-
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 -
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 -
Leukemia & Lymphoma Nov 2019Lipid rafts are ordered membrane domains, which provide an environment for the proteins participating in signal transduction. Perifosine is an alkylphospholipid (APL)...
Lipid rafts are ordered membrane domains, which provide an environment for the proteins participating in signal transduction. Perifosine is an alkylphospholipid (APL) that inhibits the AKT pathway, cytotoxic to neoplastic cells. We have shown that the lipid raft adaptor protein NTAL is a target of APLs in leukemic cells. Using human mantle cell lymphoma (MCL) Granta-519 cell line we showed here that perifosine decreased NTAL in lipid raft fractions reducing AKT phosphorylation before apoptosis. We also showed that the NTAL-knockdown by shRNA induced a state of reduced AKT activation. Experimental NTAL-knockdown in NSG mouse MCL xenografts reduced AKT activity, increased the basal apoptotic rate by 3-fold ( = 8) and decreased tumor weight by 2.7-fold ( = 5), indicating that NTAL participates in tumor growth. NTAL protein was detected by western blotting in circulating cells of 7 of 8 MCL patients in the leukemic phase, suggesting involvement in the progression of the disease.
Topics: Adaptor Proteins, Signal Transducing; Aged; Animals; Apoptosis; Biomarkers, Tumor; Cell Proliferation; Female; Gene Expression Regulation, Neoplastic; Humans; Lymphoma, Mantle-Cell; Mice; Mice, Inbred NOD; Mice, SCID; Middle Aged; Phosphorylcholine; Prognosis; Proto-Oncogene Proteins c-akt; Survival Rate; Tumor Cells, Cultured; Xenograft Model Antitumor Assays
PubMed: 31060403
DOI: 10.1080/10428194.2019.1607326 -
Experimental and Molecular Pathology Aug 2019Although tenascin-C (TNC), an extracellular matrix protein, has been shown to be widely expressed in stromal fibroblasts in various cancers, the role of its expression...
Although tenascin-C (TNC), an extracellular matrix protein, has been shown to be widely expressed in stromal fibroblasts in various cancers, the role of its expression in esophageal squamous cell carcinoma (ESCC) cells remains unclear. Using immunohistochemistry, we investigated the expression of cancer stem-like cell (CSC) markers, epithelial-to-mesenchymal transition (EMT)-related genes, and the Akt/hypoxia-inducible factor-1α (HIF1α) signal pathway in ESCC tissue specimens from 154 patients. We further addressed the effects of TNC on the Akt/HIF1α axis and its putative association with cancer stemness in several ESCC cell lines by immunofluorescence imaging and western blot analysis. Our data suggest that TNC expression was positively correlated with the expression of the CSC marker SOX2 (p = .002), and TNC-expressing cancer cells expressed SOX2 in ESCC tissues. Moreover, TNC expression was strongly associated with EMT-related gene Snail (p = .022) and positively correlated with pAkt-Ser473 (p = .004) and HIF1α (p = .003). Furthermore, TNC-silencing down-regulated the expression of CSC marker SOX2 (p < .001) and EMT-related marker Snail (p < .001). The Akt inhibitor Perifosine inhibited the protein expression of pAkt-Ser473, Akt, HIF1α, and TNC in TE10 (an ESCC cell line) cells. Short-term exposure of TE10 cells to cobalt chloride caused an increase in protein expression of HIF1α, TNC, and SOX2 in a time-dependent manner. Taken together, these results suggest that TNC may enhance the cancer stem-like properties and promote EMT-like changes via the Akt/HIF1α axis.
Topics: Biomarkers, Tumor; Cell Line, Tumor; Disease-Free Survival; Epithelial-Mesenchymal Transition; Esophageal Neoplasms; Esophageal Squamous Cell Carcinoma; Gene Expression Regulation, Neoplastic; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Immunohistochemistry; Neoplastic Stem Cells; Proto-Oncogene Proteins c-akt; RNA Interference; Signal Transduction; Tenascin
PubMed: 30904401
DOI: 10.1016/j.yexmp.2019.03.007