Theses (MSc. Molecular Medicine)

Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignancies and its recurrence, delayed diagnosis and resistance to treatments contribute to its poor survival outcomes. Epigenetic modifications such as DNA methylation significantly contribute to tumour heterogeneity, metastasis, and disease progression of PDAC. Additionally, inflammation is one of the major implications of PDAC with the nuclear factor kappa B (NF-κB) being acknowledged as its key mediator. In recent years, multiple evidence have suggested that inhibitor of kappa B kinase alpha (IKKα) plays a central role in the initiation and progression of PDAC through transcriptional activity that enhances the NF-κB-mediated gene expressions. However, there has been lack of evidence correlating the role of IKKα and its epigenetic changes in PDAC. Aim: To determine the epigenetic changes associated with IKKα in a PDAC cell line. To identify gene pathways that are regulated by IKKα in the PDAC cell line. Methodology: Methylome-wide analysis was conducted on two cell lines – the parental PANC1 cell line PANC1_WT and PANC1_33 with the IKKα knockout. Briefly, DNA extracted from both cell lines were subjected to bisulfite conversion and DNA methylation using the Illumina Infinium MethylationEPIC BeadChip array was performed to identify the differentially methylated sites between both cell lines. Pathway enrichment analysis was conducted using the identified differentially methylated regions using EnrichR to gather information on the signaling pathways and genes with altered patterns of DNA methylation in the IKKα knockout cells relative to the parental cell lines. The top 5 most significant hypomethylated and hypermethylated regions were enriched for genes involved in ERBB2 regulation. qRT-PCR was performed to confirm our methylation data and determine the ERBB2 expression in the PDAC cell line. Other confirmatory tests such as in vitro cell assays comprising of cell proliferation and cell migration were also performed to further study the characteristics of the cell lines. Statistical analysis using the EnrichR and Student’s t-test value were performed using the mean and standard deviation values. Findings were deemed statistically significant if p<0.05. Results: Genomic regions that were hypermethylated in the IKKα knockout cells relative to the parental cell lines were significantly enriched for genes involved in ERBB2-related signaling pathways such as the PTK6-ERK/MAPK, and PI3K events. The qRT-PCR confirmed the downregulation of ERBB2 expression in the IKKα knockout cell line. A decrease in the cell proliferation rate was also observed when compared to the wildtype. There were no statistically significant results obtained from the hypomethylated regions and cell migration assay. Conclusion: Perturbations to IKKα expression result in epigenetic alterations in PDAC cell line via the NF-κB dependent and/or independent pathways. Subset genes such as NRG1, NRG3, ERBB4, and EGFR were also identified to be regulated by IKKα. Key words: IKKα, NF-κB, epigenetics, DNA methylation, PDAC

Breast cancer cells often respond poorly to treatment because of their different subtypes such as low expression of the oestrogen, progesterone and receptor tyrosine-protein kinase erbB-2. Hence, there is a need to identify new molecules for the treatment. Radix Bupleurix contains glucosides, a class of oleanane derivatives called saikosaponins that have been widely used in Traditional Chinese Medicine for over a millennium. There are two main active components in Radix Bupleurix, Saikosaponin A (SSA) and Saikosaponin D (SSD), known to have a wide range of pharmacological actions. This study identifies the anti-cancer effect of SSA and SSD on breast cancer cell lines, MDA-MB-468, MCF7 and T47D. Our findings suggested that SSA and SSD can inhibit the growth of the breast cancer cell line in a dose-response-dependent manner. The IC50 for SSA and SSD ranges between 4.48 μM to 6.97 μM. SSA and SSD can inhibit cell migration and colony formation. Scratch assay results showed a significant area of closure compared to control (p < 0.05) after 120 hours. Furthermore, a combination assay showed no synergistic effect between SSA and SSD. In conclusion, these results suggest that SSA and SSD could be potential molecules for the treatment of breast cancer but further study is required to understand the pathway involved in the anti-cancer effect.

Background: Since disease states are often detected early before any symptoms are evident, the emergence and growing number of molecular diagnostic testing plays a vital role in providing essential information to patients, clinicians and public health policy makers. Quality assurance is an overall quality programme and plays a pivotal role in molecular diagnostics to ensure accurate final test results through standardisation. Even though each laboratory has its own quality assurance process, we cannot determine how effective it is. This paper aims to review the role of quality assurance in molecular diagnostic laboratory. Methods: A systematic qualitative literature review was conducted in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines for evaluating publications relating to quality assurance in molecular diagnostic laboratories. Relevant studies in health-related electronic database such as PubMed, Ovid, EBSCOHOST and Scopus databases from January 2000 to March 2023. Data from included studies were then extracted and the papers’ quality were evaluated using criteria based on Critical Appraisal Skills Programme (CASP) checklist. Results: Fifty-seven articles which were published between 2000 to 2023 were included in this review. Most of the research articles were from cancer studies which has 32 papers. Fifty-one studies were of high quality while six studies were moderate quality. A variety of molecular diagnostic fields, including cancer, infectious diseases, rare inherited diseases, and genetic disorders, have been discussed, including guidelines for conducting molecular diagnostic tests, effectiveness, future challenges, trends, and programmes aimed at improving quality assurance. including policy makers, laboratory experts and scholars should focus on development of suitable quality assurance, addressing constraints, prioritising and ensuring compliance when given the opportunities to accomplish this. Hence, molecular diagnostics laboratories that perform molecular tests in various fields must strengthen and implement quality management.

Contact dermatitis (CD) is a common skin condition characterised by inflammation caused by exposure to allergens. In Malaysia, it accounted for a significant portion of skin diseases, with irritant contact dermatitis being the most prevalent type. Ophiocordyceps sinensis, a medicinal mushroom, has been extensively studied in various disease models and has multiple therapeutic properties in treating hyperlipidemia, arrhythmias and other disorders The objective of this study was to investigate the immunomodulatory effects of O. sinensis cultivar OCS02® on sensitized NCTC 2544 keratinocytes. An in vitro assay was conducted to evaluate the cytotoxicity, expression of cytokines, ROS, and lipid peroxidation in sensitised NCTC 2544 keratinocytes exposed to OCS02® cold water extract (CWE). The results revealed OCS02®CWE were less cytotoxic to keratinocytes than paraphenylenediamine (PPD) with half maximal inhibitory concentration (IC50) 650 μg/mL and 17.7 μg/mL respectively. OCS02® CWE (at 250 μg/mL) significant down-regulated the expression of reactive oxygen species, peroxidation of membrane lipid bilayer, interleukin (IL)-18 and IL-6 cytokines in PPD-sensitised keratinocytes (p<0.05). This study reveals that O. sinensis and its extracts possess anti-inflammatory and cytoprotective properties, suggesting their potential utility in managing inflammatory conditions. Furthermore, the immunomodulatory effects observed in cytokine production indicate their promising role in alleviating diseases characterized by chronic inflammation and immune dysfunction. In conclusion, OCS02® CWE may have immunomodulatory application for the treatment of CD.

Potential cardiotoxicity remained the crucial factor limiting the clinical use of doxorubicin-based chemotherapy, despite their effectiveness in various malignancies. Combination therapy involving doxorubicin has therefore emerged as a potential therapeutic option, in an effort to enhance anticancer efficiency at the same time minimise cardiovascular side effects. Epidermal growth factor receptor (EGFR) inhibitors are a group of tyrosine kinase small molecules inhibitor recently approved for clinical use in cancer therapy. The combination of doxorubicin and EGFR inhibitors has shown great promise by demonstrating enhanced anticancer effects and ability to reverse chemoresistance associated with doxorubicin. Although EGFR inhibitor alone displayed minimal cardiotoxicity, it has raised concern that concurrent use would potentiate doxorubicin-induced cardiotoxicity. This study therefore aimed to investigate possible synergistic cardiotoxic effect of doxorubicin and EGFR inhibition. By utilising human AC16 cardiomyocytes, we first assessed the EGFR inhibitors including erlotinib, gefitinib, afatinib and lapatinib, with concentration ranging from 1-100 μM for erlotinib, gefitinib and lapatinib whereas 1-10 μM for afatinib, which revealed that all EGFR inhibitors tested elicited cytotoxic responses towards cardiomyocytes at different potency levels. When combined with doxorubicin, only erlotinib was found to synergise doxorubicin-induced cardiotoxicity. Further cell apoptotic assay confirmed this synergism, suggesting that erlotinib may enhance apoptosis induced by doxorubicin. Intriguingly, this synergism was not observed in gefitinib, afatinib and lapatinib, which proposed that synergistic cardiotoxicity may be specific to each inhibitor instead of a drug-class effect. In summary, this study suggested that the clinical use of concurrent erlotinib and doxorubicin should be approached with caution due to the possibility of increased cardiotoxicity, and this warrants further mechanistic studies for understanding the precise underlying mechanisms and developing cardioprotective agents.