Capivasertib combines with docetaxel to enhance anti-tumour activity through inhibition of AKT-mediated survival mechanisms in prostate cancer.

BACKGROUND/OBJECTIVE: To explore the anti-tumour activity of combining AKT inhibition and docetaxel in PTEN protein null and WT prostate tumours. METHODS: Mechanisms associated with docetaxel capivasertib treatment activity in prostate cancer were examined using a panel of in vivo tumour models and cell lines. RESULTS: Combining docetaxel and capivasertib had increased activity in PTEN null and WT prostate tumour models in vivo. In vitro short-term docetaxel treatment caused cell cycle arrest in the majority of cells. However, a sub-population of docetaxel-persister cells did not undergo G2/M arrest but upregulated phosphorylation of PI3K/AKT pathway effectors GSK3ß, p70S6K, 4E-BP1, but to a lesser extent AKT. In vivo acute docetaxel treatment induced p70S6K and 4E-BP1 phosphorylation. Treating PTEN null and WT docetaxel-persister cells with capivasertib reduced PI3K/AKT pathway activation and cell cycle progression. In vitro and in vivo it reduced proliferation and increased apoptosis or DNA damage though effects were more marked in PTEN null cells. Docetaxel-persister cells were partly reliant on GSK3ß as a GSK3ß inhibitor AZD2858 reversed capivasertib-induced apoptosis and DNA damage. CONCLUSION: Capivasertib can enhance anti-tumour effects of docetaxel by targeting residual docetaxel-persister cells, independent of PTEN status, to induce apoptosis and DNA damage in part through GSK3ß.

CDK7 and CDK9 inhibition interferes with transcription, translation, and stemness, and induces cytotoxicity in GBM irrespective of temozolomide sensitivity.

BACKGROUND: Glioblastoma (GBM) is refractory to current treatment modalities while side effects of treatments result in neurotoxicity and cognitive impairment. Here we test the hypothesis that inhibiting CDK7 or CDK9 would effectively combat GBM with reduced neurotoxicity. METHODS: We examined the effect of a CDK7 inhibitor, THZ1, and multiple CDK9 inhibitors (SNS032, AZD4573, NVP2, and JSH150) on GBM cell lines, patient-derived temozolomide (TMZ)-resistant and responsive primary tumor cells and glioma stem cells (GSCs). Biochemical changes were assessed by western blotting, immunofluorescence, multispectral imaging, and RT-PCR. In vivo, efficacy was assessed in orthotopic and subcutaneous xenograft models. RESULTS: CDK7 and CDK9 inhibitors suppressed the viability of TMZ-responsive and resistant GBM cells and GSCs at low nanomolar concentrations, with limited cytotoxic effects in vivo. The inhibitors abrogated RNA Pol II and p70S6K phosphorylation and nascent protein synthesis. Furthermore, the self-renewal of GSCs was significantly reduced with a corresponding reduction in Sox2 and Sox9 levels. Analysis of TCGA data showed increased expression of CDK7, CDK9, SOX2, SOX9, and RPS6KB1 in GBM; supporting this, multispectral imaging of a TMA revealed increased levels of CDK9, Sox2, Sox9, phospho-S6, and phospho-p70S6K in GBM compared to normal brains. RNA-Seq results suggested that inhibitors suppressed tumor-promoting genes while inducing tumor-suppressive genes. Furthermore, the studies conducted on subcutaneous and orthotopic GBM tumor xenograft models showed that administration of CDK9 inhibitors markedly suppressed tumor growth in vivo. CONCLUSIONS: Our results suggest that CDK7 and CDK9 targeted therapies may be effective against TMZ-sensitive and resistant GBM.

Targeting the AKT/mTOR/p70S6K Pathway for Oligodendrocyte Differentiation and Myelin Regeneration in Neurological Disorders.

BACKGROUND: The AKT/mTOR/p70S6K pathway has been shown to potentially promote spinal cord injury (SCI) repair in rats. However, its exact mechanism and beyond needs to be further explored. OBJECTIVE: This study aims to explore the AKT/mTOR/p70S6K pathway in oligodendrocyte precursor cell (OPC) differentiation, microglial polarization differentiation, and the role of these in myelin regeneration in vitro. METHODS: The isolation, induction and characterization of rat primary neuronal stem cells, OPCs and oligodendrocytes were investigated with immunofluorescence and RT-qPCR. Then, the role of AKT/mTOR/p70S6K signaling was explored using western blotting and immunofluorescence, the effect on myelination was examined with OPC-dorsal root ganglion (DRG) neurons co-culture, and the influence of M1/M2 polarization status of microglia on myelin formation was also observed by adding M1/M2 supernatants into OPC-DRG neurons co-culture. RESULTS: Activation of the AKT/mTOR/p70S6K pathway elevated the expression of oligodendrocyte differentiation markers, including MBP, PLP and MOG, which also promoted the colocalization of MBP and NFH in OPC-DRG neurons co-culture. More interestingly, stimulation of the AKT/mTOR/p70S6K pathway facilitated M2 polarization of rat microglia. M2 polarization of microglia enhanced OPC differentiation to oligodendrocytes and myelin formation. CONCLUSION: Our findings highlight the potential of targeting the AKT/mTOR/p70S6K pathway in promoting oligodendrocyte differentiation and myelin regeneration in neurological disorders such as SCI.

ABBV-744 induces autophagy in gastric cancer cells by regulating PI3K/AKT/mTOR/p70S6k and MAPK signaling pathways.

The mortality rates of gastric cancer remain high due to limited therapeutic strategies. As a highly selective inhibitor of the BD2 domain of BET family proteins, ABBV-744 has potent chemotherapeutic activity against various human solid tumors. However, whether ABBV-744 has potential anti-tumor effects in gastric cancer remain largely unknown. In this study, we evaluated the effect of ABBV-744 on gastric cancer cells and explored the possible underlying mechanisms. We found that ABBV-744 inhibited the growth of gastric cancer cells and patient-derived tumor organoids in a dose-dependent manner. Cellular experiments revealed that ABBV-744 induced mitochondria damage, reactive oxygen species accumulation, cell cycle arrest and apoptotic cell death in gastric cancer cells. Transcriptomic analysis using RNA-sequencing data identified autophagy as a crucial pathway involved in the cell death caused by ABBV-744. Mechanically, further studies showed that ABBV-744 induced autophagy flux in gastric cancer cells by inactivating PI3K/AKT/mTOR/p70S6k and activating the MAPK signaling pathways. In vivo mouse xenograft studies demonstrated that ABBV-744 significantly suppressed the growth of gastric cancer cells via inducing autophagy. Taken together, our results suggest that ABBV-744 is a novel drug candidate for gastric cancer.

Scaffold hopping of N-benzyl-3,4,5-trimethoxyaniline: 5,6,7-Trimethoxyflavan derivatives as novel potential anticancer agents modulating hippo signaling pathway.

Scaffold hopping of N-benzyl-3,4,5-trimethoxyaniline afforded 5,6,7-trimethoxyflavan derivatives that were efficiently synthesized in four linear steps. As lung cancer is the most lethal cancer, twenty-three synthesized compounds were evaluated against a panel of lung cancer cells. Amongst, compounds 8q and 8e showed interesting activity. Hence, compounds 8q and 8e were evaluated against panels of diverse cancers. Compounds 8q and 8e showed broad spectrum anticancer activity. However, compound 8q was more effective and, hence, was advanced for potency evaluation and characterization. Compound 8q showed comparable potencies to gefitinib, and oxaliplatin against lung and colorectal cancers, respectively, and superior potencies to temozolomide, dacarbazine, cisplatin, enzalutamide, methotrexate, imatinib against brain, skin, ovary, prostate, breast, and blood cancers, respectively. Compound 8q increased cleaved PARP, caspase 3, and 7 inducing apoptosis. In addition, it inhibited cyclins A, B1, H and cdc25c, and increased p53 triggering cell cycle arrest in G2/M phase. Moreover, it decreased YAP and increased LATS1 and p-mob1/mob1 activating hippo signaling. Furthermore, it decreased p-PI3K/PI3k, p-mTOR/mTOR and p-P70S6K/P70S6K inhibiting PI3k pathway. Together, these findings present compound 8q as a potential anticancer lead compound for further development of potential agents.

Diosmin nanocrystal gel alleviates imiquimod-induced psoriasis in rats via modulating TLR7,8/NF-κB/micro RNA-31, AKT/mTOR/P70S6K milieu, and Tregs/Th17 balance.

Diosmin is a flavonoid with promising anti-inflammatory and antioxidant properties. However, it has difficult physicochemical characteristics since its solubility demands a pH level of 12, which has an impact on the drug's bioavailability. The aim of this work is the development and characterization of diosmin nanocrystals using anti-solvent precipitation technique to be used for topical treatment of psoriasis. Results revealed that diosmin nanocrystals stabilized with hydroxypropyl methylcellulose (HPMC E15) in ratio (diosmin:polymer; 1:1) reached the desired particle size (276.9 ± 16.49 nm); provided promising colloidal properties and possessed high drug release profile. Additionally, in-vivo assessment was carried out to evaluate and compare the activities of diosmin nanocrystal gel using three different doses and diosmin powder gel in alleviating imiquimod-induced psoriasis in rats and investigating their possible anti-inflammatory mechanisms. Herein, 125 mg of 5% imiquimod cream (IMQ) was applied topically for 5 consecutive days on the shaved backs of rats to induce psoriasis. Diosmin nanocrystal gel especially in the highest dose used offered the best anti-inflammatory effect. This was confirmed by causing the most statistically significant reduction in the psoriasis area severity index (PASI) score and the serum inflammatory cytokines levels. Furthermore, it was capable of maintaining the balance between T helper (Th17) and T regulatory (Treg) cells. Moreover, it tackled TLR7/8/NF-κB, miRNA-31, AKT/mTOR/P70S6K and elevated the TNFAIP3/A20 (a negative regulator of NF-κB) expression in psoriatic skin tissues. This highlights the role of diosmin nanocrystal gel in tackling imiquimod-induced psoriasis in rats, and thus it could be a novel promising therapy for psoriasis.

Effects of exercise training on glucocorticoid-induced muscle atrophy: Literature review.

Glucocorticoids (GCs) administration, such as cortisol acetate (CA) and dexamethasone (DEXA), is used worldwide due to their anti-inflammatory, anti-allergic, and immunosuppressive properties. However, muscle atrophy is one of the primary deleterious induced responses from the chronic treatment with GCs since it stimulates muscle degradation inhibiting muscle protein synthesis. Animal models allow a better understanding of the molecular pathways involved in this process of gene modulation and production of hypertrophic and atrophic proteins. The treatment with GCs, such as DEXA, promotes the reduction of hypertrophic proteins such as serine/threonine tyrosine kinase (AKT), protein kinase mammalian target of rapamycin (mTOR), and ribosomal protein S6 kinase (p70S6K) and increased gene expression or production of atrophic proteins, such as myostatin, muscle atrophic F-box (atrogin-1), or muscle ring finger protein-1 (MuRF-1). In both continuous exercise (CE) and resistance exercise (RE) forms, exercise training is used to mitigate muscle atrophy induced by GCs. The CE attenuated muscle atrophy induced by CA or DEXA in the plantaris and extensor digitorum longus muscles, while RE mitigated the DEXA-induced atrophy in plantaris and flexor hallucis longus muscles. The RE response appears to have occurred by modulation of hypertrophic proteins through increased protein production or phosphorylated/total ratio of mTOR and p70S6K and decreased atrophic protein production of MuRF-1. CE needs future research to understand the molecular pathways of its protective response.

Antidepressant-like effect of CP-101,606: Evidence of mTOR pathway activation.

BACKGROUND: As a non-competitive N-methyl d-aspartate receptor antagonist, ketamine exerts rapid-onset and long-lasting antidepressant effects on depression, but some side effects limit its use. To identify a safer compound that may provide similar antidepressant effects, here we investigated whether CP-101,606, a selective NR2B receptor inhibitor, provides similar antidepressant effects and explored its underlying mechanisms. METHODS: To mimic depressive-like behavior, mice were subjected to chronic unpredictable mild stress (CUMS) for 21 days. Mice were treated with CP-101,606 at 10, 20, and 40 mg/kg doses for 7, 14, and 21 days, respectively, followed by a sucrose preference test (SPT), tail suspension test (TST), and forced swimming test (FST). Western blot analysis was performed on several targets (mTOR, p-mTOR, p70S6K, p-p70S6K, PSD-95, and GluA1), along with immunohistochemistry (GluA1) and immunofluorescence (p-mTOR) assays, using hippocampal tissue. RESULTS: CP-101,606 at 20 and 40 mg/kg doses for 7 and 14 days and fluoxetine 10 mg/kg and CP-101606 20 mg/kg for 21 days ameliorated depression-like behaviors in the SPT, TST, and FST. The effects of CP-101,606 were associated with a reversal of the CUMS-induced decrease in mTOR (Ser2448) and p70S6K (Thr389) phosphorylation and increasing PSD95 and GluA1 synthesis in the hippocampus. CONCLUSIONS: Our results demonstrate that CP-101,606 produces antidepressant effects in CUMS mice, which may be mediated by mTOR signaling cascade upregulation. Our findings suggest the possible utility of CP-101,606 as a treatment for depression.

Effect of rapamycin treatment in human seminoma TCam-2 cells through inhibition of G1-S transition.

Mammalian target of rapamycin (mTOR) is an important serine/threonine kinase that plays a critical role in several processes including cell cycle, protein synthesis, and energy metabolism. Due to its multiple roles and general dysregulation in cancer, the mTOR pathway is an important target in cancer therapy. However, studies on mTOR activity in seminoma are limited. Therefore, our aim was to investigate the expression of mTOR signaling pathway proteins in the TCam-2 cell line after rapamycin treatment. TCam-2 cells were treated with different concentrations of rapamycin (control (no rapamycin treatment), 4 nM, 20 nM, 100 nM, 500 nM, and 1000 nM rapamycin) for 48 h and 72 h. mTOR, p-mTOR, P70S6K, p-P70S6K, proliferating cell nuclear antigen (PCNA), and caspase-3 expression levels were analyzed by western blot. Apotosis and cell cycle were analyzed by flow cytometry. After 48 h of rapamycin administration, mTOR activity was significantly decreased at 1000 nM (p < 0.05). In addition, P70S6K acitivity significantly decreased in groups at all rapamycin concentrations (***p < 0.001, ****p < 0.0001). After 72 h of rapamycin administration, mTOR pathway activity were significantly decreased at 100, 500, and 1000 nM rapamycin-treated groups (p < 0.05). Moreover, P70S6K expression decreased in all treatment groups (****p < 0.0001). Caspase-3 expression were similar in all groups. While PCNA expression tended to decrease at 48 h in a dose-dependent manner, this decrease was not significant. We detected decreased PCNA expression at 1000 nM rapamycin at 72 h (p < 0.05). The rate of apoptosis increased especially at 1000 nM rapamycin at 72 h (***p < 0.001). On the other hand, according to the results of the cell cycle experiment, G1 phase arrest was detected at all rapamycin doses at 48 and 72 h (***p < 0.001). Our study indicated that 1000 nM rapamycin may inhibit TCam-2 seminoma cells growth by halting cell proliferation through inhibition of G1-S transition. Therefore, we believe that the findings obtained will contribute to the development of new treatment approaches for seminoma patients in the future and in the process of restoring testicular functions and preserving fertility.

Brucein D imparts a growth inhibitory effect in multiple myeloma cells by abrogating the Akt-driven signaling pathway.

The Akt signaling pathway is an oncogenic cascade activated in the bone marrow microenvironment of multiple myeloma (MM) cells and contributes to their uncontrolled proliferation. Abrogation of Akt signaling has been presented as one of the prime therapeutic targets in the treatment of MM. In the present report, we have investigated the effect of Brucein D (BD) on Akt-driven signaling events in MM cells. BD (300 nM) substantially inhibited cell viability and imparted growth-inhibitory effects in U266 cells as evidenced by cell viability assays and flow cytometric analysis. Effect of BD on cell viability was evaluated by MTT assay. Apoptotic cells and cell cycle arrest by BD were analyzed by flow cytometer. The results of the TUNEL assay and western blotting showed that BD induces apoptosis of MM cells by activating caspase-8 and 9 with subsequent reduction in the expression of antiapoptotic proteins (Bcl-2, Bcl-xl, survivin, cyclin D1, COX-2, VEGF, MMP-9). Analysis of activated kinases by Phospho-Kinase Array Kit revealed that Akt, p70S6K, HSP60, p53, and WNK1 were strongly expressed in untreated cells and BD treatment reversed this effect. Using transfection experiments, AKT depletion led to a decrease in phosphorylation of Akt, mTOR, p70S6K, and WNK. However, Akt overexpression led to increase in phosphorylation of these proteins. Depletion of Akt potentiated the apoptosis-inducing effect of BD whereas overexpression displayed resistance to BD-induced apoptosis suggesting the role of Akt in chemoresistance. Taken together, BD mitigates Akt-dependent signaling pathways in MM cells to impart its anticancer activity.

[Effect of moxibustion on autophagy in mice with Alzheimer's disease based on mTOR/p70S6K signaling pathway].

OBJECTIVE: To investigate the effect of moxibustion on autophagy and amyloid ß-peptide1-42 (Aß1-42) protein expression in amyloid precursor protein/presenilin 1 (APP/PS1) double-transgenic mice with Alzheimer's disease (AD). METHODS: After 2-month adaptive feeding, fifty-six 6-month-old APP/PS1 double transgenic AD mice were randomly divided into a model group, a moxibustion group, a rapamycin group and an inhibitor group, 14 mice in each group. Another 14 C57BL/6J mice with the same age were used as a normal group. The mice in the moxibustion group were treated with monkshood cake-separated moxibustion at "Baihui"(GV 20), "Fengfu" (GV 16) and "Dazhui" (GV 14) for 20 min; the mice in the rapamycin group were intraperitoneally injected with rapamycin (2 mg/kg); the mice in the inhibitor group were treated with moxibustion and injection of 1.5 mg/kg 3-methyladenine (3-MA). All the treatments were given once a day for consecutive 2 weeks. The morphology of hippocampal tissue was observed by HE staining; the ultrastructure of hippocampal tissue was observed by transmission electron microscopy; the expression of Aß1-42 protein in frontal cortex and hippocampal tissue was detected by immunohistochemistry; the expressions of mammalian target of rapamycin (mTOR), phosphorylated mTOR (p-mTOR), p70 ribosomal protein S6 kinase (p70S6K) and phosphorylated p70S6K (p-p70S6K) protein in hippocampus were detected by Western blot method. RESULTS: Compared with the normal group, the number of neuron cells was decreased, cells were necrotic and deformed, and autophagy vesicle and lysosome were decreased in the model group. Compared with the model group, the number of neuron cells was increased, cell necrosis was decreased, and autophagy vesicle and lysosome were increased in the moxibustion group and the rapamycin group. Compared with the normal group, the protein expressions of Aß1-42, mTOR, p-mTOR, p70S6K and p-p70S6K in the model group were increased (P<0.05); compared with the model group, the protein expressions of Aß1-42, mTOR, p-mTOR, p70S6K and p-p70S6K in the moxibustion group, rapamycin group and inhibitor group were decreased (P<0.05); compared with the inhibitor group, the protein expressions of Aß1-42, mTOR, p-mTOR, p70S6K and p-p70S6K in the moxibustion group and rapamycin group were decreased (P<0.05); compared with the rapamycin group, the protein expressions of mTOR, p-mTOR, p70S6K and p-p70S6K in the moxibustion group were decreased (P<0.05). CONCLUSION: Moxibustion could enhance autophagy in hippocampal tissue of APP/PS1 double transgenic AD mice and reduce abnormal Aß aggregation in brain tissue, the mechanism may be related to the inhibition of mTOR/p70S6K signaling pathway.

Urolithin A attenuates severity of chronic pancreatitis associated with continued alcohol intake by inhibiting PI3K/AKT/mTOR signaling.

Heavy alcohol consumption is the dominant risk factor for chronic pancreatitis (CP); however, treatment and prevention strategies for alcoholic chronic pancreatitis (ACP) remains limited. The present study demonstrates that ACP induction in C57BL/6 mice causes significant acinar cell injury, pancreatic stellate cell (PSC) activation, exocrine function insufficiency, and an increased fibroinflammatory response when compared with alcohol or CP alone. Although the withdrawal of alcohol during ACP recovery led to reversion of pancreatic damage, continued alcohol consumption with established ACP perpetuated pancreatic injury. In addition, phosphokinase array and Western blot analysis of ACP-induced mice pancreata revealed activation of the phosphatidylinositol 3 kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) and cyclic AMP response element binding protein (CREB) signaling pathways possibly orchestrating the fibroinflammatory program of ACP pathogenesis. Mice treated with urolithin A (Uro A, a gut-derived microbial metabolite) in the setting of ACP with continued alcohol intake (during the recovery period) showed suppression of AKT and P70S6K activation, and acinar damage was significantly reduced with a parallel reduction in pancreas-infiltrating macrophages and proinflammatory cytokine accumulation. These results collectively provide mechanistic insight into the impact of Uro A on attenuation of ACP severity through suppression of PI3K/AKT/mTOR signaling pathways and can be a useful therapeutic approach in patients with ACP with continuous alcohol intake.NEW & NOTEWORTHY Our novel findings presented here demonstrate the utility of Uro A as an effective therapeutic agent in attenuating alcoholic chronic pancreatitis (ACP) severity with alcohol continuation after established disease, through suppression of the PI3K/AKT/mTOR signaling pathway.

Neuroprotection of Triptolide against Amyloid-Beta1-42-induced toxicity via the Akt/mTOR/p70S6K-mediated Autophagy Pathway.

Triptolide is a natural active compound that has significant neuroprotective properties and shows promising effects in the treatment of Alzheimer's disease (AD). Recent studies have shown that autophagy occurs in AD. In this study, we determined whether autophagy regulated by triptolide ameliorates neuronal death caused by amyloid-Beta1-42 (Aß1-42). We examined the effects of triptolide on cell viability, autophagy, apoptosis, and the protein kinase B/mammalian target of the rapamysin/70 kDa ribosomal protein S6 kinase (Akt/mTOR/p70S6K) signaling pathway in PC12 cells. The results indicated that triptolide treatment exhibited a cytoprotective effect against cell injury induced by Aß1-42. Triptolide also reduced apoptosis and enhanced cell survival by decreasing autophagosome accumulation and inducing autophagic degradation. Furthermore, our results also showed that activating the Akt/mTOR/p70S6K mechanism was one reason for the protection of triptolide. Triptolide treatment protected against Aß1-42-induced cytotoxicity by decreasing autophagosome accumulation, and inducing autophagic degradation in PC12 cells. These findings also suggest that the reduction of autophagosome accumulation observed in triptolide-treated cells was Akt/mTOR/p70S6K pathway dependent. Overall, triptolide exhibits a neuron protective effect and this study provides new insight into AD prevention and treatment.

[Protective effect of salidroside on high fat-induced apoptosis in H9c2 cardiomyocytes through AMPK/mTOR/p70S6K pathway].

The study explored the effect of salidroside(SAL) on high fat-induced apoptosis in H9 c2 cardiomyocytes based on AMPK/mTOR/p70 S6 K pathway.H9 c2 cardiomyocytes were cultured in vitro and the lipotoxicity model of H9 c2 cardiomyocytes was constructed by 0.2 mmol·L~(-1) palmitic acid(PA) treatment for 24 hours.The cells were divided into control group, PA group, and SAL group(20 µmol·L~(-1)).Cell proliferation was detected with cell proliferation kit I(MTT) assay after SAL and PA treatment.Dihydroethidium(DHE) probe, Annexin V-FITC/PI kit, and JC-1 probe were used to estimate reactive oxygen species(ROS) level, cell apoptosis, and mitochondrial membrane potential(MMP) change, respectively.The expression levels of p-AMPK/AMPK, p-mTOR/mTOR, p-p70 S6 K/p70 S6 K and apoptosis-related proteins Bax, Bcl-2, and cleaved caspase-3 were investigated with Western blot.The mRNA levels of AMPK, mTOR and p70 S6 K were determined by quantitative reverse transcription-polymerase chain reaction(qRT-PCR).RESULTS:: showed that compared with control group, PA group had decreased cell proliferation ability, MMP, Bcl-2 protein expression and AMPK protein and mRNA expression, while increased ROS level, Bax and cleaved caspase-3 protein expression, and mTOR and p70 S6 K mRNA and protein expression, and the difference was statistically significant(P<0.05, P<0.01).Compared with PA group, SAL improved cell proliferation ability, MMP level, Bcl-2 protein expression, and AMPK mRNA and protein expression, while down-regulated ROS level, cell apoptosis, Bax and cleaved caspase-3 protein expression, and mTOR and p70 S6 K mRNA and protein expression, and the difference was statistically significant(P<0.05, P<0.01).In conclusion, SAL exerted protective effects on high fat-induced lipotoxicity of H9 c2 cardiomyocytes, alleviated the oxidative stress injury and reduced cell apoptosis via regulating AMPK/mTOR/p70 S6 K signaling pathway.

Baicalin Induced Apoptosis of Human Cholangiocarcinoma Cell through Activating AMPK/mTORC1/p70S6K Signaling Pathway.

Baicalin (naturally bioactive flavone compound isolated from Scutellaria baicalensis) has been demonstrated to exert strong anticancer activity against various tumor cells. However, the possibility of using baicalin for the treatment of cholangiocarcinoma and its effectiveness remain unstudied. The effect of baicalin on QBC939 cholangiocarcinoma cell culture was studied by assessing cell viability (CCK-8 test) and expression of the key proteins (Western blotting). Baicalin induced apoptosis of QBC939 cells in culture in a dose- and time-dependent manner. The proapoptotic effect was attributed to inhibition of the mTORC1-p70S6K signaling pathway resulting from baicalin-induced AMPK activation. These findings provide a new approach for cholangiocarcinoma treatment and serve as a basis for developing baicalin-based combination cancer therapy strategies.

Therapeutic efficacy of the novel SHP2 degrader SHP2-D26, alone or in combination, against lung cancer is associated with modulation of p70S6K/S6, Bim and Mcl-1.

SHP2, a protein tyrosine phosphatase, plays a critical role in fully activating oncogenic signaling pathways such as Ras/MAPK downstream of cell surface tyrosine receptors (e.g., EGFR), which are often activated in human cancers, and thus has emerged as an attractive cancer therapeutic target. This study focused on evaluating the therapeutic potential of the novel SHP2 degrader, SHP2-D26 (D26), either alone or in combination, against non-small cell lung cancer (NSCLC) cells. While all tested NSCLC cell lines responded to D26 with IC50s of < 8 µM, a few cell lines (4/14) were much more sensitive than others with IC50s of ≤ 4 µM. There was no clear association between basal levels of SHP2 and cell sensitivities to D26. Moreover, D26 rapidly and potently decreased SHP2 levels in different NSCLC cell lines in a sustained way regardless of cell sensitivities to D26, suggesting that additional factors may impact cell response to D26. We noted that suppression of p70S6K/S6, but not ERK1/2, was associated with cell responses to D26. In the sensitive cell lines, D26 effectively increased Bim levels while decreasing Mcl-1 levels accompanied with the induction of apoptosis. When combined with the third generation EGFR inhibitor, osimertinib (AZD9291), synergistic effects on decreasing the survival of different osimertinib-resistant cell lines were observed with enhanced induction of apoptosis. Although D26 alone exerted moderate inhibition of the growth of NSCLC xenografts, the combination of osimertinib and D26 effectively inhibited the growth of osimertinib-resistant xenografts, suggesting promising efficacy in overcoming acquired resistance to osimertinib.

The GLUT1-mTORC1 axis affects odontogenic differentiation of human dental pulp stem cells.

Human dental pulp stem cells (hDPSCs) are considered valuable for regenerative therapy. Although glucose transporter 1 (GLUT1) is known to play a critical role in cell differentiation, its mechanism of the odontogenic differentiation of hDPSCs remains unclear. This study was conducted to investigate the effect and underlying mechanisms of GLUT1 on odontogenic differentiation of hDPSCs. hDPSCs was treated with phloretin (Phl), a GLUT1 inhibitor. The impact of GLUT1 on the odontogenic differentiation of hDPSCs was analysed using quantitative real-time polymerase chain reaction, alizarin-red staining, and western blotting. Glucose uptake by hDPSCs was significantly inhibited by Phl treatment. Overall, inhibition of GLUT1 upregulated the expression of DSPP, DMP1, RUNX2, and OCN and increased the formation of mineralised nodules on odontogenic induction of hDPSCs. The levels of phosphorylated mTOR and ribosomal protein S6 kinase 1 (p70S6K) were increased after GLUT1 inhibition and decreased by an mTOR inhibitor (rapamycin, Rapa) during the odontogenic induction of hDPSCs. Moreover, mTOR suppression decreased the expression of the genes described above and formation of mineralised nodules. These results suggest that inhibition of GLUT1 promoted the odontogenic differentiation of hDPSCs via the mTORC1-p70S6K axis, providing a foundation for further application of hDPSCs in regenerative therapy.

Interleukin-10 regulates starvation-induced autophagy through the STAT3-mTOR-p70s6k axis in hepatic stellate cells.

The degree of activation of hepatic stellate cells (HSCs) is closely related to the level of autophagy in HSCs. We previously showed that interleukin-10 (IL-10) strongly inhibits HSC activation in rat fibrotic liver. However, little is known about the effect of IL-10 on HSC autophagy. For investigation of the effect of IL-10 on starvation-induced autophagy in immortal rat hepatic stellate cells (HSC-T6) and the molecular mechanism, HSC-T6 cells were incubated with serum-free DMEM for different periods and treated with IL-10 at different concentrations. Transmission electron microscopy (TEM), analysis of autophagic flux and Western blotting (WB) assays were used to observe changes in autophagosome morphology and number and autophagy-related protein expression in HSC-T6 cells and to evaluate the regulatory effect of IL-10 on starvation-induced autophagy. Cryptotanshinone (CPT) and rapamycin (Rapa) were used to block activation of the signal transducer and activator of transcription 3 (STAT3) and mTOR signaling pathways, respectively. STAT3-mTOR-p70s6k signaling pathway proteins were analyzed by WB to assess the signaling pathway by which IL-10 regulates autophagy. WB showed an increased LC3II/I ratio, increased Beclin1 expression, and decreased p62 expression in HSC-T6 cells starved for 3 h (p < 0.05). IL-10 inhibited the increases in the LC3II/I ratio and Beclin1 expression and upregulated p62 expression (p < 0.05), and the optimal IL-10 concentration was 20 ng/mL. TEM and double-labeled immunofluorescence analysis showed that IL-10 inhibited autophagosome formation and autophagic flux, as indicated by the decreased numbers of double-membrane autophagosomes and yellow autophagic puncta. Further examination of signaling pathway molecules showed that phosphorylation of the mTOR, STAT3, and p70s6k proteins was significantly decreased during starvation-induced autophagy, but IL-10 could increase mTOR, STAT3, and p70s6k protein phosphorylation (p < 0.05). Blocking either the mTOR or STAT3 pathway reversed the inhibitory effect of IL-10 on starvation-induced autophagy in HSC-T6 cells (p < 0.05). IL-10 suppresses starvation-induced autophagosome formation through activation of the STAT3-mTOR-p70s6k axis in HSC-T6 cells.

Low-frequency electroacupuncture improves disordered hepatic energy metabolism in insulin-resistant Zucker diabetic fatty rats via the AMPK/mTORC1/p70S6K signaling pathway.

BACKGROUND AND AIM: Disordered hepatic energy metabolism is found in obese rats with insulin resistance (IR). There are insufficient experimental studies of electroacupuncture (EA) for IR and type 2 diabetes mellitus (T2DM). The aim of this study was to probe the effect of EA on disordered hepatic energy metabolism and the adenosine monophosphate (AMP)-activated protein kinase (AMPK)/mammalian target of rapamycin complex 1 (mTORC1)/ribosomal protein S6 kinase, 70-kDa (p70S6K) signaling pathway. METHODS: Zucker Diabetic Fatty (ZDF) rats were randomly divided into three groups: EA group receiving EA treatment; Pi group receiving pioglitazone gavage; and ZF group remaining untreated (n = 8 per group). Inbred non-insulin-resistant Zucker lean rats formed an (untreated) healthy control group (ZL, n = 8). Fasting plasma glucose (FPG), fasting insulin (FINS), C-peptide, C-reactive protein (CRP) and homeostatic model assessment of insulin resistance (HOMA-IR) indices were measured. Hematoxylin-eosin (H&E) staining was used to investigate the liver morphologically. The mitochondrial structure of hepatocytes was observed by transmission electron microscopy (TEM). Western blotting was adopted to determine protein expression of insulin receptor substrate 1 (IRS-1), mTOR, mTORC1, AMPK, tuberous sclerosis 2 (TSC2) and p70S6K, and their phosphorylation. RT-PCR was used to quantify IRS-1, mTOR, mTORC1, AMPK and p70S6K mRNA levels. RESULTS: Compared with the ZF group, FPG, FINS, C-peptide, CRP and HOMA-IR levels were significantly reduced in the EA group (p < 0.05, p < 0.01). Evaluation of histopathology showed improvement in liver appearances following EA. Phosphorylation levels of AMPK, mTOR and TSC2 decreased, and IRS-1 and p70S6K increased, in hepatocytes of the ZF group, while these negative effects appeared to be alleviated by EA. CONCLUSIONS: EA can effectively ameliorate IR and regulate energy metabolism in the ZDF rat model. AMPK/mTORC1/p70S6K and related molecules may represent a potential mechanism of action underlying these effects.

Insulin growth factor-1 enhances proliferation and inhibits apoptosis of neural progenitor cells by phosphorylation of Akt/mTOR/p70S6K molecules and triggering intrinsic apoptosis signaling pathway.

Neural progenitor cells (NPCs) transplantation is known as a potential strategy for treating spinal cord injury (SCI). This study aimed to investigate effects of insulin growth factor-1 (IGF-I) on NPCs proliferation and clarify associated mechanisms. NPCs isolated from T8-T10 segmental spinal cord tissues of rats were cultured and identification. Then, lentivirus packing plasmids containing IGF-I was constructed and used for NPCs infection. Cell proliferation was evaluated by detecting 5-Bromodeoxyuridine (BrdU) expression in NPCs, cell differentiation was detected using double-labeling immunofluorescence staining while cell apoptosis was detected using TUNEL assay. In addition, the signal expression of Akt/mTOR/p70S6K in NPCs cells were investigated using immunofluorescence staining and western blot assay. The experimental group was defined as pCMV-IGF-I group, while the negative control group was defined as pCMV-LacZ group. Cells infected with pCMV-IGF-I lentivirus followed by addition of 100 mg/ml rapamycin were defined as pCMV-IGF-I + Rapa group. NPCs were successfully isolated, identified and cultured. IGF-I overexpression significantly inhibited cell apoptosis and enhanced cell migration. Akt/mTOR/ p70S6K signaling cascade was proved to be present in NPCs, IGF-I overexpression significantly activated Akt/mTOR/p70S6K signaling cascade, while rapamycin addition inhibited its expression. Also, the activated Akt/mTOR/p70S6K signal cascade induced by IGF-I significantly enhanced BrdU expression and inhibited cell apoptosis, and promoted the differentiation of NPC into the neuronal system. However, the rapamycin addition inhibited the cell response induced by IGF-I overexpression. IGF-I overexpression could enhance cell proliferation, inhibit cell apoptosis and promote their differentiation into neuronal systems by activating Akt/mTOR/p70S6K signaling cascade in vitro, indicating that the Akt/mTOR/p70S6K signaling cascade may be the potentially mechanism for the endogenous repair and remodeling of spinal cord after injury.