Organic cation transporter 3 mediates cisplatin and copper cross-resistance in hepatoma cells

Abstract

Platinum-based drugs are first-line compounds in the treatment of many solid cancers. Major obstacles are tumors that become resistant and toxic side effects, both largely due to the expression of transporters that mediate the cellular processing of platinum. In this study, we addressed the establishment of cisplatin resistance in the absence of copper transporter ATP7B that has been previously found to be overexpressed in various resistant cells. Cisplatin sensitivity, induction of apoptosis, drug accumulation, and transporter gene expression were determined in hepatoma cell lines. Knockout or overexpression of copper transporter ATP7B did not affect cisplatin sensitivity. Cisplatin resistant cells showed a stably reduced cisplatin accumulation and a downregulation of organic cation transporter 3 (OCT3). In contrast, OCT3 overexpression could reverse resistance. Reduced MT1 expression was detected in the resistant cell line, however transient and highly dependent on the presence of cisplatin. Cross-resistance to copper was also associated with OCT3 downregulation. Our results suggest that a decreased level of OCT3 expression results in resistance to cisplatin and copper. OCT3 may represent a novel target for improved prognosis and anticancer therapy, including HCC.

Keywords: ATP7B; MT1; OCT3; cisplatin; copper cross-resistance.

Figure 1. ATP7B expression does not affect cisplatin sensitivity in hepatoma cells

(A) Cell viability was determined by MTT assay relative to untreated cells (100%). Mean/SE are given (n = 5). (B) Intracellular cisplatin level was determined by TXRF in the soluble cellular fractions of the cells. Cells were incubated with cisplatin for 4 h. Mean/SE are given (n = 3).

Figure 2. Human hepatoma cells lacking ATP7B can adapt to proliferation in toxic cisplatin

(A) Light microscopic image of CpR cells after continuous growth in toxic cisplatin concentration for more than a year. Cell morphology was identical to parental cells. Scale bar, 50 µm. (B) Cumulative growth of CpR cells in the presence (+Cp) and absence (-Cp) of cisplatin. Growth of parental ATP7B KO cells is depicted for comparison. Mean/SE are given (n = 3). (C) Determination of apoptosis by Annexin V staining in CpR cells and ATP7B KO cells. Mean/SE are given (n = 3). (D) Intracellular cisplatin concentration was determined in nuclear and soluble fractions of CpR and ATP7B KO cells. Mean/SE are given (n = 3). ^*P < 0.05.

Figure 3. Cisplatin resistance is a stable modification of hepatoma cells

(A) mRNA expression of CpR cells relative to ATP7B KO cells was calculated using the 2^–ΔΔ method. Dotted line at a fold change of ±2 indicates significance. Mean/SE are given (n = 3). (B) Cell viability as determined by MTT assay relative to untreated cells (100%). Mean/SE are given (n = 5). ^*P < 0.05. (C) mRNA expression of CpRw cells is presented as fold change relative to ATP7B KO cells. Mean/SE are given (n = 3).

Figure 4. OCT3 mediates cisplatin resistance in hepatoma cells

(A) mRNA expression in CpR_OCT3 cells and CpR cells. Mean/SE are given (n = 3). (B) Densitometric analysis of OCT3 protein expression as determined by Western blot. ATP7B KO cells were set to 100%. Mean/SE are given (n = 3). (C) OCT3 function was assessed by ASP⁺ uptake rates. Mean/SE are given (n = 3). (D) Cell viability was determined by MTT assay relative to untreated cells (100%). Mean/SE are given (n = 5). ^*P < 0.05.

Figure 5. OCT3 confers cross-resistance

(A) Cell viability was determined by MTT assay relative to untreated cells (100%). Cells were exposed to 0.75 mM copper for 48 h. HepG2 cells expressing ATP7B are shown as control. Mean/SE are given (n = 3). (B) ATP7B KO cells were treated with siRNA directed against OCT3 and cell viability was assessed by MTT assay. Mean/SE are given (n = 3). (C) Intracellular copper concentration as determined by AAS. ATP7B KO cells served for comparison (100%). Mean/SE are given (n = 3). ^*P < 0.05.