Choon Nam Ong

Dual Role of 3-Methyladenine in Modulation of Autophagy via Different Temporal Patterns of Inhibition on Class I and III Phosphoinositide 3-Kinase

A group of phosphoinositide 3-kinase (PI3K) inhibitors, such as 3-methyladenine (3-MA) and wortmannin, have been widely used as autophagy inhibitors based on their inhibitory effect on class III PI3K activity, which is known to be essential for induction of autophagy. In this study, we systematically examined and compared the effects of these two inhibitors on autophagy under both nutrient-rich and deprivation conditions. To our surprise, 3-MA is found to promote autophagy flux when treated under nutrient-rich conditions with a prolonged period of treatment, whereas it is still capable of suppressing starvation-induced autophagy. We first observed that there are marked increases of the autophagic markers in cells treated with 3-MA in full medium for a prolonged period of time (up to 9 h). Second, we provide convincing evidence that the increase of autophagic markers is the result of enhanced autophagic flux, not due to suppression of maturation of autophagosomes or lysosomal function. More importantly, we found that the autophagy promotion activity of 3-MA is due to its differential temporal effects on class I and class III PI3K; 3-MA blocks class I PI3K persistently, whereas its suppressive effect on class III PI3K is transient. Because 3-MA has been widely used as an autophagy inhibitor in the literature, understanding the dual role of 3-MA in autophagy thus suggests that caution should be exercised in the application of 3-MA in autophagy study.

AFM indentation study of breast cancer cells.

Mechanical properties of individual living cells are known to be closely related to the health and function of the human body. Here, atomic force microscopy (AFM) indentation using a micro-sized spherical probe was carried out to characterize the elasticity of benign (MCF-10A) and cancerous (MCF-7) human breast epithelial cells. AFM imaging and confocal fluorescence imaging were also used to investigate their corresponding sub-membrane cytoskeletal structures. Malignant (MCF-7) breast cells were found to have an apparent Youngs modulus significantly lower (1.4-1.8 times) than that of their non-malignant (MCF-10A) counterparts at physiological temperature (37 degrees C), and their apparent Youngs modulus increase with loading rate. Both confocal and AFM images showed a significant difference in the organization of their sub-membrane actin structures which directly contribute to their difference in cell elasticity. This change may have facilitated easy migration and invasion of malignant cells during metastasis.

Autophagy and oxidative stress associated with gold nanoparticles.

Elemental metal nanoparticles like cadmium and silver are known to cause oxidative stress and are also highly toxic. Yet for gold nanoparticles (AuNPs), it is not well established whether these particles are biologically toxic. Here we show that AuNPs, which were taken up by MRC-5 human lung fibroblasts in vitro, induce autophagy concomitant with oxidative stress. We also observed formation of autophagosomes together with the uptake of AuNPs in the lung fibroblasts as well as upregulation of autophagy proteins, microtubule-associated protein 1 light chain 3 (MAP-LC3) and autophagy gene 7 (ATG 7) in treated samples. AuNP treated cells also generated significantly more lipid hydroperoxides (p-value<0.05), a positive indication of lipid peroxidation. Verification with western blot analysis for malondialdehyde (MDA) protein adducts confirmed the presence of oxidative damage. In addition, AuNP treatment also induced upregulation of antioxidants, stress response genes and protein expression. Exposure to AuNPs is a potential source of oxidative stress in human lung fibroblasts and autophagy may be a cellular defence mechanism against oxidative stress toxicity.

Biodistribution of gold nanoparticles and gene expression changes in the liver and spleen after intravenous administration in rats

Biodistribution of gold nanoparticles (AuNPs) in more than 25 organs were examined on 1 day, 1 week, 1 month and 2 months after a single intravenous (i.v.) injection in rats. Au was rapidly and consistently accumulated in liver (49.4+/-50.4-72.2+/-40.5 ng/g) and spleen (8.4+/-5.0-9.5+/-6.4 ng/g) throughout the entire timeframe of the study (2 months). Significant accumulation of Au in kidney (up to 5.5+/-2.5 ng/g) and testis (up to 0.6+/-0.1 ng/g) occurred from 1 month post-injection when Au level in urine and feces decreased. Significant increase of Au in blood occurred 2 months after injection, coincident with the delayed accumulation in kidney. Au accumulation in lungs was found at 1 day post-injection but decreased within a week. No accumulation of Au was found in the brain. Microarray results of liver and spleen point to significant effects on genes related to detoxification, lipid metabolism, cell cycle, defense response, and circadian rhythm. These results demonstrate that significant biodistribution of Au occurs in the body over 2 months after a single i.v. injection of AuNPs, accompanied by gene expression changes in target organs.

Microdevice for the isolation and enumeration of cancer cells from blood

Cancer metastasis is the main attribute to cancer-related deaths. Furthermore, clinical reports have shown a strong correlation between the disease development and number of circulating tumor cells (CTCs) in the peripheral blood of cancer patients. Here, we present a label-free microdevice capable of isolating cancer cells from whole blood via their distinctively different physical properties such as deformability and size. The isolation efficiency is at least 80% for tests performed on breast and colon cancer cells. Viable isolated cells are also obtained which may give further insights to the understanding of the metastatic process. Contrasting with conventional biochemical techniques, the uniqueness of this microdevice lies in the mechanistic and efficient means of isolating viable cancer cells in blood. The microdevice has the potential to be used for routine monitoring of cancer development and cancer therapy in a clinical setting.

Anti-cancer potential of sesquiterpene lactones: bioactivity and molecular mechanisms.

Sesquiterpene lactones (SLs) are the active constituents of a variety of medicinal plants used in traditional medicine for the treatment of inflammatory diseases. In recent years, the anti-cancer property of various SLs has attracted a great deal of interest and extensive research work has been carried out to characterize the anti-cancer activity, the molecular mechanisms, and the potential chemopreventive and chemotherapeutic application of SLs. In this review, we attempt to summarize the current knowledge of the anti-cancer properties of SLs by focusing on the following important issues. First, we discuss the structure-activity relationship of SLs. All SLs contain a common functional structure, an alpha-methylene-gamma-lactone group, and this important chemical characteristic means that the thiol-reactivity of SLs is an underlying mechanism responsible for their bioactivities. Second, we assess the experimental evidence for the anti-cancer function of SLs obtained from both in vitro cell culture and in vivo animal models. Various SLs have been demonstrated to execute their anti-cancer capability via inhibition of inflammatory responses, prevention of metastasis and induction of apoptosis. Thirdly, we outline the molecular mechanisms involved in the anti-cancer activity of SLs, in particular, the SL-thiols reaction, the effect of SLs on cell signaling pathways such as nuclear transcription factor-kappaB (NF-kappaB) and mitogen-activated protein kinases (MAPK). Finally, we recapitulate some important SLs with regards to their anti-cancer activities and their potential in anti-cancer drug development. Taken together, many SLs are emerging as promising anti-cancer agents with potential applications in both cancer chemotherapy and chemoprevention.

Role of oxidative stress and mitochondrial changes in cyanobacteria‐induced apoptosis and hepatotoxicity

Microcystins produced by cyanobacteria are potent and specific hepatotoxins; however, the mechanisms of microcystin-induced hepatotoxicity have not been fully elucidated. The induction of free radical formation and mitochondrial alterations are two major events found in microcystin-treated cultured rat hepatocytes. The mitochondrial alterations, i.e. loss of mitochondrial membrane potential and mitochondria permeability transition are now recognized as key steps in apoptosis. The activation of calpain and Ca(2+)/calmodulin-dependent protein kinase II is believed to be critical in the microcystin-induced apoptotic process.

DETECTION OF OXIDATIVE DNA DAMAGE IN HUMAN SPERM AND ITS ASSOCIATION WITH SPERM FUNCTION AND MALE INFERTILITY

The expanding research interest in the last two decades on reactive oxygen species (ROS), oxidative stress, and male infertility has led to the development of various techniques for evaluating oxidative DNA damage in human spermatozoa. Measurement of 8-hydroxydeoxyguanosine (8-OHdG) offers a specific and quantitative biomarker on the extent of oxidative DNA damage caused by ROS in human sperm. The close correlations of 8-OHdG level with male fertility, sperm function and routine seminal parameters indicate the potential diagnostic value of this technique in clinical applications. On the other hand, single cell gel electrophoresis (SCGE or comet assay) and terminal deoxynucleotidyl transferase (TdT) mediated dUTP nick end labeling (TUNEL) assay have also been demonstrated to be sensitive, and reliable methods for measuring DNA strand breaks in human spermatozoa. As certain technical limitations were inherent in each of these tests, it is believed that a combination of these assays will offer more comprehensive information for a better understanding of oxidative DNA damage and its biological significance in sperm function and male infertility.

Deformability study of breast cancer cells using microfluidics

Cell deformability is an important biomarker which can be used to distinguish between healthy and diseased cells. In this study, microfluidics is used to probe the biorheological behaviour of breast cancer cells in an attempt to develop a method to distinguish between non-malignant and malignant cells. A microfabricated fluidic channel design consisting of a straight channel and two reservoirs was used to study the biorheological behaviour of benign breast epithelial cells (MCF-10A) and non-metastatic tumor breast cells (MCF-7). Quantitative parameters such as entry time (time taken for the cell to squeeze into the microchannel) and transit velocity (speed of the cell flowing through the microchannel) were defined and measured from these studies. Our results demonstrated that a simple microfluidic device can be used to distinguish the difference in stiffness between benign and cancerous breast cells. This work lays the foundation for the development of potential microfluidic devices which can subsequently be used in the detection of cancer cells.

Sodium selenite‐induced oxidative stress and apoptosis in human hepatoma HepG2 cells

The mechanisms involved in the anti‐carcinogenic activity of selenium remain to be elucidated. In the present study, we examined sodium selenite‐induced oxidative stress and apoptosis in a human hepatoma cell line (HepG2). Sodium selenite (10 μM) exerted clear cytotoxic effect, as shown by the significant increase of lactate dehydrogenase leakage. Selenite‐induced DNA alterations in apoptosis were studied by: 1. comet assay; 2. TdT‐mediated dUTP nick end‐labeling assay. In addition, characteristic apoptotic morphological alterations were also observed in selenite‐treated cells. Our results clearly show that Se‐induced cell death occurs predominantly in the form of apoptosis. Selenite‐induced oxidative stress was evaluated by the measurement of reactive oxygen species production using lucigenin‐dependent chemiluminescence. The involvement of glutathione in selenite‐induced oxidative stress was further demonstrated by the concurrent decline of intracellular reduced glutathione and increase of oxidized glutathione contents in Se‐treated cells. Moreover, the finding that selenite‐induced oxidative stress and apoptosis was significantly attenuated by superoxide dismutase, catalase and deferoxamine provides additional evidence to suggest that Se‐induced oxidative stress mediates the induction of apoptosis, a mechanism related to the anti‐carcinogenic and chemopreventive effect of Se. Int. J. Cancer 81:820–828, 1999.