Modulation of P2X4/P2X7/Pannexin-1 sensitivity to extracellular ATP via Ivermectin induces a non-apoptotic and inflammatory form of cancer cell death.

Dobrin Draganov,David Ann,Sailesh Gopalakrishna-Pillai,Sara Moeller,Yun-Ru Chen,Carrie Wang,Neta Zuckerman,Peter P Lee

doi:10.1038/srep16222

Abstract

Overexpression of P2X7 receptors correlates with tumor growth and metastasis. Yet, release of ATP is associated with immunogenic cancer cell death as well as inflammatory responses caused by necrotic cell death at sites of trauma or ischemia-reperfusion injury. Using an FDA-approved anti-parasitic agent Ivermectin as a prototype agent to allosterically modulate P2X4 receptors, we can switch the balance between the dual pro-survival and cytotoxic functions of purinergic signaling in breast cancer cells. This is mediated through augmented opening of the P2X4/P2X7-gated Pannexin-1 channels that drives a mixed apoptotic and necrotic mode of cell death associated with activation of caspase-1 and is consistent with pyroptosis. We show that cancer cell death is dependent on ATP release and death signals downstream of P2X7 receptors that can be reversed by inhibition of NADPH oxidases-generated ROS, Ca2+/Calmodulin-dependent protein kinase II (CaMKII) or mitochondrial permeability transition pore (MPTP). Ivermectin induces autophagy and release of ATP and HMGB1, key mediators of inflammation. Potentiated P2X4/P2X7 signaling can be further linked to the ATP rich tumor microenvironment providing a mechanistic explanation for the tumor selectivity of purinergic receptors modulation and its potential to be used as a platform for integrated cancer immunotherapy.

Highlights

High extracellular adenosine triphosphate (ATP) is one of the major characteristics of the tumor microenvironment[1,2]
Due to its ability to stimulate P2X4/P2X7/Pannexin-1 signaling in myeloid cells[14], we investigated Ivermectin as a prototype agent to modulate purinergic signaling in breast cancer cells
Cancer cells were treated with Ivermectin and analyzed for 1) PARP cleavage; 2) activation of caspase-1-a characteristic feature of the pyroptotic cell death pathway; and 3) caspase-3 activity typically observed in classical apoptosis

Summary

Introduction

High extracellular adenosine triphosphate (ATP) is one of the major characteristics of the tumor microenvironment[1,2]. The anti-tumor activity of both Ivermectin and structurally-related avermectins has been validated in xenogeneic[8] and immune-competent syngeneic mouse models[9]; in addition, the agents demonstrated broad anti-cancer potential for various solid and hematological malignancies[9]. To explain these activities, several mechanisms have been proposed. Avermectins synergize with chemotherapeutic agents known to induce immunogenic cell death, such as doxorubicin; and Ivermectin stimulates P2X4/P2X7/Pannexin-1 signaling, which augments inflammasome activation in myeloid cells[14] Considering these findings, we hypothesized that purinergic signaling may be involved in the mechanism by which Ivermectin kills cancer cells or modulates the immune system. Inflammatory and immunogenic forms of cell death are important in the context of cancer types where tumor antigens and therapeutic targets are limited or unknown, such as TNBC, which was used as a model in our studies

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Conclusion