phalloidine and Breast-Neoplasms

Mounting evidence suggests involvement of deregulated microRNA (miRNA) expression during the complex events of tumorigenesis. Among such deregulated miRNAs in cancer, miR-125b expression is reported to be consistently low in breast cancers. In this study, we screened a panel of breast cancer cell lines (BCCLs) for miR-125b expression and detected decreased expression in 14 of 19 BCCLs. Due to the heterogeneity of breast cancers, MCF7 cells were chosen as a model system for ERBB2 independent breast cancers to restore miR-125b expression (MCF7-125b) to investigate the phenotypical and related functional changes. Earlier, miR-125b was shown to regulate cell motility by targeting ERBB2 in ERBB2 overexpressing breast cancer cells. Here we showed decreased motility and migration in miR-125b expressing MCF7 cells, independent of ERBB2. MCF7-125b cells demonstrated profoundly decreased cytoplasmic protrusions detected by phalloidin staining of filamentous actin along with decreased motility and migration behaviors detected by in vitro wound closure and transwell migration assays compared to empty vector transfected cells (MCF7-EV). Among possible numerous targets of miR-125b, we showed ARID3B (AT-rich interactive domain 3B) to be a novel target with roles in cell motility in breast cancer cells. When ARID3B was transiently silenced, the decreased cell migration was also observed. In light of these findings, miR-125b continues to emerge as an interesting regulator of cancer related phenotypes.

Topics: 3' Untranslated Regions; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Shape; DNA-Binding Proteins; Extracellular Signal-Regulated MAP Kinases; Female; Gene Expression; Gene Expression Regulation, Neoplastic; Genes, Reporter; Humans; Luciferases, Renilla; MicroRNAs; Phalloidine; Phosphorylation; Real-Time Polymerase Chain Reaction; Receptor, ErbB-2; RNA Interference

Cancer cells are characterized by a high rate of glycolysis, which is their primary energy source. Glycolysis is known to be controlled by allosteric regulators, as well as by reversible binding of glycolytic enzymes to cytoskeleton. Clotrimazole is an anti-fungal azole derivative recently recognized as a calmodulin antagonist with promising anti-cancer effect. Here, we show that clotrimazole induced morphological and functional alterations on human breast cancer derived cell line, MCF-7. The drug decreased cell viability in a dose- and time-dependent manner, exhibiting an IC50 of 88.6+/-5.3 microM and a t0.5 of 89.7+/-7.2 min, with 50 microM clotrimazole. Morphological changes were evident as observed by scanning electron microscopy, which revealed the completely loss of protrusion responsible for cell adhesion after a 180 min of treatment with 50 microM clotrimazole. Giemsa stained cells observed by optical microscopy show morphological alterations and a marked nuclear condensation. These changes occurred in parallel to the detachment of the glycolytic enzymes, 6-phosphofructo-1-kinase and aldolase, from cytoskeleton. After a 45 min treatment with 50 microM clotrimazole, the remaining activities in a cytoskeleton enriched fraction was 16.4+/-3.6% and 41.0+/-15.6% of control for 6-phosphofructo-1-kinase and aldolase, respectively. Immunocytochemistry experiments revealed a decrease in the co-localization of 6-phosphofructo-1-kinase and F-actin after clotrimazole treatment, suggesting the site of detachment of the enzymes. Altogether, our results support evidence for apoptotic events that might be started by clotrimazole involving inhibition of glycolytic flux in MCF-7 cells and makes this drug a promising agent in the fight against human breast cancer.

Topics: Actins; Breast Neoplasms; Carcinoma, Ductal, Breast; Cell Line, Tumor; Cell Survival; Clotrimazole; Cytoskeleton; Dose-Response Relationship, Drug; Female; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Fructose-Bisphosphate Aldolase; Glucose; Growth Inhibitors; Humans; Lactic Acid; Phalloidine; Phosphofructokinase-1; Rhodamines; Streptavidin

Inflammatory breast carcinoma (IBC) is a highly aggressive form of locally advanced breast cancer that has the ability to invade and block the dermal lymphatics of the skin overlying the breast. In a previous series of studies, our laboratory identified overexpression of RhoC GTPase in >90% of IBCs (K. L. van Golen et al., Clin. Cancer Res., 5: 2511-2519, 1999) and defined RhoC as a mammary oncogene involved in conferring the metastatic phenotype (K. L. van Golen et al., Cancer Res., 60: 5832-5838, 2000). RhoC GTPase is involved in cytoskeletal reorganization during cellular motility. Farnesyl transferase inhibitors (FTIs) were previously shown to be effective in modulating tumor growth in Ras-transformed tumor cells. Recently, studies have focused on RhoB as a putative non-Ras target of FTI action. In the present study, we assessed the effect of the FTI L-744,832 on RhoC-overexpressing IBC and RhoC-transfected human mammary epithelial (HME-RhoC) cells. Treatment of the SUM149 IBC cell line and HME-RhoC transfectants with the FTI L-744,832 led to reversion of the RhoC-induced phenotype, manifested by a significant decrease in anchorage-independent growth, motility, and invasion. Although RhoC expression and activation were not affected, RhoB levels were increased by FTI treatment. Transient transfection of geranylgeranylated RhoB (RhoB-GG) into the same cells reproduced the effects of the FTI, thus suggesting that FTI-induced reversion of the RhoC phenotype may be mediated by an increase in RhoB-GG levels. These data provide direct evidence that FTIs may find use in the clinic when directed against RhoC-overexpressing tumors and suggest appropriate biological markers to evaluate during FTI treatment.

Topics: Alkyl and Aryl Transferases; Apoptosis; Blotting, Western; Breast Neoplasms; Cell Division; Cell Line, Transformed; Cell Movement; Enzyme Inhibitors; Farnesyltranstransferase; Humans; Methionine; Microscopy, Electron, Scanning; Microscopy, Fluorescence; Phalloidine; Phenotype; Reverse Transcriptase Polymerase Chain Reaction; rho GTP-Binding Proteins; rhoC GTP-Binding Protein; Rhodamines; Transfection; Tumor Cells, Cultured