Antisense oligonucleotides against aldehyde dehydrogenase 3 inhibit hepatoma cell proliferation by affecting MAP kinases

Abstract

The increased activity of enzymes that eliminate anti-tumour drugs or their metabolites is one of the important limiting factors in therapeutic protocols. Among these enzymes, aldehyde dehydrogenase 3 (ALDH3) is considered a mechanism by which tumour cells evade the cytotoxic effects exerted by cyclophosphamide and drugs acting by free radical generation. It is also important in metabolising cytostatic aldehydes derived from lipid peroxidation. Therefore, ALDH3 may play a role in regulating cell proliferation in tumour cells with high activity of this enzyme. We previously reported that antisense oligonucleotides (AS-ODN) against ALDH3 strongly inhibit hepatoma cell growth, suggesting that this effect could be due to the accumulation of cytostatic aldehydes in the cells. In this research we demonstrate that AS-ODN against ALDH3 increase the quantity of malondialdehyde in the cells, and inhibit cell proliferation by affecting the MAPK pathway: a reduction of pRaf-1 and pERK1,2 was observed. These results confirm the importance of aldehydes derived from lipid peroxidation and of ALDH3 in regulating hepatoma proliferation. Moreover, the results indicate the use of AS-ODN against ALDH3 as a possible strategy to reduce growth in tumours overexpressing this enzyme.

Introduction

Resistance to anti-tumour drugs is the most important limiting factor in therapeutic protocols. It may be due to decreased drug cellular influx, or to increased cellular drug removal, or to increased activity of enzymes able to eliminate anti-tumour drugs or their active metabolites [1]. Among these enzymes, aldehyde dehydrogenase 1 (ALDH1; ALDH1A1) and 3 (ALDH3; ALDH3A1) have been correlated with drug resistance of some types of cancer. In particular, their increased expression has been identified as a mechanism by which tumour cells may evade the cytotoxic effects exerted by the alkylating drug cyclophosphamide or its analogues, as well as that of anti-tumour drugs acting by free radical generation, for example doxorubicin [2].

In hepatoma cells, cytosolic ALDH3 activity increases in parallel with the degree of deviation and has been shown to be important in metabolising cytostatic and cytotoxic aldehydes derived from lipid peroxidation [3]. These aldehydes, and in particular the most reactive product 4-hydroxynonenal (4-HNE), are known to cause a number of different effects including inhibition of cell proliferation and induction of apoptosis in both normal and tumour cells [4], [5], [6], [7], [8]. For this reason ALDH isoenzymes are important in regulating both drug resistance and cell proliferation. Their capability to induce drug resistance has been confirmed by transfection studies in both tumour and normal cells. Resistance to oxazaphosphorines has been conferred to human MCF-7 breast cancer cells by transfection with ALDH3 cDNA, and to murine L1210 hematopoietic cell line and human U937 cells by transfection with ALDH1 cDNA [9], [10]. Moreover, overexpression of ALDH1 antisense RNA has been shown to induce resistance to 4-hydroperoxycyclophosphamide, the active metabolite of cyclophosphamide, in A459 lung cancer cells and in K562 leukaemia cells [11]. As regards the importance of ALDH in regulating cell proliferation, we reported elsewhere that the restoration of lipid peroxidation in hepatoma cells reduces cell proliferation and/or decreases viability when ALDH3 activity is down-regulated by aldehydes derived from this restored lipid peroxidation [4]. Moreover, different specific inhibitors of ALDH derived from 4-amino 4-methyl 2-pentyne 1-al have been reported to induce DNA fragmentation in mouse lymphoid BAF₃ cells transfected with the bcl₂ gene, and to induce inhibition of cell proliferation in JM2 rat hepatoma cells [12], [13].

More recently, it has been demonstrated that stable transfection with class 3 ALDH, but not with class 1 ALDH, in Chinese hamster lung fibroblast V79 cells and in murine macrophage RAW 264.7 cells, confers resistance against toxicity of intermediate chain length aldehydes derived from lipid peroxidation. The protection is due to both the prevention of protein damage mediated by 4-HNE-protein adduct formation and by preventing glutathione depletion [14], [15].

With the aim of verifying the correlation between ALDH3 activity and hepatoma cell proliferation, we designed and used two antisense oligonucleotides (AS-ODNs) against ALDH3, and showed that hepatoma cell growth was strongly inhibited as a result of decreased ALDH3 mRNA content and activity of ALDH3 [16]. These initial experiments suggested a possible mechanism, which is investigated in this study.