to icity and owing to their tar get pathways may enhance the activity of currently used oncologic agents via synergism. The IC50 for pitavastatin was less than 10 uM in most of our cells tested. Similarly, the IC50 of sertraline was in the range of 3. 1 to 6. 6 uM. Predicted blood brain barrier permeation values of pitavastatin The ability of pitavastatin selleck to cross the BBB is predicted to be limited as the log BB was calculated as 0. 6499. However, the drug circulates freely in plasma and may enter the enhancing component of tumors via perme ation through typically leaky tumor microvessels. Effect of pitavastatin on GBM cells Considering the effectiveness of statins in our study, spe cifically pitavastatin in inducing cell death and owing to relatively fewer adverse effects, we decided to e plore pitavastatin in detail.

Pitavastatin induces autophagy in GBM cells Pitavastatin induced cell morphologic changes, as early as 24 hours, with adherent cells assuming a rounded configuration and detaching from the substrate. Death of tumor neurospheres was also triggered and these cells arrested in the G0 G1 phase after treatment. G0 G1 phase cells were dominant and the proportion of cells in S phase dramatically decreased. We found that pitavastatin treated GBM cells e hibited characteristics consistent with autophagy rather than apoptosis. After pitavastatin treatment, GBM cells showed e tensive vacuolization, a key feature of cellular macroautophagy. Further, pitavastatin treated cells stably e pressing the GFP LC3 fusion protein developed a punctate cytoplasmic pattern, suggesting that GFP LC3 covalently linked to phosphatidylethanolamine and was inserted into double membrane autophago somes.

Morphological observations were confirmed by Western blot analysis of LC3, which revealed a LC3 I to LC3 II transition, a hallmark of autophagy. The adherent versus sphere culture conditions did not influence AV-951 the LC3 transition, which was observed in both U87, U251 adherent stable lines and in the stem cell like SK72 cell spheres upon pitavastatin treatment. Furthermore, increasing concentrations of pitavastatin enhanced LC3 I to II transition. In addition, Anne in staining failed to detect apoptosis after pitavastatin treatment. Caspase 3 7 activity was not detectable via fluorescence or by Western blot analysis.

We could not entirely e clude the possibility that pitavastatin induced cell apoptosis by caspase independent pathways. however the cell cycle analysis shown in Figure 3B argued against this hypothesis, as it did not reveal a sub G1 population, characteristic of apoptotic cells. The mechanism of cell death induced by pitavastatin still needs Lapatinib Ditosylate more detailed investigation. Further, whether other statins can also trigger autophagy in human GBM cells remains to be determined, and this may depend, in part, on whether adherent cells or neurosphere cultures are assayed. To elucidate the possible mechanisms by which pita vastatin decreases cell survival, we also