Inhibition Of PI3K-Akt Signaling Pathway Research Articles

Conformationally regulated “nanozyme-like” cerium oxide with multiple free radical scavenging activities for osteoimmunology modulation and vascularized osseointegration

Given post-operative aseptic loosening in orthopedic disease treatment, osteointegration occurs at the bone-implant interface as a holistic process, including immunoregulation (e.g., macrophage polarization), angiogenesis and osteogenesis in sequence. In order to achieve early rapid and satisfactory osseointegration, different nano-shaped (nanocone, nanopolyhedron and nanoflower abbr. NC, NP & NF) cerium oxide (CeO2-x) coatings, endowed with “nanozyme-like” activities for multiple free radical elimination and osteoimmunology regulation, were hydrothermally synthesized on titanium alloy (TC4). In vitro cell experiments showed that nano-CeO2-x coated TC4 not only induced polarization of RAW264.7 cells toward M2 phenotype, but also promoted angiogenesis and vascularization of endothelial cells along with differentiation and mineralization of osteogenic precursor cells. Improvements in M2-polarized macrophage, angiogenesis, and bone regeneration were further confirmed in a rat femoral condyle model. Among the above three nano-morphologies, NF exhibited the best osseoinetegration. RNA sequencing and mechanism exploration suggested that the inhibition of PI3K-AKT signaling pathway was essential for immunomodulatory capacity of NF. In conclusion, it provided promising insights into the immunomodulatory exploitation of orthopedic implants.

TPN10475 alleviates concanavalin A-induced autoimmune hepatitis by limiting T cell development and function through inhibition of PI3K-AKT pathway

Autoimmune hepatitis (AIH) is an inflammatory liver disease in which the autoimmune system instigates an attack on the liver, causing inflammation and liver injury, and its incidence has increased worldwide in recent years. The mouse model of acute hepatitis established by concanavalin A (Con A) is a typical and recognized mouse model for the study of T-cell-dependent liver injury. In this study, we aimed to investigate whether the artemisinin derivative TPN10475 could alleviate AIH and its possible mechanisms. TPN10475 effectively inhibited lymphocyte proliferation and IFN-γ+ T cells production in vitro, alleviated liver injury by decreasing infiltrating inflammatory T cells producing IFN-γ in the liver and peripheral immune tissues, and demonstrated that TPN10475 weakened the activation and function of T cells by inhibiting PI3K-AKT signaling pathway. These results suggested that TPN10475 may be a potential drug for the treatment of AIH, and the inhibition of PI3K-AKT signaling pathway may provide new ideas for the study of the pathogenesis of AIH.

Analysis of gene expression profiles and experimental validations of a rat chronic cervical cord compression model

Cervical spondylotic myelopathy (CSM) is a severe non-traumatic spinal cord injury (SCI) wherein the spinal canal and cervical cord are compressed due to the degeneration of cervical tissues. To explore the mechanism of CSM, the ideal model of chronic cervical cord compression in rats was constructed by embedding a polyvinyl alcohol-polyacrylamide hydrogel in lamina space. Then, the RNA sequencing technology was used to screen the differentially expressed genes (DEGs) and enriched pathways among intact and compressed spinal cords. A total of 444 DEGs were filtered out based on the value of log2(Compression/Sham); these were associated with IL-17, PI3K-AKT, TGF-β, and Hippo signaling pathways according to the GSEA, KEGG, and GO analyses. Transmission electron microscopy indicated the changes in mitochondrial morphology. Western blot and immunofluorescence staining revealed neuronal apoptosis, astrogliosis and microglial neuroinflammation in the lesion area. Specifically, the expression of apoptotic indicators, such as Bax and cleaved caspase-3, and inflammatory cytokines, such as IL-1β, IL-6, and TNF-α, were upregulated. The activation of IL-17 signaling pathway was observed in microglia instead of neurons or astrocytes, the activation of TGF-β and inhibition of Hippo signaling pathways were detected in astrocytes instead of neurons or microglia, and the inhibition of PI3K-AKT signaling pathway was discovered in neurons rather than microglia of astrocytes in the lesion area. In conclusion, this study indicated that neuronal apoptosis was accompanied by inhibiting of the PI3K-AKT pathway. Then, the activation of microglia IL-17 pathway and NLRP3 inflammasome effectuated the neuroinflammation, and astrogliosis was ascribed to the activation of TGF-β and the inhibition of the Hippo pathway in the chronic cervical cord of compression. Therefore, therapeutic methods targeting these pathways in nerve cells could be promising CSM treatments.

Combining Radiotherapy with PI3K Isoform-Selectivity Inhibitor Increases Radiosensitivity And Suppresses Tumor Growth In Glioblastoma Cells

Glioblastoma (GBM) is the most malignant cancer that occurs within the brain and shows the dismal prognosis. The phosphoinositide 3-kinase (PI3K)-AKT signaling pathway plays a critical role in GBM. Also, the activation of Akt due to irradiation induces radioresistance. But, the pan-PI3K inhibitors have side-effects in clinical trial. Different PI3K isoforms play non-redundant roles in particular tumor. So, it is expected that selective inhibition of PI3K isoforms decrease side effects. Therefore, we investigated whether combining radiotherapy with the PI3K isoform inhibitor decreases radioresistance and tumor growth in GBM. Glioma 261 expressed luciferase (GL261-luc) cell lines were used to identify the effects of combination therapy. GL261-luc were irradiated 1Gy with or without PI3K isoform inhibitors. After irradiation, clonogenic assay was performed to confirm the GBM survival rate and observed inhibition of PI3K-AKT signaling pathway using western blot (WB) assay. In order to confirm to repair DNA double strand breaks (DSB), the expression of phospho-H2A.X was confirmed by immunofluorescence. Also, to observe the effects of combination therapy in GBM in vivo, an orthotopic mouse model was established by intracranial injection implanting GL261-luc. Irradiation was performed specifically to brain with 2Gy, 3Gy and 4Gy for 5 days with or without PI3K-α isoform inhibitor. We observed tumor growth using IVIS image and analyzed splenocyte RNA and protein level using RT-PCR and WB. According to inhibition of PI3K-AKT signaling, GL261-luc radiosensitivity was significantly increased. GL261-luc irradiated by 1Gy was suppressed cell growth about 70% compared to control (p<0.001). When irradiated with PI3K-isform inhibitor, each survival rate was about 18% (PI3K-pan, p<0.00001), 25% (PI3K-α, p<0.00001), 30% (PI3K-δ, p<0.00001), 45% (PI3K-γ/δ, p<0.00001). Also, the expression of phospho-H2A.X, DNA DSB marker, was increased by combination therapy. Also, GL261-luc irradiated with PI3K- isoform inhibitor was decreased the ability of migration. Especially, the effects of combining radiotherapy with PI3K-α inhibitor was similar as much as with PI3K-pan inhibitor (In vitro). So, we selected PI3K-α inhibitor and analyzed combination therapy effects in GBM in vivo. We demonstrated that combining radiotherapy with the PI3K-α isoform inhibitor was markedly delayed tumor growth than without inhibitor (p<0.0001). In addition, the expression of PD1, regulator of cancer immune system, in splenocyte was increased by combination therapy (p<0.001) (In vivo). Our findings demonstrate that combining radiotherapy with the PI3K-α isoform increased radiosensitivity. Also, GBM tumor growth was markedly suppressed and increased immunogenic marker such as PD1. Combining radiotherapy with the PI3K-α isoform inhibitor would be a potential strategy for overcoming radioresistance in GBM, but further study should be necessary.

18\u03b2-Glycyrrhetinic acid induces human HaCaT keratinocytes apoptosis through ROS-mediated PI3K-Akt signaling pathway and ameliorates IMQ-induced psoriasis-like skin lesions in mice

BackgroundPsoriasis is a chronic inflammatory skin disease affecting 2–3% of the population worldwide. Hyperproliferative keratinocytes were thought to be an amplifier of inflammatory response, thereby sustaining persistence of psoriasis lesions. Agents with the ability to inhibit keratinocyte proliferation or induce apoptosis are potentially useful for psoriasis treatment. 18β-Glycyrrhetinic acid (GA), an active metabolite of glycyrrhizin, exhibits diverse pharmacological activities, including anti-inflammatory, anti-bacteria and anti-proliferation. The current study aims to evaluate the effects of GA on the proliferation and apoptosis of human HaCaT keratinocytes in vitro and investigate the effects of GA on the skin lesions of imiquimod (IMQ)-induced psoriasis-like mouse model in vivo.MethodsCell viability was assayed by CCK-8. Flow cytometry was performed to measure apoptosis and reactive oxygen species (ROS), with Annexin V-FITC/PI detection kit and DCFH-DA probe respectively. Caspase 9/3 activities were measured using caspase activity assay kits. The protein levels of Akt and p-Akt were determined using Western blotting. IMQ was applied to induce psoriasis-like skin lesions in mice. The histological change in mouse skin lesions was detected using hematoxylin and eosin (H&E) staining. The severity of skin lesions was scored based on Psoriasis Area Severity Index (PASI). RT-PCR was employed to examine the relative expression of TNF-α, IL-22 and IL-17A in mouse skin lesions.ResultsGA decreased HaCaT keratinocytes viability and induced cell apoptosis in a dose-dependent manner. In the presence of GA, intracellular ROS levels were significantly elevated. NAC, a ROS inhibitor, attenuated GA-mediated HaCaT keratinocytes growth inhibition and apoptosis. In addition, GA treatment remarkably decreased p-Akt protein level, which could be restored partially when cells were co-treated with GA and NAC. LY294002 (a PI3K inhibitor) treatment significantly enhanced GA-mediated cytotoxicity. Moreover, GA ameliorated IMQ-induced psoriasis-like skin lesions in mice.ConclusionsGA inhibits proliferation and induces apoptosis in HaCaT keratinocytes through ROS-mediated inhibition of PI3K-Akt signaling pathway, and ameliorates IMQ-induced psoriasis-like skin lesions in mice.

Mechanisms of recombinant adenovirus-mediated SD-HA fusion protein proliferation inhibition and induced apoptosis of K562 cells

目的探讨融合蛋白SD-HA能否通过竞争结合BCR-ABL的第177位酪氨酸磷酸化位点（Y177p），调控与其相关的下游信号分子活性，进而抑制K562细胞增殖并诱导凋亡。方法Western blot结合免疫共沉淀技术分析融合蛋白SD-HA与BCR-ABL的Y177p的相互作用，及其对下游信号途径Ras-MAPK和磷脂酰肌醇3-激酶(PI3K)-Akt的影响；Western blot分析融合蛋白SD-HA对细胞膜受体死亡途径级联反应caspase-8、caspase-3和PRAP的影响。结果双向免疫共沉淀实验可检测到融合蛋白SD-HA能与Grb2竞争性结合BCR-ABL Y177p，形成复合物。融合蛋白SD-HA可降低活化Ras、磷酸化MAPK（p-MAPK）、p-ELK的表达水平，抑制Ras-MAPK信号途径；融合蛋白SD-HA还可降低p-Akt及Akt的底物p-GSK的表达水平，抑制PI3K-Akt信号途径，从而抑制K562细胞增殖；通过死亡效应结构域（DED）与caspases-8前体DED结合而寡聚化，激活caspases-8、caspase-3和PRAP，诱导K562细胞凋亡。结论融合蛋白SD-HA抑制BCR-ABL-Y177与Grb2结合的策略，可作为慢性髓性白血病治疗新的切入点。

Synergistic anticancer activity of 20(S)-Ginsenoside Rg3 and Sorafenib in hepatocellular carcinoma by modulating PTEN/Akt signaling pathway

Sorafenib, a multikinase inhibitor for hepatocellular carcinoma treatment, inhibits the Raf/MAPK/ERK signaling pathway. However, PI3K/Akt signaling pathway is activated by Sorafenib and cross-talks with the Raf/MAPK/ERK signaling pathway, leading to drug resistance. 20(S)-Ginsenoside Rg3 has been reported with significant anticancer effect to numerous carcinomas by inhibition of PI3K-Akt signaling pathway. Hence, we aim to examine the synergistic anticancer activity of 20(S)-Ginsenoside Rg3 and Sorafenib via modulation of PTEN/Akt signaling pathway. Human hepatocellular carcinoma cell lines HepG2 and Huh7 were used. Cell viability, clonogenic assay, apoptosis assay, western blot analysis, xenograft treatment and immunohistochemistry were carried out. The viability of hepatocellular carcinoma cells significantly decreased by the treatment of Sorafenib combined with 20(S)-Ginsenoside Rg3, as well as the enhanced apoptotic rates. The levels of PTEN, Bax and cleaved caspase-3 expression increased, while the levels of phospho-PDK1 and phospho-Akt expression decreased by the treatment of Sorafenib combined with 20(S)-Ginsenoside Rg3. In vivo, the tumor volumes and weight decreased in the Sorafenib combined with 20(S)-Ginsenoside Rg3 group. The results demonstrated the synergistic anticancer activity of 20(S)-Ginsenoside Rg3 and Sorafenib in HCC by modulating PTEN/Akt signaling pathway. These findings suggest a promising strategy for HCC treatment, which could be performed in a sufficiently frequent manner.

Systemic Codelivery of a Homoserine Derived Ceramide Analogue and Curcumin to Tumor Vasculature Inhibits Mouse Tumor Growth.

Prior studies reported significant anticancer activities of ceramides. However, anticancer activities of homoserine based ceramides have not been tested. With a view to compare the anticancer activity of ceramides and homoceramides, in the present study, we have synthesized four serine based and four homoserine based C8-ceramide analogues. Since many cancer cells have shown resistance to ceramides, curcumin is now being used in combination with ceramides because of its ability to reverse multidrug resistance. Aimed at targeting curcumin-ceramide combination to tumor endothelial cells, herein we have used a tumor vasculature targeting liposomes of a newly synthesized pegylated RGDGWK-lipopeptide. Importantly, the liposomal formulations of the homoserine based C8-ceramide analogue containing oleyl chain showed more promising antineoplastic activities under both in vitro and systemic settings than the liposomal formulations of commercially available C8-ceramide. Findings in the mouse tumor growth inhibition study revealed synergistic therapeutic benefit from simultaneous delivery of curcumin and a homoserine based ceramide containing oleyl chain to tumor vasculature. Results in RT-PCR and Western blot experiments suggest that inhibition of solid tumor growth is mediated via inhibition of PI3K-Akt signaling pathway. The present structure-activity study is the first report to demonstrate therapeutic promise of curcumin-homoserine based ceramide combination in antiangiogenic cancer therapy.

Effects of Akt inhibitor MK2206 on proliferation and apoptosis of breast cancer cells

Objective: To study the effects of Akt inhibitor MK2206 on the proliferation and apoptosis of human breast cancer cell lines T47D and MDA-MB-231, and to elucidate the possible mechanism of such effects. Methods: Human breast cancer T47D and MDA-MB-231 cells were treated with different concentrations of MK2206, respectively. The inhibitory effect of MK2206 on cell proliferation was examined by MTT assay. The apoptosis rates of T47D and MDA-MB-231 cells induced by MK2206 were detected by flow cytometry (FCM). The expression levels of caspase-3, poly (ADP-ribose) polymerase (PARP), bcl-2, bax, phosphorate-AKT (p-AKT) and total Akt (T-Akt) proteins were detected by Western blotting. Results: The proliferation of T47D and MDA-MB-231 cells were inhibited after treatment with MK2206 at 0.1, 1, 10 and 100 nmol/L for 24 h, respectively. The apoptosis rates of T47D and MDA-MB-231 cells were increased induced by MK2206. The expression levels of caspase-3, PARP and bax proteins were up-regulated, while the expression levels of bcl-2 and p-Akt proteins were down-regulated. All of these effects occurred in a dose-dependent manner. MK2206 had no significant effect on the expression of T-Akt. Conclusion: MK2206 can inhibit the proliferation and induce the apoptosis in human breast cancer cell lines T47D and MDA-MB-231. These effects may be related with the down-regulation of p-AKT and the inhibition of PI3K-Akt signaling pathway. DOI:10.3781/j.issn.1000-7431.2011.02.008

Resveratrol inhibits TNF-α-induced IL-1β, MMP-3 production in human rheumatoid arthritis fibroblast-like synoviocytes via modulation of PI3kinase/Akt pathway

Resveratrol (trans-3,4'-trihydroxystilbene), a natural phytoalexin, possesses anti-inflammatory, anti-proliferative, and immunomodulatory properties and has the potential for treating inflammatory disorders. The present study was designed to investigate the effects of resveratrol on TNF-α-induced inflammatory cytokines production of IL-1β and MMP3 in Rheumatoid arthritis (RA) Fibroblast-like synoviocytes (FLS) and further to explore the role of PI3K/Akt signaling pathway by which resveratrol modulates those cytokines production. The levels of IL-1β, MMP-3 in cultural supernatants among groups were measured by enzyme-linked immunosorbent assay. Messenger RNA expression of IL-1β and MMP-3 in RA FLS was analyzed using a reverse transcription-polymerase chain reaction. Western blot analysis was used to detect proteins expression in RA FLS intervened by resveratrol. Resveratrol inhibited both mRNA and proteins expressions of IL-1β and MMP-3 on RA FLS in a dose-dependent manner. Resveratrol also decreased significantly the expression of phosphorylated Akt dose dependently. Activation of PI3K/Akt signaling pathway exists in TNF-α-induced production of IL-1β and MMP3 on RA FLS, which is hampered by PI3K inhibitor LY294002. Immunofluorescence staining showed that TNF-α alone increased the production of P-Akt, whereas LY294002 and 50μM resveratrol suppressed the TNF-α-stimulated expression of P-Akt. Resveratrol attenuates TNF-α-induced production of IL-1β and MMP-3 via inhibition of PI3K-Akt signaling pathway in RA FLS, suggesting that resveratrol plays an anti-inflammatory role and might have beneficial effects in preventing and treating RA.