Journal: Cell Death & Disease

Article Title: Triptolide induces apoptosis in human leukemia cells through caspase-3-mediated ROCK1 activation and MLC phosphorylation

doi: 10.1038/cddis.2013.469

Figure Lengend Snippet: Triptolide induced apoptosis and mitochondrial injury in multiple leukemia cell lines. ( a ) The chemical structure of triptolide, C 20 H 24 O 6 , molecular weight: 360.4. ( b and c ) U937 cells were treated with various triptolide (TPL) concentrations for 24 h or with 40 nM triptolide for different lengths. The percentage of apoptotic cells was determined by FACS analysis using Annexin V/PI staining. Mitochondrial membrane potentials (ΔΨm) were detected by rhodamine-123 staining and flow cytometry. Values represent the mean±S.D. for five separate experiments. Total protein lysates, nuclear extracts, and cytosolic fractions were analyzed by immunoblotting using the indicated antibodies. ( d ) After triptolide treatment, cells were collected and stained with anti-AIF (green) and 4′,6-diamidino-2-phenylindole (DAPI; blue) to identify cellular nuclei. Fluorescence was visualized by a laser confocal scanning microscope. Scale bar represents 10 μ m. These data are representative of three independent experiments. ( e and f ) U937, Jurkat, and HL-60 cells were treated with or without 40 nM triptolide for 24 h, after which apoptosis was determined by FACS analysis using Annexin V/PI staining. Total protein lysates, nuclear extracts, and cytosolic fractions were analyzed by immunoblotting using the indicated antibodies. CF, cleavage fragment; C, cytosolic fractions; N, nuclear extracts

Article Snippet: Triptolide was purchased from Alexis Biochemicals (San Diego, CA, USA); z-Asp-Glu-Vad-Asp(Ome)-fluoromethylketone (z-DEVD-fmk) and z-Val-Ala-Asp(Ome)-fluoromethylketone (z-VAD-fmk) from Enzyme Systems Products (Livermore, CA, USA); exoenzyme C3 and 1-(5-iodonapthalene-1-sulfonyl) homopiperazine hydrochloride (ML-7) from Calbiochem (La Jolla, CA, USA); and Y-27632 from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Molecular Weight, Staining, Flow Cytometry, Western Blot, Fluorescence, Microscopy

Journal: Cell Death & Disease

Article Title: Triptolide induces apoptosis in human leukemia cells through caspase-3-mediated ROCK1 activation and MLC phosphorylation

doi: 10.1038/cddis.2013.469

Figure Lengend Snippet: Triptolide induced apoptosis in primary human leukemia blasts but not in normal CD34 + cells. ( a ) Primary leukemia blasts from 44 patients were treated with 40 nM triptolide for 24 or 36 h, after which apoptosis was determined by FACS analysis using Annexin V/PI staining. Upper panel: representative FACS images. Lower panel: numbers indicate the percentage of apoptotic cells; each symbol represents results from individual patients. ( b ) Total protein lysates and cytosolic fractions of the samples from four AML patients were then obtained and analyzed by immunoblotting using the indicated antibodies. ( c ) Normal CD34 + cells were isolated from 6 normal bone marrow samples and exposed to 40 nM triptolide for 24 or 36 h. The percentage of apoptotic cells was determined by FACS analysis using Annexin V/PI staining. ( d ) Two normal donors were treated with 40 nM triptolide for 24 or 36 h. Total protein lysates and cytosolic fractions of these samples were then obtained and analyzed by immunoblotting using the indicated antibodies

Article Snippet: Triptolide was purchased from Alexis Biochemicals (San Diego, CA, USA); z-Asp-Glu-Vad-Asp(Ome)-fluoromethylketone (z-DEVD-fmk) and z-Val-Ala-Asp(Ome)-fluoromethylketone (z-VAD-fmk) from Enzyme Systems Products (Livermore, CA, USA); exoenzyme C3 and 1-(5-iodonapthalene-1-sulfonyl) homopiperazine hydrochloride (ML-7) from Calbiochem (La Jolla, CA, USA); and Y-27632 from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Staining, Western Blot, Isolation

Journal: Cell Death & Disease

Article Title: Triptolide induces apoptosis in human leukemia cells through caspase-3-mediated ROCK1 activation and MLC phosphorylation

doi: 10.1038/cddis.2013.469

Figure Lengend Snippet: Triptolide induced Bax translocation in multiple leukemia cell lines. ( a ) U937 cells were treated with various triptolide concentrations for 24 h or with 40 nM triptolide for different lengths, after which cytosolic and mitochondrial fractions were isolated and subjected to immunoblot analysis by using an anti-Bax antibody. ( b ) U937 cells were treated with or without 40 nM triptolide for 24 h. Cells were collected and stained with anti-Bax (green) and MitoTracker (red) to identify mitochondria. Fluorescence was visualized by a laser confocal scanning microscope. Scale bar represents 10 μ m. These data are representative of three independent experiments. ( c ) U937, Jurkat, and HL-60 cells were treated with or without 40 nM triptolide for 24 h, after which cytosolic and mitochondrial fractions were isolated and subjected to immunoblot analysis by using an anti-Bax antibody

Article Snippet: Triptolide was purchased from Alexis Biochemicals (San Diego, CA, USA); z-Asp-Glu-Vad-Asp(Ome)-fluoromethylketone (z-DEVD-fmk) and z-Val-Ala-Asp(Ome)-fluoromethylketone (z-VAD-fmk) from Enzyme Systems Products (Livermore, CA, USA); exoenzyme C3 and 1-(5-iodonapthalene-1-sulfonyl) homopiperazine hydrochloride (ML-7) from Calbiochem (La Jolla, CA, USA); and Y-27632 from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Translocation Assay, Isolation, Western Blot, Staining, Fluorescence, Microscopy

Journal: Cell Death & Disease

Article Title: Triptolide induces apoptosis in human leukemia cells through caspase-3-mediated ROCK1 activation and MLC phosphorylation

doi: 10.1038/cddis.2013.469

Figure Lengend Snippet: Triptolide induced ROCK1 activation and MLC and MYPT phosphorylation. ( a ) U937 cells were treated with various triptolide concentrations for 24 h or with 40 nM triptolide for different lengths. Total protein lysates were analyzed by immunoblotting using the indicated antibodies. RhoA-GTP was evaluated using a Rhotekin RBD-GST pull-down. ( b ) U937, Jurkat, and HL-60 cells were treated with or without 40 nM triptolide for 24 h. Total protein lysates were analyzed by immunoblotting using the indicated antibodies. Samples from four AML patients ( c ) and two normal donors ( d ) were treated with 40 nM triptolide for 24 or 36 h. The total protein lysates of these samples were obtained and analyzed by immunoblotting using the indicated antibodies

Article Snippet: Triptolide was purchased from Alexis Biochemicals (San Diego, CA, USA); z-Asp-Glu-Vad-Asp(Ome)-fluoromethylketone (z-DEVD-fmk) and z-Val-Ala-Asp(Ome)-fluoromethylketone (z-VAD-fmk) from Enzyme Systems Products (Livermore, CA, USA); exoenzyme C3 and 1-(5-iodonapthalene-1-sulfonyl) homopiperazine hydrochloride (ML-7) from Calbiochem (La Jolla, CA, USA); and Y-27632 from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Activation Assay, Western Blot

Journal: Cell Death & Disease

Article Title: Triptolide induces apoptosis in human leukemia cells through caspase-3-mediated ROCK1 activation and MLC phosphorylation

doi: 10.1038/cddis.2013.469

Figure Lengend Snippet: Caspase-3, rather than RhoA, activated ROCK1 during triptolide-induced apoptosis. Caspase inhibitors (z-VAD-fmk or z-DEVD-fmk) and a Rho inhibitor (C3 exoenzyme) were used to explore the mechanism behind ROCK1 activation. The percentage of apoptotic cells was determined by FACS analysis using Annexin V/PI staining. Error bars represent the mean±S.D. ( n =5). Total protein lysates were analyzed by immunoblotting using the indicated antibodies. Results are representative of three independent experiments. ( a–c ) U937 cells and blasts from one AML patient were pretreated with 20 μ M z-VAD-fmk or z-DEVD-fmk for 2 h followed by treatment with or without 40 nM triptolide for 24 h. ( d – f ) U937 cells and blasts from one AML patient were transfected with 5 μ g C3 (premixed with FuGENE 6 Reagent in RPMI-1640 medium). After 5 h of incubation, cells were treated with or without 40 nM triptolide for 24 h

Article Snippet: Triptolide was purchased from Alexis Biochemicals (San Diego, CA, USA); z-Asp-Glu-Vad-Asp(Ome)-fluoromethylketone (z-DEVD-fmk) and z-Val-Ala-Asp(Ome)-fluoromethylketone (z-VAD-fmk) from Enzyme Systems Products (Livermore, CA, USA); exoenzyme C3 and 1-(5-iodonapthalene-1-sulfonyl) homopiperazine hydrochloride (ML-7) from Calbiochem (La Jolla, CA, USA); and Y-27632 from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Activation Assay, Staining, Western Blot, Transfection, Incubation

Journal: Cell Death & Disease

Article Title: Triptolide induces apoptosis in human leukemia cells through caspase-3-mediated ROCK1 activation and MLC phosphorylation

doi: 10.1038/cddis.2013.469

Figure Lengend Snippet: Inhibiting MLC phosphorylation significantly decreased triptolide-induced apoptosis. U937 cells and blasts from one AML patient were pretreated with 20 μ M ML-7, a MLC kinase inhibitor, for 2 h followed by treatment with or without 40 nM triptolide for 24 h. ( a ) The percentage of apoptotic cells was determined by FACS analysis using Annexin V/PI staining. Error bars represent the mean±S.D. ( n =5). ( b and c ) Total protein lysates and cytosolic fractions were analyzed by immunoblotting using the indicated antibodies. Results were representative of three independent experiments

Article Snippet: Triptolide was purchased from Alexis Biochemicals (San Diego, CA, USA); z-Asp-Glu-Vad-Asp(Ome)-fluoromethylketone (z-DEVD-fmk) and z-Val-Ala-Asp(Ome)-fluoromethylketone (z-VAD-fmk) from Enzyme Systems Products (Livermore, CA, USA); exoenzyme C3 and 1-(5-iodonapthalene-1-sulfonyl) homopiperazine hydrochloride (ML-7) from Calbiochem (La Jolla, CA, USA); and Y-27632 from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Staining, Western Blot

Journal: Cell Death & Disease

Article Title: Triptolide induces apoptosis in human leukemia cells through caspase-3-mediated ROCK1 activation and MLC phosphorylation

doi: 10.1038/cddis.2013.469

Figure Lengend Snippet: Inhibiting ROCK1 activation attenuated triptolide-induced apoptosis. ( a ) U937 cells and blasts from one AML patient were pretreated with 20 μ M Y-27632, a ROCK1 inhibitor, for 2 h followed by treatment with 40 nM triptolide for 24 h. The percentage of apoptotic cells was determined by FACS analysis using Annexin V/PI staining. Error bars represent the mean±S.D. ( n =5). ( b and c ) Total protein lysates and cytosolic fractions were analyzed by immunoblotting using the indicated antibodies. Results are representative of three independent experiments. ( d ) U937 cells were stably transfected with lentivirus containing ROCK1-specific siRNA (si-ROCK1) or a scrambled control siRNA. Cells were treated with or without 40 nM triptolide for 24 h, and apoptosis was measured by FACS analysis using Annexin V/PI staining. Error bars represent the mean±S.D. ( n =5). ( e and f ) Total protein lysates and cytosolic fractions were analyzed by immunoblotting using the indicated antibodies. Results are representative of three independent experiments

Article Snippet: Triptolide was purchased from Alexis Biochemicals (San Diego, CA, USA); z-Asp-Glu-Vad-Asp(Ome)-fluoromethylketone (z-DEVD-fmk) and z-Val-Ala-Asp(Ome)-fluoromethylketone (z-VAD-fmk) from Enzyme Systems Products (Livermore, CA, USA); exoenzyme C3 and 1-(5-iodonapthalene-1-sulfonyl) homopiperazine hydrochloride (ML-7) from Calbiochem (La Jolla, CA, USA); and Y-27632 from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Activation Assay, Staining, Western Blot, Stable Transfection, Transfection

Journal: Cell Death & Disease

Article Title: Triptolide induces apoptosis in human leukemia cells through caspase-3-mediated ROCK1 activation and MLC phosphorylation

doi: 10.1038/cddis.2013.469

Figure Lengend Snippet: Triptolide inhibited the growth of U937 xenografts. A total of 24 nude mice were inoculated with U937 cells and randomly divided into two groups (12 mice/group) that were treated with either vehicle or triptolide. ( a ) Tumor volumes were measured at the indicated intervals (upper panel) and images are shown for five representative tumors from each group after 40 days of treatment (lower panel). Data are shown as the mean±S.D. * P <0.05; ** P <0.01; *** P <0.001. ( b ) Changes in body weight in the mice during 40 days of triptolide treatment. ( c ) Tumors were fixed and stained with H&E to examine tumor cell morphology. TUNEL assays were used to determine the apoptotic effects of triptolide. Immunohistochemistry was used to determine the levels of cleaved caspase-3 and phospho-MLC. Scale bar represents 50 μ m. ( d ) After treatment, tumors from the vehicle and triptolide groups were lysed and subjected to immunoblotting with anti-ROCK1. Results are representative of three independent experiments. ( e ) An illustration of the molecular mechanism of triptolide-induced apoptosis. Exposure to triptolide results in activation of ROCK1 and MLC and MYPT phosphorylation, leading in turn to Bax translocation, culminating in cytochrome c release (Cyto c ), caspases activation, and apoptosis. Activated caspase-3 in turn causes activation of ROCK, thus creating a positive feedback loop

Article Snippet: Triptolide was purchased from Alexis Biochemicals (San Diego, CA, USA); z-Asp-Glu-Vad-Asp(Ome)-fluoromethylketone (z-DEVD-fmk) and z-Val-Ala-Asp(Ome)-fluoromethylketone (z-VAD-fmk) from Enzyme Systems Products (Livermore, CA, USA); exoenzyme C3 and 1-(5-iodonapthalene-1-sulfonyl) homopiperazine hydrochloride (ML-7) from Calbiochem (La Jolla, CA, USA); and Y-27632 from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Staining, TUNEL Assay, Immunohistochemistry, Western Blot, Activation Assay, Translocation Assay