Attenuation Of Akt Phosphorylation Research Articles

The AKT inhibitor MK2206 suppresses airway inflammation and the pro‑remodeling pathway in a TDI‑induced asthma mouse model.

The cellular and molecular mechanisms via which MK2206, an AKT inhibitor, prevents the activation of AKT in toluene diisocyanate (TDI)-induced asthma remain unclear. Thus, the present study aimed to evaluate the potential effects of MK2206 on airway AKT activation, inflammation and remodeling in a TDI-induced mouse model of asthma. A total of 24 BALB/c mice were selected and randomly divided into untreated (AOO), asthma (TDI), MK2206 (TDI + MK2206), and dexamethasone (TDI + DEX) groups. Phosphorylated AKT (p-AKT), total AKT, airway remodeling indices, α-smooth muscle actin (α-SMA) and collagen I levels in pulmonary tissue were measured using western blotting. Airway inflammation factors, including interleukin (IL)-4, −5, −6, and −13 in bronchoalveolar lavage fluid (BALF) and IgE in serum, were determined using ELISA. Additionally, the airway hyperresponsiveness (AHR) and pulmonary pathology of all groups were evaluated. The results of the present study demonstrated that p-AKT levels in lung protein lysate were upregulated, and neutrophil, eosinophil and lymphocyte counts were increased in the lungs obtained from the asthma group compared with the AOO group. Both MK2206 and DEX treatment in TDI-induced mice resulted not only in the attenuation of AKT phosphorylation, but also reductions in neutrophil, eosinophil and lymphocyte counts in the lungs of mice in the asthma group. Consistently, increases in the levels of the inflammatory cytokines IL-4, −5, −6 and −13 analyzed in BALF, and serum IgE in the TDI group were demonstrated to be attenuated in the TDI + MK2206 and TDI + DEX groups. Furthermore, α-SMA and AHR were significantly attenuated in the TDI + MK2206 group compared with the TDI group. These results revealed that MK2206 not only inhibited AKT activation, but also served a role in downregulating airway inflammation and airway remodeling in chemical-induced asthma. Therefore, the findings of the present study may provide important insight into further combination therapy.

Synergistic Ototoxicity of Gentamicin and Low-Dose Irradiation: Molecular Basis and Clinical Significance

Background: Inner ear structures may be included in the radiation fields when irradiation is used to treat patients with head and neck cancers. These patients may also have concurrent infections that require gentamicin treatment. Radiation and gentamicin are both potentially ototoxic, and their combined use has been shown to result in synergistic ototoxicity in animals. Objective: We aimed to confirm the synergistic ototoxicity of combined gentamicin and low-dose irradiation treatment and identify the underlying molecular mechanisms using an in vitro model. Method: We compared the ototoxic effects of gentamicin, low-dose irradiation, and their combination in the OC-k3 mouse cochlear cell line using cell viability assay, live/dead stain, apoptosis detection assay, oxidative stress detection, and studied the molecular mechanisms involved using immunoblot analysis. Results: Combined treatment led to prolonged oxidative stress, reduced cell viability, and synergistic apoptosis. Gentamicin induced the concurrent accumulation of LC3b-II and SQSTM1/p62, suggesting an impairment of autophagic flux. Low-dose irradiation induced transient p53 phosphorylation and persistent Akt phosphorylation in response to DNA damage. In combined treatment, gentamicin attenuated irradiation-induced Akt activation. Conclusions: Besides increased oxidative stress, synergistic apoptosis observed in combined treatment could be attributed to gentamicin-induced perturbation of autophagic flux and attenuation of Akt phosphorylation, which led to an impairment of radiation-induced DNA repair response.

Gomisin G Suppresses the Growth of Colon Cancer Cells by Attenuation of AKT Phosphorylation and Arrest of Cell Cycle Progression.

Colorectal cancer is one of the leading causes of cancer related death due to a poor prognosis. In this study, we investigated the effect of Gomisin G on colon cancer growth and examined the underlying mechanism of action. We found that Gomisin G significantly suppressed the viability and colony formation of LoVo cells. Gomisin G reduced the phosphorylation level of AKT implying that Gomisin G suppressed the PI3K-AKT signaling pathway. Gomisin G also induced apoptosis shown by Annexin V staining and an increased level of cleaved poly-ADP ribose polymerase (PARP) and Caspase-3 proteins. Furthermore, Gomisin G remarkably triggered the accumulation of cells at the sub-G1 phase which represents apoptotic cells. In addition, the level of cyclin D1 and phosphorylated retinoblastoma tumor suppressor protein (Rb) was also reduced by the treatment with Gomisin G thus curtailing cell cycle progression. These findings show the suppressive effect of Gomisin G by inhibiting proliferation and inducing apoptosis in LoVo cells. Taken together, these results suggest Gomisin G could be developed as a potential therapeutic compound against colon cancer.

Endocytosis is required for CXC chemokine receptor type 4 (CXCR4)-mediated Akt activation and antiapoptotic signaling.

Signaling activated by binding of the CXC motif chemokine ligand 12 (CXCL12) to its cognate G protein-coupled receptor (GPCR), chemokine CXC motif receptor 4 (CXCR4), is linked to metastatic disease. However, the mechanisms governing CXCR4 signaling remain poorly understood. Here, we show that endocytosis and early endosome antigen 1 (EEA1), which is part of the endosome fusion machinery, are required for CXCL12-mediated AKT Ser/Thr kinase (Akt) signaling selective for certain Akt substrates. Pharmacological inhibition of endocytosis partially attenuated CXCL12-induced phosphorylation of Akt, but not phosphorylation of ERK-1/2. Similarly, phosphorylation of Akt, but not ERK-1/2, stimulated by CXCL13, the cognate ligand for the chemokine receptor CXCR5, was also attenuated by inhibited endocytosis. Furthermore, siRNA-mediated depletion of the Rab5-effector EEA1, but not of adaptor protein, phosphotyrosine-interacting with PH domain and leucine zipper 1 (APPL1), partially attenuated Akt, but not ERK-1/2, phosphorylation promoted by CXCR4. Attenuation of Akt phosphorylation through inhibition of endocytosis or EEA1 depletion was associated with reduced signaling to Akt substrate forkhead box O1/3a but not the Akt substrates TSC complex subunit 2 or glycogen synthase kinase 3β. This suggested that endocytosis and endosomes govern discrete aspects of CXCR4- or CXCR5-mediated Akt signaling. Consistent with this hypothesis, depletion of EEA1 reduced the ability of CXCL12 to attenuate apoptosis in suspended, but not adherent, HeLa cells. Our results suggest a mechanism whereby compartmentalized chemokine-mediated Akt signaling from endosomes suppresses the cancer-related process known as anoikis. Targeting this signaling pathway may help inhibit metastatic cancer involving receptors such as CXCR4.

MK-2206 co-treatment with 5-fluorouracil or doxorubicin enhances chemosensitivity and apoptosis in gastric cancer by attenuation of Akt phosphorylation.

The anticancer effect of MK-2206, an Akt inhibitor, has been explored in some types of cancers, but its effect on gastric cancer is unclear. In this study, we aimed to investigate its anticancer effect in gastric cancer cells. Cell viability and colony formation assays showed that MK-2206 effectively inhibited the proliferation of SGC-7901 and MKN45 cells. The 50% inhibitory concentration values after 24, 48, and 72 hours’ treatment were 22.92, 13.68, and 8.55 μM in SGC-7901 cells and 19.21, 13.10, and 9.11 μM in MKN45 cells, respectively. Treatment with MK-2206 induced apoptosis in SGC-7901 cells as indicated by flow cytometry assay. The combination indexes of MK-2206 and doxorubicin were 0.59 in SGC-7901 cells and 0.57 in MKN45 cells, whereas for 5-fluorouracil (5-FU) the indexes were 0.17 in SGC-7901 cells and 0.73 in MKN45 cells, indicating that MK-2206 could work synergistically with doxorubicin or 5-FU to inhibit cell growth. Furthermore, a small dose (1 μM) of MK-2206 co-treatment with doxorubicin or 5-FU was sufficient for complete inhibition of chemotherapeutic alteration of phosphorylated Akt expression and significant enhancement of pro-apoptosis effect through the activation of caspase pathway. Therefore, MK-2206 effectively inhibits gastric cancer cell growth by attenuation of Akt phosphorylation and synergistically enhances the antitumor effect of doxorubicin and 5-FU via caspase-dependent apoptosis.

Tu1969 A Novel Photodynamic Therapy Targeting Tumor-Associated Macrophages

19.85, 13.14, 8.94 μmol/L in MKN45 at 24, 48, and 72 hours, respectively. MK-2206 enhanced apoptosis in SGC-7901 and increased the expression level of cleaved PARP. The combination therapy of MK-2206 and doxorubicin or 5-fluorouracil, showed a synergistic pattern of combination indexes in SGC-7901 and MKN45 cells, indicating an enhanced inhibition by MK-2206 co-treatment. Furthermore, a small dose (1 μmol/L) of MK-2206 was sufficient for complete inhibition of chemotherapeutic-alteration of pAkt expression and significant enhancement of pro-apoptosis effect through the activation of caspase pathway. Conclusion MK-2206 could effectively inhibit gastric cancer cell growth by attenuation of Akt phosphorylation. Additionally, MK-2206 could synergistically enhance the antitumor effect of doxorubicin or 5-fluorouracil via caspase-dependent apoptosis.

Knockdown of CABYR-a/b Increases Chemosensitivity of Human Non–Small Cell Lung Cancer Cells through Inactivation of Akt

CABYR is a calcium-binding tyrosine phosphorylation-regulated protein that was identified as a novel cancer testis antigen in lung cancer in our previous study. However, the role of CABYR as a driver of disease progression or as a chemosensitizer is poorly understood. This study sought to investigate the relationship between the expression levels of CABYR-a/b, which are the two predominant isoforms of the five isoform proteins encoded by CABYR, and chemosensitivity in non-small cell lung cancer cells. We found that the short hairpin RNA-mediated knockdown of CABYR-a/b significantly inhibited the proliferation of NCI-H460 and A549 cells and resulted in the attenuation of Akt phosphorylation, which is constitutively active in lung cancer cells. The silencing of CABYR-a/b expression notably impacted the downstream components of the Akt pathways: decreasing the phospho-GSK-3β (Ser9) levels and increasing the expression of the p53 and p27 proteins. Furthermore, CABYR-a/b knockdown led to a significant increase in chemosensitivity in response to chemotherapeutic drugs and drug-induced apoptosis, both in vitro and in vivo. Conversely, the transient transfection of CABYR-a/b-depleted cells with constitutively active Akt partially restored the resistance to cisplatin and paclitaxel and significantly decreased the activation of GSK-3β and cleaved PARP. Taken together, our results suggest that the inhibition of CABYR-a/b is a novel method to improve the apoptotic response and chemosensitivity in lung cancer and that this cancer testis antigen is an attractive target for lung cancer drug development. Suppression of CABYR-a/b expression increases chemosensitivity of lung cancer cells by inhibiting Akt activity.

Abstract 2136: Inhibition of autotaxin enhances radiosensitivity in human and murine glioblastoma cell lines.

Abstract Glioblastoma multiforme (GBM), a grade IV astrocytoma, is a very aggressive and prevalent primary brain tumor with a median survival rate of 12-15 months. Though the standard treatment relies on combinations of chemotherapy, radiation therapy and surgical resection, GBM remains radio-resistant with a high recurrence rate after resection. Understanding the molecular mechanisms involved in the therapeutic response could lead to the identification of novel targets to improve therapy. Radiation can trigger the activation of cytosolic phospholipase A2 (cPLA2), leading to production of lipid second-messengers such as lysophosphatidylcholine (LPC). Autotaxin (ATX), also known as lysophospholipase D (LysoPLD), is a secreted enzyme that catalyzes the production of lysophosphatidic acid (LPA) in the tumor microenvironment by cleaving the head-group of LPC. Specific G-protein coupled receptors (GPCRs) mediate the autocrine and paracrine effects of LPA such as migration, angiogenesis and proliferation in cancer. ATX is highly expressed in glioblastoma cells and is known to contribute to its invasive properties. We studied ATX as a potential target to enhance radiosensitivity in GBM by targeting ATX with PF-8380, a specific inhibitor developed by Pfizer Inc., and genetically by using shRNA. Pre-treatment with 1μM PF-8380 followed by irradiation with 4Gy resulted in decreased clonogenic survival, decreased migration and decreased invasion in mouse glioblastoma GL-261and human glioblastoma U87-MG cells. We confirmed these results by knocking down ATX with shRNA in GL-261 and U87-MG cells. Inhibition of ATX by PF-8380, or its knockdown by shRNA, lead to attenuation of Akt phosphorylation in GL-261 and U87-MG cells. Inhibition or knockdown of ATX resulted in significantly reduced radiation-induced neovascularization as assayed with GL-261 tumor vascular window model. The effect of ATX inhibition by PF-8380 in-vivo was studied using a heterotopic mouse GL-261 tumor model. Tumor bearing mice were treated with DMSO or 1 mg/kg PF-8380 followed by irradiation with 1.8Gy for 5 consecutive days. Tumor growth delay was analyzed using a non-parametric (Kruskal-Wallis) test to determine the amount of days to reach a tumor volume of 0.7 cm3 and ANOVA to determine the difference between treatments. Tumors treated with DMSO or PF-8380 took 11.2 and 12.2 days respectively to reach a volume of 0.7 cm3. Tumors treated with radiation alone took 23.3 days while the combination of PF-8380 with radiation significantly delayed tumor growth to 32 days. In this study, we found that inhibition of pro-survival cellular signaling pathways, migration, invasion and radiation-induced neovascularization can be achieved by targeting ATX. Thus, ATX could serve as a novel and effective molecular target for the development of radiosensitizers of GBM, and may provide a new approach to treating patients with this lethal form of brain cancer. Citation Format: David Y.A. Dadey, Sandeep R. Bhave, Daniel J. Ferraro, Rowan M. Karvas, Dennis E. Hallahan, Dinesh Thotala. Inhibition of autotaxin enhances radiosensitivity in human and murine glioblastoma cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2136. doi:10.1158/1538-7445.AM2013-2136

Bucillamine inhibits CD40-mediated Akt activation and antibody production in mouse B‐cell lymphoma

The improvement of rheumatoid factor titers in patients with rheumatoid arthritis is one of the significant clinical effects of bucillamine (Buc). In this study, we investigated the effects of SA981, an active metabolite of Buc, and methotrexate (MTX) on CD40-mediated antibody production using mouse B‐cell lymphoma, BCL1. SA981 significantly attenuated CD40-mediated antibody production in a concentration-dependent manner, but weakly affected cell proliferation. In contrast, MTX did not attenuate CD40-mediated antibody production until it had strongly inhibited cell proliferation at a concentration of 100nM. CD40 signaling induced protein phosphorylation, including Akt phosphorylation, p38 mitogen-activated protein kinase (p38MAPK), and IκBα. SA981 at a concentration of 30μM attenuated CD40-mediated Akt phosphorylation, but not p38MAPK or IκBα phosphorylation. MTX at a concentration of 100nM did not affect CD40-mediated Akt, p38MAPK, or IκBα phosphorylation. Commercially available Akt inhibitor VIII significantly attenuated CD40-mediated IgM production at a concentration of 100nM without significant inhibition of cell proliferation. These results suggest that SA981 inhibits CD40-mediated antibody production in mouse B‐cell lymphoma, at least in part, by attenuation of Akt phosphorylation.

Reactive Oxygen Species Signaling Facilitates FOXO-3a/FBXO-Dependent Vascular BK Channel β1 Subunit Degradation in Diabetic Mice

Activity of the vascular large conductance Ca2+-activated K+ (BK) channel is tightly regulated by its accessory β1 subunit (BK-β1). Downregulation of BK-β1 expression in diabetic vessels is associated with upregulation of the forkhead box O subfamily transcription factor-3a (FOXO-3a)–dependent F-box–only protein (FBXO) expression. However, the upstream signaling regulating this process is unclear. Overproduction of reactive oxygen species (ROS) is a common finding in diabetic vasculopathy. We hypothesized that ROS signaling cascade facilitates the FOXO-3a/FBXO-mediated BK-β1 degradation and leads to diabetic BK channel dysfunction. Using cellular biology, patch clamp, and videomicroscopy techniques, we found that reduced BK-β1 expression in streptozotocin (STZ)-induced diabetic mouse arteries and in human coronary smooth muscle cells (SMCs) cultured with high glucose was attributable to an increase in protein kinase C (PKC)-β and NADPH oxidase expressions and accompanied by attenuation of Akt phosphorylation and augmentation of atrogin-1 expression. Treatment with ruboxistaurin (a PKCβ inhibitor) or with GW501516 (a peroxisome proliferator–activated receptor δ activator) reduced atrogin-1 expression and restored BK channel-mediated coronary vasodilation in diabetic mice. Our results suggested that oxidative stress inhibited Akt signaling and facilitated the FOXO-3a/FBXO-dependent BK-β1 degradation in diabetic vessels. Suppression of the FOXO-3a/FBXO pathway prevented vascular BK-β1 degradation and protected coronary function in diabetes.

Akt and PKC are involved not only in upregulation of telomerase activity but also in cell differentiation-related function via mTORC2 in leukemia cells

We have shown previously that PI3K/Akt pathway is active after cell differentiation in HL60 cells. In the present study, we have investigated whether additional molecules, such as protein kinase C (PKC), are involved in the regulation, not only of telomerase, but also of leukemia cell differentiation. We show that PKC activates telomerase and is, itself, activated following VD3- or ATRA-induced differentiation of HL60 cells, as was observed for PI3K/Akt. To clarify the significance of PI3K/Akt and PKC pathway activation in leukemia cell differentiation, we examined the active proteins in either the downstream or upstream regulation of these pathways. In conjunction with the activation of Akt or PKC, mTOR and S6K were phosphorylated and the protein expression levels of Rictor were increased, compared with Raptor, following cell differentiation. Silencing by Rictor siRNA resulted in the attenuation of Akt phosphorylation on Ser473 and PKCα/βII phosphorylation, as well as the inhibition of Rictor itself, suggesting that Rictor is an upstream regulator of both Akt and PKC. In addition, in cells induced to differentiate by ATRA or VD3, Nitroblue-tetrazolium (NBT) reduction and esterase activity, were blocked either by LY294002, a PI3K inhibitor, or by BIM, a PKC inhibitor, without affecting cell surface markers such as CD11b or CD14. Intriguingly, the silencing of Rictor by its siRNA also suppressed the reducing ability of NBT following VD3-induced cell differentiation. Taken together, our results show that Rictor associated with mTOR (mTORC2) regulates the activity of both Akt and PKC that are involved in cell functions such as NBT reduction and esterase activity induced by leukemia cell differentiation.

HIV Protease Inhibitors Enhance the Efficacy of Irradiation

Tumor vascular endothelium is rather resistant to the cytotoxic effects of radiation. The HIV protease inhibitors (HPI) amprenavir, nelfinavir, and saquinavir have previously been shown to sensitize tumor cells to the cytotoxic effects of radiation. Additionally, this class of drug has been shown to inhibit angiogenesis and tumor cell migration. Therefore, in the current study, we wanted to determine whether HPIs could enhance the effect of radiation on endothelial function. Our study shows that HPIs, particularly nelfinavir, significantly enhance radiations effect on human umbilical vein endothelial cells (HUVEC) and tumor vascular endothelium. We show that pretreatment of HUVEC with nelfinavir results in enhanced cytotoxicity, including increased apoptosis, when combined with radiation. Moreover, using several functional assays, we show that combination treatment effectively blocks endothelial cell migration and organization. These findings were accompanied by attenuation of Akt phosphorylation, a known pathway for radioresistance. Last, in vivo analysis of tumor microvasculature destruction showed a more than additive effect for nelfinavir and radiation. This study shows that HPIs can enhance the effect of ionizing radiation on vascular endothelium. Therefore, the Food and Drug Administration-approved drug, nelfinavir, may be an effective radiosensitizer in the clinic.

5-Aminoimidazole-4-carboxamide-1-β-D-ribofuranoside Inhibits Cancer Cell Proliferation in Vitro and in Vivo via AMP-activated Protein Kinase

5-Aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR) is widely used as an AMP-kinase activator, which regulates energy homeostasis and response to metabolic stress. Here, we investigated the effect of AICAR, an AMPK activator, on proliferation of various cancer cells and observed that proliferation of all the examined cell lines was significantly inhibited by AICAR treatment due to arrest in S-phase accompanied with increased expression of p21, p27, and p53 proteins and inhibition of PI3K-Akt pathway. Inhibition in in vitro growth of cancer cells was mirrored in vivo with increased expression of p21, p27, and p53 and attenuation of Akt phosphorylation. Anti-proliferative effect of AICAR is mediated through activated AMP-activated protein kinase (AMPK) as iodotubericidin and dominant-negative AMPK expression vector reversed the AICAR-mediated growth arrest. Moreover, constitutive active AMPK arrested the cells in S-phase by inducing the expression of p21, p27, and p53 proteins and inhibiting Akt phosphorylation, suggesting the involvement of AMPK. AICAR inhibited proliferation in both LKB and LKB knock-out mouse embryo fibroblasts to similar extent and arrested cells at S-phase when transfected with dominant negative expression vector of LKB. Altogether, these results indicate that AICAR can be utilized as a therapeutic drug to inhibit cancer, and AMPK can be a potential target for treatment of various cancers independent of the functional tumor suppressor gene, LKB.