Kimberly D. Kyker

Curcumin: A new radio-sensitizer of squamous cell carcinoma cells

PURPOSE: Curcumin, a potential chemopreventive agent, was found to inhibit cancer cells in S/G2M phases of the cell cycle, when radiation is more effective. The purpose of the current study was to investigate whether curcumin can sensitize squamous cell carcinoma (SCC) cells to the ionizing effects of irradiation. METHODS: Curcumin (3.5 μM) was added for 48 hours to an SCC cell line prior to irradiation. Cell growth (counts) and colony-formation (colonogenic assay) were examined after radiation. RESULTS: Incubation with curcumin only (3.75 μM) for 48 hours did not decrease the number of cells or the ability to form colonies in the absence of radiation. However, in plates that were exposed to 1–5 Gy of radiation, cell counts dropped significantly if pretreated with curcumin with a maximal effect at 2.5 Gy (where the cell counts dropped from 1240 to 1017, P < 0.001). The colonogenic assay revealed a significant decrease in the ability to form colonies following pretreatment with curcumin in all radiation doses (P < 0.05). CONCLUSIONS: Given the appropriate doses, curcumin exhibits radio-sensitizing effects on SCC cells in vitro.

Exogenous glycosaminoglycans coat damaged bladder surfaces in experimentally damaged mouse bladder.

BackgroundInterstital cystitis is often treated with exogenous glycosaminoglycans such as heparin, chondroitin sulphate (Uracyst), hyaluronate (Cystistat) or the semi-synthetic pentosan polysulphate (Elmiron). The mechanism of action is presumed to be due to a coating of the bladder surface to replace the normally present chondroitin sulphate and heparan sulphate lost as a result of the disease. This study used fluorescent labelled chondroitin sulphate to track the distribution of glycosaminoglycans administered intravesically to mouse bladder that had been damaged on the surface.MethodsThe surfaces of mouse bladders were damaged by 3 mechanisms – trypsin, 10 mM HCl, and protamine sulphate. Texas Red-labeled chondroitin sulphate was instilled into the bladders of animals with damaged bladders and controls instilled only with saline. Bladders were harvested, frozen, and sectioned for examination by fluorescence.ResultsThe normal mouse bladder bound a very thin layer of the labelled chondroitin sulphate on the luminal surface. Trypsin- and HCl-damaged bladders bound the labelled chondroitin sulphate extensively on the surface with little penetration into the bladder muscle. Protamine produced less overt damage, and much less labelling was seen, presumably due to loss of the label as it complexed with the protamine intercalated into the bladder surface.ConclusionGlycosaminoglycan administered intravesically does bind to damaged bladder. Given that the changes seen following bladder damage resemble those seen naturally in interstitial cystitis, the mechanisms proposed for the action of these agents is consistent with a coating of damaged bladder.

Ischemic Inactivation of G Protein–Coupled Receptor Kinase and Altered Desensitization of Canine Cardiac β-Adrenergic Receptors

BackgroundG protein–coupled receptor kinases (GRKs) modulate myocardial &bgr;-adrenergic receptor (&bgr;AR) signaling. We examined whether GRK activity was altered 6, 24, and 96 hours after left anterior descending coronary artery ligation (LAD CAL) in the dog. Methods and ResultsGRK activity was measured in arrhythmogenic subepicardial border zone (EBZ) tissue overlying the infarct and from nonischemic remote-site (RS) subepicardial tissue from the same animal. GRK activity in the ischemic EBZ was 15% of RS (P =0.03, n=6) 24 hours after CAL and appeared to start as early as 6 hours through 96 hours. GRK activity and immunoblot data demonstrated a marked decrease of GRK2 but not GRK5 at 24 hours. EBZ tissue exhibited high-affinity binding for (−)-isoproterenol (Ki of 0.076±0.026 nmol/L [SEM]) at 24 hours, which was not significantly different from control tissue from nonoperated animals (1.2±0.8 nmol/L, P >0.05, n=6). A significantly lower Ki of 13.8±2.8 nmol/L (P <0.001, n=6) was observed for RS taken from the ischemic animals. This was reflected by a 4-fold increase in the EC50 of isoproterenol-stimulated adenylyl cyclase activity from 18 nmol/L in EBZ tissue to 73 nmol/L in RS (P <0.05, n=4). ConclusionsThere is a selective decrease in GRK2 activity and a loss of the ability of the arrhythmia-prone EBZ tissue to desensitize to &bgr;-adrenergic stimulation 24 hours after CAL. This correlates temporally with a second (late) peak in sudden cardiac death previously observed between 6 and 24 hours in dog and rat models of myocardial infarction.

Analysis of the interaction of extracellular matrix and phenotype of bladder cancer cells.

BackgroundThe extracellular matrix has a major effect upon the malignant properties of bladder cancer cells both in vitro in 3-dimensional culture and in vivo. Comparing gene expression of several bladder cancer cells lines grown under permissive and suppressive conditions in 3-dimensional growth on cancer-derived and normal-derived basement membrane gels respectively and on plastic in conventional tissue culture provides a model system for investigating the interaction of malignancy and extracellular matrix. Understanding how the extracellular matrix affects the phenotype of bladder cancer cells may provide important clues to identify new markers or targets for therapy.MethodsFive bladder cancer cell lines and one immortalized, but non-tumorigenic, urothelial line were grown on Matrigel, a cancer-derived ECM, on SISgel, a normal-derived ECM, and on plastic, where the only ECM is derived from the cells themselves. The transcriptomes were analyzed on an array of 1186 well-annotated cancer derived cDNAs containing most of the major pathways for malignancy. Hypervariable genes expressing more variability across cell lines than a set expressing technical variability were analyzed further. Expression values were clustered, and to identify genes most likely to represent biological factors, statistically over-represented ontologies and transcriptional regulatory elements were identified.ResultsApproximately 400 of the 1186 total genes were expressed 2 SD above background. Approximately 100 genes were hypervariable in cells grown on each ECM, but the pattern was different in each case. A core of 20 were identified as hypervariable under all 3 growth conditions, and 33 were hypervariable on both SISgel and Matrigel, but not on plastic. Clustering of the hypervariable genes showed very different patterns for the same 6 cell types on the different ECM. Even when loss of cell cycle regulation was identified, different genes were involved, depending on the ECM. Under the most permissive conditions of growth where the malignant phenotype was fully expressed, activation of AKT was noted. TGFβ1 signaling played a major role in the response of bladder cancer cells to ECM. Identification of TREs on genes that clustered together suggested some clustering was driven by specific transcription factors.ConclusionThe extracellular matrix on which cancer cells are grown has a major effect on gene expression. A core of 20 malignancy-related genes were not affected by matrix, and 33 were differentially expressed on 3-dimensional culture as opposed to plastic. Other than these genes, the patterns of expression were very different in cells grown on SISgel than on Matrigel or even plastic, supporting the hypothesis that growth of bladder cancer cells on normal matrix suppresses some malignant functions. Unique underlying regulatory networks were driving gene expression and could be identified by the approach outlined here.

A model for 3-dimensional growth of bladder cancers to investigate cell-matrix interactions.

Elucidating the mechanisms by which the phenotype of cancer cells is modulated by the extracellular matrix (ECM) potentially identifies mechanisms that could be exploited for cancer control. Three readily available bladder cancer cell lines of different aggressiveness were grown on a bioscaffold material (Small Intestine Submucosa: SIS) under a variety of media and nutrient schedules to determine the influence of epigenetic factors on phenotype. The aggressive TCCSUP and J82 lines displayed an invasive phenotype when fed twice weekly with medium containing 10% fetal calf serum (FCS), but grew as a layered, noninvasive structure when fed daily with the same nutrient. The papillary RT4 cells grew as a nearly normal appearing layered phenotype when fed with medium containing 10% FCS, regardless of the feeding schedule. The papillary phenotype appeared only when the fetal calf serum concentration was reduced to 1% and the cells were fed twice weekly. Immunohistochemical staining showed E-cadherin was detected in the nucleus of the TCCSUP line rather than on the cell periphery, thus, demonstrating that normalization of the TCCSUP line into a layered phenotype did not alter the fundamental dysregulation of the cadherin-B-catenin-cytoskeleton complex. In contrast, in the RT4 cells the biomarker was distributed discretely around the cell periphery in 10% fetal calf serum but in the nucleus in 1% fetal calf serum and twice weekly feeding. Modulating the phenotype of cancer cells from invasive to noninvasive through manipulation of matrix and nutrient components presents a model system for identifying the key factors involved in invasiveness and may identify new therapeutic targets and markers.

Systems biology approach for mapping the response of human urothelial cells to infection by Enterococcus faecalis

BackgroundTo better understand the response of urinary epithelial (urothelial) cells to Enterococcus faecalis, a uropathogen that exhibits resistance to multiple antibiotics, a genome-wide scan of gene expression was obtained as a time series from urothelial cells growing as a layered 3-dimensional culture similar to normal urothelium. We herein describe a novel means of analysis that is based on deconvolution of gene variability into technical and biological components.ResultsAnalysis of the expression of 21,521 genes from 30 minutes to 10 hours post infection, showed 9553 genes were expressed 3 standard deviations (SD) above the system zero-point noise in at least 1 time point. The asymmetric distribution of relative variances of the expressed genes was deconvoluted into technical variation (with a 6.5% relative SD) and biological variation components (>3 SD above the mode technical variability). These 1409 hypervariable (HV) genes encapsulated the effect of infection on gene expression. Pathway analysis of the HV genes revealed an orchestrated response to infection in which early events included initiation of immune response, cytoskeletal rearrangement and cell signaling followed at the end by apoptosis and shutting down cell metabolism. The number of poorly annotated genes in the earliest time points suggests heretofore unknown processes likely also are involved.ConclusionEnterococcus infection produced an orchestrated response by the host cells involving several pathways and transcription factors that potentially drive these pathways. The early time points potentially identify novel targets for enhancing the host response. These approaches combine rigorous statistical principles with a biological context and are readily applied by biologists.

Suppression and Activation of the Malignant Phenotype by Extracellular Matrix in Xenograft Models of Bladder Cancer: A Model for Tumor Cell “Dormancy”

A major problem in cancer research is the lack of a tractable model for delayed metastasis. Herein we show that cancer cells suppressed by SISgel, a gel-forming normal ECM material derived from Small Intestine Submucosa (SIS), in flank xenografts show properties of suppression and re-activation that are very similar to normal delayed metastasis and suggest these suppressed cells can serve as a novel model for developing therapeutics to target micrometastases or suppressed cancer cells. Co-injection with SISgel suppressed the malignant phenotype of highly invasive J82 bladder cancer cells and highly metastatic JB-V bladder cancer cells in nude mouse flank xenografts. Cells could remain viable up to 120 days without forming tumors and appeared much more highly differentiated and less atypical than tumors from cells co-injected with Matrigel. In 40% of SISgel xenografts, growth resumed in the malignant phenotype after a period of suppression or dormancy for at least 30 days and was more likely with implantation of 3 million or more cells. Ordinary Type I collagen did not suppress malignant growth, and tumors developed about as well with collagen as with Matrigel. A clear signal in gene expression over different cell lines was not seen by transcriptome microarray analysis, but in contrast, Reverse Phase Protein Analysis of 250 proteins across 4 cell lines identified Integrin Linked Kinase (ILK) signaling that was functionally confirmed by an ILK inhibitor. We suggest that cancer cells suppressed on SISgel could serve as a model for dormancy and re-awakening to allow for the identification of therapeutic targets for treating micrometastases.

Proteome-level display by 2-dimensional chromatography of extracellular matrix-dependent modulation of the phenotype of bladder cancer cells

BackgroundThe extracellular matrix can have a profound effect upon the phenotype of cancer cells. Previous work has shown that growth of bladder cancer cells on a matrix derived from normal basement membrane suppresses many malignant features that are displayed when the cells are grown on a matrix that has been modified by malignant tumors. This work was undertaken to investigate proteome-level changes as determined by a new commercially available proteome display involving 2-dimensional chromatography for bladder cancer cells grown on different extracellular matrix preparations that modulate the expression of the malignant phenotype.ResultsDepending on the matrix, between 1300 and 2000 distinct peaks were detected by two-dimensional chromatographic fractionation of 2.1 – 4.4 mg of total cellular protein. The fractions eluting from the reversed-phase fractionation were suitable for mass spectrometric identification following only lyophilization and trypsin digestion and achieved approximately 10-fold higher sensitivity than was obtained with gel-based separations. Abundant proteins that were unique to cells grown on one of the matrices were identified by mass spectrometry. Following concentration, peaks of 0.03 AU provided unambiguous identification of protein components when 10% of the sample was analyzed, whereas peaks of 0.05 AU was approximately the lower limit of detection when the entire sample was separated on a gel and in-gel digestion was used. Although some fractions were homogeneous, others were not, and up to 3 proteins per fraction were identified. Strong evidence for post-translational modification of the unique proteins was noted. All 13 of the unique proteins from cells grown on Matrigel were related to MYC pathway.ConclusionThe system provides a viable alternative to 2-dimensional gel electrophoresis for proteomic display of biological systems. The findings suggest the importance of MYC to the malignant phenotype of bladder cancer cells.