Application of the disk method to cultured plant cells. I. Inhibition zones

When paper disks carrying small volumes of highly concentrated drugs were placed on the suface of the medium in plant cell culture plates, diffusion of the drugs led to a circular area of non-dividing cells (an inhibition zone) around the disks. Out of 63 drugs tested 37 were inhibitory and 15 of these produced a clear inhibition zone.Drug concentrations could be estimated by measuring the inhibition zone diameter. Cell growth and drug diffusion were analysed and the influence of several variables on inhibition zone formation studied. Inhibition zones occured with cultures of Zea mays, Acer pseudoplatanus, Daucus carota and Hyoscyamus muticus and protoplast-derived cells of Rosa. Possible applications of the method in plant cell genetics and physiology are discussed.Abbreviations ETH L-ethionine - MMC methylmercury chloride - NAA l-naphthaleneacetic acid - BAP 6-benzylaminopurine - PCV Packed cell volume - PE Plating efficiency     

Promoter methylation status of VEGF receptor genes: A possible epigenetic biomarker to anticipate the efficacy of intracellular-acting VEGF-targeted drugs in cancer cells

We evaluated whether the inhibitory effects of vascular endothelial growth factor (VEGF)-targeted drugs on the proliferation of cancer cells differed according to VEGF receptor (VEGFR) genes, Flt1 and KDR, promoter methylation status. Five hyper-VEGFR-methylation and six no-VEGFR-methylation cancer cells were used for the present study, together with human umbilical endothelial cells (HUVECs) as a control. No-VEGFR-methylation cancer cells showed higher expression of Flt1 and KDR than hyper-VEGFR-methylation cancer cells. Hyper-VEGFR-methylation cancer cells only showed increased expression and protein levels of Flt1 and KDR after treatment with the demethylase 5-aza-2’-deoxycytidine. Two drugs (a VEGF-specific-antibody, bevacizumab, and a KDR-specific-antibody) targeting extracellular VEGF-VEGFR signaling and two VEGF-specific-tyrosine kinase inhibitors (PTK/ZK and sunitinib) targeting intracellular VEGFR signaling were used in the cell proliferation assay. HUVECs showed dose- and time-dependent proliferation decrease with all tested drugs over a 72 h incubation period. No- or hyper-VEGFR-methylation cancer cells showed no significant proliferation differences after treatment with VEGF-specific-antibody or VEGFR2-specific-antibody. After PTK/ZK or sunitinib treatment, no-VEGFR-methylation cancer cells showed dose- or time-dependent decreases in proliferation. Hyper-VEGFR-methylation cancer cells also showed proliferation inhibition by VEGF-specific-tyrosine kinase inhibitors after demethylation of Flt1 and KDR. Proliferation inhibition synergistically increased after combination of demethylation with PTK/ZK in hyper-VEGF-methylation cancer cells. We observed that intracellular targeting of VEGF-VEGFR signaling could be more effective than extracellular targeting of the pathway in the suppression of proliferation of some cancer cells. In particular, the efficacy of intracellular targeting of VEGF-specific-tyrosine kinase inhibitors might be influenced by the epigenetic alteration of VEGFRs.     

Toxic effects of antimalarial drugs in Paramecium : role of calcium channels

The antimalarial drugs, quinacrine, quinine and mefloquine, as well as the structurally-similar compound, W-7, inhibit calcium-dependent backward swimming and calcium currents in Paramecium calkinsi. These drugs are also toxic to paramecia at high concentrations. Therefore, one site of toxic action of the drugs may be the calcium channel. To test this hypothesis, the toxicity of the antimalarials and W-7 was compared in paramecia with and without calcium channels. Since calcium channels are located on the cilia, calcium channels were removed from the paramecia by deciliating the cells. Deciliated cells were found to be less susceptible to the lethal effects of the antimalarials and W-7 than their ciliated counterparts. Moreover, Pawns, mutants of P. tetraurelia that possess cilia but lack functional calcium channels, were also less susceptible to the antimalarials than wild-type cells. Thus, calcium channels may be one site of toxic action of the antimalarial drugs in paramecia and perhaps in other protists. Accepted: 27 December 1996     

Effects of LPA1 and LPA6 on the regulation of colony formation activity in colon cancer cells treated with anticancer drugs

Lysophosphatidic acid (LPA) is a simple physiological lipid and exhibits a variety of cellular responses via the activation of G protein-coupled transmembrane LPA receptors (LPA receptor-1 (LPA₁) to LPA₆). The aim of our study was to investigate effects of LPA receptors on soft agar colony formation in colon cancer cells treated with anticancer drugs. DLD1 cells were treated with fluorouracil (5-FU) or cisplatin (CDDP) for at least six months (DLD-5FU and DLD-CDDP cells, respectively). LPAR1 gene expression was markedly elevated in DLD-5FU cells. In contrast, DLD-CDDP cells showed the high expression of LPAR6 gene. In colony formation assay, DLD-5FU cells formed markedly large-sized colonies, while no colony formation was observed in DLD1 and DLD-CDDP cells. The large-sized colonies formed in DLD-5FU cells were suppressed by LPA₁ knockdown. In contrast, LPA₆ knockdown increased the size of colonies. In addition, DLD-5FU cells were further treated with CDDP for three months (DLD-C-F cells). DLD-CDDP cells were also treated with 5-FU (DLD-F-C cells). DLD-C-F cells formed large-sized colonies, but not DLD-F-C cells, correlating with LPAR1 and LPAR6 gene expression levels. These results suggest that LPA₁ and LPA₆ may regulate the colony formation activity in DLD1 cells treated with anticancer drugs.     

Coculture of human articular chondrocytes with peripheral blood mononuclear cells as a model to study cytokine-mediated interactions between inflammatory cells and target cells in the rheumatoid joint

Summary A model for the coculture of chondrocytes in gelified agarose with mononuclear cells was developed to serve as an in vitro equivalent for cytokine-mediated events at the cartilage-synovial pannus junction in destructive arthropathies. Chondrocytes cultured in agarose keep their phenotypic stability. They release cartilage-specific aggrecans into the surrounding artificial matrix. When activated with lipopolysaccharide for 1 h, mononuclear cells release Interleukin 1β and Tumor Necrosis Factorα, thereby stimulating the chondrocytes to produce Interleukin 6, to diminish incorporation of³⁵S into aggrecans, and to degrade these intercellular macromolecules. This coculture model is a useful tool for studying interactions between inflammatory cells and target cells. To demonstrate its usefulness, the effect of three anti-inflammatory drugs (piroxicam, sulphasalazine, and hydrocortisone) on cytokine release by mononuclear cells, and subsequently on chondrocyte aggrecan metabolism was studied. The drugs were unable to abrogate Interleukin 1 and Tumor Necrosis Factorα release by activated mononuclear cells. Therefore, these pharmacological agents did not protect the artificial target tissue against cytokine-mediated degradation.     

Optochemistry to control the microtubule cytoskeleton

Microtubule drugs have a wide range of applications in cell biology research as well as cancer therapy; however their application was so far limited to the treatment of entire cell populations and tissues. In a recent paper in Cell, Borowiak et al ( 2015 ) now describe a novel type of switchable microtubule drugs. The activity of their drugs, denoted as “photostatins”, can be switched on and off by violet and green light, respectively, which allows for the first time a precise spatial and temporal control of the microtubule cytoskeleton in single cells and tissues.     

Experimental analysis and modelling of in vitro HUVECs proliferation in the presence of various types of drugs

Objectives: This study focuses on experimental analysis and corresponding mathematical simulation of in vitro HUVECs (human umbilical vein endothelial cells) proliferation in the presence of various types of drugs. Materials and methods: HUVECs, once seeded in Petri dishes, were expanded to confluence. Temporal profiles of total count obtained by classic haemocytometry and cell size distribution measured using an electronic Coulter counter, are quantitatively simulated by a suitable model based on the population balance approach. Influence of drugs on cell proliferation is also properly simulated by accounting for suitable kinetic equations. Results and discussion: The models’ parameters have been determined by comparison with experimental data related to cell population expansion and cell size distribution in the absence of drugs. Inhibition constant for each type of drug has been estimated by comparing the experimental data with model results concerning temporal profiles of total cell count. The reliability of the model and its predictive capability have been tested by simulating cell size distribution for experiments performed in the presence of drugs. The proposed model will be useful in interpreting effects of selected drugs on expansion of readily available human cells.     

Transforming NRK cells with avian sarcoma virus reduces the extracellular Ca requirement without affecting the calcicalmodulin requirement for the G1/S transition

NRK rat cells infected with a transformation-defective, temperature-sensitive (ts) mutant of the avian sarcoma virus could not proliferate in Ca²⁺-deficient medium at a nonpermissive temperature (40 °C) that inactivated the viral pp60^v-scr-transforming product and rendered the cells phenotypically untransformed. However, these arrested cells were stimulated to initiate DNA replication with little or no delay while still in the Ca²⁺-deficient medium, either by adding Ca²⁺ or calmodulin at 40 °C or by reducing the temperature to 36 °C which restored the transformed phenotype by rapidly reactivating pp60^v-src. The G1/S transition triggered by restoring the transformed phenotype was suppressed by three different anticalmodulin drugs (R24571, trifluoperazine, W7). The suppression by one of these drugs, trifluoperazine, was overcome by adding calmodulin. Thus, neoplastic transformation by the avian sarcoma virus sharply reduces the extracellular Ca²⁺ requirement for the initiation of DNA replication without bypassing a calcical-modulin-dependent mechanism also needed for the G1/S transition.     

Immunochemotherapy of malignant glioma: synergistic activity of CD95 ligand and chemotherapeutics

 Malignant glioma cells are susceptible to CD95(Fas/APO-1)-mediated apoptosis triggered by agonistic antibody. Here we examined the proapoptotic effects of the natural CD95 ligand, a cytotoxic cytokine homologous to tumor necrosis factor, on malignant glioma cell lines LN-229, LN-308 and T98G. We assessed whether glioma cell killing is synergistically enhanced by cotreatment with CD95 ligand and chemotherapeutic agents, including doxorubicin, carmustine, vincristine, etoposide, teniposide, 5-fluorouracil and cytarabine. Synergy was examined at low concentrations of cytotoxic drugs and CD95 ligand with a defined effect level (IC₁₅). Short-term-cytotoxicity assays showed prominent killing of the glioma cells by CD95 ligand but not by the drugs at relevant concentrations. CD95 ligand-induced apoptosis in the acute toxicity paradigm was augmented by doxorubicin and vincristine. Growth-inhibition assays revealed prominent synergy between CD95 ligand and all drugs examined. The best synergy was obtained with CD95 ligand and doxorubicin, vincristine or teniposide. The strong synergistic antiproliferative effects were observed at much lower concentrations of CD95 ligand and cytotoxic drugs than the moderate synergistic acute cytotoxic effects. All cell lines examined express the Bcl-2 protein. LN-229 has partial wild-type p53 activity. T98G has mutant p53. LN-308 has a deleted p53 gene and lacks p53 protein expression. Thus, synergistic effects of CD95 ligand and cytotoxic drugs were observed in cell lines exhibiting two features thought to play a role in the chemoresistance of human malignant glioma cells: loss of wild-type p53 activity and acquisition of bcl-2 expression. Ectopic expression of murine bcl-2 conferred partial protection from CD95 ligand and drugs when administered alone but did not interfere with the mechanisms underlying the synergistic effects of CD95 ligand and chemotherapeutic drugs. Received: 31 October 1996 / Accepted: 4 January 1997     

Effects of membrane acting-drugs on plasmodium species and sickle cell erythrocytes

The effects of several membrane-acting drugs on malaria and sickle cell anemia was studied. In the initial experiments, propranolol and W-7 were shown to increase red cell density.In vitro, these drugs inhibited the growth ofP. falciparum. However,in vivo experiments using the murine malarial parasite,P. vinckei, demonstrated little, if any, anti-parasite activity with the doses of drugs employed. Subsequently, prostaglandin oligomeric derivatives were found to inhibit the growth ofP. falciparum in vitro andP. vinckei in vivo. Since prostaglandin oligomers inhibited the formation of dense, dehydrated cells (irreversible sickle cells), they may also have therapeutic efficacy in sickle cell anemia.     

Celecoxib enhanced the sensitivity of cancer cells to anticancer drugs by inhibition of the expression of P‐glycoprotein through a COX‐2‐Independent Manner

The P‐glycoprotein (p170, P‐gp) encoded by human MDR1 gene functions as a pump to extrude anticancer drugs from cancer cells. Over‐expression of p170 is closely related to primary and induced drug resistance phenotype of tumor cells. Recent studies have demonstrated that expression of cyclooxygenase‐2 (COX‐2) is positively correlated with the p170 level, suggesting a potential of COX‐2 specific inhibitors in regulation of cytotoxicity of anticancer agents. Celecoxib is one of the specific inhibitors of COX‐2 and has been widely used in clinic. However, its function in the response of cancer cells to anticancer drugs and the related mechanism are still waiting to be investigated. To explore the correlation of celecoxib and the p170‐mediated drug resistance, the role of celecoxib in drug response of cancer cells was analyzed with flow cytometry, high performance liquid chromatography (HPLC), and colony formation experiments. Celecoxib (50 µM) was found to significantly enhance the sensitivity of MCF‐7 and JAR/VP16 cells to tamoxifen and etoposide, respectively, by inhibition of p170 expression and increase in intracellular accumulation of the drugs. However, celecoxib did not affect pump function of p170. Enzyme activity and methylation analyses demonstrated that the inhibitory effect of celecoxib on p170 was independent on COX‐2 but closely related to hypermethylation of MDR1 gene promoter. Our study suggested that celecoxib was a potential agent for enhancement of the sensitivity of cancer cells to anticancer drugs. It also provided a links between epigenetic change of MDR1 and drug response of cancer cells. J. Cell. Biochem. 108: 181–194, 2009. © 2009 Wiley‐Liss, Inc.     

Glutathione is involved in the antimalarial action of chloroquine and its modulation affects drug sensitivity of human and murine species of Plasmodium

Abstract

Ferriprotoporphyrin IX (FP) is released inside the food vacuole of the malaria parasite during the digestion of host cell hemoglobin. FP is detoxified by its biomineralization to hemozoin. This process is effectively inhibited by chloroquine (CQ) and amodiaquine (AQ). Undegraded FP accumulates in the membrane fraction and inhibits enzymes of infected cells in parallel with parasite killing. FP is demonstrably degraded by reduced glutathione (GSH) in a radical-mediated mechanism. This degradation is inhibited by CQ and AQ in a competitive manner, thus explaining the ability of increased GSH levels in Plasmodium falciparum-infected cells to increase resistance to CQ and vice versa, and to render Plasmodium berghei that were selected for CQ resistance in vivo sensitive to the CQ when glutathione synthesis is inhibited. Some over-the-counter drugs that are known to reduce GSH in body tissues when used in excess were found to enhance the antimalarial action of CQ and AQ in mice infected either with P. berghei or Plasmodium vinckei. In contrast, N-acetyl-cysteine which is expected to increase the cellular levels of GSH, antagonized the action of CQ. These results suggest that some over-the-counter drugs can be used in combination with some antimalarials to which the parasite has become resistant.     

Mixed Herbs Drugs: Inhibitory Effect on Growth of the Endogenous Mycoflora and Aflatoxin Production

Twenty commercial mixed herbal drugs were examined for mycological profile. Aspergillus species were the predominant fungi found in the drugs. Other fungi harboured in the drugs with less frequency were Paecilomyces species, Eurotium species, Monascus species, Acremonium species, Penicillium species, Cladosporium species, Scopulariopsis species, Phialophora species and Fonseceae species. Fungal count was between 1.0 log₁₀ CFU and 2.4 log₁₀ CFU per gram of sample. When the drugs were incubated in 85% humidity at 25°C, fungal colonies grew on only two of the drugs. The mixed herbal drugs were extracted with water and the extracts were used to grow Aspergillus parasiticus. All extracts reduced aflatoxin B₁ and aflatoxin G₁ production by 62–97%. All but two of the extracts reduced aflatoxin B₂ and aflatoxin G₂ production by 39–95%. It can be concluded that the commercial powdered mixed herbal drugs contained low number of endogenous fungi, and these drugs are inhibitory to the growth of its endogenous fungi and aflatoxins production by aflatoxigenic fungi.     

Psychomotor stimulant- and opiate-induced c-fos mRNA expression patterns in the rat forebrain: Comparisons between acute drug treatment and a drug challenge in sensitized animals

Amphetamine-, cocaine-, and morphine-induced c-fos expression patterns were examined following an injection protocol that has previously been shown to produce behavioral sensitization and enhanced dopamine release in the striatal complex. Drug-specific c-fos patterns were observed in both acute and sensitization injection paradigms. A sensitization pretreatment schedule did, however, alter the c-fos expression patterns induced by all the drugs in the caudate putamen, nucleus accumbens, and the cerebral cortex. In some striatal and cortical regions, there was an increase or recruitment of cells expressing c-fos whereas in others there was an apparent decrease or inhibition. The somatosensory cortex was one area where pretreatment with all three drugs increased c-fos expression. The results suggest that the neuronal networks that are modulated by systemic drug injections in the sensitized animal differ from those affected by the initial drug exposure; areas of overlap may indicate common ‘sensitization’ circuits. Special issue dedicated to Dr. Eric J. Simon.     

Towards establishment of a plant-based model to assess the novel anti-cancerous lead molecule(s): An in silico,in vivo and in vitro assessment of some potential anti-cancerous drugs on Lathyrus sativus L

The drug development process is one of the important aspects of medical biology. The classical lead identification strategy in the way of drug development based on animal cell is time-consuming, expensive and involving ethical issues. The following study aims to develop a novel plant-based screening of drugs. Study shows the efficacy of certain anti-cancerous drugs (Pemetrexed, 5-Fluorouracil, Methotrexate, Topotecan and Etoposide) on a plant-based (Lathyrus sativus L.) system. Two important characteristics of cancer cells were observed in the colchicine-treated polyploid cell and the callus, where the chromosome numbers were unusual and the division of cells were uncontrolled respectively. With increasing concentration, the drugs significantly reduced the mitotic index, ploidy level and callus growth. Increasing Pemetrexed concentration decreased the plant DHFR activity. A decrease in total RNA content was observed in 5-FU and Methotrexate with increasing concentrations of the drugs. Etoposide and Topotecan inhibited plant topoisomerase II and topoisomerase I activities, which was justified through plasmid nicking and comet assay, respectively. Molecular and biochemical study revealed similar results to the animal system. The in silico study had been done, and the structural similarity of drug binding domains of L. sativus and human beings had also been established. The binding site of the selected drugs to the domains of plant target proteins was also determined. Experimental results are significant in terms of the efficacy of known anti-cancerous drugs on the plant-based system. The proposed assay system is a cost-effective, convenient and less time-consuming process for primary screening of anti-cancerous lead molecules.

     

Inhibition of Growth and of Thymidine Incorporation into DNA in Tetrahymena by Chlorpromazine,Pimozide and Penfluridol1

The antipsychotic drugs chlorpromazine, pimozide, and penfluridol caused a 50% inhibition of growth of Tetrahymena at concentrations of 4.5, 5.5, and 1.5 μM, respectively. The degree of growth inhibition was dependent on the concentration of cells; higher drug concentrations were needed to produce inhibition of denser cell cultures. Binding studies with penfluridol showed that 50% growth inhibition resulted when approximately 50 μmoles of drug were bound per 10⁶ cells. A 20-min preincubation of cells with chlorpromazine (14.7 μM) inhibited DNA synthesis by 46%, and with penfluridol (4 μM) DNA synthesis was inhibited by 27%. The incorporation of labeled thymidine into the thymidine triphosphate pool was inhibited by chlorpromazine but not by penfluridol, indicating that the drugs produce their growth inhibitory effects by different mechanisms. TDP kinase activity was demonstrated in a particle-free fraction of the cells. Its enzymatic activity was not affected by added chlorpromazine, penfluridol, or calmodulin, suggesting that inhibition of DNA synthesis by these drugs may be a consequence of growth inhibition.     

Specificity of drug transport mediated by CaMDR1: a major facilitator of Candida albicans

CaMDR1 encodes a major facilitator superfamily (MFS) protein inCandida albicans whose expression has been linked to azole resistance and which is frequently encountered in this human pathogenic yeast. In this report we have overexpressed CaMdr1p inSf9 insect cells and demonstrated for the first time that it can mediate methotrexate (MTX) and fluconazole (FLC) transport. MTX appeared to be a better substrate for CaMdr1p among these two tested drugs. Due to severe toxicity of these drugs to insect cells, further characterization of CaMdr1p as a drug transporter could not be done with this system. Therefore, as an alternative, CaMdr1p and Cdr1p, which is an ABC protein (ATP binding cassette) also involved in azole resistance inC. albicans, were independently expressed in a common hypersensitive host JG436 ofSaccharomyces cerevisiae. This allowed a better comparison between the functionality of the two export pumps. We observed that while both FLC and MTX are effluxed by CaMdr1p, MTX appeared to be a poor substrate for Cdr1p. JG436 cells expressing Cdr1p thus conferred resistance to other antifungal drugs but remained hypersensitive to MTX. Since MTX is preferentially transported by CaMdr1p, it can be used for studying the function of this MFS protein.     

Reversal of doxorubicin resistance by the amiloride analogue EIPA in multidrug resistant human colon carcinoma cells

Although multidrug resistance (mdr) may arise through a variety of mechanisms, the most widely studied and accepted form is associated with an increased concentration of P-glycoprotein (P-gp), a 170kd protein found in the membrane fraction of a number of mammalian cells. Since mdr seems to be related to the ability of resistant cells to extrude drugs and the circumvention of mdr is supposed to be due to the restored ability to accumulate drugs, membrane has been regarded as the crucial site for such a regulation and an important role for membrane ion exchangers has been suggested. The aim of this work was to elucidate whether the Na⁺/H⁺ antiporter is involved in the mechanism of regulation and circumvention of mdr and if 5-(N-ethyl-N-isopropyl) amiloride (EIPA), a selective inhibitor of the Na⁺/H⁺ exchanger, can modulate the functional expression of the mdr phenotype. The effect of EIPA on doxorubicin (DX) resistant cells (LoVo/DX) obtained from a human colon adenocarcinoma cell line (LoVo) was studied. EIPA at concentrations ranging from 10 to 50 μM was able to increase the antibiotic cytotoxicity in the resistant Lovo/DX cells. The reversal of DX resistance paralleled an increase of the ability of the cells to accumulate the drug. Both drug loading and sensitivity to the inhibitory effect of DX on cell proliferation were restored by EIPA in a dose-dependent way. These results suggest a new mechanism of mdr reversal and indicate that amiloride and its derivatives may be useful in reversing DX resistance and in enhancing the clinical effectiveness of chemotherapeutics.