Rapid and extensive lethal action of clofibrate on hepatoma cells in vitro

Abstract

Clofibrate, for a long time in use as a hypolipidemic drug, is a well known peroxisomal proliferator (PP) and hepatocarcinogen in rodents. We show here that in vitro 1 mM clofibrate induces a rapid and massive death of rat AH-130 hepatoma cells. Cell death was prominent already after 4 h of treatment, with a characteristic ;apoptotic' pattern by conventional microscopy. This was further supported by the pronounced chromatin condensation detectable on 4',6-diamine-2'-phenylindole dihydrochloride (DAPI) staining, the clearcut internucleosomal DNA fragmentation on agarose-gel electrophoresis (ladder pattern), and the accumulation of markedly hypochromic cells observed in flow cytometric DNA histograms. Consistently with the apoptotic features of the process, some parameters commonly used to detect cell death, such as plasma membrane permeabilization to trypan blue or propidium iodide, lack of mitochondrial retention of rhodamine 123, or extracellular release of lactate dehydrogenase, were all virtually negative. However, these same parameters became markedly positive after 24 h of treatment, which was suggestive for the occurrence of ;secondary' necrosis among AH-130 cells. By a combination of flow cytometric parameters, after 4 h on 1 mM clofibrate only 41% of the AH-130 cells could still be categorized as viable (i.e., non-apoptotic and non-necrotic), while 46% of cells appeared apoptotic and 13% necrotic. At 24 h, 67% of cells were necrotic, 20% apoptotic and only 13% non-apoptotic and non-necrotic. Apoptosis was also extensive in AH-130 cells treated with another PP such as nafenopin at 1 mM concentration and in human hepatoma HepG2 cells treated with clofibrate. By contrast, clofibrate did not cause apoptosis on primary rat hepatocyte cultures. These observations indicate that: (i) apart from their well-known cell growth-promoting action, PPs such as clofibrate or nafenopin may exert a substantial cytotoxic action on targets such as the AH-130 or HepG2 hepatoma cells; (ii) this cell death evolves from an initial 'apoptotic' to an eventual ;necrotic' pattern; (iii) detection of cell death requires the adoption of a full panel of tests, adequate to cover the whole evolving death pattern, while such tests may even be substantially misleading whenever applied individually; (iv) the cytotoxicity of clofibrate and similar agents on normal and, particularly, tumoural cells may deserve careful reevaluation.