LncRNA ZFAS1 regulates ATIC transcription and promotes the proliferation and migration of hepatocellular carcinoma through the PI3K/AKT signaling pathway.

PURPOSE: Long noncoding RNAs (lncRNAs) exert a significant influence on various cancer-related processes through their intricate interactions with RNAs. Among these, lncRNA ZFAS1 has been implicated in oncogenic roles in multiple cancer types. Nevertheless, the intricate biological significance and underlying mechanism of ZFAS1 in the initiation and progression of hepatocellular carcinoma (HCC) remain largely unexplored. METHODS: Analysis of The Cancer Genome Atlas Program (TCGA) database revealed a notable upregulation of lncRNA ZFAS1 in HCC tissues. To explore its function, we investigated colony formation and performed CCK-8 assays to gauge cellular proliferation and wound healing, Transwell assays to assess cellular migration, and an in vivo study employing a nude mouse model to scrutinize tumor growth and metastasis. Luciferase reporter assay was used to confirm the implicated interactions. Rescue experiments were conducted to unravel the plausible mechanism underlying the activation of the PI3K/AKT pathway by lncRNAs ZFAS1 and ATIC. RESULTS: ZFAS1 and ATIC were significantly upregulated in the HCC tissues and cells. ZFAS1 knockdown inhibited cell proliferation and migration. We observed a direct interaction between the lncRNA ZFAS1 and ATIC. ATIC knockdown also suppressed cell proliferation and migration. SC79, an activator of AKT, partially restores the effects of lncRNA ZFAS1/ATIC knockdown on cell proliferation and migration. Knockdown of lncRNA ZFAS1/ATIC inhibited tumor growth and lung metastasis in vivo. CONCLUSION: Overall, lncRNA ZFAS1 regulates ATIC transcription and contributes to the growth and migration of HCC cells through the PI3K/AKT signaling pathway.

CDK12 inhibition upregulates ATG7 triggering autophagy via AKT/FOXO3 pathway and enhances anti-PD-1 efficacy in colorectal cancer.

As the world's fourth most deadly cancer, colorectal cancer (CRC) still needed the novel therapeutic drugs and target urgently. Although cyclin-dependent kinase 12 (CDK12) has been shown to be implicated in the malignancy of several types of cancer, its functional role and mechanism in CRC remain largely unknown. Here, we found that suppression of CDK12 inhibited tumor growth in CRC by inducing apoptosis. And CDK12 inhibition triggered autophagy by upregulating autophagy related gene 7 (ATG7) expression. Inhibition of autophagy by ATG7 knockdown and chloroquine (CQ) further decreased cell viability induced by CDK12 inhibition. Further mechanism exploration showed that CDK12 interacted with protein kinase B (AKT) regulated autophagy via AKT/forkhead box O3 (AKT/FOXO3) pathway. FOXO3 transcriptionally upregulated ATG7 expression and autophagy when CDK12 inhibition in CRC. Level of CDK12 and p-FOXO3/FOXO3 ratio were correlated with survival in CRC patients. Moreover, CDK12 inhibition improved the efficacy of anti-programmed cell death 1(PD-1) therapy in CRC murine models by enhancing CD8 + T cells infiltration. Thus, our study founded that CDK12 inhibition upregulates ATG7 triggering autophagy via AKT/FOXO3 pathway and enhances anti-PD-1 efficacy in CRC. We revealed the roles of CDK12/FOXO3/ATG7 in regulating CRC progression, suggesting potential biomarkers and therapeutic target for CRC.

Akt/mTOR Pathway Agonist SC79 Inhibits Autophagy and Apoptosis of Oligodendrocyte Precursor Cells Associated with Neonatal White Matter Dysplasia.

White matter dysplasia (WMD) in preterm infants due to intrauterine inflammation is caused by excessive apoptosis of oligodendrocyte precursor cells (OPCs). In recent years, studies have found that excessive autophagy and apoptosis are highly interconnected and important in infection and inflammatory diseases in general. Therefore, in this study, we aimed to confirm whether regulation of autophagy by using the Akt phosphorylation agonist SC79 can inhibit abnormal apoptosis of OPCs and promote myelin maturation and white matter development in neonatal rats with WMD. We investigated the effect of inflammation on oligodendrocyte development in P0 neonatal rats by intracerebellar injection of LPS, and collected brain tissue at P2 and P5. Immunohistochemical and immunofluorescence staining were used to evaluate white matter damage, while immunofluorescence staining, terminal deoxynucleotidyl transferase dUTP nick end labeling analysis (TUNEL), and western blotting were used to evaluate autophagy and apoptosis. First, we observed that white matter development was arrested and white matter fiber maturation was impaired in LPS-inflicted pups compared with those in the sham-operated group. Second, treatment with SC79 reduced the levels of LC3II, caspase 3, caspase 9, and Bax/Bcl-2 and increased the levels of p62, p-Akt, and p-mTOR in the brain tissue of neonatal rats. Finally, SC79 treatment inhibited OPC apoptosis by increasing the binding of Beclin 1 to Bcl-2, which promoted OPC differentiation and maturation. However, the opposite results were observed after rapamycin administration. Taken together, our results suggest that SC79 can inhibit the abnormal apoptosis of OPCs caused by excessive autophagy through the Akt/mTOR pathway and that SC79 is a potential therapeutic agent for WMD in preterm infants.

Knockdown of PROM2 Enhances Paclitaxel Sensitivity in Endometrial Cancer Cells by Regulating the AKT/FOXO1 Pathway.

BACKGROUND: Endometrial cancer is a very common and highly lethal reproductive malignant tumour in women. Paclitaxel (PTX) is a usual drug utilized in chemotherapy for endometrial cancer. It has been uncovered that PROM2 participates in the progression of various cancers through playing a promoter. However, the regulatory function of PROM2 in PTX treatment for endometrial cancer remains unclear. METHODS: The cell viability (IC50) was examined through CCK8 assay. The mRNA and protein expressions of genes were measured through RT-qPCR and western blot. The proliferation was evaluated through colony formation and EdU assays. The cell apoptosis was assessed through flow cytometry. RESULTS: In this work, through bioinformatic analysis on online websites, it is found that the up-regulated expression of PROM2 existed in endometrial cancer. In addition, the survival probability of UCEC patients with high PROM2 expression was worse. This study adopted PTX treatment for obtaining the PTX-resistant cells (HEC-1A/PTX and KLE/PTX). Furthermore, suppression of PROM2 enhanced PTX sensitivity through decreasing IC50 and proliferation in endometrial cancer. Additionally, knockdown of PROM2 facilitated cell apoptosis in HEC-1A/PTX and KLE/PTX cells. Next, we found that silencing of PROM2 retards the AKT/FOXO1 pathway. At last, rescue assays reversed the strengthened PTX sensitivity mediated by PROM2 inhibition after SC79 treatment (AKT activator). CONCLUSION: Knockdown of PROM2 enhanced PTX sensitivity in endometrial cancer through modulating the AKT/FOXO1 pathway. This study hinted that PROM2 may be a useful therapeutic target for PTX treatment in endometrial cancer.

Chitosan Oligosaccharide Promotes Junction Barrier through Modulation of PI3K/AKT and ERK Signaling Intricate Interplay in T84 Cells.

Chitosan oligosaccharide (COS) is a breakdown product of chitin, a polymer of N-acetyl-D-glucosamine. COS promotes barrier function in intestinal epithelial cells. However, the exact mechanism of COS-induced barrier function remains unknown. This study was aimed to explore the intricate signaling cascades in the junction barrier induced by COS (100 µg/mL) in human intestinal epithelial cells (T84 cells). COS (100 µg/mL) promoted tight junction assembly and increased transepithelial electrical resistance (TEER). COS inhibited FITC-dextran flux in T84 cell monolayers at 2 h, 4 h, 6 h and 24 h post treatment. In addition, the effect of COS on TEER and FITC-dextran flux was abrogated by pre-incubation of wortmannin (2 µM), an AKT (protein kinase B) inhibitor, at 2 h and 4 h post treatment, indicating that COS-induced tight junction integrity was mediated at least in part by AKT activation. COS-induced TEER was amplified at 24 h and 48 h post treatment by pre-incubation with SC79 (2.5 µM), an AKT activator. Moreover, COS induced inhibition of extracellular signal-regulated kinase (ERK) in T84 cells. Wortmannin and SC79 pre-incubation promoted ERK activation and ERK inhibition, respectively, suggesting that COS-induced ERK inhibition was mediated by AKT. Collectively, this study reveals that COS promotes junction barrier integrity via regulating PI3K/AKT and ERK signaling intricate interplay in T84 cell monolayers. COS may be beneficial in promoting junction barrier in intestinal disorders.

Antagonistic effect of SC79 on high glucose-induced apoptosis of RPE cells and its regulatory mechanism on AKT-XIAP signaling pathway / 中华实验眼科杂志

Objective:To investigate the antagonistic effect and potential mechanism of specific AKT activator SC79 on the apoptosis of human retinal pigment epithelial (ARPE)-19 cells induced by high glucose in vitro. Methods:The ARPE-19 cells were cultured in high glucose medium (containing 30 mmol/L glucose) plus 5, 10 or 20 μg/ml SC79, respectively.After 6-, 12- and 24-hour culture, the optimal experimental concentration and timing were determined according to cell proliferation rate.Then ARPE-19 cells were divided into four groups, normal control group cultured in normal medium containing 5.6 mmol/L glucose for 48 hours, mannitol group cultured in medium containing 5.6 mmol/L glucose and 24.4 mmol/L mannitol for 48 hours, high glucose group cultured in high glucose medium for 48 hours, and high glucose+ SC79 group cultured in normal medium containing 10 μg/ml SC79 for 12 hours plus in high glucose medium for 36 hours.The proliferation rate of APRE-19 cells was detected by MTS assay.The apoptosis rate was measured by flow cytometry.The relative expression levels of phosphorylated protein kinase B (p-Akt), X-linked inhibitor of apoptosis protein (XIAP), caspase-9, caspase-3 and its active fragments (active-caspase-3) were assayed by Western blot.The ARPE-19 cells were divided into Neg-shRNA group, AKT shRNA group and blank control group and were treated with the corresponding transfection complex and serum-free medium.The AKT mRNA expression was detected by real-time PCR.The transfected ARPE-19 cells were divided into Neg-shRNA+ SC79 group and AKT shRNA+ SC79 group and were cultured according to the culturing method of high-glucose+ SC79 group.The apoptosis rate of the two groups was tested by flow cytometry.Results:Among different concentrations of SC79 and treatment times, the proliferation rate of cells treated with 10 μg/ml SC79 for 12 hours was the highest.The proliferation rate of ARPE-19 cells in high-glucose group was significantly lower than that in normal control group, mannitol group and high-glucose+ SC79 group, and the differences were statistically significant (all at P<0.01). The apoptosis rate of cells in the high-glucose group was (52.27±3.21)%, which was significantly higher than (3.90±0.71)% in normal control group and (20.70±3.62)% in high-glucose+ SC79 group (both at P<0.01). The relative expression levels of p-Akt, XIAP, caspase-9 and caspase-3 were significantly lower and the relative expression level of active-caspase-3 was significantly higher in high glucose group than those in normal control group and high-glucose+ SC79 group (all at P<0.05). The relative expression level of AKT mRNA in normal control group, Neg-shRNA group and AKT shRNA group was 0.60±0.07, 0.59±0.03 and 0.11±0.10, respectively, showing a statistically significant difference among the groups ( F=30.44, P<0.01). The apoptosis rate of cells in the AKT shRNA+ SC79 group was significantly higher than that in high-glucose+ SC79 group and Neg-shRNA+ SC79 group (both at P<0.001). Conclusions:SC79 can partially antagonize the apoptosis of ARPE-19 cells induced by high glucose, which is related to the activation of AKT/XIAP pathway and the inhibition of the caspase family.

Effects of the PI3K/Akt signaling pathway on the hair inductivity of human dermal papilla cells in hair beads.

Dermal papilla cells (DPCs), which play a central role in the regulation of hair follicle development and hair growth, are among the most promising cell sources for hair regenerative medicine. However, a critical issue in the use of DPCs is the immediate loss of hair inducing functions in typical two-dimensional (2D) culture. We have previously demonstrated that when DPCs are encapsulated in drops of collagen gel (named hair beads, HBs), the density of collagen and cells is concentrated >10-fold during 3 d of culture through the spontaneous constriction of the drops, leading to efficient hair follicle regeneration upon transplantation. However, the mechanisms responsible for the activation of the hair-inducing functions of DPCs have been poorly elucidated. Here, transcriptome comparisons of human DPCs in HB culture and in typical 2D culture revealed that the phosphoinositide 3-kinase and Akt (PI3K/Akt) signaling pathway was significantly upregulated in HB culture. Inhibition of the PI3K/Akt signaling pathway decreased the hair-inducing capability of DPCs in HBs, while the activation of the PI3K/Akt signaling pathway using an activator improved trichogenous gene expression of DPCs in 2D culture. These results suggest that the PI3K/Akt signaling pathway is crucial for the maintenance and restoration of hair inductivity of DPCs. HB culture and/or activators of the PI3K/Akt signaling pathway could be a promising strategy for preparing DPCs for hair regenerative medicine.

SLC4A4 promotes prostate cancer progression in vivo and in vitro via AKT-mediated signalling pathway.

BACKGROUND: Prostate cancer (PCa) is the second leading cause of cancer-related male deaths worldwide. The purpose of this study was to investigate the effects of homo sapiens solute carrier family 4 member 4 (SLC4A4), which encodes the electrogenic Na+/HCO3- cotransporter isoform 1 (NBCe1), in the development and progression of PCa. METHODS: The expression levels of SLC4A4 in PCa and normal prostate tissues were evaluated by immunohistochemistry. The SLC4A4 knockdown cell model was structured by lentiviral infection, and the knockdown efficiency was validated by RT-qPCR and Western blotting. The effects of SLC4A4 knockdown on cell proliferation, apoptosis and cycle, migration, and invasion were detected by Celigo cell counting assay and CCK-8 assay, flow cytometry analysis, wound-healing, and Transwell assay, respectively. Tumor growth in nude mice was surveyed by in vivo imaging and Ki-67 staining. Furthermore, underlying mechanism of SLC4A4 silence induced inhibition of PCa progression was explored by human phospho-kinase array. RESULTS: Our results revealed that SLC4A4 expression was up-regulated in PCa tissues and human PCa cell lines. High expression of SLC4A4 in tumor specimens was significantly correlated with disease progression. SLC4A4 knockdown inhibited cell proliferation, migration and invasion, while facilitated apoptosis, which was also confirmed in vivo. Moreover, SLC4A4 promoted PCa progression through the AKT-mediated signalling pathway. CONCLUSION: The results of this study indicated that SLC4A4 overexpression was closely associated with the progression of PCa; SLC4A4 knockdown suppressed PCa development in vitro and in vivo. SLC4A4 acts as a tumor promotor in PCa by regulating key components of the AKT pathway and may therefore act as a potential therapeutic target for PCa treatment.

Dezocine relieves the postoperative hyperalgesia in rats through suppressing the hyper-action of Akt1/GSK-3ß pathway.

The relieving role of dezocine in pain after surgery was previously reported, while the potential mechanism was not completely clear. Therefore, the current research probed into the regulatory mechanism of dezocine in pain after surgery. A postoperative pain model was established by performing plantar incision surgery on the juvenile Sprague-Dawley rats. After the rats were treated with dezocine or SC79 (Akt1 activator), the paw withdrawal threshold and paw withdrawal latency of rats were detected to evaluate the mechanical allodynia and thermal hyperalgesia. After the plantar tissue, dorsal root ganglions, and spinal cord of rats were collected, the expressions of Akt1, p-Akt1, GSK-3ß, and p-GSK-3ß in the tissues were determined by western blot to evaluate the activation state of the Akt1/GSK-3ß pathway. After surgery, the paw withdrawal threshold and paw withdrawal latency of rats were lessened, whereas the ratios of p-Akt1/Akt1 and p-GSK-3ß/GSK-3ß were augmented in rat plantar tissue, dorsal root ganglions, and spinal cord. After treatment with dezocine alone, the paw withdrawal threshold and paw withdrawal latency of postoperative rats were elevated, but ratios of p-Akt1/Akt1 and p-GSK-3ß/GSK-3ß were reduced. After co-treatment with dezocine and SC79, SC79 reversed the effects of dezocine on elevating the paw withdrawal threshold and paw withdrawal latency, and reducing the ratios of p-Akt1/Akt1 and p-GSK-3ß/GSK-3ß in postoperative rats. Dezocine ameliorated the postoperative hyperalgesia in rats via repressing the hyper-action of Akt1/GSK-3ß pathway.

Akt acts as a switch for GPCR transactivation of the TGF-ß receptor type 1.

Transforming growth factor (TGF)-ß signalling commences with the engagement of TGF-ß ligand to cell surface TGF-ß receptors (TGFBR) stimulating Smad2 carboxyl-terminal phosphorylation (phospho-Smad2C) and downstream biological responses. In several cell models, G protein-coupled receptors (GPCRs) transactivate the TGF-ß receptors type-1 (TGFBR1) leading to phospho-Smad2C, however, we have recently published that in keratinocytes thrombin did not transactivate the TGFBR1. The bulk of TGFBRs reside in the cytosol and in response to protein kinase B (Akt phosphorylation) can translocate to the cell surface increasing the cell's responsiveness to TGF-ß. In this study, we investigate the role of Akt in GPCR transactivation of the TGFBR1. We demonstrate that angiotensin II and thrombin do not phosphorylate Smad2C in human vascular smooth muscle cells and in keratinocytes respectively. We used Akt agonist, SC79 to sensitise the cells to Akt and observed that Ang II and thrombin phosphorylate Smad2C via Akt/AS160-dependent pathways. We show that SC79 rapidly translocates TGFBRs to the cell surface thus increasing the cell's response to the GPCR agonist. These findings highlight novel mechanistic insight for the role of Akt in GPCR transactivation of the TGFBR1.

Translocation of TMEM175 Lysosomal Potassium Channel to the Plasma Membrane by Dynasore Compounds.

TMEM175 (transmembrane protein 175) coding sequence variants are associated with increased risk of Parkinson's disease. TMEM175 is the ubiquitous lysosomal K+ channel regulated by growth factor receptor signaling and direct interaction with protein kinase B (PKB/Akt). In the present study, we show that the expression of mouse TMEM175 results in very small K+ currents through the plasma membrane in Xenopus laevis oocytes, in good accordance with the previously reported intracellular localization of the channel. However, the application of the dynamin inhibitor compounds, dynasore or dyngo-4a, substantially increased TMEM175 currents measured by the two-electrode voltage clamp method. TMEM175 was more permeable to cesium than potassium ions, voltage-dependently blocked by 4-aminopyridine (4-AP), and slightly inhibited by extracellular acidification. Immunocytochemistry experiments indicated that dyngo-4a increased the amount of epitope-tagged TMEM175 channel on the cell surface. The coexpression of dominant-negative dynamin, and the inhibition of clathrin- or caveolin-dependent endocytosis increased TMEM175 current much less than dynasore. Therefore, dynamin-independent pharmacological effects of dynasore may also contribute to the action on the channel. TMEM175 current rapidly decays after the withdrawal of dynasore, raising the possibility that an efficient internalization mechanism removes the channel from the plasma membrane. Dyngo-4a induced about 20-fold larger TMEM175 currents than the PKB activator SC79, or the coexpression of a constitutively active mutant PKB with the channel. In contrast, the allosteric PKB inhibitor MK2206 diminished the TMEM175 current in the presence of dyngo-4a. These data suggest that, in addition to the lysosomes, PKB-dependent regulation also influences TMEM175 current in the plasma membrane.

The New PI3K/mTOR Inhibitor GNE-477 Inhibits the Malignant Behavior of Human Glioblastoma Cells.

The most common primary central nervous system tumor in adults is glioblastoma multiforme (GBM). The high invasiveness of GBM cells is an important factor leading to inevitable tumor recurrence and a poor prognosis of patients. GNE-477, a novel PI3K/mTOR inhibitor, has been reported to exert antiproliferative effects on other cancer cells. However, researchers have not clearly determined whether GNE-477 produces antitumor effects on GBM. In the present study, GNE-477 significantly inhibited the proliferation, migration and invasion of U87 and U251 cells. In addition, GNE-477 also induced apoptosis of GBM cells, arresting the cell cycle in G0/G1 phase. More importantly, GNE-477 also reduced the levels of AKT and mTOR phosphorylation in the AKT/mTOR signaling pathway in a concentration-dependent manner. An increase in AKT activity induced by SC79 rescued the GNE-477-mediated inhibition of GBM cell proliferation and apoptosis. The antitumor effects of GNE-477 and the regulatory effects on related molecules were further confirmed in vivo using a nude mouse intracranial xenograft model. In conclusion, our study indicated that GNE-477 exerted significant antitumor effects on GBM cells in vitro and in vivo by downregulating the AKT/mTOR pathway.

CDDO-Me Distinctly Regulates Regional Specific Astroglial Responses to Status Epilepticus via ERK1/2-Nrf2, PTEN-PI3K-AKT and NFκB Signaling Pathways.

2-Cyano-3,12-dioxo-oleana-1,9(11)-dien-28-oic acid methyl ester (CDDO-Me) is a triterpenoid analogue of oleanolic acid. CDDO-Me shows anti-inflammatory and neuroprotective effects. Furthermore, CDDO-Me has antioxidant properties, since it activates nuclear factor-erythroid 2-related factor 2 (Nrf2), which is a key player of redox homeostasis. In the present study, we evaluated whether CDDO-Me affects astroglial responses to status epilepticus (SE, a prolonged seizure activity) in the rat hippocampus in order to understand the underlying mechanisms of reactive astrogliosis and astroglial apoptosis. Under physiological conditions, CDDO-Me increased Nrf2 expression in the hippocampus without altering activities (phosphorylations) of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), phosphatidylinositol-3-kinase (PI3K), and AKT. CDDO-Me did not affect seizure activity in response to pilocarpine. However, CDDO-Me ameliorated reduced astroglial Nrf2 expression in the CA1 region and the molecular layer of the dentate gyrus (ML), and attenuated reactive astrogliosis and ML astroglial apoptosis following SE. In CA1 astrocytes, CDDO-Me inhibited the PI3K/AKT pathway by activating PTEN. In contrast, CDDO-ME resulted in extracellular signal-related kinases 1/2 (ERK1/2)-mediated Nrf2 upregulation in ML astrocytes. Furthermore, CDDO-Me decreased nuclear factor-κB (NFκB) phosphorylation in both CA1 and ML astrocytes. Therefore, our findings suggest that CDDO-Me may attenuate SE-induced reactive astrogliosis and astroglial apoptosis via regulation of ERK1/2-Nrf2, PTEN-PI3K-AKT, and NFκB signaling pathways.

Cytisine attenuates bone loss of ovariectomy mouse by preventing RANKL-induced osteoclastogenesis.

Postmenopausal Osteoporosis (PMOP) is oestrogen withdrawal characterized of much production and activation by osteoclast in the elderly female. Cytisine is a quinolizidine alkaloid that comes from seeds or other plants of the Leguminosae (Fabaceae) family. Cytisine has been shown several potential pharmacological functions. However, its effects on PMOP remain unknown. This study designed to explore whether Cytisine is able to suppress RANKL-induced osteoclastogenesis and prevent the bone loss induced by oestrogen deficiency in ovariectomized (OVX) mice. In this study, we investigated the effect of Cytisine on RAW 264.7 cells and bone marrow monocytes (BMMs) derived osteoclast culture system in vitro and observed the effect of Cytisine on ovariectomized (OVX) mice model to imitate postmenopausal osteoporosis in vivo. We found that Cytisine inhibited F-actin ring formation and tartrate-resistant acid phosphatase (TRAP) staining in dose-dependent ways, as well as bone resorption by pit formation assays. For molecular mechanism, Cytisine suppressed RANK-related trigger RANKL by phosphorylation JNK/ERK/p38-MAPK, IκBα/p65-NF-κB, and PI3K/AKT axis and significantly inhibited these signalling pathways. However, the suppression of PI3K-AKT-NFATc1 axis was rescued by AKT activator SC79. Meanwhile, Cytisine inhibited RANKL-induced RANK-TRAF6 association and RANKL-related gene and protein markers such as NFATc1, Cathepsin K, MMP-9 and TRAP. Our study indicated that Cytisine could suppress bone loss in OVX mouse through inhibited osteoclastogenesis. All data provide the evidence that Cytisine may be a promising agent in the treatment of osteoclast-related diseases such as osteoporosis.

AKT activation by SC79 to transiently re-open pathological blood brain barrier for improved functionalized nanoparticles therapy of glioblastoma.

Glioblastoma (GBM) is one of the malignant tumors with high mortality, and the presence of the blood brain barrier (BBB) severely limits the penetration and tissue accumulation of therapeutic agents in the lesion of GBM. Active targeting nanotechnologies can achieve efficient drug delivery in the brain, while still have a very low success rate. Here we revealed a previously unexplored phenomenon that chemotherapy with active targeting nanotechnologies causes pathological BBB functional recovery through VEGF-PI3K-AKT signaling pathway inhibition, accompanied with up-regulated expression of Claudin-5 and Occludin. Seriously, pathological BBB functional recovery induces a significant decrease of intracerebral active targeting nanotechnologies transport during GBM multiple administration, leading to chemotherapy failure in GBM therapeutics. To address this issue, we chose AKT agonist SC79 to transiently re-open functional recovering pathological BBB for continuously intracerebral delivery of brain targeted nanotherapeutics, finally producing an observable anti-GBM effect in vivo, which may offer new sight for other CNS disease treatment.

PKC, AKT and ERK1/2-Mediated Modulations of PARP1, NF-κB and PEA15 Activities Distinctly Regulate Regional Specific Astroglial Responses Following Status Epilepticus.

Status epilepticus (SE, a prolonged seizure activity) leads to reactive astrogliosis and astroglial apoptosis in the regional specific manners, independent of hemodynamics. Poly(ADP-ribose) polymerase-1 (PARP1) activity is relevant to these distinct astroglial responses. Since various regulatory signaling molecules beyond PARP1 activity may be involved in the distinct astroglial response to SE, it is noteworthy to explore the roles of protein kinases in PARP1-mediated reactive astrogliosis and astroglial apoptosis following SE, albeit at a lesser extent. In the present study, inhibitions of protein kinase C (PKC), AKT and extracellular signal-related kinases 1/2 (ERK1/2), but not calcium/calmodulin-dependent protein kinase II (CaMKII), attenuated CA1 reactive astrogliosis accompanied by reducing PARP1 activity following SE, respectively. However, inhibition of AKT and ERK1/2 deteriorated SE-induced dentate astroglial loss concomitant with the diminished PARP1 activity. Following SE, PKC- and AKT inhibitors diminished phosphoprotein enriched in astrocytes of 15 kDa (PEA15)-S104 and -S116 phosphorylations in CA1 astrocytes, but not in dentate astrocytes, respectively. Inhibitors of PKC, AKT and ERK1/2 also abrogated SE-induced nuclear factor-κB (NF-κB)-S311 and -S468 phosphorylations in CA1 astrocytes. In contrast, both AKT and ERK1/2 inhibitors enhanced NF-κB-S468 phosphorylation in dentate astrocytes. Furthermore, PARP1 inhibitor aggravated dentate astroglial loss following SE. AKT inhibition deteriorated dentate astroglial loss and led to CA1 astroglial apoptosis following SE, which were ameliorated by AKT activation. These findings suggest that activities of PARP1, PEA15 and NF-κB may be distinctly regulated by PKC, AKT and ERK1/2, which may be involved in regional specific astroglial responses following SE.

SC79, a novel Akt activator, protects dopaminergic neuronal cells from MPP+ and rotenone.

In pathogenesis of Parkinson's disease (PD), mitochondrial dysfunction causes substantial reactive oxygen species (ROS) production and oxidative stress, leading to dopaminergic (DA) neuronal cell death. Mitochondrial toxins, including MPP+ (1-methyl-4-phenylpyridinium ion) and rotenone, induce oxidative injury in cultured DA neuronal cells. The current study tested the potential effect of SC79, a first-in-class small-molecule Akt activator, against the process. In SH-SY5Y cells and primary murine DA neurons, SC79 significantly attenuated MPP+- and rotenone-induced viability reduction, cell death, and apoptosis. SC79 activated Akt signaling in DA neuronal cells. Akt inhibition (by LY294002 and MK-2206) or CRISPR-Cas9-mediated Akt1 knockout completely abolished SC79-induced DA neuroprotection against MPP+. Further studies demonstrated that SC79 attenuated MPP+- and rotenone-induced ROS production, mitochondrial depolarization, and lipid peroxidation in SH-SY5Y cells and primary DA neurons. Moreover, upregulation of Nrf2-dependent genes (HO1 and NQO1) and Nrf2 protein stabilization were detected in SC79-treated SH-SY5Y cells and primary DA neurons. Together we show that SC79 protects DA neuronal cells from mitochondrial toxins possibly via activation of Akt-Nrf2 signaling.

AKT activator SC79 protects hepatocytes from TNF-α-mediated apoptosis and alleviates d-Gal/LPS-induced liver injury.

Tumor necrosis factor-α (TNF-α) is a highly pleiotropic cytokine executing biological functions as diverse as cell proliferation, metabolic activation, inflammatory responses, and cell death. TNF-α can induce multiple mechanisms to initiate apoptosis in hepatocytes leading to the subsequent liver injury. Since the phosphoinositide-3-kinase/protein kinase B (PI3K/Akt) pathway is known to have a protective role in death factor-mediated apoptosis, it is our hypothesis that activation of Akt may represent a therapeutic strategy to alleviate TNF-α-induced hepatocyte apoptosis and liver injury. We report here that the Akt activator SC79 protects hepatocytes from TNF-α-induced apoptosis and protects mice from d-galactosamine (d-Gal)/lipopolysaccharide (LPS)-induced TNF-α-mediated liver injury and damage. SC79 not only enhances the nuclear factor-κB (NF-κB) prosurvival signaling in response to TNF-α stimulation, but also increases the expression of cellular FLICE (FADD-like IL-1ß-converting enzyme)-inhibitory protein L and S (FLIPL/S), which consequently inhibits the activation of procaspase-8. Furthermore, pretreatment of the PI3K/Akt inhibitor LY294002 reverses all the SC79-induced hepatoprotective effects. These results strongly indicate that SC79 protects against TNF-α-induced hepatocyte apoptosis and suggests that SC79 is likely a promising therapeutic agent for ameliorating the development of liver injury. NEW & NOTEWORTHY SC79 protects hepatocytes from TNF-α-mediated apoptosis and mice from Gal/LPS-induced liver injury and damage. Cytoprotective effects of SC79 against TNF-α act through both AKT-mediated activation of NF-κB and upregulation of FLIPL/S.

IDH1 mutation correlates with a beneficial prognosis and suppresses tumor growth in IHCC.

BACKGROUND: Isocitrate dehydrogenase 1/2 (IDH1/2) mutations have been reported in intrahepatic cholangiocarcinoma (IHCC). However, the prognosis of a single IDH1 mutation and impact of mutant IDH1 on IHCC tumor growth remain unclear. METHODS: A total of 85 IHCC tumor samples were sequenced. Prognosis and clinicopathological correlation were analyzed. The role of mutant IDH1 in IHCC tumor growth was measured by cell proliferation assay, colony formation assay in soft agar, and xenograft tumor models. Akt, ERK, p38 MAPK, and JNK signaling, which commonly affect tumor growth, were examined by Western blotting to explore the potential mechanism. RESULTS: IDH1 mutations correlated with a beneficial prognosis and smaller tumor size. Mutant IDH1 exhibited a growth-inhibitory effect on IHCC cell lines in vitro and in vivo. Akt signaling was suppressed in IHCC cell lines expressing a mutant IDH1. The reactivation of Akt signaling by SC79 restored the inhibited growth of cell lines expressing a mutant IDH1 in IHCC. CONCLUSIONS: Collectively, we demonstrated that mutant IDH1 correlates with a beneficial prognosis and inhibits tumor growth by suppressing Akt signaling in IHCC. We suggest that patients with IDH1 mutations could be considered for both less-aggressive therapy and therapy tailored to the presence of their mutant enzyme in the future.

Activation of Akt by SC79 decreased cerebral infarct in early cerebral ischemia-reperfusion despite increased BBB disruption.

Activation of Akt has been suggested to produce neuronal protection in cerebral ischemia. Decreasing blood-brain barrier (BBB) disruption has been associated with a better neuronal outcome in cerebral ischemia. We hypothesized that activation of Akt would decrease BBB disruption and contribute to decreasing the size of infarct in the early stage of cerebral ischemia-reperfusion within the therapeutic window. Transient middle cerebral artery occlusion (MCAO) was performed in rats under isoflurane anesthesia with controlled ventilation. Rats were treated with SC79 (a selective Akt activator which is cell and BBB permeable) 0.05 mg/kg × 3 i.p. or vehicle i.p. perioperatively. After one hour of MCAO and two hours of reperfusion, the transfer coefficient (Ki) of 14C-α-aminoisobutyric acid (14C-AIB, molecular weight 104 Da) and the volume of 3H-dextran (molecular weight 70,000 Da) distribution were determined to measure the degree of BBB disruption. At the same time point, the size of infarction was determined using tetrazolium staining. In an additional group of rats, a higher dose of SC79 (0.5 mg/kg × 3) was administered to determine the size of infarct. Administration of SC79 increased the Ki in the ischemic-reperfused cortex (IR-C, +32%, p < 0.05) as well as in the contralateral cortex (CC, +35%, p < 0.05) when compared with the untreated animals with MCAO/reperfusion. The volume of dextran distribution was not significantly changed by SC79. SC79 treatment significantly produced a decrease in the percentage of cortical infarct out of total cortical area (12.7 ±â€¯1.7% vs 6.9 ±â€¯0.9%, p < 0.001). Increasing the dose of SC79 by ten times did not significantly affect the size of cortical infarct. Contrary to our hypothesis, our data demonstrated that SC79 decreased the size of the infarct in the ischemic-reperfused cortex despite an increase in BBB disruption. Our data suggest the importance of activation of Akt for neuronal survival in the early stage of cerebral ischemia-reperfusion within the therapeutic window and that the mechanism of neuroprotection may not be related to the BBB effects of SC79.