The Role of Bcl-2 and Beclin-1 Complex in "Switching" between Apoptosis and Autophagy in Human Glioma Cells upon LY294002 and Sorafenib Treatment.

BACKGROUND: Gliomas are the most malignant tumors of the central nervous system. One of the factors in their high drug resistance is avoiding programmed death (PCD) induction. This is related to the overexpression of intracellular survival pathways: PI3K-Akt/PKB-mTOR and Ras-Raf-MEK-ERK. Apoptosis and autophagy are co-existing processes due to the interactions between Bcl-2 and beclin-1 proteins. Their complex may be a molecular "toggle-switch" between PCD types. The aim of this research was to investigate the role of Bcl-2:beclin-1 complex in glioma cell elimination through the combined action of LY294002 and sorafenib. METHODS: Drug cytotoxicity was estimated with an MTT test. The type of cell death was evaluated using variant microscopy techniques (fluorochrome staining, immunocytochemistry, and transmission electron microscopy), as well as the Bcl-2:beclin-1 complex formation and protein localization. Molecular analysis of PCD indicators was conducted through immunoblotting, immunoprecipitation, and ELISA testing. SiRNA was used to block Bcl-2 and beclin-1 expression. RESULTS: The results showed the inhibitors used in simultaneous application resulted in Bcl-2:beclin-1 complex formation and apoptosis becoming dominant. This was accompanied by changes in the location of the tested proteins. CONCLUSIONS: "Switching" between apoptosis and autophagy using PI3K and Raf inhibitors with Bcl-2:beclin-1 complex formation opens new therapeutic perspectives against gliomas.

Cytological and biophysical comparative analysis of cell structures at the microsporogenesis stage in sterile and fertile Allium species.

MAIN CONCLUSION: Using a live-cell-imaging approach and autofluorescence-spectral imaging, we showed quantitative/qualitative fluctuations of chemical compounds within the meiocyte callose wall, providing insight into the molecular basis of male sterility in plants from the genus Allium. Allium sativum (garlic) is one of the plant species exhibiting male sterility, and the molecular background of this phenomenon has never been thoroughly described. This study presents comparative analyses of meiotically dividing cells, which revealed inhibition at the different microsporogenesis stages in male-sterile A. sativum plants (cultivars Harnas and Arkus) and sterile A. ampeloprasum var. ampeloprasum (GHG-L), which is phylogenetically related to garlic. Fertile species A. ampeloprasum (leek) was used as the control material, because leek is closely related to both garlic and GHG-L. To shed more light on the molecular basis of these disturbances, autofluorescence-spectral imaging of live cells was used for the assessment of the biophysical/biochemical differences in the callose wall, pollen grain sporoderm, and the tapetum in the sterile species, in comparison with the fertile leek. The use of techniques for live-cell imaging (autofluorescence-spectral imaging) allowed the observation of quantitative/qualitative fluctuations of autofluorescent chemical compounds within the meiocyte callose wall. The biophysical characterisation of the metabolic disturbances in the callose wall provides insight into the molecular basis of male sterility in A. sativum. In addition, using this method, it was possible for the first time, to determine precisely (on the basis of fluctuations of autofluorescence compounds) the meiosis stage in which normal microsporogenesis is disturbed, which was not visible using light microscopy.

Tubulin cytoskeleton during microsporogenesis in the male-sterile genotype of Allium sativum and fertile Allium ampeloprasum L.

KEY MESSAGE: Microsporogenesis in garlic. The male-sterile Allium sativum (garlic) reproduces exclusively in the vegetative mode, and anthropogenic factors seem to be the cause of the loss of sexual reproduction capability. There are many different hypotheses concerning the causes of male sterility in A.sativum; however, the mechanisms underlying this phenomenon have not been comprehensively elucidated.Numerous attempts have been undertaken to understand the causes of male sterility, but the tubulin cytoskeleton in meiotically dividing cells during microsporogenesis has never been investigated in this species. Using sterile A.sativum genotype L13 and its fertile close relative A. ampeloprasum (leek), we have analysed the distribution of the tubulin cytoskeleton during microsporogenesis. We observed that during karyokinesis and cytokinesis, in both meiotic divisions I and II, the microtubular cytoskeleton in garlic L13 formed configurations that resembled tubulin arrangement typical of monocots. However, the tubulin cytoskeleton in garlic was distinctly poorer (composed of a few MT filaments) compared with that found in meiotically dividing cells in A. ampeloprasum. These differences did not affect the course of karyogenesis, chondriokinesis, and cytokinesis, which contributed to completion of microsporogenesis, but there was no further development of the male gametophyte. At the very beginning of the successive stage of development of fertile pollen grains, i.e. gametogenesis, there were disorders involving the absence of a normal cortical cytoskeleton and dramatically progressive degeneration of the cytoplasm in garlic. Therefore,we suggest that, due to disturbances in cortical cytoskeleton formation at the very beginning of gametogenesis, the intracellular transport governed by the cytoskeleton might be perturbed, leading to microspore decay in the male-sterile garlic genotype.