prostaglandin-d2 and Osteosarcoma

Despite considerable efforts to improve treatment modalities for osteosarcoma (OS), patient survival remains poor mainly due to pro-survival pathways in OS cells. Among others, prostaglandins (PGs) are the potent regulators of bone homoeostasis and OS pathophysiology. Therefore, the present study aimed to elucidate the impact of 15-deoxy-Δ(12,14)-PGJ2 (15d-PGJ2, a stable PGD2 degradation product) on cell death/cell survival pathways in p53-deficient MG-63 OS cells. Our findings show that 15d-PGJ2 induces generation of reactive oxygen species that promote p38 MAPK activation and subsequent Akt phosphorylation. This pathway induced nuclear expression of Nrf2 and Egr1, and increased transcription of haem oxygenase-1 (HO-1) and the catalytic subunit of glutamate cysteine ligase (GCLc), catalysing the first step in GSH synthesis. Silencing of Nrf2, Egr1 and HO-1 significantly elevated 15d-PGJ2-mediated reduction of cellular metabolic activity. Activation of cell survival genes including HO-1 and GCLc inhibited 15d-PGJ2-induced cleavage of pro-caspase-3 and PARP. Annexin V/propidium iodide staining showed an increase in early/late apoptotic cells in response to 15d-PGJ2. The observed 15d-PGJ2-mediated signalling events are independent of PGD2 receptors (DP1 and DP2) and PPARγ. In addition, the electrophilic carbon atom C9 is a prerequisite for the observed activity of 15d-PGJ2. The present data show that the intracellular redox imbalance acted as a node and triggered both death and survival pathways in response to 15d-PGJ2. Pharmacological or genetic interference of the pro-survival pathway, the p38 MAPK/Akt/Nrf2-Egr1/HO-1-GCLc axis, sensitizes MG-63 cells towards 15d-PGJ2-mediated apoptosis.

Topics: Apoptosis; Catalytic Domain; Cell Line, Tumor; Cell Survival; Early Growth Response Protein 1; Gene Silencing; Glutamate-Cysteine Ligase; Heme Oxygenase-1; Humans; Mitogen-Activated Protein Kinases; NF-E2-Related Factor 2; Osteoblasts; Osteosarcoma; Oxidation-Reduction; Phosphorylation; Prostaglandin D2; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Signal Transduction

Polo-like kinase 1 (PLK1), a critical cell cycle regulator, has been identified as a potential target in osteosarcoma (OS). 15-deoxy-Δ12, 14-prostaglandin J2 (15d-PGJ2), a prostaglandin derivative, has shown its anti-tumor activity by inducing apoptosis through reactive oxygen species (ROS)-mediated inactivation of v-akt, a murine thymoma viral oncogene homolog, (AKT) in cancer cells. In the study analyzing its effects on arthritis, 15d-PGJ2 mediated shear-induced chondrocyte apoptosis via protein kinase A (PKA)-dependent regulation of PLK1. In this study, the cytotoxic effect and mechanism underlying 15d-PGJ2 effects against OS were explored using OS cell lines. 15d-PGJ2 induced significant G2/M arrest, and exerted time- and dose-dependent cytotoxic effects against all OS cell lines. Western blot analysis showed that both AKT and PKA-PLK1 were down-regulated in OS cell lines after treatment with 15d-PGJ2. In addition, transfection of constitutively active AKT or PLK1 partially rescued cells from 15d-PGJ2-induced apoptosis, suggesting crucial roles for both pathways in the anti-cancer effects of 15d-PGJ2. Moreover, ROS generation was found treatment with 15d-PGJ2, and its cytotoxic effect could be reversed with N-acetyl-l-cysteine. Furthermore, inhibition of JNK partially rescued 15d-PGJ2 cytotoxicity. Thus, ROS-mediated JNK activation may contribute to apoptosis through down-regulation of the p-Akt and PKA-PLK1 pathways. 15d-PGJ2 is a potential therapeutic agent for OS, exerting cytotoxicity mediated through both AKT and PKA-PLK1 inhibition, and these results form the basis for further analysis of its role in animal studies and clinical applications.

Topics: Apoptosis; Bone Neoplasms; Cell Cycle; Cell Cycle Proteins; Cell Growth Processes; Cell Line, Tumor; Down-Regulation; G2 Phase Cell Cycle Checkpoints; Humans; JNK Mitogen-Activated Protein Kinases; M Phase Cell Cycle Checkpoints; Osteosarcoma; Phosphorylation; Polo-Like Kinase 1; Prostaglandin D2; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species

Prostaglandins (PGs), important modulators in bone biology, may also contribute to tumor formation and progression in human osteosarcoma. 15-Deoxy-Δ(12,14)-PGJ(2) (15d-PGJ(2)), a metabolite of PGD(2) and PPARγ-ligand, exerts a panel of biological activities via receptor-dependent and -independent mechanisms. As inducible cyclooxygenase-2 (Cox-2) is a candidate inflammatory marker in human osteosarcoma and a rate-limiting enzyme in PG biosynthesis, this study aimed at investigating intracellular redox status and signaling cascades leading to Cox-2 induction in human MG-63 osteosarcoma cells. 15d-PGJ(2) induced the accumulation of reactive oxygen species (ROS) that in turn may lead to upregulation of Cox-2 via two different routes in a PPARγ-independent manner. First, phosphorylation of p38 MAPK directly enhances Cox-2 expression by promoting mRNA stability. Second, 15d-PGJ(2) induces activation of epidermal growth factor receptors and downstream activation of Cox-2 via phosphorylation of p42/44 MAPK. Glutathione precursor molecules reversed enhanced ROS levels and Cox-2 expression. Functional activity of Cox-2 expression was tested by measurement of PGE(2) and PGF(2α). The synthetic compound 9,10-dihydro-15d-PGJ(2) lacking the α,β-unsaturated carbonyl group in the cyclopentenone ring did not exhibit the cellular responses observed with 15d-PGJ(2). We conclude that the electrophilic carbon atom of 15d-PGJ(2) is responsible for alterations in intracellular redox status and Cox-2 expression.

Topics: Animals; Cell Line, Tumor; Cyclooxygenase 2; Dinoprost; Dinoprostone; ErbB Receptors; Gene Expression; Glutathione; Humans; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neoplasms; Osteosarcoma; Oxidation-Reduction; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Prostaglandin D2; Reactive Oxygen Species; RNA Stability; Signal Transduction; Up-Regulation

We examined the protective effects of cycloheximide against cytotoxicity induced by vincristine, colchicine, delta 12-prostaglandin J2, or other antitumor agents on the human osteosarcoma cell line, KSu. Vincristine at a concentration of 0.5 microgram/ml decreased the initial cell number to 34% during 4 days; however, when cycloheximide (0.5 to 10 micrograms/ml) was coexistent, the decrease of the cell number was suppressed and 68% of the initial cells remained viable at the maximum. Furthermore, 0.1 micrograms/ml of cycloheximide also reduced cytotoxicity of colchicine (0.1 to 5 microM) or delta 12-prostaglandin J2 (1 to 5 micrograms/ml) and reduced the cytotoxicity of 0.1 microgram/ml of doxorubicin or 1 micrograms/ml of mitomycin C, suggesting that protection by cycloheximide is shown against cytotoxicity of various types of antitumor agents even on human malignant cells. Next, protein synthesis was reduced to 52% of a control at 3 h by 0.1 micrograms/ml of cycloheximide, suggesting that protein synthesis inhibition precedes the protection. De novo protein synthesis analysis showed that vincristine (0.5 microgram/ml) does not induce any specific protein, whereas delta 12-prostaglandin J2 (3 or 4 micrograms/ml) induced Mr 70,000 and 90,000 proteins, and these were markedly inhibited by cycloheximide (0.1 microgram/ml). In a cell-cycle study, M-phase arrest by vincristine (0.5 microgram/ml) was inhibited in the presence of 0.1 microgram/ml of cycloheximide, suggesting that cell cycle arrest by cycloheximide may be important for protection. From these data, this protection by cycloheximide seems to be more general than expected before.

Topics: Cell Cycle; Cell Survival; Colchicine; Cycloheximide; Humans; Osteosarcoma; Prostaglandin D2; Protein Biosynthesis; Tumor Cells, Cultured; Vincristine

Effects of antineoplastic prostaglandins (PG), PGD2 and 9-deoxy-delta 9-PGD2, on colony formation of cloned Dunn osteosarcoma (TA 102), normal Swiss 3T3 and V-79 cell lines were evaluated. PGD2 significantly inhibited the colony formation of TA 102 cells in a dose-dependent manner at concentrations between 0.5 and 5 micrograms/ml. The IC50 value was calculated to be 0.72 microgram/ml. A dose-dependent inhibition of TA 102 colony formation was also observed with 9-deoxy-delta 9-PGD2 between 0.01 to 1 microgram/ml, the IC50 value being 0.22 microgram/ml. These prostaglandins did not exert cytocidal effects in vitro on Swiss 3T3 cells at concentrations between 0.01 to 1 microgram/ml. The two agents had no significant cytocidal effects on V-79 cells except for 9-deoxy-delta 9-PGD2 at a concentration of 5 ug/ml. These results suggest that PGD2 and 9-deoxy-delta 9-PGD2 are considered to have cytocidal activity on Dunn osteosarcoma cells in dosages which do not affect non-malignant cells.

Topics: Animals; Cell Division; Cell Line; Cells, Cultured; Cricetinae; Dose-Response Relationship, Drug; Mice; Osteosarcoma; Prostaglandin D2; Prostaglandins D

The cytotoxic effect of prostaglandin (PG) D2, PGE1 and PGF2 alpha was examined on human osteosarcoma cells (KSu cell line) in vitro, and PGD2 was most effective. DNA, RNA and protein syntheses of KSu cells were also found to be inhibited by PGD2 at a concentration of 5 micrograms/ml. Furthermore, the proliferation of various human malignant tumor cells was inhibited by PGD2 without exception so far. These results suggest that PGD2 shows an anti-neoplastic effect on a variety of human malignant tumor cells.

Topics: Antineoplastic Agents; Cell Division; Cell Line; Dinoprost; Dinoprostone; Humans; Neoplasm Proteins; Neoplasms; Nucleic Acids; Osteosarcoma; Prostaglandin D2; Prostaglandins D; Prostaglandins E; Prostaglandins F