ku-0063794 and temsirolimus

Autophagy is an intracellular catabolic mechanism for the degradation of cytoplasmic constituents in the autophagosomal-lysosomal pathway. This mechanism plays an important role in homeostasis and it is defective in certain diseases. Preceding studies have revealed that autophagy is developing as an important moderator of pathological responses associated to spinal cord injury (SCI) and plays a crucial role in secondary injury initiating a progressive degeneration of the spinal cord. Thus, based on this evidence in this study, we used two different selective inhibitors of mTOR activity to explore the functional role of autophagy in an in vivo model of SCI as well as to determine whether the autophagic process is involved in spinal cord tissue damage. We treated animals with a novel synthetic inhibitor temsirolimus and with a dual mTORC1 and mTORC2 inhibitor KU0063794 matched all with the well-known inhibitor of mTOR the rapamycin. Our results demonstrated that mTOR inhibitors could regulate the neuroinflammation associated to SCI and the results that we obtained evidently demonstrated that rapamycin and temsirolimus significantly diminished the expression of iNOS, COX2, GFAP, and re-established nNOS levels, but the administration of KU0063794 is able to blunt the neuroinflammation better than rapamycin and temsirolimus. In addition, neuronal loss and cell mortality in the spinal cord after injury were considerably reduced in the KU0063794-treated mice. Accordingly, taken together our results denote that the administration of KU0063794 produced a neuroprotective function at the lesion site following SCI, representing a novel therapeutic approach after SCI.

Topics: Animals; Apoptosis; Astrocytes; Cell Survival; Cyclooxygenase 2; Cytokines; Male; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Mice; Morpholines; Motor Activity; Multiprotein Complexes; Nerve Tissue; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type II; Nitrites; Pyrimidines; Signal Transduction; Sirolimus; Spinal Cord; Spinal Cord Injuries; TOR Serine-Threonine Kinases

The objective of this study was to characterise the mechanism underlying acquired resistance to temsirolimus, an inhibitor of mammalian target of rapamycin (mTOR), in renal cell carcinoma (RCC).. A parental human RCC cell line, ACHN (ACHN/P), was continuously exposed to increasing doses of up to 20 μM of temsirolimus, and a cell line resistant to temsirolimus (ACHN/R), showing a sixfold higher IC50 than that of ACHN/P, was developed.. Following treatment with temsirolimus, phosphorylation of S6 kinase in ACHN/P was markedly inhibited, whereas there was no detectable expression of phosphorylated S6 in ACHN/R before and after temsirolimus treatment. However, AKT and p44/42 mitogen-activated protein kinase (MAPK) were constitutively phosphorylated even after temsirolimus treatment in ACHN/R, but not in ACHN/P. There was no significant difference between the sensitivities of ACHN/P and ACHN/R to KU0063794, a dual inhibitor of mTOR complex 1 (mTORC1) and mTORC2. Similar sensitivities to temsirolimus in ACHN/P and ACHN/R could be achieved by additional treatment with specific inhibitors of AKT- and MAPK-signaling pathways.. The activation of signal transduction pathways via mTORC2, but not via mTORC1, may have an important role in the acquisition of a resistant phenotype to temsirolimus in RCC.

Topics: Animals; Apoptosis; Butadienes; Carcinoma, Renal Cell; Cell Line, Tumor; Chromones; Drug Resistance, Neoplasm; Humans; Kidney Neoplasms; Male; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Mice, Inbred BALB C; Mice, Nude; Mitogen-Activated Protein Kinases; Morpholines; Multiprotein Complexes; Nitriles; Phosphorylation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Pyrimidines; Ribosomal Protein S6 Kinases; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Rapamycin analogs, temsirolimus and everolimus, are approved for the treatment of advance renal cell carcinoma (RCC). Currently approved agents inhibit mechanistic target of rapamycin (mTOR) complex 1 (mTORC1). However, the mTOR kinase exists in two distinct multiprotein complexes, mTORC1 and mTORC2, and both complexes may be critical regulators of cell metabolism, growth and proliferation. Furthermore, it has been proposed that drug resistance develops due to compensatory activation of mTORC2 signaling during treatment with temsirolimus or everolimus. We evaluated Ku0063794, which is a small molecule that inhibits both mTOR complexes. Ku0063794 was compared to temsirolimus in preclinical models for renal cell carcinoma. Ku0063794 was effective in inhibiting the phosphorylation of signaling proteins downstream of both mTORC1 and mTORC2, including p70 S6K, 4E-BP1 and Akt. Ku0063794 was more effective than temsirolimus in decreasing the viability and growth of RCC cell lines, Caki-1 and 786-O, in vitro by inducing cell cycle arrest and autophagy, but not apoptosis. However, in a xenograft model there was no difference in the inhibition of tumor growth by Ku0063794 or temsirolimus. A potential explanation is that temsirolimus has additional effects on the tumor microenvironment. Consistent with this possibility, temsirolimus, but not Ku0063794, decreased tumor angiogenesis in vivo, and decreased the viability of HUVEC (Human Umbilical Vein Endothelial Cells) cells in vitro at pharmacologically relevant concentrations. Furthermore, expression levels of VEGF and PDGF were lower in Caki-1 and 786-O cells treated with temsirolimus than cells treated with Ku0063794.

Topics: Adaptor Proteins, Signal Transducing; Animals; Apoptosis; Carcinoma, Renal Cell; Cell Cycle Proteins; Cell Proliferation; Gene Expression Regulation, Neoplastic; Human Umbilical Vein Endothelial Cells; Humans; Mechanistic Target of Rapamycin Complex 1; Mechanistic Target of Rapamycin Complex 2; Mice; Morpholines; Multiprotein Complexes; Neovascularization, Pathologic; Oncogene Protein v-akt; Phosphoproteins; Pyrimidines; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transplantation, Heterologous

Mst1/Stk4, a hippo-like serine-threonine kinase, is implicated in many cancers, including prostate cancer. However, the mechanisms regulating Mst1 remain obscure. Here, we characterized the effects of phospho-Thr-120 on Mst1 in prostate cancer cells. We demonstrated that phospho-Thr-120 did not alter the nuclear localization or cleavage of Mst1 in a LNCaP or castration-resistant C4-2 prostate tumor cell model, as revealed by a mutagenesis approach. Phospho-Thr-120 appeared to be specific to cancer cells and predominantly localized in the nucleus. In contrast, phospho-Thr-183, a critical regulator of Mst1 cell death, was exclusively found in the cytoplasm. As assessed by immunohistochemistry, a similar distribution of phospho-Mst1-Thr-120/Thr-183 was also observed in a prostate cancer specimen. In addition, the blockade of PI3K signaling by a small molecule inhibitor, LY294002, increased cytoplasmic phospho-Mst1-Thr-183 without having a significant effect on nuclear phospho-Mst1-Thr-120. However, the attenuation of mammalian target of rapamycin (mTOR) activity by a selective pharmacologic inhibitor, Ku0063794 or CCI-779, caused the up-regulation of nuclear phospho-Mst1-Thr-120 without affecting cytoplasmic phospho-Mst1-Thr-183. This suggests that PI3K and mTOR pathway signaling differentially regulate phospho-Mst1-Thr-120/Thr-183. Moreover, mutagenesis and RNAi data revealed that phospho-Thr-120 resulted in C4-2 cell resistance to mTOR inhibition and reduced the Mst1 suppression of cell growth and androgen receptor-driven gene expression. Collectively, these findings indicate that phospho-Thr-120 leads to the loss of Mst1 functions, supporting cancer cell growth and survival.

Topics: Animals; Blotting, Western; Cell Line, Tumor; Cell Nucleus; Chromones; HEK293 Cells; HeLa Cells; Humans; Intracellular Signaling Peptides and Proteins; Male; Mice; Mice, Nude; Morpholines; Neoplasms, Experimental; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Prostatic Neoplasms; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Pyrimidines; RNA Interference; Signal Transduction; Sirolimus; Threonine; TOR Serine-Threonine Kinases; Transplantation, Heterologous; Tumor Burden