RM-1 Cells Research Articles

RelB regulates Bcl-xl expression and the irradiation-induced apoptosis of murine prostate cancer cells.

Apoptosis in prostate cancer (PCa) induced by ionizing radiation (IR) is believed to play a critical role in radioresistance. Bcl-xl, an important member of the anti-apoptotic Bcl-2 family, has critical roles in tumor progression and development. The aim of the present study was to investigate the association of Bcl-xl expression and radiosensitivity from murine PCa RM-1 cells. An adenovirus-mediated RNA interference technique was employed to inhibit the expression of the RelB gene. RelB proteins were detected upon irradiation following transfection with small interfering (si)RelB, as shown by western blot analysis. The radiosensitivity of the RM-1 cells was determined by clonogenic assays. The apoptosis of the RM-1 cells were detected by flow cytometry assay, then quantitative polymerase chain reaction assays were performed to determine the expression level of Bcl-xl mRNA in the RM-1 cells. Radiation treatment increased the RelB protein levels from the cytosol and nucleus in the RM-1 cells. The protein expression levels of RelB in the pLentilox-sh-RelB-transfected RM-1 cells were significantly lower than in the negative interference group following radiation treatment. The percentage of cells undergoing apoptosis in the siRelB-RM-1 group was significantly higher than that in the control group following radiation treatment. Finally, a positive link between Bcl-xl expression and RelB activity was established in the RM-1 cells. Inhibition of RelB correlates with a decrease in expression of Bcl-xl. In conclusion, adenovirus-mediated siRNA targeting RelB inhibits Bcl-xl expression, enhances radiosensitivity and regulates the irradiation-induced apoptosis of the murine PCa RM-1 cell line.

NFκB-mediated CXCL1 production in spinal cord astrocytes contributes to the maintenance of bone cancer pain in mice

BackgroundBone cancer pain (BCP) is one of the most disabling factors in patients suffering from primary bone cancer or bone metastases. Recent studies show several chemokines (for example, CCL2, CXCL10) in the spinal cord are involved in the pathogenesis of BCP. Here we investigated whether and how spinal CXCL1 contributes to BCP.MethodsMouse prostate tumor cell line, RM-1 cells were intramedullary injected into the femur to induce BCP. The mRNA expression of CXCL1 and CXCR2 was detected by quantitative real-time PCR. The protein expression and distribution of CXCL1, NFκB, and CXCR2 was examined by immunofluorescence staining and western blot. The effect of CXCL1 neutralizing antibody, NFκB antagonist, and CXCR2 antagonist on pain hypersensitivity was checked by behavioral testing.ResultsIntramedullary injection of RM-1 cells into the femur induced cortical bone damage and persistent (>21 days) mechanical allodynia and heat hyperalgesia. Tumor cell inoculation also produced CXCL1 upregulation in activated astrocytes in the spinal cord for more than 21 days. Inhibition of CXCL1 by intrathecal administration of CXCL1 neutralizing antibody at 7 days after inoculation attenuated mechanical allodynia and heat hyperalgesia. In cultured astrocytes, TNF-α induced robust CXCL1 expression, which was dose-dependently decreased by NFκB inhibitor. Furthermore, inoculation induced persistent NFκB phosphorylation in spinal astrocytes. Intrathecal injection of NFκB inhibitor attenuated BCP and reduced CXCL1 increase in the spinal cord. Finally, CXCR2, the primary receptor of CXCL1, was upregulated in dorsal horn neurons after inoculation. Inhibition of CXCR2 by its selective antagonist SB225002 attenuated BCP.ConclusionNFκB mediates CXCL1 upregulation in spinal astrocytes in the BCP model. In addition, CXCL1 may be released from astrocytes and act on CXCR2 on neurons in the spinal cord and be involved in the maintenance of BCP. Inhibition of the CXCL1 signaling may provide a new therapy for BCP management.

Anti-tumor immunological response induced by cryoablation and anti-CTLA-4 antibody in an in vivo RM-1 cell prostate cancer murine model.

Cryoablation combination therapy with blockade of the T-cell inhibitory receptor CTL-associated antigen-4 (CTLA-4) may augment the anti-tumor immune response (ATIR). It is crucial to determine the duration of ATIR after cryoablation and anti-CTLA-4 antibody therapy to determine the most appropriate treatment interval of therapy. To investigate the characteristics of ATIR induced by cryoablation and anti-CTLA-4 antibody therapy, we developed a prostate cancer model system to test the capacity of cryoablation and anti -CTLA-4 antibody to generate ATIR. Mice were randomly assigned to receive no treatment (group A), cryoablation only (group B), cryoablation plus anti-CTLA-4 antibody (group C), or anti-CTLA-4 antibody only (group D). We collected specimens on days 0, 7, 14 and 21 to study the ATIR through different techniques. Our results indicated that cryoablation induced ATIR and further enhanced this effect and reduced the number of distant metastases through combination with anti-CTLA-4 antibody. ATIR induced by cryoablation was achieved through decreasing regulatory T cell (Treg) number. The number of Tregs induced by cryoablation was lowest on day 14 but then returned to preoperative levels on day 21, indicating that ATIR induced by cryoablation was time-dependent. However, ATIR induced by anti-CTLA-4 antibody might be mainly achieved through influencing Treg function, which was exactly not by decreasing Treg number and still maintain its ATIR effect on day 21 after therapy. In conclusion, ATIR induced by cryoablation was achieved through decreasing Treg number and is time-dependent, whereas ATIR caused by anti-CTLA-4 antibody was achieved exactly not by decreasing Treg number and not time-dependent in the first 21 days after therapy.

RelB-siRNA enhanced the radiosensitivity of murine prostate cancer cell line RM-1

To explore the transfecting effects of RelB small interfering RNA (RelB-siRNA) on murine prostate cancer cell line RM-1 sensitivity of radiotherapy. The RM-1 cells were divided into RelB-siRNA vector, empty vector, non-transfection and normal groups. After transfection, the first three groups were irradiated by X ray. Clone formation array method was used to measure the surviving fraction and the relevant parameter values after 0, 2, 4, 6, 8 Gy irradiation. Apoptotic rate was measured via Annexin V/PI flow cytometry after 6 Gy irradiation. The level of RelBmRNA was estimated by real-time polymerase chain reaction (PCR) after 6 Gy irradiation. And the RelB protein of cytoplasm and nucleus was detected by Western blot after 6 Gy irradiation. Clone formation array showed that RelB group at each dose point corresponding to the survival fraction were lower that the empty vector nd non-transfection groups. The D0, Dq and SF2 values of RelB group were 1.68, 0.60, 0.43, lower than those of empty vector group 1.92, 3.08, 0.89 and non-transfection group 1.93, 2.76, 0.84. Annexin V/PI flow cytometry demonstrated that the apoptotic rate of RelB group was 15.27% ± 1.62% and it was significantly higher than the non-transfection group 7.90% ± 1.50% and the empty vector group 8.40% ± 0.69% respectively (P < 0.01) .Real-time PCR indicated that RelB-mRNA amplification of RelB group was 1.18 ± 0.03 and it was significantly lower than the non-transfection group 2.10 ± 0.61 and the empty vector group 1.97 ± 0.66 respectively (P < 0.01) .Western blot suggested that cytoplasmic gray-scale ratio of RelB group was 0.50 ± 0.08 and it was significantly lower than the non-transfection 1.77 ± 0.19 and the empty group 1.52 ± 0.12 respectively (P < 0.01) . And the nuclear gray-scale radio of the RelB group was 0.18 ± 0.03 and it was significantly lower than the non-transfection group 0.61 ± 0.12 and the empty group 0.54 ± 0.13 respectively (P < 0.01) . The RelB protein inhibition rates of cytoplasmic and nuclear RelB-siRNA were 71.8% and 70.4% respectively. RelB-siRNA can effectively inhibit the RelB expression of murine prostate cancer cell line RM-1 and significantly increase its sensitivity to radiotherapy.

Roles of microsomal prostaglandin E synthase-1 in lung metastasis formation in prostate cancer RM9 cells

Cyclooxygenase-2 (COX-2) is known to correlate with a poor prognosis of prostate cancer and contribute to tumor metastasis. However, the precise mechanism of this phenomenon remains unknown. We have previously reported that host stromal microsomal prostaglandin E synthase-1 (mPGES-1) appeared critical for tumor-associated angiogenesis and tumor growth. Here, we tested whether or not mPGES-1 has a critical role in lung metastasis formation of prostate cancer. Murine prostate cancer cells (RM9) were intravenously injected and lung metastasis was estimated by counting colonies in the lungs. Mice treated with a selective COX-2 inhibitor, celocoxib, were suppressed lung metastasis compared to vehicle mice. This lung metastasis formation was also reduced in mPGES-1 knockout (mPGES-1 KO) mice, compared with wild type (WT) mice. This was accompanied with reduced angiogenesis around the metastasized colonies of RM9. Plasma protein levels and metastasized lung tissue mRNA levels of vascular endothelial growth factor (VEGF) and stromal cell derived factor-1 (SDF-1) were significantly suppressed in mPGES-1 KO mice in comparison with WT mice. In addition, the expressions of matrix metalloproteinases (MMP)-9, and metalloproteinases (MMP)-2 were down-regulated in metastatic lungs in mPGES-1 KO mice. These results suggested that host mPGES-1 was essential for MMP-2 and MMP-9 up-regulation that enhances tumor metastasis. mPGES-1 appears to be critical for tumor metastasis in prostate cancers. mPGES-1 inhibitors may be useful to protect against prostate cancer metastasis.

OPTIMIZATION OF FERMENTATION CONDITIONS AND PURIFICATION OF CORDYCEPIN FROM Cordyceps militaris

The fermentation medium and conditions for the production of cordycepin were optimized in static culture using single-factor experiments, Placket–Burman design, a central composite design, and response surface methodology. Among seven variables including temperature, pH, and the concentrations of glucose, tryptone, yeast extract, KH2PO4, and MgSO4 · 7H2O, temperature and the concentrations of yeast extract and tryptone were found to be the important factors that significantly affected cordycepin production. The optimized medium consisted of yeast extract 9.00 g/L and tryptone 17.10 g/L, while the optimized culture conditions consisted of seed age 3 days, with an inoculum size of 10% and incubation temperature of 27.1°C. A maximum cordycepin yield of 7.35 g/L was achieved in a 5-L fermenter under the optimized conditions. Next, cordycepin was partially purified and determined. The resulting product showed 90.54% high-performance liquid chromatography (HPLC)–ultraviolet (UV) purity. Therefore, cordycepin was applied to a cell viability assay on SH-SY5Y cells and RM-1 cells. Cordycepin can inhibit the proliferation of RM-1 cells with IC50 of 133 µmol/L, but it has no inhibitory effect on SH-SY5Y cells. Supplemental materials are available for this article. Go to the publisher's online edition of Preparative Biochemistry and Biotechnology to view the supplemental file.

Monitoring luciferase-labeled human prostate stem cell antigen-expressing tumor growth in a mouse model

The aim of this study was to establish a tumor model in mice with the expression of luciferase (Luc) and human prostate stem cell antigen (PSCA), in order to evaluate the activities of anticancer drugs or vaccines for prostate cancer. RM-1 cells were stably transfected with pcDNA-Luc and pcDNA-PSCA plasmids. The Luc-expressing cells were examined using a luminometer and the PSCA-expressing cells were examined using a reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometric analysis. Male C57BL/6 mice were inoculated subcutaneously with the RM-PSCA/Luc cells, prior to the tumor growth and survival time of the mice being measured, respectively. In vivo bioluminescence imaging was used to detect Luc expression and immunohistochemical analysis was used to detect PSCA expression. Inoculation of the tumor cells into the C57BL/6 mice closely mimicked the tumor growth of prostate cancer. All of the inoculated mice exhibited a detectable tumor within two weeks. Tumor progression was able to be quantitatively monitored following the inoculation of 1×106 RM-PSCA/Luc cells. There was an excellent correlation (R2=0.9849) between the photon counts and tumor volume. The expression of PSCA in tumor tissues was confirmed using immunohistochemical analysis. The Luc and PSCA co-expression tumor model was successfully established in mice, which is likely to accelerate the understanding of the pathogenesis of prostate cancer and facilitate the development of novel antitumor drugs or vaccines for the disease.

The Ubiquitin–CXCR4 Axis Plays an Important Role in Acute Lung Infection–Enhanced Lung Tumor Metastasis

Our goals were to test the effect of acute lung infection on tumor metastasis and to investigate the underlying mechanisms. We combined bacteria-induced and lipopolysaccharide (LPS)-induced acute lung injury/inflammation (ALI) mouse models with mouse metastatic models to study the effect of acute inflammation on lung metastasis in mice. The mechanisms were investigated in ex vivo, in vitro, and in vivo studies. Both bacteria- and LPS-induced ALI significantly enhanced lung metastasis of four tail vein-injected mouse tumor cell lines. Bacteria also enhanced lung metastasis when 4T1 cells were orthotopically injected. The bronchoalveolar lavage fluid (BALF) from LPS- or bacteria-injected mice stimulated migration of tumor cells. In vivo tracking of metastatic RM-9 cells showed that bacterial injection enhanced early dissemination of tumor cells to the lung. The majority of the BALF migratory activity could be blocked by AMD3100, a chemokine receptor 4 (CXCR4) inhibitor. All tested cell lines expressed CXCR4. The levels of extracellular ubiquitin, but not stromal cell-derived factor-1, in BALF were significantly increased by LPS. Ubiquitin was able to induce AMD3100-sensitive migration of tumor cells. Finally, the antibacterial agent amoxicillin and the CXCR4 inhibitor AMD3100 blocked the enhancement effect of bacterial infection on tumor metastasis. Acute lung infection dramatically increased cancer cell homing to the lung and lung metastasis. This change may be due to an alteration of the lung microenvironment and preparation of a favorable metastatic "niche." This effect was seen in multiple cancer types and thus may have broad applications for cancer patients in prevention and/or treatment of metastasis.

Platelet‐derived growth factor‐BB accelerates prostate cancer growth by promoting the proliferation of mesenchymal stem cells

Mesenchymal stem cells (MSCs) favor cancer growth by facilitating immunosuppression status in tumor microenvironment. However, the function and mechanism of MSCs in initiating and developing prostate cancer remains to be fully understood. In this study, we first found that MSCs promoted prostate cancer (PCa) tumor growth in vivo and cell proliferation in vitro by using PCs cell strain RM-1. Both exogenous and endogenous MSCs could be recruited into the tumor microenvironment by using bone-marrow transplantation model. We further demonstrated that PDGF-BB produced by RM-1 cell promoted MSCs proliferation in vivo and in vitro, which was abrogated by Si-RNA specific to PDGF-BB. And inflammatory cytokines, such as interferon gamma, tumor necrosis factor alpha, and anti-inflammatory cytokine transformation growth factor alpha, further increased the ability of RM-1 to produce PDGF-BB. Overall, PCa cells produced PDGF-BB favors the proliferation of MSCs, which may elicit immunosuppressive function and enable PCa cells to escape from the immunity surveillance in tumor inflammatory microenvironment.

38. The use of 1,25[α] dihydroxyvitamin D3 as a cryosensitizing agent in a murine prostate cancer model

Vitamin D3 has been identified as a possible adjunctive treatment for cancer therapies due to its antiproliferative and antitumorigeneic properties. It has also been implicated in the inflammation process, which is critical to the initiation and progression of prostate cancer (CaP). Cryotherapy, the use of low temperatures to ablate tumorigenic tissues, has recently emerged as a primary treatment option for CaP. For these reasons we hypothesized that combining calcitriol with the sub lethal temperatures experienced in the periphery of the cryogenic lesion would result in an increase in apoptotic cell death as compared to either cryotherapy or calcitriol alone. This study aimed to identify the pathways activated when the biologically active metabolite of vitamin D3 (calcitriol) is combined with cryotherapy in a murine model of prostate cancer. The RM-9 cell line was treated with 1,25 α dihydroxyvitamin D3 [25–150 nM] for 24 h prior to exposure to low temperature in a temperature controlled bath. Ice nucleation was initiated by liquid nitrogen vapor and cell viability was assessed using alamarBlue for 3 days post-freeze. Data indicated that pre-treatment with calcitriol sensitizes RM-9 cells to enhanced cellular injury at low temperature. Treatment with calcitriol alone [25–400 nM] shows no effect on cell viability. Similarly, a single freeze between −5 °C and −15 °C alone had little effect on survival. However, when cells are pre-treated with calcitriol and then exposed to low temperature, there is a synergistic effect of increased efficacy of cell death. At −15 °C coupled with calcitriol [50–75 nM] cell death increased to 55% of day 1 controls following a single freeze exposure, compared to negligible cell death with freeze alone. Repeat exposure resulted in 90% cell death compared to 65% in dual freeze alone. Western blotting of signaling proteins during the calcitriol and cryotherapy sensitization regimes confirmed the activation of apoptosis. Specifically, pro-apoptotic Bid and pro-caspase-3 was found to decrease at 1 h following combination treatment, indicative of cleavage to the active form. The survival protein Akt was found to be upregulated at 4 h post-cryo at higher doses of calcitriol (150 nM), which could be indicative of pro-survival signaling through the PI3-K pathway. Additionally molecular analysis of in vivo tissue tumor samples treated with calcitriol and cryotherapy also indicated the involvement of apoptosis through caspase-3 and caspase-9 cleavage. Previous studies have elucidated the in vitro benefits of cryosensitization strategies including combining drugs such as 5-fluorouracil and taxotere with cryotherapy; however, in vivo chemotherapeutic drugs present with toxic side effects as well as the likelihood of developing resistance. The need for the development of novel, non-toxic treatments for prostate cancer is essential, as many current strategies have negative side effects. The development of an adjunctive therapy combining calcitriol and cryotherapy represents a potentially highly effective, less toxic minimally invasive alternative treatment option.

Abstract 1868: Metabolic characterization of prostate cancer cell lines.

Abstract Purpose: The tumor microenvironment has a major impact on tumor growth and progression; the composition and characteristics of the tumor microenvironment is influenced by the metabolism of both tumor and stromal cells. In preparation to determining the effects of cell metabolism on the anti-tumor T cell immune response, we studied the metabolism of two murine prostate cell lines (RM1 and Myc-Cap) under different conditions. The long-term goal of our study is to assess what features of an abnormal tumor microenvironment negatively impact T cell tumor targeting and the activation of T cells in orthotopic prostate cancer models. Methods: The metabolic profiles of the more aggressive rapidly growing RM1 and more moderate growing Myc-Cap cells were determined. A Seahorse XF96 Extracellular Flux Analyzer, which provides real-time measurements of the extracellular acidification rate (ECAR), was used to measure of glycolysis and lactate production, and the oxygen consumption rate (OCR), a measure of oxidative phosphorylation (OXPHOS). The effects of 24-hour glucose and glutamine deprivation were studied. Results: Myc-Cap cells utilized significantly less glucose, produced much less acid (lactate) and had a low proliferation rate compared to RM1 cells. RM1 cells exhibit a higher rate of glycolysis and showed higher glycolytic capacity, whereas OCR (a measure of mitochondrial respiration) was lower in RM1 compared to Myc-Cap cells. The expression of glycolytic enzymes was comparable in both cell lines; RM1 cells demonstrated higher LDHA and lower expression of mitochondrial enzymes, as measured by Western blot. For RM1, glucose withdrawal from the media resulted in reduced LDHA expression, a 77% decrease in lactate production (measured by ECAR), and a 50% decrease in OCR. Glutamine withdrawal resulted in a 35% decrease in ECAR, and an 82% decrease in OCR. Myc-Cap cells were slightly less affected by glucose withdrawal (slight change in LDHA expression, a 69% decrease in ECAR, and a 40% decrease in OCR), whereas glutamine withdrawal had a more profound effect on ECAR (75% decrease) and a similar effect on OCR (83% decrease). Glutamine withdrawal reduced the proliferation rate of both cell lines significantly more than glucose withdrawal. Conclusions: Our data demonstrate a major difference in acid production (lactate) between the two cell lines (RM1&amp;gt;Myc-Cap). RM1 cells are more glucose dependent, whereas Myc-Cap is more glutamine dependent. Understanding the metabolic differences between tumors and the impact of metabolism on the tumor microenvironment will provide specific targets that could enhance T cell therapy through a modification of the tumor microenvironment. Citation Format: Ekaterina S. Moroz, Ronald Blasberg, Inna Serganova. Metabolic characterization of prostate cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1868. doi:10.1158/1538-7445.AM2013-1868

Enhanced function of cytotoxic T lymphocytes induced by dendritic cells modified with truncated PSMA and 4-1BBL

Interactions between costimulatory molecules and their receptors are vital for Ag-presenting dendritic cells (DCs) to initiate T cells activation, expansion and their antitumor immune responses. Augmentation of costimulatory signal due to the interaction of DCs and T cells may amplify, sustain and drive diversity of cytotoxic T lymphocytes (CTLs) and consequently enhance the antitumor response. 4-1BBL/4-1BB is such a pair of costimulatory ligand and receptor, playing an important role in the co-stimulation of CTLs. Previously, we demonstrated that DCs transduced with recombinant adenovirus encoding truncated PSMA (tPSMA) and m4-1BBL could induce prostate cancer regression in mouse models. In the present study, we further explored the adjuvant role of 4-1BBL in modulating CTLs activation induced by tPSMA gene-pulsed DCs. The apoptosis and cytotoxicity against tPSMA expressing RM-1 cells of CTLs were determined. Results showed that tPSMA gene-pulsed DCs effectively induced T lymphocyte activation and cytotoxicity, which was enhanced by upregulated expression of 4-1BBL, displaying better cell viability, lower CTLs apoptosis, higher expression anti-apoptotic protein of Bcl-xL and phosphorylation of P38, enhanced NF-κB activation, as well as more IFN-γ production. These results demonstrated that 4-1BBL may play a significant role in the co-stimulation pathway for Ag-presenting DCs-mediated CTLs activity, which might be a beneficial adjuvant factor for DCs-based cancer immunotherapy.

Potent growth-inhibitory effect of a dual cancer-specific oncolytic adenovirus expressing apoptin on prostate carcinoma

Apoptin is a chicken anemia virus-derived, p53-independent, bcl-2-insensitive apoptotic protein with the ability to specifically induce apoptosis in various human tumor cells, but not in normal cells. To explore the use of apoptin in tumor gene therapy, we assessed a recombinant adenovirus expressing the apoptin protein (Ad-hTERTp-E1a-Apoptin) in order to determine its lethal and growth-inhibitory effects on PC-3 and RM-1 cells in vitro and its antitumor effect on solid tumors in vivo. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), acridine orange (AO)/ethidium bromide (EB), 4'-6-diamidino-2-phenylindole (DAPI), and Annexin V assays showed that Ad-hTERTp-E1a-Apoptin inhibited the proliferation of PC-3 and RM-1 cells in vitro by inducing apoptosis of prostate cancer cells, and that this inhibitory effect was dose and time-dependent. In the animal models, Ad-hTERTp-E1a-Apoptin significantly inhibited tumor growth and extended the lifespan of animals. Experimental results indicate that Ad-hTERTp-E1a-Apoptin has a potential application in tumor gene therapy.

Antitumor activity of tripterine via cell-penetrating peptide-coated nanostructured lipid carriers in a prostate cancer model

BackgroundThe purpose of this study was to evaluate the antitumor effect of cell-penetrating peptide-coated tripterine-loaded nanostructured lipid carriers (CT-NLC) on prostate tumor cells in vitro and in vivo.MethodsCT-NLC were developed to improve the hydrophilicity of tripterine. The antiproliferative effects of CT-NLC, tripterine-loaded nanostructured lipid carriers (T-NLC), and free tripterine in a human prostatic carcinoma cell line (PC-3) and a mouse prostate carcinoma cell line (RM-1) were evaluated using an MTT assay. The advantage of CT-NLC over T-NLC and free tripterine with regard to antitumor activity in vivo was evaluated in a prostate tumor-bearing mouse model. The induced tumor necrosis factor-alpha and interleukin-6 cytokine content was investigated by enzyme-linked immunosorbent assay to determine the effect of CT-NLC, T-NLC, and free tripterine on immune responses. Histologic and TUNEL assays were carried out to investigate the mechanisms of tumor necrosis and apoptosis.ResultsCT-NLC, T-NLC, and free tripterine showed high antiproliferative activity in a dose-dependent manner, with an IC50 of 0.60, 0.81, and 1.02 μg/mL in the PC-3 cell line and 0.41, 0.54, and 0.89 μg/mL in the RM-1 cell line after 36 hours. In vivo, the tumor inhibition rates for cyclophosphamide, high-dose (4 mg/kg) and low-dose (2 mg/kg) tripterine, high-dose (4 mg/kg) and low-dose (2 mg/kg) T-NLC, high-dose (4 mg/kg) and low-dose (2 mg/kg) CT-NLC were 76.51%, 37.07%, 29.53%, 63.56%, 48.25%, 72.68%, and 54.50%, respectively, showing a dose-dependent pattern. The induced tumor necrosis factor-alpha and interleukin-6 cytokine content after treatment with CT-NLC and T-NLC was significantly higher than that of high-dose tripterine. Moreover, CT-NLC showed the expected advantage of inducing necrosis and apoptosis in prostate tumor cells.ConclusionCT-NLC noticeably enhanced antitumor activity in vitro and in vivo and showed dramatically improved cytotoxicity in normal cells in comparison with free tripterine. In summary, CT-NLC could be used as a promising drug delivery system for the treatment of prostate cancer.

4-1BB Protects Dendritic Cells from Prostate Cancer-Induced Apoptosis

It has been shown that human prostate cancer (PCa) cells induced apoptotic death of the most potent antigen-presenting cells, dendritic cells (DCs), which are responsible for the induction of specific antitumor immune responses. Here, we investigated the function of 4-1BB on protecting DCs from prostate cancer-induced apoptosis with an agonistic mAb to 4-1BB. RM-1 cells and DCs were co-incubated for 48h and DC apoptosis was assessed by Annexin Vassay. TNF-α and IL-12 production were assessed by enzyme-linked immunosorbent assay (ELISA) and Bcl-2 and Bcl-xL on DCs were analyzed by Western blot. We have shown that co-incubation of RM-1 cells with DCs is accompanied by an increased level of DCs apoptosis. Triggering 4-1BB on DCs resulted in increased resistance of DCs to RM-1 cells-induced apoptosis, which was owing to the up-regulated expression of Bcl-2 and Bcl-xL, and increased secretion of TNF-αand IL-12. These results demonstrate that triggering 4-1BB on DCs could increased resistance of DCs to PCa-induced apoptosis.

Dendritic Cell Based PSMA Immunotherapy for Prostate Cancer Using a CD40-Targeted Adenovirus Vector

Human prostate tumor vaccine and gene therapy trials using ex vivo methods to prime dendritic cells (DCs) with prostate specific membrane antigen (PSMA) have been somewhat successful, but to date the lengthy ex vivo manipulation of DCs has limited the widespread clinical utility of this approach. Our goal was to improve upon cancer vaccination with tumor antigens by delivering PSMA via a CD40-targeted adenovirus vector directly to DCs as an efficient means for activation and antigen presentation to T-cells. To test this approach, we developed a mouse model of prostate cancer by generating clonal derivatives of the mouse RM-1 prostate cancer cell line expressing human PSMA (RM-1-PSMA cells). To maximize antigen presentation in target cells, both MHC class I and TAP protein expression was induced in RM-1 cells by transduction with an Ad vector expressing interferon-gamma (Ad5-IFNγ). Administering DCs infected ex vivo with CD40-targeted Ad5-huPSMA, as well as direct intraperitoneal injection of the vector, resulted in high levels of tumor-specific CTL responses against RM-1-PSMA cells pretreated with Ad5-IFNγ as target cells. CD40 targeting significantly improved the therapeutic antitumor efficacy of Ad5-huPSMA encoding PSMA when combined with Ad5-IFNγ in the RM-1-PSMA model. These results suggest that a CD-targeted adenovirus delivering PSMA may be effective clinically for prostate cancer immunotherapy.

Abstract A77: Preclinical multimodality imaging of metastatic prostate cancer xenograft models with 89Zr-J591.

Abstract Background: The goal of this study was to investigate the efficacy of a humanized antibody, J591, in targeting metastasized prostate cancer (PC). PC, the most common solid tumor affecting American males, is expected to be diagnosed in 241,740 men and to cause 28,170 deaths in 2012 [1]. Current imaging tools for the disease, such as positron emission tomography – computed tomography (PET/CT) with [18F]-fluorodeoxyglucose (FDG), are inadequate because they are non-specific to prostate cancer cells. PET imaging with prostate cancer specific monoclonal antibodies (J591) promises greater specificity by targeting the extracellular epitope of prostate specific membrane antigen (PSMA). PSMA is a 100 kDa protein over-expressed in prostate cancer cells and up-regulated in high-grade disease. Although, preclinical J591 ImmunoPET imaging has been successfully reported for primary tumors [2], there have been no similar reports for metastatic cancer. In this study, we investigate the performance of a radioimmunoconjugate, [89]Zr-J591, for PET/CT imaging of C4-2 (bone-seeking) and RM1 (lung-seeking) tumors on a small animal PET camera. The PET/CT images were compared to 7T magnetic resonance (MR) images of the tumors, to determine the added diagnostic value of [89]Zr-J591 imaging. Methods: [89]Zr was produced in a cyclotron using 89Y(p,n)89Zr transmutation reaction and conjugated via desferrioxamine B to the J591 monoclonal antibody (mAb). PC cell lines adapted for flank tumors in xenograft mice (LNCaP cell line), lung-seeking (RM1 cell line) and orthotopic (C4-2 cell line) were kept in culture for two weeks and then implanted into male athymic nu/nu BalbC mice, by tail intravenous or subcutaneous injection. The RM1 and C4-2 cells had previously been modified to express green fluorescent protein (GFP) and after 3 weeks, bioluminescence images were acquired to verify the growth of the tumors. MR images of selected mice were also acquired. The mice were divided into two groups, and the first was administered 200 uCi of 89Zr-J591 for imaging studies. PET/CT images of these mice were acquired 3 days post-injection. The mice in the second group were injected with 50 uCi of [89]Zr-J591 for bio-distribution studies. After 3 days, the mice were sacrificed; specific tissues were extracted, weighed and measured in a gamma counter. Results: There was excellent tumor targeting by [89]Zr-J591 with tumor-to-muscle ratios of about 10. This was confirmed by in vivo bio-distribution studies, which showed an average uptake of 22 percentage injected dose per gram (%ID/g). Standard uptake values (SUV) ranged between 3.5 and 4.7. We observed good correlation between the bioluminescence, PET/CT and MR images. We also observed that the PET/CT images provided better tumor delineation, and therefore greater diagnostic value, than MRI images Conclusions: [89]Zr-J591 shows great promise as a prostate cancer imaging biomarker. It provides high tumor to background ratio, superior quantitative properties and specificity for PSMA. Establishing an imaging biomarker is the first step toward tailoring of therapies toward individuals or specific populations at risk. [1] American Cancer Society. Cancer Facts &amp; Figures 2012. Atlanta: American Cancer Society; 2012. [2] 89Zr-DFO-J591 for immunoPET of prostate-specific membrane antigen expression in vivo. Holland JP, Divilov V, Bander NH, Smith-Jones PM, Larson SM, Lewis JS. J Nucl Med. 2010 Aug;51(8):1293-300. Epub 2010 Jul 21. Citation Format: Kofi Deh, Siddarth Chandrasekaran, Shoaib Fareedy, Joseph Osborne. Preclinical multimodality imaging of metastatic prostate cancer xenograft models with 89Zr-J591. [abstract]. In: Proceedings of the Fifth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2012 Oct 27-30; San Diego, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(10 Suppl):Abstract nr A77.

Interleukin-1α induces immunosuppression by mesenchymal stem cells promoting the growth of prostate cancer cells

Mesenchymal stem cells (MSCs) are generally used in tissue engineering, regenerative medicine and therapy for immune disorder disease. MSCs are also employed as drug carriers for tumor therapy due to their ability to migrate to tumor tissue. However, due to the immunosuppressive function of MSCs, the application of MSCs in prostate cancer therapy remains limited. In this study, we investigated the underlying mechanism by which MSCs enable prostate cancer cells to escape from immune surveillance in the inflammatory microenvironment. Firstly, we demonstrated that compared with the control groups, MSCs pretreated with IL-1α effectively promoted the growth of the mouse prostate cancer cell line RM-1 invivo. Furthermore, when RM-1 prostate cancer cells were co-injected with MSCs pretreated with IL-1α, tumor incidence significantly increased in allogeneic recipients. In addition, we investigated the mechanism through which MSCs promote the ability of RM-1 cells to escape from immune injury. The results revealed that IL-1α led to the upregulation of TGF-β in MSCs. The inflammatory cytokine-induced promotive effect of MSCs on RM-1 cells in vivo was inhibited by TGF-β siRNA. The results of our study suggest that inflammatory cytokines induce the immunosuppressive function of MSCs which enables prostate cancer cells to escape from immune injury.

Prostate-specific membrane antigen can promote in vivo osseous metastasis of prostate cancer cells in mice

Reports remain insufficient on whether and how prostate-specific membrane antigen (PSMA) can influence in vivo osseous metastasis of prostate cancer (PCa). In the present study, the authors induced stable expression of PSMA in mouse PCa cell line RM-1. In vivo osseous metastasis was induced in 37 6-week-old female C57BL/6 mice weighing 22.45 ± 0.456 g. RM-1 cells were actively injected into the femoral bone cavity, leading to bilateral dissymmetry of bone density in the femoral bone. Tumor cells were also detected in bone tissue by pathological examination. The impact on bone density was demonstrated by the significant difference between animals injected with RM-PSMA cells (0.0738 ± 0.0185 g/cm2) and animals injected with RM-empty plasmid cells (0.0895 ± 0.0241 g/cm2). The lytic bone lesion of the RM-PSMA group (68.4%) was higher than that of the control group (27.8%). Immunohistochemistry showed that the expression of both vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9) was distinctly higher in the RM-PSMA group than in the control group, while ELISA and Western blot assay indicated that VEGF and MMP-9 were higher in the RM-PSMA group compared to the control group (in vitro). Thus, the present study proposed and then confirmed for the first time that PSMA can promote in vivo osseous metastasis of PCa by increasing sclerotic destruction of PCa cells. Further analyses also suggested that PSMA functions positively on the invasive ability of RM-1 by increasing the expression of MMP-9 and VEGF by osseous metastases in vivo.

Prevalence of Prostate Cancer Metastases after Intravenous Inoculation Provides Clues into the Molecular Basis of Dormancy in the Bone Marrow Microenvironment

Bone is the preferred metastasis site of advanced prostate cancer (PCa). Using an in vivo murine model of human PCa cell metastasis to bone, we noted that the majority of animals that develop skeletal metastasis have either spinal lesions or lesions in the bones of the hindlimb. Much less frequently, lesions develop in the bones of the forelimb. We therefore speculated whether the environment of the forelimb bones is not permissive for the growth of PCa. Consequently, data on tumor prevalence were normalized to account for the number of PCa cells arriving after intravascular injection, marrow cellularity, and number of hematopoietic stem cell niches. None of these factors were able to account for the observed differences in tumor prevalence. An analysis of differential gene and protein levels identified that growth arrest specific-6 (GAS6) levels were significantly greater in the forelimb versus hindlimb bone marrow. When murine RM1 cells were implanted into subcutaneous spaces in immune competent animals, tumor growth in the GAS6-/- animals was greater than in GAS6+/+ wild-type animals. In an osseous environment, the human PC3 cell line grew significantly better in vertebral body transplants (vossicles) derived from GAS6-/- animals than in vossicles derived from GAS6+/+ animals. Together, these data suggest that the differences in tumor prevalence after intravascular inoculation are a useful model to study the molecular basis of tumor dormancy. Importantly, these data suggest that therapeutic manipulation of GAS6 levels may prove useful as a therapy for metastatic disease.