We evaluated the cytotoxicity of DT-EGF (18
) on EGFR-positive glioblastoma (U87MG and U373MG) and epithelial (HeLa and HN12) cancer cells using clonogenic survival () assays by treating with increasing doses of the targeted toxin for 24 hours, then washing away the drug and allowing surviving cells to recover and undergo clonogenic growth for up to two weeks. In all cases we obtained similar, dose-dependent cell death as demonstrated by loss of clonogenic capacity. Previous studies (19
) demonstrated that in addition to loss of metabolic activity as measured in cell viability assays, treatment with DT-EGF causes the regression of established glioblastoma tumors indicating that the drug causes bona fide glioblastoma cell death rather than growth arrest. Like many other anti cancer agents, targeted toxins usually kill tumor cells by activating caspase-dependent apoptosis (reviewed in (20
)). However, time-lapse microscopy () showed that the morphology of the dying cells was markedly different with only the epithelial cells displaying typical characteristics of caspase-dependent apoptosis such as membrane blebbing and cellular fragmentation, while dying glioma cells rounded up but showed no signs of apoptosis or membrane rupture. Western blotting of the caspase substrate PARP () and measurements of the activity of effector caspases (Supp Fig 1A
) showed that DT-EGF activated caspases in the epithelial cells but not in the glioblastoma cells. However both U87MG and HeLa cells displayed robust caspase activation after treatment with lexatumumab, an antibody that activates the TNF-related apoptosis inducing ligand (TRAIL) receptor DR5 (Supp Fig. 1B
) indicating that both cell types contain functional caspases and could undergo apoptosis. Furthermore caspase inhibition protected against DT-EGF in HeLa cells but not in U87MG cells (Supp Fig. 1C
DT-EGF kills glioblastoma and epithelial tumor cells by different mechanisms
We analyzed the proteome of dying U87MG cells treated with lexatumumab or DT-EGF. 2D-DIGE allows simultaneous analysis of different protein samples by labeling each sample with a different CyDye then combining the samples and separating them on the same gel. Lexatumumab caused numerous changes to the whole cell proteome, shown by many red and green spots that indicate differences between the samples (). Extensive changes in the proteome are expected in apoptotic cells where caspases cleave hundreds of substrates (21
). In contrast, U87MG cells dying as a result of DTEGF treatment displayed very few changes to the whole cell proteome as shown by the lack of green and red spots and the abundance of yellow spots (). When the 2D gels were quantitated using a 2 fold threshold as the cut-off for counting differences; 241 out of 2,464 (9.8%) of spots were different between the control and lexatumumab-treated samples whereas only 2.4% of proteins were different between the control and DT-EGF-treated cells. These data indicate that the U87MG cells treated with DT-EGF cells are not undergoing apoptosis or apoptosis-like death caused by other proteases that cleave many substrates. Moreover, there was no apparent loss of soluble proteins as would be expected if there was abrupt lysis of the cell membrane as in typical necrosis.
DT-EGF induces autophagy in glioblastoma cells
One notable proteomic change induced by DT-EGF involved an apparent pI shift in a protein ( boxed region) that was identified by mass spectrometry as isoforms of the translation elongation factor eEF2alpha (eEF2). This protein is the direct catalytic target of diphtheria toxin, which ADP ribosylates eEF2 on a unique dipthamide residue. However, the pI shift and western analysis (not shown) indicated that the modification caused by DT-EGF also involved phosphorylation. Because eEF2 kinase regulates autophagy in glioblastoma cells (22
), we asked if DT-EGF induces autophagy in U87MG cells. Upon induction of autophagy, the microtubule-associated light-chain 3 (LC3) is conjugated to phosphatidylethanolamine and incorporated into autophagosomes (1
). This can be visualized by looking for the formation of foci of a green fluorescent protein tagged version of LC3 (GFP-LC3) (23
). DT-EGF induced GFP-LC3 foci in glioblastoma cells () similar to that seen with the strong autophagy inducer trehalose (24
). siRNA knockdown of the autophagy regulators Beclin1, Atg 5, Atg 12 or Atg 7 (knockdown was confirmed by western blotting, Supp Fig. 2
) prevented both trehalose and DT-EGF-induced GFP-LC3 aggregation in the glioblastoma cells. However, only DT-EGF-induced autophagy was inhibited by EGFR knockdown. Autophagic flux was induced by DT-EGF in the U87MG cells as demonstrated () by increased LC3-II formation relative to the actin control when lysosomal protease inhibitors were used together with DT-EGF (25
) and processing of the autophagy cargo substrate betaine homocysteine methyltransferase (BHMT) using a GST-tagged BHMT construct (26
) (). As has been noted in other systems, detection of LC3-I was variable. At later times after DT-EGF or trehalose treatment, we detected increased numbers of mCherry but not GFP labeled structures from a tandem labeled GFP-mCherry-LC3 expression construct. This protein displays green and red fluorescence in autophagosomes but only shows red fluorescence after the autophagosomes fuse with lysosomes (27
) indicating that DT-EGF causes the maturation of autophagosomes and fusion with lysosomes (Supp Fig. 3
). DT-EGF also induced autophagy in U373MG cells () as shown using the GST-BHMT assay. DT-EGF treatment of the epithelial cell lines that we tested did not increase autophagy as indicated by LC3-II formation (), BHMT processing () or increased GFP-LC3 foci (Supp Fig. 4
). Two other DT-EGF-sensitive glioblastoma cell lines (8MGBA and SNB19) also activated processing the the autophagy cargo BHMT in response to DT-EGF treatment (Supp Fig. 5
). These data indicate that DT-EGF induces autophagosome formation and autophagic flux in some but not all tumor cells suggesting that the different characteristics of the dying cells could be related to the drug’s ability to activate autophagy in the different cell types.
Figure 3 Panel A, western analysis of LC3 processing in the presence or absence of lysosomal protease inhibitors pepstatin A and E64D. DT-EGF causes increased LC3-II, which was further stimulated by treatment with pepstatin and E64D in the glioblastoma cells but (more ...)
To test if autophagy controls the amount of DT-EGF-induced death, we performed siRNA knockdown of autophagy regulators, treated with increasing doses of DT-EGF for 24 hours, then removed the drug and cultured cells for two weeks to allow colony growth of surviving cells. Alternatively, autophagy was increased by pre-treatment with trehalose and cell death after treatment with DTEGF was monitored by time-lapse microscopy or clonogenic survival. Inhibition of autophagy increased the efficiency of DT-EGF killing in the clonogenic growth assay (). Conversely, increasing autophagy with trehalose delayed () and slightly inhibited () DT-EGF-induced glioblastoma cell death. In the U87MG cells, inhibition of autophagy by siRNA knockdown of Atg5 allowed induction of caspase 3/7 activity in response to DT-EGF (). These data indicate that the glioblastoma cells do not undergo autophagic cell death but rather are protected by the DT-EGF-induced autophagy and suggest that this occurs because autophagy prevents more efficient caspase-dependent death. Increasing autophagy prior to DT-EGF treatment also protected HN12 epithelial cells by clonogenic assay (Supp. Fig. 6A
) and time-lapse microscopy (Supp. Fig. 6B
). The protection conferred by trehalose was greater in the epithelial cells than the glioblastoma cells consistent with the idea that in glioblastoma cells, trehalose-induced autophagy was merely added to that produced by DT-EGF on its own.
Autophagy protects against DT-EGF-induced death
We next asked if the cells that activate autophagy after DT-EGF treatment released HMGB1. DT-EGF-treated U87MG cells released HMGB1 into the extracellular media as shown by time-lapse microscopy of GFP-HMGB1-transfected cells () and western blotting (). Moreover, this release was controlled by autophagy as shown by knockdown of Atg5, Atg7 or Atg12, which prevented HMGB1 release in DT-EGF-treated U87MG cells as detected by western blotting or time-lapse microscopy of GFP-HMGB1 (, Supp Fig. 8A
). These data indicate that the formation of autophagosomes is required for the release of HMGB1. Release occurred after the GFP-HMGB1 protein became localized in a punctate pattern as shown in the magnified image in . These HMGB1-positive structures, co-localized with a few of the LC3-positive dots (see arrows in Supp Fig. 7
) consistent with the idea that a subset of autophagosomal structures contains HMGB1 prior to its release from the cell.
Autophagy regulates selective release of HMGB1 from DT-EGF-treated cells
HMGB1 release usually occurs in cells that are undergoing classical necrotic cell death marked by abrupt membrane lysis and the release of soluble proteins (16
). However, unlike the necrotic, H2
-treated U87MG cells, which also released lactate dehydrogenase (LDH) () and were stained with propidium iodide () indicating that their cytoplasmic membranes were lysed, the DT-EGF-treated cells did not display other necrotic characteristics. Indeed there was greater propidium iodide staining in DT-EGF-treated cells where autophagy had been inhibited by Atg5 knockdown, due to cells in late stages of apoptosis. Together with the limited changes in the whole cell proteome in the dying DT-EGF-treated U87MG cells (), these data indicate that the HMGB1 release from these dying cells is not associated with membrane lysis and necrosis but instead represents selective release of the HMGB1 protein from the cell. Conversely, DT-EGF-treated epithelial cells (which activate caspases and undergo apoptosis) did not release significant amounts of HMGB1 ( and Supp Fig. 8
). However, when we stimulated autophagy with trehalose, then treated HN12 epithelial cells with DT-EGF, this caused HMGB1 release from the dying cells () without significant release of LDH (). This release is due to the autophagy itself because similar levels of HMGB1 release occur with treatment by trehalose alone ().
DT-EGF treatment causes HMGB1 release without causing membrane lysis and necrosis