FIN56, a novel ferroptosis inducer, triggers lysosomal membrane permeabilization in a TFEB-dependent manner in glioblastoma

Objective: To explore the anti-tumor effect of FIN56, a novel ferroptosis inducer, on glioblastoma and its underlying mechanisms. Methods: Two human glioblastoma cell lines, LN229 and U118 were applied in this study. Anti-tumor effect was measured by CCK-8 assay, EdU assay and cell cycle analysis. Fluorescent probes, immunofluorescence, plasmid transfection, shRNA knocking out, reverse transcription PCR, western blot analysis, and transmission electron microscopy were used to study the underlying mechanisms. At last, a subcutaneous nude mice model was used to study the anti-tumor effect of FIN56 in vivo. The GraphPad Prism software program was applied for statistical analysis. Results: FIN56 decreased cell viability, inhibited cell proliferation and caused cell cycle arrest on LN229 and U118 cells. Further study showed that FIN56 induced ferroptosis and induced lysosomal membrane permeabilization in a ferroptosis and transfactor EB dependent manner. Animal study demonstrated that FIN56 inhibited glioma growth and caused ferroptosis in vivo. Conclusion: FIN56 is a promising anti-tumor compound.


Introduction
Glioblastoma (GBM) is one of the most malignant primary tumors in the central nervous system [1]. Although a comprehensive treatment including surgical resection, chemo-and radiotherapy exists, the prognosis of patients with GBM still remains poor [1,2]. Apoptosis, a programmed cell death, is one of the most important signaling pathways involved in various anti-cancer treatment [3]. But tumor cells will develop resistance to apoptosis after repeated treatment [4].
Except for apoptosis, many programmed cell death modes have been discovered, including autophagy, necroptosis, pyroptosis and ferroptosis [5]. Ferroptosis, a programmed cell death denominated by Dixon in 2012, is reported to be involved in various medical diseases, including neurodegeneration, organ failure, and cancer inhibition [6]. Ferroptosis is characterized by intracellular iron accumulation and subsequent lipid peroxidation [6,7]. Morphologically, shrunken mitochondria and increased density of the mitochondrial membrane can be detected in cells with ferroptosis [5]. Cystine-glutamate antiporter (xCT), glutathione peroxidase 4 (GPX4), ferroptosis suppressor protein 1 (FSP1), and GTP cyclohydrolase-1 (GCH1) are reported to be involved in the regulation of ferroptosis by limiting the production of lipid peroxides [8].
Lysosomal membrane permeabilization (LMP), another programmed cell death mode, is a lysosome dependent cell death process [9]. In LMP, impaired lysosomal membrane induces the release of specific lysosomal enzymes into the cytoplasm, which triggers Ivyspring International Publisher a cascade of regulated cell death signaling [10]. Among all the lysosomal enzymes involved in LMP, cathepsin B and cathepsin D are the most important [11]. Recently, lysosomes are reported to be involved in the regulation of ferroptosis [12]. Gao et al. showed that erastin, a ferroptosis inhibitor, induced LMP, which contributed to ferroptosis [13]. Zhou et al reviewed that Stat3-dependent LMP facilitates the induction of ferroptosis [14]. But until now, few studies illustrated the relationship between ferroptosis and LMP. The underlying mechanisms are still unknown.
FIN56, a novel ferroptosis inducer, triggers ferroptosis by increasing the degradation of GPX4 [15]. FIN56 also activates squalene synthase, an enzyme involved in the cholesterol synthesis [16]. But whether FIN56 has anti-tumor effect is still unknown. In our study, we tried to study the potential role of FIN56 on GBM. Our results showed that FIN56 inhibited GBM cell growth both in vitro and in vivo. Besides, FIN56 increased lipid peroxidation and ROS production, which suggested that FIN56 induced ferroptosis in GBM cells. We also found that FIN56 triggered LMP. Further studies demonstrated that FIN56 induced LMP in a transfactor EB (TFEB)-dependent manner. As a result, FIN56 is a potential compound in treating GBM patients.

Cell culture
LN229 and U118, two GBM cell lines, were obtained from the Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and cultured in Dulbecco's modified Eagle's medium (DMEM, ThermoFisher Scientific; Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; ThermoFisher Scientific).

EdU assay
An EdU Assay Kit (Ribobio; Guangzhou, China) was used to test cell proliferation after different treatment. LN229 and U118 cells were seeded into 24-well, flat-bottomed plates and incubated overnight. Then cells were treated with 1 μM FIN56 or DMSO. 24 h later, cells were incubated with EdU according to the manufacturer's instructions. EdU positive cells were calculated under fluorescence microscopy (Leica DMi8; Wetzlar, Germany).

Cell cycle analysis
LN229 and U118 cells were seeded into 6-well, flat-bottomed plates and incubated overnight. After treatment with 1 μM FIN56 or DMSO for 24 h, LN229 and U118 cells were collected and fixed with 70% ethanol. Then cells were incubated with propidium iodide (PI) supplemented with RNase (Becton Dickinson, San Diego, CA). Cell cycle analysis was performed on a C6 flow cytometer (BD Biosciences; San Jose, CA, USA).

Transmission electron microscopy
The ultrastructure of glioma cells was observed under transmission electron microscopy after LN229 and U118 cells were treated with 1 μM FIN56 or DMSO. Experiments were carried out as described previously [2]. Images were obtained using a JEM-1200EX II electron microscope (JEOL; Tokyo, Japan).
After being incubated with AO (5 μg/mL) for 15 min, LN229 and U118 cells were treated with 1 μM FIN56 or DMSO. Then images were obtained by a Leica DMi8.
Lipofectamine 2000 reagent (ThermoFisher Scientific) was used for transient transfections. 48 h later, transfected cells were treated with 1 μM FIN56 or DMSO for 24 h. Images were taken by a Leica SP5 Confocal Microscope.

Animal study
In vivo study was carried out by a subcutaneous nude mouse model. Briefly, 105 LN229 cells were injected subcutaneously into the right shoulder of 4week-old nude mice (SLAC laboratory animal Center; Shanghai, China). 2 weeks later, nude mice (n = 10) were divided into two groups, control group and FIN56 treatment group. Subcutaneous tumors were harvested 30 days after treatment. Tumors were fixed and paraffin-embedded for immunohistochemistry.

Statistical analysis
Unpaired t-tests were performed using GraphPad Prism software program (Version 6.07; La Jolla, CA, USA). Results are presented as the mean ± SE. P-values < 0.05 were considered statistically significant.

FIN56 inhibits cell growth in GBM cells
Firstly, we tested the anti-tumor effect of FIN56 on GBM cells in vitro. CCK-8 was used to test the cell viability after different doses of FIN56 treatment and results showed that FIN56 decreased the cell viability of LN229 and U118 cells in a dose-dependent manner (Fig. 1A). The IC 50 for LN229 and U118 were 4.2, 2.6 µM respectively (Fig. 1A). Normal human astrocytes (NHA) were used as normal control and results showed that glioma cells were more sensitive to FIN56 than NHA (Fig. 1A). Then we used EdU assay to test the cell proliferation after FIN56 treatment. We found that FIN56 decreased cell proliferation significantly compared with control group (Fig. 1B  and 1C). Cell cycle arrest is one of the most important factors contributing to the inhibition of cell proliferation. We found that after FIN56 treatment, cell cycle of LN229 and U118 was arrested at GO/G1 phases ( Fig. 1D and 1E). All these results suggested that FIN56 had promising anti-tumor effect on GBM cells.

FIN56 induces ferroptosis in GBM cells
Previous studies recognized FIN56 as a ferroptosis inducer. In our study, we tried to figure out whether FIN56 induced ferroptosis in GBM cells. Lipid peroxidation is one of the most important features of ferroptosis. Lipid peroxidation was assessed by BODIPY 581/591 /C11, a free radical sensor. When the levels of lipid peroxidation increase, red fluorescence of BODIPY 581/591 /C11 decreases and green fluorescence shows up. FIN56 treatment increased green fluorescence and red fluorescence decayed ( Fig. 2A). Immunofluorescence revealed that FIN56 increased the expression of 4 hydroxynonenal (4-HNE), an aldehydic product of lipid peroxidation (Fig. 2B). We also found that FIN56 also increased ROS production compared with control group (Fig.  2C). The ultrastructure of GBM cell lines LN229 and U118 was studied by transmission electron microscope. Fig 2D indicated that FIN56 induced the shrink of the mitochondria and increased the density of its membrane (red arrows). To test whether the cytotoxic effect is ferroptosis-dependent, LDH release assay was carried out and results demonstrated that Ferrostatin-1 (Fer-1), a ferroptosis inhibitor, obviously inhibited FIN56-induced cell death (Fig. 2E). All these results illustrated that FIN56 induced ferroptosis in GBM cells.

FIN56 causes lysosomal membrane permeabilization in a ferroptosis-dependent manner
Our previous study showed that disulfiram, a novel ferroptosis inducer, induced LMP in GBM cells.
Here, we tried to figure out whether FIN56 had the same effect.
Acridine orange (AO), a lysosomotropic dye, is enriched in intact lysosomes, which emits red fluorescence. While in impaired lysosomes, the enrichment of AO is interfered, and red fluorescence intensity decreases. Fig. 3A illustrated that FIN56 decreased red fluorescence compared with control group.
When the membranes of lysosomes are impaired, Galectin 3, a β-galactoside binding lectin, accumulates on luminal glycoproteins and is recognized as a better method to monitor the occurrence of LMP. LN229 and U118 cells were transfected with a GFP-fused Galectin 3 (EGFP-Gal3) plasmid and results showed that FIN56 treatment increased green fluorescent dots, compared with control group (Fig. 3B and 3C). In the process of ferroptosis, levels of lipid peroxidation and ROS increase. ROS accumulation is one of the most important factors leading to LMP. Our previous study also showed that ferroptosis inducer disulfiram contributed to LMP in a ferroptosis and ROS dependent manner. Here, ferroptosis was inhibited by Fer-1 and EGFP-Gal3 dots decreased obviously in Fer-1 and FIN56 combined treatment, compared with FIN56 treatment alone ( Fig. 3D and 3E). All these data demonstrated that FIN56 triggered LMP in a ferroptosis manner in GBM cells.

FIN56 induces LMP in a TFEB dependent manner
When LMP occurs, hydrolases are released from damaged lysosomal membrane and cell death is induced. There are more than 50 acid hydrolases in the lumen of lysosomes. Among all the hydrolases involved in lysosomal cell death, cathepsin B and cathepsin D are the most important. Our previous study found that knocking down transfactor EB (TFEB), the expression of cathepsin B and cathepsin D was inhibited [11]. Besides, TFEB played an important role in the biogenesis of lysosome. As a result, we assumed that TFEB might contribute to LMP. To test our assumption, we knocked down TFEB with a specific siRNA and the knocking-down efficiency was verified by reverse transcription PCR and western blot analysis (Fig. 4A and 4B). Then we added FIN56 in control or TFEB knocking-out LN229 and U118 cells. Fig. 4C and 4D showed that after FIN56/ siramesine treatment, EGFP-Gal3 dots decreased significantly in TFEB knocking-out LN229 and U118 cells compared with control group. We also found that after knocking down TFEB, FIN56 didn't increase ROS production in LN229 and U118 cells (Fig. 4E). These data showed that FIN56 induced LMP in a TFEB dependent manner. Further study also illustrated that when cathepsin B and cathepsin D were inhibited by their inhibitor, CA-074 Me, pepstatin A, FIN56 didn't induce LMP in LN229 and U118 cells (supplementary Fig. 1), which suggested that FIN56-induced LMP was cathepsin B-and cathepsin D-dependent.

FIN56 inhibited cell growth and induced ferroptosis in vivo
Finally, we tested the anti-tumor effect of FIN56 in vivo. We implanted LN229 cell subcutaneously in nude mice and treated them with or without FIN56. 30 days later tumors were harvested and tested by immunohistochemistry. Results showed that FIN56 decreased tumor volume obviously (Fig. 5A and 5B). Ki67 is a marker to test proliferation ability and immunohistochemistry results demonstrated that FIN56 treatment decreased Ki67 positive cells (Fig. 5C  and 5D). FIN56 also significantly increased protein levels of 4-HNE (Fig. 5E and 5F). All those results illustrated that FIN56 had efficient anti-tumor effect in vivo.

Discussion
Although more and more attention has been paid to the studies of glioma, few breakthroughs have been achieved. Apoptosis is considered as the most important signaling pathway of various methods of anti-tumor treatment [4]. However, tumor cells will develop apoptotic resistance after repeated treatment, and it is one of the most important factors contributing to the relapse and poor prognosis of glioma [17]. Among many types of programmed cell death, ferroptosis is formally denominated in 2012 [18]. Until now, studies on ferroptosis keep increasing. Ferroptosis plays an important role in various types of diseases [19][20][21]. Overactive ferroptosis triggers tissue injury in diseases like cardiovascular/cerebrovascular diseases and neurodegenerative diseases [22]. While in cancer, inactivation of ferroptosis contributes to tumor growth and resistance to chemo-and radiotherapy [23]. As a result, ferroptosis inducers are promising anti-tumor reagents. Here, in our study we found that FIN56, a novel ferroptosis inducer, showed efficient anti-glioma effect both in vitro and in vivo. Firstly, FIN56 decreased cell viability in GBM cell lines LN229 and U118. FIN56 also inhibited cell proliferation by causing cell cycle arrest. As a novel ferroptosis inducer, FIN56 induced cell death in LN229 and U118 cells in a ferroptosis-dependent manner. We also found that FIN56 triggered LMP in LN229 and U118 cells. Further studies illustrated that the LMP induced by FIN56 was ROS and TFEB dependent. At last, in vivo studies showed that FIN56 had efficient anti-tumor effect in a nude mice model. But whether FIN56 will penetrate the blood brain barrier is still unknown. More studies are warranted to test the anti-tumor effect of FIN56 on orthotopic glioma xenografts.
Lysosomal membrane permeabilization (LMP) is a lysosome-dependent cell death. A study by Gao et al showed that ferroptosis is a lysosomal cell death process [13]. In our previous study, we also found that disulfiram, traditionally used for the treatment of alcoholism, induced ferroptosis and LMP in glioma cell lines [5]. As a result, ferroptosis is closely related to LMP, and lysosomes might be the key link.
Previously, lysosome is recognized as the waste bag for its role in late stage of autophagy [9]. But more and more studies demonstrate that lysosome plays an important role in regulating intracellular signaling pathways [24,25]. And TFEB, a family of basic helixloop-helix leucine zipper transcription factors, is recognized as one important regulator of lysosomal biogenesis [26][27][28]. TFEB has also been reported to be involved in clearance of oxidative stress [29]. A study by Park et al showed that TFEB activates Nrf2, an important factor in regulating oxidative stress, by increasing its stability [30]. Considering the role of TFEB in lysosomal biogenesis and oxidative stress in the initiation of LMP, we assumed that TFEB might be involved in LMP. In our study, we found that FIN56 induced LMP, and this process was inhibited by knocking down TFEB. Siramesine-induced LMP could also be inhibited by knocking down TFEB. But whether TFEB regulates ferroptosis is not known. Further studies are warranted in the future.
In summary, our study identified that FIN56, a novel ferroptosis inducer, showed significant anti-tumor effect and triggered lysosomal membrane permeabilization in a TFEB-dependent manner in glioma. FIN56 is a promising anti-tumor compound. More studies are needed on other tumor types.