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 patten), 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 nonnecrotic. 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.

Cytotoxic Properties of Clofibrate and other Peroxisome Proliferators: Relevance to Cancer Progression

“…As well as other PPs, fibrate derivatives, among which clofibrate, were largely studied as hepatocarcinogens in rodents, also with reference to their antiapoptotic action. In this regard, however, several studies reported that clofibrate treatment induces massive apoptotic death in hepatoma cells [3,4]. In different cell lines similar observation were also reported for other PPs such as nafenopin, perfluorooctanoic acid, and BR931 [3,5,6].…”

“…Such observations have been extended also to other PPs: nafenopin is able to induce apoptotic death in the AH-130 cells [3], while perfluorooctanoic acid and BR931 exert cytotoxic effects on the HepG2 hepatoma cell line [5,6]. A recent study shows that in clofibrate-treated pigs the expression of the pro-apoptotic protein Bax is up-regulated, while the levels of the antiapoptotic factor Bcl-x L are reduced, which may provide the basis for an increase of apoptotic cell death.…”

“…Finally, PPAR-dependent signal transduction pathway may be effectively modulated in the future by means of siRNA or antisense methodologies, or by selective PPAR modulators, able to exert most of the benefits, while reducing the adverse effects displayed by full PPAR agonists [136]. Wy-14,643 hepatocytes [98] perfluoroctanoic acid liver cancer cells [5] Nafenopin liver cancer cells [3] BR931 liver cancer cells [6] conjugated linoleic acid liver cancer cells [138] TDZ18 lymphocytic leukemia [139] MCC-555 colon cancer cells [140] PPAR/ GW0742 colon cancer cells [141] PPAR TDZ (troglitazone, rosiglitazone, ciglitazone, pioglitazone, TDZ18, MCC-555) osteoblasts, promyelocytic leukaemia, lymphocytic leukaemia, colon, breast, liver, thyroid, ovarian, and lung cancer [7,8,139,140,[142][143][144] 15d-PGJ2 neurons, B-lymphocytes, Blymphoma,promyelocytic leukaemia, colon, breast, thyroid and lung cancer (142,143,(145)(146)(147) DEHP Sertoli cells [148] DHEP = Di(2-ethylhexyl) phthalate; TDZ = thiazolidindione;…”

“…In this regard, however, several studies reported that clofibrate treatment induces massive apoptotic death in hepatoma cells [3,4]. In different cell lines similar observation were also reported for other PPs such as nafenopin, perfluorooctanoic acid, and BR931 [3,5,6]. PP cytotoxicity is not restricted to cells of hepatic origin, since similar effects were observed in breast or lung cancer cell lines [7,8] as well as in human keratinocytes, and normal or neoplastic T lymphocytes [9, 10; Penna et al,…”

“…A number of studies indicate that treatment with clofibrate promptly induces massive and typical apoptosis in hepatoma cells of both murine (Yoshida AH-130) and human origin (HepG2; [3]). Such observations have been extended also to other PPs: nafenopin is able to induce apoptotic death in the AH-130 cells [3], while perfluorooctanoic acid and BR931 exert cytotoxic effects on the HepG2 hepatoma cell line [5,6].…”