Original ArticleMLKL-PITPα signaling-mediated necroptosis contributes to cisplatin-triggered cell death in lung cancer A549 cells
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.
Section snippets
Reagents
Cisplatin was obtained from Qilu Pharmaceutical (Jinan, China). z-VAD(OME)-FMK was purchased from Santa Cruz Biotechnology (sc-311561; Dallas, TX, USA). Necrostatin-1 was obtained from Sigma-Aldrich (N9037; St. Louis, MO, USA). Necrosulfonamide was purchased from Abcam (ab143839; Cambridge, UK). Cholera Toxin Subunit B (Recombinant), Alexa Fluor® 594 Conjugate (CT-B) was obtained from Molecular Probes (C34777; Eugene, OR, USA). The human TNF-alpha ELISA Kit was obtained from Dakewe Biotech
Cisplatin induces both apoptosis and necroptotic-like cell death in lung cancer A549 cells
To test our hypothesis that necroptosis might contribute to cisplatin-triggered lung cancer cell death, we firstly pretreated A549 cells with zVAD-FMK, a pan-caspase inhibitor of apoptosis, before cisplatin treatment to see whether it could rescue all cells from cisplatin-induced cell death. Unexpectedly, we still noticed dead cells in A549 cells with zVAD-FMK pretreatment and the dead cells in supernatant were round and appeared to be identical, whereas, the morphology of the dead cells varied
Discussion
In many studies, the initiation of necroptosis can be ascribed to drug-induced up-regulation of RIPK3 or MLKL [1], [39]. However, in our study, the expression levels of RIPK1, RIPK3 and MLKL in A549 cells did not change after cisplatin treatment, which led us to reevaluate the mechanism and ultimately to ascribe it to autocrine TNF-α signaling. TNF-α is a classic necroptosis inducer and it triggers necroptosis especially when the capacity of caspases are inhibited [12]. Consistent with other
Funding
This work was supported by grants from the National Natural Science Foundation of China (31371405), and the National Basic Research Program of China (2015CB553701).
Acknowledgements
None.
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These two authors contributed equally to this work.