Synthesis and in-vitro study of a novel ligustrazine diselenide as a potential chemotherapy drug for lung adenocarcinoma.

A novel ligustrazine diselenide, 1,2-bis ((3,5,6-trimethylpyrazin-2-yl) methyl) diselenide (Se2), for potential treatment on adenocarcinoma of lung cancer was successfully synthesized and fully characterized by various analytical approaches. Cytotoxic, antiproliferative and apoptosis-triggering mechanism of Se2 compound have been investigated through human lung adenocarcinoma (LUAD) cell line A549. The study found that Se2 significantly inhibit the proliferation of A549 cells in a dose-dependent manner. Flow cytometry showed that Se2 induced cell arrest and apoptosis in S and G2/M phase, and the apoptotic effect of Se2 were associated with the increase of caspase 3 and PARP-1 level approved by western blot assay. Further mechanism study results suggested that Se2 suppressed the migration,invasion and colony formation of A549 cells, significantly inhibited the PI3K/Akt/m-TOR signaling pathway. The study indicated that Se2 is a bioactive substance that can induce apoptosis of A549 cells in-vitro, and it is a potent candidate drug for LUAD.

MiRNA-106a-5p Promotes Laryngeal Carcinoma Proliferation and Migration Through PI3K/AKT/m-TOR Pathway by AKTIP.

Background: Laryngeal cancer (LC) remains one of the most common tumors of the respiratory tract, the exact pathogenesis remains unclear. MiRNA-106a-5p is aberrantly expressed in a variety of cancers and plays a pro- or anti-cancer role, but is indistinct in LC. Objectives: Showing the role of miRNA-106a-5p in the development of LC. Materials and Methods: Quantitative reverse transcription-polymerase chain reaction was used for miR-106a-5p measurement in clinical samples and LC cell lines (AMC-HN8 and TU212), first. The expression of miR-106a-5p was inhibited by inhibitor, then followed clonogenic and flow cytometric assays for cell proliferation; wood healing, and Transwell assays for cell migration. Dual luciferase reporter assay was performed for interaction verification, and the activation of the signal pathway was detected by western blots. Results: MiR-106a-5p was significantly over-expressed in LC tissues and cell lines. The proliferation ability of the LC cells was significantly reduced after miR-106a-5p inhibition, and most LC cells were stagnated in the G1 phase. The migration and invasion ability of the LC cells was decreased after the miR-106a-5p knockdown. Further, we found that miR-106-5a is bound with 3'-UTR of AKT interacting protein (AKTIP) mRNA specifically, and then activate PI3K/AKT/m-TOR pathway in LC cells. Conclusions: A new mechanism was uncovered that miR-106a-5p promotes LC development via AKTIP/PI3K/AKT/m-TOR axis, which guides clinical management and drug discovery.

Bone morphogenetic protein 9 enhances osteogenic and angiogenic responses of human amniotic mesenchymal stem cells cocultured with umbilical vein endothelial cells through the PI3K/AKT/m-TOR signaling pathway.

BACKGROUND: Neovascularization plays an essential part in bone fracture and defect healing, constructing tissue engineered bone that targets bone regeneration. Bone morphogenetic protein 9 (BMP9) is a regular indicator that potentiates osteogenic and angiogenic differentiation of MSCs. OBJECTIVES: To investigate the effects of BMP9 on osteogenesis and angiogenesis of human amniotic mesenchymal stem cells (hAMSCs) cocultured with human umbilical vein endothelial cells (HUVECs) and determine the possible underlying molecular mechanism. RESULTS: The isolated hAMSCs expressed surface markers of MSCs. hAMSCs cocultured with HUVECs enhance osteogenic differentiation and upregulate the expression of angiogenic factors. BMP9 not only potentiates angiogenic signaling of hAMSCs cocultured with HUVECs also increases ectopic bone formation and subcutaneous vessel invasion. Mechanically, the coupling effect between osteogenesis and angiogenesis induced by BMP9 was activated by the BMP/Smad and PI3K/AKT/m-TOR signaling pathways. CONCLUSIONS: BMP9-enhanced osteoblastic and angiogenic differentiation in cocultivation with hAMSCs and HUVECs in vitro and in vivo also provide a chance to harness the BMP9-regulated coordinated effect between osteogenic and angiogenic pathways through BMP/Smad and PI3K/AKT/m-TOR signalings. MATERIALS AND METHODS: The ALP and Alizarin Red S staining assay to determine the effects of osteoblastic differentiation. RT-qPCR and western blot was measured the expression of angiogenesis-related factors. Ectopic bone formation was established and retrieved bony masses were subjected to histochemical staining. The angiogenesis ability and vessel invasion were subsequently determined by immunofluorescence staining. Molecular mechanisms such as the BMP/Smad and PI3K/AKT/m-TOR signaling pathways were detected by ELISA and western blot analysis.

Thymoquinone Potentiates the Effect of Phenytoin against Electroshock-Induced Convulsions in Rats by Reducing the Hyperactivation of m-TOR Pathway and Neuroinflammation: Evidence from In Vivo, In Vitro and Computational Studies.

Epilepsy is a chronic neurodegenerative disease characterized by multiple seizures, hereto 35% of patients remain poor responders. Phenytoin (PHT; 20 and 40 mg/kg) and thymoquinone (THQ; 40 and 80 mg/kg) were given alone and as a low dose combination for 14 days (p.o), prior to challenge with maximal electroshock (MES; 180 mA, 220 V, 0.2 s). Apart from observing convulsions, hippocampal mTOR, IL-1ß, IL-6 and TNF-α levels were measured. Hippocampal histomorphological analysis was also conducted. In vitro cell line studies and molecular docking studies were run in parallel. The results revealed the synergistic potential of the novel duo-drug combination regimen: PHT (20 mg/kg) and THQ (40 mg/kg) against MES-induced convulsions. MES amplified signaling through mTOR, and inflated the levels of proinflammatory markers (IL-1ß, IL-6 and TNF-α), which was significantly averted (p < 0.001) with the said drug combination. The computational studies revealed that PHT and THQ cooperatively bind the active site on Akt (upstream target of m-TOR) and establish a good network of intermolecular interactions, which indicates the sequential inhibition of PI3K/Akt/m-TOR signaling with the combination. The combination also increased cell viability by 242.81% compared to 85.66% viability from the the toxic control. The results suggest that the PHT and THQ in combination possesses excellent anticonvulsant and neuroprotective effects.

Astilbin ameliorates oxidative stress and apoptosis in D-galactose-induced senescence by regulating the PI3K/Akt/m-TOR signaling pathway in the brains of mice.

An increasing amount of evidence has shown that injection of D-galactose (D-gal) can mimic natural aging that typically is associated with brain injury. Oxidative stress and apoptosis has been shown to play an essential role in aging process. The purpose of this study was to investigate the protective effectsof astilbin (ASB) on D-Gal-induced agingin miceand to further explore the underlying mechanisms. We randomly divided 50 mice into 5 groups.To establish this model of aging, 40micewere intraperitoneally administered D-Gal (500 mg/kg). The mice in the treatmentgroupswere intragastricaly administratedASB at doses of 40 and 80 mg/kg. H&E and TUNEL staining were used to determine the effect of ASB on the number of apoptotic cells in the brain. Furthermore, biochemical indices of serum, oxidative stress factors, and apoptosis factors were determined to clarify the underlying mechanism using reagent test kits and western blotting. The results showed that varying doses of ASB could improve D-Gal-induced histopathological damageand significantly alleviatedthe aging induced by D-Galin mice. ASB remarkably decreased the activities of malondialdehyde (MDA)(p < 0.01)and Acetyl cholinesterase (AChE)(p < 0.05) and markedlyincreased the content of catalase (CAT)(p < 0.01)and superoxide dismutase (SOD)(p < 0.01), respectively. In addition, Western blotting revealed thatASB treatment (40 mg/kg)attenuated the D-gal-induced Bax and Caspase 3 protein expression(p < 0.01) and reversed the increase in Bcl-2protein expressionin brain. Moreover, ASB treatment significantly upregulated the protein expression ofp-PI3K/PI3K and altered the p-Akt/Akt ratio (p < 0.05), while inhibiting the expression of p-m-TOR relative to m-TOR(p < 0.05). Moreover, the expression of P53 tended to decreasein the low ASB treatmentgroup (40 mg/kg), whereas no change was observed in the high ASB treatmentgroup (80 mg/kg). In the intestinal flora, the richness of the normal group and the ASB group was higher than that of the D-Gal group. Heat map analysis also showed that ASB promoted Lactobacillus and other probiotics and also confirmed the advantages of ASB. The observed changes in intestinal flora further verified the efficacy of ASB.

Propofol Ameliorates ox-LDL-Induced Endothelial Damage Through Enhancing Autophagy via PI3K/Akt/m-TOR Pathway: A Novel Therapeutic Strategy in Atherosclerosis.

Objective: Atherosclerosis (AS) represents a common age-associated disease, which may be accelerated by oxidized low-density lipoprotein (ox-LDL)-induced endothelial cell injury. This study aimed to investigate the effects of Propofol on ox-LDL-induced endothelial damage and the underlying molecular mechanisms. Methods: Human umbilical vein endothelial cells (HUVECs) were exposed to ox-LDL to induce endothelial damage. HUVECs were pretreated with 0, 5, 25 and 100°µM Propofol, followed by exposure to 100°µg/ml ox-LDL for 24°h. Cell viability was assessed by cell counting kit-8 (CCK-8) assay. The expression of autophagy- and apoptosis-related proteins was detected via western blot. Autophagosome was investigated under a transmission electron microscope. After co-treatment with autophagy inhibitor Bafilomycin A1 or si-Beclin-1, cell apoptosis was detected by flow cytometry. Furthermore, under cotreatment with PI3K activator 740Y-P, PI3K/Akt/m-TOR pathway- and autophagy-related proteins were examined by western blot. Results: With a concentration-dependent manner, Propofol promoted the viability of HUVECs exposed to ox-LDL, and increased LC3-II/I ratio and Beclin-1 expression, and decreased P62 expression. The formation of autophagosome was enhanced by Propofol. Furthermore, Propofol treatment elevated Bcl-2/Bax ratio and lowered Caspase-3 expression. Bafilomycin A1 or si-Beclin-1 distinctly ameliorated the inhibitory effects of Propofol on apoptosis in ox-LDL-exposed HUVECs. Moreover, Propofol lowered the activation of PI3K/Akt/m-TOR pathway in HUVECs under exposure to ox-LDL. However, its inhibitory effects were weakened by 740Y-P. Conclusion: Collectively, this study revealed that Propofol could ameliorate ox-LDL-induced endothelial damage through enhancing autophagy via PI3K/Akt/m-TOR pathway, which might offer a novel therapeutic strategy in AS.

BW373U86 upregulates autophagy by inhibiting the PI3K/Akt pathway and regulating the mTOR pathway to protect cardiomyocytes from hypoxia-reoxygenation injury.

The purpose of this study was to explore the protective effect of BW373U86 (a δ-opioid receptor (DOR) agonist) on ischemia-reperfusion (I/R) injury in rat cardiomyocytes and its underlying mechanism. Primary rat cardiomyocytes were cultured and pretreated with BW373U86 for intervention. The cardiomyocytes were cultured under the condition of 94% N2 and 5% CO2 for 24 h to perform hypoxia culture and conventionally cultured for 12 h to perform reoxygenation culture. The cell viability of cardiomyocytes was detected by an MTT assay (Sigma-Aldrich). The autophagy lysosome levels in cardiomyocytes were evaluated by acidic vesicular organelles with dansylcadaverine (MDC) staining (autophagy test kit, Kaiji Biology, kgatg001). The protein expression levels of LC3, p62, and factors in the PI3K/Akt/mTOR signaling pathway were detected by Western blot. Pretreatment with BW373U86 could improve the cell viability of cardiomyocytes with hypoxia-reoxygenation (H/R) injury (p < 0.05). Interestingly, after coculture of BW373U86 and PI3K inhibitor (3-methyladenine), the protein expression levels of p-Akt in cardiomyocytes were markedly increased in comparison with those in the BW373U86 group (p < 0.05). However, there were no significant differences in the protein expression levels of mTOR between the coculture group and the BW373U86 group (p > 0.05). BW373U86 upregulated autophagy to protect cardiomyocytes from H/R injury, which may be related to the PI3K/Akt/m TOR pathway.

[Triptolide induces autophagy of ovarian granulosa cells via PI3K/AKT/m TOR pathway].

The aim of this paper was to observe the concentration,time and mechanism of autophagy induced by triptolide( TP) in ovarian granulosa cells( OGCs). CCK-8 method was used to compare the inhibitory effects of TP at different concentrations on primary cultured rat OGCs and IC50 was calculated. The effects of TP at different concentrations and time points on the expression of OGCs autophagy factor protein and the cascade of PI3 K/AKT/m TOR pathway were detected by Western blot. The effects of TP,autophagy inducer( brefeldin A) and PI3 K/m TOR inhibitor( NVP-BEZ235) on the expression of PI3 K/AKT/m TOR cascade and autophagy related factor protein were detected by Western blot. The results show that the IC50 of different concentrations of TP on OGCs of rat ovary was14. 65 µmol·L-1,and the minimum inhibitory concentration of TP was 0. 1 µmol·L-1( 100 nmol·L-1). Compared with the control group,the expression levels of beclin1 and LC3Ⅱ in each group were significantly higher than those in the control group( P<0. 05 or P<0. 01). After 12 hours of treatment with TP,brefeldin A and NVP-BEZ235,respectively,compared with the control group,TP could significantly promote the expression level of downstream autophagy effect or molecule beclin1,LC3Ⅱ and inhibit the expression level of LC3Ⅰ,p62 protein( P<0. 05 or P< 0. 01). Moreover,the expression of beclin1 and LC3Ⅱ/LC3Ⅰ in TP group was higher than that in brefeldin A group( P<0. 05 or P<0. 01),and the expression of p62 in TP group was lower than that in brefeldin A group( P<0. 05 or P<0. 01). At the same time,TP could significantly inhibit the expression of p-PI3 K,p-AKT,p-mTOR protein,and the inhibitory effect of TP was better than that of NVP-BEZ235 group. This study suggests that 100 nmol·L-1 TP could induce OGCs autophagy successfully in cultured rat ovary for 12 h; TP may induce OGCs autophagy by inhibiting PI3 k/Akt/m TOR signaling pathway.

Triptolide induces autophagy of ovarian granulosa cells via PI3K/AKT/m TOR pathway / 中国中药杂志

The aim of this paper was to observe the concentration,time and mechanism of autophagy induced by triptolide( TP) in ovarian granulosa cells( OGCs). CCK-8 method was used to compare the inhibitory effects of TP at different concentrations on primary cultured rat OGCs and IC50 was calculated. The effects of TP at different concentrations and time points on the expression of OGCs autophagy factor protein and the cascade of PI3 K/AKT/m TOR pathway were detected by Western blot. The effects of TP,autophagy inducer( brefeldin A) and PI3 K/m TOR inhibitor( NVP-BEZ235) on the expression of PI3 K/AKT/m TOR cascade and autophagy related factor protein were detected by Western blot. The results show that the IC50 of different concentrations of TP on OGCs of rat ovary was14. 65 μmol·L-1,and the minimum inhibitory concentration of TP was 0. 1 μmol·L-1( 100 nmol·L-1). Compared with the control group,the expression levels of beclin1 and LC3Ⅱ in each group were significantly higher than those in the control group( P<0. 05 or P<0. 01). After 12 hours of treatment with TP,brefeldin A and NVP-BEZ235,respectively,compared with the control group,TP could significantly promote the expression level of downstream autophagy effect or molecule beclin1,LC3Ⅱ and inhibit the expression level of LC3Ⅰ,p62 protein( P<0. 05 or P< 0. 01). Moreover,the expression of beclin1 and LC3Ⅱ/LC3Ⅰ in TP group was higher than that in brefeldin A group( P<0. 05 or P<0. 01),and the expression of p62 in TP group was lower than that in brefeldin A group( P<0. 05 or P<0. 01). At the same time,TP could significantly inhibit the expression of p-PI3 K,p-AKT,p-mTOR protein,and the inhibitory effect of TP was better than that of NVP-BEZ235 group. This study suggests that 100 nmol·L-1 TP could induce OGCs autophagy successfully in cultured rat ovary for 12 h; TP may induce OGCs autophagy by inhibiting PI3 k/Akt/m TOR signaling pathway.

The Effects of Autophagy and PI3K/AKT/m-TOR Signaling Pathway on the Cell-Cycle Arrest of Rats Primary Sertoli Cells Induced by Zearalenone.

A high concentration of Zearalenone (ZEA) will perturb the differentiation of germ cells, and induce a death of germ cells, but the toxic mechanism and molecular mechanism remain unclear. The Sertoli cells (SCs) play an irreplaceable role in spermatogenesis. In order to explore the potential mechanism of ZEA male reproductive toxicity, we studied the effects of ZEA on cell proliferation, cell-cycle distribution, cell-cycle-related proteins and autophagy-related pathway the PI3K/Akt/mTOR signaling in primary cultured rats SCs, and the effects of autophagy and PI3K/AKT/m TOR signaling pathway on the SCs cell-cycle arrest induced by ZEA treated with the autophagy promoter RAPA, autophagy inhibitor CQ, and the PI3K inhibitor LY294002, respectively. The data revealed that ZEA could inhibit the proliferation of SCs by arresting the cell cycle in the G2/M phase and trigger the autophagy via inhibiting the PI3K/Akt/m TOR signaling pathway. Promoting or inhibiting the level of autophagy could either augment or reverse the arrest of cell cycle. And it was regulated by PI3K/Akt/m TOR signaling pathway. Taken together, this study provides evidence that autophagy and PI3K/Akt/m TOR signaling pathway are involved in regulating rats primary SCs cell-cycle arrest due to ZEA in vitro. To some extent, ZEA-induced autophagy plays a protective role in this process.

Vascular endothelial growth factor over-expressed mesenchymal stem cells-conditioned media ameliorate palmitate-induced diabetic endothelial dysfunction through PI-3K/AKT/m-TOR/eNOS and p38/MAPK signaling pathway.

In the pathogenesis of diabetes mellitus (DM), islet microvasculares are severely damaged due to glucolipotoxicity and other reasons. Vascular endothelial growth factor (VEGF) is an indispensable and specific angiogenic factor in the pathogenesis and treatment of diabetic islet microvascular disease. Mesenchymal stem cells (MSCs) are regarded as a promising treatment of diabetes because of their immunosuppressive effect and multipotential differentiation potency. In this study, we tested whether MSCs over-expressing VEGF conditioned medium (MSC-VEGF-CM) could ameliorate pancreatic islet endothelial cells (MS-1) dysfunction induced by a common diabetic inducer palmitate (PA). We found that cell survival and migration were restrained by PA and partly repaired by the pro-protected of MSC-VEGF-CM. Meanwhile, PI-3K/AKT/m-TOR/eNOS and p38/MAPK signaling pathways were also up-regulated. Though apoptosis-related proteins, caspase-3 and caspase-9, had no significantly suppressed between MSC-VEGF-CM and MSC-CM alone, the expression levels of vascular surface factors such as CD31, VE-cadherin, occludin and ICAM-1, were remarkably up-regulated by the pro-protected of MSC-VEGF-CM. Our data suggested that MSC-VEGF-CM had therapeutic effect on the PA-induced dysfunction through the re-activation of PI-3K/AKT/m-TOR/eNOS and p38/MAPK signaling pathways.

[Study of isobutyrylshikonin inhibiting proliferation of colon carcinoma cells through PI3K/Akt/m-TOR pathway].

To investigate the inhibitory effect of isobutyrylshikonin on the growth of human colon carcinoma cells in vitro and its effect on the PI3K/Akt/m-TOR pathway. MTT assay was used to detect the inhibitory effect of different concentrations (0, 6.25, 12.5, 25, 50, 100 mg·L⁻¹) of isobutyrylshikonin on the proliferation of human colon carcinoma cell HT29 at 24, 48 h. CCK-8 method was used to detect the inhibitory effect of isobutyrylshikonin on HT29, HCT116, DLD-1 and Caco-2 at 48 h. AnnexinV/propidium iodide staining was applied in detecting the apoptoticrate of HT29 cells treated with different concentrations of isobutyrylshikonin at 24 h and 48 h. Cycletest plus DNA was employed to analyze HT29 apoptosis and cell cycle after 48 h treatment with isobutyrylshikonin at different concentrations. Western blot and RT-PCR assay were used to examine the protein and mRNA expressions of PI3K, p-PI3K, Akt, p-Akt and m-TOR. The results showed that isobutyrylshikonin inhibited the proliferation of different human colon carcinoma cells, and the inhibition rate was in a dose-dependent manner. Isobutyrylshikonin induced apoptosis mainly in the early stage and blocked cells in the G0/G1 or G2/M phase. Isobutyrylshikonin reduced the protein expressions of PI3K, p-PI3K, Akt, p-Akt, m-TOR and the mRNA expressions of PI3K, Akt, m-TOR in a dose-dependent manner. Isobutyrylshikonin can significantly inhibit the proliferation, induce the early apoptosis and change the cycle distribution in colon carcinoma cells.This biological effect may be correlated with the inhibition of PI3K/AKT/m-TOR pathway.

Study of isobutyrylshikonin inhibiting proliferation of colon carcinoma cells through PI3K/Akt/m-TOR pathway / 中国中药杂志

To investigate the inhibitory effect of isobutyrylshikonin on the growth of human colon carcinoma cells and its effect on the PI3K/Akt/m-TOR pathway. MTT assay was used to detect the inhibitory effect of different concentrations (0, 6.25, 12.5, 25, 50, 100 mg·L⁻¹) of isobutyrylshikonin on the proliferation of human colon carcinoma cell HT29 at 24, 48 h. CCK-8 method was used to detect the inhibitory effect of isobutyrylshikonin on HT29, HCT116, DLD-1 and Caco-2 at 48 h. AnnexinV/propidium iodide staining was applied in detecting the apoptoticrate of HT29 cells treated with different concentrations of isobutyrylshikonin at 24 h and 48 h. Cycletest plus DNA was employed to analyze HT29 apoptosis and cell cycle after 48 h treatment with isobutyrylshikonin at different concentrations. Western blot and RT-PCR assay were used to examine the protein and mRNA expressions of PI3K, p-PI3K, Akt, p-Akt and m-TOR. The results showed that isobutyrylshikonin inhibited the proliferation of different human colon carcinoma cells, and the inhibition rate was in a dose-dependent manner. Isobutyrylshikonin induced apoptosis mainly in the early stage and blocked cells in the G₀/G₁ or G₂/M phase. Isobutyrylshikonin reduced the protein expressions of PI3K, p-PI3K, Akt, p-Akt, m-TOR and the mRNA expressions of PI3K, Akt, m-TOR in a dose-dependent manner. Isobutyrylshikonin can significantly inhibit the proliferation, induce the early apoptosis and change the cycle distribution in colon carcinoma cells.This biological effect may be correlated with the inhibition of PI3K/AKT/m-TOR pathway.