S4 decreases glioma cell viability
We assessed the effect of S4 on the viability of glioma cell lines Hs683, LN229, U87MG and human astrocytes HA cell line by a CCK-8 assay. As shown in (Fig. 1A), S4 inhibited the growth of the three glioma cell lines in both dose- and time- dependent manner. This effect was achieved with a much lower half maximal inhibitory concentrations (IC50) values for glioma cell lines at each time point than the IC50 values for HA cells. Clonogenic growth assays showed that S4 at 60 µM decreased significantly the capability of the glioma cells to grow clonally after a 2-week treatment (Fig. 1B). Three-dimensional (3D) spheroid formation assays indicated that the LN2299 and U87MG microspheroids were substantially lessened in number after 10 days treatment with S4 (Fig. 1C). In addition, 24-hour S4 treatment caused a significant decrease in Ki67 staining in both LN229 and U87MG cells (Fig. 1D). Cumulatively, we showed that S4 decreases the viability of glioma cells in vitro.
S4 induces glioma cell death
We next determined whether the suppressed S4-induced growth inhibition in glioma cells was due to cell death. To this end, S4-treated LN229 and U87MG cells were analyzed by flow cytometry with FITC-conjugated Annexin-V and propidium iodide (PI) double staining. As illustrated in (Fig. 2A), exposure to S4 at 60 and 90 µM for 24 h significantly increased the percentage of both early and late apoptotic cells in LN229 and U87MG cell lines, suggesting an induction of apoptotic cell death. Doxorubicin (Dox) was used a positive control, which substantially increased the number of apoptotic cells as expected. In addition, we observed large amounts of the cells from S4-treated spheroids derived from glioma cells were stained with the cell-death dye PI, indicative of cell death (Fig. 2B). Furthermore, a dose-dependent cleavage of Poly (ADP-ribose) polymerase (PARP), a classical apoptosis marker, was detectable in S4-treated glioma cells (Fig. 2C). In addition, we also detected an increase in microtubule-associated protein 1 light chain 3 (LC3)-II (an autophagy marker) levels in glioma cells upon exposure to S4, suggesting that S4 might induce autophagy in these cells. No obvious change in the levels of RIP1/3, two key proteins involved in necrosis, was detected in S4-treated glioma cells (Fig. 2D).
S4 induces CRT exposure and release of HMGB1 and HSP70/90 in glioma cells
We next investigated whether S4 could trigger ICD in glioma cells by examining the ICD markers including HMGB1and HSP70/90 in cellular supernatants and CRT expression (ecto-CRT) in cell surface. Mitoxantrine (MTX), a known ICD inducer , was chosen as a positive control. As illustrated in (Fig. 3A), confocal imaging of S4-treated LN229 and U87MG cells revealed a significantly increased exposure of CRT on the cell surface compared with DMSO-treated cells. As expected, MTX treatment induced a strong exposure of CRT in both glioma cell lines. To detect the secreted DAMPs such as HMGB1 and HSP70/90 in S4-treated glioma cells, the cell culture media was collected after a 24 h exposure to S4 and concentrated supernatants were assayed by immunoblotting. As depicted in (Fig. 3B), a robust increase in protein levels of both HMGB1 and HSP70/90 was detected in concentrated supernatants of S4-treated LN229 and U87MG cells. The above findings indicated that S4 might trigger ICD in glioma cells. Given that the incidence of ICD is generally acknowledged to be tightly connected with programmed cell death such as apoptosis, autophagy and necroptosis [28–30], we then tested whether apoptosis, autophagy and necroptosis would play a role in S4-triggered ICD. To this purpose, we pretreated the cells with an autophagy inhibitor chloroquine (CQ), a necroptosis inhibitor Necrostain-1 (Nec-1), and a pan-caspase inhibitor Z-VAD-FMK (Z-VAD), respectively. The effective concentrations of these inhibitors were selected by a dose–response assay for each compound to prevent cytotoxicity (data not shown). (Fig. 3C) shows that both CQ and Z-VAD-FMK substantially blocked the release of HMGB1 and HSP70/90 in LN229 and U87MG cells upon exposure to S4, while Nec-1could not exhibit similar effects. Moreover, S4-induced translocation of CRT on cell surface in glioma cells was significantly attenuated by pretreatment with either CQ or Z-VAD-FMK, but not Nec-1 (Fig. 3D).
ER stress pathway is involved in S4-mediated immunogenic cell death
To explore the signaling pathways involved in S4-mediated ICD in glioma cells, we performed RNA sequencing (RNA-seq) analysis to compare gene expression profiles between S4-treated LN229 cells and cells treated with vehicle. The gene ontology analysis demonstrated that the differentially expressed genes regulated by S4 were largely enriched in ER stress and unfolded protein response (UPR) pathways (Fig. 4A), suggesting a role for the ER stress pathway in S4-mediated ICD. To validate this, we examined the activation of PRKR-like endoplasmic reticulum kinase (PERK)-eIF2α axis, and inositol-requiring enzyme 1 alpha (IRE1α)-X-box binding protein 1 (XBP1) axis, two major upstream players in ER stress pathways. S4 treatment caused a substantial increase in the levels of XBP1 and the phosphorylated elF2α in both LN229 and U87MG cells (Fig. 4B), confirming an induction of ER stress pathway. To ascertain the role of ER stress pathway in S4-mediaed ICD, glioma cells were pre-incubated with ER stress pathway inhibitors GSK2606414 and ISRIB ( both targeting PERK), and 4µ8C (targeting IRE1α) following S4 treatment. As shown in (Fig. 4C), pre-treatment of LN229 and U87MG cells with either GSK2606414 or ISRIB substantially reversed S4-induced secretion and release of HMGB1 and HSP70/90 while 4µ8C failed to do so, suggesting that the PERK-eIF2α axis plays a major role in S4-mediated ICD. Consistently, pre-exposure to GSK2606414 in LN229 cells or ISRIB in U87MG cells significantly blunted the translocation of CRT on cell surface induced by S4 (Fig. 4D). In addition, both GSK2606414 and ISRIB markedly reduced S4-induced LC3II expression in either LN229 or U87MG cells (Fig. 4E, upper panels). In addition, S4-iduced cleaved PARP was not affected by either GSK2606414 or ISRIB (Fig. 4E, lower panels). Altogether, these data suggest that these PERK inhibitors might antagonize S4-induced autophagy in the tested glioma cells.
S4 reduces glioma growth in mice models
To examine the effects of S4 in vivo, mice with LN229-derived tumors were injected with S4 at two different doses (10mg/kg and 50mg/kg). As illustrated in (Fig. 5A), both dose of S4 significantly reduced tumor growth without notable toxicity, while treatment with 50mg/kg dose of S4 achieved stronger effects. In addition, both Ki67 expression and CRT exposure in mice tumor tissue samples in S4-treated group and control group were assessed by immunohistochemistry. (Fig. 5B) shows both Ki67 staining and the CRT exposure was evidently observed in tumor samples of mice treated with S4 at either dose.