Timothy Turner

Enhanced noscapine delivery using uPAR-targeted optical-MR imaging trackable nanoparticles for prostate cancer therapy

The tubulin-binding anticancer activity of noscapine, an orally available plant-derived anti-tussive alkaloid, has been recently identified. Noscapine inhibits tumor growth in nude mice bearing human xenografts of hematopoietic, breast, lung, ovarian, brain and prostate origin. Despite its nontoxic attributes, significant elimination of the disease has not been achieved, perhaps since the bioavailability of noscapine to tumors saturates at an oral dose of 300 mg/kg body weight. To enable the selective and specific delivery of noscapine to prostate cancer cells, we have engineered a multifunctional nanoscale delivery vehicle that takes advantage of urokinase plasminogen activator receptor (uPAR) overexpression in prostate cancer compared to normal prostate epithelia and can be tracked by magnetic resonance imaging (MRI) and near-infrared (NIR) imaging. Specifically, we employed the human-type 135 amino-acid amino-terminal fragment (hATF) of urokinase plasminogen activator (uPA), a high-affinity natural ligand for uPAR. Noscapine (Nos) was efficiently adsorbed onto the amphiphilic polymer coating of uPAR-targeted nanoparticles (NPs). Nos-loaded NPs were uniformly compact-sized, stable at physiological pH and efficiently released the drug at pH 4 to 5 within a span of 4h. Our results demonstrate that these uPAR-targeted NPs were capable of binding to the receptor and were internalized by PC-3 cells. uPAR-targeted Nos-loaded NPs enhanced intracellular noscapine accumulation as evident by the ~6-fold stronger inhibitory effect on PC-3 growth compared to free noscapine. In addition, Nos-loaded iron oxide NPs maintained their T2 MRI contrast effect upon internalization into tumor cells owing to their significant susceptibility effect in cells. Thus, our data provide compelling evidence that these optically and magnetic resonance imaging (MRI)-trackable uPAR-targeted NPs may offer a great potential for image-directed targeted delivery of noscapine for the management of prostate cancer.

An IP-10 (CXCL10)-Derived Peptide Inhibits Angiogenesis

Angiogenesis plays a critical role in processes such as organ development, wound healing, and tumor growth. It requires well-orchestrated integration of soluble and matrix factors and timely recognition of such signals to regulate this process. Previous work has shown that newly forming vessels express the chemokine receptor CXC receptor 3 (CXCR3) and, activation by its ligand IP-10 (CXCL10), both inhibits development of new vasculature and causes regression of newly formed vessels. To identify and develop new therapeutic agents to limit or reverse pathological angiogenesis, we identified a 21 amino acid fragment of IP-10, spanning the α-helical domain residues 77–98, that mimic the actions of the whole IP-10 molecule on endothelial cells. Treatment of the endothelial cells with the 22 amino acid fragment referred to as IP-10p significantly inhibited VEGF-induced endothelial motility and tube formation in vitro, properties critical for angiogenesis. Using a Matrigel plug assay in vivo, we demonstrate that IP-10p both prevented vessel formation and induced involution of nascent vessels. CXCR3 neutralizing antibody was able to block the inhibitory effects of the IP-10p, demonstrating specificity of the peptide. Inhibition of endothelial function by IP-10p was similar to that described for IP-10, secondary to CXCR3-mediated increase in cAMP production, activation of PKA inhibiting cell migration, and inhibition of VEGF-mediated m-calpain activation. IP-10p provides a novel therapeutic agent that inhibits endothelial cell function thus, allowing for the modulation of angiogenesis.

LHRH-conjugated lytic peptides directly target prostate cancer cells.

Prostate cancer is the second leading cause of cancer deaths among men. For patients with hormone-refractory disease, few treatments are available once the tumor has metastasized beyond the prostate. In the present study, two conjugated lytic peptide sequences (named JCHLHRH and JC21LHRH) were designed to target luteinizing hormone-releasing hormone receptors (LHRH-R). Our results indicate that human prostate cancer cell lines were sensitive to both LHRH-conjugated and non-conjugated lytic peptides, with IC(50) concentrations for LNCaP cells, 4.4 and 9.1μM; for DU-145 cells, 4.8 and 5.7μM; and for PC-3 cells, 4.4 and 8.2μM, respectively. JCHLHRH and JC21LHRH were nontoxic to normal primary human prostate epithelial cells or to bone marrow stromal cells in co-culture. There were morphological changes in PC-3 cells after 3h of exposure to either peptide; after 6h, there were significant reductions in cell numbers. Exposure of PC-3 cells for 24h to either JCHLHRH or JC21LHRH blocked their growth over 3 days. Since JCHLHRH and JC21LHRH have specificity for and anti-proliferative activity against tumor cells, and low toxicity for normal prostate cells, these peptides could serve as a new type of therapy for prostate cancer.

Tumor-Stromal Interactions Influence Radiation Sensitivity in Epithelial- versus Mesenchymal-Like Prostate Cancer Cells

HS-27a human bone stromal cells, in 2D or 3D coultures, induced cellular plasticity in human prostate cancer ARCaPE and ARCaPM cells in an EMT model. Cocultured ARCaPE or ARCaPM cells with HS-27a, developed increased colony forming capacity and growth advantage, with ARCaPE exhibiting the most significant increases in presence of bone or prostate stroma cells. Prostate (Pt-N or Pt-C) or bone (HS-27a) stromal cells induced significant resistance to radiation treatment in ARCaPE cells compared to ARCaPM cells. However pretreatment with anti-E-cadherin antibody (SHEP8-7) or anti-alpha v integrin blocking antibody (CNT095) significantly decreased stromal cell-induced radiation resistance in both ARCaPE- and ARCaPM-cocultured cells. Taken together the data suggest that mesenchymal-like cancer cells reverting to epithelial-like cells in the bone microenvironment through interaction with bone marrow stromal cells and reexpress E-cadherin. These cell adhesion molecules such as E-cadherin and integrin alpha v in cancer cells induce cell survival signals and mediate resistance to cancer treatments such as radiation.

Nuclear Kaiso Indicates Aggressive Prostate Cancers and Promotes Migration and Invasiveness of Prostate Cancer Cells

Kaiso, a p120 catenin-binding protein, is expressed in the cytoplasmic and nuclear compartments of cells; however, the biological consequences and clinical implications of a shift between these compartments have yet to be established. Herein, we report an enrichment of nuclear Kaiso expression in cells of primary and metastatic prostate tumors relative to the normal prostate epithelium. Nuclear expression of Kaiso correlates with Gleason score (P < 0.001) and tumor grade (P < 0.001). There is higher nuclear expression of Kaiso in primary tumor/normal matched samples and in primary tumors from African American men (P < 0.0001). We further found that epidermal growth factor (EGF) receptor up-regulates Kaiso at the RNA and protein levels in prostate cancer cell lines, but more interestingly causes a shift of cytoplasmic Kaiso to the nucleus that is reversed by the EGF receptor-specific kinase inhibitor, PD153035. In both DU-145 and PC-3 prostate cancer cell lines, Kaiso inhibition (short hairpin RNA-Kaiso) decreased cell migration and invasion even in the presence of EGF. Further, Kaiso directly binds to the E-cadherin promoter, and inhibition of Kaiso in PC-3 cells results in increased E-cadherin expression, as well as re-establishment of cell-cell contacts. In addition, Kaiso-depleted cells show more epithelial morphology and a reversal of the mesenchymal markers N-cadherin and fibronectin. Our findings establish a defined oncogenic role of Kaiso in promoting the progression of prostate cancer.

Clinical and Biological Significance of KISS1 Expression in Prostate Cancer

For men in the United States, prostate cancer (PCa) is the most frequent malignancy and the second leading cause of cancer mortality. The metastatic spread of PCa is responsible for most deaths related to PCa. Although KISS1 functions as a metastasis suppressor in various cancers, its expression levels and functions in PCa development and progression remain undetermined. The goals of this study were to correlate the expression levels of KISS1 in PCas with clinicopathologic characteristics and to assess the biological relevance of KISS1 to the viability and motility of PCa cells. Strong KISS1 staining was detected in benign prostate tissues, but the staining was weaker in primary and metastatic PCas (both P < 0.001, t-test). Furthermore, the low expression levels of KISS1 in PCas correlated with clinical stage (P < 0.01) and with KISS1R expression (P < 0.001). Overexpression of full-length KISS1 in low KISS1-expressing PC-3M cells, but not KFMΔSS, which lacks the secretion signal sequence, induced re-sensitization of cells to anoikis, although it had no effect on either cell proliferation or apoptosis. Overexpression of KISS1 also suppressed steps in the metastatic cascade, including motility and invasiveness. Moreover, cells overexpressing KISS1 were found to enhance chemosensitivity to paclitaxel. Collectively, our data suggest that KISS1 functions as a metastasis suppressor in PCas and may serve as a useful biomarker as well as a therapeutic target for aggressive PCas.

Molecular cycloencapsulation augments solubility and improves therapeutic index of brominated noscapine in prostate cancer cells

We have previously shown that a novel microtubule-modulating noscapinoid, EM011 (9-Br-Nos), displays potent anticancer activity by inhibition of cellular proliferation and induction of apoptosis in prostate cancer cells and preclinical mice models. However, physicochemical and cellular barriers encumber the development of viable formulations for future clinical translation. To circumvent these limitations, we have synthesized EM011-cyclodextrin inclusion complexes to improve solubility and enhance therapeutic index of EM011. Phase solubility analysis indicated that EM011 formed a 1:1 stoichiometric complex with β-CD and methyl-β-CD, with a stability constant (K(c)) of 2.42 × 10(-3) M and 4.85 × 10(-3) M, respectively. Fourier transform infrared spectroscopy suggested the penetrance of either a O-CH(2) or OCH(3)-C(6)H(4)-OCH(3) moiety of EM011 in the β-CD or methyl-β-CD cavity. In addition, multifarious techniques, namely, differential scanning calorimetry, powder X-ray diffraction, scanning electron microscopy, NMR spectroscopy, and computational studies validated the cage complex of EM011 with β-CD and methyl-β-CD. Moreover, rotating frame overhauser enhancement spectroscopy showed that the H(a) proton of the OCH(3)-C(6)H(4)-OCH(3) moiety was in close proximity with H3 proton of the β-CD or methyl-β-CD cavity. Furthermore, we found that the solubility of EM011 in phosphate buffer saline (pH 7.4) was enhanced by ~11 fold and ~21 fold upon complexation with β-CD and methyl-β-CD, respectively. The enhanced dissolution of the drug CD-complexes in aqueous phase remarkably decreased their IC(50) to 28.5 μM (9-Br-Nos-β-CD) and 12.5 μM (9-Br-Nos-methyl-β-CD) in PC-3 cells compared to free EM011 (~200 μM). This is the first report to demonstrate the novel construction of cylcodextrin-based nanosupramolecular vehicles for enhanced delivery of EM011 that warrants in vivo evaluation for the superior management of prostate cancer.

Cyclodextrin Complexes of Reduced Bromonoscapine in Guar Gum Microspheres Enhance Colonic Drug Delivery

Here, we report improved solubility and enhanced colonic delivery of reduced bromonoscapine (Red-Br-Nos), a cyclic ether brominated analogue of noscapine, upon encapsulation of its cyclodextrin (CD) complexes in bioresponsive guar gum microspheres (GGM). Phase–solubility analysis suggested that Red-Br-Nos complexed with β-CD and methyl-β-CD in a 1:1 stoichiometry, with a stability constant (Kc) of 2.29 × 103 M–1 and 4.27 × 103 M–1. Fourier transforms infrared spectroscopy indicated entrance of an O–CH2 or OCH3–C6H4–OCH3 moiety of Red-Br-Nos in the β-CD or methyl-β-CD cavity. Furthermore, the cage complex of Red-Br-Nos with β-CD and methyl-β-CD was validated by several spectral techniques. Rotating frame Overhauser enhancement spectroscopy revealed that the Ha proton of the OCH3–C6H4–OCH3 moiety was closer to the H5 proton of β-CD and the H3 proton of the methyl-β-CD cavity. The solubility of Red-Br-Nos in phosphate buffer saline (PBS, pH ∼ 7.4) was improved by ∼10.7-fold and ∼21.2-fold when mixed with β-CD and methyl-β-CD, respectively. This increase in solubility led to a favorable decline in the IC50 by ∼2-fold and ∼3-fold for Red-Br-Nos−β-CD-GGM and Red-Br-Nos–methyl-β-CD-GGM formulations respectively, compared to free Red-Br-Nos−β-CD and Red-Br-Nos–methyl-β-CD in human colon HT-29 cells. GGM-bearing drug complex formulations were found to be highly cytotoxic to the HT-29 cell line and further effective with simultaneous continuous release of Red-Br-Nos from microspheres. This is the first study to showing the preparation of drug-complex loaded GGMS for colon delivery of Red-Br-Nos that warrants preclinical assessment for the effective management of colon cancer.

Predictors of 4-year retention among African American and white community-dwelling participants in the UAB study of aging.

PURPOSE To identify racial/ethnic differences in retention of older adults at 3 levels of participation in a prospective observational study: telephone, in-home assessments, and home visits followed by blood draws. DESIGN AND METHODS A prospective study of 1,000 community-dwelling Medicare beneficiaries aged 65 years and older included a baseline in-home assessment and telephone follow-up calls at 6-month intervals; at 4 years, participants were asked to complete an additional in-home assessment and have blood drawn. RESULTS After 4 years, 21.7% died and 0.7% withdrew, leaving 776 participants eligible for follow-up (49% African American; 46% male; 51% rural). Retention for telephone follow-up was 94.5% (N = 733/776); 624/733 (85.1%) had home interviews, and 408/624 (65.4%) had a nurse come to the home for the blood draw. African American race was an independent predictor of participation in in-home assessments, but African American race and rural residence were independent predictors of not participating in a blood draw. IMPLICATIONS Recruitment efforts designed to demonstrate respect for all research participants, home visits, and telephone follow-up interviews facilitate high retention rates for both African American and White older adults; however, additional efforts are required to enhance participation of African American and rural participants in research requiring blood draws.

The flavonoid quercetin transiently inhibits the activity of taxol and nocodazole through interference with the cell cycle.

Quercetin is a flavonoid with anticancer properties. In this study, we examined the effects of quercetin on cell cycle, viability, and proliferation of cancer cells, either singly or in combination with the microtubule-targeting drugs taxol and nocodazole. Although quercetin induced cell death in a dose-dependent manner, 12.5–50 μM quercetin inhibited the activity of both taxol and nocodazole to induce G2/M arrest in various cell lines. Quercetin also partially restored drug-induced loss in viability of treated cells for up to 72 h. This antagonism of microtubule-targeting drugs was accompanied by a delay in cell cycle progression and inhibition of the buildup of cyclin-B1 at the microtubule organizing center of treated cells. However, quercetin did not inhibit the microtubule targeting of taxol or nocodazole. Despite the short-term protection of cells by quercetin, colony formation and clonogenicity of HCT116 cells were still suppressed by quercetin or quercetin-taxol combination. The status of cell adherence to growth matrix was critical in determining the sensitivity of HCT116 cells to quercetin. We conclude that although long-term exposure of cancer cells to quercetin may prevent cell proliferation and survival, the interference of quercetin with cell cycle progression diminishes the efficacy of microtubule-targeting drugs to arrest cells at G2/M.