PSAT1 mediated EMT of colorectal cancer cells by regulating Pl3K/AKT signaling pathway.

Background: The metastasis of colorectal cancer (CRC) is one of the significant barriers impeding its treated consequence and bring about high mortality, less surgical resection rate and poor prognosis of CRC patients. PSAT1 is an enzyme involved in serine biosynthesis. The studies showed that PSAT1 plays the part of a crucial character in the regulation of tumor metastasis. And Epithelial-Mesenchymal Transition (EMT) is a process of cell reprogramming in which epithelialcells obtain mesenchymal phenotypes. It is a crucial course in promoting cell metastasis and the progression of malignant tumors. The relationship between PSAT1 and EMT in colorectal cancer, as well as the underlying molecular mechanisms, remains enigmatic and warrants thorough exploration. These findings suggest that PSAT1 may serve as a promising therapeutic target for mitigating colorectal cancer metastasis and holds the potential to emerge as a valuable prognostic biomarker in forthcoming research endeavors. Materials and Methods: Utilizing TCGA dataset in conjunction with clinical CRC specimens, our initial focus was directed towards an in-depth examination of PSAT1 expression within CRC, specifically exploring its potential correlation with the adverse prognostic outcomes experienced by patients. Furthermore, we conducted a comprehensive investigation into the regulatory influence exerted by PSAT1 on CRC through the utilization of siRNA knockdown techniques. In the realm of in vitro experimentation, we meticulously evaluated the impact of PSAT1 on various facets of CRC progression, including cell migration, invasion, proliferation, and colony formation. In order to elucidate the intricate effects in question, we adopted a multifaceted methodology that encompassed a range of assays and analyses. These included wound healing assays, transwell assays, utilization of the Cell Counting Kit-8 (CCK-8) assay, and colony formation assays. By employing this diverse array of investigative techniques, we were able to achieve a comprehensive comprehension of the multifaceted role that PSAT1 plays in the pathogenesis of colorectal cancer. This multifarious analysis greatly contributed to our in-depth understanding of the complex mechanisms at play in colorectal cancer pathogenesis. Using WB and PCR experiments, we found that PSAT1 has a role in regulating EMT development in CRC.In terms of mechanism, we found that PSAT1 affected EMT by Regulating Pl3K/AKT Signaling Pathway. Results: Our investigation revealed a noteworthy down-regulation of PSAT1 expression in CRC specimens. Importantly, this down-regulation exhibited a significant positive correlation with the unfavorable prognosis of patients afflicted with CRC. Functionally, our study showcased that the siRNA-mediated knockdown of PSAT1 markedly enhanced various key aspects of CRC pathogenesis in an in vitro setting. Specifically, this included a substantial promotion of CRC cell migration, invasion, proliferation, and colony formation. Moreover, the silencing of PSAT1 also demonstrated a substantial promotion of the EMT process. Intriguingly, our research unveiled a hitherto unexplored mechanism underlying the regulatory role of PSAT1 in CRC and EMT. We have established, for the first time, that PSAT1 exerts its influence by modulating the activation of the PI3K/AKT Signaling Pathway. This mechanistic insight provides a valuable contribution to the understanding of the molecular underpinnings of CRC progression and EMT induction mediated by PSAT1. Conclusions: In unison, our research findings shed light on the previously uncharted and significant role of the PSAT1/PI3K/AKT axis in the initiation of the EMT process in CRC. Furthermore, our discoveries introduce a novel biomarker with potential implications for the clinical diagnosis and treatment of CRC.

Inhibition of phosphoinositide 3-kinase activity attenuates neutrophilic airway inflammation and inhibits pyrin domain-containing 3 inflammasome activation in an ovalbumin-lipopolysaccharide-induced asthma murine model.

BACKGROUND: Existing investigations suggest that the blockade of phosphoinositide 3-kinase (PI3K) activity contributes to inflammatory solution in allergic asthma, but whether this inhibition directly attenuates neutrophilic airway inflammation in vivo is still unclear. We explored the pharmacological effects of LY294002, a specific inhibitor of PI3K on the progression of neutrophilic airway inflammation and investigated the underlying mechanism. METHODS AND RESULTS: Female C57BL/6 mice were intranasally sensitized with ovalbumin (OVA) together with lipopolysaccharide (LPS) on days 0 and 6, and challenged with OVA on days 14-17 to establish a neutrophilic airway disease model. In the challenge phase, a subset of mice was treated intratracheally with LY294002. We found that treatment of LY294002 attenuates clinic symptoms of inflammatory mice. Histological studies showed that LY294002 significantly inhibited inflammatory cell infiltration and mucus production. The treatment also significantly inhibited OVA-LPS induced increases in inflammatory cell counts, especially neutrophil counts, and IL-17 levels in bronchoalveolar lavage fluid (BALF). LY294002 treated mice exhibited significantly increased IL-10 levels in BALF compared to the untreated mice. In addition, LY294002 reduced the plasma concentrations of IL-6 and IL-17. The anti-inflammatory effects of LY29402 were correlated with the downregulation of NLRP3 inflammasome. CONCLUSIONS: Our findings suggested that LY294002 as a potential pharmacological target for neutrophilic airway inflammation.

The effect of adipose-derived mesenchymal stem cell transplantation on ovarian mitochondrial dysfunction in letrozole-induced polycystic ovary syndrome in rats: the role of PI3K-AKT signaling pathway.

OBJECTIVE: The present study aimed to elucidate how mesenchymal stem cells (MSCs) application could efficiently attenuate pathological changes of letrozole-induced poly cystic ovary syndrome (PCOS) by modulating mitochondrial dynamic via PI3K-AKT pathway. METHODS: Thirty-two female rats were randomly divided into four experimental groups: Sham, PCOS, PCOS + MSCs, and PCOS + MSCs + LY294002. The Sham group received 0.5% w/v carboxymethyl cellulose (CMC); the PCOS group received letrozole (1 mg/kg, daily) in 0.5% CMC for 21 days. Animals in the PCOS + MSCs group received 1 × 106 MSCs/rat (i.p,) on the 22th day of the study. In the PCOS + MSCs + LY294002 group, rats received LY294002 (PI3K-AKT inhibitor) 40 min before MSC transplantation. Mitochondrial dynamic gene expression, mitochondrial membrane potential (MMP), citrate synthase (CS) activity, oxidative stress, inflammation, ovarian histological parameters, serum hormone levels, homeostatic model assessment for insulin resistance (HOMA-IR), insulin and glucose concentrations, p-PI3K and p-AKT protein levels were evaluated at the end of the experiment. RESULTS: PCOS rats showed a significant disruption of mitochondrial dynamics and histological changes, lower MMP, CS, ovary super oxide dismutase (SOD) and estrogen level. They also had a notable rise in insulin and glucose concentrations, HOMA-IR, testosterone level, tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels, ovarian malondialdehyde (MDA) content as well as a notable decrease in p-PI3K and p-AKT protein levels compared to the Sham group. In the PCOS + MSCs group, the transplantation of MSCs could improve the above parameters. Administration of LY294002 (PI3K-AKT pathway inhibitor) deteriorated mitochondrial dynamic markers, oxidative stress status, inflammation markers, hormonal levels, glucose, and insulin levels and follicular development compared to the PCOS + MSCs group. CONCLUSIONS: This study demonstrated that the protective effects of MSC transplantation in regulating mitochondrial dynamics, promoting mitochondrial biogenesis, competing with redox status and inflammation response were mainly mediated through the PI3K-AKT pathway in the PCOS model.

Notch signaling regulates Th17 cells differentiation through PI3K/AKT/mTORC1 pathway and involves in the thyroid injury of autoimmune thyroiditis.

PURPOSE: Autoimmune Thyroiditis (AIT) is the most common thyroid disease; however, there were no measures to prevent the progression of the disease. The present study attempts to identify that Notch signaling regulates the differentiation of T helper 17 (Th17) cells by activating downstream Phosphatidylinositol-3 kinase/protein kinase/mechanistic target of rapamycin complex 1 (PI3K/AKT/mTORC1) pathway participating in the thyroid injury of the experimental autoimmune thyroiditis (EAT). METHODS: In vivo experiments, mice were randomly divided into 4 groups: a control group, an EAT group, and two groups with LY294002 treatment (pTg plus 25 mg/kg or 50 mg/kg LY294002, respectively). The degrees of thyroiditis were evaluated, and the percentage of Th17 cells, expression of interleukin-17A (IL-17A), and the main components of the Notch-PI3K signaling pathway were detected in different groups. In vitro experiments, two different dosages of LY294002 (25 and 50 µM) were used to intervene splenic mononuclear cells (SMCs) from EAT mice to further evaluate the regulatory effect of Notch-PI3K pathway on Th17 cells. RESULTS: Our data demonstrate that the infiltration of Th17 cells and the expressions of IL-17A, Notch, hairy and split 1 (Hes1), p­AKT (Ser473), p­AKT (Thr308), p­mTOR (Ser2448), S6K1, and S6K2 increased remarkably in EAT mice. After PI3K pathway was blocked, the degrees of thyroiditis were significantly alleviated, and the proportion of Th17 cells, the expression of IL-17A, and the above Notch-PI3K pathway-related molecules decreased in a dose-dependent manner. Additionally, the proportion of Th17 cells was positively correlated with the concentration of serum thyroglobulin antibody (TgAb), IL-17A, and Notch-PI3K pathway-related molecules mRNA levels. CONCLUSIONS: Notch signal promotes the secretion of IL-17A from Th17 cells by regulating the downstream PI3K/AKT/mTORC1 pathway through Hes-Phosphatase and tensin homolog (PTEN) and participates in thyroid autoimmune damage, and the PI3K pathway inhibitor may play important effects on AIT by affecting Th17 cells differentiation.

Furanocoumarins as Enhancers of Antitumor Potential of Sorafenib and LY294002 toward Human Glioma Cells In Vitro.

Furanocoumarins are naturally occurring compounds in the plant world, characterized by low molecular weight, simple chemical structure, and high solubility in most organic solvents. Additionally, they have a broad spectrum of activity, and their properties depend on the location and type of attached substituents. Therefore, the aim of our study was to investigate the anticancer activity of furanocoumarins (imperatorin, isoimperatorin, bergapten, and xanthotoxin) in relation to human glioblastoma multiforme (T98G) and anaplastic astrocytoma (MOGGCCM) cell lines. The tested compounds were used for the first time in combination with LY294002 (PI3K inhibitor) and sorafenib (Raf inhibitor). Apoptosis, autophagy, and necrosis were identified microscopically after straining with Hoechst 33342, acridine orange, and propidium iodide, respectively. The levels of caspase 3 and Beclin 1 were estimated by immunoblotting and for the blocking of Raf and PI3K kinases, the transfection with specific siRNA was used. The scratch test was used to assess the migration potential of glioma cells. Our studies showed that the anticancer activity of furanocoumarins strictly depended on the presence, type, and location of substituents. The obtained results suggest that achieving higher pro-apoptotic activity is determined by the presence of an isoprenyl moiety at the C8 position of the coumarin skeleton. In both anaplastic astrocytoma and glioblastoma, imperatorin was the most effective in induction apoptosis. Furthermore, the usage of imperatorin, alone and in combination with sorafenib or LY294002, decreased the migratory potential of MOGGCCM and T98G cells.

TIM-3 regulates the proliferation by BDNF-mediated PI3K/AKT axis in the process of endometriosis.

BACKGROUND: T cell immunoglobulin and mucin domain-containing molecule-3 (TIM-3) initially discovered on the surface of Th1 cells, negatively regulates immune responses and mediates apoptosis of Th1 cells. An increasing number of studies have since shown that TIM-3 is crucial in the genesis and development of immune diseases, cancers, and chronic infectious illnesses. However, the effect of TIM-3 on endometriosis is still unknown. METHODS: Quantitative real-time polymerase chain reaction, western blotting, and immunohistochemistry were used to measure TIM-3 levels in endometriosis. Cell Counting Kit-8, 5-ethynyl-2'-deoxyuridine, colony-forming, Transwell® migration, Matrigel® invasion, and flow cytometry assays were used to explore the function of TIM-3 in vitro, and xenograft experiments in nude mice were used to assess its role in vivo. According to the RNA seq, brain-derived neurotrophic factor (BDNF) was screened. The involvement of specific proliferation-related signaling molecules was determined by transfecting a plasmid and adding an inhibitor in vivo and in vitro. RESULTS: TIM-3 mRNA and protein expression levels were significantly higher in eutopic and ectopic endometrial tissues than in normal endometrial tissues. By examining the effects of TIM-3 overexpression and knockdown on cell proliferation, migration, and invasion in vitro, and lesions formation in vivo, we found that the expression of TIM-3 was positively correlated with cell proliferation and clone formation in vitro, as well as lesions growth in nude mice. By adding the phosphatidylinositol 3 kinase/protein kinase B(PI3K/AKT) pathway inhibitor LY294002 and knocking down PI3K, we further verified that TIM-3 promotes proliferation in vivo and in vitro via the PI3K pathway. By transfecting the plasmid into ESC cells and gave inhibitors to endometriotic rats models, we tested that TIM-3 regulates the proliferation by BDNF-mediated PI3K/AKT axis. CONCLUSION: TIM-3 can promote the proliferation of endometriosis by BDNF-mediated PI3K/AKT axis in vivo and in vitro, which may provide a new therapeutic target for the treatment of endometriosis.

MEK/ERK and PI3K/AKT pathway inhibitors affect the transformation of myelodysplastic syndrome into acute myeloid leukemia via H3K27me3 methylases and de­methylases.

The transformation of myelodysplastic syndrome (MDS) into acute myeloid leukemia (AML) poses a significant clinical challenge. The trimethylation of H3 on lysine 27 (H3K27me3) methylase and de­methylase pathway is involved in the regulation of MDS progression. The present study investigated the functional mechanisms of the MEK/ERK and PI3K/AKT pathways in the MDS­to­AML transformation. MDS­AML mouse and SKM­1 cell models were first established and this was followed by treatment with the MEK/ERK pathway inhibitor, U0126, the PI3K/AKT pathway inhibitor, Ly294002, or their combination. H3K27me3 methylase, enhancer of zeste homolog (EZH)1, EZH2, demethylase Jumonji domain­containing protein­3 (JMJD3) and ubiquitously transcribed tetratricopeptide repeat on chromosome X (UTX) and H3K27me3 protein levels were determined using western blot analysis. Cell viability, cycle distribution and proliferation were assessed using CCK­8, flow cytometry, EdU and colony formation assays. The ERK and AKT phosphorylation levels in clinical samples and established models were determined, and SKM­1 cell behaviors were assessed. The levels of H3K27me3 methylases and de­methylases and distal­less homeobox 5 (DLX5) were measured. The results revealed that the ERK and AKT phosphorylation levels were elevated in patients with MDS and MDS­AML, and in mouse models. Treatment with U0126, a MEK/ERK pathway inhibitor, and Ly294002, a PI3K/AKT pathway inhibitor, effectively suppressed ERK and AKT phosphorylation in mice with MDS­AML. It was observed that mice with MDS treated with U0126/Ly294002 exhibited reduced transformation to AML, delayed disease transformation and increased survival rates. Treatment of the SKM­1 cells with U0126/Ly294002 led to a decrease in cell viability and proliferation, and to an increase in cell cycle arrest by suppressing ERK/PI3K phosphorylation. Moreover, treatment with U0126/Ly294002 downregulated EZH2/EZH1 expression, and upregulated JMJD3/UTX expression. The effects of U0126/Ly294002 were nullified when EZH2/EZH1 was overexpressed or when JMJD3/UTX was inhibited in the SKM­1 cells. Treatment with U0126/Ly294002 also resulted in a decreased H3K27me3 protein level and H3K27me3 level in the DLX5 promoter region, leading to an increased DLX5 expression. Overall, the findings of the present study suggest that U0126/Ly294002 participates in MDS­AML transformation by modulating the levels of H3K27me3 methylases and de­methylases, and regulating DLX5 transcription and expression.

Corilagin attenuates intestinal ischemia/reperfusion injury in mice by inhibiting ferritinophagy-mediated ferroptosis through disrupting NCOA4-ferritin interaction.

AIMS: Intestinal ischemia reperfusion (II/R) is a common clinical emergency. Ferroptosis is reported to play a role in II/R injury. Our previous studies revealed that corilagin significantly attenuates intestinal ischemia/reperfusion injuries. However, the underlying molecular mechanism is unclear and requires further study. MATERIALS AND METHODS: DAO, GSSG/T-GSH, MDA, and Fe2+ were measured by assay kits, 4-HNE was assessed by IHC, and 15-LOX was measured by ELISA. Mitochondrial damage was observed by TEM and cellular oxidation levels were detected by C11-BODIPY 581/591 and DHE probes. LC3, p62, Beclin1, ACSL4, GPX4, NCOA4, and ferritin expression were examined by WB in vivo and in vitro. IF, co-IF, q-PCR, and constructed NCOA4-knock-down IEC-6 cells were used to evaluate the role of NCOA4 in the effect of corilagin against II/R injury. Temporal and nucleoplasmic variations with or without corilagin were observed by WB. Co-IP and molecular docking were used to investigate the NCOA4-ferritin interaction. KEY FINDINGS: Corilagin attenuated II/R-induced ferroptosis both in vitro and in vivo. Further study revealed that the anti-ferroptosis bioactivity of corilagin might be due to the modulation of iron homeostasis via inhibition of ferritinophagy in an NCOA4-dependent manner. SIGNIFICANCE: Corilagin might be a potential therapeutic agent for II/R-induced tissue injury.

The Inhibition of the FGFR/PI3K/Akt Axis by AZD4547 Disrupts the Proangiogenic Microenvironment and Vasculogenic Mimicry Arising from the Interplay between Endothelial and Triple-Negative Breast Cancer Cells.

Vasculogenic mimicry (VM), a process in which aggressive cancer cells form tube-like structures, plays a crucial role in providing nutrients and escape routes. Highly plastic tumor cells, such as those with the triple-negative breast cancer (TNBC) phenotype, can develop VM. However, little is known about the interplay between the cellular components of the tumor microenvironment and TNBC cells' VM capacity. In this study, we analyzed the ability of endothelial and stromal cells to induce VM when interacting with TNBC cells and analyzed the involvement of the FGFR/PI3K/Akt pathway in this process. VM was corroborated using fluorescently labeled TNBC cells. Only endothelial cells triggered VM formation, suggesting a predominant role of paracrine/juxtacrine factors from an endothelial origin in VM development. Via immunocytochemistry, qPCR, and secretome analyses, we determined an increased expression of proangiogenic factors as well as stemness markers in VM-forming cancer cells. Similarly, endothelial cells primed by TNBC cells showed an upregulation of proangiogenic molecules, including FGF, VEGFA, and several inflammatory cytokines. Endothelium-dependent TNBC-VM formation was prevented by AZD4547 or LY294002, strongly suggesting the involvement of the FGFR/PI3K/Akt axis in this process. Given that VM is associated with poor clinical prognosis, targeting FGFR/PI3K/Akt pharmacologically may hold promise for treating and preventing VM in TNBC tumors.

Lysophosphatidic acid is associated with oocyte maturation by enhancing autophagy via PI3K-AKT-mTOR signaling pathway in granulosa cells.

BACKGROUND: Folliculogenesis is a complex network of interacting cellular signals between somatic cells and oocytes. Many components in ovarian follicular fluid (FF) dynamically change during folliculogenesis and play a positive role in oocyte maturation. Previous studies have reported that lysophosphatidic acid (LPA) promotes cumulus cell expansion, oocyte nuclear maturation, and in vitro maturation of oocytes. RESULTS: Initially, the expression of LPA was raised in matured FF significantly (P < 0.0001). Then, 10 µM LPA treated for 24 h in human granulosa cells (KGNs) aggravated cell proliferation, with increased autophagy, and reduced apoptosis. Meanwhile, we demonstrated that LPA mediated cell function through the PI3K-AKT-mTOR signaling pathway as PI3K inhibitor (LY294002) significantly prevented LPA-induced AKT, mTOR phosphorylation and autophagy activation. Such results were also verified by immunofluorescence staining and flow cytometry. In addition, an autophagy inhibitor 3 methyladenine (3MA) could also alleviate the effects of LPA, by activating apoptosis through PI3K-AKT-mTOR pathways. Finally, we found blockade with Ki16425 or knockdown LPAR1, alleviated LPA mediated autophagy activation in KGNs, suggesting that LPA enhances autophagy through activation of the LPAR1 and PI3K-AKT-mTOR signaling pathways. CONCLUSION: This study demonstrates that increased LPA activated PI3K-Akt-mTOR pathway through LPAR1 in granulosa cells, suppressing apoptosis by enhancing autophagy, which might play a role in oocyte maturation in vivo.

Encapsulation of PI3K Inhibitor LY294002 within Polymer Nanoparticles Using Ion Pairing Flash Nanoprecipitation.

Flash nanoprecipitation (FNP) is a turbulent mixing process capable of reproducibly producing polymer nanoparticles loaded with active pharmaceutical ingredients (APIs). The nanoparticles produced with this method consist of a hydrophobic core surrounded by a hydrophilic corona. FNP produces nanoparticles with very high loading levels of nonionic hydrophobic APIs. However, hydrophobic compounds with ionizable groups are not as efficiently incorporated. To overcome this, ion pairing agents (IPs) can be incorporated into the FNP formulation to produce highly hydrophobic drug salts that efficiently precipitate during mixing. We demonstrate the encapsulation of the PI3K inhibitor, LY294002, within poly(ethylene glycol)-b-poly(D,L lactic acid) nanoparticles. We investigated how incorporating two hydrophobic IPs (palmitic acid (PA) and hexadecylphosphonic acid (HDPA)) during the FNP process affected the LY294002 loading and size of the resulting nanoparticles. The effect of organic solvent choice on the synthesis process was also examined. While the presence of either hydrophobic IP effectively increased the encapsulation of LY294002 during FNP, HDPA resulted in well-defined colloidally stable particles, while the PA resulted in ill-defined aggregates. The incorporation of hydrophobic IPs with FNP opens the door for the intravenous administration of APIs that were previously deemed unusable due to their hydrophobic nature.

LY294002 alleviates bone cancer pain by reducing mitochondrial dysfunction and the inflammatory response.

Bone cancer pain (BCP) is mainly caused by bone metastasis and markedly impairs the functional capacity and daily functions of patients. Neuroinflammation plays a pivotal role in the pathogenesis and maintenance of chronic pain. Oxidative stress in the mitochondria is a key contributor to neuroinflammation and neuropathic pain. Herein, a rat model of BCP was established which was characterized by bone destruction, pain hypersensitivity and motor disability. In the spinal cord, phosphatidylinositol 3­kinase (PI3K)/protein kinase B (Akt) signaling was activated, and the inflammatory response and mitochondrial dysfunction were also observed. The intrathecal injection of LY294002, a selective inhibitor of PI3K/Akt signaling, decreased mechanical pain sensitivity, suppressed spontaneous pain and recovered the motor coordination of rats with BCP. Second, LY294002 treatment blocked spinal inflammation by reducing astrocytic activation and downregulating the expression levels of inflammatory factors, such as NF­κB, IL­1ß and TNF­α. Moreover, LY294002 treatment recovered mitochondrial function by activating the manganese superoxide dismutase enzyme, increasing NADH:ubiquinone oxidoreductase subunit B11 expression, and decreasing BAX and dihydroorotate dehydrogenase expression. LY294002 treatment also increased the mitochondrial membrane potential and decreased the mitochondrial reactive oxygen species levels in C6 cells. On the whole, the results of the present study suggest that the inhibition of PI3K/Akt signaling by LY294002 restores mitochondrial function, suppresses spinal inflammation and alleviates BCP.

LY294002 ameliorates psoriatic skin inflammation in mice via blocking the Notch1/Hes1-PTEN/AKT/IL-17A feedback loop.

(IL)-17A, the effective factor of Th17 cells, acts an important pathological role in the pathogenesis of psoriasis. Notch1/hairy and split 1 (Hes1) and PI3K/AKT signaling pathways are interpenetrated and involved in Th17 cell differentiation and IL-17A production. In this present study, we used imiquimod (IMQ)-induced mouse psoriatic skin inflammation to explore the possible mechanism of Notch1/Hes1-PTEN/AKT/IL-17A feedback loop in psoriasis by employing AKT inhibitor LY294002 as an intervention with the methods of flow cytometry analysis, reverse transcription-quantitative polymerase chain reaction, western blot, co-immunoprecipitation, and immunofluorescence. First, LY294002 inhibition can obviously alleviate the mouse psoriatic skin inflammation both in skin structural and histopathological characteristics, which is similar to the changes found in IL-17A antibody-treated mice. Additionally, the interaction between Notch1 intracellular domain (NICD1) and nuclear factor kappa B (NF-κB) activator 1 (Act1) was demonstrated. LY294002 interruption resulted in consistent changes in expression levels of key signaling molecules both in Notch1/Hes1 and PI3K/AKT signaling pathways in a time-dependent manner. Moreover, chloroquine (CQ) can partly reverse the inhibitory effects of LY294002 on the Notch1/Hes1-PTEN/AKT/IL-17A feedback loop by affecting Notch1 ubiquitination and lysosomal degradation. The present study showed that LY294002 can exert the inhibitory effect on Notch1/Hes1-PTEN/AKT/IL-17A feedback loop to regulate Th17 cell differentiation and IL-17A function in the process of psoriasis, which provides a new possible therapeutic strategy for psoriasis.

Compound Kushen injection attenuates angiotensin II­mediated heart failure by inhibiting the PI3K/Akt pathway.

Compound Kushen injection (CKI) is a type of traditional Chinese medicine that has previously been studied for the treatment of various types of cancer. Previous studies have reported that CKI regulates cell apoptosis by downregulating the PI3K/Akt pathway. The present study aimed to determine whether CKI alleviates heart failure (HF) by attenuating cardiomyocyte apoptosis via the inhibition of the PI3K/Akt pathway. Angiotensin II (Ang II) was used to elicit HF, and osmotic minipumps with either Ang II (2 µg/kg/day) or phosphate­buffered saline (PBS; 200 µl) were subcutaneously implanted into 6­week­old male C57BL/6 mice for 3 weeks. In addition, PBS or CKI (25 mg/kg/day) were subcutaneously infused once a day for 3 weeks. Echocardiography was used to examine hemodynamics. The myocardial injury biomarkers, cardiac troponin I and N­terminal (NT)­pro hormone B­type natriuretic peptide, were assessed using enzyme­linked immunosorbent assay. Transmission electron microscopy was used to determine the morphology of the myocardium. The rate of apoptosis was detected using TUNEL staining and flow cytometry (FCM), and the expression levels of apoptosis­related proteins were measured using western blot (WB) analysis. Moreover, H9C2 cells were treated with CKI (2 mg/ml) or LY294002 (an inhibitor of the PI3K/Akt pathway; 25 µmol/l) in combination with Ang II (1 µmol/l) for 48 h. Cell Counting Kit­8 assay, FCM and WB analysis were performed in the H9C2 cells to examine cell viability, cell cycle distribution and representative signaling proteins. It was found that CKI promoted healthy cardiac function, reduced myocardial structural damage and reduced the rate of cardiomyocyte apoptosis. CKI markedly attenuated the expression of apoptosis­related proteins in the PI3K/Akt pathway. The results of the in vitro experiments indicated that CKI promoted cardiomyocyte proliferation and inhibited apoptosis, similar to LY294002. On the whole, the present study demonstrates that CKI reduces cardiomyocyte apoptosis, promotes healthy cardiac function and attenuates Ang II­mediated HF. These ameliorative effects may be associated with the inhibition of the PI3K/Akt pathway.

Identification of Autophagy as a Functional Target Suitable for the Pharmacological Treatment of Mitochondrial Membrane Protein-Associated Neurodegeneration (MPAN) In Vitro.

Mitochondrial membrane protein-associated neurodegeneration (MPAN) is a relentlessly progressive neurodegenerative disorder caused by mutations in the C19orf12 gene. C19orf12 has been implicated in playing a role in lipid metabolism, mitochondrial function, and autophagy, however, the precise functions remain unknown. To identify new robust cellular targets for small compound treatments, we evaluated reported mitochondrial function alterations, cellular signaling, and autophagy in a large cohort of MPAN patients and control fibroblasts. We found no consistent alteration of mitochondrial functions or cellular signaling messengers in MPAN fibroblasts. In contrast, we found that autophagy initiation is consistently impaired in MPAN fibroblasts and show that C19orf12 expression correlates with the amount of LC3 puncta, an autophagy marker. Finally, we screened 14 different autophagy modulators to test which can restore this autophagy defect. Amongst these compounds, carbamazepine, ABT-737, LY294002, oridonin, and paroxetine could restore LC3 puncta in the MPAN fibroblasts, identifying them as novel potential therapeutic compounds to treat MPAN. In summary, our study confirms a role for C19orf12 in autophagy, proposes LC3 puncta as a functionally robust and consistent readout for testing compounds, and pinpoints potential therapeutic compounds for MPAN.

Enhancement of the antitumor effect of 5-fluorouracil with modulation in drug transporters expression using PI3K inhibitors in colorectal cancer cells.

AIMS: 5-Fluorouracil (5-FU) represents the cornerstone for colorectal cancer therapy. However, resistance to its action is a major hindrance. This study aimed to investigate the effectiveness of suppressing the activity of PI3K/Akt/mTOR signaling pathway on the chemosensitivity of colorectal cancer cells to 5-FU, as well as to delineate the possible underlying cellular mechanisms and the expected modulation in the expression of specific ABC drug transporters. MAIN METHODS: HCT116 and Caco-2 cells were incubated with 5-FU, LY294002, or PI-103 individually or in combination. Cell viability was monitored using MTT assay. The expression of a panel of drug transporters was evaluated by RT-PCR. Immunofluorescence staining was applied to evaluate the expression pattern of phospho-AKT, phospho-mTOR, and ABGG2. HPLC evaluated the enhancement in the 5-FU cellular uptake. Cell apoptosis was detected by flow cytometry, and cell morphological changes following treatment were inspected under a fluorescence microscope. Additionally, the migration ability of cells following our suggested treatment combination was examined by wound healing assay. KEY FINDINGS: The results reveal a notable enhancement in the cytotoxicity of a low dose of 5-FU when combined with a PI3K inhibitor (LY294002 or PI-103). This enhancement was influenced by the significant reduction in the expression of p-AKT and p-mTOR and was also mediated by a significant suppression in the expression of ABCG2 and ABCC5. Consequently, we detected an increase in the cellular uptake and concentration of 5-FU in cells treated with this combination rather than a single 5-FU treatment. Our Suggested combination treatment also induced cell apoptosis and reduced the migration ability of cells. SIGNIFICANCE: Our data provide evidence that survival signaling pathways represent distinctive targets for the enhancement of chemotherapeutic sensitivity. The antitumor efficacy of 5-FU is enhanced when combined with a PI3K inhibitor, and this effect was mediated by alterations in the expression of specific drug transporters.

Cognitive improving actions of tofacitinib in a mouse model of Alzheimer disease involving TNF-α, IL-6, PI3K-Akt and GSK-3ß signalling pathway.

Aim and Objective: This current study investigated the significance of tofactinib in improving memory functions in a memory model of ß-amyloid (Aß)-induced dementia.Material and Methods: Aß1-42 was injected in the brain of mice using intracerebroventricular injection and after 14 days, the learning and memory was assessed on the Morris Water maze test. Mice were treated with tofactinib (10, 20, 30 mg/kg) two days prior to Aß1-42 injection and 14 days after Aß injection.Results: Treatment of tofactinib significantly improved the learning (decrease in day escape latency time [ELT]) and memory (increase in time spent in target quadrant). This drug also decreased the levels of T NF-α and IL-6 along with the rise in expression of p-Akt and p-GSK-3ß/GSK-3ß ratio in mice brain. Co-administration of LY294002 (P I3K inhibitor) or MK-2206 2HCl (Akt inhibitor) with tofactinib (30 mg/kg) obliterated the beneficial effects of the latter by increasing T NF-α and IL-6 levels along with decreasing the p-Akt expression and p-GSK-3ß/GSK-3ß ratio.Conclusion: It is concluded that tofactinib improves the condition of dementia of Alzheimer's type, possibly through down regulation of T NF-α and IL-6 and instigation of P I3K-Akt-p-GSK-3ß signalling system in the hippocampus of Aß-treated mice.

[Effects of matrine combined with LY294002 on proliferation, apoptosis and cell cycle of human myeloid leukemia K562 cells].

OBJECTIVE: To investigate the effects of matrine combined with LY294002 on proliferation, apoptosis and cell cycle of human myeloid leukemia K562 cells and explore the underlying mechanism. METHODS: The effects of different concentrations of matrine alone and in combination with LY294002 on the proliferation of K562 cells were examined with CCK-8 assay. The changes in morphology of K562 cells were observed following treatment for 48 h with 0.4 g/L matrine and 10 µmol/L Y294002, either alone or in combination, and cell apoptosis was detected using flow cytometry with annexin V-FITC/PI double labeling; the changes in cell cycle was detected with PI labeling. Western blotting was performed to examine the effect of matrine combined with LY294002 on expressions of p-mTOR, p-PI3K, Akt, p-Akt, cyclinD1, Bcl-2 and caspase-9 in the cells. RESULTS: Treatment with different concentrations of matrine, both alone and in combination with LY294002, inhibited the proliferation of K562 cells in a time- and concentration-dependent manner. Compared with matrine treatment alone, the combined treatment caused more obvious morphological changes of the cells, significantly increased cell apoptosis (P < 0.01), and induced cell cycle arrest in G0/G1 (P < 0.01). Western blotting showed that the protein expression levels of p-mTOR, cyclinD1, p-PI3K, p-Akt and Bcl-2 in K562 cells increased while the expression level of caspase-9 decreased significantly after the combined treatment (P < 0.01). CONCLUSION: Matrine combined with LY294002 produces a synergistic inhibitory effect on K562 cells possibly by down-regulating the p-Akt expression in PI3K/Akt signaling pathway, reducing the expressions of p-mTOR, cyclinD1 and Bcl-2, and increasing the expression of caspase-9.

Reversion of glucocorticoid-induced senescence and collagen synthesis decrease by LY294002 is mediated through p38 in skin.

Glucocorticoids (GCs) are the most common treatment for inflammatory skin disorders; however, they show several adverse side effects, including atrophy and collagen decrease following chronic treatment. In particular, transcription factors and p38 signaling for collagen synthesis have been shown to be suppressed by the active glucocorticoid receptor (GR). LY294002 (LY), a phosphoinositide 3-kinase (PI3K) inhibitor, has been reported to protect keratinocytes in epidermis against GC-induced hypoplasia; however, its protective effect in dermis remains unclear. Furthermore, clobetasol propionate (CP) is the most used commercial synthetic GC, yet studies on how CP causes side effects in dermal fibroblasts are limited. In this study, dermal atrophy was modeled using CP in human dermal fibroblasts (HDFs) and C57BL/6 mice. CP treatment significantly upregulated FK506 binding protein 5 (FKBP51), an atrophy marker (2.4 ± 0.25 and 3.3 ± 0.3 fold in in vitro and in vivo, respectively), phosphorylated GR (1.96 ± 0.08 and 2.29 ± 0.25 fold in in vitro and in vivo, respectively), decreased fibroblast proliferation (82.71 ± 1.95% in in vitro), reduced collagen synthesis (0.36 ± 0.05 and 0.3 ± 0.1 fold in in vitro and in vivo, respectively), and induced aging, all of which were reversed by LY treatment (from 1.43 ± 0.08 to 2.8 ± 0.12 fold) without showing growth inhibition and exerting the anti-inflammation of CP. Interestingly, the protective effect of LY was dose-dependently reversed by treatment with a p38 inhibitor and reached 2.9 ± 0.15 fold at dose 20 µM. Taken together, our results demonstrate that LY reduced CP-induced upregulation of the atrophy marker FKBP51, GR phosphorylation, and GR nuclear translocation via the activation of p38, whilst maintaining the anti-inflammatory effect of glucocorticoids.

Pentamidine inhibits proliferation, migration and invasion in endometrial cancer via the PI3K/AKT signaling pathway.

BACKGROUND: Pentamidine has been reported to have many pharmacological effects including anti- protozoal, anti-inflammatory, and anti-tumor activities. The aim of this study is to investigate the potential therapeutic role of Pentamidine and molecular mechanisms of Pentamidine on PI3K/AKT signaling pathway underlying the anti-tumor properties in endometrial cancer. METHODS: Our study was carried out in the central laboratory of Harbin Medical University from 2019 to 2021. Human endometrial cancer cell lines Ishikawa and HEC-1A were treated with Pentamidine. The proliferation ability of cells was investigated by MTS and colony formation assays. The cell cycle distribution was detected by flow cytometry. Cell migration and invasion were analyzed by using the wound healing assay and Transwell assay. Western blotting was performed to measure the levels of AKT, p-AKT, MMP-2, and MMP-9. RESULTS: Our results revealed that treatment of Pentamidine inhibited proliferation, migration and invasion of Ishikawa and HEC-1A endometrial cancer cells. Mechanistic investigation showed that Pentamidine inhibited PI3K/AKT signaling pathway and also reduced the expression of MMP-2 and MMP-9. In addition, co-treatment with PI3K kinase inhibitor LY294002 and Pentamidine leaded to increased repression of cell viability and the protein expression of p-AKT in Ishikawa cells. CONCLUSIONS: Pentamidine suppresses PI3K/AKT signaling pathway, and inhibits proliferation, migration and invasion of EC cells. These findings suggested that Pentamidine might be a potential candidate for treating EC through PI3K/AKT pathway.