You are hereCYTOTOXIC EFFECT OF TAXOL AND VITAMIN E SUCCINATE COMBINATION IN COMPARISON WITH TAXOL ALONE ON MCF-7 BREAST CANCER CELL CULTURE BY MTT ASSAY

CYTOTOXIC EFFECT OF TAXOL AND VITAMIN E SUCCINATE COMBINATION IN COMPARISON WITH TAXOL ALONE ON MCF-7 BREAST CANCER CELL CULTURE BY MTT ASSAY


About Author:
Brijesh Borad,
The University of London Metropolitan University,
London, UK
borad_brijesh@yahoo.com

Abstract :-
Breast cancer is widely spreaded type of cancer in women and responsible for high number of cancer death in women. Taxol has been already approved to be used for breast cancer by FDA. There have been several studies on the anti tumor activities of Vitamin E Succinate (VES) as complementary and alternative medicine. In present study, we investigated the cytotoxic effect of taxol-VES combination on MCF-7 breast cancer cell lines in comparison with taxol alone. MCF-7 cells are preffered because of higher sensitivity to estogens. MCF-7 cells were sub cultured in according to specifications of aseptic conditions by incubating at 37οC and 5%CO2. Cells were seeded in 24 well plate with culture medium and different concentration of different drug regimen ( taxol alone, VES alone, taxol-VES combination) for 72hrs. Cell viability can be checked by MTT cell viability assay. MTT assay uses a MTT dye which acts as a substrate for viable cell reductase enzyme. This enzyme reduces MTT yellow dye to purple colour formazan which is in proportional to viable cell eventually. The intensity of purple colour was measured by measuring absorbances at 570nM using spectrophotometer. The results of the study were plotted as %cell viability Vs concentration for each of three drug regimen. IC50 values for each drugs were calculated. Graphical and statistical analysis of results concluded that taxol-MCF combination has more inhibitory effect on MCF-7 breast cancer cell lines when treated for 72hrs in comparison with taxol alone and statistical analysis had concluded that results were significant enough to accept clinically to use in stratagic breast cancer therapy management.

Reference Id: PHARMATUTOR-ART-1268

1. Introduction
Breast cancer
is the second leading cause of cancer death exceeded only by lung cancer in women(Weiss M, 2008(a)).Breast cancer is an uncontrolled growth of breast cells. It is a result of mutations, or abnormal changes, in the genes responsible for healthy growth regulation of each cells. These changed cells gain the ability to keep dividing without control or order, producing more cells just like it and forming a tumour (Breast cancer research UK, 2009). A tumour can be benign (not dangerous to health) or malignant (has the potential to be dangerous).  Malignant tumours are cancerous and if they left unchecked, they spread throughout to body   (Wikipedia, 2010). Usually breast cancer either begins in the cells of the lobules which produces milk, or the ducts, the passage that passes milk from the lobules to the nipple (Merk Manual, 2010). Breast cancer is being screened by mammogram technology. Routine mamographic screening is an accepted standard for early detection of breast cancer detection (Weiss M, 2008(b)). The study of large groups of related individuals led to the identification of several breast cancer susceptibility genes, including BRCA1, BRCA2. Mutations in these genes account for all hereditary breast cancers (NCI, 2009). Now-a-days, there are many treatment options available for breast cancer like Surgery ( Lumpectomy, Mastectomy ) Radiation Therapy, Hormonal Therapy ( aromatase inhibitors, selective eastrogen receptor modulators, and estrogen receptor down regulators), targeted therapies (trastuzumab-monoclonal antibody), and at last but most widely used is chemotherapy and combination therapy of all of above (Breast cancer UK, 2009(b))Chemotherapy treatment uses medicine to weaken and destroy cancer cells in the body. Chemotherapy is used to treat early-stage invasive breast cancer to get rid of any cancer cells that may be left behind after surgery and advanced-stage breast cancer. In many cases, a combination of two or more medicines will be used as chemotherapy treatment for breast cancer. These Combinations are known as chemotherapy regimens. There is a list of drugs that are being used in chemotherapy. Standard chemotherapy regimens include Adriamycin and taxol (AT), Cytoxan, methotrexate and fluorouracil (CMF), and Flurouracil,adriamycin and cytoxan etc (Breast cancer.org, 2009) but out from these standard regimens the combination of my interest is Taxol and Vitamin-E succinate which I will discuss in later part of this study.

Paclitaxel (Taxol) is obtained from dried bark of plant Taxus brevifolia (Pacific yew tree) which is mound mostly in N.America, china, and Europe (21cecpharm, 2008) The twig of which is shown in figure 1.

( Figure1:- Twig of Plant of Taxus brevifolia ) ( Wikipedia paclitaxel, 2010)

Taxol is in top of all anticancer drugs in the battle of we humans against cancer as it constitutes about 22 percentage of all major cancer chemotherapy drugs on the world market (chemocare, 2005). It is approved by the FDA to treat ovarian camcer in 1992 , breast cancer in 1994, and AIDS-related Kaposi sarcoma(National Cancer Institute, 2008). It is best to be used together with drug called cisplatin to treat advanced ovarian cancer and breast cancer. BMS (Bristol Mayers Squibb) is the pioneer for the development of taxol to its clinical use. BMS and Ivax have made billions of dollars with taxol. (National Cancer Institute,2008).  It is clear colourless fluid that is given as as a chemotherapy infusion in early stage and metastatic breast cancer as a single or multiple drug regimen. Invivo and invitro study shows that B-subunit of microtubule heterodimer act as a target site for paclitaxel. Microtubules are a kind of cell skeleton and make up the organs of movement. However,when cell division is about to take place microtubules depolymerise back to tubulin and repolymerise to form the spindle of cell division. This spindle formation is necessary process for a cell to divide into two. In 1979, susan horwitz showed that taxol stimulates the formation of microtubules and prevents their depolymerization (Jordan M and Wilson L., April 2004) which is shown in figure2. 

(Figure-2 Mechanism of action of Taxol, as it binds with microtubule it prevents its depolymerization)  (Jordan M and Wilson L., April 2004)

So, it interrupts spindle formation process and eventually cell division process at the G2/M phase (Jordan M and Wilson L., april 2004)(Kumar N, 1981).  Thus, it acts as an anticancer agent. The adverse drug reactions associated with taxol are peripheral neuropathy, mucositis, alopecia, neutropenia, cardiotoxicity, diarrhoea, and hypotension, reduction in WBC (Wikipedia paclitaxel, 2010). Paclitaxel undergoes transesterification in methanol, and it hydrolyzes in aqueous solutions. It is available as a white powder melting point of it is 213ºC. It is solubilize in eathanol and DMSO. The storage temperature is 2-8ºC. The price for paclitaxel powder in UK is 23.50 GBP for 1MG, 74GBP for 5MG and 255GBP for 25MG (Sigma Aldrich, 2010(a)). So, it is quite expensive drug. This thing ( Quite expensive) leads me to think that it should be used with other cheaper anticancer drug with different mechanism of action and with different pharmacological properties. There is a lot research study is going on with different forms of vitamin-E for its anticancer property. So, I think why not to go in deep with vitamin-E. Eventually, I found Vitamin-E succinate as second drug in combination with taxol for my study.

Vitamin E succinate (VES, α-TOS,RRR-α-tocopherol succinate) is the most potent antitumour analogue of vitamin E  which is currently being charachterized for its chemopreventive and chemotherapeutic potential. Many groups have showed that VES inhibits cancer cell growth. The mechanism by which VES inhibits cell growth is not completely understood however it has been reported that this occurs by inducing apoptosis in cancer cells by mitochondrial destabilisation which is caused, most likely, by the detergent-like activity of VES. VES comprises a hydrophobic phytyl chain( hydrophobic domain) and the chargeable succinyl group (functional domain), separated by the bulky tocopheryl moiety(signalling domain). Due to tocopheryl moiety, the agent activates protein phosphatase-2A that, in turn, inhibits protein kinase C. This leads to hypophosphorylation of the antiapoptic protein bcl-2,with ensuing mitochondrial labilisation. The other mechanisms for inhibition of cell growth by VES includes DNA synthesis arrest, inhibition of angiogenesis, suppression of nuclear factor-kappa B activation, induced secretion and activation of transforming growth factor (TGF)-β. In addition, VES selectively damages only tumor cells without any toxic effects on normal cells or tissues For the last two decades, several studies have confirmed this observation in human breast cancer cell lines like MCF-7 cells. VES also increases the growth inhibitory effect of ionizing radiation therapy, biological modifiers and especially some chemotherapeutic agents ( may be Taxol!!!) on tumour cells, while protecting normal cells against some of their adverse reactions (Newzil J, 2003) (Albanes D and Heinonen OP et al 1996). The price for VES in UK is 21GBP for 100mg (Sigma Aldrich, 2010(b) ) (quite cheaper then taxol ).

MCF-7 is a breast cancer cell line ( as shown in figure 3 as a photograph) isolated from a 69 year old Caucasian women.

(figure 3 (A) Photograph of MCF-7 breast cancer cell lines ) ( MCF-7 cells, 2009)

(figure 3(B) photograph of MCF-7 cells taken from this research experiment from inverted microscope of science centre, London Metropolitan University)

It is the shortform of Michigan Cancer Foundation, an organization in Detroit which is the pioneer for these cell lines. Herbert soule and co-workers of this organization has established this MCF-7 cell lines in 1973. Before to this event, there was no any mammary cell line that can survive more than few months. This invention of MCF-7 cells is the source of much of current knowledge about breast cancer. MCF-7 cell line is the first hormone-responsive breast cancer cell line. It has differential sensitivities to estrogens and anti-estrogens, differential expression of estrogens receptors and progesterone receptor, and differences in tumorigenicity and proliferation rates. Clinical studies have shown that increase in estrogens activity is associated with breast cancer. The addition of extradiol which is one of the fractions of estrogen to the medium of MCF-7 cells induces a proliferative response. The characteristics of MCF-7 cells like the estradiol-dependence for growth and low metastatic potential has led to the assumption that they represent an early adrenocarcinoma of breast. Thus,MCF-7 cell line are perfect model to study the progress of malignant cells as they can be subjected to appropriate endocrinologic and physiologic selective pressures for the derivation of variants with more progressed phenotypes. MCF-7 cells have ability to go through DNA fragmentation. There are apoptotic responses of MCF-7 cell line to the apoptosis inducing agents like tumour necrosis factor and anti-Fas antibody. The MCF-7 cells are also an excellent in vitro model for studying the mechanisms of chemo-resistance because of its susceptibility to apoptosis. In short, MCF-7 cell line is another useful cell line that can be used in various cancer research especially in breast cancer research. (MCF-7 cells, 2009)

Taxol and vitamin E succinatecombination was successfully tasted and reported previously for their inducing apoptosis and chemosensitivity in human bladder cancer cell lines and the result found was quite significant.(Kunimitsu K and Eiji K et al.,March 2009).  As described earlier, taxol is quite expensive drug to be used clinically and breast cancer is widely diagnosed type of cancer amongst all in women. This certain matters make me stronger to study the cytotoxic effects of taxol-VES combination in breast cancer with the use of MCF-7 breast cancer cell lines and MTT assay.

Eventually, the aim of my study is to come out with a more cost and clinically effective drug regimen. Hypothesis for my study is “ To compare cytotoxic effects of  taxol-VES combination regimen with taxol alone in MCF-7 breast cancer cell culture by MTT assay method.” So, in the out come of this study I can report if taxol-VES combination has significant difference as compared with taxol alone or not.

The method to check my hypothesis is MTT assay with the use of MCF-7 cells. MTT assay uses a MTT dye as a substrate of viable cell reductase enzyme. Cytotoxicity is measured by measuring absorbances of converted dye and measuring IC50 for each drug or drug combination. Thus, viability of cancer cells can be measured. Then comparative drug study is based on statistical analysis which is described in later part of this study.

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