Normal Human Embryonic Lung Cells Research Articles

Solubility and biological activity enhancement of the highly lipophilic viridicatins via interaction with cyclodextrins

Viridicatin and its derivatives were found to be the potent strong inhibitors of the tumor necrosis factor-induced replication of many serious diseases. Their extremely low solubility in aqueous medium at a physiological pH compromises bioavailability and leads to poor pharmacokinetic performance and low exposure. Since the pharmacologic activation represents a valuable strategy for treatment of major diseases, we first obtained water-soluble form of viridicatin and viridicatol and realized this by encapsulation in the cyclodextrin cavity. We also investigated self-assembling behavior of resulting complexes and showed that the cyclodextrin complex is more than four times more active against diseased cells than to healthy ones. Measurement of cytotoxic activity performed with normal human embryonic lung cells and human lung carcinoma cell cultures demonstrated that the functional group in the chemical structure of viridicatin derivatives also influences its biologic properties.

Ovothiol isolated from sea urchin oocytes induces autophagy in the Hep-G2 cell line.

Ovothiols are histidine-derived thiols isolated from sea urchin eggs, where they play a key role in the protection of cells toward the oxidative burst associated with fertilization by controlling the cellular redox balance and recycling oxidized glutathione. In this study, we show that treatment of a human liver carcinoma cell line, Hep-G2, with ovothiol A, isolated from Paracentrotus lividus oocytes, results in a decrease of cell proliferation in a dose-dependent manner. The activation of an autophagic process is revealed by phase contrast and fluorescence microscopy, together with the expression of the specific autophagic molecular markers, LC3 II and Beclin-1. The effect of ovothiol is not due to its antioxidant capacity or to hydrogen peroxide generation. The concentration of ovothiol A in the culture media, as monitored by HPLC analysis, decreased by about 24% within 30 min from treatment. The proliferation of normal human embryonic lung cells is not affected by ovothiol A. These results hint at ovothiol as a promising bioactive molecule from marine organisms able to inhibit cell proliferation in cancer cells.

Molecular mechanism of a novel CD59-binding peptide sp22 induced tumor cells apoptosis

Some short peptides discovered by phage display are found to be able to inhibit cancer growth and induce cancer cell apoptosis. In this study, a novel cancer-targeting short peptide which was composed of 22 amino acids (ACHWPWCHGWHSACDLPMHPMC, abbreviated as sp22) and specifically bound to human CD59 was screened from a M13 phage display library so as to counteract tumor immune escape activity. The mechanism of exogenous sp22 peptide in inducing apoptosis of MCF-7 cells was investigated. The results suggested that sp22 could lower CD59 expression level, downregulate Bcl-2 expression, activate Fas and caspase-3, and finally increase apoptotic cell numbers of MCF-7 cells. However, sp22 had no obvious influence on normal human embryonic lung cells. In addition, the effects of endogenous sp22 gene on CD59 expression and NKM cell apoptosis were explored using the recombinant plasmid sp22-PIRES. It showed that sp22 gene was efficiently expressed in transfected NKM cells. Compared with normal NKM cells, NKM cells transfected with sp22 displayed reduced mRNA and protein expression levels of CD59, increased sensitivity to complement-mediated cytolysis, decreased cell survival ratio, changes of the expression of apoptosis associated proteins, increased number of apoptotic cells and the appearance of apoptotic morphology. The results suggested that sp22 protein could bind to CD59 and inhibit the expression of CD59. The cytolytic activity of complement on tumor cells strengthened and apoptosis signal was stepwise transferred which might be a potential way to kill tumor cells.

Determination of Cytotoxicity Attributed to Multiwall Carbon Nanotubes (MWCNT) in Normal Human Embryonic Lung Cell (WI-38) Line

Carbon nanotubes (CNT) possess beneficial physicochemical and mechanical properties; however, despite these advantages there are concerns regarding the adverse effects of CNT on lung and development of diseases, such as lung cancer and mesothelioma. According to fiber characteristics of length and diameter (aspect ratio), fibers with high aspect ratio (10–15 nm diameter and containing two different length distributions of 545 ± 230 and 10451 ± 8422 nm length) are more toxic to lung than low-aspect-ratio fibers (10–15 nm diameter and length of 192 nm). It was thus of interest to investigate the effects of multiwall carbon nanotubes (MWCNT) on the viability of normal human embryonic lung cells (WI-38) using trypan blue dye exclusion, the tetrazolium salt WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) assay, and lactate dehydrogenase (LDH) activity assay. MWCNT produced cell growth inhibition and death at 12.5–200 μg/ml after 24–72 h of incubation. In addition, high-aspect-ratio MWCNT were found to produce higher incidence of cytotoxicity than low-aspect-ratio fibers at 50–200 μg/ml concentration. In the presence of less than 10% trace element content such as iron in MWCNT, the trace element exerted no marked effect on cellular viability. Data indicate that MWCNT inhibited cell proliferation and triggered cell death, and it would appear that the MWCNT fiber characteristics rather than impurities play a predominant role in the observed the cytotoxicity attributed to MWCNT.

A Novel Approach towards Studying Non-Genotoxic Enediynes as Potential Anticancer Therapeutics

A novel uracil-containing enediyne 7 was synthesized by the fusion at N1 and N3 of uracil with an 11-membered cyclic enediyne. Compound 7 was found to be stable against cycloaromatization at 80°C. Thus, it did not cause DNA-damage. Unlike other alkylated uracil derivatives 2–6, highly strained uracil-containing enediyne 7 was reacted with methyl thioglycolate at 25°C to produce uracil (1) and linear enediyne 8. This reactivity toward a sulfhydryl group may play a significant role in the mechanism by which compound 7 directed its cytotoxicity toward tumor cell lines. Tumor cells were found to be more susceptible to enediyne 7 than normal human embryonic lung cells. A combination of 7 with adriamycin or 1-(β-d-arabinofuranosyl)cytosine resulted in synergistic anticancer activity against murine L1210 and P388 leukemias, Sarcoma 180, and human CCRF–CEM lymphoblastic leukemia. After treatment of Molt-4 cells with uracil-containing enediyne 7, light microscope examination demonstrated the presence of cell shrinkage and nuclear segmentation. Treatment of cultured Molt-4 human leukemia cells with enediyne 7 resulted in a time-dependent depletion of glutathione (GSH) whereas the exposure of the cells to the GSH precursor N-acetylcysteine (NAC) resulted in a substantial suppression of this effect. As such, involvement of GSH depletion in the process of apoptosis may explain the mechanism of action of non-genotoxic enediyne 7 against malignant tumor cell lines.

The biosynthesis of prostate-specific antigen in non prostatic cell lines

The biosynthesis of prostate specific antigen (PSA) was studied in human epidermoid carcinoma, (KB) cells and in normal human embryonic lung (WI-38)cells. The prostate carcinoma cell line, PC-35F12 was used as a control. PSA specific antibodies were used to precipitate the immunologically reactive peptides from cell extracts and conditioned media. The immunoprecipitates were analyzed by electrophoresis followed by fluorgraphy. Human PSA is initially synthesized as 32-kDa secretory glycopeptide containing one N-linked oligosaccharide and then processed to a 34-kDa secretory glycoprotein in KB cells. PSA is also expressed in normal human embryonic lung cells, WI-38. These results confirm that PSA expression is not prostate specific, but is also found in some nonprostatic cells. From these results, we conclude that PSA may play an important physiologic role in several tissues.

Design and synthesis of a cephalosporin-retinoic acid prodrug activated by a monoclonal antibody–β-lactamase conjugate

Two novel series of all- trans-β-retinoic acid derivatives were synthesized and found to possess anticancer activity. The first series, cephalosporin 3′-retinoic esters 6 and 7 were, respectively, obtained by the condensation of all- trans-β-retinoic acid ( 2) with cephalosporins 4 and 5. The second series, 7-(retinamido)cephalosporins 11 and 12, were synthesized, respectively, by the condensation of 2 with cephalosporins 9 and 10. These four heretofore undescribed compounds 6, 7, 11, and 12 showed inhibitory activity against murine leukemias (L1210 and P388), sarcoma 180, breast carcinoma (MCF7), and human T-lymphocytes (Molt4/C8 and CEM/0). They also inhibited squamous metaplasia and keratinization in tracheal organ cultures derived from vitamin-A-deficient hamsters. Moreover, cephalosporin 3′-retinoic ester 7 exhibited enhanced activity against keratinization with ED 50=3.91×10 −11 M in the presence of a β-lactamase from Staphylococcus aureus 95. A tumor targeting fusion protein (dsFv3–β-lactamase) was also used in conjunction with cephem-based retinoid 7 and the potency of 7 toward L1210, P388, and MCF7 was found to approach that of the free retinoic acid ( 2). In the presence of dsFv3–β-lactamase, tumor cells were found to be much more susceptible to retinoid 7 than normal human embryonic lung cells. These notions provide a new approach to the use of β-retinoic acid for antitumor therapy.

Expression of gap junction protein Cx43 in cultured human normal and malignant lung cells

Gap junctional intercellular communication-exchange of small molecules and ions between contiguous cells through membranous gap junctional channels-is essential for growth control and tissue homeostasis. This work concerns the functional expression of gap junction protein connexin 43 (Cx43) in normal human lung cells and the changes in lung carcinoma cells. By using Northern blot hybridization analysis and Cx43 immunocytochemical methods, it was observed that cultured normal human embryonic lung cells expressed a high level of Cx43 in both mRNA and protein levels. The Cx43 immunofluorescence was localized at cell membrane regions corresponding to the location of gap junctions. These normal lung cells were competent of intercellular communication function as detected by Lucifer yellow dye transfer. In contrast to normal cells, Cx43 mRNA and protein was not detectable in the carcinoma PG cell line. These tumor cells were defective of intercellular communication function. These results demonstrate that Cx43 is expressed in normal cultured human embryonic lung cells but not in lung tumor cells. The lack of intercellular communication in the lung tumor cell line correlates with dysfunctional intercellular communication. The suggestive role of Cx as a tumor suppersor gene is discussed.

Monoclonal Antibodies to Human Prostate and Bladder Tumor-Associated Antigens

Monoclonal antibodies to human prostate adenocarcinoma membrane antigens were produced by fusion of spleen cells from Balb/C mice immunized against the prostate cancer cells line DU145, and the P3X63/Ag8 mouse myeloma line. The hybrids were screened for antibody production using glutaraldehyde-fixed or live cells in an in vitro binding radioimmunoassay (RIA). Antibody binding specificity was also checked by quantitative adsorption studies. A hybridoma clone (83.21) was isolated that secreted antibodies which preferentially bound to prostate and bladder cancer cells but did not bind to a variety of other normal and malignant human cell lines. This antibody also reacted with a cytomegalovirus-transformed human embryonic lung cell line but not to normal human embryonic lung cells. Conventional and competitive binding studies indicate that the 83.21 monoclonal antibody does not bind to alpha-fetoprotein, carcino-embryonic antigen, prostatic acid phosphatase, HLA, β 2-microglobulin, fibronectin, prostate specific antigen or DR antigens. Freliminary immuno-precipitation data with 125I-labeled, detergent soluble, DU145 membrane proteins and the 83.21 monoclonal antibody suggest that this antibody binds two proteins with molecular weights of 210 and 97 kilodaltons. (Supported by an N.I.H. Biomedical Research Support Grant #218 and a grant from the National Prostatic Cancer Project, NCI CA-26659.)

Absence of Interferon Production in a Newly Established Human Cell Line

A cell line established from human embryonic lung, HEL-R66, was demonstrated to be highly susceptible to herpes simplex virus types 1 and 2, vaccinia virus, Newcastle disease virus (NDV), Japanese encephalitis virus (JEV), western equine encephalitis (WEE) virus, Sindbis virus, vesicular stomatitis virus (VSV), and rabies virus. The maximal yields of NDV, JEV, WEE virus, and rabies virus in this cell line exceeded by 2--4 logs those in control human embryonic lung cells. Inability of this cell line to produce interferon upon treatment with native and UV-irradiated forms of virogenic and lentogenic strains of NDV and with poly I:C was revealed. A refractory state to challenging VSV did not develop in HEL-R66 cells treated with the inducers. Furthermore, pretreatment of HEL-R66 cells with interferon did not potentiate the capacity to produce interferon in response to the addition of poly I:C, whereas the same treatment enhanced the production of interferon in normal human embryonic lung cells.