Inhibition of Human Urokinase-Type Plasminogen Activator (uPA) Enzyme Activity and Receptor Binding by DNA Aptamers as Potential Therapeutics through Binding to the Different Forms of uPA.

Urokinase-type plasminogen activator is widely discussed as a marker for cancer prognosis and diagnosis and as a target for cancer therapies. Together with its receptor, uPA plays an important role in tumorigenesis, tumor progression and metastasis. In the present study, systematic evolution of ligands by exponential enrichment (SELEX) was used to select single-stranded DNA aptamers targeting different forms of human uPA. Selected aptamers allowed the distinction between HMW-uPA and LMW-uPA, and therefore, presumably, have different binding regions. Here, uPAapt-02-FR showed highly affine binding with a KD of 0.7 nM for HMW-uPA and 21 nM for LMW-uPA and was also able to bind to pro-uPA with a KD of 14 nM. Furthermore, no cross-reactivity to mouse uPA or tissue-type plasminogen activator (tPA) was measured, demonstrating high specificity. Suppression of the catalytic activity of uPA and inhibition of uPAR-binding could be demonstrated through binding with different aptamers and several of their truncated variants. Since RNA aptamers are already known to inhibit uPA-uPAR binding and other pathological functions of the uPA system, these aptamers represent a novel, promising tool not only for detection of uPA but also for interfering with the pathological functions of the uPA system by additionally inhibiting uPA activity.

Comprehensive Evaluation of Multiple Approaches Targeting ABCB1 to Resensitize Docetaxel-Resistant Prostate Cancer Cell Lines.

Docetaxel (DTX) is a mainstay in the treatment of metastatic prostate cancer. Failure of DTX therapy is often associated with multidrug resistance caused by overexpression of efflux membrane transporters of the ABC family such as the glycoprotein ABCB1. This study investigated multiple approaches targeting ABCB1 to resensitize DTX-resistant (DTXR) prostate cancer cell lines. In DU145 DTXR and PC-3 DTXR cells as well as age-matched parental controls, the expression of selected ABC transporters was analyzed by quantitative PCR, Western blot, flow cytometry and immunofluorescence. ABCB1 effluxing activity was studied using the fluorescent ABCB1 substrate rhodamine 123. The influence of ABCB1 inhibitors (elacridar, tariquidar), ABCB1-specific siRNA and inhibition of post-translational glycosylation on DTX tolerance was assessed by cell viability and colony formation assays. In DTXR cells, only ABCB1 was highly upregulated, which was accompanied by a strong effluxing activity and additional post-translational glycosylation of ABCB1. Pharmacological inhibition and siRNA-mediated knockdown of ABCB1 completely resensitized DTXR cells to DTX. Inhibition of glycosylation with tunicamycin affected DTX resistance partially in DU145 DTXR cells, which was accompanied by a slight intracellular accumulation and decreased effluxing activity of ABCB1. In conclusion, DTX resistance can be reversed by various strategies with small molecule inhibitors representing the most promising and feasible approach.

Neuropilin-2 is an independent prognostic factor for shorter cancer-specific survival in patients with acinar adenocarcinoma of the prostate.

Neuropilin-2 (NRP2) is a member of the neuropilin receptor family and known to regulate autophagy and mTORC2 signaling in prostate cancer (PCa). Our study investigated the association of immunohistochemical NRP2 expression with clinicopathological data in PCa patients. For this purpose, we generated a tissue microarray with prostate tissue specimens from 400 PCa patients treated by radical prostatectomy. We focused on patients with high-risk factors such as extraprostatic extension (pT ≥ 3), Gleason score ≥8 and/or the presence of regional lymph node metastases (pN1). Protein levels of NRP2, the vascular endothelial growth factor C (VEGFC) and oncogenic v-ets avian erythroblastosis virus E26 oncogene homolog (ERG) gene as an indicator for TMPRSS2-ERG fusion was assessed in relation to the patients' outcome. NRP2 emerged as an independent prognostic factor for cancer-specific survival (CSS) (hazard ratio 2.360, 95% confidence interval = 1.2-4.8; p = 0.016). Moreover, the association between NRP2 expression and shorter CSS was also especially pronounced in patients at high risk for progression (log-rank test: p = 0.010). We evaluated the association between NRP2 and the TMPRSS2-ERG gene fusion status assessed by immunohistochemical nuclear ERG staining. However, ERG staining alone did not show any prognostic significance. NRP2 immunostaining is significantly associated with shorter CSS in ERG-negative tumors (log-rank test: p = 0.012). No prognostic impact of NRP2 expression on CSS was observed in ERG-positive tumors (log-rank test: p = 0.153). Our study identifies NRP2 as an important prognostic marker for a worse clinical outcome especially in patients with a high-risk PCa and in patients with ERG-negative PCa.

Urinary MicroRNAs as Potential Markers for Non-Invasive Diagnosis of Bladder Cancer.

Currently, voided urine cytology (VUC) serves as the gold standard for the detection of bladder cancer (BCa) in urine. Despite its high specificity, VUC has shortcomings in terms of sensitivity. Therefore, alternative biomarkers are being searched, which might overcome these disadvantages as a useful adjunct to VUC. The aim of this study was to evaluate the diagnostic potential of the urinary levels of selected microRNAs (miRs), which might represent such alternative biomarkers due to their BCa-specific expression. Expression levels of nine BCa-associated microRNAs (miR-21, -96, -125b, -126, -145, -183, -205, -210, -221) were assessed by quantitative PCR in urine sediments from 104 patients with primary BCa and 46 control subjects. Receiver operating characteristic (ROC) curve analyses revealed a diagnostic potential for miR-96, -125b, -126, -145, -183, and -221 with area under the curve (AUC) values between 0.605 and 0.772. The combination of the four best candidates resulted in sensitivity, specificity, positive and negative predictive values (NPV), and accuracy of 73.1%, 95.7%, 97.4%, 61.1%, and 80.0%, respectively. Combined with VUC, sensitivity and NPV could be increased by nearly 8%, each surpassing the performance of VUC alone. The present findings suggested a diagnostic potential of miR-125b, -145, -183, and -221 in combination with VUC for non-invasive detection of BCa in urine.

An orthotopic xenograft model for high-risk non-muscle invasive bladder cancer in mice: influence of mouse strain, tumor cell count, dwell time and bladder pretreatment.

BACKGROUND: Novel theranostic options for high-risk non-muscle invasive bladder cancer are urgently needed. This requires a thorough evaluation of experimental approaches in animal models best possibly reflecting human disease before entering clinical studies. Although several bladder cancer xenograft models were used in the literature, the establishment of an orthotopic bladder cancer model in mice remains challenging. METHODS: Luciferase-transduced UM-UC-3LUCK1 bladder cancer cells were instilled transurethrally via 24G permanent venous catheters into athymic NMRI and BALB/c nude mice as well as into SCID-beige mice. Besides the mouse strain, the pretreatment of the bladder wall (trypsin or poly-L-lysine), tumor cell count (0.5 × 106-5.0 × 106) and tumor cell dwell time in the murine bladder (30 min - 2 h) were varied. Tumors were morphologically and functionally visualized using bioluminescence imaging (BLI), magnetic resonance imaging (MRI), and positron emission tomography (PET). RESULTS: Immunodeficiency of the mouse strains was the most important factor influencing cancer cell engraftment, whereas modifying cell count and instillation time allowed fine-tuning of the BLI signal start and duration - both representing the possible treatment period for the evaluation of new therapeutics. Best orthotopic tumor growth was achieved by transurethral instillation of 1.0 × 106 UM-UC-3LUCK1 bladder cancer cells into SCID-beige mice for 2 h after bladder pretreatment with poly-L-lysine. A pilot PET experiment using 68Ga-cetuximab as transurethrally administered radiotracer revealed functional expression of epidermal growth factor receptor as representative molecular characteristic of engrafted cancer cells in the bladder. CONCLUSIONS: With the optimized protocol in SCID-beige mice an applicable and reliable model of high-risk non-muscle invasive bladder cancer for the development of novel theranostic approaches was established.

Development of novel radiochemotherapy approaches targeting prostate tumor progenitor cells using nanohybrids.

Many tumors including prostate cancer are maintained by cancer stem cells (CSCs), which might cause tumor relapse if not eradicated during the course of treatment. Specific targeting or radiosensitization of CSCs bear promise to improve tumor curability by synergistic effects in combination with radiotherapy. Carbon nanotubes (CNTs) can be used as promising drug delivery systems for anticancer drugs such as the flavonoid catechin. Catechin is an extensively studied active ingredient of the different plants, including green tea, and it is widely recognized as co-adjuvant in cancer therapy. Here we describe the synthesis of biocompatible, catechin-loaded and gelatin-conjugated CNTs (Gel_CT_CNTs) with anticancer properties and demonstrate their potential for the eradication of prostate CSCs in combination with X-ray irradiation. Gel_CT_CNTs showed a significant enhancement of in vitro anticancer activity as compared to catechin alone. Moreover, treatment of prostate cancer cells with Gel_CT_CNT nanohybrids inhibited the tumorigenic cell population defined by a high aldehyde dehydrogenase (ALDH) activity. A combination of X-ray irradiation and treatment with Gel_CT_CNTs caused a decrease in the protein level of stem cell-related transcription factors and regulators including Nanog, Oct4 and ß-catenin and led to an increase of cancer cell radiosensitivity as demonstrated by clonogenic and spherogenic cell survival assays. Taken together, our results suggest that a combination of irradiation and Gel_CT_CNTs can be potentially used for the radiosensitization and eradication of prostate CSC populations.

Elevated expression of prostate cancer-associated genes is linked to down-regulation of microRNAs.

BACKGROUND: Recent evidence suggests that the prostate cancer (PCa)-specific up-regulation of certain genes such as AMACR, EZH2, PSGR, PSMA and TRPM8 could be associated with an aberrant expression of non-coding microRNAs (miRNA). METHODS: In silico analyses were used to search for miRNAs being putative regulators of PCa-associated genes. The expression of nine selected miRNAs (hsa-miR-101, -138, -186, -224, -26a, -26b, -374a, -410, -660) as well as of the aforementioned PCa-associated genes was analyzed by quantitative PCR using 50 malignant (Tu) and matched non-malignant (Tf) tissue samples from prostatectomy specimens as well as 30 samples from patients with benign prostatic hyperplasia (BPH). Then, correlations between paired miRNA and target gene expression levels were analyzed. Furthermore, the effect of exogenously administered miR-26a on selected target genes was determined by quantitative PCR and Western Blot in various PCa cell lines. A luciferase reporter assay was used for target validation. RESULTS: The expression of all selected miRNAs was decreased in PCa tissue samples compared to either control group (Tu vs Tf: -1.35 to -5.61-fold; Tu vs BPH: -1.17 to -5.49-fold). The down-regulation of most miRNAs inversely correlated with an up-regulation of their putative target genes with Spearman correlation coefficients ranging from -0.107 to -0.551. MiR-186 showed a significantly diminished expression in patients with non-organ confined PCa and initial metastases. Furthermore, over-expression of miR-26a reduced the mRNA and protein expression of its potential target gene AMACR in vitro. Using the luciferase reporter assay AMACR was validated as new target for miR-26a. CONCLUSIONS: The findings of this study indicate that the expression of specific miRNAs is decreased in PCa and inversely correlates with the up-regulation of their putative target genes. Consequently, miRNAs could contribute to oncogenesis and progression of PCa via an altered miRNA-target gene-interaction.

High serum levels of Dickkopf-1 are associated with a poor prognosis in prostate cancer patients.

BACKGROUND: The Wnt inhibitor Dickkopf-1 (DKK-1) has been linked to the progression of malignant bone disease by impairing osteoblast activity. In addition, there is increasing data to suggest direct tumor promoting effects of DKK-1. The prognostic role of DKK-1 expression in prostate cancer remains unclear. METHODS: A prostate cancer tissue microarray (n = 400) was stained for DKK-1 and DKK-1 serum levels were measured in 80 patients with prostate cancer. The independent prognostic value of DKK-1 expression was assessed using multivariate analyses. RESULTS: DKK-1 tissue expression was significantly increased in prostate cancer compared to benign disease, but was not correlated with survival. However, high DKK-1 serum levels at the time of the diagnosis were associated with a significantly shorter overall and disease-specific survival. Multivariate analyses defined high serum levels of DKK-1 as an independent prognostic marker in prostate cancer (HR 3.73; 95%CI 1.44-9.66, p = 0.007). CONCLUSION: High DKK-1 serum levels are associated with a poor survival in patients with prostate cancer. In light of current clinical trials evaluating the efficacy of anti-DKK-1 antibody therapies in multiple myeloma and solid malignancies, the measurement of DKK-1 in prostate cancer may gain clinical relevance.

Prognostic impact of urokinase-type plasminogen activator system components in clear cell renal cell carcinoma patients without distant metastasis.

BACKGROUND: Members of the urokinase-type plasminogen activator (uPA) system including uPA, its receptor uPAR and the plasminogen activator inhibitor 1 (PAI-1) play an important role in tumour invasion and progression in a variety of tumour types. Since the majority of clear cell renal cell carcinoma (ccRCC) shows distant metastasis at time of diagnosis or later, the interplay of uPA, uPAR and PAI-1 might be of importance in this process determining the patients' outcome. METHODS: Corresponding pairs of malignant and non-malignant renal tissue specimens were obtained from 112 ccRCC patients without distant metastasis who underwent tumour nephrectomy. Tissue extracts prepared from fresh-frozen tissue samples by detergent extraction were used for the determination of antigen levels of uPA, uPAR and PAI-1 by ELISA. Antigen levels were normalised to protein concentrations and expressed as ng per mg of total protein. RESULTS: Antigen levels of uPA, uPAR, and PAI-1 correlated with each other in the malignant tissue specimens (rs=0.51-0.65; all P<0.001). Antigen levels of uPA system components were significantly higher in tissue extracts of non-organ confined tumours (pT3+4) compared to organ-confined tumours (pT1+2; all P<0.05). Significantly elevated levels of uPAR and PAI-1 were also observed in high grade ccRCC. When using median antigen levels as cut-off points, all three uPA system factors were significant predictors for disease-specific survival (DSS) in univariate Cox's regression analyses. High levels of uPA and uPAR remained independent predictors for DSS with HR=2.86 (95% CI 1.07-7.67, P=0.037) and HR=4.70 (95% CI 1.51-14.6, P=0.008), respectively, in multivariate Cox's regression analyses. A combination of high antigen levels of uPA and/or uPAR further improved the prediction of DSS in multivariate analysis (HR=14.5, 95% CI 1.88-111.1, P=0.010). Moreover, high uPA and/or uPAR levels defined a patient subgroup of high risk for tumour-related death in ccRCC patients with organ-confined disease (pT1+2) (HR=9.83, 95% CI 1.21-79.6, P=0.032). CONCLUSIONS: High levels of uPA and uPAR in tumour tissue extracts are associated with a significantly shorter DSS of ccRCC patients without distant metastases.

Chemosensitizing effects of carbon-based nanomaterials in cancer cells: enhanced apoptosis and inhibition of proliferation as underlying mechanisms.

Recent studies have shown that carbon nanomaterials such as carbon nanofibres (CNFs) and multi-walled carbon nanotubes (CNTs) can exert antitumor activities themselves and sensitize cancer cells to conventional chemotherapeutics such as carboplatin and cisplatin. In the present study, the chemosensitizing effect of CNFs and CNTs on cancer cells of urological origin was investigated regarding the underlying mechanisms. Prostate cancer (DU-145, PC-3) and bladder cancer (EJ28) cells were treated with carbon nanomaterials (CNFs, CNTs) and chemotherapeutics (carboplatin, cisplatin) alone as well as in combination for 24 h. Forty-eight(EJ28) or 72 h (DU-145, PC-3) after the end of treatment the effects on cellular proliferation,clonogenic survival, cell death rate and cell cycle distribution were evaluated. Depending on the cell line, simultaneous administration of chemotherapeutics and carbon nanomaterials produced an additional inhibition of cellular proliferation and clonogenic survival of up to 77% and 98%, respectively, compared to the inhibitory effects of the chemotherapeutics alone. These strongly enhanced antiproliferative effects were accompanied by an elevated cell death rate, which was predominantly mediated via apoptosis and not by necrosis. The antitumor effects of combinations with CNTs were less pronounced than those with CNFs. The enhanced effects of the combinatory treatments on cellular function were mostly of additive to partly synergistic nature. Furthermore, cell cycle analysis demonstrated an arrest at the G2/M phase mediated by a monotreatment with chemotherapeutics. Following combinatory treatments, mostly less than or nearly additive increases of cell fractions in the G2/M phase could be observed. In conclusion,the pronounced chemosensitizing effects of CNFs and CNTs were mediated by an enhanced apoptosis and inhibition of proliferation. The combination of carbon-based nanomaterials and conventional chemotherapeutics represents a novel approach in cancer therapy to bypass chemoresistance by minimizing the chemotherapeutic dosing.

PARK2 and PACRG are commonly downregulated in clear-cell renal cell carcinoma and are associated with aggressive disease and poor clinical outcome.

PARK2 is an E3 ligase, known to be involved in ubiquitination of several proteins and to play a role in neuronal protection. The gene PARK2 and its potentially co-regulated gene PACRG have been previously found to be deleted in clear-cell renal cell carcinomas (ccRCCs). The aim of our study was to evaluate the mRNA and protein expression of PARK2 and PACRG in a large cohort of ccRCC, and to investigate their association with outcome. The expression of both genes was measured by quantitative PCR in 94 primary ccRCCs and autologous nonmalignant kidney tissues. PACRG and PARK2 protein expression was determined immunohistochemically using tissue microarrays comprising 133 ccRCCs. The mRNA and protein expression of PARK2 and PACRG was significantly downregulated in ccRCCs compared with nonmalignant tissues. Low levels of PARK2 mRNA were associated with high-grade ccRCC and lymph node metastasis. Patients with low PARK2 mRNA levels showed a higher tumor-specific mortality rate and a shorter overall survival (OS) than those with high PARK2 expression. Patients without PACRG mRNA expression in the tumor had a shorter disease-free survival and OS than those with tumors expressing PACRG. In multivariate analyses, neither PARK2 nor PACRG expression were independent prognostic factors. The protein expression of PARK2 and PACRG was significantly downregulated in ccRCCs (82.8, and 96.9%, respectively), but no association with clinical outcome was noticed.

Early kinetics of C-reactive protein as prognosticator for survival in a real-world cohort of patients with metastatic renal cell cancer under first-line therapy with immune checkpoint inhibitors.

PURPOSE: This study investigated the prognostic potential of baseline C-reactive protein (CRP) levels and early CRP kinetics in a real-world cohort of patients with metastatic renal cell carcinoma (mRCC) under first-line (1L) therapy with immune checkpoint inhibitors (CPI). METHODS/PATIENTS: Analyses were performed retrospectively in a cohort of 61 mRCC patients under CPI-based 1L therapy. Patients were stratified based on baseline CRP (< 10 vs ≥ 10 mg/l) and CRP change within the initial three months of CPI therapy (normal: baseline < 10 mg/l, normalized: baseline ≥ 10 mg/l and nadir < 10 mg/l, non-normalized: baseline and nadir ≥ 10 mg/l). Finally, the association of baseline CRP and CRP change with progression-free (PFS) and overall survival (OS) was evaluated. RESULTS: Baseline CRP was not significantly associated with both PFS (p = 0.666) and OS (p = 0.143). Following stratification according to early CRP kinetics, 23, 25 and 13 patients exhibited normal, normalized and non-normalized CRP levels, respectively. Patients with normal and normalized CRP had a markedly prolonged PFS (p = 0.091) and OS (p = 0.008) compared to patients with non-normalized CRP. Consequently, significantly better PFS (p = 0.031) and OS (p = 0.002) were observed for the combined normal-normalized group. In multivariate analysis including ECOG and IMDC risk, normalized CRP kinetics alone or in combination with the normal group was identified as significant independent risk factor for OS, whereas a statistical trend was observed for PFS. CONCLUSIONS: The present study emphasizes the prognostic potential of early CRP kinetics in CPI-treated mRCC. As a standard laboratory parameter, CRP can be easily implemented into clinical routine to facilitate therapy monitoring.

Blood-Based DNA Methylation Analysis by Multiplexed OBBPA-ddPCR to Verify Indications for Prostate Biopsies in Suspected Prostate Cancer Patients.

Current prostate carcinoma (PCa) biomarkers, including total prostate-specific antigen (tPSA), have unsatisfactory diagnostic sensitivity and specificity resulting in overdiagnosis and overtreatment. Previously, we described an optimised bias-based preamplification-digital droplet PCR (OBBPA-ddPCR) technique, which detects tumour DNA in blood-derived cell-free DNA (cfDNA) of cancer patients. The current study investigated the performance of newly developed OBBPA-ddPCR-based biomarkers. Blood plasma samples from healthy individuals (n = 90, controls) and PCa (n = 39) and benign prostatic hyperplasia patients (BPH, n = 40) were analysed. PCa and BPH patients had tPSA values within a diagnostic grey area of 2-15 ng/mL, for whom further diagnostic validation is most crucial. Methylation levels of biomarkers RASSF1A, MIR129-2, NRIP3, and SOX8 were found significantly increased in PCa patients compared to controls. By combining classical PCa risk factors (percentage of free PSA compared to tPSA (QfPSA) and patient's age) with cfDNA-based biomarkers, we developed PCa risk scores with improved sensitivity and specificity compared to established tPSA and QfPSA single-marker analyses. The diagnostic specificity was increased to 70% with 100% sensitivity for clinically significant PCa patients. Thus, prostate biopsies could be avoided for 28 out of 40 BPH patients. In conclusion, the newly developed risk scores may help to confirm the clinical decision and prevent unnecessary prostate biopsy.

Immunohistochemical analysis of prognostic protein markers for primary localized clear cell renal cell carcinoma.

Recurrence is not reliably predictable in localized clear cell renal cell carcinoma. Proteinmarkers could improve predictive accuracy. Tissue-microarrays from 132 patients with primary localized ccRCC were immunohistochemically analyzed for VHL, Ki67, p53, p21, survivin, and, for microvessel-density, UEA-1. Nuclear stainings of Ki67, p21, and survivin were significantly associated with disease-specific survival and increased predictive ability from 74% to 76%, 77%, and 78%, respectively in a multivariate model including T-stage and Fuhrman grade. A score-variable, combining Ki67-, p21-, and nS-staining identified a subset of patients with high risk of disease recurrence and increased predictive ability in the multivariate model to 84%.

Serum amyloid A, phospholipase A2-IIA and C-reactive protein as inflammatory biomarkers for prostate diseases.

INTRODUCTION: Serum amyloid A (SAA), secreted group IIA phospholipase A2 (sPLA2-IIA), and C-reactive protein (CRP) are acute-phase proteins whose serum concentrations increase not only during inflammatory disorders, but also in the course of malignant diseases. MATERIALS AND METHODS: In this study we analyzed serum levels of these inflammatory markers along with prostate-specific antigens (PSA) in patients with benign prostatic hyperplasia (BPH, n = 55), localized prostate cancers (PCa, n = 55), and metastatic prostate cancers (mPCa, n = 27) using immunological assays. RESULTS: We found that in comparison to healthy individuals (n = 55), patients with BPH, PCa and mPCa have elevated serum levels of SAA, sPLA2-IIA, and CRP, in addition to elevated levels of PSA. Significant differences with respect to inflammatory biomarkers were found between localized and metastatic PCa (p < 0.001), suggesting a prognostic value of these parameters. In addition, serum concentrations of SAA and sPLA2-IIA positively correlate with CRP in BPH patients (p < 0.05) and in patients with PCa and mPCa (p < 0.001), but not with PSA levels, Gleason score, or tumor stage, emphasizing a role of SAA and sPLA2-IIA as circulating biomarkers of inflammation rather than of neoplastic transformation. In contrast to PSA, which differed significantly between BPH and localized PCa patients (p < 0.01), such a difference was not found for SAA, sPLA2-IIA, and CRP. In order to elucidate whether the elevated levels of SAA and sPLA2-IIA can be caused by cancer cell-associated synthesis, in vitro studies were performed. These analyses demonstrated the expression of SAA and sPLA2-IIA in LNCaP and PC-3 prostate cell lines, which can be further upregulated by pro-inflammatory cytokines in a cell type-dependent manner. This might suggest that, in addition to the hepatic origin, SAA and sPLA2-IIA can also be synthesized and secreted by prostatic cancer tissue itself. CONCLUSION: The results of the present study emphasize the utility of SAA, sPLA2-IIA, and CRP as circulating biomarkers of inflammation during BPH development and PCa progression.

Deregulated expression of urokinase and its inhibitor type 1 in prostate cancer cells: role of epigenetic mechanisms.

Plasminogen activator inhibitor-1 (PAI-1) and urokinase-type plasminogen activator (uPA) play a crucial role in cancer progression. In the present study we examined the regulation of PAI-1 and uPA expressions in normal prostate epithelial cells (PrEC) and the prostate cancer cell lines LNCaP, DU-145, and PC-3. The antigen and mRNA levels of PAI-1 were down-regulated in cancer cells, especially in LNCaP and DU-145. In the presence of proinflammatory cytokines, an increase of PAI-1 mRNA levels was observed in PrEC, LNCaP and PC-3, but not in DU-145 cells. Treatment with demethylating agent, 5-aza-2'-deoxycytidine increased the level of PAI-1 transcript in DU-145 cells and restored the inducing effect of cytokines on PAI-1 expression. An aberrant methylation of PAI-1 promoter in DU-145 and LNCaP cells was shown by methylation-sensitive high resolution melting (MS-HRM) analysis. PAI-1 methylation was also significantly increased in tumor samples (23.2±1.7%) in comparison to adjacent non-tumor tissue (6.0±0.8%). Furthermore, the expression of uPA was increased in high invasive cell lines DU-145 and PC-3 in comparison to PrEC and low invasive LNCaP cells. MS-HRM analysis revealed aberrant methylation of uPA promoter in LNCaP cells, but not in PrEC, DU-145 and PC-3 cells, as well as in normal and prostate cancer tissue samples. In conclusion, the study shows that PAI-1 and uPA expressions were changed in opposite directions in high invasive prostate cancer cell lines resulting in a strong decrease of PAI-1/uPA ratio, which may indicate a shift towards proteolytic activities. Methylation of the PAI-1 gene is suggested as one of the molecular mechanisms involved in the cancer-associated down-regulation of the PAI-1 expression.

Enhanced inhibition of bladder cancer cell growth by simultaneous knockdown of antiapoptotic Bcl-xL and survivin in combination with chemotherapy.

The overexpression of antiapoptotic genes, such as Bcl-xL and survivin, contributes to the increased survival of tumor cells and to the development of treatment resistances. In the bladder cancer cell lines EJ28 and J82, the siRNA-mediated knockdown of survivin reduces cell proliferation and the inhibition of Bcl-xL sensitizes these cells towards subsequent chemotherapy with mitomycin C and cisplatin. Therefore, the aim of this study was to analyze if the simultaneous knockdown of Bcl-xL and survivin might represent a more powerful treatment option for bladder cancer than the single inhibition of one of these target genes. At 96 h after transfection, reduction in cell viability was stronger after simultaneous inhibition of Bcl-xL and survivin (decrease of 40%-48%) in comparison to the single target treatments (decrease of 29% at best). Furthermore, simultaneous knockdown of Bcl-xL and survivin considerably increased the efficacy of subsequent chemotherapy. For example, cellular viability of EJ28 cells decreased to 6% in consequence of Bcl-xL and survivin inhibition plus cisplatin treatment whereas single target siRNA plus chemotherapy treatments mediated reductions down to 15%-36% only. In conclusion, the combination of simultaneous siRNA-mediated knockdown of antiapoptotic Bcl-xL and survivin-a multitarget molecular-based therapy-and conventional chemotherapy shows great potential for improving bladder cancer treatment.

Acquired resistance to irradiation or docetaxel is not associated with cross-resistance to cisplatin in prostate cancer cell lines.

PURPOSE: Platinum chemotherapy can be considered to treat metastatic castration-resistant prostate cancer (mCRPC) with features of neuroendocrine differentiation. However, platinum compounds are generally only applied after the failure of multiple prior-line treatment options. This study investigated whether acquired resistance against ionizing radiation or docetaxel chemotherapy-two commonly applied treatment modalities in prostate cancer-influences the cisplatin (CDDP) tolerance in mCRPC cell line models. METHODS: Age-matched parental as well as radio- or docetaxel-resistant DU145 and PC-3 cell lines were treated with CDDP and their sensitivity was assessed by measurements of growth rates, viability, apoptosis, metabolic activity and colony formation ability. RESULTS: The data suggest that docetaxel resistance does not influence CDDP tolerance in all tested docetaxel-resistant cell lines. Radio-resistance was associated with sensitization to CDDP in PC-3, but not in DU145 cells. In general, DU145 cells tolerated higher CDDP concentrations than PC-3 cells regardless of acquired resistances. Furthermore, non-age-matched treatment-naïve PC-3 cells exhibited significantly different CDDP tolerances. CONCLUSION: Like patients, different mCRPC cell lines exhibit significant variability regarding CDDP tolerance. The presented in vitro data suggest that previous radiation treatment may be associated with a moderate sensitization to CDDP in an isogenic and age-matched setting. Therefore, previous radiotherapy or docetaxel chemotherapy might be no contraindication against initiation of platinum chemotherapy in selected mCRPC patients.

Neuropilin-2 regulates androgen-receptor transcriptional activity in advanced prostate cancer.

Aberrant transcriptional activity of androgen receptor (AR) is one of the dominant mechanisms for developing of castration-resistant prostate cancer (CRPC). Analyzing AR-transcriptional complex related to CRPC is therefore important towards understanding the mechanism of therapy resistance. While studying its mechanism, we observed that a transmembrane protein called neuropilin-2 (NRP2) plays a contributory role in forming a novel AR-transcriptional complex containing nuclear pore proteins. Using immunogold electron microscopy, high-resolution confocal microscopy, chromatin immunoprecipitation, proteomics, and other biochemical techniques, we delineated the molecular mechanism of how a specific splice variant of NRP2 becomes sumoylated upon ligand stimulation and translocates to the inner nuclear membrane. This splice variant of NRP2 then stabilizes the complex between AR and nuclear pore proteins to promote CRPC specific gene expression. Both full-length and splice variants of AR have been identified in this specific transcriptional complex. In vitro cell line-based assays indicated that depletion of NRP2 not only destabilizes the AR-nuclear pore protein interaction but also inhibits the transcriptional activities of AR. Using an in vivo bone metastasis model, we showed that the inhibition of NRP2 led to the sensitization of CRPC cells toward established anti-AR therapies such as enzalutamide. Overall, our finding emphasize the importance of combinatorial inhibition of NRP2 and AR as an effective therapeutic strategy against treatment refractory prostate cancer.

Expression profile of WNT molecules in prostate cancer and its regulation by aminobisphosphonates.

Skeletal metastases represent a frequent complication in patients with advanced prostate cancer (PCa) and often require bisphosphonate treatment to limit skeletal-related events. Metastasized PCa cells disturb bone remodeling. Since the WNT signaling pathway regulates bone remodeling and has been implicated in tumor progression and osteomimicry, we analyzed the WNT profile of primary PCa tissues and PCa cell lines and assessed its regulation by bisphosphonates. Prostate tissue (n = 18) was obtained from patients with benign prostate hyperplasia (BPH) and PCa patients with different disease stages. Serum samples were collected from 62 patients. Skeletal metastases were present in 17 patients of whom 6 had been treated with zoledronic acid. The WNT profile and its regulation by bisphoshonates were analyzed in tissue RNA extracts and serum samples as well as in osteotropic (PC3) and non-osteotropic (DU145, LNCaP) PCa cell lines. Several members of the WNT pathway, including WNT5A, FZD5, and DKK1 were highly up-regulated in PCa tissue from patients with advanced PCa. Interestingly, osteotropic cells showed a distinct WNT profile compared to non-osteotropic cells. While WNT5A, FZD5, and DKK1 were highly expressed in PC3 cells, WNT1 and SFRP1 mRNA levels were higher in DU145 cells. Moreover, zoledronic acid down-regulated mRNA levels of WNT5A (-34%), FZD5 (-60%), and DKK1 (-46%) in PC3 cells. Interestingly, patients with skeletal metastases who received zoledronic acid had twofold higher DKK1 serum levels compared to bisphosphonate-naive patients. The WNT signaling pathway is up-regulated in advanced PCa, differentially expressed in osteotropic versus non-osteotropic cells, and is regulated by zoledronic acid.