Osteosarcoma, which has poor prognosis after metastasis, is the most common type of bone cancer in children and adolescents. Therefore, plant-derived bioactive compounds are being actively developed for cancer therapy. Artemisia apiacea Hance ex Walp. is a traditional medicinal plant native to Eastern Asia, including China, Japan, and Korea. Vitexicarpin (Vitex), derived from A. apiacea, has demonstrated analgesic, anti-inflammatory, antitumour, and immunoregulatory properties; however, there are no published studies on Vitex isolated from the aerial parts of A. apiacea. Thus, this study aimed to evaluate the antitumour activity of Vitex against human osteosarcoma cells. In the present study, Vitex (>99% purity) isolated from A. apiacea induced significant cell death in human osteosarcoma MG63 cells in a dose- and time-dependent manner; cell death was mediated by apoptosis, as evidenced by the appearance of cleaved-PARP, cleaved-caspase 3, anti-apoptotic proteins (Survivin and Bcl-2), pro-apoptotic proteins (Bax), and cell cycle-related proteins (Cyclin D1, Cdk4, and Cdk6). Additionally, a human phosphokinase array proteome profiler revealed that Vitex suppressed AKT-dependent downstream kinases. Further, Vitex reduced the phosphorylation of PRAS40, which is associated with autophagy and metastasis, induced autophagosome formation, and suppressed programmed cell death and necroptosis. Furthermore, Vitex induced antimetastatic activity by suppressing the migration and invasion of MMP13, which is the primary protease that degrades type I collagen for tumour-induced osteolysis in bone tissues and preferential metastasis sites. Taken together, our results suggest that Vitex is an attractive target for treating human osteosarcoma.

To identify the anti-fibrosis effect of PRAS40 in scar, and its potential mechanism.
We constructed a rat model of hypertrophic scarthat was locally injected the PRAS40 overexpression adenoviruses, mTORC1 inhibitor MHY1485 and activator rapamycin, and further observed the pathological changes of skin tissue and the severity of fibrosis by HE, Masson and sirius red staining, and analyzed the deposition of a-SMA and collagen I by western blot and immunofluorescence test. Meanwhile, the co-localization of KLF4 with a-SMA and type I collagen was analyzed, as well as the regulatory effect of PRAS40 on KLF4. In addition, we also verified whether the inhibition of scar fibrosis by PRAS40 is related to mTORC1, and whether the upregulation of KLF4 is related to mTORC1.
The results showed that the expression of PRAS40 was low and p-PRAS40 was high in scar skin tissue. After local injection of PRAS40 overexpression adenovirus, the expression of PRAS40 in skin tissue was increased. The overexpression of PRAS40 can inhibit scar skin fibrosis and reduce the content of a-SMA and collagen I. Further mechanism analysis confirms that the inhibitory effect of PRAS40 on skin fibrosis is related to mTORC1, and PRAS40 inhibits the activation of mTORC1. The expression of KLF4 is relatively low in scar tissue. PRAS40 administration upregulated the expression of KLF4, which is related to mTORC1 CONCLUSIONS: PRAS40 significantly improves fibrosis of scar skin tissue and increases the expression of KLF4 in scars. The anti-fibrotic effect of PRAS40 depends on mTORC1.

Objective: The PRAS40 is an essential inhibitory subunit of the mTORC1 complex, which regulates autophagy. It has been suggested that Erxian Decoction (EXD) could treat spinal cord injury (SCI) via the autophagy pathway. However, the mechanism of whether EXD acts through PRAS40 remains unclear. Methods: With the help of immobilized PRAS40, isothermal titration calorimetry (ITC) and molecular docking, the bioactive metabolites in the EXD were screened. To establish in vitro SCI models, PC12 cells were exposed to hydrogen peroxide (H2O2) and then treated with the identified EXD substances. Furthermore, Western blot assay was carried out to identify potential molecular mechanisms involved. For assessing the effect of metabolites in vivo, the SCI model rats were first pretreated with or without the metabolite and then subjected to the immunohistochemistry (IHC) staining, Basso, Beattie & Bresnahan (BBB) locomotor rating scale, and H&E staining. Results: The immobilized PRAS40 isolated indole, 4-nitrophenol, terephthalic acid, palmatine, sinapinaldehyde, and 3-chloroaniline as the potential ligands binding to PRAS40. Furthermore, the association constants of palmatine and indole as 2.84 × 106 M-1 and 3.82 × 105 M-1 were elucidated via ITC due to the drug-like properties of these two metabolites. Molecular docking results also further demonstrated the mechanism of palmatine binding to PRAS40. Western blot analysis of PC12 cells demonstrated that palmatine inhibited the expression of p-mTOR by binding to PRAS40, activating the autophagic flux by markedly increasing LC3. The injection of palmatine (10μM and 20 μM) indicated notably increased BBB scores in the SCI rat model. Additionally, a dose-dependent increase in LC3 was observed by IHC staining. Conclusion: This research proved that EXD comprises PRAS40 antagonists, and the identified metabolite, palmatine, could potentially treat SCI by activating the autophagic flux.

The mechanism by which avian reovirus (ARV)-modulated suppression of mTORC1 triggers autophagy remains largely unknown. In this work, we determined that p17 functions as a negative regulator of mTORC1. This study suggest novel mechanisms whereby p17-modulated inhibition of mTORC1 occurs via upregulation of p53, inactivation of Akt, and enhancement of binding of the endogenous mTORC1 inhibitors (PRAS40, FKBP38, and FKPP12) to mTORC1 to disrupt its assembly and accumulation on lysosomes. p17-modulated inhibition of Akt leads to activation of the downstream targets PRAS40 and TSC2, which results in mTORC1 inhibition, thereby triggering autophagy and translation shutoff, which is favorable for virus replication. p17 impairs the interaction of mTORC1 with its activator Rheb, which promotes FKBP38 interaction with mTORC1. It is worth noting that p17 activates ULK1 and Beclin1 and increases the formation of the Beclin 1/class III PI3K complex. These effects could be reversed in the presence of insulin or depletion of p53. Furthermore, we found that p17 induces autophagy in cancer cell lines by upregulating the p53/PTEN pathway, which inactivates Akt and mTORC1. This study highlights p17-modulated inhibition of Akt and mTORC1, which triggers autophagy and translation shutoff by positively modulating the tumor suppressors p53 and TSC2 and endogenous mTORC1 inhibitors. IMPORTANCE The mechanisms by which p17-modulated inhibition of mTORC1 induces autophagy and translation shutoff is elucidated. In this work, we determined that p17 serves as a negative regulator of mTORC1. This study provides several lines of conclusive evidence demonstrating that p17-modulated inhibition of mTORC1 occurs via upregulation of the p53/PTEN pathway, downregulation of the Akt/Rheb/mTORC1 pathway, enhancement of binding of the endogenous mTORC1 inhibitors to mTORC1 to disrupt its assembly, and suppression of mTORC1 accumulation on lysosomes. This work provides valuable information for better insights into p17-modulated inhibition of mTORC1, which induces autophagy and translation shutoff to benefit virus replication.

Previous studies have reported that TIFA plays different roles in various tumor types. However, the function of TIFA in colorectal cancer (CRC) remains unclear. Here, we showed that the expression of TIFA was markedly increased in CRC versus normal tissue, and positively correlated with CRC TNM stages. In agreement, we found that the CRC cell lines show increased TIFA expression levels versus normal control. The knockdown of TIFA inhibited cell proliferation but had no effect on cell apoptosis in vitro or in vivo. Moreover, the ectopic expression of TIFA enhanced cell proliferation ability in vitro and in vivo. In contrast, the expression of mutant TIFA (T9A, oligomerization site mutation; D6, TRAF6 binding site deletion) abolished TIFA-mediated cell proliferation enhancement. Exploration of the underlying mechanism revealed that the protein synthesis-associated kinase RSK and PRAS40 activation were responsible for TIFA-mediated CRC progression. In summary, these findings suggest that TIFA plays a role in mediating CRC progression. This could provide a promising target for CRC therapy.

UNASSIGNED: The circulating progenitor cells of fibroblasts (fibrocytes) have been shown to infiltrate the airway smooth muscle compartment of asthma patients; however, the pathological significance of this discovery has yet to be elucidated. This study established a co-culture model of airway smooth muscle cells (ASMCs) and fibrocytes from asthmatic or normal subjects to evaluate innate cytokine production, corticosteroid responses, and signaling in ASMCs.
UNASSIGNED: CD34+ fibrocytes were purified from peripheral blood of asthmatic (Global Initiative for Asthma treatment step 4-5) and normal subjects and cultured for 5∼7 days. In a transwell plate, ASMCs were co-cultured with fibrocytes at a ratio of 2:1, ASMCs were cultured alone (control condition), and fibrocytes were cultured alone for 48 h. Measurements were obtained of interleukin-8 (IL-8), IL-6, IL-17, thymic stromal lymphopoietin, and IL-33 levels in the supernatant and IL-33 levels in the cell lysate of the co-culture. Screening for intracellular signaling in the ASMCs after stimulation was performed using condition medium from the patients\' co-culture (PtCM) or IL-8. mRNA and western blot analysis were used to analyze AKT/mTOR signaling in ASMCs stimulated via treatment with PtCM or IL-8.
UNASSIGNED: Compared with ASMCs cultured alone, IL-8 levels in the supernatant and IL-33 levels in the ASMCs lysate were significantly higher in samples co-cultured from asthmatics, but not in those co-cultured from normal subjects. Corticosteroid-induced suppression of IL-8 production was less pronounced in ASMCs co-cultured with fibrocytes from asthma patients than in ASMCs co-cultured from normal subjects. ASMCs stimulated using PtCM and IL-8 presented elevating activated AKT substrate PRAS40. Treatment with IL-8 and PtCM increased mRNA expression of mTOR and P70S6 kinases in ASMCs. Treatment with IL-8 and PtCM also significantly increased phosphorylation of AKT and mTOR subtract S6 ribosomal protein in ASMCs.
UNASSIGNED: The interaction between ASMCs and fibrocytes from asthmatic patients was shown to increase IL-8 and IL-33 production and promote AKT/mTOR signaling in ASMCs. IL-8 production in the co-culture from asthmatic patients was less affected by corticosteroid than was that in the co-culture from normal subjects. Our results elucidate the novel role of fibrocytes and ASMCs in the pathogenesis of asthma.

Mechanistic target of rapamycin (mTOR), which functions via two multiprotein complexes termed mTORC1 and mTORC2, is positioned in the canonical phosphoinositide 3‑kinase‑related kinase (PI3K)/AKT (PI3K/AKT) pathways. These complexes exert their actions by regulating other important kinases, such as 40S ribosomal S6 kinases (S6K), eukaryotic translation initiation factor 4E (elF4E)‑binding protein 1 (4E‑BP1) and AKT, to control cell growth, proliferation, migration and survival in response to nutrients and growth factors. Glioblastoma (GB) is a devastating form of brain cancer, where the mTOR pathway is deregulated due to frequent upregulation of the Receptor Tyrosine Kinase/PI3K pathways and loss of the tumor suppressor phosphatase and tensin homologue (PTEN). Rapamycin and its analogs were less successful in clinical trials for patients with GB due to their incomplete inhibition of mTORC1 and the activation of mitogenic pathways via negative feedback loops. Here, the effects of selective ATP‑competitive dual inhibitors of mTORC1 and mTORC2, Torin1, Torin2 and XL388, are reported. Torin2 exhibited concentration‑dependent pharmacodynamic effects on inhibition of phosphorylation of the mTORC1 substrates S6KSer235/236 and 4E‑BP1Thr37/46 as well as the mTORC2 substrate AKTSer473 resulting in suppression of tumor cell migration, proliferation and S‑phase entry. Torin1 demonstrated similar effects, but only at higher doses. XL388 suppressed cell proliferation at a higher dose, but failed to inhibit cell migration. Treatment with Torin1 suppressed phosphorylation of proline rich AKT substrate of 40 kDa (PRAS40) at Threonine 246 (PRAS40Thr246) whereas Torin2 completely abolished it. XL388 treatment suppressed the phosphorylation of PRAS40Thr246 only at higher doses. Drug resistance analysis revealed that treatment of GB cells with XL388 rendered partial drug resistance, which was also seen to a lesser extent with rapamycin and Torin1 treatments. However, treatment with Torin2 completely eradicated the tumor cell population. These results strongly suggest that Torin2, compared to Torin1 or XL388, is more effective in suppressing mTORC1 and mTORC2, and therefore in the inhibition of the GB cell proliferation, dissemination and in overcoming resistance to therapy. These findings underscore the significance of Torin2 in the treatment of GB.

Numerous studies have reported that epilepsy causes memory deficits. The present study was aimed at studying the effect of rapamycin against the memory deficiency of the pentylenetetrazole (PTZ)-kindled animal model of epilepsy. In the present experiment, we randomly chose thirty male rats from the species of Wistar and categorized them in groups of control and experiment (6 for each group). The groups of experiment received the injection of rapamycin (0.5, 1 and 2 mg/kg) intraperitoneally (i.p.) and the group of control received normal saline (0.9%) treatment. Through the PTZ\'s sub-threshold dose (35 mg kg-1, i.p.), all groups were kindled 12 times. Passive avoidance test (PAT) was used for gauging the memory function and the seizure behaviors after the kindling procedure. The rodents were sacrificed at the end of the trial and their brains were scooped for measuring the expression of Gabra1 and Pras40 genes. Statistical analysis unveiled that rapamycin delayed the kindling development and the onset of seizures which are tonic-clonic. Moreover, the administration of rapamycin significantly prevented memory dysfunction in epileptic rats. Finally, it was shown that rapamycin resulted in an increase in the expression levels of Gabra1 and Pras40 genes at the brain tissues. The current research design indicated that rapamycin has beneficial effects for the prevention of memory impairment against PTZ-kindling epilepsy in rats. Such promising outcomes could be attributed to its impact on the Gabra1 and Pras40 genes.

The mechanistic target of rapamycin complex 1 (mTORC1) signaling is the prototypical pathway regulating protein synthesis and cell proliferation. The level of mTORC1 activity is high in intestinal stem cells located at the base of the crypts and thought to gradually decrease as transit-amplifying cells migrate out of the crypts and differentiate into enterocytes, goblet cells or enteroendocrine cells along the epithelium. The unknown mechanism responsible for the silencing of intestinal epithelium mTORC1 during cell differentiation was investigated in Caco-2 cells, which spontaneously differentiate into enterocytes in standard growth medium. The results show that TSC2, an upstream negative regulator of mTORC1 was central to mTORC1 silencing in differentiated Caco-2 cells. AMPK-mediated activation of TSC2 (Ser1387) and repression of Raptor (Ser792), an essential component of mTORC1, were stimulated in differentiated Caco-2 cells. ERK1/2-mediated repression of TSC2 (Ser664) seen in undifferentiated Caco-2 cells was lifted in differentiated cells. IRS-1-mediated activation of AKT (Thr308) phosphorylation was stimulated in differentiated Caco-2 cells and may be involved in cross-pathway repression of ERK1/2. Additionally, PRAS40 (Thr246) phosphorylation was decreased in differentiated Caco-2 cells compared to undifferentiated cells allowing dephosphorylated PRAS40 to displace Raptor thereby repressing mTORC1 kinase activity.

Proline-rich Akt substrate of 40 kD (PRAS40) is not only the substrate of protein kinase B (PKB/Akt), but also the binding protein of 14-3-3 protein. PRAS40 is expressed in a variety of tissues in vivo and has multiple phosphorylation sites, which its activity is closely related to phosphorylation. Studies have shown that PRAS40 is involved in regulating cell growth, cell apoptosis, oxidative stress, autophagy and angiogenesis, as well as various of signalling pathways such as mammalian target of mammalian target rapamycin (mTOR), protein kinase B (PKB/Akt), nuclear factor kappa-B(NF-κB), proto-oncogene serine/threonine-protein kinase PIM-1(PIM1) and pyruvate kinase M2 (PKM2). The interactive roles between PRAS40 and these signal proteins were analysed by bioinformatics in this paper. Moreover, it is of great necessity for analyse the important roles of PRAS40 in some human diseases including cardiovascular disease, ischaemia-reperfusion injury, neurodegenerative disease, cancer, diabetes and other metabolic diseases. Finally, the effects of miRNA on the regulation of PRAS40 function and the occurrence and development of PRAS40-related diseases are also discussed. Overall, PRAS40 is expected to be a drug target and provide a new treatment strategy for human diseases.