sirolimus and Retinoblastoma

BACKGROUND Biglycan (BGN) is an extracellular matrix (ECM) protein that regulates the growth of epithelial cells. The mammalian target of rapamycin (mTOR) inhibitor, rapamycin, is a treatment for advanced retinoblastoma. This study aimed to investigate the effects of expression of BGN on the response of human WERI-Rb-1 retinoblastoma cells to rapamycin and to investigate the associated signaling pathways. MATERIAL AND METHODS BGN gene expression was induced in human WERI-Rb-1 retinoblastoma cells, which were incubated with rapamycin at doses of 0, 5, 10, 20, 30, and 50 μg/ml. Cells were treated with the PI3K/Akt pathway inhibitor, LY294002. The MTT assay determined the rate of cell inhibition. Real-time polymerase chain reaction (RT-PCR) was performed to measure BGN gene expression using RT²-PCR. Western blot detected the protein levels of BGN, p-PI3K, p-Akt, nuclear NF-kappaB, and p65. RESULTS Rapamycin impaired cell growth, induced cell apoptosis, and suppressed the expression levels of p-PI3K, p-Akt, nuclear NF-kappaB, and p65. Overexpression of the BGN gene restored growth potential and inhibited apoptosis and was associated with the activation of the PI3K/Akt-mediated NF-kappaB pathway. In cells that overexpressed BGN, inhibition of the PI3K/Akt pathway by LY294002 increased the sensitivity of human WERI-Rb-1 retinoblastoma cells to rapamycin. CONCLUSIONS Overexpression of BGN induced rapamycin resistance in WERI-Rb-1 retinoblastoma cells by activating PI3K/Akt/NF-kappaB signaling.

Topics: Apoptosis; Biglycan; Cell Line, Tumor; Cell Proliferation; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Humans; Inhibitory Concentration 50; NF-kappa B; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Retinoblastoma; Signal Transduction; Sirolimus

Targeting the mammalian target of rapamycin (mTOR) is a promising strategy for cancer therapy. Temsirolimus, a FDA-approved anticancer drug with efficacy in certain solid tumors and hematologic malignancies, is a potent mTOR inhibitor. In this work, we are the first to provide preclinical evidence that temsirolimus is an attractive candidate for retinoblastoma treatment as a dual inhibitor of retinoblastoma and angiogenesis. We show that temsirolimus selectively inhibits growth, survival and migration of retinoblastoma cells while sparing normal retinal and fibroblast cells, with IC

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Line; Cell Line, Tumor; Cells, Cultured; Endothelial Cells; Humans; Mice; Neovascularization, Pathologic; Retinal Neoplasms; Retinoblastoma; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tumor Burden; Xenograft Model Antitumor Assays

Rapamycin is useful in the treatment of certain cancers by inhibiting mTOR(mammalian target of rapamycin) pathway. Here, anticancer activity and its acting mechanisms of rapamycin were investigated in human retinoblastoma Y79 cells. CCK-8 assay showed that the IC50 value of rapamycin against human retinoblastoma Y79 cells was 0.122±0.026 μmol/L. Flow cytometry analysis indicated that rapamycin induced G1 cell cycle arrest. Western blot assay demonstrated that the mTOR pathway in Y79 cells was blocked by rapamycin. Western blot and RT-PCR assay showed that Bmi-1 was downregulated in protein and mRNA level by rapamycin treatment. Further Western blot and RNA interference assays showed that rapamycin-mediated downregulation of Bmi-1 induced decreases of cyclin E1, which accounted for rapamycin-mediated G1 cell cycle arrest in human retinoblastoma cells. Together, all these results illustrated that rapamycin induced growth inhibition of human retinoblastoma cells, and inactive of mTOR pathway and downregulation of Bmi-1 was involved in its action mechanism.

Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Cyclin E; Dose-Response Relationship, Drug; Down-Regulation; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Humans; Inhibitory Concentration 50; Oncogene Proteins; Phosphorylation; Polycomb Repressive Complex 1; Protein Kinase Inhibitors; Retinal Neoplasms; Retinoblastoma; RNA Interference; RNA, Messenger; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transfection

We have shown that heightened AKT activity sensitized multiple myeloma cells to the antitumor effects of the mammalian target of rapamycin inhibitor CCI-779. To test the mechanism of the AKT regulatory role, we stably transfected U266 multiple myeloma cell lines with an activated AKT allele or empty vector. The AKT-transfected cells were more sensitive to cytostasis induced in vitro by rapamycin or in vivo by its analogue, CCI-779, whereas cells with quiescent AKT were resistant. The ability of mammalian target of rapamycin inhibitors to down-regulate D-cyclin expression was significantly greater in AKT-transfected multiple myeloma cells due, in part, to the ability of AKT to curtail cap-independent translation and internal ribosome entry site (IRES) activity of D-cyclin transcripts. Similar AKT-dependent regulation of rapamycin responsiveness was shown in a second myeloma model: the PTEN-null OPM-2 cell line transfected with wild-type PTEN. Because extracellular signal-regulated kinase (ERK)/p38 activity facilitates IRES-mediated translation of some transcripts, we investigated ERK/p38 as regulators of AKT-dependent effects on rapamycin sensitivity. AKT-transfected U266 cells showed significantly decreased ERK and p38 activity. However, only an ERK inhibitor prevented D-cyclin IRES activity in resistant "low-AKT" myeloma cells. Furthermore, the ERK inhibitor successfully sensitized myeloma cells to rapamycin in terms of down-regulated D-cyclin protein expression and G1 arrest. However, ectopic overexpression of an activated MEK gene did not increase cap-independent translation of D-cyclin in "high-AKT" myeloma cells, indicating that mitogen-activated protein kinase/ERK kinase/ERK activity was required, but not sufficient, for activation of the IRES. These data support a scenario where heightened AKT activity down-regulates D-cyclin IRES function in multiple myeloma cells and ERK facilitates activity.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Extracellular Signal-Regulated MAP Kinases; Humans; Male; Mice; Multiple Myeloma; Phosphorylation; Protein Biosynthesis; Protein Kinase Inhibitors; Protein Kinases; Proto-Oncogene Proteins c-akt; Retinoblastoma; Ribosomes; Sirolimus; TOR Serine-Threonine Kinases

Rapamycin and its analogues are being tested as new antitumor agents. Rapamycin binds to FKBP-12 and this complex inhibits the activity of FRAP/mammalian target of rapamycin, which leads to dephosphorylation of 4EBP1 and p70 S6 kinase, resulting in blockade of translation initiation. We have found that RAP inhibits the growth of HER-2-overexpressing breast cancer cells. The phosphorylation of mammalian target of rapamycin, p70 S6 kinase, and 4EBP1 is inhibited by rapamycin and cells are arrested in the G1 phase, as determined by growth assays, fluorescence-activated cell sorting analysis, and bromodeoxyuridine incorporation studies. Rapamycin causes down-regulation of cyclin D3 protein, retinoblastoma hypophosphorylation, loss of cyclin-dependent kinase (cdk) 4, cdk6, and cdk2 activity. The half-life of cyclin D3 protein decreases after rapamycin treatment, but not its synthesis, whereas the synthesis or half-life of cyclin D1 protein is not affected by the drug. Additionally, rapamycin caused accumulation of ubiquitinated forms of cyclin D3 protein, proteasome inhibitors blocked the effect of rapamycin on cyclin D3, and rapamycin stimulated the activity of the proteasome, showing that the effect of rapamycin on cyclin D3 is proteasome proteolysis dependent. This effect depends on the activity of HER-2 because Herceptin, a neutralizing antibody against HER-2, is able to block both the induction of proteasome activity and the cyclin D3 down-regulation due to rapamycin. Furthermore, inhibition of HER-2 gene expression by using small interfering RNA blocked the rapamycin effects on cyclin D3. These data indicate that rapamycin causes a G1 arrest in HER-2-overexpressing breast cancer cells that is associated with a differential destabilization and subsequent down-regulation of cyclin D3 protein.

Topics: Antibiotics, Antineoplastic; Breast Neoplasms; Cell Cycle; Cell Growth Processes; Cell Line, Tumor; Cyclin D3; Cyclin-Dependent Kinase 2; Cyclins; Down-Regulation; Humans; Oncogene Protein v-akt; Phosphatidylinositol 3-Kinases; Proteasome Inhibitors; Protein Kinases; Receptor, ErbB-2; Retinoblastoma; RNA, Messenger; Sirolimus; TOR Serine-Threonine Kinases; Ubiquitins