Onconase Restores Cytotoxicity in Dabrafenib-Resistant A375 Human Melanoma Cells and Affects Cell Migration, Invasion and Colony Formation Capability.

Raineri Raineri,Fasoli Fasoli,Campagnari Campagnari,Menegazzi Menegazzi,Gotte Gotte

doi:10.3390/ijms20235980

Abstract

Melanoma is a lethal tumor because of its severe metastatic potential, and serine/threonine-protein kinase B-raf inhibitors (BRAFi) are used in patients harboring BRAF-mutation. Unfortunately, BRAFi induce resistance. Therefore, we tested the activity of onconase (ONC), a cytotoxic RNase variant, against BRAFi-resistant cells to re-establish the efficacy of the chemotherapy. To do so, an A375 dabrafenib-resistant (A375DR) melanoma cell subpopulation was selected and its behavior compared with that of parental (A375P) cells by crystal violet, 5-Bromo-2’-deoxyuridine incorporation, and cleaved poly(ADP-ribose) polymerase 1 (PARP1) western blot measurements. Then, nuclear p65 Nuclear Factor kappaB (NF-κB) and IκB kinases-α/β (IKK) phosphorylation levels were measured. Gelatin zymography was performed to evaluate metalloproteinase 2 (MMP2) activity. In addition, assays to measure migration, invasion and soft agar colony formation were performed to examine the tumor cell dissemination propensity. ONC affected the total viability and the proliferation rate of both A375P and A375DR cell subpopulations in a dose-dependent manner and also induced apoptotic cell death. Among its pleiotropic effects, ONC reduced nuclear p65 NF-κB amount and IKK phosphorylation level, as well as MMP2 activity in both cell subpopulations. ONC decreased cell colony formation, migration, and invasion capability. Notably, it induced apoptosis and inhibited colony formation and invasiveness more extensively in A375DR than in A375P cells. In conclusion, ONC successfully counteracts melanoma malignancy especially in BRAFi-resistant cells and could become a tool against melanoma recurrence.

Highlights

Malignant melanoma is a highly aggressive skin cancer that has shown increasing incidence and mortality over recent decades [1]
Considering that drug resistance is one of the main factors limiting the efficacy of melanoma therapy, the BRAF-mutated-A375 human melanoma cell line was cultured with gradually increasing concentrations of dabrafenib until a cell subpopulation grew in the constant presence of 5 μM of this drug
Dabrafenib sensitivity was measured by crystal violet assay in both A375P and A375 dabrafenib-resistant (A375DR) cell subpopulations that were treated with dabrafenib concentrations ranging from 1 to 50 nM

Summary

Introduction

Malignant melanoma is a highly aggressive skin cancer that has shown increasing incidence and mortality over recent decades [1]. It was reported that, during vemurafenib treatment, resistant cells rapidly led to tumor onset in mice implanted with a mixture of sensitive and BRAFi-resistant A375 melanoma cells. Drug-resistant cells can quickly lose their adherence ability and gain both migratory and invasive capability This occurs through the penetration of the cells into the extracellular matrix (ECM), an event that leads to the formation of new tumors in other body sites [12]. Considering that ECM acts as a mechanical barrier against tumor-cell mobility, its degradation becomes crucial in supporting the tumor dissemination process In this regard, it is well known that matrix metalloproteinases (MMPs) cause ECM damage and that high levels of MMPs are secreted by melanoma cells [13,14]. The nuclear localization of β-catenin could be a specific determinant for the cell resistance to BRAFi

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Results

Conclusion