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Caenorhabditis elegans as a platform to study the mechanism of action of synthetic antitumor lipids.Cell Cycle (Georgetown, Tex.) 2014Drugs capable of specifically recognizing and killing cancer cells while sparing healthy cells are of great interest in anti-cancer therapy. An example of such a drug is...
Drugs capable of specifically recognizing and killing cancer cells while sparing healthy cells are of great interest in anti-cancer therapy. An example of such a drug is edelfosine, the prototype molecule of a family of synthetic lipids collectively known as antitumor lipids (ATLs). A better understanding of the selectivity and the mechanism of action of these compounds would lead to better anticancer treatments. Using Caenorhabditis elegans, we modeled key features of the ATL selectivity against cancer cells. Edelfosine induced a selective and direct killing action on C. elegans embryos, which was dependent on cholesterol, without affecting adult worms and larvae. Distinct ATLs ranked differently in their embryonic lethal effect with edelfosine > perifosine > erucylphosphocholine >> miltefosine. Following a biased screening of 57 C. elegans mutants we found that inactivation of components of the insulin/IGF-1 signaling pathway led to resistance against the ATL edelfosine in both C. elegans and human tumor cells. This paper shows that C. elegans can be used as a rapid platform to facilitate ATL research and to further understand the mechanism of action of edelfosine and other synthetic ATLs.
Topics: Animals; Antineoplastic Agents; Apoptosis; Caenorhabditis elegans; Cell Line, Tumor; Cholesterol; Drug Resistance; Embryo, Nonmammalian; Embryonic Development; Humans; Insulin-Like Growth Factor I; Larva; Membrane Microdomains; Phospholipid Ethers; Phosphorylcholine
PubMed: 25485582
DOI: 10.4161/15384101.2014.952183 -
BioMed Research International 2014Low-passage cancer cell lines are versatile tools to study tumor cell biology. Here, we have employed four such cell lines, established from primary tumors of colorectal...
Low-passage cancer cell lines are versatile tools to study tumor cell biology. Here, we have employed four such cell lines, established from primary tumors of colorectal cancer (CRC) patients, to evaluate effects of the small molecule kinase inhibitors (SMI) vemurafenib, trametinib, perifosine, and regorafenib in an in vitro setting. The mutant BRAF (V600E/V600K) inhibitor vemurafenib, but also the MEK1/2 inhibitor trametinib efficiently inhibited DNA synthesis, signaling through ERK1/2 and expression of genes downstream of ERK1/2 in BRAF mutant cells only. In case of the AKT inhibitor perifosine, three cell lines showed a high or intermediate responsiveness to the drug while one cell line was resistant. The multikinase inhibitor regorafenib inhibited proliferation of all CRC lines with similar efficiency and independent of the presence or absence of KRAS, BRAF, PIK3CA, and TP53 mutations. Regorafenib action was associated with broad-range inhibitory effects at the level of gene expression but not with a general inhibition of AKT or MEK/ERK signaling. In vemurafenib-sensitive cells, the antiproliferative effect of vemurafenib was enhanced by the other SMI. Together, our results provide insights into the determinants of SMI efficiencies in CRC cells and encourage the further use of low-passage CRC cell lines as preclinical models.
Topics: Aged; Aged, 80 and over; Bromodeoxyuridine; Cell Death; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genes, Neoplasm; Humans; Inhibitory Concentration 50; Male; Molecular Targeted Therapy; Protein Kinase Inhibitors; Signal Transduction; Small Molecule Libraries
PubMed: 25309914
DOI: 10.1155/2014/568693 -
PloS One 2014Schistosomiasis is a parasitic disease caused by trematodes of the genus Schistosoma. Five species of Schistosoma are known to infect humans, out of which S. haematobium... (Comparative Study)
Comparative Study
BACKGROUND
Schistosomiasis is a parasitic disease caused by trematodes of the genus Schistosoma. Five species of Schistosoma are known to infect humans, out of which S. haematobium is the most prevalent, causing the chronic parasitic disease schistosomiasis that still represents a major problem of public health in many regions of the world and especially in tropical areas, leading to serious manifestations and mortality in developing countries. Since the 1970s, praziquantel (PZQ) is the drug of choice for the treatment of schistosomiasis, but concerns about relying on a single drug to treat millions of people, and the potential appearance of drug resistance, make identification of alternative schistosomiasis chemotherapies a high priority. Alkylphospholipid analogs (APLs), together with their prototypic molecule edelfosine (EDLF), are a family of synthetic antineoplastic compounds that show additional pharmacological actions, including antiparasitic activities against several protozoan parasites.
METHODOLOGY/PRINCIPAL FINDINGS
We found APLs ranked edelfosine> perifosine> erucylphosphocholine> miltefosine for their in vitro schistosomicidal activity against adult S. mansoni worms. Edelfosine accumulated mainly in the worm tegument, and led to tegumental alterations, membrane permeabilization, motility impairment, blockade of male-female pairing as well as induction of apoptosis-like processes in cells in the close vicinity to the tegument. Edelfosine oral treatment also showed in vivo schistosomicidal activity and decreased significantly the egg burden in the liver, a key event in schistosomiasis.
CONCLUSIONS/SIGNIFICANCE
Our data show that edelfosine is the most potent APL in killing S. mansoni adult worms in vitro. Edelfosine schistosomicidal activity seems to depend on its action on the tegumental structure, leading to tegumental damage, membrane permeabilization and apoptosis-like cell death. Oral administration of edelfosine diminished worm and egg burdens in S. mansoni-infected CD1 mice. Here we report that edelfosine showed promising antischistosomal properties in vitro and in vivo.
Topics: Animals; Antineoplastic Agents; Antiparasitic Agents; Apoptosis; Female; Mice; Phospholipid Ethers; Phosphorylcholine; Schistosoma mansoni; Schistosomiasis mansoni
PubMed: 25302497
DOI: 10.1371/journal.pone.0109431 -
American Journal of Physiology.... Dec 2014Radiation-induced gastrointestinal (GI) syndrome currently has no effective prophylactic or therapeutic treatment. Previous studies and our data have demonstrated the...
Radiation-induced gastrointestinal (GI) syndrome currently has no effective prophylactic or therapeutic treatment. Previous studies and our data have demonstrated the important role of p53 in acute radiation-induced GI syndrome in mice. Many cytokines, such as tumor necrosis factor-α and fibroblast growth factor (bFGF), have been found to protect against radiation-induced intestinal injury, although the underlying mechanisms remain to be identified. Here, we report blockage of p53 through a protein kinase B (Akt) pathway in intestinal progenitor/stem cells or crypt cells as a novel molecular mechanism of growth factor-mediated intestinal radioprotection. Treatment with platelet-derived growth factor (PDGF-BB) or bFGF activated Akt phosphorylation in the intestinal crypt, lessened intestinal crypt p53 expression, decreased radiation-induced apoptosis in mouse intestinal progenitor/stem cell marker leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5)-positive cells by an average of 50%, and increased the survival rate of mice with abdominal radiation by 3 days in average. Conversely, the Akt inhibitor perifosine obstructed growth factor-simulated Akt phosphorylation while promoting radiation-induced p53 expression in intestinal crypts. Importantly, reduced Akt phosphorylation and elevated p53 expression due to the Akt inhibitor perifosine impaired intestinal progenitor/stem cells radioprotection provided by PDGF-BB and bFGF. Consistently, PDGF-BB and bFGF both upregulated Akt activation, suppressed radiation-induced p53 expression, and abrogated radiation-induced apoptosis in IEC-6 cells, although p53 overexpression in IEC-6 cells partially counteracted the radioprotection of PDGF-BB and bFGF. Our data suggest that intestinal crypt radioprotection by PDGF-BB and bFGF is dependent on regulation of Akt/p53 signaling.
Topics: Animals; Apoptosis; Becaplermin; Fibroblast Growth Factor 2; Intestines; Mice; Oncogene Protein v-akt; Proto-Oncogene Proteins c-sis; Radiation-Protective Agents; Signal Transduction; Stem Cells; Tumor Suppressor Protein p53
PubMed: 25301184
DOI: 10.1152/ajpgi.00151.2014 -
Cancer Chemotherapy and Pharmacology Nov 2014To determine the maximum tolerated dose (MTD) of perifosine (NSC 639966), an alkylphospholipid modulator of signal transduction, using different oral loading and...
PURPOSE
To determine the maximum tolerated dose (MTD) of perifosine (NSC 639966), an alkylphospholipid modulator of signal transduction, using different oral loading and maintenance regimens in an effort to avoid gastrointestinal toxicity while seeking maximal sustained plasma concentrations.
METHODS
Thirty-one patients with advanced neoplasms were treated with monthly cycles of perifosine loading doses of 300, 600, 900, 1,200 and 1,500 mg (dose levels 1 through 5, respectively) on days 1-2 depending on the actual dose of the initial cycle. For subsequent cycles, perifosine loading doses were reduced to 100, 200, 300, 400 and 1,000 mg at the respective corresponding dose levels. Daily perifosine "maintenance" doses of 50, 100, 150, 200 and 250 mg for levels 1 through 5, respectively, commenced on days 2 or 3 and continued for a total of 21 days. No treatment was given for days 22-27. The pharmacokinetics of perifosine with these schedules was characterized.
RESULTS
Dose-limiting diarrhea developed at or above dose level 4. The MTD and recommended phase II dose was dose level 3B, with a loading dose of 900 mg on day 1 divided into two doses of 450 mg administered 6 h apart and a maintenance dose of 150 mg on day 2 through 21. On subsequent cycles, the loading dose was reduced to 300 mg. Non-gastrointestinal toxicities included three episodes of gout or gout-like syndromes observed at doses above the MTD. The median peak plasma concentration of perifosine achieved at the MTD was approximately 8.3 µg/mL. Four patients had stable disease ranging from 167 to 735 days.
CONCLUSIONS
Perifosine given according to a loading and maintenance schedule can safely sustain concentrations of drug, approaching concentrations achieved in preclinical models with evidence of anti-tumor effect.
Topics: Administration, Oral; Adult; Aged; Aged, 80 and over; Anorexia; Area Under Curve; Diarrhea; Disease Progression; Dose-Response Relationship, Drug; Drug Administration Schedule; Fatigue; Female; Humans; Male; Metabolic Clearance Rate; Middle Aged; Neoplasms; Phosphorylcholine; Treatment Outcome; Young Adult
PubMed: 25183650
DOI: 10.1007/s00280-014-2569-7 -
International Journal of Molecular... Jul 2014Precartilaginous stem cells (PSCs) could self-renew or differentiate into chondrocytes to promote bone growth. In the current study, we aim to understand the role of...
Precartilaginous stem cells (PSCs) could self-renew or differentiate into chondrocytes to promote bone growth. In the current study, we aim to understand the role of transforming growth factor-β1 (TGF-β1) in precartilaginous stem cell (PSC) proliferation, and to study the underlying mechanisms. We successfully purified and primary-cultured PSCs from the neonate mice' perichondrial mesenchyme, and their phenotype was confirmed by the PSC marker fibroblast growth factor receptor-3 (FGFR-3) overexpression. We found that TGF-β1 induced Akt-glycogen synthase kinase-3β (GSK3β) phosphorylation and β-catenin nuclear translocation in the mouse PSCs, which was almost blocked by TGF-β receptor-II (TGFRII) shRNA knockdown. Further, perifosine and MK-2206, two Akt-specific inhibitors, suppressed TGF-β1-induced GSK3β phosphorylation and β-catenin nuclear translocation. Akt inhibitors, as well as β-catenin shRNA knockdown largely inhibited TGF-β1-stimulated cyclin D1/c-myc gene transcription and mouse PSC proliferation. Based on these results, we suggest that TGF-β1 induces Akt activation to promote β-catenin nuclear accumulation, which then regulates cyclin D1/c-myc gene transcription to eventually promote mouse PSC proliferation.
Topics: Active Transport, Cell Nucleus; Animals; Cell Nucleus; Cell Proliferation; Cells, Cultured; Chondrocytes; Cyclin D1; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Mesenchymal Stem Cells; Mice; Mice, Inbred C57BL; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-myc; Receptor, Fibroblast Growth Factor, Type 3; Signal Transduction; Transforming Growth Factor beta1; beta Catenin
PubMed: 25036031
DOI: 10.3390/ijms150712665 -
Oncotarget Jul 2014ARID1A mutations are observed in various tumors, including ovarian clear cell (OCCC) and endometrioid carcinomas, endometrial, and breast carcinomas. They commonly...
ARID1A mutations are observed in various tumors, including ovarian clear cell (OCCC) and endometrioid carcinomas, endometrial, and breast carcinomas. They commonly result in loss of ARID1A-protein expression and frequently co-occur with PI3K/AKT-pathway activating mechanisms. The aim of this study was to test the hypothesis as to whether PI3K/AKT-pathway activation is a critical mechanism in ARID1A-mutated tumors and if consequently ARID1A-deficient tumors show increased sensitivity to treatment with PI3K- and AKT-inhibitors. Upon ARID1A knockdown, MCF7 breast cancer cells and primary MRC5 cells exhibited a significantly increased sensitivity towards the AKT-inhibitors MK-2206 and perifosine, as well as the PI3K-inhibitor buparlisib. Knockdown of ARID1A in MCF7 led to an increase of pAKT-Ser473. AKT-inhibition with MK-2206 led to increased apoptosis and to a decrease of pS6K in ARID1A-depleted MCF7 cells but not in the controls. In five OCCC cell lines ARID1A-deficiency correlated with increased pAKT-Ser473 levels and with sensitivity towards treatment with the AKT-inhibitor MK-2206. In conclusion, ARID1A-deficient cancer cells demonstrate an increased sensitivity to treatment with small molecule inhibitors of the PI3K/AKT-pathway. These findings suggest a specific requirement of the PI3K/AKT pathway in ARID1A-deficient tumors and reveal a synthetic lethal interaction between loss of ARID1A expression and inhibition of the PI3K/AKT pathway.
Topics: Aminopyridines; Apoptosis; Breast Neoplasms; DNA-Binding Proteins; Female; Gene Knockdown Techniques; Heterocyclic Compounds, 3-Ring; Humans; MCF-7 Cells; Morpholines; Nuclear Proteins; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Phosphorylcholine; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; RNA, Small Interfering; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Transcription Factors; Transfection
PubMed: 24979463
DOI: 10.18632/oncotarget.2092 -
ACS Medicinal Chemistry Letters Dec 2013This study was aimed at investigating the antitumor activity of novel 2-oxindole derivatives against a well-characterized human nonsmall cell lung cancer (NSCLC) cell...
This study was aimed at investigating the antitumor activity of novel 2-oxindole derivatives against a well-characterized human nonsmall cell lung cancer (NSCLC) cell line. Test compounds produced an antiproliferative activity in the low micromolar/submicromolar range of concentrations and significantly induced typical apoptotic morphology with cell shrinkage, nuclear condensation and fragmentation, and rupture of cells into debris in a relatively low percentage of A549 cells. Cell cycle arrest occurred at the G1/S phase (1a and 2), and Akt phosphorylation was significantly inhibited at Thr308 and Ser473. The most active compound (1a) has an IC50 6-fold lower than the Akt inhibitor, perifosine. These data suggest that the new compounds may be cytostatic and may have maximum clinical effects in NSCLC patients who do not respond to EGFR inhibitors. These findings prompt us to further explore the oxindole structure as leading scaffold to design new molecules with potent antitumor activity against NSCLC.
PubMed: 24900620
DOI: 10.1021/ml400162g -
Cellular Logistics Jan 2014Brefeldin A (BFA) is a fungal metabolite best known for its ability to inhibit activation of ADP-ribosylation factor (Arf) and thereby inhibit secretory traffic. BFA...
Brefeldin A (BFA) is a fungal metabolite best known for its ability to inhibit activation of ADP-ribosylation factor (Arf) and thereby inhibit secretory traffic. BFA also appears to regulate the trafficking of the GLUT4 glucose transporter by inducing its relocation from intracellular stores to the cell surface. Such redistribution of GLUT4 is normally regulated by insulin-mediated signaling. Hence, we tested whether BFA may intersect with the insulin pathway. We report that BFA causes the activation of the insulin receptor (IR), IRS-1, Akt-2, and AS160 components of the insulin pathway. The response is mediated through phosphoinositol-3-kinase (PI3K) and Akt kinase since the PI3K inhibitor wortmannin and the Akt inhibitors MK2206 and perifosine inhibit the BFA effect. BFA-mediated activation of the insulin pathway results in Akt-mediated phosphorylation of the insulin-responsive transcription factor FoxO1. This leads to nuclear exclusion of FoxO1 and a decrease in transcription of the insulin-responsive gene SIRT-1. Our findings suggest novel effects for BFA in signaling and transcription, and imply that BFA has multiple intracellular targets and can be used to regulate diverse cellular responses that include vesicular trafficking, signaling and transcription.
PubMed: 24843827
DOI: 10.4161/cl.27732 -
Biochimica Et Biophysica Acta Sep 2014Centrosome amplification, which is a characteristic of cancer cells, has been understood as a driving force of genetic instability in the development of cancer. In...
Centrosome amplification, which is a characteristic of cancer cells, has been understood as a driving force of genetic instability in the development of cancer. In previous work, we demonstrated that TEIF (transcriptional element-interacting factor) distributes in the centrosomes and regulates centrosome status under both physiologic and pathologic conditions. Here we identify TEIF as a downstream effector in EGF/PI3K/Akt signaling. The addition of EGF or transfection of active Akt stimulates centrosome TEIF distribution, resulting in an increase of centrosome splitting and amplification, while inhibitors of either PI3K or Akt attenuate these changes in TEIF and the associated centrosome status. A consensus motif for Akt phosphorylation (RHRVLT) proved to be involved in centrosomal TEIF localization, and the 469-threonine of this motif may be phosphorylated by Akt both in vitro and in vivo. Elimination of this phosphorylated site on TEIF caused reduced centrosome distribution and centrosome splitting or amplification. Moreover, TEIF closely co-localized with C-NAP1 at the proximal ends of centrioles, and centriolar loading of TEIF stimulated by EGF/Akt could displace C-NAP1, resulting in centrosome splitting. These findings reveal linkage of the EGF/PI3K/Akt signaling pathway to regulation of centrosome status which may act as an oncogenic pathway and induce genetic instability in carcinogenesis.
Topics: Adaptor Proteins, Vesicular Transport; Amino Acid Sequence; Cell Line; Centrioles; Centrosome; DNA-Binding Proteins; Epidermal Growth Factor; Green Fluorescent Proteins; Humans; Molecular Sequence Data; Nuclear Proteins; Phosphatidylinositol 3-Kinases; Phosphorylation; Phosphorylcholine; Protein Transport; Proto-Oncogene Proteins c-akt; Signal Transduction; Transcription Factors
PubMed: 24769208
DOI: 10.1016/j.bbamcr.2014.04.021