Timosaponin A3 is a steroidal saponin from Anemarrhena asphodeloides that has a selective cytotoxic activity towards cancer cells
Frank King, Sylvia Fong, Isaac Cohen, Emma Shtivelman
BioNovo, Inc, Emeryville, CA
Abstract
Inhibition of caspase 4 partially protects from BN108-induced cell death
the selective cytotoxicity for the whole extract. TspA3 is a steroidal saponin whose activity against
60
BT474
50
% dead cells
UT
A3
MCF10A
BN108 UT
A3 BN108
40
UT
REDD1
30
BN108
MYC
20
BN108+ Caspase 4 inh
Hrs:
0
Du145
Silencing of REDD1 does not protect from TspA3
induced apoptosis
pS6
BT474
S6
120
Breast cancer cells sensitive to BN108 undergo apoptotic death confirmed by Annexin V staining,
caspase activation, cleavage of PARP and DNA fragmentation. Caspases 4 and 9 which are involved in
apoptosis induced by endoplasmic reticulum stress are activated by BN108. Inhibition of caspase 4
significantly inhibits BN108 induced death.
P-4eBP1
100
4eBP1
Bim
GAPDH
Timosaponin A3, a component of BN108 recapitulates the cytotoxicity
and other effects of BN108
184A1
MCF10A
MCF12A
PC3
LNCaP
Du145
MDAMB231
MDAMB361
% apoptosis
BT474
normal
fibroblasts
IMR
TspA3 (A3) and BN108 induce same changes in expression/activation of a proteins in cancer but do not
induce these changes in normal cells. Stress-response protein REDD1 and pro-apoptotic protein Bim
are induced; expression of MYC is suppressed. Activity of Akt and mTORC are inhibited in breast
cancer cells. None of these proteins are affected in normal immortalized MCF10A cells.
TspA3
Hrs:
0
1
2
BN108
231
40
231 D-1
0
UT
TspA3
BN108
4
8
16
1
2
4
8
Inhibition of REDD1 expression did not prevent inactivation of mTORC In TspA3 treated cells (not
shown). Treatment of BT474 or MM231 cells with knockdown of REDD1 did not protect them from
TspA3 cytotoxicity
16
Phospho eIF2a
Timosaponin A3 is a steroidal saponin found in BN108 extract. TspA3 appears to be the compound
responsible for the cytotoxic effect of BN108 based on the following: TspA3 is selectively cytotoxic to
same cancer cell lines as BN108 and is not cytotoxic to normal cells; expression array analysis of gene
expression changes induced by BN108 and TspA3 largely overlap; cytotoxicity of TSpA3 could be
partially inhibited in presence of caspase 4 inhibitor; time course of changes in expression of important
signaling proteins is similar in cells treated withTspA3 and BN108
eIF2a
Antiproliferative and pro-apoptotic, induced:
-REDD1 (mTORC inhibitory), p21CIP, stratifin, cyclin G2, GDF15
•BN108 induces cell death selectively in cancer cells but not in normal cells.
GAPDH
•Timosaponin A3 (TspA3) was identified as an active compound from BN108 responsible for the selective
cytotoxic activity of BN108.
•BN108/TspA3 do not induce generation of reactive oxygen species, DNA damage or mitochondrial disfunction.
Time course of changes induced by TspA3 versus BN108 in BT474 cells: practically identical
ER stress response, induced:
LRF, HEPRUD/HERP, TRIB3, GRP78
BT474
Pro-proliferative and/or anti-apoptotic, downregulated:
-Id1, Id3, Myc, Snai3, CYR61
Hrs:
0
1
2
4
MCF10A
8
16
0
1
2
4
•Induction of ER stress is a likely mechanism of cytotoxicity of BN108/TspA3 as seen from the involvement of
caspase 4 and inactivation of eIF2a kinase, as well as induction of some genes implicated in ER stress
(GRP78, ATF4 and phosphorylation of peIF2a).
•BN108/TspA3 induce rapid inactivation of AKT and mTORC selectively in breast cancer cells, which is most
likely contibutes to cell death.
8
16
Enzymes in cholesterol biosynthesis pathway (particularly in BT474)
Id-1
Pathways affected:
Akt, mTORC (inhibited), SREBP2 and cholesterol biosynthesis (moderately activated)
Summary
Id-1
C-myc
BN108 induces apoptotic death in cancer cell lines but not in non-transformed cell lines and cells.
Tumor and non-transformed cell lines and cells were treated with BN108 at 0.5 mg/ml for 24 hours. The
chart shows percentage of cells that were binding annexin V.
60
20
Genes induced or repressed in both MDA MB231 and BT474 but not in MCF10A by
BN108 and TspA3:
immortalized
mammary cells
80
REDD1 is a negative regulator of mTORC; it appeared to be a good target to silence in order to prevent
inhibition of mTORC by A3 and BN108.
REDD1 was targeted in BT474 and MDA MB 231 cells with REDD1 siRNA lentiviruses.
induces expression of proteins involved in cholesterol biosynthesis pathway and ER stress response.
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 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.
prostate cancer
cells
16
pAKT
major signaling pathways for growth and survival selectively in cancer cells (Akt and mTORC) and
breast cancer cells
8
231-REDD1 KD(D-1)
BN108 induce largely overlapping transcriptional changes in cells. Similar to BN108, TspA3 inactivates
100
80
60
40
20
0
4
AKT
those in the BN108 extract induces apoptosis in breast cancer cells but not in normal cells. TspA3 and
BN108 induces apoptosis selectively in cancer cell lines
0
231-vec
10
cancer cells remained unexplored until now. Treatment with purified TspA3 at concentrations similar to
Results
Silencing of REDD1 has no effect on inhibition of mTORC by TspA3 and
does not protect cells from death induction by TspA3
% survival
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.
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.
Expression array analysis of cells treated with BN108 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 induces 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 treated cells.
Expression array analysis also shows the induction of numerous genes encoding enzymes within the
cholesterol synthesis pathway by BN108. They are induced to various degrees in all cell lines examined.
However, the changes in total cholesterol levels are relatively minor in treated cells. In addition, BN108
also induces pattern of gene expression consistent with ER stress.
We have identified timosaponin A3 (TspA3) as an active compound from BN108 that is responsible for
BN108 and TspA3 produce similar changes in expression and activity of
certain cellular proteins
•However, silencing of REDD1, an inhibitor of mTORC activity, does not protect cells from TspA3/BN108
induced death
•TspA3 is active against breast cancer cells in low micromolar range. This, and its selectivity towards cancer
cells make it a promising candidate drug
TspA3 inhibits expression C-myc and Id-1 proteins in cancer cells but not in normal cells.
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