Decoding the Molecular Symphony: Olaparib and BRCA1 Interaction in Ovarian Cancer Cells

Introduction

Among women, the fifth most common cancer is ovarian cancer and it is also the leading death cause in the UK from gynaecological cancer. Yearly, more than 6,500 women receive an ovarian cancer diagnosis in the country with the disease being responsible for around 4,400 deaths. Seeking a healthcare dissertation help provide the most valuable insights into understanding and addressing all sorts of challenges posed by ovarian cancer in the UK.

DNA in every cell is constantly being damaged and repaired. BRCA1 (BReastCAncer 1) is an important DNA repair protein and tumour suppressor gene. Shortage of BRCA1 would actuate carcinogenesis. Mutation in BRCA1 leads to higher risks of ovarian and breast cancer development. (www.sciencedirect .com)

In this report, a chemotherapy drug: Olaparib is used as a PARP inhibitor. PARP is a DNA repair protein that allows the cancer cells to remain alive, to grow and divide. PARP inhibitor stops PARP from doing its DNA repair work.

The aim of the experiment is to investigate the impact of Olaparib on BRCA1gene expression from ovarian cancer cell lines using SNU-251 which has a BRCA1 mutation and SKVO3 cell lines which is BRCA1 wild-type. To acquire accurate and standard results mRNA quantitative methods are essential. The fold difference method enabled analyzing gene expression changes from real time quantitative PCR.

Method

The initial cell group was treated with Olaparib drug for 48hrs with the concentration of 5µM but the second cell group was not. PSB was used in washing cells and extraction of total RNA was done using the isolate II Mini Kit (Probiotek).

RLY lysis buffer was then used in lysing the cells. Extraction of RNA as well as treatment of DNAse followed guidance from the protocol of the manufacturer. The reverse -transcription of the RNA using reverse transcriptase was done to obtain cDNA samples. In order to compare and measure mRNA levels a sensitive method known as Reverse transcription -polymerase chain reaction is used (Author: A)

Quantitative Polymerase Chain Reaction (qPCR) analysis to amplify BRCA1 gene products from the cDNA samples (SNU-251 cells and SKOV3 cells) were performed.

Target DNA quantification for expression of BRCA1was done by detecting SYBR Green fluorescent dye.

The reaction is displayed below:

GAPDH was used as a housekeeping gene for normalization

The gene expression levels were quantified using the fold difference method.

Result

The result of the investigation of the expression of BRCA1 in ovarian cancer shows that the fold difference in BCRA1on SKVO3 cells and SNU-251 are both less than 1 (See table of results below). This means there is a decrease in the expression of BRCA1 when the two cells are treated with Olaparib. Therefore, Olaparib is reducing the expression of BRCA1. In other studies, the orally active inhibitor Olaparib has been seen to demonstrate anti-tumour activities in ovarian cancer and relapses.

However, the value of fold difference in BRCA1 on SKOV3 cell is 0.17, which is greater than the value on SNU-251 (0.10) . Therefore , the BRCA1 gene level on SKOV3 is higher than the BRCA1 level on SNU-251.In this report , the two different ovarian cancer behave differently as following Olaparib treatment on SKOV3 cells and on SNU251, there is a difference on the level of BRCA1 gene expression.

Table of results

Discussion and Conclusion

Chain reactions of Polymerase that are quantitative (qPCR) have become the standard method for measuring levels of gene expression (Boulter N et al 2016).Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which was at the beginning categorized as a glycolytic enzyme and associated with housekeeping genes, is utilized widely in protein experiments like DNA and mRNA as an internal control. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4383849/). GAPDH has also been implicated in various functions independent of the energy metabolism role with its expression status being deregulated in some cancer cells. The result obtained in this experiment shows that there is a decrease in BRCA1 expression when the two types of ovarian cancer cell lines are treated with Olaparib. Instructions for developing proteins that can undertake the work of suppressing tumours are provided by the BRCA1 gene. Proteins considered being tumour suppressors’ aid in the prevention of cells dividing or growing at exponential rates or in ways that cannot be controlled. They are also involved in monitoring cell proliferation through the regulation of the activities of various cellular genes that take part in cell cycle progression and growth. https://ghr.nlm.nih.gov/gene/BRCA1

Furthermore reduced expression can cause BRCA1 mutation. Increased sensitivity of mutation carriers can be reflected to the inhibitor polymerase (PARP) (Shi T et al 2017). That is why in this report the cancer cell line SNU-251 was more sensitive to Olaparib. This study has highlighted that PARP inhibition can be effective. In fact, strategies that are promising are made possible by PARP inhibitors, but the clinical efficacies as well as other many questions remain unanswered. An example is the lack of answers for the effects that are long-term regarding PARP inhibitor administration.

Overall, this new drug class of PARP inhibitors is exciting and it attracts tremendous attention while also showing to have potential that is great in developments in the future. The inhibitors are developed for various indications and this includes treating of cancers that are heritable. They are a type of targeted therapy which blocks enzymes in PARP cells therefore helping prevent cancer cells from repairing their damaged DNA.

SNU-251 and SKOV3 are all cancer cell lines. To meliorate the understanding of cancer cell biology, Cancer cell lines are extensively used for they deliver satisfactory model systems for study of mechanisms related with cancer development in biomedical research. Not only do cancer lines improve the understanding of cancer development but are also applied in the advancements and screening of novel anticancer drugs. Before use of cancer lines, careful consideration on should be given upon their accuracy by asking one question, how accurately do the cell lines garner or copy the cancer in vivo. Cell lines have usually been studied in vitro under conditions of cell cultures that are standard. Characterizing in vivo growth of different models has involved selecting cell lines that are representative of the various histological subtypes.

Nevertheless there is comparability between original tumours and cell lines regardless of their differences. For their similarity attributes, cell lines are given a higher consideration in representation of specific tumours which are displayed to valid experimental models. Due to the evidence of contamination with HeLa cells, it is crucial to work with authenticated cell lines.

Take a deeper dive into Lab Report Guidelines, UTI Diagnosis, and PDP in Biomedical Studies with our additional resources.