenger activity was confirmed in vivo and in vitro. It could act in a few different ways: as a donor of hydrogen, as an enzyme, or as an activator of enzyme activity [37-40]. Because of these properties kinetin prevents damage to DNA, proteins, and other macromolecules, avoiding the accumulation of abnormal particles in organs, tissues, and cells.

Kinetin can act as a free-radical scavenger when oxygen radicals directly abstract hydrogen from the a-carbon of the amine bond of N6-furfuryladenine [37].

The kinetin-copper complex catalyzes the dismutation of superoxide into oxygen and hydrogen peroxide at the reaction rate constants 2.3 x 10~7 M^1 s^1 at pH 9.8 and 25°C [38].

Kinetin was proved to protect DNA against the formation of 8-oxodeoxyguanine, which is the result of hydrogen peroxide generation in a Fenton reaction. Inhibition of 8-oxo-dG formation was exhibited in a dose-dependent manner with a maximum efficiency of 50% at a concentration of 100 |M [41].

Kinetin protects against oxidative and glycoxidative protein damage generated in vitro by sugars and iron/ascorbate system. Glycation is a nonenzymatic reaction of binding hexose, mostly glucose to the amine group of protein or nucleotides [42]. The products of these reactions are accumulated in cells during aging [43]. Inside the cell, due to a high concentration of glucose and other reactive sugars like pen-toses and a-oxoaldehydes, glycation/glycoxydation reactions progress fast [44,45]. Kinetin at a concentration of 50 |M exhibits an 82% inhibition of bovine serum albumin (BSA)-pentosidine formation. At 200 |M the cytokinin prevents BSA aggregation during glycation and also inhibits 59 to 68% advanced glycation end product (AGEP) development [46].

20.4.2.2 Kinetin Antiaging Properties

The antiaging properties of kinetin were shown using in vitro cell cultures, in vivo on skin, and fruit flies. The fruit fly Zaprionus, with its diet supplemented with 125 to 625 | M kinetin, prolonged life span due to a reduction in the age-specific death rates, slowed down development, and delayed maturation of insects in the larval and pupal stages. Delayed aging is reached at the cost of decreased reproductive activity and egg-laying capacity. The molecular mechanism of kinetin activity is connected with an increase in catalase activity. The enzyme belongs to the oxydoreductase group, displays strong antioxidant activity, and catalyzes the decomposition of hydrogen peroxide into water and oxygen. A concentration of 125 | M seems to be the most effective for antiaging and life-prolonging effects. A higher concentration,

500 |M and above, gives toxic and life-shortening results. The cytokinin exerts a similar effect on human cell cultures at these doses [39, 40].

Nymphs of Lipaphis erysimi fed kinetin-treated Raphanus sativus L. showed an increased activity of catalase, glutathione peroxidase, and superoxide dismutase and a decrease in the activity of APTaze [47].

In in vitro skin cultures, mammary carcinoma and cystic disease kinetin delays the onset of several morphological and biochemical processes connected with aging. During senescence in vitro cell cultures became large and flattened, full of lysosomal residual bodies and oxidation-modified macromolecules, debris, and accumulated lipofuscin with disorganized cytoskeleton and some of them contain more than one nucleus. The addition of kinetin at 40- to 200-| M concentrations in culture media prevents fibroblasts from developing these changes. In spite of avoiding age-related degeneration, kinetin can also slightly reverse the changes. Upon removal of the cy-tokinin, some of the aging characteristics reverse [48]. Some properties of kinetin were proved in vivo using aged skin of hairless dogs. After 50 d of daily application of solution containing kinetin a 0.01-, 0.1-, 1-, 10-, and 96.6-mM improvement in skin texture, wrinkling, and pigmentation was observed. After 100 d rejuvenation and depigmentation became more visible. Lower concentrations of kinetin normalized hyperpigmentation and improved the aged skin structure. Throughout the treatment no adverse effect was observed, showing that kinetin is safe for long-term therapy [49].

20.4.2.3 Kinetin Influence on Animal Cell and Tissue Cultures

Kinetin influences both the epidermis and the dermis in the skin in the same way. It stimulates keratinocyte proliferation and differentiation in the epidermis, increases the amount of laminin 5 at the dermal-epidermal junction, and influences te formation of fibrillin-1 and elastin deposition as well as their organization perpendicularly to the dermal-epidermal junction in the dermis [50]. On the other hand human ker-atinocyte culture exhibits significant growth inhibition in media containing 40 to 200 | M kinetin concentration. At the same time it stimulates the cells to differentiate, especially strongly in the presence of calcium [51]. Kinetin retards the outgrowth of epithelium skin cultures at 1 to 0.25mg/100ml and increases epithelial sheet production at 0.006 to 0.015 mg/100 ml [52]. Its riboside appears to be toxic to fibroblasts, breast carcinomas, and cystic disease cells at 1 mg/100 ml and results in reduced or no outgrowth in in vitro culture. But it is not toxic at 0.1 mg/100 ml [48].

Kinetin in high concentrations (100mg/1000ml) acts as a toxin and triggers cytoplasm vacuolization and degenerative changes in fibroblast cell cultures. At lower doses (1 mg/1000 ml, 10 mg/1000 ml) chromatin became more sensitive to acid hydrolysis, which results in higher transcription activity. DNA amounts in the fibrob-last nucleus increase after 24 and 72 h incubation with kinetin [53].

20.4.2.4 Cytokinin Influence on Cancer Cells

Cytokinins influence animal cell proliferation and differentiation, which makes them attractive as potential agents in cancer treatment. Some cytokinins, for example kinetin, isopentenyladenine, and benzyladenine, are inhibitors of ML-1, NB4, and U937 leukemia cell proliferation and stimulate the cells' mature granulocytes to differentiate as well as influence plant cancer cells [54, 55]. Also, other adenine derivatives like 6-methyladenine, 6-anilinopurine, and 2-aminopurine induce myeloid leukemia cells' HL-60 differentiation [56]. Cytokinin ribosides, including the kinetin riboside, inhibit the growth of HL-60, M4 Beu human, and B16 murine melanoma cells. Once cells are incubated with cytokinin ribosides and antioxidants, apoptosis is reduced and differentiation is increased. It triggers such a result only in the presence of antioxidants, scavengers, or the caspase inhibitor. Normally cy-tokinin ribosides reduce the intracellular ATP content and disturb the mitochondrial membrane potential and the accumulation of oxygen species, leading to apopto-sis. This shows that cytokinin ribosides can induce differentiation but first it stimulates apoptosis [56, 57]. cAMP and ATP also stimulate the differentiation of HL-60 cells but at a much lower rate than the cytokinin derivative of adenine [55]. Diverse adenine analogs display varied influences on HL-60 growth. Methyladenine (IC50 1,172-1,713 |M), adenosine (IC50 6 8 5 |M), deoxyadenosine (IC50 662 |M), and transzeatin (IC50 5 1 6 |M) appeared to be the least harmful. Other cytokinins such as kinetin (IC50 48.6 |M), benzylaminopurine (IC50 67.6 |M), and isopen-tenyloadenine (IC50 47.6 |M) display very similar abilities to inhibit growth. The benzylaminopurine riboside as well as the kinetin riboside and isopentenyladeno-sine all show a strong inhibiting influence with its IC50 at 0.706 to 0.981 | M. The mechanism of cytokinin action is connected with adenine metabolism and adenine kinase activity that converts adenine to AMP. It appeared that a cytokinin could not influence cells until it was phosphorylated and converted to its nucleotide. Adenine kinase inhibition stops kinetin-triggered HL-60 differentiation, and kinetin riboside caused apoptosis [56]. The mechanism of kinetin action in HL-60 is known to abandon the P2 receptor, which is a member of the known differentiation mechanism. It involves the induction of mitogen-activated protein kinase (MAPK) and S100P [58]. S100P belongs to the S100 binding calcium protein family and it is temporarily expressed during the early stages of differentiation in esophageal epithelial cells (EEC) [56, 59, 60]. In normal neutrophil cells, S100P is strongly expressed, while in AML expression it is weak or nondetectable [61].

20.4.2.5 Therapeutic Applications of Kinetin

Familial dysautonomia (FD) is a neurodegenerative disease caused by a mutation in the IKBKAP gene coding IKAP protein. In cytosine substitution by thymine in intron 20, tissue-specific intron skipping during alternative splicing takes place [62]. IKAP participates in transcription elongation. In the case of FD, accurate splicing is particularly ineffective in the nervous system. Kinetin stimulates the inclusion of exon 20 from an endogenous gene and from a proper IKBKAP minigene. It is known that the CAA sequence at exon 5' is necessary for kinetin to show its activity [63].

Kinetin is utilized in therapy treating Meniere's disease. A solution of 4% li-docaine and cytokinin was introduced into the tympanic cavity of patients. 87.5% of the sick reported a noticeable decrease in vestibular symptoms, and 66.7% of these patients were free of attacks for an average of 26.5 months with the same or improved hearing in 87.5% of patients [64].

20.4.2.6 Kinetin's Molecular Mechanism of Action in Animal Cells

In endothelium cells kinetin influences signaling pathways connected with the cy-toskeleton. Human dermal microvascular endothelial cells (HDMEC) between the 5th and 30th passages were treated with kinetin at 50 |M. This resulted in changes in the expression of moesin, actin, and rho GDP dissociation inhibitor (GDI) and an increased activity of rho GTPase, which influences actin. Actin is a protein that forms the cytoskeleton and is connected with the rho pathway. Moesin belongs to the ERM (ezrin/radixin/moesin) protein family that connects cell membrane proteins with actin underneath the membrane. Moesin participates in signal transduction and cytoskeleton remodeling. It is modulated by phosphorylation, the phosphoinositide pathway, and is controlled mutually with rho GTPase. Rho GDI expression is elevated in aged human umbilical vein endothelial cells (HUVEC) and it suppresses rho GTPase activity. It interacts directly with ERM protein, which reduces rho GDI activity, thereby activating rho GTPase [65, 69].

Using similar pathways kinetin takes part in the regulation of cell proliferation. Cell cycle arrest in the G1 phase is connected with senescence and leads to apop-tosis. This occurs when G1-specific cyclin D1 or cyclin E1, pRB, p16, p21, and p27 undergo changes. When cellular function decreases, p53 activation takes place. This suppresses cell cycle progression, stimulates a rise of p21 and p27, and induces G1 arrest. Kinetin decreases expression of p16, p27, and p53 and increases the amount of D1 cyclin. The rho pathway, as well as influencing cytoskeleton, supports cell cycle transition G1/S, and thus it promotes proliferation. The rho GT-Pase enhances expression of p27 and thus regulates D1 cyclin. In HDMEC treated with kinetin, rho GTPase is activated, total p16, p21, p27 is reduced, the amount of cyclin D1 is enlarged, and stimulation of G1/S transition is observed. Kinetin delays aging of endothelial cells and increases proliferation and metabolic capacity [69, 70]. Earlier experiments showed that kinetin delayed the onset of aging of fibroblasts and helped to complete cytokinesis, but it does not promote induction of the S phase. This suggests diverse activities of the cytokinin depending on the cell type [48].

There are also other cases where kinetin was proved to act through cGMP and Ca2+ connected pathways. At 50 to 150 |M, it inhibits platelet aggregation. It is supposed to stop Na+/H+ exchanger activation and phospholipase C activation and at the same time prevent phosphatidylinositol (PIP2) metabolism and lipid signaling pathways. This results in lower intracellular alkalization and Ca2+ mobilization, augmented cyclic AMP synthesis, and inhibition of thromboxane A2 formation, which is known to be responsible for platelet aggregation. cAMP stops the Na+/H+ exchanger and leads to reduced mobilization of intracellular Ca2+ and phosphorylation of P47 [immunity-related GTPases (IRG) family]. At 70|M and 150|M kinetin decreased the amount of free radicals in collagen-activated platelet. Intravenous injection of 4 to 6 mg/kg of the cytokinin into mice prolonged bleeding time by approx. 1.9 to 2.1-fold [71, 72].

6-benzylaminopurine (6-BAP), kinetin, and zeatin induce positive inotropic effects in rat atria involving P2-purinoceptors, cGMP, and intracellular calcium release but not using pathways connected with arginine/nitric oxide, cyclooxygenase, phospholipase C, or L-type calcium channels [73].

Kinetin, as well as some other cytokinins, auxins, and gibberelinins, increases rat lung, small intestine, liver, and renal cortex guanylate cyclase activity two- to fourfold. The maximal stimulation of guanylate cyclase was observed at a 1-|M concentration of the plant hormones [74].

Cytokinins inhibits muscle creatine kinase (CK-MB), activates alanine amino-tranferease (ALT) and aspartate aminotransferase (AST), and increases the level of AST, CK, and LDH, but they do not influence carbonic anhydrase and glucoses-phosphate dehydrogenase [75, 76].

Cytokinins are also incorporated into the rRNA, tRNA, and tRNA of tobacco callus, E. coli, and yeast cells. Specific incorporation of kinetin into E. coli tRNATyr at position 37 by the putative tRNA kinetin transglycosylase takes place. The exchange reaction occurs in the presence of protein from E. coli, yeast, or MRC-5V2 cell extracts. Likewise, enzymes of E. coli or MRC-5V2 are able to catalyze incorporation of kinetin into yeast tRNA [77]. This shows a relationship between tRNA and enzymes in prokaryotic and eukaryotic organisms.

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