The upsurge in Bcl 2 phosphorylation occurred despite a modest decline in total Bcl 2 levels.Treatment with bortezomib for 24 or 48 hours generated marked up-regulation of LC3 II levels in all 3 cell lines. Likewise, Beclin 1, whose appearance is known to be up-regulated during autophagy, was found to be activated following bortezomib therapy. Taken together with our fluorescence detection of autophagosome development, these data strongly indicated that bortezomib AG-1478 Tyrphostin AG-1478 induces autophagy in HNSCC cells. However, it remained possible that bortezomib may inhibit synthesis of autophogasomes with autolysosomes, or a subsequent step in the entire autophagic process. To find out whether total autophagic flux was happening in bortezomib treated cells we examined the appearance of LC3 II in cells simultaneously treated with inhibitors of lysosomal proteases. In cells undergoing full autophagic flux, induced LC3 II protein ultimately is degraded by lysosomal proteases in autolysosomes, and inhibition of these proteases results in another escalation in the degrees of cellular LC3 II. As shown in Figure 2, treatment with bortezomib in the existence of lysosomal protease inhibitors generated increased levels of erthropoyetin LC3 II relative to LC3 II levels seen in cells treated with bortezomib alone, demonstrating that bortezomib induces comprehensive autophagic flux in HNSCC cell lines. Nevertheless, despite the demonstration of total autophagic flux in bortezomib treated cells, we can not exclude the possibilities that bortezomib also may partially impair cellular LC3 degradation or partially block autophagosome fusion with lysosomes. 3To examine the mechanism of bortezomib induced HNSCC autophagy, we examined the role of JNK. Treatment of cells for 24 or 48 hours with bortezomib resulted in increased phosphorylation of JNK1 and JNK2, these phosphorylation events are considered to be associated with JNK activation. As well as examining JNK initial, we also examined the phosphorylation status supplier Dabrafenib of anti-apoptotic Bcl 2. Recent studies have shown that in cells undergoing nutrient deprivation or ceramide induced autophagy, JNK1 phosphorylates serine 70 on Bcl 2, liberating Beclin 1 to promote autophagy, and promoting disruption of Bcl 2/Beclin 1 complexes. Subsequent therapy with bortezomib, we observed a considerable increase in the phosphorylation of Bcl 2 on 70. Furthermore, even though antibody used is unique for Bcl 2 phosphorylated on serine 70, we didn’t independently verify serine 70 phosphorylation using other bio-chemical methods. Cells were treated with bortezomib in the existence of SP600125, an inhibitor of JNK activity, or SB203580, an inhibitor of p38, to find out whether bortezomib stimulated phosphorylation of Bcl 2 was determined by JNK activity. The JNK chemical eliminated bortezomib caused Bcl 2 phosphorylation, as shown in Figure 3B. Little if any effect was seen using the p38 inhibitor, while in 1483 cells p38 inhibition caused a moderate reduction in total, however not phosphorylated, Bcl 2 levels.