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)

AbstractBreast 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.

IntroductionBreast 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.

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).

Molecular & cellular

function of polyamines

Nucleic acid &

structure

Macromolecule synthesis

RNA polymerase are stimulated by polyamines

Facilitates conformational

changes in DNA

Promotes aggregation of

ribosomal subunits

Polyamines protect DNA from shear by condensing it

Polyamines influence the

rate of translation as

well as fidelity

Aminoacyl tRNA

synthetases are stimulated by polyamines

Polyamine concentration have little effect on DNA

elongation but adversely affects replicon initiation

Polyamines interact with the cell membrane and stabilizes it

Topoisomerases I is inhibited by

polyamines while topoisomerases II are stimulated by

polyamines

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 catabolismInitially 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 systemPolyamine 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 CancerResistance 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/PgpP-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).

BRCPBreast 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 MDRP-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 linesMCF-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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