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1.
Eur J Pharmacol ; 887: 173584, 2020 Nov 15.
Article in English | MEDLINE | ID: mdl-32950500

ABSTRACT

Mutations of p53 in cancer cells not only subvert its antiproliferative properties but can also promote various oncogenic responses through a gain-of-function activity. Pharmacological manipulation of the mutant p53 pathway by specific compounds could be an effective strategy for cancer therapy. We show here that gain-of-function p53 mutation in gastric cancer cells promotes tumorigenesis by enhancing p53-EGFR (epidermal growth factor receptor) signaling pathway, and such process can be blocked by small molecule NA20, a naphthalimide derivative that exhibited selective inhibition in p53 mutant gastric cancer cell lines. We found that targeting DNA and blocking the mutant p53-drived carcinogenicity accounted for the primary antitumor effect of NA20 in gastric tumor models. NA20 bound to DNA and p53 identified by a combination of drug tracking, DNA relaxation assay and coimmunoprecipitation-mass spectrometry (CoIP-MS) detection, which led to the p21 activation and the suppression of EGFR signal cascading, thereby evoking cell cycle arrest and cell apoptosis, finally leading to cancer cell inhibition both in vitro and in vivo. Taken together, these results suggest that NA20 may be a potential candidate for gastric cancer therapy.


Subject(s)
DNA/drug effects , Genes, p53/drug effects , Naphthalimides/pharmacology , Signal Transduction/drug effects , Stomach Neoplasms/prevention & control , Animals , Apoptosis/drug effects , Cell Cycle Checkpoints/drug effects , Cell Line, Tumor , Cell Survival/drug effects , ErbB Receptors/drug effects , Humans , Male , Mice , Mice, Inbred BALB C , Oncogene Protein p21(ras)/drug effects , Xenograft Model Antitumor Assays
2.
Life Sci ; 146: 81-91, 2016 Feb 01.
Article in English | MEDLINE | ID: mdl-26764233

ABSTRACT

AIMS: Among cancers, hepatocellular carcinoma is one of the commonest worldwide, and its incidence is increasing around the world. A lot of evidence underlines that natural substances usually consumed in the diet can have an important role in the prevention of cancer. In this study we investigated the molecular mechanisms underlying the antiproliferative activity of Citrus bergamia (bergamot) juice (BJ) in human hepatocellular carcinoma HepG2 cells. MAIN METHODS: HepG2 cells were exposed to BJ and then cell proliferation, cell cycle progression, apoptosis and NF-κB nuclear translocation were evaluated. KEY FINDINGS: Here we present results demonstrating that BJ reduced the growth rate of human hepatocellular carcinoma HepG2 cells in a time- and concentration-dependent manner, by a mechanism involving the activation of apoptotic machinery via both intrinsic and extrinsic pathways. Moreover, BJ increased expression of P53 and P21 proteins that may be responsible for the HepG2 cell cycle arrest in G2 phase. In addition, BJ reduced NF-κB nuclear translocation. SIGNIFICANCE: Our data demonstrate the ability of BJ in reducing the growth of HepG2 cells, revealing its mechanism of action and suggesting a promising role as anticancer drugs.


Subject(s)
Apoptosis/drug effects , Cell Proliferation/drug effects , NF-kappa B/metabolism , Plant Extracts/pharmacology , Cell Cycle/drug effects , Comet Assay , DNA Damage , DNA, Complementary/biosynthesis , DNA, Complementary/genetics , G2 Phase/drug effects , Gene Expression Regulation, Neoplastic/drug effects , Gene Expression Regulation, Neoplastic/genetics , Genes, p53/drug effects , Hep G2 Cells , Heterocyclic Compounds/chemistry , Humans , Oncogene Protein p21(ras)/drug effects , Oncogene Protein p21(ras)/genetics , RNA, Neoplasm/biosynthesis , Translocation, Genetic
3.
Surg Neurol ; 70(3): 259-66; discussion 266-7, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18295834

ABSTRACT

BACKGROUND: Activation of the p21-ras signaling pathway from aberrantly expressed receptors promotes the growth of malignant human astrocytomas. Perillyl alcohol has shown to have both chemopreventive and chemotherapeutic activities in preclinical studies. The underlying action mechanism(s) of POH has yet to be delineated but may involve effects on the TGF-beta and/or the Ras signaling pathways. The intranasal delivery allows drugs that do not cross the BBB to enter the CNS; moreover, it eliminates the need for systemic delivery, thereby reducing unwanted systemic side effects. METHODS: We are conducting a phase I/II study to evaluate the antitumoral activity of POH intranasal delivery in a 4x daily schedule in patients with recurrent MG. The objective was to determine PFS at 6 months and the safety for POH in adult patients who failed conventional treatment. Assessments were performed every 27 days. Thirty-seven patients with progressive disease after prior surgery, radiotherapy, and at least temozolomide-based chemotherapy were enrolled, 29 of whom had GBM, 5 who had anaplastic astrocytoma, and 3 had AO. RESULTS: One patient (3.4%) with GBM and 1 patient (33.3%) with AO achieved partial response; 13 patients (44.8%) with GBM, 3 patients (60%) with AA, and 1 (33.3%) with AO achieved stable disease; 15 (51.7%) patients with GBM, 2 (40%) patients with AA, and 1 (33.3%) with AO showed progressive disease. Progression-free survival (partial response and stable disease) was 48.2% for patients with GBM, 60% for patients with AA, and 66.6% for patients with AO. CONCLUSIONS: There were no toxicity events. Perillyl alcohol is well tolerated and regression of tumor size in some patients is suggestive of antitumor activity. This work discusses POH intranasal delivery as a potential adjuvant therapeutic strategy for patients with malignant gliomas.


Subject(s)
Administration, Intranasal , Antineoplastic Agents/administration & dosage , Brain Neoplasms/drug therapy , Glioma/drug therapy , Monoterpenes/administration & dosage , Antineoplastic Agents, Alkylating/administration & dosage , Blood-Brain Barrier/drug effects , Blood-Brain Barrier/physiology , Brain Neoplasms/metabolism , Brain Neoplasms/physiopathology , Dacarbazine/administration & dosage , Dacarbazine/analogs & derivatives , Disease Progression , Drug Administration Schedule , Enzyme Inhibitors/administration & dosage , Female , Glioma/metabolism , Glioma/physiopathology , Humans , Magnetic Resonance Imaging , Male , Neoplasm Recurrence, Local/drug therapy , Neoplasm Recurrence, Local/metabolism , Neoplasm Recurrence, Local/physiopathology , Oncogene Protein p21(ras)/drug effects , Oncogene Protein p21(ras)/genetics , Oncogene Protein p21(ras)/metabolism , Signal Transduction/drug effects , Signal Transduction/physiology , Survival Rate , Temozolomide , Transforming Growth Factor beta/drug effects , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/metabolism , Treatment Outcome
4.
Arterioscler Thromb Vasc Biol ; 26(11): 2454-61, 2006 Nov.
Article in English | MEDLINE | ID: mdl-16931794

ABSTRACT

OBJECTIVE: To understand the mechanism by which oxidants are linked to insulin resistance, bovine aortic endothelial cells were exposed to oxidized low-density lipoproteins (oxLDL) or peroxynitrite. METHODS AND RESULTS: OxLDL transiently increased phosphorylation of Erk and Akt within 5 minutes, but 60 minutes later, resulted in decreased insulin-induced Akt phosphorylation. OxLDL promoted a 2- to 5-fold increase in oxidant generation as measured by dihydrorhodamine or dihydroethidium oxidation that was ascribed to peroxynitrite. Exogenous peroxynitrite (25 to 100 micromol/L) or oxidized glutathione mimicked the effects of oxLDL. OxLDL increased the S-glutathiolation of p21ras, and adenoviral transfection with either a mutant p21ras (C118S) lacking the predominant site of S-glutathiolation or a dominant-negative mutant restored insulin-induced Akt phosphorylation. The requirement for oxidant-mediated S-glutathiolation and activation of p21ras in mediating insulin resistance was further implicated by showing that insulin signaling was restored by Mek inhibitors or by overexpression of glutaredoxin-1. Furthermore, oxLDL increased Erk-dependent phosphorylation of insulin receptor substrate-1 serine-616 that was prevented by inhibiting oxidant generation, Erk activation, or by the p21ras C118S mutant. CONCLUSIONS: This study provides direct evidence for a novel molecular mechanism by which oxidants can induce insulin resistance via S-glutathiolation of p21ras and Erk-dependent inhibition of insulin signaling.


Subject(s)
Endothelium, Vascular/drug effects , Endothelium, Vascular/physiology , Glutathione/metabolism , Insulin Resistance/physiology , Lipoproteins, LDL/pharmacology , Oncogene Protein p21(ras)/metabolism , Peroxynitrous Acid/pharmacology , Alprostadil/analogs & derivatives , Alprostadil/pharmacology , Animals , Aorta/cytology , Cattle , Dinoprostone/agonists , Endothelial Cells/drug effects , Endothelial Cells/physiology , Enzyme Activation , Extracellular Signal-Regulated MAP Kinases/metabolism , Glutathione Disulfide/pharmacology , Insulin/metabolism , Insulin Receptor Substrate Proteins , Lysophosphatidylcholines/pharmacology , Oncogene Protein p21(ras)/drug effects , Oxidants/pharmacology , Phosphoproteins/metabolism , Phosphorylation/drug effects , Prostaglandins E, Synthetic/pharmacology , Signal Transduction/drug effects , Signal Transduction/physiology
5.
J Thorac Cardiovasc Surg ; 129(1): 53-63, 2005 Jan.
Article in English | MEDLINE | ID: mdl-15632825

ABSTRACT

OBJECTIVE: Histone deacetylase inhibitors mediate a potent growth-inhibitory effect in cancer cells through induction of cell-cycle arrest and apoptosis. Moreover, these agents significantly induce transcriptional activation of nuclear factor kappaB, as well as p21 regulated by protein kinase C, and are thought to negatively influence the ability of histone deacetylase inhibitor to effectively mediate apoptosis. This study aimed to evaluate the effect of calphostin C (a protein kinase C inhibitor) on trichostatin A (a histone deacetylase inhibitor)-mediated upregulation of nuclear factor kappaB and p21 promotor transcriptional activity, as well as induction of apoptosis in lung and esophageal cancer cells. METHODS: Cultured lung and esophageal cancer cells were treated with calphostin C and trichostatin A. Nuclear factor kappaB transcriptional activity was quantitated by using the nuclear factor kappaB-luciferase assay. Transcription of p21 gene and p21 protein levels was evaluated by using the p21 promoter-luciferase assay and the p21 enzyme-linked immunoassay, respectively. Apoptosis was evaluated by using the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-based ApoBrdU assay. Levels of expression of nuclear factor kappaB-dependent antiapoptotic and proapoptotic proteins were evaluated by means of Western blotting. RESULTS: Exposure of lung or esophageal cancer cells to trichostatin A resulted in a dose- and cell-dependent 2-fold to greater than 20-fold increase of nuclear factor kappaB and p21 transcriptional activity. Treatment with trichostatin A and calphostin C led to a 50% to 90% decrease of trichostatin A- mediated upregulation of nuclear factor kappaB and p21 activation. Inhibition of nuclear factor kappaB activity resulted in significant reduction (30% to >99%) of trichostatin A- mediated activation of not only nuclear factor kappaB transcription but also p21 promotor activity. Importantly, 90% to 96% of thoracic cancer cells under-went apoptosis after exposure to the combination of trichostatin A plus calphostin C. CONCLUSION: Inhibition of protein kinase C abrogates trichostatin A-mediated upregulation of nuclear factor kappaB transcriptional activity and p21 expression that is associated with profound induction of apoptosis in lung or esophageal cancer cells. Protein kinase C might be a novel target for enhancing the efficacy of histone deacetylase inhibitor in cancer therapy.


Subject(s)
Apoptosis/drug effects , Hydroxamic Acids/pharmacology , NF-kappa B/metabolism , Naphthalenes/pharmacology , Oncogene Protein p21(ras)/metabolism , Blotting, Western , Cell Proliferation/drug effects , Cell Survival/drug effects , Drug Therapy, Combination , Esophageal Neoplasms/drug therapy , Esophageal Neoplasms/pathology , Histone Deacetylase Inhibitors , Humans , Lung Neoplasms/drug therapy , Lung Neoplasms/pathology , NF-kappa B/drug effects , Oncogene Protein p21(ras)/drug effects , Probability , Protein Kinase C/antagonists & inhibitors , Risk Factors , Sensitivity and Specificity , Tumor Cells, Cultured , Up-Regulation
6.
Life Sci ; 74(5): 643-50, 2003 Dec 19.
Article in English | MEDLINE | ID: mdl-14623034

ABSTRACT

Phenformin is a biguanide that has been largely used in the past for its anti-diabetic activity. A large body of evidence suggests additional effects of phenformin including antitumoral activity in different animal models in vivo. Thus, the present study has been conducted in order to elucidate possible mechanisms involved in the antitumoral effects of phenformin. In various tumoral cell lines (SH-SY5Y neuroblastoma and LNCaP prostate adenocarcinoma cells), increasing concentrations of phenformin (50-500 microM) induced a concentration-dependent inhibition of cell proliferation. This effect was not dependent on the ability of the drug to reduce glucose levels and was accompanied by induction of apoptotic cell death as measured by cytofluorometric analysis. In addition, a short-time incubation of SH-SY5Y cells with phenformin induced enhanced and transient expression of the cell cycle inhibitor p21 suggesting that phenformin causes inhibition of cell cycle progression prior to induction of apoptosis. These results demonstrate an activity at the cellular level of phenformin that supports its antitumoral effect observed in vivo.


Subject(s)
Antineoplastic Agents , Hypoglycemic Agents/pharmacology , Phenformin/pharmacology , Adenocarcinoma/pathology , Apoptosis/drug effects , Brain Neoplasms/pathology , Cell Cycle/drug effects , Cell Division/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Culture Media , Glucose/pharmacology , Humans , Male , Neuroblastoma/pathology , Oncogene Protein p21(ras)/drug effects , Prostatic Neoplasms/pathology , Tetrazolium Salts , Thiazoles
7.
J Thromb Haemost ; 1(5): 1012-8, 2003 May.
Article in English | MEDLINE | ID: mdl-12871370

ABSTRACT

BACKGROUND: In recent years it has become clear that factor (F)VIIa is not a passive mediator involved in the linear transduction of the coagulation cascade, but actively engages target cells to induce signal transduction and that this signal transduction fulfills critical functions in angiogenesis, arteriosclerosis and inflammatory processes. OBJECTIVES: The details of coagulation factor-dependent signal transduction are among the least understood in biology and thus we set out to establish the molecular events responsible for MAP kinase activation induced by the interaction of FVIIa with its cellular binding partner tissue factor (TF). METHODS: Two different TF-expressing cell types, BHKTF and HaCaT cells, were assayed for p21Ras activation using a pull-down assay that is specific for activated Ras. This activation was visualized by means of Western blotting. In addition, the upstream pathways leading to FVIIa-induced Ras activation were characterized using phosphospecific antibodies and specific inhibitors. RESULTS: We observed that in both BHKTF and HaCaT cells FVIIa-induced MAP kinase activation correlates with p21Ras activation, and that this p21Ras activation is essential for FVIIa-induced MAP kinase activation. In BHKTF cells, early p21Ras activation was mediated by the activation of protein kinase C (PKC), whereas late p21Ras activation employed alternative mechanisms. In HaCaT cells, stimulation of the Src kinase family mediated FVIIa-dependent p21Ras activation. Finally, in both cell types, Raf activity was mandatory for MAP kinase activation. CONCLUSIONS: p21Ras activation is instrumental in FVIIa signal transduction and the FVIIa-dependent activation of p21Ras involves either PKC or Src-dependent mechanisms, depending on the cell type investigated.


Subject(s)
Factor VIIa/pharmacology , MAP Kinase Signaling System/drug effects , Oncogene Protein p21(ras)/metabolism , Animals , Blotting, Western , Cell Line , Factor VIIa/physiology , Humans , Mitogen-Activated Protein Kinases/drug effects , Mitogen-Activated Protein Kinases/metabolism , Oncogene Protein p21(ras)/drug effects , Phosphorylation/drug effects , Protein Kinase C/metabolism , src-Family Kinases/metabolism
8.
Bioorg Med Chem Lett ; 12(4): 709-13, 2002 Feb 25.
Article in English | MEDLINE | ID: mdl-11844707

ABSTRACT

Metabolites of the non-steroidal anti-inflammatory drug Sulindac inhibit cell proliferation by affecting several intracellular signaling pathways including the tumorigenic Ras/Raf/MAPK pathway. Here, we report the synthesis of eight new indene derivatives derived from the Sulindac structure, and present data on their anti-proliferative properties and their effects on the p21ras protein.


Subject(s)
Antineoplastic Agents/chemical synthesis , Indenes/pharmacology , Sulindac/analogs & derivatives , Animals , Anti-Inflammatory Agents, Non-Steroidal , Antineoplastic Agents/pharmacology , Cell Division/drug effects , Combinatorial Chemistry Techniques , Cyclooxygenase Inhibitors/chemical synthesis , Cyclooxygenase Inhibitors/pharmacology , Dogs , Humans , Indenes/chemical synthesis , Inhibitory Concentration 50 , Mice , Oncogene Protein p21(ras)/antagonists & inhibitors , Oncogene Protein p21(ras)/drug effects , Prostaglandin-Endoperoxide Synthases/drug effects , Tumor Cells, Cultured/drug effects
9.
J Mol Biol ; 294(2): 403-16, 1999 Nov 26.
Article in English | MEDLINE | ID: mdl-10610767

ABSTRACT

We have previously described the rational design of mutation-selective antisense oligonucleotides targeted to codon 12 of oncogenic Ha-ras mRNA. In order to further improve the biological efficacy of these unmodified oligonucleotides, we have studied three different classes of modifications: peptide nucleic acid backbone (PNA), sugar modification (2'-O-methyl) and phosphoramidate linkage (PN). We show that PNA is unique among the investigated steric blocking agents in its ability to specifically inhibit the translation of Ha-ras mRNA in vitro. The PNA-RNA hybrid (Tm=86 degrees C), which is not dissociated by cellular proteins and resists phenol extraction and urea denaturing conditions, specifically blocks the translation of mutated Ha-ras mRNA. A PNA tridecamer which forms with wild-type Ha-ras mRNA a duplex with a central mismatch had little effect on mRNA translation. Codon 12 is located close to the translation initiation site and hybridization of the PNA at this position may interfere with the assembly of the translation initiation complex. To test whether polypeptide chain elongation can also be blocked, we have targeted PNA tridecamers to codons in the 74, 128 and 149 regions. These PNAs form equally stable duplexes as that formed by the PNA targeted to the codon 12 region (ten G.C base-pairs out of 13). We show that PNA-RNA duplexes block the progression of the 80 S ribosome. Therefore, it is possible to arrest translation with concomitant production of a truncated protein by using duplex-forming PNA oligonucleotides targeted to a G+C-rich sequences. Our data demonstrate for the first time that a non-covalent duplex can arrest the translation machinery and polypeptide chain elongation.


Subject(s)
Antisense Elements (Genetics)/pharmacology , Oncogene Protein p21(ras)/genetics , Peptide Chain Elongation, Translational/drug effects , Peptide Nucleic Acids/pharmacology , RNA, Messenger/genetics , Amides/chemistry , Animals , Antisense Elements (Genetics)/metabolism , Base Sequence , Binding Sites , Codon, Initiator/chemistry , Genes, ras , Humans , Kinetics , Nucleic Acid Conformation , Oncogene Protein p21(ras)/drug effects , Peptide Nucleic Acids/metabolism , Phosphoric Acids/chemistry , Point Mutation , Protein Biosynthesis , RNA, Messenger/chemistry , RNA, Messenger/metabolism , Rabbits
10.
Atherosclerosis ; 143(2): 307-13, 1999 Apr.
Article in English | MEDLINE | ID: mdl-10217359

ABSTRACT

Restenosis after percutaneous transluminal coronary angioplasty (PTCA) occurs due to vascular smooth muscle cell proliferation and migration. Recently, tranilast, an anti-allergic drug, has been used for the prevention of restenosis after PTCA. To determine the molecular mechanism involved, the effect of tranilast on the proliferation of human coronary smooth muscle cells (SMCs) was investigated. Tranilast arrested the proliferation of human coronary SMCs at the G0/G1 phase of the cell cycle. In association with this inhibitory effect, tranilast increased p21waf1 and p53 tumor suppressor factor, and decreased cyclin-dependent kinase 2 (CDK2) activity. These results suggest that tranilast inhibits the proliferation of human coronary SMCs during restenosis after PTCA via an induction of p21waf1 and p53. Tranilast may thus allow us to prevent restenosis after PTCA by interfering with this mechanism.


Subject(s)
Anti-Allergic Agents/pharmacology , Coronary Vessels/drug effects , Cyclin-Dependent Kinases/drug effects , Muscle, Smooth, Vascular/drug effects , Oncogene Protein p21(ras)/biosynthesis , ortho-Aminobenzoates/pharmacology , Blotting, Western , Cell Cycle/drug effects , Cell Division/drug effects , Cell Division/physiology , Cells, Cultured/drug effects , Coronary Vessels/cytology , Cyclin-Dependent Kinases/metabolism , Dose-Response Relationship, Drug , Flow Cytometry , Humans , Muscle, Smooth, Vascular/physiology , Oncogene Protein p21(ras)/drug effects , RNA, Messenger/analysis , Reference Values
11.
J Inorg Biochem ; 77(1-2): 37-42, 1999 Oct.
Article in English | MEDLINE | ID: mdl-10626351

ABSTRACT

Hitherto, it has been generally accepted as a paradigm of the biochemical pharmacology of platinum antitumor drugs that a cis configuration of the leaving groups is necessary for antitumor activity of platinum compounds. However, it has been recently observed that certain trans-platinum complexes have both in vitro and in vivo antitumor activity. We previously reported the synthesis, characterization and cytotoxic activity against ras-transformed cells of several trans-[PtCl2LL'] complexes where L and L' are asymmetric aliphatic amines (L = dimethylamine and butylamine, L' = isopropylamine). The results reported in this paper show that the compounds trans-[PtCl2(isopropylamine)(dimethylamine)] and trans-[PtCl2(isopropylamine)(butylamine)] kill Pam 212-ras cisplatin resistant cells through apoptosis induction. Moreover, Western blot data show that both compounds inhibit overexpression of H-ras oncogene in Pam 212-ras cells. Altogether, these data indicate that, in contrast with cis-DDP, the apoptotic activity of these novel trans-Pt(II) compounds in ras-transformed cells is associated with their ability to abolish ras-overexpression.


Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Genes, ras/drug effects , Organoplatinum Compounds/pharmacology , Animals , Antineoplastic Agents/chemistry , Apoptosis/genetics , Cell Line, Transformed/drug effects , Cells, Cultured , Cisplatin/pharmacology , Drug Resistance, Neoplasm , Gene Expression Regulation, Neoplastic/drug effects , Keratinocytes/drug effects , Keratinocytes/pathology , Mice , Oncogene Protein p21(ras)/drug effects , Oncogene Protein p21(ras)/genetics , Organoplatinum Compounds/chemistry
12.
J Oral Pathol Med ; 24(6): 255-60, 1995 Jul.
Article in English | MEDLINE | ID: mdl-7562661

ABSTRACT

Expression of ras p21 oncoproteins was examined in histological sections of oral squamous cell carcinoma (SCC), epithelial dysplasia, epithelial hyperkeratosis and normal oral mucosa using antibodies to ras p21 with an immunoperoxidase technique. Ras p21-positive staining was found in 47 of 51 (92.2%) cases of oral SCC, 4 of 4 (100%) cases of epithelial dysplasia, 7 of 7 (100%) cases of epithelial hyperkeratosis, and 1 of 6 (16.7%) cases of normal oral mucosa. The positive staining rate of ras p21 in oral SCC, epithelial dysplasia or epithelial hyperkeratosis was significantly higher than that in normal oral mucosa (P < 0.05). No correlation was found between ras p21 expression and patient age, tumour location, tumour size, clinical staging or histological differentiation of SCC. However, a significant positive correlation was found between ras p21 expression and patients' sex (P < 0.05) or regional lymph node status (P < 0.05). A significant positive correlation was also discovered between ras p21 expression and patients' smoking habits (P < 0.01), as well as daily or total betel quid (BQ) consumption (P < 0.05). Of the 47 immunostain-positive SCC patients, specimens from 6 patients were also obtained after chemotherapy, when ras p21 expression was found to be reduced. These results indicate that ras p21 overexpression may play an important role in the initiation and progression of oral SCCs in patients who are smokers and BQ chewers.


Subject(s)
Carcinoma, Squamous Cell/genetics , Gene Expression Regulation, Neoplastic , Mouth Neoplasms/genetics , Oncogene Protein p21(ras)/genetics , Precancerous Conditions/genetics , Adult , Age Factors , Aged , Aged, 80 and over , Antibodies , Areca , Carcinoma, Squamous Cell/drug therapy , Carcinoma, Squamous Cell/pathology , Disease Progression , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Immunoenzyme Techniques , Leukoplakia, Oral/genetics , Leukoplakia, Oral/pathology , Lymph Nodes/pathology , Male , Middle Aged , Mouth Mucosa/metabolism , Mouth Neoplasms/drug therapy , Mouth Neoplasms/pathology , Neoplasm Staging , Oncogene Protein p21(ras)/drug effects , Plants, Medicinal , Precancerous Conditions/pathology , Sex Factors , Smoking/genetics , Staining and Labeling , Taiwan
13.
Oncol Res ; 5(4-5): 161-4, 1993.
Article in English | MEDLINE | ID: mdl-8305741

ABSTRACT

The ras oncogene product (p21ras, Ras) is a GTP-binding protein and is thought to transduce signals regulating cellular proliferation and differentiation. The active form Ras-GTP is inactivated by hydrolyzing bound GTP to GDP. Tiazofurin, a specific inhibitor of IMP dehydrogenase, decreased cellular GTP pools and downregulated c-ras gene expression, leading to differentiation (Olah, E. et al., Proc. Natl. Acad. Sci. USA 85: 6533-6537, 1988; Weber et al., Cancer Commun. 3:61-66, 1991). To clarify the link between the action of tiazofurin on metabolic alterations and the induction of differentiation, we examined the effect of tiazofurin on the ratio of active Ras-GTP to total Ras in K562 cells in culture. Cells were labeled for 6 h with [32P]Pi in phosphate-free RPMI 1640. Tiazofurin (100 or 200 microM) was added to cells, and samples were taken at 0, 2, 4, 6 and 12 h of incubation. Cell lysates were immunoprecipitated with monoclonal anti-p21 antibody (Y13-259), then developed on thin layer chromatography. GTP and GDP bound to Ras were visualized by autoradiography. Tiazofurin treatment decreased Ras-GTP concentration in a time- and dose-dependent fashion. In the untreated K562 cells the Ras-GTP concentration was 26.3 +/- 1.4, and tiazofurin (200 microM) decreased it at 6 h to 16.6 +/- 2.9 and at 12 h to 10.6 +/- 2.1%. Inhibition of the GTP salvage pathway with hypoxanthine (100 or 200 microM) enhanced the tiazofurin-induced decrease of Ras-GTP, whereas addition of guanosine (100 microM) prevented the Ras-GTP decrease.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Antimetabolites, Antineoplastic/pharmacology , Guanosine Triphosphate/metabolism , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism , Oncogene Protein p21(ras)/drug effects , Ribavirin/analogs & derivatives , Cell Differentiation/drug effects , Guanosine/pharmacology , Guanosine Diphosphate/metabolism , Humans , Hypoxanthine , Hypoxanthines/pharmacology , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , Oncogene Protein p21(ras)/metabolism , Protein Binding/drug effects , Ribavirin/pharmacology , Tumor Cells, Cultured
14.
Semin Cancer Biol ; 3(6): 383-90, 1992 Dec.
Article in English | MEDLINE | ID: mdl-1286159

ABSTRACT

Numerous approaches toward anti-ras drugs are being explored. Efforts range from cell-based assays which screen fermentation products to more structural or mechanism-based in vitro screening and design. For the mechanism-based approaches, extensive research efforts on the function and regulation of the ras oncogene protein have identified potential points of intervention, including protein expression, membrane localization, guanine nucleotide activation, and interaction with effector systems.


Subject(s)
Genes, ras/drug effects , Oncogene Protein p21(ras)/drug effects , Oncogene Protein p21(ras)/physiology , Humans , Neoplasms/drug therapy
15.
Cancer Lett ; 59(2): 95-102, 1991 Aug.
Article in English | MEDLINE | ID: mdl-1884376

ABSTRACT

Normal urothelium and various lesions of the rat urinary bladder induced by the dietary administration of 0.2% N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT) (up to 77 weeks) or by the combination of 0.2% FANFT and the subsequent administration of 5% sodium saccharin or 2% DL-tryptophan (up to 104 weeks) were evaluated for immunoreactivity with monoclonal antibody to ras p21 by avidin-biotin immunohistochemistry. Seventy-one to 100% of transitional cell carcinomas showed strong reactivity to the antibody to ras p21 depending on treatment with long-term administration of FANFT or by 6 weeks administration of FANFT followed by sodium saccharin or DL-tryptophan. Focal reactivity to the ras p21 antibody was frequently observed in the hyperplastic (57-96%) or normal appearing urinary bladder epithelium (50-100%) in rats treated with FANFT (FANFT alone or in combination with sodium saccharin or tryptophan) but not in hyperplasia or normal epithelium in rats given sodium saccharin or tryptophan alone, without pretreatment with FANFT or in untreated controls. The present results show that there is a close association of enhanced immunoreactivity with ras p21 antibody in the urinary bladder epithelium to FANFT treatment, and that ras p21 is expressed in normal, hyperplastic and neoplastic lesions of the bladder of rats treated with FANFT. These results suggest that enhanced immunoreactivity with ras p21 is observed as a consequence of the treatment with FANFT but it alone does not reflect the progression from benign to malignant lesions.


Subject(s)
FANFT/pharmacology , Oncogene Protein p21(ras)/drug effects , Urinary Bladder/drug effects , Animals , Antibodies, Monoclonal , Epithelium/chemistry , Epithelium/drug effects , Epithelium/pathology , Immunoenzyme Techniques , Male , Oncogene Protein p21(ras)/analysis , Rats , Rats, Inbred F344 , Saccharin/pharmacology , Tryptophan/pharmacology , Urinary Bladder/chemistry , Urinary Bladder/pathology
16.
Biochemistry ; 30(34): 8283-6, 1991 Aug 27.
Article in English | MEDLINE | ID: mdl-1883816

ABSTRACT

Many genetic disorders result from a single point mutation, and many tumor oncogenes have been found to be altered by a point mutation. The ability to inhibit selectively the expression of the mutated form of a protein without affecting its normal counterpart is central to many therapeutic strategies, since the normal protein may serve indispensable functions. Antisense oligonucleoside methylphosphonates and their psoralen derivatives directed at either normal human Ha-ras p21 or ras p21 that is mutated at a single base in codon 61 have been examined for their efficacy and specificity as inhibitors of p21 expression. Mixed cultures of cells expressing both forms of p21 were treated with the antisense oligomer complementary to the normal p21 or with the antisense oligomer complementary to the point-mutated p21. Each of the antisense oligomers specifically inhibited expression of only the form of ras p21 to which it was completely complementary and left the other form of p21 virtually unaffected.


Subject(s)
Genes, ras , Mutation , Oligonucleotides, Antisense/pharmacology , Oncogene Protein p21(ras)/antagonists & inhibitors , Organophosphorus Compounds/pharmacology , Base Sequence , Carcinoma , Cell Line, Transformed , Gene Expression Regulation/drug effects , Genes, ras/drug effects , Humans , Lung Neoplasms , Molecular Sequence Data , Oncogene Protein p21(ras)/drug effects , Oncogene Protein p21(ras)/genetics
18.
Cell Growth Differ ; 1(10): 499-502, 1990 Oct.
Article in English | MEDLINE | ID: mdl-2278880

ABSTRACT

ras p21 must be posttranslationally processed in order to be localized to the inner plasma membrane. The first obligatory processing step is the farnesylation of ras p21. Lovastatin, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, may prevent the farnesylation of de novo synthesized ras p21. We demonstrate that N-ras oncogene-induced neuronal differentiation of UR61J rat pheochromocytoma cells is blocked by lovastatin. Our data show that this effect is due to the inhibition of ras p21 farnesylation. The results suggest that ras oncogene-induced phenotype in mammalian cells may be eliminated by preventing the proper processing of ras p21.


Subject(s)
Genes, ras/drug effects , Lovastatin/pharmacology , Neurons/drug effects , Animals , Blotting, Northern , Cell Differentiation/drug effects , Oncogene Protein p21(ras)/drug effects , Precipitin Tests , Protein Processing, Post-Translational/drug effects , Sulfur Radioisotopes , Tumor Cells, Cultured
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