Naslov
The carcinostatic and proapoptotic potential of 4-Hydroxynonenal in HeLa cells is associated with its conjugation to cellular proteins

Autori
Sović, Andrea ; Borović, Suzana ; Lončarić, Iva ; Kreuzer, Thomas ; Žarković, Kamelija ; Vuković, Tea ; Wäeg, Georg ; Hraščan, Reno ; Wintersteiger, Reinhold ; Klinger, Rainer ; Zurak, Niko ; Schaur, Rudolf Jörg ; Žarković, Neven

Izvornik
Anticancer research (0250-7005) 21 (2001); 1997-2004

Vrsta, podvrsta i kategorija rada
Radovi u časopisima, članak, znanstveni

Ključne riječi
4-hydroxynonenal; oxidative stress; lipid peroxidation; HeLa; growth modulation; serum; PCR; differential display; immunohistochemistry; apoptosis

Sažetak
Previous studies have shown that lipid peroxidation product 4-hydroxynonenal (HNE) acts as a cell growth modulator if used at low, physiological concentrations being strongly cytotoxic at higher concentrations for a number of cells. These effects of HNE also appeared to be mutually dependent on the effects of serum growth factors. The aim of this investigation was to study the concentration-dependent response of human cervical carcinoma (HeLa) cells in vitro with respect to the intracellular uptae of exogenous HNE, the cellular energy metabolism, DNA synthesys, overall gene expression and susceptibility to apoptosis. MTT assay was applied as an index of energy metabolism and the replicative activity was quantified by the 3H-thymidine incorporation assay. The occurence and intracellular distribution was studied with monoclonal antibodies directed against HNE-protein conjugates. Binding of HNE to serum proteins was determined with the same antibodes by Western-blotting. Differential gene expression was studied by differential display RT-PCR while a novel photometric assay, denoted Titer-TACS, was used for in situ detection and quantitation of apoptosis in monolayer cell cultures. A physiological concentration of HNE (1 uM) had hardly any effect on the parameters of the replicative activity and the energy metabolism. No morphological changes were observed and the number of HNE-positive cells was not significantly different compared to the untreated control cells while most of the aldehyde appeared to be bound to serum proteins (albumin fraction). A tenfold higher concentration (10 uM) was found to be cytostatic. Spindle shaped cells with a picnotic nucleus were observed occasionally, as well as membrane blebs, which were HNE-positive. The number of HNE-positive cells was significantly increased compared both to the control cells and cells treated with 1 uM HNE, but in the presence of serum the effects of 10 uM HNE were abolished due to its binding to the serum proteins. Finally, 100 uM HNE was cytotoxic for the HeLa cells. Most of the cells were picnotic, together with a few spindle shaped or oval cells. The staining for HNE was diffuse and strong (90 % of the cells were HNE-positive) while even binding of the aldehyde to serum proteins did not prevent its cytotoxic effects. This concentration of HNE caused acute stress response of the cells resulting in decreased expression of several yet unidentified genes. The altered pattern of gene expression was followed in programmed cell death, i.e. an increased number of apoptotic cells after treatment with low (1 and 10 uM) concentrations of HNE. A rebound effect was observed, i.e. a decrease of apoptotic cells after 24 hrs followed by an overshooting increase after 48 hrs. For HeLa carcinoma cells there appears to be a concentration range of HNE where it does not cause necrosis but preferentially apoptosis. In this concentration range HNE is cytochemically detectable within the cells as protein conjugate. It is proposed that a possible differential sensitivity of cancer cells and their normal counterparts to the cytostatic activity of HNE should be explored.

Izvorni jezik
Engleski

Znanstvena područja
Kemija, Biologija, Temeljne medicinske znanosti

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