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The key purpose of this research is to investigate the effect of metformin, an anti-diabetic drug, on the receptor for activated C-kinase (RACK-1) production by human breast cancer cells. Recent studies have proposed that metformin, which is used to treat Type II diabetes, decreases the risk of tumour development. This project will investigate further into the anti-tumour effects of metformin on MCF-7 breast cancer cells. Investigation of the effect of metformin on RACK-1 production will be carried out because RACK-1 has been associated with tumour development. Breast cancer is caused after the normal mammary epithelial cells sustain enough genetic damage to obtain the phenotypes characteristic of malignancy which include unregulated proliferation, resistance of cell death, and metastasis (Sachdev and Yee, 2001).
Cao et al. (2009) investigated breast carcinoma cell lines that were transfected with RACK1 and reported that RACK-1 promotes breast carcinoma proliferation and invasion/metastasis in vitro and in vivo. Furthermore, some studies have suggested that RACK-1 is capable of binding and activating the oestrogen receptor in breast cancer cells, suggestive of a role of the protein in the development of oestrogen-dependent breast cancer.
For the ethical issues, this project will focus on the potential benefits of anti-diabetic drugs on human breast cancer. It has been proposed that metformin can influence cancer cells indirectly through insulin-mediated effects or can directly affect cell proliferation and apoptosis of the cancer cells (Goodwin 2008, Hadad et al., 2008). Evans et al. (2005) also observed that the risk of diagnosis of all types of cancer including breast cancer was reduced in patients with type II diabetes who were treated with metformin.
Cell culture of breast cancer cells - (MCF-7)
This practical will begin by establishing cell culture of MCF-7 cells. All cell cultures will be done in triplicate. Below are the outlines of the experiment to be done.
MCF-7 cells will be grown in the absence (control) and presence of metformin and will be incubated for six days.
After six days, the total RNA will be extracted. The RNA will then be quantified and tested for integrity. This will be done by the following ways:
Measure optimum density of 260nm and 280nm. Run and visualise agarose gel
Following RNA quantification, equal amount (100ngs) of RNA of the control and treated cells will be reverse transcribed using the reverse transcriptase and oligo-dT primers. (Oligo-dT primers will only bind to mRNA because of its poly (A) tail). cDNA copy will be made from the RNA and somewhere, there will be RACK1 present. Real time PCR will be applied with the use of β-actin primers as control.
The results will help to find out the likely protective role and benefits of metformin in breast cancer and to determine the effect of metformin on RACK-1 production in breast cancer cells.
In this project, cell lines are going to be used so we do not need any ethical approval for this. The cells have been provided by Aston University and are properly stored in the laboratories. Although there are no likely emergencies envisaged, university protocols will be followed .To carry on this research in the future, primary cells would be needed for the experiments and have to be obtained from patients. Ethical approval will be required since it involves taking samples from patients. In this case, the project will be properly explained to the patients in a nonprofessional's language and an informed consent form will be signed. Names of participants will not be disclosed. The patients will be required to sign a consent form, fill out a general health questionnaire and provide the samples needed. The outline summary of the design will include:
Baseline physiological measures (HR, BP)
Establishing cell culture in the laboratory
Molecular analysis for RT-PCR
Interpretation of data
The experimental design will be the same as above. On collection of samples from the subjects, they will be taken to the laboratory, preserved and stored in the freezer. Ethics forms and consent forms will be completed to ensure that this research is moral and the rules and regulations for using human subjects are up to standards. For safety purposes, gloves will be worn while collecting samples and spilled samples will be cleaned with antiseptic wipes. Attached below is a copy of the consent form that will be given to the subjects and to let them know that they can withdraw from this study at any point without questions. Criteria needed in the selection of subjects include:
Disease characteristics must meet one of the following criteria:
Primary breast disease histologically or cytologically proven breast cancer at any stage of the disease.
Benign breast disease
Ages qualified for study: 18 years and above
Genders qualified for study: female (menopausal status not specified)
Females must not be pregnant
This project is being conducted to study the possible benefits of metformin, an anti-diabetic drug, on the human breast cancer cells.There are studies suggesting that metformin can reduce the risk of breast cancer so this project will look into the effects of metformin on human breast cancer to find out if this anti-diabetic drug has an anti-tumour action too. In addition, the effect of metformin on RACK1, a protein found in the cell that has been implicated in tumour development, will be determined. RACK1 has been suggested to have a function in the transformation of normal cells to cancer cells and part of this study will look at the effect of metformin on RACK1 protein.
The samples provided will be used to study the potential benefit of metformin on human breast cancer cells.
Collection of samples: the doctors will take samples during a biospy or surgery. Samples will be stored properly and taken back to the laboratory for analysis.
As this experiment involves human subjects, a consent form must be obtained from the volunteers before samples can be collected. This experiment has been designed to keep risk to the lowest amount. You should be aware that you can withdraw from this project at ANYTIME and are under no obligation to carry out the entire study. You should also be aware of what is required of you before you start this project. Questions can be asked at any point if you do not feel fully informed.
You will be asked to fill out questionnaire regarding health in general as a proceeding form. Any details you provide will be strictly monitored and treated as confidential and your personal details will NOT be included in the report of this project. Data will be anonymised and will be accessed only by the supervisor and student. Samples will be stored for five years and will not be used for anything not regarding this study.
READ THE ABOVE INFORMATION CAREFULLY AND ASK ANY QUESTIONS. IF YOU WISH TO PARTICIPATE IN THIS STUDY, PLEASE SIGN THE CONSENT FORM BELOW.
I have read the appropriate information on the page above and fully understand what is needed and expected of me. By signing this form, I have understood the information contained in the above page and I am willing to take part in the study.