INPP4B suppresses prostate cancer cell invasion

Background

INPP4B and PTEN dual specificity phosphatases are frequently lost during progression of prostate cancer to metastatic disease. We and others have previously shown that loss of INPP4B expression correlates with poor prognosis in multiple malignancies and with metastatic spread in prostate cancer.

Results

We demonstrate that de novo expression of INPP4B in highly invasive human prostate carcinoma PC-3 cells suppresses their invasion both in vitro and in vivo. Using global gene expression analysis, we found that INPP4B regulates a number of genes associated with cell adhesion, the extracellular matrix, and the cytoskeleton. Importantly, de novo expressed INPP4B suppressed the proinflammatory chemokine IL-8 and induced PAK6. These genes were regulated in a reciprocal manner following downregulation of INPP4B in the independently derived INPP4B-positive LNCaP prostate cancer cell line. Inhibition of PI3K/Akt pathway, which is highly active in both PC-3 and LNCaP cells, did not reproduce INPP4B mediated suppression of IL-8 mRNA expression in either cell type. In contrast, inhibition of PKC signaling phenocopied INPP4B-mediated inhibitory effect on IL-8 in either prostate cancer cell line. In PC-3 cells, INPP4B overexpression caused a decline in the level of metastases associated BIRC5 protein, phosphorylation of PKC, and expression of the common PKC and IL-8 downstream target, COX-2. Reciprocally, COX-2 expression was increased in LNCaP cells following depletion of endogenous INPP4B.

Conclusion

Taken together, we discovered that INPP4B is a novel suppressor of oncogenic PKC signaling, further emphasizing the role of INPP4B in maintaining normal physiology of the prostate epithelium and suppressing metastatic potential of prostate tumors.

     

Parthenolide induces apoptosis and autophagy through the suppression of PI3K/Akt signaling pathway in cervical cancer

Objective

To investigate the effect of parthenolide on apoptosis and autophagy and to study the role of the PI3K/Akt signaling pathway in cervical cancer.

Results

Parthenolide inhibits HeLa cell viability in a dose dependent-manner and was confirmed by MTT assay. Parthenolide (6 µM) induces mitochondrial-mediated apoptosis and autophagy by activation of caspase-3, upregulation of Bax, Beclin-1, ATG5, ATG3 and down-regulation of Bcl-2 and mTOR. Parthenolide also inhibits PI3K and Akt expression through activation of PTEN expression. Moreover, parthenolide induces generation of reactive oxygen species that leads to the loss of mitochondrial membrane potential.

Conclusion

Parthenolide induces apoptosis and autophagy-mediated growth inhibition in HeLa cells by suppressing the PI3K/Akt signaling pathway and mitochondrial membrane depolarization and ROS generation. Parthenolide may be a potential therapeutic agent for the treatment of cervical cancer.

     

Aggressive neuroblastomas have high p110alpha but low p110delta and p55alpha/p50alpha protein levels compared to low stage neuroblastomas

Background

The phosphoinositide 3-kinase (PI3K)/Akt pathway is involved in neuroblastoma development where Akt/PKB activation is associated with poor prognosis. PI3K activity subsequently activates Akt/PKB, and as mutations of PI3K are rare in neuroblastoma and high levels of PI3K subunit p110delta is associated with favorable disease with low p-Akt/PKB, the levels of other PI3K subunits could be important for Akt activation.

Methods

Protein levels of Type IA PI3K catalytic and regulatory subunits were investigated together with levels of phosphorylated Akt/PKB and the PI3K negative regulator PTEN in primary neuroblastoma tumors. Relation between clinical markers and protein levels were evaluated through t-tests.

Results

We found high levels of p-Akt/PKB correlating to aggressive disease and p-Akt/PKB (T308) showed inverse correlation to PTEN levels. The regulatory isomers p55alpha/p50alpha showed higher levels in favorable neuroblastoma as compared with aggressive neuroblastoma. The PI3K-subunit p110alpha was found mainly in advanced tumors while p110delta showed higher levels in favorable neuroblastoma.

Conclusions

Activation of the PI3K/Akt pathway is seen in neuroblastoma tumors, however the contribution of the different PI3K isoforms is unknown. Here we show that p110alpha is preferentially expressed in aggressive neuroblastomas, with high p-Akt/PKB and p110delta is mainly detected in favorable neuroblastomas, with low p-Akt/PKB. This is an important finding as PI3K-specific inhibitors are suggested for enrollment in treatment of neuroblastoma patients.

     

ROR2 receptor promotes the migration of osteosarcoma cells in response to Wnt5a

Background

We have reported that the phosphatidylinositol-3 kinase (PI3K)/Akt/RhoA signaling pathway mediates Wnt5a-induced cell migration of osteosarcoma cells. However, the specific receptors responding to Wnt5a ligand remain poorly defined in osteosarcoma metastasis.

Methods

Wound healing assays were used to measure the migration rate of osteosarcoma cells transfected with shRNA or siRNA specific against ROR2 or indicated constructs. We evaluated the RhoA activation in osteosarcoma MG-63 and U2OS cells with RhoA activation assay. A panel of inhibitors of PI3K and Akt treated osteosarcoma cells and blocked kinase activity. Western blotting assays were employed to measure the expression and activation of Akt. Clonogenic assays were used to measure the cell proliferation of ROR2-knockdown or ROR2-overexpressed osteosarcoma cells.

Results

Wnt5a-induced osteosarcoma cell migration was largely abolished by shRNA or siRNA specific against ROR2. Overexpression of RhoA-CA (GFP-RhoA-V14) was able to rescue the Wnt5a-induced cell migration blocked by ROR2 knockdown. The Wnt5a-induced activation of RhoA was mostly blocked by ROR2 knockdown, and elevated by ROR2 overexpression, respectively. Furthermore, we found that Wnt5a-induced cell migration was significantly retarded by RhoA-siRNA transfection or pretreatment of HS-173 (PI3Kα inhibitor), MK-2206 (Akt inhibitor), A-674563 (Akt1 inhibitor), or CCT128930 (Akt2 inhibitor). The activation of Akt was upregulated or downregulated by transfected with ROR2-Flag or ROR2-siRNA, respectively. Lastly, Wnt5a/ROR2 signaling does not alter the cell proliferation of MG-63 osteosarcoma cells.

Conclusions

Taken together, we demonstrate that ROR2 receptor responding to Wnt5a ligand activates PI3K/Akt/RhoA signaling and promotes the migration of osteosarcoma cells.

     

Akt1 inhibition promotes breast cancer metastasis through EGFR-mediated β-catenin nuclear accumulation

Background

Knockdown of Akt1 promotes Epithelial-to-Mesenchymal Transition in breast cancer cells. However, the mechanisms are not completely understood.

Methods

Western blotting, immunofluorescence, luciferase assay, real time PCR, ELISA and Matrigel invasion assay were used to investigate how Akt1 inhibition promotes breast cancer cell invasion in vitro. Mouse model of lung metastasis was used to measure in vivo efficacy of Akt inhibitor MK2206 and its combination with Gefitinib.

Results

Knockdown of Akt1 stimulated β-catenin nuclear accumulation, resulting in breast cancer cell invasion. β-catenin nuclear accumulation induced by Akt1 inhibition depended on the prolonged activation of EGFR signaling pathway in breast cancer cells. Mechanistic experiments documented that knockdown of Akt1 inactivates PIKfyve via dephosphorylating of PIKfyve at Ser³¹⁸ site, resulting in a decreased degradation of EGFR signaling pathway. Inhibition of Akt1 using MK2206 could induce an increase in the expression of EGFR and β-catenin in breast cancer cells. In addition, MK2206 at a low dosage enhance breast cancer metastasis in a mouse model of lung metastasis, while an inhibitor of EGFR tyrosine kinase Gefitinib could potentially suppress breast cancer metastasis induced by Akt1 inhibition.

Conclusion

EGFR-mediated β-catenin nuclear accumulation is critical for Akt1 inhibition-induced breast cancer metastasis.

     

Luteolin,quercetin, genistein and quercetagetin inhibit the effects of lipopolysaccharide obtained from <Emphasis Type="Italic">Porphyromonas gingivalis</Emphasis> in H9c2 cardiomyoblasts

Background

One of the microorganisms from dental plaque associated with severe inflammatory responses in infectious endocarditis is Porphyromonas gingivalis. It is a Gram-negative bacteria harvested from chronic periodontitis patients. Lipopolysaccharide (LPS) obtained from P. gingivalis promotes the expressions of interleukin-1 (IL-1), IL-6 and tumor necrosis factor alpha (TNF-α). Flavonoids are thought to participate in processes that control inflammation, such as the expression of cyclooxygenase-2 (COX-2).

Methods

We investigated the effects of luteolin, quercetin, genistein and quercetagetin on cardiomyoblasts treated with LPS alone or in combination with following inhibitors p38 (SB203580), ERK (PD98059), JNK (SP600125) and PKC (Calphostin C) for 1 h. The kinase activation and COX-2 expression levels were determined at the gene and protein levels.

Results

These flavonoids are considered to inhibit the activation of mitogen-activated protein kinase (MAPK) and the degradation of inhibitor of kappa B-alpha (IκB-α). They also play a role in COX-2 expression.

Conclusion

We conclude that the tested flavonoids inhibit inflammatory responses induced by LPS in H9c2 cells.

     

ERK-mediated autophagy promotes inactivated Sendai virus (HVJ-E)-induced apoptosis in HeLa cells in an Atg3-dependent manner

Background

Apoptosis and autophagy are known to play important roles in cancer development. It has been reported that HVJ-E induces apoptosis in cancer cells, thereby inhibiting the development of tumors. To define the mechanism by which HVJ-E induces cell death, we examined whether HVJ-E activates autophagic and apoptotic signaling pathways in HeLa cells.

Methods

Cells were treated with chloroquine (CQ) and rapamycin to determine whether autophagy is involved in HVJ-E-induced apoptosis. Treatment with the ERK inhibitor, U0126, was used to determine whether autophagy and apoptosis are mediated by the ERK pathway. Activators of the PI3K/Akt/mTOR/p70S6K pathway, 740 Y-P and SC79, were used to characterize its role in HVJ-E-induced autophagy. siRNA against Atg3 was used to knock down the protein and determine whether it plays a role in HVJ-E-induced apoptosis in HeLa cells.

Results

We found that HVJ-E infection inhibited cell viability and induced apoptosis through the mitochondrial pathway, as evidenced by the expression of caspase proteins. This process was promoted by rapamycin treatment and inhibited by CQ treatment. HVJ-E-induced autophagy was further blocked by 740 Y-P, SC79, and U0126, indicating that both the ERK- and the PI3K/Akt/mTOR/p70S6K-pathways were involved. Finally, autophagy-mediated apoptosis induced by HVJ-E was inhibited by siRNA-mediated Atg3 knockdown.

Conclusion

In HeLa cells, HVJ-E infection triggered autophagy through the PI3K/Akt/mTOR/p70S6K pathway in an ERK1/2-dependent manner, and the induction of autophagy promoted apoptosis in an Atg3-dependent manner.

     

Absence of Appl2 sensitizes endotoxin shock through activation of PI3K/Akt pathway

Background

The adapter proteins Appl1 (adaptor protein containing pleckstrin homology domain, phosphotyrosine domain, and leucine zipper motif 1) and Appl2 are highly homologous and involved in several signaling pathways. While previous studies have shown that Appl1 plays a pivotal role in adiponectin signaling and insulin secretion, the physiological functions of Appl2 are largely unknown.

Results

In the present study, the role of Appl2 in sepsis shock was investigated by using Appl2 knockout (KO) mice. When challenged with lipopolysaccharides (LPS), Appl2 KO mice exhibited more severe symptoms of endotoxin shock, accompanied by increased production of proinflammatory cytokines. In comparison with the wild-type control, deletion of Appl2 led to higher levels of TNF-α and IL-1β in primary macrophages. In addition, phosphorylation of Akt and its downstream effector NF-κB was significantly enhanced. By co-immunoprecipitation, we found that Appl2 and Appl1 interacted with each other and formed a complex with PI3K regulatory subunit p85α, which is an upstream regulator of Akt. Consistent with these results, deletion of Appl1 in macrophages exhibited characteristics of reduced Akt activation and decreased the production of TNFα and IL-1β when challenged by LPS.

Conclusions

Results of the present study demonstrated that Appl2 is a critical negative regulator of innate immune response via inhibition of PI3K/Akt/NF-κB signaling pathway by forming a complex with Appl1 and PI3K.

     

Roxarsone induces angiogenesis via PI3K/Akt signaling

Background

3-Nitro-4-hydroxy phenyl arsenic acid, roxarsone, is widely used as an organic arsenic feed additive for livestock and poultry, which may increase the level of arsenic in the environment and the risk of exposure to arsenic in human. Little information is focused on the angiogenesis roxarsone-induced and its mechanism at present. This paper aims to study the role of PI3K/Akt signaling in roxarsone-induced angiogenesis in rat vascular endothelial cells and a mouse B16–F10 melanoma xenograft model.

Results

The results showed that treatment with 0.1–10.0 µmol/L roxarsone resulted in an increase in the OD rate in the MTT assay, the number of BrdU-positive cells in the proliferation assay, the migration distance in the scratch test and the number of meshes in tube formation assay. Further, treatment with 1.0 µmol/L roxarsone was associated with significantly higher phosphorylation of PI3K/Akt and expression of VEGF than the control treatment. The PI3K inhibitor was found to significantly combat the effects of 1.0 µmol/L roxarsone. Furthermore, roxarsone treatment was observed to increase the weight and volume of B16–F10 xenografts and VEGF expression and PI3K/Akt phosphorylation in a dose-dependent manner, with the 25 mg/kg dose having significant effects.

Conclusions

These results demonstrate that roxarsone has the ability to promote growth and tube formation in vascular endothelial cells and the growth of mouse B16–F10 xenografts. Further, the findings also indicate that PI3K/Akt signaling plays a regulatory role in roxarsone-induced angiogenesis in vivo and in vitro.

     

Promigratory and proangiogenic effects of AdipoRon on bone marrow-derived mesenchymal stem cells: an in vitro study

Objectives

To investigate the effect of AdipoRon on major factors involved in survival, migration and neovascularization of rat bone marrow-derived mesenchymal stem cells.

Results

AdipoRon promoted the MSCs viability. Real-time PCR indicated that the expression of cyclooxygenase-2 (COX-2), hypoxia-inducible factor-1 (HIF-1) C-X-C chemokine receptor type 4 (CXCR4), C–C chemokine receptor type 2 (CCR2), vascular endothelial growth factor matrix metalloproteinase-2 (MMP-2) and MMP-9 were upregulated in AdipoRon-treated MSCs compared to control groups. Prostaglandin E2 (PGE2) level, as well as migration ability of MSCs (scratch assay) was enhanced by AdipoRon preconditioning.

Conclusion

Preconditioning of MSCs with AdipoRon prior to transplantation could enhance cell survival, angiogenesis and migration via activating the COX-2/PGE2/HIF-1 pathway and other contributing factors.

     

Response of Degarelix treatment in human prostate cancer monitored by HR-MAS <Superscript>1</Superscript>H NMR spectroscopy

Introduction

The androgen receptor (AR) is the master regulator of prostate cancer cell metabolism. Degarelix is a novel gonadotrophin-releasing hormone blocker, used to decrease serum androgen levels in order to treat advanced human prostate cancer. Little is known of the rapid metabolic response of the human prostate cancer tissue samples to the decreased androgen levels.

Objectives

To investigate the metabolic responses in benign and cancerous tissue samples from patients after treatment with Degarelix by using HRMAS ¹H NMR spectroscopy.

Methods

Using non-destructive HR-MAS ¹H NMR spectroscopy we analysed the metabolic changes induced by decreased AR signalling in human prostate cancer tissue samples. Absolute concentrations of the metabolites alanine, lactate, glutamine, glutamate, citrate, choline compounds [t-choline = choline + phosphocholine (PC) + glycerophosphocholine (GPC)], creatine compounds [t-creatine = creatine (Cr) + phosphocreatine (PCr)], taurine, myo-inositol and polyamines were measured in benign prostate tissue samples (n = 10), in prostate cancer specimens from untreated patients (n = 7) and prostate cancer specimens from patients treated with Degarelix (n = 6).

Results

Lactate, alanine and t-choline concentrations were significantly elevated in high-grade prostate cancer samples when compared to benign samples in untreated patients. Decreased androgen levels resulted in significant decreases of lactate and t-choline concentrations in human prostate cancer biopsies.

Conclusions

The reduced concentrations of lactate and t-choline metabolites due to Degarelix could in principle be monitored by in vivo ¹H MRS, which suggests that it would be possible to monitor the effects of physical or chemical castration in patients by that non-invasive method.

     

Non-invasive urinary metabolomic profiling discriminates prostate cancer from benign prostatic hyperplasia

Introduction

Prostate cancer (PCa) is one of the most common malignancies in men worldwide. Serum prostate specific antigen (PSA) level has been extensively used as a biomarker to detect PCa. However, PSA is not cancer-specific and various non-malignant conditions, including benign prostatic hyperplasia (BPH), can cause a rise in PSA blood levels, thus leading to many false positive results.

Objectives

In this study, we evaluated the potential of urinary metabolomic profiling for discriminating PCa from BPH.

Methods

Urine samples from 64 PCa patients and 51 individuals diagnosed with BPH were analysed using ¹H nuclear magnetic resonance (¹H-NMR). Comparative analysis of urinary metabolomic profiles was carried out using multivariate and univariate statistical approaches.

Results

The urine metabolomic profile of PCa patients is characterised by increased concentrations of branched-chain amino acids (BCAA), glutamate and pseudouridine, and decreased concentrations of glycine, dimethylglycine, fumarate and 4-imidazole-acetate compared with individuals diagnosed with BPH.

Conclusion

PCa patients have a specific urinary metabolomic profile. The results of our study underscore the clinical potential of metabolomic profiling to uncover metabolic changes that could be useful to discriminate PCa from BPH in a clinical context.

     

Lipopolysaccharide (LPS)-induced septic shock causes profound changes in myocardial energy metabolites in pigs

Introduction

Energy deficiency is a cause for myocardial dysfunction during septic shock. In rodents, septic shock decreases the oxidation of long-chain fatty acids and glucose in the myocardium causing energy deficiency. However, the effect of septic shock on myocardial energy metabolites in large animals and human is unknown.

Objectives

Investigate the effects of septic shock on myocardial energy metabolites in domestic pigs.

Methods

Seventeen female pigs divided into control and lipopolysaccharide (LPS)-induced septic shock groups. Myocardial metabolites were analyzed ex vivo by ¹H nuclear magnetic resonance spectroscopy and liquid chromatography-tandem mass spectrometry. Gene and protein expression analysis were analyzed by real-time PCR and western blot.

Results

Septic shock was associated with an increase in myocardial levels of short- and medium-chain acylcarnitines, lactate, alanine, and pyruvate dehydrogenase kinase 4 gene expression. COX-2 and prostaglandin E4 receptor gene expression also increased in the septic myocardium, although the only elevated eicosanoid in the septic animals was thromboxane B2. Myocardial levels of niacin, taurine, glutamate, glutamine, and glutathione were higher, and hypoxanthine levels lower in septic pigs than controls.

Conclusions

In pigs, septic shock induced by LPS caused myocardial changes directed to decrease the oxidation of medium- and short-chain fatty acid without an effect on long-chain fatty acid oxidation. The increase in myocardial levels of lactate, alanine, and pyruvate dehydrogenase kinase 4 gene expression suggest that septic shock decreases pyruvate dehydrogenase complex activity and glucose oxidation. Homeostasis of niacin, taurine, glutamate, glutamine, glutathione, hypoxanthine and thromboxane B2 is also affected in the septic myocardium.

     

Alpha-synuclein contributes to malignant progression of human meningioma via the Akt/mTOR pathway

Background

The aim of this study is to explore the expression of alpha-synuclein (α-synuclein) in benign, atypical, and anaplastic meningiomas and determine its role in the malignant progression of meningiomas.

Methods

Expression of α-synuclein was measured in 44 meningioma samples by real-time PCR analysis. The effects of overexpression or knockdown of α-synuclein on meningioma cell growth, invasiveness, and tumorigenicity were determined.

Results

Atypical and anaplastic meningiomas displayed significantly greater levels of α-synuclein mRNA, relative to benign tumors. Depletion of α-synuclein decreased cell proliferation and colony formation and promoted apoptosis in IOMM-Lee meningioma cells, whereas overexpression of α-synuclein facilitated cell proliferation and colony formation in CH-157MN meningioma cells. Silencing of α-synuclein attenuated IOMM-Lee cell migration and invasion. In contrast, ectopic expression of α-synuclein increased the invasiveness of CH-157MN cells. In vivo studies further demonstrated that downregulation of α-synuclein significantly retarded meningioma growth in nude mice. At the molecular level, the phosphorylation levels of Akt, mTOR, p70S6K and 4EBP were significantly decreased in α-synuclein-depleted IOMM-Lee cells.

Conclusions

In conclusion, α-synuclein upregulation contributes to aggressive phenotypes of meningiomas via the Akt/mTOR pathway and thus represents a potential therapeutic target for malignant meningiomas.

     

Hypoxia promotes the migration and invasion of human hepatocarcinoma cells through the HIF-1α–IL-8–Akt axis

Background

Hepatocellular carcinoma (HCC) is the fifth most common cancer and the third most common cause of cancer-related death worldwide. The 5-year survival rate remains low despite considerable research into treatments of HCC, including surgery, radiotherapy and chemotherapy. Many mechanisms within HCC still require investigation, including the influence of hypoxia, which has a crucial role in many cancers and is associated with metastasis. Hypoxia inducible factor-1α (HIF-1α) is known to regulate the expression of many chemokines, including interleukin-8 (IL-8), which is associated with tumor metastasis. Although many studies have reported that HIF-1α is associated with HCC migration and invasion, the underlying mechanisms remain unknown.

Methods

The expression level of HIF-1α was determined in HCC cells. The correlation of IL-8 and HIF-1α expressions was assessed via knockdown of HIF-1α. HCC cells were also used to assess the influence of HIF-1α on HCC cell migration and invasion. LY294002, an inhibitor of the Akt pathway, was used to confirm the associated signaling pathways.

Results

We observed a significant attenuation of cell migration and invasion after silencing of HIF-1α. Exogenously expressing IL-8 restored migration and invasion. Akt was found to be involved in this process.

Conclusion

Hypoxia promotes HCC cell migration and invasion through the HIF-1α–IL-8–Akt axis.

     

MiR-338-3p inhibits TNF-α-induced lipogenesis in human sebocytes

Objective

To suppress TNF-α-induced lipogenesis in sebocytes (associated with acne development) with microRNA-338-3p (miR-338-3p) and to explore the underlying mechanisms.

Results

TNF-α increased lipid droplet formation in sebocytes which were used as in vitro model of inflammation-induced acne. Flow cytometry and TLC assays validated that miR-338-3p could suppress TNF-α-induced lipid droplet formation, down-regulate the expression of PREX2a, and inactivate AKT signaling in sebocytes. In addition, suppression of AKT activity by the PI3 K and AKT inhibitors diminished TNF-α-induced lipogenesis. PREX2a siRNA mimics the effects of miR-338-3p on AKT phosphorylation and lipogenesis. PREX2a overexpression consistently restored lipogenesis and AKT phosphorylation attenuated by miR-338-3p.

Conclusions

MiR-338-3p suppresses the TNF-α-induced lipogenesis in sebocytes by targeting PREX2a and down-regulating PI3K/AKT signaling.

     

NMRProcFlow: a graphical and interactive tool dedicated to 1D spectra processing for NMR-based metabolomics

Introduction

Concerning NMR-based metabolomics, 1D spectra processing often requires an expert eye for disentangling the intertwined peaks.

Objectives

The objective of NMRProcFlow is to assist the expert in this task in the best way without requirement of programming skills.

Methods

NMRProcFlow was developed to be a graphical and interactive 1D NMR (¹H & ¹³C) spectra processing tool.

Results

NMRProcFlow (http://nmrprocflow.org), dedicated to metabolic fingerprinting and targeted metabolomics, covers all spectra processing steps including baseline correction, chemical shift calibration and alignment.

Conclusion

Biologists and NMR spectroscopists can easily interact and develop synergies by visualizing the NMR spectra along with their corresponding experimental-factor levels, thus setting a bridge between experimental design and subsequent statistical analyses.