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

Introduction

Malignant pleural mesothelioma (MPM) arises from pleural cavities and is associated with a poor prognosis (1,2). Even when treated, the median survival of patients is only about 14 months (3).

So far, multimodal treatment including chemotherapy is the therapy of choice for unresectable and advanced MPM. Standard chemotherapeutic treatment includes a platinum compound (cis- or carboplatin) combined with the antifolate pemetrexed (4,5). Cisplatin resistance is the main cause for the poor clinical outcome of patients, since the rate of response of MPM to cisplatin-based therapy averages 14% (6).

Metallothioneins (MTs) are a group of small, evolutionary highly conserved proteins able to bind heavy metals through the thiol-group of their cysteine residues, giving them an important role in zinc homeostasis and intracellular heavy metal detoxification (7). Moreover, MTs contribute to the development of drug resistance through a variety of mechanisms. In a previous study, we could observe that progression-free survival as well as overall survival is negatively correlated to MT expression in cisplatin-treated MPM samples (8). Therefore, it could be hypothesized that MTs mediate a resistance to cisplatin in MPM by its heavy metal binding capacity (8).

Zinc is an important cellular factor for cell proliferation, differentiation, and, as an antioxidant, it protects cells from free radicals and oxidative stress by mutagens (9). With their high affinity to heavy metals, MTs are involved in zinc homeostasis. Therefore, with reduced zinc intake or downregulated MT expression, cell cycle arrest or at least cell growth inhibition might be induced (10).

Zinc homeostasis is also crucial for immune system functions. It acts as a signal molecule between immune cells by regulating the activity of signaling molecules including kinases, phosphatases, and transcription factors (10).

We designed the present study to investigate the role of MTs as a potential underlying factor of platinum resistance by induction of MT expression due to supplementation of zinc in vitro. We present this article in accordance with the MDAR reporting checklist (available at https://tcr.amegroups.com/article/view/10.21037/tcr-22-2651/rc).

Methods

Study design

We analyzed the influence of zinc supplementation in different MPM cell lines and healthy lung fibroblasts serving as a control. In detail, its effect in intracellular MT expression levels was assessed via quantitative gene expression analysis of MT2A. Furthermore, the effect of zinc supplementation in combination with platin-based treatment on cellular response was analyzed. Therefore, apoptosis induction, cell viability and necrosis were quantitatively measured using an enzyme-activity-based detection system.

Cell culture

All MPM cell lines [NCI-H2052: CRL-5915; NCI-H2452: CRL-5946; MSTO-211H: CRL-2081; American Type Culture Collection (ATCC), Virginia, USA] were cultured in Roswell Park Memorial Institute 1640 medium (RPMI) (Thermo Fisher Scientific, Massachusetts, USA), supplemented with 10% fetal calf serum (FCS) and 1% penicillin and streptomycin (P/S, Thermo Fisher Scientific, Massachusetts, USA). The lung-fibroblast cell line MRC-5 (ATCC, CCL-171), was cultured in minimal essential medium (MEM; Thermo Fisher Scientific, Massachusetts, USA) plus 10% FCS and 1% P/S. Cells were incubated at 37 ℃, 5% CO₂. The immortalised cell lines used in this study were obtained from the ATCC. For passaging, cells were washed with Dulbecco’s phosphate buffered saline (DPBS) (Thermo Fisher Scientific, Massachusetts, USA). To detach cells, 3 mL (T75) and 5 mL (T175) of 0.25% Trypsin (Thermo Fisher Scientific, Massachusetts, USA) were added, respectively.

Cell state analysis

Cells were analyzed for viability, necrosis and apoptosis by using the ApoTox-Glo™ Triplex Assay according to the manufacturer instructions (Promega, Madison, WI, USA). All cell lines were treated with different concentrations of ZnSO⁴ (Sigma-Aldrich, St. Louis, Missouri, USA): 10-, 30-, 50-, 70- and 90 µM. Cells (10,000 cells per well in 50 µL) were seeded into a black-walled 96-well plate. After 6 hours of incubation, cells were subsequently treated with cisplatin (10 µM) (Selleck Chemicals, Munich, Germany) and incubated for 48 h at 37 ℃, 5% CO₂. 25 µL of Digitonin (120 µg/mL) and 25 µL Staurosporin (40 µM) were added to the cells 3 h before measurement and served as positive control for necrosis and apoptosis respectively. 25 µL of Digitonin (5 mg/mL) were added to cells 15 minutes before measurement, serving as a positive control for the decrease of cell viability. To overcome effects caused by DMSO as dissolvent of the cisplatin stock solution, the highest used concentration of DMSO (0.008 µM) served as control for determination of the baseline signal. Cell state detection was performed with the Glo Max Multi+ Detection System (Promega, Madison, WI, USA). All cell state analyses were measured in technical and biological triplicate. A technical triplicate is performed during one measurement, as the condition is measured three times. The measurement was repeated twice, to generate a biological triplicate (experiment setup and measurement were performed three times in a row).

FFPE-embedding of cell blocks

The cells were embedded in FFPE to assess gene expression of MTs via quantitative PCR. As FFPE is the most commonly available form of patient material, we performed this step, instead of isolating RNA directly from the treated cells. Therefore, cells were seeded in flasks (4 million per flask) and incubated with or without different concentrations of zinc (10-, 40-, 70 µM) for either 48- or 77 h at 37 ℃, 5% CO₂. After incubation, cells were harvested. The cells were fixed in 4.5% formalin (Otto Fischar GmbH & Co. KG, Saarbrücken, Germany) and embedded into paraffin blocks. The cells, fixed in formalin, were centrifuged at 800×g for 10 min. The pellet was moved into a 1.5 mL Eppendorf tube and staining with Eosin-G solution was performed (0.5%, Carl Roth, Karlsruhe, Germany). Subsequently, the tubes were centrifuged at 4,000×g for 2 min. The supernatant was removed by using a Pasteur pipette. The pellet was then mixed by using 1 mL of 1% agarose (Merck KGaA, Darmstadt, Germany). After solidification (ca. 10 min), the gel was divided and moved into embedding cassettes. These cassettes were stored in 4.5% buffered formalin until the dehydration process. Cassettes were embedded in paraffin after dehydration and then stored at room temperature for further analyses.

RNA-isolation, quantification and reverse transcription

Two to eight sections of FFPE samples (10 µm thickness) were used to perform RNA-isolation via the Maxwell RSC RNA FFPE Kit (Promega, Madison, WI, USA). RNA was isolated according to the manufacturers’ instructions. Isolated RNA was eluted in 50 µL RNAse-free water and stored at −80 ℃. Subsequently, 1 µL of RNA was used for RNA quantification via the Qubit™ RNA Broad Range Assay (Thermo Fisher Scientific, Massachusetts, USA). Complementary DNA (cDNA) was synthesized by using the Revert Aid First Strand cDNA Synthesis Kit via Random-Hexamer-Primer, according to the manufacturers’ instructions (Thermo Fisher Scientific, Massachusetts, USA). 11 µL of concentrated RNA (1 µg total) was applied. After cDNA-synthesis, samples were stored at −80 ℃.

Quantitative real-time PCR

For expression analyses of the metallothionein gene MT2A in treated cell lines treated with different zinc concentrations, quantitative realtime polymerase chain reaction was performed with 40 cycles according to the manufacturer’s instructions (TaqMan^® Universal PCR Master Mix User Guide, 2014, Thermo Fisher Scientific, Massachusetts, USA). For each reaction, 1 µL of cDNA (50 ng per reaction), TaqMan^® Gene Expression Assays (Thermo Fisher Scientific, Massachusetts, USA) and the TaqMan^® Universal PCR Master Mix (Thermo Fisher Scientific, Massachusetts, USA) were used. qPCR reaction was analyzed with the LightCycler 480 Instrument II (Roche, Mannheim, Germany). For each sample, expression of MT2A was measured in technical and biological triplicate. Actin-beta (ACTB) and glycerinaldehyde-3-phosphate-dehydrogenase (GAPDH) were used as reference genes. The 2^−ΔCp method served for quantification of mRNA.

All samples were analyzed by using the MIQE-guidelines (11).

Statistical analysis

For statistical and graphical analyses, the R statistical programming environment (v3.2.3) was used.

Initially, the Shapiro-Wilks-test was used to check for normal distribution of the data. Based on the results of the Shapiro-Wilks-test, either a parametric or non-parametric test was implemented. Either the two-sided students t-test (parametric) or Wilcoxon Mann-Whitney rank sum test (non-parametric) was used for analyzing ordinal versus dichotomous variables. In case of analyzing ordinal variables versus categorical variables with more than two groups, either the ANOVA (parametric) or Kruskal-Wallis test (non-parametric) was applied to identify group differences.

Double-dichotomous contingency tables were investigated by using Fisher’s Exact test. To test the dependence of ranked parameters with more than two groups, the Pearson’s Chi-squared test was implemented. We tested correlations between metric variables by using the Spearman’s-rank-correlation test as well as the Pearson’s-product-moment correlation coefficient for linear modelling.

By reason of the multiple statistical testing, all P values had to be adjusted by using the false discovery rate (FDR) which indicates the expected portion of type I errors (false positives). The level of statistical significance was defined as P≤0.05 after adjustment. FDR adjustment was performed to control the number of false positives.

Results

Induction of MTs through Heavy Metals

An induction of MT expression by zinc could already be observed after 48 h (data not shown), which further increased until 77 h (Figure 1). MSTO-211H showed highest 2^−ΔCp values (ratio of the difference in MT expression of the sample between treatment and control) of MT expression at 70 µM zinc with 2^−ΔCp =0.25 (P=0.0833) while NCI-H2052 showed higher 2^−ΔCp values of 1.2 (P=0.0833). There seems to be a critical concentration of zinc, stimulating five-fold higher expression of MT2A by 70 µM compared to 40 µM of zinc. This was observed in all MPM cell lines.

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.

Influence of zinc supplementation on the response to cisplatin

Regarding apoptotic response of cell lines to cisplatin, all cell lines showed a specific effect depending on the applied zinc concentration.

High concentrations of zinc (70–90 µM) resulted in significantly decreased apoptosis (Figure 2). Apoptosis of cells decreases proportionally to the concentration of zinc supplementation. This dose-dependent effect was observed in each tested cell line. However, MRC-5 cells showed highest changes in apoptosis rate relatively to the zinc concentration with a decline of 50%. The lowest response was found for NCI-H2452 cells with a decrease of only about 10%.

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.

In addition, the measurable effect on the apoptosis rate varies in each cell line. While MRC-5 and NCI-H2452 cells showed a nearly linear decrease of apoptotic reaction (Figure 2B,2D), MSTO-211H and NCI-H2052 require 50–70 µM of zinc to achieve a notable reduction in the apoptosis of cisplatin (Figure 2A,2C). However, the apoptosis rate was always negatively correlated with the increasing concentration of zinc. Spearman’s rank correlation test was applied to calculate P values (P<0.0001 in all cell lines) and the spearman’s correlation coefficient ρ. MRC-5 cells showed the most linear decrease of apoptosis with ρ=−0.975 (Figure 2D).

Discussion

Despite the use of platinum compounds as a standard of care for MPM, tumors lack significant therapy response rates. The underlying biological mechanism for the rather poor efficacy of platinum-compounds remains largely unknown. Against this background, we hypothesized that metallothionein overexpression might be a potent mechanism of tumor cell protection against the effects of cisplatin. MTs can be detected in the serum (9,12), and the level of MTs is positively correlated with TNM (classification system of malignant tumors) and disease stage, and the grade of cancer progression; thus, MTs act as an enriched source of biomarkers in malignant tumors (9). Modification of MT expression levels may lead to changes in cellular platinum sensitivity, opening up the chance for therapy improvement.

MTs are involved in various cellular processes and therefore its relevance in numerous cancers have been broadly studied (9,13). MTs are involved in the regulation of oxidative stress, including the inhibition of reactive oxygen species (ROS) (14). Moreover, MTs have been shown to regulate inhibition of oxidative stress-induced apoptosis, thus being important in the development of drug-resistance mechanisms (15), as they show differential expression in various tumors. MTs are downregulated in carcinomas of the lung, liver and prostate, whereas they show overexpression in carcinomas of the breast, uterus and skin (9,16).

We hereby present a study on gene expression induction of MT2A in MPM cell lines, dependent on varying zinc concentrations. In addition, we analyzed the responses of cell lines after zinc supplementation and treatment with cisplatin.

We could observe a zinc dose-dependent induction of MT2A-expression, independent of histological subtypes. In particular, strong induction of MT2A-expression was observed at 70 µM zinc, after 77 h of incubation in each MPM cell line. Hence, there seems to be a critical concentration between 40–70 µM of zinc initiating measurable induction of MT expression. This critical concentration was also observed by analyzing apoptotic rates after cisplatin treatment and supplementation of zinc after 48 h of incubation with cisplatin in NCI-H2052 and MSTO-211H cells (Figure 2A,2C). This observation leads to the suggestion of a zinc dose-dependent induction of MT2A overexpression thus generating a protective barrier against the effects of cisplatin treatment. While MT2A-expression was induced also in NCI-H2452 cells, this cell line seems to show very low apoptotic effects (10-fold lower RLU), assuming other resistance mechanisms being accountable in this cell line (17-19). However, the influence of the different zinc concentrations in H2452-cells shows a similar trend compared to the other tested cell lines.

Multiple studies have been performed investigating a potential benefit of Zn²⁺ supplementation on immune function (10,20-22). Some suggest Zn²⁺ supplementation to have a beneficial effect as a supportive cancer treatment (23-25). Kocdor et al. could show a positive effect of zinc supplementation in docetaxel treated NSCLCs (26). In our in vitro study, we were not able to assess effects of zinc supplementation on immune function. It would probably be worthwhile to evaluate immune modulation after addition of low doses of zinc in vivo.

DNA damage repair is strongly influenced by zinc homeostasis (27). The tumor suppressor TP53, an important prognostic factor in MPM itself (17-19), is a transcription factor binding DNA through a structurally complex domain stabilized by a zinc atom. Without this complex domain, it would lose its DNA binding ability and could not induce cell cycle arrest or mediate signals for apoptosis (28).

Zinc homeostasis is a complex network of processes being incompletely understood. Therefore, transferring results of in vitro to in vivo studies remain problematic. Experiments show zinc to be crucial for an adequate immune response and therefore for the defense mechanism against pathogens (10). Supplementation of an appropriate zinc quantity could therefore help to maintain an optimal function of the immune system. An over- or under supplementation may have contrarily effects. Further experiments need to clarify the complex zinc homeostasis processes paving the way for further examinations.

Since MTs show a high binding affinity to all heavy metals, additional metals beside zinc may be considered for cisplatin treatment. Cadmium for example is a heavy metal occurring in high doses in cigarette smoke (29,30). Since cadmium and other heavy metals are inhaled while cigarette smoking, they possibly also could induce MT overexpression (29,31). Therefore, it could be speculated that smoking is a crucial factor for patient outcome through cisplatin treatment. This should be considered in further treatment approaches, hopefully leading to an improved clinical management of MPM patients.

Conclusions

High expression levels of MT2A reveal a negative impact on the cellular response to platin-based chemotherapeutic agents in vitro. We could show induction of MT2A expression by supplementation of zinc in a dose-dependent manner. Supplementation of zinc showed a negative effect to sensitivity against cisplatin treatment in all MPM cell lines analyzed, independent of histological subtypes. Additional effects, including immunoregulatory processes, have to be addressed in the future and further experiments are needed to shed light upon the complex zinc homeostasis processes paving the way for clinical application.

Based on the observed effect by zinc supplementation in this study, it could be speculated to be an important issue for MPM patients smoking cigarettes, which contain heavy metals like cadmium. Smoking during a platinum-based chemotherapy thus may lead to an early therapy failure. However, this has to be validated in further studies.

Acknowledgments

The abstract has been presented on DGP2020 (104. Jahrestagung der Deutschen Gesellschaft für Pathologie).

Funding: None.

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-22-2651/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-22-2651/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-22-2651/prf

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-22-2651/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.This study is based only on commercially available cell lines and thus has been approved by the ethical committee of the University of Duisburg-Essen that no additional vote has to be obtained. The immortalised cell lines used in this study were obtained from the American Type Culture Collection (ATCC).

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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