In vitro demonstration of synergy between radionuclide and chemotherapy.

UNLABELLED: Radionuclide therapy is currently used in the treatment of some malignancies, including hepatocellular carcinoma. The effects of external beam radiotherapy are improved by combining it with chemotherapy. The aim of this study was to determine whether such a synergistic effect could be demonstrated in vitro with internal radiation therapy. METHODS: HepG2 cells were cultured from Day 0 to Day 8 under the following conditions: exposure for 4 hr on Day 2 to increasing concentrations of 5-fluorouracil (5FU), doxorubicin or cisplatin (CDDP); exposure from Day 2 to Day 8 to increasing concentrations of 131-iodide; exposure on Day 2 to low-toxicity doses of drugs for 4 hr, followed by exposure to 131I at increasing concentrations; and exposure to increasing concentrations of 131I from Day 2 to Day 8, with exposure for 4 hr on Day 6 to the drugs. Cell toxicity was assessed by enzyme release (lactate dehydrogenase and aspartate aminotransferase) in the culture medium and on cell survival (protein and tetrazolium dye test). All cultures were run in triplicate. RESULTS: A dose- and time-dependent toxicity was demonstrated with doxorubicin and CDDP but not with 5FU. When HepG2 cells were exposed to 131I, the toxicity was rather low, but significant, and was time- and dose-dependent. Treating these cells with combination radiotherapy and chemotherapy resulted in a toxicity that was significantly greater than that with 131I or chemotherapy drugs alone. CONCLUSION: The radiosensitivity of HepG2 cells is low; combining a chemotherapeutic drug with a radiotherapeutic agent improves the radiosensitivity in a synergistic fashion. This combination is thus able to strengthen the therapeutic effect of internal radiation therapy in different malignancies, particularly in hepatocellular carcinoma.

Antiproliferative effect of deferiprone on the Hep G2 cell line.

Iron is an essential element in cellular metabolism and the growth of all living species, and is involved in DNA replication. The risk of hepatocellular carcinoma development is associated with an increase in iron availability. The aim of the present work was to investigate the effect of an oral iron chelator, deferiprone (CP20), on HepG2 cell-line proliferation in culture. HepG2 cell cultures were maintained in the absence of fetal calf serum (FCS) and in the presence or not (control cultures) of CP20 at the concentrations of 50 or 100 microM; deferoxamine (DFO) was used as an iron chelator reference. Cell proliferation was investigated by the analysis of DNA synthesis using [3H] methyl-thymidine incorporation and of the cell cycle by flow cytometry. Iron chelation efficiency in the culture model was studied by analyzing the effect of CP20 on radioactive iron uptake, intracellular ferritin level, and transferrin receptor expression. CP20, at the concentration of 50 or 100 microM, inhibited DNA synthesis after 48 hr of incubation and induced an accumulation of the cells in the S phase of the cell cycle. Iron chelators inhibited cellular iron uptake, decreased intracellular ferritin level, and increased transferrin receptor protein and mRNA levels. Our results show that CP20 as well as deferoxamine inhibit HepG2 cell proliferation and block cell cycle in the S phase.

Iron may induce both DNA synthesis and repair in rat hepatocytes stimulated by EGF/pyruvate.

BACKGROUND/AIMS: Hepatocellular carcinoma develops frequently in the course of genetic hemochromatosis, and a role of iron overload in hepatic carcinogenesis is strongly suggested. METHODS: The aim of our study was to investigate the effect of iron exposure on DNA synthesis of adult rat hepatocytes maintained in primary culture stimulated or not by EGF/pyruvate and exposed to iron-citrate complex. RESULTS: In EGF/pyruvate-stimulated cultures, the level of [3H] methyl thymidine incorporation was strongly increased as compared to unstimulated cultures. The addition of iron to stimulated cultures increased [3H] methyl thymidine incorporation. The mitotic index was also significantly higher at 72 h. However, the number of cells found in the cell layer was not significantly different from iron-citrate free culture. By flow cytometry, no difference in cell ploidy was found between iron-treated and untreated EGF/pyruvate-stimulated cultures. A significant increase in LDH leakage reflecting a toxic effect of iron was found in the cell medium 48 h after cell seeding. In addition, [3H] methyl thymidine incorporation in the presence of hydroxyurea was increased in iron-treated compared to untreated cultures. CONCLUSIONS: Our results show that DNA synthesis is increased in the presence of iron in rat hepatocyte cultures stimulated by EGF/pyruvate, and they suggest that DNA synthesis is likely to be related both to cell proliferation and to DNA repair. These observations may allow better understanding of the role of iron overload in the development of hepatocellular carcinoma.

Inhibition of iron toxicity in rat and human hepatocyte cultures by the hydroxypyridin-4-ones CP20 and CP94.

The protective effect of the hydroxypyridin-4-ones (CP20 and CP94) was studied on iron-loaded rat and human hepatocytes; desferrioxamine B was used as a chelator reference. Iron load was achieved by addition of 5 up to 50 microM iron citrate to the culture medium. One day after iron treatment, an increase in lactate dehydrogenase, aspartate aminotransferase, alanine aminotransferase and malondialdehyde extracellular concentrations was measured in rat and human hepatocyte cultures. This enzyme release and the increase in free extracellular malondialdehyde were observed with 5 microM iron and high levels were obtained with 50 microM. The bidentate chelators CP20 and CP94 (150 microM) appeared to be as effective as the hexadentate chelator desferrioxamine (50 microM) in the protection of rat and human hepatocytes against the toxic effect of iron load achieved by culturing the cells for 1 day in the presence of 50 microM iron citrate. In rat and human hepatocytes cultured for 1 day in the presence of 1 microM 55Fe-50 microM iron citrate plus CP20, CP94 or desferrioxamine B, a decrease of iron uptake by the cells was observed. When the hepatocytes were cultured for 1 day in the presence of 1 microM 55Fe-50 microM iron citrate and then for a further day in the presence of CP20, CP94 or desferrioxamine B but not iron, the chelators decreased the intracellular iron level, indicating their iron releasing effect from the loaded cells. The observed effects of the hydroxypyridin-4-ones CP20 and CP94 were as potent as the effect of desferrioxamine B. This study presents new data favoring the potential clinical interest of this new class of chelating agents in the treatment of human iron overload.

Inhibition of iron toxicity in human hepatocyte cultures by pyoverdins Pa A and Pf, the peptidic siderophores of Pseudomonas aeruginosa and fluorescens.

The protective effect of pyoverdins Pa A and Pf, peptidic siderophores secreted respectively by Pseudomonas aeruginosa and fluorescens, was studied in primary cultures of human hepatocytes exposed to iron (50 or 100 microM of iron-citrate). AST, ALT and MDA releases were measured as indexes of cytotoxicity. In order to demonstrate that these chelators were able to decrease iron uptake or increase iron release from the hepatocytes, labelled cells were obtained by maintaining the cultures in the presence of 1 microM 55Fe ferric chloride plus 50 microM iron citrate. One day after iron treatment, an increase in AST, ALT and MDA release was observed with 50 or 100 microM of iron citrate; it appeared that the concentrations 50 and 100 microM of iron were highly toxic for human hepatocytes. In the presence of 50 or 100 microM of iron, the addition of 50 or 100 microM of Pa A or Pf was effective to inhibit the increase observed in the enzyme leakage and the MDA production resulting from iron exposure. In human hepatocytes cultured for 1 day in the presence of 1 microM 55Fe-50 microM iron citrate plus 50 or 100 microM Pa A or Pf, a net decrease of iron uptake by the cells was observed, as demonstrated by the low intracellular iron level. When the hepatocytes were cultured for 1 day in the presence of 1 microM 55Fe-50 microM iron citrate and then for a further day in the presence of 50 or 100 microM Pa A or Pf without additional iron, the chelators increased the extracellular iron level, indicating their iron release from the loaded cells; however, the effects of Pa A and Pf on iron release did not differ significantly. In conclusion, iron loading achieved by adding iron citrate to the culture medium is highly toxic for human hepatocytes. Pyoverdins Pa A and Pf are effective in protecting human hepatocytes against the toxic effect of iron by both decreasing the uptake of the metal and increasing its release from the loaded cells.

Deferoxamine arrests in vitro the proliferation of porcine hepatocyte in G1 phase of the cell cycle.

Iron is required for cell proliferation of all living species. Moreover, iron excess may be involved in the development of hepatocellular carcinoma. In this study we analyzed the effects of deferoxamine, an iron chelator, on normal porcine hepatocyte proliferation. We confirmed that hepatocytes isolated from young pigs proliferate in the presence of insulin and fetal calf serum as shown by [3H] methyl-thymidine incorporation, presence of mitotic figures and increase in cell number. This was paralleled by nuclear expression of p34cdc2 and its associated histone H1 kinase activity. In the presence of deferoxamine, [3H] methyl-thymidine incorporation, expression of nuclear proteins (p34cdc2 and PCNA) and H1 kinase activity were drastically reduced. In addition, in contrast with control cultures, cells in S-phase were not detected by flow cytometry. These data suggest that iron chelation by deferoxamine can arrest the progression of porcine hepatocytes in the G1 phase of the cell cycle.

Effect of ursodeoxycholic acid on liver iron stores and distribution in rats with normal or iron-supplemented diet.

Iron excess is a potential liver-damaging factor, and bile salts can increase iron digestive absorption and iron biliary excretion. The aim of this study was to investigate in rats the effect of ursodeoxycholic acid, a bile salt used in the treatment of chronic liver disease, on the hepatic iron stores in normal and iron-overload conditions. UDCA was administered by gavage to Sprague-Dawley rats. Iron hyperabsorption and overload were obtained by 5% carbonyl iron addition in diet. Hepatic iron stores and distribution were evaluated by liver iron concentration measurement and histologic assessment, respectively. Whatever the iron content of the diet, liver iron concentration was not modified by UDCA administration compared with the control groups. Iron distribution was not modified by UDCA in rats with normal diet. The total iron score was only transiently lowered by UDCA in iron supplemented rats compared with the control group at 1 month. In conclusion, chronic UDCA administration does not modify liver iron stores and distribution in rats with both normal or increased digestive iron absorption. These data suggest that UDCA is unlikely to increase hepatic iron stores in treated patients and that the benefit of UDCA treatment is probably not related to a decreasing effect of liver iron content.