Mechanisms of Growth Inhibition by Sulfasalazine and Erastin in Hepatocellular Carcinoma Cell Lines

DOI 10.17480/psk.2019.63.3.152

Mechanisms of Growth Inhibition by Sulfasalazine and Erastin in Hepatocellular Carcinoma Cell Lines

Do Hyung Kim, MD Abdullah, and Seung Jin Lee

College of Pharmacy, Chungnam National University

(Received April 9, 2019; Revised June 11, 2019; Accepted June 11, 2019)

Abstract Aberrant gene expression in the cell death pathway is a hallmark of cancers. Sulfasalazine and erastin are representative inducers of ferroptosis, which is a form of regulated cell death caused by iron-mediated accumulation of lethal lipid peroxides. This study aimed to evaluate the vulnerability of hepatocellular carcinoma cells to ferroptotic inducers. Sulfasalazine and erastin decreased cell viability with an increase of membrane permeabilization in Huh7, Huh6, and HepG2 cells. 2-Mercaptoethanol, a reducing agent, prevented cell death induced by 500 µM sulfasalazine in all tested cell lines, whereas ferrostatin-1, a ferroptosis inhibitor, restored by 57.5, 45.8, and 21.9% in Huh7, Huh6, and HepG2 cells, respectively. The cell death induced by 3 µM erastin was reversed in the presence of ferrostatin-1, by 42.9, 45.3, and 29%

in Huh7, Huh6, and HepG2 cells, respectively. Necrostatin-1, a RIPK1 inhibitor, restored sulfasalazine- or erastin-induced cell death to a similar extent as ferrostatin-1. Z-VAD-FMK, a pan-caspase inhibitor, and chloroquine, an autophagy inhibitor, failed to prevent sulfasalazine- or erastin-induced cell death. The increase of lipid peroxide by sulfasalazine was higher in Huh7 cells than in HepG2 cells, and was prevented by ferrostatin-1 and necrostatin-1 treatment of Huh7 cells.

In contrast, sulfasalazine and erastin decreased the viability of PLC/PRF/5 cells by 60% without significant morphological changes, which was not affected by any inhibitor. Together, these results showed distinct cell context-dependent mechanisms for growth inhibition by sulfasalazine and erastin in hepatocellular carcinoma.

Keywords sulfasalazine, ferroptosis, system x

, hepatocellular carcinoma

Introduction

Ferroptosis is a form of cell death distinct form other types of regulated cell death, such as apoptosis, necroptosis, and autophagic cell death. Ferroptotic cell death is caused by the iron-mediated accumulation of lipid peroxides and lethal reactive oxygen species.

Cell starvation of cysteine induces ferroptosis, which is induced by the inhibition of system x

, a transmembrane cystine- glutamate antiporter that imports cystine into cells. Cystine is reduced to cysteine and then used for the biosynthesis of glutathione (GSH). GSH is a substrate of glutathione peroxidase, which repairs lipids to avoid the accumulation of lethal lipid peroxides and consequential cell death.

Erastin and sulfasalazine are representative ferroptosis inducers, which inhibit system x

.

Erastin emerged from screening performed to identify lethal compounds for cells with oncogenic mutant HRAS, as well as the large and small T oncoproteins, and

was therefore named after its ability to eradicate Ras- and small T-transformed cells.

Erastin-induced cell death did not show any hallmark of apoptosis, such as caspase activation, annexin V staining, involvement of Bax or Bak, or morphological changes in the nucleus. Erastin acted by inhibiting system x

, leading to deregulated cellular redox homeostasis and accumulation of lethal lipid peroxide. Sulfasalazine, which is known to inhibit system x

, showed similar cellular and molecular changes as erastin. Erastin and sulfasalazine-induced lethality were potently suppressed by iron chelators (deferoxamine), lipophilic antioxidants, and ferrostatin-1, but not by small molecule inhibitors of apoptosis (Z-VAD-FMK), necroptosis (necrostatin-1), or autophagy (chloroquine) in HT1080 fibrosarcoma cells.

However, it is not clear whether inhibition of system x

induces ferroptotic cell death in other cancer cell types. Cystine starvation-induced cell death in triple-negative breast cancer cells was prevented by ferrostatin-1 and deferoxamine, as well as necrostatin-1, indicating that both necroptosis and ferroptosis were involved.

Erastin-induced mixed types of cell death have been reported to be inhibited by both ferrostatin-1 and necrostatin-1 in HL-60 leukemia cells.

Sulfasalazine activated apoptosis in malignant glioma cells by sensitizing TRAIL-induced caspase

#

Corresponding author

Seung Jin Lee, College of Pharmacy, Chungnam National University, 99 Daehak-ro, Yuseong-gu, Daejeon 34134, Republic of Korea Tel: +82-42-821-5940

E-mail: [email protected]

Mechanisms of Growth Inhibition by Sulfasalazine and Erastin 153

activation and cytochrome c release, and by inducing DNA fragmentation and annexin V exposure.

Sulfasalazine-induced cell death was prevented by an autophagy inhibitor in oral cancer cell lines.

Together, these results indicated that sulfasalazine and erastin activate various mechanisms of cell death, including necroptosis, apoptosis, autophagy, and/or ferroptosis, depending on the cell context. However, the specific mechanisms for cell death induced by sulfasalazine and erastin in hepatocellular carcinoma (HCC) cell lines are still unknown.

In the present study, we characterized the mechanisms of cell death and vulnerability to ferroptosis using chemical inhibitors of cell death in response to sulfasalazine or erastin in HCC cell lines.

We found that sulfasalazine- and erastin-induced growth inhibition was prevented by ferrostatin-1 and necrostatin-1, and the degree of reversal depended on the cell type. Our data suggested distinct mechanisms underlying regulated cell death in HCC in response to the inhibition of system x

.

Experimental Methods

Materials

Huh7 and Huh6 cell lines were obtained from the Korean Cell Line Bank (Seoul, Republic of Korea), and the PLC/PRF/5 and HepG2 cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA). Sulfasalazine, 2- mercaptoethanol, ferrostatin-1, necrostatin-1, chloroquine, and propidium iodide (PI) were purchased from MilliporeSigma (Burlington MA, USA). Z-VAD-FMK and erastin were supplied from Selleckchem (Houston, TX, USA).

Cell culture

Huh7, Huh6, and HepG2 cells were cultured in Dulbecco’s modified essential medium, and PLC/PRF/5 cells were grown in RPMI-1640 medium containing 10% fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37

C in a humidified 5% CO

incubator. Cells were maintained as described previously, and regularly checked for the absence of mycoplasma contamination.

Cell viability

Cells were seeded in fresh medium in a 96-well plate at a density of 2,000 cells per well. At 24 h after seeding, the cells were exposed to 2-mercaptoethanol, ferrostatin-1, necrostatin-1, Z- VAD-FMK, or chloroquine, 1 h before treatment with sulfasalazine or erastin for 4 days. Paired control cells were treated with dimethyl sulfoxide as a vehicle. Cell viability was measured with a CellTiter Glo

assay following the manufacturer’s instructions

(Promega, Madison, WI, USA) using a EnVision

multimode plate reader (Perkin Elmer, Waltham, MA, USA).

PI staining

After treatment with sulfasalazine as indicated, the cells were washed with phosphate-buffered saline (PBS) and incubated with 1 µg/mL PI for 30 min in the dark. Bright field and fluorescence images with excitation/emission maxima at 535/617 nm were acquired using the EVOS

FL Auto Imaging System (Thermo Fisher Scientific, Waltham, MA, USA).

Detection of lipid peroxidation

Lipid peroxide was detected using the Image-iT

Lipid Peroxidation Kit, which is based on the lipophilic BODIPY

581591 C11 probe (Thermo Fisher Scientific). After treatment as indicated, 1 µM probe was added and incubated for another 30 min at 37

C. Cells were collected by trypsinization and the fluorescence with excitation/emission at 488/525 nm was measured from 5,000 cells using a Guava

easyCyte flow cytometer and analyzed with InCyte2.6 software (MilliporeSigma).

Statistical analyses

Data are expressed as mean ± standard deviation (SD), with n indicating the number of independent in vitro experiments for a particular set of experiments. P<0.05 was considered to indicate statistical significance. Statistical analyses were performed using the two-sided Student’s t-test and one-way analysis of variance, followed by Tukey’s multiple-comparison test or repeated-measures two-way analysis of variance followed by Bonferroni’s multiple- comparison test, as applicable. All statistical analyses were conducted using SPSS statistical software for Windows (version 22; SPSS Inc., Chicago, IL, USA).

Results

Sensitivity to sulfasalazine and erastin-induced cell death in HCC cell lines

To examine the sensitivity to inhibition of system x

, dose-

dependent changes in cell viability by sulfasalazine and erastin

were monitored in four types of HCC cell lines. Sulfasalazine

significantly altered the viability of Huh7, Huh6, HepG2, and

PLC/PRF/5 cell lines (*p<0.05) (Fig. 1A). HepG2 was the cell

line most sensitive to sulfasalazine, with an IC

of 247 µM. The

Huh7 and Huh6 cell lines were moderately sensitive, with an IC

of 284 and 332 µM, respectively. The highest concentration of

sulfasalazine completely inhibited proliferation in Huh7, Huh6,

and HepG2 cells, and induced morphological changes characterized

by rounded, shrunken, and then detached cells (Fig. 1B). Staining of HepG2 and Huh7 cells with PI revealed sulfasalazine-induced cell death with increased membrane permeability and chromatin condensation, as previously reported.

However, sulfasalazine decreased the viability of PLC/PRF/5 cells only by 60%, without significant morphological changes. Next, the effect on cell viability of erastin was compared in these cell lines, which was performed with the study of Fig. 3 and displayed separately to enhance understanding (Fig. 1C). Viability of Huh6 and HepG2 cells was totally suppressed, whereas that of Huh7 and PLC/PRF/5 cells were decreased by 75 and 55%, respectively, by 3 µM erastin.

The decrease of viability of PLC/PRF/5 cells by 10 µM erastin

was similar to that by 3 µM erastin (data not shown). Together, these results showed that sulfasalazine and erastin induced growth inhibition in tested cell lines, and that Huh7, Huh6, and HepG2 cells were more sensitive than PLC/PRF/5 cell lines.

Determination of the type of cell death induced by sul- fasalazine and erastin

We then determined the type of cell death induced by sulfasalazine

in Huh7, Huh6, HepG2, and PLC/PRF/5 cell lines, using chemical

inhibitors to induce cell death. The decrease of cell viability by

500 µM sulfasalazine was prevented in all tested cells by

pretreatment with 2-mercaptoethanol, a reducing agent that may

Fig. 1. Sulfasalazine and erastin dose-dependently induced growth inhibition in HCC cells. (A) Huh7, Huh6, HepG2, and PLC/PRF/5 cells

were treated with sulfasalazine at concentrations of 62.5, 100, 125, 250, 300, 400, and 500 µM for 4 days, followed by measurement of cell

viability. Data at each dose were acquired from two to four independent analyses performed in triplicate. (B) Sulfasalazine was treated at a

concentration of 300 µM for 20 h in Huh7 cells, and at a concentration of 500 µM for 48 h in HepG2 cells. Membrane permeabilization was

detected with 0.1 µg/mL propidium iodide (PI) staining. Phase contrast (left), PI stained (middle), and merged (right) images were shown. (C)

Cells were exposed to vehicle (white bar) or 3 µM erastin (black bar) for 4 days and subjected measure viability. Three independent analyses

were performed in triplicate. Phosphate-buffered saline (PBS) and dimethyl sulfoxide were used as vehicle for sulfasalazine and erastin,

respectively, in the control group. The data are expressed as the mean±SD. **p<0.01, sulfasalazine or erastin versus the vehicle treatment.

Mechanisms of Growth Inhibition by Sulfasalazine and Erastin 155

rescue cells from L-cystine depletion caused by inhibition of system x

induced by sulfasalazine (Fig 2A). Ferrostatin-1, a lipophilic antioxidant having radical scavenging activity and preventing lipid peroxidation,

reversed sulfasalazine-induced cell death by 57.5% in Huh7 and 45.8% in Huh6 cells, whereas it reversed sulfasalazine-induced cell death by only 21.9% in

HepG2 cells. Cell death by sulfasalazine was inhibited by necrostatin- 1, a RIPK1 inhibitor, to a similar extent as ferrostatin-1 in Huh7, Huh6, and HepG2 cells. Sulfasalazine-induced cell death was prevented by ferrostatin-1 and necrostatin-1 in Huh7 and Huh6 cells, whereas partially by ferrostatin-1, necrostatin-1, and chloroquine, an autophagy inhibitor, which decreased autophagosome-lysosome Fig. 2. Determination of the type of cell death induced by sulfasalazine. Huh7 (n=4), Huh6 (n=3), HepG2 (n=3), and PLC/PRF/5 (n=4) cells were pretreated with vehicle (1) 25 µM 2-mercaptoethanol (2), 20 µM ferrostatin-1 (3), 10 µM necrostatin-1 (4), 20 µM Z-VAD-FMK (5), or 3 µM chloroquine (6) for 1 h, and then further incubated vehicle (white bar) or 500 µM sulfasalazine (black bar) for 4 days in Huh7, Huh6, HepG2, and PLC/PRF/5 cells. The cells were used for viability measurements (A), and for the capture of images (20×) (B). PBS was used as vehicle in the control group. Three to four independent analyses were performed in triplicate. The data are expressed as the mean±SD.

**p<0.01, sulfasalazine treatment in the presence versus absence of inhibitors.

fusion

in HepG2 cells. In contrast, Z-VAD-FMK, a membrane permeable pan-caspase inhibitor, did not alter sulfasalazine- induced cell death in all tested cell lines. Ferrostatin-1 and any tested inhibitor did not restore sulfasalazine-induced cell death in PLC/PRF/5 cells. The morphology of cells treated with sulfasalazine in combination with ferrostatin-1 and necrostatin-1 was similar to that of vehicle-treated cells (Fig. 2B).

Next, we assessed erastin-induced growth inhibition in the absence or presence of chemical inhibitors (Fig. 3). Treatment with 2-mercaptoethanol reversed erastin-induced growth inhibition to the greatest extent compared to the other inhibitors. Ferrostatin- 1 reversed the 3 µM erastin-induced decrease in viability by 42.9, 45.3, and 29% in Huh7, Huh6, and HepG2 cells, respectively, and necrostatin-1 restored it by similar extents. Growth inhibition by 10 µM erastin was restored by ferrostatin-1 in these cell lines, but that was inhibited by necrostatin-1 only in Huh7 cells. Z-VAD- FMK and chloroquine had little effect on erastin-induced cell death in the tested cell lines. Taken together, growth inhibition by sulfasalazine or erastin was restored by 2-mercaptoethanol, ferrostatin-1, and necrostatin-1, but the degree of reversal was cell context- and dose-dependent. None of the cell death inhibitors affected sulfasalazine or erastin-induced growth inhibition in PLC/

PRF/5 cells.

Mechanism underlying sulfasalazine and erastin-induced lipid peroxidation

Inhibition of system x

by sulfasalazine induces the accumulation of lipid peroxide, leading to ferroptotic cell death.

Because Huh7 and HepG2 cells showed differences in degree of dependency on ferrostatin-1 and necrostatin-1 for sulfasalazine-induced cell death, we assessed the amount of lipid peroxide induced by sulfasalazine at IC

dose and the effect of inhibitors in these cells.

Sulfasalazine at 300 µM significantly increased the level of lipid peroxide, by 7-fold, which was completely inhibited by pretreatment with ferrostatin-1 or necrostatin-1 in Huh7 cells. Meanwhile, sulfasalazine at 250 µM changed the level of lipid peroxide by 1.5-fold, which was partially decreased by ferrostatin-1 in HepG2 cells. Together, these results showed that equivalent dose of sulfasalazine induced different levels of lipid peroxide in a cell context-dependent manner.

Discussion

This study showed the susceptibility to cell death and ferroptosis of HCC cell lines by sulfasalazine and erastin. Huh7, Huh6, and HepG2 cells showed higher degrees of growth retardation than PLC/PRF/5 cells when treated with sulfasalazine or erastin. The Fig. 3. Determination of the type of cell death induced by erastin . Cells were pretreated with vehicle (1), 25 µM 2-mercaptoethanol (2), 20 µM ferrostatin-1 (3), 10 µM necrostatin-1 (4), 20 µM Z-VAD-FMK (5), or 3 µM chloroquine (6) for 1 h and then further incubated vehicle (white bar) or erastin at a concentration of 3 µM (black bar) for 4 days in Huh7, Huh6, HepG2, and PLC/PRF/5 cells. Dimethylsulfoxide was used as a vehicle in the control group. Three independent analyses were performed in triplicate. The data are expressed as the mean±SD.

**p<0.01, sulfasalazine treatment in the presence versus absence of inhibitors.

Mechanisms of Growth Inhibition by Sulfasalazine and Erastin 157

decreases in viability caused by sulfasalazine and erastin were significantly reversed by ferrostatin-1 and necrostatin-1 in Huh7, Huh6, and HepG2 cells, but the degree of reversal varied by cell type. Huh7 cells showed a higher level of reversal by ferrostatin- 1 against sulfasalazine-induced cell death, and greater accumulation of lipid peroxide by sulfasalazine than HepG2 cells. Sulfasalazine- and erastin-induced growth inhibition in PLC/PRF/5 cells were not affected by any of the tested chemical inhibitors.

Our data showed that the sulfasalazine- and erastin-induced decreases in viability were reversed by ferrostatin-1, but the degree of reversal was cell context-dependent. Ferrostatin-1 largely reversed sulfasalazine- and erastin-induced decreases in viability in Huh7 and Huh6 cells, but minimally restored it in HepG2 cells, indicating that the dependency on ferroptosis for sulfasalazine- and erastin-induced cell death was cell type-specific. Considering that the degree of increase in lethal lipid peroxide was higher in Huh7 cells than in HepG2 cells, HepG2 cells were probably resistant to the depletion of L-cystine caused by the inhibition of system x

. Some cells, which acquire cysteine via the metabolism of methionine through the transsulfuration pathway, can bypass

their dependence on system x

.

Although HepG2 cells might be resistant to ferroptotic cell death, they showed the lowest IC

value for sulfasalazine, and the highest degree of growth inhibition when treated with 3 µM erastin, indicating that they might be vulnerable to other types of cell death in response to sulfasalazine and erastin. Sulfasalazine is a well-known NF-kB suppressor directly inhibiting IKKa and IKKb, but is also an apoptosis inducer triggering mitochondrial accumulation of Bax, downregulation of Bcl-xL and Bcl-2, and mitochondrial-nuclear translocation of apoptosis-inducing factor.

Sulfasalazine-induced apoptosis accompanies cleavage of caspase, but Z-VAD-FMK fails to inhibit sulfasalazine-induced apoptosis.

Our results consistently showed that Z-VAD-FMK did not prevent sulfasalazine- or erastin-induced growth inhibition in any of the tested cell lines. Future studies should therefore determine whether sulfasalazine and erastin activate caspase-independent apoptosis in HCC cells, which showed resistance to the depletion of L-cystine.

Our results revealed that the sulfasalazine- and erastin-induced decreases in viability was also restored by necrostatin-1, and to a similar extent by ferrostatin-1. Necrostatin-1 blocks the kinase Fig. 4. Changes in the level of lipid peroxidation induced by sulfasalazine. After pretreatment with vehicle (1), 20 µM ferrostatin-1 (2) or 10 µM necrostatin-1 (3) for 1 h, vehicle (white bar) or 300 µM sulfasalazine (black bar) was further incubated for 20 h in Huh7 cells, and at 250 µM for 20~72 h in HepG2 cells. PBS and dimethyl sulfoxide were used as vehicle for sulfasalazine and erastin, respectively, in the control group.

Cells were further incubated with lipophilic BODIPY

581591 C11 probe and lipid peroxide was detected. Three analyses were performed

independently. The data are expressed as the mean±SD. **p<0.01, sulfasalazine versus vehicle treatment;

p<0.01, sulfasalazine treatment in

the presence versus absence of inhibitors.

activity of RIPK1, which is essential for the necroptosis and RIPK1-dependent apoptosis induced by TNFα.

RIPK1 binds to FADD and caspase-8 to form complex IIa, to promote apoptosis in apoptosis competent cells. When apoptosis is inhibited, RIPK1 forms complex IIb, consisting of RIPK1, FADD, caspase-8, and RIPK3, which in turn promotes the oligomerization of MLKL and initializes necroptosis. Loss of RIPK3 expression is a frequent reason for the escape from necroptosis of many types of cancers.

Because we found that RIPK3 was not expressed in Huh7 cells (data not shown), it was thought that necroptosis was not activated in sulfasalazine- and erastin-induced growth inhibition.

Taken together, inhibition of system x

in HCC activates RIPK1- dependent apoptosis in a caspase-independent manner.

Otherwise, RIPK1 may participate in sulfasalazine- and erastin-induced generation of lipid peroxide and ferroptotic cell death, because necrostatin-1 completely prevented the increase of lipid peroxide induced by sulfasalazine. The results showing that MPP

exhibits mixed features of non-apoptotic cell death, such as lipid peroxidation, inhibition by ferrostatin-1, and necrostatin-1, appear to support this possibility.

Conclusion

Our data characterized the distinct features of cell death induced by sulfasalazine and erastin in HCC cell lines. Sulfasalazine and erastin induced cell death in tested cell lines, but ferrostatin-1 and necrostatin-1 prevented cell death by varying degrees depending on the cell type. Sulfasalazine increased lipid peroxidation in Huh7 cells in which sulfasalazine-induced cell death was dependent on ferostatin-1. These results helped to elucidate mechanisms underlying system x

inhibition-induced cell death in a cell context-dependent manner.

Acknowledgment

This study was financially supported by a research fund of Chungnam National University (2016).

Conflict of interest

All authors declare that they have no conflict of interest.

References

1. Stockwell, B. R., Friedmann Angeli, J. P., Bayir, H., Bush, A. I., Conrad, M., Dixon, S. J., Fulda, S., Gascón, S., Hatzios, S. K., Kagan, V. E., Noel, K., Jiang, X., Linkermann, A., Murphy, M.

E., Overholtzer, M., Oyagi, A., Pagnussat, G. C., Park, J., Ran, Q.,

Rosenfeld, C. S., Salnikow, K., Tang, D., Torti, F. M., Torti, S. V., Toyokuni, S., Woerpel, K. A. and Zhang, D. D. : Ferroptosis: A Regulated Cell Death Nexus Linking Metabolism, Redox Biology, and Disease. Cell. 171, 273 (2017).

2. Hirschhorna, T. and Stockwell, B. R. : The development of the concept of Ferroptosis. Free. Radic. Biol. Med. 133, 130 (2019).

3. Dixon, S. J., Lemberg, K. M., Lamprecht, M. R., Skouta, R., Zaitsev E. M., Gleason, C. E., Patel, D. N., Bauer, A. J., Cantley, A. M., Yang, W. S., Morrison, B. 3rd. and Stockwell, B. R. : Ferroptosis: an iron-dependent form of nonapoptotic cell death.

Cell. 149, 1060 (2012).

4. Dolma, S., Lessnick, S. L., Hahn, W. C. and Stockwell, B. R. : Identification of genotype-selective antitumor agents using synthetic lethal chemical screening in engineered human tumor cells.

Cancer Cell. 3, 285 (2003).

5. Yang, W. S. and Stockwell, B. R. : Synthetic lethal screening identifies compounds activating iron-dependent, nonapoptotic cell death in oncogenic-RAS-harboring cancer cells. Chem. Biol. 15, 234 (2008).

6. Chen, M. S., Wang, S. F., Hsu, C.Y., Yin, P.H., Yeh, T. S., Lee, H. C. and Tseng, L. M. : CHAC1 degradation of glutathione enhances cystine-starvation-induced necroptosis and ferroptosis in human triple negative breast cancer cells via the GCN2-eIF2 α- ATF4 pathway. Oncotarget. 8, 114588 (2017).

7. Yu, Y., Xie, Y., Cao, L., Yang, L., Yang, M., Lotze, M. T., Zeh, H. J., Kang, R. and Tang, D. : The ferroptosis inducer erastin enhances sensitivity of acute myeloid leukemia cells to chemotherapeutic agents. Mol. Cell. Oncol. 2, e1054549 (2015).

8. Hermisson, M. and Weller, M. : NF-kappaB-independent actions of sulfasalazine dissociate the CD95L- and Apo2L/TRAIL-dependent death signaling pathways in human malignant glioma cells. Cell.

Death. Differ. 10, 1078 (2003).

9. Ignarroa, R. S., Facchinia, G., Melo, D. R., Rochac, K. J. P., Ferreira, C. V., Castilho, R. F. and Rogerioa, F. : Characteristics of sulfasalazine-induced cytotoxicity in C6 rat glioma cells. Neurosci.

Lett. 638, 189 (2017).

10. Han, H. Y., Kim, H., Jeong, S.H., Lim, D. S. and Ryu, M. H. : Sulfasalazine induces autophagic cell death in oral cancer cells via Akt and ERK pathways. Asian. Pac. J. Cancer. Prev. 15, 6939 (2014).

11. Liptay, S., Fulda, S., Schanbacher, M., Bourteele, S., Ferri, K. F., Kroemer, G., Adler, G., Debatin, K. M. and Schmid, R. M. : Molecular mechanisms of sulfasalazine-induced T-cell apoptosis.

Br. J. Pharmacol. 137, 608 (2002).

12. Tracey, I. and Dunckley P. : Importance of anti- and pronociceptive mechanisms in human disease. Gut. 53, 1553 (2004).

13. Najafov, A., Zervantonakis, I. K., Mookhtiar, A. K., Greninger, P., March, R. J., Egan, R. K., Luu, H. S., Stover, D. G., Matulonis, U. A., Benes, C. H. and Yuan, J. : BRAF and AXL oncogenes drive RIPK3 expression loss in cancer. PLoS. Biol. 16, e2005756 (2018).

14. Zilka, O., Shah, R., Li, B., Friedmann, A. J. P., Griesser, M., Conrad, M., and Pratt, D. A. : On the Mechanism of Cytoprotection by Ferrostatin-1 and Liproxstatin-1 and the Role of Lipid Peroxidation in Ferroptotic Cell Death. ACS. Cent. Sci. 3, 232 (2017).

15. Mauthe, M., Orhon, I., Rocchi, C., Zhou, X1., Luhr, M., Hijlkema, K. J., Coppes, R. P., Engedal, N., Mari, M. and Reggiori, F. : Chloroquine inhibits autophagic flux by decreasing autophagosome-lysosome fusion. Autophagy. 14, 1435 (2018).

16. Hayano, M., Yang, W. S., Corn, C. K., Pagano, N. C. and

Stockwell, B. R. : Loss of cysteinyl-tRNA synthetase (CARS)

induces the transsulfuration pathway and inhibits ferroptosis

induced by cystine deprivation. Cell. Death. Differ. 23, 270

(2016).

Mechanisms of Growth Inhibition by Sulfasalazine and Erastin 159

17. Gout, P. W., Buckley, A. R., Simms, C. R. and Bruchovsky, N. : Sulfasalazine, a potent suppressor of lymphoma growth by inhibition of the x

cystine transporter: a new action for an old drug. Leukemia. 15, 1633 (2001).

18. Wahl, C., Liptay. S., Adler, G. and Schmid, R. M. : Sulfasalazine:

a potent and specific inhibitor of nuclear factor kappa B. J. Clin.

Invest. 101, 1163 (1998).

19. Habens, F., Srinivasan, N., Oakley, F., Mann, D. A., Ganesan, A.

and Packham, G. : Novel sulfasalazine analogues with enhanced NF- κB inhibitory and apoptosis promoting activity. Apoptosis. 10, 481 (2005).

20. Weber, C. K., Liptay, S., Wirth, T., Adler, G. and Schmid, R. M. : Suppression of NF- κB activity by sulfasalazine is mediated by direct inhibition of I κB kinases α and β. Gastroenterology. 119, 1209 (2000).

21. Vanden, B.T., Linkermann, A., Jouan, L.S., Walczak, H. and Vandenabeele, P. : Regulated necrosis: the expanding network of non-apoptotic cell death pathways. Nat. Rev. Mol. Cell. Biol. 15, 135 (2014).

22. Lu, Z., Wang, H., Zhu, M., Song, W., Wang, J., Wu, C., Kong, Y., Guo, J., Li ,N., Liu, J., Li, Y. and Xu, H. : Ophiopogonin D', a Natural Product From Radix Ophiopogonis, Induces in Vitro and in Vivo RIPK1-Dependent and Caspase-Independent Apoptotic Death in Androgen-Independent Human Prostate Cancer Cells.

Front. Pharmacol. 9, 432 (2018).

23. Ito, K., Eguchi, Y., Imagawa, Y., Akai, S., Mochizuki, H. and Tsujimoto, Y. : MPP+ induces necrostatin-1- and ferrostatin-1- sensitive necrotic death of neuronal SH-SY5Y cells. Cell. Death.

Discov. 3, 17013 (2017).