Integrating cfDNA liquid biopsy and organoid-based drug screening reveals PI3K signaling as a promising therapeutic target in colorectal cancer.

BACKGROUND: The current precision medicine relies on biomarkers, which are mainly obtained through next-generation sequencing (NGS). However, this model failed to find effective drugs for most cancer patients. This study tried to combine liquid biopsy with functional drug tests using organoid models to find potential drugs for cancer patients. METHODS: Colorectal cancer (CRC) patients were prospectively enrolled and blood samples were collected from patients before the start of treatment. Targeted deep sequencing of cfDNA samples was performed using a 14-gene panel. Gastrointestinal (GI) cancer organoids were established and PI3K and mTOR inhibitors were evaluated on organoid models. RESULTS: A total of 195 mutations were detected across 58 cfDNA samples. The most frequently mutated genes were KRAS, TP53, PIK3CA, and BRAF, all of which exhibited higher mutation rates than tissue biopsy. Although 81% of variants had an allele frequency of less than 1%, certain mutations in KRAS, TP53, and SMAD4 had high allele frequencies exceeding 10%. Notably, among the seven patients with high allele frequency mutations, six had metastatic tumors, indicating that a high allele frequency of ctDNA could potentially serve as a biomarker of later-stage cancer. A high rate of PIK3CA mutation (31 out of 67, or 46.3%) was discovered in CRC patients, suggesting possible tumor progression mechanisms and targeted therapy opportunities. To evaluate the value of anti PI3K strategy in GI cancer, different lines of GI cancer organoids were established. The organoids recapitulated the morphologies of the original tumors. Organoids were generally insensitive to PI3K inhibitors. However, CRC-3 and GC-4 showed response to mTOR inhibitor Everolimus, and GC-3 was sensitive to PI3Kδ inhibitor Idelalisib. The CRC organoid with a PIK3CA mutation showed greater sensitivity to the PI3K inhibitor Alpelisib than wildtype organoids, suggesting potential treatment options for the corresponding patients. CONCLUSION: Liquid biopsy holds significant promise for improving precision treatment and tumor prognosis in colorectal cancer patients. The combination of biomarker-based drug prediction with organoid-based functional drug sensitivity assay may lead to more effective cancer treatment.

Core Target and Key Signal Pathway of Xiaoluo in the Treatment of Thyroid Cancer.

Context: At present, hormone therapy and surgery are the main treatments for thyroid cancer, and they have a quick effect but a high recurrence rate. Also, the side effects are significant. it's extremely urgent to explore treatments that can take into account both therapeutic benefits and side effects. Objective: The study intended to explore whether Xiaoluo has an inhibitory effect on the proliferation of thyroid-cancer cells in vitro and to examine the core target and key signaling pathway of Xiaoluo in the treatment of thyroid cancer, using the thyroid-cancer cell line SW579. Design: The research team performed an in-vitro study. Setting: The study took place at the College of Pharmacy at Harbin University of Commerce in Harbin, China. Outcome Measures: The research team used a Western blot analysis to detect the expression of apoptosis proteins-B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), and Caspase-3-and the activity related to the signaling pathways phosphoinositide 3-kinase (PI3K)/ protein kinase B (AKT)/ mammalian target of rapamycin 1 (mTORC1). The team measured optical densities and inhibition rates for the 1, 2, 5, 10, and 15 mg/mL Xiaokuo groups and for a negative control group. The research team measured apoptosis, expression of Bcl-2, Bax, and Caspase-3, and expression of P13K, AKT, and mTor for the 10 µmol/L LY294002, 10 mg/mL Xiaoluo, 100 ng/mL IGF-1, and 100 ng/mL IGF-1+10 mg/mL Xiaoluo groups and for the blank control group. Results: The inhibition of SW579 cell proliferation increased with each increase in the Xiaoluo concentration from 1-15 mg/mL, and the inhibition rate reached 49.63% when the concentration was 15 mg/ml. The Xiaoluo group's late and total apoptosis rates were significantly higher (both P < .01) than those of the blank control group. The Xiaoluo group's expression of the Bcl-2 protein was significantly lower (P < .05), and its expressions of Bax and Caspase-3 were significantly higher (both P < .01) than those of the blank control group. The Xiaoluo group's expressions of P-PI3K, P-Akt, and P-MTOR were significantly lower than those of the blank group (all P < .01). These findings were comparable to those that occurred with use of the PI3K/AKT/mTORC1 signaling pathway inhibitor LY294002. Conclusions: Xiaoluo exerts its antithyroid-cancer effects through the induction of apoptosis in thyroid cancer cells through the inhibition of the PI3K/AKT/mTORC1 signaling pathway. Xiaoluo may serve as a potential therapeutic agent for the treatment of thyroid cancer.

Dietary supplementation of clinically utilized PI3K p110α inhibitor extends the lifespan of male and female mice.

Diminished insulin and insulin-like growth factor-1 signaling extends the lifespan of invertebrates1-4; however, whether it is a feasible longevity target in mammals is less clear5-12. Clinically utilized therapeutics that target this pathway, such as small-molecule inhibitors of phosphoinositide 3-kinase p110α (PI3Ki), provide a translatable approach to studying the impact of these pathways on aging. Here, we provide evidence that dietary supplementation with the PI3Ki alpelisib from middle age extends the median and maximal lifespan of mice, an effect that was more pronounced in females. While long-term PI3Ki treatment was well tolerated and led to greater strength and balance, negative impacts on common human aging markers, including reductions in bone mass and mild hyperglycemia, were also evident. These results suggest that while pharmacological suppression of insulin receptor (IR)/insulin-like growth factor receptor (IGFR) targets could represent a promising approach to delaying some aspects of aging, caution should be taken in translation to humans.

Selaginella tamariscina Inhibits Glutamate-Induced Autophagic Cell Death by Activating the PI3K/AKT/mTOR Signaling Pathways.

Glutamate-induced neural toxicity in autophagic neuron death is partially mediated by increased oxidative stress. Therefore, reducing oxidative stress in the brain is critical for treating or preventing neurodegenerative diseases. Selaginella tamariscina is a traditional medicinal plant for treating gastrointestinal bleeding, hematuria, leucorrhea, inflammation, chronic hepatitis, gout, and hyperuricemia. We investigate the inhibitory effects of Selaginella tamariscina ethanol extract (STE) on neurotoxicity and autophagic cell death in glutamate-exposed HT22 mouse hippocampal cells. STE significantly increased cell viability and mitochondrial membrane potential and decreased the expression of reactive oxygen species, lactate dehydrogenase release, and cell apoptosis in glutamate-exposed HT22 cells. In addition, while glutamate induced the excessive activation of mitophagy, STE attenuated glutamate-induced light chain (LC) 3 II and Beclin-1 expression and increased p62 expression. Furthermore, STE strongly enhanced the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) phosphorylation activation. STE strongly inhibited glutamate-induced autophagy by activating the PI3K/Akt/mTOR signaling pathway. In contrast, the addition of LY294002, a PI3K/Akt inhibitor, remarkably suppressed cell viability and p-Akt and p62 expression, while markedly increasing the expression of LC3 II and Beclin-1. Our findings indicate that autophagy inhibition by activating PI3K/Akt/mTOR phosphorylation levels could be responsible for the neuroprotective effects of STE on glutamate neuronal damage.

Wild type and gain of function mutant TP53 can regulate the sensitivity of pancreatic cancer cells to chemotherapeutic drugs, EGFR/Ras/Raf/MEK, and PI3K/mTORC1/GSK-3 pathway inhibitors, nutraceuticals and alter metabolic properties.

TP53 is a master regulator of many signaling and apoptotic pathways involved in: aging, cell cycle progression, gene regulation, growth, apoptosis, cellular senescence, DNA repair, drug resistance, malignant transformation, metastasis, and metabolism. Most pancreatic cancers are classified as pancreatic ductal adenocarcinomas (PDAC). The tumor suppressor gene TP53 is mutated frequently (50-75%) in PDAC. Different types of TP53 mutations have been observed including gain of function (GOF) point mutations and various deletions of the TP53 gene resulting in lack of the protein expression. Most PDACs have point mutations at the KRAS gene which result in constitutive activation of KRas and multiple downstream signaling pathways. It has been difficult to develop specific KRas inhibitors and/or methods that result in recovery of functional TP53 activity. To further elucidate the roles of TP53 in drug-resistance of pancreatic cancer cells, we introduced wild-type (WT) TP53 or a control vector into two different PDAC cell lines. Introduction of WT-TP53 increased the sensitivity of the cells to multiple chemotherapeutic drugs, signal transduction inhibitors, drugs and nutraceuticals and influenced key metabolic properties of the cells. Therefore, TP53 is a key molecule which is critical in drug sensitivity and metabolism of PDAC.

Proangiogenesis effects of compound danshen dripping pills in zebrafish.

BACKGROUND: The compound Danshen Dripping Pill (CDDP), which is a mixture of extracts from Radix Salviae and Panax notoginseng, is a patented traditional Chinese medicine that is widely used in multiple countries for relieving coronary heart disease (CHD), but its pharmacological mechanism has not been fully elucidated. In this study, we screened the key pharmacological pathways and targets of CDDP that act on CHD using a network pharmacology-based strategy, and the angiogenic activity of CDDP was directly visually investigated in zebrafish embryos in vivo. METHODS: The potential therapeutic targets and pathways were predicted through a bioinformatics analysis. The proangiogenic effects of CDDP were examined using vascular sprouting assays on subintestinal vessels (SIVs) and optic arteries (OAs) as well as injury assays on intersegmental vessels (ISVs). Pharmacological experiments were applied to confirm the pathway involved. RESULTS: Sixty-five potential therapeutic targets of CDDP on CHD were identified and enriched in the PI3K/AKT and VEGF/VEGFR pathways. An in vivo study revealed that CDDP promoted angiogenesis in SIVs and OAs in a dose-dependent manner and relieved the impairments in ISVs induced by lenvatinib, a VEGF receptor kinase inhibitor (VRI). In addition, Vegfaa and Kdrl expression were significantly upregulated after CDDP treatment. Furthermore, the proangiogenic effect of CDDP could be abolished by PI3K/AKT pathway inhibitors. CONCLUSIONS: CDDP has a proangiogenic effect, the mechanism of which involves the VEGF/VEGFR and PI3K/AKT signaling pathways. These results suggest a new insight into the cardiovascular protective effect of CDDP.

Carbonic anhydrase IX-targeted H-APBC nanosystem combined with phototherapy facilitates the efficacy of PI3K/mTOR inhibitor and resists HIF-1α-dependent tumor hypoxia adaptation.

BACKGROUND: Non-redundant properties such as hypoxia and acidosis promote tumor metabolic adaptation and limit anti-cancer therapies. The key to the adaptation of tumor cells to hypoxia is the transcriptional and stable expression of hypoxia-inducible factor-1 alpha (HIF-1α). The phosphorylation-activated tumorigenic signal PI3K/AKT/mTOR advances the production of downstream HIF-1α to adapt to tumor hypoxia. Studies have elucidated that acid favors inhibition of mTOR signal. Nonetheless, carbonic anhydrase IX (CAIX), overexpressed on membranes of hypoxia tumor cells with pH-regulatory effects, attenuates intracellular acidity, which is unfavorable for mTOR inhibition. Herein, a drug delivery nanoplatform equipped with dual PI3K/mTOR inhibitor Dactolisib (NVP-BEZ235, BEZ235) and CAIX inhibitor 4-(2-aminoethyl) benzene sulfonamide (ABS) was designed to mitigate hypoxic adaptation and improve breast cancer treatment. RESULTS: ABS and PEG-NH2 were successfully modified on the surface of hollow polydopamine (HPDA), while BEZ235 and Chlorin e6 (Ce6) were effectively loaded with the interior of HPDA to form HPDA-ABS/PEG-BEZ235/Ce6 (H-APBC) nanoparticles. The release of BEZ235 from H-APBC in acid microenvironment could mitigate PI3K/mTOR signal and resist HIF-1α-dependent tumor hypoxia adaptation. More importantly, ABS modified on the surface of H-APBC could augment intracellular acids and enhances the mTOR inhibition. The nanoplatform combined with phototherapy inhibited orthotopic breast cancer growth while reducing spontaneous lung metastasis, angiogenesis, based on altering the microenvironment adapted to hypoxia and extracellular acidosis. CONCLUSION: Taken together, compared with free BEZ235 and ABS, the nanoplatform exhibited remarkable anti-tumor efficiency, reduced hypoxia adaptation, mitigated off-tumor toxicity of BEZ235 and solved the limited bioavailability of BEZ235 caused by weak solubility.

Management of Phosphatidylinositol-3-Kinase Inhibitor-Associated Hyperglycemia.

Phosphatidylinositol-3-kinase (PI3K) pathway hyperactivation has been associated with the development of cancer and treatment resistance. PI3K inhibitors are now used to treat hormone receptor-positive (HR+), human epidermal growth factor receptor-2-negative (HER2-), PIK3CA-mutated advanced breast cancer. Hyperglycemia, a frequently observed adverse event with PI3K inhibitors (PI3Ki), is regarded as an on-target effect because inhibition of the PI3K pathway has been shown to decrease glucose transport and increase glycogenolysis and gluconeogenesis. PI3Ki-induced hyperglycemia results in a compensatory increase in insulin release, which has been shown to reduce the efficacy of treatment by reactivating the PI3K pathway in preclinical models. Patients with an absolute or relative deficiency in insulin, and those with insulin resistance or pancreatic dysfunction, may experience exacerbated or prolonged hyperglycemia. Therefore, the effective management of PI3Ki-associated hyperglycemia depends on early identification of patients at risk, frequent monitoring to allow prompt recognition of hyperglycemia and its sequelae, and initiating appropriate management strategies. Risk factors for the development of hyperglycemia include older age (≥75 years), overweight/obese at baseline, and family history of diabetes. Consultation with an endocrinologist is recommended for patients considered high risk. The management of PI3Ki-induced hyperglycemia requires an integrative approach that combines diets low in carbohydrates and glucose-lowering medications. Medications that do not affect the PI3K pathway are preferred as the primary and secondary agents for the management of hyperglycemia. These include metformin, sodium-glucose co-transporter 2 inhibitors, thiazolidinediones, and α-glucosidase inhibitors. Insulin should only be considered as a last-line agent for PI3Ki-associated hyperglycemia due to its stimulatory effect of PI3K signaling. Clinical studies show that alpelisib-associated hyperglycemia is reversible and manageable, rarely leading to treatment discontinuation. Management of PI3Ki-associated hyperglycemia in patients with breast cancer should focus on the prevention of acute and subacute complications of hyperglycemia, allowing patients to remain on anticancer treatment longer.

Glytabastan B, a coumestan isolated from Glycine tabacina, alleviated synovial inflammation, osteoclastogenesis and collagen-induced arthritis through inhibiting MAPK and PI3K/AKT pathways.

The roots of Glycine tabacina are used to treat rheumatoid arthritis (RA) and joint infection in folk medicine. Glytabastan B (GlyB), a newly reported coumestan isolated from this species, was found to significantly attenuate IL-1ß-induced inflammation in SW982 human synovial cells at 3 and 6 µM, as evidenced by the decreased levels of pro-inflammatory mediators and matrix metalloproteinases (MMPs). GlyB also suppressed RANKL-induced osteoclastogenesis, decreased the expression of osteoclastogenic markers (NFATc1, CTSK, MMP-9) and osteoclast-mediated bone resorption. Further, GlyB administration (12.5 and 25 mg/kg) significantly inhibited inflammation, osteoclast formation and disease progression in collagen-induced arthritis (CIA) mice. Integration of network pharmacology, quantitative phosphoproteomic and experimental pharmacology results revealed that these beneficial actions were closely associated with the blockade of GlyB on the activation of MAPK, PI3K/AKT and their downstream signals including NF-κB and GSK3ß/NFATc1. Drug affinity responsive target stability (DARTS) assay, cellular thermal shift (CETSA) assay and molecular docking analysis confirmed that there were direct interactions between GlyB and its target proteins ERK2, JNK1 and class Ⅰ PI3K catalytic subunit p110 (α, ß, δ and γ), which significantly contributed to the inhibition of activation of MAPK and PI3K/AKT pathways. In conclusion, these results strongly suggest GlyB is a promising multiple-target candidate for the development of agents for the prevention and treatment of RA.

Discovery of Eucalyptin C, derived from the fruits of Eucalyptus globulus Labill., as a novel selective PI3Kγ inhibitor for immunosuppressive treatment.

The fruits of Eucalyptus globulus Labill. are known to have a plenty of medicinal properties, such as anti-tumor, anti-inflammatory, and immunosuppressive activity. Our previous study found that the phloroglucinol-sesquiterpene adducts in the fruits of E. globulus were immunosuppressive active constituents, especially Eucalyptin C (EuC). Phosphoinositide 3-kinases-γ (PI3Kγ) plays a pivotal role in T cell mediated excessive immune responses. In this study, EuC was first discovered to be a novel selective PI3Kγ inhibitor with an IC50 value of 0.9 µmol·L-1 and selectivity over 40-fold towards the other PI3K isoforms. Molecular docking, molecular dynamics simulation, and cellular thermal shift assay showed that EuC bound to PI3Kγ. Furthermore, EuC suppressed the downstream of PI3Kγ to induce the apoptosis and inhibit the activation of primary spleen cells derived from allergic contact dermatitis mice. This work highlights the role of the fruits of E. globulus as a source of bioactive plant with immunosuppressive activity.

Design, Synthesis, Biological Evaluation, and Computational Studies of Novel Fluorinated Candidates as PI3K Inhibitors: Targeting Fluorophilic Binding Sites.

Highly fluorinated candidates containing anticancer pharmacophores like thiosemicarbazone (5a-e) and its cyclic analogues hydrazineylidenethiazolidine (6a-e), 2-aminothiadiazole (7a-e), and 2-hydrazineylidenethiazolidin-4-one (8a-e) were synthesized, and their cytotoxic activity was assayed against 60 tumor cell lines. Compounds 6c, 7b, and 8b displayed the most potent activity with lower toxic effects on MCF-10a. In vitro phosphatidylinositol 3-kinase (PI3K) enzyme inhibition was performed. Compound 6c displayed half-maximal inhibitory concentration (IC50, µM) values of 5.8, 2.3, and 7.9; compound 7b displayed IC50 values of 19.4, 30.7, and 73.7; and compound 8b displayed IC50 values of 77.5, 53.5, and 121.3 for PI3Kα, ß, and δ, respectively. Moreover, cell cycle progression caused cell cycle arrest at the S phase for compounds 6c and 8b and at G1/S for compound 7b, while apoptosis was induced. In silico studies; molecular docking; physicochemical parameters; and absorption, distribution, metabolism, excretion, and toxicity (ADMET) analysis were performed. The results showed that compound 6c is the most potent one with a selectivity index (SI) of 39 and is considered as a latent lead for further optimization of anticancer agents.

Curcumol Alleviates the Inflammation of Nucleus Pulposus Cells via the PI3K/Akt/NF-κB Signaling Pathway and Delays Intervertebral Disk Degeneration.

BACKGROUND: Intervertebral disk degeneration (IVDD) is closely associated with inflammatory environments. Curcumol has been shown to alleviate inflammation in various disease models, but its effects on IVDD remain unclear. In this study, we sought to determine the mechanism of curcumol in tumor necrosis factor (TNF)-α-induced nucleus pulposus cells and a mouse IVDD model. METHODS: Nucleus pulposus cells were pretreated with curcumol and then exposed to TNF-α. Cell viability was analyzed using CCK-8, and the messenger ribonucleic acid and protein levels of inflammatory cytokines and PI3K/Akt/NF-κB-related signaling molecules were detected using real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and western blotting. The mouse IVDD model was established by puncturing the C6/7 level of the caudal spine, and then it was treated with curcumol after surgery. Alcian blue/orange G staining was performed to evaluate the severity of intervertebral disk damage, and immunohistochemistry was performed to detect the expression of TNF-α. Toxicologic effects of curcumol were measured by performing hematoxylin-eosin staining and enzyme-linked immunosorbent assay. RESULTS: Curcumol reduced IL-1ß, IL-6, and TNF-α production in NPCs, and the phosphorylation of proteins in the PI3K/Akt/NF-κB signaling pathway was also decreased. The PI3K/Akt/NF-κB-related signaling molecules decreased when TNF-α-induced NPCs were treated with a PI3K inhibitor; however, curcumol did not reverse these effects. In vivo, curcumol ameliorated the progression of IVDD at the early stage and did not exert toxicologic effects. CONCLUSIONS: These results suggest a potential therapeutic use of curcumol to alleviate inflammation via the PI3K/Akt/NF-κB signaling pathway and delay the progression of IVDD.

Inhibitory Potential of Dietary Nutraceuticals on Cellular PI3K/Akt Signaling: Implications in Cancer Prevention and Therapy.

BACKGROUND: The phosphatidyl inositol-3 kinase (PI3K)/protein kinase B (Akt)/mechanistic target of rapamycin (mTOR) signaling has been associated with many cellular physiological events, such as proliferation, maturation, survival, and metabolism. Besides its role in normal cells, the pathway is often upregulated in various cancers. Due to its prominent role in the cancer progression events, it is now being considered as a target for cancer therapy and cancer chemoprevention. OBJECTIVES: The present review provides a concise outline of the role of the PI3K/Akt/mTOR pathway in carcinogenesis and progression events, including metastasis, drug resistance and stemness. Further, emphasis needs to be placed on the PI3K/Akt/mTOR pathway inhibitory potentials of various food-derived bioactive components in cancer prevention. METHODS: Data on the PI3K/Akt/mTOR inhibiting natural products and their bioactive compounds have been obtained from PubMed/Medline, Scopus, Eurekaselect, etc. Findings from the above citation databases from 2000-2021 are included in the manuscript. RESULTS: Numerous compounds from plants have been isolated and identified as anticancer agents; among these, a predominant class is nutraceuticals. The PI3K pathway is the predominant target of these natural products, and many of these drug candidates are under various stages of drug development. These compounds have shown a significant inhibitory effect on the kinase activities of PI3K and Akt, resulting in the abrogation of cancer initiation and progression events. In addition, these compounds have been shown to reverse the resistance to chemotherapeutic drugs and also reduce the population of cancer stem cells. CONCLUSION: The nutraceuticals are promising candidates as anticancer agents by blocking PI3K signaling cascades. As the PI3K is a central pathway to various receptors signaling, the dietary intervention may prove to be highly effective.

Ziziphus jujuba Mill. leaf extract restrains adipogenesis by targeting PI3K/AKT signaling pathway.

The escalation in the global prevalence of obesity has focused attention on finding novel approaches for its management. Ziziphus jujuba Mill. (ZJL) leaf extract is reported as a traditional remedy for diverse pathological conditions, including obesity. The present study investigated whether ZJL affects adipogenic differentiation in human adipocytes. Additionally, following metabolite profiling of the extract, apigenin (APG), betulinic acid (BA) and maslinic acid (MA) were selected for biological activity evaluation. The possible interactions between APG, BA, MA and target proteins with a central role in adipogenesis were assessed through molecular docking. The potential mechanisms of ZJL, APG, BA and MA were identified using transcriptional analysis through real-time quantitative PCR and protein abundance evaluation by Western blotting. The obtained results revealed a concentration-dependent reduction of accumulated lipids after ZJL, BA and MA application. The key adipogenic transcription factors peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT-enhancer-binding protein alpha (C/EBPα) were strongly decreased at a protein level by all treatments. Moreover, the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway was found to be involved in the anti-adipogenic effect of ZJL, APG and BA. Collectively, our findings indicate that ZJL and its pure compounds hampered adipocyte differentiation through PI3K/AKT inhibition. Among the selected compounds, BA exhibits the most promising anti-adipogenic activity. Furthermore, being a complex mixture of phytochemicals, the ZJL extract could be utilized as source of yet unknown bioactive leads with potential implementation in obesity management.

Gallic acid promotes the in vitro development of sheep secondary isolated follicles involving the phosphatidylinositol 3-kinase pathway.

This study was conducted to evaluate the effect of addition of gallic acid as the single antioxidant to the base medium for in vitro culture of sheep secondary follicles and if the phosphatidylinositol 3-kinase (PI3K) pathway is involved in the action of gallic acid. Secondary follicles were isolated and cultured for 12 days in α-MEM supplemented with bovine serum albumin (BSA), insulin, glutamine, hypoxanthine, transferrin, selenium, and ascorbic acid (control medium: α-MEM+) or in α-MEM supplemented with BSA, insulin, glutamine, hypoxanthine and different concentrations of gallic acid (25, 50 or 100 µM), thus replacing transferrin, selenium and ascorbic acid in the medium. Follicle morphology, glutathione (GSH), and mitochondrial activity, and meiotic resumption were evaluated. Furthermore, inhibition of PI3K pathway was performed by pretreatment with LY294002. After 12 days of culture, the follicle survival in a medium containing 100 µM gallic acid was similar (P > 0.05) to α-MEM+ and greater (P < 0.05) compared with other gallic acid concentrations. Antrum formation, follicle diameter, GSH, and mitochondrial activity, and meiotic resumption, however, were greater (P < 0.05) when 100 µM gallic acid was included in the α-MEM+ culture medium compared with the control medium. Furthermore, LY294002 inhibited (P < 0.05) follicle survival, development, and meiotic resumption stimulated by 100 µM gallic acid. In conclusion, concentration of 100 µM of gallic acid can be a substitute for transferrin, selenium, and ascorbic acid in the base medium during in vitro culture of sheep secondary follicles, inducing follicle development likely through the PI3K pathway.

Discovery of a new series of PI3K-δ inhibitors from Virtual Screening.

PI3K-δ mediates key immune cell signaling pathways and is a target of interest for treatment of oncological and immunological disorders. Here we describe the discovery and optimization of a novel series of PI3K-δ selective inhibitors. We first identified hits containing an isoindolinone scaffold using a combined ligand- and receptor-based virtual screening workflow, and then improved potency and selectivity guided by structural data and modeling. Careful optimization of molecular properties led to compounds with improved permeability and pharmacokinetic profile, and high potency in a whole blood assay.

A network-based method for mechanistic investigation and neuroprotective effect on treatment of tanshinone â against ischemic stroke in mouse.

ETHNOPHARMACOLOGICAL RELEVANCE: Tanshinone-Ⅰ (TSNⅠ), a member of the mainly active components of Salvia miltiorrhiza Bunge (Dan Shen), which is widely used for the treatment for modern clinical diseases including cardiovascular and cerebrovascular diseases, has been reported to show the properties of anti-oxidation, anti-inflammation, neuroprotection and other pharmacological actions. However, whether TSNⅠ can improve neuron survival and neurological function against transient focal cerebral ischemia (tMCAO) in mice is still a blank field. AIM OF THE STUDY: This study aims to investigate the neuroprotective effects of TSNⅠ on ischemic stroke (IS) induced by tMCAO in mice and explore the potential mechanism of TSNⅠ against IS by combining network pharmacology approach and experimental verification. MATERIALS AND METHODS: In this study, the pivotal candidate targets of TSNⅠ against IS were screened by network pharmacology firstly. Enrichment analysis and molecular docking of those targets were performed to identify the possible mechanism of TSNⅠ against IS. Afterwards, experiments were carried out to further verify the mechanism of TSNⅠ against IS. The infarct volume and neurological deficit were evaluated by 2, 3, 5-triphenyl tetrazolium chloride (TTC) staining and Longa respectively. Immunohistochemistry was used to observe neuronal death in the hippocampus and cortical regions by detecting the change of NeuN. The predicting pathways of signaling-related proteins were assessed by Western blot in vitro and in vivo experiments. RESULTS: In vivo, TSNⅠ was found to dose-dependently decrease mice's cerebral infarct volume induced by tMCAO. In vitro, pretreatment with TSNⅠ could increase cell viability of HT-22 cell following oxygen-glucose deprivation (OGD/R). Moreover, the results showed that 125 candidate targets were identified, Protein kinase B (AKT) signaling pathway was significantly enriched by Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and mitogen-activated protein kinases 1 (MAPK1) and AKT1 could be bound to TSNⅠ more firmly by molecular docking analysis, which implies that TSNⅠ may play a role in neuroprotection through activating AKT and MAPK signaling pathways. Meanwhile, TSNⅠ was confirmed to significantly protect neurons from injury induced by IS through activating AKT and MAPK signaling pathways. CONCLUSION: In conclusion, our study clarifies that the mechanism of TSNⅠ against IS might be related to AKT and MAPK signaling pathways, which may provide the basic evidence for further development and utilization of TSNⅠ.

Akebia saponin E, as a novel PIKfyve inhibitor, induces lysosome-associated cytoplasmic vacuolation to inhibit proliferation of hepatocellular carcinoma cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Hepatocellular carcinoma (HCC) is an aggressive malignancy with increasing mortality in China. Screening and identifying effective anticancer compounds from active traditional Chinese herbs for HCC are in demand. Akebia trifoliata (Thunb) Koidz, with pharmacological anti-HCC activities in clinical, has been shown in previous research. In the present research, we elucidated a potential anticancer effect of Akebia saponin E (ASE), which is isolated from the immature seeds of Akebia trifoliata (Thunb.) Koidz, and revealed that ASE could induce severe expanded vacuoles in HCC cells. But the potential mechanism of vacuole-formation and the anti-HCC effects by ASE remain uncover. AIM OF THIS STUDY: To elucidate the potential mechanism of vacuole-formation and the proliferation inhibition effects by ASE in HCC cell lines. MATERIALS AND METHODS: MTT assay, colony formation assay and flow cytometry were performed to detect cell viability. Immunofluorescence analysis was used to examine the biomarkers of endomembrane. Cells were infected with tandem mRFP-GFP-LC3 lentivirus to assess autophagy flux. RNA-seq was conducted to analyze the genome-wide transcriptional between treatment cell groups. In vitro PIKfyve kinase assay is detected by the ADP-GloTM Kinase Assay Kit. RESULTS: ASE could inhibit the proliferation of HCC with severe expanded vacuoles in vitro, and could significantly reduce the size and weight of xenograft tumor in vivo. Further, the vacuoles induced by ASE were aberrant enlarged lysosomes instead of autophagosome or autolysosomes. With cytoplasmic vacuolation, ASE induced a mTOR-independent TFEB activation for lysosomal biogenesis and a decrement of cholesterol levels in HCC cells. Furthermore, ASE could reduce the activity of PIKfyve (phosphoinositide kinase containing a FYVE-type finger), causing aberrant lysosomal biogenesis and cholesterol dyshomeostasis which triggered the expanded vacuole formation. CONCLUSION: ASE can prospectively inhibit the kinase activity of PIKfyve to induce lysosome-associated cytoplasmic vacuolation, and may be utilized as an alternative candidate to treat human HCC.

Discovery of 2-(5-(quinolin-6-yl)-1,3,4-oxadiazol-2-yl)acetamide derivatives as novel PI3Kα inhibitors via docking-based virtual screening.

PI3Kα is an attractive target for PIK3CA mutated malignant tumor and searching for lead compounds with novel scaffold is important for the development of PI3Kα inhibitors. Therefore, the strategy of docking-based virtual screening was performed to discovery potent inhibitors. The 4L2Y_A PI3Kα crystal structure was used as the model protein receptor due to its high docking reliability. After the multistep virtual screening protocol and biological evaluation, three hits were picked up and further similarity searching led to more potent 2-(5-(quinolin-6-yl)-1,3,4-oxadiazol-2-yl)acetamide derivatives ES-25 and ES-27. In addition, the primary SAR of these novel derivatives was discussed, which provide a basis for the further structural modification.

Salvia miltiorrhiza stems and leaves total phenolic acids combination with tanshinone protect against DSS-induced ulcerative colitis through inhibiting TLR4/PI3K/AKT/mTOR signaling pathway in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Salvia miltiorrhiza Bge. as a traditional Asian medicinal plant, roots and rhizomes (Danshen) are used to treat chronic hepatitis and coronary heart disease. In recent years, the medicinal value of S. miltiorrhiza stems and leaves total phenolic acids extract (JF) similar to roots and rhizomes has received increasing attention. S. miltiorrhiza roots and rhizome tanshinone extract (DT) has a good anti-inflammatory effect. AIM OF THE STUDY: To explore the therapeutic effect and possible molecular mechanisms of JF and DT alone or in combination on dextran sulfate sodium (DSS)-induced colitis mice. MATERIALS AND METHODS: Colitis was induced by received 2% DSS in drinking water for 7 consecutive days. Then mice were administered orally for 7 days. Disease activity index (DAI) scores and body weight were recorded daily. After the end of the experiment, colon was removed, colon length was measured and histopathological analysis was performed. Inflammatory factors expression was determined by ELISA, its mRNA expression was detected by real-time quantitative PCR, and the expression of related proteins on TLR4/PI3K/AKT/mTOR signal was analyzed by Western blot. RESULTS: Treatment with JF and DT alone or in combination reduced DAI scores, increase body weight, improved colon shortening, and decreased colon histology scores. In addition, the expression level of inflammatory factors was inhibited. The combination of JF and DT had a better inhibitory effect on inflammatory factors compared to JF alone. We also found that DT alone and JF combined with DT inhibited TLR4/PI3K/AKT/mTOR signaling-related proteins expression levels (including TLR4, p-PI3K p110α/PI3K p110α, p-AKT (ser473)/AKT, mTOR, p-mTOR, NF-κB p65), showing an effective anti-inflammatory effect. CONCLUSIONS: We demonstrated for the first time that, JF and DT alone or in combination effectively ameliorated DSS-induced ulcerative colitis in mice, possibly by inhibiting the TLR4/PI3K/AKT/mTOR signaling pathway.