Cancer is a major cause of morbidity and mortality worldwide. In recent biomedical researches, the areas of cancer and infectious diseases have a leading position in utilization of medicinal plants as a source of drug discovery. Malaysia has a diversity and large quantity of underutilized fruits which are rich in phenolic compounds. The objective of study one was projected in vitro to explore natural sources of antioxidant in Artocarpus.altilis (breadfruit) extracts and antioxidant properties. The total phenolic content (TPC) was measured using the Folin-Ciocalteau method and the total flavonoid content (TFC) was determined by using aluminium chloride colorimetric method. Antioxidant properties were determined via the DPPH radical scavenging and β–carotene bleaching (βCB) assays. The various fruits parts Pulp (PU), peel (PE) and whole fruit (WF) were extracted with various solvents such as hexane, dichloromethane (DCM) and methanol. The methanol extracts obtained the highest yields among other solvents (hexane and DCM). The pulp (edible portion) had the highest yield (p<0.01). Methanol extract of pulp part revealed the highest total phenol and flavonoid content value of 781±17.32 mg (GAE)/g and 6213.33±82.24 mg (QE)/g of dry sample, respectively. IC50 values of methanol extract of pulp part in DPPH radical were obtained to be 0.05±0.00 mg/mL as compared to positive control (ascorbic acid) 0.06±0.00 and the antioxidant activity for the β-carotene bleaching assay was 88.34±0.75% of methanol extract of pulp part as compared to the positive control (Trolox) 90.02±0.87%. The objective of study two was to identify and quantify some phenolic compounds in the methanol extracts. By using the ultra high-performance liquid chromatography-tandem mass spectrometry (UHPLC/MSMS) based approach, a total of 9 compounds were detected and characterized on the basis of their chromatographic retention time, UV-vis spectra and mass spectra in the negative-ion mode and data from the literature. The results of the various parts of A. altilis fruit extracts showed promising antioxidant and potential bioactivities due to the high content of phenolic compounds. The purpose of the study three was to evaluate the cytotoxicity effects of methanol fruit extracts on four human cancer (A375, MCF-7, A549, and HT-29) cell lines. The IC50 of the samples were measured using trypan blue exclusion assay (TBEA). The methanol extract of pulp part showed the least inhibition concentration of 15.40±0.91 μg/mL on A375 cells. In the study four, the molecular mechanism of methanol extracts-induced apoptosis and cell cycle arrested in human cancer cells were investigated in a time dependent approach by using flow cytometry. The treated cells were stained with nexin to detect early and late apoptosis and with PI for cell cycle arrest associated with the DNA fragmentation, various cells arrests were occurred at G1/S, S and G2/M phases. Lastly the gene expression analysis by reverse transcription quantitative PCR (qPCR) method was carried out by analysing the expression of gene of interest for quantification of mRNA levels. Results after cells treated with IC50 were revealed by upregulating of anti-apoptotic genes/downregulated of pro-apoptotic BCL-2 gene expressions were triggered the treated cells into CASPASE-3, intrinsic and extrinsic pathways. These findings suggest that the methanol extracts of three parts of A. altilis fruit have potential chemotherapeutic activity against human cancer cell lines mainly the pulp part of the fruit.

The invasiveness of malignant melanoma is mainly attributed to the enzymatic destruction of the extracellular matrix and basement membrane components by a group of enzymes known as matrix metalloproteinases (MMPs). The expression of these proteinases is mainly regulated at the transcriptional level; therefore, high expression of MMPs is manly attributed to different transcription factors which enhance or inhibit the promoter activity of MMPs genes. Among these factors, YB-1 and CTCF proteins are transcription factors in which CTCF is mainly a tumour suppressor protein; while YB-1 is an oncogenic factor and a prognostic indicator in a wide range of tumours that regulates most of the cancer processes such as proliferation, invasion and metastasis by regulating the expression of genes related to those processes. However; the expression of these transcription factors and their potential effect on the expression of collagenases MMPs in malignant melanoma cells are not yet confirmed. Therefore, this study was conducted to determine the expression of collagenases MMPs (MMP1, MMP8 and MMP13), YB-1 and CTCF transcription factors in A375 melanoma cancer cells. In addition, the stromal effect of normal skin fibroblasts on the expression of collagenases and proliferation in A375 cell was determined. The results of this experiment demonstrated an increase in the expression of YB-1, MMP8 and MMP13 in A375 cells. Thereafter, this was followed by the establishment of YB-1 silenced strain of A375 cells using a silencing short hairpin RNA (shRNA) construct. The effect of YB-1 knockdown on the expression of collagenases MMPs was determined using reverse transcription PCR and Western blotting. In addition, the antiproliferative effect was determined using flow-cytometry, colorimetric MTT assay and cell counting; while the anti-invasive properties were determined using wound healing assay. The results of this experiment elucidated that YB-1 protein regulates the expression of MMP13, cell cycle progression, cell proliferation and cell migration of A375 cancer cells in vitro. Therefore, the direct interaction between YB-1 protein and the AP-1 promoter sequence of MMP13 was evaluated using Chromatin Immunoprecipitation assay (ChIP). The ChIP analysis has confirmed no interaction between YB-1 protein and the AP-1 promoter sequence. Finally, these experiments demonstrated YB-1, MMP8 and MMP13 were highly expressed in the A375 cancer cells. The stromal cells were found to promote A375 cell proliferation and enhance the expression of MMP1. In addition, YB-1 silencing was significantly associated with reduced expression of MMP13 enzyme, reduction in cancer cell proliferation in a cell cycle specific manner and anti-invasive properties. Therefore, YB-1, MMP13 and stromal cells are considered as promising elements that might help as a potential target in the treatment of melanoma tumour due to their roles in the processes of invasion, migration and proliferation. Further experiments are needed to demonstrate novel protein partners of YB-1 and novel binding sites within gene promoter region of MMP13 with determination of the involved signalling pathways.