Journal: Apoptosis : an international journal on programmed cell death

Article Title: PQ1, a Quinoline Derivative, Induces Apoptosis in T47D Breast Cancer Cells through Activation of Caspase-8 and Caspase-9

doi: 10.1007/s10495-013-0855-1

Figure Lengend Snippet: T47D cells were treated with DMSO and various concentrations of PQ1 for 48 hours. (a) Levels of procaspase-9 and caspase-9 p35 were examined by Western blot analysis using anti-caspase-9 p35 (H-170) antibody which detects the p35 subunit and precursor of caspase-9. Actin was used as a loading control. (b) Graphical presentation of three independent experiments shows the pixel intensities of caspase-9 p35 normalized to the controls. * P-value is <0.05 compared to control. (c) Graphical presentation of three independent experiments shows the ratio of active caspase-9 (caspase-9 p35) to pro-caspase-9. Results are normalized to the control. * P-value is <0.05 compared to control. (d) Immunofluorescence was performed using anti-cleaved caspase-9 (Asp315) antibody, a rabbit polyclonal antibody specific to the 35 kDa large fragment of caspase-9 following cleavage at aspartic acid 315. Red indicates caspase-9 p35 and blue indicates the nuclei. Percentages of cells with positive staining were labeled on top of relative images.

Article Snippet: Caspase-3 inhibitor (Ac-DMQD-CHO), caspase-8 inhibitor (Ac-IETD-CHO), and caspase-9 inhibitor (Ac-LEHD-CHO) were purchased from Enzo Life Sciences (Enzo Life Sciences, Farmingdale, NY, USA).

Techniques: Western Blot, Immunofluorescence, Staining, Labeling

Journal: Apoptosis : an international journal on programmed cell death

Article Title: PQ1, a Quinoline Derivative, Induces Apoptosis in T47D Breast Cancer Cells through Activation of Caspase-8 and Caspase-9

doi: 10.1007/s10495-013-0855-1

Figure Lengend Snippet: T47D cells were pre-treated with 20 μM caspase-3 inhibitor (Ac-DMQD-CHO), caspase-8 inhibitor (Ac-IETD-CHO), or caspase-9 inhibitor (Ac-LEHD-CHO) for 1 hour, and exposed to 500 nM PQ1 for 23 hours. Cells without treatments and cells treated with 500 nM PQ1 for 24 hours were used as controls. Cell viability was determined by trypan blue method. Data were obtained in three independent experiments and are represented as the mean ± S.D. * P-value is <0.05 compared to control. **P-value is <0.05 compared to treatment with 500 nM PQ1 for 24 hours.

Article Snippet: Caspase-3 inhibitor (Ac-DMQD-CHO), caspase-8 inhibitor (Ac-IETD-CHO), and caspase-9 inhibitor (Ac-LEHD-CHO) were purchased from Enzo Life Sciences (Enzo Life Sciences, Farmingdale, NY, USA).

Techniques:

Journal: Oncogene

Article Title: Fatty Acid Synthase inhibition engages a novel caspase-2 regulatory mechanism to induce ovarian cancer cell death

doi: 10.1038/onc.2014.271

Figure Lengend Snippet: ( A–B ) FASN inhibitor orlistat induced strong cell death (A) and effector caspase (DEVDase) activities (B) in the ovarian cancer cell line OVCA420 (420), but only had marginal effects in DOV13 (DOV) cells (* p <0.05; *** p <0.001; a.u., arbitrary units). ( C ) Activation of caspases induced by FASN inhibition was monitored at the indicated time points by the loss of full-length caspase-2, the appearance of cleaved caspase-8, -9, -3 (empty arrows), and caspase-mediated proteolysis of PARP. A rat monoclonal caspase-2 antibody (11B4) was used. ( D ) Orlistat induced the appearance of cleaved caspase-2 in 420 cells as monitored by a rabbit polyclonal caspase-2 antibody (Solid and empty arrows indicate full-length and cleaved caspase-2, respectively). ( E ) FASN inhibition activated the proteolytic cleavage of VDVAD-pNA (caspase-2 preferred substrate) in 420 (* p <0.05), but not in DOV cells (NS, not significant; p =0.08; a.u., arbitrary units). ( F ) Downregulation of caspase-2 effectively reduced cell death induced by orlistat in 420, but not in DOV cells (*** p <0.001; NS, not significant, p =0.137).

Article Snippet: Caspase-2 specific inhibitor zVDVAD-fmk (Enzo, Farmingdale, NY, USA) was utilized at 50 μM when indicated, while 50 μM pan-caspase inhibitor zVAD-fmk (Enzo) was cotreated with orlistat to examine REDD1 expression, unless otherwise specified.

Techniques: Activation Assay, Inhibition

Journal: Oncogene

Article Title: Fatty Acid Synthase inhibition engages a novel caspase-2 regulatory mechanism to induce ovarian cancer cell death

doi: 10.1038/onc.2014.271

Figure Lengend Snippet: ( A ) Cotreatment with the caspase-2 inhibitor zVDVAD-fmk prevented orlistat from inducing a decrease in full-length Bid (short exposure) and the appearance of truncated Bid (long exposure; solid and empty arrows indicate full-length and cleaved Bid, respectively). ( B) Inhibition of FASN by orlistat activated Bax and Bak. Active Bax or Bak were immunoprecipitated (IP) from the whole cell lysate (WCL) and detected as described in the Materials and Methods. ( C ) Downregulation of caspase-2 by RNAi dampened orlistat-induced Bax activation. ( D ) Inhibition of caspase-2 suppressed the activation of the caspase cascade (Solid and empty arrows indicate full-length and cleaved caspases, respectively). The loss of caspase-2 was revealed by a monoclonal caspase-2 antibody 11B4. 420 cell lysates were collected 12 h (A–C) or 24 h (D) post treatment.

Article Snippet: Caspase-2 specific inhibitor zVDVAD-fmk (Enzo, Farmingdale, NY, USA) was utilized at 50 μM when indicated, while 50 μM pan-caspase inhibitor zVAD-fmk (Enzo) was cotreated with orlistat to examine REDD1 expression, unless otherwise specified.

Techniques: Inhibition, Immunoprecipitation, Activation Assay

Journal: Oncogene

Article Title: Fatty Acid Synthase inhibition engages a novel caspase-2 regulatory mechanism to induce ovarian cancer cell death

doi: 10.1038/onc.2014.271

Figure Lengend Snippet: ( A ) REDD1 was upregulated prior to caspase-2 activation. 420 lysates were collected at the indicated time points and divided into two portions to examine cellular caspase-2-like (VDVADase) activities and REDD1 levels (* p <0.05; NS, not significant). ( B ) Caspase activities were dispensable for REDD1 induction by orlistat. REDD1 levels were monitored in 420 and DOV cells treated with DMSO or orlistat in the presence of zVAD-fmk. REDD1 is indicated by an arrow. ( C ) REDD1 siRNA prevented caspase-2 cleavage resulting from FASN inhibition in 420 cells. The loss of caspase-2 was revealed by a monoclonal caspase-2 antibody 11B4. ( D ) REDD1 deficiency suppressed caspase-2 activities induced after FASN inhibition. Orlistat induced strong caspase-2-like activities in 420 cells transfected with control siRNA, but this induction was largely suppressed in cells that received REDD1 siRNA (left panel; ** p <0.01; *** p <0.001). The same treatment with orlistat only caused negligible VDVADase activity in cells incapable of inducing REDD1 (right panel; NS, not significant, p =0.98). (E) REDD1 induction and the subsequent cell death induced by FASN inhibition was abrogated in 420 cells by siATF4 (*** p <0.001). ( F ) 420 cells were treated as in ( E ), and indicated mRNA levels were analyzed by a semi-quantitative PCR. Orlistat significantly increased REDD1 mRNA levels in cells transfected with control siRNA (black bars, siControl, DMSO vs. Orlistat), but not in ATF4 deficient cells (black bars, orlistat-treated cells, siControl vs. siATF4) (*** p <0.001).

Article Snippet: Caspase-2 specific inhibitor zVDVAD-fmk (Enzo, Farmingdale, NY, USA) was utilized at 50 μM when indicated, while 50 μM pan-caspase inhibitor zVAD-fmk (Enzo) was cotreated with orlistat to examine REDD1 expression, unless otherwise specified.

Techniques: Activation Assay, Inhibition, Transfection, Activity Assay, Real-time Polymerase Chain Reaction

Journal: Oncogene

Article Title: Fatty Acid Synthase inhibition engages a novel caspase-2 regulatory mechanism to induce ovarian cancer cell death

doi: 10.1038/onc.2014.271

Figure Lengend Snippet: (A) Orlistat caused time-dependent induction of REDD1 (indicated by an arrow; an asterisk shows a non-specific signal), decreased phosphorylated 4EBP1 at S65, and induced a downshift of the 4EBP1 proteins in control 420 cells (lane 1–4). Knockdown of REDD1 by siRNA ameliorated the dephosphorylation of 4EBP1 at S65 (lane 3 vs.7 and 4 vs. 8) and preserved the slowly migrating/hyper-phosphorylated species (arrows) detected by the 4EBP1 antibody (lane 4 vs. 8). (B–C) Downregulation of TSC2 by siRNA (B) or shRNA (C) suppressed orlistat-induced 420 cell death (* p <0.05; ** p <0.01). ( D ) Inhibition of mTOR by PP242 or rapamycin restored the sensitivity to orlistat in REDD1 deficient 420 cells (** p <0.01; *** p <0.001). ( E ) Suppression of mTOR by PP242 or rapamycin greatly enhanced DOV cell death induced by orlistat through a caspase-2-dependent pathway (* p <0.05; ***p<0.001; NS, not significant).

Article Snippet: Caspase-2 specific inhibitor zVDVAD-fmk (Enzo, Farmingdale, NY, USA) was utilized at 50 μM when indicated, while 50 μM pan-caspase inhibitor zVAD-fmk (Enzo) was cotreated with orlistat to examine REDD1 expression, unless otherwise specified.

Techniques: De-Phosphorylation Assay, shRNA, Inhibition

Journal: Oncogene

Article Title: Fatty Acid Synthase inhibition engages a novel caspase-2 regulatory mechanism to induce ovarian cancer cell death

doi: 10.1038/onc.2014.271

Figure Lengend Snippet: ( A ) Caspase-2 dimerization was monitored by a BiFC-based assay and recorded by photomicrographs taken with a confocal microscope. Paclitaxel, a known caspae-2 activator, was used as a positive control, whereas solvent (DMSO) was used as a negative control. Orlistat induced caspase-2 BiFC signals (yellow) in control BiFC cells but not in those transfected with REDD1 siRNA. DAPI (blue) stained the nucleus. ( B ) REDD1 expression was specifically induced upon FASN inhibition. BiFC cells were analyzed after indicated treatments by flow cytometry for cell death, and then lysed to measure REDD1 levels by western blotting. Actin was used as a loading control. ( C ) Orlistat induced BiFC signals in control BiFC cells, whereas this induction was largely suppressed by two different REDD1 siRNAs (middle, orlistat-treated group) or by doxycycline (Dox) (right, orlistat+Dox-treated group) (* p <0.05; ** p <0.01; *** p <0.001). The efficacy of REDD1 siRNA was examined by western blotting.

Article Snippet: Caspase-2 specific inhibitor zVDVAD-fmk (Enzo, Farmingdale, NY, USA) was utilized at 50 μM when indicated, while 50 μM pan-caspase inhibitor zVAD-fmk (Enzo) was cotreated with orlistat to examine REDD1 expression, unless otherwise specified.

Techniques: Bimolecular Fluorescence Complementation Assay, Microscopy, Positive Control, Negative Control, Transfection, Staining, Expressing, Inhibition, Flow Cytometry, Western Blot