Protein Levels Of PI3K Research Articles

Effects of Sanchinoside R1 on Growth Invasion and Migration of HL-60 Cells and Survival of AML Model Mice

To investigate the effects of sanchinoside R1 (SR1) on cell growth, invasion and migration of HL-60 cells, as well as survival of AML mice. AML mouse models were established by intravenous injection of HL-60 cells. The SR1 of 10, 25 and 50 mg/kg was intraperitoneally injected into AML models once a day for 1 week. The survival rate of mice, tumor volume and weight were measured, and protein levels of Caspase-3 and VEGF were determined by immunohistochemistry. And protein levels of p-PI3K, PI3K, p-AKT and AKT were detected by Western blot. SR1 significantly inhibited the growth of tumors (P＜0.01, r=-0.9994, -0.9952) and increased the survival rate of mice (P＜0.01). SR1 significantly increased the protein level of apoptosis-related Caspase-3(P＜0.01, r=0.9855), and decreased the protein level of migration-related protein VEGF (P＜0.01, r=-0.9848). The protein levels of p-PI3K and p-AKT were down-regulated by SR1, Thus, the relative protein levels of p-PI3K/PI3K and p-AKT/AKT were significantly decreased (P＜0.01, r=-0.9372, -0.9953). Above-mentioned effects showed a significant positive/negative correlation with the concentration of SR1. SR1 inhibits the growth, invasion and migration of tumor cells, and increas survival of mice through affecting expression of Caspase-3, VEGF, p-PI3K, and p-AKT.

Crucial role of androgen receptor in resistance and endurance trainings-induced muscle hypertrophy through IGF-1/IGF-1R- PI3K/Akt- mTOR pathway

BackgroundAndrogen receptor (AR) has been reported to play vital roles in exercise-induced increase of muscle mass in rats, but needs to be further verified and the mechanism behind remains unclear. As AR target genes, insulin growth factor-1 (IGF-1) and IGF-1 receptor (IGF-1R) promote muscle hypertrophy through activating PI3K/Akt- mammalian target of rapamycin (mTOR) pathway, a classic pathway of muscle hypertrophy. So the main purpose of this study was using AR antagonist flutamide to demonstrate AR’s effect on training-induced muscle hypertrophy and its possible mechanism: IGF-1/IGF-1R- PI3K/Akt- mTOR pathway?MethodsForty-eight Sprague Dawley male rats aged 7 weeks were randomly divided into six groups: control (C), flutamide (F), resistance training (R), resistance training plus flutamide (R + F), endurance training (E), and endurance training plus flutamide (E + F) groups. Flutamide was used to block AR in rats. Rats in R and R + F groups fulfilled 3 weeks of ladder climbing with progressively increased load, while E and E + F rats completed 3-week moderate intensity aerobic exercise on a treadmill. The relative muscle mass (muscle mass/body weight) of rats was detected. Serum levels of testosterone and IGF-1 of rats were determined by ELISA, and mRNA levels of IGF-1R and mTOR in muscles by real-time PCR. Protein levels of AR, IGF-1, IGF-1R, mTOR, PI3K, Akt, p-PI3K and p-Akt in muscles were detected by Western blot.Results(1) The training-induced rise in the relative muscle mass and the expression levels of AR were only found in the gastrocnemius of R rats and in the soleus of E rats (selective muscle hypertrophy), which were blocked by flutamide. (2) Serum testosterone in the R and E rat were increased, and flutamide exerted no effect. (3) The levels of IGF-1, IGF-1R and mTOR as well as the activities of PI3K and Akt were enhanced selectively (in the gastrocnemius of R rats and in the soleus of E rats), which were reduced by flutamide. Conclusions: AR exerted an essential role in both resistance training and endurance training-induced muscle hypertrophy, which was mediated at least partly through IGF-1/IGF-1R- PI3K/Akt- mTOR pathway.

Local Delivery of Gemcitabine Inhibits Pancreatic and Cholangiocarcinoma Tumor Growth by Promoting Epidermal Growth Factor Receptor Degradation.

Gemcitabine is clinically used to treat certain types of cancers, including pancreatic and biliary cancer. We investigated the signal transduction pathways underlying the local antitumor effects of gemcitabine-eluting membranes (GEMs) implanted in pancreatic/biliary tumor-bearing nude mice. Here, we report that GEMs increased the E3 ubiquitin ligase c-CBL protein level, leading to degradation of epidermal growth factor receptor (EGFR) in SCK and PANC-1 cells. GEMs decreased the RAS and PI3K protein levels, leading to a reduction in the protein levels of active forms of downstream signaling molecules, including PDK, AKT, and GSK3β. GEM reduced proliferation of cancer cells by upregulating cell cycle arrest proteins, particularly p53 and p21, and downregulating cyclin D1 and cyclin B. Moreover, GEMs reduced the levels of proangiogenic factors, including VEGF, VEGFR2, CD31, and HIF-1α, and inhibited tumor cell migration and invasion by inducing the expression of E-cadherin and reducing that of N-cadherin, snail, and vimentin. We demonstrated that local delivery of gemcitabine using GEM implants inhibited tumor cell growth by promoting c-CBL-mediated degradation of EGFR and inhibiting the proliferation, angiogenesis, and epithelial–mesenchymal transition of pancreatic/biliary tumors. Use of gemcitabine-eluting stents can improve stent patency by inhibiting the ingrowth of malignant biliary obstructions.

Cellulose nanocrystals/nanofibrils loaded astaxanthin nanoemulsion for the induction of apoptosis via ROS-dependent mitochondrial dysfunction in cancer cells under photobiomodulation

The present study investigated effects of low-level laser therapy with cellulose nanocrystals/cellulose nanofibrils loaded in nanoemulsion (NE) against skin cancer cells on apoptosis. The nanoemulsion was fabricated and characterized by the standard methods. The toxicity level by cytotoxicity assays, generation of reactive singlet oxygen (ROS) and antioxidant potential, cell proliferation and migration were confirmed by using standard assays. The cellular uptake efficacy was evaluated by differential staining. The protein levels of EGFR, PI3K, AKT, ERK, GAPDH, and β-actin were detected by western blot. The samples showed a spherical shaped structure with the average size confirmed strong and stable hydrogen bonding forces with high degradation temperature and endothermic transition peaks. The fabricated samples showed no toxicity and high cell proliferation by generating more singlet oxygen levels and antioxidants. The intracellular signaling pathways was regulated with high protein expression levels, which was stimulated by specific molecules for cell proliferation, migration, and differentiation in cancer cells. The results proved that combined treatment regulated the intracellular signaling pathways in cancer cells. The current study showed a novel strategy for improving therapeutic efficacy of nanoemulsion by using low-level laser therapy. Further, the current favorable outcomes will be evaluated in in vivo animal models.

Relationship between PI3K/mTOR/RhoA pathway-regulated cytoskeletal rearrangements and phagocytic capacity of macrophages.

The objective of this study was to investigate the relationship between PI3K/mTOR/RhoA signaling regulated cytoskeletal rearrangements and phagocytic capacity of macrophages. RAW264.7 macrophages were divided into four groups; blank control, negative control, PI3K-RNAi, and mTOR-RNAi. The cytoskeletal changes in the macrophages were observed. Furthermore, the phagocytic capacity of macrophages against Escherichia coli is reported as mean fluorescence intensity (MFI) and percent phagocytosis. Transfection yielded 82.1 and 81.5% gene-silencing efficiencies against PI3K and mTOR, respectively. The PI3K-RNAi group had lower mRNA and protein expression levels of PI3K, mTOR, and RhoA than the blank and negative control groups (Р<0.01). The mTOR-RNAi group had lower mRNA and protein levels of mTOR and RhoA than the blank and the negative control groups (Р<0.01). Macrophages in the PI3K-RNAi group exhibited stiff and inflexible morphology with short, disorganized filopodia and reduced number of stress fibers. Macrophages in the mTOR-RNAi group displayed pronounced cellular deformations with long, dense filopodia and an increased number of stress fibers. The PI3K-RNAi group exhibited lower MFI and percent phagocytosis than blank and negative control groups, whereas the mTOR-RNAi group displayed higher MFI and percent phagocytosis than the blank and negative controls (Р<0.01). Before and after transfection, the mRNA and protein levels of PI3K were both positively correlated with mTOR and RhoA (Р<0.05), but the mRNA and protein levels of mTOR were negatively correlated with those of RhoA (Р<0.05). Changes in the phagocytic capacity of macrophages were associated with cytoskeletal rearrangements and were regulated by the PI3K/mTOR/RhoA signaling pathway.

Cardiac Contractility Modulation Attenuates Chronic Heart Failure in a Rabbit Model via the PI3K/AKT Pathway.

The Akt plays an important role in regulating cardiac growth, myocardial angiogenesis, and cell death in cardiac myocytes. However, there are few studies to focus on the responses of the Akt pathway to cardiac contractility modulation (CCM) in a chronic heart failure (HF) model. In this study, the effects of CCM on the treatment of HF in a rabbit model were investigated. Thirty six-month-old rabbits were randomly separated into control, HF, and CCM groups. The rabbits in HF and CCM groups were pressure uploaded, which can cause an aortic constriction. Then, CCM was gradually injected to the myocardium of rabbits in the CCM group, and this process lasted for four weeks with six hours per day. Rabbit body weight, heart weight, and heart beating rates were recorded during the experiment. To assess the CCM impacts, rabbit myocardial histology was examined as well. Additionally, western blot analysis was employed to measure the protein levels of Akt, FOXO3, Beclin, Pi3k, mTOR, GSK-3β, and TORC2 in the myocardial histology of rabbits. Results showed that the body and heart weight of rabbits decreased significantly after suffering HF when compared with those in the control group. However, they gradually recovered after CCM application. The CCM significantly decreased collagen volume fraction in myocardial histology of HF rabbits, indicating that CCM therapy attenuated myocardial fibrosis and collagen deposition. The levels of Akt, FOXO3, Beclin, mTOR, GSK-3β, and TORC2 were significantly downregulated, but Pi3k concentration was greatly upregulated after CCM utilization. Based on these findings, it was concluded that CCM could elicit positive effects on HF therapy, which was potentially due to the variation in the Pi3k/Akt signaling pathway.

Panax ginseng C.A. Meyer (Rg3) Ameliorates Gastric Precancerous Lesions in Atp4a-/- Mice via Inhibition of Glycolysis through PI3K/AKT/miRNA-21 Pathway.

Gastric cancer, one of the most common types of cancers, develops over a series of consecutive histopathological stages. As such, the analysis and research of the gastric precancerous lesions (GPLs) play an important role in preventing the occurrence of gastric cancer. Ginsenoside Rg3 (Rg3), an herbal medicine, plays an important role in the prevention and treatment of various cancers. Studies have demonstrated a correlation between glycolysis and gastric cancer progression. Herein, the aim of the present study was to clarify the potential role for glycolysis pathogenesis in Rg3-treated GPL in Atp4a−/− mice. The GPL mice model showed chronic gastritis, intestinal metaplasia, and more atypical hyperplasia in gastric mucosa. According to the results of HE and AB-PAS staining, it could be confirmed that GPL mice were obviously reversed by Rg3. Additionally, the increased protein levels of PI3K, AKT, mTOR, HIF-1α, LDHA, and HK-II, which are crucial factors for evaluating GPL in the aspect of glycolysis pathogenesis in the model group, were downregulated by Rg3. Meanwhile, the miRNA-21 expression was decreased and upregulated by Rg3. Furthermore, the increased gene levels of Bcl-2 and caspase-3 were attenuated in Rg3-treated GPL mice. In conclusion, the findings of this study imply that abnormal glycolysis in GPL mice was relieved by Rg3 via regulation of the expressions of PI3K, AKT, mTOR, HIF-1α, LDHA, HK-II, and miRNA-21. Rg3 is an effective supplement for GPL treatment and can be harnessed to inhibit proliferation and induce apoptosis of GPL cells.

Narrow-Leafed Lupin (Lupinus angustifolius L.) Seeds Gamma-Conglutin is an Anti-Inflammatory Protein Promoting Insulin Resistance Improvement and Oxidative Stress Amelioration in PANC-1 Pancreatic Cell-Line.

(1) Background: Inflammation molecular cues and insulin resistance development are some of the main contributors for the development and advance of the pathogenesis of inflammatory-related diseases; (2) Methods: We isolated and purified γ-conglutin protein from narrow-leafed lupin (NLL or blue lupin) mature seeds using affinity-chromatography to evaluate its anti-inflammatory activities at molecular level using both, a bacterial lipopolysaccharide (LPS)-induced inflammation and an insulin resistance pancreatic cell models; (3) Results: NLL γ-conglutin achieved a plethora of functional effects as the strong reduction of cell oxidative stress induced by inflammation through decreasing proteins carbonylation, nitric oxide synthesis and inducible nitric oxide synthase (iNOS) transcriptional levels, and raising glutathione (GSH) levels and modulation of superoxide dismutase (SOD) and catalase enzymes activities. γ-conglutin induced up-regulated transcriptomic and protein levels of insulin signalling pathway IRS-1, Glut-4, and PI3K, improving glucose uptake, while decreasing pro-inflammatory mediators as iNOs, TNFα, IL-1β, INFγ, IL-6, IL-12, IL-17, and IL-27; (4) Conclusion: These results suggest a promising use of NLL γ-conglutin protein in functional foods, which could also be implemented in alternative diagnosis and therapeutic molecular tools helping to prevent and treat inflammatory-related diseases.

Trastuzumab in combination with AT-101 induces cytotoxicity and apoptosis in Her2 positive breast cancer cells.

Aim: AT-101 is a polyphenolic compound with potent anti-apoptotic effects in various cancers. In this study, the possible synergistic cytotoxic and apoptotic effect of trastuzumab/AT-101 combination was investigated in HER2-positive breast cancer cell lines. Materials & methods: SKBR-3, MDA-MB-453 and MCF-10A cell lines were treated with a trastuzumab/AT-101 combination. Synergistic cytotoxicity and apoptosis effects were shown and then PI3K and Akt protein levels were studied. Result: The trastuzumab/AT-101 combination induced synergistic cytotoxicity and apoptosis in both breast cancer cells but not in MCF-10A cells. Combination treatment induced cytotoxicity via inhibiting PI3K/AKT but not the MAPK/ERK pathway. Conclusion: The trastuzumab/AT-101 combination may be a good candidate for patients with trastuzumab-resistant Her2-positive breast cancer and inhibition of the PI3K/AKT pathway may be one of the underlying mechanisms.

Long Non-Coding RNA HOTAIR Modulates KLF12 to Regulate Gastric Cancer Progression via PI3K/ATK Signaling Pathway by Sponging miR-618.

PurposeLong non-coding RNA (lncRNA) HOX transcript antisense RNA (HOTAIR) has been reported to dysregulate in many tumors. However, the mechanism of HOTAIR was rarely reported in GC.MethodsThe levels of HOTAIR, microRNA-618 (miR-618) and Krueppel-like factor 12 (KLF12) in GC tissues and cells were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The cell viability and apoptotic rate were assessed via cell counting kit-8 (CCK-8) assay and flow cytometry, respectively. The migrating and invading abilities were tested by Transwell assay. The protein levels of KLF12, p-PI3K, PI3K, p-ATK and ATK were measured by Western blot assay. These interactions between miR-618 and HOTAIR or KLF12 were predicted by DIANA tools, and then, dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were conducted to validate these interactions. Besides, the xenograft tumor experiment was performed to further verify the roles of HOTAIR in GC.ResultsThe levels of HOTAIR and KLF12 were significantly upregulated and the level of miR-618 was strikingly downregulated in GC tissues and cells. miR-618 was verified as a direct target of HOTAIR or KLF12. HOTAIR silencing blocked GC progression and PI3K/ATK signaling pathway by sponging miR-618 and also restrained xenograft tumor growth in vivo. miR-618 inhibited GC progression and PI3K/ATK signaling pathway by targeting KLF12. Mechanistically, HOTAIR modulated KLF12 expression by sponging miR-618 in GC cells.ConclusionThese data unraveled that HOTAIR promoted GC progression through PI3K/ATK signaling pathway via miR-618/KLF12 axis.

Protective effects of Weipixiao decoction against MNNG-induced gastric precancerous lesions in rats

Gastric cancer is recognized as one of the most common cancer. In-depth research of gastric precancerous lesions (GPL) plays an important role in preventing the occurrence of gastric cancer. Meanwhile, traditional treatment provides a novel sight in the prevention of occurrence and development of gastric cancer. The current study was designed to assess the effects of therapy with Weipixiao (WPX) decoction on N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced GPL rats and the underlying molecular mechanisms. After 10-weeks treatment, all rats were sacrificed. Histopathological changes of gastric tissue were assessed via hematoxylin-eosin (HE) and High-iron diamine-Alcian blue-Periodic acid-Schiff (HID-AB-PAS) staining. To be fully evidenced, RT-qPCR, Western blot and immunohistochemistry were used to detect the expressions of LDHA, CD147, HIF-1α, MCT4, PI3K, AKT, mTOR and miRNA-34a, which were crucial factors for evaluating GPL in the aspect of glycolysis pathogenesis. According to the results of HE and HID-AB-PAS staining, it could be confirmed that MNNG-induced GPL rats were obviously reversed by WPX decoction. Additionally, the increased gene levels of LDHA, CD147, MCT4, PI3K, AKT, mTOR and HIF-1α in model group were down-regulated by WPX decoction, while miRNA-34a expression was decreased and up-regulated by WPX decoction. The significantly increased protein levels of LDHA, CD147, MCT4, PI3K, AKT, mTOR and HIF-1α induced by MNNG were attenuated in rats treated with WPX decoction. In brief, the findings of this study imply that abnormal glycolysis in MNNG-induced GPL rats was relieved by WPX decoction via regulation of the expressions of LDHA, CD147, HIF-1α, MCT4, PI3K, AKT, mTOR and miRNA-34a.

Anti-Inflammatory Effects of Licania macrocarpa Cuatrec Methanol Extract Target Src- and TAK1-Mediated Pathways.

In this study, we investigated the anti-inflammatory effects of Licania macrocarpa Cuatrec methanol extract (Lm-ME) in vitro and in vivo and found pharmacological target proteins of Lm-ME in TLR4-mediated inflammatory signaling. This extract reduced NO production and mRNA expression of inflammatory cytokines such as iNOS, COX-2, IL-6, and IL-1β. In the NF-κB- and AP-1-mediated luciferase reporter gene assay, transcription factor activities decreased under cotransfection with MyD88 or TRIF. Phosphorylated protein levels of Src, PI3K, IKKα/β, and IκBα as well as p50 and p65 in the NF-κB signal pathway were downregulated, and phosphorylation of TAK1, MEK1/2, MKK4/7, and MKK3/6 as well as ERK, JNK, and p38 was decreased in the AP-1 signal pathway. Through overexpression of HA-Src and HA-TAK1, respectively, Lm-ME inhibited autophosphorylation of overexpressed proteins and thereby activated fewer downstream signaling molecules. Lm-ME also attenuated stomach ulcers in an HCl/EtOH-induced acute gastritis model mice, and COX-2 mRNA expression and phosphorylated TAK1 levels in gastric tissues were diminished. The flavonoids kaempferol and quercetin were identified in the HPLC analysis of Lm-ME; both are actively anti-inflammatory. Therefore, these results suggest that Lm-ME can be used for anti-inflammatory remedy by targeting Src and TAK1.

COX‑2 promotes epithelial‑mesenchymal transition and migration in osteosarcoma MG‑63 cells via PI3K/AKT/NF‑κB signaling.

The present study aimed to investigate the mechanism by which cyclooxygenase-2 (COX-2) promotes the metastasis of MG-63 osteosarcoma cells through the PI3K/AKT/NF-κB pathway. To achieve this, a recombinant lentivirus containing the COX-2 gene was constructed in order to overexpress COX-2; a recombinant lentivirus containing a control sequence was also constructed. A Transwell chamber migration assay was performed to quantify the migration of the COX-2-transduced cells, and of cells treated with a COX-2 inhibitor (NS398) or a PI3K inhibitor (LY294002). Immunofluorescence assays were performed to determine changes in E-cadherin, vimentin and NF-κB expression levels. ELISAs were performed to quantify the levels of matrix metallopeptidase (MMP)-2, MMP-9 and vascular endothelial growth factor (VEGF) in the culture medium. Western blot analysis was conducted to measure the protein expression levels of MMP-2, MMP-9, PI3K, phosphorylated (p-) PI3K, AKT, p-AKT, inhibitor of NF-κΒ kinase (IKK) and p-IKK. The results demonstrated that the migration ability of the COX-2-overexpressing MG-63 cells was significantly increased compared with the control cells. The migration ability of cells treated with NS398 or LY294002 was significantly decreased. Compared with the control cells, E-cadherin expression was significantly decreased in COX-2-overexpressing cells, while the expression levels of vimentin, MMP-2, MMP-9, VEGF, p-PI3K, p-AKT and p-IKK were significantly increased. Compared with the control cells, E-cadherin expression was significantly increased in cells treated with NS398 or LY294002, while the expression levels of vimentin, MMP-2, MMP-9, VEGF, p-PI3K, p-AKT, and p-IKK were significantly decreased. The total protein levels of PI3K, AKT and IKK were not changed among the treatment groups. In summary, COX-2 overexpression decreased the expression levels of the epithelial protein E-cadherin and increased the expression levels of the mesenchymal proteins vimentin, MMP-2 and MMP-9, as well as promoted cell migration, by activating the PI3K/AKT/NF-κB signaling pathway.

Inhibitory Effect of Hydroxysafflor Yellow B on the Proliferation of Human Breast Cancer MCF-7 Cells.

A recent patent has been issued for hydroxysafflor yellow A (HSYA) as a drug to prevent blood circulation disorders. Hydroxysafflor yellow B (HSYB), an isomer of HSYA with antioxidative effects, has been isolated from the florets of Carthamus tinctorius. The effects of HSYB on the proliferation of cancer cells and its mechanism of action have not been investigated. The aims of this study were to investigate the anti-cancer effects and the molecular mechanism of HSYB for breast cancer MCF-7 cells. MTT assays and colony formation assays were used to assess the survival and proliferation of MCF-7 cells, respectively. Hoechst 33258 and flow cytometry were used to measure cell apoptosis and flow cytometry to determine effects on the cell cycle. Western blots were used to measure protein levels. Treatment with HSYB reduced survival and proliferation of human breast cancer MCF-7 cells in a dose-dependent manner. Furthermore, HSYB arrested the MCF-7 cell cycle at the S phase and downregulated cyclin D1, cyclin E, and CDK2. Compared with a control group, HSYB suppressed the protein levels of p-PI3K, PI3K, AKT, and p-AKT in MCF-7 cells. In addition, HSYB decreased the levels of Bcl- 2, increased the levels of Bax, cleaved caspase-3 and caspase-9, and subsequently induced MCF-7 cell apoptosis. These data demonstrate that HSYB arrests the MCF-7 cell cycle at the S phase and induces cell apoptosis. Patent US20170246228 indicates that HSYB can be potentially used for the prevention and treatment of human breast cancer.

< Omega >-3 PUFA Attenuates LPS-Induced Neuro-Injury of Neonatal Rats through the PI3K/AKT Pathway.

Inflammation may result in periventricular leukomalacia, which is the leading cause of preterm brain encephalopathy. Moreover, <omega>-3 polyunsaturated fatty acids (<omega>-3 PUFAs) play a pivotal role against central nervous system injury, which is likely related to its anti-inflammatory effect. However, the mechanism regarding the remedial effects of <omega>-3 PUFA for LPS-induced neuro-injury has remained unclear. In this study, newborn SD rats were intraperitoneally injected with LPS or < omega>-3 PUFA, and the proliferation and apoptosis of neurocytes in the hippocampus were measured by TUNEL and BrdU. Quantitative real-time PCR (qPCR) and Western blot assay were used to analyze the mRNA and protein levels of PI3K, AKT and β-catenin in vitro and in vivo. We found that <omega>-3 PUFA promoted the proliferation and migration of neurocytes in vitro and in vivo and inhibited apoptosis. Furthermore, we confirmed that <omega>-3 PUFA through the PI3K/AKT signaling pathway positively regulated the expression of PI3K and further caused the phosphorylation of AKT activation, followed by the upregulation of β-catenin expression. Interestingly, this phenomenon became more noticeable with the combined application of <omega>-3 PUFA and a PI3K/AKT agonist. In conclusion, we confirm that <omega>-3 PUFA plays an important role in neuroprotection by activating the PI3K/AKT/β-catenin pathway. It may be a promising strategy against brain injury.

Effect of Ligustrazine on Endometrium Injury of Thin Endometrium Rats.

The purpose of this experiment is to establish a rat model of thin endometrium and to explore the effect of ligustrazine on the thin endometrium of rats. The thin endometrium model was made by using infusing absolute ethyl alcohol into the uterine cavity. The thickness of endometrium was measured. Hematoxylin-Eosin (HE) staining was used to observe the histopathological changes of endometrium. The mRNA levels of VEGF, VEGFR-2, PI3K, and AKT were detected by RT-PCR. Western blotting was used to detect the levels of VEGF, VEGFR-2, PI3K, and AKT in endometrial tissue. The thickness of endometrium in the model group was significantly thinner than that in the control group. Compared with the model group, the thickness of endometrium in ligustrazine group was increased. HE staining shown that ligustrazine restored the histopathological changes of endometrium. RT-PCR and Western Blotting results showed that the mRNA and protein levels of VEGF, VEGFR-2, PI3K, and AKT in the model group were significantly decreased compared with the control group, while ligustrazine restored the changes. Ligustrazine can improve the morphology of endometrium, can promote the growth of endometrium, and has obvious therapeutic effect. Its mechanism is related to the activation of PI3K/Akt signaling pathway through upregulation of VEGF and VEGFR-2 expression to induce the repair of thin endometrium in rats.

The Small GTPase ARF6 Activates PI3K in Melanoma to Induce a Prometastatic State.

Melanoma has an unusual capacity to spread in early-stage disease, prompting aggressive clinical intervention in very thin primary tumors. Despite these proactive efforts, patients with low-risk, low-stage disease can still develop metastasis, indicating the presence of permissive cues for distant spread. Here, we show that constitutive activation of the small GTPase ARF6 (ARF6Q67L) is sufficient to accelerate metastasis in mice with BRAFV600E/Cdkn2aNULL melanoma at a similar incidence and severity to Pten loss, a major driver of PI3K activation and melanoma metastasis. ARF6Q67L promoted spontaneous metastasis from significantly smaller primary tumors than PTENNULL, implying an enhanced ability of ARF6-GTP to drive distant spread. ARF6 activation increased lung colonization from circulating melanoma cells, suggesting that the prometastatic function of ARF6 extends to late steps in metastasis. Unexpectedly, ARF6Q67L tumors showed upregulation of Pik3r1 expression, which encodes the p85 regulatory subunit of PI3K. Tumor cells expressing ARF6Q67L displayed increased PI3K protein levels and activity, enhanced PI3K distribution to cellular protrusions, and increased AKT activation in invadopodia. ARF6 is necessary and sufficient for activation of both PI3K and AKT, and PI3K and AKT are necessary for ARF6-mediated invasion. We provide evidence for aberrant ARF6 activation in human melanoma samples, which is associated with reduced survival. Our work reveals a previously unknown ARF6-PI3K-AKT proinvasive pathway, it demonstrates a critical role for ARF6 in multiple steps of the metastatic cascade, and it illuminates how melanoma cells can acquire an early metastatic phenotype in patients. SIGNIFICANCE: These findings reveal a prometastatic role for ARF6 independent of tumor growth, which may help explain how melanoma spreads distantly from thin, early-stage primary tumors.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/11/2892/F1.large.jpg.

Reduction of the PI3K/Akt related signaling activities in skeletal muscle tissues involves insulin resistance in intrauterine growth restriction rats with catch-up growth.

Individuals with intrauterine growth retardation (IUGR) are prone to insulin resistance, whereas the underlying molecular mechanisms remain unclear. In this study, we investigated if the PI3K/Akt signaling pathway in skeletal muscle tissues involves insulin resistance in IUGR offsprings, particularly ones with catch-up growth. An IUGR rat model was established by feeding rats an isocaloric (30.50 Kcal/g) diet containing 8% protein (low-protein diet) from day 1 of pregnancy until the birth of their pups. Glucometabolic parameters were measured and compared. Quantitative PCR and western blot were performed to assess mRNA and protein expression changes of the PI3K/Akt related signals in skeletal muscle tissues. Pearson analysis was used to assess the correlation of the PI3K/Akt signaling level and catch-up growth with the insulin resistance index (IRI). The values of fasting plasma glucose, fasting insulin and IRI were significantly higher, whereas insulin sensitivity index was significantly lower in IUGR offsprings than those in the controls. The PI3K mRNA and protein levels as well as the phospho-AktSer473 levels were significantly lower in IUGR offsprings compared to the controls. Reductions of GLUT4 as well as increases of PTEN and nuclear fractional PPARγ were detected in IUGR offsprings. Catch-up growth IUGR rats were positively correlated with insulin resistance and underwent more remarkable alterations of the PI3K, PTEN and GLUT4 expressions. Our results demonstrated that rats born IUGR developed insulin resistance later in life, which was likely mediated by reductions of the PI3K/Akt related signaling activities, particularly in those with excess catch-up growth.

Effect of BDNF-TrKB pathway on apoptosis of retinal ganglion cells in glaucomatous animal model.

The aim of this paper is to investigate the effect of BDNF-TrKB pathway on AOH by accessing its regulatory role in retinal ganglion cell apoptosis. Acute ocular hypertension (AOH) model in rats was established by anterior chamber perfusion to increase intraocular tension. Rats were randomly divided into AOH group, control group and k252a group, with ten rats in each group. Rats were sacrificed 72 h after animal procedures and eyeballs were harvested. HE staining was used to observe retinal structural changes at different time points. Immunohistochemical staining was used to observe the BDNF-positive cells in retinal tissues. TUNEL staining was conducted to measure apoptosis of retinal ganglion cells. Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) and Western blot were performed to detect mRNA and protein levels of BDNF, TrKB, PI3K and ERK1 in retinal tissues, respectively. HE and TUNEL staining showed significant pathological changes and abundant apoptotic cells in rat retina of AOH group and k252a group compared with those of the control group (p<0.05). The number of survived retinal ganglion cells in the AOH group was lower than that of the control group (p<0.05). K252a group had the lowest number of survived retinal ganglion cells. Immunohistochemical results showed that BDNF was rarely expressed in rat retinal tissues of the control group, which was remarkably pronounced in the AOH group and k252a group. The number of BDNF-positive cells in the k252a group was higher than that in the AOH group (p<0.05). RT-PCR and Western blot indicated that mRNA and protein levels of relative genes in BDNF-TrKB and PI3K/ERK1 pathways were upregulated in AOH group (p<0.05), but were significantly downregulated in k252a group (p<0.05). BNDF-TrKB pathway exerts a protective effect on retina against acute ocular hypertension by reducing retinal cell apoptosis.

Research on miR-126 in glioma targeted regulation of PTEN/PI3K/Akt and MDM2-p53 pathways.

To study the correlations of micro ribonucleic acid (miR)-126 expression with pathogenesis and prognosis of glioma, and to screen potential biological targets for the diagnosis, treatment and prognosis of glioma. miR-126 expression in cancer tissues, normal brain tissues, U87MG cells and normal astrocytes in glioma patients was quantitatively analyzed via quantitative reverse transcription-polymerase chain reaction (qRT-PCR). U87MG cells were transfected with miR-126 mimics or miR-126 inhibitor, followed by verification via qRT-PCR. The cell proliferation, apoptosis, migration and invasion after transfection were analyzed using methyl thiazolyl tetrazolium (MTT) assay, Annexin V/propidium iodide (PI) assay, wound healing assay and transwell assay, respectively. The expression levels of proteins related to phosphatase and tensin homolog deleted on chromosome ten/phosphatidylinositol 3-kinase/protein kinase B (PTEN/PI3K/Akt) pathway and double mouse minute 2 homolog (MDM2)-p53 pathway were detected via Western blotting. Moreover, the prognostic analysis was performed using the Kaplan-Meier method and log-rank test. Results of qRT-PCR showed that the miR-126 expression in highly malignant glioma tissues and U87MG cells were significantly lower than those in normal brain cells, and its expression level was significantly higher or lower than that in negative control group after transfection with miR-126 mimics or inhibitor. Analyses of cell proliferation, apoptosis, migration and invasion revealed that the up-regulation of miR-126 could remarkably inhibit the in-vitro proliferation, migration and invasion and promote apoptosis of glioma cells, and vice versa. Results of Western blotting manifested that after overexpression of miR-126, PI3K, p-Akt and MDM2 protein levels in U87MG cells were significantly decreased compared with those in control group, but PTEN and p53 protein expressions were significantly increased, and vice versa. Besides, according to prognostic analysis, the prognosis of patients with a low miR-126 level was poorer. The miR-126 expression is abnormally low in glioma cells, and miR-126 inhibits the course of glioma through targeted regulation of PTEN/PI3K/Akt and MDM2-p53 pathways, which, therefore, can be used as a new potential biomarker for the diagnosis, treatment and prognosis of glioma.