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Delta-aminolevulinic acid cytotoxic effects on human hepatocarcinoma cell lines.

De Siervi A, Vazquez ES, Rezaval C, Rossetti MV, del Batlle AM - BMC Cancer (2002)

Bottom Line: Because apoptosis is usually associated with DNA fragmentation, the DNA of ALA treated and untreated cells were analyzed.To elucidate the mechanisms of ALA induced apoptosis, we examined its effect on p53 expression.CDK2 and CDK4 protein levels were reduced after ALA treatment at physiological concentrations.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centro de Investigaciones sobre Porfirinas y Porfirias, Argentine National Research Council (CONICET), Department of Biological Chemistry, FCEN, University of Buenos Aires, Argentina. desiervi@qb.fcen.uba.ar

ABSTRACT

Background: Acute Intermittent Porphyria is a genetic disorder of heme metabolism, characterized by increased levels of porphyrin precursors, delta-aminolevulinic acid (ALA) and porphobilinogen (PBG). ALA has been reported to generate reactive oxygen species and to cause oxidative damage to proteins, subcellular structures and DNA. It is known that oxidative stress can induce apoptosis. The aim of this work was to study the cytotoxic effect of ALA on two hepatocarcinoma cell lines.

Results: We have determined the impact of ALA on HEP G2 and HEP 3B hepatocarcinoma cell lines survival as measured by the MTT assay. ALA proved to be cytotoxic in both cell lines however; HEP G2 was more sensitive to ALA than HEP 3B. Addition of hemin or glucose diminished ALA cytotoxicity in HEP G2 cells; instead it was enhanced in HEP 3B cells. Because apoptosis is usually associated with DNA fragmentation, the DNA of ALA treated and untreated cells were analyzed. The characteristic pattern of DNA fragmentation ladders was observed in ALA treated cells. To elucidate the mechanisms of ALA induced apoptosis, we examined its effect on p53 expression. No changes in p53 mRNA levels were observed after exposure of both cell lines to ALA for 24 h. CDK2 and CDK4 protein levels were reduced after ALA treatment at physiological concentrations.

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DNA fragmentation analysis in human hepatocarcinoma cells undergoing ALA-induced apoptosis. HEP G2 (a) or HEP 3B (b) cells were treated with ALA (0; 1; 2 or 5 mM) and DNA fragmentation was examined 24 h later. HEP 3B cells were also treated with Etoposide (Eto, 5 μM), used as positive control.
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Figure 4: DNA fragmentation analysis in human hepatocarcinoma cells undergoing ALA-induced apoptosis. HEP G2 (a) or HEP 3B (b) cells were treated with ALA (0; 1; 2 or 5 mM) and DNA fragmentation was examined 24 h later. HEP 3B cells were also treated with Etoposide (Eto, 5 μM), used as positive control.

Mentions: As shown in Figure 4a, after 24 h exposure of HEP G2 cells to ALA (1–5 mM) both attached and unattached cells were harvested. In each case, nucleosomal DNA ladders, typical of apoptosis, were visible on agarose gel after staining with ethidium bromide. When HEP 3B cells were treated under the same conditions, nucleosomal DNA ladders were much less visualized (Figure 4b). Apoptosis was also corroborated morphologically by phase contrast microscopy in both cell lines. The decrease in viability was due to cytotoxicity as observed under phase contrast microscopy. ALA caused hepatoma cells first to shrink and then gradually to become dettached, starting about 14 hours after treatment as observed by phase contrast microscopy (data not shown).


Delta-aminolevulinic acid cytotoxic effects on human hepatocarcinoma cell lines.

De Siervi A, Vazquez ES, Rezaval C, Rossetti MV, del Batlle AM - BMC Cancer (2002)

DNA fragmentation analysis in human hepatocarcinoma cells undergoing ALA-induced apoptosis. HEP G2 (a) or HEP 3B (b) cells were treated with ALA (0; 1; 2 or 5 mM) and DNA fragmentation was examined 24 h later. HEP 3B cells were also treated with Etoposide (Eto, 5 μM), used as positive control.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC101407&req=5

Figure 4: DNA fragmentation analysis in human hepatocarcinoma cells undergoing ALA-induced apoptosis. HEP G2 (a) or HEP 3B (b) cells were treated with ALA (0; 1; 2 or 5 mM) and DNA fragmentation was examined 24 h later. HEP 3B cells were also treated with Etoposide (Eto, 5 μM), used as positive control.
Mentions: As shown in Figure 4a, after 24 h exposure of HEP G2 cells to ALA (1–5 mM) both attached and unattached cells were harvested. In each case, nucleosomal DNA ladders, typical of apoptosis, were visible on agarose gel after staining with ethidium bromide. When HEP 3B cells were treated under the same conditions, nucleosomal DNA ladders were much less visualized (Figure 4b). Apoptosis was also corroborated morphologically by phase contrast microscopy in both cell lines. The decrease in viability was due to cytotoxicity as observed under phase contrast microscopy. ALA caused hepatoma cells first to shrink and then gradually to become dettached, starting about 14 hours after treatment as observed by phase contrast microscopy (data not shown).

Bottom Line: Because apoptosis is usually associated with DNA fragmentation, the DNA of ALA treated and untreated cells were analyzed.To elucidate the mechanisms of ALA induced apoptosis, we examined its effect on p53 expression.CDK2 and CDK4 protein levels were reduced after ALA treatment at physiological concentrations.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centro de Investigaciones sobre Porfirinas y Porfirias, Argentine National Research Council (CONICET), Department of Biological Chemistry, FCEN, University of Buenos Aires, Argentina. desiervi@qb.fcen.uba.ar

ABSTRACT

Background: Acute Intermittent Porphyria is a genetic disorder of heme metabolism, characterized by increased levels of porphyrin precursors, delta-aminolevulinic acid (ALA) and porphobilinogen (PBG). ALA has been reported to generate reactive oxygen species and to cause oxidative damage to proteins, subcellular structures and DNA. It is known that oxidative stress can induce apoptosis. The aim of this work was to study the cytotoxic effect of ALA on two hepatocarcinoma cell lines.

Results: We have determined the impact of ALA on HEP G2 and HEP 3B hepatocarcinoma cell lines survival as measured by the MTT assay. ALA proved to be cytotoxic in both cell lines however; HEP G2 was more sensitive to ALA than HEP 3B. Addition of hemin or glucose diminished ALA cytotoxicity in HEP G2 cells; instead it was enhanced in HEP 3B cells. Because apoptosis is usually associated with DNA fragmentation, the DNA of ALA treated and untreated cells were analyzed. The characteristic pattern of DNA fragmentation ladders was observed in ALA treated cells. To elucidate the mechanisms of ALA induced apoptosis, we examined its effect on p53 expression. No changes in p53 mRNA levels were observed after exposure of both cell lines to ALA for 24 h. CDK2 and CDK4 protein levels were reduced after ALA treatment at physiological concentrations.

Show MeSH
Related in: MedlinePlus