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Abstract # 551708
Molecular analysis of the selective pro-apoptotic effect of BN107 on estrogen receptor negative
breast cancer cells
Ruth Chu, Xiaoyue Zhao, Chandi Griffin, Mark Shoemaker, Mary Tagliaferi, Dale Leitman, Isaac Cohen, Emma Shtivelman, and Sylvia Fong
Bionovo Inc., Emeryville, CA
PI
–
BN107 appears to exert its growth inhibition properties on breast
cancer cells via the mitochondrial apoptotic machinery.
–
BN107 induces rapid alterations in mTOR signaling pathway and in
cholesterol (CHL) metabolism. Co-administering CHL and BN107
abolishes its pro-apoptotic effect, as well as restores the mTOR
signaling pathway.
– The
disruption of CHL-rich lipid raft might be mediating the proapoptotic effect of BN107.
– Absence
of estrogen receptor (ER) in the cells correlates with
sensitivity to BN107. Introduction of ERa expression into a breast
cancer line results in protection from the pro-apoptotic effect of BN107.
Consistently, treating ER- breast cancer cells with a differentiating agent
(trichostatin A), also confers resistance to BN107. Transcriptomic
analysis comparing sensitive (ER-) and insensitive (ER+) lines treated
with BN107 reveals distinct patterns of gene expression that might be
responsible for the differential sensitivity.
References
1. Rugo et al. (2006) Breast Cancer Res Treat, 105(1):17-28.
2. Fong et al. (2008) Cancer Biol Ther,7(4): 577-586.
3. Campbell et al. (2002) Anticancer research, 22, 3843-3853.
+
PI
Annexin V
C.
0 4
D.
8 16 BN107 Rx (hr)
Cytochrome C
GAPDH
•
Here, we present results of analysis on the mechanism of action of BN107,
another anti-breast cancer product in development, ,for its pro-apoptotic
effect on breast cancer cells. BN107 is an aqueous extract of the fruit of
Gleditsia sinensis Lam. It has been shown to exhibit strong growth inhibition
across various breast cancer cell lines (Campbell et al. 2002).
-
BN107
•
Bionovo Inc. has a pipeline of anti-breast cancer products in development.
We have recently completed a phase 1/1B clinical trial for BZL101, a drug
candidate for metastatic breast cancer, in a heavily pretreated patient
population with metastatic breast cancer and showed that it had a favorable
toxicity profile and demonstrated encouraging clinical efficacy (Rugo et al.
2006). We are currently enrolling patients for phase 2 clinical testing.
Analysis of mechanism of action of BZL101 demonstrated the selectivity of
BZL101 in targeting breast cancer cells while sparing non-transformed cells
(Fong, et al. 2008).
+
Annexin V
•
Botanical medicines are frequently sought and used by cancer patients,
yet few studies have evaluated their safety and efficacy as well as their
mechanisms of action.
-
Control
BN107
80
Caspase 3
++
-
+
Hs578T
++++
-
+
++++
-
0
1
3
6 16
0
1
3
Caspase 9
MDA-MB-453
++++
-
+
MCF10A
+++
-
-
IMR90
++++
-
-
MDA-MB-361
-/+
+
+
BT474
-/+
+
+
MCF7
-
+
-
Table 1. Cells without ER are more sensitive to BN107 induced
apoptosis, while Her2 status appears not correlative with BN107
sensitivity. Cells were treated with BN107 and harvested after 18 hours for
analysis of Annexin V/PI binding. The summary shown is a result of 3
independent experiments.
BN107
MCF7 BN107
Non LR plasma
membrane
Cont
GM-1
D.
Non-LR PM
MCF7 Cont
MCF7 BN107
BN107+CHL
LacZ
ERa
+
+
BN107
E2 (10 nM)
ERa
MCF7
0 1 3 6 16
MDA-MB-231
0 1 3 6 16
BN107 Rx (hr)
TR
Transferrin Receptor
90
60
30
0
ERa
BN107
CD44
LR
WISP2
Total
GM-1
CD44
MDA231 BN107+CHL
CAV1
-
+
-
+
+
BN107 Rx
CHL
LR
4EBP
Flotillin 1
Non-LR PM
TR
C.
120
Control
100
DMSO
80
TsA
60
40
20
0
Figure 5. ERa expression rescues MDA-MB231 cells from BN107
induced apoptosis. A. MDA-MB231 cells infected with LacZ or ERa virus
were treated with BN107 for 18 hrs in the presence of estrogen (E2, 10 nM)
and analyzed with Annexin/PI binding. The chart shows percentage of
Annexin- PI- (live) cells. B. Real time qRTPCR analysis of WISP2 expression,
a downstream target of ERa, indicating functional ERa status. C. MDA-MB231 cells were treated with a differentiating histone deacetylase inhibitor,
tricostatin A (TsA, 50nM ) or DMSO for 2 days. The cells were then treated
with BN107 and analyzed with Annexin/PI binding as in A.
p-4EBP (Ser65)
Figure 4 . In ER – cell line, the cholesterol content and the localization
BN107+LDL
of lipid raft (LR) resident proteins, as well as LR-mediated mTOR
60
BN107+GOH
signaling are disrupted by BN107 treatment. A. CHL content in LR, NonBN107+FOH
p4EBP (Ser65)
40
LR plasma membrane (PM) fractions and total lysate. B..
Taxol
Total
4EBP
20
Taxol+CHL
Immunofluorescence staining of caveolin 1 (CAV1) and CD44 (green) in cells
treated with 70 mg/ml of BN107 for 4 hrs and fixed in 4% paraformaldehyde.
0
% Survival
C. Dot plot analysis of GM-1 (marker for LR fractions) and transferrin
Figure 3. Cholesterol specifically protects cells from BN107 induced receptor (TR, marker for non-LR plasma membrane fractions). D. Western
death and restores mTOR signaling. A. MDA-MB231 cells were treated analysis of total lysate (upper panel) and LR or non-LR PM fractions (lower
with 70 mg/ml BN107 alone or with 500 mM cholesetrol (CHL), 50 mg/ml LDL, panel). MDA-MB231 or MCF7 cells were treated with 60 mg/ml BN107 (± 500
50 mM of isoprenoid precursors (geranylgeraniol, GOH or farnesol, FOH) for mM CHL) for 3 hr. Various fractions were collected after separation of triton18 hours. The chart shows percentage of live cells (annexin V-, PI-). B. X100 soluble and insoluble proteins with density gradient centrifugation (A, C,
Western blot analysis of mTOR downstream substrates in cells treated with D). Fractions were spotted directly from fractions (C), or were precipitated to
BN107 and +/- LDL.
load the same amount of protein (D).
80
LacZ
GAPDH
MDA231 BN107
pS6K (Thr389)
Total S6K
BN107
CAV1
GAPDH
- - + + BN107, 4hr
- + - + LDL (50 mg/ml)
Control
120
100
80
60
40
20
0
+
+
MDA231 Cont
Exogenous cholesterol protects cells from BN107 induced
apoptosis and restores mTOR signaling pathway
A.
B.
Control
MCF7 Cont
Total 4EBP
Figure 2. Analysis of proteins involved in mTOR signaling pathway.
MDA231 (sensitive) and MCF7 (insensitive) cells were treated with BN107 (70
mg/ml) and harvested at indicated time points.
100
MDA231 BN107
B.
C.
p-4EBP (Ser65)
120
450
400
350
300
250
200
150
100
50
0
BN107 Rx (hr)
p-mTOR (Ser2448)
mTOR
RICTOR
RAPTOR
p-S6K (Thr389)
S6K
-
6 16
MDA231 Cont
LR
Table 2. Cellular/signaling pathways induced by BN107 treatment, based
on Ingenuity Pathway Analysis (IPA) of microarray data generated using
BN107 sensitive (Hs578T) and insensitive (MCF7) cells treated with BN107
for 4 hr.
MCF7
MDA-MB231
0
SKBr3
MDA-MB-231
- Ah receptor signaling
- IGF1 receptor signaling
- MAPK signaling
- Cell growth
- Acute stress response
20
Her2
-
- Apoptosis
- Cell cycle
- Cholesterol synthesis
- Oxidative response
- MAPK signaling
- Acute stress response
40
Annexin V ER
PI staining
++
MCF7 (ER+)
60
Figure 1. BN107 induces apoptosis via mitochondrial machinery in
breast cancer cells, assessed by A. Annexin V-PI staining, B. DNA
fragmentation, C. Western blot showing Cytochrome C released in cytosol,
and, D. Activation of caspases 3 and 9. Treatment with BN107 also leads to
dissipation of mitochondrial transmembrane potential (data not shown).
Hs578T cells were treated with BN107 (70 mg/ml) and harvested after 18 (A),
8 (B), or 3 (D) hours of treatment.
MDA-MB-468
Hs578T (ER-)
A.
Expression of ER in ER- breast cancer cell line confers
resistance to BN107
A.
B.
% Live cells, % Control
Control
BN107
BN107 depletes CHL and disrupts lipid raft (LR) and LRmediated mTOR signaling
Relative expression
MDA231 Hs578T
BN107 induces expression of distinct sets of genes and
alters mTOR signaling in sensitive vs insensitive breast
cancer cells
Live Cells, % Control
BN107 induces apoptosis in breast cancer cells
A.
B.
Results
Results
m g of CHL in fractions
Despite advances in early detection and adjuvant therapy have made a
favorable impact on survival, current regimens lead to toxic side effects and
are mostly ineffective against metastatic breast cancer. Currently, patients
with estrogen receptor (ER)-negative/progesterone receptor (PR)negative/HER-2-negative tumors still present a therapeutic challenge for the
oncologists. Therefore, novel and effective therapies with minimal toxicities
are urgently needed for this patient population.
Results
Results
% Live cells
•
Results
% cells with activated caspase
Background
Summary
•
Mitochondrial-mediated apoptosis appears to be the major cellular
pathway mediating the growth inhibitory effect of BN107.
•
Cholesterol depletion and disruption of lipid raft-mediated signaling might
be responsible for the pro-apoptotic effect of BN107.
•
Estrogen receptor status correlates with BN107 sensitivity.
Specifically, ER- breast cancer lines that have mesenchymal
characateristics, are highly sensitive to BN107. Conversely, when ERa
expression is exogenously introduced or induced, the cells exhibit far less
sensitivity to BN107.
•
Studies are underway to investigate the protective role of ERa in
lipid-raft disruption induced by BN107; as well as to identify active
components in the extract responsible for its anti-tumor effect.