Showing papers in "Journal of Experimental Therapeutics and Oncology in 2008"

Noninvasive radiofrequency field-induced hyperthermic cytotoxicity in human cancer cells using cetuximab-targeted gold nanoparticles.

Abstract: Shortwave (MHz range) radiofrequency (RF) energy is nonionizing, penetrates deeply into biologic tissues with no adverse side effects, and heats gold nanoparticles efficiently. Targeted delivery of gold nanoparticles to cancer cells should result in hyperthermic cytotoxicity upon exposure to a focused, noninvasive RF field. In this report we demonstrate that gold nanoparticles conjugated with cetuximab (C225) are quickly internalized by Panc-1 (pancreatic adenocarcinoma) and Difi (colorectal adenocarcinoma) cancer cells overexpressing epidermal growth factor receptor (EGFR). Panc-1 or Difi cells treated with naked gold nanoparticles or nonspecific IgG-conjugated gold nanoparticles demonstrated minimal intracellular uptake of gold nanoparticles by transmission electron microscopy (TEM). In contrast, there were dense concentrations of cytoplasmic vesicles containing gold nanoparticles following treatment with cetuximab-conjugated gold nanoparticles. Exposure of cells to a noninvasive RF field produced nearly 100% cytotoxicity in cells treated with the cetuximab-conjugated gold nanoparticles, but significantly lower levels of cytotoxicity in the two control groups (P < 0.00012). Treatment of a breast cancer cell line (CAMA-1) that does not express EGFR with cetuximab-conjugated gold nanoparticles produced no enhanced cytotoxicity following treatment in the RF field. Conjugation of cancer cell-directed targeting agents to gold nanoparticles may represent an effective and cancer-specific therapy to treat numerous types of human malignant disease using noninvasive RF hyperthermia.

Nrf2-dependent induction of human ABC transporter ABCG2 and heme oxygenase-1 in HepG2 cells by photoactivation of porphyrins: biochemical implications for cancer cell response to photodynamic therapy.

Abstract: Photodynamic therapy is a recently developed anticancer treatment that utilizes the generation of singlet oxygen and other reactive oxygen species in cancer tissue. Nrf2, an NF-E2-related transcription factor, plays a pivotal role in transcriptional upregulation of many target genes, including those for metabolizing enzymes and transporters essential for cellular defense in response to oxidative stress. In the present study, we examined the potential involvement of Nrf2 in the induction of human ABC transporter ABCG2 and heme oxygenase-1 (HO-1). When HepG2 cells were incubated with non-toxic concentrations of delta-aminolevulinic acid, protoporphyrin IX, or pheophorbide a and then exposed to visible light for 90 min, the mRNA level of HO-1 began increasing markedly, reaching the maximal level in 4 h. Following the transient induction of HO-1, the mRNA level of ABCG2 gradually increased in a time-dependent manner, whereas the ABCB6 mRNA level was little affected. Nrf2-specific siRNA treatments suppressed the induction of both ABCG2 and HO-1 after the photoactivation of porphyrins, suggesting that Nrf2 is a common regulator for transcriptional activation of the ABCG2 and HO-1 genes. On the other hand, the mRNA level of HO-1 was remarkably enhanced by Zn(2+)-protoporphyrin IX or hemin even in the absence of light. This induction may be attributed to inactivation of Bach1, a repressor for the HO-1 gene, by those compounds. Since patients have demonstrated individual defferences in their response to photodynamic therapy, transcriptional activation of the ABCG2 and HO-1 genes in cancer cells may affect patients' responses to photodynamic therapy.

Molecular and pharmacological blockade of the EP4 receptor selectively inhibits both proliferation and invasion of human inflammatory breast cancer cells.

Abstract: Inflammatory breast cancer (IBC) is the most aggressive form of locally advanced breast cancer (LABC) characterized by rapid growth and aggressive invasion with no selective therapies developed to treat IBC. Cyclooxygenase-2 (Cox-2), which produces prostaglandin E2 (PGE2) is known to be upregulated in primary IBC tumors and metastatic lesions, however the use of selective Cox-2 inhibitors has diminished due to cardiovascular side effects. One alternative approach to targeting Cox-2 enzyme activity is to block binding of the PGE2 ligand to its prostanoid (EP) receptors, which are designated as EP1, EP2, EP3, and EP4 and are members of a subfamily of G protein coupled receptors (GPCRs). While SUM149 IBC tumor cells and MCF-7 non-IBC breast tumor cells produce both EP2 and EP4 receptors, the invasive MDA-MB-231 non-IBC breast tumor cells produced low but detectable levels of these receptors. PGE2 and the EP4 agonist, PGE2 alcohol, stimulated significantly increased (p < 0.05) levels of proliferation and invasion by SUM149 IBC tumor cells, with no effect on proliferation of either of the two non-IBC breast tumor cell lines. In contrast, the EP2 agonist butaprost had no effect on proliferation or invasion of any cell line examined. The selective EP4 antagonist, GW627368X, induced inhibition of proliferation and invasion of human SUM149 IBC tumor cells beginning at 0.1 microM, with inhibition of proliferation and invasion by MDA-MB-231 non-IBC cells at higher concentrations of GW627368X. Molecular knockdown of the EP4 receptor was accomplished by stable transfection of an EP4 short hairpin RNA (shRNA) construct, with a clonally derived cell line designated as SUM149/Clone 1 exhibiting significantly slowed proliferation and diminished invasion compared to SUM149/Vector 5 which contained a scrambled shRNA control vector. This is the first report using both a selective pharmacologic inhibitor and a molecular shRNA knockdown approach to demonstrate that EP4 is directly involved in regulation of proliferation and invasion of IBC cells.

CDDO-Me inhibits proliferation, induces apoptosis, down-regulates Akt, mTOR, NF-kappaB and NF-kappaB-regulated antiapoptotic and proangiogenic proteins in TRAMP prostate cancer cells.

Abstract: Chemoprevention represents a promising strategy to reducing the incidence of prostate cancer which afflicts more than 240,000 males annually in the U.S. 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO) and its C-28 methyl ester (CCDO-Me) and C-28 imidazole (CDDO-Im) derivatives are synthetic oleanane triterpenoids that exhibit several-fold more potent antiinflammatory activity than naturally occurring oleanolic acid, but have not been investigated for prevention of the prostate. In order to evaluate the anticancer activity of CDDOs for prostate cancer, we have investigated the effect of synthetic oleanane triterpenoids on molecular targets relevant to the chemoprevention and treatment of prostate cancer in vitro in TRAMPC-1 cells derived from the primary tumor in the prostate of a transgenic adenocarcinoma of the mouse prostate (TRAMP) mouse. Data demonstrate that CDDOs strongly inhibit the proliferation of TRAMPC-1 cells with a potency order of CDDO-Me>CDDO-Im>CDDO. Because CDDO-Me showed the most growth inhibitory activity it was further analyzed for the anticancer activity. CDDO-Me induced apoptosis in TRAMPC-1 cells as shown by the increased binding of annexin V-FITC and cleavage of procaspases 3, -8, and -9. It effectively inhibited the molecular targets such as p-Akt, NF-kappaB, and p-mTOR and downstream effectors of mTOR (p-S6K1, cyclin-D1, and cdk4). Further, CDDO-Me inhibited NF-kappaB-regulated antiapoptotic Bcl-2, Bcl-xL, and XIAP and proangiogenic VEGF. Taken together, these data demonstrate that CDDO-Me is potentially a potent chemopreventive agent that inhibits several molecular targets that are known to play critical roles in the development and progression of prostate cancer.

Bone morphogenetic proteins 1 to 7 in human breast cancer, expression pattern and clinical/prognostic relevance.

Abstract: BACKGROUND AND AIMS: Bone morphogenetic proteins (BMPs) have a diverse role and they act in a time, concentration and cell type specific manner. They regulate cellular motility and the cells ability to invade. Recently, BMP molecules have further been shown to have an impact on the biological behaviour of breast cancer cells. In this study, we looked, for the first time, at the expression of a panel of BMPs in breast carcinomas. METHOD: Fresh frozen primary human breast cancer tissues (n = 120) and non-neoplastic mammary tissues (n = 32) were used. The distribution and location of BMPs was assessed using immunohistochemical methods and the transcript levels of BMPs (BMP-1, -2, -3, -4, -5, -6, and -7) were determined using quantitative RT-PCR. The results were analysed against the clinical, pathological and follow-up (10 years) data. RESULTS: BMP-2 and BMP-7 exhibited contrasting patterns of expression in normal and tumour tissues, wherein BMP-2 transcript level was lower and BMP-7 was higher in breast tumours than normal tissues. BMP-2 transcript was also significantly lower in tumours from patients with a moderate and poor prognosis than from those with a good prognosis (p = 0.04). BMP-2 and BMP-7 also showed a significant difference between node positive and node negative tumours (p = 0.033 and p = 0.031 respectively). BMPs 1, 3, 4, 5 and 6 showed an inconsistent variation in transcript levels within the cancer subgroups with no statistically significant results. CONCLUSION: This study has demonstrated a differential pattern of expression of BMP molecules in breast cancer and reveals a potential prognostic value of BMP-2 and BMP-7 for patients. The findings also suggest that these BMPs may be potential therapeutic targets.

Glutathione S-transferase M1, T1 and P1 polymorphisms: susceptibility and outcome in lung cancer patients.

Abstract: The glutathione S-transferases (GSTs) are a superfamily of genes whose products are phase II enzymes, catalyzing the conjugation of reactive intermediates to soluble glutathione. Some of the GSTs are polymorphic and may play a role in lung cancer susceptibility. We investigated whether genetic polymorphisms of GSTM1, GSTP1 and GSTT1 genes modulated lung cancer risk and affect survival among lung cancer patients. We determined the GST genotypes in 422 study subjects, using polymerase chain reaction (PCR) and reverse PCR and restriction fragment length polymorphism (RFLP). Logistic Regression analysis was carried out to find the association of various polymorphisms and GSTs and lung cancer. The influence of the genetic polymorphisms on patient survival was estimated using the method of Kaplan-Meier survival function. Cox Proportional Hazard models were used to estimate hazard ratios (HR) for deaths. GSTT1 -/- genotype conferred a higher odds ratio of 2.9 (P = 0.001) compared to the GSTT1+/+. So also, the GSTP1 GG genotype too had higher risk compared to the GSTP1 AA genotype (OR = 2.3, P = 0.033). When the combined GST M1, GSTT1 and GSTP1 genotypes were examined, patients with the combinations GSTM1 null and GSTT1 null had a significant OR of 3.6. So also the combinations GSTT1-/- GSTP1 AA (P = 0.005) and GSTT1-/- GSTP1 AG/GG (P = 0.001) came out to be significant. There were some significant interactions between GST genotypes with tobacco smoking and also for clinicopathological factors. Regarding survival analysis, no association of GSTM1 or GSTP1 genes with survival was noted. The GSTT1 -/- genotype along with stage was significantly associated with overall survival and found to be an independent prognostic factors for shorter lung cancer survival.

Vesicular flavonoid in combating diethylnitrosamine induced hepatocarcinoma in rat model.

Abstract: Reactive oxygen species (O2(*-), OH(-), H2O2) are known to play an important role in tumor initiation in hepatocarcinoma. Hepatocarcinoma was developed in the Swiss Albino rats by administration three doses of diethylnitrosamine (DEN) (200 mg/kg b. wt.) (i.p.) at 15 days interval. Quercetin (QC), herbal polyphenolic compound, is a potent anticancer drug. Clinical trials are difficult for its hydrophobic nature. To overcome this problem, our study was aimed to formulate soluble liver specific, galactosylated liposomal QC and to investigate its efficacy against hepatocarcinoma in rat model. Galactosylated liposomal QC was formulated and the suspension was introduced intravenously to rats (8.98 microM/kg) once in a week for 16 weeks. Hepatocarcinoma in rat model and its pathological improvement were evaluated histopathologically, histochemically and electron microscopically. Severe oxidative damage was noticed in the whole liver and its microsomal fraction of DEN treated rats. Huge numbers of hyperplastic nodules (HNs) with pre-neoplastic lesions appeared in rat liver by DEN administration. Galactosylated liposomal QC injections prevented DEN mediated development of hepatocarcinoma and oxidative damage in rat liver. Quercetin in liver specific galactosylated liposomal drug delivery system may be recommended as a potent therapeutic formulation against DEN-induced hepatocarcinoma.

Amentoflavone stimulates apoptosis in B16F-10 melanoma cells by regulating bcl-2, p53 as well as caspase-3 genes and regulates the nitric oxide as well as proinflammatory cytokine production in B16F-10 melanoma cells, tumor associated macrophages and peritoneal macrophages.

Abstract: Polyphenolic compounds present in fruits, vegetables, herbs, roots and leaves act as bioactive components and has been recognized as cancer chemopreventive agents. The present study is focused on the regulatory effect of amentoflavone, a biflavonoid from Biophytum sensitivum on the apoptotic process in B16F-10 melanoma cells as well as nitric oxide (NO) and cytokine production in B16F-10 cells, Tumor associated macrophages (TAMs) and peritoneal macrophages. Amentoflavone at a concentration of 10 microg/mL could significantly (p < 0.001) inhibit NO and proinflammatory cytokine (IL-1beta, IL-6, GM-CSF and TNF-alpha) production in B16F-10 cells, TAMs and peritoneal macrophages. Incubation of B16F-10 cells with amentoflavone showed the presence of apoptotic bodies and induced DNA fragmentation. Furthermore, amentoflavone showed inhibitory effect on bcl-2 expression and upregulated p53 and caspase-3 gene expression in B16F-10 melanoma cells. In conclusion, the observed results suggests that, amentoflavone stimulates apoptosis by regulating bcl-2, Caspase-3 and p53 genes in B16F-10 melanoma cells and regulates nitric oxide and proinflammatory cytokine production in B16F-10 cells, TAMs and peritoneal macrophages.

Bortezomib as a therapeutic candidate for neuroblastoma.

Abstract: Outcomes remain poor in neuroblastoma despite intensive treatment. Agents with potential efficacy can be drawn from anti-neoplastic drugs introduced for other malignancies. Bortezomib, a proteasome inhibitor, modulates cell-signaling molecules leading to apoptosis. Bortezomib, alone and in combination with other agents, was tested across an in vitro panel of neuroblastic, stromal, and chemo-resistant neuroblastoma cell types to determine its effect on cell viability and to assess for interactions between bortezomib and other chemotherapeutic agents that either limit or increase overall response. Each subtype of neuroblastoma was sensitive to bortezomib and killing occurred with EC50 values of approximately 50 nM. When bortezomib was combined with other agents (doxorubicin, etoposide, SN-38, carboplatin, or cisplatin), no antagonism was observed. The bortezomib-doxorubicin combination was especially effective, demonstrating synergy on isobolographic analysis and resulting in a decrease in EC50 from 50 ng/mL with doxorubicin alone to 5 ng/mL with 25 nM bortezomib. Interestingly, the different cell types exhibited varying response patterns to treatment with bortezomib alone and in combination with other drugs suggesting different mechanisms may be engaged. A decision analysis, incorporating these results showing efficacy in all cell types, the synergy obtained in combination, and the available toxicity data, supports a phase II clinical trial of bortezomib in neuroblastoma.

Determinants of human and mouse melanoma cell sensitivities to oleandrin.

Abstract: Oleandrin, a cardiac glycoside component of Nerium oleander, has been shown to induce apoptosis in malignant cells. While human tumor cells are very sensitive to growth inhibition by oleandrin, murine tumor cells are extremely resistant. Using human BRO and mouse B16 melanoma cell lines, we explored several possible determinants of cell sensitivity to oleandrin and compared with ouabain. The studies include Na+, K(+)-ATPase activity and its isoforms as well as the cellular uptake of these cardiac glycosides. Oleandrin and ouabain induced apoptosis was detected in BRO cells while no evidence of cell death was observed in B16 cells even at concentrations 1000-fold higher than that used for BRO cells. Cellular uptake of oleandrin and ouabain was 3-4 fold greater in human BRO tumor cells than murine tumor cells. Partially purified Na+, K(+)-ATPase from human BRO cells was inhibited at a concentration that was 1000-fold less than that was required to inhibit mouse B16 enzyme to the same extent. Using Western blot analyses, human BRO cells were found to express both the sensitive alpha3 isoform and the less sensitive alpha1 isoform of Na+, K(+)-ATPase while mouse B16 cells expressed only the alpha1 isoform. These data suggest that differential expressions of Na+, K(+)-ATPase activities and its isoforms in BRO and B16 cells as well as cellular drug uptake may be important determinants of tumor cell sensitivity to cardiac glycosides.

Organic extracts of Larrea divaricata Cav. induced apoptosis on tumoral MCF7 cells with an higher cytotoxicity than nordihydroguaiaretic acid or paclitaxel.

Abstract: The methanolic and methane dichloride extracts of aerial parts of Larrea divaricata Cay. (Jarilla) plants exhibited a pronounced cytotoxic effect by arresting cell viability at a level of 35% against the MCF-7 cell line, a human breast adenocarcinoma, while only a weak cytotoxic effect was observed for the aqueous extract. Apoptosis was detected by flow cytometry, using annexin V and by cytological detection with fluorescein diacetate / propidium iodide staining. The annexin V, as well as the uptake of fluorescein diacetate/propidium iodide staining, revealed that methanolic and methane dichloride extracts of L. divaricata treatment of cells resulted in a rapid plasma membrane perturbation and triggered cellular death (>70%). Methanolic extracts were submitted to further treatment by bioassay-guided purification, involving fractionation and chromatography. 0.1% (w/w) nordihydroguaiaretic acid (NDGA) was found, which displayed weak citotoxic activity.

Mushroom polysaccharide extracts delay progression of carcinogenesis in mice

Abstract: Hepatocellular carcinoma results when cancerous cells are localized in the liver. It is distributed globally with high prevalence in sub-Saharan African, southern Asia, China and Japan. Diagnosis is experimental and in many cases inaccurate due to unreliability of markers. Prognosis is poor and the cost of treatment prohibitive. Conventional radiation and chemotherapy lead to loss of hair, fertility and general weakening of the body's immune system increasing a patient's risk to infection. These observations underscore the need for improved, or additional methods of cancer diagnosis and management. We investigated the effect of polysaccharide rich Pleurotus pulmonarius fruit body extracts on progression of chemically induced hepatocellular carcinoma in CBA mice. Addition of Pleurotus pulmonarius extracts in diet delayed progression of carcinogenesis suggesting that these extracts may be useful as adjuvants to conventional cancer therapies.

13 cis-retinoic acid regulates cytokine production and inhibits angiogenesis by disrupting endothelial cell migration and tube formation.

Abstract: Cancer prevention using natural products has become an integral part of cancer control. In this study we investigated the effect of 13-cis-retinoic acid on the inhibition of angiogenesis using in vivo as well as in vitro models. Our studies using animal model reveled that 13-cis-retinoic acid could significantly (p < 0.001) inhibit the tumor directed capillaries. The cytokine profile in the serum of these animals showed a drastically increased level of proinflammatory cytokines such as IL-1beta, TNF-alpha, IL-6, GM-CSF and the direct endothelial cell proliferating agent, VEGF during the onset of angiogenesis. Administration of 13-cis-retinoic acid could differentially regulate these cytokine's elevation. The differential elevation is further evidenced by the increased production of IL-2 and tissue inhibitor of metalloprotease-1 (TIMP-1) in the 13-cis-retinoic acid treated animals. Aortic ring assay for in vitro angiogenesis revealed that 13-cis-retinoic acid could markedly inhibit the microvessel sprouting. Moreover, 13-cis-retinoic acid was able to inhibit vascular endothelial cell proliferation, migration and tube formation. Furthermore, 13-cis-retinoic acid treatment could inhibit the activation and nuclear translocation of p65, p50, c-Rel subunits of nuclear factor-KB, and other transcription factors such as c-fos, activated transcription factor-2, and cyclic adenosine monophosphate response element-binding protein in B16F-10 melanoma cells. These findings suggest that the anti-angiogenic potential of 13-cis-retinoic acid is mediated through inhibition of endothelial cell migration and tube formation and altered cytokine production during the onset of angiogenesis.

Inhibition of progression of erythroleukemia induced by Friend virus in BALB/c mice by natural products--berberine, curcumin and picroliv.

Abstract: The infection with Friend murine leukemia virus (FMuLv) is being used as a retrovirus infection model for searching the potential anti-viral medicinal preparations or establishing new treatment strategies. In the present study we have evaluated the inhibitory effect of three non-toxic antiviral natural compounds namely berberine, curcumin or picroliv against FMuLv induced erythroleukemia in BALB/c mice. To understand the effect of these compounds in the initiation and progression of leukemia we did a series of analysis, which include hematological and biochemical parameters, histopathological evaluations of the liver and the spleen and expression analysis of selected genes such as Bcl-2, p53, p45NFE2, Raf-1, Erk-1, IFNgamma receptor and erythropoietin in spleen. The treatment with berberine, curcumin or picroliv were found to (a) elevate the life span of leukemia harboring animals by more than 60 days; (b) decreased the anemic condition which was highly prevalent in FMuLv alone treated group; (c) histopathological evaluations showed that the compounds tested here inhibited the massive leukemic cell infiltrations to sinusoidal spaces in spleen; (d) decrease the expression of Bcl-2, Raf-1, Erk-1 IFNgamma receptor and erythropoietin; (e) induce the expression of p53. Overall, our results suggest that berberine, curcumin and picroliv were able to suppress the progression of leukemia induced by FMuLv and further support their chemopreventive potential against virally induced cancers.

Anti-metastatic effect of Biophytum sensitivum is exerted through its cytokine and immunomodulatory activity and its regulatory effect on the activation and nuclear translocation of transcription factors in B16F-10 melanoma cells.

Abstract: The major clinical challenge for cancer therapy remains the eradication or prevention of metastatic process. This study was an investigation of the antimetastatic activity of Biophytum sensitivum, using B16F-10 melanoma-induced experimental lung metastasis in C57BL/6 mice. B. sensitivum treatment significantly reduced lung tumor nodule formation accompanied by reduced lung collagen hydroxyproline, hexosamine, and uronic acid levels. Serum sialic acid and gamma-glutamyl transpeptidase levels were also significantly inhibited after B. sensitivum treatment. B. sensitivum treatment down-regulated the expression of matrix metalloprotease-2 and -9 and at the same time upregulated the lung tissue inhibitor of metalloprotease-1 and -2 expression. The cytokine profile and growth factors such as interleukin-1beta, interleukin-6, tumor necrosis factor-alpha, granulocyte monocyte-colony stimulating factor, vascular endothelial growth factor, interleukin-2 and tissue inhibitor of metalloprotease-1 in the serum of these animals were markedly altered after B. sensitivum treatment. This altered level of cytokines after B. sensitivum treatment was also accompanied by enhanced natural killer cell and antibody-dependent cellular cytotoxicity. The study reveals that B. sensitivum treatment could alter proinflammatory cytokine production and could inhibit the activation and nuclear translocation of p65, p50, c-Rel subunits of nuclear factor-kappaB, and other transcription factors such as c-fos, activated transcription factor-2, and cyclic adenosine monophosphate response element-binding protein in B16F-10 melanoma cells.

Cytotoxic and apoptotic effects of bortezomib and gefitinib compared to alkylating agents on human glioblastoma cells.

Abstract: Glioblastoma is a malignant astrocytic tumor with a median survival of about 12 months for which new therapeutic strategies are required. We therefore examined the cytotoxicity of anticancer drugs with different mechanisms of action on two human glioblastoma cell lines expressing various levels of EGFR (epidermal growth factor receptor). Apoptosis induced by these anticancer agents was evaluated by flow cytometry. The cytotoxicity of alkylating drugs followed a dose-effect curve and cytotoxicity index values were lower with carboplatin than with BCNU and temozolomide. Anti-EGFR gefitinib (10 microM) cytotoxicity on DBTRG.05-MG expressing high levels of EGFR was significantly higher than on U87-MG expressing low levels of EGFR. Carboplatin and temozolomide cytotoxicity was potentiated with the addition of gefitinib on DBTRG.05-MG. Among the anticancer agents tested, the proteasome inhibitor bortezomib was the most cytotoxic with very low IC50 on the two cell lines. Moreover, all anticancer drugs tested induced apoptosis in a concentration-dependent manner. Bortezomib proved to be a more potent inductor of apoptosis than gefitinib and alkylating agents. These results show the efficacy of bortezomib and of the association between conventional chemotherapy and gefitinib on glioblastoma cells and therefore suggest the interest of these molecules in the treatment of glioblastoma.

FTY720 treatment in experimentally urethane-induced lung tumors

Abstract: FTY720 has been shown to prevent cancer development in experimental models but there is no report whether this beneficial effect is associated with the time point of the drug administration. Lung adenoma was induced in mice by urethane injection followed by different periods of FTY720 administration in order to evaluate lung tumor development. BALB/c mice received two doses (1, 5 g/kg) of urethane intraperitoneally and were submitted to five daily doses of FTY720 (1 mg/kg/day) starting just after urethane injection (G2 n=5), 4 weeks after urethane injection (G3 n=10), 8 weeks after urethane injection (G4 n=10) and no FTY720 administration (G1 n=5). Twenty-four weeks after urethane administration mice were evaluated for leukocyte numbers in blood, lymphocytes in spleen, and lungs were evaluated for changes in histology and PCNA expression. Lung nodules were present in higher numbers both in non treated (G1; 0.0-7.0) and FTY720 treated 8 weeks after urethane injection (G4; 0.0-6.0). G4 Group also presented the highest number of papillary nodules. There was a decrease in PCNA staining in early time FTY720 treated mice. Therefore, our data suggest that FTY720 treatment in early periods after lung tumor induction is beneficial and impairs adenoma development.

Boerhaavia diffusa stimulates cell-mediated immune response by upregulating IL-2 and downregulating the pro-inflammatory cytokines and GM-CSF in B16F-10 metastatic melanoma bearing mice.

Abstract: Effect of Boerhaavia diffusa extract on the Cell Mediated Immune (CMI) response in metastatic condition was studied using C57BL/6 mice model. Administration of Boerhaavia diffusa enhanced Natural Killer (NK) cell activity, antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent complement mediated cytotoxicity (ACC) and the activity was observed in treated group much earlier compared to the metastatic tumor bearing control. Production of the cytokine IL-2 was significantly enhanced by the administration of Boerhaavia diffusa compared to the untreated metastatic tumor bearing control. Levels of GM-CSF and pro-inflammatory cytokines such as IL-1beta, IL-6 and TNF-a were significantly lowered by Boerhaavia diffusa administration compared to metastatic control. The gene expression level of IL-2, IL-1beta, IL-6, TNF-alpha and GM-CSF in B16F-10 cells also correlate the above result. These results indicate Boerhaavia diffusa could enhance the immune response against metastatic progression of B16F-10 melanoma cells in mice.

Anti-thrombin as a prognostic biomarker candidate for patients with recurrent glioblastoma multiform under treatment with perillyl alcohol.

Abstract: Perillyl alcohol (POH) is a naturally occurring monoterpene with antiangiogenic and anti-tumoral properties. This chemotherapeutic agent has proven effectiveness in several clinical trials, including an ongoing phase I, comprising patients with recurrent glioblastoma multiform (GBM) under treatment with POH by intranasal administration. Proteomics offers tools to distinguish states of biological systems according to protein expression differences and therefore, can be used to gain pathological insights and to search for disease follow-up biomarkers. In this work, a differential gel electrophoresis (DIGE) proteomic approach was used to search for plasma proteins that correlated with the disease progression in 10 of these patients. Our results pointed antithrombin (down) and fibrinogen (up) regulated after a four months treatment deserving to be further verified as prognostic markers for this treatment. Possible links between tumor progression and anti-thrombin expression level are also discussed.

Analysis of human serum from women affected by cervical lesions.

Abstract: Cervical cancer is one of the first causes of death in Mexican women population. The plasma proteome has a wide dynamic range concentrations of different protein and their alterations reflect the physiological state of the individual's health. The aim of this study was to characterize the 2D-PAGE serum patterns from healthy women and with different levels of cervical lesions. Changes in haptoglobin, apolipoproteins, and transthyretin, when comparing the serum from healthy women and serum from patients with different levels of cervical lesion were found. The Western blot analysis showed increasing concentrations of metalloproteinases (MMP's), proteins with important biological roles in tumor development and metastasis. Protein profiles in conjunction with MS, bioinformatics, and Western blot analysis, allow us to compile information for the acquisition of results to proposed candidates biomarkers of cervical cancer among Mexican women population.

Antitumor activity of two derivatives from 2-acylamine-1, 4-naphthoquinone in mice bearing S180 tumor.

Abstract: Drugs containing a quinone moiety, such as anthracyclines, mitoxantrones and lapachol, show excellent anticancer activity. In this study, 2-butanoylamine-1,4-naphthoquinone (1) and 2-propanoylamine-1,4-naphthoquinone (2) derivatives from 2-amine-1 ,4-naphthoquinone were synthesized, and their antitumor activity in mice bearing Sarcoma 180 tumor were examined. In addition, hematology and biochemistry analyses, as well as, histopathological and morphological analyses were performed in order to evaluate the toxicological aspects of the naphthoquinones treatment. Both naphthoquinones showed potente antitumor activity. The inhibition rates were 33.48 and 42.35% for (1) and 37.65 and 55.24% for (2) at the dose of 25 and 50 mg/kg/day, respectively. In the histopathological analysis, the naphthoquinones showed only weak toxicity. Neither enzimatic activity of transaminases (aspartate aminotransferase-AST nor alanine aminotransferase-ALT), urea level nor hematological paramenter were significantly modified after naphthoquinones treatment. These data reinforce the anticancer potential of naphthoquinones derivatives.

Schedule shifts, cancer and longevity: good, bad or indifferent?

Abstract: Prompted by a recent report of the possible carcinogenic effect of shiftwork focusing on the disruption of circadian rhythms, we review studies involving shifts in schedule implemented at varying intervals in unicells, insects and mammals, including humans. Results indicate the desirability to account for a broader-than-circadian view. They also suggest the possibility of optimizing schedule shifts by selecting intervals between consecutive shifts associated with potential side-effects such as an increase in cancer risk. Toward this goal, marker rhythmometry is most desirable. The monitoring of blood pressure and heart rate present the added benefit of assessing cardiovascular disease risks resulting not only from an elevated blood pressure but also from abnormal variability in blood pressure and/or heart rate of normotensive as well as hypertensive subjects.

Antitumor and apoptosis promoting properties of atorvastatin, an inhibitor of HMG-CoA reductase, against Dalton's Lymphoma Ascites tumor in mice.

Abstract: Epidemiological and experimental data indicate that high body fat or high dietary fat can be ascribed to the induction of human cancers. Increased level of products of lipid peroxidation and cholesterol-enriched lipid domains in the plasma membrane can favorthe malignant transformation of cells. An effective chemopreventive agent with hypolipaedemic effect will be worthwhile to intervene early in the process of carcinogenesis to eliminate the pre-malignant cells. Apoptotic promoting and antitumor activities of a HMG-Co A reductase inhibitor, atorvatstain were investigated. The antitumor activity was evaluated using Daltons' Lymphoma Ascites (DLA) cell line transplanted ascites tumor model in mice. Proapoptotic activity was evaluated in DLA cell line induced ascites animals after the treatment of atorvastatin (4 and 16 mg/kg, i.p). Apoptosis was analyzed morphologically by staining with giemsa and biochemically by observing the laddering of DNA in agarose gel electrophoresis. In vitro short term cytotoxic activity of atorvatstain was studied by trypan blue dye exclusion method. Doxorubicin was used as the reference standard. Atorvastatin significantly (P < 0.01) inhibited the ascites tumor growth at 16 mg/kg body wt (i.p). The percent increase in life span (%ILS) in the 16 mg/kg treated group was 41.1%. Single dose of atorvastatin (16 mg/kg body wt) was also effective to promote the apoptosis of DLA cells in the ascites tumor bearing mice that was evident from the multiple fragmentation of DNA in agarose gel electrophoresis. Further the morphological analysis of DLA cells aspirated from the atorvastatin treated ascites tumor bearing animals showed 36.34 +/- 6.78% apoptotic cells compared to the control animals (10.50 +/- 3.53%). Concentration of atorvastatin required for the 50% of the cytotoxicity was 30 +/- 2 microg/ml. Results of the study concluded that the antitumor activity of atorvastatin may be due to its proapoptotic and cytotoxic activities. These pleiotropic activities of the hypolipedaemic drug atorvastatin suggest its possible use as a cancer chemopreventive agent.

Interferon-alpha plus capecitabine and thalidomide in patients with metastatic renal cell cancer.

Abstract: Thirty patients with progressive metastatic renal cell carcinoma were treated. Eleven patients received 2 or more prior systemic therapies; 2 had a complete response, 7 had a partial response, and 11 had stable disease. The complete responders are off therapy and remain without disease recurrence. The median duration of response was 3.8 months (range 1 - 48+ months). Therapy was well tolerated; predominant toxicities were fatigue, paraesthesias and hand/foot syndrome. The data suggest that the combination of interferon-alpha, thalidomide and capecitabine has anti-tumor activity in previously treated patients with progressive metastatic renal cell carcinoma. A prospective investigation of this combination warranted.

VEGF165 is necessary to the metastatic potential of Fas− osteosarcoma cells but will not rescue the Fas+ cells.

Abstract: Our previous studies showed that Fas expression correlates inversely with the metastatic potential of osteosarcoma (OS) cells and that the manipulation of Fas expression alters the lung metastatic phenotype. However, the role of VEGF in the growth and metastases of OS is unclear. The purpose of this study was to determine whether altering VEGF expression affects lung metastatic potential. LM7 metastatic OS cells were transfected with a small interfering RNA targeting human VEGF165 (LM7/siVEGF165) or a pcDNA4 plasmid expressing human VEGF165 (LM7/VEGF). We confirmed that VEGF165 expression was decreased in LM7/siVEGF165 cells and was increased in LM7/VEGF clones compared with control transfected clones. Fas expression was not altered in these transfected clones. We also transfected LM7 cells with Fas (LM7/Fas) or Fas together with VEGF165 (LM7/Fas/VEGF) to determine whether the overexpression of VEGF165 could enhance the metastatic potential of LM7 OS cells with high Fas expression (Fas(+)). LM7/siVEGF165 and LM7/Fas cells showed decreased lung metastatic potential. In addition, the overexpression of VEGF had no effect on the ability of LM7/Fas cells to form lung metastases. We therefore concluded that VEGF165 is critical to lung metastatic potential but is not sufficient to allow Fas(+) OS cells to survive in the Fas ligand lung microenvironment.

Dynamic heterogeneity of proteomic expression in human cancer cells does not affect Cdk1/Cdk4 co-expression.

Abstract: Tumour heterogeneity is becoming increasingly important as an obstacle to genomic and proteomic technologies designed to improve the diagnosis and treatment of human cancer. In a panel of 19 human in-vitro cancer cell lines, we show marked heterogeneity of proteomic expression of key genes responsible for the control of cell division and death. Patterns of expression of these proteins were unique for each cell line. In addition, dynamic heterogeneity of proteomic expression of Cyclin D1, Cdk1, Cdk4 and even actin was detected. The relative levels of each protein fluctuated independently from experiment to experiment separated only by short passages in tissue culture. Cdk1 and Cdk4 proteomic co-expression (Seabra, 2007) was not, however, affected by dynamic heterogeneity, or, in 4 cell lines, by treatment with D0.1 doses of CDDP. Cdk1/Cdk4 may thus provide a complex molecular target for anti-cancer drug development which is unaffected by tumour heterogeneity and is not disrupted by conventional chemotherapy.

Effects of Hsp90 inhibition in neuroblastoma: analysis of drug sensitivity, target modulation and the influence of bone marrow microenvironment.

Abstract: Heat shock protein 90 (Hsp90) safeguards the structural integrity and function of many of the key growth regulatory proteins found in malignant cells. Consequently, among the new generation targeted therapeutics, heat shock protein inhibitors have the unique property of being able to target an expansive array of divergent molecular mechanisms involved in cancer growth and metastasis. 17-N-Allylamino-17-demethoxygeldanamycin (17-AAG) is one such agent that has been shown to bind to Hsp90 and thus reduce the stability and activity of many key growth regulatory molecules and pathways. A number of recent clinical trials have investigated the maximum tolerated dose, toxicity and pharmacokinetic profiles of 17-AAG in pediatric patients with recurrent tumors. In this study, we describe the effects of 17-AAG against a panel of neuroblastoma (NB) cell lines with respect to cytotoxicity, target modulation and inhibition of vascular endothelial growth factor (VEGF) expression. 17-AAG was found to inhibit the growth of all NB cell lines tested, though effective inhibitory concentrations varied among cell lines. 17-AAG also suppressed the expression of VEGF. The cytotoxic effect of 17-AAG on tumor cells was diminished when co-cultured with bone marrow stromal cells suggesting a potential role for the microenvironment in tumor drug interactions. Findings from target modulation analysis as well as drug combination assays provide a frame-work to formulate effective protocols for the treatment of NB.

Hereditary breast/ovarian cancer: clinicopathological characteristics and survival of BRCA2 positive and negative cases.

Abstract: The clinical and pathological characteristics and prognostic outcome of patients with hereditary breast/ovarian cancer and BRCA2 mutations are poorly known. Hence, the present study aimed to correlate the BRCA2 mutation status with clinical characteristics and overall survival of 102 breast/ovarian cancer patients in Kerala, South India. All the coding regions of BRCA2 genes were PCR amplified and analyzed for mutations employing Conformation Sensitive Gel Electrophoresis and characterized by sequencing. The ORs with 95% Cls was computed to assess the association between BRCA2 gene mutation status and clinicopathologic characteristics of breast cancer patients. Survival curves were generated according to Kaplan-Meier method using Log Rank test and Cox proportional hazards regression method. Out of the 102 breast/ovarian cancer patients with known BRCA2 status, 19 were BRCA2 mutation positive. In survival analysis, BRCA2 gene mutation status (P = 0.02) and clinicopathologic parameters such as tumour size (p = 0.01), metastasis (P = 0.01), disease stage (P = 0.03) and laterality (P = 0.02) were significantly associated with poor prognosis of breast cancer patients. Patients with hereditary breast/ovarian cancer resulting from a BRCA2 mutation have been conclusively shown to have a worse survival prognosis compared to the non mutated group of patients.