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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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Western blot analysis of CDK4 and CDK2. Proteins were isolated after treatment of cells with ALA (0–5 mM) during 24 h. Proteins were normalized to 50 μg per lane and the amount of CDK2 and CDK4 was detected using polyclonal antibodies and visualized using ECL reagents. Intensity of bands was analysed with ImageMaster.
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Figure 6: Western blot analysis of CDK4 and CDK2. Proteins were isolated after treatment of cells with ALA (0–5 mM) during 24 h. Proteins were normalized to 50 μg per lane and the amount of CDK2 and CDK4 was detected using polyclonal antibodies and visualized using ECL reagents. Intensity of bands was analysed with ImageMaster.

Mentions: When CDK2 and CDK4 protein levels were analyzed in HEP G2 cells after exposure to ALA for 24 h (Figure 6), both kinases decreased with increased ALA concentrations, up to 2 mM. At concentrations above 2 mM protein content increased reaching basal levels at 5 mM (Figure 6).


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)

Western blot analysis of CDK4 and CDK2. Proteins were isolated after treatment of cells with ALA (0–5 mM) during 24 h. Proteins were normalized to 50 μg per lane and the amount of CDK2 and CDK4 was detected using polyclonal antibodies and visualized using ECL reagents. Intensity of bands was analysed with ImageMaster.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC101407&req=5

Figure 6: Western blot analysis of CDK4 and CDK2. Proteins were isolated after treatment of cells with ALA (0–5 mM) during 24 h. Proteins were normalized to 50 μg per lane and the amount of CDK2 and CDK4 was detected using polyclonal antibodies and visualized using ECL reagents. Intensity of bands was analysed with ImageMaster.
Mentions: When CDK2 and CDK4 protein levels were analyzed in HEP G2 cells after exposure to ALA for 24 h (Figure 6), both kinases decreased with increased ALA concentrations, up to 2 mM. At concentrations above 2 mM protein content increased reaching basal levels at 5 mM (Figure 6).

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