Haloperidol, a sigma receptor 1 antagonist, promotes ferroptosis in hepatocellular carcinoma cells
Abstract
Ferroptosis is a novel form of cell death, which is characterized by accumulation of reactive oxygen species (ROS). Sigma 1 receptor (S1R) has been suggested to function in oxidative stress metabolism. Both erastin and sorafenib significantly induced S1R protein expression. Haloperidol strongly promoted erastin- and sorafenib-induced cell death, which was blocked by ferrostatin-1 but not ZVAD-FMK or necrosulfonamide. During ferroptosis, haloperidol substantially increased the cellular levels of Fe2+, GSH and lipid peroxidation. Furthermore, several ferroptosis-related protein targets were up-regulated in the absence of haloperidol. Thus, Our study identified an association between haloperidol and ferroptosis for the first time. Our analyses of a combination of drugs may provide a novel strategy of hepatocellular carcinoma (HCC) therapy.
1. Introduction
Ferroptosis was recently identified as a new form of regulated cell death (RCD) by Brent R. Stockwell’s laboratory in 2012. Fer- roptosis differs from apoptosis and other major forms of RCD (e.g., necroptosis and autophagic cell death) in many aspects. For example, it is characterized by cell volume shrinkage and increased mitochondrial membranes and is mediated by iron-dependent lipid peroxide accumulation [1]. Ferroptosis may occur in both physiological and pathological processes and was specifically found in several types of tumor cells [2e4]. The iron-dependent accu- mulation of ROS finally induces ferroptosis through inactivation of glutathione peroxidase 4 (GPX4) or glutathione (GSH) deficiency [2]. To date, several ferroptosis-related targets have been identified, including nuclear factor erythroid 2-related factor 2 (NRF2) [5], haeme oxygenase 1 (HO-1) [6], GPX4 and the well-known p53 [7]. However, detailed signal transduction pathways and key tran- scriptional regulators of ferroptosis remain unknown.
S1R is a protein modulator that is associated with a variety of neurological diseases and ischaemia-reperfusion injury [8]. S1R is generally localized at the endoplasmic reticulum (ER), mitochon- drial membrane [9] and the plasma membrane [10]. Recently, it was observed at the nuclear envelope [11]. However, its localization was altered following intracellular oxidative stress [12]. S1R agonists have been shown to contribute to cellular protection by suppress- ing ischaemia-induced ROS production. In contrast, S1R antagonists decreased tumor cell survival [9,13,14]. Furthermore, a recent metabolic study showed an increase in oxidative stress markers (including oxidized GSH and GSH) in the liver of S1R-knockout (S1RKO) mice compared to those of wild-type (WT) mice [15]. Meanwhile, another study reported elevated ROS levels and decreased GSH levels in Müller cells harvested from S1RKO mice compared with those of cells from WT mice [16]. Although the exact role of S1R has not been confirmed, these findings strongly suggest that S1R plays an important role in oxidative stress metabolism.
S1Rs are found in many tissues in humans, rats and mice, but their expression is generally concentrated in the central nervous system. S1R is highly abundant in the liver as well. Various classes of psychotropic drugs bind to S1R, including haloperidol, which has a relatively high affinity for S1R [17]. In this study, we identified haloperidol as a promoter of ferroptosis in hepatocellular carci- noma (HCC) cells, in both erastin- and sorafenib-induced condi- tions. Furthermore, we demonstrated that haloperidol increases iron accumulation and lipid peroxidation in ferroptosis and con- tributes to this process by inducing the expression of S1R and other regulators involved in ferroptosis. Collectively, our results indicate the novel pharmacological action of haloperidol in promoting cellular ferroptosis.
2. Methods
2.1. Regents
Antibodies to NRF2 (ab62352), S1R (ab151288), HO-1 (ab68477) and GPX4 (ab125066) were all obtained from Abcam (Shanghai, China). Erastin (No. S7242), sorafenib (No. S7397), ZVAD-FMK (No. S7023), and ferrostatin-1 (No. S7243) were obtained from Selleck (Shanghai, China). Haloperidol (H1512) and necrosulfonamide (ab143839) were obtained from Sigma Aldrich (Shanghai, China) and Abcam (Shanghai, China), respectively.
2.2. Cell culture
The human HCC cell lines Hep G2 and Huh-7, maintained at our institute, were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (HyClone) in a hu- midified incubator with 5% CO2 and 95% air.
2.3. Cell viability assay
Cell viability was evaluated with a Cell Counting Kit-8 (CCK-8) (Dojindo Laboratories, Shanghai, China) according to the manu- facturer’s instructions. WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4- nitrophenyl)-5(2,4-disulfophenyl)-2H-tetrazolium monosodium salt], which is a highly water-soluble tetrazolium salt, was used to evaluate cell viability by CCK-8 assays. WST-8 is a next-generation, sensitive reagent that serves as a chromogenic indicator for NADH. Following the reaction, NADH can reduce WST-8 at neutral pH values in the presence of 1-methoxy PMS to produce a water- soluble formazan dye, which is used as a cell viability indicator in cell proliferation and death assays by measuring absorption at 450 nM.
2.4. Western blot
Cells were lysed with radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitor complexes to harvest proteins. Then, the proteins in the supernatants of each sample were sepa- rated by 8%e12% SDS poly-acrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (pore size 0.45 mm). After the membrane was blocked (5% milk) for approxi- mately 1 h, it was incubated overnight at 4 ◦C with each primary antibody, followed by incubation with secondary antibody for 2 h at room temperature. Finally, we visualized the blots using an enhanced chemiluminescence system.
2.5. Iron assay
The intracellular iron concentration in cell lysates was assessed using an iron colorimetric assay kit purchased from Biovision (Milpitas, California, USA) according to the manufacturer’s in- structions. In this assay, ferric carrier proteins dissociate ferric iron
into solution in the presence of acid buffer. After reduction to the ferrous form (Fe2+), iron reacts with Ferene S to produce a stable colored complex. This kit can thus measure iron level in samples.
2.6. Lipid peroxidation assay
The intracellular malondialdehyde (MDA) concentration in cell lysates was assessed using a lipid peroxidation colorimetric assay kit purchased from Biovision (Milpitas, California, USA) according to the manufacturer’s instructions. Lipid peroxidation, which pro- duces MDA as an end product, occurs as a result of oxidative damage and is a marker for oxidative stress.
2.7. GSH assay
The intracellular GSH level in cell lysates was assessed using a GSH colorimetric assay kit purchased from Biovision (Milpitas, California, USA) according to the manufacturer’s instructions.
2.8. Statistical analysis
All data are expressed as the mean ± SD. Data were analyzed using two-tailed Student’s t tests for comparison of two groups or ANOVA LSD tests for comparisons among multiple groups. Signifi- cance was defined as p < 0.05. 3. Results 3.1. Identification of haloperidol as a promoter of erastin- and sorafenib-induced cell death As previously described [15,16], S1R is involved in oxidative stress metabolism. To determine whether inducing ferroptosis by erastin and sorafenib affects S1R, we analyzed the protein levels of S1R in two HCC cell lines. The results indicated that both erastin and sorafenib significantly induced S1R protein expression (Fig. 1A and B). Next, we examined the effect of haloperidol (an S1R antagonist) on ferroptosis. Cell viability was measured in Hep G2 and Huh-7 cell lines following treatment with 10 mM erastin or 5 mM sorafenib in the absence or presence of haloperidol. Indeed, haloperidol promoted erastin- and sorafenib-induced cell death in a dose- (Fig. 1C and D) and time- (Fig. 1E and F) dependent manner in both HCC cell lines. These findings suggest that haloperidol promotes both erastin- and sorafenib-induced cell death. 3.2. Haloperidol promotes cellular ferroptosis but not necroptosis or apoptosis Next, we added ferrostatin-1 (a strong ferroptosis inhibitor), ZVAD-FMK (a strong apoptosis inhibitor) and necrosulfonamide (a strong necroptosis inhibitor) to address the mechanism of halo- peridol in ferroptosis. Ferrostatin-1 inhibits ferroptosis by depressing cellular lipid peroxidation. Similar to results from pre- vious studies [1,2,18e20], erastin- and sorafenib-induced cell death in both HCC cell lines was blocked by ferrostatin-1 but not ZVAD- FMK or necrosulfonamide. As expected, in the presence of 10 mM haloperidol, the aggravated erastin- and sorafenib-induced cell death was also blocked by ferrostatin-1 in both HCC cell lines, but the other inhibitors had no significant effects (Fig. 2A and B). These findings indicate that haloperidol at 10 mM promotes cellular fer- roptosis but not necroptosis or apoptosis.
3.3. Haloperidol accelerates iron accumulation and lipid peroxidation, while facilitating GSH depletion, in ferroptosis
Accumulation of ferrous iron and lipid peroxidation products were reported to participate in erastin- and sorafenib-induced ferroptosis [5]. Thus, we analyzed MDA and Fe2+ levels to determine whether haloperidol-promoted ferroptosis is associated with the regulation of iron and lipid peroxidation. MDA, an end product of lipid peroxidation, was substantially elevated in the presence of haloperidol compared to that with only erastin or sorafenib in the two HCC cell lines (Fig. 3C and D). Fe2+ participates in the Fenton reaction, which, along with lipids, produces ROS. In the present iron assays, haloperidol also aggravated the accumulation of cellular Fe2+ compared to that of only erastin or sorafenib in both HCC cell lines (Fig. 3E and F). According to previous studies, GSH depletion plays an important role in ferroptosis because of the consequent GPX4 degradation, which is the core step of the whole process [2].
As expected, treatment with haloperidol accelerated erastin- and sorafenib-induced GSH depletion in both HCC cell lines (Fig. 3A and B). Thus, haloperidol augments three major biochemical events during ferroptosis.
3.4. Haloperidol influences many ferroptosis-related targets
NRF2 is an important antioxidative transcription factor, regu- lating a variety of cytoprotective genes in antioxidant process and drug metabolism by binding to antioxidant response elements (AREs). HO-1, which has been shown to be up-regulated in numerous cancers, is a major source of intracellular iron. GPX4 is a unique member of the GPXs. In contrast to other proteins in this group, GPX4 catalyzes the reduction of intracellular lipid peroxides, protecting cells from their destructive effects. All the above can significantly influence ferroptosis. We confirmed that protein levels of these enzymes were up-regulated during ferroptosis in the HCC cell lines, similar to results from previous studies [2,5,6]. Following haloperidol treatment, we observed even higher protein levels compared with those following treatment with only erastin and sorafenib (Fig. 4A), especially for HO-1, which can accelerate fer- roptotic cell death (Fig. 4B and C). The expression of S1R was elevated as well, demonstrating that haloperidol induces higher S1R expression in the process of ferroptosis (Fig. 4A). Collectively, these findings indicate that haloperidol promotes erastin- and sorafenib-induced ferroptosis by influencing related targets.
4. Discussion
In this study, we confirmed that haloperidolda psychotropic drugdaugments the effect of erastin- and sorafenib-induced fer- roptosis, while any single use of these three drugs at low doses did not have the same effect. Haloperidol not only plays a role in cellular metabolism but also influences a variety of targets involved in ferroptosis.
Ferroptosis is a novel form of RCD. The number of identified types of RCD is still increasing and currently includes apoptosis, necroptosis, pyroptosis, anoikis, entosis, cornification, netosis, parthanatosis, mitotic catastrophe, autophagy and the recently discovered ferroptosis [21]. Ferroptosis does not cause the chro- matin condensation that occurs in apoptosis, the destruction of plasma membrane integrity that occurs in necrosis, or the double membrane-layered vacuoles in autophagy; uniquely, it results in mitochondrial shrinkage and increased mitochondrial membrane density [1]. Initially, relevant studies focused on RAS-selective cell death with erastin. Recently, ischaemia reperfusion diseases and various types of cancers were shown to involve ferroptosis as well [22]. Thus, it is important to elucidate the signalling pathways and metabolic characteristics of ferroptosis and related pharmacolog- ical agents.
Human HCC is the second leading cause of cancer-related death worldwide [23]. Currently, no clinical treatments, including sur- gery, result in satisfactory outcomes. Sorafenib, a multi-kinase in- hibitor, is the first drug approved by the U.S. Food and Drug Administration for patients with advanced HCC [24,25]. However, drug resistance to sorafenib is a problem [26]. Many studies have focused on other targets and new drugs [27,28]. Other reports investigated its ability to induce apoptosis [29,30]. Recently, several studies demonstrated that sorafenib induced ferroptosis via non- kinase targets in HCC cell lines [4,5,18,31]. Furthermore, another study found that the serum concentration of oxidative stress response markers was related to progression-free survival in a portion of HCC patients undergoing sorafenib treatment [32], suggesting the important role sorafenib plays in the survival of HCC patients by inducing ferroptosis. In this study, we demonstrated that haloperidol strengthened sorafenib-induced ferroptosis in HCC cell lines for the first time, even with both drugs at relatively lower doses, indicating that haloperidol may help HCC patients treated with sorafenib by reducing the dosage or reinforcing the effectiveness of this drug or both.
S1R is a special receptor chaperone, which can be activated or deactivated by specific ligands. Manipulation of S1R was either cytoprotective or cytotoxic in tumor cells [12,15,33,34]. However, some normal cell types, even those with high levels of S1R, are resistant to the cytotoxic effects of S1R antagonists [35]. Thus although S1R is abundant in tumors and rapidly dividing normal tissues, S1R antagonist-induced cell death only occurs in the former [36,37]. Many previous studies explored the apoptotic effects of S1R antagonists. Our current study demonstrated for the first time that haloperidol has a close relationship with ferroptosis in cellular metabolism and lipid peroxidation. Additionally, haloperidol induced the expression of HO-1. In a recent study, erastin was shown to induce HO-1 expression in HT-1080 fibrosarcoma cells, and overexpression of HO-1 accelerated erastin-triggered ferrop- totic cell death [6]. Combined with our results, these findings indicate that haloperidol promotes ferroptosis partly by inducing HO-1 expression.
Previous studies strongly suggested that GPX4 protected cells from detrimental effects of lipid peroxides [2,38,39]. GPX4 is believed to play a central role in ferroptosis. Ferroptotic cell death can be induced by two classes of small-molecule compounds. By depleting the intracellular GSH pool, class 1 compounds such as erastin and buthionine sulfoximine can reduce GPX4 activity and increase ROS levels. Class 2 compounds, such as Ras selective lethal 3 (RSL3), can directly inhibit GPX4 and cause accumulation of lipid peroxides. Both pathways are finally mediated by GPX4 [40,41]. A recent study demonstrated that erastin induced GPX4 degradation in PANC1 cells [42]. Interestingly, erastin- and sorafenib-induced ferroptosis led to up-regulation of GPX4 in HCC cells in the pre- sent study. Analogous differences were found in another research team’s studies as well [5,42]. They observed up-regulation of NRF2 through ferroptosis in HCC cells, similar to our results, and a degradation of NRF2 through ferroptosis in pancreatic cancer cells. The differences between ferroptosis in various cancer cells thus require further studies for clarification.Here, we identified a connection between haloperidol and fer- roptosis for the first time. By assessing a combination of drugs, our findings may provide a novel strategy of HCC therapy. Furthermore, these results increased our understanding of ferroptosis as well.