Inhibition of bladder cancer cell proliferation by allyl isothiocyanate (mustard essential oil)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Fibronectin glycation increases IGF-I induced proliferation of human aortic smooth muscle cells

The advanced glycation end products, namely AGEs, contribute to long-termed complications of diabetes mellitus, including macroangiopathy, where smooth muscle cells (SMC) proliferation stimulated by platelet-derived growth factor (PDGF) isoforms and insulin-like growth factor-I (IGF-I) plays an important role. The objective of the present study was to investigate the effect of an AGE-modified extracellular matrix protein on IGF-I induced SMC proliferation and on the IGF-I-IGF binding protein 4 (IGFBP-4) axis under basal conditions and after stimulation with PDGF-BB. IGF-I resulted in significantly higher thymidine incorporation in SMC seeded on AGE-modified fibronectin (AGE-FN) in comparison to cells seeded on fibronectin (FN). This augmented proliferation could not be accounted for by increased expression of IGF-IR, by decreased secretion of IGFBP-4, a binding protein that inhibits IGF-I mitogenic effects or by increased IGF-IR autophosphorylation. PDGF-BB did not modulate IGF-IR and IGFBP-4 mRNA expression in any of the substrata, however, this growth factor elicited opposite effects on the IGFBP-4 content in the conditioned media, increasing it in cells plated on FN and diminishing it in cells plated on AGE-FN. These findings suggest that one mechanism by which AGE-modified proteins is involved in the pathogenesis of diabetes-associated atherosclerosis might be by increasing SMC susceptibility to IGF-I mitogenic effects.

Mesenchymal stem cells with rhBMP-2 inhibits the growth of canine osteosarcoma cells

Background: The bone morphogenetic proteins (BMPs) belong to a unique group of proteins that includes the growth factor TGF-beta. BMPs play important roles in cell differentiation, cell proliferation, and inhibition of cell growth. They also participate in the maturation of several cell types, depending on the microenvironment and interactions with other regulatory factors. Depending on their concentration gradient, the BMPs can attract various types of cells and act as chemotactic, mitogenic, or differentiation agents. BMPs can interfere with cell proliferation and the formation of cartilage and bone. In addition, BMPs can induce the differentiation of mesenchymal progenitor cells into various cell types, including chondroblasts and osteoblasts. The aim of this study was to analyze the effects of treatment with rhBMP-2 on the proliferation of canine mesenchymal stem cells (cMSCs) and the tumor suppression properties of rhBMP-2 in canine osteocarcoma (OST) cells. Osteosarcoma cell lines were isolated from biopsies and excisions of animals with osteosarcoma and were characterized by the Laboratory of Biochemistry and Biophysics, Butantan Institute. The mesenchymal stem cells were derived from the bone marrow of canine fetuses (cMSCs) and belong to the University of Sao Paulo, College of Veterinary Medicine (FMVZ-USP) stem cell bank. After expansion, the cells were cultured in a 12-well Transwell system; cells were treated with bone marrow mesenchymal stem cells associated with rhBMP2. Expression of the intracytoplasmic and nuclear markers such as Caspase-3, Bax, Bad, Bcl-2, Ki-67, p53, Oct3/4, Nanog, Stro-1 were performed by flow citometry. Results: We evaluated the regenerative potential of in vitro treatment with rhBMP-2 and found that both osteogenic induction and tumor regression occur in stem cells from canine bone marrow. rhBMP-2 inhibits the proliferation capacity of OST cells by mechanisms of apoptosis and tumor suppression mediated by p53. Conclusion: We propose that rhBMP-2 has great therapeutic potential in bone marrow cells by serving as a tumor suppressor to increase p53 and the pro-apoptotic proteins Bad and Bax, as well as by increasing the activity of phosphorylated caspase 3. Study design: Canine bone marrow mesenchymal stem cells associated with rhBMP2 in canine osteosarcoma treatment: "in vitro" study

Treatment With Methotrexate Inhibits Atherogenesis in Cholesterol-Fed Rabbits

A decrease in the number of cardiovascular events in patients with rheumatoid arthritis or psoriasis treated with methotrexate (MTX) has been observed in the literature. The aim of this study was to test whether MTX could promote anti-inflammatory effects and reduce the atherosclerotic lesions in rabbits with atherosclerosis induced by cholesterol feeding. Twenty male New Zealand rabbits were fed a 1% cholesterol diet for 60 days. Starting from day 30 of cholesterol feeding, 10 animals were treated with 4 weekly intravenous injections of MTX (4 mg/kg) and 10 with 4 weekly saline solution injections for 30 days. MTX reduced the size of the lesion areas of cholesterol-fed animals by 75% and intima-media ratio 2- fold. The drug inhibited macrophage migration into the intima by 50% and the presence of apoptotic cells by 84% but did not inhibit the intimal proliferation of smooth muscle cells. MTX treatment also diminished the positive staining area of metalloproteinase 9 in the intima, which is probably beneficial. In the tumor necrosis factor-alpha-treated human umbilical vein endothelial cell line, incubation with MTX led to downregulation of 5 pro-inflammatory genes, TNF-alpha, VAP-1, IL-1 beta, CXCL2, and TLR2, and upregulation of the antiinflammatory TGF-beta 1 gene, thus showing endothelium-protective properties. In conclusion, MTX showed direct in vivo anti-atherosclerotic action and may have potential in the treatment of this disorder.

The role of cytoskeleton, components of inositol phospholipid signaling pathway and iron in Ehrlichia canis in vitro proliferation

Ehrlichia canis, etiologic agent of Canine Monocytic Ehrlichiosis, is an obligatory intracellular bacterium that parasitizes monocytes and macrophages. In this study we analyzed the role of the cytoskeleton specifically actin microfilaments and microtubules, components of inositol phospholipid signaling pathway such as phospholipase C (PLC), protein kinase (PTK) and calcium channels as well as the role of iron in the E. canis proliferation in DH82 cells. Different inhibitory compounds were used for each component: Cytochalasin D (inhibits actin polymerization), Nocodazole (inhibits microtubule polymerization), Neomycin (PLC inhibitor), Genistein (PTK inhibitor), Verapamil (calcium channel blocker) and Deferoxamine (iron chelator). We observed a significant decrease in the total number of bacteria in infected cells treated suggesting that these cellular components analized are essentials to E. canis proliferation.

Serum amyloid A is a growth factor for 3T3-L1 adipocytes, inhibits differentiation and promotes insulin resistance

BACKGROUND/OBJECTIVES: Serum amyloid A (SAA) is an acute-phase protein that has been recently correlated with obesity and insulin resistance. Therefore, we first examined whether human recombinant SAA (rSAA) could affect the proliferation, differentiation and metabolism of 3T3-L1 preadipocytes. DESIGN: Preadipocytes were treated with rSAA and analyzed for changes in viability and [H-3-methyl]-thymidine incorporation as well as cell cycle perturbations using flow cytometry analysis. The mRNA expression profiles of adipogenic factors during the differentiation protocol were also analyzed using real-time PCR. After differentiation, 2-deoxy-[1,2-H-3]-glucose uptake and glycerol release were evaluated. RESULTS: rSAA treatment caused a 2.6-fold increase in cell proliferation, which was consistent with the results from flow cytometry showing that rSAA treatment augmented the percentage of cells in the S phase (60.9 +/- 0.54%) compared with the control cells (39.8 +/- 2.2%, ***P<0.001). The rSAA-induced cell proliferation was mediated by the ERK1/2 signaling pathway, which was assessed by pretreatment with the inhibitor PD98059. However, the exposure of 3T3-L1 cells to rSAA during the differentiation process resulted in attenuated adipogenesis and decreased expression of adipogenesis-related factors. During the first 72 h of differentiation, rSAA inhibited the differentiation process by altering the mRNA expression kinetics of adipogenic transcription factors and proteins, such as PPAR gamma 2 (peroxisome proliferator-activated receptor gamma 2), C/EBP beta (CCAAT/enhancer-binding protein beta) and GLUT4. rSAA prevented the intracellular accumulation of lipids and, in fully differentiated cells, increased lipolysis and prevented 2-deoxy-[1,2-H-3]-glucose uptake, which favors insulin resistance. Additionally, rSAA stimulated the secretion of proinflammatory cytokines interleukin 6 and tumor necrosis factor alpha, and upregulated SAA3 mRNA expression during adipogenesis. CONCLUSIONS: We showed that rSAA enhanced proliferation and inhibited differentiation in 3T3-L1 preadipocytes and altered insulin sensitivity in differentiated cells. These results highlight the complex role of SAA in the adipogenic process and support a direct link between obesity and its co-morbidities such as type II diabetes.

Retroviral transfer of the p16INK4a cDNA inhibits C6 glioma formation in Wistar rats

Abstract Background The p16INK4A gene product halts cell proliferation by preventing phosphorylation of the Rb protein. The p16INK4a gene is often deleted in human glioblastoma multiforme, contributing to unchecked Rb phosphorylation and rapid cell division. We show here that transduction of the human p16INK4a cDNA using the pCL retroviral system is an efficient means of stopping the proliferation of the rat-derrived glioma cell line, C6, both in tissue culture and in an animal model. C6 cells were transduced with pCL retrovirus encoding the p16INK4a, p53, or Rb genes. These cells were analyzed by a colony formation assay. Expression of p16INK4a was confirmed by immunohistochemistry and Western blot analysis. The altered morphology of the p16-expressing cells was further characterized by the senescence-associated β-galactosidase assay. C6 cells infected ex vivo were implanted by stereotaxic injection in order to assess tumor formation. Results The p16INK4a gene arrested C6 cells more efficiently than either p53 or Rb. Continued studies with the p16INK4a gene revealed that a large portion of infected cells expressed the p16INK4a protein and the morphology of these cells was altered. The enlarged, flat, and bi-polar shape indicated a senescence-like state, confirmed by the senescence-associated β-galactosidase assay. The animal model revealed that cells infected with the pCLp16 virus did not form tumors. Conclusion Our results show that retrovirus mediated transfer of p16INK4a halts glioma formation in a rat model. These results corroborate the idea that retrovirus-mediated transfer of the p16INK4a gene may be an effective means to arrest human glioma and glioblastoma.

Drugs down-regulating E2F-1 expression hinders cell proliferation through a p53-indipendent mechanism

E2F-1 is a transcription factor that plays a key role in cell-cycle control at G1/S check-point level by regulating the timely expression of many target genes whose products are required for S phase entry and progression. In mammalian cells, E2F-1 is negatively regulated by hypo-phosphorylated Retinoblastoma protein (pRb) whereas it is protected against degradation by its binding to Mouse Double Minute 2 protein (MDM2). In this study we experimented a drug combination in order to obtain a strong down-regulation of E2F-1 by acting on two different mechanisms of E2F-1 regulation mentioned above. This was achieved by combining drugs inhibiting the phosphorylation of pRb with drugs inactivating the MDM2 binding capability. The mechanism of action of these drugs in down-regulating E2F-1 level and activity is p53 independent. As expected, when combined, these drugs strongly inhibits E2F-1 and hinder cell proliferation in p53-/- and p53-mutated cells by blocking them in G1 phase of cell cycle, suggesting that E2F-1 down-regulation may represent a valid chemotherapeutic approach to inhibit proliferation in tumors independently of p53 status.

Deletion of CD39 inhibits development of metastatic liver cancer

New vessel formation and tumor infiltrating lymphocytes (TIL) influence host responses to malignant tissues. Extracellular adenosine-mediated pathways promote both vascular endothelial cell proliferation and inhibit cytotoxic T cells, thereby potentiating cancer growth. CD39 is the dominant ectonucleotidase of vascular and T regulatory cells and has the potential to generate high levels of adenosine locally. We have previously shown that deletion of Cd39 results in angiogenic failure and T regulatory cell dysfunction with loss of immune suppressive functions. Aim: Investigate impact of CD39 upon development of hepatic metastases. Methods and Results: We studied the development of metastatic liver deposits following portal vein infusion of 1.5x105 melanoma B16/F10 cells, with luciferase expression, in wild type and Cd39-null C57BL/6 mice (n=24). Tumor formation in liver was directly examined and animals imaged at days 7-17 after tumor cell implantation. As predicted, the formation of hepatic malignant foci was markedly suppressed in Cd39-null mice, at all time points examined. To test whether the major impact of Cd39-deletion was upon neovasculature formation or immune responsiveness, adoptive transfer experiments were conducted. Bone marrow transplants (BMT) from Cd39-null or wild type BL/6 mice were placed in lethally irradiated control and/or null mice, in a crossover manner (total n=24 for each group, respectively). Eight weeks postadoptive transfer, melanoma cells were infused via portal vein as before and tumor growth studied. The Cd39-null mice that received wild type BMT mirrored the wild type phenotype with progressive tumor growth observed (n=8 per time point; p=0.015). In contrast, metastases were significantly inhibited in both number and size and ultimately became necrotic in the wild type mice that had received Cd39-null BMT. Conclusions: Bone marrow derived cells mediate the major inhibitory effects of CD39 deletion on tumor growth. Pharmacological inhibition of CD39 may find utility as an adjunct therapy in the management of hepatic malignancy.

AMR-Me inhibits PI3K/Akt signaling in hormone-dependent MCF-7 breast cancer cells and inactivates NF-κB in hormone-independent MDA-MB-231 cells

AMR-Me, a C-28 methylester derivative of triterpenoid compound Amooranin isolated from Amoora rohituka stem bark and the plant has been reported to possess multitude of medicinal properties. Our previous studies have shown that AMR-Me can induce apoptosis through mitochondrial apoptotic and MAPK signaling pathways by regulating the expression of apoptosis related genes in human breast cancer MCF-7 cells. However, the molecular mechanism of AMR-Me induced apoptotic cell death remains unclear. Our results showed that AMR-Me dose-dependently inhibited the proliferation of MCF-7 and MDA-MB-231 cells under serum-free conditions supplemented with 1 nM estrogen (E2) with an IC50 value of 0.15 µM, 0.45 µM, respectively. AMR-Me had minimal effects on human normal breast epithelial MCF-10A + ras and MCF-10A cells with IC50 value of 6 and 6.5 µM, respectively. AMR-Me downregulated PI3K p85, Akt1, and p-Akt in an ERα-independent manner in MCF-7 cells and no change in expression levels of PI3K p85 and Akt were observed in MDA-MB-231 cells treated under similar conditions. The PI3K inhibitor LY294002 suppressed Akt activation similar to AMR-Me and potentiated AMR-Me induced apoptosis in MCF-7 cells. EMSA revealed that AMR-Me inhibited nuclear factor-kappaB (NF-κB) DNA binding activity in MDA-MB-231 cells in a time-dependent manner and abrogated EGF induced NF-κB activation. From these studies we conclude that AMR-Me decreased ERα expression and effectively inhibited Akt phosphorylation in MCF-7 cells and inactivate constitutive nuclear NF-κB and its regulated proteins in MDA-MB-231 cells. Due to this multifactorial effect in hormone-dependent and independent breast cancer cells AMR-Me deserves attention for use in breast cancer prevention and therapy

Dasatinib inhibits the growth of molecularly heterogeneous myeloid leukemias.

PURPOSE: Dasatinib is a dual Src/Abl inhibitor recently approved for Bcr-Abl+ leukemias with resistance or intolerance to prior therapy. Because Src kinases contribute to multiple blood cell functions by triggering a variety of signaling pathways, we hypothesized that their molecular targeting might lead to growth inhibition in acute myeloid leukemia (AML). EXPERIMENTAL DESIGN: We studied growth factor-dependent and growth factor-independent leukemic cell lines, including three cell lines expressing mutants of receptor tyrosine kinases (Flt3 or c-Kit) as well as primary AML blasts for responsiveness to dasatinib. RESULTS: Dasatinib resulted in the inhibition of Src family kinases in all cell lines and blast cells at approximately 1 x 10(-9) mol/L. It also inhibited mutant Flt3 or Kit tyrosine phosphorylation at approximately 1 x 10(-6) mol/L. Mo7e cells expressing the activating mutation (codon 816) of c-Kit were most sensitive to growth inhibition with a GI(50) of 5 x 10(-9) mol/L. Primary AML blast cells exhibited a growth inhibition of <1 x>10(-6) mol/L. Cell lines that showed growth inhibition at approximately 1 x 10(-6) mol/L showed a G(1) cell cycle arrest and correlated with accumulation of p21 and p27 protein. The addition of rapamycin or cytotoxic agents enhanced growth inhibition. Dasatinib also caused the apoptosis of Mo7e cells expressing oncogenic Kit. CONCLUSIONS: Although all of the precise targets for dasatinib are not known, this multikinase inhibitor causes either growth arrest or apoptosis in molecularly heterogeneous AML. The addition of cytotoxic or targeted agents can enhance its effects.

Survival in patients with high-risk prostate cancer is predicted by miR-221, which regulates proliferation, apoptosis, and invasion of prostate cancer cells by inhibiting IRF2 and SOCS3

A lack of reliably informative biomarkers to distinguish indolent and lethal prostate cancer is one reason this disease is overtreated. miR-221 has been suggested as a biomarker in high-risk prostate cancer, but there is insufficient evidence of its potential utility. Here we report that miR-221 is an independent predictor for cancer-related death, extending and validating earlier findings. By mechanistic investigations we showed that miR-221 regulates cell growth, invasiveness, and apoptosis in prostate cancer at least partially via STAT1/STAT3-mediated activation of the JAK/STAT signaling pathway. miR-221 directly inhibits the expression of SOCS3 and IRF2, two oncogenes that negatively regulate this signaling pathway. miR-221 expression sensitized prostate cancer cells for IFN-γ-mediated growth inhibition. Our findings suggest that miR-221 offers a novel prognostic biomarker and therapeutic target in high-risk prostate cancer.

HRGbeta1 inhibits metastasis of breast cancer cell lines, one of which exhibits lineage infidelity

Recent publications have questioned the origin of the MDA-MB-435 breast cancer cell line and have suggested that it is of melanocyte origin rather than breast epithelial origin. The data presented herein show unequivocally that MDA-MB-435 does express breast epithelial markers and produces milk-specific lipids. The data also indicated that MDA-MB-435 does express some melanocyte proteins but this expression occurs in the same MDA-MB-435 cells that express breast epithelial proteins. Although MDA-MB-435 does not strictly adhere to a breast lineage, it does retain breast specific markers and is thus valid as an experimental cell line in breast cancer studies. ^ Heregulinβ1 (HRGβ1) has been shown to both stimulate and inhibit breast tumorigenic and metasastasic phenotypes. Some studies used only the EGF-like domain of the extracellular domain of HRGβ1 while others used bacterially-expressed HRGβ1. Our in vitro data demonstrated that the full-length extracellular domain of human HRGβ1 reduced clonal growth of MDA-MB-435 breast cancer cells but stimulated apoptosis in MDA-MB-435 and MCF-7 breast cancer cells. In addition, mammalian-expressed HRGβ1 did not dramatically affect matrix metalloproteinase-9 activity but did inhibit cell motility of MDA-MB-435 and MCF-7 cells. Taken together, the in vitro data indicated that HRGβ1 inhibits metastasis-associated properties. ^ The in vivo data demonstrated that inducible expression of the full-length extracellular domain of human HRGβ1 in MDA-MB-435 cells reduced tumor volume and cell proliferation but increased apoptosis of cells injected at the mammary fat pad in nude mice. More importantly, HRGβ1 reduced the number of metastases observed by a spontaneous metastasis assay. Taken together, these data indicate that the full-length extracellular domain of human HRGβ1 has the net effect of inhibiting breast cancer metastasis. ^

Endothelin ETA receptor blockade restores NO-mediated endothelial function and inhibits atherosclerosis in apolipoprotein E-deficient mice

This study investigated whether endothelin-1 (ET-1), a potent vasoconstrictor, which also stimulates cell proliferation, contributes to endothelial dysfunction and atherosclerosis. Apolipoprotein E (apoE)-deficient mice and C57BL/6 control mice were treated with a Western-type diet to accelerate atherosclerosis with or without ETA receptor antagonist LU135252 (50 mg/kg/d) for 30 wk. Systolic blood pressure, plasma lipid profile, and plasma nitrate levels were determined. In the aorta, NO-mediated endothelium-dependent relaxation, atheroma formation, ET receptor-binding capacity, and vascular ET-1 protein content were assessed. In apoE-deficient but not C57BL/6 mice, severe atherosclerosis developed within 30 wk. Aortic ET-1 protein content (P < 0.0001) and binding capacity for ETA receptors was increased as compared with C57BL/6 mice. In contrast, NO-mediated, endothelium-dependent relaxation to acetylcholine (56 ± 3 vs. 99 ± 2%, P < 0.0001) and plasma nitrate were reduced (57.9 ± 4 vs. 93 ± 10 μmol/liter, P < 0.01). Treatment with the ETA receptor antagonist LU135252 for 30 wk had no effect on the lipid profile or systolic blood pressure in apoE-deficient mice, but increased NO-mediated endothelium-dependent relaxation (from 56 ± 3 to 93 ± 2%, P < 0.0001 vs. untreated) as well as circulating nitrate levels (from 57.9 ± 4 to 80 ± 8.3 μmol/liter, P < 0.05). Chronic ETA receptor blockade reduced elevated tissue ET-1 levels comparable with those found in C57BL/6 mice and inhibited atherosclerosis in the aorta by 31% without affecting plaque morphology or ET receptor-binding capacity. Thus, chronic ETA receptor blockade normalizes NO-mediated endothelial dysfunction and reduces atheroma formation independent of plasma cholesterol and blood pressure in a mouse model of human atherosclerosis. ETA receptor blockade may have therapeutic potential in patients with atherosclerosis.

Interferon-β gene therapy inhibits tumor formation and causes regression of established tumors in immune-deficient mice

Despite the potential of type 1 interferons (IFNs) for the treatment of cancer, clinical experience with IFN protein therapy of solid tumors has been disappointing. IFN-β has potent antiproliferative activity against most human tumor cells in vitro in addition to its known immunomodulatory activities. The antiproliferative effect, however, relies on IFN-β concentrations that cannot be achieved by parenteral protein administration because of rapid protein clearance and systemic toxicities. We demonstrate here that ex vivo IFN-β gene transduction by a replication-defective adenovirus in as few as 1% of implanted cells blocked tumor formation. Direct in vivo IFN-β gene delivery into established tumors generated high local concentrations of IFN-β, inhibited tumor growth, and in many cases caused complete tumor regression. Because the mice were immune-deficient, it is likely that the anti-tumor effect was primarily through direct inhibition of tumor cell proliferation and survival. Based on these studies, we argue that local IFN-β gene therapy with replication-defective adenoviral vectors might be an effective treatment for some solid tumors.