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Life Sciences Jun 2020Pulmonary arterial hypertension (PAH) is a severe pulmonary vascular disease characterized by unbalanced proliferation and apoptosis of pulmonary arterial smooth muscle...
BACKGROUND
Pulmonary arterial hypertension (PAH) is a severe pulmonary vascular disease characterized by unbalanced proliferation and apoptosis of pulmonary arterial smooth muscle cells (PASMCs). Prohibitin 1 (PHB1) is known for its significant anti-proliferative activity. However, the role of PHB1 in PASMCs and PAH have not been elucidated.
METHODS
Monocrotaline (MCT 60 mg/kg) was used to build a PAH model in SD rats. Right ventricular systolic pressure (RVSP) and right ventricle (RV) hypertrophy were measured. Morphology of pulmonary vessels was observed by Hematoxylin-Eosin (HE) staining. Expression of PHB1 in pulmonary arteries and PASMCs was determinated by immunoblot and immunofluorescence. Cell proliferation was detected by CCK8 and EDU when PASMCs were stimulated by PDGF-BB (20 ng/mL). Furthermore, siRNA for PHB1 and Akt inhibitor were conducted to investigate the mechanism behind the role of PHB1 and AKT signaling pathway in PASMCs proliferation and apoptosis.
RESULTS
The protein expression of PHB1 in PAH rats lung tissue was significantly up-regulated accompanied by elevated RVSP and enhanced RV hypertrophy. Immunohistochemistry showed that PHB1 was mainly localized in the pulmonary vascular smooth muscle layer. PDGF-BB significantly up-regulated the expression of PHB1 in rat primary PASMCs in a time- and dose-dependent manner. After PHB1 knock down, PASMCs proliferation was significantly suppressed while apoptosis was significantly recovered. Meanwhile the level of proliferating cell nuclear antigen (PCNA) and P-Akt were significantly down-regulated. Perifosine (Akt inhibitor) also significantly inhibit proliferation of PASMCs.
CONCLUSION
PHB1 contributes to pulmonary vascular remodeling by accelerating proliferation of PASMCs which involves AKT phosphorylation.
Topics: Animals; Apoptosis; Cell Proliferation; Disease Models, Animal; Gene Silencing; Heart Ventricles; Hemodynamics; Hypertension, Pulmonary; Lung; Male; Monocrotaline; Myocytes, Smooth Muscle; Phosphatidylinositol 3-Kinases; Prohibitins; Pulmonary Artery; RNA, Small Interfering; Rats; Rats, Sprague-Dawley; Repressor Proteins
PubMed: 32173312
DOI: 10.1016/j.lfs.2020.117548 -
Current Topics in Medicinal Chemistry 2020Cancer is a devastating disease that has plagued humans from ancient times to this day. After decades of slow research progress, promising drug development, and the... (Review)
Review
Cancer is a devastating disease that has plagued humans from ancient times to this day. After decades of slow research progress, promising drug development, and the identification of new targets, the war on cancer was launched, in 1972. The P13K/Akt pathway is a growth-regulating cellular signaling pathway, which in many human cancers is over-activated. Studies have demonstrated that a decrease in Akt activity by Akt inhibitors is associated with a reduction in tumor cell proliferation. There have been several promising drug candidates that have been studied, including but not limited to ipatasertib (RG7440), 1; afuresertib (GSK2110183), 2; uprosertib (GSK2141795), 3; capivasertib (AZD5363), 4; which reportedly bind to the ATP active site and inhibit Akt activity, thus exerting cytotoxic and antiproliferative activities against human cancer cells. For most of the compounds discussed in this review, data from preclinical studies in various cancers suggest a mechanistic basis involving hyperactivated Akt signaling. Allosteric inhibitors are also known to alter the activity of kinases. Perifosine (KRX- 0401), 5, an alkylphospholipid, is known as the first allosteric Akt inhibitor to enter clinical development and is mechanistically characterized as a PH-domain dependent inhibitor, non-competitive with ATP. This results in a reduction in Akt enzymatic and cellular activities. Other small molecule (MK- 2206, 6, PHT-427, Akti-1/2) inhibitors with a similar mechanism of action, alter Akt activity through the suppression of cell growth mediated by the inhibition of Akt membrane localization and subsequent activation. The natural product solenopsin has been identified as an inhibitor of Akt. A few promising solenopsin derivatives have emerged through pharmacophore modeling, energy-based calculations, and property predictions.
Topics: Antineoplastic Agents; Benzylamines; Cell Line, Tumor; Diamines; Drug Design; Heterocyclic Compounds, 3-Ring; Humans; Molecular Docking Simulation; Phosphatidylinositol 3-Kinases; Phospholipids; Piperazines; Protein Conformation; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Pyrazoles; Pyrimidines; Pyrroles; Quinoxalines; Signal Transduction; Structure-Activity Relationship; Sulfonamides; Thiadiazoles; Thiophenes
PubMed: 32091335
DOI: 10.2174/1568026620666200224101808 -
Biology Open Jan 2020In clinical practice, some breast cancer (BC) patients carry a rare ERBB2 in-frame insertion (p. Pro780_Tyr781insGlySerPro) and are resistant to anti-ERBB2 therapy. To...
In clinical practice, some breast cancer (BC) patients carry a rare ERBB2 in-frame insertion (p. Pro780_Tyr781insGlySerPro) and are resistant to anti-ERBB2 therapy. To explore the potential procarcinogenic role of this ERBB2 mutation, we conducted the present study using BC cells overexpressing wild-type (WT) ERBB2 or P780-Y781 ERBB2 [mutated (MT)]. MDA-MB-231 and MCF-7 cells were transfected with the following plasmids using a lentivirus system: negative control (ERBB2-NC), WT ERBB2 overexpression (ERBB2-WT), and P780-Y781 ERBB2 overexpression (ERBB2-MT). P780-Y781 ERBB2 conferred significant resistance to lapatinib, as assessed by cell viability and colony counts. Analysis of the cell cycle showed that the P780-Y781 ERBB2 group showed an elevated proportion of cells in S, G2, and M phases compared with WT ERBB2 when exposed to lapatinib. Following lapatinib treatment, phosphorylated AKT (p-AKT) was strongly upregulated in the P780-Y781 ERBB2 group. Among ERBB2+ patients, the P780-Y781 ERBB2 group showed increased levels of p-AKT. Furthermore, the AKT inhibitor perifosine effectively suppressed lapatinib resistance, as indicated by the lapatinib inhibition curve and results of the colony formation assay, and decreased AKT phosphorylation. Altogether, we discovered a procarcinogenic mutation of ERBB2 that enhances BC cell growth through AKT signaling and causes resistance to lapatinib. Patients with this in-frame insertion mutation of ERBB2 should be recommended other therapeutic strategies apart from ERBB2 tyrosine kinase inhibitors, in particular lapatinib.
Topics: Alleles; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Biomarkers, Tumor; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Drug Resistance, Neoplasm; Female; Humans; Lapatinib; Middle Aged; Mutagenesis, Insertional; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Receptor, ErbB-2; Signal Transduction; Tomography, X-Ray Computed; Treatment Outcome
PubMed: 31980423
DOI: 10.1242/bio.047662 -
Analytical Chemistry Nov 2019In this paper, we present an easy-to-follow procedure for the analysis of tissue sections from 3D cell cultures (spheroids) by matrix-assisted laser...
In this paper, we present an easy-to-follow procedure for the analysis of tissue sections from 3D cell cultures (spheroids) by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) and laser scanning confocal microscopy (LSCM). MALDI MSI was chosen to detect the distribution of the drug of interest, while fluorescence immunohistochemistry (IHC) followed by LSCM was used to localize the cells featuring specific markers of viability, proliferation, apoptosis and metastasis. The overlay of the mass spectrometry (MS) and IHC spheroid images, typically without any morphological features, required fiducial-based coregistration. The MALDI MSI protocol was optimized in terms of fiducial composition and antigen epitope preservation to allow MALDI MSI to be performed and directly followed by IHC analysis on exactly the same spheroid section. Once MS and IHC images were coregistered, the quantification of the MS and IHC signals was performed by an algorithm evaluating signal intensities along equidistant layers from the spheroid boundary to its center. This accurate colocalization of MS and IHC signals showed limited penetration of the clinically tested drug perifosine into spheroids during a 24 h period, revealing the fraction of proliferating and promigratory/proinvasive cells present in the perifosine-free areas, decrease of their abundance in the perifosine-positive regions, and distinguishing between apoptosis resulting from hypoxia/nutrient deprivation and drug exposure.
Topics: Animals; Cell Culture Techniques; Fiducial Markers; Fluorescent Antibody Technique; HT29 Cells; Humans; Imaging, Three-Dimensional; Microscopy, Confocal; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
PubMed: 31584797
DOI: 10.1021/acs.analchem.9b02462 -
Anticancer Research Oct 2019The P13K/Akt signaling pathway is a growth-regulating cellular pathway that is constitutively activated in a variety of human cancers. In previous studies, we reported...
BACKGROUND/AIM
The P13K/Akt signaling pathway is a growth-regulating cellular pathway that is constitutively activated in a variety of human cancers. In previous studies, we reported that a solenopsin analog, compound (MU-06-SC-608-7), shows inhibitory effects on Akt phosphorylation at a key activation site, as well as on proliferation of tumorigenic cells at sub-micromolar concentrations. The purpose of this study was to evaluate the effect of compound on downstream effectors of Akt kinase, phosphorylation of Akt at a second activation site, Akt kinase activity in vitro, tumorigenic cell viability and other signaling pathways.
MATERIALS AND METHODS
Western blot analyses were performed using WBras1 epithelial and H2009 human carcinoma cells and cell viability assays were performed on H2009 cells. In vitro Akt kinase assays were performed using a commercially available kit.
RESULTS
Compound decreased the phosphorylation of Akt at the Thr308 activation site and key downstream effectors of Akt kinase, but did not directly inhibit Akt kinase. Substantial decreases in cell viability were observed at concentrations above 5 μM. No effect was seen on ERK or JNK pathways.
CONCLUSION
The results earmark this compound for further studies as a potential targeted cancer therapy.
Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Survival; Enzyme Inhibitors; Humans; MAP Kinase Signaling System; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Pyrimidines; Signal Transduction
PubMed: 31570426
DOI: 10.21873/anticanres.13725 -
Advances in Experimental Medicine and... 2019Diabetic neuropathy (DN) is the most common chronic complication of DM and its major pathological changes show axonal dysfunction, atrophy and loss. However, there are...
Diabetic neuropathy (DN) is the most common chronic complication of DM and its major pathological changes show axonal dysfunction, atrophy and loss. However, there are few reports that taurine promotes neurite growth of dorsal root ganglion (DRG) cells. In current study, DRG neurons were exposed to high glucose (HG) with or without taurine. The neurite outgrowth of DRG neurons was observed by fluorescent immunohistochemistry method. Expression of Gap-43, Akt, phosphorylated Akt, mTOR and phosphorylated mTOR was determined by Western blot assay. Our results showed that HG significantly decreased the neurite outgrowth and expression of Gap-43 in DRG neurons. Moreover, phosphorylated levels of Akt and mTOR were downregulated in DRG neurons exposed to HG. On the contrary, taurine supplementation significantly reversed the decreased neurite outgrowth and Gap-43 expression, and the downregulated phosphorylated levels of Akt and mTOR. However, the protective effects of taurine were blocked in the presence of PI3K antagonists LY294002 or Akt antagonists Perifosine. These results indicate that taurine promotes neurite outgrowth of DRG neurons exposed to HG via activating Akt/mTOR signal pathway.
Topics: Cells, Cultured; GAP-43 Protein; Ganglia, Spinal; Glucose; Humans; Neurites; Neurons; Proto-Oncogene Proteins c-akt; TOR Serine-Threonine Kinases; Taurine
PubMed: 31468457
DOI: 10.1007/978-981-13-8023-5_77 -
Advances in Experimental Medicine and... 2019Diabetes causes memory loss. Hippocampus is responsible for memory and increased apoptosis was found in diabetes patients. Taurine improved memory in diabetes condition....
Diabetes causes memory loss. Hippocampus is responsible for memory and increased apoptosis was found in diabetes patients. Taurine improved memory in diabetes condition. However, mechanism is unclear. In current study, hippocampal cell line HT-22 cells were subjected to analysis as five groups i.e. Control, High glucose (HG) at concentration of 150 mM, HG + 10 mM (T1), 20 mM (T2) and 40 mM (T3) taurine solution. TUNEL assay showed that HG increased the number of apoptotic cell significantly while taurine reduced apoptosis. Taurine increased phosphorylation of Akt in HT-22 cell treated with HG, and increased phosphorylation of Bad (p-Bad) was seen suggesting involvement of Akt/Bad signaling pathway. Expression of Bcl-2 was reduced in HG group but taurine improved this. Bax expression showed opposite trend. This indicated that taurine may reduce apoptosis by controlling balance of Bcl-2 and Bax. When the activation of Akt was blocked by using of perifosine, the effect of taurine disappears either partially or altogether. Thus, it was clear that taurine reduces apoptosis via Akt/Bad pathway in HT-22 cells exposed to HG which further improves downstream balance of Bcl-2 and Bax. This mechanism may be involved in apoptosis of hippocampus cells in diabetic condition.
Topics: Animals; Apoptosis; Cell Line; Glucose; Hippocampus; Mice; Neurons; Phosphorylation; Signal Transduction; Taurine
PubMed: 31468455
DOI: 10.1007/978-981-13-8023-5_75 -
Journal of Neuro-oncology Sep 2019Perifosine (PRF) is an oral alkylphospholipid with antineoplastic effects and reasonable tolerability. It inhibits signaling through the PI3/AKT axis and other cascades...
PURPOSE
Perifosine (PRF) is an oral alkylphospholipid with antineoplastic effects and reasonable tolerability. It inhibits signaling through the PI3/AKT axis and other cascades of biologic importance in glioblastoma, and has promising pre-clinical activity in vitro and in vivo. Therefore, we conducted a phase II open-label single-arm clinical trial of perifosine for patients with recurrent glioblastoma (GBM).
METHODS
We planned to accrue up to 30 adults with recurrent GBM with a minimum Karnofsky Performance Status of 50 following radiotherapy but without other restrictions on the number or types of prior therapy. Concurrent p450 stimulating hepatic enzyme inducing anticonvulsants were prohibited. Patients were treated with a loading dose of 600 mg PRF (in 4 divided doses on day 1) followed by 100 mg daily until either disease progression or intolerable toxicity. The primary endpoint was the 6-month progression free survival (PFS6) rate, with at least 20% considered promising. Accrual was continuous but if 0 of the first 12 patients with GBM reached PFS6, then further accrual would terminate for futility. Patients with other high grade gliomas were accrued concurrently to an exploratory cohort.
RESULTS
Treatment was generally well tolerated; gastrointestinal toxicities were the most common side effects, although none resulted in treatment discontinuation. However, there was limited to no efficacy in GBM (n = 16): the PFS6 rate was 0%, median PFS was 1.58 months [95% CI (1.08, 1.84)], median overall survival was 3.68 months [95% CI (2.50, 7.79)], with no radiographic responses. There was a confirmed partial response in one patient with anaplastic astrocytoma (n = 14).
CONCLUSIONS
PRF is tolerable but ineffective as monotherapy for GBM. Preclinical data suggests synergistic effects of PRF in combination with other approaches, and further study is ongoing.
Topics: Adult; Aged; Brain Neoplasms; Female; Follow-Up Studies; Glioblastoma; Humans; Male; Middle Aged; Neoplasm Recurrence, Local; Phosphorylcholine; Prognosis; Prospective Studies; Proto-Oncogene Proteins c-akt; Survival Rate; Young Adult
PubMed: 31325145
DOI: 10.1007/s11060-019-03243-7 -
Handbook of Experimental Pharmacology 2020Synthetic antitumor lipids are metabolically stable lysophosphatidylcholine derivatives, encompassing a class of non-mutagenic drugs that selectively target cancerous... (Review)
Review
Synthetic antitumor lipids are metabolically stable lysophosphatidylcholine derivatives, encompassing a class of non-mutagenic drugs that selectively target cancerous cells. In this chapter we review the literature as relates to the clinical efficacy of these antitumor lipid drugs and how our understanding of their mode of action has evolved alongside key advances in our knowledge of membrane structure, organization, and function. First, the history of the development of this class of drugs is described, providing a summary of clinical outcomes of key members including edelfosine, miltefosine, perifosine, erufosine, and erucylphosphocholine. A detailed description of the biophysical properties of these drugs and specific drug-lipid interactions which may contribute to the selectivity of the antitumor lipids for cancer cells follows. An updated model on the mode of action of these lipid drugs as membrane disorganizing agents is presented. Membrane domain organization as opposed to targeting specific proteins on membranes is discussed. By altering membranes, these antitumor lipids inhibit many survival pathways while activating pro-apoptotic signals leading to cell demise.
Topics: Antineoplastic Agents; Apoptosis; Humans; Lipids; Membrane Microdomains; Neoplasms
PubMed: 31302758
DOI: 10.1007/164_2019_222 -
PloS One 2019Protein kinase B (AKT) is a serine/threonine kinase that functions as an important downstream effector of phosphoinositide 3-kinase. We have recently shown that MK-2206...
Protein kinase B (AKT) is a serine/threonine kinase that functions as an important downstream effector of phosphoinositide 3-kinase. We have recently shown that MK-2206 and triciribine, two highly selective AKT inhibitors increase the level of low density lipoprotein receptor (LDLR) mRNA which leads to increased amount of cell-surface LDLRs. However, whereas MK-2206 induces transcription of the LDLR gene, triciribine stabilizes LDLR mRNA, raising the possibility that the two inhibitors may actually affect other kinases than AKT. In this study, we aimed to ascertain the role of AKT in regulation of LDLR mRNA expression by examining the effect of five additional AKT inhibitors on LDLR mRNA levels. Here we show that in cultured HepG2 cells, AKT inhibitors ARQ-092, AKT inhibitor VIII, perifosine, AT7867 and CCT128930 increase LDLR mRNA levels by inducing the activity of LDLR promoter. CCT128930 also increased the stability of LDLR mRNA. To study the role of AKT isoforms on LDLR mRNA levels, we examined the effect of siRNA-mediated knockdown of AKT1 or AKT2 on LDLR promoter activity and LDLR mRNA stability. Whereas knockdown of either AKT1 or AKT2 led to upregulation of LDLR promoter activity, only knockdown of AKT2 had a stabilizing effect on LDLR mRNA. Taken together, these results provide strong evidence for involvement of AKT in regulation of LDLR mRNA expression, and point towards the AKT isoform specificity for upregulation of LDLR mRNA expression.
Topics: Aminopyridines; Animals; Benzimidazoles; CHO Cells; Cricetinae; Cricetulus; Hep G2 Cells; Heterocyclic Compounds, 3-Ring; Humans; Imidazoles; Phosphorylcholine; Piperidines; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Pyrazoles; Pyrimidines; Pyrroles; Quinoxalines; RNA Stability; RNA, Messenger; Receptors, LDL; Ribonucleosides; Transcriptional Activation
PubMed: 31216345
DOI: 10.1371/journal.pone.0218537