Percentage Of Surviving Cells Research Articles

Synthesis of CNS active thyrotropin-releasing hormone (TRH)-like peptides: Biological evaluation and effect on cognitive impairment induced by cerebral ischemia in mice

Thyrotropin-releasing hormone (TRH)-like peptides were synthesized by replacing critical histidine and pGlu residues in the native peptide. The peptides were evaluated in vitro for receptor binding activity assay and in the cell functional assay; the peptides exhibit selective basal signaling agonist behavior toward TRH-R2. For example, peptides 8a, 8b, 8c, 8f, 8h, 8l and 12d activated TRH-R2 with potency (EC50) of 0.53μM, 0.048μM, 0.05μM, 0.006μM, 0.31μM, 0.034μM and 0.004μM, respectively. In contrast for signaling activation of TRH-R1, the same peptide required higher concentration of 19.35μM, 3.98μM, 2.54μM, 0.287μM, 11.28μM, 0.986μM and 0.944μM, respectively. The results showed that peptides were 36.5, 82.9, 50.8, 47.8, 36.3, 32.6 and 235-fold selective to TRH-R2 receptor subtype. The peptides were investigated for CNS activity at 10μmol/kg in pentobarbital-induced sleep assay study. Peptides 8c (16.5±1.4min) and 8l (16.5±2.1min) displayed excellent CNS activity. In an in vivo study, peptide 8c did not cause significant change in the rat plasma TSH levels. The peptide 8c was further investigated for neuroprotective potential, and significantly reduced infracts volume and neurological score in the focal cerebral ischemia model in mice. Peptide 8c also significantly lowered MDA levels, indicating reduction of oxidative and enhanced percentage cell survival in CA1 region, when compared to ischemic brain.

Assessment of spermicidal activity of the antimicrobial peptide sarcotoxin Pd: A potent contraceptive agent

Objectives In searching for new spermicidal microbicides for use in the prevention of unplanned pregnancy and sexually transmitted infections (STIs) we investigated the spermicidal and cytotoxicity activities of the antimicrobial peptide sarcotoxin Pd.Methods Washed sperm from 10 healthy, normal volunteers was treated with different concentrations of sarcotoxin Pd. Sperm motility and morphology were assessed at 0, 0.3, 5, 10 and 15 min. The cytotoxicity of sarcotoxin Pd in normal human cervical HeLa cells was measured. Percentage cell survival was expressed as the number of live cells in the test group.Results The cytotoxic effect of sarcotoxin Pd was concentration-dependent. Significant cytotoxicity was observed at concentrations above 24 μg/ml. Sarcotoxin Pd immobilised 100% of spermatozoa at a dose of 90 and 80 μg/ml after 0.3 and 5 min, respectively, and immobilised 50% of spermatozoa after 15 min at lower doses. Sarcotoxin Pd inhibited sperm motility in a dose-dependent manner. The peptide immobilised sperm within 20 s at its maximal effective concentration of 90 μg/ml.Conclusions Sarcotoxin Pd appears to be a good candidate for a contraceptive agent in the prevention of unplanned pregnancy and STIs.

Evaluating LRRK2 genetic variants with unclear pathogenicity.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) have been known to be a major genetic component affecting Parkinson's disease (PD). However, the pathogenicity of many of the LRRK2 variants is unclear because they have been detected in single patients or also in patients and controls. Here, we selected 5 exonic variants (L1165P, T1410M, M1646T, L2063X, and Y2189C) from each of the protein domain of LRRK2 and analysed their possible association with pathogenicity using in vitro functional assays. Point mutations representing each of these variants were incorporated into the LRRK2 gene, and functional aspects such as the percentage of cell survival upon application of stress and kinase activity were measured. Our results showed that all 5 variants had a significantly negative effect on the survival of cells, in both presence and absence of stress, as compared to the wild-type. In addition, there was also a slight increase in kinase activity in most of the variants in comparison to the wild-type. A negative correlation between cell survival and kinase activity was observed. These data suggest that most of the variants despite being located in different domains of LRRK2 appear to exert a potential pathogenic effect possibly through an increased kinase activity, supporting a gain of function mechanism.

Anti-proliferative and pro-apoptotic activities of two marine sponge-derived fungi extracts in HepG2, HCT116 and A375 cancer cell lines

The marine biome is a thriving resource for the search for bioactive compounds, and may be key in the quest for more efficient and less toxic anti-neoplastic drugs for cancer therapy [1 – 2]. The aim of this study was to assess the antitumor effect of crude extracts of sponge-derived fungi Neosartorya paulistensis KUFC 7897 (E1) and N. siamensis KUFA 0017 (E2) on a panel of seven cancer cell lines. The anti-proliferative effect of the extracts (0.1 – 500 µg/ml) was assessed by an MTT assay for 48h, with determination of IC50 per cell line. Ensuing assays were based on 48h of cell exposure to respective IC50. Long-term effect on cell proliferation was determined by a clonogenic assay. In order to observe potential cell death mechanisms, induction of apoptosis was assessed by nuclear condensation and DNA damage was evaluated by comet assay. E1 and E2 extracts decreased the proliferation of HepG2 (liver), HCT116 (colon) and A375 (skin) cell lines. Selected IC50 values ranged from 124 – 197 µg/ml. E1 extract induced a significant decrease in the percentage of clonogenic cell survival of HepG2, A375 and HCT116, as did E2 extract in HepG2, thus reducing indefinite proliferation. Nuclear condensation results showed that E1 and E2 extracts induced a 9% increase of apoptosis in HepG2, whereas E1 exhibited a 24% increase and E2 a 16% increase of apoptosis in HCT116. Yet, the induction of apoptosis was not due to DNA damage. Our results suggest that these extracts show an interesting anti-proliferative and apoptotic activity and thus are contenders for further isolation of specific compounds with chemotherapeutic potential.

In vitro cytotoxicity of 4'-OH-tamoxifen and estradiol in human endometrial adenocarcinoma cells HEC-1A and HEC-1B.

Tamoxifen (TAM), used to treat estrogen receptor (ER)-positive breast cancer, is a well known estrogen antagonist in the breast, but a partial estrogen agonist in the endometrium. In addition, TAM metabolites, such as 4'-hydroxy-tamoxifen (4-OH-TAM), have been shown to be more potent than the parent compound. The objective of this study was to determine the effects of 4-OH-TAM and estradiol (E2) on two human endometrial adenocarcinoma cell lines, HEC-1B and HEC-1A. When HEC-1B cells were treated with lower concentrations (10-1,000 nM) of 4-OH-TAM or E2 for 1-3 days, no significant difference in the percentage of cell survival was observed among the varying concentrations. At higher 4-OH-TAM or E2 concentrations (1-100 µM), HEC-1B and HEC-1A cells responded similarly with an obvious decrease in cell growth noted at 10 and 100 µM 4-OH-TAM and 100 µM E2. In order to address the observed cell death, DNA laddering was performed at various time intervals with 4-OH-TAM (10 µM) or E2 (10 or 50 µM). DNA gel electrophoresis failed to show the typical laddering pattern (180-200 bp) observed in apoptosis. Furthermore, western blot analysis of caspase-8 and -3 failed to demonstrate caspase activity. These results suggest that apoptosis was not the underlying cellular mechanism of cell death. Due to the lack of apoptotic markers, a cytotoxic (cell death) effect was differentiated from a cytostatic (growth inhibition) effect confirming that cell death had occurred. In summary, micromolar concentrations of 4-OH-TAM induced a non-apoptotic cytotoxic effect in the endometrium; however further studies are needed to elucidate the cytotoxic pathway being utilized.

Abstract 861: Survivin-mediated adaptive response: a risk factor for IGRT

Abstract Exposure of cells to very low doses of ionizing radiation can induce an enhanced resistance or adaptive response to a subsequent larger radiation dose as demonstrated by an increase in cell survival. Expression of a radio-adaptive response has been attributed to pro-survival signaling processes induced by very low radiation doses in the range of 5 to 100 mGy. The radiation-induced adaptive response is gaining considerable attention due in part to the expanding use of imaging technologies such as computerized axial tomography and portal imaging to monitor tumor response and positioning during multi-dose standard radiation therapy protocols. The focus of this study was to investigate whether this process could occur when the very small imaging radiation dose was administered prior to or following the administration of the first of two typically used radiation therapy level doses. SA-NH mouse sarcoma cells and SA-NH cells stably transfected with mutated IκBα (SA-NH+mIκBα1) to inhibit the activation of NFκB were grown to confluence in vitro or as tumors in the right hind leg of C3H mice. Percentage of cell survival, apoptosis frequencies and survivin protein levels were determined as a function of radiation conditions and transfection of cells with survivin siRNA. A significant radio-adaptive response was observed in cells exposed to 5 mGy - 100 mGy 15 min to 6 h prior to or after irradiation with the first of two 2 Gy therapeutic radiation doses and was associated with a concomitant reduction in apoptosis frequencies and a rapid elevation in survivin protein levels. The very low dose radiation-induced elevation in survivin levels, the reduction in apoptosis frequencies and enhanced cell survival were not observed in SA-NH+mIκBα1 cells or SA-NH cells transfected with survivin siRNA. We have identified a very low radiation dose-induced survivin-mediated adaptive response that could compromise therapeutic outcomes associated with the increased usage of imaging procedures in radiation therapy. This work was supported by NIH/NCI grant R01 CA132998 and DOE Low Dose Program/Project Grant DE-SC0001271 (D.J.G.) Citation Format: David J. Grdina, Jeffrey S. Murley, Richard C. Miller, Gayle E. Woloschak, Jian Jian Li, Ralph R. Weichselbaum. Survivin-mediated adaptive response: a risk factor for IGRT. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 861. doi:10.1158/1538-7445.AM2014-861

Pyroelectrically Generated Very Low Dose Ionizing Radiation Enhances chemopreventive and Chemotherapeutic Effects of Genistein Isoflavone in Human Prostate Cancer Cells

An estimated 233,000 American men will be diagnosed with new cases of prostate cancer with 29,480 men expected to die from the disease in 2014. These statistics will regrettably add to the burden of about 2,617,682 men currently living with the disease in the United States. The development of resistance by cancer cells to radiation and chemotherapy has limited the efficacy of the current therapeutic interventions, thereby informing the need for a novel effective and safer alternative therapeutic regimen. In this study, we exploited the role of low dose Hyperradiosensitivity (LDHRS) in enhancing the chemopreventive and chemotherapeutic effects of Genistein by pretreatment exposure to very low dose ionizing radiation (VLDR, 20mGy/hr) using a Pyroelectrically generated X-ray source (non-radioactive) in two human prostate cancer cell lines in vitro. The presence of HRS was assessed using the MTT assay. Treatment-induced intracellular levels of ROS and SOD were assessed using Nitro blue tetrazolium assay. Adaptive response mediated by VLDR following delayed drug treatments was also investigated and found to be both time-dependent and dose-dependent in LNCaP cell line. LNCaP cells were found to be slightly more radiosensitive compared to PC3 cells after exposure to 20mGy/hr VLDR. Both cell lines were found to induce some form of HRS. VLDR alone or in combination with a low dose of Genistein (10µM) was found to proffer the highest treatment induced chemoprevention by elevating the levels of cellular anti-oxidant enzymes (SOD). High doses of Genistein increased intracellular levels of ROS and growth inhibition, thereby, favoring the chemotherapeutic effects of Genistein. In all treatment groups, apoptosis tends to be the most common mechanism of cell death. These findings indicate that chemo enhancement by VLDR can be associated with modulation of the percentage cell survival rate (CSR), apoptosis, HRS, and SOD or ROS levels in prostate cancer cells.

Dynamic rearrangement of F-actin is required to maintain the antitumor effect of trichostatin A.

Actin plays a role in various processes in eukaryotic cells, including cell growth and death. We investigated whether the antitumor effect of trichostatin A (TSA) is associated with the dynamic rearrangement of F-actin. TSA is an antitumor drug that induces hyper-acetylation of histones by inhibiting histone deacetylase. HeLa human cervical cancer cells were used to measure the antitumor effect of TSA. The percent cell survival was determined by an MTT assay. Hypodiploid cell formation was assessed by flow cytometry. Collapse of the mitochondrial membrane potential (MMP) was identified by a decrease in the percentage of cells with red MitoProbe J-aggregate (JC-1) fluorescence. Cell survival was reduced by treatment with TSA, as judged by an MTT assay and staining with propidium iodide, FITC-labeled annexin V, or 4′,6-diamidino-2-phenylindole (DAPI). TSA also induced an MMP collapse, as judged by the measurement of intracellular red JC-1 fluorescence. In addition, the F-actin depolymerizers cytochalasin D (CytoD) and latrunculin B (LatB) induced an MMP collapse and increased apoptotic cell death in HeLa cells. However, our data show that apoptotic cell death and the MMP collapse induced by TSA were decreased by the co-treatment of cells with CytoD and LatB. These findings demonstrate that the dynamic rearrangement of F-actin might be necessary for TSA-induced HeLa cell apoptosis involving a TSA-induced MMP collapse. They also suggest that actin cytoskeleton dynamics play an important role in maintaining the therapeutic effects of antitumor agents in tumor cells. They further suggest that maintaining the MMP could be a novel strategy for increasing drug sensitivity in TSA-treated tumors.

Application of response surface method to evaluate the cytotoxic potency of Ulva fasciata Delile, a marine macro alga

Bioprospecting of marine natural products has recently produced a substantial number of drug candidates. Ulva fasciata Delile, belonging to the family Ulvaceae, is a green marine macro alga that grows profusely on the coastal seashore of South India. In the present study, we investigated the in vitro cytotoxic potential of a methanolic extract of U.fasciata Delile (MEUF) using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay against human colon carcinoma (HT-29), human hepatocyte carcinoma (Hep-G2), and human breast carcinoma (MCF-7) cell lines. Response surface methodology (RSM) was applied using central-composite experimental design (CCD) to obtain optimum combined effect of concentration and cancer cells with highest cytotoxicity. The effect of concentration, cancer cell lines as independent variables on absorbance (OD), percent cell survival and percent cell inhibition as dependent variables was investigated. Maximum cytotoxic activity of MEUF was established for Hep-G2 with lowest OD or percent cell survival; highest percent cell inhibition with significant difference (p>0.05) was compared to HT-29 and MCF-7.

English

Objectives: Multi-drug resistance (MDR) is a major hurdle in treatment of cancer, contributing to the failure of chemotherapy. Drug resistance is found to be linked to the overexpression of ATP-binding cassette (ABC) drug transporter proteins that include P-glycoprotein (P-gp), causing a reduction in drug accretion inside the cancer cells. In the present study, the effect of the extracts from the fruit peel and pulp of Momordica charantia (MCFPE) fruit in modulating the function of P-gp in human smallcell lung cancer (SCLC) cell lines was assessed. Methods: The effects of MCFPE were tested on drug-sensitive (H69) and multi-drug resistant (H69/LX4) human SCLC cells. The cell survival percentage was assessed by MTT cytotoxicity assay. The percentage of drug accumulation and drug efflux were assessed by using [3H]-paclitaxel. The expression of MDR1 gene was analysed by reverse transcription polymerase chain reaction (RT-PCR), and P-gp by western blot analysis. Results: The extract was able to induce death of cancer cells as measured by cell survival percentage as well as improve drug accumulation, as evidenced by intracellular paclitaxel retention. Prior exposure of cells to MCFPE reversed resistance to paclitaxel. Treatment with MCFPE was found to have a significant impact on MDR 1 gene expression in H69/LX4 cell line by decreasing its expression. The extract had no influence on expression of MDR 1 gene in the drug-sensitive SCLC cell lines. Western blot analysis of P-gp protein in H69 and H69/LX4 cells revealed that the treatment with the extract modulates the expression of MDR 1 in H69/LX4 and had negligible effect on H69 cells. Conclusion: The results indicate that MCFPE was able to effectively reverse multi-drug resistance and improve cancer chemotherapy.

Synergistic Effects of Tamoxifen and Tranilast on VEGF and MMP-9 Regulation in Cultured Human Breast Cancer Cells

Vascular endothelial growth factor and matrix metalloproteinases are two important factors for angiogenesis associated with breast cancer growth and progression. The present study was aimed to examine the effects of tamoxifen and tranilast drugs singly or in combination on proliferation of breast cancer cells and also to evaluate VEGF and MMP-9 expression and VEGF secretion levels. Human breast cancer cell lines, MCF-7 and MDA-MB-231, were treated with tamoxifen and/or tranilast alone or in combination and percentage cell survival and proliferative activity were evaluated using LDH leakage and MTT assays. mRNA expression and protein levels were examined by real-time RT-PCR and ELISA assay, respectively. LDH and MTT assays showed that the combined treatment of tamoxifen and tranilast resulted in a significant decrease in cell viability and cell proliferation compared with tamoxifen or tranilast treatment alone, with significant decrease in VEGF mRNA and protein levels. We also found that tamoxifen as a single agent rarely increased MMP-9 expression. A decrease in MMP-9 expression was seen after treatment with tranilast alone and in the combined treatment MMP-9 mRNA level was decreased. This combination treatment can able to inhibit growth, proliferation and angiogenesis of breast cancer cells.

Development, characterization, and photocytotoxicity assessment on human melanoma of chloroaluminum phthalocyanine nanocapsules

In this work we have developed nanocapsules containing chloroaluminum phthalocyanine (ClAlPc) and assessed their phototoxic action on WM1552C, WM278, and WM1617 human melanoma cell lines. The ClAlPc-loaded nanocapsules were prepared by the nanoprecipitation method and optimized by means of a 23 full factorial design. The ClAlPc nanocapsules were characterized by particle size and distribution, zeta potential, morphology, encapsulation efficiency, singlet oxygen production, stability, and phototoxic action on melanoma cells. Both the development and optimization studies revealed that stable colloidal formulations could be obtained by using 1.75% (w/v) soybean lecithin, 1.25% (w/v) Poloxamer 188, 2.5% (v/v) soybean oil, and 0.75% (w/v) poly(D,L-lactide-co-glycolide). The nanocapsules had a mean diameter of 230nm, homogeneous size distribution (polydispersity index<0.3), and negative zeta potential (about −30mV). Their morphology was spherical, with evident polymer membrane coating droplet. The encapsulation efficiency was 70%, as expected for hydrophobic drugs, and the nanoencapsulated ClAlPc was able to produce high singlet oxygen quantum yield. ClAlPc nanocapsules exhibited good physical stability over a 12-month period. WM1552C primary melanoma cells were more sensitive (p<0.05) to the phototoxic effect elicited by ClAlPc nanocapsules (0.3μgml−1) under light irradiation at 20mJcm−2. On the other hand, the cell survival percentage for all the melanoma cell lines treated with the highest light dose (150mJcm−2) was lower than 10%. In summary, ClAlPc nanoencapsulation could enable application of this hydrophobic photosensitizer in the treatment of malignant melanoma with the use of both low sensitizer drug concentration and light dose.

Air-Suspension Fluidized-Bed Microencapsulation of Probiotics

In the present research, an air-suspension fluidized-bed technique for generation of core and shell microcapsules containing probiotic Lactobacillusparacasei cells was evaluated. The air-suspension process was performed in a Würster coater system with a bottom-spraying atomizer. In the first stage, a solution containing trehalose, maltodextrin, and probiotic cells was spray-coated onto and absorbed by the inert carrier microcrystalline cellulose to produce nonagglomerating dry coated particles with high probiotic cell viability (109 colony-forming units [cfu]/g particles). The effect of inlet air temperature, spray flow rate, solids concentration, cell concentration, and encapsulation formulation on survival was investigated. The inlet air temperature had the most pronounced effect; a 15°C increase in inlet air temperature led to a 250-fold decrease in survival percentage. There was no agglomeration of the coated adsorbed particles at spray flow rates of 1 to 3.5 mL/min. Spraycoating was performed with both laboratory- and pilot-scale Würster systems. Scaling up the equipment from the lab scale to the pilot scale did not affect cell survival percentage. Low solids concentration (<20%) and low probiotic cell concentration (6%) resulted in a fair survival percentage (35–40). Spraying a solution formulation of trehalose–maltodextrin with dextrose equivalent (DE) 3 at a 1:1 ratio provided the best protection in terms of cell viability during the spray-coating process as well during storage. Layering with ethylcellulose (ETHOCEL) provided some protection in a low acidic environment (12%); however, the nonuniform coating and cracks detected by scanning electron microscopy (SEM) imaging were the main reasons for the layering's relatively poor protection of the cells against humidity, oxygen, and acid environment.

Neuroprotective effect of an angiotensin receptor type 2 agonist following cerebral ischemia in vitro and in vivo

BackgroundIntracerebral administration of the angiotensin II type 2 receptor (AT2R) agonist, CGP42112, is neuroprotective in a rat model of ischemic stroke. To explore further its possible cellular target(s) and therapeutic utility, we firstly examined whether CGP42112 may exert direct protective effects on primary neurons following glucose deprivation in vitro. Secondly, we tested whether CGP42112 is effective when administered systemically in a mouse model of cerebral ischemia.MethodsPrimary cortical neurons were cultured from E17 C57Bl6 mouse embryos for 9 d, exposed to glucose deprivation for 24 h alone or with drug treatments, and percent cell survival assessed using trypan blue exclusion. Ischemic stroke was induced in adult male C57Bl6 mice by middle cerebral artery occlusion for 30 min, followed by reperfusion for 23.5 h. Neurological assessment was performed and then mice were euthanized and infarct and edema volume were analysed.ResultsDuring glucose deprivation, CGP42112 (1x10-8 M and 1x10-7 M) reduced cell death by ~30%, an effect that was prevented by the AT2R antagonist, PD123319 (1x10-6 M). Neuroprotection by CGP42112 was lost at a higher concentration (1x10-6 M) but was unmasked by co-application with the AT1R antagonist, candesartan (1x10-7 M). By contrast, Compound 21 (1x10-8 M to 1x10-6 M), a second AT2R agonist, had no effect on neuronal survival. Mice treated with CGP42112 (1 mg/kg i.p.) after cerebral ischemia had improved functional outcomes over vehicle-treated mice as well as reduced total and cortical infarct volumes.ConclusionsThese results indicate that CGP42112 can directly protect neurons from ischemia-like injury in vitro via activation of AT2Rs, an effect opposed by AT1R activation at high concentrations. Furthermore, systemic administration of CGP42112 can reduce functional deficits and infarct volume following cerebral ischemia in vivo.

In vitro survival of MCF-7 breast cancer cells following combined treatment with ionizing radiation and mitoxantrone-mediated photodynamic therapy

This study evaluated the effects of mitoxantrone (MX) as a sensitizer following combined treatment with ionizing radiation and photodynamic therapy in the MCF-7 human breast cancer cell line. Cells were incubated with MX at different concentrations for 90min and exposed to different fluence rates of non-coherent light and different dose rates of ionizing X-ray radiation in independent treatment groups. Additionally, the combined effects of chemotherapy, phototherapy, and radiotherapy were evaluated. The percent cell survival was investigated using the MTT assay. MX acted as both a photosensitizer and radiosensitizer. Furthermore, the use of 1μM MX in combination with PDT at 10J/Cm(2) and 4Gy of X-ray radiation strongly resulted in the death of cancer cells and reduced the percentage of viable cancer cells to 2.4±1.15. Our data demonstrated that the adverse effects of MX in combination with radiotherapy were partially abated, without a reduction in the efficacy of treatment. This new therapeutic avenue for breast cancer therapy merits further investigation using in vivo models for application in humans.

Adenovirus-mediated gene transfer of human D2S combined with bromocriptine inhibits proliferation of GH3 rat anterior pituitary cells

Objective To study the effects of adenovirus - mediated D2S gene combined with bromocriptine therapy on proliferation of GH3 rat anterior pituitary cells.Methods GH3 cells was transfected with adenovirus containing dopamine 2 receptor short isoform gene(D2S) and confirmed by real time RT - PCR,Western - blot.Cell cytotoxicity assays were performed using a Cell Counting Kit - 8 ( CCK -8).Results Expression levels of D2S mRNA and protein increased in GH3 cells transfected with adenovirus containing D2S gene.The percentage of cell survival of GH3 cells transfected with adenovirus containing [2S gene significantly decreased after bromocriptine treatment,compared with the control group and the pAd - EGFP transfection group ( 32.76 ± 3.88 ) ％,( 101.78 ± 2.05 ) ％,( 91.79 ± 3.80 ) ％,respectively,P ＜ 0.01.Conclusion Adenovirus - mediated D2S gene combined with bromocriptine therapy effectively inhibits GH3 cells growth. Key words: Adenovirus vectors; GH3 cells; Gene therapy; Bromocriptine; Dopamine D2 receptor short isoform

Cytotoxic Effects of two Iranian Scorpions Odontobuthusdoriae and Bothutus saulcyi on Five Human Cultured Cell lines and Fractions of Toxic Venom.

Scorpion venom toxicity is of major concern due to its influence on human activities and public health. We investigated the in-vitro process of cell death caused by two Iranian scorpions Odontobuthus doriae and Bothutus salceyi venom on human cell lines. The aim of this study was to provide further information about triggering cell death and suggestion of methods for the elimination of unwanted cells such as tumor cells. The cytotoxicity and apoptosis induced by effect of scorpion venoms on five established eukaryotic cell lines are analyzed on different human cell lines. All cultured cell lines were incubated with varying doses of scorpion venom for 24 h at 37°C. Control culture was treated with an equal amount of SFM. The percentage of cell survival was measured using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium (MTT) colorimetric assay. Our data demonstrated that Bothutus saulcyi, does not show cytotoxic effect on any of the used cell lines. Odontobuthus doriae, however, has resulted a dose dependent cytotoxic effect with maximum at 1 ug/mL on 1321N1 glioma like cell line. Then the cytotoxic venom of O. doriae was fractionated using Sephadex G50 gel chromatography. The toxic fractions on mouse used to Cytotoxicity assay on 1321 N1 cell line and data demonstrated that, the fraction F3 showed a dose dependent Cytotoxicity assay. Further studies to explode the mode of action of these venoms are recommended and purification of the toxic fraction should be done.

Phosphodiesterase inhibitors control A172 human glioblastoma cell death through cAMP-mediated activation of protein kinase A and Epac1/Rap1 pathways

AimsWe investigated whether cAMP-mediated protein kinase A(PKA) and Epac1/Rap1 pathways differentially affect brain tumor cell death using 4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone(rolipram), specific phosphodiesterase type IV(PDE IV) inhibitor. Main methodsA172 and U87MG human glioblastoma cells were used. Percentage of cell survival was determined by MTT assay. PKA and Epac1/Rap1 activation was determined by western blotting and pull-down assay, respectively. Cell cycle and hypodiploid cell formation were assessed by flow cytometry analysis. Key findingsNon-specific PDE inhibitors, isobutylmethylxanthine(IBMX) and theophylline reduce survival percentage of A172 and U87MG cells. The expression of PDE4A and PDE4B was detected in A172 and U87MG cells. Rolipram-treated A172 or U87MG cell survival was lower in the presence of forskolin, adenylate cyclase activator, than that in its absence. Co-treatment with rolipram and forskolin also enhanced CREB phosphorylation on serine 133 that was inhibited by H-89, PKA inhibitor and cAMP-responsive guanine nucleotide exchange factor 1(Epac1), a Rap GDP exchange factor-mediated Rap1 activity in A172 cells. When A172 cells were treated with cell-permeable dibutyryl-cAMP(dbcAMP), PKA activator or 8-(4-chloro-phenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate(CPT), Epac1 activator, basal level of cell death was increased and cell cycle was arrested at the phase of G2/M. Rolipram-induced A172 cell death was also increased by the co-treatment with dbcAMP or CPT, but it was inhibited by the pre-treatment with H-89. SignificanceThese findings demonstrate that PKA and Epac1/Rap1 pathways could cooperatively play a role in rolipram-induced brain tumor cell death. It suggests that rolipram might regulate glioblastoma cell density through dual pathways of PKA- and Epac1/Rap1-mediated cell death and cell cycle arrest.

Estrogen-like activities and cytotoxicity effects of Thai herbal medicines as natural ingredients in anti-ageing

The objective of the study was to search for the appropriate herbal extracts by comparative analysis of their estrogenic and cytotoxic activities. Some potentially estrogenic activity of herbal extracts in the management of female disorder symptoms was investigated by E-screen assay. The extracts having a promising activity were further evaluated in vitro for their cytotoxic activity. Of 13 herbal extracts tested, 10 showed interesting estrogenic properties. These were Pueraria candollei var mirifica, Linum usitatissimum, Glycine max, Curcuma aeruginosa, Cissus quadrangularis, Tadehagi godefroyanum, Curcuma comosa, Butea superba, Trigonella foenum-graecum and Punica granatum. The proliferative activity of those extracts could be completely inhibited by the addition of an estrogen receptor antagonist. The extract of T. foenum-graecum exhibited the strongest cytotoxicity on mouse fibroblast cells (cell viability <80% at 100 µg/ml) while a growth-promoting effect could be observed for P. candollei var mirifica, C. aeruginosa, C. quadrangularis and C. comosa. Pre-treatment of those 10 extracts to the mouse fibroblast cells prior to the addition of H2O2 reduced the apoptotic cells as well as increased the percentage of cell survival. Nine (9) extracts (not T. foenum-graecum) were selected as potentially active ingredients for the treatment of skin-ageing in post-menopausal women.

Cancer cell response to nanoparticles: criticality and optimality

A stochastic variation in size and electrical parameters is common in nanoparticles. Synthesizing gold nanoparticles with a varying range of size and zeta potential, we show that there is clustering at certain regions of hydrodynamic diameter and zeta potentials that can be classified using k-clustering technique. A cluster boundary was observed around 50 nm, a size known for its optimal response to cells. However, neither size nor zeta potential alone determined the optimal cellular response (e.g., percentage cell survival) induced by such nanoparticles. A complex interplay prevails between size, zeta potential, nature of surface functionalization, and extent of adhesion of the cell to a solid matrix. However, it follows that the ratio of zeta potential to surface area, which scales as the electrical field (by Gaussian law), serves as an appropriate indicator for optimal cellular response. The phase plot spanned by fractional survival and effective electric field (charge density) indicates a positive correlation between mean cell survival and the magnitude of the electric field. The phase plot spanned by fractional survival and effective electric field (charge density) associated with the nanosurface shows a bifurcation behavior. Wide variation of cell survival response is observed at certain critical values of the surface charge density, whereas in other ranges the cellular response is well behaved and more predictable. Existence of phase points near the critical region corresponds to wide fluctuation of nanoparticle-induced response, for small changes in the nanosurface property. Smaller nanoparticles with low zeta potential (e.g., those conjugated with arginine) can have such an attribute (i.e., higher electrical field strength), and eventually they cause more cell death. The study may help in optimal design of nanodrugs. From the Clinical EditorIn this paper, important aspects of the background theory of optimizing gold nanoparticles for cancer therapy is discussed. Such nanoparticles have an enormous potential in future human applications once we fully understand the design parameters.