Loss of soluble N-ethylmaleimide-sensitive factor attachment protein α (αSNAP) induces epithelial cell apoptosis via down-regulation of Bcl-2 expression and disruption of the Golgi.

Intracellular trafficking represents a key mechanism that regulates cell fate by participating in either prodeath or prosurvival signaling. Soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein α (αSNAP) is a well known component of vesicle trafficking machinery that mediates intermembrane fusion. αSNAP increases cell resistance to cytotoxic stimuli, although mechanisms of its prosurvival function are poorly understood. In this study, we found that either siRNA-mediated knockdown of αSNAP or expression of its dominant negative mutant induced epithelial cell apoptosis. Apoptosis was not caused by activation of the major prodeath regulators Bax and p53 and was independent of a key αSNAP binding partner, NSF. Instead, death of αSNAP-depleted cells was accompanied by down-regulation of the antiapoptotic Bcl-2 protein; it was mimicked by inhibition and attenuated by overexpression of Bcl-2. Knockdown of αSNAP resulted in impairment of Golgi to endoplasmic reticulum (ER) trafficking and fragmentation of the Golgi. Moreover, pharmacological disruption of ER-Golgi transport by brefeldin A and eeyarestatin 1 or siRNA-mediated depletion of an ER/Golgi-associated p97 ATPase recapitulated the effects of αSNAP inhibition by decreasing Bcl-2 level and triggering apoptosis. These results reveal a novel role for αSNAP in promoting epithelial cell survival by unique mechanisms involving regulation of Bcl-2 expression and Golgi biogenesis.

PPARgamma Ligand as a Promising Candidate for Colorectal Cancer Chemoprevention: A Pilot Study.

Activating synthetic ligands for peroxisome proliferator-activated receptor gamma (PPARgamma), such as pioglitazone, are commonly used to treat persons with diabetes mellitus with improvement of insulin resistance. Several reports have clearly demonstrated that PPARgamma ligands could inhibit colorectal cancer cell growth and induce apoptosis. Meanwhile, aberrant crypt foci (ACF) have come to be established as a biomarker of the risk of CRC in azoxymethane-treated mice and rats. In humans, ACF can be detected using magnifying colonoscopy. Previously, CRC and adenoma were used as a target for chemopreventive agents, but it needs a long time to evaluate, however, ACF can be a surrogate marker of CRC even for a brief period. In this clinical study, we investigated the chemopreventive effect of pioglitazone on the development of human ACF as a surrogate marker of CRC. Twenty-nine patients were divided into two groups, 20 were in the endoscopically normal control group and 9 were in the pioglitazone (15 mg/day) group, and ACF and adenoma were examined before and after 1-month treatment. The number of ACF was significantly decreased (5.8 +/- 1.1 to 3.3 +/- 2.3) after 1 month of pioglitazone treatment, however, there was no significant change in the number of crypts/ACF or in the number and size of adenomas. Pioglitazone may have a clinical application as a cancer-preventive drug. This investigation is just a pilot study, therefore, further clinical studies are needed to show that the PPARgamma ligand may be a promising candidate as a chemopreventive agent for colorectal carcinogenesis.

Mitigation effect of an FGF-2 peptide on acute gastrointestinal syndrome after high-dose ionizing radiation.

PURPOSE: Acute gastrointestinal syndrome (AGS) resulting from ionizing radiation causes death within 7 days. Currently, no satisfactory agent exists for mitigation of AGS. A peptide derived from the receptor binding domain of fibroblast growth factor 2 (FGF-P) was synthesized and its mitigation effect on AGS was examined. METHODS AND MATERIALS: A subtotal body irradiation (sub-TBI) model was created to induce gastrointestinal (GI) death while avoiding bone marrow death. After 10.5 to 16 Gy sub-TBI, mice received an intramuscular injection of FGF-P (10 mg/kg/day) or saline (0.2 ml/day) for 5 days; survival (frequency and duration) was measured. Crypt cells and their proliferation were assessed by hematoxylin, eosin, and BrdU staining. In addition, GI hemoccult score, stool formation, and plasma levels of endotoxin, insulin, amylase, interleukin (IL)-6, keratinocyte-derived chemokine (KC) monocyte chemoattractant protein 1 (MCP-1) and tumor necrosis factor (TNF)-alpha were evaluated. RESULTS: Treatment with FGF-P rescued a significant fraction of four strains of mice (33-50%) exposed to a lethal dose of sub-TBI. Use of FGF-P improved crypt survival and repopulation and partially preserved or restored GI function. Furthermore, whereas sub-TBI increased plasma endotoxin levels and several pro-inflammation cytokines (IL-6, KC, MCP-1, and TNF-alpha), FGF-P reduced these adverse responses. CONCLUSIONS: The study data support pursuing FGF-P as a mitigator for AGS.

The microtubule-targeting agent T0070907 induces proteasomal degradation of tubulin.

Current microtubule-targeting agents interfere with the regulated assembly of microtubules from alpha/beta tubulin heterodimers but do not markedly alter tubulin levels. Previously, we showed that the compound T0070907 interferes with microtubule function by reversibly decreasing alpha and beta tubulin protein levels by more than 50% in multiple CRC cell lines. Since tubulin levels are generally relatively stable, and cells lack regulatory networks to respond to decreased tubulin levels by increasing synthesis, our result suggested the possibility of cancer therapies that act directly on tubulin homeostasis. The aim of this study was to determine whether T0070907 caused tubulin loss by increasing the degradation rate, and determine the proteases responsible for any increased degradation. T0070907 increased tubulin degradation rates in HT-29 cells. The proteasomal inhibitors MG132, epoxomicin, lactacystin, and ALLN suppressed T0070907-mediated tubulin loss, although epoxomicin and lactacystin were less effective than MG132, even at concentrations that completely inhibited TNFalpha-induced IkappaBalpha degradation. Inhibitors of lysosomal, aggresomal, and calpain-mediated degradation, as well as the caspase inhibitor zVAD-fmk had no effect on tubulin loss, and the cathepsin and calpain inhibitor E64d was unable to increase epoxomicin's ability to suppress tubulin loss. We conclude that T0070907-induced tubulin degradation proceeds through a proteasome-dependent pathway.

PPARgamma Inhibitors as Novel Tubulin-Targeting Agents.

The microtubule-targeting agents (MTAs) are a very successful class of cancer drugs with therapeutic benefits in both hematopoietic and solid tumors. However, resistance to these drugs is a significant problem. Current MTAs bind to microtubules, and/or to their constituent tubulin heterodimers, and affect microtubule polymerization and dynamics. The PPARgamma inhibitor T0070907 can reduce tubulin levels in colorectal cancer cell lines and suppress tumor growth in a murine xenograft model. T0070907 does not alter microtubule polymerization in vitro, and does not appear to work by triggering modulation of tubulin RNA levels subsequent to decreased polymerization. This observation suggests the possible development of antimicrotubule drugs that work by a novel mechanism, and implies the presence of cancer therapeutic targets that have not yet been exploited. This review summarizes what is known about PPARgamma inhibitors and cancer cell death, with emphasis on the tubulin phenotype and PPAR-dependence, and identifies potential mechanisms of action.

PPAR-gamma inhibitors as novel tubulin-targeting agents.

The microtubule-targeting agents are one of the most successful classes of cancer therapeutics. All known antimicrotubule drugs bind to microtubules, or to their constituent tubulin heterodimers, and affect microtubule polymerization and dynamics. Recently, PPAR-gamma inhibitors were shown to reduce tubulin levels without affecting the polymerization of tubulin in vitro. This observation suggests the possible development of antimicrotubule drugs that target tubulin itself, rather than the equilibrium between tubulin and microtubules.

PPARgamma inhibitors reduce tubulin protein levels by a PPARgamma, PPARdelta and proteasome-independent mechanism, resulting in cell cycle arrest, apoptosis and reduced metastasis of colorectal carcinoma cells.

The nuclear transcription factor peroxisome proliferator-activated receptor-gamma (PPARgamma) has been identified as an important therapeutic target in murine models of colorectal cancer (CRC). To examine whether PPARgamma inhibition has therapeutic effects in late-stage CRC, the effects of PPARgamma inhibitors on CRC cell survival were examined in CRC cell lines and a murine CRC model. Low doses (0.1-1 microM) of PPARgamma inhibitors (T0070907, GW9662 and BADGE) did not affect cell survival, while higher doses (10-100 microM) of all 3 PPARgamma inhibitors caused caspase-dependent apoptosis in HT-29, Caco-2 and LoVo CRC cell lines. Apoptosis was preceded by altered cell morphology, and this alteration was not prevented by caspase inhibition. PPARgamma inhibitors also caused dual G and M cell cycle arrest, which was not required for apoptosis or for morphologic alterations. Furthermore, PPARgamma inhibitors triggered loss of the microtubule network. Notably, unlike other standard antimicrotubule agents, PPARgamma inhibitors caused microtubule loss by regulating tubulin post-transcriptionally rather than by altering microtubule polymerization or dynamics. Proteasome inhibition by epoxomicin was unable to prevent tubulin loss. siRNA-mediated reduction of PPARgamma and PPARdelta proteins did not replicate the effects of PPARgamma inhibitors or interfere with the inhibitors' effects on apoptosis, cell cycle or tubulin. PPARgamma inhibitors also reduced CRC cell migration and invasion in assays in vitro and reduced both the number and size of metastases in a HT-29/SCID xenograft metastatic model of CRC. These results suggest that PPARgamma inhibitors are a novel potential antimicrotubule therapy for CRC that acts by directly reducing microtubule precursors.

Peroxisome proliferator-activated receptor gamma inhibition prevents adhesion to the extracellular matrix and induces anoikis in hepatocellular carcinoma cells.

Activation of the nuclear transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma) inhibits growth and survival of hepatocellular carcinoma (HCC) cell lines. To further investigate the function of PPARgamma in HCC, PPARgamma expression patterns in primary tumors were examined, and the responses of two HCC cell lines to PPARgamma activation and inhibition were compared. PPARgamma expression was increased in HCC and benign-appearing peritumoral hepatocytes compared with remote benign hepatocytes. Both compound PPARgamma inhibitors and PPARgamma small interfering RNAs prevented HCC cell lines from adhering to the extracellular matrix. Loss of adhesion was followed by caspase-dependent apoptosis (anoikis). PPARgamma inhibitors had no effect on initial beta1 integrin-mediated adhesion, or on total focal adhesion kinase levels but did reduce focal adhesion kinase phosphorylation. The PPARgamma inhibitor T0070907 was significantly more efficient at causing cancer cell death than the activators troglitazone and rosiglitazone. T0070907 caused cell death by reducing adhesion and inducing anoikis, whereas the activators had no direct effect on adhesion and caused cell death at much higher concentrations. In conclusion, PPARgamma overexpression is present in HCC. Inhibition of PPARgamma function causes HCC cell death by preventing adhesion and inducing anoikis-mediated apoptosis. PPARgamma inhibitors represent a potential novel treatment approach to HCC.

Intestinal antiinflammatory effects of thiazolidenedione peroxisome proliferator-activated receptor-gamma ligands on T helper type 1 chemokine regulation include nontranscriptional control mechanisms.

Crohn's disease is associated with an excessive T helper (TH) type 1 inflammatory immune response. Reducing the influx of disease-associated CD4+ TH1 cells into the inflamed intestine is likely to be beneficial in preventing a disease flare-up and even possibly in reducing the effect of acute disease. Thiazolidenedione (TZD) ligands, which activate peroxisome proliferator-activated receptor-gamma (PPARgamma), have been shown to reduce TH1 inflammation in murine models of colitis, primarily in a preventative fashion. To determine whether PPARgamma ligands reduce this inflammation in part by reducing TH1 chemoattractant levels in vivo, the TZD pioglitazone was tested for its effects on a TH1 chemokine (CXCL10) in 2 models of colitis (i.e., dextran sodium sulfate and 2,4,6-dinitrobenzene sulfonic acid-mediated colitis). In both models, CXCL10 levels were significantly reduced by pioglitazone. Because TZDs can affect gene expression either directly, by regulating the binding of PPARgamma to consensus promoter elements, or indirectly, by modulating other signaling pathways that can affect gene transcription, the regulation of CXCL10 by TZDs was investigated in vitro in both HT-29 colon epithelial cells and THP-1 monocyte/macrophage cells. TZDs significantly reduced CXCL10 protein levels from activated HT-29 cells and THP-1-derived macrophages in a dose-dependent manner at nanomolar concentrations. However, TZDs did not affect messenger RNA levels or nuclear factor-kappaB activation at these concentrations in these cells. These findings imply the existence of a novel posttranscriptional regulatory antiinflammatory mechanism by TZDs that is not associated with reductions in nuclear factor-kappaB activation.

Compartmentalized Eph receptor and ephrin expression in the thymus.

The maturation of T cells is an intricate process involving the interaction of developing thymocytes with discrete microenvironments within the thymus. Numerous studies have indicated that distinct thymic compartments provide signals required for each stage of thymocyte maturation. In this study we performed a comprehensive analysis of the expression patterns of Eph-A receptors and ephrins-A in the thymus using in situ hybridization and reverse transcription-polymerase chain reaction, and show that expression of these molecules is highly compartmentalized. Based on these expression patterns and the known mechanisms of action of Eph receptor/ephrin interactions in other organs, these data suggest that differential Eph receptor expression on discrete subsets of thymic stromal cells may be important in establishing compartment boundaries and preventing intermingling of stromal cell subtypes. Further, together with chemotactic signals such as those provided by chemokines, regulated Eph receptor/ephrin expression on thymocytes may play a role in thymocyte migration.

Compartmentalized Eph receptor and ephrin expression in the thymus.

The maturation of T cells is an intricate process involving the interaction of developing thymocytes with discrete microenvironments within the thymus. Numerous studies have indicated that distinct thymic compartments provide signals required for each stage of thymocyte maturation. In this study we performed a comprehensive analysis of the expression patterns of Eph-A receptors and ephrins-A in the thymus using in situ hybridization and reverse transcription-polymerase chain reaction, and show that expression of these molecules is highly compartmentalized. Based on these expression patterns and the known mechanisms of action of Eph receptor/ephrin interactions in other organs, these data suggest that differential Eph receptor expression on discrete subsets of thymic stromal cells may be important in establishing compartment boundaries and preventing intermingling of stromal cell subtypes. Further, together with chemotactic signals such as those provided by chemokines, regulated Eph receptor/ephrin expression on thymocytes may play a role in thymocyte migration.