Lectins are highly specific proteins that bind to carbohydrates and are found in many plants, animals, and bacteria. Phytohaemagglutinin (PHA) is the lectin of kidney beans (Phaseolus vulgaris) and it is one of the most common and widely studied lectins. Mitogenic activity of PHA is known since 60th of the XX century. This lectin has an tetrameric molecular structure, namely PHA is a mixture of different isolectins, including erythroagglutinin (PHA-E) and leukoagglutinin (PHA-L). However, proapoptotic mechanisms of PHA and its isolectins activity still remain unclear and should be elucidated. In this study we addressed to analyze the influence of PHA and its isolectins on apoptosis in cultivated cells in vitro and to clarify possible signalling pathways endorsed by lectins activity. In our study we have analyzed the influence of the commercial preparation of P. vulgaris lectins, meanly PHA, PHA-E and PHA-L. We have used three different mammalian cell lines as a test system. The frequency of live, dead and apoptotic cells has been analyzed using acridine orange and ethidium bromide staining. The changes of caspase-3, caspase-8 and Bax have been inspected by Western blot analysis. Gene expression was analysed using real-time PCR. For protein-protein docking analyses we used 3D structures obtained from Protein Data Bank. It was shown the significant influence of all lectins have been studied on cell culture proliferation in concentration-dependent manner. In non-tumor 4BL cell line all isolectines have been studied were shown to enhance the cell culture growth in concentration 0,01-10 ug/ml; and in concentration 100< ug/ml the inhibition of culture growth was observed. In malignant Hep-2 cell line the mitogenic properties of PHA isolectines was much less displayed then in 4BL cells, and PHA-L was shown to be cytotoxic even in minimal concentration has been studied (0,01 ug/ml). The influence of total PHA preparation differs significantly from theoretically calculated synergic interaction. Apoptosis induction by total PHA preparation and its isolectins in cell cultures of tumour and non-tumour origin under the experimental conditions was demonstrated too in all concentrations of all isolectines have been studied. The differences between pro-apoptotic effect of PHA isolectines in tumour and non-tumour human cell was discovered in prolonged studies. Thus, in non-tumor 4BL cells the PHA-E was shown to cause the severe apoptotic changes in cell population, whereas in carcinoma Hep-2 cells the massive apoptotic damage was discovered in PHA-L-treated cell population. Chinese hamster cells were shown to be more resistant to PHA-induced apoptotic damage in all experimental conditions have been studied. It was shown that both total PHA preparation and PHA isolectins induces the cell apoptosis due to caspase-3, caspase-8 and pro-apoptotic Bax protein induction. All analyzed PHA lectins increased the level of Bax gene expression compared to controls. Regarding caspases, PHA-E was shown to be the most powerful inductor of these proteins expression, but other isolectins were able to induce caspases expression too. The differences between tumor and non-tumor cell lines were also revealed in this case. Thus, using Western blot analysis the PHA-E was shown to be the most effective Cas8 inductor in 4BL cells, whereas in Hep-2 cell line the strongest effect has been shown in total PHA-treated cell culture. These data were supported by RT-PCR: it was shown that both PHA-L and PHA-E cause the significant enhancing of Bax mRNA expression. Another apoptosis-associated protein coding gene, Bcl-2, have been shown to significantly reduce its expression on mRNA level under the PHA-E treatment, but not in PHA-L- and total PHA-treated cells. These data are strongly consistent, because Bcl-2 is anti-apoptotic protein. We obtained the computational model of leukoagglutinin complexes with pro-apoptotic receptors (FasR and TNFR-1) and anti-apoptotic receptors (IGF-1 and EGFR). We revealed that leukoagglutinin is able to interact with these important receptors through protein-protein binding with high energy. Thus, for FasR receptor the Gibbs binding energy was appreciated to be -620,0 kJ/M. for TNFR-1 it was -863,8 kJ/m, for IGF-1 - -567,4 kJ/M, and for EGFR it has been valued as -609,4 kJ/M. These calculations supports our hypothesis that PHA isolectins could be the modulators of apoptosis, mainly, pro-apoptotic agents, due to their involvement in regulatory pathways and via both receptor-dependent and mitochondrial mechanism of apoptosis induction.
