Human Prostate Tumor Cells Research Articles

Comparison between endothelial and tumor cells in the response to verteporfin-photodynamic therapy and a PI3K pathway inhibitor

BackgroundPhotodynamic therapy (PDT) is an established cancer treatment. Molecular-targeted agents targeting phosphatidylinositol 3-kinase (PI3K) pathway is showing great promise as anticancer drugs. This study compared SVEC mouse endothelial and PC-3 human prostate tumor cells in the response to verteporfin-mediated photodynamic therapy (PDT) and a pan-PI3K pathway inhibitor LY294002. MethodsVerteporfin cellular uptake and intracellular localization was determined by spectrofluorometry and confocal fluorescence microscopy, respectively, in the SVEC and PC-3 cells. Cytotoxicity induced by LY294002 and verteporfin-PDT was assessed by the MTS assay. Effects of treatments on cell survival and death signaling were examined by Western blot analysis. ResultsPC-3 cells had a higher cellular uptake of verteporfin than SVEC cells at 15min after incubation with verteporfin. Verteporfin was mainly localized in mitochondria in both SVEC and PC-3 cells. Verteporfin-PDT alone as well as PDT in combination with LY294002 induced more apoptosis and caused more reduction in cell viability in SVEC cells than in PC-3 cells. PC-3 cells exhibited a higher level of anti-apoptotic Bcl-2 family proteins than SVEC cells. ConclusionsSVEC cells were more responsive to verteporfin-PDT and PI3K pathway inhibitor LY294002 than PC-3 cells. Such differences in response were likely due to differences in Bcl-2 family protein level. These results support tumor vascular targeting by verteporfin-PDT and its therapeutic enhancement by PI3K pathway inhibition.

Phytochemicals in Cancer Prevention and Therapy.

Despite advances in modern medicine, cancer is still the major cause of mortality in both developing and developed countries. Search for safer and more effective chemoprevention and treatment strategy is a need for the improvement of patient care in the field. Prevention may be more effective and less costly because cancer is largely a preventable disease which could be attributed to a greater extent to lifestyle. Dietary phytochemicals have been used for the treatment of cancer throughout history due to their safety, low toxicity, and general availability. Population based studies suggest that a reduced risk of cancer is associated with high consumption of vegetables and fruits. Promising phytochemicals not only disrupt aberrant signaling pathways leading to cancer but also synergize with chemotherapy and radiotherapy. Thus, the cancer chemoprevention and therapeutic potential of naturally occurring phytochemicals are of great interest. In this special issue we have collected many interesting original research articles and reviews that provide solid evidence to support the application of phytochemicals or dietary agents in prevention and treatment of cancer. This special issue contains 3 review articles and 9 original peer-reviewed papers. A. M. Harrison et al. performed a systematic review of the biomedical literature for the use of phytochemicals for management of cancer therapy pain in human subjects; X.-Y. Chen et al. explored the molecular mechanisms underlying the antimetastatic activity of black rice anthocyanins and identified its molecular targets in HER2+ breast cancer cells; the study by Y. Zeng et al. reports that Southwest China (especially Yunnan and Tibet) is the center of lowest mortality of cancers in China based on coevolution between human's anticancer activities and functional foods from crop origin center; M. Sugata et al. studied the anti-inflammatory and anticancer activities of Taiwanese purple-fleshed sweet potatoes (Ipomoea batatas L. Lam.) extracts; R. Moo-Puc et al. investigated the antiproliferative activity of bonediol, an alkyl catechol isolated from the Mayan medicinal plant Bonellia macrocarpa against human prostate tumor cells; B. Moyo and S. Mukanganyama demonstrate the antiproliferative potential of T. welwitschii extract on Jurkat T cells; C.-J. Tai et al. report the potential of ethanolic extract of Taiwanofungus camphoratus (Antrodia camphorata) to enhance the cytotoxicity of cisplatin and doxorubicin on human hepatocellular carcinoma cells; G. Wang et al. explored the molecular mechanism of total flavonoids extracted from Cotinus coggygria against glioblastoma cancer in vitro and in vivo; M. N. Mallick et al. studied the anticancer activity of hydroalcoholic extract of Picrorhiza kurroa and its fractions; M. F. Abu Bakar et al. demonstrate that the Garcinia dulcis fruit extract induced cytotoxicity and apoptosis in HepG2 liver cancer cells; G. Weng et al. reported the curcumin enhanced busulfan-induced apoptosis in leukemia stem-like KG1a cells via downregulating the expression of survivin; S. Kumar and J. Kim in their review discuss potency and selectivity of PLK-1-targeted inhibitors and their molecular interactions with PLK-1 domains. In conclusion, this special issue discussed the potential anticancer phytochemicals and dietary agents, their molecular targets, and their mechanisms of actions. The understanding of molecular mechanism of a specific plant derived compound against a particular type of cancer will lead to the invention of novel drug and drug targets for therapeutic intervention.

Antiproliferative and Antiestrogenic Activities of Bonediol an Alkyl Catechol from Bonellia macrocarpa.

The purpose of this study was to investigate antiproliferative activity of bonediol, an alkyl catechol isolated from the Mayan medicinal plant Bonellia macrocarpa. Bonediol was assessed for growth inhibition of androgen-sensitive (LNCaP), androgen-insensitive (PC-3), and metastatic androgen-insensitive (PC-3M) human prostate tumor cells; toxicity on normal cell line (HEK 293) was also evaluated. Hedgehog pathway was evaluated and competitive 3H-estradiol ligand binding assay was performed. Additionally, antioxidant activity on Nrf2-ARE pathway was evaluated. Bonediol induced a growth inhibition on prostate cancer cell lines (IC50 from 8.5 to 20.6 µM). Interestingly, bonediol binds to both estrogen receptors (ERα (2.5 µM) and ERβ (2.1 µM)) and displaces the native ligand E2 (17β-estradiol). No significant activity was found in the Hedgehog pathway. Additionally, activity of bonediol on Nrf2-ARE pathway suggested that bonediol could induce oxidative stress and activation of detoxification enzymes at 1 µM (3.8-fold). We propose that the compound bonediol may serve as a potential chemopreventive treatment with therapeutic potential against prostate cancer.

The systemic delivery of an oncolytic adenovirus expressing decorin inhibits bone metastasis in a mouse model of human prostate cancer.

In an effort to develop a new therapy for prostate cancer bone metastases, we have created Ad.dcn, a recombinant oncolytic adenovirus carrying the human decorin gene. Infection of PC-3 and DU-145, the human prostate tumor cells, with Ad.dcn or a non-replicating adenovirus Ad(E1-).dcn resulted in decorin expression; Ad.dcn produced high viral titers and cytotoxicity in human prostate tumor cells. Adenoviral-mediated decorin expression inhibited Met, the Wnt/β-catenin signaling axis, vascular endothelial growth factor A, reduced mitochondrial DNA levels, and inhibited tumor cell migration. To examine the anti-tumor response of Ad.dcn, PC-3-luc cells were inoculated in the left heart ventricle to establish bone metastases in nude mice. Ad.dcn, in conjunction with control replicating and non-replicating vectors were injected via tail vein. The real-time monitoring of mice, once a week, by bioluminescence imaging and X-ray radiography showed that Ad.dcn produced significant inhibition of skeletal metastases. Analyses of the mice at the terminal time point indicated a significant reduction in the tumor burden, osteoclast number, serum TRACP 5b levels, osteocalcin levels, hypercalcemia, inhibition of cancer cachexia, and an increase in the animal survival. Based on these studies, we believe that Ad.dcn can be developed as a potential new therapy for prostate cancer bone metastasis.

Androgen deprivation therapy increases the sensitivity of human prostate carcinoma cells to T cell-mediated lysis through an androgen receptor dependent mechanism

Despite recent advances in diagnosis and therapy, prostate cancer remains the most frequently diagnosed non-skin cancer in the United States and the third leading cause of cancer deaths. Failure of chemotherapies and hormone-deprivation therapies is the major cause of death in patients with castration-resistant prostate cancer (CRPC). Currently, the androgen inhibitors Enzalutamide and Abiraterone are approved for treatment of metastatic CRPC. Enzalutamide is a second-generation androgen antagonist with no agonist activity. Abiraterone is a cytochrome P450 inhibitor that blocks adrenal and intratumoral androgen production. Here we show for the first time that both Enzalutamide and Abiraterone induce immunogenic modulation in human prostate tumor cells, rendering them more sensitive to T cell-mediated lysis, and that these immunomodulatory activities are androgen receptor (AR)-dependent. In studies reported here, exposure in vitro and in vivo of human prostate carcinoma cells to Enzalutamide significantly down-regulated the anti-apoptotic NAIP gene. Functional analysis revealed that NAIP played a critical role in inducing CTL sensitivity. Amplification of AR is a major mechanism of resistance to androgen-deprivation therapy (ADT). Here, we show that Enzalutamide enhances sensitivity to immune-mediated killing of prostate tumor cells that overexpress AR. The immunomodulatory properties of Enzalutamide and Abiraterone provide a rationale for their use in combination with immunotherapeutic agents in CRPC, particularly for patients with minimal response to Enzalutamide or Abiraterone alone, or for patients who have developed resistance to ADT.

Pretargeted dual-modality immuno-SPECT and near-infrared fluorescence imaging for image-guided surgery of prostate cancer.

Radical removal of malignant lesions may be improved using tumor-targeted dual-modality probes that contain both a radiotracer and a fluorescent label to allow for enhanced intraoperative delineation of tumor resection margins. Because pretargeting strategies yield high signal-to-background ratios, we evaluated the feasibility of a pretargeting strategy for intraoperative imaging in prostate cancer using an anti-TROP-2 x anti-HSG bispecific antibody (TF12) in conjunction with the dual-labeled diHSG peptide (RDC018) equipped with both a DOTA chelate for radiolabeling purposes and a fluorophore (IRdye800CW) to allow near-infrared optical imaging. Nude mice implanted s.c. with TROP-2-expressing PC3 human prostate tumor cells or with PC3 metastases in the scapular and suprarenal region were injected i.v. with 1 mg of TF12 and, after 16 hours of tumor accumulation and blood clearance, were subsequently injected with 10 MBq, 0.2 nmol/mouse of either (111)In-RDC018 or (111)In-IMP288 as a control. Two hours after injection, both microSPECT/CT and fluorescence images were acquired, both before and after resection of the tumor nodules. After image acquisition, the biodistribution of (111)In-RDC018 and (111)In-IMP288 was determined and tumors were analyzed immunohistochemically. The biodistribution of the dual-label RDC018 showed specific accumulation in the TROP-2-expressing PC3 tumors (12.4 ± 3.7% ID/g at 2 hours postinjection), comparable with (111)In-IMP288 (9.1 ± 2.8% ID/g at 2 hours postinjection). MicroSPECT/CT and near-infrared fluorescence (NIRF) imaging confirmed this TROP-2-specific uptake of the dual-label (111)In-RDC018 in both the s.c. and metastatic growing tumor model. In addition, PC3 metastases could be visualized preoperatively with SPECT/CT and could subsequently be resected by image-guided surgery using intraoperative NIRF imaging, showing the preclinical feasibility of pretargeted dual-modality imaging approach in prostate cancer.

Abstract 2092: Elevated PKCα expression and its association with Notch signaling in localized and metastatic prostate cancers

Abstract Background: Conventional protein Kinase C alpha (PKCα) is linked to the regulation of cell proliferation, differentiation, motility, survival, and apoptosis. Elevated PKCα expression has been reported in human early prostate cancer (PCa) and rat prostatic tumor cell lines. In this study, the expression level of PKCα was evaluated by immunohistochemistry (IHC) and correlated with the expression levels of Notch1 and Jagged1 in localized and metastatic PCa. Methods: : IHC for PKCα was performed on tissue microarray (TMA) slides containing 156 formalin-fixed and paraffin-embedded (FFPE) prostatic tissues specimens, including 39 benign prostatic changes, 97 localized prostate cancers and 20 metastatic prostate cancers. The expression level of PKCα was reported as a final IHC score calculated as staining extent score (0-3) multiplied by intensity score (0-3) with a maximal score of 9. ANOVA was employed to reveal the differences in IHC scores among pathologic categories, followed by Tukey-adjusted pairwise comparisons. Spearman correlation coefficient was evaluated to measure correlation between expression levels of PKCα, Notch1, and Jagged1. Results: IHC staining for PKCα was predominantly observed in cytoplasm (in 94% specimens). The score of PKCα cytoplasmic staining in metastatic PCa (5.48 ± 1.84, Mean ±SD) was significantly higher than those in localized PCa (3.30 ± 2.10, P = 2.95E-05) and in benign prostatic tissue (1.92 ± 1.53, P = 6.45E-09). Similarly, IHC score in localized PCa was significantly higher than that observed in benign prostatic changes (P = 0.001). Correlation analysis revealed that the expression level of cytoplasmic PKCα was significantly correlated with the expression levels of cytoplasmic Notch1 (r = 0.396, P = 5.85E-05), cytoplasmic Jagged1 (r = 0.610, P = 3.42E-11), and membranous Jagged1 (r = 0.354, P = 3.76E-04, respectively) in localized and metastatic prostate cancers. Conclusions: Our results indicate that up-regulation of PKCα plays a role not only in PCa formation and progression, but also in PCa metastasis. The evidence of significant correlation between PKCα and Jagged1-Notch1 signaling pathway supports the notion that synergy of PKCα with Jagged1-Notch1 signaling may play an important role in modulating the tumorgenesis and metastasis in human PCa. Citation Format: Xinchun Zhou, He Zhu, Xu Zhang, Jack Lewin, Lucio Miele. Elevated PKCα expression and its association with Notch signaling in localized and metastatic prostate cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2092. doi:10.1158/1538-7445.AM2014-2092

Abstract 1566: Notch-1 regulation of the PTEN - mTOR axis in prostate

Abstract Prostate cancer affects one in three men over the age of 60. Loss of expression or function of PTEN is the most commonly observed molecular defect in human prostate cancer. Thirty to 70% of clinical cases exhibit loss of this critical tumor suppressor. PTEN is an essential negative regulator of PI3K/Akt signaling. Together with the mTOR pathway, the PTEN/PI3K/Akt cascade forms a network for cellular responses to growth factors and nutrients. Studies in mice have demonstrated that small changes in PTEN dose can influence cancer development. Thus, understanding the regulation of PTEN expression and function is key to understanding tumor progression. We have previously reported that signaling through the Notch-1 receptor pathway results in increased transcriptional expression of the tumor suppressor PTEN. We further reported that Notch-1 signaling was lost in the tumor foci of clinical prostate cancer cases as compared to the surrounding benign tissue. Herein, we report that Notch-1 signaling alters the pool of active, unphosphorylated PTEN in the human metastatic prostate tumor cell line DU145. Retroviral transduction was used to generate DU145 cells that express constitutively active Notch-1 (DU/ICN1). In cells with constitutively active Notch-1 (DU/ICN1), the pool of unphosphorylated PTEN was increased roughly 2.5 fold compared to the vector only control. Constitutive Notch-1 signaling also resulted in decreased mTOR signaling as determined by a decrease in phosphorlyated 4E-BP1 and phosphorylated S6 ribosomal protein. Decreased expression of Raptor and Rictor and decreased phosphorylation of mTOR were also observed in the presence of constitutively active Notch-1. To test if Notch-1 signaling influences tumor engraftment and growth in a syngeneic model, the tumorigenic C2 cell line, derived from TRAMP mice, was transduced with constitutively active Notch-1. C2/ICN1 and parental C2 cells were engrafted into C57/BL6 mice. All recipients of the parental C2 cell line developed tumors within 46 days, whereas none that received cells expressing constitutively active Notch-1 (C2/ICN1) had developed tumors. Only after 61 days did one of the four mice that received C2/ICN1 cells develop tumors. We next tested if loss of Notch-1 expression promotes characteristics associated with tumorigenicity by using lentiviral transduction to knock down endogenous Notch-1 expression in DU145 cells (DU/shN1). DU145 cells with loss of Notch-1 (DU/shN1) exhibited decreased expression of PTEN protein and a decreased ability to migrate in transwell experiments as compared with control cells expressing endogenous Notch-1. Collectively, these data indicate a role for Notch-1 receptor signaling in modulating the activity of PI3K/Akt and mTOR axis through regulation of the PTEN tumor suppressor, and suggest a mechanistic basis for Notch-1 tumor suppressive activity in prostate cells. Citation Format: Jennifer M. Nutter, C William Angus, Fred E. Bertrand. Notch-1 regulation of the PTEN - mTOR axis in prostate. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1566. doi:10.1158/1538-7445.AM2014-1566

Androgen deprivation therapy sensitizes prostate cancer cells to T-cell killing through androgen receptor dependent modulation of the apoptotic pathway.

Despite recent advances in diagnosis and management, prostrate cancer remains the second most common cause of death from cancer in American men, after lung cancer. Failure of chemotherapies and hormone-deprivation therapies is the major cause of death in patients with castration-resistant prostate cancer (CRPC). Currently, the androgen inhibitors enzalutamide and abiraterone are approved for treatment of metastatic CRPC. Here we show for the first time that both enzalutamide and abiraterone render prostate tumor cells more sensitive to T cell-mediated lysis through immunogenic modulation, and that these immunomodulatory activities are androgen receptor (AR)-dependent. In studies reported here, the NAIP gene was significantly down-regulated in human prostate tumor cells treated in vitro and in vivo with enzalutamide. Functional analysis revealed that NAIP played a critical role in inducing CTL sensitivity. Amplification of AR is a major mechanism of resistance to androgen-deprivation therapy (ADT). Here, we show that enzalutamide enhances sensitivity to immune-mediated killing of prostate tumor cells that overexpress AR. The immunomodulatory properties of enzalutamide and abiraterone provide a rationale for their use in combination with immunotherapeutic agents in CRPC, especially for patients with minimal response to enzalutamide or abiraterone alone, or for patients who have developed resistance to ADT.

Protein kinase C-delta inactivation inhibits the proliferation and survival of cancer stem cells in culture and in vivo

BackgroundA subpopulation of tumor cells with distinct stem-like properties (cancer stem-like cells, CSCs) may be responsible for tumor initiation, invasive growth, and possibly dissemination to distant organ sites. CSCs exhibit a spectrum of biological, biochemical, and molecular features that are consistent with a stem-like phenotype, including growth as non-adherent spheres (clonogenic potential), ability to form a new tumor in xenograft assays, unlimited self-renewal, and the capacity for multipotency and lineage-specific differentiation. PKCδ is a novel class serine/threonine kinase of the PKC family, and functions in a number of cellular activities including cell proliferation, survival or apoptosis. PKCδ has previously been validated as a synthetic lethal target in cancer cells of multiple types with aberrant activation of Ras signaling, using both genetic (shRNA and dominant-negative PKCδ mutants) and small molecule inhibitors. In contrast, PKCδ is not required for the proliferation or survival of normal cells, suggesting the potential tumor-specificity of a PKCδ-targeted approach.MethodsshRNA knockdown was used validate PKCδ as a target in primary cancer stem cell lines and stem-like cells derived from human tumor cell lines, including breast, pancreatic, prostate and melanoma tumor cells. Novel and potent small molecule PKCδ inhibitors were employed in assays monitoring apoptosis, proliferation and clonogenic capacity of these cancer stem-like populations. Significant differences among data sets were determined using two-tailed Student’s t tests or ANOVA.ResultsWe demonstrate that CSC-like populations derived from multiple types of human primary tumors, from human cancer cell lines, and from transformed human cells, require PKCδ activity and are susceptible to agents which deplete PKCδ protein or activity. Inhibition of PKCδ by specific genetic strategies (shRNA) or by novel small molecule inhibitors is growth inhibitory and cytotoxic to multiple types of human CSCs in culture. PKCδ inhibition efficiently prevents tumor sphere outgrowth from tumor cell cultures, with exposure times as short as six hours. Small-molecule PKCδ inhibitors also inhibit human CSC growth in vivo in a mouse xenograft model.ConclusionsThese findings suggest that the novel PKC isozyme PKCδ may represent a new molecular target for cancer stem cell populations.

Pharmacokinetic and Biodistribution Assessment of a Near Infrared-Labeled PSMA-Specific Small Molecule in Tumor-Bearing Mice.

Prostate cancer is the most frequently diagnosed cancer in men and often requires surgery. Use of near infrared (NIR) technologies to perform image-guided surgery may improve accurate delineation of tumor margins. To facilitate preclinical testing of such outcomes, here we developed and characterized a PSMA-targeted small molecule, YC-27. IRDye 800CW was conjugated to YC-27 or an anti-PSMA antibody used for reference. Human 22Rv1, PC3M-LN4, and/or LNCaP prostate tumor cells were exposed to the labeled compounds. In vivo targeting and clearance properties were determined in tumor-bearing mice. Organs and tumors were excised and imaged to assess probe localization. YC-27 exhibited a dose dependent increase in signal upon binding. Binding specificity and internalization were visualized by microscopy. In vitro and in vivo blocking studies confirmed YC-27 specificity. In vivo, YC-27 showed good tumor delineation and tissue contrast at doses as low as 0.25 nmole. YC-27 was cleared via the kidneys but bound the proximal tubules of the renal cortex and epididymis. Since PSMA is also broadly expressed on the neovasculature of most tumors, we expect YC-27 will have clinical utility for image-guided surgery and tumor resections.

PTK6 Activation at the Membrane Regulates Epithelial–Mesenchymal Transition in Prostate Cancer

The intracellular tyrosine kinase protein tyrosine kinase 6 (PTK6) lacks a membrane-targeting SH4 domain and localizes to the nuclei of normal prostate epithelial cells. However, PTK6 translocates from the nucleus to the cytoplasm in human prostate tumor cells. Here, we show that while PTK6 is located primarily within the cytoplasm, the pool of active PTK6 in prostate cancer cells localizes to membranes. Ectopic expression of membrane-targeted active PTK6 promoted epithelial-mesenchymal transition in part by enhancing activation of AKT, thereby stimulating cancer cell migration and metastases in xenograft models of prostate cancer. Conversely, siRNA-mediated silencing of endogenous PTK6 promoted an epithelial phenotype and impaired tumor xenograft growth. In mice, PTEN deficiency caused endogenous active PTK6 to localize at membranes in association with decreased E-cadherin expression. Active PTK6 was detected at membranes in some high-grade human prostate tumors, and PTK6 and E-cadherin expression levels were inversely correlated in human prostate cancers. In addition, high levels of PTK6 expression predicted poor prognosis in patients with prostate cancer. Our findings reveal novel functions for PTK6 in the pathophysiology of prostate cancer, and they define this kinase as a candidate therapeutic target. Cancer Res; 73(17); 5426-37. ©2013 AACR.

SPARCL1 suppresses metastasis in prostate cancer

PurposeMetastasis, the main cause of death from cancer, remains poorly understood at the molecular level. Experimental designBased on a pattern of reduced expression in human prostate cancer tissues and tumor cell lines, a candidate suppressor gene (SPARCL1) was identified. We used in vitro approaches to determine whether overexpression of SPARCL1 affects cell growth, migration, and invasiveness. We then employed xenograft mouse models to analyze the impact of SPARCL1 on prostate cancer cell growth and metastasis in vivo. ResultsSPARCL1 expression did not inhibit tumor cell proliferation in vitro. By contrast, SPARCL1 did suppress tumor cell migration and invasiveness in vitro and tumor metastatic growth in vivo, conferring improved survival in xenograft mouse models. ConclusionsWe present the first in vivo data suggesting that SPARCL1 suppresses metastasis of prostate cancer.

A rat model of metastatic spinal cord compression using human prostate adenocarcinoma: histopathological and functional analysis

Background contextCancer is a major global public health problem responsible for one in every four deaths in the United States. Prostate cancer alone accounts for 29% of all cancers in men and is the sixth leading cause of death in men. It is estimated that up to 30% of patients with cancer will develop metastatic disease, the spine being one of the most frequently affected sites in patients with prostate cancer. PurposeTo study this condition in a preclinical setting, we have created a novel animal model of human metastatic prostate cancer to the spine and have characterized it histologically, functionally, and via bioluminescence imaging. Study designTranslational science investigation of animal model of human prostate cancer in the spine. MethodsLuciferase-positive human prostate tumor cells PC3 (PC3-Luc) were injected in the flank of athymic male rats. PC3-Luc tumor samples were then implanted into the L5 vertebral body of male athymic rats (5 weeks old). Thirty-two rats were randomized into three surgical groups: experimental, control, and sham. Tumor growth was assessed qualitatively and noninvasively via bioluminescence emission, upon luciferin injection. To determine the functional impact of tumor growth in the spine, rats were evaluated for gait abnormalities during gait locomotion using video-assisted gait analysis. Rats were euthanized 22 days after tumor implantation, and spines were subjected to histopathological analyses. ResultsTwenty days after tumor implantation, the tumor-implanted rats showed distinct signs of gait disturbances: dragging tail, right- or left–hind limb uncoordination, and absence of toe clearance during forward limb movement. At 20 days, all rats experienced tumor growth, evidenced by bioluminescent signal. Locomotion parameters negatively affected in tumor-implanted rats included stride length, velocity, and duration. At necropsy, all spines showed evidence of tumor growth, and the histological analysis found spinal cord compression and peritumoral osteoblastic reaction characteristic of bony prostate tumors. None of the rats in the sham or control groups demonstrated any evidence of bioluminescence signal or signs of gait disturbances. ConclusionsIn this project, we have developed a novel animal model of metastatic spine cancer using human prostate cancer cells. Tumor growth, evaluated via bioluminescence and corroborated by histopathological analyses, affected hind limb locomotion in ways that mimic motor deficits present in humans afflicted with metastatic spine disease. Our model represents a reliable method to evaluate the experimental therapeutic approaches of human tumors of the spine in animals. Gait locomotion and bioluminescence analyses can be used as surrogate noninvasive methods to evaluate tumor growth in this model.

β-Ionone Induces Cell Cycle Arrest and Apoptosis in Human Prostate Tumor Cells

3-Hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase is the rate-limiting activity in the mevalonate pathway that provides essential intermediates for posttranslational modification of growth-associated proteins. Assorted dietary isoprenoids found in plant foods suppress HMG CoA reductase and have cancer chemopreventive activity. β-Ionone, a cyclic sesquiterpene and an end-ring analog of β-carotene, induced concentration-dependent inhibition of the proliferation of human DU145 (IC50 = 210 μmol/L) and LNCaP (IC50 = 130 μmol/L) prostate carcinoma cells and PC-3 prostate adenocarcinoma cells (IC50 = 130 μmol/L). Concomitantly, β-ionone-induced apoptosis and cell cycle arrest at the G1 phase in DU145 and PC-3 cells were shown by fluorescence microscopy, flow cytometry, and TUNEL reaction, and downregulation of cyclin-dependent kinase 4 (Cdk4) and cyclin D1 proteins. Growth suppression was accompanied by β-ionone-induced downregulation of reductase protein. A blend of β-ionone (150 μmol/L) and trans, trans-farnesol (25 μmol/L), an acyclic sesquiterpene that putatively initiates the degradation of reductase, suppressed the net growth of DU145 cells by 73%, an impact exceeding the sum of those of β-ionone (36%) and farnesol (22%), suggesting a synergistic effect. β-ionone, individually or in combination with other HMG CoA reductase suppressors, may have potential in prostate cancer chemoprevention and/or therapy.

Distribution of Fe, Cu and Zn in Cellular Spheroid Derived Human Prostate Tumor Cells by Synchrotron X-Ray Fluorescence

The formation of three-dimensional cell microspheres such as spheroids has attracted attention as a useful culture technique. In this study, we investigated the distribution (mapping) of Fe, Cu and Zn in cellular spheroid derived human prostate tumor cells. The measurements were performed in standard geometry of 45° incidence, exciting with a white beam collimated with the help of a 20 μm diameter optical capillary in the XRF beam line at the Synchrotron Light National Laboratory (Campinas, Brazil). The results showed that all the elements analyzed presented non-uniform distribution. The spheroids analyzed showed different elemental distribution of Fe and Cu. Zinc concentrations were higher in the central regions of the spheroids analyzed.

Abstract No. 263 - In situ nanocarbon assisted microwave therapy (NAMT) for application of targeting human prostate tumor cells in nude mice causing cytotoxic thermal ablation using extremely short cycle microwave energy as primary therapy

Purpose Evaluate use of spherical nanocarbon (Grafex) injected into known Human Prostatic carcinoma to increase absorption of microwave energy, specifically into tumor cells. This study was selected to evaluate if nanocarbon and microwave can be used as primary treatment and selected to study heat sink associated with microwave treatment. This specific nanocarbon has demonstrated a range of energy absorption capabilities superior to other non-metallic materials. Materials and Methods 10 Nude nu/nu mice were injected with DU145 (ATC#HTB-81) 1x 10^7 Human Prostate carcinoma cells introducted into the dermis and allowed to grow to >1cm. One control mouse received no treatment. One control mouse received only an injection of nanocarbon. Remaining 8 mice received treatment with the microwave and the nanocarbon. Medwave and BSD magnetron generators with temperature probes were used. Nanocarbon and a viscous carrier were injected into the tumors. Short cycle power using 10 watts at 15 seconds used as baseline settings. Target temperature within the tumor was 60 degrees C. Results The control non treated mouse was euthanized due to metastatic prostate cancer 3 weeks after initial injection. Seven of the original treated mice have resolved their prostate tumors with no apparent ill effects from the nanocarbon. One mouse had a non treated skin ulcer from the non cooled microwave probe during the treatment process and was also euthanized ,however, was responding to treatment. The one mouse that received just the nanocarbon treatment, is alive, however the tumor growth has not receded or advanced. All mice were kept in isolation and were observed to have no side effects from the nanocarbon. Conclusion Nanocarbon assisted microwave therapy maximizes energy transfer. Conversion of microwave energy causes thermal ablation to the cancer cells. This reduces heat sink effect and surrounding tissue damage by using much shorter treatment times and lower power output wattage. This suggests Grafex nanocarbon causes enhancement of the dielectric properties of the tumor causing cytotoxic heating. To date, there is no observed toxic side effects in mice from this small study.

α-santalol inhibits the angiogenesis and growth of human prostate tumor growth by targeting vascular endothelial growth factor receptor 2-mediated AKT/mTOR/P70S6K signaling pathway.

BackgroundVEGF receptor 2 (VEGFR2) inhibitors, as efficient antiangiogenesis agents, have been applied in the cancer treatment. However, recently, most of these anticancer drugs have some adverse effects. Discovery of novel VEGFR2 inhibitors as anticancer drug candidates is still needed.MethodsWe used α-santalol and analyzed its inhibitory effects on human umbilical vein endothelial cells (HUVECs) and Prostate tumor cells (PC-3 or LNCaP) in vitro. Tumor xenografts in nude mice were used to examine the in vivo activity of α-santalol.Resultsα-santalol significantly inhibits HUVEC proliferation, migration, invasion, and tube formation. Western blot analysis indicated that α-santalol inhibited VEGF-induced phosphorylation of VEGFR2 kinase and the downstream protein kinases including AKT, ERK, FAK, Src, mTOR, and pS6K in HUVEC, PC-3 and LNCaP cells. α-santalol treatment inhibited ex vivo and in vivo angiogenesis as evident by rat aortic and sponge implant angiogenesis assay. α-santalol significantly reduced the volume and the weight of solid tumors in prostate xenograft mouse model. The antiangiogenic effect by CD31 immunohistochemical staining indicated that α-santalol inhibited tumorigenesis by targeting angiogenesis. Furthermore, α-santalol reduced the cell viability and induced apoptosis in PC-3 cells, which were correlated with the downregulation of AKT, mTOR and P70S6K expressions. Molecular docking simulation indicated that α-santalol form hydrogen bonds and aromatic interactions within the ATP-binding region of the VEGFR2 kinase unit.Conclusionα-santalol inhibits angiogenesis by targeting VEGFR2 regulated AKT/mTOR/P70S6K signaling pathway, and could be used as a potential drug candidate for cancer therapy.

6r, a novel oxadiazole analogue of ethacrynic acid, exhibits antitumor activity both in vitro and in vivo by induction of cell apoptosis and S-phase arrest

This study investigated the in vitro and in vivo antitumor effects of 5-[2,3-Dichloro-4-(2-methylene-1-oxobutyl) phenoxymethyl]-3-methyl-1,2,4- oxadiazole (6r), a novel ethacrynic acid (EA) derivative. The in vitro effect of 6r on cell proliferation of human colon, leukemia, prostate, lung, breast, ovarian and cervical tumor cell lines was assessed using MTT assay and the in vivo effect was determined with an SW620 xenografts nude mice model. The effect of 6r on expressions of GST P1-1 and apoptosis-related proteins were measured by western blotting and the effect on cell apoptosis was analysed by Hoechst 33258 nuclear staining as well as by cell surface staining of annexin V/propidium iodide. The effect on cell cycle was assessed by flow cytometry. Results showed that 6r inhibit proliferation of a range of human cancer cells in vitro and growth of SW620 tumor xenografts in vivo. The anti-proliferative effect of 6r is associated with cell apoptosis as a result of increased ratio of cellular Bax/bcl-2 expression and subsequent cytochrome-c and caspase-3 activation. Unlike EA, 6r did not show any influence on cellular GST P1-1 expression and its anti-proliferative action was associated with cell cycle arrest in G1/S-phase. In conclusion, 6r has the potential to be developed as a chemotherapeutic agent by induction of cell apoptosis but not regulating GST P1-1.

RNAi-mediated knockdown of pituitary tumor- transforming gene-1 (PTTG1) suppresses the proliferation and invasive potential of PC3 human prostate cancer cells

Pituitary tumor-transforming gene-1 (PTTG1) is a proto-oncogene that promotes tumorigenesis and metastasis in numerous cell types and is overexpressed in a variety of human tumors. We have demonstrated that PTTG1 expression was up-regulated in both human prostate cancer specimens and prostate cancer cell lines. For a more direct assessment of the function of PTTG1 in prostate tumorigenesis, RNAi-mediated knockdown was used to selectively decrease PTTG1 expression in PC3 human prostate tumor cells. After three weeks of selection, colonies stably transfected with PTTG1-targeted RNAi (the knockdown PC3 cell line) or empty vector (the control PC3 cell line) were selected and expanded to investigate the role of PTTG1 expression in PC3 cell growth and invasion. Cell proliferation rate was significantly slower (28%) in the PTTG1 knockdown line after 6 days of growth as indicated by an MTT cell viability assay (P < 0.05). Similarly, a soft agar colony formation assay revealed significantly fewer (66.7%) PTTG1 knockdown PC3 cell colonies than control colonies after three weeks of growth. In addition, PTTG1 knockdown resulted in cell cycle arrest at G1 as indicated by fluorescence-activated cell sorting. The PTTG1 knockdown PC3 cell line also exhibited significantly reduced migration through Matrigel in a transwell assay of invasive potential, and down-regulation of PTTG1 could lead to increased sensitivity of these prostate cancer cells to a commonly used anticancer drug, taxol. Thus, PTTG1 expression is crucial for PC3 cell proliferation and invasion, and could be a promising new target for prostate cancer therapy.