TRAF6-mediated ubiquitination of AKT1 in the nucleus occurs in a ß-arrestin2-dependent manner upon insulin stimulation.

AKT, also known as protein kinase B (PKB), serves as a crucial regulator of numerous biological functions, including cell growth, metabolism, and tumorigenesis. Increasing evidence suggests that the kinase activity of AKT is regulated via ubiquitination by various E3 ligase enzymes in response to different stimuli. However, the molecular mechanisms underlying insulin-induced AKT ubiquitination are not yet fully understood. Here, we show that activation of the insulin receptor (IR) leads to enhanced ubiquitination of AKT1 at K8 and K14 residues, facilitated by the cytosolic E3 ubiquitin ligase enzyme, TRAF6. Further investigation using AKT1 mutants with modified nucleocytoplasmic shuttling properties reveals that TRAF6-mediated AKT1 ubiquitination occurs within the nucleus in a ß-Arr2-dependent manner. The nuclear entry of TRAF6 depends on importin ß1, while ß-Arr2 regulates this process by facilitating the interaction between TRAF6 and importin ß1. Additionally, the ubiquitination of AKT1 is essential for its translocation to the activated IR on the plasma membrane, where it plays a functional role in recruiting Glut4 and facilitating glucose uptake. This study uncovers the cellular components and processes involved in insulin-induced ubiquitination and activation of AKT1, providing insights and detailed strategies for manipulating AKT1.

Bellidifolin from Gentianella acuta (Michx.) Hulten protects H9c2 cells from hydrogen peroxide-induced injury via the PI3K-Akt signal pathway.

Cardiovascular disease is the most common disease in the world and the first among the causes of human death. Its morbidity and mortality increase annually, but no effective treatment is available. Therefore, new drugs should be developed to treat cardiovascular disease. Gentianella acuta (Michx.) Hulten (G. acuta) is an important Mongolian medicine in China and elicits protective effects on cardiovascular health. In this study, liquid chromatography-mass spectrometry (LC-MS) combined with network pharmacology was used to screen the main active ingredients and confirm that bellidifolin was one of the main components for the treatment of ischemic heart disease. Then, rat myocardial (H9c2) cells injury model induced by hydrogen peroxide (H2O2) in vitro was established to verify the effect of bellidifolin on oxidative stress stimulation, including determination of antioxidant enzyme activity and apoptosis. Transcriptome sequencing, qRT-PCR, and western blot were performed to further verify the antioxidant stress mechanism of bellidifolin. Results showed that bellidifolin pretreatment decreased the rate of apoptosis and the levels of lactate dehydrogenase (LDH), creatine kinase (CK), and alanine aminotransferase (ALT). Conversely, it increased the contents of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in a dose-dependent manner, indicating that bellidifolin caused a protective effect on cardiomyocyte injury. Bellidifolin minimized the H2O2-induced cell injury by activating the PI3K-Akt signal pathway and downregulating glycogen synthase kinase-3ß (GSK-3ß) and p-Akt1/Akt1. Therefore, this work revealed that G. acuta has a good development prospect as an edible medicinal plant in cardiovascular disease. Its bellidifolin component is a potential therapeutic agent for cardiovascular disease induced by oxidative stress damage.

PI3K/AKT1 Signaling Pathway Mediates Sinomenine-Induced Hepatocellular Carcinoma Cells Apoptosis: An in Vitro and in Vivo Study.

Hepatocellular carcinoma (HCC) is one of the most frequent cancers. Sinomenine (SIN) is a compound derived from Sinomenium acutum. Our previous investigations have found that SIN inhibited protein kinase B (AKT) signaling to induce autophagic death of tumor cells. However, whether inhibition of this pathway by SIN could impact the proliferation of HCC cells is unknown. Thus, we applied SIN to SK-Hep-1 cells and used cell counting kit 8 (CCK8), lactate dehydrogenase (LDH), colony formation and 5-ethynyl-20-deoxyuridine (EdU) incorporation experiments to detect cell viability. Then, staining with annexin V/propidium iodide (PI) coupled with terminal deoxynucleotidyl transferase-mediated biotinylated uridine 5'-triphosphate (UTP) nick end labeling (TUNEL) staining were utilized to monitor apoptosis. Changes in cell mitochondrial membrane capacity were explored via 5,5',6,6'-Tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) staining, whilst Western blot or immunohistochemistry was applied to evaluate the expression levels of key proteins, consisting of Cleaved Caspase 3, AKT1, B-cell leukemia/lymphoma 2 (BCL-2), phosphatidylinositol 3-kinase (PI3K) p85α, and Cleaved Caspase 9 etc. The Balb/c nude mice were utilized to establish HCC xenograft tumor model, administered by SIN. After treatments, the tumor volume along with weight were measured. The results illustrated that SIN suppressed SK-Hep-1 HCC cells' proliferation, enhanced the collapse of potential of the mitochondrial membrane, triggered cell apoptosis, down-regulated PI3K p85α, AKT1, BCL-2, Pro-Caspase 9, Pro-Caspase 3 expressions, and up-regulated Cleaved Caspase 9 and Cleaved Caspase 3 expressions in vitro and in vivo. Meanwhile, SIN reduced the tumor volume along with weight of mice. In addition, insulin-like growth factor-1 (IGF-1), a powerful activator of the PI3K/AKT pathway, could reverse the high apoptosis of SK-Hep-1 HCC cells induced by SIN. Overall, inhibition of PI3K/AKT1 signaling cascade by SIN induced HCC cells apoptosis.

Co-expression of plasmid-based protein kinase Bl-specific small interfering RNA and P53 synergistically inhibits proliferation, migration and invasion of hepatocellular carcinoma cells / 解剖学报

Objective To investigate the effect of the dual expression plasmid of protein kinase B1 (Akt 1)-specific siRNA and P53 on the proliferation, migration, invasion and apoptosis of hepatocellular carcinoma (HCC) cells. Methods We constructed a dual expression plasmid that co-expressed Akt 1-specific siRNA and wild-type p53 gene (pSi-Aktl-P53). The dual expression plasmid pSi-Aktl-P53 was transfected into HepG2 cells of HCC,The expression of Aktl and P53 was detected by Real-time PCR and Western blotting. Then, the dual expression plasmid was transfected into HepG2 cells, sh- Aktl plasmid and P53 plasmid were used as control. The effects of the dual plasmid on the proliferation, migration, invasion and apoptosis of HepG2 cells were detected by CCK-8 and 5-ethynyl-2'-deoxyuridine (EdU) experiments, Wound scratch experiment, Transwell chamber experiment and flow cytometry, respectively. Results After the dual plasmid was transfected into HepG2 cells, the expression of Aktl protein was significantly reduced and the expression of P53 protein was significantly increased in HepG2 cells. Compared with the shAktl and P53 plasmids, the dual expression plasmid pSi-Aktl-P53 significantly inhibited the proliferation N migration and invasion of HepG2 cells and significantly increased the apoptosis of HepG2 cells. Conclusion The dual expression plasmid pSi-Aktl-P53 can synergistically inhibit the proliferation, migration and invasion of HepG2 cells, significantly increased the apoptosis of HepG2 cells.

[DeSUMOylation of protein kinase B1 inhibits cell proliferation and metastasis of hepatocellular carcinoma].

Objective: To investigate the effect of AKT1 deSUMOylation induced by Ubc9 silencing on the proliferation and metastasis of hepatocellular carcinoma (HCC) cells. Methods: The Ubc9 gene was silenced using RNA interference, and the expression levels of Ubc9, SUMO1 and AKT1 protein were detected by Western blot. Cell proliferation and cell cycle was analyzed by MTT and flow cytometry. Wound healing and transwell assays were used to detect the cell migration ability. Furthermore, the xenograft model was established, and tumor growth curves were drawn. The in situ apoptotic rates was measured using TUNEL Apoptosis Assay. The expression of proliferating cell nuclear antigen (PCNA), matrix metalloproteinase (MMP)-2 and MMP-9 were evaluated by immunohistochemical staining. Results: Knockdown of Ubc9 gene significantly decreased the protein expression levels of Ubc9, conjugated SUMO1, free SUMO1 and AKT1 in HCC cells (P<0.05 for all). In control, siR-neg and siR-Ubc9 groups, the cell proliferation indexes were 53.19%, 54.25% and 39.17%, respectively. Moreover, cell migration distance and migrating cells per low power field for all these three groups were (59.47±4.66) µm and 89.44±8.36, (56.56±5.37) µm and 93.84±8.79, as well as (34.57±6.61) µm and 41.67±5.39, respectively. In the xenograft model, the weights of subcutaneous tumors for these three groups were (3.78±0.69) g, (3.72±0.72) g and (2.09±0.61) g, respectively. The corresponding apoptotic cell rates were (7.79±2.21)%, (6.45±2.48)% and (33.59±5.44)%, respectively. The expression levels of PCNA, MMP-2 and MMP-9 protein were significantly decreased in siR-Ubc9 group (P<0.05). Conclusions: Ubc9 silencing in HCC cells induces AKT1 deSUMOylation, and then inhibits the proliferation and metastasis. These results provide a new therapeutic strategy for liver cancer in the future.

DeSUMOylation of protein kinase B1 inhibits cell proliferation and metastasis of hepatocellular carcinoma / 中华肿瘤杂志

Objective@#To investigate the effect of AKT1 deSUMOylation induced by Ubc9 silencing on the proliferation and metastasis of hepatocellular carcinoma (HCC) cells.@*Methods@#The Ubc9 gene was silenced using RNA interference, and the expression levels of Ubc9, SUMO1 and AKT1 protein were detected by Western blot. Cell proliferation and cell cycle was analyzed by MTT and flow cytometry. Wound healing and transwell assays were used to detect the cell migration ability. Furthermore, the xenograft model was established, and tumor growth curves were drawn. The in situ apoptotic rates was measured using TUNEL Apoptosis Assay. The expression of proliferating cell nuclear antigen (PCNA), matrix metalloproteinase (MMP)-2 and MMP-9 were evaluated by immunohistochemical staining.@*Results@#Knockdown of Ubc9 gene significantly decreased the protein expression levels of Ubc9, conjugated SUMO1, free SUMO1 and AKT1 in HCC cells (P<0.05 for all). In control, siR-neg and siR-Ubc9 groups, the cell proliferation indexes were 53.19%, 54.25% and 39.17%, respectively. Moreover, cell migration distance and migrating cells per low power field for all these three groups were (59.47±4.66) μm and 89.44±8.36, (56.56±5.37) μm and 93.84±8.79, as well as (34.57±6.61) μm and 41.67±5.39, respectively. In the xenograft model, the weights of subcutaneous tumors for these three groups were (3.78±0.69) g, (3.72±0.72) g and (2.09±0.61) g, respectively. The corresponding apoptotic cell rates were (7.79±2.21)%, (6.45±2.48)% and (33.59±5.44)%, respectively. The expression levels of PCNA, MMP-2 and MMP-9 protein were significantly decreased in siR-Ubc9 group (P<0.05).@*Conclusions@#Ubc9 silencing in HCC cells induces AKT1 deSUMOylation, and then inhibits the proliferation and metastasis. These results provide a new therapeutic strategy for liver cancer in the future.

Expressions and clinical significance of pAkt1 and hTERT in epithelial ovarian carcinoma / 国际肿瘤学杂志

Objective To evaluate the expressions and prognostic significance of phosphorylation of protein kinase B1 (pAkt1) and human telomere reverse transcriptase (hTERT) in epithelial ovarian carcinoma.Methods The expressions of pAkt1 and hTERT were examined by immunohistochemical SP method in 72 cases of epithelial ovarian carcinoma and 10 normal endometrial tissues to analyze the correlation between pAkt1 and hTERT and their relationships with clinic-pathological features and prognosis.Results The positive expression rate of pAkt1 in epithelial ovarian carcinoma was 73.6％,and the positive expression rate of hTERT was 56.9％,compared with normal ovarian tissues,with statistical significance (x2 =20.814,P ＜ 0.001;x2 =11.3g9,P ＜ 0.001).The expression of pAkt1 was not associated with hTERT expression (r =0.075,P =0.532) in epithelial ovarian carcinoma.The expressions of pAkt1 and hTERT in epithelial ovarian carcinoma were correlated with tumor differentiation status (x2=6.475,P =0.011;x2 =1.370,P =0.001).The disease-free survival of patients with pAkt1 and hTERT positive coexpression was significantly shorter than others (8.585 months vs.11.227 months,x2 =4.361,P =0.037),but there was no significant difference in overall survival (11.107 months:11.695 months,x2 =0.394,P =0.530).Conclusion pAkt1 and hTERT are highly expressed in epithelial ovarian carcinoma,and high coexpression of pAkt1 and hTERT indicates poor prognosis in epithelial ovarian carcinoma.

Pleiotropic effects of methionine adenosyltransferases deregulation as determinants of liver cancer progression and prognosis.

Downregulation of liver-specific MAT1A gene, encoding S-adenosylmethionine (SAM) synthesizing isozymes MATI/III, and upregulation of widely expressed MAT2A, encoding MATII isozyme, known as MAT1A:MAT2A switch, occurs in hepatocellular carcinoma (HCC). Being inhibited by its reaction product, MATII isoform upregulation cannot compensate for MATI/III decrease. Therefore, MAT1A:MAT2A switch contributes to decrease in SAM level in rodent and human hepatocarcinogenesis. SAM administration to carcinogen-treated rats prevents hepatocarcinogenesis, whereas MAT1A-KO mice, characterized by chronic SAM deficiency, exhibit macrovesicular steatosis, mononuclear cell infiltration in periportal areas, and HCC development. This review focuses upon the pleiotropic changes, induced by MAT1A/MAT2A switch, associated with HCC development. Epigenetic control of MATs expression occurs at transcriptional and post-transcriptional levels. In HCC cells, MAT1A/MAT2A switch is associated with global DNA hypomethylation, decrease in DNA repair, genomic instability, and signaling deregulation including c-MYC overexpression, rise in polyamine synthesis, upregulation of RAS/ERK, IKK/NF-kB, PI3K/AKT, and LKB1/AMPK axis. Furthermore, decrease in MAT1A expression and SAM levels results in increased HCC cell proliferation, cell survival, and microvascularization. All of these changes are reversed by SAM treatment in vivo or forced MAT1A overexpression or MAT2A inhibition in cultured HCC cells. In human HCC, MAT1A:MAT2A and MATI/III:MATII ratios correlate negatively with cell proliferation and genomic instability, and positively with apoptosis and global DNA methylation. This suggests that SAM decrease and MATs deregulation represent potential therapeutic targets for HCC. Finally, MATI/III:MATII ratio strongly predicts patients' survival length suggesting that MAT1A:MAT2A expression ratio is a putative prognostic marker for human HCC.

Effects of Buyang Huanwu decoction on protein kinase B1 and c-Jun amino terminal kinase 1/2 in rats after ;focal cerebral ischemia / 中国中西医结合急救杂志

Objective To explore the effect of Buyang Huanwu decoction(BYHWD)on protein kinase B1 (AKT1)and c-Jun amino terminal kinase 1/2(JNK1/2)in rats after focal cerebral ischemia. Methods According to the random number table method,48 Sprague-Dawley(SD)rats were randomly allocated to four groups:normal control group,sham-operated group,model group,traditional BYHWD group(each n=12). The rat model of right focal cerebral ischemia was established by the method of middle cerebral artery occlusion(MCAO). The rats in BYHWD group were ingested with the decoction of BYHWD 14.2 g/kg after 2 hours of the operation(the main ingredients of BYHWD including astragalus mongholicus 120 g,Chinese angelica 6 g,radix paeoniae rubra 4.5 g, rhizoma ligustici wallichii 3 g,safflower 3 g,peach kernel 3 g,earthworm 3 g),once a day for 7 days. Other groups of animals were given the same amount of normal saline orally. After operation,on the 7th day,the animals were killed,and their brains were taken out. The reverse transcription-polymerase chain reaction(RT-PCR)assay was used to detect AKT1 mRNA expression,and immunohistochemical method was applied to measure JNK1/2 protein expression. Results Compared with normal control and sham-operated groups,the level of AKT1 mRNA expression〔absorbance(A)〕was decreased obviously(0.48±0.08 vs. 0.63±0.11,0.61±0.09,both P<0.05),and the number of JNK1/2 positive cells(cell/mm2)was increased significantly(34.13±4.57 vs. 16.15±1.09,16.23±2.05,both P<0.05)in model group;compared with model group,the AKT1 mRNA expression in brain tissue(0.93±0.11)and the number of JNK1/2 positive cells(45.04±5.68)was increased significantly in BYHWD group,the differences being statistically significant(P<0.05 or P<0.01). Conclusion BYHWD can up-regulate expressions of AKT1 mRNA and JNK1/2 positive cells in ischemic brain tissue that is one of the mechanisms in the protection of brain.

Association of PKB1 gene polymorphisms and event-related potentials in major depressive disorder / 中华行为医学与脑科学杂志

Objective To explore the relationship of protein kinase B1 ( PKB1 ) gene polymorphisms in PI3-K pathway of BDNF and event-related potentials in depression.Methods The design of case-control research was used ,and 91 major depressive patients and 65 normal controls who were made in age and gender matched with patients were measured auditory event-related potential P300 and contingent negative variation ( CNV ) in the day when two groups were collected.Polymerase chain reaction (PCR) and direct DNA sequencing technology were used to detect PKB1 gene polymorphisms.Three SNPs that named rs3001371 ,rs2494738 ,rs1130214 were selected from 3 representative BLOCK Districts of PKB1.Two independent samples t test was used to analysis P300 and CNV between two groups,and the same way to analysis the average level of P300 and CNV and PKB1 SNP genolatency of P2(P＜0.05) and lower amplitude of P3a(P＜0.01 ) ,P3b(P＜0.01 ) and P3 (P＜0.01 ) ;CNV had der had statistical difference (P＜ 0.05 )in PKB1 rs3001371 gene between C/C and C/T genotype combined which included C allele, and T/T genotype.The amplitude of P3a( (5.93 ± 2.35 ) μV, P3b(6.51 ± 3.00) μV, P3 (6.27±2.43) μV) were lower than TT Genotype ( (7.45 ±2.19)μV, (8.63 ±3.57)μV,(8.04 ±2.57)μV,respectively).The mean of CNV indicators were not found different in statistics among the rs3001371 genotypes.Conclusions PKB1 gene rs3001371 polymorphism is associated with the principal component of P300 amplitude in patients with Major depressive disorder which suggest that genetic factors may have a certain impact on cognitive function in the patients with Major depressive disorder.

Inhibitory effect of adenovirus-mediated short hairpin RNA targeting P85 and Akt1 on growth of human gastric adenocarcinoma cell / 中华内科杂志

Objective To construct a short hairpin RNA (shRNA) adenovirus vector targeting P85 and protein kinase B1 (PKB1/Akt1) and study its effects on the growth of SGC-7901 human gastric adenocareinoma cells. Methods P85 and Aktl shRNA expression frames were subcloned to pGSadeno adenovirus vector with homologous recombination technology to construct pGSadeno-P85 + Akt1 (rAd5-P + A) vector. After screening and amplification, the recombinant adenovirus vector was digested with PacI and transfected into SGC-7901 cells and then its titer and transfection efficiency were detected with fluorescent microscope. P85 and Akt1 mRNA protein expression was identified with real-time PCR and Western blot. The proliferative activity of tumor cells was evaluated with MTr assay and flow cytometry in vitro, rAd5-HK and rAd5-P + A mediated by adenovirus were injected into the established subcutancous SGC-7901 gastric adenocarcinoma in nude mice. During the observation period of 21 days, tumor volume was measured every 3 days to further testify the anti-tumor effect of rAd5-P + A on the SGC-7901 gastric adenocarcinoma cells and cell in situ apoptosis was detected with TUNEL assay. Results The adenovirus vector rAd5-P + A was successfully constructed and it dramatically downregulated P85 and Akt1 mRNA expression in SGC-7901 gastric adenocarcinoma cells. Compared with a control group of SGC-7901 cells and cells transfected with general adenovirus rAd5-HK as control, P85 and Akt1 protein expression 48 h and 72 h after rAd5-P + A transfection was decreased by 57.5% and 63. 7%, 67. 8% and 75.6% with statistical significance(P = 0. 005, P = 0. 003). Cell proliferative activity in rAd5-P + A transfected cells was suppressed from the second day (P <0. 001) and the decreased P85 and Akt1 expression was accompanied by 5.9% -7. 1% decrease of S phase fraction and 12. 1% - 13.7% increase of G0/G1 phase. The tumor volume of rAd5-P + A treated group was smaller than that of the control and rAd.5-HK group with statistical significance (F = 9. 871, P = 0. 025) . Moreover, rAd5-P + A could induce cell in situ apoptosis. Conclusions Adenovirus-mediated targeting P85 and Akt1 shRNA can inhibit the growth of SGC-7901 human gastric adenocarcinoma cells and this may provide a new strategy of combination gene therapy in gastric adenocarcinoma.