formazans and Osteosarcoma

Cyclooxygenase2 (COX-2), one isoform of cyclooxygenase proinflammatory enzymes, is responsible for tumor development, invasion and metastasis. Due to its role and frequent overexpression in a variety of human malignancies, including osteosarcoma, COX-2 has received considerable attention. However, the function of COX-2 in the pathogenesis of cancer is not well understood. We examined the role of COX-2 in osteosarcoma.. We employed lentivirus mediated-RNA interference technology to knockdown endogenous gene COX-2 expression in human osteosarcoma cells (SaOS2) and analyzed the phenotypical changes. The effect of COX-2 treatment on the proliferation, cell cycle, invasion and migration of the SaOS2 cells were assessed using the MTT, flow cytometry, invasion and migration assays, respectively. COX-2, vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) mRNA and protein expression were detected by RT-PCR and western blotting.. Our results indicate that a decrease of COX-2 expression in human osteosarcoma cells significantly inhibited the growth, decreased the invasion and migration ability of SaOS2 cells. In addition, it also reduced VEGF, EGF and bFGF mRNA and protein expression.. The COX-2 signaling pathway may provide a novel therapeutic target for the treatment of human osteosarcoma.

Topics: Bone Neoplasms; Case-Control Studies; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cyclooxygenase 2; Flow Cytometry; Formazans; Gene Knockdown Techniques; Humans; Neoplasm Invasiveness; Osteosarcoma; RNA Interference; Tetrazolium Salts

To introduce a rapid colorimetric method for assessing the viability of osteosarcoma cells after chemotherapy.. Colorimetric assay and automatic microplate scanning spectrophotometer were used for assaying the viability of osteosarcoma cells.. Close correlation was found between the absorbance at 570 nm of the formazan products and the number of viable osteosarcoma cells.. An effective, sensitive and convenient colorimetric assay has been established to assess the survival of osteosarcoma cells following chemotherapy.

Topics: Antineoplastic Agents; Bone Neoplasms; Cell Survival; Colorimetry; Formazans; Humans; Osteosarcoma; Sensitivity and Specificity; Tetrazolium Salts

To characterize and compare the different biological behaviors of 2 novel human osteosarcoma cell lines, Zos and Zos-M, established respectively from the primary tumor and the skip metastasis of an osteosarcoma patient.. In vitro studies included morphological observations, karyotype analysis, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell proliferation assay, and cell sensitivity to chemotherapeutic drugs. Subcutaneous and intravenous inoculations into nude mice were carried out to study the tumorigenicity and the metastatic potential. RT-PCR was performed to assess the expression of the osteoblastic markers and some metastasis-related genes.. Both cell lines remained stable for more than 100 passages in vitro without interruption. The RT-PCR examination indicated that they retained the molecular characteristics of an osteoblastic lineage. The karyotype analysis displayed aneuploidy and various structural abnormalities. Both cell lines are tumorigenic; Zos-M differs from Zos by the former's ability to develop lung metastasis after intravenous injection. The comparison of the expression patterns of some metastasis-related genes revealed that the decreased expression of cadherin-11 in Zos-M may correlate with a high potential of metastases. Moreover, both cell lines are less sensitive to the current chemotherapy protocols.. The establishment of osteosarcoma cell lines, Zos and Zos-M, and related animal models provide a useful resource for studying the aggressive behavior of osteosarcoma and will be helpful for screening effective treatment strategies.

Topics: Adolescent; Animals; Antimetabolites, Antineoplastic; Biomarkers, Tumor; Cell Line, Tumor; Cell Lineage; Cell Proliferation; Collagen; Drug Combinations; Extracellular Matrix; Formazans; Humans; Inhibitory Concentration 50; Karyotyping; Laminin; Male; Methotrexate; Mice; Mice, Nude; Neoplasm Metastasis; Osteosarcoma; Proteoglycans; Tetrazolium Salts; Xenograft Model Antitumor Assays

Statin, a HMG-CoA reductase inhibitor, was shown to increase BMP-2 gene expression for bone formation, by blocking the mevalonate pathway in cholesterol production. We investigated the effect of naringin, a flavonoid available commonly in citrus fruits, which was also a HMG-CoA reductase inhibitor, in UMR 106 osteoblastic cell line in vitro. The control group consisted of cells cultured without any intervention for different time intervals (24 h, 48 h, and 72 h), whereas the experimental (naringin) group consisted of cells cultured with naringin of different concentrations (0.001 micromol/L, 0.01 micromol/L, and 0.1 micromol/L) for the same time intervals of the control. Colorimetric Tetrazolium (MTT) assay, total protein content assay, and alkaline phosphatase activity were used to measure the cellular activities. Results for the naringin group showed an increase in MTT assay compared with the control and the effect was dose dependent. At high concentration (0.1 micromol), the increases ranged from 60% to 80%. In the total protein content assay, naringin also showed an increase compared with control and the effect was also dose dependent. At high concentration (0.1 micromol), the increases ranged from 9% to 20%. In the alkaline phosphatase activity assay, naringin at high concentration (0.1 micromol) significantly increased the activity up to 20%. In conclusion, naringin significantly increased bone cell activities in vitro. This is the first study specifically attempted to investigate the effect of naringin on bone cell activities. Besides statin, this provided another example of mevalonate pathway blockage in the cholesterol production pathway by HMG-CoA reductase inhibition will increase the bone cell activities.

Topics: Alkaline Phosphatase; Animals; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Flavanones; Formazans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Osteoblasts; Osteosarcoma; Proteins; Rats; Tetrazolium Salts

To determine whether exposure of canine osteosarcoma cells to deracoxib or piroxicam results in decreased viability, whether the cytotoxic effects of deracoxib and piroxicam involve induction of apoptosis, and whether deracoxib is a more potent inhibitor of osteosarcoma cell growth than piroxicam.. 1 fibroblast and 3 osteosarcoma cell lines.. Cell counts and viability assays were performed using osteosarcoma cells (POS, highly metastatic POS, and canine osteosarcoma cell 31) and fibroblasts after 72 hours of incubation with deracoxib at concentrations of 0.5 microM to 500 microM or piroxicam at concentrations of 1 microM to 1,000 microM. Percentage viability was determined for each concentration. A DNA fragmentation analysis was performed to assess drug-induced apoptosis.. Concentration of deracoxib required for 50% inhibition of cell viability (IC50) was reached in all 3 osteosarcoma cell lines and ranged from 70 to 150 microM, whereas the IC50 for piroxicam was only reached in the POS cell line at 500 microM. Neither deracoxib nor piroxicam induced sufficient toxicity in fibroblasts to reach an IC50. Exposure of osteosarcoma cells to cytotoxic concentrations of deracoxib and piroxicam did not result in DNA fragmentation.. Intermediate and high concentrations of deracoxib and high concentrations of piroxicam were cytotoxic to osteosarcoma cells; neither drug inhibited cell viability at typical plasma concentrations in dogs. Deracoxib inhibited viability of cells at concentrations that did not affect fibroblast viability. There was no evidence of apoptosis induction for either drug; however, only 1 cell line was evaluated for apoptosis induction and only for a limited selection of drug concentrations.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Bone Neoplasms; Cell Count; Cell Line, Tumor; Cell Survival; DNA Fragmentation; Dog Diseases; Dogs; Electrophoresis, Agar Gel; Formazans; Inhibitory Concentration 50; Osteosarcoma; Piroxicam; Sulfonamides; Tetrazolium Salts

Using a model with external ligation of the thigh, the effect of ischemia-reperfusion injury on tumor growth and the activity of lung metastasis was investigated in mice inoculated a spontaneous murine osteosarcoma cell line (POS-1) in vivo. POS-1 cell suspension was inoculated into the right hind footpad of 70 mice. Four weeks after inoculation, the ipsilateral thigh was ligated for 3 h in 15 mice and the contralateral thigh in 15 mice. Another ten mice were inoculated with POS-1 without ligating the thigh. The number of metastatic foci on the lung surface 6 weeks after inoculation was 2.29+/-0.98 (mean+/-SE) foci/lungs in mice with ipsilateral ligation and 6.25+/-2.41 in mice with contralateral ligation, which were significantly lower than control (13.40+/-1.42 in mice no ligation) (P<0.01). The number of metastatic foci on the lung surface in mice with intraperitoneal injection of superoxide dismutase (SOD) and catalase was 3.25+/-0.65 (mean+/-SE) foci/lungs in mice with ligation which was significantly greater than that in mice without SOD and catalase injection 1.29+/-0.97 (P=0.04). Cell viability was 9.12+/-4.07% with 100 microM H(2)O(2) in 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. It revealed that at concentrations of 100 microM H(2)O(2) or higher was cytotoxic to POS-1. In cell invasion assay, the number of invading cells with 10 microM H(2)O(2) was 2.80+/-0.53 cells/field, which was significantly lower than control (5.93+/-0.18) (mean+/-SE), indicating that low-dose H(2)O(2) suppressed invasion of POS-1. These results suggested that reperfusion injury had selective cytotoxicity to POS-1 through producing reactive oxygen species. Activated oxygen was considered to inhibit the regional growth and the ability of lung metastasis of POS-1 cells.

Topics: Animals; Bone Neoplasms; Catalase; Cell Division; Cell Survival; Disease Models, Animal; Formazans; Hydrogen Peroxide; Ligation; Lung Neoplasms; Male; Mice; Mice, Inbred C3H; Neoplasm Transplantation; Osteosarcoma; Reactive Oxygen Species; Reperfusion Injury; Superoxide Dismutase; Tetrazolium Salts; Thigh; Tumor Cells, Cultured