INVESTIGATION OF OXYRESVERATROL CYTOTOXICITY

Oxyresveratrol (OXY) belongs to the group of phytochemicals known as hydroxystilbenoids, and has a molecular structure similar to the well-known phytochemical resveratrol. It is found to be particularly concentrated in the heartwood of Artocarpus lakoocha Wall. ex Roxb. (Family: Moraceae), an indigenous plant in Thailand. This species has been used as a Thai traditional medicine for the treatment of various parasitic diseases. Previous studies have also reported that it may exhibit pro-oxidative properties, which associated with the amount of the main compound in the extracts. OXY may share biological activities with resveratrol and be developed as an anticancer agent. This thesis aimed to investigate the cytotoxicity of OXY, consequently. The heartwood was ground, dried and extracted. The chromatographic and spectroscopic analyses were carried out and OXY was identified as the major component in the extracts (33.55%, 37.81% and 64.23 % w/w in water, ethanol and ethyl acetate, respectively). Resveratrol was quantified as a minor compound, (0.37%, 0.45% and 0.65% w/w in water, ethanol and ethyl acetate extracts, respectively). The pro-oxidant activity was investigated using DNA–nick, reactive oxygen species (ROS), copper reducing and glutathione depletion assays. The results showed that OXY induced DNA damage dose-dependently in the presence of copper (II) ions. It was also found to generate ROS in a dose-dependent manner, reduce copper (II) to copper (I) and depleting glutathione. OXY was able to show higher capability than resveratrol at the same doses, while the trans–stilbene did not show the activities. The three A. lakoocha extracts also showed damaged DNA consistent with the amount of OXY presented. The cytotoxicity of OXY to different kinds of cancer cell lines including Human Caucasian lung carcinoma cells (A549), human colorectal cancer cells (CACO–2), Human Caucasian hepatocyte carcinoma cells (HepG2), Human breast cancer cells (MCF7), Caucasian prostate adenocarcinoma cells (PC–3), Mouse macrophage cells (RAW 264.7) and the two non-transformed cell lines including Human foetal lung cells (MRC–5) and normal human breast cells (MCF10A), was investigated using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). Cells were either pre-treated with 50µM Cu(OAc)2 for 24 h, or OXY in the medium supplemented with 50µM Cu(OAc)2 then exposed to OXY for 48 h compared to the control with no Cu(OAc)2. More than 80% of the cells treated with OXY in the presence of copper showed less toxicity than the condition without copper. MCF7 cell was the most susceptible to the compound and therefore chosen to be a model studying molecular effects. The cells that OXY caused toxicity (IC50) to were MCF7 (30.64±4.79 µM), HepG2 (104.47±0.82 µM), PC–3 (106.90±8.63 µM), RAW 264.7 (115.95±11.28 µM) and A549 (148.63±4.48 µM). Whereas, the compound was not toxic to non-transformed cells (MRC–5 and MCF10A). Moreover, OXY and OXY–copper (II) did not increase intracellular ROS and damage the DNA in MCF7 at the concentration investigated. MCF7 was chosen to be a cell line model for gene expression investigation. The gene-level expression of more than 20,000 human well-annotated genes was determined using Clariom Affymetrix microarray under two different OXY treatments (50 μM and 100 μM) for 24 h. A total of 686 genes were found to have altered mRNA expression levels of two-fold or more in the 50 μM OXY-treated group (262 upregulated and 424 downregulated genes). Total 2,338 genes were differentially expressed in the 100 µM-treated group (907 upregulated and 1,431 downregulated genes). The relevant visualized global expression patterns of genes and pathways were generated; genes involved in cell cycle control, apoptosis and autophagy, as well as DNA repair, showed the greatest differences in expression relative to controls. Gene expression was validated by quantitative PCR and western blot protein analysis. OXY induced apoptosis in MCF7 and HepG2 cells. The cascade events were activated through extrinsic and intrinsic pathways through various genes including caspase 3. The final stage was activated resulting in losing cell membrane integrity, losing mitochondrial membrane potential, chromatin condensation and eventually cell death. However, OXY did not cause TP53 activation, ROS generation and severe cellular DNA breakage. OXY also down-regulated genes controlling cell migration and metastasis, especially genes in the non–canonical pathway (SMAD–independent) such as PI3K/AKT, CXCR4 and its ligand (CXCL12) which may consequently affect cell growth, proliferation and cell survival. Interestingly, OXY inhibited the gene expression in the cancer DNA repair pathway; the most significantly affected were RAD51 (P < 0.0001) and genes in homologous recombination. These results indicated that OXY could overcome drug resistance, enhance the efficacy of chemotherapy drugs and could be developed as chemotherapy or chemosensitising agent