Article Figures & Data
Figures
- Figure 1.
MEK1/2 inhibitors and 17AAG synergize to kill human hepatoma cells in vitro. A, human hepatoma cells (HuH7, HEPG2, and HEP3B) were treated 24 h after triplicate plating with vehicle (VEH), PD184352 (PD; 1 μmol/L), 17AAG (1 μmol/L), or both drugs combined. Cell viability was determined 48 h after drug treatment by trypan blue exclusion visible light microscopy assays in triplicate using a hemacytometer (± SE; n = 3 independent studies; *, P < 0.05, value greater amount of cell killing compared with vehicle-treated cells). B, human hepatoma cells (HEP3B) were treated 24 h after triplicate plating with vehicle, PD184352 (1 μmol/L), 17AAG (1 μmol/L), or both drugs combined. Cell viability was determined 24 or 48 h after drug treatment, as indicated, by Annexin V/propidium iodide flow cytometric assay and terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling staining and fluorescent microscopy (± SE; n = 3 independent studies; *, P < 0.05, value greater amount of cell killing compared with vehicle-treated cells). C, human hepatoma cells (HEPG2 and HEP3B) were treated 24 h after triplicate plating with vehicle (DMSO) or the caspase-8 inhibitor IETD (50 μmol/L). Thirty minutes after caspase inhibitor treatment, cells were treated with vehicle, PD184352 (1 μmol/L), 17DMAG, or both drugs combined. Cell viability was determined 48 h after drug treatment as indicated by trypan blue exclusion visible light microscopy assays in triplicate using a hemacytometer (± SE; n = 2 independent studies; #, P < 0.05, value lower amount of cell killing compared with vehicle-treated cells). D, HEP3B cells were plated as single cells (250-1,500 per well) in sextuplicate and 12 h after plating treated with vehicle (DMSO), MEK1/2 inhibitors PD184352 (0.5-2.0 μmol/L) or AZD6244 (200-400 nmol/L), a geldanamycin (17AAG; 0.25-1.00 μmol/L), or both drugs combined as indicated at a fixed concentration ratio to perform median dose-effect analyses for synergy determination. After drug exposure (48 h), the medium was changed and cells were cultured in drug-free medium for 10 to 14 d. Cells were fixed and stained with crystal violet and colonies of >50 cells per colony were counted. Colony formation data were entered into the Calcusyn program and combination index values were determined. A combination index value of <1.00 indicates synergy. Mean ± SE combination index values from three separate studies.
- Figure 2.
MEK1/2 inhibitors and 17AAG kill hepatoma cells via caspase-8- and caspase-9-dependent pathways. A, human hepatoma cells (HEPG2 and HEP3B) were infected 12 h after plating with either a control empty vector recombinant adenovirus [cytomegalovirus (CMV)], a recombinant virus to express CRM A, or a virus to express dominant-negative caspase-9. Twenty-four hours after virus infection, cells were treated with vehicle, PD184352, 17AAG, or both drugs combined. Cell viability was determined 48 h after drug treatment as indicated by trypan blue exclusion visible light microscopy assays in triplicate using a hemacytometer (± SE; n = 2 independent studies; #, P < 0.05, value lower amount of cell killing compared with vehicle-treated cells). Topmost inset blotting, cells were infected with CRM A and treated with drugs. Cells were isolated 48 h after exposure and SDS-PAGE and immunoblotting was done to determine the cleavage status of pro-caspase-3. B, SV40 large T antigen transformed mouse embryonic fibroblasts (wild-type) or deleted for both alleles of specific survival regulatory proteins 24 h after triplicate plating were treated with vehicle, PD184352, 17AAG, or both drugs combined. Cell viability was determined 48 h after drug treatment as indicated by trypan blue exclusion visible light microscopy assays in triplicate using a hemacytometer (± SE; n = 2 independent studies; #, P < 0.05 value lower amount of cell killing compared with vehicle-treated cells).
- Figure 3.
MEK1/2 inhibitors and 17AAG promote cell killing via a caspase-8-dependent pathway that requires suppression of c-FLIP-s expression. A, human hepatoma cells (HEPG2 and HEP3B) were infected 12 h after plating with either a control empty vector recombinant adenovirus (CMV), a recombinant virus to express c-FLIP-s, or a virus to express XIAP or BCL-XL. Twenty-four hours after virus infection, cells were treated with vehicle, PD184352, 17AAG, or both drugs combined. Cell viability was determined 48 h after drug treatment as indicated by trypan blue exclusion visible light microscopy assays in triplicate using a hemacytometer (± SE; n = 2 independent studies; #, P < 0.05, value lower amount of cell killing compared with vehicle-treated cells). B, top immunoblotting section, HEP3B cells were infected 12 h after plating with either a control empty vector recombinant adenovirus (CMV) or a recombinant virus to express c-FLIP-s. Twenty-four hours after virus infection, cells were treated with vehicle, PD184352, 17AAG, or both drugs combined. The expression/integrity of pro-caspase-8, BID, c-FLIP-s, and GAPDH was determined 24 h after drug treatment as indicated after SDS-PAGE and immunoblotting assays (representative of three studies). Bottom graphical section, human hepatoma cells (HEP3B) were infected 12 h after plating with either a control empty vector recombinant adenovirus (CMV) or a recombinant virus to express c-FLIP-s. Twenty-four hours after virus infection, cells were treated with vehicle, PD184352, 17AAG, or both drugs combined. Cell viability was determined 48 h after drug treatment as indicated by Annexin V/propidium iodide flow cytometric assay (± SE; n = 2 independent studies; #, P < 0.05, value lower amount of cell killing compared with vehicle-treated cells).
- Figure 4.
MEK1/2 inhibitors and 17AAG kill hepatoma cells in a CD95-FADD-caspase-8-BID-dependent manner. A and B, HEPG2 and HEP3B cells were transfected with either a scrambled siRNA molecule (siSCR) or validated siRNA molecules to knockdown the expression of CD95, FADD, BID, or c-FLIP-s. Twelve hours after transfection, cells were replated. Twenty-four hours after replating, cells were treated with vehicle, PD184352, 17AAG, or both drugs combined. Cell viability was determined 48 h after drug treatment as indicated by terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling assay and fluorescent light microscopy or by trypan blue exclusion visible light microscopy assays in triplicate using a hemacytometer as indicated (± SE; n = 2 independent studies for each method; #, P < 0.05, value lower amount of cell killing compared with vehicle-treated cells). A, inset immunoblotting sections, 36 h after siRNA transfection to knockdown the expression of CD95 and FADD, cells were isolated and subjected to immunoblotting to determine the expression of CD95 and FADD and GAPDH loading control (n = 2). Data for knockdown of c-FLIP-s and BID are not shown. C, top, HEP3B cells 24 h after plating were treated with vehicle, PD184352, 17AAG, or both drugs combined. Six hours after drug exposure, cells were lysed and CD95 was immunoprecipitated as described in Materials and Methods based on established protocols. SDS-PAGE and immunoblotting was done to determine the levels of pro-caspase-8 and c-FLIP isoforms in the immunoprecipitates and the level of GAPDH in the cell lysates following immunoprecipitation. Bottom, HEP3B cells were transfected with either a scrambled siRNA molecule or a validated siRNA molecule to knockdown the expression of CD95. Twelve hours after transfection, cells were replated. Twenty-four hours after replating, cells were treated with vehicle, PD184352, 17AAG, or both drugs combined. Twelve hours after drug treatment, cells were isolated and the cytosolic and membrane/mitochondrial fractions of the cells were purified as described in Materials and Methods. The amount of cytosolic and mitochondrial-associated cytochrome c was determined by SDS-PAGE followed by immunoblotting (n = 3). D, HEP3B cells 24 h after plating into four-well glass slides were treated with vehicle, PD184352, 17AAG, or both drugs combined. Six hours after drug exposure, cells were fixed in situ, but were not permeabilized, and stained with a FITC-labeled anti-CD95 antibody. Studies with a nonspecific identical subtype antibody as a control showed the observed effects were CD95 dependent. Identical analyses were also done on any new batch of antibody used over the course of the studies. Cells were visualized under fluorescent light. Representative field from four separate/independent experiments. To mathematically prove that our visual observation was actually significant, the mean increase in CD95 surface fluorescence was determined by analyzing 5 cells from vehicle or 17AAG + PD184352 conditions with 20 individual random areas of fluorescent density determination per cell (100 density determinations in total ± SE) with at least one cell per experiment, and this value is presented below the pictorial data in the figure. *, P < 0.05, value greater amount of cell surface CD95 compared with vehicle-treated cells.
- Figure 5.
Combined exposure to MEK1/2 inhibitor and 17AAG results in prolonged suppression of ERK1/2 and AKT phosphorylation and prolonged suppression of c-FLIP-s, BCL-XL, and XIAP levels. A, HEP3B cells 24 h after plating were treated with vehicle, PD184352, 17AAG, or both drugs combined. At the indicated times after drug exposure, cells were isolated and subjected to SDS-PAGE followed by immunoblotting to determine the phosphorylation of ERK1/2, AKT (S473), and p38 MAPK and the expression of FLIP-l, FLIP-s, BCL-XL, XIAP, and GAPDH (n = 3-4). B, HEPG2 and HEP3B cells 12 h after plating in triplicate were infected with either a control empty vector recombinant adenovirus (CMV), a recombinant virus to express constitutively active MEK1 EE, a recombinant virus to express constitutively active AKT, or both viruses to express activated AKT and MEK1 EE. Twenty-four hours after infection, as indicated, cells were treated with either vehicle, PD98059, or both drugs combined. Bottom graphical sections, HEPG2 and HEP3B cells were isolated 48 h after drug exposure and cell viability was determined as indicated by trypan blue exclusion visible light microscopy assays in triplicate using a hemacytometer (± SE; n = 3). #, P < 0.05, value less lethality compared with corresponding value in CMV-infected cells; ##, P < 0.05, value less lethality compared with corresponding value in constitutively activated AKT-infected and constitutively activated MEK1-infected cells. Inset, HEP3B cells expressing activated AKT and MEK1 EE and treated with PD98059 and 17AAG, or their appropriate parallel control cells, were isolated 24 h after drug exposure and following SDS-PAGE immunoblotting done to determine the expression of c-FLIP-s, BCL-XL, and XIAP (n = 2 independent studies). C, blotting to left, HEP3B cells expressing either a control empty vector recombinant adenovirus (CMV), a recombinant virus to express dominant-negative MEK1, a recombinant virus to express dominant-negative AKT, or both viruses to express dominant-negative AKT and dominant-negative MEK1 were isolated 48 h after infection and processed for SDS-PAGE to determine the expression of c-FLIP-s, BCL-XL, and XIAP and the phosphorylation of ERK1/2, AKT (S473), and p38 MAPK (n = 2 independent studies). Blotting to right, HEP3B cells were treated with either vehicle (PBS) or with N-acetylcysteine (20 mmol/L) 30 min before treatment with vehicle (DMSO) or with MEK1/2 inhibitor PD184352 (1 μmol/L) and geldanamycin (17AAG, 1 μmol/L). Cells were isolated 24 h after drug exposure and the phosphorylation of ERK1/2, AKT (S473), and p38 MAPK was determined after SDS-PAGE by immunoblotting (n = 2 independent studies).
- Figure 6.
CD95 activation by MEK1/2 inhibitors and 17AAG is p38 MAPK dependent. A, section (i), HEP3B cells 12 h after plating were infected with either a control empty vector recombinant adenovirus (CMV), a recombinant virus to express dominant-negative p38α MAPK, or viruses to express dominant-negative forms of MKK3 and MKK6. Twenty-four hours after infection, as indicated, cells were treated with either vehicle, PD184352, or PD98059 as indicated, 17AAG, or the drugs in combination. Six hours after drug exposure, cells were lysed and CD95 was immunoprecipitated as described in Materials and Methods. SDS-PAGE and immunoblotting were done to determine the levels of pro-caspase-8 in the immunoprecipitates and the level of pro-caspase-8 and GAPDH in the cell lysates following immunoprecipitation (n = 3 independent studies). Section (ii), HEP3B cells 12 h after plating were infected with either a control empty vector recombinant adenovirus (CMV) or recombinant adenoviruses to express constitutively active forms of MKK1 EE and AKT. Twenty-four hours after infection, as indicated, cells were treated with either vehicle (DMSO) or the combination of PD184352 and 17AAG. Six hours after drug exposure, cells were lysed and CD95 was immunoprecipitated as described in Materials and Methods. SDS-PAGE and immunoblotting was done to determine the levels of pro-caspase-8 in the immunoprecipitates and the level of pro-caspase-8 and GAPDH in the cell lysates following immunoprecipitation (n = 2 independent studies). B, HEP3B cells 12 h after plating into four-chamber glass slides were infected with either a control empty vector recombinant adenovirus (CMV) or a recombinant virus to express dominant-negative p38α MAPK. Twenty-four hours after infection, as indicated, cells were treated with either vehicle or the combination of PD98059 and 17AAG. Six hours after drug exposure, cells were fixed in situ, but were not permeabilized, and stained with a FITC-labeled anti-CD95 antibody. Studies with a nonspecific identical subtype antibody as a control showed that the observed effects were CD95 dependent. Identical analyses were also done on any new batch of antibody used over the course of the studies. Cells were visualized under fluorescent light. Representative field from four separate/independent experiments. To mathematically prove that our visual observation was actually significant, the mean increase in CD95 surface fluorescence was determined by analyzing 5 cells from vehicle or 17AAG + PD184352 conditions with 20 individual random areas of fluorescent density determination per cell (100 density determinations in total ± SE) with at least one cell per experiment, and this value is presented below the pictorial data in the figure. *, P < 0.05, value greater amount of cell surface CD95 compared with vehicle-treated cells. C, HEP3B cells 12 h after plating were infected with either a control empty vector recombinant adenovirus (CMV) or a recombinant virus to express dominant-negative p38α MAPK. Twenty-four hours after infection, as indicated, cells were pretreated with vehicle (DMSO) or a JNK inhibitory peptide (JNK-IP; 10 μmol/L) followed 30 min later by treatment with either vehicle, PD184352, 17AAG, or both drugs combined. Cells were isolated 48 h after drug exposure and cell viability was determined as indicated by trypan blue exclusion visible light microscopy assays in triplicate using a hemacytometer (± SE; n = 3 independent studies). #, P < 0.05, value less than amount of killing compared with CMV-infected cells. Top inset, HEP3B cells 12 h after plating were infected with either a control empty vector recombinant adenovirus (CMV) or a recombinant virus to express dominant-negative p38α MAPK. Twenty-four hours after infection, as indicated, cells were treated with either vehicle, PD184352, 17AAG, or both drugs combined. Cells were isolated 6 h after drug exposure and immunoprecipitations were done to isolate activated forms of BAX and of BAK from each treatment condition. SDS-PAGE was done on immunoprecipitates and on total cell lysates to determine the total and activated levels of BAX and BAK in treated cells (n = 2 independent studies). D, HEP3B cells (107) were injected into the rear right flanks of athymic mice and tumors were permitted to form (∼150 mm3). Animals were treated with 17AAG and MEK1/2 inhibitor PD184352 as described in Materials and Methods for 30 h after which tumors were isolated, sectioned, portions macerated, and digested to obtain individual tumor cells for ex vivo colony formation assays. Bottom numerical section, true percentage plating efficiency of tumor cells in ex vivo colony formation assays from two tumors plated each in sextuplicate at multiple cell dilutions (2,000-4,000 cells per well) ± SE. In parallel, on sectioned portions of the isolated tumors, staining was done to determine H&E (morphology) and terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (apoptosis) as well as immunohistochemistry with 4′,6-diamidino-2-phenylindole counterstaining (dark blue/mauve nuclei); the cleavage status of caspase-3 (cherry red); the expression of c-FLIP-s (green); and the phosphorylation status of ERK1/2 (green), p38 MAPK (green), and AKT (S473; cherry red). Representative of two independent experiments.
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Volume 7, Issue 9
