MSLN Gene Silencing Has an Anti-Malignant Effect on Cell Lines Overexpressing Mesothelin Deriving from Malignant Pleural Mesothelioma

Genes involved in the carcinogenetic mechanisms underlying malignant pleural mesothelioma (MPM) are still poorly characterized. So far, mesothelin (MSLN) has aroused the most interest. It encodes for a membrane glycoprotein, frequently over-expressed in various malignancies such as MPM, and ovarian and pancreatic cancers. It has been proposed as a diagnostic and immunotherapeutic target with promising results. However, an alternative therapeutic approach seems to rise, whereby synthetic molecules, such as antisense oligonucleotides, could be used to inhibit MSLN activity. To date, such a gene-level inhibition has been attempted in two studies only, both on pancreatic and ovarian carcinoma cell lines, with the use of silencing RNA approaches. With regard to MPM, only one cell line (H2373) has been employed to study the effects of MSLN depletion. Indeed, the knowledge on the role of MSLN in MPM needs expanding. Accordingly, we investigated the expression of MSLN in a panel of three MPM cell lines, i.e. NCI-H28, Mero-14, and IstMes2; one non-MPM cell line was used as reference (Met5A). MSLN knock-down experiments on MSLN-overexpressing cells were also performed through silencing RNA (siRNA) to verify whether previous findings could be generalized to a different set of cell cultures. In agreement with previous studies, transient MSLN-silencing caused decreased proliferation rate and reduced invasive capacity and sphere formation in MSLN-overexpressing Mero-14 cells. Moreover, MSLN-siRNA combined with cisplatin, triggered a marked increase in apoptosis and a decrease in proliferation as compared to cells treated with each agent alone, thereby suggesting a sensitizing effect of siRNA towards cisplatin. In summary, our findings confirm that MSLN should be considered a key molecular target for novel gene-based targeted therapies of cancer.     

Induction of hsp 72/73 by herbimycin A, an inhibitor of transformation by tyrosine kinase oncogenes

Herbimycin A, which has been known to inactivate and degrade p60v-src tyrosine kinase, induced an elevated synthesis of a protein with a molecular size of 70 kDa in A431 human epidermoid carcinoma cells. This protein showed the same migration distance on SDS-polyacrylamide gel electrophoresis as that of the protein induced in the cells by heat shock treatment, and this 70-kDa protein was identified as a member of the heat shock protein 70 family (hsp70) through immunoprecipitation with anti-hsp72/73 antibody and partial digestion with V8 protease. The induced level of the 70-kDa protein was dependent on the length of period and the concentration of herbimycin A treatment. Cellular fractionation and indirect immunofluorescence analyses revealed that the 70-kDa protein induced by herbimycin A was localized in the cytoplasm, in contrast to the nuclear distribution of hsp70 induced by heat treatment. Induction of hsp70 by herbimycin A was also observed in several other cells, including HeLa S3 cells, chicken embryo fibroblasts, NIH3T3 cells, and Rous sarcoma virus-transformed NIH3T3 cells.     

Dexamethasone suppressible hypergonadotropism in an adolescent patient with Prader-Willi syndrome

We report an adolescent patient with Prader-Willi syndrome accompanying suppressible hypergonadotropism. The subject is an 18-year-old female. She was obese (body mass index: 35.7) and hypomyotonic with mental retardation. On endocrinological examination, a high serum LH concentration and hyperresponsiveness of luteinizing hormone (LH) to intravenously administered LH-Releasing Hormone (LH-RH) were observed, while the basal follicle stimulating hormone level was within the normal range. In addition, serum dehydroxyepiandrosterone sulfate (DHEA-S) was also increased. Following 2 mg dexamethasone administration for 7 days, serum LH and DHEA-S were almost normalized and hyperresponse of LH to LH-RH completely disappeared. The present study provides evidence that altered responsiveness to adrenal steroid may be involved in the establishment of hypergonadotropinism in an adolescent patient with Prader-Willi syndrome.     

Interactions of organic calcium channel antagonists with calcium channels in single frog atrial cells

Inhibition of whole-cell calcium currents in enzymatically dispersed frog atrial myocytes by D-600, diltiazem, and nifedipine was studied using a single-micropipette voltage-clamp technique. The objective of these experiments was to test the applicability of a modulated-receptor hypothesis similar to that proposed for local anesthetic interactions with sodium channels to account for the tonic and frequency-dependent interactions of these organic compounds with myocardial calcium channels. Data consistent with such a hypothesis include: (a) prominent use-dependent block of iCa by D-600 and diltiazem, which are predominantly charged at physiological pH; (b) iCa block by an externally applied, permanently charged dihydropyridine derivative is greatly attenuated; (c) all three antagonists produce large negative shifts in the voltage dependence of iCa availability; (d) block of iCa by these compounds is state-dependent; (e) reactivation of iCa in the presence of all three antagonists is biexponential, which suggests that drug-free channels recover with a normal time course and drug-bound channels recover more slowly; and (f) the kinetics of the drug-induced slow iCa recovery process may be determined largely by factors such as size and molecular weight, in addition to lipid solubility of the compounds. Experiments in which the pH was modified, however, reveal some important differences for the interaction of organic calcium antagonists with myocardial calcium channels. Acidification, in addition to changing the proportion of charged and neutral antagonist in solution, was found to selectively antagonize tonic inhibition of iCa by diltiazem and nifedipine, without changing the kinetics of the drug-induced slow iCa reactivation process. It is concluded that two distinct receptor sites may be involved in block of iCa by some of these compounds: a proton-accessible site and a proton-inaccessible site.     

Antitumor polysaccharides from P. ostreatus (Fr.) Quél.: isolation and structure of a beta-glucan

We isolated an antitumor glucan (HA beta-glucan) from the neutral polysaccharide fraction (A3) of a hot-water extract of the edible mushroom P. ostreatus (Fr.) Quél. Purification was accomplished by extractions with 20% sodium chloride solution saturated with thymol and by precipitations with ethanol from dimethyl sulfoxide solution. The glucan showed marked antitumor activity at a dose of 0.1 mg/kg. It is a highly branched (1----3)-beta-glucan having an average structure represented by a pentasaccharide segment consisting of one nonreducing terminal, one 3,6-di-O-substituted, and three 3-mono-O-substituted beta-D-glucopyranosyl residues. This structure was confirmed by examining 13C-n.m.r. spectra taken at 75.46 MHz.     

Differential expression of apoptosis-related Fas antigen on lymphocyte subpopulations in human peripheral blood.

The Fas Ag is a newly defined cell-surface molecule that may mediate apoptosis. The antibody against Fas Ag can induce the apoptotic cell death in cell lines expressing this Ag. PBL subpopulations at various ages were here examined for Fas expression by two-or three-color flow-cytometric analyses using anti-Fas mAb. It was found that Fas Ag was appreciably detected on a proportion of T and B cells, whereas its expression was absent for NK cells. For CD4+ and CD8+ T cells, Fas Ag was expressed preferentially on CD45RO+ (memory or previously activated) populations, but not on CD45RO- naive ones. TCR-gamma/delta+ T cells, especially their CD45RO+ subsets, also expressed Fas Ag. Expectably, neonatal T cell subpopulations, most of which had the naive (CD45RO-) phenotype, expressed little Fas Ag. Fas-expressing B cells dominated in surface(s) IgD- populations, but neonatal B cells as well as adult sIgD+ B cells had little Fas Ag. The Fas Ag was inducible after in vitro mitogenic stimulation of naive T and B cells from neonatal blood. These observations suggested that expression of Fas Ag on T and B cells in the peripheral blood might reflect their in vivo Ag-activated status. In contrast to Fas-expressing cultured cell lines, however, viability of in vitro stimulated T and B cells as well as freshly isolated CD45RO+ T cells was not significantly changed after the treatment with anti-Fas mAb, indicating that additional cellular conditions to Fas expression might be required for anti-Fas-induced cell death.     

Aggregation of bacteriochlorophyll c homologs to dimers,tetramers, and polymers in water-saturated carbon tetrachloride

Three homologs of BChl c, 2-(R)-(1-hydroxyethyl)-4-n-propyl-5-ethyl-farnesyl BChl c (PEF-BChl c), 2-(R)-(1-hydroxyethyl)-4-ethyl-5-ethyl-farnesyl BChl c (EEF-BChl c), and 2-(S)-(1-hydroxyethyl)-4-isobutyl-5-methyl/ethyl-farnesyl BChl c (iBM/EF-BChl c), formed aggregates in water-saturated carbon tetrachloride (H₂O-satd CCl₄). The water content was about 100 times higher than that of the dried CCl₄ previously used. Absorption spectra were recorded for 8 concentrations for the three homologs of BChl c and were deconvoluted in terms of standard spectra of monomer, dimer, tetramer and polymer (747-nm aggregate, Olson and Pedersen (1990) Photosynthe Res 25: 25). PEF- and EEF-BChl c formed dimers (680 nm maximum) and tetramers (705–710 nm maximum), but iBM/EF-BChl c formed polymers. Inhibition of dimer formation by water faciliated the study of the initial stages of the polymerization of BChl c. When the logarithm of polymer concentration was plotted versus the logarithm of the monomer concentration for iBM/EF-BChl c, the initial slope was 30±10 and indicated the cooperation of 20–40 BChl c molecules to form a polymer from a seed. Circular dichroism spectra of the polymers with positive and negative bands at 743 and 760 nm, respectively, were similar to those for chlorosomes (Brune et al. (1990) Photosynth Res 24: 253).Abbreviations BChl bacteriochlorophyll - CD circular dichroism - EEF 4-ethyl-5-ethyl farnesyl - iBM/EF 4-isobutyl-5-methyl/ethyl farnesyl - H₂O-satd CCl₄ water saturated carbon tetrachloride - PEF 4-n-propyl-5-ethyl farnesyl