sirolimus and perifosine

Malignant glioma (MG) is the most deadly primary brain cancer. Signaling though the PI3K/AKT/mTOR axis is activated in most MGs and therefore a potential therapeutic target. The mTOR inhibitor temsirolimus and the AKT inhibitor perifosine are each well-tolerated as single agents but with limited activity reclinical data demonstrate synergistic anti-tumor effects from combined treatment. Therefore, we initiated a phase I trial of combined therapy in recurrent MGs to determine safety and a recommended phase II dose.. Adults with recurrent MG, Karnofsky Performance Status ≥ 60 were enrolled, with no limit on the number of prior therapies. Temsirolimus dose was escalated using standard 3 + 3 design from 15 mg to 170 mg administered once weekly. Perifosine was fixed as a 600 mg load on day 1 followed by 100 mg nightly (single agent MTD) until dose level 7 when the load increased to 900 mg.. We treated 35 patients with with glioblastoma (17) or other MGs (18; including nine anaplastic astrocytoma, nine anaplastic oligodendroglioma, one anaplastic oligoastrocytoma, and two low grade astrocytomas with radiographic transformation to MG). We observed five dose-limiting toxicities (DLTs): one at dose level 3 (50mg temsirolimus), then two at dose level 7 expansion (170 mg temsirolimus), and then two more at dose level 6 expansion (170 mg temsirolimus). DLTs included thrombocytopenia (n = 3), intracerebral hemorrhage (n = 1) and lung infection (n = 1).. Combining the mTOR inhibitor temsirolimus dosed at 115 mg weekly and the AKT inhibitor perifosine dosed at 100 mg daily (following 600 mg load) is tolerable in heavily pretreated adults with recurrent MGs.

Topics: Adult; Aged; Antineoplastic Agents; Brain Neoplasms; Drug Therapy, Combination; Female; Glioblastoma; Humans; Male; Middle Aged; Neoplasm Recurrence, Local; Phosphorylcholine; Prospective Studies; Proto-Oncogene Proteins c-akt; Sirolimus; TOR Serine-Threonine Kinases; Young Adult

Convection-enhanced delivery (CED), a local drug delivery technique, is typically performed as a single session and drug concentrations therefore decline quickly post CED. Prolonged CED (pCED) overcomes this problem by performing a long-term infusion to maintain effective drug concentrations for an extended period. The purpose of the current study was to assess the toxicity of using pCED to deliver single and multi-drug therapy in naïve rat brainstem.. Sixteen rats underwent pCED of three small-molecule kinase inhibitors in the pons. Single and multi-drug combinations were delivered continuously for 7 days using ALZET mini-osmotic pumps (model 2001, rate of 1 μl/h). Rats were monitored daily for neurological signs of toxicity. Rats were sacrificed 10 days post completion of infusion, and appropriate tissue sections were analyzed for histological signs of toxicity.. Two rats exhibited signs of neurological deficits, which corresponded with diffuse inflammation, necrosis, and parenchymal damage on histological analysis. The remaining rats showed no neurological or histological signs of toxicity.. The neurological deficits in the two rats were likely due to injury from physical force, such as cannula movement post insertion and subsequent encephalitis. The remaining rats showed no toxicity and therefore brainstem targeting using pCED to infuse single and multi-drug therapy was well tolerated in these rats.

Topics: Animals; Antineoplastic Agents; Brain Stem; Convection; Dasatinib; Drug Delivery Systems; Everolimus; Female; Infusions, Intraventricular; Phosphorylcholine; Protein Kinase Inhibitors; Pyrimidines; Rats; Rats, Sprague-Dawley; Sirolimus; Thiazoles

1. This study investigated the alteration of carboxylesterases in type 2 diabetes. We found that the carboxylesterase 1d (Ces1d) and carboxylesterase 1e (Ces1e) expression and the capacity of hydrolytic activity of liver and intestine decreased, whereas the Akt/mTOR/HIF-1α/ Stra13 (DEC1) signaling was activated in T2D mice. Consistently, high insulin could give rise to the same results in the high-glucose DMEM condition, which mimicked T2D, in primary mouse hepatocytes. 2. Perifosine or rapamycin almost abolished the decrease of the Ces1d and Ces1e expression and the hydrolytic activity induced by the insulin in the primary mouse hepatocytes. 3. The responsiveness of human hepatoma (HepG2) cells to high insulin in high-glucose condition was similar to that of primary mouse hepatocytes in terms of the altered expression of carboxylesterases. 4. The knockdown of HIF-1α or DEC1 with shRNA construct abrogated the decrease of the CES1 and CES2 expression induced by the insulin in high glucose condition in HepG2 cells. 5. Taken together, the decreased carboxylesterases expression and hydrolytic activity in T2D mice are through the Akt/mTOR/HIF-1α/Stra13 (DEC1) pathway.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Blood Glucose; Carboxylic Ester Hydrolases; Diabetes Mellitus, Type 2; Disease Models, Animal; Gene Knockdown Techniques; Glucose; Hep G2 Cells; Hepatocytes; Homeodomain Proteins; Humans; Hydrolysis; Hypoxia-Inducible Factor 1, alpha Subunit; Insulin; Intestines; Liver; Male; Mice, Inbred C57BL; Overweight; Phosphorylcholine; Proto-Oncogene Proteins c-akt; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Inhibition of the mammalian target of rapamycin (mTOR), a regulator of hypoxia inducible factor (HIF), is an established therapy for advanced renal cell cancer (RCC). Inhibition of mTOR results in compensatory AKT activation, a likely resistance mechanism. We evaluated whether addition of the Akt inhibitor perifosine to the mTOR inhibitor rapamycin would synergistically inhibit RCC.. Select RCC cell lines were studied [786-O, A498 (VHL mutant), CAKI-1 (VHL wild type), and 769-P (VHL methylated)] with single agent and combination therapy. Growth inhibition was assessed by MTT and cell cycling by flow cytometry. Phospho-AKT (S473) and HIF-2α were assessed by Western blot. Total RNA was isolated from 786-O cells subjected to single agent and combination treatments. In these cells, genome-wide expression profiles were assessed, and real-time PCR was used to confirm a limited set of expression results.. Three out of four cell lines (CAKI-1, 769-P, and 786-O) were sensitive to single-agent perifosine with 50% inhibitory concentrations ranging from 5 to 10 μM. Perifosine blocked phosphorylation of AKT induced by rapamycin and inhibited HIF-2α expression in 786-O and CAKI-1. Combined treatment resulted in sub-additive growth inhibition. GeneChip analysis and pathway modeling revealed inhibition of the IL-8 pathway by these agents, concomitant with up-regulation of the KLF2 gene, a known suppressor of HIF1α.. Perifosine is active in select RCC lines, abrogating the induction of AKT phosphorylation mediated by mTOR inhibition. Combined mTOR and AKT inhibition resulted in the modulation of pro-angiogenesis pathways, providing a basis for future investigations.

Topics: Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Renal Cell; Cell Line, Tumor; Drug Resistance, Neoplasm; Drug Synergism; Gene Expression Regulation, Neoplastic; Humans; Inhibitory Concentration 50; Interleukin-8; Kidney Neoplasms; Neovascularization, Pathologic; Oligonucleotide Array Sequence Analysis; Phosphorylation; Phosphorylcholine; Proto-Oncogene Proteins c-akt; Sirolimus; TOR Serine-Threonine Kinases

mTORC1 inhibitors, including rapamycin and its analogs, have been actively studied both pre-clinically and clinically. However, the single treatment of mTORC1 inhibitors has been modest in most cancer types. We have previously demonstrated that the activation of PI3K/Akt and MEK/ERK signaling pathways attenuates the anticancer efficacy of mTORC1 inhibitors. In this study, we report that mTORC1 inhibition also phosphorylates and inactivates GSK3β, which is a tumor suppressor in lung cancer. Moreover, we show that perifosine, as an Akt inhibitor, decreases rapamycin-induced phosphorylation of GSK3β and elevated p-GSK3β levels in rapamycin-resistant cell lines. Combination of perifosine with mTORC1 inhibitors showed enhanced anticancer efficacy both in cell cultures and in a xenograft mouse model. In addition, perifosine inhibits the growth of both rapamycin sensitive and resistant A549 cells. However, inhibition of GSK3β by a selective inhibitor- LiCl, or downregulation of GSK3β expression by siRNA, reverses the growth inhibitory effects of perifosine on rapamycin resistant cells, suggesting the important role of GSK3β activation in enhancing mTORC1 inhibitors efficacy by perifosine. Thus, our results provide a potential therapeutic strategy to enhance mTORC1-targeted cancer therapy by using perifosine or targeting GSK3β.

Topics: Animals; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Female; Gene Knockdown Techniques; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Lung Neoplasms; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Nude; Molecular Targeted Therapy; Multiprotein Complexes; Phosphorylation; Phosphorylcholine; Proteins; RNA, Small Interfering; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

Mantle cell lymphoma (MCL) is a distinct subtype of B-cell lymphoma; the complete response rate for standard therapies in use today is 85 - 90%. NVP-BEZ235 inhibits the PI3K/Akt/mTOR signaling axis at the level of both PI3K and mTOR. In this study, we analyzed the inhibitory effects of NVP-BEZ235 on mantle cell lines and its effects in combination with enzastaurin, everolimus and perifosine.. The effects of NVP-BEZ235 on cell proliferation and apoptosis were evaluated using MTT assay and flow cytometry analysis. The cell cycle analysis was performed applying BrdU incorporation. Western blot analysis was utilized for phosphorylation status evaluation of protein kinases. The interaction between NVP-BEZ235 and enzastaurin, everolimus and perifosine was examined by Chou-Talalay method.. NVP-BEZ235 induced significant increase of apoptosis, both via intrinsic and extrinsic pathways. We found that NVP-BEZ235 inhibited mantle cells growth by induction of G1 arrest. NVP-BEZ235 exerts its antitumor activity even when mantle cells were in contact with bone marrow microenvironment. Enzastaurin, everolimus and perifosine enhanced the cytotoxicity triggered by NVP-BEZ235.. The above results encourage clinical development of NVP-BEZ235 in combination and the possible inclusion of patients with mantle lymphoma in Phase I/II clinical trials.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Blotting, Western; Bone Marrow; Cell Line, Tumor; Cell Proliferation; Everolimus; Flow Cytometry; G1 Phase Cell Cycle Checkpoints; Humans; Imidazoles; Indoles; Lymphoma, Mantle-Cell; Phosphorylation; Phosphorylcholine; Protein Kinases; Quinolines; Sirolimus

Platelet derived growth factor receptor (PDGFR) activity is deregulated in human GBM due to amplification and rearrangement of the PDGFR-alpha gene locus or overexpression of the PDGF ligand, resulting in the activation of downstream kinases such as phosphatidylinositol 3-kinase (PI3K), Akt, and mammalian target of rapamycin (mTOR). Aberrant PDGFR signaling is observed in approximately 25-30% of human GBMs, which are frequently molecularly classified as the proneural subclass. It would be valuable to understand how PDGFR driven GBMs respond to Akt and mTOR inhibition.. Using genetically engineered PTEN-intact and PTEN-deficient PDGF-driven mouse models of GBM that closely mimic the histology and genetics of the human PDGF subgroup, we investigated the effect of inhibiting Akt and mTOR alone or in combination in vitro and in vivo. We used perifosine and CCI-779 to inhibit Akt and mTOR, respectively. Here, we show in vitro data demonstrating that the most effective inhibition of Akt and mTOR activity in both PTEN-intact and PTEN-null primary glioma cell cultures is obtained when using both inhibitors in combination. We next investigated if the effects we observed in culture could be duplicated in vivo by treating mice with gliomas for 5 days. The in vivo treatments with the combination of CCI-779 and perifosine resulted in decreased Akt and mTOR signaling, which correlated to decreased proliferation and increased cell death independent of PTEN status, as monitored by immunoblot analysis, histology and MRI.. These findings underline the importance of simultaneously targeting Akt and mTOR to achieve significant down-regulation of the PI3K pathway and support the rationale for testing the perifosine and CCI-779 combination in the human PDGF-subgroup of GBM.

Topics: Animals; Antineoplastic Agents; Cell Death; Cell Line, Tumor; Cell Proliferation; Down-Regulation; Drug Synergism; Glioblastoma; Mice; Phosphatidylinositol 3-Kinases; Phosphorylcholine; Platelet-Derived Growth Factor; Protein Kinase Inhibitors; PTEN Phosphohydrolase; Sirolimus

The phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway mediates multiple myeloma (MM) cell proliferation, survival, and development of drug resistance, underscoring the role of mTOR inhibitors, such as rapamycin, with potential anti-MM activity. However, recent data show a positive feedback loop from mTOR/S6K1 to Akt, whereby Akt activation confers resistance to mTOR inhibitors. We confirmed that suppression of mTOR signaling in MM cells by rapamycin was associated with upregulation of Akt phosphorylation. We hypothesized that inhibiting this positive feedback by a potent Akt inhibitor perifosine would augment rapamycin-induced cytotoxicity in MM cells. Perifosine inhibited rapamycin-induced phosphorylated Akt, resulting in enhanced cytotoxicity in MM.1S cells even in the presence of interleukin-6, insulin-like growth factor-I, or bone marrow stromal cells. Moreover, rapamycin-induced autophagy in MM.1S MM cells, as evidenced by electron microscopy and immunocytochemistry, was augmented by perifosine. Combination therapy increased apoptosis detected by Annexin V/propidium iodide analysis and caspase/poly(ADP-ribose) polymerase cleavage. Importantly, in vivo antitumor activity and prolongation of survival in a MM mouse xenograft model after treatment was enhanced with combination of nanoparticle albumin-bound-rapamycin and perifosine. Utilizing the in silico predictive analysis, we confirmed our experimental findings of this drug combination on PI3K, Akt, mTOR kinases, and the caspases. Our data suggest that mutual suppression of the PI3K/Akt/mTOR pathway by rapamycin and perifosine combination induces synergistic MM cell cytotoxicity, providing the rationale for clinical trials in patients with relapsed/refractory MM. Mol Cancer Ther; 9(4); 963-75. (c)2010 AACR.

Topics: Albumins; Animals; Apoptosis; Autophagy; Bone Marrow Cells; Caspases; Cell Line, Tumor; Cell Proliferation; Cell Survival; Down-Regulation; Drug Screening Assays, Antitumor; Drug Synergism; Enzyme Activation; Humans; Insulin-Like Growth Factor I; Interleukin-6; Intracellular Signaling Peptides and Proteins; Mice; Multiple Myeloma; Nanoparticles; Phosphorylation; Phosphorylcholine; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Xenograft Model Antitumor Assays

We investigated the genotype-dependent therapeutic potential of targeting the phosphoinositide 3-kinase (PI3K)/Akt pathway for thyroid cancer. Proliferation of TPC1, Hth7, FTC133, OCUT1, K1, and BCPAP cells that harbored PI3K/Akt-activating genetic alterations was potently inhibited by the Akt inhibitor perifosine, whereas SW1736, Hth74, WRO, KAT18, and TAD2 cells that harbored no genetic alterations had no or only modest responses. Inhibition of Akt phosphorylation by perifosine was seen in these cells. Genetic-dependent apoptosis was induced by perifosine in cells selectively tested. Similarly, potent inhibition of cell proliferation by the mammalian target of rapamycin (mTOR) inhibitor temsirolimus occurred in virtually all the cells harboring genetic alterations, whereas modest inhibition was seen in some of the cells not harboring genetic alterations. Temsirolimus inhibited the phosphorylation of p70S6K, a substrate of mTOR. Knockdown of Akt1/2 or mTOR by shRNA approach inhibited the proliferation and colony formation of FTC133 and OCUT1 cells that harbored genetic alterations in the PI3K/Akt pathway but had no effect on SW1736 and KAT18 cells that did not. Transfection with PIK3CA mutants greatly sensitized SW1736 cells to perifosine and temsirolimus. Growth of xenograft tumors derived from FTC133 cells but not SW1736 cells in nude mice was dramatically inhibited by perifosine. Thus, this work for the first time shows that genetic alterations in the PI3K/Akt pathway confer thyroid cancer cells addiction to this pathway and their sensitivity to inhibition by targeting Akt and mTOR. This genotype-based targeting of the PI3K/Akt pathway using Akt and mTOR inhibitors may offer an effective therapeutic strategy for thyroid cancer and warrants further studies.

Topics: Animals; Cell Growth Processes; Cell Line, Tumor; Class I Phosphatidylinositol 3-Kinases; Humans; Mice; Mice, Nude; Oncogene Protein v-akt; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylcholine; Protein Kinases; RNA, Small Interfering; Signal Transduction; Sirolimus; Thyroid Neoplasms; TOR Serine-Threonine Kinases; Transfection; Xenograft Model Antitumor Assays