Possible involvement of TRPM2 activation in 5-fluorouracil-induced myelosuppression in mice

Abstract

Transient receptor potential melastatin 2 (TRPM2) is an oxidative stress-sensitive Ca²⁺-permeable channel. The activation of TRPM2 by H₂O₂ causes cell death in various types of cells. 5-Fluorouracil (5-FU) is an important anticancer drug, but myelosuppression is one of the most frequent adverse effects. The involvement of oxidative stress in 5-FU-induced myelosuppression has been reported, and bone marrow cells are known to express TRPM2. The aim of this study was to investigate whether TRPM2 is involved in 5-FU-induced myelosuppression. Enhancement of H₂O₂-induced intracellular Ca²⁺ concentration ([Ca²⁺]_i) increase by 5-FU treatment was observed in human embryonic kidney 293 (HEK) cells stably expressing TRPM2 but not in HEK cells, indicating that 5-FU stimulates TRPM2 activation. In CD117 positive cells from wild type (WT) mouse bone marrow, 5-FU also enhanced the H₂O₂-induced [Ca²⁺]_i increases, but not in cells from Trpm2 knockout (KO) mice. In the CFU-GM colony assay, the 5-FU-induced reduction of colony number was alleviated by Trpm2 deficiency. Moreover, the reduction of leukocytes in blood by administration with 5-FU in WT mice was also alleviated in Trpm2 KO mice. The activation of TRPM2 in bone marrow cells seems to be involved in 5-FU-induced myelosuppression.

Introduction

5-Fluorouracil (5-FU) is one of the pyrimidine antimetabolite drugs and is widely used in the treatment of cancers such as colorectal cancer, esophageal cancer, pancreas cancer, and breast cancer (Conroy et al., 2011; Namer et al., 2006; Saltz et al., 2019; Shinoda et al., 2015). 5-FU exerts its antitumor activity through the inhibition of thymidylate synthase leading to cancer cell death, accompanied with the arrest of the cell cycle (Copur et al., 1995; Hartmann et al., 1961; Sawyer et al., 1984). The major adverse effect of 5-FU is myelosuppression due to cell cycle arrest, and myelosuppression is a dose-limiting factor for 5-FU-based chemotherapy (Conroy et al., 2011). Notably, the decrease in white blood cells (WBC) resulting from myelosuppression is a risk factor that readily causes infection and febrile neutropenia (Bucaneve et al., 2014; Kuderer et al., 2007). Therefore, minimizing the risk for myelosuppression is important for 5-FU-based chemotherapy. On the other hand, previous studies have suggested that the production of intracellular reactive oxygen species (ROS) is involved in 5-FU-induced cell toxicity and cell death in cancer cells (Aresvik et al., 2010; Pan et al., 2013). Furthermore, Numazawa et al. reported the possible involvement of oxidative stress in 5-FU-induced myelotoxicity (Numazawa et al., 2011). These studies suggest that not only cell cycle arrest but also oxidative stress-induced myelotoxicity contribute to 5-FU-induced myelosuppression. However, the mechanisms by which oxidative stress induces myelotoxicity leading to myelosuppression remain unclear.

Transient receptor potential melastatin 2 (TRPM2) is a Ca²⁺-permeable channel that is activated by oxidative stress induced by ROS such as H₂O₂ (Hara et al., 2002). The expression of TRPM2 has been observed in the brain, hematopoietic stem cells, pancreatic β-cells, lungs, and leucocytes (Hara et al., 2002; Nazıroğlu et al., 2012; Perraud et al., 2001; Togashi et al., 2006; Wehage et al., 2002).

In various types of cells, the activation of TRPM2 channels induced by ROS has been known to mediate cell death through an intracellular Ca²⁺ overload (Ishii et al., 2014; Zhang et al., 2003). Therefore, TRPM2 activation leading to cell death may be involved in 5-FU-induced myelotoxicity. However, there is no report investigating TRPM2 activation in 5-FU-induced myelotoxicity leading to myelosuppression.

In the present study, we firstly investigated the effect of 5-FU on TRPM2 activation, and found that 5-FU enhances TRPM2 activation induced by H₂O₂ in TRPM2-expressed human embryonic kidney 293 (HEK) cells, hematopoietic stem cells (HSCs), and hematopoietic progenitor cells (HPCs). In addition, the reduction of colony number and leukocytes in blood by 5-FU administration was alleviated by Trpm2 deficiency. Our finding suggested that TRPM2 contributes to 5-FU-induced myelotoxicity leading to myelosuppression.