Today in the western world one of the leading causes of death is cancer(WHO Media centre, 2010). To start off with I will quote some facts. One in every three people worldwide will develop some form of cancer during their lifetime and in the UK one in every four people die due to cancer. In the year 2007 there were just under 300,000 new cases of cancer diagnosed in the UK alone
As health care professionals we would be very interested in finding ways to treat and prevent cancer, as this would lead to a longer and better quality of life for the patient.
Cancer is a name given when an abnormal cell grows out of control. The cell is said to be immortal as it does not follow the normal path of programmed cell death (apoptosis). Cancer usually start of in a particular area it can easily spread to neighbouring cells and to other organs throughout the body.
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Due to the facts mentioned it would be a great benefit to mankind to be able to treat, cure or even prevent cancer. Cancer itself is not caused by a single cause but is multifactorial. There are many theories as to what causes cancer but one that I am going to look into is the free radical theory of cancer.
Free Radical Theory
Free radicals are molecules or atoms that contain unpaired electrons and therefore are unstable and very reactive. There are many ways which free radicals can be produced in the body but most of them are produced as a by-product of respiration (Fang, 2002).
During respiration steps glycolysis, link reaction and the Krebs cycle, reduced NAD and FAD are produced. The reduced coenzymes pass through electron transport chains where they are oxidised and the by-products are hydrogen atoms. The hydrogen atom is then split to produce an electron and a proton. The proton goes onto help produce ATP (energy) which is the end required result of respiration(Baker, 2007).
After ATP has been produced the electrons are accepted by oxygen along with the protons to produce water. For every oxygen acceptor molecule (O), four electrons and four protons are required to produce two molecules of water. As shown below
However is not always the case. Sometimes the electrons leak prematurely onto the oxygen before the protons arrive, which produces free radicals such as superoxide, hydrogen peroxide and also hydroxyl radicals depending on the number of electrons that are leaked (as shown below).
There are many types of free radicals, but in the body the ones we are most interested in are superoxides, hydroxyl, peroxyl radicals and non radicals such as hydrogen peroxide (Yan, 2006). As the free radicals mentioned have oxygen at its centre they are known as reactive oxygen species (ROS).
Other than this free radical intermediate hydrogen peroxide is produced in the thyroid gland in order to produce thyroxine. Hydrogen peroxide acts as a catalyst in the reaction which involves attaching iodine to thyroglobulins.
Superoxide in the body is not always a bad thing as phagocytes produce superoxide's using NADPH Oxidase (enzyme), which is used to kill pathogens invading the body.
Oxidative stress can cause cancer (Gate, 1999). Oxidative stress can be categorised as endogenous and/or exogenous. We will first consider endogenous.
Free radicals are thought to cause cancer, inflammation, disease and old age but before I go into that I must explain the mechanism as to how they upset the natural order of the body.
As stated previously free radicals are highly unstable and reactive and they will try to stabilise as soon as possible by stealing an electron from somewhere. This could mean that that if a hydroxyl radical were to come in contact with a cell wall which is made from a phospholipids bi-layer it would try and steal and electron from it. As an electron is removed from the lipid cell wall the cell wall becomes the radical, and the image below shows how it eventually become lipid peroxidise, which would distort the original bilayer structure.
Lipid peroxide formation means the cell wall is damaged which leads to cell wall leakage and the end result of lipid peroxides may even be carcinogenic (Marnett, 1999)
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Another situation can occur when free radicals can damage the DNA itself (Aust, 1999) which may not necessarily kill the cell but the free radical interacting with DNA may alter the DNA replication or cause mutations and produce abnormal proteins which in turn causes cancer (Dreher, 1996) (Valco, 2004) (Nakab, 2006).
Mutations themselves can be substitution, addition or deletion, and can result in inactivation of tumour suppressor genes (Mates, 2000).
Free radicals are normally at a state of homeostasis in the body when produced moderately. The free radicals produces are used for many physiological processes as mentioned before and also for vascular tone (NO) - (Dr, 2002).
Exogenous oxidative stress can arise from many external environmental causes such as smoking, pollution, ionising radiation and UV light etc.
Cigerettes contain tar which contains free radicals such as quinine and hydroquinone. The free radicals in the tar are capable of reducing oxygen to produce superoxide leading to hydroxyl radicals and we already know what they are capable of doing in the body (Church, 1985).
Pollution in the atmosphere has broken down the ozone layer which has allowed harmful UV light to come through. UV light hitting the skin can produce free radicals such as hydroxyl which cause DNA damage leading to cancer (Autier, 1995)
Ionising radiation directly act on the body such as alpha (from the inside), beta and gamma (from the outside) radiation. They directly ionise the atoms in the body producing free radicals, which can eventually cause cancer.
Inflammation is also a known to produce free radicals under oxidative stress. Oxidative stress can activate transcription factors which lead to an increase in gene expression of certain cytokines. This may later cause chronic inflammation which based on observational data is known to cause cancer (Reuter, 2010).
The diagram below summarised free radical production naturally and via oxidative stress.
Antioxidant Activity Test
As discussed previously antioxidants work as free radical scavengers. In order to compare different antioxidant and to see which works better we must have means to compare them.
There are a number of ways in which we can measure the capacity of antioxidants, they include Oxygen Radical Absorbance Capacity (ORAC), 2,2-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. The most commonly used method is using the DPPH assay (Sharma and Bhat, 2009)
DPPH is a stable free radical with an unpaired electron on the outer shell of the nitrogen atom. Antioxidants can be used to try and scavenge the free radicals and the results can be found using one of two methods. The first method is by using of electron paramagnetic resonance (EPR) which measures the spin of atoms, the second is by measuring optical absorbance of DPPH between 515nm - 520nm (mostly around 517nm) UV Spectroscopy. The One I will concentrate on is the UV Spectroscopy method.
I will now briefly discuss how DPPH is produced for use.
DPPH comes in crystal form, and a stock solution is produced by mixing DPPH into methanol and buffer The DPPH solution produced is deep violet in colour, but when the free radicals become neutralised it becomes pale yellow in colour. As we can see the colour changes that occur with DPPH that tells us it occurs within the visible spectrum which is between 390nm and 750nm (oxygen dependence), so if we ran a scan of DPPH using this spectrum it should give a peak absorbance at around 517nm. The reason we measure absorbance is because from it we are finding out the change in concentration as per the Beer Lambert Law which is absorbance is directly proportional to concentration. This makes sense when we add the antioxidant to DPPH it start reducing the free radical concentration which will be shown by a reduction in absorbance. Now the better the antioxidant and its ability to scavenge free radicals the lower the absorbance will become. To prepare the antioxidant it is dissolved in the same medium as DPPH (methanol and buffer) on molar basis with DPPH, but is made more concentrated (normally 10 times the strength of DPPH solution). This is done so we can add the minimum volume of antioxidant to the DPPH solution so the DPPH solution does not become dilute as this would reduce the absorbance, hence the concentration, before we even start the experiment which would skewer our results (Sharma, 2009).
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Now that we are able to tell by what extent different antioxidant reduce DPPH we need to be able to compare them against each other and against what other people have produced. This is done by using a reference standard, to which all antioxidant activity can be compared to and in the field the standard that is used is Trolox (soluble vitamin E Derivative). The difference between the standard absorption of DPPH and with Trolox is compared to the differences produced by other antioxidant compounds, and the better the antioxidant the higher the numerical value of difference, relative to Trolox (Re, 1999).
Other things to note about DPPH is that although it is a stable free radical it can be affected by light and heat so care must be taken that it is stored in a cold and dark environment such as a freezer. Due to the methanol in DPPH it shouldn't freeze but should stay perfectly preserved giving accurate results every time. DPPH is also affected by pH that is why we add buffer to the DPPH stock solution, to ensure stable pH (Sharma, 2009).
When free radicals are normally produced, natural antioxidant enzymes such as superoxide dismutase and glutathione peroxidise catalase mop them up in the body. This leads to a homeostatic balance in the body. Now when the balance is overturned and free radicals are produced at a faster rate than they can be eliminated due to oxidative stress, we get excess free radicals being produced in the body. Below is a picture of how natural antioxidants eliminate free radicals in the body.
From the above picture we can tell that antioxidants scavenge free radicals. As discussed previously excess free radicals can cause cancer, so if we could eliminate and neutralise the excess free radicals in the body it would be a move back towards the homeostasis of free radicals in the body.
Glutathione is sometimes called the 'master antioxidant' in the body. The reason for this is that in most cases it is glutathione that reduces the major free radicals produced in the body. Glutathione has other roles other than just an antioxidant.
Its other roles include possible repairing DNA which may have been damaged when it came into contact with free radicals (Pujari, 2009).
It makes the immune system stronger by increasing the amount of T cells and natural killer cells produced (Chang, 2002)
It regulates the nitric oxide cycle (Ha, 1999)
Through direct conjugation it detoxifies xenobiotics and carcinogens (Wadleigh, 1988).
Our body also produces other natural antioxidants such as Ubiquinol which is a natural lipid soluble antioxidant, made in most cells (Jezek, 2005). Blood plasma contains uric acid, and uric acid is known as a potent reducing agent and antioxidant (Maxwell, 1997)
Other than our own naturally produced antioxidants we can gain a lot of antioxidants by having the right diet. By eating the right plants we can gain antioxidants such as vitamin C and E.
One of the main groups of natural antioxidants are the flavonoids. Flavonoids are potent antioxidants found in food such as plants and vegetables and also in bewerages such as wine, beer and tea (Ishige, 2001).
As explained previously flavonoid is an umbrella term but underneath it there are many different phenolic compounds which are categorised by their structure which include flavones, flavonols, flavanones, isoflavones, anthocyanidins, catechins and chalcones. Other than flavonoids antioxidant affects, other benefits of flavonoids include antiviral, anti-allergic, anti-platelet, anti-inflammatory, and antitumor activities (Buhler, 2000).
Flavonoids differ in structure to each other because differences in structure result in differences in activity. A popular structural change done to flavonoids to change its activity is changing the number and position of hydroxy or methoxyl groups. It is the hydroxyl or methoxyl group that provide the electrons for antioxidant effects but more doesn't necessarily mean better. Research has shown that by attaching 3 hydroxy groups to flavonoid it results in an increase in activity while if 5 or 7 hydroxyl groups are added it increases cytotoxicity. As for methoxyl it has shown to yield good results with 3 or 5 groups but shown cytotoxicity with 6 groups attached (Jeong, 2007)(Heim, 2002)(Rice-Evans, 1996).
There are other potential mechanisms by which flavonoids show potential for cancer management other than just as antioxidants
Anti oestrogenic mechanisms - by competitively inhibiting the oestrogen receptor (Burt, 2001).
Anti proliferation mechanism - this is done by inducing apoptosis of cancer cell and stopping the cell cycle and growth of the cell by working at the chekpoints G2/M and G1/S (Simoni, 2006) (Lee, 2004) (Burt, 2001).
As stated previously by detoxification, Immune responce and DNA repair
From the different type of flavonoids the ones I am most interested in are the stilbene's and the chalcones.
Stilbenes such as Resveratrol have been used in animal trials, already and showed a lot of promise. In animal testing where it showed supplementation of Resveratrol were given it prevented mice from getting skin cancer when carcinogens were introduced into them (Jang, 1997). On another occasion when Resveratrol given to mice orally at a dose of 1mg/kg it reduced the size and number of the tumours which had formed in the oesophagus (Li, 2002). Another study looking into Resveratrol and its derivatives and showed there effects on cancer cells (HL60 leukaemia). The study concluded that a certain derivative they called 6b induced apoptosis, and it looked promising for future research (Simoni, 2006).
The second flavonoid group of flavonoids I will look at are chalcones.
Chalcones are flavonoids that lack a C ring. There has been research done into chalcones but not a great deal, but new interest has been shown to these compounds as the potential benefits of chalcones are coming to light.
A study was conducted where chalcone sydnone-substitution took place. The resulting chalcones were found to be cytotoxic to tumour cells and reduced the activity and size of the tumours. Some of the chalcones were also found to inhibit lipid peroxide and were able to mop up free radicals such as superoxides and hydroxyl radicals. The results also showed an interesting discovery that the ability of the derivatives to suppress tumour growth was not dependent on just the antioxidant activity, which would mean that there are potentially other mechanisms at work other than just free radical moping (Ruby, 1994).
A different study was done into chalcones and there antioxidant properties. The results showed that the synthetic chalcones they produced were moderately good antioxidants but were very good at inhibiting hydroperoxide (Gacche, 2008).
More recently a relatively new compound Garcinol has been isolated. It is a very potent antioxidant and very potent anticancer compound. It has also shown to inhibit NF-kB which is a protein that controls transcription in DNA. NF-kB is known to be quite active in cancer cells and is known as a potential site of action when it comes to controlling cancer, as suppression of NF-kB is shown to reduce proliferation of cancer cells. NF-kB levels increase under oxidative stress, so by mopping up free radicals in the nuclear area Garcinol has shown to be a very interesting therapeutic drug (Padhye, 2009).
Cancer today is one of the biggest killers. We know that antioxidants are known to suppress tumour growth, and based on past studies that have already been conducted, we know that more research is needs to be done in this area as the potential prospects look promising. Due to this I will be further researching the antioxidant affects of different chalcones, as I believe they have the potential to save millions of lives.