MLKL-PITPα signaling-mediated necroptosis contributes to cisplatin-triggered cell death in lung cancer A549 cells

Highlights

• Autocrine TNF-α contributes to the initiation of cisplatin-triggered necroptosis of lung cancer cells.

• PITPα interacts with MLKL.

• PITPα is involved in the function of MLKL in necroptosis.

Abstract

Necroptosis has been reported to be involved in cisplatin-induced cell death, but the mechanisms underlying the occurrence of necroptosis are not fully elucidated. In this study, we show that apart from apoptosis, cisplatin induces necroptosis in A549 cells. The alleviation of cell death by two necroptosis inhibitors—necrostatin-1 (Nec-1) and necrosulfonamide (NSA), and the phosphorylation of mixed lineage kinase domain-like protein (MLKL) at serine 358, suggest the involvement of receptor-interacting protein kinase 1 (RIPK1)-RIPK3-MLKL signaling in cisplatin-treated A549 cells. Additionally, the initiation of cisplatin-induced necroptosis relies on autocrine tumor necrosis factor alpha (TNF-α). Furthermore, we present the first evidence that phosphatidylinositol transfer protein alpha (PITPα) is involved in MLKL-mediated necroptosis by interacting with the N terminal MLKL on its sixth helix and the preceding loop, which facilitates MLKL oligomerization and plasma membrane translocation in necroptosis. Silencing of PITPα expression interferes with MLKL function and reduces cell death. Our data elucidate that cisplatin-treated lung cancer cells undergo a new type of programmed cell death called necroptosis and shed new light on how MLKL translocates to the plasma membrane.

Introduction

The core of cancer therapy is to remove cancer cells from normal tissue. Apart from surgery, chemotherapy is the strategy that best manifests this idea. Cisplatin (cis-diamminedichloroplatinum(II), DDP), a chemotherapeutic drug targeting DNA, is still incorporated in a broad range of combined antineoplastic chemotherapy regimens for various solid tumors. Inasmuch as cisplatin remains a first-line agent in lung cancer chemotherapy, it is pivotal to fully understand its cytotoxic mechanisms in lung cancer cells. Generally, apoptosis is widely believed to be the fate of the cisplatin-treated cells. Recent researches have shown that cisplatin may cause a new type of programmed cell death known as necroptosis [1], [2], [3]. Necroptosis is substantially involved in pathological states such as organ inflammation [4], [5], arteriosclerosis [6], cerebral ischemia [7], and antiviral responses [8]. Mechanisms that initiate necroptosis differ. Chemical compounds (MNNG, shikonin), protein ligands (TNF-α, TRAIL), and analog of nucleic acids (poly (I:C)) are suggested to trigger necroptosis under certain circumstances [9], [10], [11], [12], [13]. Several antineoplastic drugs (5-fluorouracil, Obatoclax, and cisplatin) are demonstrated to kill tumor cells through necroptosis as well [14], [15], [16].

Interactions between RIPK1-RIPK3 or RIPK3-RIPK3 and the ensuing recruitment and phosphorylation of MLKL are widely acknowledged necroptosis pathways [17], [18], [19]. Phosphorylated MLKL then forms oligomers and translocates to the plasma membrane [20], [21]. It has been demonstrated that to kill cells, MLKL needs to bind on some species of phosphatidylinositol phosphates (PIPs) at the plasma membrane, and interfering with the synthesis of PIPs inhibits its killing ability [22], [23], [24]. However, the underlying mechanisms are not fully explored. PITPα belongs to the family of PITPs. It participates in the transfer of phosphatidylinositol (PI) between membranes, as well as in the regulation of PIPs synthesis [25]. Thus, we hypothesized that PITPα might assist in the function of MLKL.

In this study, we show that apart from apoptosis, cisplatin-treated A549 cells undergo necroptosis. We show that MLKL forms oligomers and shifts from the cytosol to the plasma membrane during necroptosis, and its oligomerization and membrane translocation partially depend on PITPα. Additionally, autocrine TNF-α signaling contributes to the initiation of cisplatin-induced necroptosis.