sirolimus and thiazolyl-blue

Despite careful patient selection, hepatocellular carcinoma (HCC) recurs in 10-20% of cases after liver transplantation, and the use of potent adjuvant anticancer drugs would be welcome. The aim of this study was to evaluate the efficiency of a combined therapy of rapamycin (sirolimus) and anti-death receptor (DR)5 monoclonal antibody (mAb) on HCC.. We first assessed the side effects of anti-DR5 mAb administration in vivo by giving various doses of anti-DR5 mAb. Cell proliferation assays were then performed using mouse Hepa1-6 cells or human Huh7 cells to quantify the relative cell viability under various concentrations of sirolimus, anti-DR5 mAb or a combination. Next, one million Hepa1-6 cells were transplanted into C.B17-SCID-beige mice subcutaneously, and four groups were created: (1) untreated, (2) anti-DR5 mAb alone, (3) sirolimus alone and (4) anti-DR5 mAb + sirolimus.. Anti-DR5 mAb (200 and 300 μg/day) induced liver dysfunction with partial necrosis of the liver, but 100 μg/day was well tolerated with transaminitis, but normal bilirubin and only minor histological liver damage. In vitro, anti-DR5 mAb lysed Hepa1-6 and Huh7 cells in a dose-dependent manner, and combinations of sirolimus and anti-DR5 mAb demonstrated an additive effect. In vivo studies demonstrated that tumour sizes were significantly smaller in the combined therapy group than in the monotherapy groups.. Combining sirolimus and low-dose anti-DR5 mAb has a significant effect against HCC. This strategy represents a potential novel approach for the management of HCC.

Topics: Analysis of Variance; Animals; Antibodies, Monoclonal; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Hepatocellular; Cell Line; Cell Proliferation; Cell Survival; Dose-Response Relationship, Drug; Drug Synergism; Humans; Liver Neoplasms; Mice; Mice, Inbred C57BL; Receptors, TNF-Related Apoptosis-Inducing Ligand; Sirolimus; Tetrazolium Salts; Thiazoles

Trimethyltin (TMT) is a triorganotin compound which determines neurodegeneration of specific brain areas particularly damaging the limbic system. Earlier ultrastructural studies indicated the formation of autophagic vacuoles in neurons after TMT intoxication. However, no evaluation has been attempted to determine the role of the autophagic pathway in TMT neurotoxicity. To assess the contribution of autophagy to TMT-induced neuronal cell death, we checked the vulnerability of neuronal cultures to TMT after activation or inhibition of autophagy. Our results show that autophagy inhibitors (3-methyladenine and L-asparagine) greatly enhanced TMT neurotoxicity. Conversely, known activators of autophagy, such as lithium and rapamycin, displayed neuroprotection against this toxic compound. Due to its diverse targets, the action of lithium was complex. When lithium was administered according to a chronic treatment protocol (6 days pretreatment) it was able to rescue both hippocampal and cortical neurons from TMT (or from glutamate toxicity used as reference). This effect was accompanied by an increased phosphorylation of glycogen synthase kinase 3 which is a known target for lithium neuroprotection. If the pre-incubation time was reduced to 2 h (acute treatment protocol), lithium was still able to counteract TMT toxicity in hippocampal but not in cortical neurons. The neuroprotective effect of lithium acutely administered against TMT in hippocampal neurons can be completely reverted by an excess of inositol and is possibly related to the inactivation of inositol monophosphatase, a key regulator of autophagy. These data indicate that TMT neurotoxicity can be dramatically modified, at least in vitro, by lithium addition which seems to act through different mechanisms if acutely or chronically administered.

Topics: Adenine; Adjuvants, Immunologic; Aldehydes; Analysis of Variance; Animals; Asparagine; Autophagy; Brain; Cell Count; Cells, Cultured; Dose-Response Relationship, Drug; Embryo, Mammalian; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; L-Lactate Dehydrogenase; Lithium Chloride; Mice; Mice, Inbred C57BL; Microscopy, Electron, Transmission; Mitochondria; Neurons; Phosphorylation; Serine; Sirolimus; Tetrazolium Salts; Thiazoles; Trimethyltin Compounds; Vacuoles

Recent evidence has suggested that breast cancer contains a rare population of cells called cancer stem cells (CSCs), which have an extensive self-renewal ability and contribute to metastasis and therapeutic resistance. This study evaluated the in vitro and in vivo effects of RAD001 (Everolimus) alone or in combination with docetaxel on stem cells from primary breast cancer cells and two breast cancer cell lines (MCF-7 and MDA-MB-231). In In vitro studies, we sorted ESA(+)CD44(+)CD24(-/low) cells as stem cells using flow cytometry from primary breast cancer cells, MCF-7 and MDA-MB-231 cell lines. MTT assays were used to quantify the inhibitory effect of the drugs on total cells and stem cells. Apoptosis and the cell cycle distributions of stem cells were examined by flow cytometry. The tumourigenicity of stem cells after treatment was investigated by soft agar colony formation assays. In In vivo studies, the BALB/c mice were injected with MDA-MB-231 stem cells and the different treatments were administered. After necropsy, the expression of Ki67, CD31, AKT1, and phospho-AKT (Thr308) was analysed by immunohistochemistry. In In vitro studies, all three populations of stem cells were resistant to the standard treatment doses of docetaxel compared with total cells treated with the same drug. Treatment with RAD001 resulted in growth inhibition of all stem cells in a dose-dependent manner. An additive growth inhibitory effect of the combination treatment on the three stem cells was observed in in vitro compared with treatment with RAD001 alone (P<0.001). In addition, an increase in G2/M cell cycle arrest and an increased population of cells in early apoptosis were seen in the combination treatment group compared with either single-agent group (P<0.01). In vivo, the volumes of the xenograft tumours significantly decreased in RAD001 alone group compared to control group (P=0.008), and RAD001 plus docetaxel therapy was much more effective at reducing tumour volume in mice compared with either single-agent alone (P<0.05). Compared with RAD001 alone, the combination of RAD001 and docetaxel reduced the expression of Ki67, CD31, AKT1 and phospho-AKT (Thr308) (P<0.05). We conclude that the combination treatment of RAD001 and docetaxel can inhibit the growth of stem cells in vitro and in vivo by inhibiting cell proliferation, inducing apoptosis, cell cycle arrest and reducing the expression of Ki67, CD31, AKT1 and phospho-AKT (Thr308). These data indicate that combinati

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Docetaxel; Dose-Response Relationship, Drug; Everolimus; Female; Humans; Immunohistochemistry; Immunosuppressive Agents; Mice; Mice, Inbred BALB C; Mice, Nude; Sirolimus; Stem Cells; Taxoids; Tetrazolium Salts; Thiazoles

Rapamycin is an attractive approach for the treatment and prevention of HCC recurrence after liver transplantation. However, the objective response rates of rapamycin achieved with single-agent therapy were modest, supporting that rapamycin resistance is a frequently observed characteristic of many cancers. Some studies have been devoted to understanding the mechanisms of rapamycin resistance, however, the mechanisms are cell-type-dependent and studies on rapamycin resistance in HCC are extremely limited.. The anti-tumor sensitivity of rapamycin was modest in vitro and in vivo. In both human and rat HCC cells, rapamycin up-regulated the expression and phosphorylation of PDGFRβ in a time and dose-dependent manner as assessed by RT-PCR and western blot analysis. Using siRNA mediated knockdown of PDGFRβ, we confirmed that subsequent activation of AKT and ERK was PDGFRβ-dependent and compromised the anti-tumor activity of rapamycin. Then, blockade of this PDGFRβ-dependent feedback loop by sorafenib enhanced the anti-tumor sensitivity of rapamycin in vitro and in an immunocompetent orthotopic rat model of HCC.. Activation of PI3K/AKT and MAPK pathway through a PDGFRβ-dependent feedback loop compromises the anti-tumor activity of rapamycin in HCC, and blockade of this feedback loop by sorafenib is an attractive approach to improve the anti-tumor effect of rapamycin, particularly in preventing or treating HCC recurrence after liver transplantation.

Topics: Animals; Blotting, Western; Carcinoma, Hepatocellular; Cell Line, Tumor; Drug Resistance, Neoplasm; Enzyme Activation; Feedback, Physiological; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Immunohistochemistry; Liver Neoplasms; Male; Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Rats; Receptor, Platelet-Derived Growth Factor beta; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Signal Transduction; Sirolimus; Tetrazolium Salts; Thiazoles

mTOR (mammalian target of rapamycin) inhibitors were recently found to be effective in the treatment of various human non-Hodgkin's lymphomas (NHLs). We recently reported that RAD001, an mTOR inhibitor, suppressed the growth of lymphoma cells at concentrations much lower than those required for carcinomas. However, the basis for the enhanced sensitivity to RAD001 is unknown. Seven aggressive NHL cell lines and seven carcinoma cell lines were used in this study. Cell cycle was analysed by flow cytometry. pAKT (phosphorylated AKT) (Ser(473) and Thr(308)), p-p70S6K, p-4E-BP1, p-mTOR, p-eIF4E (phosphorylated eIF4E), cyclin A, cyclin E, cyclin D3, c-Myc and insulin receptor substrate-1 (IRS-1) protein expression were assessed by immunoblotting. The PI3K/AKT/mTOR (phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin) signalling pathway was constitutively expressed in all seven lymphoma cell lines. RAD001 down-regulated p-mTOR, p-p70S6K, p-4E-BP1, cyclin A, cyclin E, cyclin D3, and c-Myc, but did not affect IRS-1. In parallel with RAD001-induced inhibition of cell viability, a dose- and schedule- dependent down-regulation of pAKT and p-eIF4E expressions was demonstrated. In contrast, a compensatory activation of pAKT and p-eIF4E, was observed in seven carcinoma cells. These findings indicate that the basis for enhanced activity of mTOR inhibitors in NHL may be the lack of compensatory activation of AKT and eIF4E phosphorylation in lymphoma cells.

Topics: Apoptosis; Carcinoma; Cell Cycle; Cell Line, Tumor; Eukaryotic Initiation Factor-4E; Everolimus; Gene Expression Regulation, Neoplastic; Humans; Immunosuppressive Agents; Jurkat Cells; Lymphoma; Models, Biological; Phosphorylation; Proto-Oncogene Proteins c-akt; Sirolimus; Tetrazolium Salts; Thiazoles; TOR Serine-Threonine Kinases

Rapamycin (sirolimus) is an antibiotic that inhibits protein synthesis through mammalian targeting of rapamycin (mTOR) signaling, and is used as an immunosuppressant in the treatment of organ rejection in transplant recipients. Rapamycin confers preconditioning-like protection against ischemic-reperfusion injury in isolated mouse heart cultures. Our aim was to further define the role of rapamycin in intracellular Ca(2+) homeostasis and to investigate the mechanism by which rapamycin protects cardiomyocytes from hypoxic damage.. We demonstrate here that rapamycin protects rat heart cultures from hypoxic-reoxygenation (H/R) damage, as revealed by assays of lactate dehydrogenase (LDH) and creatine kinase (CK) leakage to the medium, by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) measurements, and desmin immunostaining. As a result of hypoxia, intracellular calcium levels ([Ca(2+)](i)) were elevated. However, treatment of heart cultures with rapamycin during hypoxia attenuated the increase of [Ca(2+)](i). Rapamycin also attenuated (45)Ca(2+) uptake into the sarcoplasmic reticulum (SR) of skinned heart cultures in a dose- and time-dependent manner. KB-R7943, which inhibits the "reverse" mode of Na(+)/Ca(2+) exchanger (NCX), protected heart cultures from H/R damage with or without the addition of rapamycin. Rapamycin decreased [Ca(2+)](i) following its elevation by extracellular Ca(2+) ([Ca(2+)](o)) influx, thapsigargin treatment, or depolarization with KCl.. We suggest that rapamycin induces cardioprotection against hypoxic/reoxygenation damage in primary heart cultures by stimulating NCX to extrude Ca(2+) outside the cardiomyocytes.. According to our findings, rapamycin preserves Ca(2+) homeostasis and prevents Ca(2+) overload via extrusion of Ca(2+) surplus outside the sarcolemma, thereby protecting the cells from hypoxic stress.

Topics: Animals; Calcium; Cell Hypoxia; Cells, Cultured; Creatine Kinase; Dose-Response Relationship, Drug; Homeostasis; L-Lactate Dehydrogenase; Rats; Reperfusion Injury; Sarcoplasmic Reticulum; Sirolimus; Sodium-Calcium Exchanger; Tetrazolium Salts; Thiazoles; Thiourea; Time Factors

Metastatic squamous cell carcinoma (SCC) of the head and neck expresses chemokine receptor 7 (CCR7), which activates phosphoinositide-3 kinase (PI3K) to promote invasion and survival of SCC cells in the head and neck. We hypothesised that mammalian target of rapamycin (mTOR) may be the downstream molecule of the CCR7-PI3K pathway. Results have shown that interaction between CCR7 and its ligand CCL19 induces the phosphorylation of mTOR and its target p70s6k. This phosphorylation is abolished by inhibition of CCR7 and PI3K/Akt, indicating that mTOR is involved in the CCR7-PI3K cascade. The inhibitors of mTOR and CCR7-PI3K also lead to a significant increase in CCL19-induced death, apoptosis, and cell-cycle arrest of metastatic SCC cells in the head and neck. Taken together, our data indicate the important part played by mTOR in CCR7-induced survival of such SCC cells.

Topics: Antibodies, Monoclonal; Antineoplastic Agents; Apoptosis; Blotting, Western; Carcinoma, Squamous Cell; Cell Cycle; Cell Death; Cell Line, Tumor; Cell Survival; Chemokine CCL19; Chromones; Cisplatin; Coloring Agents; Enzyme Inhibitors; Flow Cytometry; Head and Neck Neoplasms; Humans; Intracellular Signaling Peptides and Proteins; Morpholines; Neoplasm Invasiveness; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Polymerase Chain Reaction; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Receptors, CCR7; Ribosomal Protein S6 Kinases, 70-kDa; Sirolimus; Tetrazolium Salts; Thiazoles; TOR Serine-Threonine Kinases

We have previously reported an aberrant accumulation of activated protein kinase B (PKB), glycogen synthase kinase (GSK)-3beta, extracellular signal-regulated kinase (ERK1/2), c-Jun N-terminal kinase (JNK), p38 and p70 S6 kinase (p70S6K) in neurons bearing neurofibrillary tangles (NFTs) in Alzheimer's disease (AD). However, the mechanism by which these tau candidate kinases are involved in the regulation of p70S6K and GSK-3beta phosphorylation is unknown. In the current study, 100 microM zinc sulfate was used, and influences of various components of phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways on p70S6K and GSK-3beta phosphorylation have been investigated in serum-deprived SH-SY5Y neuroblastoma cells. We found that zinc could induce an increase of phosphorylated (p) p70S6K, p-PKB, p-GSK-3beta, p-ERK1/2, p-JNK and p-p38, especially in long-term treatment (4-8 h). Treatment with different inhibitors including rapamycin, wortmannin, LY294002, and U0126, and their combinations, indicated that phosphorylation of p70S6K and GSK-3beta is regulated by rapamycin-dependent, PI3K and MAPK pathways. Furthermore, phosphorylation of p70S6K and GSK-3beta affected levels of tau unphosphorylated at the Tau-1 site and phosphorylated at the PHF-1 site, and p70S6K phosphorylation affected the total tau level. Thus, 100 microM zinc might activate PKB, GSK-3beta, ERK1/2, JNK, p38 and p70S6K, that are consequently involved in tau changes in SH-SY5Y cells.

Topics: Analysis of Variance; Animals; Antibiotics, Antineoplastic; Blotting, Western; Brain; Cell Line, Tumor; Cell Survival; Culture Media, Serum-Free; Cytoplasm; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Immunohistochemistry; In Vitro Techniques; Microscopy, Immunoelectron; Models, Biological; Neuroblastoma; Phosphorylation; rab5 GTP-Binding Proteins; Rats; Ribosomal Protein S6 Kinases, 70-kDa; Sirolimus; tau Proteins; Tetrazolium Salts; Thiazoles; Time Factors; Zinc; Zinc Sulfate

FK506, CsA, and rapamycin are potent inhibitors of T lymphocyte activation; relatively little is known of their effects on cells of the monocyte/macrophage lineage. Studies were undertaken to determine the effects of these drugs on the proliferative response of bone marrow-derived mononuclear phagocytes (BMMP) to CSFs. Rapamycin inhibited the proliferation of BMMP cultured in the presence of 10% L cell-conditioned medium, used as a source of macrophage CSF. The inhibition by rapamycin was dose dependent and apparent at concentrations of 0.1 nM or greater. In a similar fashion, rapamycin inhibited the proliferation of BMMP stimulated by the recombinant forms of murine IL-3 and murine granulocyte-macrophage CSF, and human macrophage CSF. In contrast, neither FK506 nor CsA at concentrations as high as 1000 nM diminished the proliferation of BMMP cultured under identical conditions. FK506, but not CsA, blocked the inhibitory effects of rapamycin on the response of BMMP to CSFs. In summary, these data indicate that rapamycin inhibits the proliferation of BMMP in response to CSFs. These results imply that patients receiving rapamycin, but not FK506 or CsA, may have an impaired ability to generate a functional mononuclear phagocyte population.

Topics: Animals; Bone Marrow Cells; Cell Count; Cell Division; Colony-Stimulating Factors; Concanavalin A; Culture Media, Conditioned; Cyclosporine; Female; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Phagocytes; Polyenes; Sirolimus; Spleen; T-Lymphocytes; Tacrolimus; Tetrazolium Salts; Thiazoles; Thymidine; Tritium