Molecular Mechanisms Regulating Resistance to the Akt Inhibitor Perifosine in Waldenstrom’s Macroglobulinemia, the Role of the ERK and PKC Pathways.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2416-2416
Author(s):  
Xavier Leleu ◽  
Hai Ngo ◽  
Xiaoying Jia ◽  
Anne-Sophie Moreau ◽  
Evdoxia Hatjiharisi ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Mek Inhibitor ◽  
Pi3k Inhibitor ◽  
Erk Pathway ◽  
Waldenström’S Macroglobulinemia ◽  
Akt Inhibitor ◽  
Erk Activation ◽  
Pi3k Inhibitor Ly294002 ◽  
Akt Phosphorylation ◽  
Waldenström's Macroglobulinemia

Abstract Background: We have previously demonstrated activity of the new Akt inhibitor perifosine (NSC 639966; Keryx, NY) in Waldenstrom’s Macroglobulinemia (WM). Perifosine induced complete inhibition of Akt phosphorylation along with induction of apoptosis in WM cells. However, MAPK pathways and PKC proteins were activated in response to perifosine. MAPK and PKC pathways are known to promote cell proliferation. Therefore, understanding the mechanism by which perifosine induces MEK/ERK and PKC activation is important to better understand the mechanisms of response/resistance to this novel agent in ongoing clinical trials. Methods: WM cell lines (BCWM.1, WM-WSU) were treated with perifosine or with the specific Akt inhibitor Triciribine (Biomol, PA). In addition, knockdown of Akt was performed using shRNA silencing techniques (lentivirus shRNA vector, Invitrogen, Ca). The following inhibitors were also used: PI3K inhibitor LY294002 (Calbiochem, CA) and MEK inhibitor (U0126, Calbiochem, CA). Inhibition of proliferation was measured using the MTT assay. Immunoblotting was performed at different time points. Results: Perifosine induced cytotoxicity in WM cells and induced MEK/ERK activation and pPKC activation in a dose and time dependent fashion. We then treated WM cells with perifosine in the presence or absence of the MEK inhibitor U0126 and demonstrated that the combination of the two agents induced significant synergistic activity. We sought to identify the molecular mechanism by which perifosine induces MEK/ERK activation. We demonstrated that the specific AKT inhibitor Triciribine inhibited AKT and induced cytotoxicity in WM cells in a similar fashion to perifosine. However, unlike perifosine, it did not enhance MEK/ERK activity. Similarly, using Akt shRNA, we demonstrated that, despite inhibition of Akt activation, MEK/ERK was not activated. These data indicate that the effect of perifosine on MEK/ERK pathway is not through a compensatory feedback mechanism of Akt inhibition as previously thought. Therefore, we hypothesized that the effect of perifosine on the MEK/ERK pathway is through modulation of upstream pathways, specifically PI3K, PKC and c-Raf/MEK pathways. We first demonstrated that the specific PI3K inhibitor LY294002 (25mM for 15 minutes) completely abrogated Akt phosphorylation, while inducing significant ERK activation, indicating that the effect of perifosine on MEK/ERK may be similar to that of LY294002. We also demonstrated that perifosine and LY294002 activated c-Raf and pan-pPKC at 4 hrs. Conclusion: Based on this, we believe that in the presence of perifosine, growth receptor stimulation leads to PLC and RTK activation, which induces PIP2 stimulation. PIP2 is upstream of PI3K and PKC. Given that PI3K is blocked by perifosine, PIP2 leads to activation of PKC, which then induces growth stimulation, and activation of c-Raf and downstream MEK/ERK. In addition, growth receptors may also activate Raf through the Ras/Raf/MEK pathway, independent of PKC. These studies provide a better understanding of molecular mechanisms that regulate resistance to perifosine. Future combinations of perifosine with MEK inhibitors or PKC inhibitors such as AZD6244 and Enzastaurin may overcome this resistance and induce significant activity in WM.

scholarly journals Monophosphoryl lipid A induces protection against LPS in medullary thick ascending limb through a TLR4-TRIF-PI3K signaling pathway

2017 ◽  
Vol 313 (1) ◽  
pp. F103-F115 ◽  
Author(s):  
Bruns A. Watts ◽  
Thampi George ◽  
Edward R. Sherwood ◽  
David W. Good
Keyword(s):  
Bacterial Infections ◽  
Lipid A ◽  
Thick Ascending Limb ◽  
Akt Inhibitor ◽  
Erk Activation ◽  
Akt Phosphorylation ◽  
Medullary Thick Ascending Limb ◽  
Monophosphoryl Lipid A ◽  
Monophosphoryl Lipid

Monophosphoryl lipid A (MPLA) is a detoxified derivative of LPS that induces tolerance to LPS and augments host resistance to bacterial infections. Previously, we demonstrated that LPS inhibits [Formula: see text] absorption in the medullary thick ascending limb (MTAL) through a basolateral Toll-like receptor 4 (TLR4)-myeloid differentiation factor 88 (MyD88)-ERK pathway. Here we examined whether pretreatment with MPLA would attenuate LPS inhibition. MTALs from rats were perfused in vitro with MPLA (1 µg/ml) in bath and lumen or bath alone for 2 h, and then LPS was added to (and MPLA removed from) the bath solution. Pretreatment with MPLA eliminated LPS-induced inhibition of [Formula: see text] absorption. In MTALs pretreated with MPLA plus a phosphatidylinositol 3-kinase (PI3K) or Akt inhibitor, LPS decreased [Formula: see text] absorption. MPLA increased Akt phosphorylation in dissected MTALs. The Akt activation was eliminated by a PI3K inhibitor and in MTALs from TLR4−/−or Toll/IL-1 receptor domain-containing adaptor-inducing IFN-β (TRIF)−/−mice. The effect of MPLA to prevent LPS inhibition of [Formula: see text] absorption also was TRIF dependent. Pretreatment with MPLA prevented LPS-induced ERK activation; this effect was dependent on PI3K. MPLA alone had no effect on [Formula: see text] absorption, and MPLA pretreatment did not prevent ERK-mediated inhibition of [Formula: see text] absorption by aldosterone, consistent with MPLA's low toxicity profile. These results demonstrate that pretreatment with MPLA prevents the effect of LPS to inhibit [Formula: see text] absorption in the MTAL. This protective effect is mediated directly through MPLA stimulation of a TLR4-TRIF-PI3K-Akt pathway that prevents LPS-induced ERK activation. These studies identify detoxified TLR4-based immunomodulators as novel potential therapeutic agents to prevent or treat renal tubule dysfunction in response to bacterial infections.

scholarly journals PI3K inhibitor LY294002, as opposed to wortmannin, enhances AKT phosphorylation in gemcitabine-resistant pancreatic cancer cells

2016 ◽  
Vol 50 (2) ◽  
pp. 606-612 ◽  
Author(s):  
Yufeng Wang ◽  
Yasuhiro Kuramitsu ◽  
Byron Baron ◽  
Takao Kitagawa ◽  
Kazuhiro Tokuda ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Cancer Cells ◽  
Pi3k Inhibitor ◽  
Pi3k Inhibitor Ly294002 ◽  
Akt Phosphorylation ◽  
Pancreatic Cancer Cells

scholarly journals PI3K/Akt inhibitor partly decreases TNF-α-induced activation of fibroblast-like synoviocytes in osteoarthritis

2019 ◽  
Vol 14 (1) ◽  
Author(s):  
Songyang Liu ◽  
Chenxi Cao ◽  
Yujun Zhang ◽  
Guangyu Liu ◽  
Weixia Ren ◽  
...  
Keyword(s):  
Cartilage Damage ◽  
Inhibitory Effect ◽  
Pi3k Inhibitor ◽  
Akt Pathway ◽  
Akt Inhibitor ◽  
Pi3k Inhibitor Ly294002 ◽  
Tnf Α ◽  
Invasive Capacity ◽  
And Cartilage ◽  
Fibroblast Like Synoviocytes

Abstract Background The Cadherin-11 and PI3K/Akt pathway are increasingly recognized as the potential therapeutic target of osteoarthritis (OA) synovitis. The study aimed to investigate the role of PI3K/Akt signaling pathway in the expression of Cadherin-11 and migration and invasive capacity of fibroblast-like synoviocytes (FLS) of OA patients under stimulation of TNF-α and to explore the effect of the PI3K/Akt inhibitor and Cadherin-11 antibody in the therapy of the collagenase-induced osteoarthritis (CIOA) mice. Methods FLS were primarily cultured from synovium of osteoarthritic patients during total knee arthroplasty. Under the simulation of TNF-α, with or without PI3K/Akt inhibitor LY294002, Cadherin-11 expression was detected by real-time PCR and Western blot, as well as the migration and invasive capacity changes of OA FLS. Cadherin-11 antibody was injected intraarticularly or LY294002 was injected intraperitoneally in CIOA mice to evaluate the changes of synovitis score, cartilage damage, and Cadherin-11 expression. Results TNF-α stimulation increased Cadherin-11 expression at mRNA and protein level in OA FLS and also increased the phosphorylation-dependent activation of Akt. PI3K inhibitor LY294002 attenuated TNF-α-induced overexpression of Cadherin-11 and decreased the invasive capacity of OA FLS. Intraperitoneal injection of PI3K inhibitor LY294002 could decrease the Cadherin-11 protein expression in synovium of CIOA mice, although it has no significant inhibitory effect on synovitis and cartilage damage. Intraarticular injection of Cadherin-11 antibody attenuated the synovitis and cartilage damage in the CIOA joints and decreased Cadherin-11 expression in the synovial lining. Conclusions PI3K/Akt pathway was associated with TNF-α-induced activation of OA FLS, which may involve in the pathogenesis of osteoarthritis. Anti-Cadherin-11 therapy in CIOA mice could attenuate the pathological changes of OA joints.

scholarly journals FcγR-induced production of superoxide and inflammatory cytokines is differentially regulated by SHIP through its influence on PI3K and/or Ras/Erk pathways

Blood ◽  
2006 ◽  
Vol 108 (2) ◽  
pp. 718-725 ◽  
Author(s):  
Latha P. Ganesan ◽  
Trupti Joshi ◽  
Huiqing Fang ◽  
Vijay Kumar Kutala ◽  
Julie Roda ◽  
...  
Keyword(s):  
Inflammatory Cytokines ◽  
Clustering Analysis ◽  
Molecular Mechanisms ◽  
Erk Pathway ◽  
Erk Activation ◽  
Coated Particles ◽  
Inositol Phosphatase ◽  
Enhanced Production ◽  
Gtp Binding

Phagocytosis of IgG-coated particles via FcγR is accompanied by the generation of superoxide and inflammatory cytokines, which can cause collateral tissue damage in the absence of regulation. Molecular mechanisms regulating these phagocytosis-associated events are not known. SHIP is an inositol phosphatase that downregulates PI3K-mediated activation events. Here, we have examined the role of SHIP in FcγR-induced production of superoxide and inflammatory cytokines. We report that primary SHIP-deficient bone marrow macrophages produce elevated levels of superoxide upon FcγR clustering. Analysis of the molecular mechanism revealed that SHIP regulates upstream Rac-GTP binding, an obligatory event for superoxide production. Likewise, SHIP-deficient macrophages displayed enhanced IL-1β and IL-6 production in response to FcγR clustering. Interestingly, whereas IL-6 production required activation of both PI3K and Ras/Erk pathways, IL-1β production was dependent only on Ras/Erk activation, suggesting that SHIP may also regulate the Ras/Erk pathway in macrophages. Consistently, SHIP-deficient macrophages displayed enhanced activation of Erk upon FcγR clustering. Inhibition of Ras/Erk or PI3K suppressed the enhanced production of IL-6 in SHIP-deficient macrophages. In contrast, inhibition of Ras/Erk, but not PI3K, suppressed IL-1β production in these cells. Together, these data demonstrate that SHIP regulates phagocytosis-associated events through the inhibition of PI3K and Ras/Erk pathways.

Akt Inhibitor Perifosine-Induced Cytotoxicity Is Associated with Significant Downregulation of Survivin in Human Multiple Myeloma (MM) Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3410-3410
Author(s):  
Teru Hideshima ◽  
Hiroshi Yasui ◽  
Laurence Catley ◽  
Noopur Raje ◽  
Dharminder Chauhan ◽  
...  
Keyword(s):  
Cell Lines ◽  
Molecular Mechanisms ◽  
Caspase 3 ◽  
Single Agent ◽  
Survivin Expression ◽  
Mek Inhibitor ◽  
Parp Cleavage ◽  
Caspase 8 ◽  
Akt Inhibitor ◽  
Apoptotic Proteins

Abstract Perifosine (NSC 639966; Keryx Biopharmaceuticals, New York, NY) is a synthetic novel alkylphospholipid, a new class of anti-tumor agents which potently inhibits Akt (PKB) activity. Our previous studies have shown that Perifosine induces significant cytotoxicity in MM cells triggered by c-Jun NH2-terminal kinase (JNK) activation followed by caspase-8, caspase-9, and PARP cleavage even in the presence of cytokines (ie, IL-6 and IGF-1) or bone marrow stromal cell (BMSCs). Importantly, MEK inhibitor and bortezomib enhance Perifosine-induced cytotoxicity. It has also shown significant anti-tumor activity in a human MM cell xenograft mouse model (Hideshima et al. Blood2006, 107:4053–4062). In this study, we further delineated molecular mechanisms whereby Perifosine triggers cytotoxicity as a single agent and in combination with bortezomib in MM cells. In most MM cell lines, the IC50 for Perifosine-induced cytotoxicity is 5–10 μM range assessed by MTT assay at 24h; however, apoptosis assessed by APO2.7 staining, varied in each cell line. Moreover, neither the degree of JNK phosphorylation nor caspase-8/9/PARP cleavage correlated with Perifosine-induced cytotoxicity. Therefore we further examined expression level of anti-apoptotic proteins in MM cell lines and found that survivin, which has a crucial role in regulation of caspase-3 activity, was markedly downregulated by Perifosine treatment in a time- and dose-dependent fashion, without affecting expression of other anti-apoptotic proteins (ie, cIAP, XIAP, Bcl-2, Bcl-xL). Since survivin is a known downstream protein of β-catenin/TCF-4 cascade, we next hypothesized that Perifosine may inhibit β-catenin activity. As expected, Perifosine significantly downregulated both phosphorylation and protein expression of β-catenin, associated with downregulation of survivin and enhanced caspase-3 cleavage. Real-time PCR confirmed that gene expression of survivin was suppressed 35% and 55% after 3h and 6h Perifosine treatment, respectively. Since β-catenin is a substrate of proteasomes, we further examined whether bortezomib could augment survivin expression by blocking its degradation. Importantly, bortezomib significantly upregulated β-catenin and survivin, which was blocked in the presence of Perifosine. These results suggest that inhibition of bortezomib-induced survivin expression, at least in part, accounts for enhanced bortezomib-induced cytotoxicity by Perifosine. Based upon these preclinical studies, a rational combination trial of bortezomib with Perifosine to treat relapsed refractory MM is currently ongoing.

scholarly journals Adenovirus Type 7 Induces Interleukin-8 Production via Activation of Extracellular Regulated Kinase 1/2

2001 ◽  
Vol 75 (14) ◽  
pp. 6450-6459 ◽  
Author(s):  
M. J. Alcorn ◽  
J. L. Booth ◽  
K. M. Coggeshall ◽  
J. P. Metcalf
Keyword(s):  
Epithelial Cells ◽  
Neutrophil Infiltration ◽  
Adenovirus Type ◽  
Mek Inhibitor ◽  
Interleukin 8 ◽  
Erk Pathway ◽  
Erk Activation ◽  
Extracellular Regulated Kinase ◽  
Rapid Activation

ABSTRACT Infection with adenovirus serotype 7 (Ad7) frequently causes lower respiratory pneumonia and is associated with severe lung inflammation and neutrophil infiltration. Earlier studies indicated release of proinflammatory cytokines, specifically interleukin-8 (IL-8), by pulmonary epithelial cells following infection by Ad7. However, the mechanism of IL-8 induction by Ad7 is unclear. We have explored the role of the Ras/Raf/MEK/Erk pathway in the Ad7-associated induction of IL-8 using a model system of A549 epithelial cells. We found that Ad7 infection induced a rapid activation of epithelial cell-derived Erk. The MEK-specific inhibitors PD98059 and U0126 blocked Erk activation and release of IL-8 following infection with Ad7. Treatment with PD98059 is cytostatic and not cytotoxic, as treated cells regain the ability to phosphorylate Erk and secrete IL-8 after removal of the drug. The expression of a mutated form of Ras in A549 epithelial cells blocked the induction of IL-8 promoter activity, and MEK inhibitor blocked induction of IL-8 mRNA. These results suggest that the Ras/Raf/MEK/Erk pathway is necessary for the Ad7 induction of IL-8 and that induction occurs at the level of transcription. Further, the kinetics of Erk activation and IL-8 induction suggest that an early viral event, such as receptor binding, may be responsible for the observed inflammatory response.

scholarly journals Testosterone induces cardiomyocyte hypertrophy through mammalian target of rapamycin complex 1 pathway

2009 ◽  
Vol 202 (2) ◽  
pp. 299-307 ◽  
Author(s):  
Francisco Altamirano ◽  
César Oyarce ◽  
Patricio Silva ◽  
Marcela Toyos ◽  
Carlos Wilson ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Mammalian Target Of Rapamycin ◽  
Cardiac Cells ◽  
Initiation Factor ◽  
Target Of Rapamycin ◽  
Cardiomyocyte Hypertrophy ◽  
Akt Inhibitor ◽  
Mtor Inhibition ◽  
Akt Phosphorylation ◽  
Mtorc1 Signaling

Elevated testosterone concentrations induce cardiac hypertrophy but the molecular mechanisms are poorly understood. Anabolic properties of testosterone involve an increase in protein synthesis. The mammalian target of rapamycin complex 1 (mTORC1) pathway is a major regulator of cell growth, but the relationship between testosterone action and mTORC1 in cardiac cells remains unknown. Here, we investigated whether the hypertrophic effects of testosterone are mediated by mTORC1 signaling in cultured cardiomyocytes. Testosterone increases the phosphorylation of mTOR and its downstream targets 40S ribosomal protein S6 kinase 1 (S6K1; also known as RPS6KB1) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). The S6K1 phosphorylation induced by testosterone was blocked by rapamycin and small interfering RNA to mTOR. Moreover, the hormone increased both extracellular-regulated kinase (ERK1/2) and protein kinase B (Akt) phosphorylation. ERK1/2 inhibitor PD98059 blocked the testosterone-induced S6K1 phosphorylation, whereas Akt inhibition (Akt-inhibitor-X) had no effect. Testosterone-induced ERK1/2 and S6K1 phosphorylation increases were blocked by either 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid-acetoxymethylester or by inhibitors of inositol 1,4,5-trisphosphate (IP3) pathway: U-73122 and 2-aminoethyl diphenylborate. Finally, cardiomyocyte hypertrophy was evaluated by, the expression of β-myosin heavy chain, α-skeletal actin, cell size, and amino acid incorporation. Testosterone increased all four parameters and the increase being blocked by mTOR inhibition. Our findings suggest that testosterone activates the mTORC1/S6K1 axis through IP3/Ca2+ and MEK/ERK1/2 to induce cardiomyocyte hypertrophy.

scholarly journals IGF1-induced AKT phosphorylation and cell proliferation are suppressed with the increase in PTEN during luteinization in human granulosa cells

Reproduction ◽  
2009 ◽  
Vol 137 (5) ◽  
pp. 835-842 ◽  
Author(s):  
Maki Goto ◽  
Akira Iwase ◽  
Toko Harata ◽  
Sachiko Takigawa ◽  
Kyosuke Suzuki ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Granulosa Cells ◽  
Inverse Relationship ◽  
Mek Inhibitor ◽  
Pten Expression ◽  
Follicular Growth ◽  
Pi3k Inhibitor Ly294002 ◽  
Akt Phosphorylation ◽  
Human Granulosa Cells ◽  
Phosphatase And Tensin Homologue

Granulosa cells proliferate and then undergo differentiation; an inverse relationship between these processes is observed during terminal follicular growth. During terminal follicular growth and initial luteinization, there is a necessary transition of granulosa cells to a less proliferative and highly steroidogenic form in response to LH. Although the expression of several molecules has been reported to be up-regulated by LH, proliferation/differentiation transition is not fully understood. Here, we show that the expression of a tumor suppressor, phosphatase and tensin homologue deleted on chromosome 10 (PTEN) was induced with human chorionic gonadotropin (hCG) treatment in human luteinized granulosa cells. Pretreatment with hCG attenuated insulin-like growth factor (IGF)-1-induced phosphorylation of AKT and cell proliferation, not phosphorylation of ERK1/2. Moreover, suppression of hCG-induced PTEN expression with siRNA increased AKT phosphorylation and cell proliferation in response to IGF1. We also demonstrate that a PI3K inhibitor, LY294002, not a MEK inhibitor, PD98059, inhibited IGF1-induced cell proliferation. In conclusion, PTEN induced to express by hCG in luteinized granulosa cells that inactivates AKT, not ERK, and attenuates IGF1-induced cell proliferation. PTEN expression may be a trigger for proliferation/differentiation transition in human granulosa cells.

scholarly journals Curcuma amarissima Extract Activates Growth and Survival Signal Transduction Networks to Stimulate Proliferation of Human Keratinocyte

Biology ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 289
Author(s):  
Wutigri Nimlamool ◽  
Saranyapin Potikanond ◽  
Jirapak Ruttanapattanakul ◽  
Nitwara Wikan ◽  
Siriporn Okonogi ◽  
...  
Keyword(s):  
Wound Healing ◽  
Cell Viability ◽  
Molecular Mechanisms ◽  
Cell Monolayer ◽  
Mek Inhibitor ◽  
Convincing Evidence ◽  
Cell Viability Assay ◽  
Pi3k Inhibitor Ly294002 ◽  
Viability Assay ◽  
Human Keratinocyte

Many medicinal plants have been used to treat wounds. Here, we revealed the potential wound healing effects of Curcuma amarissima (CA). Our cell viability assay showed that CA extract increased the viability of HaCaT cells that were cultured in the absence of serum. This increase in cell viability was proved to be associated with the pharmacological activities of CA extract in inducing cell proliferation. To further define possible molecular mechanisms of action, we performed Western blot analysis and immunofluorescence study, and our data demonstrated that CA extract rapidly induced ERK1/2 and Akt activation. Consistently, CA extract accelerated cell migration, resulting in rapid healing of wounded human keratinocyte monolayer. Specifically, the CA-induced increase of cell monolayer wound healing was blocked by the MEK inhibitor (U0126) or the PI3K inhibitor (LY294002). Moreover, CA extract induced the expression of Mcl-1, which is an anti-apoptotic protein, supporting that CA extract enhances human keratinocyte survival. Taken together, our study provided convincing evidence that Curcuma amarissima can promote proliferation and survival of human keratinocyte through stimulating the MAPK and PI3K/Akt signaling cascades. These promising data emphasize the possibility to develop this plant as a wound healing agent for the potential application in regenerative medicine.

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