Citation

  • Authors: Pellavio G. et al.
  • Year: 2022
  • Journal: Cells 11 1892
  • Applications: in vitro / DNA / jetOPTIMUS
  • Cell types:
    1. Name: Malignant pleural mesothelioma cells
    2. Name: Mesothelial cells

Method

Mesothelial and MPM cells were seeded into 2 mL plastic dishes, and HyPer7-NES transfection (3 µg DNA / dish) was performed when cells reached the 60–70% confluency using the JetOPTIMUS DNA transfection reagent (# 117-15, Polyplus transfection, IllkirchGraffenstaden, France) according to the manufacturer’s instructions. Briefly, the medium was removed and substituted with Opti-MEM containing the plasmid DNA and the transfection reagent. Plasmid DNA (3 µg) was diluted in JetOPTIMUS Buffer (# 717-60, Polyplus transfection, Illkirch-Graffenstaden, France) and then mixed with JetOPTIMUS reagent according to the suggested ratio 1:1 between µg of DNA and µL of transfection reagent. After 10 min incubation at room temperature, the solution containing the DNA was added dropwise to the cells and incubated at 37 ◦C for 4 h. Then, the media were exchanged for fresh culture media. All the experiments were performed 24 h after transfection.

Abstract

Malignant pleural mesothelioma (MPM) is an aggressive cancer of the pleural surface and is associated with previous asbestos exposure. The chemotherapy drug is one of the main treatments, but the median survival ranges from 8 to 14 months from diagnosis. The redox homeostasis of tumor cells should be carefully considered since elevated levels of ROS favor cancer cell progression (proliferation and migration), while a further elevation leads to ferroptosis. This study aims to analyze the functioning/role of aquaporins (AQPs) as a hydrogen peroxide (H2O2) channel in epithelial and biphasic MPM cell lines, as well as their possible involvement in chemotherapy drug resistance. Results show that AQP-3, -5, -6, -9, and -11 were expressed at mRNA and protein levels. AQP-6 was localized in the plasma membrane and intracellular structures. Compared to normal mesothelial cells, the water permeability of mesothelioma cells is not reduced by exogenous oxidative stress, but it is considerably increased by heat stress, making these cells resistant to ferroptosis. Functional experiments performed in mesothelioma cells silenced for aquaporin-6 revealed that it is responsible, at least in part, for the increase in H2O2 efflux caused by heat stress. Moreover, mesothelioma cells knocked down for AQP-6 showed a reduced proliferation compared to mock cells. Current findings suggest the major role of AQP-6 in providing mesothelioma cells with the ability to resist oxidative stress that underlies their resistance to chemotherapy drugs.

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