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.

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.

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.

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.

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.

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.

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.

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.

Mechanism of macrophage activation by (1,4)-alpha-D-glucan isolated from Tinospora cordifolia.

The signaling mechanism of the novel (1,4)-alpha-D-glucan (RR1) isolated from the medicinal plant Tinospora cordifolia was investigated in macrophages to evaluate its immunostimulating properties. When RAW264.7 macrophages were incubated with RR1 at 4 degrees C, the novel glucan inhibited the phagocytosis of unopsonized zymosan A bioparticles in a dose-dependent manner. RR1 also inhibited the binding and internalization of opsonized zymosan A bioparticles, although at a lower level than laminarin. Incubation of macrophages with anti-CD11b mAb followed by RR1 failed to show any inhibitory effect on RR1-induced TNF-alpha synthesis confirming that complement receptor 3 (CR3) is not involved in the opsonic binding and internalization of RR1 in macrophages unlike zymosan A. The anti-CD11b mAb has significant inhibitory effect on the zymosan A-induced tumor necrosis factor (TNF)-alpha synthesis. RR1 induced TNF-alpha synthesis in macrophages in a dose-dependent manner which can be completely inhibited by the NF-kappaB inhibitor caffeic acid phenethyl ester (CAPE) or curcumin. RR1 activated NF-kappaB in a time- and dose-dependent manner and this modulation of nuclear NF-kappaB activity is associated with the degradation of I-kappaB alpha thus facilitating the translocation of NF-kappaB into the nucleus. RR1-induced NF-kappaB activity peaks at 8 h of RR1 stimulation while I-kappaB alpha degradation occurred within 1 h of stimulation. RR1-induced NF-kappaB activation occurred through TLR6 signaling as evidenced by the synthesis of IL-8 in TLR6-transfected HEK293 cells. These results show that the novel (1,4)-alpha-D-glucan from Tinospora cordifolia activates the immune system through the activation of macrophages that occurs through TLR6 signaling, NF-kappaB translocation and cytokine production.

Expression profiles of apoptotic genes induced by curcumin in human breast cancer and mammary epithelial cell lines.

Curcumin (diferuloyl methane), the yellow-colored dietary pigment from the rhizomes of turmeric, has been recognized as a chemopreventive agent because of its antitumor, antioxidant and antiproliferative effects. The cytotoxic, apoptotic and gene regulatory effects of both turmeric and curcumin were investigated in the MCF-7 human breast cancer carcinoma cell line and compared with the effects in MCF-10A human mammary epithelial cells. MCF-7 cells were more sensitive to turmeric and curcumin than MCF-10A cells. MCF-10A cells retained comparatively less curcumin in the medium than MCF- 7 cells after 24 h, thereby reducing the cytotoxic effect. Curcumin induced a significantly higher percentage of apoptosis in MCF-7 than MCF-10A cells at all doses. Microarray hybridization of Clonetech apoptotic arrays with labeled first-strand probes of total RNA was performed to identify and characterize the genes regulated by curcumin in tumor cells. Of the 214 apoptosis-associated genes in the array, the expression of 104 genes was altered by curcumin treatment. The gene expression was altered up to 14-fold levels in MCF-7 as compared to only up to 1.5-fold in the MCF-10A cell line by curcumin. Curcumin up-regulated (>3 fold) 22 genes and down-regulated (<3-fold) 17 genes at both 25 microg/ml and 50 microg/ml doses in the MCF-7 cell line. The up-regulated genes include HIAP1, CRAF1, TRAF6, CASP1, CASP2, CASP3, CASP4, HPRT, GADD45, MCL-1, NIP1, BCL2L2, TRAP3, GSTP1, DAXX, PIG11, UBC, PIG3, PCNA, CDC10, JNK1 and RBP2. The down-regulated genes were TRAIL, TNFR, AP13, IGFBP3, SARP3, PKB, IGFBP, CASP7, CASP9, TNFSF6, TRICK2A, CAS, TRAIL-R2, RATS1, hTRIP, TNFb and TNFRSF5. While a dose-dependent gene expression change was noticed in some genes, opposite regulatory effects were induced by different curcumin doses in three apoptotic genes. These results suggest that curcumin induces apoptosis in breast cancer cells by regulation of multiple signaling pathways, indicating its potential use for prevention and treatment of cancer.

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.

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.

Curcumin inhibits telomerase activity through human telomerase reverse transcritpase in MCF-7 breast cancer cell line.

The inhibitory effect of curcumin, the yellow-colored pigment from turmeric, on telomerase activity was analyzed in human mammary epithelial (MCF-10A) and breast cancer (MCF-7) cells. Telomerase activity in MCF-7 cells is 6.9-fold higher than that of human mammary epithelial cells. In MCF-7 cells, telomerase activity decreased with increasing concentrations of curcumin, inhibiting about 93.4% activity at 100 microM concentration. The inhibition of telomerase activity in MCF-7 cells may be due to down-regulation of hTERT expression. Increasing concentrations of curcumin caused a steady decrease in the level of hTERT mRNA in MCF-7 cells whereas the level of hTER and c-myc mRNAs remained the same. Our results suggest that curcumin inhibits telomerase activity by down-regulating hTERT expression in breast cancer cells and this down-regulation is not through the c-myc pathway.

Immune stimulating properties of a novel polysaccharide from the medicinal plant Tinospora cordifolia.

An alpha-D-glucan (RR1) composed of (1-->4) linked back bone and (1-->6) linked branches with a molecular mass of >550 kDa and exhibiting unique immune stimulating properties is isolated and characterized from the medicinal plant Tinospora cordifolia. This novel polysaccharide is noncytotoxic and nonproliferating to normal lymphocytes as well as tumor cell lines at 0-1000 microg/ml. It activated different subsets of the lymphocytes such as natural killer (NK) cells (331%), T cells (102%), and B cells (39%) at 100 microg/ml concentration. The significant activation of NK cells is associated with the dose-dependent killing of tumor cells by activated normal lymphocytes in a functional assay. Immune activation by RR1 in normal lymphocytes elicited the synthesis of interleukin (IL)-1beta (1080 pg/ml), IL-6 (21,833 pg/ml), IL-12 p70 (50.19 pg/ml), IL-12 p40 (918.23 pg/ml), IL-18 (27.47 pg/ml), IFN- gamma (90.16 pg/ml), tumor necrosis factor (TNF)-alpha (2225 pg/ml) and monocyte chemoattractant protein (MCP)-1 (2307 pg/ml) at 100 microg/ml concentration, while it did not induce the production of IL-2, IL-4, IL-10, interferon (IFN)-alpha and TNF-beta. The cytokine profile clearly demonstrates the Th1 pathway of T helper cell differentiation essential for cell mediated immunity, with a self-regulatory mechanism for the control of its overproduction. RR1 also activated the complements in the alternate pathway, demonstrated by a stepwise increase in C3a des Arg components. Incidentally, RR1 stimulation did not produce any oxidative stress or inducible nitric oxide synthase (iNOS) in the lymphocytes or any significant increase in nitric oxide production. The water solubility, high molecular mass, activation of lymphocytes especially NK cells, complement activation, Th1 pathway-associated cytokine profile, together with a low level of nitric oxide synthesis and absence of oxidative stress confer important immunoprotective potential to this novel alpha-D-glucan.

Molecular studies in pediatric medulloblastomas.

Ten pediatric medulloblastoma patients were analyzed for DNA content, cell cycle, expression of drug resistance, apoptosis, cell proliferation, and N-myc genes to determine their prognostic significance. Medulloblastoma patients with progressive disease had fourth ventricle foraminal extension and larger tumors in the imaging studies. Patients with aneuploid tumors responded well to treatment regimens as compared with those with diploid tumors. Cell cycle analysis showed that the patients with progressive disease had a high S-phase fraction in the tumor cell population as compared with patients with favorable response to treatment. The correlation coefficients between Bcl-2 and MRP, Bcl-2 and Bax, p53 and p21, as well as Ki67 and PCNA were positive and significant, indicating their possible coregulated expression. The relationship between these markers indicates their relative and cumulative effect on cellular drug resistance, apoptosis, and/or cell proliferation in pediatric medulloblastomas.

Effect of MDR1 phosphorothioate antisense oligodeoxynucleotides in multidrug-resistant human tumor cell lines and xenografts.

The effect of MDR1 antisense phosphorothiate oligodeoxynucleotides (S-ODNs) on resistant phenotype was investigated in multidrug-resistant human colon carcinoma and breast carcinoma cells in vitro and in vivo. Drug resistance in human colon carcinoma (SW620 Ad300) and breast carcinoma (MCF-7/INT500, MCF-7/AD150 and MCF-7/TH) cell lines is predominantly due to overexpression of P-glycoprotein (P-gp) resulting in decreased daunorubicin (DNR) accumulation. Two MDR1 antisense S-ODNs, one complementary to the initial 15 bases of first exon (S-ODN I) and the other a loop forming sequence (S-ODN II) complementary to bases from 993-1007 of MDR1 gene, were tested for enhancing the doxorubicin (DOX) cytotoxicity in vitro and the efficiency of chemotherapy in human tumor xenografts. MDR1 antisense S-ODN I reduced the DOX IC50 value 9-fold in multidrug-resistant SW620 Ad300 human colon carcinoma cells and 7 to 10-fold in breast carcinoma cells in vitro. The increase in DOX cytotoxicity correlated with a significant reduction of MDR1 mRNA in antisense S-ODN I-treated SW620 Ad300 cells. Even though the P-gp level was reduced at the end of the third day in antisense S-ODN I-treated cells, the rate of reduction was only partial compared to mRNA. The combination treatment of MDR1 antisense S-ODN I or II for three days and DOX for four days significantly controlled tumor growth rate in human tumors developed in nude mice. Our results suggest that MDR1 antisense S-ODN treatment can increase the efficiency of chemotherapy by suppressing gene expression and resistant phenotype.

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.