The 4-N-acyl and 4-N-alkyl gemcitabine analogues with silicon-fluoride-acceptor: Application to 18F-Radiolabeling.

The coupling of gemcitabine with functionalized carboxylic acids using peptide coupling conditions afforded 4-N-alkanoyl analogues with a terminal alkyne or azido moiety. Reaction of 4-N-tosylgemcitabine with azidoalkyl amine provided 4-N-alkyl gemcitabine with a terminal azido group. Click reaction with silane building blocks afforded 4-N-alkanoyl or 4-N-alkyl gemcitabine analogues suitable for fluorination. RP-HPLC analysis indicated better chemical stability of 4-N-alkyl gemcitabine analogues versus 4-N-alkanoyl analogues in acidic aqueous conditions. The 4-N-alkanoyl gemcitabine analogues showed potent cytostatic activity against L1210 cell line, but cytotoxicity of the 4-N-alkylgemcitabine analogues was low. However, 4-N-alkanoyl and 4-N-alkyl analogues had comparable antiproliferative activities in the HEK293 cells. The 4-N-alkyl analogue with a terminal azide group was shown to be localized inside HEK293 cells by fluorescence microscopy after labelling with Fluor 488-alkyne. The [18F]4-N-alkyl or alkanoyl silane gemcitabine analogues were successfully synthesized using microscale and conventional silane-labeling radiochemical protocols. Preliminary positron-emission tomography (PET) imaging in mice showed the biodistribution of [18F]4-N-alkyl to have initial concentration in the liver, kidneys and GI tract followed by increasing signal in the bone.

Synergistic Antioxidant and Anti-Inflammatory Effects between Modified Citrus Pectin and Honokiol.

Inflammation is a normal physiological process; however, dysregulation of this process may contribute to inflammatory-based chronic disorders and diseases in animals and humans. Therefore, the antioxidant and anti-inflammatory properties of natural products, often recognized in traditional medicine systems, represent therapeutic modalities to reduce or prevent uncontrolled inflammatory processes which in turn potentially ameliorate or prevent sequelae of inflammatory-based symptoms of chronic diseases. We have investigated the antioxidant and anti-inflammatory effects of honokiol (HNK) and modified citrus pectin (MCP) in vitro and examined whether the MCP : HNK combination has synergistic effects on antioxidant and anti-inflammatory properties. Although both HNK and MCP induced a dose-dependent increase in antioxidant activity, the latter has a consistently higher antioxidant effect. The MCP : HNK (9 : 1) combination induced a synergistic effect on antioxidant activity suggesting that the combination is significantly more efficacious than individual compounds. In mouse monocytes, the lipopolysaccharide- (LPS-) induced tumor necrosis-α (TNF-α) synthesis was significantly inhibited by HNK and the MCP : HNK combination in a dose-dependent manner and synergistic effects were clearly demonstrated with the combination on TNF-α inhibition. This combination effect was also evident on inhibition of nuclear factor-kappa B activity, cyclooxygenase-II activity, and lipid peroxidation in mouse monocytes. Further research into the combination is warranted.

In Vivo Antitumor Effect of Supercritical CO2 Extract of Mango Ginger ( Curcuma amada Roxb) in U-87MG Human Glioblastoma Nude Mice Xenografts.

Glioblastoma multiforme (GBM) is one the most aggressive and lethal human neoplasms with poor prognosis and very limited positive treatment options. The antitumor effect of supercritical CO2 extract of mango ginger ( Curcuma amada Roxb) (CA) with and without irinotecan (IR) was analyzed in U-87MG human glioblastoma multiforme (GBM) cells in vitro and in nude mice xenografts. CA is highly cytotoxic to GBM cells and is synergistic with IR as indicated by the combination index values of <1 in the CompuSyn analysis. CA inhibits tumor growth rate in GBM xenografts, the inhibition rate being higher than in IR treated group. GBM xenograft mice treated with IR + CA combination showed almost complete inhibition of tumor growth rate. Gene expression analysis of xenograft tumors indicated that IR + CA treatment significantly downregulated anti-apoptotic (Bcl-2 and mutant p53), inflammation-associated (COX-2) and cell division-associated (CCNB2) genes and upregulated pro-apoptotic genes (p21 and caspase-3). These results confirmed the therapeutic efficiency of IR + CA combination against GBM and the need for further clinical investigations.

Inhibition of AKT signaling by supercritical CO2 extract of mango ginger (Curcuma amada Roxb.) in human glioblastoma cells.

BACKGROUND: Mango ginger (Curcuma amada Roxb.) is a less-investigated herb for anticancer properties than other related Curcuma species. AKT (a serine/threonine protein kinase B, originally identified as an oncogene in the transforming retrovirus AKT8) plays a central role in the development and promotion of cancer. In this investigation, we have analyzed the effect of supercritical CO2 extract of mango ginger (CA) on the genetic pathways associated with AKT signaling in human glioblastoma cells. METHODS: The inhibitory effect of supercritical CO2 extract of mango ginger (Curcuma amada) on AKT signaling was investigated in U-87MG glioblastoma cells. RESULTS: CA was highly cytotoxic to glioblastoma cell line (IC50=4.92±0.81 µg/mL) compared to mHypoE-N1 normal mouse hypothalamus cell line (IC50=40.57±0.06 µg/mL). CA inhibits AKT (protein Kinase B) and adenosine monophophate -activated protein kinase α (AMPKα) phosphorylation significantly in a dose-dependent manner. The cell migration which is necessary for invasion and metastasis was also inhibited by CA treatment, with about 43% reduction at 20 µg/mL concentration. Analysis of mRNA and protein expression of genes associated with apoptosis, cell proliferation and angiogenesis showed that CA modulates expression of genes associated with apoptosis (Bax, Bcl-2, Bcl-X, BNIP3, caspase-3, mutant p53 and p21), cell proliferation (Ki67) and angiogenesis vascular endothelial growth factor (VEGF). Additionally, heat shock protein 90 (HSP90) and AMPKα genes interacting with the AKT signaling pathway were also downregulated by CA treatment. CONCLUSIONS: These results indicate the molecular targets and mechanisms underlying the anticancer effect of CA in human glioblastoma cells.

Therapeutic Effect of Supercritical CO2 Extracts of Curcuma Species with Cancer Drugs in Rhabdomyosarcoma Cell Lines.

Synergistic effect of supercritical CO2 extracts of Curcuma species with conventional chemotherapeutic drugs was investigated in human alveolar (SJRH30) and embryonal (RD) rhabdomyosarcoma cell lines. The Curcuma amada (mango ginger) (CA) extract showed the highest levels of cytotoxicity with inhibitory concentration IC50 values of 7.133 µg/ml and 7.501 µg/ml for SJRH30 and RD cell lines, respectively, as compared with Curcuma longa (turmeric) and Curcuma xanthorrhiza (Javanese turmeric) extracts. CA showed synergistic cytotoxic effects with vinblastine (VBL) and cyclophosphamide (CP) as indicated by the combination index values of <1 for VBL + CA, CP + CA, and VBL + CP + CA combinations in both embryonal and alveolar rhabdomyosarcomas. When lower doses of CA (0.1-0.2 µg/ml) were combined with cancer drugs like CP and VBL, caspase-3 activity increased significantly compared with individual agents and correlated with the percentage of apoptotic cells. CA in combination with VBL and CP induced a higher percentage of apoptosis than single agents in both cell lines. CA also modulated the expression of genes associated with intrinsic pathway of apoptosis (Bcl-2, Bax, Bak, and p53) and also inhibited the expression of genes associated with inflammation such as COX-2 and NF-κB. Xenograft studies with SJRH30 tumors in nude mice showed that CA treatment inhibited tumor growth rate with and without VBL and increased the survival rate significantly. These results suggest that CA can be evaluated further as an adjuvant with cancer drugs for the treatment of rhabdomyosarcoma patients. Copyright © 2015 John Wiley & Sons, Ltd.

Anticancer potential and mechanism of action of mango ginger (Curcuma amada Roxb.) supercritical CO2 extract in human glioblastoma cells.

Mango ginger (Curcuma amada Roxb.) is among the less-investigated species of Curcuma for anticancer properties. We have investigated the anticancer potential and the mechanism of action of a supercritical CO2 extract of mango ginger (CA) in the U-87MG human glioblastoma cell line. CA demonstrated higher cytotoxicity than temozolomide, etoposide, curcumin, and turmeric force with IC50, IC75, and IC90 values of 4.92 µg/mL, 12.87 µg/mL, and 21.30 µg/mL, respectively. Inhibitory concentration values of CA for normal embryonic mouse hypothalamus cell line (mHypoE-N1) is significantly higher than glioblastoma cell line, indicating the specificity of CA against brain tumor cells. CompuSyn analysis indicates that CA acts synergistically with temozolomide and etoposide for the cytotoxicity with combination index values of <1. CA treatment also induces apoptosis in glioblastoma cells in a dose-dependent manner and downregulates genes associated with apoptosis, cell proliferation, telomerase activity, oncogenesis, and drug resistance in glioblastoma cells.

Improved neuroprotective effects by combining Bacopa monnieri and Rosmarinus officinalis supercritical CO2 extracts.

Ethnobotanical evidence suggests that herbs such as brahmi (Bacopa monnieri) and rosemary (Rosmarinus officinalis) may possess antioxidant and neuroprotective properties. We compared the antioxidant and neuroprotective effects of supercritical extract of Bacopa monnieri and rosemary antioxidant extract obtained from Rosmarinus officinalis as well as their combination to examine the effects on human glial (U-87 MG) and embryonic mouse hypothalamus cells. Bacopa monnieri extract, rosemary antioxidant extract, and their combination (1:1) are not cytotoxic in both glial and embryonic mouse hypothalamus cell lines up to 200 µg/mL concentration. The combination of extracts of Bacopa monnieri + rosemary antioxidant has better antioxidant potential and antilipid peroxidation activity than either agent alone. Although the extract of Bacopa monnieri + rosemary antioxidant showed almost similar inhibition of phospho tau expression as Bacopa monnieri or rosemary antioxidant extract alone, the combination has better inhibitory effect on amyloid precursor protein synthesis and higher brain-derived neurotrophic factor production in hypothalamus cells than single agents. These results suggest that the extract of Bacopa monnieri + rosemary antioxidant is more neuroprotective than Bacopa monnieri or rosemary antioxidant extract.

Synergistic cytotoxicity of red beetroot (Beta vulgaris L.) extract with doxorubicin in human pancreatic, breast and prostate cancer cell lines.

Although a wide variety of cytotoxic plant extracts and phytochemicals are known to act synergistically with anticancer drug doxorubicin (D), their clinical application is hindered by safety concerns of such combination therapy. Our earlier studies showed that red beetroot (Beta vulgaris L.) extract (B), approved by Food and Drug Administration and European Union as red food color E162, reduced multi-organ tumor formations in various animal models when administered in drinking water. This led us to postulate that a long-term daily exposure to low doses of B through diet might be safe and sufficient to produce cancer chemopreventive effect in humans. Further, our recent comparative cytotoxic investigation with B and D in several human cancer cell lines indicated their potential for synergistic activity. Since B is considered safe for human use with no known toxicity, we conducted the present study to evaluate its synergistic antiproliferative activity with D against pancreatic (PaCa), breast (MCF-7) and prostate (PC-3) tumor cells of human origin. Different concentrations of B and D (0.29-290 µg/ml) and in various combinations (B:D ratio = 1:0, 1:1, 5:1, 1:5 and 0:1) were tested for cytotoxic effects against the three cancer cells. The viability of cells was assessed after 72 h incubation with various combinations of B and D using the trypan-blue staining method. The cytotoxic data were analyzed by the combination index method of Chou and Talalay to establish synergy between B and D. The results indicated that an overall positive reduction in drug concentration was achieved by D when combined with B in its cytotoxicity profile in the three human cancer cells tested. The synergistic cytotoxicity was best when the B:D ratio of 1:5 was used in PaCa cells at IC50, IC75 and IC90 dose levels and in MCF-7 cells at IC90 dose level. These results warrant further studies on the potential of red beetroot extract-doxorubicin combination in treating human cancers.

Immunomodulatory effects of the botanical compound LCS101: implications for cancer treatment.

OBJECTIVE: To examine the effects of LSC101, a botanical compound, on adaptive and innate immunity. MATERIALS AND METHODS: LCS101 preparations were tested for batch-to-batch consistency using high-performance liquid chromatography. T-cell activation was quantified in murine spleen cells using 3H-thymidine incorporation, and cytokine production analyzed with enzyme-linked immunosorbent assay. Natural killer cell activity was tested on human blood cells using flow cytometry, and cytotoxicity measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and apoptosis using a FACSCalibur. Effects on interferon-γ production in fluorouracil/doxorubicin-treated mice were tested with enzyme-linked immunosorbent assay. RESULTS: High-performance liquid chromatography analysis demonstrated batch-to-batch consistency. T-cell proliferation was increased, and a dose-dependent activation of natural killer cells and macrophage tumor necrosis factor-α secretion were observed with LCS101 treatment. Interferon-γ levels, reduced following fluorouracil treatment, were corrected in treated animals. No toxicity or compromised treatment outcomes were associated with LCS101 exposure. CONCLUSIONS: LCS101 demonstrated significant effects on a number of immune processes. Further research is needed in order to understand the molecular immunomodulatory pathways affected by this compound, as well as clinical implications for treatment.

Potentiation of etoposide and temozolomide cytotoxicity by curcumin and turmeric force™ in brain tumor cell lines.

We have investigated on the potentiation of etoposide (ETP) and temozolomide (TMZ) cytotoxicity in U-87MG glioblastoma and D283 medulloblastoma cell lines by curcumin (CUR) and turmeric force (TF), a nutraceutical formulation of turmeric, with the objective of assessing the potential for their adjuvant use in brain tumor chemotherapy. While U-87MG cell line was generally resistant to TMZ, IC50 values for CUR and TF were 37.33 and 30.75 µg/ml, respectively. TF is the only agent that demonstrated efficacy at the IC90 level. When CUR or TF was combined with ETP and TMZ, increased chemotherapeutic efficiency in the U-87MG cells was observed. TF is highly cytotoxic to D283 Med cell line compared to curcumin with an IC50 value of 1.55 ug/ml. Although both CUR and TF potentiated ETP and TMZ cytotoxicity, TF is more efficient than CUR in both U-87MG and D283 Med cell lines. Treatment of U-87MG cells with the triple combination of TMZ+ETP+TF induced a high percentage of apoptotic cells. Potential mechanisms that may explain evidence of synergy include down regulation of p10 and p53 mRNAs and increase in BAX/Bcl-2 mRNA ratio. These pre-clinical results suggest that TF may be useful as an adjuvant with ETP and TMZ for brain tumor chemotherapy.

Inhibition of nitric oxide synthesis in mouse macrophage cells by feverfew supercritical extract.

Feverfew is the most commonly used medicinal herb against migraine headache. The antimigraine mechanism of feverfew supercritical extract was investigated in vitro using the mouse macrophage cell line (RAW 264.7). Mouse macrophage cells were treated with lipopolysaccharide in the presence and absence of feverfew extracts. Inhibition of lipopolysaccharide-induced nitric oxide and TNF-α synthesis were quantified by ELISA. The mRNA and protein expression of iNOS and eNOS genes were analysed by RT-PCR and western blot analysis, respectively. The feverfew extract inhibited both nitric oxide (NO) and TNF-α production in a dose-dependent manner with complete inhibition of NO occurring at 5 µg/mL of feverfew extract. Both eNOS and iNOS mRNA levels were unchanged with the feverfew treatment. However, eNOS and iNOS proteins were significantly down-regulated by the feverfew extract. Feverfew inhibition of NO is due to the down-regulation of both eNOS and iNOS enzymes at the translational and/or post-translational level.

Activation of human T-helper/inducer cell, T-cytotoxic cell, B-cell, and natural killer (NK)-cells and induction of natural killer cell activity against K562 chronic myeloid leukemia cells with modified citrus pectin.

BACKGROUND: Modified citrus pectin (MCP) is known for its anti-cancer effects and its ability to be absorbed and circulated in the human body. In this report we tested the ability of MCP to induce the activation of human blood lymphocyte subsets like T, B and NK-cells. METHODS: MCP treated human blood samples were incubated with specific antibody combinations and analyzed in a flow cytometer using a 3-color protocol. To test functionality of the activated NK-cells, isolated normal lymphocytes were treated with increasing concentrations of MCP. Log-phase PKH26-labeled K562 leukemic cells were added to the lymphocytes and incubated for 4 h. The mixture was stained with FITC-labeled active form of caspase 3 antibody and analyzed by a 2-color flow cytometry protocol. The percentage of K562 cells positive for PKH26 and FITC were calculated as the dead cells induced by NK-cells. Monosaccharide analysis of the MCP was performed by high-performance anion-exchange chromatography with pulse amperometric detection (HPAEC-PAD). RESULTS: MCP activated T-cytotoxic cells and B-cell in a dose-dependent manner, and induced significant dose-dependent activation of NK-cells. MCP-activated NK-cells demonstrated functionality in inducing cancer cell death. MCP consisted of oligogalacturonic acids with some containing 4,5-unsaturated non-reducing ends. CONCLUSIONS: MCP has immunostimulatory properties in human blood samples, including the activation of functional NK cells against K562 leukemic cells in culture. Unsaturated oligogalacturonic acids appear to be the immunostimulatory carbohydrates in MCP.

Potentiation of gemcitabine by Turmeric Force in pancreatic cancer cell lines.

Gemcitabine is a first line cancer drug widely used for the treatment of pancreatic cancer. However, its therapeutic efficiency is significantly limited by resistance of pancreatic cancer cells to this and other chemotherapeutic drugs. We have investigated the cytotoxic effect of Turmeric Force (TF), a supercritical and hydroethanolic extract of turmeric, alone and in combination with gemcitabine in two pancreatic carcinoma cell lines (BxPC3 and Panc-1). TF is highly cytotoxic to BxPC3 and Panc-1 cell lines with IC50 values of 1.0 and 1.22 microg/ml, respectively with superior cytotoxicity than curcumin. Gemcitabine IC50 value for both of these cell line is 0.03 microg/ml; however, 30-48% of the pancreatic cancer cells are resistant to gemcitabine even at concentrations >100 microg/ml. In comparison, TF induced cell death in 96% of the cells at 50 microg/ml. The combination of gemcitabine and TF was synergistic with IC90 levels achieved in both pancreatic cancer cell lines at lower concentrations. CalcuSyn analysis of cytotoxicity data showed that the Gemcitabine + Turmeric Force combination has strong synergism with combination index (CI) values of 0.050 and 0.183 in BxPC3 and Panc-1 lines, respectively at IC50 level. This synergistic effect is due to the increased inhibitory effect of the combination on nuclear factor-kappaB activity and signal transducer and activator of transcription factor 3 expression as compared to the single agent.

Immunological response to (1,4)-alpha-D-glucan in the lung and spleen of endotoxin-stimulated juvenile rats.

We investigated the effects of (1,4)-alpha-D-glucan (alpha-DG), a novel immune stimulatory drug from Tinospora cordifolia, on the concentration of pro- and anti-inflammatory cytokines (interleukin [IL]-1beta, IL-6, tumour necrosis factor-alpha [TNF-alpha], gamma-interferon [IFN-gamma] and IL-10) in the lung and spleen of endotoxin-stimulated juvenile rats. Experimental groups (n = 16/group) included controls with an intraperitoneal injection of saline, endotoxaemic rats with a non-lethal dose of 10 mg/kg Escherichia coli endotoxin, and endotoxaemic rats treated with two doses of 10 mg/kg alpha-DG, intraperitoneally, 2 and 4 hr after endotoxin injection. At 24 hr of treatment, rats were euthanized and lungs and spleen were removed for cytokines determination and lung injury. Endotoxaemia increased IL-1beta concentration by fivefold in both organs, while creating a moderate pulmonary hypercellularity (demonstrated by about 11% increase in the alveolar-septal thickening and 11% decrease in the alveolar-interstitial space ratio). In the lung, alpha-DG treatment reduced concentrations of IL-1beta by 30% (p > 0.05), IL-6 by 43% (p < 0.01), IFN-gamma by 46% (p < 0.01) and the anti-inflammatory cytokine, IL-10, by 31% (p > 0.05) compared to endotoxaemia. In the spleen, alpha-DG treatment decreased the ratio of IL-1beta to IL-10 by 55% (p < 0.05), demonstrating an anti-inflammatory trend. These data suggest that alpha-DG differentially modulates cytokine response in the lung and spleen and modifies the pro- and anti-inflammatory balance during an early period of endotoxaemia in juvenile rats.

Isolation and characterization of an anticancer catechol compound from Semecarpus anacardium.

ETHNOPHARMACOLOGICAL RELEVANCE: The fruits and seeds of Semecarpus anacardium are used widely for the treatment of human cancers and other diseases in the Ayurvedic and Sidda systems of medicine in India. AIM OF THE STUDY: The principal aim of this investigation was to isolate and characterize the anticancer compound from the kernel of Semecarpus anacardium nut. MATERIALS AND METHODS: The bioactivity-tailored isolation and detailed chemical characterization were used to identify the active compound. Cytotoxicity, apoptosis, cell cycle arrest as well as synergism between the identified anticancer compound and doxorubicin in human tumor cell lines were analyzed. RESULTS: GC/MS, IR, proton NMR, carbon NMR and collisionally induced dissociation (CID) spectra analysis showed that the isolated active compound is 3-(8'(Z),11'(Z)-pentadecadienyl) catechol (SA-3C). SA-3C is cytotoxic to tumor cell lines with IC(50) values lower than doxorubicin and even multidrug resistant tumor cell lines were equally sensitive to SA-3C. SA-3C induced apoptosis in human leukemia cell lines in a dose-dependent manner and showed synergistic cytotoxicity with doxorubicin. The cell cycle arrest induced by SA-3C at S- and G(2)/M-phases correlated with inhibition of checkpoint kinases. CONCLUSION: SA-3C isolated from the kernel of Semecarpus anacardium can be developed as an important anticancer agent for single agent and/or multiagent cancer therapy.

Physiological effects of a novel immune stimulator drug, (1,4)-α-D-glucan, in rats.

The (1,4)-α-D-glucan (α-D-glucan), derived from medicinal plant, Tinospora cordifolia, activates human lymphocytes with downstream synthesis of the pro- and anti-inflammatory cytokines, in vitro. We investigated physiological and immunological effects of a low and a high dose of α-D-glucan (0.5 and 10 mg/kg), in vivo, testing the hypothesis that intravenous administration of α-D-glucan does not affect haemodynamic, respiratory, haematological, and immune responses in normal rats. Male rats (300-400 g) were anaesthetized, tracheostomized, and catheterized in one femoral artery and vein. The mean arterial blood pressure and heart rate were continuously recorded. The baselines for gas exchange, differential blood cell count, and plasma concentration of TNF-α, IL-1ß, IL-4, IL-6, and IFN-γ were determined. Rats were then randomly assigned to controls (n = 7), a low dose (0.5 mg/kg; n = 10), and a high dose (10 mg/kg; n = 7) of α-D-glucan for a six 6 hr study period. Gas exchange, differential cell count, plasma concentration of TNF-α, IL-1ß, IL-4, IL-6, and IFN-γ, and mean arterial blood pressure values remained within physiological range. Intravenous administration of 10 mg/kg α-D-glucan created tachycardia, associated with hyperventilation, and significant reductions in the blood haemoglobin and haematocrit concentrations. We suggest that these in vivo effects of α-D-glucan should be considered for future clinical and/or experimental trials.

The effects of CO2 on cytokine concentrations in endotoxin-stimulated human whole blood.

OBJECTIVES: Hypercapnia is known to modulate inflammation in lungs. However, the effect of hypocapnia and hypercapnia on blood cytokine production during sepsis is not well understood. We hypothesized that CO2 modulates ex vivo inflammatory cytokine production during endotoxin stimulation. To test this hypothesis, we measured the production of pro- and anti-inflammatory cytokines in endotoxin-stimulated human whole blood cultures under hypercapnic, normocapnic, and hypocapnic conditions. DESIGN: Prospective randomized study. SETTING: Basic research laboratory. SUBJECTS: Ten male and 10 female volunteers. INTERVENTIONS: Venous blood samples, taken from volunteers were cultured at 37 degrees C, under hypocapnic (2% CO2), normocapnic (5% CO2), and hypercapnic (7% CO2) conditions, with and without endotoxin stimulation. After 24 hrs of incubation, each culture's supernatant was analyzed for tumor necrosis factor-alpha, interleukin-1beta, interleukin-6, interleukin-10, and interferon-gamma concentrations by enzyme-linked immunosorbent assay. Data were analyzed using nonparametric repeated measures of analysis of variance followed by Dunn's multiple comparisons test. Analysis of variance with Bonferroni correction was used to compare gender differences in cytokine concentrations. The Pearson test was used to estimate correlation between hydrogen ion and individual cytokine concentrations. MEASUREMENTS AND MAIN RESULTS: Concentrations of the proinflammatory cytokines tumor necrosis factor-alpha, interleukin-1beta and of the anti-inflammatory cytokine interleukin-10 under hypercapnic condition were significantly decreased (p < 0.05, 0.01, and 0.001, respectively) for both genders when compared with either normocapnic or hypocapnic conditions. Concentrations of tumor necrosis factor-alpha and interleukin-1beta were significantly higher in men. In women, concentrations of interleukin-6 were significantly decreased under hypercapnic condition when compared with hypocapnic condition. An inverse relationship was found between hydrogen ion concentration and concentrations of tumor necrosis factor-alpha and interleukin-10. CONCLUSIONS: Our results are consistent with the hypothesis that CO2 can affect the production of pro- and anti-inflammatory cytokines after ex vivo stimulation with endotoxin.

Invasion suppressor cystatin E/M (CST6): high-level cell type-specific expression in normal brain and epigenetic silencing in gliomas.

DNA hypermethylation-mediated gene silencing is a frequent and early contributor to aberrant cell growth and invasion in cancer. Malignant gliomas are the most common primary brain tumors in adults and the second most common tumor in children. Morbidity and mortality are high in glioma patients because tumors are resistant to treatment and are highly invasive into surrounding brain tissue rendering complete surgical resection impossible. Invasiveness is regulated by the interplay between secreted proteases (eg, cathepsins) and their endogenous inhibitors (cystatins). In our previous studies we identified cystatin E/M (CST6) as a frequent target of epigenetic silencing in glioma. Cystatin E/M is a potent inhibitor of cathepsin B, which is frequently overexpressed in glioma. Here, we study the expression of cystatin E/M in normal brain and show that it is highly and moderately expressed in oligodendrocytes and astrocytes, respectively, but not in neurons. Consistent with this, the CST6 promoter is hypomethylated in all normal samples using methylation-specific PCR, bisulfite genomic sequencing, and pyrosequencing. In contrast, 78% of 28 primary brain tumors demonstrated reduced/absent cystatin E/M expression using a tissue microarray and this reduced expression correlated with CST6 promoter hypermethylation. Interestingly, CST6 was expressed in neural stem cells (NSC) and markedly induced upon differentiation, whereas a glioma tumor initiating cell (TIC) line was completely blocked for CST6 expression by promoter methylation. Analysis of primary pediatric brain tumor-derived lines also showed CST6 downregulation and methylation in nearly 100% of 12 cases. Finally, ectopic expression of cystatin E/M in glioma lines reduced cell motility and invasion. These results demonstrate that epigenetic silencing of CST6 is frequent in adult and pediatric brain tumors and occurs in TICs, which are thought to give rise to the tumor. CST6 methylation may therefore represent a novel prognostic marker and therapeutic target specifically altered in TICs.

Anticancer effects of Annona glabra plant extracts in human leukemia cell lines.

Annona glabra (pond apple), a tropical tree growing wild in the Americas and Asia, is used in traditional medicine against several human ailments, including cancer. To validate the ethnopharmacological claims against cancer, the anticancer effects of alcoholic extracts prepared from pond apple leaves, pulp and seed, were investigated in human leukemia cell lines. The alcoholic extracts were not cytotoxic to normal human lymphocytes. However, extracts were highly cytotoxic to drug sensitive (CEM) and multidrug-resistant leukemia (CEM/VLB) cell lines. The seed extract was more potent than leaf and pulp extracts, and the cytotoxicity values were significantly lower than that for adriamycin. The seed extract caused a concentration-dependent increase in the percentage of the sub G0/G1, as well as G0/G1 cell population, contributing to the cytotoxicity. The sub G0/G1 population increased from 2.2 to 7.0% in CEM and from 1.0 to 10.7% in CEM/VLB cell lines, when the cells were treated with 0-10 Bg/ml seed extract. Treatment of CEM and CEM/VLB cells with seed extract induced apoptosis and necrosis in both sensitive and resistant leukemia cells in a concentration-dependent manner. The seed extract at 2 and 5 Bg/ml enhanced cellular daunorubicin accumulation, indicating the competitive P-glycoprotein binding ability and drug-resistance reversal effect. Treatment of CEM and CEM/VLB cells with seed extracts also up-regulated the expression of cyclin kinase inhibitor (WAF1/p21) contributing to the arrest of cells at the G0/G1 phase of the cell cycle. These results support the traditional use of A. glabra and the alcoholic seed extract is a potent source of anticancer compounds that could be utilized pharmaceutically.

Investigation of cytokine expression in human leukocyte cultures with two immune-modulatory homeopathic preparations.

BACKGROUND: The efficacy of homeopathic medicines for maintaining human health and treating disease has been extensively examined in clinical trials. However, there is a paucity of preclinical evaluations of the effects of homeopathic medicinal preparations on cellular signaling pathways relevant to the applications of these preparations. MATERIALS AND METHODS: In this study, the immune-modulatory effects of Phase 6 (for the stimulation of the nonspecific defense system) and Flu Terminator (for influenza and viral diseases) (Be Well Homeopathics Inc. Miami, FL), two homeopathic preparations developed for the purpose, were evaluated in normal human leukocyte cultures in vitro. RESULTS: Both Phase 6 and Flu Terminator stimulated the production of pro-and anti-inflammatory cytokines by human leukocytes, although higher doses often produced a weaker response than lower doses. The carrier solvent (20% ethanol) failed to elicit any cytokine synthesis. CONCLUSIONS: The results of the in vitro studies suggested that ultralow concentrations of ingredients in Phase 6 and Flu Terminator were capable of eliciting a human immune response.