[show abstract][hide abstract] ABSTRACT: Glucose-regulated protein 78 (GRP78) is an endoplasmic reticulum (ER)-resident chaperone and a major regulator of the unfolded protein response (UPR). Accumulating evidence indicate that GRP78 is overexpressed in many cancer cell lines, and contributes to the invasion and metastasis in many human tumors. Besides, GRP78 upregulation is detected in response to different ER stress-inducing anticancer therapies, including photodynamic therapy (PDT). This study demonstrates that GRP78 mRNA and protein levels are elevated in response to PDT in various cancer cell lines. Stable overexpression of GRP78 confers resistance to PDT substantiating its cytoprotective role. Moreover, GRP78-targeting subtilase cytotoxin catalytic subunit fused with epidermal growth factor (EGF-SubA) sensitizes various cancer cells to Photofrin-mediated PDT. The combination treatment is cytotoxic to apoptosis-competent SW-900 lung cancer cells, as well as to Bax-deficient and apoptosis-resistant DU-145 prostate cancer cells. In these cells, PDT and EGF-SubA cytotoxin induce protein kinase R-like ER kinase and inositol-requiring enzyme 1 branches of UPR and also increase the level of C/EBP (CCAAT/enhancer-binding protein) homologous protein, an ER stress-associated apoptosis-promoting transcription factor. Although some apoptotic events such as disruption of mitochondrial membrane and caspase activation are detected after PDT, there is no phosphatidylserine plasma membrane externalization or DNA fragmentation, suggesting that in DU-145 cells the late apoptotic events are missing. Moreover, in SW-900 cells, EGF-SubA cytotoxin potentiates PDT-mediated cell death but attenuates PDT-induced apoptosis. In addition, the cell death cannot be reversed by caspase inhibitor z-VAD, confirming that apoptosis is not a major cell death mode triggered by the combination therapy. Moreover, no typical features of necrotic or autophagic cell death are recognized. Instead, an extensive cellular vacuolation of ER origin is observed. Altogether, these findings indicate that PDT and GRP78-targeting cytotoxin treatment can efficiently kill cancer cells independent on their apoptotic competence and triggers an atypical, non-apoptotic cell death.
Cell Death & Disease 01/2013; 4:e741. · 6.04 Impact Factor
[show abstract][hide abstract] ABSTRACT: The origin of cardiac lymphatics from venous endothelial cells or from scattered lymphangioblasts has been discussed in the literature. We aimed to establish the stage when lymphatic vessels appear in the developing mouse heart, the location of the first lymphatics, and to define cellular phenotypes of growing lymphatics.
We found that scattered Lyve-1-positive cells located in the subepicardial area of developing heart expressed CD45, CD68, F4/80, or CD11b but not CD31. Prox-1(+)/Lyve-1(+) cellular cords or vessels were found to invade 12.5-13.5-dpc hearts via two routes: from the venous pole, i.e., dorsal atrioventricular sulcus, or on the dorsal atrial surface from mediastinum and from the arterial pole, i.e., along the great arteries. The Prox-1(+)/Lyve-1(+) vessels were located among the Prox-1(+)/Lyve-1(-) cords and among the scattered Prox-1(-)/Lyve-1(+) cells. The Prox-1(+)/Lyve-1(-) cellular cords/tubules dominate initially at the arterial pole whereas Lyve-1(+)/Prox-1(-) cellular cords/tubules dominate initially on the venous pole, i.e., dorsal atrioventricular sulcus. The Lyve-1(+)/CD45(+), Lyve-1(+)/CD11b(+), Lyve-1(+)/F4/80(+) and Lyve-1(+)/CD68(+) cells were subsequently found to be co-opted to the wall of the developing lymphatic vessels while gaining Flk-1.
Lymphatic primordia exhibit different cellular phenotypes and different spatiotemporal pattern on the venous pole as compared with the arterial pole of the heart.
[show abstract][hide abstract] ABSTRACT: Bronchial remodeling is currently known to affect not only patients with asthma, but also COPD patients. Some studies have demonstrated that basement membrane thickening and destruction of the bronchial epithelium are also found in COPD. The aim of the study was to compare the basement membrane thickness (BMT) and epithelial damage in biopsy specimens from patients with asthma and COPD.
The study was performed in 20 subjects with asthma and 12 subjects with COPD, who had not been treated with corticosteroids for at least 3 months before study enrollment. Subjects' characteristics were based on the results of clinical assessment, allergic skin-prick tests, lung function testing, and methacholine bronchial challenge. All subjects underwent bronchoscopy with forceps biopsies of bronchial mucosa. Light-microscope and semi-automatic software were used to measure BMT in hematoxylin-eosin stained sections. Total (denudation) and partial epithelial damage were assessed independently by 2 pathologists.
The mean BMT in subjects with asthma was 12.54 ± 2.8 μm, and only 7.81 ± 2.0 μm in COPD patients (P < .001). Overall percentage of the basement membrane length lined with damaged epithelium was 45 ± 20% in the asthma group and 47 ± 22% in the COPD group (difference not significant). Complete and partial epithelial damage did not differ between the groups.
BMT might be a histopathological parameter helpful in distinguishing asthma and COPD patients, whereas the extent and pattern of epithelial damage is not.
Respiratory care 10/2011; 57(4):557-64. · 2.03 Impact Factor
[show abstract][hide abstract] ABSTRACT: Recent case reports provided alarming signals that treatment with bortezomib might be associated with cardiac events. In all reported cases, patients experiencing cardiac problems were previously or concomitantly treated with other chemotherapeutics including cardiotoxic anthracyclines. Therefore, it is difficult to distinguish which components of the therapeutic regimens contribute to cardiotoxicity. Here, we addressed the influence of bortezomib on cardiac function in rats that were not treated with other drugs. Rats were treated with bortezomib at a dose of 0.2 mg/kg thrice weekly. Echocardiography, histopathology, and electron microscopy were used to evaluate cardiac function and structural changes. Respiration of the rat heart mitochondria was measured polarographically. Cell culture experiments were used to determine the influence of bortezomib on cardiomyocyte survival, contractility, Ca(2+) fluxes, induction of endoplasmic reticulum stress, and autophagy. Our findings indicate that bortezomib treatment leads to left ventricular contractile dysfunction manifested by a significant drop in left ventricle ejection fraction. Dramatic ultrastructural abnormalities of cardiomyocytes, especially within mitochondria, were accompanied by decreased ATP synthesis and decreased cardiomyocyte contractility. Monitoring of cardiac function in bortezomib-treated patients should be implemented to evaluate how frequently cardiotoxicity develops especially in patients with pre-existing cardiac conditions, as well as when using additional cardiotoxic drugs.
American Journal Of Pathology 06/2010; 176(6):2658-68. · 4.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: Photodynamic therapy (PDT) is an approved therapeutic procedure that exerts cytotoxic activity toward tumor cells by inducing production of reactive oxygen species such as singlet oxygen. PDT leads to oxidative damage of cellular macromolecules, including proteins that undergo multiple modifications such as fragmentation, cross-linking, and carbonylation that result in protein unfolding and aggregation. Because the major mechanism for elimination of carbonylated proteins is their degradation by proteasomes, we hypothesized that a combination of PDT with proteasome inhibitors might lead to accumulation of carbonylated proteins in endoplasmic reticulum (ER), aggravated ER stress, and potentiated cytotoxicity toward tumor cells. We observed that Photofrin-mediated PDT leads to robust carbonylation of cellular proteins and induction of unfolded protein response. Pretreatment of tumor cells with three different proteasome inhibitors, including bortezomib, MG132, and PSI, gave increased accumulation of carbonylated and ubiquitinated proteins in PDT-treated cells. Proteasome inhibitors effectively sensitized tumor cells of murine (EMT6 and C-26) as well as human (HeLa) origin to PDT-mediated cytotoxicity. Significant retardation of tumor growth with 60% to 100% complete responses was observed in vivo in two different murine tumor models (EMT6 and C-26) when PDT was combined with either bortezomib or PSI. Altogether, these observations indicate that combination of PDT with proteasome inhibitors leads to potentiated antitumor effects. The results of these studies are of immediate clinical application because bortezomib is a clinically approved drug that undergoes extensive clinical evaluations for the treatment of solid tumors.
Cancer Research 06/2009; 69(10):4235-43. · 8.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Airway remodeling is a characteristic feature of asthma. It is believed that airway remodeling affects lung function and bronchial hyper-responsiveness. Therefore, the relationship between remodeling and lung function is still a matter of extensive research. However, the results of many studies are inconsistent. The aim of the study was to assess the relationship between lung function parameters and basement membrane (BM) thickness in patients with asthma.
Twenty asthma patients were chosen for the study (ten male, ten female, mean age 37 +/- 15 yrs). Ten were newly diagnosed, steroid-naive patients and the other ten were patients known to have asthma who had not been treated with steroids for at least three months. The study group was selected based on the results of: clinical assessment, allergic skin-prick tests, lung function testing and bronchial challenge with methacholine. Nine (45%) patients had chronic mild, nine (45%) had moderate and two (10%) had intermittent asthma. Mean FEV1% pred. was 83 +/- 18, mean FEV1%VC 69 +/- 9, mean FVC% pred. 101 +/- 14. All patients underwent research fiberoptic bronchoscopy with BAL and bronchial mucosal biopsies. Light-microscopic measurements of BM thickness were performed in hematoxylin-eosin stained slides of bronchial wall specimens with semi-automatic software analysis MultiScan Base 08.98.
Mean BM thickness was 12.8 +/- 2.8 microm (range: 8.5-20.7 microm). No significant correlations between BM thickness and FEV1% pred., FEV1%VC, FVC% pred., RV% pred., TLC% pred., Raw (pre- and post-bronchodilator) and PC20 were observed.
In our group of asthma patients, mean BM was significantly thickened. No relationship between BM thickness and lung function tests, including hyper-responsiveness, was found.
Pneumonologia i alergologia polska: organ Polskiego Towarzystwa Ftyzjopneumonologicznego, Polskiego Towarzystwa Alergologicznego, i Instytutu Gruzlicy i Chorob Pluc 02/2009; 77(3):256-63.
[show abstract][hide abstract] ABSTRACT: HeLa cells stably expressing the alpha chain of T-cell receptor (alphaTCR), a model substrate of ER-associated degradation (ERAD), were used to analyze the effects of BiP/Grp78 depletion by the SubAB cytotoxin. SubAB induced XBP1 splicing, followed by JNK phosphorylation, eIF2alpha phosphorylation, upregulation of ATF3/4 and partial ATF6 cleavage. Other markers of ER stress, including elements of ERAD pathway, as well as markers of cytoplasmic stress, were not induced. SubAB treatment decreased absolute levels of alphaTCR, which was caused by inhibition of protein synthesis. At the same time, the half-life of alphaTCR was extended almost fourfold from 70 min to 210 min, suggesting that BiP normally facilitates ERAD. Depletion of p97/VCP partially rescued SubAB-induced depletion of alphaTCR, confirming the role of VCP in ERAD of alphaTCR. It therefore appears that ERAD of alphaTCR is driven by at least two different ATP-ase systems located at two sides of the ER membrane, BiP located on the lumenal side, while p97/VCP on the cytoplasmic side. While SubAB altered cell morphology by inducing cytoplasm vacuolization and accumulation of lipid droplets, caspase activation was partial and subsided after prolonged incubation. Expression of CHOP/GADD153 occurred only after prolonged incubation and was not associated with apoptosis.
The International Journal of Biochemistry & Cell Biology 07/2008; 40(12):2865-79. · 4.15 Impact Factor
[show abstract][hide abstract] ABSTRACT: We previously investigated the biological, non-antibacterial effects of bacteriophage T4 in mammals (binding to cancer cells in vitro and attenuating tumour growth and metastases in vivo); we selected the phage mutant HAP1 that was significantly more effective than T4. In this study we describe a non-sense mutation in the hoc gene that differentiates bacteriophage HAP1 and its parental strain T4. We found no substantial effects of the mutation on the mutant morphology, and its effects on electrophoretic mobility and hydrodynamic size were moderate. Only the high ionic strength of the environment resulted in a size difference of about 10 nm between T4 and HAP1. We compared the antimetastatic activity of the T2 phage, which does not express protein Hoc, with those of T4 and HAP1 (B16 melanoma lung colonies). We found that HAP1 and T2 decreased metastases with equal effect, more strongly than did T4. We also investigated concentrations of T4 and HAP1 in the murine blood, tumour (B16), spleen, liver, or muscle. We found that HAP1 was rapidly cleared from the organism, most probably by the liver. Although HAP1 was previously defined to bind cancer cells more effectively (than T4), its rapid elimination precluded its higher concentration in tumours.
Archives of Microbiology 07/2007; 187(6):489-98. · 1.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: Airway inflammation and remodeling are well recognized features of asthma. Remodeling is usually regarded as a consequence of chronic inflammation, however there are also data suggesting that remodeling is a relatively independent process in asthma. Neither inflammation nor remodeling is a uniform process. Thus the precise relationship between markers of inflammation and different patterns of remodeling are still matter of investigations. The aim of the study was to assess the relationship between total and differential cell count in induced sputum (IS) and BALF, and thickness of the basement membrane (BM) in patients with stable asthma.
18 patients with asthma (M/F 9/9, mean age 36 +/- 15 yrs). Duration of symptoms amounted to 12.7 +/- 11.5 years. Patients who have not been treated with steroids for at least 3 months were enrolled to the study. All patients underwent sputum induction and fiberoptic bronchoscopy with BAL and bronchial biopsies. Total and differential cell counts were measured in induced sputum and BALF. Light-microscopic measurements of BM thickness were performed in hematoxylin-eosin stained slides of bronchial wall specimens with semi-automatic software analysis.
Mean BM thickness was 12.9 +/- 2.8 microm (range: 8.5-20.7 microm). Total sputum cell count was 3.4 +/- 2.7 x 106 cells/ml, whereas in BALF 9.7 +/- 10.2 x 106 cells/ml. There was no correlation between differential cell count in induced sputum and BALF. No significant correlations between BM thickness and total and differential cell count in IS and BALF were observed. There also was no correlation between BM thickness and length of asthma duration or degree of the disease.
There was no relationship between BM thickness and total number of cells nor number of eosinophils in BALF and/or IS.
Pneumonologia i alergologia polska: organ Polskiego Towarzystwa Ftyzjopneumonologicznego, Polskiego Towarzystwa Alergologicznego, i Instytutu Gruzlicy i Chorob Pluc 02/2007; 75(4):363-9.
[show abstract][hide abstract] ABSTRACT: Valosin-containing protein (VCP; p97; cdc48 in yeast) is a hexameric ATPase of the AAA family (ATPases with multiple cellular activities) involved in multiple cellular functions, including degradation of proteins by the ubiquitin (Ub)-proteasome system (UPS). We examined the consequences of the reduction of VCP levels after RNA interference (RNAi) of VCP. A new stringent method of microarray analysis demonstrated that only four transcripts were nonspecifically affected by RNAi, whereas approximately 30 transcripts were affected in response to reduced VCP levels in a sequence-independent manner. These transcripts encoded proteins involved in endoplasmic reticulum (ER) stress, apoptosis, and amino acid starvation. RNAi of VCP promoted the unfolded protein response, without eliciting a cytosolic stress response. RNAi of VCP inhibited the degradation of R-GFP (green fluorescent protein) and Ub-(G76V)-GFP, two cytoplasmic reporter proteins degraded by the UPS, and of alpha chain of the T-cell receptor, an established substrate of the ER-associated degradation (ERAD) pathway. Surprisingly, RNAi of VCP had no detectable effect on the degradation of two other ERAD substrates, alpha1-antitrypsin and deltaCD3. These results indicate that VCP is required for maintenance of normal ER structure and function and mediates the degradation of some proteins via the UPS, but is dispensable for the UPS-dependent degradation of some ERAD substrates.
Molecular Biology of the Cell 12/2006; 17(11):4606-18. · 4.60 Impact Factor
[show abstract][hide abstract] ABSTRACT: ABC transporters pump out from cells a large number of endo- and xenobiotics including signal molecules and toxins; they are molecular markers of stem/progenitor cells as well. Here, we present the study of temporal/spatial patterns of Abcb1 isoforms and Abcg2 transporter expression and efflux activity in pre- and early postimplantation murine embryos. We found in 2-cell embryos abcb1a, abcb1b and abcg2 mRNAs which were believed to be maternally inherited. The expression of abcb1b and abcg2 genes was found in blastocysts and in 7 days postcoitum (dpc) embryos, while in 9dpc embryos beside of abcb1b/abcg2, the abcb1a gene was expressed. The abcb2 mRNA was detectable neither in pre- nor in postimplantation embryos. Moreover, we analysed temporal/spatial patterns of rhodamine 123/Hoechst 33342 efflux, which mirrors the ABC transporter phenotype, from individual cells of pre- and postimplantation murine embryos. The blastomeres of 2-, 4- and 8-cell embryos had efflux-inactive phenotype. Single, efflux-active cells emerged first in the morulae and their number increased in blastocyst inner cell mass. In 6 and 7 dpc embryos, all embryonic cells hold the efflux-active phenotype. Proximal embryonic endoderm of 6-8 dpc embryos contained two sub-domains: one consisted of efflux-active cells and another one of efflux-inactive cells reflecting polarity of an embryo. Between 7 and 8 dpc, at the onset of organogenesis, the vehement surge of efflux-inactive embryonic cells occurred, and their number increased in 9 dpc embryos, which consequently contained few efflux-active cells.
[show abstract][hide abstract] ABSTRACT: Quantitative ultrastructural evaluation of satellite cells (SCs) of rats immobilized for 7, 14, and 36 days after soleus muscle denervation was performed. Alterations in SCs of experimental animals concerned mainly the decrease in the size of cells and their nuclei, in the volume fractions of the nucleus and nucleolus, as well as in the number of ribosome-like structures. They suggest that immobilization which proceeded denervation caused a decline in cell activity. An increase in the volume fraction and number of endosome/lysosome-like structures, suggesting elevated processes of degradation was also observed. The changes occurred mainly in the period between 7 and 14 days after muscle denervation and immobilization. In all groups of experimental animals an increase in number of caveolae-like structures on both, inner (muscle fiber-facing) and outer (basal lamina-facing) sides of SCs was found. Thus, it is likely that SCs of denervated and immobilized rats are affected by signal molecules released by muscle fibers and/or other cell types present in muscle. A tendency in changes in SCs, observed in the present study, was similar to those which we noticed previously in denervated soleus muscle. However, immobilization after denervation aggravated some of the ultrastructural alterations or the changes appeared earlier.
Experimental and Molecular Pathology 03/2005; 78(1):78-85. · 2.13 Impact Factor
[show abstract][hide abstract] ABSTRACT: The aim of this work was to study the influence of hypokinetic conditions on the ovary and corpus luteum of pregnant rats. The rats were kept in hypokinetic conditions for 5 days in the period between the 13th and 18th days of pregnancy. A three-dimensional reconstruction of the ovary and corpora lutea and also a stereological evaluation of the luteal cells and their nuclei were performed using serially cut material. Hypokinesia caused a decrease in the mean volume of the ovary and individual corpus luteum and in the total volume of corpora lutea per ovary in immobilised animals as compared to the control. Moreover, a decrease was observed in the mean number of luteal cells and an increase in the size of these cells, as well as in the mean volume fraction of their nuclei. These results indicate that immobilisation of pregnant rats for 5 days considerably influences the morphology of the corpus luteum and luteal cells.
[show abstract][hide abstract] ABSTRACT: Morphometric analysis of the ultrastructural changes of satellite cells (SCs) of rat soleus muscle 7, 14, and 36 days post denervation was performed. Denervation caused decrease of surface area cross-section of SCs and their nuclei, volume fractions of nucleus and Golgi complex elements and ribosomes number. In contrast, the increase of surface area/volume ratio of SCs and nucleus, volume fraction and number of endosome/lysosome-like structures, and number of caveolae-like structures was noticed. Ultrastructural changes of SCs in denervated muscles strongly suggest decline of cell activity accompanied by increased processes of degradation of material of endo-, and/or egzogenous origin.
Experimental and Molecular Pathology 05/2004; 76(2):166-72. · 2.13 Impact Factor
[show abstract][hide abstract] ABSTRACT: Bacteriophages (phages) as bacterial viruses are generally believed to have no intrinsic tropism for mammalian cells. In this study the interactions between phages and various eukaryotic cells were investigated. Binding of phages to the membranes of cancer and normal blood cells was observed. Moreover, it was shown that the wild-type phage T4 (wtT4) and its substrain HAP1 with enhanced affinity for melanoma cells inhibit markedly and significantly experimental lung metastasis of murine B16 melanoma cells by 47% and 80%, respectively. A possible molecular mechanism of these effects, namely a specific interaction between the Lys-Gly-Asp motif of the phage protein 24 and beta3-integrin receptors on target cells is proposed. It was also shown that anti-beta3 antibodies and synthetic peptides mimicking natural beta3 ligands inhibit the phage binding to cancer cells. This is in line with the well-described beta3 integrin-dependent mechanism of tumor metastasis. It is concluded that the blocking of beta3 integrins by phage preparations results in a significant decrease in tumor invasiveness.