Novel immunoaffinity purification approach for isolation of new cytokinin based anticancer drug and its identification in plant tissues

Cytokinins [1] are plant hormones that trigger a wide range of biological processes in plants. Among their effects the control of cell division is the crucial function. Ribosides are one of the forms in which cytokinins naturally occur. In addition to their actions in plants, it has been discovered that these substances are also very effective inhibitors of cancer cell proliferation and very potent inducers of apoptosis in several human leukemia cell lines with typical morphological and biochemical hallmarks [2,3,4]. We have been studying cytotoxic effect of several synthetic cytokinin ribosides against various leukaemia cell lines. Surprisingly, we have proved considerable cytotoxicity of 6-(2-hydroxy-3-methoxybenzylamino)purine riboside (20H3MeOBAPR) toward CEM and HL-60 leukaemia cell lines with IC₅₀ values of 0.3µM and 0.15µM, respectively [5]. In order to study this interesting compound in more details, we prepared monoclonal antibodies against 20H3MeOBAPR and we developed effective batch immunoaffinity extraction technique for its purification from various biological materials [6]. Consequently, we performed a study on per oral bioavailability [6] and pharmacokinetic quantitative analyses of this compound and its proposed pro-drug form in murine blood samples by high performance liquid chromatography-mass spectrometry (HPLC/MS) measurement. Further, we undertook a general screening for new di- and trisubstituted cytokinin ribosides as endogenous cytokinins in various biological materials, namely in Agrobacterium tumefaciens and Arabidopsis thaliana tissue. By the use of high resolution measurement, on a hybrid quadrupole-time of flight (Q-TOF) mass analyzer with electrospray ionization technique connected to capillary liquid chromatography (capLC), we were able to identify two new disubstituted benzyladenosines in both investigated biological materials [5].

Acknowledgement: The work was supported by the Ministry of Education, Youth, and Sports of the Czech Republic (MSM 6198959216).

References: 1. Mok, D.W.S., Mok, M.C. (2001) Annu. Rev. Plant Physiol. Plant Mol. Biol. 52. 2. Mlejnek, P., Kuglik, P. (2000)J. Cell. Biochem. 77:6–17. 3. Ishii, Y. et al. (2002) Cell Growth Differ. 13:19–26. 4. Ishii, Y. et al. (2003) Biochim. Biophys. Acta-Mol. Cell Res. 1643:11–24.

5. Dolezal, K. et al. (2007) Bioorg. Med. Chem. 15: 3737–3747. 6. Hauserova, E. (2005)J. Chromatogr. A 1100:116–125.