High incidence of breast cancer

Abstract:

Breast cancer is the most popular cancer in industrialised nations and the second top reason of death in female population. Although breast cancer incidence is high, there have been some decrease in recent years due to earlier detection. The evidence shows that Prostaglandins (PGs) play an important role in the development of cancer as well as cyclooxygenase (COX). From two isomers of COX, COX-2 is highly involved in cancerous cells and it is a good target molecule to track cancer progression. COX-2 inhibitors allow us to investigate the properties of COX-2 that have an effect on breast cancer. Studies by US Women's Health initiative supports the idea that NSAIDs reduce breast cancer incidence. Other studies show that selective aromatase regulators share the same properties with COX-2 inhibitors. Also recent development in anti-cancer cobalt complexes that are the most potent COX-2 inhibitors so far.

Ovarian cancer has been drawn to attention recently because it is one of the most common cancers in the western world. Studies revealed that ovarian cancer cells are associated with the over-expression of COX-1 and COX-2 enzyme and identified potential targets for its treatment.

Laryngeal cancer is one of the largest head and neck cancers and is due to over-expression of COX-2 enzyme. In addition, it was shown that epidermal growth factor receptor (EGFR) and COX-2 pathways are connected to each other forming a positive feedback loop. Therefore, different anti-tumor strategies were proposed relevant to that positive feedback loop. Pancreatic cancer is another type of cancer and it is of major importance because of absence of its symptoms. The over-expression of peroxisome proliferator activated receptor gamma (PPAR-?) has been associated with pancreatic cancer. Also it was shown that PPAR-? and COX-2 pathways are connected. As a consequence PPAR-? COX-2 pathways are considered as potential target for inhibiting the progression of pancreatic cancer.

NSAIDs such as celecoxib selectively inhibit COX-2 enzymes and are effective in treatment of RA. They have fewer adverse effects than non-selective NSAIDS, especially gastric complications. Although celecoxib is a selective COX-2 inhibitor but it has anti-cancer activity in tissues in which COX-2 is not present. This indicates that celecoxib is an anticancer agent which is independent of COX-2. Another non-selective NSAID, Rofecoxib, despite of having a potential role in the treatment of lung carcinoma, RA and OA, was withdrawn from market in 2004 because of an increased risk of heart attacks and strokes.

Neurodegeneration in Alzheimer's disease indicates inflammation which could be a potential target to be treated by NSAIDs. Epidemiological studies indicate that COX-2 inhibitors may possess an advantage over non-selective NSAIDs as a potential treatment. Studies showed that over-expression of COX-2 is evident in brains affected by the disease and there are similarities between neuronal and inflammatory cells which suggests that NSAIDs can protect patients against Alzheimer's disease. There is now evidence that COX-inhibitors reduce incidence of cardiovascular diseases. Low-dose aspirin can be used for the prophylactic treatment of myocardial infarction (MI) and thromboembolic disease.

Introduction:

Breast cancer is the most popular malignant growth in women in industrialised nations and the second top reason of female cancer-related death. Statistics estimate a figure of 40000 women develop breast cancer in the UK each year. Unfortunately, there has been an increase in breast cancer incidence by two-thirds in recent years but the good news is that mortality rates have decreased by one-third due to earlier detection of breast cancer. Prognosis of the breast cancer is extremely important and strongly related to initial diagnosis. The majority of patients die due to breast cancer which causes malignant growth in their bones. The evidence from clinical studies shows that Prostaglandins (PGs) play an important role in the growth and development of cancer. Cyclooxygenase (COX) is a rate-limiting enzyme that transforms arachdonic acid into prostaglandin-H2. PGH2 is a precursor for several molecules such as PGE2, prostacyclin and thromboxane. There are two isomers of the COX enzyme: COX-1 and COX-2 which are found in human tissues. COX-1 expression is continuous and at low levels, whereas COX-2 is expressed in response to various stimuli. New blood vessels surrounding cancerous cells have COX-2 within themselves. Consequently, by inhibiting COX-2 enzyme, not only the epithelial cells of tumours but also the endothelial cells are targeted (Bundred & Barnes, 2005).

Main body:

As noted above, COX-2 is highly involved in malignant growth which makes it a good choice to be used as a reference molecule to track the progression of cancer. In addition, understanding of different roles of COX-2 in tumour formation and progression can assist in tackling the issue of mortality from breast cancer. For metastasis to manifest itself, development of new blood cells, lysis of matrix and motility are needed.

Many studies showed that COX-2 is particularly expressed in different types of cancers in human including colon, rectal, breast and prostate. Experiments carried out by scientists also proved that COX-2 is highly expressed in oestrogen independent breast cancer cell lines. Namely these cell lines consist of MDA- MB 231, Hs578T and 12, 0-tetradecanoyl- phorbol-13-acetate (TPA) whereas COX-2 is not expressed in poorly and oestrogen dependant cell lines such as MCF7. Ristamaki et al (2002) verified that high expression of COX-2 in breast cancers is linked with a large tumour size, high tumour grade, negative oestrogen status and the likely course of cancer.

Many of the critical steps in formation of invasive tumours are associated with COX-2 expression. These steps include cell proliferation, cell resistance to self-destruction, stimulating the development of new blood vessels, increasing cell movement and suppressing immunity. Prostaglandins (PG) and thromboxanes are the final products of COX-2 activity and may trigger cancer cell progression by causing these changes. Increased levels of PGs, significantly PGE2 have been noticed in breast cancer lines and in invasive breast cancer. To prove this theory, Gihooly et al used TPA to induce activity of COX-2 in breast cancers. The result was production of PGE2 which stimulated cell multiplication by elevating oestrogen levels and activating the aromatase enzyme.

Mechanisms of action of COX-2 inhibitors:

Bundred & Barnes (2005) pointed out three potential antitumor mechanisms;

1. inhibiting multiplication in epithelial cells

Studies by Boland et al (2004) showed that COX-2 expression and multiplication of epithelial cells are strongly correlated.

2. increasing apoptosis

COX-2 inhibition affects intrinsic mitochondrial pathway which increases apoptosis in the breast.

3. reducing angiogenesis

Celecoxib is a potent COX-2 inhibitor which indicates antiangiogenic properties. According to Woods et al (2003) celecoxib reduced secretion of vascular growth factor in vivo and endothelial tube formation in vitro. Also studies showed that COX-2 regulates new blood vessel production in normal mammary tissue by producing PGE2. Therefore, inhibiting COX-2 enzyme could have a potential to prevent breast cancer.

Studies on COX-2 inhibitors:

A recent study has suggested that in order for cancer cells to invade and migrate, cells must dissolve all extracellular matrix components (e.g. laminin, collagen, entacin, growth factors and cytokines) which requires matrix metalloproteinase (MMP) enzyme. MMPs are activated and expressed by overexpressed COX-2 in tumour cells. Studies by Sivula et al (2005) proved that increase in expression of COX-2 in breast cancer cells elevates the expression of MMPs.

In the study carried out by Larkins et al (2006), eight MMPs were screened in breast cancer cells while they were treated with and without COX-2 inhibitors. The results of clinical and laboratory based studies indicated that there is a reduction in incidence of breast cancer when COX-2 inhibitors (e.g. NSAIDS such as aspirin and indometacin) are administered. Therefore, COX-inhibitors allow us to further investigate the properties of COX-2 that have an effect on breast cancer development. Also it was proved that low concentrations of specific COX-2 inhibitors were adequate to minimise the proliferation, invasion and migration of COX-2 responsible for expression of breast cancer cells. Motility of a carcinogenic cell is one of the last steps of metastasis and it is important for a cell as it moves through the extracellular matrix and enters the systemic circulation to travel to another site. This study showed that the expression of NS-398 (a COX-2 inhibitor) caused a decline in the cell motility of the MDA-231 cells. This finding confirmed that COX-2 activity facilitates the movement of breast cancer cells across the membrane towards a chemoattractant.

As elevated levels of PGs, especially PGE2 is related to many different types of invasive cancers, it is important to prove that NS-398 down-regulates the production of PGs which results in malfunction of COX-2. PGE2 stimulates transcription of aromatase which leads to high concentrations of oestrogens. As a result of overexpressed COX-2, PGE2 levels increase and leads to oestrogen-dependant disease. Inhibition of COX-2 activity, reduces the endogenous PGE2 production levels by 75%. Therefore, inhibiting of PGE2 synthesis by COX-2 inhibitors may inhibit activity of aromatase enzyme. When COX-2 inhibitors are combined with aromatase inhibitors, they can inhibit a common target. A preclinical evidence confirms that administration of celecoxib combined with exemestane notably prevents the growth of mammary tumours. The antitumor effects of NSAIDs and selective COX-2 inhibitors may require other mechanisms than COX-2 inhibition (Mazhar et al 2005). For instance, Hwang et al (2002) suggested that NSAIDs and selective COX-2 inhibitors suppress the growth of cells in vitro that do not express COX-2 enzyme.

Epidemiology of NSAIDs use and breast cancer incidence

The evidence from cell lines and animal studies substantiates the idea that COX-2 may be involved in breast cancer development. Furthermore it suggests that COX-2 inhibition could play a role in prevention or treatment of the disease.

Two different types of studies carried out to find a possible link between COX-2 inhibitors and breast cancer. Firstly, by Gonzalez-Parez et al (2003) that showed a significant reduction in breast cancer incidence among those used aspirin and other NSAIDs. Secondly, by Harris et al (2003) which provided evidence of significant decline in risk in subjects used NSAIDs.

Similar evidence can be found from a study by the US Women's Health Initiative to support the view that NSAIDs reduce breast cancer risk factors. Results were as the following:

• 21% reduction in the risk for women used NSAID tablets regularly for 5-9 years
• 28% reduction for those used over 10 years
• The reduction was greater with Ibuprofen than with aspirin.
• For aspirin, frequency of use had stronger effects than duration of use.
• For ibuprofen, frequency and duration did not decrease risk factors.

According to Su et al (2006), 60% of premenopausal and 75% of postmenopausal breast cancers are developed via oestrogen-dependant pathways. As breast tumors require oestrogen hormone for their growth, they express high quantity of oestrogen receptors (ORs). Consequently, blocking oestrogen action by antagonising ORs or decreasing oestrogen production could be strategies to battle hormone-dependant breast cancer. This first strategy involves oestrogen antagonist molecules to compete with oestrogen for binding to ORs. The second approach uses aromatase inhibitors (AIs) that interfere in the final rate-limiting step of oestrogen biosynthesis. Researches have developed molecules that can selectively antagonise the oestrogen effects in breast tissue without inhibiting the functions of oestrogen in other tissues. These agents that show such a specific activity were termed selective oestrogen receptor modulators (SERMs). For example, tamoxifen is one the most widely used SERMs and has made a considerable contribution in reducing mortality rate since 1990.

Anastrozole is an aromatase inhibitor that significantly decreases plasma oestrogen levels. Aromatase inhibitors exhibit high clinical efficacy and favourable safety profile which make them more preferable alternatives to tamoxifen for the treatment of hormone-dependant breast cancer. However, AIs significantly inhibit aromatase enzyme which leads to a reduction in the bone density and therefore, osteoporosis develops.

As mentioned in the preceding paragraphs, COX enzyme product, PGE2 can induce expression of aromatase by stimulating adenylate cyclase in adipose cells. This biochemical mechanism may explain the benefits of using NSAIDs in the treatment of breast cancer. COX-2 inhibitors, N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide and N-(2-phenoxy-4-nitrophenyl)methanesulfonamide (nimesulide), suppress aromatase activity in breast cancer cell by inhibiting aromatase transcription. These two agents share very similar structure as shown (Figure 1). Interestingly, derivatives of N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide show COX-2 inhibition activity whereas derivatives of nimesulide exhibit no COX-2 inhibition.

Su et al (2006) carried out the synthesis of the target compounds as shown below, in which R represents benzyl moieties and X represents chloride or bromide.

The ability of synthesised compounds were tested and showed that the same concentration from each compound was required to exhibit aromatase suppression activity. The results confirm that there should be multiple pathways in which compounds are involved in to exert anti-aromatase activity. Also the results of cellular aromatase assays showed that adding one carbon at the 2-position of nimesulide, significantly increases the suppression of aromatase in breast cancers compared with nimesulide. Compound 4c showed the best IC50 value which is the most bulky compound. Overall, the results indicated that a few agents selectively inhibit aromatase activity and enzyme expression at very low concentrations in breast cancers. These agents are 10- to 80- fold more active than nimesulide. In addition, this suppression of aromatase activity takes place at the transcriptional level. In the study by Su et al (2006), several compounds were developed by modifying COX-2 inhibitor, nimesulide, which could be potentially used as selective aromatase expression regulators. Unfortunately, it is difficult to deduce an extensive structure-activity relationships for these compounds as their effectiveness is limited. However, these initial findings will lead to the discovery of novel drug in question.

Anticancer drugs containing platinum are commonly used in the treatment of human cancers. Many efforts have been made to discover new compounds that inhibit cell growth and division, however, with no great success. Only little is known cytostatic properties of elements such as copper, zinc or cobalt. Recently, there have been reports about cytotoxic cobalt-alkyne complexes. These complexes consist of six carbonyl groups, two cobalt elements attached to an alkyne ligand. Some of these complexes are found to be active against a variety of cancers especially breast cancer. There is only one lead compound among these complexes which is 2-acetoxy-(2-propynyl)benzoate]hexacarbonyl- dicobalt (Co-ASS). To further investigate the structural requirements of Co-ASS for high anti-tumor properties, the molecule was modified with different groups and atoms as shown below (Figure 1).

Subsequently, DNA binding studies were performed. Observations showed that the reduced form of glutathione is in the mM-range and the glutathione reductase system is upregulated in human breast cancer. Cobalt-containing drugs interfere with this cellular redox system. As Co-ASS is a derivative of NSAIDs, similar pharmacological properties are expected to be present in Co-ASS. Recently, COX enzymes have become targets in the treatment of cancer. Currently, a few NSAIDs are studied in clinics for tumor therapy and prophylaxis. As the most active compounds (Co-ASS, Co-SAL) are derivatives of the NSAIDs, it might be the fact that the NSAID-character of the compounds is necessary to inhibit malignant growth of cells. Co-ASS is a more effective COX-inhibitor than the parent compound aspirin. As mentioned above, the compounds formed by complexation of NSAIDs with copper show positive effects. They exhibit stronger or similar anti-inflammatory properties compared to NSAIDs. The cox inhibitory potency of the cobalt-alkyne complexes are strongly dependant on the alkyne chain. The order of COX-inhibitory activity (Co-ASS > Co-SAL > Co-Benz > Co-Prop) corresponds to their order of cytotoxicity. As a result, Co-ASS is the most cytotoxic drug among the COX-inhibiting anti-tumor drugs so far. It can be said that Co-ASS is derived from aspirin and propargylic alcohol after complexation with Co2(CO)6 which is a more potent COX-inhibitor and exhibits a higher cytotoxic activity.

Synthesis and Biological Evaluation of Selective Aromatase Expression Regulators in Breast Cancer Cells

Glaucoma is another condition in which COX-2 enzyme is invloved. COX-2 triggers the apoptotic death of neurons. Brust et al (2008) convincingly argued that COX-2 inhibition significantly prevented cells from apoptosis and reduced PGE2 concentrations. They also suggested that COX-2 inhibitors are more potent than non-selective or COX-1 inhibitors.

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