Role of polyamines in Breast cancer and evaluation of

toxicity of Polyamine conjugate in four different cell lines

(MCF-7, MDA-MB-231, MCF-7 TAX 30, MDA TAX 30)

Abstract
Breast cancer is the most common form of cancer afflicting women
worldwide. Even with the availability of highly potent and effective
drugs chemotherapy has failed to provide effective cancer
treatment due to 2 major problems. First, since cancer cells are
similar to normal cells problem arises in selective delivery of the
drugs to cancer cells. Second problem is multidrug resistance
(MDR). A molecule with novel mechanism or selective delivery
would greatly increase the therapeutic efficacy of the drugs and
would drastically reduce the associated toxicities. As we know that
cancer cells have increased demand for polyamines. Polyamines can
act as vectors to which cytotoxic drugs could be coupled and
selectively target cancer cells by utilizing upregulated polyamine
transport system. Various polyamine analogues and conjugates
have been synthesized till now, which are similar in structure to
natural polyamine. As polyamine transport system cannot
distinguish between natural and synthetic polyamines, analogues
and conjugates can specifically target cancer cells without affecting
normal cells.
_____________________________________________________________________

Keywords: Polyamines, toxicity, breast cancer cells, MDR.

Introduction
Breast cancer is the 2nd major cause of death in Western world and
most prevalent form of invasive non-skin cancer among women.
Reports of Breast Cancer UK suggest that breast cancer is most
common form of cancer in UK. In 2007, 45,972 new cases of breast
cancer were detected. Each year, 120 in 100,000 women screened
for risk of breast cancer have developing cancer and 30% possibility
of diagnosed breast cancer to become metastatic during their
lifetime.

Treatment of breast cancer involves multiple approach of surgery,

radiation therapy, chemotherapy, hormone replacement therapy or
biotherapy. Biggest hurdle in the effective treatment with these
approaches is that initially tumours are responds well to both
hormone replacement therapy as well as to chemotherapy but later
becomes aggressive and unresponsive to both the ways of
treatment.

Also the current approaches are associated with morbidity, have

limited clinical benefits and lack of target specificity causes toxicity
in the host. The narrow therapeutic index of the existing anticancer
drugs has created an intense interest in developing new anti-
neoplastic drugs which have novel mechanism to overcome drug
resistance in tumours. With so many difficulties to overcome for an
effective cancer treatment, polyamines offer an interesting
approach which was recently explored. The characteristic of a
cancer cell are higher than in normal intracellular concentration of
polyamine, deregulated polyamine metabolism and also
upregulated polyamine transport system for uptake of polyamines
(Wallace et al., 2009). These characteristic features of the cancer
cells could be utilized for delivering anticancer drug to the tumour.

What are polyamines?

Polyamines are chemically active aliphatic amines commonly found
in foods, but most polyamines are synthesized intracellularly by
microflora. Mammalian polyamines (putrescine, spermidine,
spermine) are aliphatic cation and are ubiquitous in nature.
Polyamines at physiological pH carry positive charge and are
supercations carrying positive. Spermine has 4 units of positive
charge followed by spermidine, 3 then putrescine which has 2 units
of positive charge (Yuan et al., 2001) (refer to fig 1). The positive
charge on the polyamines enables them to interact with DNA, RNA,
phospholipids of the membrane and form complexes with it (Wallace
et al. 2009). Polyamines are also involved in cell growth specifically
in proliferation, differentiation and transformation. Processes like
DNA replication, transcription and translation requires polyamines
(Canizares et al. 1999). Polyamines can regulate activity of many
enzymes such as adenylate cyclase, tissue transglutaminase.
Polyamines interact with DNA and cause conformational changes in
DNA (Luk and Casero, 1987).

Fig 1. Charge distribution of along the length of carbon chain on Polyamines

(Image adapted from Cell proliferation group, 2008)

Role of polyamine in cell cycle could be linked with its active

biosynthesis at G1 to S phase transition. Research published by
Lund University using Chinese hamster ovary cells has suggested
that polyamine pool depletion by inhibition of polyamine synthesis
by an inhibitor downregulates the DNA replication process by
reduction in DNA elongation rate (Oredsson et al 2008). It has been
suggested by Oredsson et al. in their findings that in presence of a
polyamine inhibitor which decreased intracellular polyamine levels,
lengthened S phase, and affected the G1 to S phase transition and
G2 phase progression were observed.

In programmed cell death activated endonucleases causes

fragmentation of chromosomal DNA. Interesting fact observed in
polyamine depleted cells is that these cells have unstable DNA
which is susceptible to nuclease attack and could be easily
degraded (Oredsson et al 2008). The role that polyamine play in this
process is still under investigation.
 Molecular &
cellular
function of
polyamines

Topoisomerases I is
Polyamines inhibited by RNA
Nucleic acid polyamines while
interact with the Macromolecule polymerase ar
& topoisomerases II
cell membrane are stimulated by synthesis stimulated by
structure
and stabilizes it polyamines polyamines

Polyamines Aminoacyl
Promotes Polyamine
Polyamines influence the tRNA
Facilitates aggregation of concentration ha
protect DNA rate of synthetases are little effect on DN
conformational ribosomal stimulated by elongation but
from shear by translation as
changes in DNA subunits polyamines adversely affect
condensing it well as fidelity replicon initiatio

Luk and Casero suggested that putrescine is necessary for nucleolar

formation in early development of embryos and inhibitors of ODC
stop the development of embryo. Increase in the ODC activity is
observed in maturation during meiosis. This fact suggests that
polyamine depletion inhibits cell proliferation and movement thus
affecting embryo development whereas excess polyamine
concentration causes apoptosis and transformation of the cell (Luk
and Casero, 1987 ).
Biosynthesis of polyamines

Intracellular polyamine concentration is derived both from dietary

supply of polyamines and biosynthesis in the cell. Gut microflora
produces polyamines by utilizing amino acids obtained from dietary
sources (Eliassen et al. 2002). Bacterias such as Clostridium,
Bacuillus, Proteus etc are capable of producing polyamines.

Intracellular production of polyamines initiates by the enzymatic

action of ornithine decarboxylase enzyme (ODC) on amino acid l-
ornithine. L-ornithine is a non-essential amino acids synthesized by
the ornithine cycle (Adamo, 2006). L-ornithine is converted to
Putrescine which serves as a precursor of Spermidine (refer fig 2).

Spermidine is produced by enzymatically by spermidine synthase

which attaches a propyl group to putrescine. Propyl groups are
produced as a result of enzymatic action on L-methionine which
gets converted to S-adenosylmethionine which is further acted on
by S-adenosylmethionine decarboxylase to yield decarboxylated S-
adenosylmethionine (Wallace et al. 2003) (refer fig 2).

On addition of another propyl group to spermidine, spermine is

produced. Addition of another propyl group is catalyzed by spermine
synthase (Wallace et al. 2003) (refer fig 2). Putrescine synthesis is
dependent only on the availability of the enzyme ODC whereas
synthesis of spermidine and spermine are dependent on the propyl
group provided by catalytic conversion of L-methionine to
Decarboxylated S- adenosylmethionine (Wallace et al. 2003).

Fig 2. Showing polyamine biosynthesis (adapted from Wallace et al. 2003)

ODC is the most important enzyme in the synthesis of polyamine. It

is present in very low concentration in non-dividing cells (or
quiescent cells). In response to external stimuli such as tumour
promoter, growth factors and hormone ODC is highly expressed and
its activity increases many folds (Canizares, 1999). ODC limited
activity time from few minutes to hours. Antizyme , a protein
inhibitor regulates the activity of ODC (Wallace et al. 2003).

Polyamine catabolism
Initially it was believed that polyamine biosynthesis is a
unidirectional process but it has a separate pathway which allows
conversion back. Aminoxidases and acetyltransferases are involved
in this pathway which catalyzes the retroconversion.

Of conversion of spermine back to spermidine, reaction needs to be

catalyzed by spermine/spermidine acetyltransferase. In this step an
intermediate is produced, N1 acetylspermine which could undergo
further oxidation or could be transported out of the cell by
polyamine transport system. N1 acetylspermine is further catalyzed
by polyamine oxidase (Seiler, 1990) (refer fig 2). Similarly,
spermidine can be converted back to putrescine by
spermine/spermidine acetyltransferase and by polyamine oxidase.

During the catalyzed retroconversion of acetylated polyamines, 3-

acetamidopropanol and hydrogen peroxide is produced. Hydrogen
peroxide is a strong inducer of SSAT as well as induces apoptosis.
This pathway is important for cells that are not involved in cell cycle
and cell division to lower their cellular polyamine content (Wallace
et al. 2003). This indicates that induction of SSAT can cause damage
to DNA and lead to apoptosis (Seiler, 1990).

Fig 3 showing the death cycle and induction of SSAT (adapted from
Wallace et al, 2003).

Polyamine transport system

Polyamine transport system imports and exports polyamine of the
cell and maintains the cellular concentration of polyamines. When
intracellular concentration decreases transporter increases cellular
uptake of polyamines. Many cells have single transporter for all the
polyamines while some have 2 transporters (Wallace at el. 2003).
Thus making PAT an ideal process to introduce polyamine linked
drugs into the cell. The mechanism of transport is by receptor
mediated endocytosis (Wallace at el. 2003). Antizyme which
modulates the activity of ODC also inhibits polyamine transport
system (refer fig 2). The non-selective transport of PAT has led to
design of polyamine conjugated drug which targets only cancer cells
and normal cells are not affected.

Role of Polyamines in cancer cells

In breast cancer, polyamines are present in 3-4 times of their

normal levels (Wallace et al. 2000). It is known that polyamines are
involved in growth processes but actual mechanism by which they
affect growth is not known. It is believed that polyamines can
modulate the growth promoting genes and they increase the
synthesis of protein involved in growth (Adamo, 2006).

It is believed that there exists a close association between RNA,

polyamines and hormone Insulin. Once inside the cell, insulin
stimulates the growth by activating ribosomes (refer fig 4).
Ribosomes diligently codes for amino acids which forms proteins
and polyamine enhances the efficiency of this association by
stabilizing the mRNA so that more amount of protein could be
produced from mRNA (Adamo, 2006).

Breast cancer cells have higher than normal concentration of acetyl

polyamines indicating increased synthesis, decreased degradation,
increased uptake of polyamines by transporters and decreased
export. They have decreased activity of PAO enzymes which
correlates to aggressiveness of the tumour (Wallace et al. 2000).

Fig 4. showing role of polyamines, mRNA and insulin in promotion

of cell growth (adapted from Adamo, 2006).

As already discussed, polyamines are essential for the growth of the

cells and in actively dividing cells polyamine requirement goes up.
Similar phenomenon is seen in cancer cells which have very high
demand for polyamines (Canizares, 1999). So blocking polyamine
biosynthesis by inhibiting ODC (by α DMFO) activity depletes
polyamine pool and affects cancer cell growth and metastases.
High polyamine level have another deleterious effect, it inhibits the
body’s immune response against tumour cells by blocking the action
of natural killer cells (NK cells) (Shah et al. 1999). So we can say
that polyamines not only have growth promoting effects but also
have immunosuppressive action.

Observational studies in colon cancer patients have revealed that

these patients had increased ODC activity, increased levels of free
polyamines and altered blood group antigen expression (Adamo,
2006). These changes can serves as biomarkers for early detection
of cancer.

Moshier et al.have suggested that for a cell to transform into

malignant cell, ODC undergoes overexpression by increased
translation of ODC mRNA (Moshier et al. 1993). This suggests that
ODC gene is an oncogene and its altered expression is related to
invasiveness and angiogenesis of tumour. In breast cancer, besides
over expression of oncogenes chromosomal deletion of tumour
suppressor genes is observed (Canizares, 1999).

Drug resistance in Cancer

Resistance developed by cancer cells to multiple chemotherapeutic
drugs is termed as multi drug resistance (MDR). MDR is major hurdle
in the effective treatment of breast carcinoma. MDR was initially
linked to P-glycoprotein (Pgp/MDR1) over expression, which includes
multidrug resistance gene (MDR1) which codes for transmembrane
xenobiotics transporter protein P-glycoprotein (P-gp) and causes
MDR (Liu et al. 2008). Later it was clear that only a single protein
could not cause drug resistance in multiple independent drug
resistant cells, then multidrug resistance protein (MRP1/ABCC1) and
breast cancer resistance protein (BCRP/ ABCG2) were discovered
and their over expression was linked to MDR (Kuo, 2000). MRP1 and
BCRP cause multidrug resistance independent of the P-gp protein
(Stein et al. 2002).
Fig structures of P-gp,MRP1 and BCRP (image adapted from
Morrow and Cowan 2005)(BCRP Image adapted from Kuo, 2000)

MDR1/Pgp
P-gp is encoded by MDR1 and causes multidrug resistance in cells
that has been exposed to anti-cancer agents like doxorubicin,
taxanes, etoposide and vincristine (Kuo, 2000). These drugs enter
into the cell by passive diffusion as they are hydrophobic in nature.
But, P-gp transports drugs out of the cells against the concentration
gradient by utilizing energy in the form of ATP (Stein et al. 2002). It
is still uncertain how structurally dissimilar drugs act as substrate
for one protein.

MRP
In total there are 9 MRP’s, MRP1- MRP9. Resistance to wide range of
anticancer agents is due to the overexpression of MRP1. Since
substrates of MRP1 and Pgp are different, one drug cannot cause
equal expression of both the protein (Kuo, 2000).

MRP1 is unable to carry out efflux of drugs independently as it

requires glutathione, glucuronic acid or sulphate as cofactor to
transport drugs out of the cells (Yamane et al. 1998).

Yamane et al. exposed the cells to a variety of anti-cancer drugs,

heavy metals, nitric oxides and results revealed that MRP1
expression in exposed cells increased as compared to unexposed
cells. This indicated that MRP1 is inducible by a variety of agents.
But the mechanism of induction of MRP1 was unclear until Kuo’s
findings suggested that, it is the enzyme γ-glutamylcysteine
synthetase (rate-limiting enzyme) which synthesizes glutathione
which is induced by cytotoxic agents and these agents also
increases the expression of MRP1. In many cancers both the genes
for MRP1 and γ-glutamylcysteine synthetase were found to be
upregulated indicating a common mechanism for both the genes
(Yamane et al.1998).

BRCP
Breast Cancer Resistance Protein belongs to transporter family ATP
binding cassette. BCRP is half the size of MDR1 and MRP protein
(Yamane et al. 1998). Cell lines which are resistant to
chemotherapeutic drugs like mitoxantrone, topotecan and
doxorubicin are drug resistance due to over expression of MDR1 and
also have overexpressed BCRP. Unlike MRP, BCRP does not require
cofactor. Studies in mouse on BCRP have revealed that bcrp (-/-)
mouse exhibited normal physiology suggesting that it is non-
essential in wild type mouse but have crucial role in development of
resistance to multiple drugs (Kuo 2000)
Mechanism of MDR
P-glycoprotein is an ATP dependent pump which transports drug in
unidirection. It transports drug out of the cells and prevent drug
accumulation to lethal concentration intracellularly (Xu et al. 2006).
In this way it helps cancer cell to evade effective attack of anti-
cancer drugs.

In breast cancer, breast cancer resistance protein/ mitoxantrone

resistance associated transporter (BCRP/MXR) are associated with
drug resistance. BCRP/MXR proteins are members of ATP-binding
cassette family and are often overexpressed in dug resistance
cancer cells (Stein, et al. 2002).

Resistant breast cancer cell line MCF7/AdVp3000 exhibited over

expression of ABC transporters like ABCG2 and explains the cause
of high degree of resistance. Genomic and proteomic profiling of
ABC proteins showed that the MCF-7/AdVp3000 over expresses ABC
transport protein and along with it several other proteins like 14-3-
3σ are overexpressed as well (Liu et al. 2008).

While searching elements responsible for drug resistance scientists

have stumbled upon a fact that drug resistant cancer cells have
over expression of Fatty acid synthase (FASN). It has now been
experimentally proved that increased levels of FASN leads to
resistance to Adriamycin and mitoxantrone in MCF-7/AdVp 3000 and
in MDA cell lines (Liu et al. 2008)

Mechanism of how polyamine concentration affects cancer cell

growth is not yet known and is under investigation. But some of the
studies conducted have revealed that there exists a strong
relationship between estrogen, growth factors and polyamines
(Canizares et al. 1999)

Classical multidrug resistance mediated by MDR1/P-gp could be

overcome by either blocking the function of P-gp by P-gp blocker
also known as MDR reversal agents or by down regulating its
expression (Xu et al. 2006). Some of P-gp inhibitors are P-gp
substrates like Verampamil and curcuminoids. These agents are
competitive inhibitor of P-gp mediated efflux of drugs hence the
transport action of P-gp gets suppressed (Kuo, 2000).

For the evaluation of cytotoxicity of the novel polyamine conjugates

four cell lines were used 2 were drug sensitive MCF-7 and MDA-MB-
231 and 2 were drug resistant MCF-7 TAX30 and MDA TAX 30.

Characteristics of breast cancer cell lines

MCF-7: These cells are estrogen dependent hence have estrogen
receptor (ER α positive) and are weakly invasive.
MDA-MB-231: These cells are estrogen independent hence do not
express estrogen receptor (ER α negative) and phenotypically these
are spindle shape highly invasive and causes aggressive forms of
cancer.

MCF-7 TAX 30 and MDA TAX 30 are cell lines which are resistant
to taxane class of anticancer drug such as docetaxel, paclitaxel.

Role of Polyamines in breast cancer cells

Increased polyamine concentration is essential for tumour cell for

both development and its maintenance of proliferative
characteristics. Polyamine acts as a positive regulator of the
neoplastic phenotype of the tumour cells. They interact with
promoter sequences and transcription factors and regulate gene
expression.

NF-κB is a nuclear factor that belongs to rel family of transcription

factor. It is present in an inactive form as a bound to IκBS. Stumuli
like oxidative stress, cytokines and several anti-cancer drugs causes
activation of NF-κB (Shah et al.1999). Upon activation Nf-κB moves
to nucleus and binds to NRE sequences, which causes expression of
proteins involved in cell proliferation and thus prevents apoptosis.
This apoptosis preventive action is similar to polyamine action of
decreasing apoptosis by over expressing proteins for cell
proliferation. In human breast tumour cells, over expression of NF-
κB is observed and inhibition of NF-κB activity induced apoptosis in
them (Shah et al.1999).

Nakshatri et al. suggested that NF-κB has high constitutive binding

to the response elements in ER-negative breast cancer cell lines
(Nakshatri et al. 1997).

Polyamines have a stimulatory effect on the NF-κB-NRE binding in

breast cancer cells. Thus it is clear that NF-κB act as anti-apoptotic
factor so by an effective approach would be to block the NF-κB
activation by inhibition of polyamines, thereby making cells
susceptible to apoptosis (Shah et al.1999) (Davidson et al. 1999).

In breast cancer, hormone independent cells become aggressive

and are difficult to treat. Hormone independence of breast cancer
cells is attributed to over expression of oncogenes by amplification.
Over expression of oncogenes induces ODC and as a result increase
in polyamine level is observed (Nishioka, 1996). Increased
polyamine levels helps breast cancer cells to get rid of hormone
dependence.

Polyamine Analogues & conjugates

Fig 5. Showing potential targets in polyamine metabolism pathway.

Polyamine conjugates like anthracene 4 are new molecules and are

still being tested for their cytotoxicity. Anthracene 4 is an
topoisomerases II inhibitor. While testing Ant 4, it was observed that
the polyamine component of the molecule serves it function of
selectively and effectively delivery the conjugate into the cancer by
utilizing Polyamine transport system. By blocking the action of ODC,
cell polyamine import could be increased and thereby increased
concentration of Ant 4 could be created inside the cell (Wallace et
al. 2009). It was observed that Ant 4 not only causes polyamine
depletion but also lead induction of apoptosis by loss of
mitochondrial membrane potential and release of cytochrome c,
indicating cytotoxicity of Ant 4(Wallace et al. 2009). Since this
conjugates was designed to deliver the drug into the cell but
exhibited an additional function of depletion of polyamine content
and induction of apoptosis (Wallace et al. 2009).

Recently focus has been given on polyamine metabolic pathway

steps as potential targets. One such analogue is N,N’-bis(ethyl)
spermine. It exhibits downregulation of ODC, depletes polyamine
pool and leads to arrest of cell growth. It causes growth arrest by
inducing the enzyme spermidine/spermine N1-acetyltransferase
(SSAT) (Davidson et al. 1999). These analogues readily accumulate
inside the cell imported by polyamine transport system, they do not
substitute for natural polyamine hence normal cell are not affected
(Davidson et al. 1999).

In clinical trials with polyamine analogues BENSpm, gastrointestinal

and neurological toxicity was of concern and could be controlled by
reducing dose and no haematological toxicity was observed
(Davison et al. 1999). Breast cancer cell lines are used to evaluate
the combination of polyamine analogues and conventional cytotoxic
agents used to treat breast cancer and keen interest is to see
whether polyamine analogues and cytotoxic agents show synergism
in their action.
Conclusion

Polyamines are the only approach which offers effective and

selective treatment of cancer. But as new and new molecules are
being synthesized scientists try to achieve perfection. Polyamine
analogues have wide range of effects but new conjugates are more
specific and selective in their approach. As we establish the
cytotoxicity levels of drugs we can be able to use then with more
accuracy. In current era of highly regulated world where scientists
are working hard in labs to find a more efficacious drugs, better
methods to introduce drug to the cancer cell and tumour specific
drugs ready to come in, the question to ask is that the incidence of
cancer has increased by 50% over last 25 years in spite of so much
measure and effective treatments in place. Causes of cancer are
being blamed on the patients as genetic link, lifestyle and food
habits but why environmental risk factors haven’t been considered
to which a person is exposed to constantly.

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