Andras Szigeti

Inhibition of cell death by a novel 16.2 kD heat shock protein predominantly via Hsp90 mediated lipid rafts stabilization and Akt activation pathway

AlphaB-crystallin homology, heat stress induction and chaperone activity suggested that a previously encloned gene product is a novel small heat shock protein (Hsp16.2). Suppression of Hsp16.2 by siRNA sensitized cells to hydrogen peroxide or taxol induced cell-death. Over-expressing of Hsp16.2 protected cells against stress stimuli by inhibiting cytochrome c release from the mitochondria, nuclear translocation of AIF and endonuclease G, and caspase 3 activation. Recombinant Hsp16.2 protected mitochondrial membrane potential against calcium induced collapse in vitro indicating that Hsp16.2 stabilizes mitochondrial membrane systems. Hsp16.2 formed self-aggregates and bound to Hsp90. Inhibition of Hsp90 by geldanamycin diminished the cytoprotective effect of Hsp16.2 indicating that this effect was Hsp90-mediated. Hsp16.2 over-expression increased lipid rafts formation as demonstrated by increased cell surface labeling with fluorescent cholera toxin B, and increased Akt phosphorylation. The inhibition of PI-3-kinase—Akt pathway by LY-294002 or wortmannin significantly decreased the protective effect of the Hsp16.2. These data indicate that the over-expression of Hsp16.2 inhibits cell death via the stabilization of mitochondrial membrane system, activation of Hsp90, stabilization of lipid rafts and by the activation of PI-3-kinase—Akt cytoprotective pathway.

Induction of necrotic cell death and mitochondrial permeabilization by heme binding protein 2/SOUL

We found that heme‐binding protein 2/SOUL sensitised NIH3T3 cells to cell death induced by A23187 and etoposide, but it did not affect reactive oxygen species formation. In the presence of sub‐threshold calcium, recombinant SOUL provoked mitochondrial permeability transition (mPT) in vitro that was inhibited by cyclosporine A (CsA). This effect was verified in vivo by monitoring the dissipation of mitochondrial membrane potential. Flow cytometry analysis showed that SOUL promoted necrotic death in A23187 and etoposide treated cells, which effect was prevented by CsA. These data suggest that besides its heme‐binding properties SOUL promotes necrotic cell death by inducing mPT.

Potentiation of paclitaxel-induced apoptosis by galectin-13 overexpression via activation of Ask-1-p38-MAP kinase and JNK/SAPK pathways and suppression of Akt and ERK1/2 activation in U-937 human macrophage cells.

Galectin-13 transcripts have been identified in several normal and malignant tissues, but the physiological function of galectin-13 is still poorly understood. Here, we present evidence for its possible role in promoting cell death in the U-937 human macrophage cell line. Transfection of U-937 human macrophages by a galectin-13 cDNA-containing mammalian expression vector increased the galectin-13 level and sensitized the cells to stress stimuli. Galectin-13 overexpression facilitated paclitaxel-induced cell death and nuclear translocation of apoptosis-inducing factor (AIF) and endonuclease-G without inducing mitochondrial cytochrome-c release or caspase-3 activation. Immunoblot and immunofluorescence data showed that overexpression of galectin-13 induced long-term activation of c-Jun N-terminal kinase (JNK) and p38-mitogen-activated protein kinase (MAPK) pathways, as well as activation of apoptosis signal-regulating kinase-1 (Ask-1) kinase while it suppressed paclitaxel-induced long-term activation of the phosphatidilylositol-3-kinase (PI-3K)-Akt and extracellular signal-regulated kinase (ERK1/2) cytoprotective pathways. In addition, pharmacological inhibition of JNK and p38-MAPK pathways protected the cells from paclitaxel-induced cell death. All this data indicate that galectin-13 overexpression promoted apoptosis presumably by activating the Ask-1 kinase-JNK and p38-MAPK pro-apoptotic pathways and by suppressing the PI-3K-Akt and ERK1/2 cytoprotective pathways.

TIP47 protects mitochondrial membrane integrity and inhibits oxidative‐stress‐induced cell death

We found that overexpression of tail interacting protein of 47 kDa (TIP47), but not its truncated form (t‐TIP47) protected NIH3T3 cells from hydrogen‐peroxide‐induced cell death, prevented the hydrogen‐peroxide‐induced mitochondrial depolarization determined by 5,5′,6,6′‐tetrachloro‐1,1′,3,3′‐tetraethyl‐benzimidazolylcarbocyanine iodide (JC1), while suppression of TIP47 in HeLa cells facilitated oxidative‐stress‐induced cell death. TIP47 was located to the cytoplasm of untreated cells, but some was associated to mitochondria in oxidative stress. Recombinant TIP47, but not t‐TIP47 increased the mitochondrial membrane potential (Δψ), and partially prevented Ca2+ induced depolarization. It is assumed that TIP47 can bind to mitochondria in oxidative stress, and inhibit mitochondria mediated cell death by protecting mitochondrial membrane integrity.

Facilitation of Mitochondrial Outer and Inner Membrane Permeabilization and Cell Death in Oxidative Stress by a Novel Bcl-2 Homology 3 Domain Protein

We identified a sequence homologous to the Bcl-2 homology 3 (BH3) domain of Bcl-2 proteins in SOUL. Tissues expressed the protein to different extents. It was predominantly located in the cytoplasm, although a fraction of SOUL was associated with the mitochondria that increased upon oxidative stress. Recombinant SOUL protein facilitated mitochondrial permeability transition and collapse of mitochondrial membrane potential (MMP) and facilitated the release of proapoptotic mitochondrial intermembrane proteins (PMIP) at low calcium and phosphate concentrations in a cyclosporine A-dependent manner in vitro in isolated mitochondria. Suppression of endogenous SOUL by diced small interfering RNA in HeLa cells increased their viability in oxidative stress. Overexpression of SOUL in NIH3T3 cells promoted hydrogen peroxide-induced cell death and stimulated the release of PMIP but did not enhance caspase-3 activation. Despite the release of PMIP, SOUL facilitated predominantly necrotic cell death, as revealed by annexin V and propidium iodide staining. This necrotic death could be the result of SOUL-facilitated collapse of MMP demonstrated by JC-1 fluorescence. Deletion of the putative BH3 domain sequence prevented all of these effects of SOUL. Suppression of cyclophilin D prevented these effects too, indicating that SOUL facilitated mitochondrial permeability transition in vivo. Overexpression of Bcl-2 and Bcl-xL, which can counteract the mitochondria-permeabilizing effect of BH3 domain proteins, also prevented SOUL-facilitated collapse of MMP and cell death. These data indicate that SOUL can be a novel member of the BH3 domain-only proteins that cannot induce cell death alone but can facilitate both outer and inner mitochondrial membrane permeabilization and predominantly necrotic cell death in oxidative stress.

Correlation between the progressive cytoplasmic expression of a novel small heat shock protein (Hsp16.2) and malignancy in brain tumors

BackgroundSmall heat shock proteins are molecular chaperones that protect proteins against stress-induced aggregation. They have also been found to have anti-apoptotic activity and to play a part in the development of tumors. Recently, we identified a new small heat shock protein, Hsp16.2 which displayed increased expression in neuroectodermal tumors. Our aim was to investigate the expression of Hsp16.2 in different types of brain tumors and to correlate its expression with the histological grade of the tumor.MethodsImmunohistochemistry with a polyclonal antibody to Hsp16.2 was carried out on formalin-fixed, paraffin-wax-embedded sections using the streptavidin-biotin method. 91 samples were examined and their histological grade was defined. According to the intensity of Hsp16.2 immunoreactivity, low (+), moderate (++), high (+++) or none (-) scores were given.Immunoblotting was carried out on 30 samples of brain tumors using SDS-polyacrylamide gel electrophoresis and Western-blotting.ResultsLow grade (grades 1–2) brain tumors displayed low cytoplasmic Hsp16.2 immunoreactivity, grade 3 tumors showed moderate cytoplasmic staining, while high grade (grade 4) tumors exhibited intensive cytoplasmic Hsp16.2 staining. Immunoblotting supported the above mentioned results. Normal brain tissue acted as a negative control for the experiment, since the cytoplasm did not stain for Hsp16.2. There was a positive correlation between the level of Hsp16.2 expression and the level of anaplasia in different malignant tissue samples.ConclusionHsp16.2 expression was directly correlated with the histological grade of brain tumors, therefore Hsp16.2 may have relevance as becoming a possible tumor marker.

Effects of Pituitary Adenylate Cyclase Activating Polypeptide on the Survival and Signal Transduction Pathways in Human Choriocarcinoma Cells

The pituitary adenylate cyclase activating polypeptide (PACAP) has several effects in endocrine and reproductive organs, including the placenta. PACAP is generally known as a survival‐promoting peptide acting on divergent signal transduction pathways. However, its effects on the survival and signaling mechanisms of trophoblast cells are not known. In the present study we found that 1‐h pretreatment with PACAP38 did not significantly influence the survival of JAR cytotrophoblast cells. However, the survival rate of cells exposed to oxidative stress or CoCl2‐induced in vitro hypoxia showed a significant further decrease in PACAP‐treated cells, implying that PACAP sensitizes the cells to these stressors. This was not observed in the case of lipopolysaccharide or ethanol treatment. Western blot data revealed that, in cells exposed to oxidative stress, PACAP treatment decreased phosphorylation of all extracellular signal‐regulated kinase (ERK), phospho‐jun N‐terminal kinase (JNK), protein kinase B, p38 mitogen‐activated protein kinase (MAPK), and phospho‐glycogen synthase kinase (GSK) and the expression of bax. The overall effect seems to be a sensitizing effect in almost all examined pathways when oxidative stress was applied, which may explain the enhancing effect of PACAP on cell death in contrast to most other cell types examined so far. Our data show that the signaling mechanism of PACAP may be different in trophoblast cells to that observed in other cell lines.

TIP47 confers resistance to taxol-induced cell death by preventing the nuclear translocation of AIF and Endonuclease G

Tail-interacting protein (TIP47, also named PP17) has been implicated in lipid droplet metabolism and in the development of late endosomes, to date however, no data about its possible role in regulating cell death processes has been available. Here, we provide evidence for the role of TIP47 in the regulation of mitochondrial membrane stability and cell death. Overexpression of TIP47 protected NIH3T3 cells from taxol-induced cell death, while suppression of TIP47 by siRNA facilitated cell death. TIP47, but not its truncated form, t-TIP47, decreased taxol-induced cell death as determined by propidium iodide and fluorescent Annexin V staining. Recombinant TIP47, but not t-TIP47, partially prevented taxol-induced depolarization of mitochondria in vitro. Overexpression of TIP47, but not its truncated form, prevented the taxol-induced nuclear and cytoplasmic translocation of AIF and Endonuclease G, as well as the taxol-induced depolarization of mitochondria in NIH3T3 cells. Furthermore, overexpression of TIP47 facilitated Bcl-2 expression and suppressed Bax expression in taxol-treated cells. These data show that besides its previously known functions, TIP47 is involved in the regulation of mitochondria-related cell death by directly stabilizing the mitochondrial membrane system and by favorably affecting the expression of Bcl-2 homologues. Since TIP47 is overexpressed in certain tumors, it is possible that TIP47 contributes to the development of cytostatic resistance.

Chemo-radiotherapy in locally advanced squamous cell oesophageal cancer--are upper third tumours more responsive?

Before neoadjuvant therapy was widely applied, the prognosis of oesophageal cancer had been considered dependent on the location of the tumor, i.e. upper third cancers had had the worst prognosis. The aim of this retrolective study was to prove the efficiency of the neoadjuvant treatment, and to compare the response of esophageal cancer in different locations. Between January 1998 and September 2005, 102 patients with locally advanced squamous cell oesophageal cancer received preoperative chemo-radiotherapy. In 40 cases the tumor was located in the upper third and in 62 cases in the middle third of the oesophagus. After a four-week-long treatment free period restaging was carried out and patients considered resectable were submitted to surgery. From 40 patients with upper third oesophageal cancer 28 underwent oesophageal resection or pharyngo-laryngectomy. Thiry-five percent a complete histopathological remission was observed. From 62 patients with middle third oesophageal cancer 43 underwent oesophageal resection. Histological examination of the resected specimens documented complete response only in three patients. The median survival and the R0 resection rate were similar in the two groups. Although the resection rate, perioperative morbidity, mortality and the median survival were similar in the two groups, a significantly higher rate of complete response (p < 0,05) was observed in patients with upper third oesophageal cancer compared to patients with middle third oesophageal cancer. It seems that upper third oesophageal cancer has superior sensitivity to multimodal treatment therefore our results may support that upper third location is not an unfavorable prognostic factor any more.

Conkiss: conformal kidneys sparing 3D noncoplanar radiotherapy treatment for pancreatic cancer as an alternative to IMRT.

When treating pancreatic cancer using standard (ST) 3D conformal radiotherapy (3D-CRT) beam arrangements, the kidneys often receive a higher dose than their probable tolerance limit. Our aim was to elaborate a new planning method that--similarly to IMRT--effectively spares the kidneys without compromising the target coverage. Conformal kidneys sparing (CONKISS) 5-field, noncoplanar plans were compared with ST plans for 23 consecutive patients retrospectively. Optimal beam arrangements were used consisting of a left- and right-wedged beam-pair and an anteroposterior beam inclined in the caudal direction. The wedge direction determination (WEDDE) algorithm was developed to adjust the adequate direction of wedges. The aimed organs at risk (OARs) mean dose limits were: kidney <12 Gy, liver <25 Gy, small bowels <30 Gy, and spinal cord maximum <45 Gy. Conformity and homogeneity indexes with z-test were used to evaluate and compare the different planning approaches. The mean dose to the kidneys decreased significantly (p < 0.05): left kidney 7.7 vs. 10.7 Gy, right kidney 9.1 vs. 11.7 Gy. Meanwhile the mean dose to the liver increased significantly (18.1 vs. 15.0 Gy). The changes in the conformity, homogeneity, and in the doses to other OARs were not significant. The CONKISS method balances the load among the OARs and significantly reduces the dose to the kidneys, without any significant change in the conformity and homogeneity. Using 3D-CRT the CONKISS method can be a smart alternative to IMRT to enhance the possibility of dose escalation.