Induction of metallothionein expression by supplementation of zinc induces resistance against platinum-based treatment in malignant pleural mesothelioma

Abstract

Background: Malignant pleural mesothelioma (MPM) is an aggressive tumor with a dismal prognosis. Currently, multimodality treatment including chemotherapy with cisplatin or carboplatin in combination with pemetrexed offers the best options. Detoxification of heavy metals in the cell by metallothioneins (MT) is associated with early failure to platin-based chemotherapy. The induction of MTs gene expression or its enzyme results in saturation by exposure to metal ions such as zinc or cadmium. Its therapeutically effect is still not analyzed in depth.

Methods: In our study, we investigated three MPM cell lines and one fibroblast cell line in the course of cisplatin treatment and supplementation of zinc. Cell state analyses via an enzyme-activity based assay were performed. With this, we were able to analyze apoptosis, necrosis and viability of cells. Additionally, we tested treated cells for changes in metallothionein IIA (MT2A) expression by using quantitative realtime polymerase chain reaction.

Results: Zinc supplementation induces gene expression of MT2A. Overall, a zinc dose-dependent induction of apoptosis under platin-based treatment could be observed. This effect could be verified in all analyzed cell lines in varying intensity.

Conclusions: MT expression is induced by zinc in a dose-dependent manner and inhibits a successful cisplatin therapy. Therefore, heavy metal exposure during cisplatin therapy, e.g., via cigarette smoke, might be an important factor. This should be considered in further therapeutic approaches.

Keywords: Malignant pleural mesothelioma (MPM); cisplatin; metallothionein; zinc.

Figure 1

Gene expression of MT2A after supplementation of different zinc concentrations. The data were measured 77 h after supplementation of zinc. The difference in MT2A expression of the sample between zinc-treatment and control is shown on the y-axis (2^−ΔCp) (Cp means the crossing point describing the cycle at which the fluorescence first rises significantly above the background fluorescence). All MPM cell lines showed elevated expression of MT2A by adding higher concentrations of zinc [(A) MSTO211H (P=0.0833), (B) NCI-H2452 (P=1), and (C) NCI-H2052 (P=0.0833)]. P values were calculated by using the Spearman’s rank correlation test. MT2A, metallothionein IIA; MPM, malignant pleural mesothelioma.

Figure 2

Apoptotic response of cell lines (A) NCI-H2052, (B) NCI-H2452, (C) MSTO211H, and (D) MRC5 to cisplatin (10 µM) after addition of varying zinc concentrations. Cells were seeded into the 96-well plate with varying zinc concentrations. Six h after incubation, cisplatin was added to the cells. Measurements were performed after 48 h of treatment with cisplatin. On the y-axis RLU are shown. RLU and increasing apoptosis-rates show a direct correlation. The x-axis depicts zinc concentrations being applied on cells. Each cell line showed linear decrease in apoptosis with increasing zinc concentrations. All cell lines showed significant linear decrease in apoptosis when zinc concentration was elevated. P values and correlation-coefficient (ρ) were calculated by using the Spearman’s rank correlation test. RLU, relative luminescence units.