Adenine Combined with Cisplatin Promotes Anticancer Activity against Hepatocellular Cancer Cells through AMPK-Mediated p53/p21 and p38 MAPK Cascades.

Cisplatin has been widely used in cancer treatments. Recent evidence indicates that adenine has potential anticancer activities against various types of cancers. However, the effects of the combination of adenine and cisplatin on hepatocellular carcinoma (HCC) cells remain sketchy. Here, our objective was to elucidate the anticancer activity of adenine in combination with cisplatin in HCC cells and its mechanistic pathways. Cell viability and cell cycle progression were assessed by the SRB assay and flow cytometry, respectively. Apoptosis was demonstrated by PI/annexin V staining and flow cytometric analysis. Protein expression, signaling cascade, and mRNA expression were analyzed by Western blotting and quantitative RT-PCR, respectively. Our results showed that adenine jointly potentiated the inhibitory effects of cisplatin on the cell viability of SK-Hep1 and Huh7 cells. Further investigation showed that adenine combined with cisplatin induced higher S phase arrest and apoptosis in HCC cells. Mechanically, adenine induced AMPK activation, reduced mTOR phosphorylation, and increased p53 and p21 levels. The combination of adenine and cisplatin synergistically reduced Bcl-2 and increased PUMA, cleaved caspase-3, and PARP in HCC cells. Adenine also upregulated the mRNA expression of p53, p21, PUMA, and PARP, while knockdown of AMPK reduced the increased expression of these genes. Furthermore, adenine also induced the activation of p38 MAPK through AMPK signaling, and the inhibition of p38 MAPK reduced the apoptosis of HCC cells with exposure to adenine combined with cisplatin. Collectively, these findings reveal that the combination of adenine and cisplatin synergistically enhances apoptosis of HCC cells, which may be attributed to the AMPK-mediated p53/p21 and p38 MAPK cascades. It suggests that adenine may be a potential adjuvant for the treatment of HCC in combination with cisplatin.

ENERGI-F703 gel, as a new topical treatment for diabetic foot and leg ulcers: A multicenter, randomized, double-blind, phase II trial.

Background: Diabetic foot and leg ulcers are a major cause of disability among patients with diabetes mellitus. A topical gel called ENERGI-F703, applied twice daily and with adenine as its active pharmaceutical ingredient, accelerated wound healing in diabetic mice. The current study evaluated the safety and efficacy of ENERGI-F703 for patients with diabetic foot and leg ulcers. Methods: This randomized, double-blind, multicenter, phase II trial recruited patients from eight medical centers in Taiwan. Patients with intractable diabetic foot and leg ulcers (Wagner Grade 1-3 without active osteomyelitis) were randomly assigned (2:1) to receive topical ENERGI-F703 gel or vehicle gel twice daily for 12 weeks or until complete ulcer closure. The investigator, enrolled patients and site personnel were masked to treatment allocation. Intention to treat (ITT) population and safety population were patient to primary analyses and safety analyses, respectively. Primary outcome was complete ulcer closure rate at the end of treatment. This trial is registered with ClinicalTrials.gov, number NCT02672436. Findings: Starting from March 15th, 2017 to December 26th, 2019, 141 patients were enrolled as safety population and randomized into ENERGI-F703 gel (n = 95) group or vehicle gel (n = 46) group. In ITT population, ENERGI-F703 (n = 90) and vehicle group showed ulcer closure rates of 36.7% (95% CI = 26.75% - 47.49%) and 26.2% (95% CI = 13.86% - 42.04%) with difference of 9.74 % (95 % CI = -6.74% - 26.23%) and 25% quartiles of the time to complete ulcer closure of 69 days and 84 days, respectively. There were 25 (26.3%) patients in ENERGI-F703 group and 11 (23.9%) patients in vehicle group experiencing serious adverse events and five deaths occurred during the study period, none of them related to the treatment. Interpretation: Our study suggests that ENERGI-F703 gel is a safe and well-tolerated treatment for chronic diabetic foot and leg ulcers. Further studies are needed to corroborate our findings in light of limitations. Funding: Energenesis Biomedical Co., Ltd.

The Cell Protective Effect of Adenine on Hypoxia-Reoxygenation Injury through PPAR Delta Activation.

Ischemia followed by blood supply reperfusion in cardiomyocytes leads to an overproduction of free radicals and a rapid decrease of adenosine triphosphate concentration. The cardioprotective effect of a potential drug, adenine, was evaluated using H9c2 rat cardiomyoblasts. After hypoxia-reoxygenation (HR) treatment consisting of hypoxia for 21 h followed by reoxygenation for 6 h, it was revealed that pretreatment with 200 µM adenine for 2 h effectively prevented HR-induced cell death. Adenine also significantly decreased the production of reactive oxygen species and reduced cell apoptosis after HR injury. The antioxidant effect of adenine was also revealed in this study. Adenine pretreatment significantly reduced the expression of activating transcription factor 4 (ATF4) and glucose-regulated protein 78 (GRP78) proteins, and protein disulfide isomerase induced a protective effect on mitochondria after HR stimulation. Intracellular adenosine monophosphate-activated protein kinase, peroxisome proliferator-activated receptor delta (PPARδ), and perilipin levels were increased by adenine after HR stimulation. Adenine had a protective effect in HR-damaged H9c2 cells. It may be used in multiple preventive medicines in the future.

Adenine Inhibits the Invasive Potential of DLD-1 Human Colorectal Cancer Cell via the AMPK/FAK Axis.

Tumor metastasis is a major cause of death of patients with colorectal cancer (CRC). Our previous findings show that adenine has antiproliferation activity against tumor cells. However, whether adenine reduces the invasiveness of DLD-1 and SW480 CRC cells has not been thoroughly explored. In this study, we aimed to explore the effects of adenine on the invasion potential of DLD-1 cells. Our findings showed that adenine at concentrations of ≤200 µM did not influence the cell viability of DLD-1 and SW480 CRC cells. By contrast, adenine reduced the migratory potential of the CRC cells. Moreover, it decreased the invasion capacity of the CRC cells in a dose-dependent manner. We further observed that adenine downregulated the protein levels of tissue plasminogen activator, matrix metalloproteinase-9, Snail, TWIST, and vimentin, but upregulated the tissue inhibitor of metalloproteinase-1 expression in DLD-1 cells. Adenine decreased the integrin αV level and reduced the activation of integrin-associated signaling components, including focal adhesion kinase (FAK), paxillin, and Src in DLD-1 cells. Further observations showed that adenine induced AMP-activated protein kinase (AMPK) activation and inhibited mTOR phosphorylation in DLD-1 cells. The knockdown of AMPK restored the reduced integrin αV level and FAK/paxillin/Src signaling inhibited by adenine in DLD-1 cells. Collectively, these findings reveal that adenine reduces the invasion potential of DLD-1 cells through the AMPK/integrin/FAK axis, suggesting that adenine may have anti-metastatic potential in CRC cells.

Modulation of adenine phosphoribosyltransferase-mediated salvage pathway to accelerate diabetic wound healing.

Adenine phosphoribosyltransferase (APRT) is the key enzyme involved in purine salvage by the incorporation of adenine and phosphoribosyl pyrophosphate to provide adenylate nucleotides. To evaluate the role of APRT in the repair processes of cutaneous wounds in healthy skin and in diabetic patients, a diabetic mouse model (db/db) and age-matched wild-type mice were used. Moreover, the topical application of adenine was assessed. In vitro studies, analytical, histological, and immunohistochemical methods were used. Diabetic mice treated with adenine exhibited elevated ATP levels in organismic skin and accelerated wound healing. In vitro studies showed that APRT utilized adenine to rescue cellular ATP levels and proliferation from hydrogen peroxide-induced oxidative damage. HPLC-ESI-MS/MS-based analysis of total adenylate nucleotides in NIH-3T3 fibroblasts demonstrated that adenine addition enlarged the cellular adenylate pool, reduced the adenylate energy charge, and provided additional AMP for the further generation of ATP. These data indicate an upregulation of APRT in skin wounds, highlighting its role during the healing of diabetic wounds through regulation of the nucleotide pool after injury. Furthermore, topical adenine supplementation resulted in an enlargement of the adenylate pool needed for the generation of ATP, an important molecule for wound repair.

Adenine inhibits growth of hepatocellular carcinoma cells via AMPK-mediated S phase arrest and apoptotic cascade.

Background: Adenine exhibits potential anticancer activity against several types of malignancies. However, whether adenine has anticancer effects on hepatocellular carcinoma (HCC) cells is incompletely explored. Methods: Human HCC cell lines HepG2 and SK-Hep-1 (p53-wild type) and Hep3B (p53-deficient) were used as cell model. Cell growth and cell cycle distribution were determined using MTT assay and flow cytometric analysis, respectively. Protein expression and phosphorylation were assessed by Western blot. Involvement of AMP-activated protein kinase (AMPK) was evaluated using specific inhibitor and small inhibitory RNA (siRNA). Results: Adenine treatments (0.5 - 2 mM) clearly decreased the cell growth of Hep G2 and SK-Hep-1 cells to 72.5 ± 3.4% and 71.3 ± 4.6% of control, respectively. In parallel, adenine also induced sub-G1 and S phase accumulation in both HCC cells. However, adenine did not affect the cell growth and cell cycle distribution of Hep3B cell. Western blot analysis showed that adenine reduced expression of cyclin A/D1 and cyclin-dependent kinase (CDK)2 and upregulated p53, p21, Bax, PUMA, and NOXA in HepG2 cell. Moreover, adenine induced AMPK activation that was involved in the p53-associated apoptotic cascade in HepG2 cells. Inhibition of AMPK activation or knockdown of AMPK restored the decreased cell growth of HepG2 and SK-Hep-1 cells in response to adenine. Conclusions: These findings reveal that adenine reduces the cell growth of HepG2 and SK-Hep-1 but not Hep3B cells, attributing to the AMPK/p53-mediated S phase arrest and apoptosis. It suggests that adenine has anticancer potential against p53-wild type HCC cells and may be beneficial as an adjuvant for HCC treatment.

Deregulated HOXB7 expression predicts poor prognosis of patients with malignancies of digestive system.

INTRODUCTION: Numerous studies have investigated the relationship between deregulated HOXB7 expression with the clinical outcome in patients with digestive stem cancers, HOXB7 has showed negative impacts but with varying levels. We aimed to comprehensively evaluate the prediction and prognostic value of HOXB7 in digestive stem cancers. EVIDENCE ACQUISITION: Electronic databases updated to December 1st, 2016 were retrieved to collect relevant eligible studies to quantitatively explore the potential roles of HOXB7 as a prognostic indicator in digestive system cancers. EVIDENCE SYNTHESIS: A total of 9 studies (N.=1298) was included in this synthetical meta-analysis. The pooled hazard ratios suggested that high expression of HOXB7 protein was associated with poor prognosis of OS in patients with digestive system cancers (HR=1.97, 95% CI: 1.65-2.28, P=0.000), and HOXB7 protein could act as an independent prognostic factor for predicting OS of patients with digestive system cancers (HR=2.02, 95% CI: 1.69-2.36, P=0.000). Statistical significance was also observed in subgroup meta-analysis based on the cancer type, histology type, country, sample size and publication date. Furthermore, we examined the correlations between HOXB7 protein and clinicopathological features. It showed that altered expression of HOXB7 protein was correlated with tumor invasion (P=0.000), lymph node status (P=0.000), distant metastasis (P=0.001) and TNM stage (P=0.000). However, the expression of HOXB7 protein was not associated with age (P=0.64), gender (P=0.40) or levels of differentiation (P=0.19). CONCLUSIONS: High expression of HOXB7 protein was associated with poor prognosis of patients with digestive system cancers, as well as clinicopathologic characteristics, including the tumor invasion, lymph node status, distant metastasis and TNM stage. The expression of HOXB7 protein was not associated with age, gender or levels of differentiation. HOXB7 protein expression level in tumor tissue might serve as a novel prognostic marker for digestive system cancers.

Adenine causes cell cycle arrest and autophagy of chronic myelogenous leukemia K562 cells via AMP-activated protein kinase signaling.

AMP-activated protein kinase (AMPK) is known as a pivotal regulator of cellular metabolism. Mounting evidences have demonstrated that AMPK activation exerts tumor suppressive activity on leukemia cells. The present study reported that adenine, an AMPK activator, triggers cell cycle arrest and autophagy of human chronic myelogenous leukemia K562 cells consequently suppressing cell viability. The present findings revealed that adenine treatment (4.0-8.0 mM) significantly inhibited the viability of K562 cells to 69.3±2.5% (24 h) and 53.4±2.1% (48 h) of the control. Flow cytometric analysis revealed that there was a significant accumulation in G2/M phase, but not sub-G1 phase K562 cells following exposure to adenine. Additional investigation demonstrated that adenine treatments significantly increased the number of acidic vesicular organelles and the level of autophagosomal microtubule associated protein 1 light chain 3 α (LC3) marker. By contrast, cleavage of caspase-9, caspase-3 and poly-ADP-ribose polymerase was insignificantly affected in K562 cells following adenine treatment. In K562 cells, adenine was able to markedly promote the phosphorylation of AMPKα and suppress the phosphorylation of mammalian target of rapamycin (mTOR), a downstream target of AMPK. In addition, inhibiting AMPK phosphorylation using dorsomorphin restored mTOR phosphorylation, inhibited the accumulation of LC3 and significantly recovered the suppressed cell viability in response to adenine. Taken together, the present results demonstrated that adenine induced G2/M phase arrest and autophagic cell death, consequently suppressing the viability of K562 cells, which may attribute to the AMPK activation triggered by adenine. These findings provide evidence that adenine may be beneficial to chronic myelogenous leukemia therapy by suppressing excessive cell proliferation.

Evaluation of disulfide scrambling during the enzymatic digestion of bevacizumab at various pH values using mass spectrometry.

Disulfide linkages play an important role in protein stability and activity. Thus, it is critical to characterize disulfide bonds to ensure the quality and function of protein pharmaceuticals. There are, however, problems associated with maintaining disulfide linkages in the conventional procedures that are used to digest a protein. In order to preserve enzyme activity during the digestion of a protein, it is commonly carried out at neutral to basic environment which increases the possibilities of disulfide bond scrambling. However, it is not easy to differentiate whether the scrambled disulfide linkages are initiated by the sample itself or whether they are induced during the protease digestion process. In this study, the optimum pH for minimizing disulfide bond rearrangements during the digestion process was determined. Three sets of proteases, trypsin plus Glu-C, Lys-C and thermolysin were used, followed by dimethyl labeling and mass spectrometry for a bevacizumab (Avastin) disulfide linkage analysis. No disulfide linkage scrambling was detected at pH6 when Lys-C or trypsin plus Glu-C were used as enzymes. When thermolysin was applied, some scrambled disulfide bonds were identified at pH5, 6 and 7. Nevertheless, there was less disulfide bond scrambling at a lower pH. All correct disulfide bonds on bevacizumab could be identified using this approach. The results demonstrated that by choosing the proper enzymes, using a lower pH environment for the digestion could reduce the degree of artifact disulfide scrambling.

Gold Nanoparticles Compromise TNF-α-Induced Endothelial Cell Adhesion Molecule Expression Through NF-κB and Protein Degradation Pathways and Reduce Neointima Formation in A Rat Carotid Balloon Injury Model.

The aim of this study was to investigate the anti-inflammatory effects and mechanism of action of the gold nanoparticles (AuNPs) on vascular injury. In vitro vascular endothelial cell (EC) inflammation and in vivo rat carotid balloon injury models were used. The expression of TNF-α-induced cell adhesion molecules (CAMs) was suppressed by the AuNPs in human umbilical vein ECs and aortic ECs. The AuNPs reduced TNF-α-induced intracellular ROS production and NF-κB signaling pathways and enhanced CAM protein degradation by increasing their ubiquitination. However, they did not interfere with the mTOR pathway for protein synthesis and TNF-αbinding to ECs. These effects led to a reduction of monocyte adhesion to EC monolayers in vitro and endothelial CAM expression and monocyte/macrophage level in the vascular injured areas, contributing to a substantial decrease of arterial neointima formation in the rat carotid balloon injury model. The serum gold concentration was 99.5±18 ng/ml after three-day oral administration. Moreover, incubation of the AuNPs with serum and albumin led to an increase of particle sizes of the AuNPs. Collectively, we provide the first evidence that demonstrates that AuNPs possess anti-inflammatory bioactivity on vascular ECsin vitro and can reduce arterial neointima hyperplasia during vascular injury in vivo.

Activation of AMP-Activated Protein Kinase by Adenine Alleviates TNF-Alpha-Induced Inflammation in Human Umbilical Vein Endothelial Cells.

The AMP-activated protein kinase (AMPK) signaling system plays a key role in cellular stress by repressing the inflammatory responses induced by the nuclear factor-kappa B (NF-κB) system. Previous studies suggest that the anti-inflammatory role of AMPK involves activation by adenine, but the mechanism that allows adenine to produce these effects has not yet been elucidated. In human umbilical vein endothelial cells (HUVECs), adenine was observed to induce the phosphorylation of AMPK in both a time- and dose-dependent manner as well as its downstream target acetyl Co-A carboxylase (ACC). Adenine also attenuated NF-κB targeting of gene expression in a dose-dependent manner and decreased monocyte adhesion to HUVECs following tumor necrosis factor (TNF-α) treatment. The short hairpin RNA (shRNA) against AMPK α1 in HUVECs attenuated the adenine-induced inhibition of NF-κB activation in response to TNF-α, thereby suggesting that the anti-inflammatory role of adenine is mediated by AMPK. Following the knockdown of adenosyl phosphoribosyl transferase (APRT) in HUVECs, adenine supplementation failed to induce the phosphorylation of AMPK and ACC. Similarly, the expression of a shRNA against APRT nullified the anti-inflammatory effects of adenine in HUVECs. These results suggested that the role of adenine as an AMPK activator is related to catabolism by APRT, which increases the cellular AMP levels to activate AMPK.

Combination of temozolomide and Taxol exerts a synergistic inhibitory effect on Taxol­resistant glioma cells via inhibition of glucose metabolism.

Malignant gliomas, which comprise the most common type of primary malignant brain tumor, are associated with a poor prognosis and quality of life. Paclitaxel (Taxol) and temozolomide (TMZ) are Food and Drug Administration­approved anticancer agents, which are known to have therapeutic applications in various malignancies. However, similar to other chemotherapeutic agents, the development of resistance to TMZ and Taxol is common. The aim of the present study was to investigate the regulation of glucose metabolism by TMZ and Taxol in glioma cells. The results demonstrated that glioma cells exhibit decreased glucose uptake and lactate production in response to treatment with TMZ; however, glucose metabolism was increased in response to Taxol treatment. Following analysis of TMZ­ and Taxol­resistant cell lines, it was reported that glucose metabolism was decreased in the TMZ­resistant cells, but was increased in the Taxol­resistant cells. Notably, a combination of TMZ and Taxol exerted synergistic inhibitory effects on Taxol­resistant glioma cells. However, the synergistic phenotype was not observed following treatment with a combination of 5­fluorouracil and Taxol. Furthermore, restoration of glucose metabolism by overexpression of glucose transporter 1 in Taxol­resistant cells resulted in regained resistance to Taxol. Therefore, the present study proposes a novel mechanism accounting for the synergistic effects of Taxol and TMZ co­treatment, which may contribute to the development of therapeutic strategies for overcoming chemoresistance in patients with cancer.

Purine analogue ENERGI-F706 induces apoptosis of 786-O renal carcinoma cells via 5'-adenosine monophosphate-activated protein kinase activation.

Purine compounds are known to activate 5'-adenosine monophosphate-activated protein kinase (AMPK), which has important roles in treatments for renal cell carcinoma. The present study was aimed to investigate the effects of the purine analogue ENERGI­F706 on the human renal carcinoma cell line 786­O and the underlying mechanisms. The results revealed that ENERGI­F706 (0.2­0.6 mg/ml) significantly decreased the cell viability to up to 36.4±2.4% of that of the control. Compared to 786­O cells, ENERGI­F706 exerted less suppressive effects on the viability of the human non­tumorigenic renal cell line HK­2. Flow cytometric analysis showed that ENERGI­F706 contributed to cell cycle arrest at S­phase and triggered apoptosis of 786­O cells. Immunoblot analysis revealed that anti­apoptotic B­cell lymphoma 2 (Bcl­2) levels were reduced and pro­apoptotic Bcl­2­associated X protein levels were diminished. In addition, activation of caspase­9, caspase­3 and poly(adenosine diphosphate ribose) polymerase (PARP) was promoted in 786­O cells in response to ENERGI­F706. Effects of ENERGI­F706 on AMPK cascades were investigated and the results showed that ENERGI­F706 enhanced phosphorylation of AMPKα (T172) and p53 (S15), a downstream target of AMPK. In addition, the AMPK activation, p53 (S15) phosphorylation, reduction of Bcl­2, cleavage of caspase­3 and PARP as well as suppressed cell viability induced by ENERGI­F706 were reversed in the presence of AMPK inhibitor compound C (dorsomorphin). In conclusion, the findings of the present study revealed that ENERGI­F706 significantly suppressed the viability of 786­O cells via induction of cell cycle arrest and apoptosis, attributing to AMPK and p53 activation and subsequent cell cycle regulatory and apoptotic signaling. It was therefore indicated that ENERGI­F706 may be suitable for the treatment of renal cell carcinoma.

The neuroprotective role of metformin in advanced glycation end product treated human neural stem cells is AMPK-dependent.

Diabetic neuronal damage results from hyperglycemia followed by increased formation of advanced glycosylation end products (AGEs), which leads to neurodegeneration, although the molecular mechanisms are still not well understood. Metformin, one of the most widely used anti-diabetic drugs, exerts its effects in part by activation of AMP-activated protein kinase (AMPK). AMPK is a critical evolutionarily conserved enzyme expressed in the liver, skeletal muscle and brain, and promotes cellular energy homeostasis and biogenesis by regulating several metabolic processes. While the mechanisms of AMPK as a metabolic regulator are well established, the neuronal role for AMPK is still unknown. In the present study, human neural stem cells (hNSCs) exposed to AGEs had significantly reduced cell viability, which correlated with decreased AMPK and mitochondria associated gene/protein (PGC1α, NRF-1 and Tfam) expressions, as well as increased activation of caspase 3 and 9 activities. Metformin prevented AGEs induced cytochrome c release from mitochondria into cytosol in the hNSCs. Co-treatment with metformin significantly abrogated the AGE-mediated effects in hNSCs. Metformin also significantly rescued hNSCs from AGE-mediated mitochondrial deficiency (lower ATP, D-loop level, mitochondrial mass, maximal respiratory function, COX activity, and mitochondrial membrane potential). Furthermore, co-treatment of hNSCs with metformin significantly blocked AGE-mediated reductions in the expression levels of several neuroprotective genes (PPARγ, Bcl-2 and CREB). These findings extend our understanding of the molecular mechanisms of both AGE-induced neuronal toxicity, and AMPK-dependent neuroprotection by metformin. This study further suggests that AMPK may be a potential therapeutic target for treating diabetic neurodegeneration.

Quantitative analysis of prostate specific antigen isoforms using immunoprecipitation and stable isotope labeling mass spectrometry.

Prostate specific antigen (PSA) is a widely used serum marker for prostate cancer (PCa), but has limited specificity for distinguishing early PCa from benign prostatic hyperplasia (BPH). Recently, proPSAs, comprised of native proPSA, as well as truncated proPSA forms, [-2] proPSA, [-5] proPSA, and [-7] proPSA, have been shown to be better diagnostic targets than PSA for PCa. Stable isotope labeling-multiple reaction monitoring mass spectrometry (SIL/MRM-MS) has been frequently used to measure low-abundance biomarkers in tissues and biofluids, owing to its high sensitivity and specificity, simplicity, and multiplexing capability. In this study, we have developed and optimized a strategy using immunoprecipitation in conjunction with SIL/MRM-MS assay which is capable of sensitive and accurate quantification of proPSA in serum. Since serum and plasma are by far the most complex biological fluids, the immunoprecipitation workflow was optimized to achieve sufficient sensitivity, efficiencies of protein purification with immunoaffinity depletion were determined. The developed strategy can detect proPSA and PSA with a limit of detection (LOD) and limit of quantitation (LOQ) at nanogram per milliliter levels, corresponding to a concentration 6 orders-of-magnitude lower than the most abundant serum proteins. Furthermore, the simultaneous measurement of multiple biomarkers, including the mature and precursor forms of PSA, can be achieved in a single multiplexed analysis using LC/MRM-MS. The strategy demonstrated here provides an attractive alternative to ELISAs or RIAs for the reliably measurement of proPSA to improve the specificity of PCa diagnosis.

AMPK activation inhibits expression of proinflammatory mediators through downregulation of PI3K/p38 MAPK and NF-κB signaling in murine macrophages.

Adenosine monophosphate (AMP)-activated protein kinase (AMPK) plays a central role in energy homeostasis and regulation of inflammatory responses. The present study is aimed to investigate the anti-inflammatory effects of ENERGI-F704, a nucleobase analogue isolated from bamboo leaves, on expression of proinflammatory mediators in murine macrophage RAW264.7 in response to lipopolysaccharide (LPS). ENERGI-F704 enhanced phosphorylation of AMPK(T172) but insignificantly affected the viability of RAW264.7 cells. Further investigation showed that ENERGI-F704 decreased mRNA expression of interleukin (IL)-6, IL-8, tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX2), and inducible nitric oxide synthase (iNOS) induced by LPS, as well as suppressed the production of prostaglandin E2 (PGE2) and nitric oxide (NO). Additionally, the inhibitory effects of ENERGI-F704 on the LPS-induced proinflammatory mediators were diminished by pretreatment of AMPK inhibitor Compound C. ENERGI-F704 also inhibited LPS-triggered activation of nuclear factor kappa B (NF-κB), phosphatidylinositol 3-kinase (PI3K), and p38 mitogen-activated protein kinase (p38), whereas extracellular signal-regulated kinase (Erk)1/2 and c-Jun N-terminal kinase (JNK) were insignificantly influenced. Our findings indicate that ENERGI-F704 may exert anti-inflammatory activity on RAW264.7 cells in response to LPS through the activation of AMPK and suppression of PI3K/P38/NF-κB signaling and the consequent decreased expression of proinflammatory mediators, suggesting that ENERGI-F704 is beneficial to the amelioration of inflammatory disorders.

Lutein inhibits the migration of retinal pigment epithelial cells via cytosolic and mitochondrial Akt pathways (lutein inhibits RPE cells migration).

During the course of proliferative vitreoretinopathy (PVR), the retinal pigment epithelium (RPE) cells will de-differentiate, proliferate, and migrate onto the surfaces of the sensory retina. Several studies have shown that platelet-derived growth factor (PDGF) can induce migration of RPE cells via an Akt-related pathway. In this study, the effect of lutein on PDGF-BB-induced RPE cells migration was examined using transwell migration assays and Western blot analyses. We found that both phosphorylation of Akt and mitochondrial translocation of Akt in RPE cells induced by PDGF-BB stimulation were suppressed by lutein. Furthermore, the increased migration observed in RPE cells with overexpressed mitochondrial Akt could also be suppressed by lutein. Our results demonstrate that lutein can inhibit PDGF-BB induced RPE cells migration through the inhibition of both cytoplasmic and mitochondrial Akt activation.

Amelioration of LPS-induced inflammation response in microglia by AMPK activation.

Adenosine 5'-monophosphate-activated protein kinase (AMPK) is a key regulator of cellular energy homeostasis via modulating metabolism of glucose, lipid, and protein. In addition to energy modulation, AMPK has been demonstrated to associate with several important cellular events including inflammation. The results showed that ENERGI-F704 identified from bamboo shoot extract was nontoxic in concentrations up to 80 µM and dose-dependently induced phosphorylation of AMPK (Thr-172) in microglia BV2 cells. Our findings also showed that the treatment of BV2 with ENERGI-F704 ameliorated the LPS-induced elevation of IL-6 and TNF-α production. In addition, ENERGI-F704 reduced increased production of nitric oxide (NO) and prostaglandin E2 (PGE2) via downregulating the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2), respectively. Moreover, ENERGI-F704 decreased activated nuclear translocation and protein level of NF-κB. Inhibition of AMPK with compound C restored decreased NF-κB translocation by ENERGI-F704. In conclusion, ENERGI-F704 exerts inhibitory activity on LPS-induced inflammation through manipulating AMPK signaling and exhibits a potential therapeutic agent for neuroinflammatory disease.

AMP-activated protein kinase activators in diabetic ulcers: from animal studies to Phase II drugs under investigation.

INTRODUCTION: Diagnosed cases of diabetes have gradually increased year by year, and research on diabetes mellitus (DM) has attracted greater attention from the medical profession. Diabetic ulcers present persistent pain and the risk of bacterial infection. However, no promising treatment methods have been found. As a regulator of cellular energy balance, 5' adenosine monophosphate-activated protein kinase (AMPK) has been suggested as a drug target for DM, including such drugs as metformin. AREAS COVERED: This review summarizes the current research and clinical trials of AMPK activators on diabetic wound healing and diabetic ulcers. Furthermore, it discusses the feasibility of AMPK activators in the treatment of diabetic wounds. EXPERT OPINION: Animal studies have demonstrated that AMPK activators are a potential treatment for diabetic ulcers. AMPK activators alleviate tissue inflammation and promote re-epithelialization in diabetic wounds. However, due to the complicated pathological mechanism of diabetic foot ulcers, AMPK activators should be combined with other approaches. The new strategies for combination therapy with AMPK activator may provide a therapeutic advantage for patients with diabetic ulcers.

Rosiglitazone promotes neurite outgrowth and mitochondrial function in N2A cells via PPARgamma pathway.

Several pieces of evidence indicate that peroxisome proliferator-activated receptor gamma (PPARγ) stimulation promotes neuronal differentiation. However, to date, the effects of a synthetic PPARγ agonist (Rosiglitazone, Rosi) on neurite outgrowth have not yet been well described. Here we have evaluated the effects of Rosi on neurite outgrowth and mitochondrial function in the mouse neuroblastoma Neuro 2a (N2A) cell line. Our results show that Rosi promotes neurite outgrowth of N2A cells and significantly increases the population of neurite-bearing cells, with apparent increase of intracellular calcium and the expression of calmodulin-dependent kinase I (CaMKI). Rosi also increases the intracellular cAMP and expression of both protein kinase A (PKA) and cAMP response element binding protein (CREB). Phosphorylation of CREB was also detected in the Rosi treated N2A cells. Moreover, Rosi significantly increases the transcription of AMP-activated kinase (AMPK) and Sirtuin 1 (SIRT1). Besides, the expression of PPAR coactivator 1α (PGC1α), as well as the mRNA level its downstream genes, including nuclear respiratory factors 1 and 2 (NRF1 and NRF2) and mitochondrial transcription factor A (Tfam) were induced by Rosi treatments. Furthermore, Rosi increases the level of ATP, D-loop, and mitochondrial mass in N2A cells. Collectively, these findings provide an array of evidence that PPARγ activation provides beneficial neuronal networks within neurite outgrowth.