Akt inhibitor augments anti-proliferative efficacy of a dual mTORC1/2 inhibitor by FOXO3a activation in p53 mutated hepatocarcinoma cells

AbstractHepatocellular carcinoma (HCC) is one of the most common malignancy-related deaths. p53 mutation in HCC associates with worse clinicopathologic features including therapeutic limitation. A combination of targeted therapy may have some advantages. Akt/mTOR signaling contributes to the regulation of cell proliferation and cell death. Akt inhibitor (AZD5363) and mTORC1/2 dual inhibitor (AZD8055) are in a clinical trial for HCC and other cancers. In this study, we examined whether these inhibitors successfully induce antiproliferative activity in p53 mutant HCC cells, and the underlying mechanisms. We observed that a combination of AZD5363 and AZD8055 treatment synergizes antiproliferative activity on p53 mutated or wild-type HCC cell lines and induces apoptotic cell death. Mechanistic insights indicate that a combination of AZD5363 and AZD8055 activated FOXO3a to induce Bim-associated apoptosis in p53 mutated HCC cells, whereas cells retaining functional p53 enhanced Bax. siRNA-mediated knock-down of Bim or Bax prevented apoptosis in inhibitor-treated cells. We further observed a combination of treatment inhibits phosphorylation of FOXO3a and protects FOXO3a from MDM2 mediated degradation by preventing the phosphorylation of Akt and SGK1. FOXO3a accumulates in the nucleus under these conditions and induces Bim transcription in p53 mutant HCC cells. Combination treatment in the HCC cells expressing wild-type p53 causes interference of FOXO3a function for direct interaction with functional p53 and unable to induce Bim-associated cell death. On the other hand, Bim-associated cell death occurs in p53 mutant cells due to uninterrupted FOXO3a function. Overall, our findings suggested that a combined regimen of dual mTORC1/2 and Akt inhibitors may be an effective therapeutic strategy for HCC patients harboring p53 mutation.

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AKT is involved in granulosa cell autophagy regulation via mTOR signaling during rat follicular development and atresia

Reproduction ◽

10.1530/rep-13-0386 ◽

2014 ◽

Vol 147 (1) ◽

pp. 73-80 ◽

Cited By ~ 43

Author(s):

JongYeob Choi ◽

MinWha Jo ◽

EunYoung Lee ◽

DooSeok Choi

Keyword(s):

Cell Death ◽

Granulosa Cell ◽

Granulosa Cells ◽

Apoptotic Cell ◽

Follicular Development ◽

Negative Regulator ◽

Metabolic Clearance ◽

Akt Inhibitor ◽

Western Blots

In this study, we examined whether granulosa cell autophagy during follicular development and atresia was regulated by the class I phosphoinositide-3 kinase/protein kinase B (AKT) pathway, which is known to control the activity of mammalian target of rapamycin (mTOR), a major negative regulator of autophagy. Ovaries and granulosa cells were obtained using an established gonadotropin-primed immature rat model that induces follicular development and atresia. Autophagy was evaluated by measuring the expression level of microtubule-associated protein light chain 3-II (LC3-II) using western blots and immunohistochemistry. The activity of AKT and mTOR was also examined by observing the phosphorylation of AKT and ribosomal protein S6 kinase (S6K) respectively. After gonadotropin injection, LC3-II expression was suppressed and phosphorylation of AKT and S6K increased in rat granulosa cells. By contrast, gonadotropin withdrawal by metabolic clearance promoted LC3-II expression and decreased phosphorylation of AKT and S6K. In addition,in-vitroFSH treatment of rat granulosa cells also indicated inhibition of LC3-II expression accompanied by a marked increase in phosphorylation of AKT and S6K. Inhibition of AKT phosphorylation using AKT inhibitor VIII suppressed FSH-mediated phosphorylation of S6K, followed by an increase in LC3-II expression. Furthermore, co-treatment with FSH and AKT inhibitor increased the levels of apoptosis and cell death of granulosa cells compared with the single treatment with FSH. Taken together, our findings indicated that AKT-mediated activation of mTOR suppresses granulosa cell autophagy during follicular development and is involved in the regulation of apoptotic cell death.

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Identification and Characterization of NTB451 as a Potential Inhibitor of Necroptosis

Molecules ◽

10.3390/molecules23112884 ◽

2018 ◽

Vol 23 (11) ◽

pp. 2884 ◽

Cited By ~ 4

Author(s):

Eun-Jung In ◽

Yuno Lee ◽

Sushruta Koppula ◽

Tae-Yeon Kim ◽

Jun-Hyuk Han ◽

...

Keyword(s):

Cell Death ◽

Md Simulation ◽

Ischemia Reperfusion Injury ◽

Apoptotic Cell ◽

Ischemia Reperfusion ◽

Direct Interaction ◽

Inhibitory Effect ◽

Therapeutic Implications ◽

Pathological Conditions ◽

Tnf Α

Necroptosis, or caspase-independent programmed cell death, is known to be involved in various pathological conditions, such as ischemia/reperfusion injury, myocardial infarction, atherosclerosis, and inflammatory bowel diseases. Although several inhibitors of necroptosis have been identified, none of them are currently in clinical use. In the present study, we identified a new compound, 4-({[5-(4-aminophenyl)-4-ethyl-4H-1,2,4-triazol-3-yl]sulfanyl}methyl)-N-(1,3-thiazol-2-yl) benzamide (NTB451), with significant inhibitory activity on the necroptosis induced by various triggers, such as tumor necrosis factor-α (TNF-α) and toll-like receptor (TLR) agonists. Mechanistic studies revealed that NTB451 inhibited phosphorylation and oligomerization of mixed lineage kinase domain like (MLKL), and this activity was linked to its inhibitory effect on the formation of the receptor interacting serine/threonine-protein kinase 1 (RIPK1)-RIPK3 complex. Small interfering RNA (siRNA)-mediated RIPK1 knockdown, drug affinity responsive target stability assay, and molecular dynamics (MD) simulation study illustrated that RIPK1 is a specific target of NTB451. Moreover, MD simulation showed a direct interaction of NTB451 and RIPK1. Further experiments to ensure that the inhibitory effect of NTB451 was restricted to necroptosis and NTB451 had no effect on nuclear factor-κB (NF-κB) activation or apoptotic cell death upon triggering with TNF-α were also performed. Considering the data obtained, our study confirmed the potential of NTB451 as a new necroptosis inhibitor, suggesting its therapeutic implications for pathological conditions induced by necroptotic cell death.

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The AKT Inhibitor, A443654, Induces Cell Cycle Arrest, Apoptosis and Synergizes with Chemotherapeutic Drugs in Multi-Drug Resistant T-Cell Acute Lymphoblastic Leukemia - A Novel Agent for Therapy of Drug Resistant ALL.

Blood ◽

10.1182/blood.v110.11.3344.3344 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3344-3344

Author(s):

James A. McCubrey ◽

William L. Blalock ◽

Pier Luigi Tazzari ◽

Alessandra Cappellini ◽

Ilaria Iacobucci ◽

...

Keyword(s):

Cell Death ◽

Caspase 3 ◽

Apoptotic Cell ◽

Apoptotic Cell Death ◽

Jurkat Cells ◽

Drug Resistant ◽

Akt Inhibitor ◽

Cycle Arrest ◽

Hematopoietic Precursor ◽

Dose Dependent

Abstract The PI3K/Akt pathway is a key mediator of proliferation and survival; its constitutive activation is implicated in pathogenesis and progression of a wide variety of hematological malignancies. Constitutively-activated Akt is a common feature of the T-ALL cell lines (MOLT-4, Jurkat, CEM). Hyperactivity of Akt leads not only to their growth and resistance to apoptosis but also to drug-resistance. In this study, we sought to examine the potential of a novel Akt inhibitor, A443654, in the therapy of T-ALL. A443654 led to rapid cell death of CEM, Jurkat, and MOLT-4. All three lines were sensitive to nanomolar (nM) doses of A443654 (IC50=60 nM, 120 nM and 900 nM for MOLT-4, CEM, and Jurkat, respectively). Effects were dose dependent and resulted in G2/M arrest. Indeed, approximately 39% of Jurkat cells treated with A443654 were in G2/M whereas only 11% of the untreated cells were in G2/M. Cell cycle arrest was followed by apoptotic cell death as determined by annexin V-PI and trypan blue staining. Treatment of CEM and Jurkat cells led to de-phosphorylation of the downstream Akt substrate GSK-3beta. Treatment of Jurkat cells with A443654 resulted in activation of caspase-2, -3, -8, and -9. Apoptotic cell death was greatly reduced by caspase-3 and -9 selective inhibitors. Additionally, A443654 was shown to be highly effective against the drug-resistant cell line CEM-VBL100 (CEM-R), which overexpresses Pgp. This Akt inhibitor initially (1 to 4 hour treatments) decreased Pgp activity, but not protein levels, further documenting a link between Akt and Pgp activity. While CEM-R cells displayed decreased sensitivity to A443654, treatment of CEM-R cells with sub-lethal doses of A443654 for 24 hours, reduced the surface expression of Pgp. Moreover, A443654 synergized with the DNA damaging agent etoposide, a substrate of Ppg, in both drug sensitive and resistant lines. At etoposide concentrations between 25 mM to 100 mM, A443654 enhanced the extent of cell killing from 25% to 45%. We then confirmed the effect of A443654 on human leukemia samples using blasts from 6 patients with T-ALL, all of whom displayed constitutive Akt activation. In a dose dependent fashion, A443654 was able to induce apoptotic cell death of T-ALL blast cells, as indicated by flow cytometric analysis of samples immunostained for active (cleaved) caspase-3. In contrast, this Akt inhibitor was determined to be minimally cytotoxic on normal CD34+ hematopoietic precursor cells isolated from cord blood. Taken together, our findings indicate that the Akt inhibitor, A443654, either alone or in combination with existing drugs, may in the future be a useful therapeutic option for primary and refractory T-ALL displaying activated Akt signaling. Furthermore, this novel Akt inhibitor was effective in suppressing the growth of multidrug resistant ALL cells while having minimal effects on normal hematopoietic precursor cells documenting its poteintial in the treatment of drug resistant leukemias.

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The Akt-inhibitor Erufosine induces apoptotic cell death in prostate cancer cells and increases the short term effects of ionizing radiation

Radiation Oncology ◽

10.1186/1748-717x-5-108 ◽

2010 ◽

Vol 5 (1) ◽

pp. 108 ◽

Cited By ~ 41

Author(s):

Justine Rudner ◽

Carola-Ellen Ruiner ◽

René Handrick ◽

Hans-Jörg Eibl ◽

Claus Belka ◽

...

Keyword(s):

Prostate Cancer ◽

Cell Death ◽

Ionizing Radiation ◽

Cancer Cells ◽

Apoptotic Cell ◽

Apoptotic Cell Death ◽

Prostate Cancer Cells ◽

Akt Inhibitor ◽

Short Term ◽

Short Term Effects

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The MDM2 Antagonist Nutlin-3 Is Lethal to Mantle Cell Lymphoma with Wild Type p53.

Blood ◽

10.1182/blood.v110.11.1382.1382 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1382-1382

Author(s):

Yoko Tabe ◽

Denise Sebasigari ◽

Martina Rudelius ◽

Stefania Pittaluga ◽

Mark Raffel

Keyword(s):

Cell Proliferation ◽

Cell Death ◽

Mantle Cell Lymphoma ◽

Cell Lines ◽

Target Genes ◽

Cell Lymphoma ◽

Apoptotic Cell ◽

P53 Mutation ◽

Mantle Cell ◽

P53 Target Genes

Abstract Mantle cell lymphoma (MCL) has one of the poorest overall prognoses of the non-Hodgkin lymphomas, and is resistant to standard chemotherapy. P53 mutations are rare in typical mantle cell lymphoma and occur in only about 1/3 of the less common blastoid variant. The relatively low overall rate of p53 mutation in MCL suggests that this lymphoma may be a good candidate for biologic therapies that upregulate p53 and lead to cell death. Nutlin-3 is a novel small-molecule antagonist of MDM2 that efficiently activates p53 by blocking the MDM2-p53 interaction. In this study, we investigated the effects of nutlin-3 in 5 MCL cell lines with known p53 mutation status (wt-p53: REC-1, Z138, Granta-519, JVM-2; mt-p53: Jeko-1). IC50s for all cell lines were determined, and the activation of p53 and multiple p53 target genes were studied by western blot analysis. Cell proliferation and apoptosis were assessed using the MTT test and flow cytometry. Treatment with Nutlin-3 resulted in a marked reduction in cell proliferation/viability, and an increase in the apoptotic fraction, in a time and concentration-dependent manner in wt-p53 cells (IC50 at 48 hrs; 7.5 uM for Granta-519, 9.2 uM for REC-1, 0.5 uM for Z138, and 5.7 uM for JVM-2), while mt-p53 Jeko was resistant to nutlin-3 treatment (IC50>60 uM), and showed no increase in the apoptotic cell fraction. In the wt-p53 MCL cell lines, nutlin-3 treatment increased the cellular levels of p53, and several p53 dependent proteins including p21, MDM2 itself and the proapoptotic BH3-only protein Puma, while there was no change in the levels of these proteins in Jeko-1. Recently, attention has been focused on novel p53 target genes that function to inhibit cell growth and proliferation, rather than by inducing apoptosis. These include the AKT-mTOR regulators PTEN, TSC2 and AMPKβ1. We saw no increase in these novel p53 targets in any of the cell lines analyzed, suggesting that nutlin-3-activated p53 activates only a subset of its possible targets. These findings demonstrate that nutlin-3 successfully activates wt-p53 in mantle cell lymphoma leading to the upregulation of traditional targets such as p21 and proapoptotic proteins including Puma, and result in apoptotic cell death. The data suggest that p53 activators such as nutlin-3 may be effective agents in the treatment of mantle cell lymphoma.

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Berberine Induces Apoptotic Cell Death via Activation of Caspase-3 and -8 in HL-60 Human Leukemia Cells: Nuclear Localization and Structure–Activity Relationships

The American Journal of Chinese Medicine ◽

10.1142/s0192415x17500811 ◽

2017 ◽

Vol 45 (07) ◽

pp. 1497-1511 ◽

Cited By ~ 18

Author(s):

Shinya Okubo ◽

Takuhiro Uto ◽

Aya Goto ◽

Hiroyuki Tanaka ◽

Tsuyoshi Nishioku ◽

...

Keyword(s):

Cell Death ◽

Dna Fragmentation ◽

Antiproliferative Activity ◽

Caspase 3 ◽

Apoptotic Cell ◽

Apoptotic Cell Death ◽

Leukemia Cells ◽

Caspase 8 ◽

Human Leukemia ◽

Caspase 9

Berberine (BBR), an isoquinoline alkaloid, is a well-known bioactive compound contained in medicinal plants used in traditional and folk medicines. In this study, we investigated the subcellular localization and the apoptotic mechanisms of BBR were elucidated. First, we confirmed the incorporation of BBR into the cell visually. BBR showed antiproliferative activity and promptly localized to the nucleus from 5[Formula: see text]min to 15[Formula: see text]min after BBR treatment in HL-60 human promyelocytic leukemia cells. Next, we examined the antiproliferative activity of BBR (1) and its biosynthetically related compounds (2-7) in HL-60 cells. BBR exerted strongest antiproliferative activity among 1-7 and the results of structures and activity relation suggested that a methylenedioxyl group in ring A, an [Formula: see text]-alkyl group at C-9 position, and the frame of isoquinoline may be necessary for antiproliferative activity. Moreover, BBR showed the most potent antiproliferative activity in HL-60 cells among human cancer and normal cell lines tested. Next, we examined the effect of BBR on molecular events known as apoptosis induction. In HL-60 cells, BBR induced chromatin condensation and DNA fragmentation, and triggered the activation of PARP, caspase-3 and caspase-8 without the activation of caspase-9. BBR-induced DNA fragmentation was abolished by pretreatment with inhibitors against caspase-3 and caspase-8, but not against caspase-9. ERK and p38 were promptly phosphorylated after 15 min of BBR treatment, and this was correlated with time of localization to the nucleus of BBR. These results demonstrated that BBR translocated into nucleus immediately after treatments and induced apoptotic cell death by activation of caspase-3 and caspase-8.

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Aggregates from mutant and wild-type α-synuclein proteins and NAC peptide induce apoptotic cell death in human neuroblastoma cells by formation of β-sheet and amyloid-like filaments

FEBS Letters ◽

10.1016/s0014-5793(98)01418-5 ◽

1998 ◽

Vol 440 (1-2) ◽

pp. 71-75 ◽

Cited By ~ 257

Author(s):

Omar M.A El-Agnaf ◽

Ross Jakes ◽

Martin D Curran ◽

Derek Middleton ◽

Raffaele Ingenito ◽

...

Keyword(s):

Cell Death ◽

Apoptotic Cell ◽

Neuroblastoma Cells ◽

Apoptotic Cell Death ◽

Wild Type ◽

Human Neuroblastoma Cells ◽

Human Neuroblastoma ◽

Synuclein Proteins ◽

Β Sheet

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In self-defence: hexokinase promotes voltage-dependent anion channel closure and prevents mitochondria-mediated apoptotic cell death

Biochemical Journal ◽

10.1042/bj20031465 ◽

2004 ◽

Vol 377 (2) ◽

pp. 347-355 ◽

Cited By ~ 259

Author(s):

Heftsi AZOULAY-ZOHAR ◽

Adrian ISRAELSON ◽

Salah ABU-HAMAD ◽

Varda SHOSHAN-BARMATZ

Keyword(s):

Cell Death ◽

Cell Survival ◽

Molecular Mechanisms ◽

Apoptotic Cell ◽

Direct Interaction ◽

Anion Channel ◽

Mitochondrial Outer Membrane ◽

Voltage Dependent Anion Channel ◽

Metabolic Intermediate ◽

Voltage Dependent

In tumour cells, elevated levels of mitochondria-bound isoforms of hexokinase (HK-I and HK-II) result in the evasion of apoptosis, thereby allowing the cells to continue proliferating. The molecular mechanisms by which bound HK promotes cell survival are not yet fully understood. Our studies relying on the purified mitochondrial outer membrane protein VDAC (voltage-dependent anion channel), isolated mitochondria or cells in culture suggested that the anti-apoptotic activity of HK-I occurs via modulation of the mitochondrial phase of apoptosis. In the present paper, a direct interaction of HK-I with bilayer-reconstituted purified VDAC, inducing channel closure, is demonstrated for the first time. Moreover, HK-I prevented the Ca2+-dependent opening of the mitochondrial PTP (permeability transition pore) and release of the pro-apoptotic protein cytochrome c. The effects of HK-I on VDAC activity and PTP opening were prevented by the HK reaction product glucose 6-phosphate, a metabolic intermediate in most biosynthetic pathways. Furthermore, glucose 6-phosphate re-opened both the VDAC and the PTP closed by HK-I. The HK-I-mediated effects on VDAC and PTP were not observed using either yeast HK or HK-I lacking the N-terminal hydrophobic peptide responsible for binding to mitochondria, or in the presence of an antibody specific for the N-terminus of HK-I. Finally, HK-I overexpression in leukaemia-derived U-937 or vascular smooth muscle cells protected against staurosporine-induced apoptosis, with a decrease of up to 70% in cell death. These results offer insight into the mechanisms by which bound HK promotes tumour cell survival, and suggests that its overexpression not only ensures supplies of energy and phosphometabolites, but also reflects an anti-apoptotic defence mechanism.

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Comparison of Apoptosis in Wild-Type and Fas-Resistant Cells: Chemotherapy-Induced Apoptosis Is Not Dependent on Fas/Fas Ligand Interactions

Blood ◽

10.1182/blood.v90.3.935 ◽

1997 ◽

Vol 90 (3) ◽

pp. 935-943 ◽

Cited By ~ 178

Author(s):

Christine M. Eischen ◽

Timothy J. Kottke ◽

Luis M. Martins ◽

Guriqbal S. Basi ◽

Jay S. Tung ◽

...

Keyword(s):

Cell Death ◽

Fas Ligand ◽

Apoptotic Cell ◽

Chemotherapeutic Agents ◽

Jurkat Cells ◽

Wild Type ◽

Γ Irradiation ◽

Human T Cell ◽

Apoptotic Pathways ◽

Induced Apoptosis

Abstract The Fas/Fas ligand (FasL) pathway is widely involved in apoptotic cell death in lymphoid and nonlymphoid cells. It has recently been postulated that many chemotherapeutic agents also induce cell death by activating the Fas/FasL pathway. In the present study we compared apoptotic pathways induced by anti-Fas or chemotherapeutic agents in the Jurkat human T-cell leukemia line. Immunoblotting showed that treatment of wild-type Jurkat cells with anti-Fas or the topoisomerase II-directed agent etoposide resulted in proteolytic cleavage of precursors for the cysteine-dependent aspartate-directed proteases caspase-3 and caspase-7 and degradation of the caspase substrates poly(ADP-ribose) polymerase (PARP) and lamin B1 . Likewise, affinity labeling with N-(Nα-benzyloxycarbonylglutamyl-Nε-biotinyllysyl)aspartic acid [(2,6-dimethyl-benzoyl)oxy]methyl ketone [Z-EK (bio)D-amok] labeled the same five active caspase species after each treatment, suggesting that the same downstream apoptotic pathways have been activated by anti-Fas and etoposide. Treatment with ZB4, an antibody that inhibits Fas-mediated cell death, failed to block etoposide-induced apoptosis, raising the possibility that etoposide does not initiate apoptosis through Fas/FasL interactions. To further explore the relationship between Fas- and chemotherapy-induced apoptosis, Fas-resistant Jurkat cells were treated with various chemotherapeutic agents. Multiple independently derived Fas-resistant Jurkat lines underwent apoptosis that was indistinguishable from that of the Fas-sensitive parental cells after treatment with etoposide, doxorubicin, topotecan, cisplatin, methotrexate, staurosporine, or γ-irradiation. These results indicate that antineoplastic treatments induce apoptosis through a Fas-independent pathway even though Fas- and chemotherapy-induced pathways converge on common downstream apoptotic effector molecules.

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