Initial experiments focused on defining potential mechanisms by which GST-MDA-7 promotes cell killing in four independent primary human glioma cell populations: GBM5, GBM6, GBM12, and GBM14 (23
). Treatment of GBM5, GBM6, GBM12, and GBM14 cells with GST-MDA-7 was first noted to induce cell killing ~48 h after exposure and caused significant cell killing within 72 h (; ; Supplementary Fig. S1
Cell killing was blocked by a pan-caspase inhibitor and an inhibitor of caspase-9 but not by an inhibitor of caspase-8. Cell killing correlated with increased release of cytochrome c
into the cytosol of GBM6 cells and with the cleavage of caspase-3 (, top
). In GBM6 cells, GST-MDA-7 treatment caused inactivation of ERK1/2 that correlated with dephosphorylation of BAD S112 and dephosphorylation and increased expression of BIM (). In GBM6 cells, GST-MDA-7 treatment caused activation of JNK1-3 that was causal in the activation of BAX and the cleavage of BID and caspase-3 (). However, the decrease in BCL-xL
protein expression caused by GST-MDA-7 treatment was noted to be JNK independent. In all four glioma isolates, inhibition of caspase-8 function (IETD; expression of CRM A or c-FLIP-s) did not alter GST-MDA-7 lethality, whereas inhibition of caspase-9 function (LEHD; expression of dominant-negative caspase-9 or XIAP or BCL-xL
) significantly reduced GST-MDA-7 lethality as judged by cells becoming PI positive or retaining trypan blue dye (; Supplementary Fig. S2
; data not shown).9
GST-MDA-7 lethality correlated with caspase-9-dependent cleavage of pro-caspase-3 (, top inset
Figure 1 GST-MDA-7 causes a caspase-9-dependent induction of primary human glioma cell death. A, primary human glioma cells (GBM6) were treated 24 h after plating with GST-MDA-7 (30 nmol/L). At the indicated time points after GST-MDA-7 treatment, GBM6 cells were (more ...)
GST-MDA-7 causes a caspase-9-dependent induction of primary human glioma cell death
To further investigate mechanisms of GST-MDA-7-induced mitochondrial dysfunction, we used SV40 large T antigen transformed mouse embryonic fibroblasts (MEF) devoid of expression of defined proapoptotic genes. Loss of BIM or BAK function had a modest but significant effect on GST-MDA-7 lethality, whereas loss of BAX and loss of BAX and BAK expression profoundly reduced toxicity (). In agreement with data in , genetic manipulation to delete expression of BID also significantly reduced GST-MDA-7 toxicity. Loss of BAX and BAK function or loss of BID function reduced the ability of GST-MDA-7 to promote cytochrome c release into the cytosol (, top immunoblotting). In agreement with data showing that BAX activation and BID cleavage were JNK dependent, inhibition of JNK1-3 with JNK inhibitory peptide treatment suppressed the toxicity of GST-MDA-7 in GBM6 cells from 28.5 ± 1.3% above the GST control value with vehicle treatment to 7.6 ± 0.8% above the GST control value (±SE; n = 3). Collectively, these findings argue that GST-MDA-7 promoted activation of multiple proteins, which act to induce mitochondrial dysfunction, and that activation of the intrinsic mitochondrial pathway represents an important apoptotic mechanism for this cytokine in transformed cells.
In , we noted that BID function, but not caspase-8 function, correlated with GST-MDA-7-induced lethality. BID is a substrate for both caspase-8 and cathepsin proteases, and in glioma cells, cathepsin enzymes are overexpressed and play a key role in tumor invasion and angiogenesis (29
). GST-MDA-7 toxicity was nearly abolished by the loss of cathepsin B expression comparing appropriate matched immortalized rodent fibroblast cells, of a different lineage to those used in , which correlated with a reduction in the GST-MDA-7-induced release of cytochrome c
into the cytosol of these cells (). Combined inhibition of caspase-9 and cathepsin function was required to suppress GST-MDA-7 lethality in transformed fibroblasts and in GBM6 and GBM12 cells (). Loss of cathepsin B function suppressed the GST-MDA-7-induced degradation of BID and caspase-3 in transformed fibroblasts, and inhibition of cathepsin B function suppressed the GST-MDA-7-induced degradation of BID in GBM6 cells . The cleavage of p43 cathepsin B after GST-MDA-7 treatment was suppressed by inhibition of JNK1-3 . Collectively, these findings suggest that GST-MDA-7 induces multiple parallel proapoptotic pathways in transformed cells that converge to cause mitochondrial dysfunction: a JNK1-3-dependent activation of BAX; a JNK1-3-dependent activation of cathepsin B, leading to a cathepsin B–dependent cleavage of BID; and increased activity of BAD and BIM.
Figure 2 Cathepsin B – dependent cleavage of BID plays an important role in GST-MDA-7 toxicity in transformed cells. A, mouse immortalized embryonic fibroblasts (WT; cathepsin B−/−) were cultured for 36 h and then treated with GST or GST-MDA-7 (more ...)
Cathepsin B plays an important role in GST-MDA-7 toxicity in transformed cells
MDA-7/IL-24 has been suggested to promote ER stress signaling and cell death by binding to and inactivating the PERK-binding protein BiP/GRP78 (19
). Treatment of transformed fibroblasts that lacked expression of PERK (PERK−/−) with GST-MDA-7 caused significantly less cell killing than observed in their isogenic matched WT counterparts (). This correlated with reduced release of cytochrome c
into the cytosol of GST-MDA-7 treated PERK−/− cells; cytochrome c
release into the cytosol was JNK dependent (, top blotting
). Of note, this observation was the opposite to treating these cells with an established inducer of ER stress, thapsigargin (Supplementary Fig. S3
Surprisingly, based on known downstream targets of PERK signaling, expression of a dominant-negative eIF2α S51A protein only modestly modified the survival response of GBM6 cells treated with GST-MDA-7 (Supplementary Fig. S4
GST-MDA-7-induced BID cleavage, cathepsin B cleavage, suppression of BCL-xL
expression, inhibition of ERK1/2 phosphorylation, and increased eIF2α S51 phosphorylation in transformed fibroblasts were PERK dependent ().
Figure 3 GST-MDA-7 promotes transformed cell killing through a PERK-dependent pathway. A, transformed MEFs: WT or lacking expression of PERK (PERK−/−) were cultured for 36 h and then treated with GST or GST-MDA-7 (0–60 nmol/L, as indicated). (more ...)
Considering published reports indicating that ER stress signaling is linked to the activation of the JNK pathway, and our present studies showing that GST-MDA-7-induced toxicity is JNK1-3 dependent and that inhibition of JNK signaling block cytochrome c
release, we determined whether loss of PERK expression altered the activation of JNK1-3 following GST-MDA-7 exposure (35
). Treatment of WT-transformed fibroblasts with GST-MDA-7 promoted JNK1/2 activation, predominantly JNK1, which was causal in cell killing (). In PERK−/− cells, JNK1/2 was very weakly activated by GST-MDA-7, whereas, of note, loss of cathepsin B function did not alter JNK1/2 activation. Hence, GST-MDA-7 induces a PERK-dependent form of ER stress that promotes JNK pathway-dependent activation of BAX and mitochondrial dysfunction as well as promoting a JNK-dependent activation of cathepsin B that acts to cleave and activate BID, thereby likely promoting further BAX activation and mitochondrial dysfunction.
ER stress–induced cell killing can also be mediated by caspase-2 and caspase-4 that can cause mitochondrial dysfunction as well as initiate cell killing directly (39
). Knockdown via small interfering RNA (siRNA) of caspase-2 or caspase-4 expression in GBM6 glioma cells partially, albeit significantly, reduced GST-MDA-7 toxicity in this cell line (; see also inset
on top right
, confirming siRNA knockdown); GST-MDA-7 caused pro-caspase-2 and pro-caspase-4 cleavage that was JNK dependent (, bottom right
Because GST-MDA-7 promotes ER stress signaling, we investigated whether cell killing was associated with lysosomal vacuolization and whether any processes known to be associated with autophagy occurred. Treatment of transformed MEFs with GST-MDA-7 caused vacuolization of acidic compartments within 12 h, as judged by Lyso-tracker Red staining, an effect that was not observed in PERK−/− cells at either 12 or 24 h after GST-MDA-7 treatment [; data not shown]. As noted previously, 24 h after GST-MDA-7 exposure, relatively little cell induction of cell killing was observed (data not shown). In MEFs expressing a bona fide dominant-negative form of one downstream substrate of PERK, eIF2α S51A, vacuolization of acidic compartments was observed at 12 and 24 h, albeit to a lesser extent than that noted in WT cells. The acidic compartment vacuolization effect in transformed MEFs, and in GBM6 and U251 cells, was suppressed by a nonspecific inhibitor of autophagy, 3MA, suggestive that cells may be undergoing autophagy . GST-MDA-7-induced vacuolization of acidic compartments was not blocked by inhibition of JNK1-3 or p38 mitogen-activated protein kinase (data not shown).
Figure 4 GST-MDA-7 causes vacuolization in transformed fibroblasts in a PERK-dependent and eIF2α-independent manner. A, (i ), left, transformed MEFs (WT; deleted for PERK, PERK−/−, expressing dominant-negative eIF2α S51A, eIF2α (more ...)
Based on findings showing 3MA-dependent GST-MDA-7-induced vacuolization of acidic compartments, we determined whether the vacuoles also contain a marker for autophagy, LC3. Cells were transfected with a GFP-tagged form of LC3, treated with GST-MDA-7, and the vacuolization of LC3-GFP into punctuate bodies was determined by fluorescent microscopy. In U251 cells, GST-MDA-7 caused vacuolization of LC3-GFP within 24 h, effects that were also 3MA dependent (; ). Identical data to that in U251 cells were obtained in GBM6 cells with respect to GST-MDA-7- and 3MA-dependent vacuolization of LC3-GFP (; ). Transfection with GFP alone did not generate punctuate bodies after GST-MDA-7 treatment (data not shown).
dnPERK or 3MA treatment block LC3-GFP vesicle formation by GST-MDA-7
Considering that GST-MDA-7-induced cell killing appeared to cause vacuolization, which also contained putative autophagic vacuoles, we explored whether these events were dependent on the function of PERK. GST-MDA-7 induced punctuate staining of GFP-LC3 vacuoles in U251 and GBM6 cells within 24 h that were blocked by transient transfection of dnPERK [; ]. Based on these findings, we determined whether knockdown of ATG5 or Beclin-1, proteins that are known to play a regulatory role in autophagy, altered GST-MDA-7-induced vacuole formation. The ATG12-ATG5 and the ATG8 (LC3)-PE conjugation systems are interdependent, and a disruption in one system has a direct negative effect on the autophagic process (30
). Beclin-1 is a functional component of the lipase signaling complex, which is essential for the induction of autophagy (30
). Therefore, perturbation of the levels of ATG5 or Beclin-1 should result in reduced autophagy and the attenuation of the biological effects of GST-MDA-7. To test this, RNA interference was used to specifically suppress ATG5 or Beclin-1 protein levels in tumor cells. Knockdown of ATG5 or Beclin-1 expression significantly suppressed GST-MDA-7-induced GFP-LC3 vacuolization in U251 cells [; ]. In agreement with these findings, treatment of U251 cells with GST-MDA-7 caused increased expression of ATG5 and Beclin-1 within 24 h as well as the cleavage of endogenous LC3 protein . In transformed fibroblasts, treatment with GST-MDA-7 also caused increased expression of ATG5, a more modest increase in Beclin-1 levels, and modification of endogenous LC3 protein, effects that were abolished in PERK−/− cells (). Collectively, these data argue that GST-MDA-7 causes an initial autophagic response in human glioma cells and transformed rodent fibroblasts in vitro
Knockdown of Beclin-1 or ATG5 expression blocks LC3-GFP vesicle formation by GST-MDA-7
Based on the findings in , we determined whether modulation of PERK function or ER stress signaling altered GBM cell survival after GST-MDA-7 exposure. Overexpression of the MDA-7/IL-24 and PERK-binding protein BiP/ GRP78 significantly suppressed LC3-GFP vacuolization after GST-MDA-7 exposure and suppressed GST-MDA-7 toxicity by 64 ± 4.9% (±SE; n = 3; , top), that is, overexpressed exogenous BiP/GRP78 bound to GST-MDA-7 inside the cell, and thus lowered the free intracellular concentration of GST-MDA-7, reducing the overall level of cell killing.
Figure 5 GST-MDA-7 causes LC3-GFP vacuolization that is blocked by overexpression of BiP/ GRP78. A, bottom, U251 and GBM6 cells were plated and 24 h after plating were treated with vehicle (PBS, vehicle) or with 3MA (5 mmol/L), followed 30 min later by treatment (more ...)
In concordance with our data showing that 3MA blocked GST-MDA-7-induced LC3-GFP vacuolization, treatment of GBM6 and U251 cells with 3MA also significantly reduced the toxicity of GST-MDA-7 (, bottom
). Knockdown of either Beclin-1 or ATG5 expression in U251 and/or GBM6 cells suppressed GST-MDA-7 lethality (; Supplementary Fig. S5
Collectively, these results suggest a direct link between toxicity induction by GST-MDA-7 and promotion of autophagy by GST-MDA-7 in human GBM cells.
The induction of BiP/GRP78 expression is considered as one classic sign of ER stress. GST-MDA-7 rapidly increased expression of BiP/GRP78, and in a delayed fashion decreased expression of the protective protein HSP70, in U251 and GBM6 cells (). Overexpression of HSP70 reduced GST-MDA-7-induced LC3-GFP vacuolization, particularly at times where HSP70 expression had been suppressed by GST-MDA-7 treatment, and overexpression of HSP70 suppressed GST-MDA-7 toxicity ( and ). Overexpression of HSP70 did not, however, abolish GST-MDA-7 toxicity. Collectively, our data suggest that GST-MDA-7 induces parallel cytotoxic death signals and cytoprotective survival signals in GBM cells.
Figure 6 GST-MDA-7 causes cell killing by promoting PERK-dependent vacuolization and JNK pathway activation in transformed cells. A, U251 cells were plated and 12 h after plating infected at a multiplicity of infection of 50 to express no gene or HSP70. Twenty-four (more ...)