Epi-reevesioside F inhibits Na+/K+-ATPase, causing cytosolic acidification, Bak activation and apoptosis in glioblastoma.

Epi-reevesioside F, a new cardiac glycoside isolated from the root of Reevesia formosana, displayed potent activity against glioblastoma cells. Epi-reevesioside F was more potent than ouabain with IC50 values of 27.3±1.7 vs. 48.7±1.8 nM (P < 0.001) and 45.0±3.4 vs. 81.3±4.3 nM (P < 0.001) in glioblastoma T98 and U87 cells, respectively. However, both Epi-reevesioside F and ouabain were ineffective in A172 cells, a glioblastoma cell line with low Na+/K+-ATPase α3 subunit expression. Epi-reevesioside F induced cell cycle arrest at S and G2 phases and apoptosis. It also induced an increase of intracellular concentration of Na+ but not Ca2+, cleavage and exposure of N-terminus of Bak, loss of mitochondrial membrane potential, inhibition of Akt activity and induction of caspase cascades. Potassium supplements significantly inhibited Epi-reevesioside F-induced effects. Notably, Epi-reevesioside F caused cytosolic acidification that was highly correlated with the anti-proliferative activity. In summary, the data suggest that Epi-reevesioside F inhibits Na+/K+-ATPase, leading to overload of intracellular Na+ and cytosolic acidification, Bak activation and loss of mitochondrial membrane potential. The PI3-kinase/Akt pathway is inhibited and caspase-dependent apoptosis is ultimately triggered in Epi-reevesioside F-treated glioblastoma cells.

Calanquinone A induces anti-glioblastoma activity through glutathione-involved DNA damage and AMPK activation.

Glioblastoma, a highly malignant glioma, is resistant to both radiation and chemotherapy and is an intractable problem in clinical treatment. New therapeutic approaches are in urgent need. Calanquinone A, an herbal constituent, displayed anti-proliferative activity against glioblastoma cells, including A172, T98 and U87. Flow cytometric analysis showed an S phase arrest and a subsequent apoptosis to calanquinone A action. Further identification demonstrated a rapid increase of γH2A.X formation at S phase. The data together with comet tail formation and Chk1 activation indicated DNA damage response. N-acetyl cysteine (an antioxidant and a glutathione precursor) and exogenously applied glutathione, but not trolox (an antioxidant), completely abolished calanquinone A-induced effects. Immunofluorescence assay revealed that calanquinone A decreased the intracellular glutathione levels in both A172 and T98 cells. However, calanquinone A, by itself, did not conjugate glutathione. The data suggested that the decrease of cellular glutathione predominantly contributed to the anticancer mechanism. Furthermore, calanquinone A induced the activation of AMP-activated protein kinase (AMPK) and the inhibition of p70S6K activity. Rhodamine efflux assay showed that calanquinone A did not block efflux activity, indicating that calanquinone A was not a P-glycoprotein substrate. In summary, the data suggest that calanquinone A displays anti-glioblastoma activity through a decrease of cellular glutathione levels that subsequently induces DNA damage stress and AMPK activation, leading to cell cycle arrest at S-phase and apoptotic cell death. Furthermore, calanquinone A does not serve as a P-glycoprotein substrate, suggesting a potential for further development in anti-glioblastoma therapy.

FKBP12 regulates the localization and processing of amyloid precursor protein in human cell lines.

One of the pathological hallmarks of Alzheimer's disease is the presence of insoluble extracellular amyloid plaques. These plaques are mainly constituted of amyloid beta peptide (A beta), a proteolytic product of amyloid precursor protein (APP). APP processing also generates the APP intracellular domain (AICD). We have previously demonstrated that AICD interacts with FKBP12, a peptidyl-prolyl cis-trans isomerase (PPIase) ubiquitous in nerve systems. This interaction was interfered by FK506, a clinically used immunosuppressant that has recently been reported to be neuroprotective. To elucidate the roles of FKBP12 in the pathogenesis of Alzheimer's disease, the effect of FKBP12 overexpression on APP processing was evaluated. Our results revealed that APP processing was shifted towards the amyloidogenic pathway, accompanied by a change in the subcellular localization of APP, upon FKBP12 overexpression. This FKBP12-overexpression-induced effect was reverted by FK506. These findings support our hypothesis that FKBP12 may participate in the regulation of APP processing. FKBP12 overexpression may lead to the stabilization of a certain isomer (presumably the cis form) of the Thr668-Pro669 peptide bond in AICD, therefore change its affinity to flotillin-1 or other raft-associated proteins, and eventually change the localization pattern and cause a shift in the proteolytic processing of APP.

The intracellular domain of amyloid precursor protein interacts with FKBP12.

To elucidate the roles of the APP intracellular domain (AICD) in the development of Alzheimer's disease, a yeast two-hybrid system was used to screen for AICD-interacting proteins. Our result revealed that FKBP12, an immunophilin with a peptidyl-prolyl cis-trans isomerase (PPIase) activity, may interact with AICD. This interaction was confirmed by coimmunoprecipitation studies. FKBP12 has been shown to be expressed at a higher level in areas of pathology of patients with neurodegenerative diseases. In addition, Pin1, a member of another PPIase family, has been suggested to be involved in the amyloidogenic APP processing and Abeta production. The interaction between FKBP12 and AICD might hint at a possible role FKBP12 plays, probably in a fashion similar to Pin1, in the amyloidogenesis of APP. We also found that the interaction was interfered, in a dose-dependent manner, by FK506, whose neuroprotective effect has been suggested to be correlated with its PPIase inhibitory activity.