Increase In Number Of Viable Cells Research Articles

Involvement of the excretory/secretory and surface-associated antigens of Dirofilaria immitis adult worms in the angiogenic response in an in-vitro endothelial cell model

Angiogenesis is a process by which new vessels are formed from pre-existing ones when the physiological conditions of the vascular endothelium are altered. Heartworm disease, caused by Dirofilaria immitis, causes changes in the vascular endothelium of the pulmonary arteries due to obstruction, friction, and hypoxia. The aim of this study was to analyze whether the excretory/secretory and surface-associated antigens of adult worms interact and modulates the angiogenic mechanism, viable cell number and cell migration, as well as the formation of pseudo-capillaries. Cultures of human vascular endothelial cells (HUVECs) stimulated with excretory/secretory antigens (DiES), surface-associated antigens (Cut) from D. immitis adult worms, VEFG-A (Vascular Endothelial Growth Factor A), as well as DiES+VEFG-A and Cut+VEFG-A were used. The production of VEFG-A and other proangiogenic [soluble VEFGR-2 (sVEFGR-2), membrane Endoglin (mEndoglin)] and antiangiogenic [VEFGR-1/soluble Flt (sFlt), soluble Endoglin (sEndoglin)] molecules was assessed using commercial ELISA kits. Cell viability was analyzed by live cell count and cytotoxicity assays by a commercial kit. In addition, viable cell number by MTT-based assay, cell migration by wound-healing assay carrying out scratched wounds, and the capacity of pseudo-capillary formation to analyze cell connections and cell groups in Matrigel cell cultures, were evaluated. In all cases, non‑stimulated cultures were used as controls. DiES+VEFG-A and Cut+VEFG-A significantly increased the production of VEFG-A and sVEFGR-2, and only Cut+VEFG-A significantly increased the production of VEFGR-1/sFlt compared to other groups and non-stimulated cultures. Moreover, only DiES+VEFG-A produced a significant increase in viable cell number and significant decrease cell migration, as well as in the organization and number of cell connections. Excretory/secretory and surface-associated antigens of adult D. immitis activated the angiogenic mechanism by mainly stimulating the synthesis of proangiogenic factors, and only excretory/secretory antigens increased viable cell number, activated cell migration and the formation of pseudo-capillaries. These processes could lead to vascular endothelial remodeling of the infected host and favor the long-term survival of the parasite.

Abstract 4683: Developing human Ewing sarcoma in vitro models to prioritise new treatments

Abstract Models that more accurately reflect Ewing sarcoma (ES) will enable the prioritization of novel targeted agents from bench to clinic. To date, development of an ES mouse model have been unsuccessful1 and engraftment of human ES in mice is variable2. Coupled with a moral obligation to minimize the use of mammals, we have established and characterized models using patient-derived ES cultures in vitro and in larval zebrafish. The transcriptomes of ES cell lines and patient-derived ES cultures3 were analyzed and compared using total RNAseq followed by DESeq2 and Gene Set Enrichment analysis. Cells in 2D, as 3D spheroids, in combination with cells of the tumor microenvironment and in larval zebrafish (Danio rerio) were characterized. Six cell lines clustered independently from patient-derived ES cultures (n=20), reflecting differential expression of 15,709 RNAs (p<0.01). Interrogation of the differentially expressed genes using Reactome, KEGG and Gene Ontology identified up regulation of genes associated with mitotic spindle and G2/M G1/S DNA damage spindle checkpoints, homologous recombination and the cell cycle in the cell lines compared to patient-derived cultures (p<0.01). These data are consistent with the quicker doubling time (DT) and increased sensitivity of ES cell lines to chemotherapy (actinomycin D, doxorubicin, vincristine, etoposide; DT range=19-33h, EC50 range=4-34nM) compared to patient-derived cultures (DT range=55-197h, EC50>10μM, p<0.05). All patient-derived ES cultures formed spheroids of similar diameter (range 105-258µm) at 96h (p>0.05), although there was heterogeneity in spheroid weight (range 205-3639ng, p<0.05) and migration index (MI; range 30-170, p<0.05). Treatment with actinomycin D (1-10 µM) significantly increased spheroid diameter (p<0.05), whereas 4-hyperoxyifosfamide (active metabolite of ifosfamide, 7-30µM) decreased spheroid diameter (p<0.05). Doxorubicin did not significantly change the diameter of spheroids. When propagated in combination with mesenchymal stem cells there was a 2-fold increase in viable cell number (p<0.0001) compared to ES cultures alone. Patient-derived ES cultures in zebrafish were observed at the injection site (perivitelline space). Some cells were detected in circulation 3 days post injection. This is consistent with heterogeneity and the high MI of patient-derived ES cultures. The transcriptome of ES cell lines is significantly different from that of ES patient-derived cultures and is associated with increased doubling times and response to chemotherapies. This highlights the importance of developing preclinical models using patient-derived ES cultures. Patient-derived ES cultures in 2D and 3D models in vitro and in zebrafish may provide a reliable cost-effective preclinical pipeline 1Minas et al. 2017.Oncotarget,8(21):34141-34163.2Nanni et al. 2019.Sci Rep,9(1):12174 3Roundhill et al. 2021.Cell Oncol,44(5):1065-1085. Citation Format: Elizabeth A. Roundhill, Elton J. Vasconcelos, David R. Westhead, Sarah Grissenberger, Martin Distel, Susan A. Burchill. Developing human Ewing sarcoma in vitro models to prioritise new treatments. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4683.

Hypoxic pulmonary endothelial cells release epidermal growth factor leading to vascular smooth muscle cell arginase-2 expression and proliferation.

The hallmark of pulmonary hypertension (PH) is vascular remodeling. We have previously shown that human pulmonary microvascular endothelial cells (hPMVEC) respond to hypoxia with epidermal growth factor (EGF) mediated activation of the receptor tyrosine kinase, EGF receptor (EGFR), resulting in arginase‐2 (Arg2)‐dependent proliferation. We hypothesized that the release of EGF by hPMVEC could result in the proliferation of human pulmonary arterial smooth muscle cells (hPASMC) via activation of EGFR on the hPASMC leading to Arg2 up‐regulation. To test this hypothesis, we used conditioned media (CM) from hPMVEC grown either in normoxia (NCM) or hypoxia (HCM). Human PASMC were incubated in normoxia with either HCM or NCM, and HCM caused significant induction of Arg2 and viable cell numbers. When HCM was generated with either an EGF‐neutralizing antibody or an EGFR blocking antibody the resulting HCM did not induce Arg2 or increase viable cell numbers in hPASMC. Adding an EGFR blocking antibody to HCM, prevented the HCM‐induced increase in Arg2 and viable cell numbers. HCM induced robust phosphorylation of hPASMC EGFR. When hPASMC were transfected with siRNA against EGFR the HCM‐induced increase in viable cell numbers was prevented. When hPASMC were treated with the arginase antagonist nor‐NOHA, the HCM‐induced increase in viable cell numbers was prevented. These data suggest that hypoxic hPMVEC releases EGF, which activates hPASMC EGFR leading to Arg2 protein expression and an increase in viable cell numbers. We speculate that EGF neutralizing antibodies or EGFR blocking antibodies represent potential therapeutics to prevent and/or attenuate vascular remodeling in PH associated with hypoxia.

E-cigarette synthetic cooling agent WS-23 and nicotine aerosols differentially modulate airway epithelial cell responses

Electronic cigarette (e-cig) aerosol exposures are strongly associated with pulmonary dysfunctions, and the airway epithelial cells (AECs) of respiratory passages play a pivotal role in understanding this association. However, not much is known about the effect of synthetic cooling agents such as WS-23 on AECs. WS-23 is a synthetic menthol-like cooling agent widely used to enhance the appeal of e-cigs and to suppress the harshness and bitterness of other e-cig constituents. Using primary human AECs, we compared the effects of aerosolized WS-23 with propylene glycol/vegetable glycerin (PG/VG) vehicle control and nicotine aerosol exposures. AECs treated with 3 % WS-23 aerosols showed a significant increase in viable cell numbers compared to PG/VG-vehicle aerosol exposed cells and cell growth was comparable following 2.5 % nicotine aerosol exposure. AEC inflammatory factors, IL-6 and ICAM-1 levels were significantly suppressed by WS-23 aerosols compared to PG/VG-controls. When differentiated AECs were challenged with WS-23 aerosols, there was a significant increase in secretory mucin MUC5AC expression with no discernible change in airway inflammatory SCGB1A1 expression. Compared to PG/VG-controls, WS-23 or nicotine aerosols presented with increased MUC5AC expression, but there was no synergistic effect of WS-23 + nicotine combination exposure. Thus, WS-23 and nicotine aerosols modulate the AEC responses and induce goblet cell hyperplasia, which could impact the airway physiology and susceptibility to respiratory diseases.

Levels of Some Polyaromatic Hydrocarbons, Heavy Metals and Capacity of Microbes Isolated from Soil Contaminated with Diesel to Utilize Hexadecane

Soil samples contaminated with diesel were analysed to determine the bacterial and fungal diversity together with the concentration of polyaromatic hydrocarbons (PAHs) and some selected metals which are often targets in environmental checks. Bacteria and fungi were isolated and identified using standard microbiological methods. Identification and quantification of PAH, metal was accomplished using standard methods. A total of fifteen fungal species were isolated and identified. Enterobacter xiangfangensis isolated was identified using molecular tools. Degradation test using Rhizopus stolonifer and Enterobacter xiangfangensis separately was done. The fungal growth pattern revealed that there was an increase in viable cell number from 0.05 to 0.25 (80%) at 84ppm of nickel nitrate (highest). The highest bacterial count was 18.0× 10 6 cfu/ml. The results showed that six of the USEPA target PAH assayed were detected. The concentrations of the priority pollutants in the soil samples varied from 2.08 mg/kg to 1.28 mg/kg for site 1 and 1.99 mg/kg to 1.03 mg/kg in site 2. Cd was found below detection limit. These results are valuable in addition to other reports on decontamination of sites co-contaminated. Future efforts will be aimed at determining the extent that microorganisms can utilise both heavy metals and hydrocarbons.

Hypoxic Pulmonary Endothelial Cells Release Epidermal Growth Factor which Results in Vascular Smooth Muscle Cell Arginase 2 Expression and Proliferation

The hallmark of pulmonary hypertension (PH) is vascular remodeling leading to alterations in hemodynamics and vasoreactivity. Endothelial cells (EC) are central in the response of a vessel to stimuli associated with PH. EC can proliferate contributing to vascular remodeling, but likely a more important role of EC is the release of factors that induce proliferation in the underlying smooth muscle cells (SMC) leading to intimal thickening. Hypoxia is associated with the development of PH in certain human diseases. We have previously shown that human pulmonary microvascular EC (hPMVEC) respond to hypoxia with proliferation that is dependent on arginase 2 up‐regulation. This arginase 2 up‐regulation is mediated by the release of epidermal growth factor (EGF) and resultant activation of EGF receptors (EGFR) on hPMVEC. We hypothesized that the release of EGF by hPMVEC would also result in proliferation of human pulmonary arterial SMC (hPASMC) via EGFR‐mediated arginase 2 up‐regulation. To test this hypothesis, we used conditioned media (CM) from hPMVEC grown either in normoxia or hypoxia to study the role of EGF released by hPMVEC on the regulation of arginase 2 and proliferation in hPASMC. When hPASMC were grown with CM from hypoxic hPMVEC there was a 9‐fold induction of arginase 2 protein levels and more than a doubling of viable hPASMC numbers compared to hPASMC grown with CM from normoxic hPMVEC. In another experiment, hPVMEC were transfected with a scramble siRNA (scramble) or an siRNA against EGF (siEGF) and then the hPMVEC placed in hypoxia to generate conditioned media. In the hPASMC treated with the CM from siEGF‐treated hypoxic hPMVEC there were lower levels of arginase 2 protein than in the hPASMC grown in CM from hPMVEC grown in normoxia; and substantially less arginase 2 than in hPASMC treated with CM from hypoxic hPMVEC treated with scramble. Furthermore, hPASMC treated with hypoxic CM from siEGF‐treated hPMVEC had substantially lower viable cell numbers than in hPASMC treated with hypoxic CM from scramble‐treated hPMVEC. When hPASMC were treated with hypoxic CM from hPMVEC the addition of an EGF neutralizing antibody to the CM prevented the increase in viable cell numbers seen with hypoxic CM. When hPASMC were transfected with siRNA against EGFR and then treated with hypoxic CM from hPMVEC the hypoxia‐induced increase in arginase 2 protein levels and viable cell numbers were prevented. These data demonstrate that hPMVEC release EGF, which can activate EGFR on hPASMC to induce arginase 2 protein expression and increase viable cell numbers. We speculate that EGF neutralizing antibodies or EGFR blocking antibodies are potential therapeutics to prevent and/or attenuate the vascular remodeling seen in PH associated with hypoxia.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 112: Tumor-associated macrophages promote enzalutamide resistance in microscale castrate-resistant prostate cancer models

Abstract Background: Despite recent advances in hormonal therapy for men with advanced prostate cancer, the development of therapeutic resistance continues to limit the efficacy of hormone-directed agents. This includes resistance to second-generation anti-androgens, such as enzalutamide, that can improve both morbidity and overall survival in men with castrate-resistant prostate cancer (CRPC). Tumor-associated macrophages (TAMs) are myeloid immune cells within the tumor microenvironment (TME), which have been shown to promote resistance to hormone therapies in prostate cancer. While they are known to have a role in the development of castrate resistance, their role in enzalutamide resistance is not yet understood. Methods: We have developed a novel microscale cell culture platform that enables efficient multi-culture of >5 primary cell populations along with multiplexed analysis of cell viability, migration, mRNA and protein expression, and secretion of secretory factors. Utilizing this platform, we cultured androgen-sensitive prostate cancer cells (LNCaPs) with primary monocyte-derived macrophages (MDMs) as well as additional primary stromal and immune cell populations (T cells, fibroblasts). Cultures were treated with enzalutamide therapy and analyzed for cell viability, mRNA and protein expression, and cytokine secretion. Results: After enzalutamide treatment, LNCaPs in co-culture with primary MDMs had an increase in viable cell number compared to LNCaPs in monoculture. CSF-1 expression was elevated in LNCaPs cultured with MDMs, and this expression was increased even further with enzalutamide treatment. In MDMs cultured with LNCaPs, there was a significant increase in expression of the CSF-1-responsive genes IL-10 and CCL18. This effect was observed consistently in MDMs from multiple patients. Conclusions: Within microscale tumor models, we have demonstrated that primary, patient-derived MDMs promote resistance to enzalutamide therapy in androgen-sensitive prostate cancer cells. An increase in CSF-1 expression by LNCaPs in our tumor models was associated with an increase in CSF-1 responsive genes within the MDMs, including IL-10 and CCL18. Elevated expression of both of these cytokines has been associated with more aggressive prostate cancers in patients. IL-10 and CCL18 have also been shown to protect against apoptosis in vitro as well as promote immune suppression in vivo. Our findings suggest that CSF-1 secretion by tumor cells may promote hormone resistance in prostate cancer through TAM-mediated secretion of IL-10 and CCL18. These pathways represent potentially high-value targets to limit or prevent the development of castrate resistance in men with advanced prostate cancer. Further studies targeting the secretion of CSF-1, IL-10, and CCL18 in combination with enzalutamide treatment are ongoing. Citation Format: David Kosoff, Jiaquan Yu, Vikram Suresh, David J. Beebe, Joshua M. Lang. Tumor-associated macrophages promote enzalutamide resistance in microscale castrate-resistant prostate cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 112.

Improving phloroglucinol tolerance and production in Escherichia coli by GroESL overexpression

BackgroundPhloroglucinol is an important chemical which has been successfully produced by engineered Escherichia coli. However, the toxicity of phloroglucinol can enormously inhibit E. coli cell growth and viability, and the productivity is still too low and not economically feasible for industrial applications. Therefore, strain tolerance to toxic metabolites remains a key issue during the production of chemicals using biological processes.ResultsIn the present work, we examined the impact of the native GroESL chaperone system with different overexpression levels on phloroglucinol tolerance and production in E. coli. The groESL gene was cloned into an expression vector, of which expression level was regulated by three different promoters (natural, tac and T7 promoter). Strain tolerance was evaluated employing viable cell counts and phloroglucinol production. In comparison with the control strain, all GroESL overexpressing strains showed good characteristics in cell viability and phloroglucinol synthesis. Strain which overexpressed GroESL under tac promoter was found to show the best tolerance in all of those tested, resulting in a 3.19-fold increase in viable cell numbers compared with control strain of agar-plate culture under the condition of 0.7 g/L phloroglucinol, and a 39.5% increase in phloroglucinol production under fed-batch fermentation. This engineered strain finally accumulated phloroglucinol up to 5.3 g/L in the fed-batch cultivation 10 h after induction, and the productivity was 0.53 g/L/h. To date, the highest phloroglucinol production was achieved in this work compared with the previous reports, which is promising to make the bioprocess feasible from the economical point.ConclusionsThe data show that appropriate expression level of GroESL plays a critical role in improving phloroglucinol tolerance and production in E. coli, and maybe involve in controlling some aspects of the stress response system through upregulation of GroESL. GroESL overexpression is therefore a feasible and efficient approach for improvement of E. coli tolerance.

Dual action of NSC606985 on cell growth and apoptosis mediated through PKCδ in prostatic cancer cells.

Chemotherapy is a vital therapeutic strategy for castration-resistant prostate cancer (CRPC). We have previously shown that NSC606985 (NSC), a camptothecin (CPT) analog, induced cell apoptosis via interacting with topoisomerase I (Topo I) in prostate cancer cells. In the present study, the effect and mechanism of CPT analogs in LAPC4 cells were investigated. LAPC-4 cells were treated with NSC, CPT, and topotecan. Cell proliferation, apoptosis, and protein kinase Cδ (PKCδ) subcellular activation were measured at different doses and time-points, with or without PKCδ inhibition or knockdown of PKCδ expression. NSC at doses ranging from 10 to 100 nM induced a dose-dependent increase in viable cell number and DNA biosynthesis with mild cell apoptosis, whereas, at doses ranging from 500 nM to 5 mM, NSC produced a dose-dependent decrease in cell proliferation and DNA biosynthesis with a significant induction of cell apoptosis. Both NSC-induced cell proliferation and apoptosis were blocked by knockdown of PKCδ with a specific RNAi, or by the co-administration of rottlerin, a PKCδ inhibitor. Moreover, NSC produced a dose-dependent subcellular activation of PKCδ. The dose-dependent dual action of NSC is mediated at least in part through the differential subcellular activation of PKCδ in LAPC4 cells. The demonstration of a differential cell response to camptothecin analogs would facilitate the identification of biomarker(s) to CPT sensitivity and promote the personalization of CPT chemotherapy in CRPC.

Boosting the growth of the probiotic strain Lactobacillus paracasei ssp. paracasei F19.

Single so-called booster substances were added to the fermentation medium of the probiotic strain Lactobacillus (L.) paracasei ssp. paracasei F19 to enhance its growth. A wide screening was carried out in microtiter plates and a statistical analysis of the growth parameters was performed. CFU counts were used to correlate the increase in OD590nm with the increase in viable cell number. Sodium ascorbate, sodium pyruvate, manganese sulfate and cysteine had a remarkable boosting effect on the growth of L. paracasei F19. Three of the boosters increased the growth rate of the strain and led to a higher cell density and biomass yield in laboratory conditions. Cysteine significantly shortened the lag phase, therefore reducing the fermentation times. The boosters were tested on four additional Lactobacillus species and their growth boosting activity was retained. To investigate whether the growth boosters could improve the tolerance of L. paracasei F19 to the adverse condition in the GI tract, additional tests were performed. Sodium ascorbate and sodium pyruvate exerted a certain antioxidant effect, as they improved the tolerance of L. paracasei F19 to H2O2. Sodium ascorbate enhanced the growth of the strain in low pH.

Hypoxia-induced proliferation of HeLa cells depends on epidermal growth factor receptor-mediated arginase II induction.

Solid tumors can often be hypoxic in regions, and cancer cells can respond to hypoxia with an increase in proliferation and a decrease in apoptosis, leading to a net increase in viable cell numbers. We have recently found that in an osteosarcoma cell line, hypoxia‐induced proliferation depends on arginase II induction. Epidermal growth factor receptor (EGFR) has been shown to mediate the hypoxia‐induced cellular proliferation in some cancer cell lines. We hypothesized that hypoxia‐induced proliferation of HeLa cells would depend on arginase II induction and that this induction of arginase II would occur through EGFR activation. Exposure of HeLa cells to hypoxia resulted in an upregulation of arginase II mRNA and protein levels, with no effect on arginase I expression. Hypoxia also resulted in significantly greater viable cell numbers than did normoxia. The hypoxia‐induced increase in viable cell numbers was prevented by either a small molecule inhibitor of arginase or siRNA targeting arginase II. Overexpression of arginase II resulted in an increase in viable cell numbers both in normoxia and hypoxia. Hypoxia caused a substantial induction of both epidermal growth factor (EGF) and EGFR. Preventing hypoxia‐induced EGFR expression using siRNA abolished hypoxia‐induced arginase II expression and the increase in viable cell numbers. Treatment with EGF in normoxia not only induced arginase II expression but also resulted in an increase in viable cell numbers. Blocking EGF interactions with EGFR using either an EGF neutralizing antibody or an EGFR antibody prevented the hypoxia‐induced increase in viable cell numbers. These results demonstrate an EGF/EGFR/arginase II pathway that is necessary for hypoxic proliferation in HeLa cells.

Hypoxic proliferation requires EGFR-mediated ERK activation in human pulmonary microvascular endothelial cells.

We have previously shown that hypoxic proliferation of human pulmonary microvascular endothelial cells (hPMVECs) depends on epidermal growth factor receptor (EGFR) activation. To determine downstream signaling leading to proliferation, we tested the hypothesis that hypoxia-induced proliferation in hPMVECs would require EGFR-mediated activation of extracellular signal-regulated kinase (ERK) leading to arginase II induction. To test this hypothesis, hPMVECs were incubated in either normoxia (21% O2, 5% CO2) or hypoxia (1% O2, 5% CO2) and Western blotting was performed for EGFR, arginase II, phosphorylated-ERK (pERK), and total ERK (ERK). Hypoxia led to greater EGFR, pERK, and arginase II protein levels than did normoxia in hPMVECs. To examine the role of EGFR in these hypoxia-induced changes, hPMVECs were transfected with siRNA against EGFR or a scrambled siRNA and placed in hypoxia. Inhibition of EGFR using siRNA attenuated hypoxia-induced pERK and arginase II expression as well as the hypoxia-induced increase in viable cell numbers. hPMVECs were then treated with vehicle, an EGFR inhibitor (AG1478), or an ERK pathway inhibitor (U0126) and placed in hypoxia. Pharmacologic inhibition of EGFR significantly attenuated the hypoxia-induced increase in pERK level. Both AG1478 and U0126 also significantly attenuated the hypoxia-induced increase in viable hPMVECs numbers. hPMVECs were transfected with an adenoviral vector containing arginase II (AdArg2) and overexpression of arginase II rescued the U0126-mediated decrease in viable cell numbers in hypoxic hPMVECs. Our findings suggest that hypoxic activation of EGFR results in phosphorylation of ERK, which is required for hypoxic induction of arginase II and cellular proliferation.

Impact of carrier material on fermentative activity of encapsulated yoghurt culture in whey based substrate

The main objectives of this paper were to study the influence of the carrier material used for encapsulation and of bead size to fermentative activity and viability of the dairy starter culture ?Lactoferm ABY 6?. Encapsulation of yoghurt culture in beads with diameter of 1mm provides better results than encapsulation in beads with larger diameter. Alginate beads and chitosan coated beads have proved to be a strong barrier for nutrients from substrate, so samples with those beads have lower viable cell count, lower titratable acidity and higher pH value after 5h of fermentation at 42?C, than samples with WPC-alginate beads. Also those beads have significantly (P < 0.05) lower cell leaking, than WPC-alginate beads and lower antioxidant capacity. Encapsulation of yoghurt culture in WPC-alginate carrier with diameter of approximately 1mm provided the best characteristics for fermented product. Samples with these beads have significantly (P < 0.05) higher increase of viable cell number after fermentation, despite of major cell leaking (19.7 %). Moreover, sample with these beads have the highest titratable acidity, the lowest pH value after fermentation (the best fermentative activity) and the best antioxidant characteristics.

Low-concentration vemurafenib induces the proliferation and invasion of human HaCaT keratinocytes through mitogen-activated protein kinase pathway activation.

Cutaneous squamous cell carcinomas and keratoacanthomas commonly occur in patients treated with BRAF inhibitors. We investigated the effect of the BRAF inhibitor vemurafenib on normal immortalized human HaCaT keratinocytes to explore the mechanism of hyperproliferative cutaneous neoplasia associated with the use of BRAF inhibitors. Vemurafenib induced an increase in viable cell number in BRAF wild-type cell lines (SK-MEL-2 and HaCaT) but not in BRAF mutant cell lines (SK-MEL-24 and G361). In HaCaT keratinocytes, a low concentration (2μmol/L) of vemurafenib increased cell proliferation and activated mitogen-activated protein kinase kinase/extracellular signal-regulated kinase in a CRAF-dependent manner. Invasiveness of HaCaT cells in a Matrigel assay significantly increased upon cultivation of cells with 2μmol/L vemurafenib for 24h. Gelatin zymography, reverse transcription polymerase chain reaction and western blot results revealed that 2μmol/L vemurafenib treatment increased matrix metalloproteinase (MMP)-2 and MMP-9 expressions and activities in HaCaT cells. These results offer additional insight into the complex mechanism of paradoxical mitogen-activated protein kinase signaling involved in hyperproliferative cutaneous neoplasias that arise after BRAF inhibition and suggest a possible role for MMP in tumor progression and invasion.

Betaine stabilizes cell volume and protects against apoptosis in human corneal epithelial cells under hyperosmotic stress

Elevated tear osmolarity is one of the key pathological factors in dry eye leading to ocular discomfort associated with damage to the ocular surface and inflammation. The aim of this study was to determine the capacity of the organic osmolyte, betaine, to act as an osmoprotectant against hypertonic stress-induced human corneal epithelial cell shrinkage and apoptosis using in vitro cell culture models. Human corneal limbal epithelial (HCLE) cells exposed to culture medium for 16 h at 300 mOsm (isotonic) or 500 mOsm (hyperosmotic) in the presence or absence of betaine (5 or 10 mM) were evaluated for cell volume changes; cell viability; and apoptosis. Betaine (10 mM) ameliorated hyperosmotically induced reduction of cell volume (from 27% reduction to 11%) and resulted in increased mitochondrial activity (by 17%) and an increase in viable cell numbers (by 12%) compared to controls (exposure to hyperosmotic medium without betaine). Hyperosmotically shocked HCLE cells in the presence of betaine (10 mM) halved the number of damaged cells (apoptotic/necrotic) compared to cells in the absence of betaine. The presence of betaine (at 5 or 10 mM) significantly reduced the activity of caspase-8, -9 and -3/7 and release of TNF-α was also reduced by 34% or 55% after exposure of HCLE to 500 mOsm in the presence of 5 or 10 mM betaine, respectively. Using polyclonal antibody against Betaine/GABA transporter 1 (BGT-1), we detected the presence of BGT-1 in HCLE. We demonstrated that the transport of betaine was facilitated by increased osmolarity. In conclusion, betaine stabilized corneal epithelial cell volume under hyperosmotic stress and limited hyperosmotic stress-induced HCLE apoptosis.

Molecular Mechanisms of Pro-Survival and Differentiating Function of Bone Marrow-Derived Adipocytes On Acute Monoblastic Leukemia Cells.

AbstractAbstract 2582Survival of adult patients with acute monocytic leukemia remains dismal. We proposed the hypothesis that bone marrow (BM) adipocytes abundant in aging marrow microenvironment play a role in chemoresistance of AML cells. We have reported that BM adipocytes promote monocytic differentiation, activation, and survival of monoblastic leukemia cells (ASH, 2012).In this study, we examined the molecular mechanism of BM adipocytes causing survival and differentiation of monoblastic leukemia cells. Monoblastic leukemia cell line U937 and primary samples from acute monoblastic leukemia patients (n=6) were co-cultured with BM-derived marrow stromal cells (MSC) and MSC-differentiated adipocytes. Under limiting serum conditions, MSCs and adipocytes inhibited spontaneous cell death of U937 cells with decrease in subG1 fractions, more prominently in co-cultures with adipocytes than with MSCs (% subG1 fraction; cultured alone 23.9+9.0, co-cultured with MSC 15.7+1.9%, co-cultured with adipocytes 11.9+1.2%, p=0.04). Adipocytes but not MSC moderately reduced spontaneous apoptosis in primary leukemic blasts (n=6, p=0.02).We next focused on adipocytokines, lepin and plasminogen activator inhibitor 1(PAI-1), both implicated in cancer progression, as potential mediators of the protective effects by adipocytes. mRNA levels of leptin, leptin receptor and PAI-1 were up-regulated in MSC-derived adipocytes during adipocytic differentiation (relative expression to GAPDH, leptin: MSC 0, adipocyte 123.3+35.0; leptin receptor: MSC 0.16+0.05, adipocyte 4.47+0.37; PAI-1: MSC 314.9+46.5, adipocyte 3766.1+656.2). High level of secreted leptin was confirmed by ELISA in adipocyte conditioning medium (MSC 23.1+2.9, adipocyte 110.0+4.6 pg/mL). In serum-starved conditions, human recombinant leptin (5ng/mL) stimulated cell growth of monoblastic U937 cells known to express leptin receptor (35.8+7.9% increase of viable cell number at 48 hours, p=0.02) and inhibited apoptosis (%subG1 fraction; control 26.2+3.9, leptin16.4+4.1%, p=0.04).Human recombinant PAI-1 inhibited spontaneous and ara-C-induced apoptosis in U937 cells (11.2+1.1% and 15.1+1.7% decrease of Annexin V posivitity p=0.01). These results suggest that BM adipocytes produced leptin and PAI-1contribute to support myeloid leukemia cells survival within BM microenvironment.We next focused on molecular changes in U937 cells co-cultured with adipocytes. We found up-regulation of monocyte/macrophage differentiation gene PPARg and of PPARg target genes, CD36 and FABP4, by quantitative RT-PCR (relative expression to GAPDH, control vs co-culture with adipocytes: PPARg: 8.8+0.9 v.s.18.8+2.6, CD36; 105.0+11.8v.s.425.7+35.6; FABP4: 0.27+0.03v.s.153.7+12.8). The purity of U937 cells separated from adipocytes was confirmed by lack of CD90 mRNA expression by PCR. PPARγ is known to promote monocytic differentiation and uptake of oxidized cholesterol through the induction of scavenger receptor CD36, a protein marker of mature monocytes/macrophages. The up-regulation of cell surface CD36 expression by co-culture with adipocytes was indeed found in U937 cells and in 4 of the 6 primary AML samples by flow cytometory (increase in CD36 MFI: U937; 230.28, Pt samples; 46.4+20.2). These data suggest that BM adipocytes promote monocytic differentiation of monoblastic leukemia cells.In summary, our findings indicate that secretion of leptin and PAI-1 by BM adipocytes supports leukemic cell survival, while PAI-1 primarily and directly inhibits apoptosis of AML cells. It is conceivable that increased adipocyte content of BM in elderly AML patients may negatively affect the responsiveness of monocytic leukemia cells to chemotherapy. Disclosures:No relevant conflicts of interest to declare.

Analysis of CD20-Mediated Signals in Refractory Diffuse Large B-Cell Lymphoma Cells.

Abstract 2684Loss of CD20 expression isimportant not only because of the loss of a therapeutic target, but because it is often associated with a poor prognosis of the patient with DLBCL at relapse. In newly diagnosed cases of DLBCL, reduced expression of CD20 at diagnosis is also associated with poor prognosis. CD20-mediated signaling pathway(s) and how rituximab affect the phenotype trough CD20 in refractory B-cell lymphoma remains to be studied.We analyzed CD20-mediated signals in refractory DLBCL using a CD20-negative DLBCL cell line, SD07 as a model. SD07 was established from a patient with DLBCL at CD20-negative relapse after receiving immuno-chemotherapy that included rituximab. We transiently transfected expression vector(pIRES2-GFP) carrying CD20 cDNA into SD07 cells. CD20 gene was successfully re-expressed in SD07 cells. By immunoblot, CD20 protein was detectable in SD07 cells for 72 hours after transfection. By flowcytometry (FCM), expression levels of cell surface CD20 protein was even more than those in Daudi B-cell lymphoma cells, which is sensitive to growth inhibition of rituximab. Expression of CD20 gene in SD07 produced increased viable cell number through 6 days’ culture compared with those in transfected with vector alone(SD07=4.9±0.1(d1), 4.8±0.2(d3), 4.5±0.1(d6) and SD07CD20=5.6±0.3(d1), 5.7±0.1(d3), 5.9±0.4(d6), x105cells/ml, P<0.05). Increase of number of cells transfected CD20 gene was accompanied with rapid increase of expression of c-myc mRNA analyzed by RT-PCR. c-myc mRNA was detectable 24hrs and remained to be detected up to 6days after transfection, while those was undetectable in cells tranfected with vector alone. By FCM, increase of c-myc protein was also detected 24 hours after transfection (SD07=5.9±0.2, SD07CD20=8.2±0.4, mean signal intensity(MSI), p<0.01). Incubation with rituximab (20μg/ml), unexpectedly, even stimulated the increase of viable cell number of CD20-tranfected SD07 cells (SD07CD20=5.7±0.1(d1), 5.9±0.3(d3), 6.1±0.2(d6), x105cells/ml). c-myc protein of CD20-transfected SD07 remained to be up-regulated in the presence of rituximab (SD07CD20=7.6±0.4, MSI). Accelerated phosphphorylation of retinoblastoma protein was observed in CD20-transfected SD07 cells. By immunoblot, expression levels of either of p-Src(Tyr416), p-Akt(Ser473), Bcl2 or BclxL protein was not affected by the transfection of CD20 into SD07 cells for 6 days’ culture in the presence or absence of rituximab. Those results suggest that growth-promoting effect are involved in CD20-mediated signals in some types of B-cell lymphoma.We hypothesized that oncogenic activation of PI3K-AKT pathway might be involved in the lack of rituximab-induced anti-lymphoma effects in CD20-tranfected SD07 cells in culture, because SD07 shows a homozygous deletion of PTEN and constitutive Akt Ser473 phosphorylation. Rituximab showed significant growth inhibition on CD20-transfected SD07 cells in combination with suboptimal concentration of various chemotherapeutic drugs only in the presence of LY294002 (LY, 10μM), a selective PI3K inhibitor, while rituximab without LY rather stimulated cell growth of those cells(medium=85±0.1%, cisplatin(1μg/ml)=71±0.3%, doxorubicin(100ng/ml)=78±0.2%, bendamustine(10μM)=71±0.1%, etoposide(2μM)= 61±0.3%, Ara-C(10μM)=70±0.5%, % of cell number of CD20-transfected SD07 incubated without LY at day 3, P<0.01) Moreover, FCM analysis of cells stained with PI showed that rituximab significantly increased of subG1 DNA content portion in CD20-transfected SD07 cells in the presence of both LY and chemotherapeutic drugs (medium=5.1±0.8%, cisplatin=6.7±0.2%, doxorubicin =7.6±0.1%, bendamustine=9.0±0.2%, etoposide=9.0±0.2%, Ara-C=11.3±0.2%). Those suggest that deregulated PI3K-AKT pathway may contribute, at least in part, to defects of growth-inhibitory/pro-apoptotic effect of rituximab on SD07 cells.In summary, although a limited in a DLBCL cell line, we showed expression of CD20 is involved in cell growth and cell cycle progression accompanied with rapid induction of c-myc in refractory DLBCL cells. In chemo-refractory lymphoma cells, accumulating genetic alteration(s) of signal transduction pathways downstream of CD20 might impair rituximab-induced anti-lymphoma effects. CD20-mediated signaling pathways are possible therapeutic target(s) to overcome refractory B- cell lymphoma. Disclosures:No relevant conflicts of interest to declare.

Abstract 3256: Sphingolipid regulation by sphingosine kinase anchoring protein (SKAP) and its implication in cancer

Abstract Background: Sphingolipids are important in cancer cell signalling. Sphingosine 1 phosphate (S1P) promotes cell survival and resistance to apoptosis, while S1P precursors ceramide (CER) and sphingosine (SPH), mediate antiproliferative and apoptotic responses. S1P is generated from SPH by sphingosine kinase (SK) enzymes (SK1 and SK2), with SK activity and localisation regulated by other proteins, including PKC, PKA and a SK anchoring protein (SKAP) that has been reported to negatively regulate SK1 activity in fibroblasts. S1P localisation is thought to play an important role in its function. Based on our preliminary observation in primary AML cells that SKAP expression resulted in an increase in S1P, we have investigated the effect of SKAP transfection on S1P production and localisation. Methods: K562, (and for some confirmatory experiments MCF-7), cells were transfected with the SKAP gene using standard techniques. SKAP is normally silenced in both cell lines. Transfection was confirmed by RNA expression. Intracellular and extracellular S1P and SPH, and intracellular SK activity (based on the production of C17 S1P from C17 SPH, an unnatural SPH that is a SK substrate) in intact cells were measured by LC-MS/MS. Phorbol 12-myristate 13-acetate (PMA) was used to induce membrane associated SK function, and MK-571 and fumitremorgen C (FTC) were used to block S1P efflux through ABCC1 and ABCG2 efflux pumps, respectively. Chemosensitivity to doxorubicin and imatinib in transfected cells was also studied. Results: K562 cells transfected with the SKAP gene showed a 2.5 fold increase in intracellular and extracellular levels of basal S1P compared to vector alone control. (In MCF-7 cells SKAP transfection resulted in an almost 10-fold increase in S1P). Further studies in K562 cells confirmed a significant increase in intracellular SK activity in SKAP transfected compared to vector alone cells, based on C17 S1P production (8.8 ± 2.6 vs 1.4 ± 0.4 ng/106 cells respectively after 24 hrs, p&amp;lt; 0.05). This increase was also observed, though to a lesser extent, in extracellular C17 S1P (678 ± 50 in SKAP vs 462 ± 47 pg/ml in vector alone, p&amp;lt; 0.05). In a proliferation assay this increase in SK activity was associated with a 25% increase in viable cell number (p&amp;lt;0.01 after 3 days). SK activity could be induced further in SKAP cells by the PKC activator PMA, although to a lesser extent than in vector alone cells, while the addition of MK-571 and FTC resulted in a marked increase in intracellular S1P in SKAP and vector alone cells, with a decrease in extracellular S1P levels. These experiments confirm the membrane localisation of SK1 in SKAP transfected cells. SKAP transfection did not affect sensitivity to imatinib or doxorubicin compared to vector alone. Conclusion: These data suggest that SKAP may act as a positive regulator of SK1 activity in cancer cells, an observation that has implications in carcinogenesis and chemosensitivity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3256. doi:1538-7445.AM2012-3256

Differential Effect of Nimodipine in Attenuating Iron-Induced Toxicity in Brain- and Blood–Brain Barrier-Associated Cell Types

Metal homeostasis is increasingly being evaluated as a therapeutic target in stroke and neurodegenerative diseases. Metal dysregulation has been shown to lead to protein aggregation, plaque formation and neuronal death. In 2007, we first reported that voltage-gated calcium channels act as a facile conduit for the entry of free ferrous (Fe(2+)) ions into neurons. Herein, we evaluate differential iron toxicity to central nervous system cells and assess the ability of the typical L-type voltage-gated calcium channel blocker nimodipine to attenuate iron-induced toxicity. The data demonstrate that iron sulfate induces a dose-dependent decrease in cell viability in rat brain endothelial cells (RBE4; LC(50) = 150 μM), neuronal cells (Neuro-2α neuroblastoma; LC(50) = 400 μM), and in astrocytes (DI TNC1; LC(50) = 1.1 mM). Pre-treatment with nimodipine prior to iron sulfate exposure provided a significant (P < 0.05) increase in viable cell numbers for RBE4 (2.5-fold), Neuro2-α (~2-fold), and nearly abolished toxicity in primary neurons. Astrocytes were highly resistant to iron toxicity compared to the other cell types tested and nimodipine had no (P > 0.05) protective effect in these cells. The data demonstrate variable susceptibility to iron overload conditions in different cell types of the brain and suggest that typical L-type voltage-gated calcium channel blockers (here represented by nimodipine), may serve as protective agents in conditions involving iron overload, particularly in cell types highly susceptible to iron toxicity.

Induction and Resuscitation of the Viable but Nonculturable State in a Cyanobacteria-Lysing Bacterium Isolated from Cyanobacterial Bloom

The viable but nonculturable (VBNC) state has been found to be a growth strategy used by many aquatic pathogens; however, few studies have focused on VBNC state on other aquatic bacterial groups. The purpose of this study was to explore the VBNC state of cyanobacteria-lysing bacteria and the conditions that regulate their VBNC state transformation. Three cyanobacteria-lysing heterotrophic bacterial strains (F1, F2 and F3) were isolated with liquid infection method from a lake that has experienced a cyanobacterial bloom. According to their morphological, physiological and biochemical characteristics and results of 16SrDNA sequence analysis, F1, F2 and F3 were identified as strains of Staphylococcus sp., Stappia sp. and Microbacterium sp., respectively. After being co-cultured with the axenic cyanobacterium, Microcystis aeruginosa 905, for 7days, strains F1, F2 and F3 exhibited an inhibition effect on cyanobacterial growth, which was expressed as a reduction in chlorophyll concentration of 96.0%, 94.9% and 84.8%, respectively. Both autoclaved and filtered bacterial cultures still showed lytic effects on cyanobacterial cells while centrifuged pellets were less efficient than other fractions. This indicated that lytic factors were extracelluar and heat-resistant. The environmental conditions that could induce the VBNC state of strain F1 were also studied. Under low temperature (4°C), distilled deionized water (DDW) induced almost 100% of F1 cells to the VBNC state after 6days while different salinities (1%, 3% and 5% of NaCl solution) and lake water required 18days. A solution of the cyanobacterial toxin microcystin-LR (MC-LR) crude extract also induced F1 to the VBNC state, and the effect was stronger than DDW. Even the lowest MC-LR concentration (10μgL(-1)) could induce 69.7% of F1 cells into VBNC state after 24h. On the other hand, addition of Microcystis aeruginosa cells caused resuscitation of VBNC state F1 cells within 1day, expressed as an increase of viable cell number and a decrease of VBNC ratio. Both VBNC state and culturable state F1 cells showed lytic effects on cyanobacteria, with their VBNC ratio varying during co-culturing with cyanobacteria. The findings indicated that VBNC state transformation of cyanobacteria-lysing bacteria could be regulated by cyanobacterial cells or their toxin, and the transformation may play an important role in cyanobacterial termination.