Inhibition of 5-lipoxygenase downregulates stemness and kills prostate cancer stem cells by triggering apoptosis via activation of c-Jun N-terminal kinase.

The cancer stem cell (CSC) concept suggests that neoplastic clones are maintained exclusively by a rare group of cells possessed with stem cell properties. CSCs are characterized by features that include self-renewal, pluripotency and tumorigenicity, and are thought to be solely responsible for tumor recurrence and metastasis. A hierarchically organized CSC model is becoming increasingly evident for various types of cancer, including prostate cancer. The CD44 (+), CD133 (+) cell subpopulations were isolated from human prostate tumors which exhibit stem-like properties showing therapeutic-resistance, capacity of self-renewal, and exact recapitulation of the original tumor in vivo. Thus, an important challenge is to find measures to eliminate these cancer stem cells, which will stop tumor growth and prevent disease-recurrence. However, knowledge about molecular features critical for the survival of prostate cancer stem cells (PCSC) is meager. Here we report that inhibition of 5-lipoxygenase (5-Lox) by shRNA or MK591 dramatically kills PCSC by inducing apoptosis, suggesting that 5-Lox plays an essential role in the survival of PCSC. Interestingly, MK591 treatment decreases protein levels and inhibits transcriptional activities of Nanog and c-Myc. Since Nanog and c-Myc play important roles as stemness factors, our findings indicate that the 5-Lox activity plays a causal role in maintaining prostate cancer stemness via regulation of Nanog and c-Myc, and suggest that further exploration of 5-Lox-mediated signaling in PCSC may lead to development of novel, target-based, durable strategies to effectively block development and growth of prostate tumors, and prevent prostate cancer recurrence.

Concentration-independent MRI of pH with a dendrimer-based pH-responsive nanoprobe.

The measurement of extracellular pH (pHe ) has significant clinical value for pathological diagnoses and for monitoring the effects of pH-altering therapies. One of the major problems of measuring pHe with a relaxation-based MRI contrast agent is that the longitudinal relaxivity depends on both pH and the concentration of the agent, requiring the use of a second pH-unresponsive agent to measure the concentration. Here we tested the feasibility of measuring pH with a relaxation-based dendritic MRI contrast agent in a concentration-independent manner at clinically relevant field strengths. The transverse and longitudinal relaxation times in solutions of the contrast agent (GdDOTA-4AmP)44 -G5, a G5-PAMAM dendrimer-based MRI contrast agent in water, were measured at 3 T and 7 T magnetic field strengths as a function of pH. At 3 T, longitudinal relaxivity (r1 ) increased from 7.91 to 9.65 mM(-1) s(-1) (on a per Gd(3+) basis) on changing pH from 8.84 to 6.35. At 7 T, r1 relaxivity showed pH response, albeit at lower mean values; transverse relaxivity (r2 ) remained independent of pH and magnetic field strengths. The longitudinal relaxivity of (GdDOTA-4AmP)44 -G5 exhibited a strong and reversible pH dependence. The ratio of relaxation rates R2 /R1 also showed a linear relationship in a pH-responsive manner, and this pH response was independent of the absolute concentration of (GdDOTA-4AmP)44 -G5 agent. Importantly, the nanoprobe (GdDOTA-4AmP)44 -G5 shows pH response in the range commonly found in the microenvironment of solid tumors.

Effect of Curcumin on Pro-angiogenic Factors in the Xenograft Model of Breast Cancer.

The formation of a new blood vessel is stimulated by angiogenic factors. Curcumin, which is the active ingredient of the spice plant Curcuma longa L and is used as food and traditional medicine, has shown anticancer effects against different types of cancers. We evaluated the effects of curcumin on angiogenesis/pro-angiogenic factors in a mouse model of human breast cancer. Cell viability was measured by the MTT assay after curcumin treatment in triple-negative breast cancer cells (MDA-MB-231). For the in vivo study, human breast cancer was induced in athymic mice and treated with 300 mg/kg/day of curcumin administered intraperitoneally. Tumor size was measured weekly, and the animals underwent single photon emission computed tomography (SPECT) scanning with Tc-99m tagged VEGF-c to detect the in vivo expression of VEGFR2/3. In addition, the expression of proangiogenic/ growth factors in the tumor extracts was evaluated by a membrane antibody array. Histological analysis was performed to confirm the effect of curcumin on neovascularization. The MTT assay showed that curcumin significantly reduced the cell viability of MDA-MB-231 cells. In breast cancer xenografts, curcumin treatment led to a decrease in tumor volume and cell proliferation (Ki-67) compared with the vehicle treated group. Tc-99m-HYNIC-VEGF-c-SPECT imaging showed decreased uptake to the tumor, which may indicate a lower expression of VEGFR2/3 in curcumin treated tumors; however, a statistically significant difference was not achieved (p>0.05). Additionally, curcumin treatment showed a significantly low level of expression of pro-angiogenic factors (p<0.05) and a decrease in micro-vessel density (vWF) in animals compared with that of vehicle treated tumors. In conclusion, curcumin treatment showed effectiveness in reducing tumor growth and cell proliferation, as well as in the inhibition of angiogenesis.

Effect of melatonin on tumor growth and angiogenesis in xenograft model of breast cancer.

As neovascularization is essential for tumor growth and metastasis, controlling angiogenesis is a promising tactic in limiting cancer progression. Melatonin has been studied for their inhibitory properties on angiogenesis in cancer. We performed an in vivo study to evaluate the effects of melatonin treatment on angiogenesis in breast cancer. Cell viability was measured by MTT assay after melatonin treatment in triple-negative breast cancer cells (MDA-MB-231). After, cells were implanted in athymic nude mice and treated with melatonin or vehicle daily, administered intraperitoneally 1 hour before turning the room light off. Volume of the tumors was measured weekly with a digital caliper and at the end of treatments animals underwent single photon emission computed tomography (SPECT) with Technetium-99m tagged vascular endothelial growth factor (VEGF) C to detect in vivo angiogenesis. In addition, expression of pro-angiogenic/growth factors in the tumor extracts was evaluated by membrane antibody array and collected tumor tissues were analyzed with histochemical staining. Melatonin in vitro treatment (1 mM) decreased cell viability (p<0.05). The breast cancer xenografts nude mice treated with melatonin showed reduced tumor size and cell proliferation (Ki-67) compared to control animals after 21 days of treatment (p<0.05). Expression of VEGF receptor 2 decreased significantly in the treated animals compared to that of control when determined by immunohistochemistry (p<0.05) but the changes were not significant on SPECT (p>0.05) images. In addition, there was a decrease of micro-vessel density (Von Willebrand Factor) in melatonin treated mice (p<0.05). However, semiquantitative densitometry analysis of membrane array indicated increased expression of epidermal growth factor receptor and insulin-like growth factor 1 in treated tumors compared to vehicle treated tumors (p<0.05). In conclusion, melatonin treatment showed effectiveness in reducing tumor growth and cell proliferation, as well as in the inhibition of angiogenesis.

Subcurative radiation significantly increases cell proliferation, invasion, and migration of primary glioblastoma multiforme in vivo.

Tumor cell proliferation, infiltration, migration, and neovascularization are known causes of treatment resistance in glioblastoma multiforme (GBM). The purpose of this study was to determine the effect of radiation on the growth characteristics of primary human GBM developed in a nude rat. Primary GBM cells grown from explanted GBM tissues were implanted orthotopically in nude rats. Tumor growth was confirmed by magnetic resonance imaging on day 77 (baseline) after implantation. The rats underwent irradiation to a dose of 50 Gy delivered subcuratively on day 84 postimplantation (n = 8), or underwent no radiation (n = 8). Brain tissues were obtained on day 112 (nonirradiated) or day 133 (irradiated). Immunohistochemistry was performed to determine tumor cell proliferation (Ki-67) and to assess the expression of infiltration marker (matrix metalloproteinase-2, MMP-2) and cell migration marker (CD44). Tumor neovascularization was assessed by microvessel density using von-Willebrand factor (vWF) staining. Magnetic resonance imaging showed well-developed, infiltrative tumors in 11 weeks postimplantation. The proportion of Ki-67-positive cells in tumors undergoing radiation was (71 +/- 15)% compared with (25 +/- 12)% in the nonirradiated group (P = 0.02). The number of MMP-2-positive areas and proportion of CD44-positive cells were also high in tumors receiving radiation, indicating great invasion and infiltration. Microvessel density analysis did not show a significant difference between nonirradiated and irradiated tumors. Taken together, we found that subcurative radiation significantly increased proliferation, invasion, and migration of primary GBM. Our study provides insights into possible mechanisms of treatment resistance following radiation therapy for GBM.

Inhibition of telomerase activity by oleanane triterpenoid CDDO-Me in pancreatic cancer cells is ROS-dependent.

Methyl-2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oate (CDDO-Me) is a synthetic derivative of oleanolic acid, a triterpene, with apoptosis-inducing activity in a wide range of cancer cells. Induction of apoptosis by CDDO-Me is associated with the generation of reactive oxygen species (ROS) and inhibition of telomerase activity. In the present study, we investigated the role of ROS in inhibition of telomerase by CDDO-me. Treatment of MiaPaCa-2 and Panc-1 pancreatic cancer cell lines with CDDO-Me induced the production of hydrogen peroxide and superoxide anions and inhibited the telomerase activity. Pretreatment of cells with N-acetylcycsteine, a general purpose antioxidant or overexpression of glutathione peroxidase (GPx) or superoxide dismutase-1 (SOD-1) blocked the telomerase inhibitory activity of CDDO-Me. Furthermore, blocking ROS generation also prevented the inhibition of hTERT gene expression, hTERT protein production and expression of a number of hTERT-regulatory proteins by CDDO-Me (e.g., c-Myc, Sp1, NF-κB and p-Akt). Data also showed that Akt plays an important role in the activation of telomerase activity. Together, these data suggest that inhibition of telomerase activity by CDDO-Me is mediated through a ROS-dependent mechanism; however, more work is needed to fully understand the role of ROS in down-regulation of hTERT gene and hTERT-regulatory proteins by CDDO-Me.

Effects of tyrosine kinase inhibitors and CXCR4 antagonist on tumor growth and angiogenesis in rat glioma model: MRI and protein analysis study.

The aim of the study was to determine the antiangiogenic efficacy of vatalanib, sunitinib, and AMD3100 in an animal model of human glioblastoma (GBM) by using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and tumor protein expression analysis. Orthotopic GBM-bearing animals were randomly assigned either to control group or vatalanib, sunitinib, and AMD3100 treatment groups. Following 2 weeks of drug treatment, tumor growth and vascular parameters were measured using DCE-MRI. Expression of different angiogenic factors in tumor extracts was measured using a membrane-based human antibody array kit. Tumor angiogenesis and invasion were determined by immunohistochemistry. DCE-MRI showed a significant increase in tumor size after vatalanib treatment. AMD3100-treated group showed a significant decrease in a number of vascular parameters determined by DCE-MRI. AMD3100 significantly decreased the expression of different angiogenic factors compared to sunitinib or vatalanib; however, there were no significant changes in vascular density among the groups. Sunitinib-treated animals showed significantly higher migration of the invasive cells, whereas in both vatalanib- and AMD3100-treated animals the invasive cell migration distance was significantly lower compared to that of control. Vatalanib and sunitinib resulted in suboptimal therapeutic effect, but AMD3100 treatment resulted in a significant reduction in tumor growth, permeability, interstitial space volume, and invasion of tumor cells in an animal model of GBM.

MRI to assess chemoprevention in transgenic adenocarcinoma of mouse prostate (TRAMP).

BACKGROUND: The current method to determine the efficacy of chemoprevention in TRAMP mouse model of carcinoma of prostate (CaP) is by extracting and weighing the prostate at different time points or by immunohistochemistry analysis. Non-invasive determination of volumes of prostate glands and seminal vesicles before, during and after treatment would be valuable in investigating the efficacy of newer chemopreventive agents in CaP. The purpose of this study was to determine whether in vivo magnetic resonance imaging (MRI) using a 3 tesla clinical MRI system can be used to follow the effect of chemoprevention in TRAMP model of mouse CaP. METHODS: Mice were randomized into control and treated groups. The animals in treated group received 10 µmol/kg of CDDO, 5 days a week for 20 weeks. Animals underwent in vivo MRI of prostate gland and seminal vesicles by a clinical 3 Tesla MRI system just before (at 5 weeks), during and at the end of treatment, at 25 weeks. T1-weighted and fat saturation (FATSAT) multiecho fast spin echo T2-weighted images (T2WI) were acquired. Volume of the prostate glands and seminal vesicles was determined from MR images. T2 signal intensity changes in the seminal vesicles were determined by subtracting higher echo time (TE) from lower TE T2WI. Following treatments all animals were sacrificed, prostate and seminal vesicles collected, and the tissues prepared for histological staining. All data were expressed as mean ± 1 standard deviation. Two-way or multivariate analysis of variance followed by post-hoc test was applied to determine the significant differences. A p-value of <0.05 was considered significant. RESULTS: Histological analysis indicated tumor in 100% of control mice, whereas 10% of the treated mice showed tumor in prostate gland. Both MRI and measured prostate weights showed higher volume/weight in control mouse group. MRI showed significantly higher volume of seminal vesicles in control animals and T2 signal intensity changes in seminal vesicles of control mice indicating higher number of tumor foci, which was also proven by histology. CONCLUSIONS: In vivo MRI is helpful in determining the efficacy of chemoprevention of prostate cancer in TRAMP mice.

Differentiation of glioma and radiation injury in rats using in vitro produce magnetically labeled cytotoxic T-cells and MRI.

BACKGROUND: A limitation with current imaging strategies of recurrent glioma undergoing radiotherapy is that tumor and radiation injury cannot be differentiated with post contrast CT or MRI, or with PET or other more complex parametric analyses of MRI data. We propose to address the imaging limitation building on emerging evidence indicating that effective therapy for recurrent glioma can be attained by sensitized T-cells following vaccination of primed dendritic cells (DCs). The purpose of this study was to determine whether cord blood T-cells can be sensitized against glioma cells (U-251) and if these sensitized cytotoxic T-cells (CTLs) can be used as cellular magnetic resonance imaging probes to identify and differentiate glioma from radiation necrosis in rodent models. METHODOLOGY/PRINCIPAL FINDINGS: Cord blood T and CD14+ cells were collected. Isolated CD14+ cells were then converted to dendritic cells (DCs), primed with glioma cell lysate and used to sensitize T-cells. Phenotypical expression of the generated DCs were analyzed to determine the expression level of CD14, CD86, CD83 and HLA-DR. Cells positive for CD25, CD4, CD8 were determined in generated CTLs. Specificity of cytotoxicity of the generated CTLs was also determined by lactate dehydrogenase (LDH) release assay. Secondary proliferation capacity of magnetically labeled and unlabeled CTLs was also determined. Generated CTLs were magnetically labeled and intravenously injected into glioma bearing animals that underwent MRI on days 3 and 7 post- injection. CTLs were also administered to animals with focal radiation injury to determine whether these CTLs accumulated non-specifically to the injury sites. Multi-echo T2- and T2*-weighted images were acquired and R2 and R2* maps created. Our method produced functional, sensitized CTLs that specifically induced U251 cell death in vitro. Both labeled and unlabeled CTLs proliferated equally after the secondary stimulation. There were significantly higher CD25 positive cells (p = <0.006) in CTLs. In addition, T2- and T2*-weighted MR images showed increased low signal intensity areas in animals that received labeled CTLs as compared to the images from animals that received control cells. Histological analysis confirmed the presence of iron positive cells in sites corresponding to MRI low signal intensity regions. Significant differences (p = <0.001) in tumor R2 and R2* values were observed among the groups of animals. Animals with radiation injury exhibited neither MRI hypointense areas nor presence of iron positive cells. CONCLUSION: Our results indicate that T-cells can be effectively sensitized by in vitro methods and used as cellular probes to identify and differentiate glioma from radiation necrosis.

Changes in vascular permeability and expression of different angiogenic factors following anti-angiogenic treatment in rat glioma.

BACKGROUND: Anti-angiogenic treatments of malignant tumors targeting vascular endothelial growth factor receptors (VEGFR) tyrosine kinase are being used in different early stages of clinical trials. Very recently, VEGFR tyrosine kinase inhibitor (Vetanalib, PTK787) was used in glioma patient in conjunction with chemotherapy and radiotherapy. However, changes in the tumor size, tumor vascular permeability, vascular density, expression of VEGFR2 and other angiogenic factors in response to PTK787 are not well documented. This study was to determine the changes in tumor size, vascular permeability, fractional plasma volume and expression of VEGFR2 in PTK787 treated U-251 glioma rat model by in vivo magnetic resonance imaging (MRI) and single photon emission computed tomography (SPECT). The findings were validated with histochemical and western blot studies. METHODOLOGIES AND PRINCIPAL FINDINGS: Seven days after implantation of U251 glioma cells, animals were treated with either PTK787 or vehicle-only for two weeks, and then tumor size, tumor vascular permeability transfer constant (K(trans)), fractional plasma volume (fPV) and expression of VEGFR2 and other relevant angiogenic factors were assessed by in vivo MRI and SPECT (Tc-99-HYNIC-VEGF), and by immunohistochemistry and western blot analysis. Dynamic contrast-enhanced MRI (DCE-MRI) using a high molecular weight contrast agent albumin-(GdDTPA) showed significantly increased K(trans) at the rim of the treated tumors compared to that of the central part of the treated as well as the untreated (vehicle treated) tumors. Size of the tumors was also increased in the treated group. Expression of VEGFR2 detected by Tc-99m-HYNIC-VEGF SPECT also showed significantly increased activity in the treated tumors. In PTK787-treated tumors, histological staining revealed increase in microvessel density in the close proximity to the tumor border. Western blot analysis indicated increased expression of VEGF, SDF-1, HIF-1alpha, VEGFR2, VEGFR3 and EGFR at the peripheral part of the treated tumors compared to that of central part of the treated tumors. Similar expression patters were not observed in vehicle treated tumors. CONCLUSION: These findings indicate that PTK787 treatment induced over expression of VEGF as well as the Flk-1/VEGFR2 receptor tyrosine kinase, especially at the rim of the tumor, as proven by DCE-MRI, SPECT imaging, immunohistochemistry and western blot.