Ascites Tumor Cells Research Articles

Profiling signal transduction pathways in ascites tumor cells of gastric cancer.

e14583 Background: Malignant ascites is a manifestation of end stage events in a variety of cancers and associated with a poor prognosis with limited options for therapies. We evaluated the potential use of tumor cells isolated in ascites collected from gastric cancer (GCA) patients. Here we demonstrate successful profiling of signal transduction pathway proteins in isolated ascites-tumor cells (ATCs) and their modulations in response to drug treatments. Methods: The collaborative enzyme enhanced reactive-immunoassay (CEER) is a multiplexed protein microarray platform requiring co-localization of two detector enzyme-conjugated-antibodies. Once target proteins are captured on the microarray-surface, channeling events between two detector enzymes in proximity enable the profiling of the target proteins with high sensitivity. In this study, ATCs were isolated from ascites fluid obtained from advanced GCA patients and analyzed for HER1, HER2, HER3, cMET, cKIT, IGF-1R, PI3K, and other pathway proteins for their expression/activation and the degree of pathway modulation in response to targeted drug treatments. Results: Here, we report successful profiling of activation and expression of proteins in RTK pathway in ATCs of GCA patients. Varying levels of HER2 expression was detected in some patients and with significant levels of activation in subset of these patients. A complete in vitro ablation of phosphorylation of HER2 was observed when EpCAM positive ATCs were treated with HER2 targeted inhibitor in one GCA patient. Differential patterns of other RTKs profile and downstream pathway proteins were also observed. Conclusions: The ATCs from GCA are suitable resource for real-time profiling for the status of proteins in RTK and downstream proteins in signal transduction pathways. Monitoring the profile shift of pathway proteins over time, either treatment-related or due to the natural history of disease in GCA patients, may allow more effective selection and adjustment of targeted therapy for an individual patient. As CEER requires limited amounts of sample, real-time biopsy can be performed on tumor cells obtained from ascites, FNA or CTCs for therapy optimization (combination/sequencing) during the course of treatments.

CD38 expression as response of hematopoietic system to cancer

Erythrocyte and lymphocyte NAD(+) glycohydrolase levels were previously found to be elevated in cancer patients. These results were confirmed in an animal model. The administration of live Ehrlich ascites tumor cells to BALB/c mice led to increases in erythrocyte and lymphocyte NAD(+) glycohydrolase, along with tumor development. Serum samples, ascites fluid from mice with developed tumors, serum samples from cancer patients and Ehrlich cell supernatants had a similar stimulatory effect when administered to mice or when incubated with peripheric lymphocytes in culture. These increases were accompanied by the appearance of an anti-CD38 reactive band of 45 kDa in SDS-PAGE/Western blot analyses of erythrocyte ghost and lymphocyte membrane proteins. The results, supported by flow cytometry data, support previous clinical findings that an enhancement in CD38 expression occurs in the hematopoietic system during proliferative processes. Moreover, they suggest that CD38 expression is triggered at least in part by a certain cytokine(s) secreted by cancer cells. Finally, the results emphasize the prospective use of CD38 expression as a marker of tumor development and progression.

Up-regulation of mitochondrial antioxidation signals in ovarian cancer cells with aggressive biologic behavior

Recently, a high frequency of mutations in mitochondrial DNA (mtDNA) has been detected in ovarian cancer. To explore the alterations of proteins in mitochondria in ovarian cancer, a pair of human ovarian carcinoma cell lines (SKOV3/SKOV3.ip1) with different metastatic potentials was examined. Cancer cells SKOV3.ip1 were derived from the ascitic tumor cells of nude mice bearing a tumor of ovarian cancer cells SKOV3. SKOV3.ip1 exhibited a higher degree of migration potential than its paired cell line SKOV3. The proteins in the mitochondria of these two cells were isolated and separated by 2-D gel electrophoresis. The differently expressed proteins were extracted and identified using matrix assisted laser desorption ionisation/time-of-flight/time-of-flight (MALDI-TOF/TOF), and finally a selected protein candidate was further investigated by immunohistochemistry (IHC) method in nude mice bearing tumor tissues of these two cells. A total of 35 spots with different expressions were identified between the two cells using 2D-polyacrylamide gel electrophoresis (PAGE) approach. Among them, 17 spots were detected only in either SKOV3 or SKOV3.ip1 cells. Eighteen spots expressed different levels, with as much as a three-fold difference between the two cells. Twenty spots were analyzed using MALDI-TOF/TOF, and 11 of them were identified successfully; four were known to be located in mitochondria, including superoxide dismutase 2 (SOD2), fumarate hydratase (FH), mitochondrial ribosomal protein L38 (MRPL38), and mRNA turnover 4 homolog (MRTO4). An increased staining of SOD2 was observed in SKOV3.ip1 over that of SKOV3 in IHC analysis. Our results indicate that the enhanced antioxidation and metabolic potentials of ovarian cancer cells might contribute to their aggressive and metastatic behaviors. The underlying mechanism warrants further study.

Investigation of Ehrlich ascites tumor cell death mechanisms induced by Synadenium umbellatum Pax.

Ethnopharmacological relevanceSynadenium umbellatum Pax. is widely found in South America and empirically used in Brazil for the treatment of several diseases, mainly cancer. The aim of the study was to investigate cell death mechanisms induced by Synadenium umbellatum Pax. using Ehrlich ascites tumor (EAT) cells, as well as the myelotoxicity potential of this plant. Materials and methodsS. umbellatum cytotoxicity was evaluated in EAT cells by trypan blue exclusion and MTT reduction test and the mechanisms involved in EAT cell death were investigated by light and fluorescence microscopy, flow cytometry and immunocytochemistry. Investigation of S. umbellatum myelotoxicity was performed by clonogenic assay of colony forming unit- granulocyte macrophage (CFU-GM). Results and ConclusionOur results demonstrated that S. umbellatum decreased the viability of EAT cells using both methods. Morphological analyses revealed that S. umbellatum-treatment induced EAT cell death by apoptotic pathway. We demonstrated the occurrence of reactive oxygen species (ROS) overgeneration, increased intracellular Ca2+ concentration, alteration in mitochondrial membrane potential, phosphatydylserine externalization, and activation of caspases 3, 8, and 9. However, S. umbellatum produced myelotoxicity in bone marrow cells in a concentration-dependent manner. In comparison to EAT cells, the effects of S. umbellatum in bone marrow cells were 8-fold lower. Taken together, our results showed that S. umbellatum induced apoptosis in EAT cells at several levels and seems more toxic to tumor cells than to normal bone marrow cells.

Abstract 4712: Evidence of clinical efficacy of the combination of flavopiridol (Alvocidib) and cisplatin in platin-resistant ovarian and primary peritoneal carcinoma: Phase 2 trial MC0261

Abstract Background: Based upon preclinical synergy and prior phase 1 study results, the clinical efficacy of flavopiridol combined with cisplatin was assessed in patients with recurrent ovarian and primary peritoneal cancers. Methods: A two cohort phase 2 trial of cisplatin (60 mg/m2 IV) followed by flavopiridol (100 mg/m2 IV, 24 h continuous infusion; 21 day cycles) was undertaken in patients with recurrent platin-sensitive or platin-resistant ovarian/primary peritoneal cancers (defined by disease progression > vs. <6 months following platin-based therapy). Measurable disease (RECIST criteria) – or evaluable disease plus CA125 >2X the post-treatment nadir – was required, as was ECOG performance <2 and exposure to only one prior treatment regimen. Results: Forty-five patients were enrolled between April 20, 2004 and March 4, 2010 – 40 platin-resistant patients (Group 1), and 5 platin-sensitive patients (Group 2). In Group 1, the median number of treatment cycles was 3 (range 2-12); 39 of the 40 eligible patients have now discontinued treatment. While only 10% of all patients incurred grade 4 toxicities, grade 3 toxicities were seen in the majority (65%). The most frequent grade 3 and 4 toxicities were neutropenia (all grade 3, 17.5%); nausea (12.5%); vomiting, fatigue, thrombosis, anemia (10% each). Sensory neuropathy, grade 1 or 2, was observed in 75% of all patients – with grade 3 and 4 neuropathy not observed primarily due to pre-specified aggressive dose reductions. Six patients (15%) in Group 1 achieved a confirmed response (1 CR, 5 PR), with a median response duration of 119 days (range 84-212). Ten additional Group 1 patients (32.5%) experienced maintained stable disease. Median Group 1 overall time to progression was 3.7 months; overall survival was 17.2 months. Pilot assessment of attained ascites flavopiridol level and sensitivity of patient ascitic tumor cells to flavopiridol confirmed that patient flavopiridol levels were consistent with observed clinical antitumor efficacy. In Group 2, although 2 of 5 patients also responded (40%; 2 PR), the cohort was closed due to poor accrual. Conclusions: The combination of flavopiridol and cisplatin has promising clinical activity in both platin-sensitive and platin-resistant ovarian and primary peritoneal cancers. Supported in part by NCI CA097129, CA15083 and CM62205; clinicaltrials.gov identifier NCT00083122 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4712. doi:10.1158/1538-7445.AM2011-4712

Stability of Artemisinin in Aqueous Environments: Impact on its Cytotoxic Action to Ehrlich Ascites Tumour Cells

We have recently shown artemisinin to be cytotoxic against Ehrlich ascites tumour cells. The aim of this study was to investigate the stability of this compound in the aqueous environment of the in-vitro Ehrlich ascites tumour cell system (RPMI 1640 cell culture medium supplemented with 10% foetal bovine serum (RPMI/FBS) with reference to its cytotoxic action. Literature data show that artemisinin can react with Fe2+ yielding reactive intermediates leaving artemisinin G as a major end-product. The current study showed that only excess addition of Fe2+ to artemisinin in distilled water, phosphate-buffered saline (PBS) and RPMI/FBS and incubation for 24 h led to degradation of artemisinin and yielded artemisinin G. If Fe2+ was not added results from HPLC analysis were indicative of complete recovery of artemisinin from distilled water and RPMI/FBS, with or without cells, at 37 degrees C for at least 24 h. In addition, incubation of artemisinin in RPMI/FBS with or without cells at 37 degrees C for 24 h before cytotoxicity assay did not change its cytotoxic action. On the basis of these results, we suggest that cytotoxicity to tumour cells was caused by unchanged artemisinin. This is not so for the antimalarial activity of artemisinin and derivatives, for which the presence of a pool of (haem) Fe2+ is a prerequisite resulting in free radicals or electrophilic intermediates or both.

Novel Glycolipoproteins from Ginseng

Ginseng has been used as a general tonic agent to invigorate human body. In the present study, we isolated novel glycolipoproteins from ginseng that activate Ca²?-activated Cl- channel (CaCC) in Xenopus oocytes and transiently increase intracellular free Ca²? concentration ([Ca²?] i ) in mouse Ehrlich ascites tumor cells. We named the active ingredients as gintonin. Gintonin exists in at least six different forms. The native molecular weight of gintonin is about 67 kDa but its apparent molecular weight is about 13 kDa, indicating that gintonin might be a pentamer. Gintonin is rich in hydrophobic amino acids. Its main carbohydrates are glucose and glucosamine. Its lipid components are linoleic, palmitic, oleic, and stearic acids. Gintonin actions were blocked by U73122, a phospholipase C inhibitor, 2-aminoethxydiphenyl borate, an inositol 1,4,5-trisphosphate receptor antagonist, or bis (o-aminophenoxy) ethane-N,N,N0,N0-tetracetic acid acetoxymethyl ester, a membrane permeable Ca²? chelator. In the present study, we for the fi rst time isolated novel gintonin and showed the signaling pathways on gintonin-mediated CaCC activations and transient increase of [Ca²?] i . Since [Ca²?] i as a second messenger plays a pivotal role in the regulation of diverse Ca²?-dependent intracellular signal pathways, gintonin-mediated regulations of [Ca²?] i might contribute to biological actions of ginseng.

Antioxidant and cytoprotective properties of 2-(hydroxymethyl)-3-methoxybenzaldehyde

Currently there is a great deal of interest in the study of natural compounds with free radical scavenging activity because of their potential role in maintaining human health and preventing diseases. In this paper, we report the antioxidant and cytoprotective properties of 2-(hydroxymethyl)-3-methoxybenzaldehyde (HMMB) isolated from the aqueous extract of Decalepis hamiltonii roots. Our results show that HMMB is a potent scavenger of superoxide (O2-), hydroxyl (OH), nitric oxide (NO), and lipid peroxide (LOO) physiologically relevant free radicals with IC50 values in the nmolar (5–214) range. HMMB also exhibited concentration dependent secondary antioxidant activities, such as reducing power, metal chelating activity, and inhibition of protein carbonylation. Further, HMMB at nmolar concentration prevented CuSO4-induced human LDL oxidation. Apart from the in vitro free radical scavenging activity, HMMB demonstrated cytoprotective activity in primary hepatocytes and Ehrlich Ascites Tumour (EAT) cells against oxidative stress inducing xenobiotics. The mechanism of cytoprotective action involved maintaining the intracellular glutathione (GSH), scavenging of reactive oxygen species (ROS), and inhibition of lipid peroxidation (LPO). Based on the results it is suggested that HMMB is a novel bioactive molecule with health implications in both prevention and amelioration of diseases involving oxidative stress, as well as in the general well being.

Stimulation of glucose uptake byMusasp. (cv. elakki bale) flower and pseudostem extracts in Ehrlich ascites tumor cells

Glucose uptake study plays a major role in diabetes research. Impaired glucose uptake has been implicated in the development of hyperglycemia during diabetes. Banana plant is known for its anti-diabetic properties and our earlier report revealed that banana flower and pseudostem of Musa sp. cv. elakki bale is beneficial during diabetes in rat models. The present study was designed to evaluate the potential effect of banana flower and pseudostem extracts on glucose uptake in Ehrlich ascites tumor (EAT) cells using 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG), a fluorescent analogue of 2-deoxyglucose. Methanol and aqueous extracts of banana flower and pseudostem were more potent in promoting glucose uptake in EAT cells, in comparison to acetone and ethanol extracts. At 20 µg dosage, highest net glucose uptake was observed in aqueous extracts of banana flower (18.17 ± 0.43 nmol L⁻¹) and pseudostem (19.69 ± 0.41 nmol L⁻¹). Total polyphenol content was higher in methanol (9.031 ± 0.036 g kg⁻¹) and aqueous (6.862 ± 0.024 g kg⁻¹) extracts of banana flower compared to pseudostem, which were 0.442 ± 0.006 and 0.811 ± 0.011 g kg⁻¹, respectively. Banana flower and pseudostem extracts are able to promote glucose uptake into the cells, presumably through glucose transporters 1 and 3, which could be beneficial in diabetes. Glucose uptake is likely promoted by phenolic acids besides other bioactives. It can be hypothesized that consumption of nutraceutical-rich extract of banana flower and pseudostem could replace some amount of insulin being taken for diabetes.

In vivo peritoneal antiangiogenesis and in vitro antiproliferative properties of some bischalcone derivatives

In this study, three, bisarylidene cyclopentanones (curcumin analogs) 3a–c are synthesized by Claisen Schmidt condensation reaction. Antiangiogenic effects of the compounds were studied in Ehrlich ascites tumor (EAT) cells transplanted mouse in vivo. Antiangiogenic effect of 3a–c showed reduction in ascites volume, cell number, and induced apoptotic bodies in EAT cells in tested animals. The antiproliferative effects of the 3a–c were determined at different concentrations by MTT assay on HepG2 and HeLa cells. The compounds showed significant antiproliferative activity at 40 μM concentration. Compound 3a (fluoro derivative) showed highest antiproliferative effect with the IC50 value 39 and 48 μm for HeLa and HepG2, respectively. Growth inhibition of the HeLa cells and antiangiogenesis was mediated by promoting apoptosis which was confirmed by DNA fragmentation study.

SFE from Bidens pilosa Linné to obtain extracts rich in cytotoxic polyacetylenes with antitumor activity

Bidens pilosa L. is a plant considered medicinal by some South American cultures. It contains polyacetylenes which may be the constituents responsible for its antitumor activity. Extracts obtained by hydroethanol maceration (HCE) and supercritical fluid extraction (SFE) were monitored for antitumor activity and the presence of polyacetylenes in the constitution. Both extracts killed concentration-dependently the MCF-7 cells in culture, although the SFE extract presented superior cytotoxic activity. The SFE presented IC50=437 (428–446)μg/mL in 24h of incubation, decreasing to IC50=291 (282–299)μg/mL at 48h. The HCE started causing DNA cleavage at 160μg/mL while the SFE extract started at 40μg/mL, a concentration enough to initiate the in vitro cleavage. The presence of polyacetylenes as the major compounds in SFE was confirmed by TL chromatography combined with UV–vis analyses. Ehrlich ascites carcinoma-bearing mice were used for the antitumor study. Animals were divided in five groups: normal, negative control, positive control (Doxorubicin 0.06mg/kg), test group HCE and test group SFE (100mg/kg b.w. per day). After 9 days of treatment, 50% of randomly chosen animals from each group were sacrificed for the study. The parameters evaluated were: body weight, abdominal circumference, volume of ascitic fluid and tumor cells, viable and nonviable tumor cell count, determination of mean survival time and increased life-span. Both extracts presented antitumor activity, but SFE reduced more the volumes of ascites fluid and the tumor cells (4±1 and 1±0.4mL, respectively), while caused higher mean survival time (17 days) and increased life span (∼31%). The results suggest the importance of the polyacetylenes from B. pilosa as leader molecules to contribute to a new anticancer drug by using the supercritical technology.

Inhibition of ascites tumor growth in vivo by sTie‐2 is potentiated by a combinatorial therapy with sFLT‐1

Inhibition of tumor angiogenesis is a promising approach for cancer therapy and the Tie-2/angiopoietin pathway appears to play an important role. In the present study, we have developed strategies to explore the therapeutic potential of blocking the Tie-2/angiopoietin pathway by sTie-2. Ehrlich ascites tumor (EAT) cells were stably transfected to overexpress a truncated form of sTie-2. Transfectants were characterized for their in vitro growth behavior and transplanted into nude mice. Furthermore, recombinant sTie-2 produced by the baculovirus expression system was used to sequester angiopoietins in the murine ascites carcinoma model. The effect of sTie-2 treatment alone or in combination with sFLT-1 on the weight of the animal, ascites cell number and volume was studied. EAT cells stably transfected with a truncated form of sTie-2 showed no change in cell proliferation in vitro and colony forming in soft agar compared to control cells. However, sTie-2 transfected EAT cells transplanted into nude mice reduced tumor burden and demonstrated a reduction in ascites formation and peritoneal angiogenesis. Recombinant sTie-2 showed angiogenic activity in the tube formation and wound healing assay in vitro. sTie-2 treatment alone or in combination with sFLT-1 in an ascites tumor mouse model resulted in reduced peritoneal angiogenesis, with a concomitant decrease in tumor cell number, volume of ascites and the number of invasive tumor cells, as assayed by CD31 staining. The findings of the present study demonstrate an important role for the Tie-2/angiopoietin pathway in the formation of tumor vasculature and suggest that sTie-2 might yield useful anticancer therapy.

Efficacy of LY2784544, a Small Molecule Inhibitor Selective for Mutant JAK2 Kinase, In JAK2 V617F-Induced Hematologic Malignancy Models

AbstractAbstract 4087 Introduction:The JAK2-V617F mutation is the most common molecular abnormality in the BCR/ABL-negative myeloid proliferative neoplasms (MPNs) and is present in approximately 95% of patients with polycythemia vera (PV) and roughly 60% of patients with either essential thrombocythemia (ET) or primary myelofibrosis (PMF). The JAK2 V617F kinase is constitutively active, oncogenic and recapitulates MPN in murine models. Therefore, mutant JAK2 is a potential therapeutic target for MPNs. Here, we describe the efficacy of the clinical candidate LY2784544; a novel small molecule inhibitor selective for JAK2 V617F mutant, in JAK2 V617F-induced in vitro and in vivo models. Methods:Assessment of the ability of LY2784544 to inhibit the signaling of the V617F mutant or wild type JAK2 was evaluated in Ba/F3 cells expressing V617F or wild type JAK2, whereby the cellular level of phospho-STAT5 (pSTAT5) was measured by a high content imaging (Cellomics) assay. The antiproliferative activity of LY2784544 and its ability to induce apoptosis were examined by Celltiter-Glo Cell Viability and Caspase-Glo 3/7 Apoptosis Assays, respectively. The effect of LY2784544 on JAK3 was investigated in IL-2 stimulated NK-92 (nature killer) cells by measuring the inhibition of JAK3-STAT5 signaling with the Cellomics assay. To examine the in vivo target inhibition, a JAK2 V617F-induced ascites tumor model was established by implanting Ba/F3-JAK2 V617F-GFP cells into the intraperitoneal cavity of severe combined immunodeficiency (SCID) mice. After orally administering LY2784544, the inhibition of pSTAT5 was measured in Ba/F3-JAK2 V617F-GFP ascites tumor cells by fluorescence activated cell sorting (FACS) assay. The anti-tumor efficacy of LY2784544 was investigated in a SCID mouse model of MPN developed by intravenously implanting Ba/F3-JAK2 V617F-GFP cells. After oral treatment for 7 or 14 days, twice daily (BID), tumor burden reduction and the effect on erythroid progenitor cells were determined by measuring the GFP positive tumor cells and CD71/Ter119 positive cells in the spleens with FACS assays, respectively. Results:In the in vitro tests using Ba/F3 cells expressing either wild type or V617F mutant JAK2, LY2784544 potently inhibited the JAK2 V617F-STAT5 signaling at a concentration that was 41-fold lower than that required to inhibit IL-3-activated wild type JAK2-STAT5 signaling (IC50=0.055 μ M for JAK2 V617F vs. 2.26 μ M for WT JAK2). Similarly in the proliferation assay, LY2784544 selectively inhibited the JAK2 V617F-driven cell proliferation (IC50= 0.068 μ M). Inhibition of JAK2 V617F signaling correlated well with the induction of apoptosis (EC50= 0.113 μ M) in Ba/F3 cell model. Consistent with the observed apparent selectivity for the V617F mutant and reduced sensitivity against wild type JAK2, a lower potency was observed with LY2784544 in the IL-3-dependent cell proliferation assay (IC50=1.356 μ M) as well as less effective inhibition of IL-2 dependent JAK3-STAT5 signaling in NK-92 cells (IC50=0.94 μ M). In vivo, LY2784544 effectively inhibited STAT5 phosphorylation in Ba/F3-JAK2 V617F-GFP ascites tumor cells with a Threshold Effective Dose 50 (TED50) of 12.7 mg/kg. Consistent with this effective inhibition of JAK2V617F, LY2784544 also significantly reduced Ba/F3-JAK2 V617F-GFP tumor burden in the JAK2 V617F-induced MPN model with a TED50 of 13.7 mg/kg after oral treatment (BID) for 14 days, indicating that the efficacy of LY2784544 is mediated by pharmacological inhibition of JAK2 V16F-STAT5 pathway. Furthermore, LY2784544 selectively reduced Ba/F3-JAK2 V617F-GFP tumor cells with no effect on CD71/Ter119 positive erythroid progenitors in spleens of SCID mice after oral treatment (BID) for seven days. Conclusions:The in vitro data suggest that constitutively active JAK2 V617F is more sensitive to LY2784544 than cytokine-activated wild type JAK2. Consistent with this observation, in MPN disease model testing, LY2784544 selectively reduces JAK2 V617F tumor cell burden with no effect on erythroid progenitor cells. These findings suggest that as a JAK2 V617F selective targeted therapy, LY2784544 has the potential to induce apoptosis in JAK2 V617F malignant clones while potentially minimizing unintended effects on the normal hematopoietic progenitor cells. These results helped to support the advancement of LY2784544 into an ongoing phase I trial in the MPNs (I3X-MC-JHTA, NCT01134120). Disclosures:Ma:Eli Lilly and Company: Employment. Zhao:Eli Lilly and Company: Employment. Walgren:Eli Lilly and Company: Employment. Clayton:Eli Lilly and Company: Employment. Blosser:Eli Lilly and Company: Employment. Burkholder:Eli Lilly and Company: Employment. Smith:Eli Lilly and Company: Employment.

Relationship between Proliferation of Ehrlich Ascitic Tumor Cells and Status of the Chalone System under Conditions of Modified Photoregimen

The effects of permanent darkness on proliferation of Ehrlich ascitic tumor cells and status of the chalone system in the tumor were studied. Chalone-containing preparations from animals exposed to different light conditions exhibited different biological effects on cell proliferation in this tumor. A relationship between biological activity of chalone-containing preparations and sensitivity of tumor cells to these preparations under conditions of modified photoregimen was revealed.

Antitumor effects of nano-bubble hydrogen-dissolved water are enhanced by coexistent platinum colloid and the combined hyperthermia with apoptosis-like cell death

In order to erase reactive oxygen species (ROS) related with the proliferation of tumor cells by reducing activity of hydrogen, we developed functional water containing nano-bubbles (diameters: <900 nm for 71%/population) hydrogen of 1.1-1.5 ppm (the theoretical maximum: 1.6 ppm) with a reducing ability (an oxidation-reduction potential -650 mV, normal water: +100-200 mV) using a microporous-filter hydrogen-jetting device. We showed that hydrogen water erased ROS indispensable for tumor cell growth by ESR/spin trap, the redox indicator CDCFH-DA assay, and was cytotoxic to Ehrlich ascites tumor cells as assessed by WST-8 assay, crystal violet dye stain and scanning electron microscopy, after 24-h or 48-h incubation sequent to warming at 37°C or 42°C. Hydrogen water supplemented with platinum colloid (0.3 ppm Pt in 4% polyvinylpyrrolidone) had more antitumor activity than hydrogen water alone, mineral water alone (15.6%), hydrogen water plus mineral water, or platinum colloid alone as observed by decreased cell numbers, cell shrinkage and pycnosis (nuclear condensation)/karyorrhexis (nuclear fragmentation) indicative of apoptosis, together with cell deformation and disappearance of microvilli on the membrane surface. These antitumor effects were promoted by combination with hyperthermia at 42°C. Thus, the nano-bubble hydrogen water with platinum colloid is potent as an anti-tumor agent.

Regulation of taurine homeostasis by protein kinase CK2 in mouse fibroblasts

Increased expression of the ubiquitous serine/threonine protein kinase CK2 has been associated with increased proliferative capacity and increased resistance towards apoptosis. Taurine is the primary organic osmolyte involved in cell volume control in mammalian cells, and shift in cell volume is a critical step in cell proliferation, differentiation and induction of apoptosis. In the present study, we use mouse NIH3T3 fibroblasts and Ehrlich Lettré ascites tumour cells with different CK2 expression levels. Taurine uptake via the Na(+) dependent transporter TauT and taurine release are increased and reduced, respectively, following pharmacological CK2 inhibition. The effect of CK2 inhibition on TauT involves modulation of transport kinetics, whereas the effect on the taurine release pathway involves reduction in the open-probability of the efflux pathway. Stimulation of PLA(2) activity, exposure to exogenous reactive oxygen species as well as inhibition of protein tyrosine phosphotases (PTP) potentiate the swelling-induced taurine loss. Inhibition of PI3K and PTEN reduces and potentiates swelling-induced taurine release, respectively. Inhibition of CK2 has no effect on PLA(2) activity and ROS production by NADPH oxidase, whereas it lifts the effect of PTEN and PTP inhibition. It is suggested that CK2 regulates the taurine release downstream to known swelling-induced signal transducers including PLA(2), NADPH oxidase and PI3K.

Development and characterization of hyaluronic acid decorated PLGA nanoparticles for delivery of 5-fluorouracil

The present investigation was aimed to develop and explore the prospective of engineered PLGA nanoparticles as vehicles for targeted delivery of 5-fluorouracil (5-FU). Nanoparticles of 5-FU-loaded hyaluronic acid-poly(ethylene glycol)-poly(lactide-co-glycolide) (HA-PEG-PLGA-FU) copolymer were prepared and characterized by FTIR, NMR, transmission electron microscopy, particle size analysis, DSC, and X-ray diffractometer measurement studies. The nanoparticulate formulation was evaluated for in vitro release, hemolytic toxicity, and hematological toxicity. Cytotoxicity studies were performed on Ehrlich ascites tumor (EAT) cell lines using MTT cell proliferation assay. Biodistribution studies of 99mTc labeled formulation were conducted on EAT-bearing mice. The in vivo tumor inhibition study was also performed after i.v. administration of HA-PEG-PLGA-FU nanoparticles. The HA conjugated formulation was found to be less hemolytic but more cytotoxic as compared to free drug. The hematological data suggested that HA-PEG-PLGA-FU formulation was less immunogenic compared to plain drug. The tissue distribution studies displayed that HA-PEG-PLGA-FU were able to deliver a higher concentration of 5-FU in the tumor mass. In addition, the HA-PEG-PLGA-FU nanoparticles reduced tumor volume significantly in comparison with 5-FU. Thus, it was concluded that the conjugation of HA imparts targetability to the formulation, and enhanced permeation and retention effect ruled out its access to the non-tumor tissues, at the same time favored selective entry in tumors, thereby reducing the side-effects both in vitro and in vivo.

Robert Kellogg Crane: A scientist remembers

Robert Kellogg Crane: A scientist remembers

Activation of the TASK-2 channel after cell swelling is dependent on tyrosine phosphorylation

The swelling-activated K(+) currents (I(K,vol)) in Ehrlich ascites tumor cells (EATC) has been reported to be through the two-pore domain (K(2p)), TWIK-related acid-sensitive K(+) channel 2 (TASK-2). The regulatory volume decrease (RVD), following hypotonic exposure in EATC, is rate limited by I(K,vol) indicating that inhibition of RVD reflects inhibition of TASK-2. We find that in EATC the tyrosine kinase inhibitor genistein inhibits RVD by 90%, and that the tyrosine phosphatase inhibitor monoperoxo(picolinato)-oxo-vanadate(V) [mpV(pic)] shifted the volume set point for inactivation of the channel to a lower cell volume. Swelling-activated K(+) efflux was impaired by genistein and the Src kinase family inhibitor 4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) and enhanced by the tyrosine phosphatase inhibitor mpV(pic). With the use of the TASK-2 inhibitor clofilium, it is demonstrated that mpV(pic) increased the volume-sensitive part of the K(+) efflux 1.3 times. To exclude K(+) efflux via a KCl cotransporter, cellular Cl(-) was substituted with NO(3)(-). Also under these conditions K(+) efflux was completely blocked by genistein. Thus tyrosine kinases seem to be involved in the activation of the volume-sensitive K(+) channel, whereas tyrosine phosphatases appears to be involved in inactivation of the channel. Overexpressing TASK-2 in human embryonic kidney (HEK)-293 cells increased the RVD rate and reduced the volume set point. TASK-2 has tyrosine sites, and precipitation of TASK-2 together with Western blotting and antibodies against phosphotyrosines revealed a cell swelling-induced, time-dependent tyrosine phosphorylation of the channel. Even though we found an inhibiting effect of PP2 on RVD, neither Src nor the focal adhesion kinase (FAK) seem to be involved. Inhibitors of the epidermal growth factor receptor tyrosine kinases had no effect on RVD, whereas the Janus kinase (JAK) inhibitor cucurbitacin inhibited the RVD by 40%. It is suggested that the cytokine receptor-coupled JAK/STAT pathway is upstream of the swelling-induced phosphorylation and activation of TASK-2 in EATC.

Evaluation of morphine effect on tumour angiogenesis in mouse breast tumour model, EATC

Breast cancer is the leading cause of death among women, and morphine is used to relieve the pain of patients with cancer. The data on the effects of morphine on tumour growth and angiogenesis are contradictory. We determined in mouse breast cancer model whether analgesic doses of morphine would affect tumour angiogenesis, and then the correlation between microvessel density (MVD), Doppler sonography (DS) and 99mTc-Tetrofosmin (TF) uptake. Ehrlich ascites tumour cell xenografts, Pgp-negative tumour were divided into two groups: (a) Morphine sulphate [0.714 mg/kg/day (equivalent to 50 mg per day for a 70 kg human)], (b) no-morphine. For the determination of angiogenesis in mice tumour tissue, TF scintigraphy, microvessel density and DS were done. MVD was significantly different between groups (49.4±1.8 vs. 41.8±1.9, morphine and no-morphine groups, respectively, P<0.001). A strong correlation was found between late uptakes of mass at scintigraphy and degree of angiogenesis in histopathologic examination (r=0.52, P<0.01). There was statistically significant inverse correlation between degree of angiogenesis in histopathologic examination and washout ratio of TF (r=0.40, P<0.05). The higher values for angiogenesis are related to higher TF reuptake. There was no statistically significant correlation between DS and TF. A strong correlation was found between MVD and grade of DS (r=0.51, P<0.01). Our preclinical mice study indicates that morphine at clinically relevant doses stimulates angiogenesis, and angiogenesis triggered of morphine is demonstrated with MVD and DS, but not TF. However, uptake and washout of TF are compared with immunohistochemically assessed morphine-stimulated angiogenesis in tumour tissue.