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1
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
Results
Results
Results
Results
Results
Background
Results
BN107 depletes CHL and disrupts lipid raft (LR)
and LR-mediated mTOR signaling A.


B. C.

D. Figure 4 . In ER cell line,
the cholesterol content and the localization of
lipid raft (LR) resident proteins, as well as
LR-mediated mTOR signaling are disrupted by BN107
treatment. A. CHL content in LR, Non-LR plasma
membrane (PM) fractions and total lysate. B..
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. C. Dot plot analysis of GM-1
(marker for LR fractions) and transferrin
receptor (TR, marker for non-LR plasma membrane
fractions). D. Western analysis of total lysate
(upper panel) and LR or non-LR PM fractions
(lower panel). MDA-MB231 or MCF7 cells were
treated with 60 mg/ml BN107 ( 500 mM CHL) for 3
hr. Various fractions were collected after
separation of triton-X100 soluble and insoluble
proteins with density gradient centrifugation (A,
C, D). Fractions were spotted directly from
fractions (C), or were precipitated to load the
same amount of protein (D).
Expression of ER in ER- breast cancer cell line
confers resistance to BN107 A. B.
C. 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-MB-231 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.

• 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.
• 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.
• 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).
• 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 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 pro-apoptotic 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.

• BN107 induces apoptosis in breast cancer cells
• A.
  B.
• 
  D.

BN107 induces expression of distinct sets of
genes and alters mTOR signaling in sensitive vs
insensitive breast cancer cells 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.
MDA231 Hs578T
Control
BN107 - -
PI
Hs578T (ER-) MCF7 (ER)
Apoptosis Cell cycle Cholesterol synthesis Oxidative response MAPK signaling Acute stress response Ah receptor signaling IGF1 receptor signaling MAPK signaling Cell growth Acute stress response
Relative expression
Annexin V
BN107
PI
Cont BN107
BN107
E2 (10 nM)
Annexin V
CAV1 CD44
ERa
MCF7
MDA-MB231
0 1 3 6 16 0
1 3 6 16 BN107 Rx (hr)
0 4 8 16 BN107 Rx
(hr)
GAPDH
p-mTOR (Ser2448)
mTOR
Cytochrome C
RICTOR RAPTOR
GAPDH
p-S6K (Thr389)
S6K
MCF7 MDA-MB-231
LR Non-LR PM GM-1 TR
0 1 3 6 16 0 1 3 6 16
BN107 Rx (hr)
p-4EBP (Ser65)
GM-1 CD44 CAV1
Total 4EBP
MDA231 Cont
MDA231 BN107
GAPDH
MDA231 BN107CHL
Transferrin Receptor
MCF7 Cont
MCF7 BN107
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.
- BN107 Rx
- - CHL
Annexin V PI staining ER Her2
SKBr3 -
Hs578T -
MDA-MB-468 - -
MDA-MB-231 - -
MDA-MB-453 -
MCF10A - -
IMR90 - -
MDA-MB-361 -/
BT474 -/
MCF7 - -
Exogenous cholesterol protects cells from BN107
induced apoptosis and restores mTOR signaling
pathway A.
B. Figure 3.
Cholesterol specifically protects cells from
BN107 induced death and restores mTOR signaling.
A. MDA-MB231 cells were treated with 70 mg/ml
BN107 alone or with 500 mM cholesetrol (CHL), 50
mg/ml LDL, 50 mM of isoprenoid precursors
(geranylgeraniol, GOH or farnesol, FOH) for 18
hours. The chart shows percentage of live cells
(annexin V-, PI-). B. Western blot analysis of
mTOR downstream substrates in cells treated with
BN107 and /- LDL.
Summary
p-4EBP (Ser65) 4EBP
LR Non-LR PM

• 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.

Flotillin 1 TR
- - BN107, 4hr - -
LDL (50 mg/ml)
pS6K (Thr389)
Total S6K
p4EBP (Ser65)
Total 4EBP
References

• Rugo et al. (2006) Breast Cancer Res Treat,
  105(1)17-28.
• Fong et al. (2008) Cancer Biol Ther,7(4)
  577-586.
• Campbell et al. (2002) Anticancer research, 22,
  3843-3853.