sirolimus and Leukemia--Promyelocytic--Acute

Despite the high efficacy and safety of arsenic trioxide (ATO) in treating acute promyelocytic leukemia (APL) and eradicating APL leukemia-initiating cells (LICs), the mechanism underlying its selective cytotoxicity remains elusive. We have recently demonstrated that APL cells undergo a novel cell death program, termed ETosis, through autophagy. However, the role of ETosis in ATO-induced APL LIC eradication remains unclear. For this study, we evaluated the effects of ATO on ETosis and the contributions of drug-induced ETosis to APL LIC eradication. In NB4 cells, ATO primarily increased ETosis at moderate concentrations (0.5-0.75 μM) and stimulated apoptosis at higher doses (1.0-2.0 μM). Furthermore, ATO induced ETosis through mammalian target of rapamycin (mTOR)-dependent autophagy, which was partially regulated by reactive oxygen species. Additionally, rapamycin-enhanced ATO-induced ETosis in NB4 cells and APL cells from newly diagnosed and relapsed patients. In contrast, rapamycin had no effect on apoptosis in these cells. We also noted that PML/RARA oncoprotein was effectively cleared with this combination. Intriguingly, activation of autophagy with rapamycin-enhanced APL LIC eradication clearance by ATO in vitro and in a xenograft APL model, while inhibition of autophagy spared clonogenic cells. Our current results show that ATO exerts antileukemic effects at least partially through ETosis and targets LICs primarily through ETosis. Addition of drugs that target the ETotic pathway could be a promising therapeutic strategy to further eradicate LICs and reduce relapse.

Topics: Adolescent; Adult; Animals; Apoptosis; Arsenic Trioxide; Autophagy; Cell Line, Tumor; Disease Models, Animal; Drug Synergism; Female; Humans; Leukemia, Promyelocytic, Acute; Male; Mice, SCID; Middle Aged; Neoplasm Recurrence, Local; Neoplastic Stem Cells; Reactive Oxygen Species; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tumor Burden; Young Adult

We aimed at investigating the effects of rapamycin on apoptosis and autophagy of human acute promyelocytic leukemia cell line HL-60, and to preliminarily explore the mechanism of extra medullary infiltration of leukemia cells with human acute promyelocytic leukemia cell line HL-60 as the object of study, providing a theoretical basis for the clinical treatment of leukemia.. After HL-60 cells were cultured in vitro, the effect of rapamycin on proliferation ability of HL-60 cells was determined by methyl thiazolyl tetrazolium (MTT) method, the cell apoptosis ratio was detected by flow cytometer, the change of autophagy after HL-60 cells acted by rapamycin was tested by monodansylcadaverine (MDC) fluorescence staining, the mRNA expression of autophagy-related molecule was detected by polymerase chain reaction (PCR), and the expressions of apoptosis-related protein and autophagy-related protein were determined by Western blotting (WB).. HL-60 cell proliferation could be significantly inhibited by rapamycin (80 μg/mL-640 μg/mL), which was in a dose-dependent manner. HL-60 cell apoptosis ratio and apoptosis-related protein expression were distinctly improved by rapamycin. Cell autophagy level, mRNA expression of autophagy-related molecule and autophagy-related protein expression were remarkably induced by rapamycin.. Rapamycin can induce HL-60 cell apoptosis, which is produced mainly by inducing cell autophagy.

Topics: Apoptosis; Autophagy; Autophagy-Related Protein 5; Caspase 3; Cell Proliferation; HL-60 Cells; Humans; Leukemia, Promyelocytic, Acute; Microtubule-Associated Proteins; Sirolimus

Topics: Antineoplastic Agents; Arsenic Trioxide; Arsenicals; Brain Neoplasms; Drug Resistance, Neoplasm; ErbB Receptors; Erlotinib Hydrochloride; Glioblastoma; Humans; Leukemia, Promyelocytic, Acute; Oxides; Quinazolines; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases

Autophagy is a survival pathway required for cellular viability during starvation through catabolic self-digestion of damaged proteins and organelles; however, autophagy may result in cell death if it proceeds to completion. Although the exact mechanism of this process is not clear, it seems that proper regulation of autophagy can potentially contribute to the therapeutics of cancers. This study was designed to examine the role of autophagy in the death of human acute promyelocytic leukemia NB4 cells initiated by arsenic trioxide. Furthermore, the effects of autophagy inhibition and augmentation on cell viability were also compared. Our data suggested that both augmentation and suppression of autophagy could enhance the treatment effects while the latter was preferable. This study indicated that autophagy regulation augmented the treatment effects initiated by arsenic trioxide in NB4 cells, and that the selection of regulator should be precisely considered.

Topics: Adenine; Antibiotics, Antineoplastic; Apoptosis; Arsenic Trioxide; Arsenicals; Autophagy; Cell Line, Tumor; Cell Survival; Flow Cytometry; Humans; Leukemia, Promyelocytic, Acute; Oxides; Sirolimus

We previously showed that the MEK inhibitor AZD6244 induced apoptosis in acute myelogenous leukemia (AML) HL60 cells. However, the mechanisms of AZD6244 to induce apoptosis remain to be fully elucidated. This study found that exposure of HL60 cells to AZD6244 down-regulated the levels of phosphor (p)-4E-binding protein 1 (4E-BP1), a substrate of mammalian target of rapamycin complex 1 (mTORC1), and anti-apoptotic protein Mcl-1. On the other hand, exposure of EOL-1 and MOLM13 cells to AZD6244 failed to induce apoptosis and levels of p-4E-BP1 and Mcl-1 were not down-regulated in these cells. These observations prompted us to hypothesize that down-regulation od 4E-BP1 and Mcl-1 might play an important role in AZD6244-mediated apoptosis. As expected, down-regulation of 4E-BP1 by an siRNA sensitized EOL-1 cells to AZD6244-mediated apoptosis in parallel with down-regulation of Mcl-1. Moreover, we found that blockade of mTORC1 by RAD001 synergistically enhanced the action of AZD6244 in leukemia cells.

Topics: Adaptor Proteins, Signal Transducing; Adult; Aged; Apoptosis; Benzimidazoles; Cell Cycle; Cell Cycle Proteins; Cell Proliferation; Down-Regulation; Everolimus; Extracellular Signal-Regulated MAP Kinases; Female; HL-60 Cells; Humans; Leukemia, Promyelocytic, Acute; Male; MAP Kinase Signaling System; Mechanistic Target of Rapamycin Complex 1; Middle Aged; Mitogen-Activated Protein Kinase Kinases; Multiprotein Complexes; Myeloid Cell Leukemia Sequence 1 Protein; Phosphoproteins; Protein Kinase Inhibitors; Proteins; Proto-Oncogene Proteins c-bcl-2; Sirolimus; TOR Serine-Threonine Kinases; Transcription Factors

We have investigated the role of phosphoinositide 3-kinases (PI3Ks) in the in vitro pathophysiology of acute promyelocytic leukemia (APL) and in the response to treatment with all-trans-retinoic-acid (ATRA), utilizing a range of novel inhibitors that target individual or all catalytic class I isoforms of PI3K (p110alpha, p110beta, p110delta, and p110gamma). ATRA-induced phosphorylation of the Akt kinase and ribosomal S6 protein in APL cells was sensitive to class I PI3K, and p110beta or p110delta inhibitors, and to the mammalian target of rapamycin (mTOR) inhibitor rapamycin. In primary APL, inhibition of p110beta or p110delta triggered apoptosis in the absence or presence of ATRA. Class I PI3K inhibition could also reverse ATRA-induced protection of these cells against doxorubicin and arsenic trioxide, correlating with impaired induction of the antiapoptotic MCL-1 protein. The differentiation-inducing effects of ATRA were not dependent on class I PI3K/mTOR. In summary, class I PI3K signaling, mediated by p110beta and p110delta, plays an important role in basal and ATRA-induced cell survival mechanisms in APL. Addition of PI3K inhibitors to induction treatment regimens may provide therapeutic benefit.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Differentiation; Cell Growth Processes; Humans; Isoenzymes; Leukemia, Promyelocytic, Acute; Myeloid Cell Leukemia Sequence 1 Protein; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; Protein Kinases; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Sirolimus; Substrate Specificity; TOR Serine-Threonine Kinases; Tretinoin; Up-Regulation

This study found that MS-275, a novel synthetic benzamide histone deacetylase inhibitor (HDACI), blocked Akt/mammalian target of rapamycin (mTOR) signaling in acute myelogenous leukemia (AML) HL60 and acute promyelocytic leukemia (APL) NB4 cells, as assessed by decreased levels of the phosphorylated (p)-Akt, p-p70 ribosomal S6 kinase (p70S6K) and p-S6K by western blot analysis. Interestingly, further inactivation of mTOR by rapamycin analog RAD001 (everolimus) significantly enhanced MS-275-mediated growth inhibition and apoptosis of these cells in parallel with enhanced upregulation of p27(kip1) and downregulation of c-Myc. In addition, RAD001 potentiated the ability of MS-275 to induce differentiation of HL60 and NB4 cells, as measured by the expression of CD11b cell surface antigens, as well as reduction of nitroblue tetrazolium. Importantly, RAD001 potentiated the ability of MS-275 to induce the expression of the myeloid differentiation-related transcription factor, CCAAT enhancer-binding protein-epsilon, in these cells in association with enhanced acetylation of histone H3 on its promoter. Furthermore, RAD001 (5 mg/kg) significantly enhanced the effects of MS-275 (10 mg/kg) to inhibit proliferation of HL60 tumor xenografts in nude mice without adverse effects. Taken together, concomitant administration of an HDACI and an mTOR inhibitor may be a promising treatment strategy for the individuals with a subset of human leukemia.

Topics: Acetylation; Animals; Apoptosis; Benzamides; CCAAT-Enhancer-Binding Proteins; Cell Differentiation; Cell Division; Drug Synergism; Everolimus; Female; Histone Deacetylase 1; Histone Deacetylase Inhibitors; Histone Deacetylases; Histones; HL-60 Cells; Humans; Immunosuppressive Agents; Leukemia, Myeloid, Acute; Leukemia, Promyelocytic, Acute; Mechanistic Target of Rapamycin Complex 1; Mice; Mice, Inbred BALB C; Mice, Nude; Multiprotein Complexes; Promoter Regions, Genetic; Proteins; Proto-Oncogene Proteins c-akt; Pyridines; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transcription Factors; Xenograft Model Antitumor Assays

Although the mechanisms by which all-trans-retinoic acid (RA) regulates gene transcription are well understood, very little is known on the signaling events regulating RA-dependent initiation of mRNA translation. We examined whether the mammalian target of rapamycin (mTOR)/p70 S6 kinase pathway is activated by RA. RA treatment of sensitive cell lines resulted in phosphorylation/activation of mTOR and downstream induction of p70 S6 kinase activity. Such phosphorylation/activation of p70 S6 kinase was inducible in primary acute promyelocytic leukemia (APL) blasts and RA-sensitive NB-4 cells, but was defective in an NB-4 variant cell line (NB-4.007/6) that is resistant to the biologic effects of RA. The RA-dependent activation of p70 S6 kinase was also phosphatidylinositol 3' kinase (PI3'K)-dependent, and resulted in downstream phosphorylation of the S6 ribosomal protein on Ser235/236 and Ser240/244, events important for initiation of translation for mRNAs with oligopyrimidine tracts in their 5' untranslated region. RA treatment of leukemia cells also resulted in an mTOR-mediated phosphorylation of the 4E-BP1 repressor of mRNA translation, to induce its deactivation and dissociation from the eukaryotic initiation factor-4E (eIF-4E) complex. Altogether, these findings provide evidence for the existence of a novel RA-activated cellular pathway that regulates cap-dependent translation, and strongly suggest that this cascade plays a role in the induction of retinoid responses in APL cells.

Topics: Cell Differentiation; Cell Line, Tumor; Enzyme Activation; Eukaryotic Initiation Factor-4E; Growth Inhibitors; Humans; Leukemia, Promyelocytic, Acute; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Kinases; Ribosomal Protein S6 Kinases, 70-kDa; RNA Caps; Serine; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Tretinoin

Synthetic analogs of vitamin D for potential use in differentiation therapy should selectively regulate genes necessary for differentiation without inducing any perturbations in calcium homeostasis. PRI-1906, an analog of vitamin D2, and PRI-2191, an analog of vitamin D3 bind nuclear vitamin D receptor (nVDR) with substantially lower affinity than 1,25-dihydroxyvitamin D3 (1,25-D3), but have higher differentiation-inducing activity as estimated in HL-60 leukemia cellmodel. To examine how their increased differentiation-inducing activity is regulated we tested the hypothesis that membrane-mediated events, unrelated to nVDR, take part in the differentiation in response to PRI-1906 and PRI-2191. The induction of leukemia cell differentiation in response to the analogs of vitamin D was inhibited by LY294002 (phosphatidylinositol 3-kinase inhibitor), PD98059 (inhibitor of MEK1,2, an upstream regulator of extracellular-signal regulated kinase) and rapamycin (p70S6K inhibitor) pointing out that activation of signal transduction pathways unrelated to nVDR is necessary for differentiation. On the other hand, inhibition of cytosolic phospholipase A2 accelerated the differentiation of HL-60 cells induced by either 1,25-D3 or by the vitamin D analogs suggesting possible existence of a feedback loop between extracellular-signal regulated kinases and phospholipase A2.

Topics: Cell Cycle; Cell Differentiation; Enzyme Activation; HL-60 Cells; Humans; Leukemia, Promyelocytic, Acute; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Monocytes; Phosphatidylinositol 3-Kinases; Sirolimus; Vitamin D

Apoptosis can be regulated in a number of different systems by the actions of cytokines. Rapamycin has been shown to exert its effects on growth factor-induced cell proliferation, at least in part, by blocking the activation of the p70 S6 kinase and thus preventing the downstream signaling process, such as the activation of the members of the cdk family. To determine whether this pathway plays a role in the regulation of apoptosis, we assessed the effect of rapamycin on apoptosis induced by interleukin 2 deprivation in murine T-cell lines, by T-cell receptor ligation in a murine T-cell hybridoma, by enforced c-myc expression in murine fibroblasts, and by corticosteroids in murine T-lymphoma cell lines. Although rapamycin did not induce apoptosis on its own, rapamycin augmented apoptosis in each of the cell lines used as indicated by increased genomic DNA fragmentation, decreased cell viability, and characteristic apoptotic changes in morphology. These results suggest that a signal transduction pathway(s) inhibited by rapamycin plays an important role in the susceptibility of cells to apoptosis. Many chemotherapeutic agents kill cancer cells through the induction of apoptosis. Strikingly, rapamycin increased the ability of the alkylating agent, cisplatin, to induce apoptosis in the human promyelocytic leukemia cell line HL-60 and the human ovarian cancer cell line SKOV3. These data suggest that a signal transduction pathway, likely related to p70 S6 kinase, inhibited by rapamycin may be an important component of the pathway which prevents cell death in many cell lineages and also indicate that rapamycin has the potential to augment the efficacy of selected anticancer therapies.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cisplatin; Cricetinae; Drug Synergism; Female; Humans; Immunosuppressive Agents; Leukemia, Promyelocytic, Acute; Lymphocyte Activation; Mice; Ovarian Neoplasms; Polyenes; Sensitivity and Specificity; Sirolimus; T-Lymphocytes; Tumor Cells, Cultured