Journal: Cell Death & Disease

Article Title: Withanolide E sensitizes renal carcinoma cells to TRAIL-induced apoptosis by increasing cFLIP degradation

doi: 10.1038/cddis.2015.38

Figure Lengend Snippet: Withanolide E enhances TRAIL-induced extrinsic apoptotic pathway in ACHN cells. Cells were treated without (open bars) or with (filled bars) withanolide E for 3–4 h followed by agonistic anti-DR4 ( a ) or DR5 ( b ) antibodies for 20–24 h. For panels c – f , cells were treated 4 h±withanolide E (WE) then 4 h (for a total of 8 h) or 24 h±TRAIL in the continued presence of WE. Open bars: withanolide E only; black: withanolide E followed by TRAIL. ( c ) Cells were pretreated with ZVAD-FMK to block caspase activation followed by WE, then TRAIL. Caspase 8 ( d ) and caspase 3 ( e ) activities, and DNA fragmentation ( f ) were assessed using commercial kits. ( g ) Caspase cleavage and cFLIP degradation were assessed by immunoblot (total treatment time, 8 h). ( h ) TRAIL-dependent DISC formation was assessed in withanolide E- or bortezomib (Bzb – control with known activity)-treated ACHN cells (overnight treatment followed by biotinylated TRAIL at 4 °C, cell lysis, DISC precipitation and immunoblot). Error bars represent S.D. ( n =3)

Article Snippet: To assess death receptor utilization, agonistic anti-DR4 (Alexis/Enzo, Farmingdale, NY, USA) or DR5 (R&D Systems, Minneapolis, MN, USA) replaced TRAIL.

Techniques: Blocking Assay, Activation Assay, Western Blot, Activity Assay, Lysis

Journal: Cell Death & Disease

Article Title: Withanolide E sensitizes renal carcinoma cells to TRAIL-induced apoptosis by increasing cFLIP degradation

doi: 10.1038/cddis.2015.38

Figure Lengend Snippet: Withanolide E effects on TRAIL sensitization mechanisms. ( a ) Effects of withanolide E on expression of pro- and antiapoptotic proteins. ACHN cells were treated 24 h with bortezomib (20 nM) or withanolide E, then±TRAIL (50 ng/ml) and immunoblot. ( b ) ACHN cells were treated±bortezomib (Bzb, positive control) or withanolide E (WE) followed by±TRAIL (total treatment time, 8 h) and immunoblot analysis of ER stress markers. ( c ) Cells were treated 4 h±withanolide E then 4 h (for a total of 8 h) or 24 h±TRAIL in the continued presence of WE. Open bars: withanolide E only; black: withanolide E followed by TRAIL. Mitochondrial potential was assessed (JC-1 assay). ( d ) Cells were treated with withanolide E (solid symbols) or withanolide A (inactive control, open symbols) and ROS generation estimated with DCFDA. ( e ) ACHN cells were pretreated with NAC (10 mM), Trolox (200 μ M) or vitamin C (vitC, 200 μ M) followed by withanolide E (2 h), then TRAIL (24 h). Open bars: withanolide E only; gray bars: TRAIL only; black bars: withanolide E+TRAIL. ( f ) ACHN cells were treated for 8 h or 24 h with 10 μ M withanolide E and assessed for expression of TRAIL receptors 1 and 2 by FACS (FACS Caliber, BD Biosciences). Antibodies used were (anti-TRAIL-R1 (human), mAb (HS101) (ATTO 488) or anti-TRAIL-R2 (human), mAb (HS201) (ATTO 647N) or mouse IgG1 atto488 or mouse IgG1atto 647 (Adipogen)). Dashed line represents isotype control, solid line DMSO-treated cells, and filled line withanolide E-treated cells. Error bars represent S.D. ( n =3–4)

Article Snippet: To assess death receptor utilization, agonistic anti-DR4 (Alexis/Enzo, Farmingdale, NY, USA) or DR5 (R&D Systems, Minneapolis, MN, USA) replaced TRAIL.

Techniques: Expressing, Western Blot, Positive Control

Journal: Cell Death & Disease

Article Title: Withanolide E sensitizes renal carcinoma cells to TRAIL-induced apoptosis by increasing cFLIP degradation

doi: 10.1038/cddis.2015.38

Figure Lengend Snippet: Withanolide E enhances death receptor-induced apoptosis in vivo . Effects of withanolide E and drozitumab (DR5 agonist antibody) on growth of ACHN cell-derived tumors in athymic mice was assessed as described in the text. ( a ) Tumor size at 75 days (intratumor), ( b ) survival up to 150 days (intraperitoneal) – pooled from two separate experiments with similar findings. Numbers of mice were: vehicle control (●, n =10), withanolide E (▴, n =10), drozitumab (▪, n =14), withanolide E plus drozitumab (□, n =16). * P <0.05

Article Snippet: For the generation of subcutaneous tumors, mice were injected subcutaneously with 1 × 10 6 ACHN cells that had been adapted for in vivo passage in a volume of 100 μ l. After tumor volumes reached ~100 mm 3 (~14 days post injection) therapy was initiated - either direct injection of withanolide E and Drozitumab (agonist antibody to TRAIL death receptor DR5, kindly provided by Dr. Avi Ashkenazi, Genentech, South San Francisco, CA, USA , ) into the tumor, or intraperitoneal injection of both agents.

Techniques: In Vivo, Derivative Assay, Control