Several small molecules such as erastin and RSL are reported

Several small molecules, such as erastin and RSL3, are reported to induce ferroptosis in both normal and cancer TH-302 [21,22]. We confirmed that erastin-induced growth inhibition is an ideal ferroptosis model for cardiomyocytes. The erastin inhibits the growth of H9c2 cells in a Fe-dependent manner, because antioxidants Fer-1 could significantly rescue it. Whereas the H2O2-induced toxicity of H9c2 cells, most caused through apoptosis and necrosis, could not be rescued by Fer-1 [23]. Furthermore, the erastin-induced ferroptosis exhibited morphologically distinct and disparate from H2O2-induced cell death. Ferroptosis is mediated through the redoxactive metal Fe and ROS generation. We further confirmed that erastin-induced the ROS increase in erastin-treated H9c2 cells. All these observations suggested that erastin-induced ferroptosis is an ideal ferroptosis model of cardiomyocytes. The mechanisms that ENPP2/LPA signal in suppressing ferroptosis were further investigated. H9c2 cardiomyocytes express the LPA receptor, LPAR1, LPAR3, LPAR4 and LPAR5. Adenovirus mediated ENPP2 overexpression leads to increase LPA level in cell supernatants. And the supernatants from H9c2 ENPP2 transduced cells exhibit suppressive effect on erastin-induced ferroptosis. It is suggested that autocrine ENPP2/LPA might play a central role in regulation cardiomyocytes ferroptosis. Several genes such as GPX4, ACSL4 and NRF2 have recently been identified to regulate ferroptosis by directly or indirectly targeting iron metabolism and lipid peroxidation. GPX4 is an antioxidant enzyme which prevents the iron-mediated reactions of peroxides and induces ferroptotic cell death [24]. ENPP2 overexpression causes upregulation of GPX4 in H9c2 cells. The tumor suppressor p53 has been demonstrated to promote ferroptosis via a transcription-dependent mechanism [25]. NRF2 is a major target of ARF in p53-independent tumor suppression and considered as an executive molecule in ferroptosis [26]. NRF2 inhibition reverses the resistance of cancer cells to artesunate-induced ferroptosis [26,27]. It is indicated that NRF2 is as major regulator in ferroptosis. ACSL4 acts as an essential component for ferroptosis execution [28,29]. In erastin-induced ferroptosis of H9c2 cells, both NRF2 and ACSL4 are increased, whereas ENPP2 overexpression reduces their expression in erastin-treated H9c2 cells. The α6β4 integrin has been reported to protect adherent epithelial and carcinoma cells from ferroptosis induced by erastin [5]. It also had been reported to promote expression of ENPP2 autocrine and cooperates with LPA to activate multiple signals such as Rho and Rac small GTPases [30,31]. The collaboration with integrins may contribute to the inhibitory effect of ENPP2/LPA on H9c2 cell ferroptosis.
Conflicts of interest statement
Introduction
Regulated cell death (RDC) and ROS For decades, apoptosis was considered the only form of regulated cell death (RDC), as opposed to necrosis, which was regarded as an accidental and uncontrollable form of cell death, independent of any specific genetic program. However, accumulating evidence in the previous years showed that many forms of non-apoptotic, RDC exist [42], [43], [44], [45]. Nowadays, these RDC forms are regarded as a class of genetically regulated type of cell death, each one impinging on different (although overlapping) signaling pathways. Morphologically, non-apoptotic RDC are characterized by cellular swelling, plasma membrane damage, and cell content leakage [45]; however, peculiar morphological features exist, as evidenced by ferroptosis (see dedicated paragraph). Although differing in the molecular pathways that elicit them, non-apoptotic RDC share some downstream execution mechanisms. In this regard, bioenergetics and redox metabolism disruption have been proposed as a common basis in their progression and execution [45]. Among the non-apoptotic RDC forms described thus far, necroptosis is the best characterized. Other recently discovered forms include pyroptosis, parthanatos, NETotic cell death, and ferroptosis [43], [44], [45], [46]. We will focus below on redox involvement in two of these forms, necroptosis, as forerunner of the group and for which the role of ROS has been long investigated, and ferroptosis, an oxidative form of cell death for which alterations in redox metabolism constitute the main deciphered mechanism of action (Fig. 2).