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Abstract # 2666
Timosaponin A3 is a steroidal saponin from Anemarrhena asphodeloides that has a selective
cytotoxic activity towards cancer cells
Sylvia Fong, Frank King, Xiaoyue Zhao, Isaac Cohen, and Emma Shtivelman
Bionovo Inc., Emeryville, CA
In addition, BN108 and TspA3 induce pattern of gene expression consistent
with induction of ER stress and authophagy.
Inhibition of mTORC1 and induction of ER stress by TspA3 are most likely
contributing to the activation of autophagy, which plays a protective role in in
TA3 induced cell death.
In conclusion, a component of BN108 extract, TspA3 is selectively cytotoxic
for cancer versus normal cells. The selective cytotoxic properties of TspA3
could be related to the inhibition of major oncogenic pathways and induction
of References
ER stress. Future studies will be aimed at understanding the relationship
between the effect of TspA3 on these pathways and induction of apoptosis,
which may give rise to a unique pathway for targeting tumor cells.
40
AKT
p4eBP1
AKT
4eBP1
GAPDH
1 2.5 5 7.5 10
(M)
TspB2
GAPDH
Figure 2. TspA3 and BN108 selectively inhibit phosphorylation of
mTORC1 targets in breast cancer cells. A. Western blot analysis of
phosphorylation of s6 ribosomal protein and 4eBP, as well as AKT kinase, in
BT474 or MCF10A cells treated with 2.5 M TspA3 or 0.5 mg/ml BN108 for 16
hours. B. Time course of mTORC1 inhibition by TspA3 treatment (2.5 M) in
BT474 cells.
TspA3 and BN108 induce ER stress
A.
E.
40
30
UT
TspB2
20
lam
TspB2+lam
10
E.
Grp78
BT474
TspA3 appears to be the major cytotoxic compound in BN108 that
selectively induces apoptosis in cancer cells while sparing normal cells.
•
60
UT
40
TspA3
20
MG132
0
grp78
GAPDH
B.
250
Control
BN108
200
150
100
50
0
BT474
IMR90
BN108 and TspA3 induce gene expression changes that inhibit the major
survival signaling pathway, namely mTORC1, in breast cancer cells.
•
BN108 and TspA3 induce ER stress selectively in breast cancer cells,
evidenced by increase in phosphorylation of eIF2α and activation of caspase
4.
•
MDA-MB-231
Figure 1. BN108 or TspA3 selectively induces apoptosis in cancer cells.
A. The cells were treated with 0.5 mg/ml of BN108 for 24 hours. Cells were
analyzed for apoptosis by flow cytometry of Annexin V/PI stained cells.
Percentage of Annexin V-binding cells is shown. B. Western blot analysis of
PARP cleavage. C. Cells were treated with various concentrations of TspA3
for 24 hours, and cell viability was analyzed as in A. D. Structures of TspA3
and timosaponin B2 (TspB2). E. Cell lines were treated for 24 hours with
TspB2 at 40 M, heat-inactivated laminarinase alone (lam, 0.5 g/ml), or a
mixture of both (TspB2+lam) after incubation for 30 minutes at 500C and heatinactivation of the enzyme.
MCF10A
•
GAPDH
vec
MDA-MB-231
Summary
eIF2α
0
CQ
TspA3
CQ+TspA3
Figure 4 . Inhibition of autophagy augments apoptosis induced by TspA3 in
MDA-MB-231 and MCF10A cell lines but not in BT474 cells. Cells were
pretreated for 3 hours with 20 M of chloroquine (CQ), an autophagy inhibitor,
after which TspA3 (4 M for BT474, 5 M for MDA-MB-231 and 7.5 M for
MCF10A) was added for 24 hours. Cells were analyzed as in Figure 1A.
Vec Grp78
Vec Grp78
F.
TspA3
BN108
0 1 2 4 8 16 1 2 4 8 16 (hrs)
peIF2α
TspB2 + lam→ TspA3
60
50
40
30
20
10
0
TspA3+LEVD
pAKT
MCF10A
0
D.
90
80
70
60
50
40
30
20
10
0
BT474
TspA3
S6
Fibroblast
MDA-MB-231
BN108, 6hr BN108, 24hr BN108, 24hr
+ LEVD
pS6
4eBP1
TspA3
BN108, 3hr
BN108+LEVD
.
BT474
0
Control
p4eBP1
D.
100
80
60
40
20
0
400
% apoptotic cells
Normal
Breast
800
% apoptotic cells
IMR90
184A1
MCF10A
MCF12A
PC3
LNCaP
MDAMB231
Du145
Prostate
Cancer
1200
S6
PARP-1
MDAMB361
0
Caspase 4
Caspase 9
BN108
20
Autophagy plays a protective role in TspA3-induced
apoptosis
% apoptotic cells
pAKT
pS6
C.
% apoptosis
0 1 4 8 16 (Hrs)
1600
UT
BN108
Results
ER stress is partially responsible for BN108- and TspA3induced apoptosis
C.
Caspase activity, % Control
BT474
MCF10A
UT A3 BN UT A3 BN
60
Breast
Cancer
BN108 and TspA3 induce rapid inactivation of AKT and mTOR kinases in
breast cancer but not in non-transformed cells. The well-defined targets of
mTORC1, S6kinase, S6 ribosomal protein and 4eBP1 are inactivated in
BN108- and TspA3-treated cells.
Expression array analysis of cells treated with BN108 or TspA3 shows
induction of expression of several known pro-apoptotic and anti-proliferative
genes such as REDD1, p21CIP, cyclin G2, stratifin and more. None are
affected in normal mammary cells. BN108 and TA3 also induced numerous
genes encoding enzymes within the cholesterol synthesis pathway in all cell
lines examined, including non-transformed cells. However, the changes in
total cholesterol levels are relatively minor in treated cells.
80
MCF10A
+
Results
TspA3 and BN108 inhibit major pro-survival signals
selectively in breast cancer cells.
A.
B.
Caspase 4 activity, %
control
We have identified timosaponin A3 (TspA3), a steroidal saponin, as an active
compound from BN108 that is responsible for the selective cytotoxicity of
BN108. Treatment with purified TspA3 at concentrations similar to those in
the BN108 extract induces apoptosis in breast cancer cells but not in normal
cells.
BT474
+
BT474
BN108, aqueous extract of Anemarrhena asphodeloides Bunge, induces cell
death selectively in breast cancer lines and tumor cells of various origins but
not in normal mammary epithelial cells and fibroblasts. Breast cancer cells
sensitive to BN108 undergo apoptotic death, confirmed by Annexin V staining,
caspase activation, cleavage of PARP and DNA fragmentation. In particular,
caspases 4 and 9, whose activation is observed during endoplasmic
reticulum (ER) stress induced apoptosis, are proteolytically activated.
Inhibition of caspase 4 partially protects breast cancer cells from death
induced by BN108.
Results
Results
TspA3 is the cytotoxic compound in BN108 that
selectively induces apoptosis in cancer cells.
A.
B.
% apoptotic cells
Botanical medicine is one of the most popular complementary medical
approaches, and herbal therapies are frequently sought and used by breast
cancer patients. However, the molecular mechanisms through which certain
herbal extracts exert growth inhibitory activity on breast cancer cells remain
largely unknown.
Results
% viable cells
Abstract
Figure 3 . A. Western blot analysis of the levels of phosphorylated translation
initiation factor eIF2α, in BT474 cells treated with 2.5 M TspA3 or 0.5 mg/ml
BN108. B. BT474 and IMR90 were treated for 24 hours with BN108, and
analyzed for caspase 4 activity, involved in ER stress-induced apoptosis.
C. BT474 treated with BN108 (± 8 M caspase 4 inhibitor LEVD-fmk) for
indicated times were analyzed for caspase-4 and caspase-9 activities.
D. BT474 cells were treated for 24 hours with BN108 (0.5 mg/ml) or TspA3
(2.5 M) in the presence of 10 M of caspase 4 inhibitor LEVD-fmk.
Apoptotic cells were detected as described in Figure 1A.. E. Western blot
analysis showing Grp78 overexpression in MDA-MB-231 cells as compared
to vector (vec) transfected cells. F. Expression of exogenous Grp78 in MDAMB-231 cells offers partial protection against apoptosis induced by TspA3 (5
 M) or MG132 (250 nM).
ER-stress mediated apoptosis appears to be partially responsible for
BN108- and TspA3- induced cytotoxicity. Specifically, inhibition of ER stress
response by co-incubation with caspase 4 inhibitor or by exogenously
expressing chaperone Grp78 protects breast cancer cells from the proapoptotic effect of BN108 or TspA3.
•
Autophagy plays a protective role in TspA3-induced apoptosis. Induction
of autophagy by TspA3 is likely a consequence of inhibition of mTORC1 and
ER stress. Cancer cells that fail to activate autophagy in response to TspA3
are more sensitive to it.