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

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

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

234 S. Basu et al.

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

TGF-βSmad

BreastTumor

Epithelial Cancer Cells

Mesenchymal Cancer Cells

Pulmonary caner Cells

Blood vessel

Pulmonary epithelial Cells

Fig. 16.1 A model for metastasis of breast cancer cell

23516 Apoptosis of Breast Cancer Cells…

least 1,000 reports regarding apoptosis in breast cancer cells. Induction of apoptosis can be classi fi ed 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 m M 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 m M) (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

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

236 S. Basu et al.

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 ) .

GSL (GD3/GD1b)cis-Platin?

Fas/TNFGlc-Gal

SASA

(GD3)

??

P-Choline

Sphase

Mitochondrion

FADDPTP

CERAMIDEL-/D-PPMP

Caspase-3

Caspase-8

Caspase-9

IKKs

Active Casp-3(p17/12)

Cyto-C

Apaf-1Sphingosine

GSLIKKs

IkBp53

DFF/ICAD/PARP etc.

Sph-1-P

PKC

NF-kBUbi

NF-kB P

DNAFLIP,cIAP2BFL1,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 m M/2h ERK ↑ ↑ ↓ JNK/SAPK ↑ ↓ ↓ p38 ↑ ↓ ↓

Cisplatin 100 m M/2

ERK − or ↓ − − JNK/SAPK ↓ ↓ or − ↑ p38 − − −

↑: Increased phosphorylation ↓: Decreased phosphorylation −: No notable change

23716 Apoptosis of Breast Cancer Cells…

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 unde fi ned. 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. 2010 a; Mullauer et al. 2011a ; Kessler et al. 2007a ; Laszczyk 2009 ; Bzesk et al. 2006a ; Goldoni et al. 2008 a; 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. 1997 a; 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 signi fi cantly 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 m g/mL.

238 S. Basu et al.

It also showed a minor growth inhibitor in human erythroleukimic K562 cells (IC50 > 100 m g/mL). Activation of mitochondria and release of mitochondrial apop-togenic factors by betulinic acid (BA), a melanoma-speci fi c cytotoxic agent in neu-roectodermal tumors such as neuroblastoma, medulloblastoma, and Ewing’s sarcoma, was fi rst 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 fi brillogenesis 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

23916 Apoptosis of Breast Cancer Cells…

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 lea fl et 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.

Ceramide

GlcTD-/L-PDMPD-/L-PPMP Galα1- 3Galβ1- 4GlcNAcβ1-3Lc2

Galβ1-4Glc-Cerα2-3

SA

Galβ cANclGreC-clG4-1 β1-3Lc2SAT-1 GlcNAcT- 1

Glc-Cer

GalT-2

GM3 Lc3GalT-4SAT- 2

Lc2

G lT 5’

GalT-5

Galα1- 3Galβ1- 4GlcNAcβ1-3Lc2

nLc5

GalNAc β1-4Gal-Glc-Cer

SA

GalNAcT-1

GM2

Galβ1- 4GlcNAcβ1-3Lc2nLc4

SAT-3

G lβ1 4Gl NA β1 3L 2

Galβ1-4Glc-CerGD3SA

α2-8SA

SAT- 2

GalNAcT-1

GlcNAcT -2

Galα1-4Lc2

GalT-5’

GalNAcT-2Gb3

Galβ1- 3GalNAc-Gal-Glc-Cer

SA GM1

GalT-3

SAT-4

Galβ1- 4GlcNAcβ1-3Lc2α2-3

SALM1

FucT-3

GalNAc β1-4Gal-Glc-Cer

SA GD2

SAGalT-3

GlcNAcT 2

GlcNAcβ1-3nLc4

inLc5

GlcNAcT-3Galβ1- 4GlcNAcβ1-3Lc2

GalNAc β1-3Gb3

GbOsc4Cer

Gal-GalNAc-Gal-Glc-Cer

SAα2-3

SAGD1a

Galβ1-3GalNAc-Gal-Glc-Cer

GD1b

SA

SA nLc4

Ii

GlcNAcβ1-3

GlcNAcβ1-6

Galβ1- 4GlcNAcβ1-3Lc2

SA

SA-LeX

α1-3Fuc i

I

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

240 S. Basu et al.

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 unidenti fi ed 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

PCPS

PS

APOPTOTICREAGENTS

PS2-48hrs

Nucleus

NON-APOPTOTICCARCINOMA CELL

Nucleus

APOPTOTIC/DEAD CELL

PS = DG-O-P-O-CH2-CH

I O-

=O NH3

+

COO-

=O

1) PSS-380 DyeBlue Fluorescence (Apoptotic Cells) 2) Propidium IodideRed Fluorescence (Permeable Dead Cells)

APOPTOTIC REAGENTS

i) L/D-PPMPii) GD3/GD1biii) cis-Platiniv) Betulinic Acidv) Mephalan, etc

Me

PC

PE = DG-O-P-O-CH2-CH2- NH3+

= DG-O-P-O-CH2-CH2- N+ - MeMe

IO-

=IO

-=

O

AKS-0 Smith, B. et.al. (Patent Pending)

Smith, B. et.al. (Patent Pending)

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

24116 Apoptosis of Breast Cancer Cells…

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 fl uorescent 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.

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 speci fi cally 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)

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) → Gal b –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) → Gal a –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)

24316 Apoptosis of Breast Cancer Cells…

(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

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

244 S. Basu et al.

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 ) .

Table 16.4 Transcriptional regulation of glyco-related genes in breast cancer cells (treated with 2 m M 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

Table 16.3 Transcriptional regulation of glycogenes in apoptotic breast cancer cells (treated with 2 m M l -PPMP for 24 h) (Ma et al. 2009 ; 2011 ; Kessler et al. 2007b )

Cell line GLT gene name Linkage formed

Fold change

MCF-7 ST8SIA4 NeuAc a 2–8NeuAc-R1 1.39

GCNT2 GlcNAc b 1-3nLc4 → GlcNAc b 1-3 nLc4 1.39 GlcNAc b 1-6

B3GALT2 Lc3 → Gal b 1–3Lc3 1.36 B3GALNT2 Gb3 → GalNAc b 1–3Gb3 1.31 B3GALT5 Lc3 → Gal b 1–3Lc3 1.16 UGT8 Gal b 1–1Cer −1.14

MDA-468 GCNT2 GlcNAc b 1-3nLc4→ GlcNAc b 1-3 nLc4 −1.11 GlcNAc b 1-6

SKBR-3 GCNT1 Gal b 1-3GalNAc a -R2 → Gal b 1-3

GalNAc a 1-R2 −1.19 GlcNAc b 1-6

24516 Apoptosis of Breast Cancer Cells…

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 targeted only to a cancer cell avoiding any normal cell.

Fig. 16.8 An ideal drug delivery system

Novel Drug Delivery Systems for Breast Cancer Treatments

The tissues in human bodies contain 70–90% water. Drug molecules (soluble or suspended fi ne 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 coef fi cient of 10 −7 cm 2 /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 fi eld. 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 ef fi cient 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.

DIFFERENT DRUG DELIVERY SYSTEMSUNDER 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

Fig. 16.9 Different drug delivery systems under study

246 S. Basu et al.

Electrically Enhanced Chemo-drug Delivery to Human Breast Cancer Cells

Human MCF-7 BCC was used to evaluate the treatment ef fi cacy. 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 ef fi ciency 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 ) .

SA-Lex

SA-Lea/or b (or any antigen-

specific antibody)

Synthetic Liposomes

Metastatic Cancer Cells(Colon, Breast,Neuronal, Prostate, )

Containing: Apoptotic/Anti-cancer Drugs/

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

24716 Apoptosis of Breast Cancer Cells…

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 speci fi c 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 fl uid technologies. Supercritical fl uids 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 fl uids 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 fi eld 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 in fl ammation 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 m M) 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 m M) 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 speci fi c antibodies (CSLEX monoclonal antibody binds to SA-LeX) against cell surface-speci fi c 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.

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