Laboratory of Biochemistry
Research Activities
Our research interests at the Laboratory of Biochemistry are to study diseases arising from abnormalities of proteins, both in terms of changes in their amounts or modifications of their structure which affect their ability to perform their function. Major research areas include:
This work has been published as numerous articles in international journals and researchers in the laboratory have received several awards, such as the Outstanding Scientist of Thailand Award and the Outstanding Researcher Award.
Cancer Biomarkers
Cancer is initiated by accumulated mutations and proliferation to form a tumor. Often cancers may be cured if diagnosis and treatment can be made at the early stages, but there are still few biomarkers that will distinguish cancer tissues from normal tissues or that are specific for a particular cancer. Our research group was the first in Thailand to use the technique of proteomics, where the proteome patterns (or total protein present at any tissue under any given condition) are compared, such as by two-dimensional electrophoresis. Initial studies, in collaboration with Phramongkutklao Hospital, showed that increased expression of proteins, cathepsin B and prohibitin, was observed in neoplastic thyroid diseases compared to non-neoplastic diseases, such as goiter or nodular hypoplasia, while high levels of expression of galectin-3 appeared to correlate with metastatic potential.
Since liver cancer is prevalent in North Thailand and bile duct cancer is prevalent in Northeast Thailand, extensive proteomic studies have been performed on hepatocellular carcinoma (HCC-S102, HepG2) and cholangiocarcinoma (HuCCA-1) cell lines. Comparison of proteins from these cell lines, suggest various potential biomarkers for distinguishing the two cancer types. Furthermore, study using patient tissues confirmed that the protein lipocalin 2 was detectable in all cancer tissues, but not in the normal tissues of cholangiocarcinoma patients, indicating its potential as a biomarker. Further studies on hepatocellular and cholangiocarcinoma cells focus on using three-dimensional culture techniques to mimic natural tumour growth and microenvironment, as well as studying subcellular fractions, such as secretomes, and improving culture conditions to increase protein yield for proteomics.
Another interest is the role of post-translational modification in cancer, particularly O-GlcNAc (O-linked N-acetylglucosamine), a single sugar molecule attachment occurring mainly on serine and threonine residues of cytoplasmic and nuclear proteins. This GlcNAc modification may act as a nutrient sensor through the hexosamine biosynthesis pathway, a minor branch of glycolysis, and may also affect cellular regulation, since it occurs on amino acids that may be phosphorylated. Study of O-GlcNAc modified proteins in breast and colon cancer tissues compared to adjacent normal tissues indicate increased GlcNAcylation in both cancers and indicated possible biomarkers. RNA interference of OGT, an enzyme catalyzing O-GlcNAcylation, decreased global O-GlcNAc levels and reduced colony formation of breast (MDA-MB-231 and MCF-7) and colon (HT29, SW480, and SW620) cancer cells, thus suggesting that O-GlcNAcylation plays important roles in cancer progression. Other studies on breast and colon cancer cells showed more GlcNAc modified proteins in the extracellular compartments of breast and colon cancer cells compared to their normal cells.
Our current interest has expanded to explore abnormality of extracellular glycoproteins from serum/plasma of cancer patients. Proteins in blood circulation system may contain some important biomolecules which reflect the pathological and physiological states of diseases including cancer. Aberrant O-linked and N-linked glycosylation of serum proteins has been examined in many types of cancer. Indeed, research on glycoproteomics, glycosylation patterns and site specific glycans on proteins are being applied to discover the heterogeneity of glycan structures on glycoproteins. Thus, we recently found that several glycoforms of serum glycoproteins were altered in patients with colorectal cancer. This indicates that abnormality of glycoforms on glycoproteins may provide a novel frontier for cancer biomarker discovery.∧Top
Anti-Cancer Agents and Tumor Microenvironment
As the tumor grows, it becomes hypoxic, inducing the tumor to form new blood vessels (angiogenesis) to provide oxygen and nutrients. Cancer cells can also acquire invasiveness, leading to metastasis or spread of cancer to other tissues, which makes treatment even more difficult. This can occur anytime, while cancer cells are in the body. Cancer cells may develop resistance to drugs, either before or during chemotherapeutic treatment. Two major problems of cancer therapy, namely metastasis and drug resistance, are associated with adaptation of cancer cells to the microenvironment, such as hypoxia, growth factors and cytokines, extracellular matrix changes, and loss of cell attachment. Research in our laboratory covers many of these problems and often uses different cell culture conditions to provide models of these problems and cellular processes, as well as to explore natural products or synthetic agents for development of novel anti-cancer drugs.
Anti-cancer Agents
Conventional chemotherapy can cause toxicity, and is typically administered for intermittent short periods. An alternative approach is to use non-toxic agents which may be used for long-term treatment to prevent or suppress cancer cell invasion. We have screened many plant extracts for non-toxic compounds, which inhibit invasion of cancer cells, since they may have potential as anti-metastatic drugs. Extracts of Helixanthera parasitica and Eclipta prostrata inhibited invasion and migration of cancer cells in vitro, but could not be further purified. Later, vanillin, the flavoring agent in vanilla, was found to inhibit invasion of 4T1 mouse breast cancer cells in vitro. Vanillin also reduced in vivo metastasis of the breast cancer cells in BALB/c mice, when orally administered for one month. Further study using human A549 lung cancer cells showed that vanillin disturbed signal transduction regulating invasion, by selectively inhibiting Akt phosphorylation, as well as inhibiting the enzymatic activity of phosphoinositide 3-kinase (PI3K). Metastatic growth requires VEGF to induce angiogenesis to support oxygen and nutrients. Another compound, chrysin, a flavonoid isolated from Thai propolis, also suppressed metastasis progression of breast cancer cells in mice. Chrysin retarded the growth of metastatic colonies of breast cancer in lungs by inhibiting hypoxia-induced STAT3 activation which is necessary for VEGF gene expression.
Photodynamic therapy: Photodynamic therapy (PDT) has emerged as an alternative chemotherapy that uses energy of visible light to trigger activation of photosensitizer for killing cancer cells. PDT uses a photosensitizer, which by itself has little or no cytotoxicity, but upon irradiation with light, generates reactive oxygen species (ROS) to destroy cells. The advantages of PDT compared to conventional therapies are the minimally invasive, local destruction of tumors, and repeatability for treatment of cancer recurrence. However, a limitation of PDT is the lower water solubility of almost photosensitizers. Therefore, polymeric nanoparticles have been used to improve delivery of the hydrophobic sensitizers to tumors. We showed that photosensitizer-encapsulated polymeric nanoparticles exhibited no cytotoxicity in the dark, but effectively killed HT-29 colon cancer cells and FTC-133 thyroid cancer cells in vitro when irradiated with light.
Drug Resistance
We have established two different models for studying the development of drug resistance in cancer cells. To mimic drug resistance during chemotherapy, a repeated drug treatment approach was used to establish a multidrug resistant lung cell line A549RT-eto, which exhibited 28-, 5-, and 3-fold higher IC50 for etoposide, doxorubicin, and paclitaxel, respectively, compared to parental A549 cells. Real-time PCR, Western blot analysis, and flow cytometry indicated that the cell line showed increased expression of a multidrug transporter, P-glycoprotein (P-gp/MDR1), which pumps out anti-cancer drugs. Interestingly, screening of synthetic compounds led to the discovery of three benzo[α]quinolizin-4-one derivatives, which decreased drug resistance.
Drug resistance is also frequently observed in advanced stage cancer patients, who have never previously received chemotherapy. We established a metastasis-associated drug resistance model by culturing H460 lung cancer cells as floating cells under non-adherent conditions that mimic floating of cancer cells during metastasis through blood or lymphatic circulations. The H460 floating cells rapidly acquired paclitaxel resistance, with 15-fold increase in IC50 for paclitaxel, with the underlying mechanism involving alteration of beta-tubulin isotype expression. Change in microenvironment: As the tumor grows, changes occur in tumor microenvironment, including growth factors, cytokines, and extracellular matrix, which can affect the behavior of cancer cells. We have shown that hepatocyte growth factor enhanced the aggressiveness of HepG2 liver cancer cells, so that they undergo “vasculogenic mimicry” (VM), whereby tumor cells can form blood channels in the absence of endothelial cells. The creation of VM capillaries in vitro occurs through the induction of a cellular change from epithelial phenotype to mesenchymal phenotype, known as the “epithelial-mesenchymal transition” (EMT). Aggressive tumor cells such as SK-Hep-1 liver cancer cells can spontaneously form VM channels without additional stimuli, and this VM confers resistance to conventional anti-angiogenic therapy, and can promote spreading of cancer cells. Our studies suggest that VM is decreased by compounds inhibiting invasion, such as curcumin. We are also studying the influence of extracellular matrix components on the resistance of cancer cells to drugs. Thus, we found that A549 cells exposed to extracellular matrix proteins exhibited some characteristics similar to those found in dormant cancer cells, including a decrease in cell proliferation, cell motility and invasion, and an increase in chemoresistance for the drugs that target actively proliferating cells.
Loss of cell attachment: Cancer cells in in vitro culture typically grow as monolayers attached to the culture plate. Normal epithelial cells die after detachment from extracellular matrix or culture plate in a phenomenon named anoikis. However, cancer cells can develop “anoikis resistance” and this property can enable cancer cells to survive when floating in the blood or lymphatic circulation during metastasis. Prevention of cell attachment to the culture plate, achieved by using polymer-coated culture plates, allows us to study anoikis resistance in cancer cells. Proteomic studies of anoikis resistant HuCCA-1 cholangiocarcinoma cells show up-regulation of certain proteins, which may be of interest as potential biomarkers. Involvement of specific proteins has also been shown to be important for the progression of metastastic thyroid cancer cells. ∧Top
Genetic diseases
Abnormal hemoglobins and thalassemia
Our early studies on genetic diseases concerned the hemoglobinopathies, which consist of two types. Thalassemia results from the lack or decreased synthesis of hemoglobin chains, with α-thalassemia and β-thalassemia occurring with high frequency in Thailand, at 20% and 10% respectively. Abnormal hemoglobins have mutations altering the structure of a globin chain: they are usually rare, but some occur at high frequency, such as Hb E [β26 Glu-Lys] which can rise to 53% frequency in the Northeast. We have studied both thalassemias and abnormal hemoglobins in collaboration with Professor Suthat Fucharoen at Mahidol University, and characterized 10 out of the 30 or more abnormal hemoglobins found in Thailand. Since hemoglobinopathies are now well characterized in Thailand, we turned our attention to other genetic diseases.
Inborn Errors of Metabolism
Inborn errors of metabolism arise from deficiencies of enzymes in metabolic pathways, such as amino acid metabolism and macromolecule degradation. They are often detected by an accumulation of the substrate and/or a decrease in the metabolic products of the reaction. Typically, each inborn error of metabolism occurs with low frequency, but there are many defects, so cumulatively, inborn errors of metabolism are significant problems. Some cause severe clinical manifestations, such as mental retardation or developmental abnormalities, but sometimes, devastating effects can be avoided through proper treatment, so early detection is important.
We collaborate with pediatricians in Bangkok, including Siriraj Hospital and Ramathibodi Hospital at Mahidol University, to analyze the levels of deficient enzymes and detect mutant genes causing the diseases. A major interest includes lysosomal storage diseases, such as Pompe disease, Fabry disease, Gaucher disease, Hurler Syndrome and Hunter Syndrome, where enzyme deficiency causes accumulation of substances leading to organ damage and physical defects. Information on mutations are useful for genetic counseling, and assist in early diagnosis, which is important in enabling treatment to be carried out early enough to prevent the effects of the disease. We also study methylmalonic acidemia (MMA), caused by defective function of vitamin B12-dependent enzyme methylmalonyl CoA mutase, due to defects in mutase enzyme itself or in coenzyme metabolism. In both cases, many mutations are unique to Thailand, so need to be studied locally. Growing evidence reveals that some patients with Parkinson’s disease carry a heterozygous state of glucocerebrosidase (GBA) gene mutation (Gaucher disease carrier). This leads to the hypothesis that GBA mutations in the heterozygous state may contribute as a genetic risk factor for sporadic Parkinson’s disease. We are also studying the GBA mutations in association with Parkinson’s disease using the proteomic approach. ∧Top
Action of environmental contaminants on human health
Exposure to environmental contaminants, including pesticides and xenobiotics, is a risk factor for the development of diseases, such as cancer and neurodegenerative disorders. As pesticide use increases in Thailand, risk of exposure increases, even though new generation pesticides are supposedly safer, because farmers and/or general public are not trained on proper handling. We have used proteomics to elucidate the modes of action of pesticides and their effects, using human cancer cell lines as model systems. Exposure of high concentrations of atrazine, a triazine herbicide, on hepatocellular carcinoma cells implicated its possible involvement in causing cardiovascular diseases. On the other hand, fipronil, a broad spectrum phenylpyrazole insecticide, affected neuronal cells by altering the expression of proteins involved in protein biosynthesis, mitochondrial function, and glucose metabolism. Proteomic studies in plants in relation to pesticides are also interesting due to their importance in agriculture sector, and chlorpyrifos, an organophosphate insecticide, showed effects on proteins involved in growth and photosynthetic activity in the leaves of Chinese kale. Moreover, the cellular and molecular effects of xenobiotics that persist in the environment, such as perfluorinated compounds, are also of interest. ∧Top