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The effect of zinc and D-penicillamine in a stable human hepatoma ATP7B knockout cell line.

Chandhok G, Schmitt N, Sauer V, Aggarwal A, Bhatt M, Schmidt HH - PLoS ONE (2014)

Bottom Line: Induction of metallothionein (MT1X) after Cu exposure was significantly reduced in KO cells.D-penicillamine treatment had a minor effect on the viability of KO cells whereas the parental cell line showed a pronounced improvement.Combined treatment displayed a highly synergistic effect in KO cells.

View Article: PubMed Central - PubMed

Affiliation: Clinic for Transplantation Medicine, Münster University Hospital, Münster, Germany; Wilson Disease Clinic, Kokilaben Dhirubhai Ambani Hospital and Medical Research Institute, Mumbai, India.

ABSTRACT
Mutations in the copper (Cu) transporter gene ATP7B, the primary cause of Wilson disease (WD), result in high liver Cu and death of hepatocytes. Cu chelators and zinc salts are the two most important drugs used in the treatment of WD patients; however, the molecular mechanisms of the drugs with regard to ATP7B expression have not been determined. A targeted knockout of ATP7B (KO) was established in the most widely used human hepatoma cell line, HepG2 for molecular studies of the pathogenesis and treatment of the disease. KO cells showed similar growth, Cu uptake, release, and gene expression as compared to parental cells. However, in the presence of Cu, morphological changes, oxidative stress, apoptosis, and loss of viability were observed. Induction of metallothionein (MT1X) after Cu exposure was significantly reduced in KO cells. Following zinc treatment, MT1X expression was strongly induced and a high percentage of KO cells could be rescued from Cu induced toxicity. D-penicillamine treatment had a minor effect on the viability of KO cells whereas the parental cell line showed a pronounced improvement. Combined treatment displayed a highly synergistic effect in KO cells. The data suggest that zinc has a previously unrecognized effect on the viability of hepatocytes that lack ATP7B due to a high induction of MT1X expression that compensates low gene expression after Cu exposure. A combination therapy that simultaneously targets at MT1X induction and Cu chelation improves the overall survival of hepatocytes for most efficient therapy of patients having WD.

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Oxidative stress and viability of KO cell line.(A) Oxidative stress following Cu exposure at 0.1 mM Cu for 1 h. The four histograms represent fluorescence of viable cells as obtained by flow cytometry after staining with H2DCFDA dye. A shift of the two histograms appears in KO cells after Cu exposure (shaded) relative to untreated control (open) indicating induction of OS. (B) Viability of KO (circles) and HepG2 cells (triangle) relative to untreated control was determined by MTT assay after 48 h of Cu exposure. (C) Induction of apoptosis was determined after 24 h of 0.1 mM Cu exposure using Annexin-V staining followed by flow cytometry analysis. (D) Cell viability of knockin versus KO cell is shown. Viability was determined at 0.5 mM Cu by MTT assay after 48 h. Data is represented as mean±SE of three independent experiments. Asterisks indicate significance (p<0.05).
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pone-0098809-g003: Oxidative stress and viability of KO cell line.(A) Oxidative stress following Cu exposure at 0.1 mM Cu for 1 h. The four histograms represent fluorescence of viable cells as obtained by flow cytometry after staining with H2DCFDA dye. A shift of the two histograms appears in KO cells after Cu exposure (shaded) relative to untreated control (open) indicating induction of OS. (B) Viability of KO (circles) and HepG2 cells (triangle) relative to untreated control was determined by MTT assay after 48 h of Cu exposure. (C) Induction of apoptosis was determined after 24 h of 0.1 mM Cu exposure using Annexin-V staining followed by flow cytometry analysis. (D) Cell viability of knockin versus KO cell is shown. Viability was determined at 0.5 mM Cu by MTT assay after 48 h. Data is represented as mean±SE of three independent experiments. Asterisks indicate significance (p<0.05).

Mentions: The induction of oxidative stress (OS) was determined in KO cells using staining with H2DCFDA indicator dye followed by flow cytometry analysis. Addition of Cu to standard cell culture medium provoked a shift of histograms in KO cells but not in parental cells suggesting OS induction (Fig. 3A). Viability of KO cells was assessed in the presence of various Cu concentrations by MTT assay (Fig. 3B). KO cells rapidly (<24 h) underwent a significant change of cell morphology including cell rounding and shrinkage indicated by flow cytometry analysis (Figure S1). Significant apoptosis was induced in KO cells after Annexin-V staining (Fig. 3C). In order to confirm that the knockout is the cause of the observed Cu sensitivity, ATP7B was reintroduced in KO cells by lentiviral vector. The viability of KO cells in the presence of high Cu concentrations was restored to a similar range as observed in HepG2 cells (Fig. 3D). ATP7B knockin cells also restored resistance to Cu induced OS stress (data not shown).


The effect of zinc and D-penicillamine in a stable human hepatoma ATP7B knockout cell line.

Chandhok G, Schmitt N, Sauer V, Aggarwal A, Bhatt M, Schmidt HH - PLoS ONE (2014)

Oxidative stress and viability of KO cell line.(A) Oxidative stress following Cu exposure at 0.1 mM Cu for 1 h. The four histograms represent fluorescence of viable cells as obtained by flow cytometry after staining with H2DCFDA dye. A shift of the two histograms appears in KO cells after Cu exposure (shaded) relative to untreated control (open) indicating induction of OS. (B) Viability of KO (circles) and HepG2 cells (triangle) relative to untreated control was determined by MTT assay after 48 h of Cu exposure. (C) Induction of apoptosis was determined after 24 h of 0.1 mM Cu exposure using Annexin-V staining followed by flow cytometry analysis. (D) Cell viability of knockin versus KO cell is shown. Viability was determined at 0.5 mM Cu by MTT assay after 48 h. Data is represented as mean±SE of three independent experiments. Asterisks indicate significance (p<0.05).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4044041&req=5

pone-0098809-g003: Oxidative stress and viability of KO cell line.(A) Oxidative stress following Cu exposure at 0.1 mM Cu for 1 h. The four histograms represent fluorescence of viable cells as obtained by flow cytometry after staining with H2DCFDA dye. A shift of the two histograms appears in KO cells after Cu exposure (shaded) relative to untreated control (open) indicating induction of OS. (B) Viability of KO (circles) and HepG2 cells (triangle) relative to untreated control was determined by MTT assay after 48 h of Cu exposure. (C) Induction of apoptosis was determined after 24 h of 0.1 mM Cu exposure using Annexin-V staining followed by flow cytometry analysis. (D) Cell viability of knockin versus KO cell is shown. Viability was determined at 0.5 mM Cu by MTT assay after 48 h. Data is represented as mean±SE of three independent experiments. Asterisks indicate significance (p<0.05).
Mentions: The induction of oxidative stress (OS) was determined in KO cells using staining with H2DCFDA indicator dye followed by flow cytometry analysis. Addition of Cu to standard cell culture medium provoked a shift of histograms in KO cells but not in parental cells suggesting OS induction (Fig. 3A). Viability of KO cells was assessed in the presence of various Cu concentrations by MTT assay (Fig. 3B). KO cells rapidly (<24 h) underwent a significant change of cell morphology including cell rounding and shrinkage indicated by flow cytometry analysis (Figure S1). Significant apoptosis was induced in KO cells after Annexin-V staining (Fig. 3C). In order to confirm that the knockout is the cause of the observed Cu sensitivity, ATP7B was reintroduced in KO cells by lentiviral vector. The viability of KO cells in the presence of high Cu concentrations was restored to a similar range as observed in HepG2 cells (Fig. 3D). ATP7B knockin cells also restored resistance to Cu induced OS stress (data not shown).

Bottom Line: Induction of metallothionein (MT1X) after Cu exposure was significantly reduced in KO cells.D-penicillamine treatment had a minor effect on the viability of KO cells whereas the parental cell line showed a pronounced improvement.Combined treatment displayed a highly synergistic effect in KO cells.

View Article: PubMed Central - PubMed

Affiliation: Clinic for Transplantation Medicine, Münster University Hospital, Münster, Germany; Wilson Disease Clinic, Kokilaben Dhirubhai Ambani Hospital and Medical Research Institute, Mumbai, India.

ABSTRACT
Mutations in the copper (Cu) transporter gene ATP7B, the primary cause of Wilson disease (WD), result in high liver Cu and death of hepatocytes. Cu chelators and zinc salts are the two most important drugs used in the treatment of WD patients; however, the molecular mechanisms of the drugs with regard to ATP7B expression have not been determined. A targeted knockout of ATP7B (KO) was established in the most widely used human hepatoma cell line, HepG2 for molecular studies of the pathogenesis and treatment of the disease. KO cells showed similar growth, Cu uptake, release, and gene expression as compared to parental cells. However, in the presence of Cu, morphological changes, oxidative stress, apoptosis, and loss of viability were observed. Induction of metallothionein (MT1X) after Cu exposure was significantly reduced in KO cells. Following zinc treatment, MT1X expression was strongly induced and a high percentage of KO cells could be rescued from Cu induced toxicity. D-penicillamine treatment had a minor effect on the viability of KO cells whereas the parental cell line showed a pronounced improvement. Combined treatment displayed a highly synergistic effect in KO cells. The data suggest that zinc has a previously unrecognized effect on the viability of hepatocytes that lack ATP7B due to a high induction of MT1X expression that compensates low gene expression after Cu exposure. A combination therapy that simultaneously targets at MT1X induction and Cu chelation improves the overall survival of hepatocytes for most efficient therapy of patients having WD.

Show MeSH
Related in: MedlinePlus