Our early studies on genetic diseases involved the hemoglobinopathies, which occur with rather high frequency in Thailand, and consist of two types. Thalassemia results from lack or decreased synthesis of one or more of the hemoglobin chains. alpha-Thalassemia results from absence or decreased levels of a chains and occurs with a frequency of about 20% in Thailand, while beta-thalassemia is due to absence or decreased levels of b-chains and occurs with a frequency of about 10% in Thailand.  Abnormal hemoglobin or hemogobin variants are hemoglobins which have mutations altering the structure of one of the globin chains, typically the alpha or the beta chains.  If the mutation occurs in a region of functional importance, this can affect the physiological function or the stability of the hemoglobin, so studies on abnormal hemoglobins have provided important information about the rela­tionship between protein structure and function.

     More than 800 abnormal hemoglo­bins have now been characterized world-wide, and some 30 abnormal Hb have also been detected in Thailand over the last 40 years. We have collaborated with researchers at Mahidol University for 20 years, and have characterised 10 of these variants, including Hb Lepore Washington Boston. Of the variants found in Thailand, 9 are point mutations in alpha chains, 15 are point mutations in beta chains, 3 are C-terminal elongations, 1 is alpha deletion, 1 is a frameshift mutation, and 1 is a delta-beta hybrid hemoglobin resulting from crossing-over. Most abnormal Hb are rare except for Hb E [beta26 Glu-Lys], which can rise to a frequency of 53% in the Northeast, and Hb Constant Spring, an alpha-chain extension occurring with a frequency of 1-8 %.  Moreover, since Thailand has high incidence of Hb E, alpha-thalassemia and beta-thalassemia, abnormal Hb can often be found in compound heterozygosity in Thailand, unlike in most countries, so that the effect of such associations on the hematological and clinical profile is of interest. Since many hemoglobinopathies have now been well characterized in Thailand, we have turned our interests to other genetic diseases, as outlined below.

     There are many inborn errors of metabolism, which can cause severe clinical manifestations, such as mental retardation or developmental abnormalities.  In general, inborn errors arise from deficiencies in enzymes of various metabolic pathways, such as the urea cycle, pathways for degradation or synthesis of specific amino acids, or mucopolysaccharide degradation.  Such disorders may be due to mutations leading to dysfunctional or poorly functioning enzyme, or may result from lowered expression or absence of these enzymes.  Such enzyme deficiencies are typically detected by an accumulation of the substrate of the enzyme reaction and/or a decrease in the level of metabolites, which occur after the enzyme 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.  In many cases, the devastating effects can be avoided through proper treatment, e.g. in phenylketonuria, which may be treated with diets low in phenylalanine.  So it is important to detect inborn errors early, and in many countries, newborn screening for selected inborn errors or metabolism is routine. 

     Many inborn errors of metabolism have also been found in Thailand.  We began our studies of inborn errors of metabolism about 10 years ago, in collaboration with various medical doctors. At that time, analytical facilities in Thailand were still limited.  Thus we established the use of HPLC methods for determining the amino acid composition of blood and urine, for use in diagnosing amino acid disorders and urea cycle disorders.  Plasma amino acid levels in normal Thai children of different age ranges were established, in collaboration with the Faculty of Medicine Siriraj Hospital, Mahidol University and with the Queen Sirikit National Institute of Child Health, Ministry of Public Health.  Amino acid analyses were also used in diagnosing various disorders.  This led to the establishment of facilities for amino acid analyses at Siriraj Hospital, relieving us of the need to perform routine analyses.

     In the more recent years, we have been able to study selected cases of inborn errors of metabolism in more detail, in collaboration with the Faculty of Medicine Siriraj Hopsital and the Faculty of Medicine Ramathibodi Hospital at Mahidol University, and the Faculty of Medicine, Chulalongkorn Hospital.   Typically, this involves analysing the levels of enzymes suspected of being deficient, using leukocytes or cultured fibroblasts from patients.  Then, we design primers for preparing cDNA or genomic fragments by RT-PCR or PCR, and then perform automated sequence of the cDNA to determine the mutation.  However, each disease represents a whole research field, so we have limited our work to a number of inborn errors of metabolism, such as methylmalonic acidemia, Gaucher disease, Hurler Syndrome and Hunter Syndrome.  Interestingly, in many cases, the mutations found in Thailand are novel mutations, not previously found in other populations, stressing the need to continue such studies locally.

      Cancer is a disorder resulting from autonomous, uncontrolled cell growth and differentiation, and with malignant behavior, is capable of invasion and metastasis.  Carcinogenesis is initiated by non-lethal genetic damage, followed by a multi-step process involving both the phenotypic and genetic levels. Regulatory genes such as the proto-oncogenes, the tumor suppressor genes, and genes regulating apoptosis are important targets of genetic damage, as well as the DNA repair genes. Mutational damage to these genes will result in activation or inactivation of the functions of their gene products, resulting in uncontrolled proliferation with abnormal differentiation and acquisition of the capability for invasion or metastasis. In addition, tumors undergo various interactions involving a variety of adhesion molecules for detachment and attachment, such as the E-cadherins, laminin or the integrin family; the cytoskeletal proteins such as catenins; and the proteases and anti-proteases such as matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs).

      Because of our longstanding interests in proteins, we are exploring the protein changes that occur in human cancer. Initially, we collaborated with the Department of Pathology, Phramongkutklao Hospital, Bangkok to analyze changes in protein in human cancer tissues. Surgical specimens of tumor tissue and normal tissue from the same patient have been collected from many types of cancer and characterized in terms of pathology.  Our group is the first research group in Thailand to use the proteomic approach, where the proteome patterns (or the total protein present at any tissue at any given time) are studied.  In this case, the proteome patterns in tumor tissue are compared with normal tissue from the same patient using the powerful technique of two-dimensional electrophoresis (2-DE). The proteins of interest are positively identified by peptide mass fingerprinting, ESI/MS/MS or automated protein sequence analysis.  We have had some success in the study of thyroid disease, where we could demonstrate the increased expression of certain proteins, such as cathepsin B and prohibitin, in neoplastic thyroid diseases compared to non-neoplastic diseases, such as goiter or nodular hypoplasia.  Immunoblotting studies also showed that   metastatic potential appeared to be correlated with high levels of expression of galectin-3.

      This collaboration with Phramongkutklao Hospital is continuing, and other tissues and body fluids are being studied in relation to cancer, using both 2-dimensional electrophoresis and mass spectrometry, as well as immunoblotting techniques.  The Laboratory is also involved in the Asian Oceanian Human Proteome Organization’s (AOHUPO) Membrane Proteome Initiative (MPI), which aims at standardizing the methodologies for separation and identification of membrane proteins.

     Our laboratory is also studying human cancer cell lines as possible models of cancer.  Special emphasis is placed on the cholangiocarcinoma cell line, HuCCA-1, and the hepatocellular cell line, HCC-S102 cell line, since both were originated from Thai patients. Cholangiocarcinoma is found with high frequency in Northeast Thailand, often associated with liver fluke infection, while hepatocellular carcinoma is frequent in North Thailand, often associated with hepatitis infection.  We have compared the proteomic patterns of HuCCA-1 and HCC-S102 cell lines to each other, to define proteins differing between cholangiocarcinoma and hepatocellular carcinoma cells, and found notable differences in the cytokeratins and other cytoskeletal proteins, which may useful as potential biomarkers.

     We are also using the Thai cholangiocarcinoma (HuCCA-1) and hepatocellular carcinoma (HCC-S102) cell lines to screen medicinal plants for anti-cancer activities, in collaboration with the Chemistry Laboratories of the Chulabhorn Research. Thailand has abundant resources of medicinal plants and is rich in knowledge of folk medicine employing such plants. Historically, many important anti-cancer drugs, such as taxol, podophyllotoxin, vinblastine and vincristine, originate from plants. Since cancer cells are characterized by rapid and unrestrained growth, cytotoxic compounds have been used as lead compounds for the development of novel anti-cancer chemotherapeutic drugs. The effect of bioactive compounds on cancer cells is also being studied by proteomics to identify the proteins changed as a result of treating cancer cells with the bioactive compound.  Thus, work involves not only screening for cytotoxic anti-cancer agents, but also studying how these potential therapeutic compounds affect the expression of proteins in the cancer cells.

     Apart from searching for cytotoxic compounds, we are also interested in searching for natural products, which may be used for other chemotherapeutic approaches.  One target is metastasis, whereby cancer cells invade surrounding tissues, enter the blood or lymphatic vessels, and form metastatic colonies at distant sites.  Thus, we screen medicinal plants or marine organisms for bioactive compounds, which are not cytotoxic to cells, but which inhibit the ability of cancer cells to invade extracellular matrix in vitro.   Such compounds may be of interest as potential lead compounds for anti-metastatic therapy. Interestingly the vanillin, a well-known, generally regarded as safe, food flavouring, was found to inhibit invasion of cancer cells and to reduce metastasis of adenocarcinoma in a mouse model. 

     We are also developing cell line models to screen for other types of anti-cancer activity. One such target is angiogenesis, since growth of a solid tumor beyond the size of 1-2 mm³ requires the formation of new blood vessels for providing oxygen and nutrients. Since conventional in vivo models for angiogenesis are often complex, we are trying to develop cellular models for screening for anti-angiogenic compounds by studying tube formation in liver cancer cell lines.  We are also trying to develop cell line models to study drug-resistance and to screen for compounds that will reverse drug resistance.  This is an important, since a major problem in cancer therapy is that cancer cells become resistant to the drug.

     Thus, the use of human cancer cell lines in our laboratory covers many aspects, including defining novel biomarkers for liver cancer, screening for cytotoxic and anti-metastatic compounds, as well as the development of cell line models for screening for compounds, which inhibit angiogenesis or can reverse drug-resistance. Cell lines are also being used to elucidate the molecular mechanisms of the effect of these various bioactive compounds.

     More recent studies in our laboratory have expanded the use of proteomics to other systems, particularly to study the effect of bioactive compounds.  This includes study of protein changes occurring when pesticides are used on vegetables and the changes in plasma proteins when when the  bioactive compound curcurmin is used to treat thalassemia patients.