The overall issue being raised in the paper is to investigate the upregulation of the phosphoinositide-3-kinase/Akt pathways to see if it displays similar characteristics in local breast cancer(LBC) recurrence as it does in recurrent colorectal tumours.(Myles J. Smith et al) A comparison between primary and recurrent breast cancer 4T1 tumours which are dangerous in the fact that they can grow at an accelerated rate, due to the ability of PI3-k to transform certain mammalian cells to uncontrolled growth(Lodish,2008) They can then be compared by a process of gene expression which when carried out can identify there mechanisms and way in which they operate. The main aim of this experiment is in order to see if there any biological therapies besides chemoherapy such as vaccinations that are capable of targeting survival pathways such as the PI3K/Akt pathway which is known to increase chemotherapeutic drug and hormonal resistance in breast cancer(McCubrey et all,2008) and be used as future treatment for breast cancer in order to ensure survival rates of patients, which have not changed significantly over the years unfortunately despite modern researching technologies..(F.Macdonald and C.H.J Ford)
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Many breast cancers develop in women aged 30 years plus and often arise either in the upper outer part of the breast with 76% of all tumours belonging to the invasive ductal breast cancers group. (F. Macdonald and C.H.J Ford,1997) It is very common in developed countries and accounts for 20% of all cancers in these countries (F.Macdonald and C.H.J Ford,1997) and according to statistics affects on in every 12 of the female population (Rubens,1992) while displaying rapid growth in females rather than males.(King,1996) Cancer of the breast is the most important malignant tumour in women; however the cause for it is regrettably unknown. At first, the tumour cells grow entirely within the ducts of the breast. This is similar for most cancers as they are most common in epithelial cells with initiation being very difficult to define(King,1996) it is a layer of cells which line these small ducts branching from the nipple that give rise to almost all breast cancers known. Many hormones are known to influence the division of the lining cells especially estrogens and hence stimulating cell growth which is experienced at a rapid rate in breast cancer tumours (Snell,Richard S,1992) along with the altered rate combined with resistance to apoptosis which is the death of many cells is due to the upregulation of the PI3/Akt pathway which signals to cancer cells. The PI3K/Akt pathway interacts with several genes and receptors involved in metal-induced carcinogenesis, including VEGF, HIF-1, P53, and src, influencing cell survival, proliferation, and perhaps even migration to other nearby tissues. (Leonard et al,2004) This signal causes cells to display unusual characteristics but the PI3K-Akt pathway provides an attractive target for therapies based on small molecule inhibitors.(Alexandre Arcaro et al,2007) The PI3-K pathway is known to be activated by the protein kinase B also known in this case as Akt. PI3-K is activated by the platelet-derived growth factor receptor (PDGFR) which also in turn inhibits PDGFBB. (Arcaro and Guerreiro,2007) The cancer is operable once no signs of LBC is not displayed in the patient. (Rubens,1992) Many pathways are also believed to compliment one another or to activate another pathway like a cascade of signalling according to (Vestey et all,2005) On the other hand the Akt (serine/threonine kinase) is well known for causing resistance to drug and hormonal therapies as they may be activated within minutes but have a long lasting effect..
Many techniques have been created and designed to aid the identification of different cancer genes such as databases like FatiGO1 which is a functional profiling onto-tool for genomic data. (Al-Shahrour at al, 2007) and qRT-PCR. Western Blotting plays a big role as it assists in the detection of proteins and glycoprotein's which are proteins of interest in Breast Cancer research and is used as a basic protocol as suggested by (Adrian,2005). Many current techniques which have been solely used for research purposes have now also found a place for practical clinical use in recent years.(De Cecco,2009)
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One of the biggest problems with conservation is relapse which is has a higher risk of happening to younger women, however after breast conservation many women do happen to remain free from breast recurrence after surgery with only 3% experiencing relapse after surgery.(Rubens,1992) As the PI3K-Akt pathway undergoes drastic alterations it makes it difficult to treat it as a result.( Arcaro and Guerreiro,2007) Other pathways such as Ras co-operates with the PI3K-Akt pathway as they are both activated by polyoma middle T oncoprotein (PyMT) allowing a tumour to maintain its malignant properties powerfully. (Pylayeva et al,2009) As treatments include radiotherapy, chemotherapy and hormonal therapy. Many treatments 20-30 years ago focused on the removal of tissue by surgery but newer treatments are being sought after strongly while many antibodies and drugs are being tested to block signal pathways to cancer cells.(Snell,1992) Breast cancer can be treated safely but risk needs to be taken into account also when discussing suitable treatment. Conservation techniques happen to be more favourable for operable tumours which are <4 cm or less in diameter (Rubens,1992) Patients then with larger tumours who wish to have breast conserving surgery may get chemotherapy in order to reduce tumour size.(Rubens,1992) Biological therapies seem to becoming a very popular means of treatment as the 'use of biological materials such as cells or cell products that will either effect the tumour proliferation and differentiation directly or enhance the biological response to the disease.'(Perren & Selby,1992) The vaccination tested in this experiment is a good example of this as it is loaded with dendritic cells (DC's) which themselves 'are specialized antigen presenting cells that acquire, process, and present tumour-associated antigens to T-cells' and in turn induce tumour stability.(Gottfried et al, 2008)The interesting thing is how they are so effective in stimulating the response to T-cells when used as a component of the vaccination despite being functionally defective in cancer tumours leading to immunodeficiency responses within the cell.(Gervaiswhilst et all,2005) also proving to have anti-tumour responses in-vivo as tested by(Shilling et al,2007)
All results obtained were done so under a controlled environment in order to succeed in obtaining accurate results. Animal models were used although being an expensive form of testing but an easy way of gathering vital information. Many techniques such as gene analysis and experiments such as western blotting was carried out in order to confirm the conformational changes in the P13K/Akt pathway and to examine how effective an activated HSPPC loaded with DC's would have on the growth of a recurrent 4T1 tumour.
4T1 Recurrent Tumours versus Primary Tumours
Mice were used as models for this experiment as they are suitable in the way that they display similar responses to that of humans when examining tumour growth but also proving to be expensive for lab research also. As expected, recurrent 4T1 tumours when inoculated grew significantly faster in volume than primary tumours. This also supports the idea that recurrent dynamics does not depend on the recurrent site itself or occur at this site specifically.(Demicheli et al,2008) The growth of all these tumour cells were examined over a period of 18 days with the recurrent tumour cells growing twice the size of both primary and control tumours who's growth remains ~230mm3 compared to that of the tumour ~470mm3.
The gene expression can reveal a lot of information about genes which are either underexpressed or overexpressed in 4T1 tumours which allow us to identify what exactly occurs in the tumour as it progresses and also allows us to understand its behavioral patterns. This way of profiling is also a great way of predicting a local recurrence after breast-conserving therapy. By using profiles with known gene expressions it allows to predict LBC recurrence. (Nuytenet al,2006)
From the results table of overexpressed genes it's evident that there is a notable high fold change in Granzymes (Gzm). This is due to the fact that Granzymes play a key role in cell death (possibly three different pathways). They are released by a distinct process of granule exocytosis once specific cells have been targeted and so they are eliminated by cytoxic granules knows as cytotoxic-T lymphocytes and other natural killer cells i.e. NK cells (Chowdhury and Lieberman,2008) Granzymes such as GzmB were believed to be only ever expressed by NK and CTL's but thanks to further research it has been discovered that Granzymes B are found in human dendritic cells also. (Dipanjan Chowdhury and Judy Lieberman,2008) This is relevant to this experiment as granzyme B is grown by gene duplications to encode other multiple granzymes for example, GzmD, E, F, G, L and N,with G,D,E and F all showing a significant fold change. GzmB is known to be expressed in both normal and as well as abnormal mast cells both in vitro and in vivo and is secreted upon activation as it is in cytoxic T-cells (CTL) which make them easy enough to measure as a result.( Chowdhury and Lieberman,2008)
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In the table of underexpressed genes it's noticed that DC's inflammatory protein 1 changes in fold by 4.4 which would support the idea that Granzymes down-regulates the activity of DC's.
Gene expression microarray with quantitive real time PCR
The graph shows the fold change of specific genes whilst not presenting any suprises for increased fold change but attention is drawn to the negative fold change of Platelet derived growth factor (PDGFBB) this result is linked to that of the neuroactive ligand-receptor interaction and it's low impact factor which is due to PDGFBB not being expressed as PDGFR is clearly known for inhibiting its actions in the PI3K-Akt pathway.(LI et al,1994)
FatiGO analysis was also used as a technique as it is an interaction of data with microarray experiments combined and is a very valid and important online tool in terms of gene expression research.(Al-Shahrour et al,2007) It would of been anticipated that signal transduction and cell growth would have been upregulated at a higher percentage as well as the rapid cell growth.
PI3K/Akt Pathway is one of the major pathways unregulated in recurrent 4T1 tumours with an impact factor of 28.5 being at least 6 times the impact factor of the neuroactive ligand-receptor interaction has. The neuroactive ligand-receptor interaction is linked to the fact that PDGFBB can be partially blocked by PDGFR making it unable to carry out its duties and allow for something as simple as co-expression to occur.(Chayeb et al,2006) This could be largely due to the fact that AKt induces many cellular responses such a pro-apoptic proteins by phosphorylation whilst PI3-k is activated, it goes to the plasma membrane and acts as a docking site for several signal-transducing proteins so they can it can be as fast acting as possible when in terms of communication and develpoment.
From the table of results it's clear that the activity of Cyotoxic T lymphocyte(CTL) cells is increased while the activity of the DC's is decreased, as represented by the fold change. This supports the idea that DC function is suppressed by the PI3K-Akt pathway and how CTL cells are increased dramatically due to activation. It could be due to the fact that they are closely linked to Granzymes such as (G,D,E,F and G). This supports the idea of disorder and apoptosis that is expected to be occurring during tumour growth. It also explains how DC's are under expressed ie. being down-regulated. There appears to be no balance between either cell's and makes it impossible for DC's to take action as antigen presenting cell's due to granzmyes being produced in large quantities.
The main purpose of western blotting is to gain a greater understanding of how cell signaling occurs exactly and to increase our knowledge on the topic of gene expression as well as to monitor post-translocational modifications.
In the first slide of results it presents the Beta-actin in both primary and recurrent tumours, being more dominant in primary tumours than recurrent tumour sample. This demontstrates how the cells ability to carry out mechanical procedures such as phagocytosis is more possible in primary than recurrent tumours. The AKT and phospho-AKT results are placed purposely alongside each other to demonstrate the significance of the results. In both samples, it is evident that neither AKT nor phospho-Akt is equally expressed in either primary or recurrent tumours. The fact that the bands are darker for phospho-Akt and lighter for Akt in recurrent tumours allows one to believe that a dramatic change has occurred in the cell cycle pathway. The dark bands presented in the third slide prove the upregulation of phospho-Akt due to something such as stress activation eg. Heat shock. The second slide presents the levels of Akt expressed is generally much higher in primary tumours while phosphor-Akt is expressed at greater levels in recurrent tumours. The slide truly represents that the activation of Akt influences cell cycle progression through dysregulation of the cell cycle checkpoint, hence the reason why Akt is more so expressed in primary tumours than recurrent ones but its expression in early recurrenct tumours is detectable due to the light band present in the second slide.(Vestey et all,2005)
HSPCC loaded DC vaccination
The last graph summarizes the impact of the vaccination used in this experiment raises a number of interesting questions. Firstly it shows how with just surgery alone that tumour growth volume of 4T1 tumours rose to ~450mm3 in a period of 14 to 16 days, whilst when surgery was accompanied with a vaccination a dramatic growth decrease was noticed as within the same period it only grew to ~100 mm3. the most. This confirms also how tumour growth leads to inactivated DC's leading to immunsuppression, the graph supports this hypothesis being proposed in the paper and showing the possibility of a purpose for this form of biological therapy in the future. The result gained from surgery and the vaccination is an indication of the stimulatory function DC has and suggests DC do have anti-tumour immune response against T-cells.(Gottfried et all,2007)
This paper is a novel paper as it highlights many fresh and original ideas for the near future in terms of treating LBC recurrence. All necessary approaches were taken when it came to exploring tumour growth patterns in ways to narrow down specific pathways involved. By doing so it allows to determine what the target would be and what pathway would be targeted exactly. The vaccination applied, which has been proven to be anti-tumour in previous experiments, in this experiment had a high impact on tumour growth by decreasing it drastically hence, showing its effectiveness. This new development is great in terms of biological therapies which are being greatly sought after as an alernative means of therapy in the modern world of science. As already discussed in the introduction chemotherapy is widely used as a means to reduce tumour size in local breast cancer patients in order for the tumour to be operable, the vaccination tested in this experiment could be another option perhaps. It is over the last decade or so that novel modalities of therapy have appeared on the scene and seem to create a great deal of interest. The over-all result is satisfactory which allows further research to be carried out on this solid foundation of results. The likes of this therapy needs to be compared to previous forms of therapy such as chemotherapy to allow to see if this form of therapy can be developed further and to test for the likely hood of relapse occurring after its application in patients etc. This discovery is exciting as biological treatments have proved to have been successful to date such as DNA recombinant cytokines which have established a place in the management of some human cancers according to (Perren and Selby,1992).
The overall result from all steps carried out in the experiment, display a comprehensible clear results and hat is,it is certain that DC supression and CTL activation combined with the upregulation of P13K-Akt pathway which will allow for further potential studies to be used to develop new therapies for cancer patients in the near future.