Cucurbitacin D Is a Disruptor of the HSP90 Chaperone Machinery

Article abstract of DOI:10.1021/acs.jnatprod.5b00054

Heat shock protein 90 (Hsp90) facilitates the maturation of many newly synthesized and unfolded proteins (clients) via the Hsp90 chaperone cycle, in which Hsp90 forms a heteroprotein complex and relies upon cochaperones, immunophilins, etc., for assistance in client folding. Hsp90 inhibition has emerged as a strategy for anticancer therapies due to the involvement of clients in many oncogenic pathways. Inhibition of chaperone function results in client ubiquitinylation and degradation via the proteasome, ultimately leading to tumor digression. Small molecule inhibitors perturb ATPase activity at the N-terminus and include derivatives of the natural product geldanamycin. However, N-terminal inhibition also leads to induction of the pro-survival heat shock response (HSR), in which displacement of the Hsp90-bound transcription factor, heat shock factor-1, translocates to the nucleus and induces transcription of heat shock proteins, including Hsp90. An alternative strategy for Hsp90 inhibition is disruption of the Hsp90 heteroprotein complex. Disruption of the Hsp90 heteroprotein complex is an effective strategy to prevent client maturation without induction of the HSR. Cucurbitacin D, isolated from Cucurbita texana, and 3-epi-isocucurbitacin D prevented client maturation without induction of the HSR. Cucurbitacin D also disrupted interactions between Hsp90 and two cochaperones, Cdc37 and p23.

Full text of DOI:10.1021/acs.jnatprod.5b00054

Article
pubs.acs.org/jnp
Cucurbitacin D Is a Disruptor of the HSP90 Chaperone Machinery
Jessica A. Hall,† Sahithi Seedarala,† Nichole Rice,‡ Lucas Kopel,‡ Fathi Halaweish,‡ and Brian S. J. Blagg*,†
†Department of Medicinal Chemistry, The University Of Kansas, 1251 Wescoe Hall Drive, 4070 Malott Hall, Lawrence, Kansas
66045, United States
‡Department of Chemistry & Biochemistry, South Dakota State University, Brookings, South Dakota 57007, United States
S
* Supporting Information
ABSTRACT: Heat shock protein 90 (Hsp90) facilitates the
maturation of many newly synthesized and unfolded proteins
(clients) via the Hsp90 chaperone cycle, in which Hsp90 forms
a heteroprotein complex and relies upon cochaperones,
immunophilins, etc., for assistance in client folding. Hsp90
inhibition has emerged as a strategy for anticancer therapies
due to the involvement of clients in many oncogenic pathways.
Inhibition of chaperone function results in client ubiquitiny-
lation and degradation via the proteasome, ultimately leading
to tumor digression. Small molecule inhibitors perturb ATPase activity at the N-terminus and include derivatives of the natural
product geldanamycin. However, N-terminal inhibition also leads to induction of the pro-survival heat shock response (HSR), in
which displacement of the Hsp90-bound transcription factor, heat shock factor-1, translocates to the nucleus and induces
transcription of heat shock proteins, including Hsp90. An alternative strategy for Hsp90 inhibition is disruption of the Hsp90
heteroprotein complex. Disruption of the Hsp90 heteroprotein complex is an effective strategy to prevent client maturation
without induction of the HSR. Cucurbitacin D, isolated from Cucurbita texana, and 3-epi-isocucurbitacin D prevented client
maturation without induction of the HSR. Cucurbitacin D also disrupted interactions between Hsp90 and two cochaperones,
Cdc37 and p23.
sp90 is a molecular chaperone responsible for the correct
Hconformation of many newly synthesized polypeptides
and the rematuration of denatured proteins termed “clients” via
the Hsp90 chaperone cycle. Hsp90 functions as a homodimer
and requires the formation of the heteroprotein complex
composed of several immunophilins, cochaperones, and partner
proteins for assistance in chaperone activity. Several conforma-
tional transitions of the Hsp90 heteroprotein−client complex,
coupled to Hsp90 ATPase activit,y results in correct folding
and release of client protein.1,2 Inhibition of the chaperone
Figure 1. Structures of geldanamycin (GDA) (1), celastrol (2), and
gedunin (3).
cycle leads to client protein ubiquiltinylation and subsequent
degradation via the proteasome.3−5 Hsp90 inhibition has
emerged as a strategy for anticancer chemotherapeutics due
shock response (HSR).13−16 Upon displacement, HSF-1
to the involvement of Hsp90-dependent clients in a variety of
oncogenic signaling pathways.6−11 Many clients (i.e., ErbB2, B-
trimerizes, translocates to the nucleus, and induces tran-
scription of the heat shock element. This leads to increases in
the cellular concentration of heat shock proteins, including
Hsp90, and resulted in dosing and scheduling problems during
clinical trials of N-terminal inhibitors. Therefore, anticancer
Hsp90 inhibitors that avoid induction of the HSR are needed.
Alternative strategies to inhibit the chaperone cycle include
targeting the Hsp90 C-terminus and disruption of the
heteroprotein complex. C-Terminal inhibitors include deriva-
tives of the coumarin-containing natural product novobio-
cin.17−20 These inhibitors prevent cancer cell proliferation at
Raf, Akt, steroid hormone receptors, mutant p53, HIF-1,
survivin, telomerase, etc.) are contributors to the six hallmarks
of cancer. Oncogenic client degradation via Hsp90 inhibition
ultimately halts cancer progression and has been observed
during clinical trials of Hsp90 inhibitors.
Classic small molecule Hsp90 inhibitors are designed to
perturb N-terminal ATPase activity and include derivatives of
geldanamycin (1) (Figure 1), radicicol, and several purine
analogs.12 Although Hsp90 N-terminal inhibitors are effective
at inhibiting Hsp90 function and lead to tumor digression
through client degradation, N-terminal inhibition also leads to
displacement of the Hsp90-bound transcription factor, heat
shock factor-1 (HSF-1), and induction of the pro-survival heat
Received: January 19, 2015
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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concentrations similar to N-terminal inhibitors and destabilize
Hsp90−client protein interactions without induction of the
HSR. Maturation of all clients requires additional proteins that
interact with Hsp90 and modulate ATPase activity and/or
assist in correct client folding.21,22 For example, the
cochaperone Hsp90−Hsp70 organizing protein (HOP) asso-
ciates with the Hsp90 dimer during the initial stages of the
chaperone cycle and facilitates the delivery of certain clients
from Hsp70 to Hsp90. Peptidyl−prolyl isomerase cochaper-
ones (PPIases) assist in the correct folding and 3-diminsional
structure of clients, and the cochaperone p23 promotes the
optimal Hsp90 conformation for ATP binding and hydrolysis,
ultimately resulting in client protein release.23 In addition,
classes of clients require the assistance of specific cochaperones
for maturation (e.g. all Hsp90-dependent kinases require the
assistance of the cochaperone cell division cycle 37 (Cdc37) for
maturation). Disruption of interactions between Hsp90 and
client-specific cochaperones may allow for select degradation of
clients and avoid systemic client protein degradation, a
consequence of Hsp90 inhibitors under current clinical
investigation, and may be the cause of certain side effects.
The cucurbitacin class of natural products is a group of
triterpenoids that are present in the Cucurbitaceae and other
closely related families. More than 50 cucurbitacins have been
identified and exhibit a wide variety of biological activities that
include, but are not limited to, cytotoxicity, antiproliferation,
anti-inflammation, antioxidant, antihepatotoxicity, antibacterial,
and antiviral properties, as well as antimetastatic properties and
improved anticancer activity when combined with current
chemotherapies.24−26 Despite the presence of an α-, β-
unsaturated ketone (Michael acceptor; a well-established and
nonspecific disruptor of general cellular processes via electro-
philic chemical reactivity) located within many of the
cucurbitacin compounds, several cucurbitacins interact with
specific cellular targets involved in gene regulation, tran-
scription, and overall cellular fate. Cucurbitacin D (4) reduced
the proliferation of T-cell leukemia cells and ultimately led to
cancer cell apoptosis.27 This cucurbitacin was shown to affect
the NF-κB pathway and led to the accumulation of NF-κB and
IκB-α within the cytosol. Interestingly, a decrease in the cellular
levels of Bcl-xL and Bcl-2 was also observed. Bcl-xL and Bcl-2
levels are regulated by Hsp90 clients that depend on the
chaperone cycle for correct folding and function. It has been
reported that incubation with N-terminal inhibitors led to
cellular decreases in Bcl-xL and Bcl-2 levels.28,29 Furthermore,
4, as well as other cucurbitacins, share structural similarities to
the natural products celastrol (2) and gedunin (3), which are
known inhibitors of client maturation via disruption of the
Hsp90 heteroprotein complex (Figure 1). However, these
natural products inhibit chaperone function by different
mechanisms. 2 covalently binds the Hsp90 N-terminus and
disrupts the interaction between Hsp90 and Cdc37, ultimately
resulting in kinase client degradation.30 3 binds the
cochapreone p23 and disrupts Hsp90-cochaperone interactions,
leading to client degradation across many different cancer cell
lines.31 Given certain structural similarities between the
cucurbitacins, 2 and 3, as well as decreased levels of proteins
that depend on functional clients after exposure to
cucurbitacins, it was hypothesized that 4, and other
cucurbitains, may inhibit the Hsp90 chaperone cycle in a
manner similar to 2 and/or 3.
complex. With the exception of 23,24-dihydrocucurbitacin D
(9) and 23,24-dihydrocucurbitacin B (10), all cucurbitacins
exhibited antiproliferative IC50 values at nanomolar concen-
trations against the MCF7 breast cancer cell line. Western blot
analysis for client levels in the presence of high and low
concentrations of each cucurbitacin revealed that incubation
with 4 and 3-epi-isocucurbitacin D (6) led to client degradation.
4 also disrupted the interaction between Hsp90 and the
cochaperones Cdc37 and p23; however, 6 did not disrupt these
Hsp90-co-chaperone interactions components and caused client
degradation through alternative mechanism(s). Furthermore, 4
did not induce the HSR, as no increases in the levels of heat
shock proteins Hsp90, Hsp70, or Hsp27 was observed. Client
protein degradation, disruption of Hsp90-Cdc37 and Hsp90-
p23 interactions as well as a lack of heat shock protein
induction, notably Hsp27, has previously been observed after
incubation with 3 and suggested 4 inhibited cancer cell growth
through Hsp90 client degradation via disruption of Hsp90-co-
chaperone interactions by a mechanism comparable to 3.
RESULTS AND DISCUSSION
■
Cucurbitacin D (4) and cucurbitacin B (5), along with isomeric
3-epi-isocucurbitacin D (6), were isolated from the fruits of
Cucurbita texana (Cucurbitaceae) following previously de-
scribed protocols (Figure 2).32−34 Two additional oxidized
Figure 2. Chemical structures of cucurbitacins isolated from Cucurbita
texana (Cucurbitaceae) [cucurbitacin D (4) and B (5); 3-epi-
isocucurbitacin D (6); cucurbitacin I (7) and E (8)] and the
structures of the semisynthesized natural products [3-epi-cucurbitacin
D (6), cucurbitacin I (7), 23,24-dihydrocucurbitacin D (9)
(cucurbitacin R) and B (10)].
derivatives, cucurbitacin I (7) and E (8), were obtained as well.
These latter compounds along with 6 were obtained in reduced
quantities compared to that of 4 and 5. Fortunately,
semisynthetic approaches to these particular compounds have
been investigated in the literature and can be seen as a solution
to the lack of material for these natural products. In addition, 9,
10, and other reduced C23−24 derivatives have routinely been
acquired via hydrogenation conditions. Structures of the
isolated and semisynthesized cucurbitacins of interest are
shown in Figure 2.
The isomerization of the α-hydroxyketone at C2 and C3 of
some cucurbitacins has been determined to take place under
both acidic and basic conditions, with the most thorough
Herein, we report that 4 prevented the maturation of Hsp90-
dependent clients by disruption of the Hsp90 heteroprotein
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investigation having been performed by Galindo and co-
workers.35,36 However, an alternative to Galindo’s procedure
was chosen instead, one similar to that reported by Sneden et
al. that takes advantage of the slightly acidic nature of silica
gel.37 Heating a CH2Cl2 solution of 4 and silica gel to 40 °C
provided useful quantities of 6 for biological evaluation and the
ability to recover unreacted 4 (Scheme 1).
for each cucurbitacin can be found in Table 1 along with values
for 1, an Hsp90 N-terminal inhibitor, 2, and 3.
Table 1. Calculated IC50 Values for the Controls
Geldanamycin (GDA) (1), Celastrol (2), Gedunin (3), and
Select Cucurbitacins against the MCF7 Breast Cancer Cell
Line
compound
MCF7 IC50 values (μM)
Scheme 1. Semisynthesis of Cucurbitacins: 3-epi-
Isocucurbitacin D (6), Cucurbitacin I (7), 23,24-
Dihydrocucurbitacin D (9) (Cucurbitacin R), and 23,24-
GDA (1)
0.0427 0.018
0.153 0.004
10.9 0.9
celastrol (2)
a
gedunin (3)
Dihydrocucurbitacin B (10)
cucurbitacin D (4)
0.598 0.001
0.0413 0.003
0.718 0.009
0.0607 0.012
0.055 0.006
5.13 0.01
cucurbitacin B (5)
3-epi-isocucurbitacin D (6)
cucurbitacin I (7)
cucurbitacin E (8)
23,24-dihydrocucurbitacin D (9)
23,24-dihydrocucurbitacin B (10)
46.3 0.1
Hsp90 inhibitors and disruptors of the heteroprotein
complex prevent cancer cell proliferation at concentrations
near the IC50 value by depleting levels of clients necessary for
cancer cell growth. To correlate client degradation via
inhibition of the chaperone cycle to the IC50 value for each
cucurbitacin, client levels from MCF7 cell lysates dosed with
high and low concentrations (5 times the IC50 value and half of
the IC50 value, respectively) of each cucurbitacin were
dectected by Western blot analysis. The levels of heat shock
proteins were also detected to account for induction of the
HSR as well as the levels of p23 and Cdc37. Incubation with
high concentrations of the control compounds 1, 2, and 3 led
to client protein degradation and little change in the levels of
Cdc37 and p23 (Figure 3A). Consistent with previous reports,
incubation with GDA led to increased levels of Hsp90, Hsp70,
and Hsp27, indicating induction of the HSR (Figure 3A).
Minimal induction of the HSR was observed at the
concentrations of 2 and 3 tested; incubation with 3 caused
only a slight increase in Hsp70 levels, and the levels of Hsp90
and Hsp27 were comparable to vehicle control upon incubation
with 2 and 3 (Figure 3A).
Levels of clients and heat shock proteins after incubation
with high and low concentrations of each cucurbitacin can be
found in Figure 3B and within the Supporting Information
(Figures S1 and S2). Incubation with high concentrations of 4
and 6 led to a decrease in client levels (Supporting Information,
Figure S3), indicating disruption of client maturation. These
cucurbitacins also decreased client levels in a dose-dependent
manner at and around their IC50 values (Figure 3B). No
changes in Hsp90 or Hsp70 levels were observed; however,
Hsp27 levels were increased at high concentrations of 6. The
levels of Cdc37 and p23 remained constant at all concen-
trations (Supporting Information, Figure S3, and Figure 3B).
Client and heat shock protein levels were unchanged after
incubation with high and low concentrations of the remaining
cucurbitacins (Supporting Information, Figure S1 and S2). No
client degradation at high concentrations of compound and
constant heat shock protein levels at high and low
concentrations indicates the mechanism(s) by which these
compounds inhibited MCF7 cancer cell growth is unrelated to
Hsp90 inhibition.
a
Reagents and conditions: (a) silica gel, CH2Cl2, 40 °C, 25% (30%
conversion); (b) Pd(OAc)2, PhBr, NaOAc, DMA, 80 °C, 38% (60%
conversion); (c) H2 (1 atm), Pd/C 5%, EtOH, 88%.
While attempting to form cucurbitacin derivatives via a Heck
reaction,38 in an effort to improve their biological activity, an
undesired product was isolated and later determined by
spectroscopic analysis to be 7. Even though this is not the
first report of the oxidation of the α-hydroxyketone to a
diosphenol moiety for cucurbitacins,39−41 this is the first
example of the transformation via a Saegusa oxidation (Scheme
1).42−45 This result can be rationalized via the ability of
cucurbitacins to isomerize to form the intermediate enediol,
which can be trapped and oxidized to the final diosphenol
product. This is the same enediol intermediate that is necessary
for the formation of isocucurbitacins. While 7 was not the
initially desired product, the resulting extra quantity was now
available for biological testing.
To date, Cucurbita texana has not produced any 9 or 10 via
natural product isolation. However, hydrogenation of the C23−
24 olefin of 4 and 5 has allowed for the ability to obtain useful
quantities of the semisynthesized 9 and 10 from a plant species
that does not routinely provide them through natural product
isolation (Scheme 1).35,40,46−52
Select Cucurbitacins Exhibit Potent Antiproliferative
Activity and Decreased Hsp90-Dependent Client Pro-
tein Levels without Induction of the HSR. Consistent with
previous data against several cancer cell lines, many of these
cucurbitacins exhibited nanomolar IC50 values against the
MCF7 breast cancer cell line. The decrease in potency observed
for 9 and 10 may be explained by reduction of the α-, β-
unsaturated ketone. This would reduce the potential for
nonspecific, attack by cellular nucleophiles. The IC50 values
Cucurbitacin D, But Not 3-epi-Isocucurbitacin D,
Caused Client Protein Degradation through Disruption
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Figure 3. (A) Western blot for the levels of Hsp90 clients (pAkt, Her2, Cdk6, and Raf), heat shock proteins (Hsp90, Hsp70, and Hsp27),
cochaperones (Cdc37 and p23), and actin from MCF7 cell lysates treated for 24 h with vehicle (0.25% DMSO) or high concentrations (5 times the
IC50 value) of the controls geldanamycin (GDA) (1), celastrol (2), gedunin (3). (B) Levels of Hsp90 clients, heat shock proteins and cochaperones
from cell lysates treated with vehicle or increasing concentrations of cucurbitacin D (4) or 3-epi-isocucurbitacin D (6).
of Hsp90-cochaperone Interactions. Concentration-de-
pendent client degradation void of induction of the HSR
indicates an alternative mechanism to traditional N-terminal
inhibitors for preventing client maturation. Compounds such as
2 and 3 disrupt the Hsp90 heteroprotein complex, thereby
preventing cochaperone assistance during the chaperone cycle.
In addition, concentrations of these compounds that destabilize
the heteroprotein−client complex do not induce the HSR.
There are several reports of 2 and 3 as Hsp90 modulators
present in literature. These natural products disrupt the
interactions between Hsp90 and cochaperones involved in
both early and late stages of the chaperone cycle. Celastrol (2)
was shown to covalently bind the Hsp90 N-terminus, thereby
disrupting Hsp90 from interacting with Cdc37, a cochaperone
involved during the early stages of the chaperone cycle.53
Gedunin (3) binds the cochaperone p23 and prevents p23 from
facilitating progression of the Hsp90 chaperone cycle.31 This
ultimately results in improper trafficking and localization of
clients, such as the steroid receptor, glucocorticoid receptor.
Consistent with previous studies, Figure 4A confirmed that
high concentrations of 2 and 3 disrupted components of the
heteroprotein complex, while the N-terminal inhibitor 1 did not
disrupt these Hsp90-co-chaperone interactions. Co-immuno-
precipitation of Cdc37 and p23 was disrupted in the presence
of high concentrations of 2 and 3 when using an antibody
targeting Hsp90. A high concentration of 4 also disrupted these
heteroprotein complex components from Hsp90 (Figure 4B).
Incubation with a high concentration of 4 completely disrupted
Hsp90−Cdc37 and Hsp90−p23 interactions from MCF7 cell
lysates. Interestingly, a high concentration of 6 had no effect on
the interactions between Hsp90 and these cochaperones;
Hsp90−Cdc37 and Hsp90−p23 interactions remained intact at
high and low concentrations of 6 (Figure 4B).
These data suggested that, although 4 and 6 both resulted in
client protein degradation, the mechanisms by which these
compounds disrupted client maturation are different. 4
disrupted Hsp90−Cdc37 and Hsp90−p23 complexes, which
ultimately led to a cellular decrease of client levels. 6 also
reduced client levels but did not affect these Hsp90-
cochaperone interactions. This cucurbitacin may directly inhibit
Hsp90 and bind an alternative site to the N-terminal ATP-
binding pocket (e.g., C-terminal inhibitors, sansalvamide A,
D
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Figure 5. Western blot for Hsp27 and actin levels from MCF7 cell
lysates treated for 24 h with vehicle (0.25% DMSO), 5 times (5×) ,or
10 times (10×) the IC50 value of the gedunin (final 5× concentration
= 54.5 μM; final 10× concentration = 109.0 μM) (3), cucurbitacin D
(final 5× concentration = 0.765 μM; final 10× concentration = 5.980
μM) (4) and 3-epi-isocucurbitacin D (final 5× concentration = 3.590
μM; final 10× concentration = 7.180 μM) (6).
increased levels of Hsp27 were detected at high concentrations
of 6, further supporting alternative mechanisms of client
degradation for these cucurbitacins. In conclusion, the
curcurbitacins have been shown to affect multiple cellular
pathways and exhibit potent inhibitory activity against many
cancer cell lines. Here we report the antiproliferative activity of
cucurbitacin D and 3-epi-isocucurbitacin D is partly attributed
to disruption of Hsp90 client protein maturation and
cucurbitacin D destabilizes components of the Hsp90
heteroprotein complex, ultimately resulting in client degrada-
tion.
Figure 4. (A) Co-immunoprecipitation of Cdc37 and p23 using an
anti-Hsp90 antibody (abcam) from MCF7 cell lysates treated for 24 h
with vehicle (0.25% DMSO) or high concentrations (5 times the IC50
value) of the controls geldanamycin (GDA; final concentration =
0.2135 μM) (1), celastrol (final concentration = 0.765 μM) (2),
gedunin (final concentration = 54.5 μM) (3). (B) Co-immunopreci-
pitation of Cdc37 and p23 using an anti-Hsp90 antibody from cell
lysates treated with high and low concentrations (5 times the IC50
value and half the IC50 value, respectively) of cucurbitacin D (final [H]
concentration = 0.765 μM; final [L] concentration = 0.299 μM) (4) or
3-epi-isocucurbitacin D (final [H] concentration = 3.590 μM; final [L]
concentration = 0.359 μM) (6).
EXPERIMENTAL SECTION
■
General Experimental Procedures. Experimental data for each
isolated compound was compared to previously reported literature
data. NMR spectra were acquired on a Bruker AVANCE-400 MHz
NMR spectrometer and compared to previously published data. High-
resolution mass spectrometry (HRMS) data was obtained using either
electron ionization (EI) on a ThermoFinnigan MAT 95 XL mass
spectrometer or electrospray ionization (ESI) on a ThermoFinnigan
LCQ Advantage ion trap liquid chromatography−mass spectrometer
(LC/MS). Melting points were determined on a Vernier Melt Station
melting point apparatus. TLC analysis was performed using precoated
silica gel PE sheets (UV254, 250 μm layer). Compounds were purified
via column chromatography using silica gel 40−63 μm (230−400
mesh), preparative normal phase-TLC (silica gel, UV254, 2 μm layer),
and reverse phase HPLC using a Dynamax liquid chromatograph
(Varian Chromatography Systems) with a PDA-2 photodiode array
UV detector (Alltech preparative column, Econosil C18 10 μm, length
250 mm, ID 22 mm). Gradients of MeOH/H2O or ACN/H2O were
utilized with a 9 or 13 mL/min flow rate. Analytical separations were
optimized on an Alltima C18 (Alltech analytical column, 5 μm, length
250 mm, ID 4.6 mm) HPLC column using a flow rate of 1 mL/min
then transferred to preparative scale. All reagents and solvents were
obtained from commercial suppliers and used as received.
etc.), bind and inhibit the function of proteins important for
client maturation (e.g., efrapeptins, cruentaren A, etc.), or
disrupt interactions of other protein with the Hsp90
heteroprotein complex.54−56
High Concentrations of Cucurbitacin D Did Not
Increase Cellular Levels of Hsp27. Patwardhan et al.
reported that 3 does not induce the HSR in several different
cancer cell lines, including the cervical cancer cell line HeLa and
the breast cancer cell lines Hs578T and MCF7.31 More
specifically, no increases in the levels of Hsp90, Hsp70, or
Hsp27 were observed after incubation with concentrations up
to 160 μM of 3. To corroborate Western blot and co-
immunoprecipitation data reported here and further verify 4
disrupted client protein maturation in a manner comparable to
3, Hsp27 levels were detected after incubation with high
concentrations of 3, 4, and 6. MCF7 cells incubated with 5 and
10 times the IC50 value of each compound, and levels of Hsp27
were detected via Western blot analysis (Figure 5). The levels
of Hsp27 were unchanged after incubation with high
concentrations of 3 and 4 and comparable to vehicle control;
however, increased Hsp27 levels were observed at high
concentrations of 6 and are consistent with Figure 3B.
Collectively, these data suggest that 4 destabilized client
maturation through similar mechanisms to 3 by disruption of
Cdc37 and p23 from the Hsp90 heteroprotein complex;
however, 4 exhibited a dramatic 18-fold increase in potency
when compared to 3. 6 also led to client degradation at
nanomolar concentrations, but did not disrupt Hsp90−Cdc37
or Hsp90−p23 interactions and is presumed to cause
degradation by alternative mechanisms. Like 3, no increase in
Hsp27 levels was observed at high concentrations of 4, yet
Isolation and Semisynthesis of Cucurbitacins. Fruits from
Cucurbita texana (Cucurbitaceae) were cut into pieces and
homogenized with MeOH and filtered, and the solvent was removed
under reduced pressure. The residue was subjected to flash column
chromatography with gradient elution (hexane/EtOAc and then
EtOAc/MeOH of increasing polarity). The fractions containing the
same compounds as determined by TLC, were combined, and
subjected to further purification by one of three methods: normal
phase column chromatography, preparative normal phase TLC, or
preparative reverse phase HPLC.32−35 The spectroscopic data for the
pure isolated compounds were compared to published literature data:
(1) cucurbitacin D (4);57−59 (2) cucurbitacin B (5);52,60 (3) 3-epi-
isocucurbitacin D (6);57,61,62 (4) cucurbitacin I (7);60,63 (5)
cucurbitacin E (8);60,63 (6) 23,24-dihydrocucurbitacin D (9);64 (7)
23,24-dihydrocucurbitacin B (10);52,65 (8) data for multiple
cucurbitacins.66−69
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Preparation of 3-epi-Isocucurbitacin D (6). Cucurbitacin D (4)
(50 mg, mmol) was added to a suspension of silica gel (3 g) in CH2Cl2
(5 mL). The reaction was heated to 40 °C for 72 h. The solution was
allowed to cool, followed by dilution with EtOAc and filtration
through plug of silica gel. The crude mixture was purified via
preparative normal phase TLC to provide 3-epi-isocucurbitacin D (6)
(13 mg) as a white solid in 25% yield.
Preparation of Cucurbitacin I (8). Palladium acetate (5 mg, 0.022
mmol, 0.3 equiv) was added to a vial containing cucurbitacin D (4)
(44 mg, 0.085 mmol, 1 equiv), NaOAc (4 mg, 0.043 mmol, 0.5 equiv),
and bromobenzene (0.01 mL, 0.095 mmol, 1.1 equiv) dissolved in
dimethylacetamide (DMA) (0.40 mL). The vial was sealed with a
Teflon lined cap, and the reaction was stirred at 80 °C for 24 h. The
reaction was allowed to cool and diluted with EtOAc (10 mL) and
H2O (5 mL), followed by extracting the aqueous layer three times with
EtOAc. The combined organic layers were washed with brine and
dried over anhydrous Na2SO4. The crude product was initially purified
via column chromatography using hexane/EtOAc to remove residual
DMA. Minor impurities were further removed via preparative reverse
phase HPLC using MeOH/H2O, resulting in the isolation of
cucurbitacin I (8) (16 mg) as a white solid in 38% yield (unoptimized
reaction conditions).
Preparation of 23,24-Dihydrocucurbitacin D (10) (Cucurbitacin
R) and 23,24-Dihydrocucurbitacin B (11). First, 5% palladium on
carbon (16 mg) was added to a 25 mL round-bottom flask containing
the desired cucurbitacin starting material (44.0 mg) dissolved in EtOH
(5 mL). Then a septum was added to the flask, the headspace was
flushed with H2, and the reaction was stirred for 4 h at 1 atm H2. Upon
completion, the reaction was filtered through a plug of silica gel using
EtOAC. The crude product was purified using column chromatog-
raphy (hexanes/EtOAc) to provide the desired dihydro compounds as
white solids in 76−88% yield.
Antibodies and Reagents. Antibodies targeting Her2, Cdk6,
phospho-Akt (pAkt), and Hsp27 were purchased from Cell Signaling
Technology. Antibodies targeting Raf and actin were purchased from
Santa Cruz Biotechnology. The remaining antibodies are listed and
were purchased from the indicated vendors: Hsp90 (Enzo Life
Sciences), Hsp70 (Assay Designs), and Cdc37 and p23 (Abcam).
Gedunin was isolated in house. Celastrol was purchased from Cayman
Chemical, and geldanamycin was purchased from Sigma-Aldrich.
Cell Culture. MCF7 cells were maintained in Advanced DMEM/
F12 (1:1; Gibco) supplemented with streptomycin (500 μg/mL;
Corning Cellgro), penicillin (100 units/mL; Corning Cellgro), L-
glutamine (2 mM; Corning), and 10% FBS (Atlanta Biologicals). Cells
were grown in a humidified atmosphere (37 °C, 5% CO2) and
passaged when confluent.
conditions (10% and 12% acrylamide gels; made in house), transferred
to PVDF (0.45 μm; Thermo Scientific), and immunoblotted with the
corresponding antibody. Membranes were incubated with an
appropriate horseradish peroxidase-labeled secondary antibody
(Santa Cruz Biotechnology and GE Healthcare), developed with a
chemiluminescent substrate, and visualized.
Co-immunoprecipitation. MCF7 cells were grown to confluence
and seeded at 2 × 106 cells/5 mL in 10 cm dishes. Cells were
incubated for 24 h and then treated with compound in DMSO (0.25%
DMSO final concentration) or vehicle (DMSO) for 24 h. Media and
cells were collected with DPBS and centrifuged at 200g for 5 min at 4
°C. Supernatant was aspirated, and pellets were washed one time with
cold DPBS and centrifuged. Supernatant was aspirated, and cell pellets
were subsequently suspended in nondenaturing lysis buffer (20 mM
Tris-HCl at pH 7.5, Fisher Scientific; 25 mM KCl, Sigma-Aldrich; 2
mM MgCl, Fisher Scientific; 2 mM DTT, Sigma-Aldrich; 20 mM
sodium molybdate, Sigma-Aldrich; 2 mM ATP, Sigma-Aldrich; and
0.1% NP-40, US Biological) supplemented with protease and
phosphatase inhibitors (Roche) and incubated on ice for 2 h. Lysates
were clarified at 14000g for 10 min at 4 °C. Protein concentrations
were determined using the Pierce BCA protein assay kit per the
manufacturer’s instructions. Equal protein (400 μg) was incubated
with 1 μg of Hsp90 antibody (H90.10; abcam) in 500 μL total volume
of lysis buffer for approximately 2 h with rocking at 4 °C. Following
incubation, 30 μL of resuspended MagBeads Protein G (GenScript)
were added to each sample and incubated with rocking for 1.5 h at 4
°C. Protein G beads were washed 3 times with lysis buffer (500 μL),
suspended in Laemmli sample buffer (15 μL), and boiled at 70 °C for
15 min to dissociate proteins from beads. Samples were electro-
phoresed under reducing conditions (12% acrylamide gels; made in
house), transferred to PVDF, and immunoblotted with the indicated
antibodies. Membranes were incubated with a species-appropriate
horseradish peroxidase-labeled secondary antibody, developed with a
chemiluminescent substrate, and visualized.
ASSOCIATED CONTENT
* Supporting Information
■
S
Western blot for Hsp90 client proteins (pAkt, Her2, and Cdk6)
and heat shock proteins (Hsp70 and Hsp90) from cell lysates
dosed for 24 hours with high and low concentrations of each
1
cucurbitacin; H NMR spectra of cucurbitacins 4−10; electron
impact HR-MS for semisynthesized cucurbitacin I (7). This
material is available free of charge via the Internet at http://
pubs.acs.org.
Antiproliferation. Cells were grown to confluence, seeded (2000
cells/well, 100 μL total media) in clear, flat-bottom 96-well plates and
allowed to attach overnight. Varying concentrations of compound in
DMSO (1% DMSO final concentration; Sigma-Aldrich) was added.
Cells were returned to the incubator for an additional 72 h. After 72 h,
cell growth was determined using an MTS/PMS cell proliferation kit
(Promega) per the manufacturer’s instructions. Cells that incubated in
1% DMSO were used as 100% proliferation (i.e., DMSO = 100%
growth), and the relative growth for each compound concentration
was compared to 1% DMSO. IC50 values were calculated from two
separate experiments performed in triplicate using GraphPad Prism 6.0
(GraphPad Software).
Western Blot. MCF7 cells were grown to confluence and seeded at
0.4 × 106 cells/well/2 mL. Cells were incubated for 24 h and treated
with varying concentrations of compound in DMSO (0.25% DMSO
final concentration) or vehicle (DMSO) for 24 h. Cells were harvested
in cold DPBS (Corning Cellgro) and lysed using MPER (Thermo
Scientific) supplemented with protease and phosphatase inhibitors
(Roche) according to the manufacturer’s directions. Lysates were
clarified at 14000g for 10 min at 4 °C. Protein concentrations were
determined using the Pierce BCA protein assay kit per the
manufacturer’s instructions. Equal amounts of protein (20 μg) were
suspended in Laemmli sample buffer (15 μL; BioRad) and boiled at 70
°C for 15 min. Samples were then electrophoresed under reducing
AUTHOR INFORMATION
Corresponding Author
*Phone: (785) 864-2288. E-mail: bblagg@ku.edu.
Notes
■
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the NIH grant CA109265 and the
NIGMS Biotechnology Predoctoral Training Grant Program.
We would like to thank South Dakota Board of Regents-2010
Biological Control and Analysis by Applied Photonics
(BCAAP) for financial support.
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