Integrated structural and functional analysis of the protective effects of kinetin against oxidative stress in mammalian cellular systems

Abstract

Metabolism and signaling of cytokinins was first established in plants, followed by cytokinin discoveries in all kingdoms of life. However, understanding of their role in mammalian cells is still scarce. Kinetin is a cytokinin that mitigates the effects of oxidative stress in mammalian cells. The effective concentrations of exogenously applied kinetin in invoking various cellular responses are not well standardized. Likewise, the metabolism of kinetin and its cellular targets within the mammalian cells are still not well studied. Applying vitality tests as well as comet assays under normal and hyper-oxidative states, our analysis suggests that kinetin concentrations of 500 nM and above cause cytotoxicity as well as genotoxicity in various cell types. However, concentrations below 100 nM do not cause any toxicity, rather in this range kinetin counteracts oxidative burst and cytotoxicity. We focus here on these effects. To get insights into the cellular targets of kinetin mediating these pro-survival functions and protective effects we applied structural and computational approaches on two previously testified targets for these effects. Our analysis deciphers vital residues in adenine phosphoribosyltransferase (APRT) and adenosine receptor (A2A-R) that facilitate the binding of kinetin to these two important human cellular proteins. We finally discuss how the therapeutic potential of kinetin against oxidative stress helps in various pathophysiological conditions. The small-molecule adenosine N 6-furfuryladenine (N6FFA: kinetin) is commonly used by the plant community as a low-priced proxy for the natural cytokinins (CKs) in plant tissue-culture experiments 1. CKs are a group of phytohormones influencing the entire bauplan of plants; ranging from seed germination, cell division, flowering, organogenesis, immunity, and communication until senescence of the plant 1,2. In plants, kinetin binds to almost all known CKs canonical pathway receptors and invokes analogous physiological responses as many more specific CK-types 2. The naturally occurring CKs in plants are isoprenoid-type CKs, for instance, isopentenyl adenine (iP), trans-zeatin (tZ), cis-zeatin (cZ), and dihydrozeatin (DZ) are the most common forms 3. The majority of naturally occurring CKs exist as free (active forms) bases. Cytokinins conjugate with sugars or amino acid residues and thus form inactive forms 4,5. Previously, CKs were assumed to be exclusively present in the kingdom Plantae; however, their discovery in all forms of life except Archaea, have changed the former notion 6. Likewise, land plants are considered to be the only eukaryotes that harbor two-components system (TCS) that senses and transduces the signal of CKs 7. No such CKs-sensing circuitry has ever been reported for mammalian cells. More intriguingly, many human pathogens such as Mycobacterium tuberculosis 8 and rodent malarial parasites such as apicomplexan

Modulatory and Toxicological Perspectives on the Effects of the Small Molecule Kinetin

“…Previous studies have demonstrated the anti-ageing, anti-inflammatory, immunomodulatory, and neuro-protective effects of kinetin in mammalian cells [ 20 , 21 , 22 , 23 ]. Despite the lack of understanding of its complete detailed biogenesis [ 22 ] or its targets in human cellular proteome and functional implications for mammalian cells [ 26 ], kinetin caught much attention recently for its potential therapeutic applications. One of the putative kinetin binding targets (KBP) is the adenosine receptor.…”

“…These concordant results from docking, binding energy, and crystal structure validate the docking output and the notion that the adopted computational approach is working here correctly. In a previous study, we found multiple kinetin binding residues on the A2a-R receptor in a non-adenosine binding socket [ 26 ]. To get insight into the functional homology between kinetin and adenosine, we specifically focused on the adenosine binding sockets of the A2a-R receptor.…”

“…The in vitro effects of kinetin in terms of protection against oxidative stress have been extensively analyzed for the first time in a report by our group in various cellular systems with diverse potencies such as a human promyelocytic cell line (HL-60), a human keratinocyte cell line (HaCaT), an epithelial rat kidney cell line (NRK), and even in freshly isolated human lymphocytes [ 25 , 26 ]. It was demonstrated that kinetin concentrations below 100 nM can protect mammalian cells against genotoxicity induced cell death, as well as apoptosis [ 25 ].…”

“…Likewise, the exogenous application of 1–10 µM concentrations of kinetin has shown no significant toxicity effects on the nerve cells [ 22 ]. More recently, it has been demonstrated that human promyelocytic HL-60 cells treated with less than 500 nM kinetin did not show a significant reduction in cell viability and genotoxicity [ 26 ]. However, higher kinetin concentrations beyond 500 nM induced both cytotoxicity and genotoxicity in the treated cells.…”

“…Moreover, the H 2 O 2 treated cells were protected from oxidative burst when simultaneously treated with a low concentration of kinetin (100 nM). However, higher kinetin concentrations (more than 500 nM) failed to rescue cells from the oxidative burst [ 25 , 26 ]. It is noteworthy to mention that all these previous studies pertinent to the rescuing effects of kinetin have been conducted on diverse cell lines.…”