The transsulfuration pathway converts homocysteine to cysteine and represents the metabolic link between antioxidant and methylation metabolism. The first and committing step in this pathway is catalyzed by cystathionine ␤-synthase (CBS), which is subject to complex regulation, including allosteric activation by the methyl donor, S-adenosylmethionine (AdoMet). In this study, we demonstrate that methionine restriction leads to a >10-fold decrease in CBS protein levels, and pulse proteolysis studies reveal that binding of AdoMet stabilizes the protein against degradation by Ϸ12 kcal͞mol. These observations predict that under pathological conditions where AdoMet levels are diminished, CBS, and therefore glutathione levels, will be reduced. Indeed, we demonstrate this to be the case in a mouse model for spontaneous steatohepatitis in which the gene for the MAT1A isoenzyme encoding AdoMet synthetase has been disrupted, and in human hepatocellular carcinoma, where MAT1A is silenced. Furthermore, diminished CBS levels are associated with reduced cell viability in hepatoma cells challenged with tert-butyl hydroperoxide. This study uncovers a mechanism by which CBS is allosterically activated by AdoMet under normal conditions but is destabilized under pathological conditions, for redirecting the metabolic flux toward methionine conservation. A mechanistic basis for the coordinate changes in redox and methylation metabolism that are a hallmark of several complex diseases is explained by these observations. glutathione ͉ liver disease C ellular methylation and antioxidant metabolism are linked by the transsulfuration pathway, which converts the methionine cycle intermediate, homocysteine, to cysteine, the limiting reagent in glutathione synthesis. The balance between conserving methionine via transmethylation under conditions of methionine restriction and committing it to transsulfuration under conditions of plenty is regulated at two key control points, methionine adenosyltransferase (MAT) and cystathionine ␤-synthase (CBS) (Fig. 1). Aberrations in methylation and redox homeostasis are common to a number of chronic diseases including pathologies of the liver. In alcoholic liver disease and in hepatocellular carcinoma an increase in markers of oxidative stress is observed (1, 2). Furthermore, there is a switch in the expression of MAT genes from MAT1A to MAT2A in liver cancer, which correlates with lower S-adenosylmethionine (AdoMet) levels (3).Under normal conditions, coordinate regulation of methylation and antioxidant metabolism is achieved by the allosteric activation of CBS by AdoMet (Fig. 1). AdoMet is a V-type allosteric effector that increases CBS activity 2-to 3-fold (4, 5). Under conditions of plenty, methionine is directed toward cysteine synthesis via the transsulfuration pathway for use in glutathione and other cellular functions or directed toward catabolism. Cysteine is the limiting reagent in glutathione synthesis and in liver; Ϸ50% of the cysteine in glutathione is derived from methionine via the transsulf...

Methionine metabolism provides two key cellular reagents: S-adenosylmethionine and glutathione, derived from the common intermediate, homocysteine. A majority of cancer cells exhibit a methionine-dependent phenotype whereby they are unable to grow in medium in which methionine is replaced by its precursor, homocysteine. Additionally, CpG island hypermethylation of tumor suppressor gene promoters is observed in a background of global hypomethylation in cancerous cells. In this study, we have profiled the expression levels of the homocysteine junction enzymes, methionine synthase (MS), MS reductase (MSR), and cystathionine B-synthase (CBS) in the NCI60 panel of cancer cell lines. The doubling time of non-small lung cell cancer lines, which exhibit the lowest levels of MS within the panel, was significantly correlated with expression of MS. The ratio of MS to MSR varied over a 5-fold range in the different cell types, which may modulate methionine synthesis. Interestingly, markedly reduced CBS expression was seen in the methionine-dependent prostate cancer cell line, PC-3, but not in the methionine-independent cell line, DU-145. However, neither provision of the transsulfuration pathway product, cysteine, nor overexpression of CBS rescued the growth impairment, indicating that reduced CBS was not responsible for the methionine-dependent phenotype in this cell line. (Cancer Res 2005; 65(4): 1554-60)

Cancer such as breast carcinoma is a major public health problem worldwide. The actual cause of death is the formation of metastasis which often occurs in the lung. Among the cell types present in the tumor microenvironment, macrophages have been proven to be the dominant leukocyte population with high macrophage density correlating to poor patient prognosis. Thus, these cells are of high interest as targets for cancer therapeutics, making them a valuable research topic. We utilize an innovative organotypic tumor invasion model, using living human Precision-Cut Lung Slices (PCLS) and cancer cells to focus on the local immunological response during early metastasis formation. An AdGFP-transduced human breast cancer cell line MDA-MB-231 was added to human PCLS over a period of one week. Viability assays such as LIVE/DEAD® staining and LDH measurements were performed to assure intact human tissue throughout the experimental procedure. Tissue immune staining methods against CD68 and Ki67 were used to visualize locations of macrophages and proliferating cells, respectively. Immune response and neoangiogenesis were determined by cytokines IL-10 and IL-1beta and tumor markers VEGF and GM-CSF. An in vitro neoangiogenesis assay was performed to observe whether the tissue is able to attract endothelial cells through a BME-coated membrane. Surrounding parenchyma remains viable during infection and invasion with cancer cells. Colocalization of CD68-positive macrophages and MDA-MB-231 was found during the entire invasion period, visualizing expansion and proliferation of the cancer cells within the organotypic model. The VEGF/GM-CSF release correlated with the MDA-MB-231 growth curves. HUVEC cell invasion shows a 5-fold increase in the PCLS that had been treated with MDA-MB-231 in relation to untreated controls. Here we mimic cancer cell proliferation and immune responses in the native microenvironment of human lung tissue which will be used for testing of anti-tumor drugs in the near future