(A) Representative immunostaining for endothelial marker CD31 (upper row), hypoxia markers HIF-1α (second row; blue=Hoescht nuclear staining; green=HIF-1α), and CA9 (third row), and autophagy-mediating BNIP3 (lower row; blue=Hoescht nuclear staining; green=BNIP3) in this glioblastoma resistant to bevacizumab (right) compared to paired specimens from the same patient before treatment (left). CA9 and BNIP3 stainings are from adjacent sections. (B) Vessel density decreased (P
Figure 2. Hypoxia causes autophagy-associated protein changes…
Figure 2. Hypoxia causes autophagy-associated protein changes in GBM cells
Culturing U87MG (A) and T98G…
Figure 2. Hypoxia causes autophagy-associated protein changes in GBM cells Culturing U87MG (A) and T98G (B) GBM cells in hypoxia for up to 24 hours increased, relative to normoxia degradation of p62 and total LC3 (C), with T98G cells also showing increased conversion of LC3-I to LC3-II in hypoxia (B). Similar findings in 3 other glioma cell lines (U251, U138, A172, and G55) are shown at 24 hours (D). All cell lines also showed hypoxia-induced increased expression of autophagy-mediating BNIP3 (E). (F) Culturing primary glioma cells SF8167, SF8106, SF7796, and SF8244 led to the same hypoxia-induced autophagy-associated changes (p62 degradation, LC3-I to LC3-II conversion, and degradation of total LC3) after 24 hours.
Figure 3. Autophagy inhibitors block the hypoxia-induced…
Figure 3. Autophagy inhibitors block the hypoxia-induced expression of autophagy mediators in glioma cells
Figure 3. Autophagy inhibitors block the hypoxia-induced expression of autophagy mediators in glioma cells (A) After 24 hours, 3- MA (1 mM) and BafA1 (1 nM) blocked hypoxia-induced p62 degradation in cultured U87MG cells. 3-MA inhibited conversion of LC3-I to LC3-II, while BafA1 increased LC3-I to LC3-II conversion. (B) After 24 hours of hypoxia, U373/GFP-LC3 cells exhibited more punctate green fluorescent staining, consistent with autophagy, which decreased after 3-MA treatment, but remained high after BafA1 treatment. 40x magnification, scale bar=200 µm. (C) 3-MA reduced the percent of cells with over 10 punctate green fluorescent dots (P=0.01).
Figure 4. Inhibiting hypoxia-induced autophagy increases cell…
Figure 4. Inhibiting hypoxia-induced autophagy increases cell death
(A) U87MG and T98G cells in hypoxia…
Figure 4. Inhibiting hypoxia-induced autophagy increases cell death (A) U87MG and T98G cells in hypoxia for 48 hours exhibited decreased cell numbers, as assessed by absorbance at 490 nm (reflecting number of cells) minus background measured in the MTS assay, when treated with 3-MA (1 mM) or bafilomycin A1 (BafA1; 1 nM) (P<0.05). (B) U87MG cells in hypoxia for 24 hours exhibited an increased percent of AnnexinV+PI+ cells (permeabilized near death cells, leftmost bars) in the presence of 3-MA (P<0.05) or BafA1 (P<0.01) relative to the presence of these inhibitors in normoxia, whilethese inhibitors did not change the percent of AnnexinV+PI− cells (early apoptosis, rightmost bars) in hypoxia (P>0.05). (C) Western blotting revealed increased cleavage of PARP, indicating apoptosis, in hypoxic cells treated with 3-MA or BafA1 for 24 hours, suggesting that autophagy inhibitors were promoting apoptosis.
Figure 5. Hypoxia upregulates HIF-1α expression and…
Figure 5. Hypoxia upregulates HIF-1α expression and AMPK phosphorylation, which contribute to some aspects of…
Figure 5. Hypoxia upregulates HIF-1α expression and AMPK phosphorylation, which contribute to some aspects of hypoxia-induced autophagy (A) U87MG and T98G cells cultured in hypoxia exhibited time-dependent activation of HIF-1α and AMPK (with AMPK activation assessed by detecting phosphorylated AMPK in the first row), with HIF-1α activation occurring before AMPK activation. (B) siRNA-mediated knockdown of AMPK and HIF-1α in hypoxic U87MG cells exhibited reduced LC3-I to LC3-II conversion and reduced total LC3 degradation but neither siRNA affected hypoxia-mediated p62 degradation, while only HIF-1α siRNA reduced hypoxia-induced BNIP3 expression. (C) Similarly, YC-1 (a HIF-1α inhibitor) blocked hypoxia-mediated LC3-I to LC3-II conversion and total LC3 degradation without affecting hypoxia-mediated p62 degradation in T98G cells. (D) In U87MG cells cultured for 24 hours in hypoxia, YC-1 blocked hypoxia-mediated BNIP3 upregulation.
Figure 6. Autophagy inhibitor chloroquine combined with…
Figure 6. Autophagy inhibitor chloroquine combined with bevacizumab inhibits GBM39 tumor growth in vivo
Figure 6. Autophagy inhibitor chloroquine combined with bevacizumab inhibits GBM39 tumor growth in vivo (A) Subcutaneous tumors in athymic mice were treated with PBS, chloroquine, bevacizumab, and chloroquine plus bevacizumab. After 4 weeks, there was significantly different tumor volumes amongst groups (P