Oxidative metabolic events in liver disease and cancer: from mechanisms to therapeutic approaches
Dr. Walee Chamulitrat
Research opportunities for students:
Medical students who are interested in pursuing MD/PhD in experimental medicine are welcomed to our laboratory. Our cell biology and biochemistry techniques include tissue culture, use of siRNA, RT-PCR, Western analysis, indirect immunofluorescence and various activity assays. In some studies, experiments using animal models are available for students.
Generation of free radicals including reactive oxygen species (ROS) is important in normal physiological functions, such as, cell proliferation, and differentiation. Hyper-generation of ROS known as oxidative stress is known to be associated with disease states as manifested by cell death, apoptosis and inflammation. Hence, activation and inactivation of ROS-generating enzymes, such as, mitochondrial oxidoreductases, NADPH oxidase isoforms, lipoxygenases, myeloperoxidase and nitric oxide oxidoreductase may dictate cellular fates and phenotypes. Consequently, modification of cellular free fatty acids, phospholipids, proteins, and DNA by ROS may influence cellular metabolisms and signaling involving transcription factors. These events can be used as targets to suppress injury and inflammation associated with changes in oxidative and metabolic events. Our research projects involve cell biology and experimental Hepatology designed to understand mechanistic insights as well as to suppress the progression and development of liver disease and cancer. Three research projects are currently active in our laboratory.
Bile acid-phospholipid based therapy for treatment of steatohepatitis
It is known that mitochondrial damage by an apoptosis stimulus by either extrinsic or intrinsic pathways generate excessive ROS, and this event can be inhibited by non-redox mitochondrial stabilizers, such as, hydrophilic bile acids and phospholipids. Apoptosis is the major feature of non-alcoholic steatohepatitis (NASH), and thus a compound designed to stabilize mitochondria may represent a strategy for treatment of NASH which is urgently needed. A novel compound ursodeoxycholyl phosphatidylethanolamide (UDCA-LPE) was designed and recently shown to inhibit apoptosis (Hepatology 2009). Mechanistic studies were designed to shed lights into cytoprotective effects of UDCA-LPE. These involve signaling pathways related to activation of growth factor receptor/PI3K/Akt and caspase8/cFLIP. High fat intake in NASH and subsequent lipotoxicity causes apoptosis with concomitant accumulation of triglycerides, and lyso-phosphatidylcholine (LPC). Anita Pathil-Warth has shown that alterations of hepatic LPC/PC pools and metabolic parameters in animal models of NASH were reversed UDCA-LPE treatment. Further lipodomic studies for molecular species of oxidized and unoxidized phospholipids and fatty acids will be investigated. Furthermore, current studies indicate that UDCA-LPE has an anti-inflammatory, anti-cancer, anti-fibrotic and fat-lowering capacity. Mechanisms involving transcription factors subsequent to oxidized (phospho)lipids will be investigated. This project will be funded by the DFG under Prof. W. Stremmel’s leadership.
Role of gp91phox homolog Nox1 in cell transformation of human epithelial cells
Among NADPH oxidase isoforms, the first gp91phox homolog Nox1 was discovered in 1999 and was shown to induce tumorigenic conversion of mouse fibroblasts. Such conversion is not possible for human epithelial cells. Our data have indicated that Nox1 may accelerate neoplastic progression of benign human epithelial cells towards tumorigenic conversion. We showed that Nox1 expression is associated with fibroblast-like epithelial cells which generate increased ROS generation and exhibit anchorage-independent growth (Oncogene 2003). Overexpression with Nox1 in human epithelial cells induces the generation of progenitor cells which exhibit resistance against calcium-induced differentiation (JID 2007). Current experiments reveal that Nox1 regulates the expression of fetal epithelial keratin 18 at post-translational level. In light of the role of ROS-generating Nox1 in neoplastic progression by inducing fetal phenotype keratins, we are extending these studies to myogenic proteins. In addition, Nox1 partitioning into specialized microdomains or lipid rafts is found in cancer cells more than non-transformed cells. Nox1 partitioning out of lipid rafts occurs during apoptosis of cancer cells indicating that Nox1 may activate growth factor receptors localized in lipid rafts. This project has been funded by the DFG (CH288 3-3) and Thailand Research Fund/DAAD.
Development of antioxidant 2,3-dehydrosilybin as a template molecule for treatment of liver disease and cancer
Naturally occurring antioxidants such as flavonoids are known to prevent cancer development. Flavonoid silybin is the major compound in silymarin extracts which has been used to treat jaundice and gallstones since the ancient times. We showed that the oxidized form of silybin called 2,3-dehydrosilybin (DHS) is superior to silybin in scavenging ROS as well as protecting ROS-induced cell death and apoptosis (BBA 2008). This property is a prerequisite for treatment of oxidative injury during liver disease. In addition, DHS induces apoptosis either directly or under sensitization with TNF-a in cancer cells more than non-transformed cells (Chemotherapy 2008). TNF-a-sensitization is explained by DHS’s ability to inhibit topoisomerase I activity better than silybin (Chemotherapy 2009). DHS will be used as a template molecule for further design for treatment of liver disease and cancer. This project was funded by commission of grants from Medical Faculty Heidelberg and Paul-Erhlich-Society for Chemotherapy.
- Anita Pathil, Arne Warth, Walee Chamulitrat, Wolfgang Stremmel. The Synthetic Bile acid-Phospholipid Conjugate Ursodeoxycholyl Lysophosphatidylethanolamide Suppresses TNFα-induced Liver Injury . J. Hepatology, in press
- Chamulitrat W. Role of gp91phox homolog nox1 in induction of premalignant spindle phenotypes of HPV 16 E6/E7-immortalized human keratinocytes. ScientificWorldJournal. 2010;10:1435-49.
- Chamulitrat W, Burhene J., Rehlen T., Pathil-Warth A., Stremmel W: The bile salt-phospholipid conjugate ursodeoxycholyl lysophosphatidylethanolamide as a hepatoprotective agent. Hepatology 2009, ;50(1):143-54
- Chamulitrat W, Sattayakhom A, Herold-Mende C, Stremmel W. Human papillomavirus 16 E6/E7-immortalized human gingival keratinocytes with epithelial mesenchymal transition acquire increased expression of cIAP-1, Bclx and p27(Kip1).Exp Dermatol. 2009;18(12):1067-9.
- Chamulitrat W, Sattayakhom A, Herold-Mended C, Stremmel W: Human papillomavirus 16 E6/E7-immortalized human gingival keratinocytes with epithelial mesenchymal transition acquire increased expression of cIAP-1, Bclx and p27kip1. Exp. Dermatol. 2009 in press.
- Thongphasuk P, Stremmel W, Chamulitrat W: 2,3-dehydrosilybin is a better DNA topoisomerase I inhibitor than its parental silybin. Chemotherapy. 55(1):42-48, 2009.
- Huber, A., Thongphasuk, P., Stremmel, W., Chamulitrat, W.: Significant greater antioxidant anticancer activities of novel dehydrosilybin than silybin. Biochim. Biophys. Acta. Biochim Biophys Acta. 1780:837-847, 2008.
- Müssig E, Steinberg T, Kohl A, Chamulitrat W, Komposch G, Tomakidi P.: Discrimination of epithelium-like and fibroblast-like phenotypes derived from ethanol-treated immortalised human gingival keratinocytes in epithelial equivalents. Cell Tissue Res. 332:57-71, 2008.
- Thongphasuk P., Stremmel W., Chamulitrat W.: Potent direct or TNF-alpha promoted anticancer effects of 2,3-dehydrosilybin: comparison study with silybin'. Chemotherapy 54:23-30, 2008
- Chamulitrat, W.,Huber, A., Riedel, H., Stremmel, W.: Nox1 induces differentiation resistance of immortalized human keratinocytes generating cells that express simple epithelial keratins. J. Invest. Dermatol. 127:2171-2183, 2007.
- Chamulitrat, W., Stremmel, W., Kawahara, T., Rokutan, K., Fujii, H., Wingler, K., H. H. W. Schmidt, Schmidt, R. A constitutive NADPH oxidase-like system containing gp91phox homologs in human keratinocytes. J. Invest. Dermatol. 122: 1-10, 2004.
- Chunglok, W., Ittarat, W., Tomakidi, P., Schmidt, R., Stremmel, W., Chamulitrat, W. Phenotypic stability during human keratinocyte transformation is associated with inducible nitric oxide synthase (iNOS): Evidence for iNOS regulation by MAP kinases. Nitric Oxide: Biology and Chemistry 11: 237-246, 2004.
- Chamulitrat, W., Schmidt, R., Chunglok, W., Tomakidi, P., Kawahara, T., Rokutan, K.: Association of gp91phox Homolog Nox1 with anchorage independent growth and MAP Kinase-activation of transformed human keratinocytes. Oncogene 22: 6045-6054, 2003.
- Chamulitrat, W., Schmidt, R., Chunglok, W., Kohl, A., Tomakidi, P.: Epithelium-like and fibroblast-like phenotypes derived from HPV16 E6/E7-immortalized human gingival keratinocytes following chronic ethanol treatment. Eur. J. Cell Biol. 82: 313-322, 2003.
Accomplishment of group members
2008|Contributed oral presentation at the American liver meeting (AASLD)
2009|Contributed oral presentation at the German liver meeting (GASL) with best presentation award given to Anita Pathil-Warth
2009|Contributed oral presentation at the European liver meeting (EASL) and travel award given to Anita Pathil-Warth
2009|Best Poster Price at Liver and Metabolic Syndrome, Falk Symposium, 2009 given to Dr. W. Chamulitrat
2010|DAAD student award to Apsorn Sattayakohm
2010|Contributed oral presentation at 'Kongress der Deutschen Gesellschaft für Innere Medizin (DGIM)', 2010 given by Anita Pathil-Warth
2010|Contributed oral presentation at 65. Jahrestagung der Deutschen Gesellschaft für Verdauungs- und Stoffwechselkrankheiten (DGVS) 2010 given by Anita Pathil-Warth