Chapter 16

Apoptosis of Breast Cancer Cells:

Modulation of Genes for Glycoconjugate
Biosynthesis and Targeted Drug Delivery*

Subhash Basu, Rui Ma, Joseph R. Moskal, Manju Basu, and Sipra Banerjee

Introduction

Approximately 250,000 women are diagnosed with breast cancer every year in the
United States (American Cancer Society). Almost 10% (25,000) die of the disease.
Luminal A- and luminal B-type breast cancers are characterized by a low chance of
metastasis and relatively good medical prognoses. On the other hand, basal-like
breast cancers (BLBC) are highly invasive and migrate aggressively to distal organs,
and are untreatable medically either by chemotherapy or through combined chemo-
radiation treatments (Sorlie et al. 2001; Kalluri and Weinberg 2009). Breast cancer
metastasis is dependent on EMT (epithelial to mesenchymal transition). The EMT
process is believed to take place through TGF-beta genes during progression of
breast cancers (Kalluri and Weinberg 2009; Xu et al. 2009; Sato et al. 2010). During
this process, epithelial cancer cells acquire more mesenchymal cell-like properties.
Recent studies have revealed in the role of TGF-beta genes in the suppression of
other epithelial genes and the activation of mesenchymal genes to produce highly
motile mesenchymal-like breast cancer cells (Fig. 16.1). These cells invade pulmo-
nary epithelial cells and proliferate as secondary tumor cells (Takahashi et al. 2010;
Vincent et al. 2010; Santibanez et al. 2010). However, the precise signaling cascades

* This is the IXth article in this series of publications from our laboratory
S. Basu (*) • M. Basu
Department of Chemistry and Biochemistry and Cancer Drug Delivery Research Foundation,
University of Notre Dame, P.O. Box-752, Notre Dame, IN 46556, USA
e-mail: sbasu@nd.edu
R. Ma
Siemens, P.O Box 2297, Elkhart, IN 46515, USA
J.R. Moskal
The Falk Center for Molecular Therapeutics, Northwestern University, Evanston, IL 60201, USA
S. Banerjee
Department of Cancer Biology, Cleveland Clinic Foundation, Cleveland, OH 44129, USA

P.R. Sudhakaran and A. Surolia (eds.), Biochemical Roles of Eukaryotic Cell Surface 233
Macromolecules, Advances in Experimental Medicine and Biology 749,
DOI 10.1007/978-1-4614-3381-1_16, © Springer Science+Business Media, LLC 2012
234 S. Basu et al.

TGF-β
Smad

Breast
Tumor

Epithelial Cancer Cells

Pulmonary caner Cells

Mesenchymal Cancer Cells

Pulmonary epithelial Cells

Blood vessel

Fig. 16.1 A model for metastasis of breast cancer cell

involved in this process of metastasis (Fig. 16.1) of breast cancer cells are not
completely understood (Lee et al. 2010; Deng et al. 2010; de Graauw et al. 2010;
Araki et al. 2010; Singh et al. 2010). Analysis of Hunk (−/−) mice revealed that this
Kinase is required (Yeh et al. 2011) for metastasis of c-myc-induced mammary
tumors, but not c-myc-induced primary transformed cells.

Induction of Apoptosis in Breast Cancer Cells by Novel Agents

Apoptosis is a naturally occurring process by which normal cells are directed to

programmed death activating several signaling pathways by some inducer mole-
cules from outside. It is a distinctive mode of cell death (through self-destruction)
with characteristic changes in morphologic features which is regulating the size of
animal tissues. In contrast, the process of necrosis in cancer cells is a progressive
disintegration of cells, which affect nearby viable cells to disintegrate. Viable cells
discriminate apoptosis and necrosis targets via distinct cell surface receptors
(Weedon et al. 1979; Kostrzewa 2000; Blagosklonny 2000; Patel et al. 2009).
Unlike necrosis, apoptotic cell death is less damaging in the patients carrying
highly metastatic breast carcinomas. Studies of induction (or initiation) and regula-
tion of apoptotic cascades in breast and colon cancer cells are of prime interest in
anticancer drug discovery. If we search this topic in the PubMed, we will obtain at
16 Apoptosis of Breast Cancer Cells… 235

Fig. 16.2 Structures of five different apoptotic agents under study with breast carcinoma cells
(SKBR-3, MDA-468, and MCF-7)

least 1,000 reports regarding apoptosis in breast cancer cells. Induction of apoptosis
can be classified into four categories: (1) Internal mitochondrial-caspase activation
pathway (IMCAP), (2) External Bad receptor activating pathway (EBRAP),
(3) NFkappaB activation pathway (NFkBP), and (4) Cascades of protein kinase
activation Pathways (CPKAP).
Using highly metastatic breast carcinoma cells (SKBR-3, MDA-468, and
MCF-7), we have reported in recent years (Basu et al. 2004a, b, c; Ma et al. 2004;
Boyle et al. 2006) apoptotic induction by d-/l-PPMP, d-PDMP (inhibitors of GSL
biosynthesis), cisplatin (inhibitor of DNA biosynthesis), betulinic acid (a triterpe-
noid used as an anticancer agent in China as an alternative medicine), tamoxifen (a
common anticancer agent used for the treatment of breast cancers today), GD3,
GD1b, and melphalan (a scrambler for Golgi bodies) in the range of 2–16 mM con-
centrations (Fig. 16.2) (Basu et al. 2004a, b, c; Ma et al. 2004; Boyle et al. 2006).
Inhibition of cell growth (IC-50) was different with three different metastatic breast
cancer cell lines when tested with a single apoptotic agent (l-PPMP) for 24 h (Boyle
et al. 2006). All these chemicals (Fig. 16.2) induce apoptosis by activating IMCAP
(Fig. 16.3). On the other hand, cisplatin (used in the treatment of testicular cancers)
induced apoptosis (Fig. 16.3) (Basu et al. 2004a) occurs in those cell lines at much
higher concentrations (50–150 mM) (Boyle et al. 2006) by EBRAP followed by
activation of caspase-8 (Fig. 16.3) Several new chemicals have been tested in recent
years for induction of the apoptotic process by activating IMCAP (internal mito-
chondrial caspase activating pathway), NF-kappaB (NFKBAP), EBRAP (external
Bad-receptor activating pathway), or CPKAP pathway (Yuan et al. 2011; Leung
et al. 2011; Ullah et al. 2011; Marchetti et al. 2011; Kim et al. 2010; Laezza et al.
2010; Wesierska et al. 2011; Chou et al. 2010; Zhang et al. 2010; Patil et al. 2010;
Banerjee et al. 2010; Shirure et al. 2011; Cazet et al. 2010). We also tested activation
236 S. Basu et al.

cis-Platin
GSL (GD3/GD1b)
?
P-Choline
Fas/TNF
Glc-Gal (GD3)
? Sphase ? SA
Caspase-8 SA
L-/D-PPMP
FADD CERAMIDE
PTP

IKKs Caspase-3 Mitochondrion GSL

Cyto-C
Sphingosine
Apaf-1
p53
IkB Active Casp-3
NF-kB (p17/12) Caspase-9
Sph-1-P
Ubi
PKC
NF-kB P DFF/ICAD/PARP etc.

DNA
FLIP,cIAP2
BFL1,BCL-XL DNA Pol-α
cis-Platin
?

Fig. 16.3 Apoptosis signal pathways (extrinsic and intrinsic) induced by novel anticancer agents

Table 16.1 Modulations of MAPKinases in human breast cancer cells by 2 h treatment with
l-PPMP and cisplatin
MDA-468 MCF-7 SK-BR-3
l-PPMP 10 mM/2h
ERK ↑ ↑ ↓
JNK/SAPK ↑ ↓ ↓
p38 ↑ ↓ ↓
Cisplatin 100 mM/2
ERK − or ↓ − −
JNK/SAPK ↓ ↓ or − ↑
p38 − − −
↑: Increased phosphorylation
↓: Decreased phosphorylation
−: No notable change

of apoptosis via all other pathways given in Table 16.1 in the above mentioned cell
lines using those apoptotic agents (Fig. 16.2).
Human MCF-7 breast cancer cells are resistant to proapoptotic stimuli due to
caspase-3 inactivation (Ito et al. 2009). However, reconstitution of human MCF-7
breast cancer cells with caspase-3 gene does not sensitize these cells to the inhibi-
tory action of ROSC (roscovitine) and OLO (Olomoucine) (Wesierska et al. 2011).
16 Apoptosis of Breast Cancer Cells… 237

However, apoptotic mechanism of MCF-7 cell by cisplatin or l-PPMP is still

unknown (Basu et al. 2004b, c; Boyle et al. 2006; Yuan et al. 2011; Leung et al.
2011; Ullah et al. 2011; Marchetti et al. 2011; Kim et al. 2010; Laezza et al. 2010;
Wesierska et al. 2011; Chou et al. 2010; Zhang et al. 2010; Patil et al. 2010).
Quercetin (a natural polyphenolic compound) induced apoptosis in many human
cancer cells, including MCF-7 human breast cancer cells (Chou et al. 2010). The
involvement of possible signaling pathways and the roles of quercetin in the apop-
tosis of other cancer cells remain undefined. However, the recent results (Chou et al.
2010) suggest quercetin may induce apoptosis by direct activation of caspase
cascade through IMCAP bypassing the caspase-3 activation process without involv-
ing caspase-3.

Apoptotic Agents as a New Generation of Anticancer Drugs

In cancer cells, alterations of mitochondrial structure and function have been recog-
nized for quite some time. Targeting mitochondria as a cancer therapeutic strategy
is of interest in recent years. Besides recognition of inhibitors of GSL biosynthesis
(l-PPMP, d-PPMNP, and d-PDMP) and DNA biosynthesis (cisplatin) in our labora-
tory (Basu et al. 2004a, b, c; Ma et al. 2004; Boyle et al. 2006) several other differ-
ent chemicals have been tested as apoptosis inducing agents (via intrinsic
mitochondrial or extrinsic receptor pathways) in breast cancer cells (Zhang et al.
2010; Patil et al. 2010; Banerjee et al. 2010, 2011; Shirure et al. 2011; Cazet et al.
2010; Jada et al. 2008; Domingo-Domenech et al. 2008; Nakagawa et al. 2007;
Nakajima et al. 2007; Sato-Cerrato et al. 2005; Balabhadrapatharuni et al. 2005;
Hatasukari et al. 2003; Chung et al. 2002; Hansen et al. 2000; Fromigue et al. 2003;
Elton et al. 2000; Distefano et al. 1998; Han et al. 1998; Schwartz et al. 1997; Sarkar
et al. 2007; Singh et al. 2009; Lu et al. 2006; Somers-Edgar and Rosegren 2009;
Neuzil et al. 2007; Fulda et al. 1997; Li et al. 2010a; Chaouki et al. 2010a; Mullauer
et al. 2011a; Kessler et al. 2007a; Laszczyk 2009; Bzesk et al. 2006a; Goldoni et al.
2008a; Ellerby et al. 1999).

Betulin and Betulinic Acid as Apoptotic Agents in Cancer Cells

Several reports are available where betulin (lup-20)-ene-3beta28-diol, the natural

occurring triterpene, trigger apoptosis (Fulda et al. 1997a; Li et al. 2010b; Chaouki
et al. 2010b; Mullauer et al. 2011b; Kessler et al. 2007b; Ma 2008) in human cancer
cells through IMCAP (intrinsic mitochondrial caspase activation pathways). The
results showed betulin significantly inhibited cell viability in cervix carcinoma
HeLa cells, hepatoma HepG2 cells, lung adenocarcinoma A549 cells, prostate car-
cinoma PC3, and lungs carcinoma NCI-H460, with IC50 values ranging 20–60 mg/mL.
238 S. Basu et al.

It also showed a minor growth inhibitor in human erythroleukimic K562 cells

(IC50 > 100 mg/mL). Activation of mitochondria and release of mitochondrial apop-
togenic factors by betulinic acid (BA), a melanoma-specific cytotoxic agent in neu-
roectodermal tumors such as neuroblastoma, medulloblastoma, and Ewing’s
sarcoma, was first recognized by Fulda and his associates (Li et al. 2010b). In recent
years, we have demonstrated that BA activates the apoptotic pathway via IMCAP in
human breast carcinoma cells: SKBR-3, MDA-468, and MCF-7 also (Basu et al.
2004b; Ma et al. 2009, 2011; Kessler et al. 2007b). Whether these cells also regulate
cell surface GSL biosynthesis is not known and is under investigation. Apoptotic
cell death by membrane phosphatidylserine translocation was observed. However,
exact mechanism of apoptosis induced by betulinic acid is not understood as yet
(Laszczyk 2009).

Proteins and Peptides

Decorin (a protein core that directly modulates collagen fibrillogenesis and matrix
assembly) is a member of the small leucine-rich proteoglycan gene family (Bzesk
et al. 2006b). It downregulates members of the ErbB-receptor, tyrosine kinase fam-
ily and regulates their signaling pathway, leading to growth inhibition. The effect of
decorin on the overexpression ErbB2 in mammary carcinoma cells suggests that it
is an effective therapeutic agent against tumor growth of breast cancer and its meta-
static spreading to other organs. Decorin inhibits MTLn3 cell proliferation, in a
dose-dependent manner, as well as anchorage-independent cell growth and colony
formation (Bzesk et al. 2006b). Decorin also slows cell motility and stops cell inva-
sion through a three-dimensional extracellular matrix formation. Anticancer activ-
ity of targeted proapoptotic peptides has also been reported (Goldoni et al. 2008b).
An inhibitor of glycoprotein biosynthesis, tunicamycin (an apoptotic agent) pro-
duce unfolded protein also inhibited in Nu/Nu mice microvasculature is suggested
for human breast cancer treatment (Ellerby et al. 2009).

Disialogangliosides

A short chain ganglioside, GD3 (Sialic-alpha2–8Sialic-alpha2–3Galactose-beta1–

4Glc-beta1–1ceramide) occurs in the central nervous system (CNS) and in cancer
cells (Banerjee et al. 2011; Nohara et al. 1998; Basu et al. 1973, 1987, 1991, 1999,
2000; Basu and Basu 1972, 1973, 1982; Higashi et al. 1985; Basu 1991; Keenan
et al. 1974). It is an intermediate in the long chain ganglioside biosynthesis
(Fig. 16.4) (Basu 1991; Basu et al. 1999, 2000). It also occurs in the optic nerve
(Drazba et al. 1991; Holm et al. 1972). It is a minor ganglioside in the normal tissue
16 Apoptosis of Breast Cancer Cells… 239

Ceramide
D-/L-PDMP
GlcT
D-/L-PPMP
Galα1- 3Galβ1- 4GlcNAcβ1-3Lc2
Glc-Cer
GalT-2 nLc5
SAT-1 GlcNAcT- 1
Galβ1-4Glc-Cer Galβ 1-4Gl c -Cer Gl c NA cβ1-3Lc2
α2-3 Lc2 Lc3
GalT-5
GM3 GalT-4
SA SAT- 2 GalT-5’
G lT 5’
Galβ1-4Glc-Cer
GalNAcT-1
SA GD3 Galβ1- 4GlcNAcβ1-3Lc2
Galα1-4Lc2
α2-8 nLc4
GalNAc β1-4Gal-Glc-Cer SA GalNAcT-1 Gb3 SAT-3
GalNAcT-2
GM2 GlcNAcT -22
SA 4Gl NA β1 3L 2
G lβ1 4GlcNAcβ1-3Lc2
Galβ1-
GalT-3 GalNAc β1-4Gal-Glc-Cer α2-3 LM1
SA GlcNAcβ1-3nLc4
SA GD2 GalNAc β1-3Gb3
Galβ1- 3GalNAc-Gal-Glc-Cer FucT-3
GalT-3 GbOsc4Cer inLc5
GM1 SA
SAT-4 SA GlcNAcT-3
Galβ1- 4GlcNAcβ1-3Lc2
Galβ1-3GalNAc-Gal-Glc-Cer α1-3
SA Fuc
Gal-GalNAc-Gal-Glc-Cer GD1b GlcNAcβ1-3 i
α2-3 SA nLc4
GD1a SA-Le X
SA SA SA GlcNAcβ1-6 I
Ii

Fig. 16.4 Biosynthetic pathways of ganglio-, globo-, and lacto-family glycosphingolipids (Chung
et al. 2002)

also (Daniotti et al. 2002). It has been detected as a major GSL in meningiomas
(Fredman et al. 1990), gliomas (Wikstrand et al. 1994), melanomas (Thomas et al.
1995), colorectal carcinomas (Fredman et al. 1983), and breast cancer cells
(Marquina et al. 1996). GD3 also sensitizes human hepatoma cells to cancer therapy
(Paris et al. 2002). GD3 is released by Microglia and induces okigodendrocyte
apoptosis (Simon et al. 2002). An early increase in the GD3 contributes to the devel-
opment of neuronal apoptosis (Melchiorri et al. 2002). The pathways for biosynthe-
sis of GD3 (Fig. 16.4) (Basu 1991; Basu et al. 1999, 2000; Kaufman et al. 1968) and
LD1a (Basu and Basu 1973) were established before in embryonic chicken brain
cells. GD3 ganglioside as a proapoptotic agent has been established in recent years
(Ma et al. 2004, 2009, 2011; Basu et al. 2004b; Kessler et al. 2007b; Malisan and
Testi 2002a, b). We have employed the disialosyl gangliosides (GD3 and GD1b) to
induce apoptosis (Ma et al. 2004, 2009, 2011; Basu et al. 2004b; Kessler et al.
2007b) in human breast cancer cells, SKBR-3 grown in culture. Apoptosis induc-
tion was monitored by the concomitant appearance of activated caspase-3 and by
binding of PS-380 to the outer leaflet of phosphatidyl serine (Fig. 16.5) (Ma et al.
2009, 2011; Kessler et al. 2007b). These results indicated that, in addition to many
unknown GSLs on the cancer cell surfaces disialosylgangliosides (GD3 or GD1b)
could be employed as a breast cancer killing therapeutic agent (Kessler et al. 2007b).
Posttranslational and transcriptional regulation of GSL biosynthesizing genes dur-
ing the induction of apoptosis by l-PPMP in breast cancer cells has been published
in recent years (Ma et al. 2009, 2011; Kessler et al. 2007b). However, exact regula-
tions of GLT-genes in disialosylganglioside induced apoptosis of breast cancer cells
are not known yet.
240 S. Basu et al.

PC
PS

PS 2-48hrs
PS
APOPTOTIC
REAGENTS

Nucleus
Nucleus

NON-APOPTOTIC
CARCINOMA CELL APOPTOTIC/DEAD CELL

1) PSS-380 Dye
=O

NH3+
PS = DG-O-P-O-CH -CH Blue Fluorescence (Apoptotic Cells)
2
2) Propidium Iodide
=O IO-

COO-
Red Fluorescence (Permeable Dead Cells)
=

PE = DG-O-P-O-CH2-CH2- NH3+
APOPTOTIC REAGENTS
=O IO-

Me
PC = DG-O-P-O-CH2-CH2- N+ - Me i) L/D-PPMP
AKS-0 Smith, B. et.al.
IO-

Me (Patent Pending)
ii) GD3/GD1b
iii) cis-Platin
iv) Betulinic Acid
Smith, B. et.al. v) Mephalan, etc
(Patent Pending)

Fig. 16.5 A schematic presentation for detection of phosphatidylserine (by PSS-380) and the
damage of mitochondrial membranes (by AKS(0)) fluorescent dyes during apoptosis

Glycosphingolipid Expression in Breast Cancer Cells

Breast cancer cell adhesion to vascular endothelium is a critical process in metastasis.

MDA-468 and BT-20 breast cancer cells (BCC) adhered to cytokine-activated human
umbilical cord vein endothelial cells (HUVECVs). The same is not true for anti-E
selectin monoclonal antibody-treated HUVECs: BT. It is suggested that BT-20 cells
express sialosyl-LewisX (SA-LeX) and sialosyl Lewis A (SA-Lea), but MDA-MB-468
BCC has novel unidentified E-selectin-binding epitopes (Shirure et al. 2011).
The disialoganglioside GD3 (Fig. 16.2) is overexpressed in 50% of invasive duc-
tal breast carcinoma, and the SAT-2 (or ST8SIAT) (Fig. 16.4) displays higher
expression among estrogen receptor-negative breast cancer tumors, associated with
a decreased survival of BC patients (336). It has been shown previously that overex-
pression of SAT-2 in MDA-MB-231 acquires a proliferative phenotype in the
absence of serum when grown in culture. Using two animal models (leghorn chicken
and C57BL/6 mice) in human breast cancer cells, increased NeuGcGM3 expression
has also been reported. SAT-2 or GD3 synthase overexpression enhances prolifera-
tion and migration of MDA-MB-231 breast cancer cells (Banerjee et al. 2011).
Analysis of glycosphingolipid composition of MDA-MB-231 and MCF-7 human
BCCs showed abundant presence of GM3, GM2, GM1, and GD1a in both the cell
lines (Banerjee et al. 2011). The 18-fold increased amount of GM3 ganglioside sug-
gests some role for this simple ganglioside in the growth regulation in MDA-MB-231
16 Apoptosis of Breast Cancer Cells… 241

BCCs. However, insertion of GM3 ganglioside into the plasma membrane of MCF-7
cells blocked the growth stimulatory effect of EGF. Biosynthesis of glycosphingo-
lipids in both Ganglio (Gg)- and Lacto (Lc)-series pathways (Fig. 16.4) is catalyzed
by at least 18 different glycolipid glycosyltransferases (GLTs) expressed in normal
embryonic tissues and cancer cells have been characterized in last three decades
(Fig. 16.4) (Nohara et al. 1998; Basu et al. 1973, 1987, 1991, 1999, 2000; Basu and
Basu 1972, 1973; Higashi et al. 1985; Basu 1991; Keenan et al. 1974; Basu and
Basu 1982) and have also been cloned in recent years by many laboratories. GLTs
involved in the syntheses of sialo-LeX in the nonapoptotic breast cancer cells have
also been investigated in different laboratories (Fig. 16.4) (Matsuura et al. 1998;
Ugorski and Laskowska 2002; Kikuchi and Narimatsu 2006).
Functions of glycosphingolipids on the eukaryotic cell plasma membrane during
the onset of oncogenic processes and cell death are not well understood. Several
Inhibitors of glycosphingolipid biosynthesis were recently found to trigger apopto-
sis in many carcinoma cells including breast cancer SKBR-3, MCF-7, and MDA-
468 cells through either intrinsic or extrinsic apoptotic pathways (Fig. 16.3), as we
have previously reported (Basu et al. 2004a, b, c; Ma et al. 2004, 2009, 2011; Boyle
et al. 2006; Kessler et al. 2007b). These inhibitors of glucosylceramide biosynthesis
(Radin 1999) could increase ceramide concentration (Basu et al. 2004a) by blocking
the functions of glycolipid glycosyltransferases (GLTs; Fig. 16.4). Using two novel
fluorescent dyes PSS-380 (Koulov et al. 2003) and ASK-0 (Arunkumar et al. 2006)
(Fig. 16.5), our recent studies have revealed (Ma et al. 2009, 2011; Kessler et al.
2007b) the damage of cell organelle membranes during apoptosis by the inhibitor of
glucosylceramide biosynthesis (l-PPMP). Inhibition of GalT-2 (Fig. 16.4) by l- and
d-PDMP has also been reported (Chatterjee et al. 1996). The drug- and cell-depen-
dent regulation of MAPKs was also found by cisplatin and l-PPMP when inducing
apoptosis in SKBR-3, MCF-7, and MDA-468 cells (Ma et al. 2009, 2011; Kessler
et al. 2007b). A summary of our protein kinase studies with the apoptotic BCCs is
given in Table 16.1. In the presence of l-PPMP, all three pathways (ERK, JNK/
SAPK, and p38) were activated in both MDA-468 and MCF-7 cell lines, whereas in
SKBR-3 cell lines these pathways were inhibited. Further study is needed to impli-
cate these pathways in the apoptotic breast cancer cells (MDA-468, MCF-7, and
SKBR-3) induced by cisplatin.

Regulation of Glycosphingolipid Biosynthetic Genes

in Apoptotic Breast Cancer Cells

During process of normal growth, differentiation, or metastasis, the cell surface

glycosphingolipids (GSLs) are believed to be regulated by the interaction of small
molecules to the cell signaling systems. External chemicals, which induce apopto-
sis, may regulate expression of macromolecules on the cell surfaces and may con-
trol directly at the gene level in the production of catalytic proteins such as
glycosyltransferases, which in turn regulate expression of cell surface GSLs.
242 S. Basu et al.

Table 16.2 Summary of posttranslational regulation of glycosphingolipid: glycosyltransferase activities

after 48 h treatment with l-PPMP (Ma et al. 2009; 2011; Kessler et al. 2007b)
GSL-GLT Catalyzed reaction Enzymatic activity
GalT-4 Lc3 (GlcNAc–Gal–Glc–Cer) → Galb–Lc3 Decrease
(MCF-7/l-PPMP-2 h,6 h; SKBR-3/cisP,
l-PPMP and MDA-468/l-PPMP, 48 h)
GalT-5 Lc4 (Gal–GlcNAc–Gal–Glc–Cer) → Gala–Lc4 Decrease
(SKBR-3/l-PPMP-2 h,6 h; MCF-7,
MDA-468/l-PPMP-6 h; SKBR-3/cisP
and MDA-468/L-PPMP-48 h)
SAT-2 GM3 → GD3 Decrease
(MCF-7/cisP,l-PPMP-48 h)
SAT-4 GM1 → GD1a Decrease
(MCF-7/l-PPMP, SKBR-3/cisP
and MDA-468/l-PPMP-48 h)
SAT-4¢ Gg4 (Gal–GalNAc–Gal–Glc–Cer) → GM1b Decrease
(SKBR-3/cisP and MDA-468/l-PPMP-48 h)
FucT-3 LM1 → SA-LeX Decrease
(SKBR-3/l-PPMP-48 h)

The glycosyltransferases (GLTs) (Fig. 16.4) (Basu 1991; Basu et al. 1999, 2000)
catalyzing their synthesis have been characterized in Golgi bodies (Keenan et al.
1974). Very little is known about gene-regulation of these GLTs either during
embryonic development (Basu and Basu 1982) or during metastatic processes. We
know the complete biosynthetic pathways of GSLs during embryonic development
or onset of oncogenic processes, but its regulation during apoptosis is unknown (Ma
et al. 2009, 2011; Kessler et al. 2007b; Matsuura et al. 1998; Ugorski and Laskowska
2002; Kikuchi and Narimatsu 2006). Inhibitors of GLTs (l-PPMP and d-PDMP)
and DNA (cisplatin) trigger apoptosis in Colo-205, SKBR-3, MCF-7, and MDA-
468 through either intrinsic or extrinsic apoptotic pathways. These inhibitors regu-
late GLT gene expression posttranslationally as well as posttranscriptionally (Ma
et al. 2009, 2011; Kessler et al. 2007b). Apoptotic effects initiate activation of cas-
pases (-3, -8, and -9) (Basu et al. 2004a, b, c; Ma et al. 2004, 2009, 2011; Boyle
et al. 2006; Kessler et al. 2007b). Using novel DNA-microarrays specifically
designed for screening over 340 Glyco-related genes, transcriptional-regulation of
several glycosyltransferases involved in the biosyntheses of Sialo-LeX and Sialo-
Lea (cancer cell surface antigens) was observed with l-PPMP (Ma et al. 2009, 2011;
Kessler et al. 2007b). Downregulation of GLT activities and upregulation of some
GLT mRNA suggest a tight regulation of these enzymes by signal transduction
pathways. These apoptotic agents could be employed as a new generation of anti-
cancer drugs. Proper drug delivery system (Liposome Magic Bullet containing cis-
platin or betulinic acid) is discussed in the last chapters.
A dose- and time-dependent downregulation of GLTs was investigated by GLT
enzymatic assay (Table 16.2) and DNA microarray analyses (Ma et al. 2009, 2011;
Kessler et al. 2007b). These GLTs are involved in biosynthesis of LeX and sialosyl-
LeX (neolactosyl-ceramide series) such as GalT-4 (UDP-Gal: LcOse3cer beta-
galactosyltransferase), GalT-5 (UDP-Gal: nLcOse4Cer 1, 3galactosyltransferase)
16 Apoptosis of Breast Cancer Cells… 243

Fig. 16.6 Posttranslational downregulation of GALT-5 during apoptosis in breast carcinoma cells
in the presence of cisplatin and l-PPMP

Fig. 16.7 Posttranslational downregulation of SAT-2 during apoptosis in MCF breast carcinoma
cells in the presence of cisplatin and l-PPMP

(Fig. 16.5), and FucT-3 (GDP-Fucose: LM1 alpha1, 4fucosyltransferase) (Kessler

et al. 2007b). A similar effect was observed with the GLTs involved in the biosyn-
theses of Gg-series gangliosides, such as SAT-4 (CMP-NeuAc: GgOse4Cer alpha2,
3sialyltransferase), Fig. 16.6 and SAT-2 (CMP-NeuAc: GM3 alpha2, 8sialyltrans-
ferase) (Fig. 16.7) (Ma et al. 2009, 2011; Kessler et al. 2007b). The glyco-related
244 S. Basu et al.

Table 16.3 Transcriptional regulation of glycogenes in apoptotic breast cancer cells (treated with
2 mM l-PPMP for 24 h) (Ma et al. 2009; 2011; Kessler et al. 2007b)
GLT gene Fold
Cell line name Linkage formed change
MCF-7 ST8SIA4 NeuAca2–8NeuAc-R1 1.39
GlcNAcb1-3 nLc4 1.39
GCNT2 GlcNAcb1-3nLc4 →
GlcNAcb1-6
B3GALT2 Lc3 → Galb1–3Lc3 1.36
B3GALNT2 Gb3 → GalNAcb1–3Gb3 1.31
B3GALT5 Lc3 → Galb1–3Lc3 1.16
UGT8 Galb1–1Cer −1.14
GlcNAcb1-3 nLc4
MDA-468 GCNT2 GlcNAcb1-3nLc4→ −1.11
GlcNAcb1-6
Galb1-3
SKBR-3 GCNT1 Galb1-3GalNAca-R2 → GalNAca1-R2 −1.19
GlcNAcb1-6

Table 16.4 Transcriptional regulation of glyco-related genes in breast cancer cells (treated with
2 mM l-PPMP for 2 and 24 h) (Ma et al. 2009)
Gene Name 2 h 24 h Symbol Gene Name
MCF-7
NM_000188 1.20 0.67 HK1 HEXOKINASE 1
NM_000194 1.19 0.60 HPRT1 HYPOXANTHINE
PHOSPHORIBOSYLTRANSFERASE 1
(LESCH-NYHAN SYNDROME)
NM_001069 1.45 0.38 TUBB2A TUBULIN, BETA 2A
NM_002629 1.11 1.20 PGAM1 PHOSPHOGLYCERATE MUTASE 1 (BRAIN)
NM_005573 1.40 0.46 LMNB1 LAMIN B1
NM_033170 1.19 1.16 B3GALT5 UDP-Gal:betaGlcNAc beta 1,3-galactosyltrans-
ferase, polypeptide 5 (B3GALT5), transcript
variant 2
NM_170707 1.35 0.68 LMNA LAMIN A/C
SKBR-3
NM_152932 1.35 0.87 GLT8D1 GLYCOSYLTRANSFERASE 8 DOMAIN
CONTAINING 1
MDA-468
NM_006082 0.78 0.77 TUBA6 TUBULIN, ALPHA, UBIQUITOUS

gene DNA-microarrays (Kroes et al. 2011), containing more than 300 different
genes, also suggested (Tables 16.3 and 16.4) modulation of the transcriptional regu-
lation (many were stimulated) of several GLTs involved in the biosynthesis of
neolactosylceramide containing cell-surface antigens in these apoptotic breast car-
cinoma cells. In the early apoptotic stages (2–6 h after l-PPMP treatment) in addi-
tion to the GlcT-1 gene, several genes (betaGalTs and betaGlcNAcTs) in the SA-Lea
pathway were stimulated (Tables 16.3 and 16.4). Transcriptional regulation of dif-
ferent glycogenes during apoptosis of breast cancer cells is also reported in recent
years (Oskouian and Saba 2010; Saltzman 2001).
16 Apoptosis of Breast Cancer Cells… 245

Novel Drug Delivery Systems for Breast Cancer Treatments

The tissues in human bodies contain 70–90% water. Drug molecules (soluble or
suspended fine nanostructure) can be introduced into the body of patients in a vari-
ety of ways: topical, intravenous injection, intravenous infusion, subcutaneous
injection, submuscular injection, or controlled release from any transplant. The
effectiveness of a drug therapy depends on the rate and extent to which drug mole-
cules can move through structures to their targeted site of action (e.g., breast cancer
tumors). Diffusion is the basic process by which migration of drug molecule occurs
in the cells (normal or carcinoma). The rate of diffusion (i.e., a diffusion constant)
depends on the structure of the diffusing molecules. An average diffusion coefficient
of 10−7 cm2/s is desirable for an effective therapeutic drug (Saltzman 2001).
However, this diffusion process can be enhanced when a therapeutic drug is tar-
geted by the aid of a special molecule present on the cancer cell surfaces. The search
for better therapeutics (e.g., apoptotic agents) includes search for its proper strate-
gies to cross the cancer cell surfaces without damaging normal cells by simple
diffusion process (Fig. 16.8). Modern anticancer therapeutic science is a developing
field. Properties of the lipid membranes are critically important in regulating the
movement of the molecules between these aqueous spaces, from blood to the intra-
cellular space of cancer cells. The relationship between liposome structure, stability,
and penetration through plasma membranes is an area of active, ongoing study in
our present research also. Much of the effort in drug design and drug delivery is
devoted to overcoming the membrane diffusional barriers of the cancer cells
(Masserini et al. 2002; Pecheur and Hoekstra 2002; Ghosh and Bell 2002; Basu
and Basu 2002) could be adopted as an efficient drug delivery system (Fig. 16.9).
A tentative model of targeted drug delivery system (Fig. 16.10) is under study in our
laboratory using advantage of the antigens of cancer cell surfaces.

AN IDEAL DRUG DELIVERY SYSTEM

1.The Physicochemical properties of a drug
must be well controlled during the delivery
inside a cancer cell

2. An ideal drug delivery system should be

Fig. 16.8 An ideal drug targeted only to a cancer cell avoiding any
delivery system normal cell.

DIFFERENT DRUG DELIVERY SYSTEMS

Fig. 16.9 Different drug
delivery systems under study
UNDER STUDY
1. Electrically-Enhanced Chemodrug Delivery to
Human Breast Cancer cells. of natural
phospholipids, synthetic phospholipids or
2. Micellular Spheres (composed detergents).
3. Liposomes, Nanoliposomes, and Other lipid –based
carriers.
4. Carbon Nano-Tubes
246 S. Basu et al.

SA-Lex
a/or b (or any antigen-
SA-Le specific antibody)

Synthetic Liposomes
Containing: Apoptotic/Anti-
cancer Drugs/
Metastatic Cancer Cells
(Colon, Breast,Neuronal,
Prostate, )

Fig. 16.10 Targeted delivery of drugs (apoptotic agents) by liposome bullets under study

Electrically Enhanced Chemo-drug Delivery

to Human Breast Cancer Cells

Human MCF-7 BCC was used to evaluate the treatment efficacy. The electroparam-
eters included 200 v/cm, 20 ms and 40 ms pulses. Recent results suggest that appli-
cations of electrical pulses along with chemo treatment to breast cancer cells grown
in culture enhanced the drug transport across the plasma membranes (Camarillo
et al. 2008). However, the method will have limitations when one would like to
apply it to the breast cancer patients.

Liposome and Lipid-Based Different Delivery Systems

Several reports are available on encapsulation of anticancer agents in liposomal bi-

or multilamellar membranes with the intension of protecting healthy tissues from its
cytotoxic effects (Smith et al. 2011). Estrogen receptor-targeted liposomes (Smith
et al. 2011) were designed to enhance the efficiency of delivery to its destination sites
containing metastasized breast cancer cells. Estrogen receptor (ER)-targeted formu-
lation of apoptotic agents could be potentially useful for ER-positive breast cancer
tumors. Stealth nanoliposomes (100 nm) were used to encapsulate daunorubicin and
tamoxifen and tested for inhibition effects on breast cancer cells (Rai et al. 2011;
Guo et al. 2010). When these encapsulated nanoliposomes were applied to the
MCF-7 Xenograft in mice, they showed antitumor activity (Li et al. 2011). Liposomes
loaded with histone deacetylase showed as inhibitory for breast cancer therapy
(Urbinati et al. 2010). Liposomes containing pactitaxel also showed antiangiogenic
in MDA-MB-231 tumor-xenograft growth in Balb/c nude mice (Heney et al. 2010).
16 Apoptosis of Breast Cancer Cells… 247

It is therapeutically desirable to effectively deliver inorganic or hydrophobic

drugs and at the same time as apoptotic agents across the bilayer membranes of the
plasma membranes. Different Sphingolipid intermediates are shown to control
between mutagenesis and apoptosis. These lipids serve both the structural role on
the plasma membranes and an intracellular signaling role within a cell. C6 ceramide
is one of many naturally occurring long chain ceramides. Use of uni- or bilamellar
phospholipid liposomes might be suitable to deliver hydrophobic apoptotic com-
pounds such as triterpenoid (e.g., betulinic acid) to the breast cancer cells or tumors.
However, a targeted delivery of these liposome bullets could be guided by putting a
cancer cell surface specific antibody on the surfaces of these liposome bullets.
Several reports are available on encapsulation of anticancer agents in liposomal bi-
or multilamellar membranes with intension of protecting healthy tissues from its
cytotoxic effects.
Phospholipid nanosomes are small, uniform liposomes manufactured utilizing
supercritical fluid technologies. Supercritical fluids are solvated, and then decom-
pressed to form nanosomes that can encapsulate hydrophilic molecules such as cis-
platin, proteins, or nucleic acids. Hydrophobic therapeutics (e.g., l/d-PPMP,
betulinic acid, tamoxifen, etc.) are co-solvated with phospholipids in supercritical
fluids that when decompressed, form phospholipid nanosomes encapsulating these
drugs in their lipid bilayers (Castor 2011).

Carbon Nanotubes as Transporter of Drugs

In recent years, carbon nanotubes (CNTs) have attracted researchers in the field of
cancer-cure drug (hydrophobic) delivery (Arsawang et al. 2010) through cancer cell
plasma membranes. Single-walled CNTs (SWCNT) have been used as daunorubi-
cin drug carriers in lymphoblastic leukemic T-cells (Singh 2010). The drug mole-
cules were located inside the SWCNTs (Singh 2010). Recent studies also show that
CNTs are toxic and the toxicity depends on the properties of the CNTs, such as their
structures (single wall or multiwalls), length, surface area, degree of aggregation,
and extent of oxidation. Prominent pulmonary inflammation and apoptosis in non-
cancerous cells are reported when CNTs are used as aerosol form for drug delivery.
However, use of CNTs or SWCNTS as carriers of apoptotic agent for treatment of
breast cancers is not available. Exclusion of CNTs or SWCNTs from the patients’
bodies is of primary importance (Thakare et al. 2010; Beq et al. 2011).

Conclusion

TGF-beta signaling has been studied in many development contexts, which is the
ability to induce transition of epithelial cells to mesenchymal cells (EMT). EMTs
play crucial roles during embryonic development and also believed to be occurring
248 S. Basu et al.

during metastasis of breast cancer cells (Walsh and Damianovski 2011). Our present
observations suggest induced apoptosis in highly metastatic breast carcinoma (Taner
et al. 2004; Zhang et al. 2007; Kruger and Reddy 2003; Huang et al. 2011) cells:
SKBR-3 (Taner et al. 2004; Zhang et al. 2007), MDA-468 (Kruger and Reddy
2003), MCF-3 (Kruger and Reddy 2003), and Colo-205 (Huang et al. 2011). GSL
biosynthesis inhibitors (l-PPMP/or d-PDMP), DNA-biosynthesis inhibitor (cispla-
tin), disialosyl-gangliosides (GD3 or GD1b), betulinic acid (an herbal origin triter-
penoid used as an alternate medicine against cancer in China), tamoxifen (established
anti-breast-cancer drug), and melphalan (a Golgi membrane scrambler). All of these
apoptotic agents (2–16 mM) activate caspase-3 and -9 and also modulate genes for
glycosyltransferases within 2–6 h, as is evidenced by DNA-Microarray studies.
However, cisplatin (a common inhibitor of DNA biosynthesis) with higher doses
(50–100 mM) induces caspase-8 activation. Use of synthetic liposomes to encage
inorganic cisplatin or betulinic acid (a triterpenoid) to deliver inside the cancer cells
is under study. Use of the specific antibodies (CSLEX monoclonal antibody binds
to SA-LeX) against cell surface-specific glycoantigens could be one of the targeted
drug deliveries and will be the answer to effective cancer chemotherapy (Shishido
et al. 2010; Sanchez-Navarro and Rojo 2010; Sanchez-Martin et al. 2011; Takahashi
et al. 2011).

Acknowledgment We thank Mrs. Doris Ann Nielsen and Mr. Eric Kuehner for their help during
preparation of this manuscript.

References

American Cancer Society http://www.cancer.org/Research/CancerFactsFigures

Araki S, Eitel JA, Batuello CN, Bijangi-Vishehsaraei K, Xie XJ, Danielpour D, Pollok KE,
Boothman DA, Mayo LD (2010) TGF-beta1-induced expression of human Mdm2 correlates
with late-stage metastatic breast cancer. J Clin Invest 120(1):290–302
Arsawang U, Saengsawang O, Rungrotmongkol T, Sornmee P, Remsungnen T, Hannongbua S
(2010) How do carbon nanotubes serve as carriers for gemcitabine transport in a drug delivery
system? J Mol Graph Model 29(1–8):591–598
Arunkumar E, Fu N, Smith BD (2006) Squarine-derived rotaxanes: highly stable, fluorescent near-
IR dyes. Chemistry 12(17):4684–4690
Balabhadrapatharuni S, Santhakumaran LM, Tjomas TJ, Shirahata A, Gallo MA, Thomas T (2005)
Bis(ethyl)norspermine potentiate4s the apoptotic activity of the pure antieestrogen ICI 182780
in breast cancer cells. Oncol Rep 13(1):101–108
Banerjee M, Singh P, Panda D (2010) Curcumin suppresses the dsynamic instability of micro-
tubles activates the mitotic checkpoint and induces apoptosis in MCF-7 cells. FEBS J 277(16):
3437–3448
Banerjee A, Lang JY, Hung M-C, Sengupta K, Banerjee SK, Bakshi K, Banerjee DK (2011)
Unfolded protein response is required in Nu/Nu mice microvasculature for treating breast
tumor with Tunicamycin. J Biol Chem 286(33):29127–29138
Basu SC (1991) The serendipity of ganglioside biosynthesis: pathway to CARS and HY-CARS
glycosyltransferases. Glycobiology 1(5):469–475
Basu M, Basu S (1972) Enzymatic synthesis of a tetraglycosylceramide by a galactosyltransferase
from rabbit bone marrow. J Biol Chem 247(5):1489–1495
16 Apoptosis of Breast Cancer Cells… 249

Basu M, Basu S (1973) Enzymatic synthesis of a blood group B related pentaglycosyl -ceramide
by an alpha-galactosyltransferase from rabbit bone marrow. J Biol Chem 248(5):1700–1706
Basu S, Basu M (1982) Expression of glycolipid glycosyltransferases in development and transfor-
mation. In: Horowitz M (ed) Glycoconjugates, vol 3. Academic, New York, pp 265–285
Basu M, Basu S (2002) Micelles and liposomes in metabolic enzymes and glycolipid glycosyl-
transferase assays. In: Basu SC, Basu M (eds) Liposome methods and protocols. Humana,
Totowa, NJ, pp 107–130
Basu S, Kaufman B, Roseman S (1973) Enzymatic synthesis of glucocerebroside by a glucosyl-
transferase from embryonic chicken brain. J Biol Chem 248(4):1388–1394
Basu M, De T, Das K, Kyle JW, Chon HC, Schaeper RJ, Basu S (1987) Glycosyltransferases
involved in glycolipid biosynthesis. In: Ginsburg V (ed) Methods in enzymology. Academic
Press, New York, pp 575–607
Basu M, Hawes JW, Li Z, Ghosh S, Khan FA, Zhang BJ, Basu S (1991) Biosynthesis in vitro of
SA-LeX and SA-diLeX by alpha 1-3 fucosyltransferases from colon carcinoma cells and
embryonic brain tissues. Glycobiology 1(5):527–535
Basu S, Basu M, Dastgheib S, Hawes JW (1999) Biosynthesis and regulation of glycosphingolip-
ids. In: Meth-Cohn O, Pinto BM, Barton DHR, Nakanishi K (eds) Comprehensive natural
products chemistry. Pergamon, New York, pp 107–128
Basu S, Das K, Basu M (2000) Glycosyltransferase in glycosphingolipid biosynthesis. In: Ernst B,
Sinay P, Hart G (eds) Oligosaccharides in chemistry and biology—a comprehensive handbook.
Wiley-VCH Verlag Gmbh, Germany, pp 329–347
Basu S, Ma R, Mikulla B, Bradley M, Moulton C, Basu M, Banerjee S, Inokuchi J (2004) Apoptosis
of human carcinoma cells in the presence of inhibitors of Glycosphingolipid biosynthesis: I.
Treatment of Colo-205 and SKBR3 cells with isomers of PDMP and PPMP. Glycoconj J
20(3):157–168
Basu S, Ma R, Boyle PJ, Mikulla B, Bradley M, Smith B, Basu M, Banerjee S (2004b) Apoptosis
of human carcinoma cells in the presence of potential anti-cancer drugs: III. Treatment of Colo-
205 and SKBR3 cells with: cis -Platin, Tamoxifen, Melphalan, Betulinic acid, L-PDMP,
L-PPMP, and GD3 ganglioside. Glycoconj J 20(9):563–577
Basu S, Ma R, Basu M, Goodson H, Smith B, Banerjee S (2004) In: Haldar Das SK (ed)
Glycosphingolipid metabolism and signaling in apoptotic cancer cells, lipids: sphingolipid
metabolizing enzymes. Research Signpost, India, pp 81–100
Beq S, Rizwan M, Sheikh AM, Hasnain MS, Anwer K, Kohli K (2011) Advancement in carbon
nanotubes: basics, biomedical applications and toxicity. J Pharm Pharmacol 63(2):141–163
Blagosklonny MV (2000) Cell death beyond apoptosis. Leukemia 14(8):1502–8
Boyle PJ, Ma R, Tuteja N, Banerjee S, Basu S (2006) Apoptosis of human breast carcinoma cells
in the presence of cis-platin and L-/D-PPMP: IV. Modulation of replication complexes and
glycolipid: glycosyltransferases. Glycoconj J 23(3–4):175–187
Bzesk W, Stepulak A, Szymanski M, Sifringer M, Welkksza K Zdzisinska B, Kandefer-Szerszen
M (2006) Betulinic acid decreases expression of bcl-2 and cyclin D1. Inhibhits proliferation,
migration, and induces apoptosis in cancer cells. Nauyn Schmiedebergs Arch Pharmacol
374(1):11–20
Bzesk W, Stepulak A, Szymanski M, Sifringer M, Welkksza K Zdzisinska B, Kandefer-Szerszen
M (2006) Betulinic acid decreases expression of bcl-2 and cyclin D1 inhibits proliferation,
migration, and induces apoptosis in cancer cells. Nauyn Schmiedebergs Arch Pharmacol
374(1):11–20
Camarillo IG, Nichol M, Zheng M, Sonnenburg D, Sundarajan R (2008) Electro-endocrine com-
bination therapy for aggressive breast tumors. J Electrostat 66:99–106
Castor TP (2011) Phospholipid nanosomes. Curr Drug Deliv 2(4):1567–2018
Cazet A, Lefebvre J, Adriaenssens E, Julien S, Bobowski H, Grigoriads A, Tutt A, Tulasne D,
LeBourhis X, Delannoy P (2010) GD3 synthase expression enhances proliferation and tumor
growth of MDA-MB-231 breast cancer cells through c-met activation. Mol Cancer Res
8(11):1526–35
250 S. Basu et al.

Chaouki W, Leger DY, Eliastimi JL, Hmamouchi M (2010) Antiproliferative effect of extracts
from Aristolochia baetica and Origanum compactumm on human breast cancer cell line MCF-
7. Pharm Biol 48(3):269–74
Chatterjee S, Cleveland T, Shi WY, Inokuchi J, Radin NS (1996) Studies of the action of ceramide-like
substances (D- and LPDMP) on sphingolipid glycosyltransferases and purified lactosylceramide
synthase. Glycoconj J 13(3):481–486
Chou CC, Yang JS, Lu HF, Ip SW, Lo C, Wu CC, Lin JP, Tang NNY, Chung JG, Chou MJ, Teng
YH, Chen DR (2010) Quercetin-mediated cell cycle arrest and apoptosis involving activation
of a Caspase cascade through mitochondrial pathway in human breast cancer MCF-7 cells.
Arch Pharm Res 33(8):1181–91
Chung YL, Sheu ML, Yang SC, Lin CH, Yen SH (2002) Resistance to tamoxifen-induced apopto-
sis is associated with direct interaction between Her2/neu and cell membrane estrogen receptor
in breast cancer. Int J Cancer 97(3):306–312
Daniotti JL, Zurita AR, Trindade VM, Maccioni HJ (2002) GD3 expression in CHO-K1 cells
increases growth rate, induces morphological changes, and affects cell-substrate interactions.
Neurochem Res 27:1421–9
de Graauw M, van Miltenburg MH, Schmidt MK, Pont C, Lalai R, Kartopawiro J, Pardali E, Le
Devedec SE, Smit VT, van der Wal A, Van’t Veer LJ, Cleton-Jansen AM, ten Dijke P, van de
Water B (2010) Annexin A1 regulates TGF-beta signaling and promotes metastasis formation
of basal-like breast cancer cells. Proc Natl Acad Sci USA 107(14):6340–6345
Deng B, Yang X, Liu J, He F, Zhu Z, Zhang C (2010) Focal adhesion kinase mediates TGF-beta1-
induced renal tubular epithelial-to-mesenchymal transition in vitro. Mol Cell Biochem
340(1–2):21–9
Distefano M, Scambia G, Ferini C, Gallo D, De Vincenzo R, Fillippini P, Riva A, Bombardelli E,
Mancuso S (1998) Antitumor activity of paclitaxel (taxol) analogues on MDR-positive human
cancer cells. Anticancer Drug Des 13(5):489–499
Domingo-Domenech J, Pippa R, Tapia M, Gascon P, Bachs O, Bosch M (2008) Inactivation of
NF-kappaB by proteasome inhibition contributes to increased apoptosis induced by histone
deacetylase inhibitors in human breast cancer cells. Breast Cancer Res Treat 112(1):53–62
Drazba J, Pierce M, Lemmon V (1991) Studies of the developing chick retina using monoclonal
antibody 8A2 that recognizes a novel set of gangliosides. Dev Biol 145(1):154–163
Ellerby HM, Arap W, Ellerby LM, Kain R, Andrusiak R, Rio GD, Krajewski S, Lomkbardo CR,
Rao R, Ruosalahti, Bredessen DE, Pasqualini R (1999) Anti-cancer activity of targeted
proapoptotic peptides. Nat Med 5(9i):1032–1038
Ellerby HM, Arap W, Ellerby LM, Kain R, Andrusiak R, Rio GD, Krajewski S, Lomkbardo CR,
Rao R, Ruosalahti, Bredessen DE, Pasqualini R (2009) Anti-cancer activity of targeted
proapoptotic Peptides. Nat Med 5(9i):1032–1038
Elton GF, Gu J, Slater LM, Hara K, Jacobs JW (2000) Tumor apoptosis induced by epoxide-con-
taining piperazines a new class of anti-cancer agents. Cancer Chemother Pharmacol
45(3):183–191
Fredman P, Nilsson O, Svennerholm L, Myrvold H, Persson B, Pettersson S, Holmgren J, Lindhom
L (1983) Colorectal carcinomas have a characteristic ganglioside pattern. Med Biol 61(1):45–8
Fredman P, Dumanski J, Davidsson P, Svennerholm L, Collins VP (1990) Expression of the
ganglioside GD3 in human meningiomas is associated with monosomy of chromorome 22.
J Neurochem 55:1838–1840
Fromigue O, Lagneaux L, Body JJ (2003) Bisphosphonates induce breast cancer cell death in vitro.
J Bone Miner Res 15(11):2211–2221
Fulda S, Friesen C, Los M, Mier W, Benedict M, Nunez G, Krammer PH, Peter ME, Debatin KM
(1997) Betulinic acid triggers CD95 (APO-1/Fas)- and p53- independent apoptosis via activa-
tion of caspases in neurroectodermal tumors. Cancer Res 37:4956–4964
Ghosh S, Bell R (2002) Liposomes: applications and protein-lipid interaction studies. In: Basu SC,
Basu M (eds) Liposome methods and protocols. Humana, Totowa, NJ, pp 49–60
Goldoni S, Seidler DG Health J, Fassan M, Baffa R, Thakur ML, Owens RT, McQuillan DJ, Lozzo
R (2008) An antimetastatic role for Decorin in breast cancer. Am J Pathol 173(3):844–855
16 Apoptosis of Breast Cancer Cells… 251

Guo J, Zhou J, Ying X, Men Y, Li RJ, Zhang Y, Du J, Tian W, Yao HJ, Wang XX, Ju RJ, Lu WL
(2010) Effects of stealth liposomal daunorubicin plus tanmoxifen on the breast cancer and
cancer stem cells. J Pharm Sci 13(2):136–151
Han EK, Aber N, Yamamoto H, Lim JT, Delohery T, Pamukcu R, Piazza GA, Xing WQ, Weynstein
IB (1998) Effects of sulindac and its metabolites on growth and apoptosis in human mammary
epithelial and breast carcinoma cell lines. Breast Cancer Res Treat 48(3):195–203
Hansen CM, Hansen D, Holm PK, Larson R, Binderup L (2000) Cyanoguanidine CHS 828 induces
programmed cell death with apoptotic feature in human breast cancer cells in vitro. Anticancer
Res 20(6B):4211–4220
Hatasukari I, Hitosugi N, Matsumoto I, Nagasaka H, Sakagami I (2003) Induction of early apop-
tosis marker by morphine in human lung and breast carcinoma cell lines. Anticancer Res
239(2B):2413–2417
Heney M, Alipour M, Vergidis D, Omri A, Mugabe C, Th’ng J, Suntres Z (2010) Effectiveness of
liposomal pacletaxel against MCF-7 breast cancer cells. Can J Physiol Pharmacol 88(12):
1172–1180
Higashi H, Basu M, Basu S (1985) Biosynthesis in vitro of disialosylneolacto- tetraosyl- ceramide
by a solubilized sialyltransferase from embryonic chicken brain. J Biol Chem 260(2):
824–828
Holm M, Mansson JE, Vanier MT, Svennerholm L (1972) Gangliosides of human, bovine and rab-
bit retina. Biochim Biophys Acta 280:356–364
Huang WS, Chin CC, Chen CN, Kuo YH, Chen TC, Yu HR, Tung SY, Shen CH, Hsieh YY, Guo
SE, Shi CS, Liu TJ, Kuo HC (2011) Stromal cell-derived factor-1/CXC receptor 4 and b1 inte-
grin interaction regulates urokinase-type plasminogen activator expression on human colorec-
tal cancer cells. J Cell Physiol. doi:10.1002/jcp. 22831
Ito H, Murakami M, Furuhata A, Gao S, Yoshida K, Sobue S, Hagiwara K, Takagi A, Kojima T,
Banno Y, Tanaka K, Tamiya-Koizumi K, Kyogashima M, Nozawa Y, Murate T (2009)
Transcriptional regulation of neutral sphingomyelinase 2 gene expression of a human breast
cancer cell line, MCF-7 induced by the anti-cancer drug, daunorubicin. Biochim Biophys Acta
1789(11–12):681–690
Jada SR, Mathews C, Saad MS, Hamzah AS, Lajis NH, Stevens MF, Stanslas J (2008) Benzylidine
derivatives of andrographolide inhibit growth of breast and colon cancer cells in vitro by induc-
ing G(1) arrest and apoptosis. Br J Pharmacol 155(5):641–654
Kalluri R, Weinberg RA (2009) The basics of epithelial-mesenchymal transition. J Clin Invest
119(6):1420–8
Kaufman B, Basu S, Roseman S (1968) Enzymatic synthesis of disialogangliosidees from mono-
sialogangliosides by sialyltransferases from embryonic chicken brain. J Biol Chem
243:5804–5806
Keenan TW, Morre JD, Basu S (1974) Ganglioside biosynthesis: concentration of glycolipid gly-
cosyltransferases in Golgi apparatus from rat liver. J Biol Chem 249:410–416
Kessler JH, Mullauer FB, de Roo GM, Medema JP (2007a) Broad in vitro efficacy of plant-derived
Betulinic acid against cell lines derived from the most prevent human cancer types. Cancer Lett
251(1):132–145
Kessler JH, Mullauer FB, de Roo GM, Medema JP (2007b) Broad in vitro efficacy of plant-derived
Betulinic acid against cell lines derived from the most prevent human cancer types. Cancer Lett
251(1):132–145
Kikuchi N, Narimatsu H (2006) Bioinformatics for comprehensive finding and analysis of glyco-
syltransferases. Biochim Biophys Acta 1760(4):578–583
Kim DY, Kang SH, Ghil SH (2010) Circium japonicum extract induces apoptosis and anti-prolif-
eration in the human breast cancer cell line MCF-. Mol Med Rep 3(3):427–432
Kostrzewa RM (2000) Review of apoptosis vs. necrosis of substantia nigra pars compacta in
Parkinson’s disease. Neurotox Res 2(2–3):239–50
Koulov AV, Stucker KA, Lakshmi C, Robinson JP, Smith BD (2003) Detection of apoptotic cells
using a synthetic fluorescent sensor for membrane surfaces that contain phosphatidylserine.
Cell Death Differ 10(12):1357–1359
252 S. Basu et al.

Kroes RA, He H, Emmett MR, Nilsson CL, Leach III FE, Amster IJ, Marshall AG, Moskal JR
(2011) Overexpression of ST6GalNAcV, a gangliposide-specific alpha-2,6-sialyltransferase,
inhibits glioma growth in vivo. Proc Nartl Acad Sci USA 107(28):12646–12651
Kruger JS, Reddy KB (2003) Distinct mechanisms mediate the initial and sustained phases of cell
migration in epidermal growth factor receptor-overexpressing cells. Mol Cancer Res 11:801–809
Laezza C, Malfitano AM, DiMatola T, Ricchi P, Bifulco M (2010) Involvement of Akt/NF-kappaB
pathway in N6-isopentenyladenosine-induced apoptosis in human breast cancer cells. Mol
Carcinog 49(10):892–901
Laszczyk MN (2009) Pentacyclic triterpenes of the lupine, oleanane and ursane group as tools in
cancer therapy. Planta Med 75(15):1549–1560
Lee J, Moon HJ, Lee JM, Joo CK (2010) Smad3 regulates Rho signaling via NET1 in the trans-
forming growth factor-beta-induced epithelial-mesenchymal transition of human retinal pig-
ment epithelial cells. J Biol Chem 285(34):26618–27
Leung E, Kim JE, Rewcastle GW, Finlay GJ, Baguley BC (2011) Comparison of the effects of the
P13K/mTOR inhibitors NVP-BEZ235 and GSK2126458 on tamoxifen-resistant breast cancer
cells. Cancer Biol Ther 11(11):938–46
Li Y, He K, Huang Y, Zheng D, Gap C, Jin YH (2010a) Betulin induces mitochondrial cytochrome
c release associated apoptosis in human cancer cells. Mol Carcinog 49(7):630–640
Li Y, He K, Huang Y, Zheng D, Gap C, Jin YH (2010b) Betulin induces mitochondrial cytochrome
c release associated apoptosis in human cancer cells. Mol Carcinog 49(7):630–640
Li S, Goins B, Phillips WT, Saenz M, Otto PM, Bao A (2011) Post-humpectomy intracavitary
retenation and lymph node targeting of (99m)Tc-encapsulated liposomes in nude rats with
breast cancer xenograft. Breast Cancer Res Treat 130(1):97–107
Lu PH, Kung FL, Kug SC, Chueh SC, Guh JH (2006) Investigation of anti-tumor mechanisms of
k2154:characterization of tubulin isotypes, mitotic arrest and apoptotic machinery. Nauyn
Schmiedebergs Arch Pharmacol 374(3):223–233
Ma R (2008) Apoptosis of breast and colon carcinoma cells by inhibitors of glycolipid and DNA
biosynthesis. Ph.D. Thesis, University of Notre Dame, Notre Dame, p. 272
Ma R, Koulov A, Moulton C, Basu M, Banerjee S, Goodson H, Basu S (2004) Apoptosis of human
breast carcinoma cells in the presence of disialosyl gangliosides: II. Treatment of SKBR3 cells
with GD3 and GD1b gangliosides. Glycoconj J 20(5):319–330
Ma R, Decker NM, Anilas V, Moskal JR, Burgdor J, Johnson JR, Basu M, Banerjee S, Basu S
(2009) Post-translational and transcriptional regulation of glycolipid glycosyltransferase genes
in apoptotic breast carcinoma cells: VII. Studied by DNA-microarray after treatment with
L-PPMP. Glycoconj J 26:647–661
Ma R, Hopp EA, Decker MN, Loucks AJ, Johnson JR, Moskal JR, Basu M, Banerjee S, Basu S
(2011) Regulation of glycosyltransferase genes in apoptotic breast cancer cells induced by
L-PPMP and cis-Platin. In: Proceedings of the molecular immunology cancer congress (MICC-
3). Advances in experimental biology. Springer, New York, pp 699–714
Malisan F, Testi R (2002a) GD3 ganglioside and apoptosis. Biochim Biophys Acta 1585:179–87
Malisan F, Testi R (2002b) GD3 in cellular ageing and apoptosis. Exp Gerontol 37:1273–82
Marchetti M, Russo L, Balducci D, Falanga A (2011) All trans-retinoic acid modulates the proco-
agulant activity of human breast cancer cells. Thromb Res 128(4):368–74
Marquina G, Waki H, Fernandez LE, Kon K, Carr A, Valiente O, Perez R, Ando S (1996)
Gangliosides expressed in human breast cancer. Cancer Res 56:5165–71
Masserini M, Palestini P, Pitto M, Chigormo, Sonnino S (2002) Preparation and use for the study
of sphingolipid segregation in membrane model systems. In: Basu SC, Basu M (eds) Liposome
methods and protocols. Humana, Totowa, NJ, pp 17–27
Matsuura N, Narita T, Hiraiwa N, Murai H, Iwase T, Funahashi H, Imai T, Takagi H, Kannagi R
(1998) Gene expression of fucosyl-and sialyl-transferases which synthesize sialyl LewisX, the
carbohydrate ligands for E-selectin, in human breast cancer. Int J Oncol 12(5):1157–1164
Melchiorri D, Martini F, Lococo E, Gradini R, Barletta E, De Maria R, Caricasole A, Nicoletti F,
Lenti L (2002) An early increase in the disialoganglioside GD3 contributes to the development
of neuronal apoptosis in culture. Cell Death Differ 9:609–15
16 Apoptosis of Breast Cancer Cells… 253

Mullauer EB, van Bloois L, Daalhusin JB, Ten Brink MS, Storm G, Medema JP, Schiffelers RM,
Kesseler JH (2011a) Betulinic acid delivered in liposomes reduces growth of human lung and
colon cancers in mice without causing systemic toxicity. Anticancer Drugs 22(3):223–233
Mullauer EB, van Bloois L, Daalhusin JB, Ten Brink MS, Storm G, Medema JP, Schiffelers RM,
Kesseler JH (2011b) Betulinic acid delivered in liposomes reduces growth of human lung and
colon cancers in mice without causing systemic toxicity. Anticancer Drugs 22(3):223–233
Nakagawa A, Sawada TM, Okada T, Ohsawa T, Adachi M, Kubota K (2007) New antineoplastic
agent, MK635 from UME (a variety of) Japanese apricot inhibits growth of breast cancer cells
in vitro. Breast J 13(1):44–49
Nakajima H, Sakeguchi K, Mizuta N, Fujiwara I, Hayakawa A, Magae J (2007) Apoptotic mecha-
nisms induced in breast cancer cells by vinorelbine. Gan To Kagaku Ryoho (Japanese)
34(4):583–588
Neuzil J, Dong LF, Ramanathapurram L, Hahn T, Chladova M, Wang XF, Zobalova R, Prochazaka
L, Gold M, Freeman R, Turaneek J, Akporiaye ET, Dyason JC, Ralph SJ (2007) Vitamin E
analogues as a novel group of mitocans:anti-cancer agents that act by targeting mitochondria.
Mol Aspects Med 28(5–6):607–645
Nohara K, Wang F, Spiegel S (1998) Glycosphingolipid composition of MDA-MB-231 and MCF-7
human breast cancer cells. Breast Cancer Res Treat 48(2):149–157
Oskouian B, Saba JD (2010) Cancer treatment strategies targeting sphingolipid metabolism. Adv
Exp Med Biol 688:185–205
Paris R, Morales A, Coll O, Sanchez-Reyes A, Garcia-Ruiz C, Fernandez-Checa JC (2002)
Ganglioside GD3 sensitizes human hepatomacells to cancer therapy. J Biol Chem 277: 49870–6
Patel VA, Lee DJ, Longacre-Antoni A, Feng L, Lieberthal W, Rauch J, Ucker DS, Levine JS
(2009) Apoptotic and necrotic cells as sentinels of local tissue stress and inflammation: response
pathways initiated in nearby viable cells. Autoimmunity 42(4):317–21
Patil JB, Kim J, Jayprakasha GK (2010) Berbarine induces apoptosis in breast cancer cells (MCF-
7) through mitochondrial-dependent pathway. Eur J Pharmacol 645(1–3):70–78
Pecheur E-I, Hoekstra (2002) Peptide-induced fusion of liposomes. In: Basu SC, Basu M (eds)
Liposome methods and protocols. Humana, Totowa, NJ, pp 31–48
Radin NS (1999) Chemotherapy by slowing glucosphingolipid synthesis. Biochem Pharmacol
57(6):0589–595
Rai S, Paliwal R, Vyas SP (2011) Doxorubicin encapsulated nanocarriers for targeted delivery to
estrogen responsive breast cancer. J Biomed Nanotechnol 7(1):121–122
Saltzman WM (2001) Diffusion and drug dispersion. In: Drug delivery: engineering principles for
drug therapy. Oxford University Press, Oxford, pp 23–48
Sanchez-Martin D, Cuesta AM, Fogal V, Ruosslahti E, Alvarez-Vallina (2011) The multicompart-
mental p32/ggClgR as a new target for antibody-based tumor targeting strategies. J Biol Chem
286(7):5197–5203
Sanchez-Navarro M, Rojo J (2010) Targeting DC-SIGN with carbohydrate multivalent systems.
Drug News Prospect 23(9):557–572
Santibanez JF, Kocic J, Fabra A, Cano A, Quintanilla M (2010) Rac1 modulates TGF-beta1-
mediated epithelial cell plasticity and MMP9 production in transformed keratinocytes. FEBS
Lett 584(11):2305–10
Sarkar FH, Adsule S, Li Y, Padhye S (2007) Back to the future: COX-2 inhibitors for chemopre-
vention and cancer therapy. Mini Rev Med Chem 7(6):599–608
Sato Y, Harada K, Itatsu K, Ikeda H, Kakuda Y, Shimomura S, Shan Ren X, Yoneda NM, Sasaki M,
Nakanuma Y (2010) Epithelial-mesenchymal transition induced by transforming growth factor-
{beta}1/Snail activation aggravates invasive growth of cholangiocarcinoma. Am J Pathol
177(1):141–52
Sato-Cerrato V, Montaner BM, Martaner B, Martinell M, Vilaseca M, Giiralt E, Perez-Tomas
(2005) Cell cycle and proapoptotic effects of the anticancer cyclodepsipeptide serratamolide
(AT514) are independent of p53 status in breast cancer cells. Biochem Pharmacol
7(1–2):32–41
254 S. Basu et al.

Schwartz GK, Farsi K, Masalak P, Kelsen DP, Spriggs D (1997) Potentiation of apoptosis by
flavopiridol in mitomycin-C-treated gastric and breast cancer cells. Clin Cancer Res 3(9):
1467–72
Shirure VS, Henson KA, Schnaar RL, Nimrichter L, Burdick MH (2011) Gangliosides expressed
on breast cancer cells are E-selectin ligands. Biochem Biophys Res Commun 406(3):423–429
Shishido T, Mieda H, Hwang SY, Nishimura Y, Tanaka T, Ogino C, Fukuda H, Kondo A (2010)
Affibody-displaying bionanocapsules for specific drug delivery to HER2-expressing cancer
cells. Bioorg Med Chem Lett 20(19):5726–31
Simon BM, Malisan F, Testi R, Nicotera P, Leist M (2002) Disialoganglioside GD3 is released by
microglia and induces oligodendrocyte apoptosis. Cell Death Differ 9:758–67
Singh S (2010) Nanomedicine-nanoscale drugs and delivery systems. J Nanosci Nanotechnol
10(12):7906–18
Singh N, Nigam M, Ranjan V, Sharma R, Balapure AK, Rath SK (2009) Caspase mediated enhanced
apoptotic action of cyclophosphamide- and resveratol-treated MCF-7 cells. J Pharmacol Sci
109(4):473–485
Singh G, Singh SK, Konig A, Reutlinger K, Nye MD, Adhikary T, Eilers M, Gress TM, Fernandez-
Zapico ME, Ellenrieder V (2010) Sequential activation of NFAT and c-Myc transcription fac-
tors mediates the TGF-beta switch from a suppressor to a promoter of cancer cell proliferation.
J Biol Chem 285(35):27241–27250
Smith B, Lyaskhov I, Loomis K, Needle D, Baxa U, Yavlovich A, Capala J, Blumenthal R, Puri A
(2011) Hyperthermia- triggerd intercellular delivery of anticancer agent to HER2(+) cells by
HER2-specific antibody (ZHER2-GS-Cvs)-conjugated thermosensitive liposomes (HER2(+)
affisomes). J Control Release 153(2):187–94
Somers-Edgar TJ, Rosegren RJ (2009) Coenzyme Q0 induces apoptosis and modulates the cell
cycle in estrogen receptor negative breast cancer cells. Anticancer Drugs 20:33–40
Sorlie T, Perou CM, Tibshirani R, Aas T, Geisler S, Johnsen, Hastie HT, Eisen MB, van de Rijn M,
Jeffrey SS, Thorsen T, Quist H, Matese JC, Brown PO, Botstein D, Eystein P, Lonnin, Borresen-
Dale AL (2001) Gene expression patterns of breast carcinomas distinguish tumor subclasses
with clinical implications. Proc Natl Acad Sci USA 98(19):10869–10874
Takahashi E, Nagano O, Ishimoto T, Yae T, Suzuki Y, Shinoda T, Nakamura S. Niwa S, Ikeda S,
Koga H, Tanihara H, Saya H (2010) Tumor necrosis factor-alpha regulates transforming growth
factor-beta-dependent epithelial-mesenchymal transition by promoting hyaluronan-CD44-
moesin interaction. J Biol Chem 285(6):4060–4073
Takahashi S, Kato K, Nakamura K, Nakano R, Kuboto K, Hamada H (2011) Neuronal cell adhe-
sion molecule 2 as a target molecule for prostate and breast cancer gene therapy. Cancer Sci
10(4):808–814
Taner M, Kapanen AL, Junttila T, Raheem O, Grenman S, Elo J, Elinus K, Isola J (2004)
Characterization of a novel cell line established from a patient with Herceptin breast cancer.
Mol Cancer Sci 12:1285–1292
Thakare VS, Das M, Jain AK, Patil S, Jain S (2010) Carbon nanotubes in cancer theragnosis.
Nanomedicine (Lond) 5(8):1277–1301
Thomas CP, Buronfosse A, Fertil B, Portoulalian J (1995) Surface expression of GD3 disialogan-
gliosides in human melanoma cells is correlated to both metastatic potential in vivo and radio-
sensitivity in vitro. Cr Acad Sci III 318(12):1233–8
Ugorski M, Laskowska A (2002) Sialyl Lewis(a): a tumor-associated carbohydrate antigen involved
in adhesion and metastatic potential of cancer cells. Acta Biochim Pol 49(2):303–311
Ullah MF, Ahmad A, Zubair H, Khan HY, Wang Z, Sarkar FH, Hadi SM (2011) Soy isoflavone
genisten induces cell death in breast cancer cells through mobilization of endogenous copper
ions and generation of reactive oxygen species. Mol Nutr Food Res 55(4):553–559
Urbinati G, Marsoud V, Plassat V, Fattal E, Lesieur S, Renoir JM (2010) Lopisomes loaded with
histone deacetylase inhibitors for breast cancer therapy. Int J Pharm Biopharm 397(1–2):
184–193
16 Apoptosis of Breast Cancer Cells… 255

Vincent T, Neve EP, Johnson JR, Kukalev A, Rojo F, Albanel J, Pietras IK, Virtanen I, Philipson
L, Leopold PL, Crystal RG, de Herreros AG, Moustakas A, Pettersson RF, Fuxe J (2010) A
SNAIL1-SMAD3/4 transcriptional repressor complex promotes TGF-beta mediated epithelial-
mesenchymal transition. Nat Cell Biol 11(8):943–50
Walsh LA, Damianovski S (2011) IGF-1 increases invasive potential of MCF-7 breast cancer cells
and induces activation of latent TGF-beta-1 resulting in epithelial mesenchymal transition. Cell
Commun Signal 9(1):10
Weedon D, Searle J, Kerr JF (1979) Apoptosis. Its nature and implications for dermatopathology.
Am J Dermatopathol 1(2):133–144
Wesierska J, Hacki S, Zulehner N, Maurer M, Komina O (2011) Reconstitution of human MCF-7
breast cancer cells with Caspase-3 does not sensitize them to action of CDK inhibitor. J Cell
Biochem 112(1):273–288
Wikstrand CJ, Fredman P, McLendon RR, Svennerholm L, Bigner DD (1994) Altered expression
of ganglioside phenotypes of human gliomas in vivo and in vitro. Mol Chem Neuropathol
21:129–38
Xu J, Lamouille S, Derynck R (2009) TGF-beta-induced epithelial to mesenchymal transition.
Cell Res 19(2):156–72
Yeh ES, Yan TW, Jung JJ, Gardner HP, Cardiff RD, Chodosh LA (2011) Hunk is required for
HER2/Neu-induced mammary tumorigenesis. J Clin Invest 121(3):866–79
Yuan J, He Z, Wu J, Lin Y, Zhu X (2011) A novel adriamycin analogue derived from marine
microbes induces apoptosis by blocking Akt activation in human breast cancer cells. Mol Med
Rep 4(2):261–265
Zhang GH, Yang WT, Zhou XY, Zeng Y, Lu HF, Shi DR (2007) Study of correlation between
HER-2 gene and Lymphangiogenesis and their prognostic significance in hunan breast cancer.
Zhonghua Yi Xue Za Zhi 87(3):155–60
Zhang N, Kong X, Yan S, Yuan C, Yang Q (2010) Huair aqueous extract inhibits proliferation of
breast cancer cells by inducing apoptosis. Cancer Sci 101(11):2375–2383