Neonatal Lethality in Knockout Mice Expressing the Kinase-Dead Form of the Gefitinib Target GAK Is Caused by Pulmonary Dysfunction

Gefitinib (Iressa) is an inhibitor of the epidermal growth factor receptor (EGFR) that has shown promising activity in the treatment of patients with non-small cell lung cancer (NSCLC). However, adverse side effects of gefitinib treatment, such as respiratory dysfunction, have limited the therapeutic benefit of this targeting strategy. The present results show that this adverse effect can be attributed to the inhibition of the novel gefitinib target GAK (Cyclin G-associated kinase), which is as potently inhibited by the drug as the tyrosine kinase activity of EGFR. Knockout mice expressing the kinase-dead form of GAK (GAK-kd) died within 30 min after birth primarily due to respiratory dysfunction. Immunohistochemical analysis revealed that surfactant protein A (SP-A) was abundant within alveolar spaces in GAK-kd+/+ mice but not in GAK-kd-/- pups. E-cadherin and phosphorylated EGFR signals were also abnormal, suggesting the presence of flat alveolar cells with thin junctions. These results suggest that inhibition of GAK by gefitinib may cause pulmonary alveolar dysfunction, and the present study may help prevent side effects associated with gefitinib therapy in NSCLC patients

Withaferin A Induces Cell Death Selectively in Androgen-Independent Prostate Cancer Cells but Not in Normal Fibroblast Cells

<div><p>Withaferin A (WA), a major bioactive component of the Indian herb <i>Withania somnifera</i>, induces cell death (apoptosis/necrosis) in multiple types of tumor cells, but the molecular mechanism underlying this cytotoxicity remains elusive. We report here that 2 μM WA induced cell death selectively in androgen-insensitive PC-3 and DU-145 prostate adenocarcinoma cells, whereas its toxicity was less severe in androgen-sensitive LNCaP prostate adenocarcinoma cells and normal human fibroblasts (TIG-1 and KD). WA also killed PC-3 cells in spheroid-forming medium. DNA microarray analysis revealed that WA significantly increased mRNA levels of c-Fos and 11 heat-shock proteins (HSPs) in PC-3 and DU-145, but not in LNCaP and TIG-1. Western analysis revealed increased expression of c-Fos and reduced expression of the anti-apoptotic protein c-FLIP(L). Expression of HSPs such as HSPA6 and Hsp70 was conspicuously elevated; however, because siRNA-mediated depletion of HSF-1, an HSP-inducing transcription factor, reduced PC-3 cell viability, it is likely that these heat-shock genes were involved in protecting against cell death. Moreover, WA induced generation of reactive oxygen species (ROS) in PC-3 and DU-145, but not in normal fibroblasts. Immunocytochemistry and immuno-electron microscopy revealed that WA disrupted the vimentin cytoskeleton, possibly inducing the ROS generation, c-Fos expression and c-FLIP(L) suppression. These observations suggest that multiple events followed by disruption of the vimentin cytoskeleton play pivotal roles in WA-mediated cell death.</p></div

Cell viability of TIG-1, LNCaP, DU-145, and PC-3 cells after WA treatment.

<p><b>(A, B)</b> Cell viability was measured at 4, 8, and 24 h after 2 μM (A) or 4 μM (B) WA treatment. NT, non-treated. Green and blue arrows indicate bars for surviving cells, whereas pink and red arrows indicate bars for cells that died under the same conditions. Yellow arrows indicate the samples used for DNA microarray analysis. Bars represent means ± SEM for three independent experiments. Purple arrows indicate significant reductions in cell viabilities (*, <i>P</i> < 0.05; **, <i>P</i> < 0.01).</p

WA induces BAG3-mediated autophagy in PC-3 cells.

<p>A) Observation of EGFP and EGFP-LC3 signals by immunofluorescence microscopy. Bar, 10 μm. (B) Bar graphs showing the percentage of cells containing punctate EGFP-LC3; arrows show that the values gradually increased under the indicated conditions. Data are represented as the means ± SEM of three independent experiments; green arrows indicate statistically significant increases (**, <i>P</i> < 0.01). (C) Western blot analysis to detect LC-3 and GAPDH (loading control) in PC-3 under the indicated conditions. (D) Bar graphs showing cell viability (%) under the indicated conditions. (E) Western blot analysis to detect BAG3, LC-3, and GAPDH (loading control) in PC-3 cells in the presence of the indicated conditions of siBAG3 or siGL2 (negative control). (F) Bar graphs showing cell viability (%) at the indicated conditions. (D, F) Data are represented as means ± SEM of three independent experiments; red arrows indicate statistically significant reductions in cell viability (**, <i>P</i> < 0.01). (A–F) Samples were collected at 4 h after WA treatment. (G) Expression profiles of the autophagy-related proteins BAG3 and LC3 in PC-3 cells at 4, 8, and 24 h after treatment with 4 μM WA. NT: non-treated.</p

c-Fos and FLIP play a role in induction of cell death.

<p>(A, B) Western blot analysis to detect c-Fos (FosB) in TIG-1, LNCaP, DU-145, and PC-3 cells at 4, 8, and 24 h after treatment with 4 μM (A) or 2 μM (B) WA. NT, non-treated. (C) Western blot analysis to detect c-Fos, PARP, FLIP and GAPDH in PC-3 cells at 12 h after 4 μM WA treatment in the presence (+) or absence (-) of three different siRNAs (X–Z) from OriGene. (D) Viability of PC-3 cells after siFos treatment. Data are represented as means ± SEM of three independent experiments; pink arrows indicate a statistically significant reduction in cell number following siFos treatment (**, <i>P</i> < 0.01). (E) Population of apoptotic, necrotic, and live cells distinctly stained with Annexin V–EnzoGold, 7-AAD-Red, and GFP. Data are represented as means ± SEM of three independent experiments; red, blue, and green arrows indicate statistically significant changes (**, <i>P</i> < 0.01). (F) Western blot analysis of c-Fos, PARP, FLIP, and GAPDH in DU-145 at 12 h after 4 μM WA treatment in the presence (+) or absence (-) of siRNAs; siFos or siGL2 (siControl). (G) Viability of DU-145 cells after exogenous overexpression of pcDNA3-FLIP or vector alone in the presence of DMSO (solvent) or 4 μM WA.</p

Expression of HSP genes is up-regulated after WA treatment.

<p>(A) Western blot analysis to detect HSPA6, HSP40, HSPA70, EGR-1, PAR-4, and GAPDH (loading control) in TIG-1, LNCaP, DU-145, and PC-3 cells at 0, 4, 8 and 24 h after 4 μM WA treatment. Arrow indicates the band for bona fide EGR-1. (B) Influence of siHSPA6 expression on PC-3 cell growth. (i) Schematic for this experiment. (ii) Western blot analysis to confirm the knockdown of HSPA6 protein by siHSPA6, relative to cells treated with siGL2 (negative control). (iii) Influence of siHSPA6 and siGL2 on PC-3 cell growth, with or without WA treatment. Arrow highlights the reduction in PC-3 cell growth. (C) Influence of siHSF-1 expression on PC-3 cell growth. (i) Schematics for this experiment. (ii) Western blot analysis to confirm the knockdown of HSP protein by siHSF-1, relative to cells treated with siGL2 (negative control), and to determine whether HSF-1 knockdown affected HSP protein levels by detection of HSPA6, HSP40, HSPA70, and GAPDH in PC-3 cells. (iii) Influence of siHSF-1 and siGL2 on PC-3 cell growth, with or without WA treatment. Arrow highlights the reduction in PC-3 cell growth. Data are represented as means ± SEM of three independent experiments; pink, green, and blue arrows indicate significant decreases in cell viabilities (*, <i>P</i> < 0.05; **, <i>P</i> < 0.01).</p

Sphere-forming PC3 and SAS cells had higher resistance to cisplatin, but not to WA, than adherent cells.

<p>(A) Frequency of spheres larger than 100 μm (i) and typical images (ii) after incubation of TIG-1, LNCaP, PC-3, DU-145, and SAS cells in sphere-formation medium for 10 days. (B) Viability of SAS cells after WA treatment. Bars represent means ± SEM of three independent experiments. Cell viability was measured at 4, 8, and 24 h after treatment with 2 μM (blue bars) or 4 μM (red bars) WA. NT, non-treated. Data are represented as means ± SEM of three independent experiments; red arrows indicate statistically significant reductions in cell viability (**, <i>P</i> < 0.01). (C) Western blot analysis to detect c-Fos, HSPA6, HSP40, HSP70, PARP, and GAPDH (loading control) in SAS cells at 4, 8, and 24 h after treatment with 2 μM or 4 μM WA. NT: non-treated. (D) Comparison of sensitivity to cisplatin between parental cells and spheres of PC-3 (i and ii) and SAS (iii and iv) after 48 h of incubation following treatment with indicated cisplatin concentrations.</p

Protein levels and localizations of vimentin and F-actin following WA treatment.

<p>(A) Western blot analysis to detect vimentin and actin in TIG-1, LNCaP, PC-3, and DU-145 at 4, 8, and 24 h after treatment with 2 μM or 4 μM WA. NT: non-treated. (B) Typical images of TIG-1 (i) and PC-3 (ii) stained with anti-vimentin antibody, phalloidin (for F-actin), and Hoechst 33342 (for DNA) at 2, 4, and 6 (h) after WA treatment. NT: non-treated. Pink and yellow arrows indicated colocalized vimentin and F-actin aggregates. Merged images are shown in the bottom row. Bar, 10 μm. (C) Percentage of all observed cells containing dots of aggregated or non-aggregated vimentin are shown for TIG-1 (i) and PC-3 (ii). Cells harboring over 10 vimentin aggregates were scored as “aggregated”, whereas cells with non-aggregated vimentin were scored as “non-aggregated”. Data are represented as means ± SEM of three independent experiments; asterisks indicate statistically significant changes in cell viability (**, <i>P</i> < 0.01; ***, <i>P</i> < 0.001). (D) Jasplakinoloide (Jsp) did not influence PC-3 cell viability. (i) Schematic of the experimental design. (ii) Bar graphs showing cell viability (%) in the presence (+) or absence (-) of 4 μM WA or Jasp 8 h after the addition of WA. NT: non-treated. Bar graphs represent means ± SEM for three independent experiments. Pink, blue, and green arrows indicate statistically significant reductions in cell viability (*, <i>P</i> < 0.05; **, <i>P</i> < 0.01).</p

Expression profiles of ER stress–related proteins following WA treatment.

<p>(A) Western blot analysis to detect IRE-1α, PERK, pS51-eIF2α, CHOP, caspase 3, PARP, BiP, and GAPDH (loading control) in TIG-1, LNCaP, DU-145, and PC-3 cells at 4, 8, and 24 h after 4 μM WA treatment. NT: non-treated. (B) Schematic representation of the analyzed proteins in the apoptotic pathway induced by ER stress. (C) Typical fluorescence images of TIG-1 (i), LNCaP (ii), PC-3 (iii), and DU-145 (iv) that were treated with 4 μM WA for 24 h and subsequently treated with ROS detection reagents using the Image-iT LIVE Green ROS Detection Kit. Merged images are shown of cytoplasmic ROS signals (green) and nuclear DNA signals stained with Hoechst33342 (blue). Green bar, 100 μm. Black bar, 10 μm.</p