ABSTRACT
Fibronectin (FN) expressed by tumor cells has been known to be tumor suppressive but the pericellular FN (periFN) assembled on circulating tumor cells appears to evidently promote distant metastasis. Whereas the regulation of periFN assembly in suspended cells has currently been under investigation, how it is regulated in adherent tumor cells and the role of periFN in primary tumor growth remain elusive. Techniques of RNAi, plasmid transfections, immunoblotting, fluorescence/immunohistochemistry staining, cell proliferation assays, and primary tumor growth in C57BL6 mice and Fischer 344 rats were employed in this study. We found that endogenously synthesized FN in adherent tumor cells was required for periFN assembly which was aligned by RhoA-organized actin stress fiber (SF). Depleting periFN on adherent tumor cells congruently promoted in vivo tumor growth but surprisingly did not autonomously impact on in vitro tumor cell proliferation and apoptosis, suggestive of a non-autonomous role of periFN in in vivo tumor growth. We showed that the proliferative ability of shFN-expressing tumor cells was higher than shScramble cells did in the presence of fibroblasts. Altogether, these results suggested that depriving RhoA/SF-regulated periFN matrices non-autonomously promotes fibroblast-mediated tumor cell growth.
Subject(s)
Extracellular Matrix/metabolism , Fibroblasts/physiology , Fibronectins/metabolism , Neoplasms/pathology , Stress Fibers/metabolism , rhoA GTP-Binding Protein/metabolism , Animals , Cell Adhesion/genetics , Cell Proliferation/genetics , Extracellular Matrix/pathology , Fibroblasts/pathology , Fibronectins/genetics , Mice , Mice, Inbred C57BL , Neoplasm Metastasis , Neoplasms/metabolism , Rats , Rats, Inbred F344 , Stress Fibers/pathology , Tumor Burden/physiology , Tumor Cells, Cultured , rhoA GTP-Binding Protein/geneticsABSTRACT
Metastasis is a major obstacle that must be overcome for the successful treatment of lung cancer. Proteins secreted by cancer cells may facilitate the progression of metastasis, particularly within the phases of migration and invasion. To discover metastasis-promoting secretory proteins within cancer cells, we used the label-free quantitative proteomics approach and compared the secretomes from the lung adenocarcinoma cell lines CL1-0 and CL1-5, which exhibit low and high metastatic properties, respectively. By employing quantitative analyses, we identified 660 proteins, 68 of which were considered to be expressed at different levels between the two cell lines. High levels of A1AT were secreted by CL1-5, and the roles of A1AT in the influence of lung adenocarcinoma metastasis were investigated. Molecular and pathological confirmation demonstrated that altered expression of A1AT correlates with the metastatic potential of lung adenocarcinoma. The migration and invasion properties of CL1-5 cells were significantly diminished by reducing the expression and secretion of their A1AT proteins. Conversely, the migration and invasion properties of CL1-0 cells were significantly increased through the overexpression and secretion of A1AT proteins. Furthermore, the assembly levels of the metastasis-promoting pericellular fibronectin (FN1), which facilitates colonization of lung capillary endothelia by adhering to the cell surface receptor dipeptidyl peptidase IV (DPP IV), were higher on the surfaces of suspended CL1-5 cells than on those of the CL1-0 cells. This discovery reflects previous findings in breast cancer. In line with this finding, FN1 assembly and the lung colonization of suspended CL1-5 cells were inhibited when endogenous A1AT protein was knocked down using siRNA. The major thrust of this study is to demonstrate the effects of coupling the label-free proteomics strategy with the secretomes of cancer cells that differentially exhibit invasive and metastatic properties. This provides a new opportunity for the effective identification of metastasis-associated proteins that are secreted by cancer cells and promote experimental metastasis.
Subject(s)
Adenocarcinoma/metabolism , Adenocarcinoma/pathology , Cell Movement , Fibronectins/metabolism , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Lung/pathology , Neoplasm Proteins/metabolism , alpha 1-Antitrypsin/metabolism , Adenocarcinoma of Lung , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Culture Media, Conditioned/pharmacology , Humans , Immunohistochemistry , Lung/metabolism , Male , Middle Aged , Neoplasm Invasiveness , Neoplasm Metastasis , Polymerization/drug effects , Reproducibility of ResultsABSTRACT
We report the fluorescent labeling of osteoblast cells using the biocompatible hydroxyapatite (HA) grown with nucleating seed of hydrophilic CdSe/ZnS quantum dots (QDs) allowing the real-time observation of cell under confocal microscope. We found that the MC3T3-E1 osteoblast cells can engulf HA with surface-tailored QDs showing fluorescent spots in the cytoplasm, while HA and QDs nanoparticles were not engulfed. It is interesting to see that the fluorescence was only displayed in the cytoplasm of MC3T3-E1 osteoblast cells. It can be envisioned that the nano-sized hydroxyapatite bearing fluorescent QD can only be internalized in the cytoplasm. Therefore, it is worth utilizing these composite particles to observe cellular physiology with minimal toxicity to the osteoblast cells.
Subject(s)
Cadmium Compounds/metabolism , Durapatite/chemistry , Nanoparticles/chemistry , Osteoblasts/cytology , Quantum Dots , Selenium Compounds/metabolism , Sulfides/metabolism , Zinc Compounds/metabolism , 3T3 Cells , Animals , Biological Transport , Cadmium Compounds/chemistry , Cell Survival , Durapatite/metabolism , Fluorescence , Mice , Microscopy, Confocal , Nanoparticles/ultrastructure , Osteoblasts/metabolism , Selenium Compounds/chemistry , Staining and Labeling/methods , Sulfides/chemistry , Zinc Compounds/chemistryABSTRACT
Eight new xenia diterpenoids, blumiolide-A (1) (novel carbon skeleton), blumiolide-B (2), 9-deoxy-isoxeniolide-A (3), 9-deoxy-7,8-epoxy-isoxeniolide-A (4), 9-deacetoxy-7,8-epoxy-13-epi-xenicin (5), 9-deoxy-7,8-epoxy-xeniolide-A (6), blumiolide-C (7), and blumicin-A (8), were isolated from the methylene chloride solubles of the Formosan soft coral Xenia blumi. Their structures were elucidated by extensive spectroscopic analysis, and their cytotoxicity against selected cancer cells was measured in vitro.
