MEK Inhibition Sensitizes Precursor B-Cell Acute Lymphoblastic Leukemia (B-ALL) Cells to Dexamethasone through Modulation of mTOR Activity and Stimulation of Autophagy
Fig 5
MEK1/2 inhibitor, selumetinib, intensifies DEX induced LC3 conversion, MDC staining and GFP-LC3 relocalization in GC-resistant SEMK2 ALL cells.
(A) GC-sensitive (RS4;11) and GC-resistant (SEMK2) cells were incubated with DEX (0.05 μg/mL) in the presence or absence of MEK1/2 inhibitor, selumetinib (SEL, 200 nM) for 24h. When indicated, cells were pretreated for 3 h with 50 μM or 100 μM of chloroquine (CQ). Thereafter, LC3 processing was assessed by immunoblotting. Densitometric analyses of LC3II/I are indicated below the blots. (B) SEMK2 and RS4;11 cells were cultured as described above for 24h, stained with MDC (50 μM) and analyzed by fluorescence microscopy. (C) SEMK2 cells were stably transduced with GFP-LC3 and incubated with DEX, SEL or combination of DEX+SEL for 24h. GFP-LC3 relocalization from diffuse cytoplasmic in control cells to a massive dotty pattern in DEX+SEL treated cells indicates LC3 recruitment to autophagosome membranes. In the lower panel, the percentage of cells with GFP-LC3 dots was quantified by counting the number of cells with > 3 dots and divided by a total number of GFP positive cells in 5 random non-overlapping fields. Pictures were taken at 630 × magnification. P value was calculated using Student’s t-test. (D) Induction of autophagy markers by the DEX and SEL co-treatment involves mTOR suppression. SEMK2 and RS4;11 cells were incubated with DEX in the presence or absence of SEL and lysed. 4E-BP1 phoshorylation status was assessed by immunoblotting. (E) GC-resistant (SEMK2) and—sensitive (RS4;11) cells were incubated with mTOR inhibitor rapamycin (100nM) in the presence or absence of DEX (0.05 μg/mL) for 24h. Thereafter, LC3 processing was assessed by immunoblotting and quantified. Densitometric analyses of LC3II/I are indicated below the blots. (F) Cells were treated as in (E) for 72h. Thereafter, cell numbers were assessed by counting 6 independent fields in Burker’s chamber. Data represent two independent experiments. P-values were calculated using 2-sided Student’s t-test.