Investigations on Zinc Isotope Fractionation in Breast Cancer Tissue Using in vitro Cell Culture Uptake-Efflux Experiments.

Kathrin Schilling,Helen Sheldon,Fiona Larner,Syed Haider,Adrian L Harris,Alex N Halliday,Christopher J Schofield

doi:10.3389/fmed.2021.746532

Abstract

Zinc (Zn) accumulates in breast cancer tumors compared to adjacent healthy tissue. Clinical samples of breast cancer tissue show light Zn isotopic compositions (δ66Zn) relative to healthy tissue. The underlying mechanisms causing such effects are unknown. To investigate if the isotopic discrimination observed for in vivo breast cancer tissue samples can be reproduced in vitro, we report isotopic data for Zn uptake-efflux experiments using a human breast cancer cell line. MDA-MB-231 cell line was used as a model for triple receptor negative breast cancer. We determined Zn isotope fractionation for Zn cell uptake (Δ66Znuptake) and cell efflux (Δ66Znefflux) using a drip-flow reactor to enable comparison with the in vivo environment. The MDA-MB-231 cell line analyses show Zn isotopic fractionations in an opposite direction to those observed for in vivo breast cancer tissue. Uptake of isotopically heavy Zn (Δ66Znuptake = +0.23 ± 0.05‰) is consistent with transport via Zn transporters (ZIPs), which have histidine-rich binding sites. Zinc excreted during efflux is isotopically lighter than Zn taken up by the cells (Δ66Znefflux = −0.35 ± 0.06‰). The difference in Zn isotope fractionation observed between in vitro MDA-MB-231 cell line experiments and in vivo breast tissues might be due to differences in Zn transporter levels or intercellular Zn storage (endoplasmic reticulum and/or Zn specific vesicles); stromal cells, such as fibroblasts and immune cells. Although, additional experiments using other human breast cancer cell lines (e.g., MCF-7, BT-20) with varying Zn protein characteristics are required, the results highlight differences between in vitro and in vivo Zn isotope fractionation.

Highlights

Over the last decade analysis of natural metal isotopes has emerged as an interdisciplinary field of substantial biomedical potential, including diagnosis and defining disease mechanisms [1,2,3,4,5,6,7,8,9,10,11,12,13,14]
Two recently published breast cancer studies found that Zn dyshomeostasis linked to carcinogenesis is reflected in enrichment of light Zn isotopes in malignant breast tissue compared to adjacent histologically healthy tissue [3, 5]
The Zn blank for the experimental setup ranged between 0.5 and 2.7 ng Zn ml−1 after 1 h effluent collection and decreased to 0.6–1.4 ng Zn ml−1 after 2.5 h (Table 2). This corresponds to 0.7–4.8% of Zn collected in the effluent of the cell line efflux experiment

Summary

Introduction

Over the last decade analysis of natural metal isotopes has emerged as an interdisciplinary field of substantial biomedical potential, including diagnosis and defining disease mechanisms [1,2,3,4,5,6,7,8,9,10,11,12,13,14]. Two recently published breast cancer studies found that Zn dyshomeostasis linked to carcinogenesis is reflected in enrichment of light Zn isotopes in malignant breast tissue compared to adjacent histologically healthy tissue [3, 5]. Zinc homeostasis in humans is maintained by multiple proteins which tightly regulate intracellular Zn concentrations. Among these proteins are influx-controlling Zn importers (SLC39A; ZIP1-ZIP14), efflux-controlling Zn transporters (SLC30A; ZnT1-ZnT10), and Zn-sequestering proteins (e.g., metallothionein) [e.g., [15,16,17,18,19]]. Cancer-induced Zn dyshomeostasis has been related to up- or down-regulation of Zn proteins [23] and changes in their coordination and ligand chemistry [24]. Upregulation of ZIP6, ZIP7, ZIP10, ZnT2 and overexpression of metallothionein have been proposed to implicate higher Zn levels in malignant relative to healthy tissue [25,26,27,28,29]

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Conclusion