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1.  Anti-cancer oncolytic activity of respiratory syncytial virus 
Cancer gene therapy  2009;16(12):923-935.
Oncolytic virotherapy is an emerging bio-therapeutic platform for cancer treatment, which is based on selective infection/killing of cancer cells by viruses. Herein we identify the human respiratory syncytial virus (RSV) as an oncolytic virus. Using prostate cancer models, we show dramatic enhancement of RSV infectivity in vitro in the androgen-independent, highly metastatic PC-3 human prostate cancer cells compared to the non-tumorigenic RWPE-1 human prostate cells. The oncolytic efficiency of RSV was established in vivo using human prostate tumor xenografts in nude mice. Intra-tumoral and intra-peritoneal injections of RSV led to a significant regression of prostate tumors. Furthermore, enhanced viral burden in PC-3 cells led to selective destruction of PC-3 cancer cells in vitro and in xenograft tumors in vivo due to apoptosis triggered by the down-regulation of NF-κB activity (and the resulting loss of anti-apoptotic function of NF-κB) in RSV-infected PC-3 cells. The intrinsic (mitochondrial) pathway constitutes the major apoptotic pathway; however, the death-receptor-dependent extrinsic pathway, mediated by the paracrine/autocrine action of tumor necrosis factor-α produced from infected cells, also partly contributed to apoptosis. Thus, the oncolytic property of RSV can potentially be exploited to develop targeted therapeutics for the clinical management of prostate tumors.
doi:10.1038/cgt.2009.34
PMCID: PMC2813688  PMID: 19444304
Oncolytic; respiratory syncytial virus; anti-cancer; prostate cancer; apoptosis
2.  Switch from antagonist to agonist of the androgen receptor blocker bicalutamide is associated with prostate tumour progression in a new model system 
British Journal of Cancer  1999;81(2):242-251.
Advanced prostate cancer is treated by androgen ablation and/or androgen receptor (AR) antagonists. In order to investigate the mechanisms relevant to the development of therapy-resistant tumours, we established a new tumour model which closely resembles the situation in patients who receive androgen ablation therapy. Androgen-sensitive LNCaP cells were kept in androgen-depleted medium for 87 passages. The new LNCaP cell subline established in this manner, LNCaP-abl, displayed a hypersensitive biphasic proliferative response to androgen until passage 75. Maximal proliferation of LNCaP-abl cells was achieved at 0.001 nM of the synthetic androgen methyltrienolone (R1881), whereas 0.01 nM of this compound induced the same effect in parental cells. At later passages (> 75), androgen exerted an inhibitory effect on growth of LNCaP-abl cells. The non-steroidal anti-androgen bicalutamide stimulated proliferation of LNCaP-abl cells. AR protein expression in LNCaP-abl cells increased approximately fourfold. The basal AR transcriptional activity was 30-fold higher in LNCaP-abl than in LNCaP cells. R1881 stimulated reporter gene activity in LNCaP-abl cells even at 0.01 nM, whereas 0.1 nM of R1881 was needed for induction of the same level of reporter gene activity in LNCaP cells. Bicalutamide that acts as a pure antagonist in parental LNCaP cells showed agonistic effects on AR transactivation activity in LNCaP-abl cells and was not able to block the effects of androgen in these cells. The non-steroidal AR blocker hydroxyflutamide exerted stimulatory effects on AR activity in both LNCaP and LNCaP-abl cells; however, the induction of reporter gene activity by hydroxyflutamide was 2.4- to 4-fold higher in the LNCaP-abl subline. The changes in AR activity were associated neither with a new alteration in AR cDNA sequence nor with amplification of the AR gene. Growth of LNCaP-abl xenografts in nude mice was stimulated by bicalutamide and repressed by testosterone. In conclusion, our results show for the first time that the non-steroidal anti-androgen bicalutamide acquires agonistic properties during long-term androgen ablation. These findings may have repercussions on the natural course of prostate cancer with androgen deprivation and on strategies of therapeutic intervention. © 1999 Cancer Research Campaign
doi:10.1038/sj.bjc.6690684
PMCID: PMC2362859  PMID: 10496349
prostate cancer; androgen ablation; LNCaP cells; androgen receptor; bicalutamide; tumour progression
3.  A Probasin Promoter, Conditionally Replicating Adenovirus that Expresses the Sodium Iodide Symporter (NIS) for Radiovirotherapy of Prostate Cancer 
Gene therapy  2010;17(11):1325-1332.
The sodium iodide symporter (NIS) directs the uptake and concentration of iodide in thyroid cells. We have extended the use of NIS-mediated radioiodine therapy to other types of cancer, we transferred and expressed the sodium-iodide symporter (NIS) gene into prostate, colon, and breast cancer cells using adenoviral vectors. To improve vector efficiency we have developed a conditionally replicating adenovirus (CRAd) in which the E1a gene is driven by the prostate specific promoter, Probasin and the cassette RSV promoter-human NIScDNA-bGH polyA replaces the E3 region (CRAd Ad5PB_RSV-NIS). In vitro infection of the prostate cancer cell line LnCaP resulted in virus replication, cytolysis, and release of infective viral particles. Conversely, the prostate cancer cell line PC-3 (androgen receptor negative) and the pancreatic cancer cell line Panc-1 were refractory to the viral cytopathic effect and did not support viral replication. Radioiodine uptake was readily measurable in LnCaP cells infected with Ad5PB_RSV-NIS 24 hours post-infection, confirming NIS expression. In vivo, LnCaP tumor xenografts in nude mice injected intratumorally with Ad5PB_RSV_NIS CRAd expressed NIS actively as evidenced by 99Tc uptake and imaging. Administration of therapeutic 131I after virus injection significantly increased survival probability in mice carrying xenografted LnCaP tumors compared to virotherapy alone. The data indicate that Ad5PB_RSV_NIS replication is stringently restricted to androgen positive prostate cancer cells and results in effective NIS expression and uptake of radioiodine. This construct may allow multimodal therapy, combining cytolytic virotherapy with radioiodine treatment, to be developed as a novel treatment for prostate cancer.
doi:10.1038/gt.2010.63
PMCID: PMC2914818  PMID: 20428214
prostate cancer; probasin; adenovirus; sodium iodide symporter; virotherapy; gene therapy
4.  Human Prostate Fibroblasts Induce Growth and Confer Castration Resistance and Metastatic Potential in LNCaP Cells 
European urology  2009;58(1):162-172.
Background
The tumor microenvironment is important for progressive and metastatic disease.
Objective
To study the hypothesis that prostate fibroblasts have differential ability to induce castration-resistant prostate cancer (PCa) and metastatic progression and whether this effect might vary depending on the zonal origin of the fibroblast.
Design, setting, and participants
Human prostate fibroblasts from the peripheral (PZ), transition (TZ) and central (CZ) zones of radical prostatectomy specimens (n = 13) were isolated and compared for their ability to promote androgen independence and metastatic progression in androgen-responsive PCa lymph node carcinoma of the prostate (LNCaP) cells in vivo.
Interventions
By coinoculating marginally tumorigenic LNCaP cells with PZ or TZ and by altering host hormonal milieu, a series of tumorigenic and metastatic LNCaP epithelial sublines–P4, P4-2 (derivatives from interaction with PZ), T4, and T4-2 (derivatives from interaction with TZ)–were established and characterized.
Measurements
In vivo and in vitro evaluation of induction of tumor growth and metastatic potential.
Results and limitations
1) LNCaP sublines were permanently altered in their cytogenetic and biologic profiles after cellular interaction with prostate stromal fibroblasts. LNCaP sublines grew faster under anchorage-dependent and -independent conditions, expressed 1–12-fold more prostate-specific antigen in vitro than LNCaP cells, and gained metastatic potential; 2) zonal differences of stromal fibroblasts in their ability to induce the growth and progression of LNCaP tumors as xenografts in mice may exist but need further analysis; 3) PZ-conditioned medium induced more anchorage-independent growth of LNCaP cells in vitro. TZ had a higher growth rate and were more sensitive to dihydrotestosterone.
Conclusions
We demonstrate that prostate fibroblasts have growth inductive potential on PCa cells and affect their subsequent progression to castration resistance and development of a metastatic phenotype.
doi:10.1016/j.eururo.2009.08.026
PMCID: PMC2889152  PMID: 19747763
Androgen-independence; Animal model; Bone metastasis; LNCaP; Prostate cancer; Prostate fibroblasts; Stromal-epithelial Interaction; Transition; peripheral and central zones; Tumor microenvironment
5.  RhoGDIα suppresses growth and survival of prostate cancer cells 
The Prostate  2011;72(4):392-398.
Background
Treatment for primary prostate cancer (CaP) is the withdrawal of androgens. However, CaP eventually progresses to grow in a castration-resistant state. The mechanisms involved in the development and progression of castration-resistant prostate cancer (CRPC) remain unknown. We have previously generated LNCaP-IL6+ cells by treating LNCaP cells chronically with interleukin-6 (IL-6), which have acquired the ability to grow in androgen-deprived conditions.
Methods
We compared the protein expression profile of LNCaP and LNCaP-IL6+ cells using two-dimensional gel electrophoresis. The gels were then silver stained in order to visualize proteins and the differentially expressed spots were identified and characterized by micro sequencing using MALDI-PMF mass spectrometry.
Results
In this study, we have identified RhoGDIα (GDIα) as a suppressor of prostate cancer growth. Expression of GDIα was reduced in LNCaP-IL6+ cells and was downregulated in more aggressive prostate cancer cells compared to LNCaP cells. Over expression of GDIα inhibited the growth of prostate cancer cells and caused LNCaP-IL6+ cells reversal to androgen-sensitive state, while down regulation of GDIα enhanced growth of androgen-sensitive LNCaP prostate cancer cells in androgen-deprived conditions. In addition, GDIα suppressed the tumorigenic ability of prostate tumor xenografts in vivo.
Conclusions
These results demonstrate that loss of GDIα expression promotes the development and progression of prostate cancer.
doi:10.1002/pros.21441
PMCID: PMC3961824  PMID: 21681778
Prostate cancer; RhoGDIα; IL-6
6.  Early Steps of the Virus Replication Cycle Are Inhibited in Prostate Cancer Cells Resistant to Oncolytic Vesicular Stomatitis Virus ▿  
Journal of Virology  2008;82(24):12104-12115.
Vesicular stomatitis virus (VSV) is currently being studied as a candidate oncolytic virus for tumor therapies due to its potent tumoricidal activity. Previous studies have demonstrated that VSV selectively infects tumor cells due to defects in their antiviral pathways. These defects make them more susceptible to VSV-induced killing than normal cells. However, some cancer cells display differential sensitivity to VSV. Specifically, LNCaP prostate cancer cells are sensitive to infection with VSV, while PC3 prostate cancer cells are relatively resistant to VSV. This suggests that tumor cells vary in the extent to which they develop defects in antiviral pathways and, thus, permit virus replication. The goal of these studies was to identify the step(s) of the viral replication cycle that is inhibited in PC3 cells. Results showed that although attachment of VSV was not significantly different among cell types, penetration was delayed by 10 to 30 min in PC3 cells relative to LNCaP cells. Primary transcription was delayed by 6 to 8 h in PC3 cells relative to LNCaP cells. Similarly, both secondary transcription and viral protein synthesis rates were delayed by about 6 to 8 h. The progressively increasing delay suggests that more than one step is affected in PC3 cells. Analysis of cellular gene expression showed that in contrast to LNCaP cells, PC3 cells constitutively expressed numerous antiviral gene products, which may enhance their resistance to VSV. These data indicate that the use of VSV for oncolytic virus therapy for prostate tumors may require prescreening of tumors for their level of susceptibility.
doi:10.1128/JVI.01508-08
PMCID: PMC2593309  PMID: 18829743
7.  Demethylation of the miR-146a promoter by 5-Aza-2’-deoxycytidine correlates with delayed progression of castration-resistant prostate cancer 
BMC Cancer  2014;14:308.
Background
Androgen deprivation therapy is the primary strategy for the treatment of advanced prostate cancer; however, after an initial regression, most patients will inevitably develop a fatal androgen-independent tumor. Therefore, understanding the mechanisms of the transition to androgen independence prostate cancer is critical to identify new ways to treat older patients who are ineligible for conventional chemotherapy.
Methods
The effects of 5-Aza-2’-deoxycytidine (5-Aza-CdR) on the viability and the apoptosis of the androgen-dependent (LNCaP) and androgen-independent (PC3) cell lines were examined by MTS assay and western blot analysis for the activation of caspase-3. The subcutaneous LNCaP xenografts were established in a nude mice model. MiR-146a and DNMTs expressions were analyzed by qRT-PCR and DNA methylation rates of LINE-1 were measured by COBRA-IRS to determine the global DNA methylation levels. The methylation levels of miR-146a promoter region in the different groups were quantified by the bisulfite sequencing PCR (BSP) assay.
Results
We validated that 5-Aza-CdR induced cell death and increased miR-146a expression in both LNCaP and PC3 cells. Notably, the expression of miR-146a in LNCaP cells was much higher than in PC3 cells. MiR-146a inhibitor was shown to suppress apoptosis in 5-Aza-CdR-treated cells. In a castrate mouse LNCaP xenograft model, 5-Aza-CdR significantly suppressed the tumors growth and also inhibited prostate cancer progression. Meanwhile, miR-146a expression was significantly enhanced in the tumor xenografts of 5-Aza-CdR-treated mice and the androgen-dependent but not the androgen-independent stage of castrated mice. In particular, the expression of miR-146a was significantly augmented in both stages of the combined treatment (castration and 5-Aza-CdR). Additionally, the methylation percentage of the two CpG sites (−444 bp and −433 bp), which were around the NF-κB binding site at miR-146a promoter, showed the lowest methylation levels among all CpG sites in the combined treatment tumors of both stages.
Conclusion
Up-regulating miR-146a expression via the hypomethylation of the miR-146a promoter by 5-Aza-CdR was correlated with delayed progression of castration-resistant prostate cancers. Moreover, site-specific DNA methylation may play an important role in miR-146a expression in androgen-dependent prostate cancer progression to androgen-independent prostate cancer and therefore provides a potentially useful biomarker for assessing drug efficacy in prostate cancer.
doi:10.1186/1471-2407-14-308
PMCID: PMC4024097  PMID: 24885368
Prostate cancer; 5-Aza-2’-deoxycytidine (5-Aza-CdR); DNA methyltransferases (DNMTs); miR-146a; Castration
8.  Piperine, a Bioactive Component of Pepper Spice Exerts Therapeutic Effects on Androgen Dependent and Androgen Independent Prostate Cancer Cells 
PLoS ONE  2013;8(6):e65889.
Prostate cancer is the most common solid malignancy in men, with 32,000 deaths annually. Piperine, a major alkaloid constituent of black pepper, has previously been reported to have anti-cancer activity in variety of cancer cell lines. The effect of piperine against prostate cancer is not currently known. Therefore, in this study, we investigated the anti-tumor mechanisms of piperine on androgen dependent and androgen independent prostate cancer cells. Here, we show that piperine inhibited the proliferation of LNCaP, PC-3, 22RV1 and DU-145 prostate cancer cells in a dose dependent manner. Furthermore, Annexin-V staining demonstrated that piperine treatment induced apoptosis in hormone dependent prostate cancer cells (LNCaP). Using global caspase activation assay, we show that piperine-induced apoptosis resulted in caspase activation in LNCaP and PC-3 cells. Further studies revealed that piperine treatment resulted in the activation of caspase-3 and cleavage of PARP-1 proteins in LNCaP, PC-3 and DU-145 prostate cancer cells. Piperine treatment also disrupted androgen receptor (AR) expression in LNCaP prostate cancer cells. Our evaluations further show that there is a significant reduction of Prostate Specific Antigen (PSA) levels following piperine treatment in LNCaP cells. NF-kB and STAT-3 transcription factors have previously been shown to play a role in angiogenesis and invasion of prostate cancer cells. Interestingly, treatment of LNCaP, PC-3 and DU-145 prostate cancer cells with piperine resulted in reduced expression of phosphorylated STAT-3 and Nuclear factor-κB (NF-kB) transcription factors. These results correlated with the results of Boyden chamber assay, wherein piperine treatment reduced the cell migration of LNCaP and PC-3 cells. Finally, we show that piperine treatment significantly reduced the androgen dependent and androgen independent tumor growth in nude mice model xenotransplanted with prostate cancer cells. Taken together, these results support further investigation of piperine as a potential therapeutic agent in the treatment of prostate cancer.
doi:10.1371/journal.pone.0065889
PMCID: PMC3688824  PMID: 23824300
9.  PKA Knockdown Enhances Cell Killing In Response To Radiation And Androgen Deprivation 
The therapeutic efficacy of Gem®231, a second generation antisense molecule targeted to the RIα subunit of PKARIα (AS-PKA), administered in combination with androgen deprivation (AD) and radiation therapy (RT), was examined in androgen sensitive (LNCaP) and insensitive (PC3) cell lines.
Apoptosis was assayed by Caspase 3+7 activity and AnnexinV binding. AS-PKA significantly increased apoptosis in vitro from RT (both lines), with further increases in LNCaP cells grown in AD medium. In LNCaP cells, AD increased phosphorylated map-kinase (pMAPK), which was reduced by AS-PKA relative to the MM controls. AS-PKA also reduced pMAPK levels in PC3 cells. Cell death was measured by clonogenic survival assays.
In vivo, LNCaP cells were grown orthotopically in nude mice. Tumor kinetics were measured by magnetic resonance imaging and serum prostate-specific antigen. PC3 cells were grown subcutaneously and tumor volume assessed by caliper measurements. In PC3 xenografts, AS-PKA caused a significant increase in tumor doubling time relative to MM controls as a monotherapy or in combination with RT. In orthotopic LNCaP tumors, AS-PKA was ineffective as a monotherapy; however it caused a statistically significant increase in tumor doubling time relative to MM controls when used in combination with AD, with or without RT. PKARIα levels in tumors were quantified via immunohistochemical (IHC) staining and image analysis. IHC measurements in LNCaP cells showed that AS-PKA reduced PKARIα levels in vivo.
We demonstrate for the first time that AS-PKA enhances cell killing androgen sensitive prostate cancer cells to AD±RT and androgen insensitive cells to RT.
doi:10.1002/ijc.25634
PMCID: PMC3391603  PMID: 20960462
Antisense; Protein kinase A; prostate cancer; radiation; androgen deprivation
10.  Targeting of Interferon-Beta to Produce a Specific, Multi-Mechanistic Oncolytic Vaccinia Virus 
PLoS Medicine  2007;4(12):e353.
Background
Oncolytic viruses hold much promise for clinical treatment of many cancers, but a lack of systemic delivery and insufficient tumor cell killing have limited their usefulness. We have previously demonstrated that vaccinia virus strains are capable of systemic delivery to tumors in mouse models, but infection of normal tissues remains an issue. We hypothesized that interferon-beta (IFN-β) expression from an oncolytic vaccinia strain incapable of responding to this cytokine would have dual benefits as a cancer therapeutic: increased anticancer effects and enhanced virus inactivation in normal tissues. We report the construction and preclinical testing of this virus.
Methods and Findings
In vitro screening of viral strains by cytotoxicity and replication assay was coupled to cellular characterization by phospho-flow cytometry in order to select a novel oncolytic vaccinia virus. This virus was then examined in vivo in mouse models by non-invasive imaging techniques. A vaccinia B18R deletion mutant was selected as the backbone for IFN-β expression, because the B18R gene product neutralizes secreted type-I IFNs. The oncolytic B18R deletion mutant demonstrated IFN-dependent cancer selectivity and efficacy in vitro, and tumor targeting and efficacy in mouse models in vivo. Both tumor cells and tumor-associated vascular endothelial cells were targeted. Complete tumor responses in preclinical models were accompanied by immune-mediated protection against tumor rechallenge. Cancer selectivity was also demonstrated in primary human tumor explant tissues and adjacent normal tissues. The IFN-β gene was then cloned into the thymidine kinase (TK) region of this virus to create JX-795 (TK−/B18R−/IFN-β+). JX-795 had superior tumor selectivity and systemic intravenous efficacy when compared with the TK−/B18R− control or wild-type vaccinia in preclinical models.
Conclusions
By combining IFN-dependent cancer selectivity with IFN-β expression to optimize both anticancer effects and normal tissue antiviral effects, we were able to achieve, to our knowledge for the first time, tumor-specific replication, IFN-β gene expression, and efficacy following systemic delivery in preclinical models.
Stephen Thorne and colleagues describe, in a mouse model, an oncolytic vaccinia virus with interferon-dependent cancer selectivity that allows tumor-specific replication; it also expresses the IFN-β gene and hence has efficacy against tumors.
Editors' Summary
Background.
Normally, throughout life, cell division (which produces new cells) and cell death are carefully balanced to keep the body in good working order. But sometimes cells acquire changes (mutations) in their genetic material that allow them to divide uncontrollably to form cancers—disorganized masses of cells. Cancers can develop anywhere in the body and, as they develop, their cells acquire other genetic changes that enable them to move and start new tumors (metastases) elsewhere. Chemotherapy drugs kill rapidly dividing cancer cells but, because some normal cells are also sensitive to these drugs, it is hard to destroy the cancer without causing serious side effects. Consequently, researchers are trying to develop “targeted” therapies that attack the changes in cancer cells that allow them to divide uncontrollably but leave normal cells unscathed. One promising class of targeted therapies is oncolytic viruses. These viruses make numerous copies of themselves inside cancer cells (but not inside normal cells). Eventually the cancer cell bursts open (lyses), releases more of the therapeutic agent, and dies.
Why Was This Study Done?
Existing oncolytic viruses have two major disadvantages: they have to be injected directly into tumors, and therefore they can't destroy distant metastases; and they don't kill cancer cells particularly efficiently. In this study, the researchers have tried to adapt vaccinia virus (a virus that infects humans and which has recently been shown to kill tumor cells when injected into the bloodstream) in two ways: to both infect cancer cells selectively and then to kill them effectively.
They hypothesized that putting a gene that causes expression of a protein called interferon-beta (IFN-β) in a particular virus strain that is itself incapable of responding to IFN-β might achieve these aims. Human cells infected with viruses usually release IFNs, which induce an antiviral state in nearby cells. But vaccinia virus makes anti-IFN proteins that prevent IFN release. If the viral genes that encode these proteins are removed from the virus, the virus cannot spread through normal cells. However, many cancer cells have defective IFN signaling pathways so the virus can spread through them. IFN-β expression by the virus, however, should improve its innate anticancer effects because IFN-β stops cancer cells dividing, induces an antitumor immune response, and stops tumors developing good blood supplies.
What Did the Researchers Do and Find?
The researchers selected a vaccinia virus strain called WR-delB18R in which the B18R gene, which encodes an anti-IFN protein, had been removed from the virus. (WR is a wild-type virus.) In laboratory experiments, IFN treatment blocked the spread of WR-delB18R in normal human cells but not in human tumor cells. After being injected into the veins of tumor-bearing mice, WR-delB18R was rapidly cleared from normal tissues but persisted in the tumors. A single injection of WR-delB18R directly into the tumor killed most of the tumor cells. A similar dose injected into a vein was less effective but nevertheless increased the survival time of some of the mice by directly killing the tumor cells, by targeting the blood supply of the tumors, and by inducing antitumor immunity. Finally, when the researchers inserted the IFN-β gene into this WR-delB18R, the new virus—JX-795—was much better at killing tumors after intravenous injection than either WR or WR-delB18R.
What Do These Findings Mean?
These findings indicate that the vaccinia virus can be adapted so that it replicates only in tumor cells and kills these cells effectively after intravenous injection. In particular, they show that the strategy adopted by the researchers both optimizes the anticancer effects of the virus and minimizes viral replication in normal tissues. JX-795 is a promising oncolytic virus, therefore, particularly since vaccinia virus has been safely used for many years to vaccinate people against smallpox. Nevertheless, it will be some years before JX-795 can be used clinically. Vaccinia virus constructs like this need to be tested extensively in the laboratory and in animals before any attempt is made to test them in people and, even if they passes all these preclinical tests with flying colors, only clinical trials will reveal whether they can treat human cancer. Several related strains of vaccinia virus are currently undergoing clinical testing.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/:10.1371/journal.pmed.0040353.
The US National Cancer Institute provides information on all aspects of cancer (in English and Spanish)
CancerQuest, from Emory University, provides information on all aspects of cancer (in several languages)
The UK charity Cancerbackup also provides information on all aspects of cancer
Wikipedia has a page on oncolytic viruses (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
A short interview about oncolytic viruses with researcher Dr. John Bell is available on the Insidermedicine Web site
The Oncolytic virus Web page provides lists of oncolytic viruses classified by type
doi:10.1371/journal.pmed.0040353
PMCID: PMC2222946  PMID: 18162040
11.  ADENOVIRAL E2F1 OVEREXPRESSION SENSITIZES LNCAP AND PC3 PROSTATE TUMOR CELLS TO RADIATION IN VIVO 
Purpose
We previously showed that E2F1 overexpression radiosensitizes prostate cancer cells in-vitro. Here, we demonstrate the radiosensitization efficacy of Ad-E2F1 in growing LNCaP (orthotopically) and PC3 (subcutaneously) nude mice xenograft tumors.
Methods and Materials
Adenoviral E2F1 was injected intra-tumorally in LNCaP (3×108 PFU) and PC3 (5×108 PFU) tumors treated with or without radiation. Tumor volumes (TV) were measured by MRI in LNCaP tumors, calipers in PC3 tumors and serum PSA levels by ELISA in LNCaP tumors. Apoptosis was measured by TUNEL staining and key proteins involved in cell death signaling were analyzed by Western blot.
Results
Intracellular overexpression of Ad-E2F1 had significant effect in the regression of TV and reducing the PSA relative to adenoviral luciferase (Ad-Luc) control. The in-vivo regressing effect of Ad-E2F1 on LNCaP tumor growth was significant (PSA-34 ng/ml/TV-142 mm3) compared to Ad-Luc control (PSA-59 ng/ml/TV-218 mm3; p<0.05). This effect was significantly enhanced by radiation therapy (PSA-16 ng/ml/TV-55 mm3 compared to Ad-Luc/PSA-42 ng/ml/TV-174 mm3; p<0.05). For PC3 tumors, the greatest effect was observed with Ad-E2F1 alone, there was little or no effect when RT was combined. However, addition of RT enhanced the level of in-situ apoptosis in PC3 tumors. Molecularly, Ad-E2F1 in a combination setting abrogated radiation induced BCL-2 protein and was associated with an increase in activated BAX, together caused a potent radiosensitizing effect irrespective of p53 and AR functional status.
Conclusions
We show here for the first time that ectopic overexpression of E2F1 in-vivo using an adenoviral vector significantly inhibits orthotopic p53wild-type LNCaP and subcutaneous p53null PC3 tumors in nude mice. Furthermore, we demonstrate that E2F1 strongly sensitizes LNCaP tumors to RT. These findings suggest that E2F1 overexpression can sensitize prostate tumor cells in-vivo independent of p53 or androgen receptor status.
doi:10.1016/j.ijrobp.2010.08.013
PMCID: PMC4299462  PMID: 21195876
Ad-E2F1; Prostate cancer; Radiation; LNCaP; PC3
12.  Effect of Small Molecules Modulating Androgen Receptor (SARMs) in Human Prostate Cancer Models 
PLoS ONE  2013;8(5):e62657.
The management of hormone-refractory prostate cancer represents a major challenge in the therapy of this tumor, and identification of novel androgen receptor antagonists is needed to render treatment more effective. We analyzed the activity of two novel androgen receptor antagonists, (S)-11 and (R)-9, in in vitro and in vivo experimental models of hormone-sensitive or castration-resistant prostate cancer (CRPC). In vitro experiments were performed on LNCaP, LNCaP-AR, LNCaP-Rbic and VCaP human prostate cancer cells. Cytotoxic activity was assessed by SRB and BrdU uptake, AR transactivation by luciferase reporter assay and PSA levels by Real Time RT-PCR and ELISA assays. Cell cycle progression-related markers were evaluated by western blot. In vivo experiments were performed on SCID mice xenografted with cells with different sensitivity to hormonal treatment. In hormone-sensitive LNCaP and LNCaP-AR cells, the latter expressing high androgen receptor levels, (R)-9 and (S)-11 exhibited a higher cytotoxic effect compared to that of the reference compound ((R)-bicalutamide), also in the presence of the synthetic androgen R1881. Furthermore, the cytotoxic effect produced by (R)-9 was higher than that of (S)-11 in the two hormone-resistant LNCaP-AR and VCaP cells. A significant reduction in PSA levels was observed after exposure to both molecules. Moreover, (S)-11 and (R)-9 inhibited DNA synthesis by blocking the androgen-induced increase in cyclin D1 protein levels. In vivo studies on the toxicological profile of (R)-9 did not reveal the presence of adverse events. Furthermore, (R)-9 inhibited tumor growth in various in vivo models, especially LNCaP-Rbic xenografts, representative of recurrent disease. Our in vitro results highlight the antitumor activity of the two novel molecules (R)-9 and (S)-11, making them a potentially attractive option for the treatment of CRPC.
doi:10.1371/journal.pone.0062657
PMCID: PMC3648536  PMID: 23667504
13.  Inhibition of Aberrant Androgen Receptor Induction of Prostate Specific Antigen Gene Expression, Cell Proliferation and Tumor Growth by 17α-Estradiol in Prostate Cancer 
The Journal of urology  2011;185(1):305-314.
Purpose
Androgen independent prostate cancer growth and metastasis are a major cause of prostate cancer death. Aberrant androgen receptor activation due to androgen receptor mutation is an important mechanism of androgen independence. We determined the effectiveness and mechanism of 17α-estradiol (Sigma®) in blocking aberrant androgen receptor activation due to androgen receptor mutation.
Materials and Methods
We used LNCaP and MDA Pca-2b prostatic tumor cells (ATCC®) containing a mutated androgen receptor and WT estrogen receptor β to test 17α-estradiol inhibition of aberrant androgen receptor activation of prostate specific antigen gene expression and cell growth. Cotransfection analysis was used to further elucidate the mechanism of 17α-estradiol action. Xenograft animals with an LNCaP prostate tumor were prepared to study the in vivo effect of 17α-estradiol on tumor growth inhibition.
Results
In LNCaP cells 17α-estradiol produced a dose dependent inhibition of cyproterone acetate (Sigma) or dihydrotestosterone induced prostate specific antigen gene expression. In MDA Pca-2b cells 17α-estradiol inhibited cortisol (Sigma) induced prostate specific antigen expression and blocked dihydrotestosterone and cortisol induced cell proliferation in LNCaP and MDA Pca-2b cells, respectively. Cotransfection analysis showed that 17α-estradiol inhibition of aberrant androgen receptor activation of prostate specific antigen gene expression was medicated via estrogen receptors. In xenograft mice with LNCaP prostate cancer 17α-estradiol but not 17β-estradiol (Sigma) significantly inhibited tumor growth, although each estrogen tended to decrease tumor growth.
Conclusions
Results suggest that 17α-estradiol with less classic estrogenic activity is a potential therapeutic agent for androgen independent prostate cancer due to androgen receptor mutation.
doi:10.1016/j.juro.2010.09.008
PMCID: PMC3039213  PMID: 21075385
prostate; receptors; androgen; mutation; estradiol; prostatic neoplasms
14.  A Hyperfusogenic F Protein Enhances the Oncolytic Potency of a Paramyxovirus Simian Virus 5 P/V Mutant without Compromising Sensitivity to Type I Interferon ▿  
Journal of Virology  2008;82(19):9369-9380.
Viral fusogenic membrane proteins have been proposed as tools to increase the potency of oncolytic viruses, but there is a need for mechanisms to control the spread of fusogenic viruses in normal versus tumor cells. We have previously shown that a mutant of the paramyxovirus simian virus 5 (SV5) that harbors mutations in the P/V gene from the canine parainfluenza virus (P/V-CPI−) is a potent inducer of type I interferon (IFN) and apoptosis and is restricted for spread through normal but not tumor cells in vitro. Here, we have used the cytopathic P/V-CPI− as a backbone vector to test the hypothesis that a virus expressing a hyperfusogenic glycoprotein will be a more effective oncolytic vector but will retain sensitivity to IFN. A P/V mutant virus expressing an F protein with a glycine-to-alanine substitution in the fusion peptide (P/V-CPI−-G3A) was more fusogenic than the parental P/V-CPI− mutant. In two model prostate tumor cell lines which are defective in IFN production (LNCaP and DU145), the hyperfusogenic P/V-CPI−-G3A mutant had normal growth properties at low multiplicities of infection and was more effective than the parental P/V-CPI− mutant at cell killing in vitro. However, in PC3 cells which produce and respond to IFN, the hyperfusogenic P/V-CPI−-G3A mutant was attenuated for growth and spread. Killing of PC3 cells was equivalent between the parental P/V-CPI− mutant and the hyperfusogenic P/V-CPI−-G3A mutant. In a nude mouse model using LNCaP cells, the hyperfusogenic P/V-CPI−-G3A mutant was more effective than P/V-CPI− at reducing tumor burden. In the case of DU145 tumors, the two vectors based on P/V-CPI− were equally effective at limiting tumor growth. Together, our results provide proof of principle that a cytopathic SV5 P/V mutant can serve as an oncolytic virus and that the oncolytic effectiveness of P/V mutants can be enhanced by a fusogenic membrane protein without compromising sensitivity to IFN. The potential advantages of SV5-based oncolytic vectors are discussed.
doi:10.1128/JVI.01054-08
PMCID: PMC2546974  PMID: 18667520
15.  Radioiodine therapy for castration-resistant prostate cancer following prostate-specific membrane antigen promoter-mediated transfer of the human sodium iodide symporter 
Asian Journal of Andrology  2013;16(1):120-123.
Radioiodine therapy, the most effective form of systemic radiotherapy available, is currently useful only for thyroid cancer because of the thyroid-specific expression of the human sodium iodide symporter (hNIS). Here, we explore the efficacy of a novel form of gene therapy using prostate-specific membrane antigen (PSMA) promoter-mediated hNIS gene transfer followed by radioiodine administration for the treatment of castration-resistant prostate cancer (CRPC). The androgen-dependent C33 LNCaP cell line and the androgen-independent C81 LNCaP cell line were transfected by adenovirus. PSMA promoter-hNIS (Ad.PSMApro-hNIS) or adenovirus.cytomegalovirus–hNIS containing the cytomegalovirus promoter (Ad.CMV-hNIS) or a control virus. The iodide uptake was measured in vitro. The in vivo iodide uptake by C81 cell xenografts in nude mice injected with an adenovirus carrying the hNIS gene linked to PSMA and the corresponding tumor volume fluctuation were assessed. Iodide accumulation was shown in different LNCaP cell lines after Ad.PSMApro-hNIS and Ad.CMV-hNIS infection, but not in different LNCaP cell lines after adenovirus.cytomegalovirus (Ad.CMV) infection. At each time point, higher iodide uptake was shown in the C81 cells infected with Ad.PSMApro-hNIS than in the C33 cells (P < 0.05). An in vivo animal model showed a significant difference in 131I radioiodine uptake in the tumors infected with Ad.PSMApro-hNIS, Ad.CMV-hNIS and control virus (P < 0.05) and a maximum reduction of tumor volume in mice infected with Ad.PSMApro-hNIS. These results show prostate-specific expression of the hNIS gene delivered by the PSMA promoter and effective radioiodine therapy of CRPC by the PSMA promoter-driven hNIS transfection.
doi:10.4103/1008-682X.122354
PMCID: PMC3901869  PMID: 24369144
genetic therapy; prostate-specific membrane antigen (PSMA); prostatic neoplasms; sodium-iodide symporter
16.  Androgen Suppresses Proliferation of Castration-Resistant LNCaP 104-R2 Prostate Cancer Cells via Androgen Receptor, Skp2, and c-Myc 
Cancer science  2011;102(11):2022-2028.
Summary
Androgen ablation therapy is the primary treatment for metastatic prostate cancer. However, this therapy is associated with several undesired side-effects, including increased risk of cardiovascular diseases. To study if termination of long-term androgen ablation and restoring testosterone level may suppress the growth of relapsed hormone-refractory prostate tumors, we implanted testosterone pellets in castrated nude mice carrying androgen receptor (AR)-positive LNCaP 104-R2 cells, which relapsed from androgen-dependent LNCaP 104-S cells after long-term androgen deprivation. 104-R2 tumor xenografts regressed after testosterone pellets implant. 24 out of 33 tumors adapted to elevation of testosterone level and relapsed as androgen-insensitive tumors. Relapsed tumors (R2Ad) expressed less AR and prostate-specific antigen (PSA). We then study the molecular mechanism lying underneath the androgenic regulation of prostate cancer cell proliferation. Androgen suppresses proliferation of 104-R2 by inducing G1 cell cycle arrest via reduction of Skp2 and c-Myc, and induction of p27Kip1. 104-R2 cells adapted to androgen treatment and the adapted cells, R2Ad, were androgen-insensitive cells with slower growing rate and low protein level of AR, high levels of c-Myc and Skp2, and low levels of p27Kip1. Nuclear AR and PSA expression is present in 104-R2 cells but not R2Ad cells when androgen is absent. Overexpression of AR in R2Ad cells regenerated an androgen-repressed phenotype, while knockdown of AR in 104-R2 cells generated an androgen-insensitive phenotype. Overexpression of Skp2 and c-Myc in 104-R2 cells blocked the growth inhibition caused by androgens. We concluded that androgens cause growth inhibition in LNCaP 104-R2 prostate cancer cells via AR, Skp2, and c-Myc.
doi:10.1111/j.1349-7006.2011.02043.x
PMCID: PMC3200457  PMID: 21781227
17.  Aberrant activation of androgen receptor in a new neuropeptide-autocrine model of androgen-insensitive prostate cancer1 
Cancer research  2009;69(1):151-160.
Treatments of advanced prostate cancer with androgen deprivation therapy inevitably render the tumors to become castration resistant and incurable. Under these conditions, neuroendocrine differentiation of prostate cancer (CaP) cells is often detected and neuropeptides released by these cells may facilitate the development of androgen independence. Exemplified by GRP (gastrin-releasing peptide), these neuropeptides transmit their signals through G-protein coupled receptor (GPCRs), which are often overexpressed in prostate cancer, and aberrantly activate androgen receptor (AR) in the absence of androgen. We developed an autocrine neuropeptide model by overexpressing GRP in LNCaP cells and the resultant cell line, LNCaP-GRP, exhibited androgen-independent growth with enhanced motility in vitro. When orthotopically implanted in castrated nude mice, LNCaP-GRP produced aggressive tumors, which express GRP, prostate-specific antigen, and nuclear-localized AR. Chromatin immunoprecipitation studies of LNCaP-GRP clones suggest that GRP activates and recruits AR to the cognate promoter in the absence of androgen. A Src family kinase (SFK) inhibitor, AZD0530 inhibits androgen-independent growth and migration of the GRP-expressing cell lines, and blocks the nuclear transloation of AR, indicating the involvement of SFK in the aberrant activation of AR and demonstrating the potential use of SFK inhibitor in the treatment of castration resistant CaP. In vivo study showed AZD0530 profoundly inhibits tumor metastasis in severe combined immunodeficient (SCID) mice implanted with GRP-autocrine LNCaP cells. This xenograft model demonstrates autocrine, neuropeptide- and Src kinase-mediated progression of androgen-independent CaP post-castration, and is potentially useful for testing novel therapeutic agents.
doi:10.1158/0008-5472.CAN-08-0442
PMCID: PMC2626435  PMID: 19117998
GPCR; neuroendocrine; neuropeptides; androgen-independence; Src kinase; prostate cancer
18.  Enhanced sensitivity to androgen withdrawal due to overexpression of interleukin-6 in androgen-dependent human prostate cancer LNCaP cells 
British Journal of Cancer  2009;101(10):1731-1739.
Background:
The objective of this study was to investigate the effects of interleukin-6 (IL-6) overexpression in androgen-dependent prostate cancer LNCaP cells on their phenotype under an androgen-deprived condition.
Methods:
We established IL-6-overexpressing LNCaP (LNCaP/IL-6) by introducing the expression vector containing IL-6 cDNA. Changes in the phenotype in LNCaP/IL-6 were compared with that in LNCaP transfected with control vector alone (LNCaP/Co).
Results:
In vitro, the growth of LNCaP/IL-6 was significantly inferior to that of LNCaP/Co under an androgen-deprived condition. Similarly, LNCaP/IL-6 tumour in nude mice rapidly regressed after castration; however, LNCaP/Co tumour growth was transiently inhibited after castration and then continuously accelerated. After androgen withdrawal, expression levels of phosphorylated p44/42 mitogen-activated protein kinase (MAPK) and Akt in LNCaP/IL-6 were markedly upregulated compared with those in LNCaP/Co; however, additional treatment with specific inhibitor of the MAPK or Akt signalling pathway significantly inhibited the growth of LNCaP/IL-6 compared with that of LNCaP/Co. Furthermore, gene microarray analyses showed that androgen deprivation resulted in differential expression of genes involved in growth, apoptotsis and tumorigenesis between LNCaP/Co and LNCaP/IL-6.
Conclusion:
Excessive secretion of IL-6 by LNCaP cells in an autocrine manner may have a suppressive function in their growth and acquisition of androgen-independent phenotype under an androgen-deprived condition.
doi:10.1038/sj.bjc.6605358
PMCID: PMC2778549  PMID: 19844233
prostate cancer; interleukin-6; androgen-independent progression; androgen withdrawal; signal transduction
19.  Characterization of Infectivity-Enhanced Conditionally Replicating Adenovectors for Prostate Cancer Radiovirotherapy 
Human Gene Therapy  2012;23(9):951-959.
Abstract
Prostate cancer (PCa) is the second most commonly diagnosed and sixth leading cause of cancer death in American men and one for which no curative therapy exists after metastasis. To meet this need for novel therapies, our laboratory has previously generated conditionally replicating adenovirus (CRAd) vectors expressing the sodium iodide symporter (hNIS). This virus transduced PCa cells and induced functional NIS expression, allowing for noninvasive tumor imaging and combination therapy with radioiodide, referred to as radiovirotherapy. We have now generated two new modified vectors to further improve efficacy. Ad5/3PB-ADP-hNIS and Ad5/3PB-hNIS include a hybrid Ad5/3 fiber knob to improve transduction efficiency, and express NIS from the endogenous major late promoter to restrict NIS expression to target cells. Additionally, Ad5/3PB-ADP-hNIS includes the adenovirus death protein (ADP), which hastens the release of viral particles after assembly. These two vectors specifically induce radioisotope uptake, cytopathic effect, and viral replication in androgen receptor–expressing PCa cell lines with Ad5/3PB-ADP-hNIS showing earlier 131I uptake and cytolysis at low multiplicity of infection. SPECT-CT imaging of xenograft tumors infected with Ad5/3PB-hNIS showed steady uptake, whereas infection with Ad5/3PB-ADP-hNIS led to increasing uptake, indicating viral spread. Radiovirotherapy of xenograft LNCaP tumors with Ad5/3PB-ADP-hNIS showed the most significant survival extension versus control tumors (p=0.001), but the benefit of radiovirotherapy was not statistically significant compared with virotherapy alone in this model. These results show the potential of Ad5/3PB-ADP-hNIS as a vector for treatment of prostate cancer.
Oneal and colleagues report on two novel conditionally replicating adenoviral vectors that combine 131I therapy with conventional virotherapy, a treatment termed radiovirotherapy. They show that these vectors can specifically induce radioisotope uptake, cytopathic effect, and viral replication in androgen receptor-expressing prostate cancer cell lines. In vivo efficacy studies show that treatment of tumor-bearing xenograft mice with these vectors results in a significant oncolytic effect.
doi:10.1089/hum.2012.047
PMCID: PMC3440023  PMID: 22694073
20.  Natura-Alpha Targets Forkhead Box M1 and Inhibits Androgen-Dependent and -Independent Prostate Cancer Growth and Invasion 
Purpose
The development of new effective therapeutic agents with minimal side effects for prostate cancer treatment is much needed. Indirubin, an active molecule identified in the traditional Chinese herbal medicine – Qing Dai (Indigo Naturalis), has been used to treat leukemia for decades. However, the anti-cancer properties of Natura-alpha, an indirubin derivative, are not well studied in solid tumors, particularly in prostate cancer.
Experimental Design
Human prostate cancer cell lines were treated with or without Natura-alpha followed by cell growth and invasion assays measured. The anti-tumor effects of Natura-alpha were examined in nude mice tumor xenograft models, as well as in a patient with advanced hormone refractory metastatic prostate cancer. Signal network proteins targeted by Natura-alpha were analyzed using Proteomic Pathway Array Analysis (PPAA) on xenografts.
Results
Natura-alpha inhibited the growth of both androgen-dependent (LNCaP), and androgen-independent (LNCaP-AI, PC-3, and DU145) prostate cancer cells with IC50 between 4 to 10 Μm, also inhibits invasion of androgen-independent prostate cancer cells. Its anti-tumor effects were further evident in vivo tumor reduction in androgen-dependent and -independent nude mice tumor xenograft models as well as reduced tumor volume in the patient with hormone refractory metastatic prostate cancer. PPAA revealed that anti-proliferative and anti-invasive activities of Natura-alpha on prostate cancer might primarily be through its down-regulation of Forkhead box M1 (FOXM1) protein. Forced over-expression of FOXM1 largely reversed the inhibition by Natura-alpha.
Conclusion
Natura-alpha could serve as a novel and effective therapeutic agent for treatment of both hormone sensitive and hormone refractory prostate cancer with minimal side effects.
doi:10.1158/1078-0432.CCR-11-0431
PMCID: PMC3196615  PMID: 21606178
21.  Effectiveness of the Histone Deacetylase Inhibitor (S)-2 against LNCaP and PC3 Human Prostate Cancer Cells 
PLoS ONE  2013;8(3):e58267.
Histone deacetylase inhibitors (HDACi) represent a promising class of epigenetic agents with anticancer properties. Here, we report that (S)-2, a novel hydroxamate-based HDACi, shown previously to be effective against acute myeloid leukemia cells, was also a potent inducer of apoptosis/differentiation in human prostate LNCaP and PC3 cancer cells. In LNCaP cells (S)-2 was capable of triggering H3/H4 histone acetylation, H2AX phosphorylation as a marker of DNA damage and producing G0/G1 cell cycle arrest. Consistently, (S)-2 led to enhanced expression of both the protein and mRNA p21 levels in LNCaP cells but, contrary to SAHA, not in normal non-tumorigenic prostate PNT1A cells. Mechanistic studies demonstrated that (S)-2-induced apoptosis in LNCaP cells developed through the cleavage of pro-caspase 9 and 3 and of poly(ADP-ribose)-polymerase accompanied by the dose-dependent loss of mitochondrial membrane potential. Indeed, the addition of the pan-caspase inhibitor Z-VAD-fmk greatly reduced drug-mediated apoptosis while the antioxidant N-acetyl-cysteine was virtually ineffective. Importantly, preliminary data with nude mice xenografted with LNCaP cells showed that (S)-2 prompted a decrease in the tumor volume and an increase in H2AX phosphorylation within the cancer cells. Moreover, the highly metastatic prostate cancer PC3 cells were also sensitive to (S)-2 that: i) induced growth arrest and moderate apoptosis; ii) steered cells towards differentiation and neutral lipid accumulation; iii) reduced cell invasiveness potential by decreasing the amount of MMP-9 activity and up-regulating TIMP-1 expression; and iv) inhibited cell motility and migration through the Matrigel. Overall, (S)-2 has proven to be a powerful HDACi capable of inducing growth arrest, cell death and/or differentiation of LNCaP and PC3 prostate cancer cells and, due to its low toxicity and efficacy in vivo, might also be of clinical interest to support conventional prostate cancer therapy.
doi:10.1371/journal.pone.0058267
PMCID: PMC3587597  PMID: 23469273
22.  ADAMTS1 alters blood vessel morphology and TSP1 levels in LNCaP and LNCaP-19 prostate tumors 
BMC Cancer  2010;10:288.
Background
Decreased expression of the angiogenesis inhibitor ADAMTS1 (ADAM metallopeptidase with thrombospondin type 1 motif, 1) has previously been reported during prostate cancer progression. The aim of this study was to investigate the function of ADAMTS1 in prostate tumors.
Methods
ADAMTS1 was downregulated by shRNA technology in the human prostate cancer cell line LNCaP (androgen-dependent), originally expressing ADAMTS1, and was upregulated by transfection in its subline LNCaP-19 (androgen-independent), expressing low levels of ADAMTS1. Cells were implanted subcutaneously in nude mice and tumor growth, microvessel density (MVD), blood vessel morphology, pericyte coverage and thrombospondin 1 (TSP1) were studied in the tumor xenografts.
Results
Modified expression of ADAMTS1 resulted in altered blood vessel morphology in the tumors. Low expression levels of ADAMTS1 were associated with small diameter blood vessels both in LNCaP and LNCaP-19 tumors, while high levels of ADAMTS1 were associated with larger vessels. In addition, TSP1 levels in the tumor xenografts were inversely related to ADAMTS1 expression. MVD and pericyte coverage were not affected. Moreover, upregulation of ADAMTS1 inhibited tumor growth of LNCaP-19, as evidenced by delayed tumor establishment. In contrast, downregulation of ADAMTS1 in LNCaP resulted in reduced tumor growth rate.
Conclusions
The present study demonstrates that ADAMTS1 is an important regulatory factor of angiogenesis and tumor growth in prostate tumors, where modified ADAMTS1 expression resulted in markedly changed blood vessel morphology, possibly related to altered TSP1 levels.
doi:10.1186/1471-2407-10-288
PMCID: PMC2894797  PMID: 20546609
23.  Resveratrol-Loaded Nanoparticles Based on Poly(epsilon-caprolactone) and Poly(d,l-lactic-co-glycolic acid)–Poly(ethylene glycol) Blend for Prostate Cancer Treatment 
Molecular pharmaceutics  2013;10(10):3871-3881.
Nanoencapsulation of antiproliferative and chemopreventive phytoalexin trans-resveratrol (RSV) is likely to provide protection against degradation, enhancement of bioavailability, improvement in intracellular penetration and control delivery. In this study, polymeric nanoparticles (NPs) encapsulating RSV (nano-RSV) as novel prototypes for prostate cancer (PCa) treatment were designed, characterized and evaluated using human PCa cells. Nanosystems, composed of a biocompatible blend of poly(epsilon-caprolactone) (PCL) and poly(d,l-lactic-co-glycolic acid)-poly(ethylene glycol) conjugate (PLGA-PEG-COOH), were prepared by a nanoprecipitation method, and characterized in terms of morphology, particle size and zeta potential, encapsulation efficiency, thermal analyses, and in vitro release studies. Cellular uptake of NPs was then evaluated in PCa cell lines DU-145, PC-3, and LNCaP using confocal fluorescence microscopy, and antiproliferative efficacy was assessed using MTT assay. With encapsulation efficiencies ranging from 74% to 98%, RSV was successfully loaded in PCL:PLGA-PEG-COOH NPs, which showed an average diameter of 150 nm. NPs were able to control the RSV release at pH 6.5 and 7.4, mimicking the acidic tumoral microenvironment and physiological conditions, respectively, with only 55% of RSV released within 7 h. In gastrointestinal simulated fluids, NPs released about 55% of RSV in the first 2 h in acidic medium, and their total RSV content within the subsequent 5 h at pH 7.4. Confocal fluorescence microscopy observations revealed that NPs were efficiently taken up by PCa cell lines. Furthermore, nano-RSV significantly improved the cytotoxicity compared to that of free RSV toward all three cell lines, at all tested concentrations (from 10 µM to 40 µM), proving a consistent sensitivity toward both the androgen-independent DU-145 and hormone-sensitive LNCaP cells. Our findings support the potential use of developed nanoprototypes for the controlled delivery of bioactive RSV for Pca chemoprevention/chemotherapy.
doi:10.1021/mp400342f
PMCID: PMC4100701  PMID: 23968375
resveratrol; nanoparticles; poly(epsilon-caprolactone); poly(d,l-lactic-co-glycolic acid)-poly(ethylene glycol) conjugate; prostate cancer
24.  PROGRESSION TO ANDROGEN-INDEPENDENT LNCAP HUMAN PROSTATE TUMORS: CELLULAR AND MOLECULAR ALTERATIONS 
Lethal phenotypes of human prostate cancer are characterized by progression to androgen-independence and metastasis. For want of a clinically relevant animal model, mechanisms behind this progression remain unclear. Our study used an in vivo model of androgen-sensitive LNCaP human prostate cancer cell xenografts in male SCID mice to study the cellular and molecular biology of tumor progression. Primary tumors were established orthotopically, and the mice were then surgically castrated to withdraw androgens. Five generations of androgen-independent tumors were developed using castrated host mice. Tumor samples were used to determine expressions of cellular and molecular markers. Androgen-independent tumors had increased proliferation and decreased apoptosis compared to androgen-sensitive tumors, outcomes associated with elevated expression of p53, p21/waf1, bcl-2, bax and the bcl-2/bax ratio. Blood vessel growth in androgen-independent tumor was associated with increased expression of vascular endothelial growth factor. Overexpression of androgen receptor mRNA and reduced expression of androgen receptor protein in androgen-independent tumors suggest that the androgen receptor signaling pathway may play an important role in the progression of human prostate cancer to androgen-independence. The in vivo orthotopic LNCaP tumor model described in our study mimics the clinical course of human prostate cancer progression. As such, it can be used as a model for defining the molecular mechanisms of prostate cancer progression to androgen-independence and for evaluating the effect of preventive or therapeutic regimens for androgen-independent human prostate cancer.
doi:10.1002/ijc.20206
PMCID: PMC2683255  PMID: 15170660
prostate cancer; androgen-sensitive; androgen-independent; orthotopic model
25.  Enhanced detection of metastatic prostate cancer cells in human plasma with lipid bodies staining 
BMC Cancer  2014;14:91.
Background
Reprogramming of energy metabolism of malignant cancer cells confers competitive advantage in growth environments with limited resources. However, not every process of cancer development is associated with competition for resources. During hematogenous transport, cancer cells are exposed to high levels of oxygen and nutrients. Does energy metabolism of cancer cells change as a function of exposure to the bloodstream? Could such changes be exploited to improve the detection of circulating tumor cells (CTC)? These questions have clinical significance, but have not yet been sufficiently examined.
Methods
The energy metabolism was examined as a function of incubation in nutrient-rich plasma in prostate metastatic cancer cells LNCaP and non-transformed prostate epithelial cells RWPE1. Uptake kinetics of a fluorescent glucose analog (2-NBD) and lipophilic dyes (DiD & Bodipy) were measured in both cell lines, as well as in peripheral blood mononuclear cells (PBMC).
Results
LNCaP cells exhibited hyper-acetylation of low molecular weight proteins compared to RWPE1 cells. Following plasma incubation, protein lysine acetylation profile was unchanged for LNCaP cells while significantly altered for RWPE1 cells. O-linked glycosylated protein profiles were different between LNCaP and RWPE1 cells and varied in both cell lines with plasma incubation. Maximal respiration or glycolytic capacities was unchanged in LNCaP cells and impaired in RWPE1 cells following plasma incubation. However, the uptake rates of 2-NBD and DiD were insufficient for discrimination of LNCaP, or RWPE1 cells from PBMC. On the other hand, both RWPE1 and LNCaP cells exhibited intracellular lipid bodies following plasma incubation; whereas, PBMC did not. The presence of lipid bodies in LNCaP cells permitted retention of Bodipy dye and allowed discrimination of LNCaP cells from PBMC with flow cytometry.
Conclusions
Despite clear differences in energy metabolism, metastatic prostate cancer cells could not be efficiently distinguished from non-transformed prostate epithelial cells using fluorescent glucose or lipid uptake kinetics. However, metastatic prostate cancer cells in plasma could be clearly distinguished from blood nucleated cells due to the presence of intracellular lipid bodies. Fluorescent labeling of lipid bodies permitted a simple and sensitive means for high throughput detection of metastatic prostate cancer cells in human plasma.
doi:10.1186/1471-2407-14-91
PMCID: PMC3931481  PMID: 24528787
Cancer energy metabolism; Coherent anti-Stokes Raman microscopy; Circulating tumor cell; Flow cytometry; Lipid bodies; Prostate cancer; Protein lysine acetylation; Protein O-linked glycosylation; Proteomics

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