RAS-RAF-MEK-dependent oxidative cell death involving voltage-dependent anion channels.
ABSTRACT Therapeutics that discriminate between the genetic makeup of normal cells and tumour cells are valuable for treating and understanding cancer. Small molecules with oncogene-selective lethality may reveal novel functions of oncoproteins and enable the creation of more selective drugs. Here we describe the mechanism of action of the selective anti-tumour agent erastin, involving the RAS-RAF-MEK signalling pathway functioning in cell proliferation, differentiation and survival. Erastin exhibits greater lethality in human tumour cells harbouring mutations in the oncogenes HRAS, KRAS or BRAF. Using affinity purification and mass spectrometry, we discovered that erastin acts through mitochondrial voltage-dependent anion channels (VDACs)--a novel target for anti-cancer drugs. We show that erastin treatment of cells harbouring oncogenic RAS causes the appearance of oxidative species and subsequent death through an oxidative, non-apoptotic mechanism. RNA-interference-mediated knockdown of VDAC2 or VDAC3 caused resistance to erastin, implicating these two VDAC isoforms in the mechanism of action of erastin. Moreover, using purified mitochondria expressing a single VDAC isoform, we found that erastin alters the permeability of the outer mitochondrial membrane. Finally, using a radiolabelled analogue and a filter-binding assay, we show that erastin binds directly to VDAC2. These results demonstrate that ligands to VDAC proteins can induce non-apoptotic cell death selectively in some tumour cells harbouring activating mutations in the RAS-RAF-MEK pathway.
- SourceAvailable from: Darpan Patel[Show abstract] [Hide abstract]
ABSTRACT: Exchange of extracellular cystine for intracellular glutamate by the antiporter system xc- is implicated in numerous pathologies. Pharmacological agents that inhibit system xc- activity have long been sought, but have remained elusive. Here, we report that the small molecule erastin is a potent, selective inhibitor of system xc-. RNA sequencing revealed that inhibition of cystine-glutamate exchange leads to activation of an ER stress response and upregulation of CHAC1, providing a pharmacodynamic marker for system xc- inhibition. We also found that the clinically approved anti-cancer drug sorafenib, but not other kinase inhibitors, inhibits system xc- function and can trigger ER stress and ferroptosis. In an analysis of hospital records and adverse event reports, we found that patients treated with sorafenib exhibited unique metabolic and phenotypic alterations compared to patients treated with other kinase-inhibiting drugs. Finally, using a genetic approach, we identified new genes dramatically upregulated in cells resistant to ferroptosis.eLife Sciences 05/2014; · 8.52 Impact Factor
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ABSTRACT: Glutamate-induced oxidative stress is a major contributor to neurodegenerative diseases. Here, we identify small-molecule inhibitors of this process. We screen a kinase inhibitor library on neuronal cells and identify Flt3 and PI3Kα inhibitors as potent protectors against glutamate toxicity. Both inhibitors prevented reactive oxygen species (ROS) generation, mitochondrial hyperpolarization and lipid peroxidation in neuronal cells, but they do so by distinct molecular mechanisms. The PI3Kα inhibitor protects cells by inducing partial restoration of depleted glutathione levels and accumulation of intracellular amino acids, whereas the Flt3 inhibitor prevents lipid peroxidation, a key mechanism of glutamate-mediated toxicity. We also demonstrate that glutamate toxicity involves a combination of ferroptosis, necrosis and AIF-dependent apoptosis. We confirm the protective effect by using multiple inhibitors of these kinases and multiple cell types. Our results not only identify compounds that protect against glutamate-stimulated oxidative stress, but also provide new insights into the mechanisms of glutamate toxicity in neurons.Nature Communications 01/2014; 5:3672. · 10.74 Impact Factor
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ABSTRACT: Activated RAS GTPase signalling is a critical driver of oncogenic transformation and malignant disease. Cellular models of RAS-dependent cancers have been used to identify experimental small molecules, such as SCH51344, but their molecular mechanism of action remains generally unknown. Here, using a chemical proteomic approach, we identify the target of SCH51344 as the human mutT homologue MTH1 (also known as NUDT1), a nucleotide pool sanitizing enzyme. Loss-of-function of MTH1 impaired growth of KRAS tumour cells, whereas MTH1 overexpression mitigated sensitivity towards SCH51344. Searching for more drug-like inhibitors, we identified the kinase inhibitor crizotinib as a nanomolar suppressor of MTH1 activity. Surprisingly, the clinically used (R)-enantiomer of the drug was inactive, whereas the (S)-enantiomer selectively inhibited MTH1 catalytic activity. Enzymatic assays, chemical proteomic profiling, kinome-wide activity surveys and MTH1 co-crystal structures of both enantiomers provide a rationale for this remarkable stereospecificity. Disruption of nucleotide pool homeostasis via MTH1 inhibition by (S)-crizotinib induced an increase in DNA single-strand breaks, activated DNA repair in human colon carcinoma cells, and effectively suppressed tumour growth in animal models. Our results propose (S)-crizotinib as an attractive chemical entity for further pre-clinical evaluation, and small-molecule inhibitors of MTH1 in general as a promising novel class of anticancer agents.Nature 04/2014; · 38.60 Impact Factor
RAS–RAF–MEK-dependent oxidative cell death
involving voltage-dependent anion channels
Nicholas Yagoda1*, Moritz von Rechenberg3*, Elma Zaganjor1*, Andras J. Bauer1, Wan Seok Yang1,
Daniel J. Fridman1, Adam J. Wolpaw1, Inese Smukste1, John M. Peltier3, J. Jay Boniface3, Richard Smith4,
Stephen L. Lessnick4,5, Sudhir Sahasrabudhe3& Brent R. Stockwell1,2
Therapeutics that discriminate between the genetic makeup of
normal cells and tumour cells are valuable for treating and under-
may reveal novel functions of oncoproteins and enable the cre-
ation of more selective drugs1.Here wedescribe the mechanism of
action of the selective anti-tumour agent erastin, involving the
RAS–RAF–MEK signalling pathway functioning in cell prolifera-
in human tumour cells harbouring mutations in the oncogenes
HRAS, KRAS or BRAF. Using affinity purification and mass spec-
trometry, we discovered that erastin acts through mitochondrial
voltage-dependent anion channels (VDACs)—a novel target for
anti-cancer drugs. We show that erastin treatment of cells har-
bouring oncogenicRAScausestheappearance ofoxidativespecies
and subsequent death through an oxidative, non-apoptotic mech-
anism. RNA-interference-mediated knockdown of VDAC2 or
VDAC3 caused resistance to erastin, implicating these two VDAC
isoforms in the mechanism of action of erastin. Moreover, using
purified mitochondria expressing a single VDAC isoform, we
found that erastin alters the permeability of the outer mitochon-
drial membrane. Finally, using a radiolabelled analogue and a fil-
ter-binding assay, we show that erastin binds directly to VDAC2.
These results demonstrate that ligands to VDAC proteins can
induce non-apoptotic cell death selectively in some tumour cells
harbouring activating mutations in the RAS–RAF–MEK pathway.
In a screen of about 24,000 compounds, we discovered erastin,
which induces rapid death in engineered human tumour cells (BJ-
TERT/LT/ST/RASV12cells, ref.2) with oncogenic v-Ha-ras Harvey
rat sarcoma viral oncogene homologue (HRAS)V12, but not in iso-
genic, non-tumorigenic cells lacking oncogenic RAS (BJ-TERT/LT/
not dependent on the rate of cell division, nor was it idiosyncratic to
these cells (Fig. 1a and Supplementary Fig.2), because cell lines engi-
neered in a similar way responded similarly.
We found that erastin-treated cells did not display changes in nuc-
lear morphologycharacteristic ofapoptosis (Fig. 1c,ref.3).However,
imaging by electron microscopy did reveal changes in mitochondrial
chondrial morphological changes were not observed in response to
staurosporine, hydrogen peroxide or rapamycin—compounds that
induce apoptosis, necrosis and autophagy, respectively (Fig. 1b and
data not shown).
Given that erastin is a new compound found in a cell-based
screen, we had no insight into its mechanism of action. We used a
two-pronged approach to define the mechanism, involving, first, a
suppressor screen to identify annotated compounds that prevent
erastin-induced cell death and, second, an affinity purification
approach to identify proteins that mediate the activity of erastin.
First, we performed a suppressor screen using a library of about
2,000 biologically active compounds4and found that antioxidants
(a-tocopherol, butylated hydroxytoluene and b-carotene) prevent
erastin-induced death (Fig. 1d, Supplementary Fig.3). Moreover,
we detected generation of an oxidizing species in response to erastin
treatment in BJ-TERT/LT/ST/RASV12cells, but not in isogenic BJ-
TERT cells (Fig. 1e, Supplementary Fig.4). Finally, we found that
erastin-induced death intheHT-1080 fibrosarcoma celllinewas also
suppressed by antioxidants (Supplementary Fig.5).
To characterize the mode of erastin-induced cell death, we looked
the oxidizing species generated in BJ-TERT/LT/ST/RASV12cells in
the presence of erastin emanate from mitochondria (see Supple-
mentary Discussion), consistent with the perturbation in mitochon-
drial morphology. We discovered that the oxidizing species do not
or pro-caspase-3 cleavage (Fig. 1h)3,5,6—all of which are hallmarks of
apoptosis, a stereotypical form of cell death activated by many anti-
tumour agents3,7–10as well as by staurosporine (Fig. 1f–h). Moreover,
we looked for these hallmarks in four different cell lines sensitive to
erastin; none showed activation of these markers (Fig. 1f,h). Other
canonical hallmarks of apoptosis were similarly absent (see Supple-
mentaryInformation). Insummary, theseinitialstudiesrevealedthat
erastin induces rapid, oxidative, non-apoptotic death in tumour cells
with oncogenic HRAS.
To define the genetic basis of the selective lethality of erastin, we
used a lentiviral-based RNA interference system11. We originally
discovered erastin in a screen for oncogenic-HRAS-selective lethal
compounds. However, v-Ki-ras2 Kirsten rat sarcoma viral oncogene
homologue (KRAS) is more frequently mutated in human cancers
than HRAS12. Selective toxicity in mutant-KRAS-expressing cell
lines would broaden the applicability of erastin as a therapeutic.
Thus, we tested whether erastin was selectively lethal to tumour
cells harbouring oncogenic KRAS, and whether this lethality
could be arrested by knockdown of KRAS. We found that a lung
carcinoma cell line (Calu-1) with an activating mutation in KRAS
was sensitive to erastin (half-maximal inhibitory concentration,
IC5054mM; Fig. 2a); when infected with lentiviral constructs
expressing two different short hairpin RNAs (shRNAs) targeting
*These authors contributed equally to this work.
1Department of Biological Sciences, Fairchild Center, 1212 Amsterdam Avenue, MC 2406,2Department of Chemistry, Columbia University, New York, New York 10027, USA.
3Prolexys Pharmaceuticals, 2150West DauntlessAvenue,SaltLakeCity, Utah84116,USA.4CenterforChildren,Huntsman Cancer Institute,2000CircleofHopeSaltLakeCity,Utah
84112, USA.5Division of Pediatric Hematology/Oncology, the Department of Oncological Sciences.
Vol 447|14 June 2007|doi:10.1038/nature05859
KRAS (Supplementary Table4), these cells exhibited resistance to
erastin (Fig. 2a,b).
signalling, we sought evidence that erastin acts in a manner that is
specific to cells with activated RAS–RAF–MEK signalling (Supple-
mentary Table1 and Supplementary Discussion). One cell line with
moderate sensitivity to erastin was A-673 (Supplementary Table1),
containing an activating V600E mutation in v-raf murine sarcoma
viral oncogene homologue B1 (BRAF)—a direct target of RAS13. To
determine whether the activating mutation in BRAF influences era-
stin sensitivity, we created two different shRNAs targeting BRAF
messenger RNA(Supplementary Table4).Wefound that A-673cells
containing either of these constructs were resistant to erastin (Fig.
2c–e). Moreover, co-expression of a non-targetable V600E mutant
BRAF partially restored sensitivity of these cells to erastin (Sup-
tumour cells sensitive to erastin, we examined the effect of mitogen-
(ERK)kinase1/2 (MEK1/2) inhibitorsonerastinsensitivity. Allthree
inhibitors caused erastin resistance in both BJ-TERT/LT/ST/RASV12
and HT-1080 cells, with activating mutations in HRAS and NRAS,
shows the effect of these MEK inhibitors alone on cell viability).
Finally, we found a modest correlation between ERK1/2 phosphory-
lation status and erastin sensitivity in 12 sarcoma cell lines (Table 1).
In summary, KRAS and BRAF knockdown, MEK inhibition, and
analysis of ERK1/2 phosphorylation status together suggest that era-
stin contains a degree of selectivity for cells in which the RAS–RAF–
MEK pathway is constitutively activated.
For the second prong of our approach to defining the mechanism
of action of erastin (affinity-based target identification), we synthe-
sized erastin analogues that could be linked to a solid-phase resin for
biochemical purification of potential targets. We found that replace-
ment of the p-chloro substituent in erastin with the aminomethyl
A6, Fig. 2g) that, though less potent, retained the ability to kill BJ-
TERT/LT/ST/RASV12cells, but not BJ-TERT cells (Supplementary
Fig.9). We also identified a suitable analogue (erastin B2, Supple-
mentary Information) that lacked activity (Fig. 2g and Supplemen-
tary Fig.9), and thus could serve as a negative control for target
We immobilized erastin A6 and erastin B2 on solid-phase resin
and sought proteins that interact with A6, but not B2. Using BJ-
TERT/LT/ST/RASV12cell lysates, we identified all three isoforms
of the human mitochondrial voltage-dependent anion channels
(VDAC1, VDAC2 and VDAC3) on the A6 resin, but only VDAC1
on the B2 resin (Supplementary Table3,7–14). Using BJ-TERT cell
lysates, we identified a small amount of VDAC1 on the A6 resin, but
none of the VDACs on the B2 resin. Thus, it appears that erastin A6
isolates VDAC2 and VDAC3 more efficiently than does erastin B2.
Furthermore, the finding that erastin pulls down a mitochondrial
protein (VDAC) is consistent with observations that erastin induces
mitochondria-driven oxidative death.
VDACs (also known as eukaryotic porins) are membrane-
spanning channels that facilitate transmembrane transport of ions
24 h erastin, light
Erastin + DMSO
Erastin + BHT
No treatment, EM
10 h erastin, EM
5 h STS, EM
6 h 8 h
6 h 10 h
6 h 8 h
NT STS Erastin
6 h 8 h
NTNTSTS STS Erastin NT STS Erastin
6 h 8 h
6 h 8 h
6 h 8 h
6 h 8 h
2468 10 12
Duration of treatment (h)
Percentage in M1
Figure 1 | Erastin activates a rapid, oxidative, non-apoptotic cell death
process. a, HRASV12-expressing cell lines are sensitive to erastin, whereas
isogenic lines lacking HRASV12are resistant, as determined by Trypan blue
exclusion; the graph is a representative outcome of multiple independent
experiments. b, Transmission electron microscopy images (320,000) of BJ-
TERT/LT/ST/RASV12mitochondria after cells were treated with nothing,
erastin (37mM for 10h) or staurosporine (STS, 1mM for 5h). c, Phase-
treatment indicates that nuclei are intact after cell death. d, Anti-oxidants
suppress erastin-induced death in BJ-TERT/LT/ST/RASV12cells. BHT,
butylated hydroxytoluene; DMSO, dimethylsulphoxide. e, Level of
intracellularoxidative species in BJ-TERT/LT/ST/RASV12(black line) or BJ-
TERT (grey line) cells treated with 4.6mM erastin. yaxis, percentage of cells
exhibiting dichlorofluorescein (DCF) fluorescencewithin region of interest,
M1, as measured by flow cytometry; error bars, one standard deviation,
n52. f, PARP1 cleavage is not seen during erastin-induced cell death in A-
673, HT-1080 and HeLa cells. NT, no treatment. g, STS, but not erastin,
induces cytochrome c (cyt c) release from BJ-TERT/LT/ST/RASV12
mitochondria; mitochondrial fraction, mito; cytosolic fraction, cyto. h, Pro-
caspase-3 is not cleaved in response to erastin.
NATURE|Vol 447|14 June 2007
and metabolites14,15, most notably across the outer mitochondrial
membrane16. It has been demonstrated that VDACs are gated by
membrane voltage, at least invitro (Supplementary Discussion): in
the closed state, ions, but not small molecule metabolites, can pen-
etrate VDAC pores17; in the open state, both ions and metabolites
pass through VDAC channels. In addition, the closed state is cation-
selective, whereas the open state is anion-selective.
evidence that altered expression of VDACs contributes to altered
erastin sensitivity. To determine whether VDACs are upregulated
in response to oncogenic HRAS, we measured VDAC abundance in
ering and genetics of BJ cell series). In BJ-TERT/LT/ST/RASV12cells,
the total amount of VDAC protein is increased (Fig. 3a and Sup-
plementary Fig.10). These results suggest that erastin acts by a gain-
of-function mechanism, and that cells with more VDAC protein are
more sensitive to erastin, although other aspects of cellular physi-
ology may also be relevant.
Next, using two-dimensional gel electrophoresis to evaluate pro-
tein expression in BJ-TERT/LT/ST/RASV12cells (Supplementary
Fig.11), we found that, after 8h of erastin treatment, VDAC3 was
no longer detectable, and, after 10h, VDAC2 became undetectable
act by a gain-of-function mechanism on their molecular targets,
topoisomerase IIa and topoisomerase I, respectively18. Itmay be that
a cellular response to erastin is downregulation of VDAC2/3 after
lethal oxidative species have been generated, as occurs with camp-
tothecin and topoisomeraseI following DNA damage. The fact that
VDAC1 is still present at later time points suggests that the loss of
VDAC2/3 is not simply caused by loss of mitochondria.
To test the gain-of-function hypothesis, we reduced VDAC pro-
tein levels using lentiviral shRNA expression11. We created multiple
shRNA constructs targeting each VDAC isoform (Supplementary
Table4 and Supplementary Fig.12) and tested their effects on cell
sensitivity to erastin (Supplementary Figs13–15). We found that
knockdown of VDAC3 caused significant resistance to erastin (Fig.
3c,d and Supplementary Fig.15). We also observed some degree of
1.5 µM erastin
20 µM U0126
10 µM MEK inhibitor I
50 µM MEK1/2 inhibitor
1 2.5510 20
Erastin B1, R2 = F
Erastin B2, R2 = CH2NH2
Erastin, R1 = Cl
Erastin A6, R1 = CH2NH2
Figure 2 | Erastin lethality is dependent on the RAS–RAF–MEK pathway.
a, Calu-1 cells infected with lentivirus containing shRNAs targeting KRAS
coding sequence. b, KRAS knockdown was confirmed by western blot
analysis. c, d, A-673 cells were infected with lentivirus expressing indicated
shRNAs or a control GFP plasmid. Cells were treated with erastin for 24h;
percentage inhibition of viability (yaxis) was measured using c, Alamar blue
and d, Trypan blue. Luc, luciferase; BRAFex5, exon 5 of BRAF transcript.
withMEKinhibitors(U0126,Sigma;MEK inhibitor 1,Calbiochem;MEK1/2
inhibitor, Calbiochem) prevents erastin-induced lethality in BJ-TERT/LT/
ST/RASV12cells; percentage viability (yaxis) was determined using Trypan
blue. g, Structures of erastin and related analogues. All error bars in Fig. 2
represent one standard deviation; n52 or 3.
Table 1 | Correlation between erastin sensitivity and phospho-ERK level
Human cell lineErastin sensitivity (%)Phospho-ERK1/2
level of phospho-ERK1/2 as quantified by western blot (arbitrary units). The correlation is 0.41.
NATURE|Vol 447|14 June 2007
erastin resistance when VDAC2 was knocked down (Fig. 3c,e and
Supplementary Fig.14).Incontrast, overexpression ofVDAC3alone
in BJ-TERT cells yielded no increase in sensitivity to erastin
(Supplementary Fig.16), suggesting that VDAC3, and to some
also needed. Overall, these results are consistent with a gain-of-func-
tion mechanism involving erastin and VDAC2/3. Furthermore, this
effect is specific to erastin, and not to other lethal compounds:
VDAC2-deficient embryonic stem cells have been shown to be more
sensitive, not less sensitive, to staurosporine and etoposide19.
To test the hypothesis that erastin alters mitochondrial outer
purified mitochondria from yeast engineered to express a single
mouse VDAC isoform in place of yeast VDAC20. A previous report
demonstrated that the rate of NADH uptake through the outer
membrane of such mitochondria is dependent on the specific
VDAC expressed in yeast. We found that erastin treatment yielded
a decrease in the rate of NADH oxidation, suggesting a reduced
permeability of these mitochondria to NADH when mouse
VDAC1 or VDAC2 were expressed (Fig. 3f and Supplementary
Fig.17). We found little NADH oxidation in mitochondria expres-
sing VDAC3, suggesting minimal intrinsic membrane permeability
(Supplementary Fig.17); this is consistent with previous reports that
no such effect (Fig. 3f and Supplementary Fig.17). These results
suggest that erastin affects VDAC gating, possibly switching its ion
selectivity and allowing cationic species into mitochondria.
Having demonstrated interactions between erastin and VDAC
using affinity-based target identification and VDAC functional
assays, we explored the direct binding of erastin to VDACs. Using
modified versions of previously reported protocols, we isolated
ment21,22using a radiolabelled analogue (erastin A9). The results
demonstrate that the cold RAS-selective lethal erastin A9 (IC505
1.9mM, Supplementary Fig.9), unlike inactive erastin A8, directly
binds to VDAC2 (dissociation constant, Kd5112nM; Fig. 3g), in
the process competing off radiolabelled erastin A9.
The data presented herein are consistent with a model in which
erastin interacts with VDAC proteins to induce mitochondrial dys-
function, release of oxidative species and, ultimately, non-apoptotic,
oxidative cell death. This process has a degree of selectivity for cells
that it is feasible to discover oncogene-selective compounds and to
use them to clarify oncogene-related cell death mechanisms.
TERT cell lysate and washed before protein was eluted and then digested as
described23. Reverse-phase high-performance liquid chromatography was per-
formed before samples were analysed using both a 4700 Proteomics Analyser
matrix-assisted laser desorption/ionization-time of flight (TOF)/TOF (TOF/
TOF; Applied Biosystems) and a Q Trap (AB/MDS Sciex). Tandem mass spec-
protein sequence database or the HumanNR database (http://www.ncbi.nlm.
nih.gov/). GPS Explorer (Applied Biosystems) was used for submitting data
acquired from the TOF/TOF for database searching.
Knockdown using lentiviral shRNAs. HT-1080 and Calu-1 cells were infected
with lentiviruses expressing shRNAs targeting VDAC and KRAS, respectively
(Supplementary Table4). A-673 cells were infected with retroviruses24expres-
sing shRNAs against either BRAF or luciferase (Supplementary Table4).
resuspending mitochondria isolated from yeast in 25mM NADH, with or with-
out erastin, and monitoring absorbance at l5340nm (ref.20). As a control for
mitochondrial intactness, parallel assays were run using hypotonically shocked
VDAC2 binding assay. VDAC2 protein was isolated from E.coli using a modi-
fied version of a previously described protocol22. To assay direct binding of
erastin analogues, 40mg of purified VDAC2 was incubated in the presence of
20mM radiolabelled erastin A9 and serial dilutions of unlabelled erastin A9 or
erastin A8. The mixture was then deposited onto a binding filter using vacuum
filtration. After rinsing, radioactivity was detected on a liquid scintillation
counter (LKB Wallac 1,211 RACKBETA).
Full protocols. For detailed methods, which additionally include cell culture,
western blotting, cell viability assays, PARP cleavage, cytochrome c release, oxi-
sion, PCR with reverse transcription and quantitative PCR, see Methods.
Full Methods and any associated references are available in the online version of
the paper at www.nature.com/nature.
Received 17 November 2006; accepted 17 April 2007.
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Figure 3 | Erastin compounds act through VDACs. a, VDAC/eukaryotic
initiation factor 4E (eIF4E) protein ratio in engineered BJ-derived cells as
quantified using western blot. b, BJ-TERT/LT/ST/RASV12cells were treated
with erastin and harvested at indicated time points for quantitative two-
separated by two-dimensional gel electrophoresis. c, Isoform-specific
knockdown of VDAC in HT-1080 cells infected with virus expressing either
VDAC3- or VDAC2-targeted shRNA plasmid(shVDAC3or shVDAC2)was
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treated with erastin dilutions, and viability relative to no treatment (y axis)
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unlabelled erastin A9 (red) or erastin A8 (black). All error bars in Fig. 3
represent one standard deviation, n52 or 3.
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Supplementary Information is linked to the online version of the paper at
Acknowledgements We thank S. Flaherty and S. Dolma for supporting
experiments, H. Widlund for supplying the BRAF shRNA construct, R. Becklin and
J. Savage for help with the analysis of the pull-down data, P. Robbins for help with
the pull-down experiments, K. Brown for assistance with transmission electron
microscopy, and M. Colombini for supplying engineered yeast and for discussions.
Welcome Fund and by the National Cancer Institute. S.L.L. is supported by an NCI
grant,an American Cancer Society Research Scholars Grant,the TerriAnnaPerine
Sarcoma Fund, a Primary Children’s Medical Center Foundation Innovative
Research Grant, a Hope Street Kids grant and a Catalyst Grant from the University
of Utah School of Medicine.
Author Contributions N.Y. designed and performed the RNAi and VDAC
overexpression, quantitative PCR, erastin analogue viability and chemical
characterization experiments. E.Z. performed two-dimensional western analysis,
PARP-1 and pro-caspase-3 cleavage, and cytochrome c release experiments. E.Z.
and N.Y. performed transmission electron microscopy experiments. A.J.B., D.J.F.
and N.Y. performed the NADH oxidation and direct binding experiments. W.S.Y.
characterized sensitivity to erastin in the BJ-derived cell series. A.J.W. performed
and S.L.L. provided BRAF shRNAs, analysis of BRAF knockdown and the
phospho-ERK western analysis. J.M.P., J.J.B. and S.S. were responsible for setting
up the technology platform to pull down proteins binding to small molecule
compounds. M.v.R. and J.M.P. performed the pull-down experiments. J.J.B., J.M.P.
and S.S. designed, reviewed and supervised the pull-down experiments, and
contributed to the analysis of the data. B.R.S. conceived of and supervised the
project, designed and analysed experiments, and performed the anti-oxidant
studies. B.R.S. and N.Y. prepared the manuscript.
Author Information Reprints and permissions information is available at
www.nature.com/reprints. The authors declare no competing financial interests.
Correspondence and requests for materials should be addressed to B.R.S.
NATURE|Vol 447|14 June 2007
Cell culture and western blotting. BJ-TERT/LT/ST/RASV12cells were cultured
as described in ref.3. Other cell lines were grown according to specifications of
and each dish was washed twice with 10ml ice-cold PBS. Cells were lysed with
200ml buffer (50mM HEPES, 40mM NaCl, 2mM EDTA, 0.5% Triton-X,
1.5mM sodium orthovanadate, 50mM NaF, 10mM sodium pyrophosphate,
10mM sodium b-glycerophosphate and protease inhibitor tablet (Roche),
(pH7.4)). For one-dimensional western blots, samples were separated using
SDS–polyacrylamide gel electrophoresis; for two-dimensional western blots,
samples were prepared using the ZOOM IPGRunner System (Invitrogen).
ide membrane, blocked for 1h at room temperature in Licor Odyssey Blocking
VDAC1 (Abcam, ab3434), anti-VDAC1 (Calbiochem, 529534), anti-VDAC2
(Abcam, ab22170), anti-eIF4E (Santa Cruz Biotechnology, sc-9976), anti-a-
tubulin (Sigma, T6199), anti-actin (Santa Cruz Biotechnology, 1616R), IRDye
800 goat anti-rabbit antibody (Rockland Immunochemicals, 611-132-122),
Alexa Fluor 680 goat anti-mouse (Molecular Probes, A21058), PathScan
Multiplex Western Cocktail I Kit (Cell Signalling Technology) and anti-PARP
(Abcam, ab105). Membranes were scanned using the Licor Odyssey Imaging
PARP cleavage and cytochrome c release. BJ-TERT/LT/ST/RASV12cells were
seeded in polystyrene 100320mm dishes (Falcon, 353003) in 10ml medium.
33106cells were seeded in each dish. After overnight incubation at 37uC with
12.5, 13, 14, 18 or 26h, and prepared for western blotting. For the cytochrome c
release assay, cells were washed with 10ml ice-cold PBS, suspended in 120ml
buffer (300mM sucrose, 0.1% BSA, 10mM HEPES (pH7.5), 10mM KCl,
1.5mM MgCl2, 1mM EGTA, 1mM EDTA, 1mM dithiothreitol (DTT), 2mM
phenylmethanesulphonyl fluoride and one protease inhibitor tablet (Roche))
and incubated on ice for 15min. Cells were lysed by passing them through a
to remove the nuclear fraction. Mitochondria were removed from the soluble
cytosolic fractionby pelleting at 10,500g. Supernatant andmitochondrial pellets
were solubilized in SDS–polyacrylamide gel electrophoresis loading buffer and
analysed by western blotting.
(H2DCF-DA, Molecular Probes) was used to measure oxidative species by flow
cytometry. Non-fluorescent H2DCF-DA was cleaved by endogenous esterases
and then was oxidized to generate fluorescent DCF. BJ-TERT/LT/ST/RASV12
and BJ-TERT cells were seeded at 33105cells per dish in 60mm dishes and
and 12h. For each time point, controls were maintained for untreated cells and
also for positive control (treated directly with 500mM hydrogen peroxide for
5min). Cells were incubated with 10mM H2DCF-DA for 10min, harvested by
trypsinization, washed twice with cold PBS, resuspended in 100ml PBS and
incubated with 5ml of 50mgml–1propidium iodide for 10min. 400ml PBS was
added and the solution was analysed by flow cytometry (FACSCalibur, Becton-
Dickinson). FL1-H indicates arbitrary units of DCF fluorescence detected.
150-mm plates) were washed with PBS, lysed in 25mM HEPES (pH7.5),
150mM NaCl, 1% NP-40, 10mM MgCl2, 1mM EDTA, 10% glycerol, 1mM
DTT and protease inhibitor cocktail. Total protein concentration was deter-
mined using a Bradford colorimetric assay.
Erastin A6 and B2 were dissolved in DMSO at 10mgml–1. 100ml AffiGel-10
(BioRad) was washed and resuspended in 400ml DMSO. 10ml compound and
3ml 1:100 dilution of triethylamine in DMSO were added. The suspension was
incubated at room temperature for 14h, washed (1ml per wash) five times in
DMSO,threetimes inPBS,resuspended in3M ethanolamineinPBS,incubated
1h at room temperature, washed five times in PBS and resuspended in 200ml
with binding buffer (0.1M KCl, 20mM HEPES (pH7.6), 0.1mM EDTA, 10%
glycerol, 0.1% NP-40, 1mM DTT, 0.25mM phenylmethylsulphonyl fluoride)
and incubated with 1ml cell lysate (2mgml–1for 1.5h at 4uC), washed with
binding buffer, washed three times with high salt buffer (0.35M KCl, 20mM
HEPES (pH7.6), 0.1mM EDTA, 10% glycerol, 0.1% NP-40, 1mM DTT,
0.25mM phenylmethylsulphonyl fluoride), washed with binding buffer, and
sine). Proteinsfrom the supernatant were precipitatedwith 400ml ethanol,sedi-
mented by centrifugation (16,800g) and digested as described23. Reverse-phase
high-performance liquid chromatography was performed using a nano LC sys-
tem (Dionex): a 75mm3150mm column, a Famos autosampler, a Switchos II
system and an UltiMate binary pumping module. Samples were analysed using
both a 4700 Proteomics Analyser MALDI-TOF/TOF (TOF/TOF; Applied
Biosystems) and a Q Trap (AB/MDS Sciex), and the peptide level data were
combined. To construct the databases used for protein identification, the fol-
lowing steps were performed: the NCBInr protein sequence FASTA file was
downloaded, the GI numbers (sequence version identification) were updated,
and the missing or incorrectly annotated taxonomies were fixed by referencing
them to the NCBI taxonomy index (index of GI number versus species). The
human subset of proteins in the database was extracted into a separate database
(HumanNR). All protein sequences in HumanNR were matched to the corres-
searches were performed against either the corrected NCBInr protein sequence
database or the HumanNR database. GPS Explorer (Applied Biosystems) was
used for submitting data acquired from the TOF/TOF for database searching.
The Mascot-based search was performed using the default settings for the spe-
cific instrument type as supplied by Matrix Science, except that ions with scores
below ten were excluded from the results.
The spectra of the peptides identified in the automatic data analysis were
manually inspected for the quality of the corresponding spectra and for consist-
ency with the obtained results. Only high quality spectra and results with a
peptide score of 20 or higher were accepted and used for the identification of
Transmission electron microscopy of BJ-TERT/LT/ST/RASV12cells treated
or nothing, fixed with 2.5% glutaraldehyde in 0.1M Sorenson’s buffer (0.1M
H2PO4, 0.1M HPO4(pH7.2))for atleast1h,andthentreatedwith1% OsO4in
0.1M Sorenson’s buffer for 1h. Enblock staining used 1% tannic acid. After
dehydration through an ethanol series, cells were embedded in Lx-112 (Ladd
Research Industries) and Embed-812 (EMS). Thin sections were cut on an MT-
7000 ultramicrotome, stained with 1% uranyl acetate and 0.4% lead citrate, and
examined under a Jeol JEM-1200 EXII electron microscope. Pictures were taken
on an ORCA-HR digital camera (Hamamatsu) at ,20,000-fold magnification,
and measurements were made using the AMT Image Capture Engine.
Knockdown using lentiviral shRNAs. VDACs, KRAS and BRAF were knocked
down in HT-1080, Calu-1 and A-673 cells, respectively, using shRNA lentiviral
vectors (Supplementary Table4). On day1, 293T cells were seeded in 10cm
(pLKO.1 vector for VDAC- and KRAS-targeting constructs; pSIRIPP24for
BRAF- or luciferase-targeting constructs; Supplementary Table4) and the
FuGENE 6 Transfection Reagent (Roche); on day3, the medium was changed;
on day4, the supernatant, containing virus, was transferred to target cells in
10cm tissue culture dishes (13106cells per dish); on day5, cells were trans-
ferred to 175cm2flasks and medium was supplemented with puromycin
(1.5mgml–1); on days6 and 7, medium was changed and again supplemented
with puromycin; on day8, samples were harvested for western blot and quant-
per well), in duplicate, and treated with erastin dilutions. All cells were cultured
at 37uC, 5% CO2, in growth media as recommended by ATCC (American Type
Overexpression of VDAC3 using lentiviral constructs. VDAC3 was over-
expressed in BJ-TERT cells using a human ORF clone (Invitrogen) recombined
into the pLENTi6/V5-DEST lentiviral vector (Invitrogen). On day1, 293T cells
were seeded in 10cm tissue culture dishes (23106cells per dish); on day2, the
VDAC3 construct and the pD8.9 and pVSV-G helper plasmids were co-trans-
3, the medium was changed; on day4, the supernatant, containing virus, was
transferred to BJ-TERT cells in 10cm tissue-culture dishes (13106cells per
dish); on day5, cells were transferredto 175cm2flasks and medium was supple-
mented with blasticidin (5mgml–1); on days6 and 7, medium was changed and
again supplemented with blasticidin. These cells were maintained in selection
medium for 12days before samples were harvested for future assay.
Reverse transcription and quantitative PCR. Total RNA was isolated from
cells using RNeasy Mini Kit (Qiagen). Reverse transcription was performed on
2mg isolated RNA using Taqman Reverse Transcription Reagents (Applied
Biosystems). The ABI Prism 7,300 was then used for quantitative PCR. 20ng
complementary DNA product was mixed with Power SYBR Green PCR Master
Mix (Applied Biosystems) and each of the appropriate forward/reverse primer
set (Supplementary Table5). Relative mRNA expression levels were quantified
with Sequence Detection Software v1.3.1 (Applied Biosystems).
resuspending mitochondria isolated from yeast in R-buffer (0.65M sucrose,
10mM HEPES (pH7.5), 10mM KH2PO4, 5mM KCl, 5mM MgCl2) to a final
concentration of 100mgml–1. Mitochondrial concentration was measured by
dissolving mitochondria in 0.6% SDS and reading absorbance at l5280nm.
The mitochondrial suspension was then incubated with 25mM NADH, and the
absorbance at l5340nm monitored over a 15min period. The assay was
repeatedin thepresenceof erastin.To assessmitochondrial intactness,a parallel
assay was run in which mitochondria were hypotonically shocked before addi-
tion of NADH. The mitochondrial pellet was resuspended in distilled H2O and
incubated on ice for 3min to disrupt the outer mitochondrial membrane;
23R-buffer was then added to restore normal osmotic conditions.
Cell viability assays. Trypan blue exclusion: cells were trypsinized, pelleted and
resuspended in 1ml growth media. Trypan blue exclusion analysis was per-
formed using the Vi-CELL Series Cell Viability Analyser 1.01 (Beckman
Coulter). Alamar blue metabolism: 10% Alamar blue was added to assay plates,
which were then incubated for an additional 16h. Red fluorescence, resulting
from reduction of Alamar blue, was detected on a Victor3 platereader (excita-
VDAC2 binding assay. VDAC2 protein was isolated from E.coli using a modi-
grown in Luria-Bertani broth containing 50mgl–1ampicillin to an absorbance
of 0.6 (l5600nm), and induced using 0.4mM isopropylthiogalactoside over-
night. Cultures were harvested by centrifugation at 6,000g for 10min. The pellet
was then washed with distilled H2O and resuspended in buffer (20% sucrose,
20mM Tris buffer (pH8.0), 50mMml–1lysozyme) and incubated at 25uC for
10min. The lysate was then sonicated for 2330s and centrifuged at 15,000g for
20min. The pellet was resuspended in resuspension buffer (4.5M guanidine-
HCl, 0.1M NaCl, 20mM Tris (pH8.0)) and incubated for 1h at 25uC. The
suspension was then centrifuged (20min, 15,000g), and the supernatant was
loaded on a Ni-NTA Superflow column (Qiagen) pre-equilibrated with five
volumes of resuspension buffer. The column was washed with five column
volumes of resuspension buffer containing 10mM imidazole. The protein was
eluted using resuspension buffer containing 225mM imidazole. The eluate was
then dialysed against 0.1M NaCl, 20mM Tris (pH8.0) and 2% LDAO (Fluka)
overnight, and then concentrated by means of centrifugation to 4mgml–1. To
assay direct binding of erastin analogues, 40mg of purified VDAC2 was resus-
pended in 100ml of binding buffer (25mM HEPES (pH8.0), 0.1% BSA, 7mM
vacuum filtration. The filter was rinsed 5 times with 1ml wash buffer (25mM
HEPES, 0.1% BSA), and incubated in 5ml scintillation liquid (Cytoscint, MP
biomedicals). Radioactivity was detected on a LKB Wallac 1211 RACKBETA
liquid scintillation counter.
25. Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. Basic local
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