p-Akt Levels Research Articles

The protective effect of Macrostemonoside T from Allium macrostemon Bunge against Isoproterenol-Induced myocardial injury via the PI3K/Akt/mTOR signaling pathway

Myocardial injury (MI) signifies a pathological aspect of cardiovascular diseases (CVDs) such as coronary artery disease, diabetic cardiomyopathy, and myocarditis. Macrostemonoside T (MST) has been isolated from Allium macrostemon Bunge (AMB), a key traditional Chinese medicine (TCM) used for treating chest stuffiness and pains. Although MST has demonstrated considerable antioxidant activity in vitro, its protective effect against MI remains unexplored. To investigate MST's effects in both in vivo and in vitro models of isoproterenol (ISO)-induced MI and elucidate its underlying molecular mechanisms. This study established an ISO-induced MI model in rats and assessed H9c2 cytotoxicity to examine MST's impact on MI. Various assays, including histopathological staining, TUNEL staining, immunohistochemical staining, DCFH-DA staining, JC-1 staining, ELISA technique, and Western blot (WB), were utilized to explore the potential molecular mechanisms of MI protection. In vivo experiments demonstrated that ISO caused myocardial fiber disorders, elevated cardiac enzyme levels, and apoptosis. However, pretreatment with MST significantly mitigated these detrimental changes. In vitro experiments revealed that MST boosted antioxidant enzyme levels and suppressed malondialdehyde (MDA) production in H9c2 cells. Concurrently, MST inhibited ISO-induced reactive oxygen species (ROS) production and mitigated the decline in mitochondrial membrane potential, thereby reducing the apoptosis rate. Moreover, pretreatment with MST elevated the expression levels of p-PI3K, p-Akt, and p-mTOR, indicating activation of the PI3K/Akt/mTOR signaling pathway and consequent protection against MI. MST attenuated ISO-induced MI in rats by impeding apoptosis through activation of the PI3K/Akt/mTOR signaling pathway. This study presents potential avenues for the development of precursor drugs for CVDs.

Dynamic RGD ligands derived from highly mobile cyclodextrins regulate spreading and proliferation of endothelial cells to promote vasculogenesis

A thiolated RGD was incorporated into the threaded allyl-β-cyclodextrins (Allyl-β-CDs) of the polyrotaxane (PR) through a thiol-ene click reaction, resulting in the formation of dynamic RGD ligands on the PR surface (dRGD-PR). When maintaining consistent RGD density and other physical properties, endothelial cells (ECs) cultured on dRGD-PR exhibited significantly increased cell proliferation and a larger cell spreading area compared to those on the non-dynamic RGD (nRGD-PCL). Furthermore, ECs on dRGD-PR demonstrated elevated expression levels of FAK, p-FAK, and p-AKT, along with a larger population of cells in the G2/M stage during cell cycle analysis, in contrast to cells on nRGD-PCL. These findings suggest that the movement of the RGD ligands may exert additional beneficial effects in promoting EC spreading and proliferation, beyond their essential adhesion and proliferation-promoting capabilities, possibly mediated by the RGD-integrin-FAK-AKT pathway. Moreover, in vitro vasculogenesis tests were conducted using two methods, revealing that ECs cultured on dRGD-PR exhibited much better vasculogenesis than nRGD-PCL in vitro. In vivo testing further demonstrated an increased presence of CD31-positive tissues on dRGD-PR. In conclusion, the enhanced EC spreading and proliferation resulting from the dynamic RGD ligands may contribute to improved in vitro vasculogenesis and in vivo vascularization.

Scutellarin alleviates microglia-mediated neuroinflammation and apoptosis after ischemic stroke through the PI3K/AKT/GSK3β signaling pathway.

Microglia are resident immune cells in the central nervous system that are rapidly activated to mediate neuroinflammation and apoptosis, thereby aggravating brain tissue damage after ischemic stroke (IS). Although scutellarin has a specific therapeutic effect on IS, the potential target mechanism of its treatment has not been fully elucidated. In this study, we explored the potential mechanism of scutellarin in treating IS using network pharmacology. Lipopolysaccharide (LPS) was used to induce an in vitro BV-2 microglial cell model, while middle cerebral artery occlusion (MCAO) was used to induce an in vivo animal model. Our findings indicated that scutellarin promoted the recovery of cerebral blood flow in MCAO rats at 3days, significantly different from that in the MCAO group. Western blotting and immunofluorescence revealed that scutellarin treatment of BV-2 microglial cells resulted in a significant reduction in the protein expression levels and incidence of cells immunopositive for p-NF-κB, TNF-α, IL-1β, Bax, and C-caspase-3. In contrast, the expression levels of p-PI3K, p-AKT, p-GSK3β, and Bcl-2 were further increased, significantly different from those in the LPS group. The PI3K inhibitor LY294002 had similar effects to scutellarin by inhibiting neuroinflammation and apoptosis in activated microglia. The results of the PI3K/AKT/GSK3β signaling pathway and NF-κB pathway in vivo in MCAO models induced microglia at 3days were consistent with those obtained from in vitro cells. These findings indicate that scutellarin plays a neuroprotective role by reducing microglial neuroinflammation and apoptosis mediated by the activated PI3K/AKT/GSK3β/NF-κB signaling pathway.

The protective effect of Astaxanthin on scopolamine - induced Alzheimer's model in mice.

To investigate the fundamental mechanisms of the neuroprotective impact of Astaxanthin (AST) in a mouse model of Alzheimer's disease (AD) induced by scopolamine. This research constituted an in vivo animal study encompassing 36 adult male mice, divided into 6 groups: Control, 100mg/kg AST, 2mg/kg scopolamine (AD group), 100mg/kg AST+2mg/kg scopolamine, 3mg/kg galantamine+2mg/kg scopolamine, and 100mg/kg AST+3mg/kg galantamine+2mg/kg scopolamine. After 14 days, the mice's short-term memory, hippocampus tissue, oxidative and inflammatory markers were evaluated. The AST demonstrated a beneficial influence on short-term memory and a reduction in acetylcholinesterase activity in the brain. It exhibited neuroprotective and anti-amyloidogenic properties, significantly decreased pro-inflammatory markers and oxidative stress, and reversed the decline of the Akt-1 and phosphorylated Akt pathway, a crucial regulator of abnormal tau. Furthermore, AST enhanced the effect of galantamine in reducing inflammation and oxidative stress. The findings indicate that AST may offer therapeutic benefits against cognitive dysfunction in AD. This is attributed to its ability to reduce oxidative stress, control neuroinflammation, and enhance Akt-1 and pAkt levels, thereby underscoring its potential in AD treatment strategies.

Anticancer potential of grifolin in lung cancer treatment through PI3K/AKT pathway inhibition

ObjectiveGrifolin is a natural secondary metabolite isolated from edible fruiting bodies of the mushroom Albatrellus confluens. Grifolin has antitumor activities in several types of cancer. We aimed to determine the effects of grifolin on lung cancer. MethodsWe determined the proliferation, migration, invasion, and apoptosis of lung cancer cells using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Ethynyl deoxyuridine, colony formation, wound scratch, transwell, flow cytometry, and xenograft mouse assays. Molecular docking evaluated the binding relation between grifolin and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA). The levels of PIK3CA, AKT, and p-AKT were measured by western blot. ResultsGrifolin (10, 20, or 40 μM) inhibited the proliferation, migration, and invasion of lung cancer cells, and induced cell cycle arrest and apoptosis. Grifolin also decreased CDK4, CDK6, and CyclinD1 expression and significantly decreased PIK3CA and p-AKT expression in lung cancer cells. These anticancer effects were abolished by 740Y-P. ConclusionsGrifolin regulates the PI3K/AKT pathway, thus inhibiting lung cancer progression.

Reversal Effect of NVP-BEZ235 on Doxorubicin-Resistance in Burkitt Lymphoma RAJI Cell Line

To study the reversal effect of NVP-BEZ235 on doxorubicin resistance in Burkitt lymphoma RAJI cell line. The doxorubicin-resistant cell line was induced by treating RAJI cells with a concentration gradient of doxorubicin. The levels of Pgp, p-AKT, and p-mTOR in cells were detected by Western blot. Cell viability was detected by MTT assay. IC50 was computed by SPSS. The doxorubicin-resistant Burkitt lymphoma cell line, RAJI/DOX, was established successfully. The expression of Pgp and the phosphorylation levels of AKT and mTOR in RAJI/DOX cell line were both higher than those in RAJI cell line. NVP-BEZ235 downregulated the phosphorylation levels of AKT and mTOR in RAJI/DOX cell line. NVP-BEZ235 inhibited the proliferation of RAJI/DOX cell line, and the effect was obvious when it was cooperated with doxorubicin. The constitutive activation of PI3K/AKT/mTOR pathway of RAJI/DOX cell line was more serious than RAJI cell line. NVP-BEZ235 reversed doxorubicin resistance of RAJI/DOX cell line by inhibiting the PI3K/AKT/mTOR signal pathway.

Mechanism of Notoginseng Radix et Rhizoma in regulating PI3K/Akt/mTORC1-mitochondrial energy metabolism to treat chronic renal failure in rats with blood stasis syndrome

This study observed the effects of Notoginseng Radix et Rhizoma on the phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)/mammalian target of rapamycin complex 1(mTORC1) signaling pathway and mitochondrial energy metabolism in the rat model of adriamycin-induced renal fibrosis with blood stasis syndrome to explore the mechanism of Notoginseng Radix et Rhizoma in protecting the kidney. Thirty male rats with adriamycin-induced renal fibrosis were randomized into model, low-, medium-, and high-dose Notoginseng Radix et Rhizoma, and positive control groups(n=6). Six clean SD male rats were selected into the normal group. The normal group and model group were administrated with normal saline, and other groups with corresponding drugs. After 8 weeks of treatment, the renal function, renal pathology, adenosine triphosphate(ATP) levels, Na~+-K~+-ATPase and Ca~(2+)-Mg~(2+)-ATPase activities, and the protein levels of ATP5B, mTORC1, 70 kDa ribosomal protein S6 kinase(P70S6K), P85, Akt, p-Akt, and SH2-containing inositol phosphatase(SHIP2) in the renal tissue were determined. Compared with the normal group, the model group showed elevated levels of blood urea nitrogen(BUN) and serum creatinine(SCr)(P<0.01). Compared with the model group, Notoginseng Radix et Rhizoma and the positive control lowered the levels of BUN and SCr, which were significant in the medium-and high-dose Noto-ginseng Radix et Rhizoma groups and the positive control group(P<0.05). Compared with the model group, Notoginseng Radix et Rhizoma and the positive control alleviated the pathological changes in the renal tissue, such as vacuolar and fibroid changes, glomerulus atrophy, cystic expansion of renal tubules, and massive infiltration of inflammatory cells. Compared with the normal group, the model group showed decreased mitochondrial ATP content and Na~+-K~+-ATPase and Ca~(2+)-Mg~(2+)-ATPase activities in the renal tissue(P<0.05), and medium-and high-dose Notoginseng Radix et Rhizoma and positive control mitigated such decreases(P<0.05). Compared with the model group, medium-and high-dose Notoginseng Radix et Rhizoma and the positive control up-regulated the protein levels of ATP5B and SHIP2 and down-regulated the protein levels of mTORC1, P70S6K, P85, Akt, and p-Akt(P<0.05 or P<0.01 or P<0.001). Notoginseng Radix et Rhizoma may exert an anti-fibrosis effect by inhibiting the activation of the PI3K/Akt/mTORC1 pathway to restore mitochondrial energy metabolism, thus protecting the kidney.

Synergistic effects of the combination of trametinib and alpelisib in anaplastic thyroid cancer with BRAF and PI3KCA co-mutations

BackgroundAnaplastic thyroid cancer (ATC), a rare and aggressive malignancy with a poor prognosis, has shown promise with the approved dabrafenib/trametinib combination for BRAFV600E mutation. Co-occurring PI3KCA mutations, identified as negative prognostic factors in lung cancer with BRAFV600E mutation, emphasize the need to target both pathways. Exploring trametinib and alpelisib combination becomes crucial for ATC. MethodsA patient-derived xenograft (PDX) and primary cell line were obtained from an ATC patient with BRAF and PI3KCA co-mutation. Individual testing of targeted therapies against BRAF, MEK, and PI3KCA was followed by a combination treatment. Synergistic effects were evaluated using the combination index. Immunoblotting assessed the efficacy, with validation performed using a PDX model. ResultsIn this study, the ATC0802 cell line and PDX were established from a refractory ATC patient. NGS revealed BRAF and PI3KCA co-mutations pre- and post-dabrafenib/trametinib treatment. Trametinib/alpelisib combination showed synergy, suppressing both pERK and pAKT levels, unlike monotherapies or BRAF knockdown. The combination induced apoptosis and, in the PDX model, demonstrated superior tumor growth inhibition compared to monotherapies. ConclusionsThe combination of trametinib and alpelisib showed promise as a strategy for treating ATC with co-mutations in BRAF and PI3KCA, both in vitro and in vivo. This combination offers insights into overcoming resistance to BRAF-targeted treatments in ATC with mutations in BRAF and PI3KCA.

Gemcitabine elaidate and ONC201 combination therapy for inhibiting pancreatic cancer in a KRAS mutated syngeneic mouse model

Approximately 90% of pancreatic cancer (PC) contain KRAS mutations. Mutated KRAS activates the downstream oncogenic PI3K/AKT and MEK signaling pathways and induces drug resistance. However, targeting both pathways with different drugs can also lead to excessive toxicity. ONC201 is a dual PI3K/AKT and MEK pathway inhibitor with an excellent safety profile that targets death receptor 5 (DR5) to induce apoptosis. Gemcitabine (GEM) is a first-line chemotherapy in PC, but it is metabolically unstable and can be stabilized by a prodrug approach. In this study, phospho-Akt, phospho-mTOR, and phospho-ERK protein expressions were evaluated in patient PDAC-tissues (n = 10). We used lipid-gemcitabine (L_GEM) conjugate, which is more stable and enters the cells by passive diffusion. Further, we evaluated the efficacy of L_GEM and ONC201 in PC cells and “KrasLSL-G12D; p53LoxP; Pdx1-CreER (KPC) triple mutant xenograft tumor-bearing mice. PDAC patient tissues showed significantly higher levels of p-AKT (Ser473), p-ERK (T202/T204), and p-mTOR compared to surrounding non-cancerous tissues. ONC201 in combination with L_GEM, showed a superior inhibitory effect on the growth of MIA PaCa-2 cells. In our in-vivo study, we found that ONC201 and L_GEM combination prevented neoplastic proliferation via AKT/ERK blockade to overcome chemoresistance and increased T-cell tumor surveillance. Simultaneous inhibition of the PI3K/AKT and MEK pathways with ONC201 is an attractive approach to potentiate the effect of GEM. Our findings provide insight into rational-directed precision chemo and immunotherapy therapy in PDAC.

The identification of a novel shared therapeutic target and drug across all insulin-sensitive tissues under insulin resistance.

To identify key and shared insulin resistance (IR) molecular signatures across all insulin-sensitive tissues (ISTs), and their potential targeted drugs. Three datasets from Gene Expression Omnibus (GEO) were acquired, in which the ISTs (fat, muscle, and liver) were from the same individual with obese mice. Integrated bioinformatics analysis was performed to obtain the differentially expressed genes (DEGs). Weighted gene co-expression network analysis (WGCNA) was carried out to determine the "most significant trait-related genes" (MSTRGs). Enrichment analysis and PPI network were performed to find common features and novel hub genes in ISTs. The shared genes of DEGs and genes between DEGs and MSTRGs across four ISTs were identified as key IR therapeutic target. The Attie Lab diabetes database and obese rats were used to verify candidate genes. A medical drug-gene interaction network was conducted by using the Comparative Toxicogenomics Database (CTD) to find potential targeted drugs. The candidate drug was validated in Hepa1-6 cells. Lipid metabolic process, mitochondrion, and oxidoreductase activity as common features were enriched from ISTs under an obese context. Thirteen shared genes (Ubd, Lbp, Hp, Arntl, Cfd, Npas2, Thrsp., Tpx2, Pkp1, Sftpd, Mthfd2, Tnfaip2, and Vnn3) of DEGs across ISTs were obtained and confirmed. Among them, Ubd was the only shared gene between DEGs and MSTRGs across four ISTs. The expression of Ubd was significantly upregulated across four ISTs in obese rats, especially in the liver. The IR Hepa1-6 cell models treated with dexamethasone (Dex), palmitic acid (PA), and 2-deoxy-D-ribose (dRib) had elevated expression of Ubd. Knockdown of Ubd increased the level of p-Akt. A lowing Ubd expression drug, promethazine (PMZ) from CTD analysis rescued the decreased p-Akt level in IR Hepa1-6 cells. This study revealed Ubd, a novel and shared IR molecular signature across four ISTs, as an effective biomarker and provided new insight into the mechanisms of IR. PMZ was a candidate drug for IR which increased p-Akt level and thus improved IR by targeting Ubd and downregulation of Ubd expression. Both Ubd and PMZ merit further clinical translational investigation to improve IR.

Abstract 1978: The combination of neratinib with sotorasib is synergistic in models of HER2-amplified and KRASG12C-mutated cancer

Abstract Introduction: Constitutive activation of KRAS through gain-of-function mutations, such as KRASG12C, is thought to reduce sensitivity to HER-targeted TKIs like neratinib (N), lapatinib (L) and tucatinib (T). Sotorasib (S) is a KRASG12C specific inhibitor clinically approved for the treatment of advanced KRASG12C-mutated non-small cell lung cancer. One identified resistance mechanism to sotorasib is the increase of multiple phosphorylated receptor tyrosine kinases, including EGFR and HER2. The objective of this study was to assess the in vitro efficacy of the single agents N, L, T and S, and the combination of N and S in HER2-amplified, KRASG12C-mutated cancer cell lines. Methods: The antiproliferative effects of N, L, T and S were assessed in the HER2-amplified and KRASG12C-mutated KYSE-410 (esophageal) and NCI-H2030 (lung) cancer cell lines, and KRASG12C-mutated MiaPaca-2 (pancreatic) cell line by 5-day acid phosphatase assay. IC50 values were calculated using CalcuSyn software. To assess the synergy between N and S, matrix assays were performed and analyzed using Combenefit software. To further determine the efficacy of the drugs, changes in signaling pathways were assessed by Western blotting. Results: N displayed nanomolar IC50 values (< 100 nM) in all three cell lines, while nanomolar IC50 values with L and T were only achieved in KYSE-410 (IC50 = 426.84 nM and IC50 = 363.38 nM respectively). S was most potent in MiaPaca-2 (IC50 = 6.04 nM), followed by NCI-H2030 (IC50 = 270.60 nM) and KYSE-410 (IC50 = 5.46 µM). Matrix assays showed that NS was broadly additive in MiaPaca-2 cells, and synergistic in both NCI-H2030 and KYSE-410 cell lines. N and NS numerically decreased HER2 levels compared to control in NCI-H2030 after 24h of treatment (6.4-fold, p = 0.58 and 2.5-fold, p = 0.79 respectively) but not in KYSE-410. KRAS levels were significantly increased (1.89-fold, p ≤ 0.05) after treatment with NS in NCI-H2030. No significant effects were observed on the activation of HER2 and EGFR in either cell line at 24h. In NCI-H2030, NS had a significantly stronger inhibitory effect on pERK compared to N (p ≤ 0.0001) and S (p = 0.012) alone. In KYSE-410, pERK levels were also slightly decreased with NS, but not significantly different from N (p = 0.12). However, pAkt levels in KYSE-410 were significantly reduced by N (1.3-fold, p = 0.009) and NS (1.7-fold, p ≤ 0.0001) compared to control, with NS showing a significantly greater decrease than N (p = 0.026). It thus appears that the synergy between N and S occurs through a stronger inhibition of ERK activity in NCI-H2030, while it occurs through a stronger inhibition of Akt activity in KYSE-410. Conclusion: NS is additive or synergistic in the three KRASG12C-mutated cancer cell lines examined. Further work is planned to investigate the basis of the synergy observed. Citation Format: Myra E. Castel, Neil T. Conlon, Naomi Walsh, Georg F. Bischof, John P. Crown, Denis M. Collins. The combination of neratinib with sotorasib is synergistic in models of HER2-amplified and KRASG12C-mutated cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1978.

Abstract 7596: (TTFields) and imipridone ONC206 co-treatment inhibits p-AKT and spheroid growth and upregulates caspase-10 in human GBM cell lines

Abstract Background: Glioblastoma (GBM) is the most common primary brain malignancy with dismal prognosis. Tumor Treating Fields (TTFields) therapy is available for the treatment of both newly diagnosed and recurrent glioblastoma and represents a new category of treatment modalities in oncologic therapy. Despite decades of study, few FDA approved modalities are available for GBM and provide limited survival improvements for patients. AKT functions as a key serine/threonine kinase in the RTK/PTEN/PI3K pathway, and extensive genomic analysis of GBM has revealed that this pathway is mutated in a significant majority of GBM cases. Activation of this pathway ultimately leads to the activation of AKT, and p-AKT levels are elevated in the majority of GBM tumor samples and cell lines. Studies have shown that increased p-AKT levels contribute to uncontrolled growth, resistance to apoptosis, and enhanced invasive capabilities of glioma cells, which are characteristics of tumor progression in GBM. AKT serves as a critical hub in this pathway, enabling the amplification of growth signals, thereby making the inhibition of AKT an appealing and promising therapeutic target for treating GBM. Imipridone ONC206 is under clinical development for treatment of pediatric and adult patients with primary brain tumors (NCT04732065 and NCT04541082). Here we show inhibition of p-AKT as well as upregulation of CHOP and caspase-10 following cotreatment of ONC206 and TTFields. Materials & Methods: We investigated the effect of ONC206 (at IC50s) plus TTFields in U251 and T98G human GBM cell lines at different time points. Effect of treatment on cPARP, BCL-2, CHOP, caspase-10, and p-AKT were investigated using western blot. Neurospheres were generated using mentioned cell lines and used for investigating effect of co-treatment on 3D structure. Results: Treatment with ONC206 at the IC50 in T98G cells with TTFields at 24h showed synergistic upregulation of cPARP as well as inhibition of BCL-2. At 96hour time point, inhibition of p-AKT was observed following all treatments (ONC206, TTFields, and co-application compared to control. In U251 cells, following treatment of ONC206 and TTFields, inhibition of BCL-2 was observed at 24h time point at all treatment conditions. Upregulation of CHOP and Casapse-10 at 96h time point was seen, as well as inhibition of p-AKT. Spheroid models showed structure disruption and growth arrest following combination treatment. Conclusion: Here we showed cotreatment results in inhibition of p-AKT, the key node in GBM growth pathway. Also, the upregulation of CHOP and caspase-10 that has not been discussed before can be the key to focus on novel therapeutic targets for GBM More studies are needed to elaborate the exact mechanism of synergy but these new findings can be used to develop novel treatment combination Citation Format: Vida Tajiknia, Praveen Srinivasan, Maximilian Pinho-Schwermann, William MacDonald, Connor Purcell, Wafik El-Deiry. (TTFields) and imipridone ONC206 co-treatment inhibits p-AKT and spheroid growth and upregulates caspase-10 in human GBM cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7596.

The role of TIM3+ NK and TIM3- NK cells in the immune pathogenesis of severe aplastic anemia.

Natural killer (NK) cells play important immunoregulatory roles in the immune pathogenesis of severe aplastic anemia (SAA). Our previous research showed that SAA caused a decrease in T cell immunoglobulin mucin-3 (TIM3) expression on NK cells. Here we investigated the expression of surface receptors, and the cytotoxicity of peripheral TIM3+ NK and TIM3- NK cells in patients with SAA. The expressions of surface receptors and cytoplasmic protein of TIM3+ NK and TIM3- NK cells from peripheral blood were detected by FCM. The functions of mDCs, and apoptosis rate of K562 cells after co-culture with TIM3+ NK and TIM3- NK cells were maesured by FCM. Westren-blot was used to detect the changes of TIM3+ NK and TIM3- NK signaling pathway proteins (AKT, P-AKT) and compare the functional activity of the two groups. Activating receptors NKG2D and Granzyme B were higher, while inhibiting receptors NKG2A, CD158a and CD158b were lower on TIM3- NK cells compared with TIM3+ NK cells in patients with SAA. In SAA, the expression of CD80 and CD86 on mDCs (Myeloid dendritic cells) was significantly decreased after incubation with TIM3- NK cells. The apoptosis rate (AR) of K562 cells was significantly increased after being incubated with TIM3- NK cells in SAA. The level of signal pathway protein AKT of TIM3- NK cells in SAA was similar to that of TIM3+ NK cells, and the levels of P-AKT and P-AKT/AKT ratio of TIM3- NK cells were significantly higher than those of TIM3+ NK cells. Therefore, TIM3 exerts its inhibitory effect on NK cells and participates in the immune pathogenesis of SAA. Low expression of TIM3 contributes to the enhancement of NK cell activity which in turn inhibits the immune activation state of SAA and improves the disease state. Our research may aid the development of new therapeutic strategies based on TIM3-NK cells infusion for the treatment of SAA.

Mathematical Modeling of PI3K/Akt Pathway in Microglia.

The motility of microglia involves intracellular signaling pathways that are predominantly controlled by changes in cytosolic Ca2+ and activation of PI3K/Akt (phosphoinositide-3-kinase/protein kinase B). In this letter, we develop a novel biophysical model for cytosolic Ca2+ activation of the PI3K/Akt pathway in microglia where Ca2+ influx is mediated by both P2Y purinergic receptors (P2YR) and P2X purinergic receptors (P2XR). The model parameters are estimated by employing optimization techniques to fit the model to phosphorylated Akt (pAkt) experimental modeling/in vitro data. The integrated model supports the hypothesis that Ca2+ influx via P2YR and P2XR can explain the experimentally reported biphasic transient responses in measuring pAkt levels. Our predictions reveal new quantitative insights into P2Rs on how they regulate Ca2+ and Akt in terms of physiological interactions and transient responses. It is shown that the upregulation of P2X receptors through a repetitive application of agonist results in a continual increase in the baseline [Ca2+], which causes the biphasic response to become a monophasic response which prolongs elevated levels of pAkt.

KAT7/HMGN1 signaling epigenetically induces tyrosine phosphorylation-regulated kinase 1A expression to ameliorate insulin resistance in Alzheimer's disease.

Epidemiological studies have revealed a correlation between Alzheimer's disease (AD) and type 2 diabetes mellitus (T2D). Insulin resistance in the brain is a common feature in patients with T2D and AD. KAT7 is a histone acetyltransferase that participates in the modulation of various genes. To determine the effects of KAT7 on insulin patients with AD. APPswe/PS1-dE9 double-transgenic and db/db mice were used to mimic AD and diabetes, respectively. An in vitro model of AD was established by Aβ stimulation. Insulin resistance was induced by chronic stimulation with high insulin levels. The expression of microtubule-associated protein 2 (MAP2) was assessed using immunofluorescence. The protein levels of MAP2, Aβ, dual-specificity tyrosine phosphorylation-regulated kinase-1A (DYRK1A), IRS-1, p-AKT, total AKT, p-GSK3β, total GSK3β, DYRK1A, and KAT7 were measured via western blotting. Accumulation of reactive oxygen species (ROS), malondialdehyde (MDA), and SOD activity was measured to determine cellular oxidative stress. Flow cytometry and CCK-8 assay were performed to evaluate neuronal cell death and proliferation, respectively. Relative RNA levels of KAT7 and DYRK1A were examined using quantitative PCR. A chromatin immunoprecipitation assay was conducted to detect H3K14ac in DYRK1A. KAT7 expression was suppressed in the AD mice. Overexpression of KAT7 decreased Aβ accumulation and MAP2 expression in AD brains. KAT7 overexpression decreased ROS and MDA levels, elevated SOD activity in brain tissues and neurons, and simultaneously suppressed neuronal apoptosis. KAT7 upregulated levels of p-AKT and p-GSK3β to alleviate insulin resistance, along with elevated expression of DYRK1A. KAT7 depletion suppressed DYRK1A expression and impaired H3K14ac of DYRK1A. HMGN1 overexpression recovered DYRK1A levels and reversed insulin resistance caused by KAT7 depletion. We determined that KAT7 overexpression recovered insulin sensitivity in AD by recruiting HMGN1 to enhance DYRK1A acetylation. Our findings suggest that KAT7 is a novel and promising therapeutic target for the resistance in AD.

Prognosis significance and potential association between ALDOA and AKT expression in colorectal cancer.

Colorectal cancer (CRC) is one of the most common malignant tumors of the digestive tract and a leading cause of cancer-related death worldwide. Since many CRC patients are diagnosed already in the advanced stage, and traditional chemoradiotherapy is prone to drug resistance, it is important to find new therapeutic targets. In this study, the expression levels of ALDOA and p-AKT were detected in cancer tissues and paired normal tissues, and it was found that they were significantly increased in CRC tissues, and their high expression indicated poor prognosis. Moreover, a positive correlation between the expression of ALDOA and p-AKT was found in CRC tissues and paired normal tissues. In addition, the Kaplan-Meier analysis revealed that the group with both negative of ALDOA/p-AKT expression had longer five-year survival rates compared with the other group. Besides, the group with both high expression of ALDOA/p-AKT had a worse prognosis compared with the other group. Based on the expression of ALDOA and p-AKT in tumor tissues, we can effectively distinguish tumor tissues from normal tissues through cluster analysis. Furthermore, we constructed nomograms to predict 3-year and 5-year overall survival, showing that the expression of ALDOA/p-AKT plays a crucial role in predicting the prognosis of CRC patients. Therefore, ALDOA/p-AKT may act as a crucial role in CRC, which may provide new horizons for targeted therapies for CRC.

Isoquercitrin alleviates pirarubicin-induced cardiotoxicity in vivo and in vitro by inhibiting apoptosis through Phlpp1/AKT/Bcl-2 signaling pathway.

Introduction: Due to the cardiotoxicity of pirarubicin (THP), it is necessary to investigate new compounds for the treatment of THP-induced cardiotoxicity. Isoquercitrin (IQC) is a natural flavonoid with anti-oxidant and anti-apoptosis properties. Thus, the present study aimed to investigate the influence of IQC on preventing the THP-induced cardiotoxicity in vivo and in vitro. Methods: The optimal concentration and time required for IQC to prevent THP-induced cardiomyocyte damage were determined by an MTT assay. The protective effect was further verified in H9c2 and HCM cells using dichlorodihydrofluorescein diacetate fluorescent probes, MitoTracker Red probe, enzyme-linked immunosorbent assay, JC-1 probe, and real time-quantitative polymerase chain reaction (RT-qPCR). Rats were administered THP to establish cardiotoxicity. An electrocardiogram (ECG) was performed, and cardiac hemodynamics, myocardial enzymes, oxidative stress indicators, and hematoxylin-eosin staining were studied. Voltage-dependent anion channel 1 (VDAC1), adenine nucleotide translocase 1 (ANT1), and cyclophilin D (CYPD) were detected by qRT-PCR, and the Phlpp1/AKT/Bcl-2 axis proteins were detected by western blot, confirming that IQC markedly increased cell viability and superoxide dismutase (SOD) levels, diminished the levels of ROS and MDA, and elevated mitochondrial function and apoptosis in vivo and in vitro. Results: Results showed that IQC reduced THP-induced myocardial histopathological injury, electrocardiogram (ECG) abnormalities, and cardiac dysfunction in vivo. IQC also decreased serum levels of MDA, BNP, CK-MB, c-TnT, and LDH, while increasing levels of SOD and GSH. We also found that IQC significantly reduced VDAC1, ANT1, and CYPD mRNA expression. In addition, IQC controlled apoptosis by modulating Phlpp1/AKT/Bcl-2 signaling pathways. IQC markedly increased H9c2 and HCM cell viability and SOD levels, diminished the levels of ROS and MDA, and elevated mitochondrial function in H9c2 and HCM cells to defend against THP-induced cardiomyocyte apoptosis in vitro. The AKT inhibitor IMQ demonstrated that IQC lacked antioxidant and anti-apoptotic properties. Moreover, our data showed that IQC regulates Phlpp1 expression, thereby influencing the expression levels of p-AKT, cytochrome c, caspase-3, caspase-9, Bcl-2, and Bax. Discussion: In conclusion, our results indicate that IQC protects the changes in mitochondrial membrane permeability in cardiomyocytes by regulating the Phlpp1/AKT/Bcl-2 signaling pathway, inhibits the release of cytc from the mitochondrial inner membrane to the cytoplasm, forms apoptotic bodies, induces cell apoptosis, and reduces THP induced cardiotoxicity.

Liraglutide Attenuates Diabetic Cardiomyopathy via the ILK/PI3K/AKT/PTEN Signaling Pathway in Rats with Streptozotocin-Induced Type 2 Diabetes Mellitus.

One of the possible candidates for the treatment of diabetic cardiomyopathy is liraglutide, a glucagon-like peptide-1 receptor (GLP1R) agonist. In this study, the impacts of liraglutide on the integrin-linked kinase (ILK)-related PI3K/AKT axis in rats with type 2 diabetes induced via streptozotocin were examined. Twenty-four Wistar albino rats were distributed in four different groups, and a high-fat diet and streptozotocin were used to induce type 2 in two groups. Rats in the untreated control groups were administered 0.9% NaCl solution over a 6-week period, and those in the treatment groups were administered 0.9% NaCl for 3 weeks, followed by subcutaneous injection of liraglutide (150 μg/kg) for an additional 3 weeks. In the liraglutide-treated diabetic group, the heart-to-body weight ratio was significantly reduced, levels of cardiac biomarkers, troponin I and creatine-kinase-MB, were improved; activities of antioxidant enzymes, glutathione peroxidase and superoxide dismutase, were increased; and levels of malondialdehyde were decreased. Western blotting and immunohistochemical studies revealed increased levels of ILK, P-PI3K, P-AKT, and BCL2, as well as those of caspase 3, BAX, and P-PTEN, indicating mitigation of cardiomyocyte apoptosis. Our results show that liraglutide, by targeting GLP1Rs, enhances the expression of proteins in the ILK/PI3K/AKT/PTEN pathway and thereby exerts its cardioprotective effects in rats with DCM.

Targeting CAMK2N1/CAMK2 inhibits invasion, migration and angiogenesis of non-small cell lung cancer by promoting autophagy and apoptosis via AKT/mTOR signaling pathway

Deregulation of calcium/calmodulin-dependent protein kinase II (CAMK2) inhibitor 1 (CAMK2N1) has been reported to be associated with the development of several malignancies. To date, there have been few studies on the role of CAMK2N1 in lung cancer. This study aimed to investigate the relationship between CAMK2N1 and the progression of non-small cell lung cancer (NSCLC). Methodological quality was assessed using the ARRIVE guidelines. CAMK2N1 was expressed at low levels in NSCLC tissues. Overexpression of CAMK2N1 in NSCLC cell lines resulted in changes such as proliferation inhibition, metastasis inhibition, autophagy increase, and apoptosis. Mechanistic studies revealed the regulatory role of CAMK2N1/CAMK2 in AKT/mTOR signaling. Upregulation of CAMK2N1 decreased the expression levels of phosphorylated calmodulin kinase 2 (p-CaMK2), phosphorylated Akt (p-Akt), and phosphorylated-mTOR (p-mTOR). In contrast, CAMK2 overexpression increased p-AKT and p-mTOR levels. Inhibition of autophagy or activation of AKT signaling reduced CAMK2N1-mediated tumor suppression. The tumorigenic ability of CAMK2N1 overexpressing cells significantly diminished in nude mice. In conclusion, this study demonstrated the cancer suppressive function of CAMK2N1 in NSCLC and showed that CAMK2N1/CAMK2 exerted anti-cancer effects by inhibiting the AKT/mTOR signaling pathway to promote autophagy.

High expression of CD9 and Epidermal Growth Factor Receptor promotes the development of tongue cancer.

Tongue cancer is distinguished by aggressive behavior, a high risk of recurrence, lymph, and distant metastases. Hypoxia-Induced Factor 1 α functions as a CD9 transcription factor. CD9 is a transmembrane protein that may be found on the cell membrane. It can modulate the expression of the Epidermal Growth Factor Receptor (EGFR) pathway. ELISA was used to measure serum CD9, p-EGFR, and p-Akt levels in 70 tongue cancer patients and 35 healthy controls. RT-PCR was used to analyze the gene expression of the related genes. The gene as well as protein expression of CD9, EGFR/p-EGFR, and Akt/p-Akt was significantly higher in case subjects when compared with the controls. The expression of CD9 was higher in case subjects who were smokers/alcoholics when to control subjects who were smokers/alcoholics. Overexpression of CD9 due to hypoxic conditions leads to the activation of EGFR-signaling pathway resulting in cancer progression, resistance to chemotherapy. Hence, CD9 could be a potential target to suppress cancer progression.