Cryptocaryol Structure-Activity Relationship Study of Cancer Cell Cytotoxicity and Ability to Stabilize PDCD4

Article abstract of DOI:10.1021/ml4005039

The synthetic cryptocaryols A and B and a series of their analogues have been evaluated for their cytotoxicity and their ability to stabilize the tumor suppressor PDCD4. Cytotoxicities in the 3 to 30 μM range were found. Both the cytotoxicity and PDCD4 stabilizing ability were tolerant of large stereochemical changes to the molecule. Co-dosing studies with cryptocaryols A and B and several known cancer drugs showed no measuable enhancement in cancer drug cytotoxicity.

Full text of DOI:10.1021/ml4005039

Letter
pubs.acs.org/acsmedchemlett
Cryptocaryol Structure−Activity Relationship Study of Cancer Cell
Cytotoxicity and Ability to Stabilize PDCD4
Michael F. Cuccarese,† Yanping Wang,† Penny J. Beuning,* and George A. O’Doherty*
Department of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts 02115, United States
S
* Supporting Information
ABSTRACT: The synthetic cryptocaryols A and B and a series of
their analogues have been evaluated for their cytotoxicity and their
ability to stabilize the tumor suppressor PDCD4. Cytotoxicities in
the 3 to 30 μM range were found. Both the cytotoxicity and
PDCD4 stabilizing ability were tolerant of large stereochemical
changes to the molecule. Co-dosing studies with cryptocaryols A
and B and several known cancer drugs showed no measuable
enhancement in cancer drug cytotoxicity.
KEYWORDS: PDCD4, PDCD4 stabilizer, cryptocaryols, cryptocaryol SAR
Loss of PDCD4 is observed in lung, breast, colon, and
prostate cancers.12 Similarly, in a panel of 124 lung cancer
patients, expression of PDCD4 in tumor cells was inversely
related to poor prognosis.13 Also, expression of PDCD4 has
been shown to confer increased sensitivity to some anticancer
drugs14 and reduce the malignancy of ovarian cancer cells.
Thus, PDCD4 is a target for the development of novel
antineoplastic agents.15
PDCD4 is degraded within the cell via a discrete pathway.
Phosphorylation of PDCD4 by Akt leads to ubiquitination and
proteasomal degradation.16 This degradation (aka, destabiliza-
tion) appears to be increased in some tumors.17 Given the
benefits of PDCD4 expression, stabilization of PDCD4 is an
attractive way to elevate PDCD4 levels and holds the potential
to increase cancer cell sensitivity to chemotherapy. Rapamycin
is known to both stabilize PDCD4 and synergize with
anticancer drugs;18 unfortunately, the use of rapamycin
(RAP) in cancer treatment is hindered by its immunosup-
pressive effects.19 In an effort to find compounds that sensitize
cells to cancer drugs, we have developed a synthesis of
cryptocaryols A and B (CTCA and CTCB) as well as a series of
analogues (Scheme 2).20,21
ince the discovery of protein kinase C (PKC) as a potential
Scancer target, there has been a search for related
downstream kinase targets (e.g., mTOR, Akt).1 It is believed
that modulation of these kinases will lead to selective tumor
suppression.2,3 Mouse epidermal cells that were resistant to
tumor promotion were discovered to have elevated levels of
programmed cell death 4 (PDCD4).4 PDCD4 is regulated by
mTOR5 and Akt6 and regulates protein synthesis by binding to
translation initiation factor eIF4A.7−9 Down-regulation of
PDCD4 has been shown to increase translation and in turn
tumor cell transformation and invasion (Scheme 1).10,11
Scheme 1. Tumor-Suppressive Effects of PDCD4/Inhibition
a
of eIF4A
Cryptocaryols A and B are a class of natural products that
share a 5,6-dihydro-α-pyranone and a 1,3-polyol segment. They
were isolated from Cryptocarya spp. and identified by Gustafson
in a high-throughput assay to stabilize PDCD4.22 They are
structurally interesting compounds in that the C-2 symmetry of
the polyol can be leveraged to simplify the synthesis of
cryptocaryol analogues, their enantiomers, and other analogues.
Our interest in the cryptocaryols was three-fold. First, we were
interested in elucidating their 3D structure by means of
a
PDCD4 is an inhibitor of protein synthesis via inhibition of initiation
factor eIF4A (red line). The E3 ubiquitin ligase β-TrCP binds to
phosphorylated PDCD4, targeting it for ubiquitination and degrada-
tion via the 26S proteasome. TPA activates PKC, which initiates
phosphorylation and degradation of PDCD4 via PI3K, Akt, or mTOR.
Stabilizers of PDCD4 interfere with the degradation of PDCD4 (i.e.,
rapamycin inhibits mTOR, inhibiting PDCD4 phosphorylation).
Received: December 9, 2013
Accepted: February 17, 2014
Published: February 17, 2014
© 2014 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
Scheme 2. Retrosynthetic Analysis for Cryptocaryols A/B
and Analogues
Table 1. PDCD4 Stabilization and Cytotoxicity Data
a
b
[rel.]
cell line, IC50 (μM)
a
compd (1 μM)
PDCD4
MCF-7
HT-29
H460
CTCA (1)
3.6
4.5
3.8
3.6
1.9
1.8
2.8
8.1
4.2
5.4
CTCB (2)
5.8
2.9
3.8
ent-CTCA (3)
ent-CTCB (4)
6-epi-ent-CTCB (5)
hexaol (6)
25.8
9.0
4.1
7.9
2.4
4.0
13.3
>500
162
4.9
7.8
>1500
1278
>1500
>1000
hexaol Ac (7)
a
PDCD4 stabilization is presented as a relative value over cells treated
b
only with TPA (see Figure 1). IC50 was determined via MTT
colorimetric analysis and curve fitting in Graphpad Prism. The most
cytotoxic cryptocaryol analogue CTCB is also the best PDCD4
stabilizer.
a
The seven cryptocaryol analogues were elucidated from the key
intermediate 8. In turn protected pentaol 8 with pseudo-Cs symmetry
was synthesized from dienoate 9 with iterative asymmetric hydration
(see Supporting Information).
asymmetric synthesis. Second, we were interested in determin-
ing the structure−activity relationship (SAR) as it relates to
cancer cell cytotoxicity. Third, we were interested in studying
their cytotoxicity and relating it to their ability to stabilize
PDCD4. Recently, we disclosed our successful synthetic efforts
at elucidating the structure of the cryptocaryols and providing
material for its initial SAR studies. This effort led to the
synthesis of cryptocaryols A (1) and B (2), their enantiomers 3
and 4, and a diastereomer of cryptocaryol B (5), where all but
one of the stereocenters were inverted. In addition, two
analogue structures of cryptocaryols A and B, 6 and 7, were
prepared that lacked the pyranone ring. Herein, we detail our
efforts to determine the cytotoxicity in three cell lines (MCF-7,
HT-29, and H460), demonstrate/quantify their ability to
stabilize PDCD4, and explore their potential to sensitize cancer
cells to anticancer agents (camptothecin (CPT), digitoxin
(DIG),23−26 etoposide (ETO), 5-fluorouracil (5FU), and
oxaliplatin (OXA)).
While other PDCD4 stabilizers are known to be cytotoxic,
there is very little data to correlate their cytotoxicity to PDCD4
stabilization.27 We chose three cancer cell lines to evaluate
cytotoxicity for the seven polyols. Three cell lines selected for
study were chosen based on their basal PDCD4 content as
measured by RNA microarray and protein content analysis by
immunoblot. The first cell line studied was MCF-7, which has
been shown to have high expression levels of PDCD4. The
second cell line chosen was HT-29, which has a medium
expression level of PDCD4. The third cell line studied was
H460, which has very low expression levels of PDCD4.14
Both cryptocaryols A and B possessed growth inhibitory
activity against the three cell lines in the micromolar range. The
relative cytotoxicity of the cryptocaryols was consistent with
their PDCD4 stabilizing activity (i.e., 2 slightly more active than
1) for each cell line; however, the cell line sensitivity to a given
compound did not correlate with the cell line’s PDCD4
expression levels. That is to say, HT-29 cell lines, with the
medium level of PDCD4 expression, were the most sensitive
(e.g., 4.2 μM for 1); whereas MCF-7 cells, with the highest
level of PDCD4 expression, were the least sensitive (e.g., 8.1
Figure 1. MCF-7 cells were seeded at 100,000 cells per well in 12-well
plates. After 24 h, cells were treated with RAP (10 nM) or
cryptocaryol analogues (1 μM). After 30 min, cells were treated
with TPA (20 nM). After an additional 6 h, cells were harvested by
scraping and lysed with RIPA buffer. Gel electrophoresis was carried
out with 16 μL of cell lysate on a 12% polyacrylamide gel and
transferred to a PVDF membrane. Western analysis was performed
with antibodies for PDCD4 (Abcam ab87678) and β-actin (Abcam
ab8226), and visualized with chemiluminescence. Contrast was
adjusted uniformly over the image for clarity. Band density
measurements (Imagestudio) were made prior to contrast-adjustment.
Bars are band density relative to TPA-only, which was set to 1.
μM for 1). This trend held true for cryptocaryols A and B (1/
2) as well as the diastereomers 3−5. The pyranone
functionality was identified to be an important pharmacophore,
as the two analogues, 6 and 7, without a pyranone ring lost
cytotoxicity (>30-fold). The stereochemistry of the pyranone
ring has some importance for cytotoxicity, as the diastereomer
5 (with only the C-6 pyrano-stereocenter retained) had a small
loss in cytotoxicity (∼2-fold). The effect of C-16 acylation
could be seen in the comparison between cryptocaryols A and
B (1/2) and 6/7, which lacked the pyranone ring. Surprisingly,
the stereochemistry of natural products did not have a
significant effect on cytotoxicity as ent-cryptocaryol B (3) and
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ACS Medicinal Chemistry Letters
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the C16 acetate was a better stabilizer than the one without the
acetate.
Encouraged by the significant PDCD4 stabilization, we
further explored the potential use of cryptocaryols in
combination with other anticancer drugs to determine if
PDCD4 stabilization could result in an enhanced anticancer
effect. These studies were carried out in both MCF-7 (Figures 2
and 3) and HT-29 (Figure 4) cell lines. The co-dosing studies
were performed first with just the combination of cryptocaryol
and cancer drug and later with the addition of TPA, which
might better mimic the tumor environment where PDCD4 is
more rapidly degraded.
Figure 2. Effect of CTCA and CTCB on the cytotoxicity of CPT and
DIG against MCF-7 cells. Cells were pretreated with 1 nM to 1 μM
CTCA or CTCB followed by CPT or DIG, and cytotoxicity was
measured by MTT after 72 h. No enhancement of anticancer activity
was observed following treatment with cryptocaryols.
Figure 3. Changes in CTC/anticancer drug profiles depending on the
presence of 20 nM of TPA. When MCF-7 cells are treated with 1 μM
CTCA or CTCB and anticancer drugs, the presence of TPA has no
effect, indicating that the degradation of PDCD4 with TPA and
recovery with CTCB does not play a role in cytotoxicity.
Table 2. Co-dosing Effects of the Cryptocaryols in MCF-7
Cells
a
IC50 (102 nM)
co-drug
drug
CTCA
CTCB
Table 3. Co-Dosing Effect of CTCB and CPT in the
Presence of TPA in MCF-7
CPT
DIG
CPT
DIG
a
0 nM
1.39
1.24
1.33
1.08
0.81
0.78
3.6
3.27
4.35
3.06
4.74
4.86
1.34
0.99
0.86
1.05
1.38
1.13
1.12
4.5
2.04
3.46
2.75
2.86
2.99
1.04
treatment
CPT IC50 (102 nM)
1 nM
10 nM
100 nM
1 μM
no combination
20 nM TPA
1.41
2.14
2.02
1 μM CTCB + TPA
b
CI @ ED75
a
An increase in cell viability was observed in the presence of TPA,
c
Rel. PDCD4
whereas pretreatment with CTCB had no further effect on cytotoxicity
of CPT. Drugs with lower cytotoxicity (ETO, 5FU, and OXA; see
Supporting Information) were also tested, but accurate IC50
measurements could not be calculated.
a
b
IC50 for CPT and DIG in combination with cryptocaryols. CI @
ED75 was calculated via Chou−Talalay (Calcusyn). No synergistic
relationship was observed. Cryptocaryols with the highest PDCD4
c
stabilization activity were chosen. PDCD4 stabilization activity does
not enhance the cytotoxicity of CPT or DIG in MCF-7.
ent-cryptocaryol A (4) had only a 2- to 3-fold loss in
cytotoxicity.
In addition, the seven compounds were also evaluated for
their ability to stabilize PDCD4. This analysis by immunoblot
followed the protocol described by Tobias Schmid,27 which
uses TPA to initiate PDCD4 degradation, with the known
PDCD4 stabilizer, rapamycin, as the positive control. The high
expression level of MCF-713 made it the ideal cell line for
PDCD4 stabilization studies. These results are outlined in
Table 1 and Figure 1. Cryptocaryols A and B both showed
significant ability to stabilize PDCD4 levels, with cryptocaryol B
being a slightly better stabilizer than A, which was in line with
the results found by Gustafson’s high throughput screen. To
our surprise, the cryptocaryol diastereomers also showed the
ability to stabilize PDCD4 levels. It is also noteworthy that both
hexaol 6 and hexaol acetate 7, which lack the pyranone ring,
retained significant PDCD4 stabilizing ability, while essentially
losing all cytotoxicity (>10-fold). Once again, the polyol with
Figure 4. HT-29 cells were pretreated with PDCD4 stabilizers CTCA,
CTCB (1 μM), and rapamycin (10 nM) followed by DIG or CPT.
We initially studied whether simple co-dosing of cryptocar-
yols A and B would reveal a synergistic effect in MCF-7 cells.
Adopting a drug-combination strategy from our gentamicin-
induced sensitization studies,28 our results are outlined in
Figure 2 and Table 2. MCF-7 cells were treated with several
concentrations of CTCA and CTCB followed by CPT and
DIG. Similar results were observed with HT-29 cells (Figure 4).
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ACS Medicinal Chemistry Letters
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cryptocaryols A and B, no sensitization effects could be seen
in co-dosing studies with several cancer drugs (CPT, DIG, and
5FU) in two different cancer cell lines (MCF-7 and HT-29).
Further efforts to elucidate the mechanism of action for this
class of natural products are ongoing.
Table 4. Perturbation in IC50 for CPT and DIG in the
Presence of CTCA, CTCB or Rapamycin; No Significant
Change in IC50 was Observed
drug, IC50 (102 nM)
co-drug
PDCD4 stabil.
CPT
DIG
ASSOCIATED CONTENT
* Supporting Information
Assay protocols, statistical analysis data, synthetic procedures,
characterization data, and NMR spectra. This material is
available free of charge via the Internet at http://pubs.acs.org.
no co-drug
0
2.07
1.87
2.14
1.84
1.52
2.04
2.24
2.00
■
1 μM CTCA (1)
1 μM CTCB (2)
10 nM rapamycin
3.6
4.5
6.6
S
No enhancement of cytotoxicity for either anticancer drug was
observed. Chou−Talalay analysis of this data gave CI @ ED75
values in the range of 1.3 to 0.8, which are consistent with no
synergistic relationship between the two drugs.
AUTHOR INFORMATION
Corresponding Authors
*(G.A.O.) E-mail: g.odoherty@neu.edu.
*(P.J.B.) E-mail: p.beuning@neu.edu.
■
To further probe for a sensitization for the PDCD4
stabilizers we decided to perform the co-dosing studies in the
presence of TPA, which reduces PDCD4 levels by interaction
with PKC. This experiment was performed for four cancer
drugs (CPT, ETO, 5FU, and OXA), but only clean dose−
response curves could be obtained for camptothecin (Figure 3).
Thus, MCF-7 cells were exposed to a range of camptothecin
doses in the presence of TPA (20 nM) and CTCB (1 μM).
Using similar conditions to our Western blot studies (i.e.,
where PDCD4 stabilization was observed), the cells were dosed
with CTCB 30 min prior to the addition of TPA. After an
additional 8 h, cells were treated with a range of CPT
concentrations. Under these conditions, no sensitization effect
could be seen. In fact, TPA had a larger protective effect than
cryptocaryol B (Table 3).
Using the optimized conditions we found for MCF-7 (Table
2, entry 5), we also screened for a sensitizing effect in HT-29
cells. With 12 h pretreatment with PDCD4 stabilizers,
sensitivity of HT-29 cells to 5FU and CPT was unchanged
and appeared to have an antagonistic effect with DIG. The HT-
29 cell line was the most sensitive to the cryptocaryols (Figure
4 and Table 4) and expressed a moderate level of PDCD4.
These studies were conducted with 1 μM cryptocaryols A or B
and at a range of cancer drug doses (camptothecin and
digitoxin) without the use of TPA. As was observed for MCF-7,
no enhancement in cytotoxicity between either cryptocaryols
(CTCA and CTCB) and anticancer drugs (CPT and DIG)
could be observed. This dose−response assay was also
performed for CPT with CTCB in the presence of TPA,
once again no significant enhancement in cytotoxicity was
observed (see Supporting Information). Similar studies were
also carried out with 5FU, but these gave inconclusive dose−
response curves without observable IC50s.21 Interestingly,
rapamycin, a well-known PDCD4 stabilizer, also showed no
significant enhancement in this co-dosing cytotoxicity assay.
In summary, we have determined the cytotoxicity of both
cryptocaryols A and B in three cell lines as well as for several
analogues. In addition, the ability to stabilize the tumor
suppressor PDCD4 was also determined for these compounds.
Rudimentary structure−activity relationships could be drawn as
structures with the best PDCD4 stabilizing ability tended to be
the most cytotoxic. However, changes to the structures that
removed cytotoxicity (e.g., 6 and 7) did not completely remove
the PDCD4 stabilizing activity. To our surprise, both the
cytotoxicity and PDCD4 stabilizing ability were tolerant to
changes in stereochemistry, as enantiomers (e.g., 3 and 4) and
diastereomers (e.g., 5) retained significant activity in both
assays. For the two most potent PDCD4 stabilizers,
Author Contributions
†Co-first authors, the order is alphabetical. The manuscript was
written with contributions from all authors. M.F.C. and Y.W.
performed all experiments. P.J.B. and G.A.O. developed the
hypotheses and aims.
Funding
We thank NIH (GM090259), NSF (CHE-1213596), and the
American Cancer Society (RSG-12-161-01-DMC) for their
support of our research.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful to Prof. Yon Rojanasakul (West Virginia
University) and Dr. Todd A. Stueckle (West Virginia
University) for the cancer cell lines (i.e., NCI-H460, A549,
and MCF-7).
ABBREVIATIONS
■
FBS, fetal bovine serum; MTT, 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide; TPA, 12-O-tetradecanoyl-
phorbol-13-acetate; PVDF, polyvinylidene fluoride
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