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Breast cancer is the most frequent type of cancer among women which accounts for 32% of the cancer burden in the UK.(W2009F)(U2009B) Around one-in-nine women in the UK are diagnosed with breast cancer at some point in their life.(S2008S) Statistics for 2004-2006 show that 45,524 women per year are diagnosed with breast cancer with 12,429 cases die from the disease.(U2009B) Breast cancer incidence increases with age, oestrogen exposure, significant radiation exposure, family history and other specific conditions.(M2008B)
The vast majority of breast cancers arise from epithelial cells lining the terminal duct lobular unit. Some tumors do show distinct patterns of growth and cellular morphology, and on this basis certain types of breast cancer can be identified. Approximately 20% of invasive breast cancer with special types can be classified into tubular, cribriform, medullary-like, mucoid, papillary and classic lobular. However, another majority cases are of no special type.(M2008B) Breast cancer can be treated or cured depending on different stages and types. The mainstay of breast cancer treatment is surgery before tumor metastasis (new tumors appear far from the original site), associated with possible adjuvant therapies including radiotherapy, chemotherapy, and recently, biotherapy.(W2003G)
Ion channels are integral membrane proteins that open in response to a depolarizing stimulus, thereby allowing ion travel between extracellular space and cell interior. Ion channels are major signaling complexes expressed in many tissues, where they have diverse involvement in cellular activity.(W2007T) Most channels are specific for one ion such as potassium, calcium and sodium.
Sodium channels conducting sodium ions (Na+) through the cell plasma membrane. Channel opening is transient, allowing the flow of sodium ions down their concentration gradient, thus generating an inward current, and most channels rapidly inactivate, within milliseconds of opening, and the undergo conformational changes to recover form inactivation. Sodium ion channels are responsible for the rising phase of action potentials in excitable cells, for example, neurons and myocytes.(V2009S)
There are two types of sodium ion channels: voltage-gated Na+ channels (VGSCs) and ligand-gated Na+ channels. Voltage-gated Na+ channels are heteromultimers of α and β protein subunits. The α subunit is sufficiently to form the core of a functional channel when expressed by a cell, whereas modulatory β subunit is on auxiliary status. The α subunits of sodium channels (VGSCα) are large polypetides that are organized into four homologous domains (DI - DIV), each consisting of six transmembrane segments that are connected by intra- and extracellular linkers (Figure 1).(V2009S) VGSCs are expressed in a wide variety of excitable and non-excitable tissues, and display a range of functional forms.(A2005A)
Figure 1. Structure of sodium ion channels
Sodium pump isoform expression is specifically altered in a tissue-specific manner in diseases such as hyper- and hypothyroidism, hypokalemia, hypertension, heart failure and also in cancer cells and tissues.(T2007C) Ion channels are increasingly being suggested to be involved in different stages of cancer process including cell proliferation, migration and survival. According to the recent investigation, voltage-gated ion channels are widely expressed in a range of cancer cells.(W2007T) Moreover, increasing evidence suggests that expression of voltage-gated ion channels become epigenetically abnormal in metastatic cancer cells of epithelial origin.(R2009M) As such, these channels are becoming the targets of significant drug development efforts to block or reduce voltage-gated ion channel activity in order to prevent or combat malignant disease. Components with specific VGSCs inhibition property may reduce cellular behaviors and may have anti-tumor, indeed anti metastatic breast cancer potential.
Voltage-gated Na+ channels have been detected in different cell lines including human breast cancer. Importantly, upregulation of functional voltage-gated Na+ channels has been found in metastatic human breast cancer in vitro and in vivo.(W2007T) Nav1.5 expression strongly correlated with lymph node metastasis in breast cancer in vivo shows that Nav1.5 may play a key role in metastasis.
W. J. Brackenbury et al has clarified the specific involvement of 'neonatal' splice variant of Nav1.5 (nNav1.5) in migrated MDA-MB-231 cells. That study tested the migrated cell line with RNA small interference (siRNA) and a polyclonal antibody (NESO-pAb). The results of westen blotting and immunocytochemistry suggested that nNav1.5 was indeed highly expressed in the plasma membrane of MDA-MB-231 (highly metastatic breast cancer cell line) compare to the lowly metastatic MCF-7 cells. nNav1.5 is proved to be the primarily responsible for the VGSC-depedent invasive behaviour in MDA-MB-231 cell line according to the investigation. Specifically targeting of nNav1.5 expression considered to be significant in clinical management of metastatic breast cancer as novel therapy.(W2007T)(R2009M)
Docosahexaenoic acid, also known as DHA, is an omega-3 polyunsaturated long chain fatty acid naturally sufficient in marine food. Previous work of Banu Isbilen er al has tested the MDA-MB-231 human breast cancer cells treated with DHA in vitro. The results proved that long-term treated with docosahexaenoic acid down-regulated mRNA and plasma membrane protein levels of neonatal Nav1.5 voltage-gated Na+ channel which known to be predominant in migration MDA-MB-231 cell line. Also, DHA helps to reduced the migration of cancer cell to the same extent as tetrodotoxin (Figure 2) (a specific VGSCs inhibitor) by suppressed VGSCs activity.(B2006D)
Figure 2. Tetrodotoxin
Cardiotonic steroids (CS) or cardiotonic glycosides represent a group of compounds target the sodium pump. Those drugs have been widely used for the treatment of heart failure. Epidemiological studies reported that the tumour cell populations from breast cancer patients on CSs medication for cardiac problems appeared to have a lower proliferative capacity than tumour cells from patients not on CSs treatment. Such matter of fact interests the researchers to develop CSs as potential anti-cancer agents over the last 10 years.(T2007C)
Generally, the main structure of CS is an 4 ring system with a sugar moiety at position 3 and a R group at position 17.(Figure 3) Different cis or trans fused of A/B and C/D rings gives different dimensional structures. Several plants, more particularly those belonging to Asclepiadacea, Apocynaceae, Ranunculaceae and Scrophulariaceae families, are recognized to contain CSs. Many medicinal plants containing CSs have been used in the treatment of heart diseases. Clinically used drug digoxin (Figure 4), extracted from the most well-known plant Digitalis from Scrophulariaceae family, was proved to have cytotoxicity effect in human MCF-7 breast cancer cells. G-strophantin (ouabain) (Figure 5), a poisonous cardiac glycoside isolated from the seeds of Strophanthus gratus and the bark of Acokanthera in the family Apocynaceae, was tested in human breast cancer cell line. At concentrations <100 nM, ouabain can produce anti-proliferative effects. The induction of apoptosis was only at concentrations µM. Several lines of evidence indicated that CSs possess potent anti-breast cancer activity.(T2007C)
Figure 3. The main structure of cardiotonic steroids
Figure 4. Digoxin
Figure 5. Ouabain
Vinca alkaloids is a group of compounds isolated from plants or synthetically produced with indole (Figure 6) in the structure. Vincristine (brand name Oncovin®) (Figure 7) is a mitotic inhibitor approved by the United States Food and Drug Administration (FDA) in 1963 as a cancer chemotherapy to treat acute lymphoblastic leukemia and breast cancer. Vinflunine (brand name Javlor®) (Figure 8) is a novel fluorinated derivative of vinca alkaloid.(Y2009N) There are two indole moieties involved in both vincristine and vinflunine structure, such group of vinca alkaloids is called bisindole alkaloids. Recently, vinflunine has been launched in the UK as monotherapy for the treatment of adult patients with advanved or metastatic transitional cell carcinoma of the urothelial tract with a recommended dose of 320mg/m2 IV every 3 weeks.(WV)(SH) The anti-breast cancer effect of vinflunine is currently evaluated in clinical trials in combination with other agents.(Y2009N)
Figure 6. The structure of indole
Figure 7. Vincristine
Figure 8. Vinflunine
There has been a growing interest in the use of natural products, especially extract from herbs, as a potent source of new therapeutic anticancer drugs. A variety of plant secondary metabolites have been used as agents for chemoprevention of cancer. Such as several known natural aromatase inhibitors based on the flavonoid skeletons. However, little is known about the natural anti-cancer products targeting the ion channels on cellular level.
Recent researches are starting to screening natural compounds from plants as potential ion channels blocker. Some herbal alkaloids, for example tetrandrine has been tested to determine the efficient target of its cell proliferation inhibite function. Though no evidence shows that ion channels were involved in that test, further work on natural substances targeting ion channels is the novel area to be investigated.(G2004H)
Since both cardiotonic steroids and bisindole alkaloids have the potential to become anti-breast cancer agents, it is important to find some candidate plants contain both kinds of compounds. Apocynaceae family contains 200 genera and over 2000 species of trees, shrubs, herbs, and lianas. Many researched plants belong to Apocynaceae family are rich in cardiotonic steroids and/or bisindole alkaloids. This study intends to investigate the cardiotonic steroids and bisindole alkaloids in Apocynaceae family as anti-breast cancer agents especially target the sodium ion channels.
Aim of the investigation
To prove and investigate the status and mechanisms of (sodium) ion channels involved in breast cancer cell line.
To examine the anti breast cancer activity of ion channels targeting components.
To establish and evaluate a suitable method including extraction and isolation techniques, to get pure compounds from selected families.
To evaluate the extract and separated compounds from selected herb and compare the activity with clinic used medicines to treat breast cancer.
To determine the chemical structure of the isolated compounds.
To modify the chemical structures of the isolated natural products to improve the activity of preventing and/or anti breast cancer.