ABSTRACT
Epithelial-to-mesenchymal transition (EMT) gives rise to cells with properties similar to cancer stem cells (CSCs). Targeting the EMT program to selectively eliminate CSCs is a promising way to improve cancer therapy. Salinomycin (Sal), a K+/H+ ionophore, was identified as highly selective towards CSC-like cells, but its mechanism of action and selectivity remains elusive. Here, we show that Sal, similar to monensin and nigericin, disturbs the function of the Golgi. Sal alters the expression of Golgi-related genes and leads to marked changes in Golgi morphology, particularly in cells that have undergone EMT. Moreover, Golgi-disturbing agents severely affect post-translational modifications of proteins, including protein processing, glycosylation and secretion. We discover that the alterations induced by Golgi-disturbing agents specifically affect the viability of EMT cells. Collectively, our work reveals a novel vulnerability related to the EMT, suggesting an important role for the Golgi in the EMT and that targeting the Golgi could represent a novel therapeutic approach against CSCs.
Subject(s)
Epithelial-Mesenchymal Transition , Pyrans , Cell Line, Tumor , Epithelial-Mesenchymal Transition/genetics , Pyrans/pharmacology , Pyrans/metabolism , Pyrans/therapeutic use , Golgi Apparatus , Neoplastic Stem Cells/metabolismABSTRACT
AIM: To evaluate the effectiveness of laser fenestration of intravesical ureterocele in prevention of vesicoureteral reflux. MATERIAL AND METHODS: The result of intravesical ureterocele holmium laser fenestration (LF) was retrospectively analyzed in 29 neonatal patients (mean age 8.1 days, range 3-28) in comparison with 38 neonates (mean age 9.6 days, range 5-28) treated with electrosurgical incision (ES). Patients' records were reviewed for preoperative findings, endoscopic procedure description and postoperative outcomes. RESULTS: Vesicoureteral reflux (VUR) was found in two patients (5.6%) in LF and in 25 patients (65.8%) in ES group after six months (P=0.000). The patients with VUR in LF group had reflux grade III. In ES group six patients (15.8%) had reflux grade III, ten (26.3%) grade IV and nine (23.7%) grade V. DISCUSSION: In our study we found that de novo VUR is several times more common in patients treated with electrosurgical incision. This is the main difference between two described endoscopic procedures. Although this is a relatively new surgical technique, and other authors had similar results, which implies the importance of laser fenestration in the prevention of VUR in neonates with ureterocele. CONCLUSION: The occurrence of VUR is significantly lower in neonatal patients treated with holmium-laser fenestration, compared to standard electrosurgical incision, although both techniques are highly effective in relieving the obstruction. Since the use of this technique reduces the incidence of VUR, the need for subsequent surgery is lower in patients treated with holmium-laser. KEY WORDS: Laser Reflux Prevention, Ureterocele.
Subject(s)
Lasers, Solid-State , Surgical Wound , Ureterocele , Vesico-Ureteral Reflux , Infant, Newborn , Humans , Infant , Vesico-Ureteral Reflux/complications , Vesico-Ureteral Reflux/prevention & control , Holmium , Lasers, Solid-State/therapeutic use , Ureterocele/complications , Ureterocele/surgery , Retrospective Studies , Treatment OutcomeABSTRACT
Considering the enormous importance of protein turns as participants in various biological events, such as protein-protein interactions, great efforts have been made to develop their conformationally and proteolytically stable mimetics. Ferrocene-1,1'-diamine was previously shown to nucleate the stable turn structures in peptides prepared by conjugation with Ala (III) and Ala-Pro (VI). Here, we prepared the homochiral conjugates of ferrocene-1,1'-diamine with l-/d-Phe (32/35), l-/d-Val (33/36), and l-/d-Leu (34/37) to investigate (1) whether the organometallic template induces the turn structure upon conjugation with amino acids, and (2) whether the bulky or branched side chains of Phe, Val, and Leu affect hydrogen bonding. Detailed spectroscopic (IR, NMR, CD), X-ray, and DFT studies revealed the presence of two simultaneous 10-membered interstrand hydrogen bonds, i.e., two simultaneous ß-turns in goal compounds. A preliminary biological evaluation of d-Leu conjugate 37 showed its modest potential to induce cell cycle arrest in the G0/G1 phase in the HeLa cell line but these results need further investigation.
Subject(s)
Diamines , Dipeptides , Humans , Metallocenes/chemistry , Hydrogen Bonding , HeLa Cells , Crystallography, X-Ray , Stereoisomerism , Dipeptides/chemistry , Peptides/chemistry , Amino Acids/chemistry , Protein ConformationABSTRACT
ETHNOPHARMACOLOGICAL RELEVANCE: Arctostaphylos uva-ursi (L.) Spreng. (bearberry) is a well-known traditional herbal plant used as a urinary tract disinfectant. Its antiseptic and diuretic properties can be attributed to hydroquinone, obtained by hydrolysis of arbutin. AIM OF THE STUDY: This study aimed to determine the toxic profile of free hydroquinone on urinary bladder cells (T24) as a target of therapeutic action. MATERIALS AND METHODS: Quantitative and qualitative analysis of the extract and the digestive stability and bioavailability of arbutin and hydroquinone were performed by HPLC assay and simulated in vitro digestion, respectively. Cytotoxic effect, reactive oxygen species induction and proteome changes in T24 cells after hydroquinone treatment were determined using Neutral red assay, 2',7'-dichlorofluorescein-diacetate (DCFH-DA) assay and mass spectrometry, respectively. RESULTS: Through in vitro digestion, arbutin was stable, but hydroquinone increased after pepsin treatment (109.6%) and then decreased after the small intestine phase (65.38%). The recommended doses of Uva-ursi had a cytotoxic effect on T24 cells only when all hydroquinone conjugates were converted to free hydroquinone (320 and 900 µg/mL) and the toxic effect was enhanced by recovery. One cup of the therapeutic dose had a prooxidative effect after 4 h of incubation. Shorter time of cell exposure (2 h) to hydroquinone did not have any impact on reactive oxygen species induction. Proteomic analysis found 17 significantly up-regulated proteins compared to control. Hydroquinone activated proteins related to oxidative stress response, stress-adaptive signalling, heat shock response and initiation of translation. CONCLUSIONS: Despite the therapeutic properties of bearberry, up-regulated T24 cell proteins are evidence that plant compounds, although from a natural source, may exhibit negative properties.
Subject(s)
Arctostaphylos/chemistry , Hydroquinones/toxicity , Plant Extracts/toxicity , Urinary Bladder/drug effects , Arbutin/chemistry , Arbutin/isolation & purification , Caco-2 Cells , Cell Line, Tumor , Chromatography, High Pressure Liquid , Humans , Hydroquinones/isolation & purification , Oxidative Stress/drug effects , Plant Extracts/chemistry , Proteome , Proteomics , Urinary Bladder/cytologyABSTRACT
A phalloplasty is a complex genital reconstruction procedure of creating a neophallus. Several techniques to create a neophallus are described, based on different vascularized flaps, and each of them has its advantages and drawbacks. The aim of this study is to present musculocutaneous latissimus dorsi (MLD) flap as a viable option for total phalloplasty, with an interest in clinical outcomes and complications. A comprehensive literature review of all available reports about MLD flap phalloplasty was made. The following keywords were used on PubMed: latissimus dorsi musculocutaneous/myocutaneous free flap and phalloplasty. Research criteria revealed five articles and the results of 182 patients were analyzed. A total number of the patients, indications, operative technique, follow-up period, postoperative results, and complications were presented. In conclusion, MLD free flap presents a good choice for phalloplasty providing sufficient amount of tissue for safe implantation of penile prosthesis and successful penetrative sexual intercourse. The erogenous sensitivity is preserved with clitoris or glans penis incorporated into the base of the neophallus, and voiding in a standing position is achievable after urethral reconstruction. The main drawback is the lack of tactile sensation of the neophallus and the significant advantage is a well-concealed donor site.
Subject(s)
Penile Prosthesis , Plastic Surgery Procedures , Female , Humans , Male , Penis/surgery , Surgical Flaps , UrethraABSTRACT
Multidrug resistance (MDR) is a widespread phenomenon exhibited by many cancers and represents a fundamental obstacle for successful cancer treatments. Tumour cells commonly achieve MDR phenotype through overexpression and/or increased activity of ABC transporters. P-glycoprotein transporter (P-gp, ABCB1) is a major cause of MDR and therefore represents a valuable target for MDR reversal. Several naturally occurring potassium ionophores (e.g. salinomycin) were shown to inhibit P-gp effectively. We have previously shown antitumour activity of a number of 18-crown-6 ether compounds that transport potassium ions across membranes. Here we present data on P-gp inhibitory activity of 16 adamantane-substituted monoaza- and diaza-18-crown-6 ether compounds, and their effect on MDR reversal in model cell lines. We show that crown ether activity depends on their lipophilicity as well as on the linker to adamantane moiety. The most active crown ethers were shown to be more effective in sensitising MDR cells to paclitaxel and adriamycin than verapamil, a well-known P-gp inhibitor. Altogether our data demonstrate a novel use of crown ethers for inhibition of P-gp and reversal of MDR phenotype.
Subject(s)
Crown Ethers , Drug Resistance, Multiple/drug effects , Drug Resistance, Neoplasm/drug effects , Neoplasms/metabolism , ATP Binding Cassette Transporter, Subfamily B/antagonists & inhibitors , ATP Binding Cassette Transporter, Subfamily B/metabolism , Animals , Cell Line, Tumor , Cell Membrane/metabolism , Cell Membrane/pathology , Crown Ethers/chemistry , Crown Ethers/pharmacology , Dogs , Doxorubicin/pharmacology , Humans , Ion Transport/drug effects , Madin Darby Canine Kidney Cells , Neoplasms/drug therapy , Neoplasms/pathology , Paclitaxel/pharmacology , Potassium/metabolism , Vincristine/pharmacologyABSTRACT
The generation of multiciliated cells (MCCs) is required for the proper function of many tissues, including the respiratory tract, brain, and germline. Defects in MCC development have been demonstrated to cause a subclass of mucociliary clearance disorders termed reduced generation of multiple motile cilia (RGMC). To date, only two genes, Multicilin (MCIDAS) and cyclin O (CCNO) have been identified in this disorder in humans. Here, we describe mice lacking GEMC1 (GMNC), a protein with a similar domain organization as Multicilin that has been implicated in DNA replication control. We have found that GEMC1-deficient mice are growth impaired, develop hydrocephaly with a high penetrance, and are infertile, due to defects in the formation of MCCs in the brain, respiratory tract, and germline. Our data demonstrate that GEMC1 is a critical regulator of MCC differentiation and a candidate gene for human RGMC or related disorders.
Subject(s)
Carrier Proteins/metabolism , Cell Differentiation , Cilia/genetics , Cilia/physiology , Growth Disorders/genetics , Growth Disorders/pathology , Animals , Carrier Proteins/genetics , Cell Cycle Proteins , Mice , Mice, KnockoutABSTRACT
CEP63 is a centrosomal protein that facilitates centriole duplication and is regulated by the DNA damage response. Mutations in CEP63 cause Seckel syndrome, a human disease characterized by microcephaly and dwarfism. Here we demonstrate that Cep63-deficient mice recapitulate Seckel syndrome pathology. The attrition of neural progenitor cells involves p53-dependent cell death, and brain size is rescued by the deletion of p53. Cell death is not the result of an aberrant DNA damage response but is triggered by centrosome-based mitotic errors. In addition, Cep63 loss severely impairs meiotic recombination, leading to profound male infertility. Cep63-deficient spermatocytes display numerical and structural centrosome aberrations, chromosome entanglements and defective telomere clustering, suggesting that a reduction in centrosome-mediated chromosome movements underlies recombination failure. Our results provide novel insight into the molecular pathology of microcephaly and establish a role for the centrosome in meiotic recombination.
Subject(s)
Cell Cycle Proteins/genetics , Centrosome/metabolism , Dwarfism/genetics , Homologous Recombination/genetics , Meiosis/genetics , Microcephaly/genetics , Spermatocytes/metabolism , Tumor Suppressor Protein p53/genetics , Animals , DNA Damage , Facies , Immunohistochemistry , Male , Mice , Real-Time Polymerase Chain Reaction , Recombination, Genetic/genetics , Sperm Count , Spermatocytes/pathologyABSTRACT
Centrosomes consist of two centrioles embedded in pericentriolar material and function as the main microtubule organising centres in dividing animal cells. They ensure proper formation and orientation of the mitotic spindle and are therefore essential for the maintenance of genome stability. Centrosome function is crucial during embryonic development, highlighted by the discovery of mutations in genes encoding centrosome or spindle pole proteins that cause autosomal recessive primary microcephaly, including Cep63 and Cep152. In this study we show that Cep63 functions to ensure that centriole duplication occurs reliably in dividing mammalian cells. We show that the interaction between Cep63 and Cep152 can occur independently of centrosome localisation and that the two proteins are dependent on one another for centrosomal localisation. Further, both mouse and human Cep63 and Cep152 cooperate to ensure efficient centriole duplication by promoting the accumulation of essential centriole duplication factors upstream of SAS-6 recruitment and procentriole formation. These observations describe the requirement for Cep63 in maintaining centriole number in dividing mammalian cells and further establish the order of events in centriole formation.
Subject(s)
Cell Cycle Proteins/metabolism , Centrioles/metabolism , Neoplasm Proteins/metabolism , Animals , Cell Cycle Proteins/genetics , Cell Line , Centrioles/genetics , Centrosome/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Humans , Mice , Neoplasm Proteins/genetics , Protein Binding , Protein TransportABSTRACT
The DNA damage response (DDR) rapidly recognizes DNA lesions and initiates the appropriate cellular programs to maintain genome integrity. This includes the coordination of cell cycle checkpoints, transcription, translation, DNA repair, metabolism, and cell fate decisions, such as apoptosis or senescence (Jackson and Bartek, 2009). DNA double-strand breaks (DSBs) represent one of the most cytotoxic DNA lesions and defects in their metabolism underlie many human hereditary diseases characterized by genomic instability (Stracker and Petrini, 2011; McKinnon, 2012). Patients with hereditary defects in the DDR display defects in development, particularly affecting the central nervous system, the immune system and the germline, as well as aberrant metabolic regulation and cancer predisposition. Central to the DDR to DSBs is the ataxia-telangiectasia mutated (ATM) kinase, a master controller of signal transduction. Understanding how ATM signaling regulates various aspects of the DDR and its roles in vivo is critical for our understanding of human disease, its diagnosis and its treatment. This review will describe the general roles of ATM signaling and highlight some recent advances that have shed light on the diverse roles of ATM and related proteins in human disease.
ABSTRACT
Six recently synthesized cyano-substituted heteroaryles, which do not bind to DNA but are highly cytotoxic against the human tumor cell line HeLa, were analyzed for their antitumor mechanisms of action (MOA). They did not interfere with the expression of human papillomavirus oncogenes integrated in the HeLa cell genome, but they did induce strong G1 arrest and result in the activation of caspase-3 and apoptosis. A computational analysis was performed that compared the antiproliferative activities of our compounds in 13 different tumor cell lines with those of compounds listed in the National Cancer Institute database. The results indicate that interference with cytoskeletal function and inhibition of mitosis are the likely antitumor MOA. Furthermore, a second in silico investigation revealed that the tumor cells that are sensitive to the cyano-substituted compounds show differences in their expression of locomotion genes compared with that of insensitive cell lines, thus corroborating the involvement of the cytoskeleton. This MOA was also confirmed experimentally: the cyano-substituted heteroaryles disrupted the actin and the tubulin networks in HeLa cells and inhibited cellular migration. However, further analysis indicated that multiple MOA may exist that depend on the position of the cyano-group; while cyano-substituted naphthiophene reduced the expression of cytoskeletal proteins, cyano-substituted thieno-thiophene-carboxanilide inhibited the formation of cellular reactive oxygen species.
Subject(s)
Cell Proliferation/drug effects , Heterocyclic Compounds/pharmacology , Uterine Cervical Neoplasms/pathology , Apoptosis/drug effects , Caspase 3/metabolism , Cell Cycle/drug effects , Cell Movement/drug effects , Computer Simulation , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Cyclin-Dependent Kinase Inhibitor p27/metabolism , Cytoskeletal Proteins/metabolism , Cytoskeleton/drug effects , Cytoskeleton/metabolism , Dose-Response Relationship, Drug , Female , Gene Expression Regulation, Viral/drug effects , HCT116 Cells , HL-60 Cells , HT29 Cells , HeLa Cells , Heterocyclic Compounds/chemical synthesis , Humans , Mitosis/drug effects , Papillomaviridae/genetics , Poly (ADP-Ribose) Polymerase-1 , Poly(ADP-ribose) Polymerases/metabolism , Reactive Oxygen Species/metabolism , Time Factors , Transcription, Genetic/drug effects , Tumor Suppressor Protein p53/metabolism , Uterine Cervical Neoplasms/genetics , Uterine Cervical Neoplasms/metabolism , Uterine Cervical Neoplasms/virologyABSTRACT
A series of (1-adamantyl)phthalimides, 1-4, and (2-adamantyl)phthalimides, 5-8, characterized by different chain length between the adamantyl and the phthalimide moiety were synthesized, as well as 1- and 2-adamantylphthalimides substituted by nitro 9, 10, and amino group 11, 12, and phthalimides bearing homoadamantyl 13 and protoadamantyl substituent 14 and 15. The compounds were tested for antiproliferative activity in vitro on a series of five human cancer lines: MCF-7 (breast carcinoma), SW 620 (colon carcinoma), HCT 116 (colon carcinoma), MOLT-4 (acute lymphoblastic leukemia), H 460 (lung carcinoma), and a non-tumor cell line HaCaT (human keratinocytes). All compounds except nitro derivatives 9 and 10 exhibited antiproliferative activity. The activity was generally better in the 2-adamantyl series 5-8 and in the compounds having the longest alkyl spacers as in 4 and 8, or with an amino group as in 9 and 10. The most active compounds with the propylene spacer 4 and 8 showed the highest selectivity toward tumor cells. The activity was found to be due to a delay in the progress through the cell cycle at G1/S phase.
Subject(s)
Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Cell Proliferation/drug effects , Phthalimides/chemistry , Phthalimides/pharmacology , Adamantane/chemistry , Adamantane/pharmacology , Cell Cycle/drug effects , Cell Line, Tumor , Drug Design , Drug Screening Assays, Antitumor , Humans , Neoplasms/drug therapy , Structure-Activity RelationshipABSTRACT
18-crown-6 ethers are known to exert their biological activity by transporting K(+) ions across cell membranes. Using non-linear Support Vector Machines regression, we searched for structural features that influence antiproliferative activity in a diverse set of 19 known oxa-, monoaza- and diaza-18-crown-6 ethers. Here, we show that the logP of the molecule is the most important molecular descriptor, among â¼1300 tested descriptors, in determining biological potency (R(2)(cv) = 0.704). The optimal logP was at 5.5 (Ghose-Crippen ALOGP estimate) while both higher and lower values were detrimental to biological potency. After controlling for logP, we found that the antiproliferative activity of the molecule was generally not affected by side chain length, molecular symmetry, or presence of side chain amide links. To validate this QSAR model, we synthesized six novel, highly lipophilic diaza-18-crown-6 derivatives with adamantane moieties attached to the side arms. These compounds have near-optimal logP values and consequently exhibit strong growth inhibition in various human cancer cell lines and a bacterial system. The bioactivities of different diaza-18-crown-6 analogs in Bacillus subtilis and cancer cells were correlated, suggesting conserved molecular features may be mediating the cytotoxic response. We conclude that relying primarily on the logP is a sensible strategy in preparing future 18-crown-6 analogs with optimized biological activity.
Subject(s)
Adamantane/chemistry , Antineoplastic Agents/chemical synthesis , Bacillus subtilis/drug effects , Cell Cycle/drug effects , Cell Survival/drug effects , Crown Ethers/chemical synthesis , Hydrophobic and Hydrophilic Interactions , Algorithms , Antineoplastic Agents/pharmacology , Bacillus subtilis/growth & development , Cell Line, Tumor , Crown Ethers/pharmacology , Drug Design , Drug Screening Assays, Antitumor , Escherichia coli/drug effects , Escherichia coli/growth & development , Ethers/chemistry , Female , Humans , Inhibitory Concentration 50 , Models, Molecular , Neoplasms/drug therapy , Neoplasms/pathology , Quantitative Structure-Activity Relationship , Software , Species Specificity , Structure-Activity RelationshipABSTRACT
Novel amidino-substituted conformationally restricted derivatives of pentamidine were synthesized and their antiproliferative activity against several human cancer cell lines determined. It was found that introduction of furandicarboxamide core moiety (9, 10) increases antiproliferative activity as well as selectivity against certain tumor cell lines in comparison with amidino-substituted furan-mono-carboxamide (5, 6). Unlike the furan series where iso-propyl substituted amidine (10) exhibits more potent overall antiproliferative activity and selectivity toward certain cell lines, the same was found for unsubstituted amidines in pyridine series. Amongst all tested compounds the compound 10 is the only one that possesses antiproliferative activity against SW 620 cell line (4 µM). Spectroscopic studies of the interactions of prepared diamidines with double-stranded DNA and RNA polynucleotides show that all compounds preferentially bind into the minor groove of DNA, while most of them intercalate into RNA. The structure-dependant biological activity and the lack of DNA/RNA selective binding suggest that the mechanism of action of the here-presented compounds is controlled not only by the interactions with cellular nucleic acids, but also with other more specific protein targets.
Subject(s)
Amidines/chemistry , Antineoplastic Agents/chemical synthesis , Intercalating Agents/chemical synthesis , Pentamidine/chemical synthesis , Polynucleotides/chemistry , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Survival/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Humans , Inhibitory Concentration 50 , Intercalating Agents/pharmacology , Pentamidine/pharmacology , Structure-Activity RelationshipABSTRACT
Two novel guanidiniocarbonyl pyrrole-pyrene conjugates 3 and 4 as spectroscopic probes for ds-polynucleotides were synthesized and their interaction with different ds-DNAs/RNAs studied. Compared to a previously reported first set of conjugates (1 and 2) the significant extension and increased rigidity of the central part of the structure resulted in a switch of DNA binding mode from intercalative (previously studied derivatives 1 and 2 with a nonbinding and flexible linker) to minor groove binding of the two novel guanidiniocarbonyl-pyrrole-pyrene conjugates 3 and 4. These two compounds interact strongly with ds-DNAs, but only weakly with ds-RNA. The newly incorporated heterocyclic moieties within the central part of the structure of 3 and 4 were able to control by steric and hydrogen-bonding effects the alignment of the molecules within various, structurally different forms of DNA minor grooves, whereby even small differences in the position of the attached pyrene within the groove were reflected in different fluorimetric responses. In addition, 3 and 4 revealed intriguing in vitro selectivity among various human tumour cell lines.
Subject(s)
DNA/chemistry , Pyrroles/chemistry , RNA/chemistry , Cell Line, Tumor , Circular Dichroism , Humans , Models, Molecular , Nucleic Acid Conformation , Solutions/chemistry , Spectrometry, Fluorescence , TemperatureABSTRACT
A synthesis and biological evaluation of new benzimidazole derivatives, related to 2,3-disubstituted acrylonitriles, benzimidazo[1,2-a]quinoline-6-carbonitriles and heteroaromatic fluorenes was described. The molecular and crystal structures of three compounds 4, 16 and 17 reveal that non-fused fluoro derivative, 4, deviates from planarity by 13.11(2) degrees, while fused methyl, 16, and fluoro, 17, derivatives are planar within 4 degrees exhibiting a planar aromatic surface capable to intercalate into double-stranded DNA. Compound 4 exists as E-isomer. The crystal structures confirmed that hydrogen bonding patterns are characterized dominantly by the weak C-H...N(F) bonds, except in the case of 4 where the presence of ethanol molecule of crystallization resulted in the N-H...O and O-H...N hydrogen bonds formation. In the crystal structures of 16 and 17 cyano group participates in hydrogen bonding formation, while in 4 this is not the case. All compounds, except 16 and 14 exerted pronounced antiproliferative activity on five tumor cell lines, whereby 2-benzimidazolyl-3-N-methylpyrolyl-acrylonitrile 13 and its fused analogue 23 exerted the highest activity on all cell lines (IC50=0.8-30 microM) and showed a special selectivity toward HeLa cells. There is no major difference in the biological activity between non-fused and fused analogues. Similarly, all compounds showed significant interaction with ct-DNA, supporting the fact that their antitumor activity could partially be the consequence of DNA-binding. The cyano moiety is important for the activity, but not the selectivity of tested compounds.
Subject(s)
Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Benzimidazoles/chemistry , Benzimidazoles/pharmacology , Animals , Antineoplastic Agents/metabolism , Benzimidazoles/metabolism , Cattle , Cell Line, Tumor , Crystallography, X-Ray , DNA/metabolism , Fluorenes/chemistry , Humans , Inhibitory Concentration 50 , Models, Molecular , Molecular Conformation , Nitriles/chemistry , Quinolines/chemistryABSTRACT
We present a systematic study of different guanidiniocarbonylpyrrole-aryl derivatives designed to interact with DNA or RNA both through intercalation of an aromatic moiety into the base stack of the nucleotide and through groove binding of a guanidiniocarbonylpyrrole cation. We varied 1) the size of the aromatic ring (benzene, naphthalene, pyrene and acridine), 2) the length and flexibility of the linker connecting the two binding groups, and 3) the total number of positive charges present at different pH values. The compounds and their interactions with DNA and RNA were studied by UV/Vis, fluorescence and CD spectroscopy. Antiproliferative activities against human tumour cell lines were also determined. Our studies show that efficient interaction with, for example, DNA requires a significantly large aromatic ring (pyrene) connected through a flexible linker to the pyrrole moiety. However, a positive charge, as in 12, is also needed. Compound 12 allows for base-pair-selective recognition of ds-DNA at physiological pH values. The antiproliferative activities of these compounds correlate with their binding affinities towards DNA, suggesting that their biological effects are most probably due to DNA binding.
Subject(s)
DNA/chemistry , Guanidines/chemistry , Guanidines/pharmacology , Pyrenes/chemistry , Pyrroles/chemistry , RNA/chemistry , Base Pairing , Base Sequence , Binding Sites , Cell Line, Tumor , Cell Proliferation , DNA/metabolism , Humans , Models, Molecular , Molecular Sequence Data , Molecular Structure , Nucleic Acid Conformation , RNA/metabolism , Spectrometry, Fluorescence , Spectrophotometry , Spectrophotometry, Ultraviolet , Structure-Activity RelationshipABSTRACT
PURPOSE: p53 gene plays a crucial role in the response to therapy. Since it is inactivated in the majority of human cancers, it is strongly believed that the p53 mutations confer resistance to therapeutics. In this paper we analyzed the influence of two mechanistically diverse antitumor agents--cisplatin and methotrexate on the proliferation and cell cycle of two head and neck squamous cancer cell lines HEp-2 (wild type p53 gene, but HPV 18/E6-inactivated protein) and CAL 27 (mutated p53 gene), along with the influence of adenovirally mediated p53 overexpression in modulation of cisplatin and methoterexate effects, whereby subtoxic vector/compound concentrations were employed. METHODS: p53 gene was introduced into tumor cells using adenoviral vector (AdCMV-p53). The cell cycle perturbations were measured by two parameter flow cytometry. The expression of p53, p21(WAF1/CIP1) and cyclin B1 proteins was examined using immunocytochemistry and western blot methods. RESULTS: In CAL 27 cells overexpression of p53 completely abrogated high S phase content observed in methotrexate-treated cells into a G1 and slight G2 arrest, while it sustained G2 arrest of the cells treated with cisplatin, along with the reduction of DNA synthesis and cyclin B1 expression. On the other hand, in HEp-2 cell line p53 overexpression slightly slowed down the progression through S phase in cells treated with methotrexate, decreased the cyclin B1 expression only after 24 h, and failed to sustain the G2 arrest after treatment with cisplatin alone. Instead, it increased the population of S phase cells that were not actively synthesizing DNA, sustained cyclin B1 expression and allowed the G2 cells to progress through mitosis. CONCLUSIONS: This study demonstrates that adenovirally mediated p53 overexpression at sub-cytotoxic levels enhanced the activity of low doses of cisplatin and methotrexate in HEp-2 and CAL 27 cells through changes in the cell cycle. However, the mechanisms of these effects differ depending on the genetic context and on the chemotherapeutics' modality of action.
Subject(s)
Adenoviridae/physiology , Cell Cycle/drug effects , Cell Line, Tumor , Cisplatin/pharmacology , Genes, p53 , Methotrexate/pharmacology , Adenoviridae/genetics , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/pharmacology , Carcinoma, Squamous Cell/drug therapy , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/pathology , Cell Cycle/genetics , Cell Proliferation/drug effects , Cells, Cultured , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , Gene Transfer Techniques , Head and Neck Neoplasms/drug therapy , Head and Neck Neoplasms/genetics , Head and Neck Neoplasms/pathology , Humans , Methotrexate/administration & dosageABSTRACT
Synthesis of a series of novel cyano- and amidinobenzothiazole derivatives 3-31 is described. All studied amidino derivatives showed noticeable antiproliferative effect on several tumor cell lines. Cyano derivatives 11-17 showed considerably less pronounced activity because of their poor solubility in aqueous cell culture medium, which was confirmed by the principal components (PC) analysis. Compounds 21, 22, 28, and 29 were tested for their effects on the cell cycle and apoptosis, whereby 22 and 29, having methyl group at the C-6 position in pyridine ring, showed drastic cell cycle perturbations that were both concentration- and time-dependent and induced apoptosis. The QSAR modeling, based on the physicochemical descriptors and on the measured biological activities, indicated the relevance of molecular polarizability and particular distribution of pharmacophores on the molecular surface for activity. In conclusion, benzothiazoles containing either isopropylamidino or imidazolyl groups will be considered as starting compounds for further investigation on lead identification.