RNA expression induced by cisplatin in an organ of Corti-derived immortalized cell line.
Cisplatin [cis-diamminedichloroplatinum(II)] (CDDP) is an organic compound that is widely used for the treatment of a large number of tumors. Its clinical use is limited by the presence of some undesired side effects, like as oto- and nephro toxicity, whose mechanisms of action are not understood. One of the possible CDDP toxicity mechanism seems to involve the generation of reactive oxygen species (ROS), that can impair morphology and function of hair cells (HC) in the organ of Corti. To test this hypothesis we evaluated the effect of CDDP treatment on RNA steady-state levels of 15,000 genes by microarray analysis, using, as a experimental model, the OC-k3 cell line, obtained from the organ of Corti of transgenic mice and constitutively expressing the large SV40 T antigen. We have found overexpression of several genes related to arachidonate mobilization including phospholipase A2, group IV and V, phospholipase A2 activating protein and lysophospholipase I and III, as well as lipoxygenation like arachidonate 12-lipoxygenase and arachidonate 5-lipoxygenase activating protein. In addition, we found significant transcription of genes regulating cell respiration, including cyt c oxidase, as well as genes involved in xenobiotic detoxification and lipid peroxidation such as cyt P450, and other oxidases including spermine oxidase and monoamine oxidase. As a whole, overexpression of the group of different genes seems to indicate that an oxidative burst could take place during cisplatin administration. We therefore searched for evidences of superoxide anion and hydrogen peroxide by means of electron paramagnetic resonance (EPR) spectroscopy and flow cytometry, but failed to detect them. On the other hand, we found an increase of malondialdehyde (MDA) synthesis and protein carbonylation products, indicating the occurence of lipid peroxidative degradation. When we tested the effectiveness of butylated hydroxytoluene (BHT), dithiothreitol (DTT) and N-acetylcysteine (N-Ac) as cytoprotectants, all of them reduced protein carbonylation to control levels and significantly protected OC-k3 from CDDP-induced cell death, with an higher protection when using the lipophylic antioxidant BHT. The same antioxidants prevented also the onset of protein carbonylation, which extent was decreased to basal levels. These data indicate that CDDP is able to stimulate gene expression up to 12 h after the beginning of the treatment. This increase in gene transcription involves a large number of genes potentially able to increase the level of cell ROS. Consistently, cells survival is improved by cotreatment with antioxidants, in particular lipophilics.
Department of Morphology and Embryology, Human Anatomy Division, Ferrara University Via Fossato di Mortara 66, 44100 Ferrara, Italy. prm@unife.it
Maurizio
M
Previati
Irene
I
Lanzoni
Elisa
E
Corbacella
Sara
S
Magosso
Sarah
S
Giuffrè
Francesca
F
Francioso
Diego
D
Arcelli
Stefano
S
Volinia
Andrea
A
Barbieri
Stavros
S
Hatzopoulos
Silvano
S
Capitani
Alessandro
A
Martini
D000111:Acetylcysteine / Q000494:pharmacology; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D000975:Antioxidants / Q000494:pharmacology; D002084:Butylated Hydroxytoluene / Q000494:pharmacology; D002461:Cell Line, Transformed; D002470:Cell Survival / Q000187:drug effects; D002945:Cisplatin / Q000494:pharmacology*; D019610:Cytoprotection; D004229:Dithiothreitol / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D008315:Malondialdehyde / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D020411:Oligonucleotide Array Sequence Analysis; D009925:Organ of Corti / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism* / Q000502:physiology; D012313:RNA / Q000378:metabolism*; D013997:Time Factors
D000970:Antineoplastic Agents; D000975:Antioxidants; D002084:Butylated Hydroxytoluene; D008315:Malondialdehyde; D012313:RNA; D002945:Cisplatin; D004229:Dithiothreitol; D000111:Acetylcysteine