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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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Drug induced tolerance of copper treated KO cells.Cells were treated with Zn, DPA and Zn+DPA and viability was determined by MTT assay. Zn pretreatment was for 2 h. DPA and Cu exposure was for 48 h. The gain of viability following treatment (black) is given relative to the viability of untreated KO (open) and HepG2 cells (shadow). Significance (p<0.05) is indicated: *treated vs untreated; †gain by treatment in KO vs. gain in HepG2 cells; aZn+DPA treatment vs. Zn; bZn+DPA treatment vs. DPA.
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pone-0098809-g005: Drug induced tolerance of copper treated KO cells.Cells were treated with Zn, DPA and Zn+DPA and viability was determined by MTT assay. Zn pretreatment was for 2 h. DPA and Cu exposure was for 48 h. The gain of viability following treatment (black) is given relative to the viability of untreated KO (open) and HepG2 cells (shadow). Significance (p<0.05) is indicated: *treated vs untreated; †gain by treatment in KO vs. gain in HepG2 cells; aZn+DPA treatment vs. Zn; bZn+DPA treatment vs. DPA.

Mentions: The impact of zinc (Zn), D-penicillamine (DPA), and combined treatment (Zn+DPA) on the viability of the KO cells following Cu exposure was determined by MTT assay in the presence of different Cu concentrations (Fig. 5). Of note, Zn treatment (Fig. 5A) was reduced throughout the study to a 2 h pre-treatment period [12]. DPA was present for 48 h. After Zn treatment of KO cells, a high viability was gained (range 33.8% to 70.3%; mean 54.6±4%) up to a Cu concentration of 0.75 mM, whereas at higher Cu concentrations (≥ 1.0 mM) the improvement by Zn was lower (<20%) suggesting that a saturation of the effect is achieved. In HepG2 cells, the gain of viability by Zn treatment was generally lower (range 5.1% to 23.2%; mean 11.8±1%) suggesting that hepatocytes expressing ATP7B benefit less from zinc treatment. The effect of Zn treatment was not due to an overall higher toxicity of the KO cells, since HepG2 cells showed high rates of toxicity (>1.0 mM Cu) but relative low improvement. HepG2 cells had improved viability after DPA treatment (Fig. 5B) at Cu concentration ≥ 1.0 mM (range 43.3% to 80.1%; mean 59.3±3%). The effect of DPA on KO cells was significantly lower (range 0.2% to 25.9%; mean 13.2±6%) and further decreased (<10%) at higher Cu concentrations (>1.0 mM).Combined treatment (Zn+DPA) showed a highly synergistic effect in KO cells as compared to single drug treatment (Fig. 5C). Viability of KO cells was restored to high levels (mean 62.5±10%) at all Cu concentrations suggesting that combined Zn and DPA treatment has an advantage for the survival of human hepatocytes that lack ATP7B. HepG2 cells did not significantly improve viability by combined treatment as compared to single DPA treatment.


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)

Drug induced tolerance of copper treated KO cells.Cells were treated with Zn, DPA and Zn+DPA and viability was determined by MTT assay. Zn pretreatment was for 2 h. DPA and Cu exposure was for 48 h. The gain of viability following treatment (black) is given relative to the viability of untreated KO (open) and HepG2 cells (shadow). Significance (p<0.05) is indicated: *treated vs untreated; †gain by treatment in KO vs. gain in HepG2 cells; aZn+DPA treatment vs. Zn; bZn+DPA treatment vs. DPA.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0098809-g005: Drug induced tolerance of copper treated KO cells.Cells were treated with Zn, DPA and Zn+DPA and viability was determined by MTT assay. Zn pretreatment was for 2 h. DPA and Cu exposure was for 48 h. The gain of viability following treatment (black) is given relative to the viability of untreated KO (open) and HepG2 cells (shadow). Significance (p<0.05) is indicated: *treated vs untreated; †gain by treatment in KO vs. gain in HepG2 cells; aZn+DPA treatment vs. Zn; bZn+DPA treatment vs. DPA.
Mentions: The impact of zinc (Zn), D-penicillamine (DPA), and combined treatment (Zn+DPA) on the viability of the KO cells following Cu exposure was determined by MTT assay in the presence of different Cu concentrations (Fig. 5). Of note, Zn treatment (Fig. 5A) was reduced throughout the study to a 2 h pre-treatment period [12]. DPA was present for 48 h. After Zn treatment of KO cells, a high viability was gained (range 33.8% to 70.3%; mean 54.6±4%) up to a Cu concentration of 0.75 mM, whereas at higher Cu concentrations (≥ 1.0 mM) the improvement by Zn was lower (<20%) suggesting that a saturation of the effect is achieved. In HepG2 cells, the gain of viability by Zn treatment was generally lower (range 5.1% to 23.2%; mean 11.8±1%) suggesting that hepatocytes expressing ATP7B benefit less from zinc treatment. The effect of Zn treatment was not due to an overall higher toxicity of the KO cells, since HepG2 cells showed high rates of toxicity (>1.0 mM Cu) but relative low improvement. HepG2 cells had improved viability after DPA treatment (Fig. 5B) at Cu concentration ≥ 1.0 mM (range 43.3% to 80.1%; mean 59.3±3%). The effect of DPA on KO cells was significantly lower (range 0.2% to 25.9%; mean 13.2±6%) and further decreased (<10%) at higher Cu concentrations (>1.0 mM).Combined treatment (Zn+DPA) showed a highly synergistic effect in KO cells as compared to single drug treatment (Fig. 5C). Viability of KO cells was restored to high levels (mean 62.5±10%) at all Cu concentrations suggesting that combined Zn and DPA treatment has an advantage for the survival of human hepatocytes that lack ATP7B. HepG2 cells did not significantly improve viability by combined treatment as compared to single DPA treatment.

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