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

Background: Previous studies have shown that the lipid peroxidation product 4-hydroxynonenal (HNE) acts as a cell growth modulator if used at low, phy siological concentrations being strongly cytotoxic at higher concentrations for a number of cells. These effects of HNE also appeared to be mutually d ependent on the effects of serum growth factors. The aim of this investigat ion was to study the concentration-dependent response of human cervical car cinoma (HeLa) cells in vitro with respect to the intracellular uptake of ex ogenous HNE, the cellular energy metabolism, DNA synthesis, overall gene ex pression and susceptibility to apoptosis. Materials and Methods: MTT assay was applied as an index of energy metabolism and the replicative activity w as quantitated by the H-3-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 determ ined with the same antibodies by Western blotting. Differential gene expres sion was studied by differential display RT-PCR while a novel photometric a ssay, denoted Titer-TACS, was used for in situ detection and quantitation o f apoptosis in monolayer cell cultures. Results: A physiological concentrat ion of HNE (1 muM) had hardly any effect on the parameters of the replicati ve activity and the energy metabolism. No morphological changes were observ ed and the number of HNE positive cells was not significantly different whe n compared to the untreated control cells, while most of the aldehyde appea red to be bound to serum proteins (albumin fraction). A ten fold higher con centration (10 muM) was found to be cytostatic. Spindle-shaped cells with a picnotic nucleus were observed occasionally, as well as membrane blebs, wh ich were HNE positive. The number of HNE positive cells was significantly i ncreased compared both to the control cells and cells treated with 1 muM HN E, but in the presence of serum the effects of 10 muM HNE were negated due to its binding to the serum proteins. Finally, 100 muM HNE was cytotoxic fo r the HeLa cells. Most of the cells were picnotic, together with a few spin dle-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 seru m proteins did riot prevent its cytotoxic effects. This concentration of HN E caused acute stress response of the cells resulting in the decreased expr ession of several as yet unidentified genes. The altered pattern of gene ex pression was followed by programmed cell death, i.e. an increased number of apoptotic cells after treatment with low (1 and 10 muM) concentrations of HNE. A rebound effect was observed, i.e. a decrease of apoptotic cells afte r 24 hours followed by an overshooting increase after 48 hours. Conclusions : For HeLa carcinoma cells there appears to be a concentration range of HNE where it does not cause necrosis but preferentially apoptosis. At this con centration. range HNE is cytochemically detectable within the cells as a pr otein conjugate. It is proposed that a possible differential sensitivity of cancer cells and their normal counterparts to the cytostatic activity of H NE should be explored.