Androgen-Regulated Expression of Arginase 1, Arginase 2 and Interleukin-8 in Human Prostate Cancer

Background

Prostate cancer (PCa) is the most frequently diagnosed cancer in North American men. Androgen-deprivation therapy (ADT) accentuates the infiltration of immune cells within the prostate. However, the immunosuppressive pathways regulated by androgens in PCa are not well characterized. Arginase 2 (ARG2) expression by PCa cells leads to a reduced activation of tumor-specific T cells. Our hypothesis was that androgens could regulate the expression of ARG2 by PCa cells.

Methodology/Principal Findings

In this report, we demonstrate that both ARG1 and ARG2 are expressed by hormone-sensitive (HS) and hormone-refractory (HR) PCa cell lines, with the LNCaP cells having the highest arginase activity. In prostate tissue samples, ARG2 was more expressed in normal and non-malignant prostatic tissues compared to tumor tissues. Following androgen stimulation of LNCaP cells with 10 nM R1881, both ARG1 and ARG2 were overexpressed. The regulation of arginase expression following androgen stimulation was dependent on the androgen receptor (AR), as a siRNA treatment targeting the AR inhibited both ARG1 and ARG2 overexpression. This observation was correlated in vivo in patients by immunohistochemistry. Patients treated by ADT prior to surgery had lower ARG2 expression in both non-malignant and malignant tissues. Furthermore, ARG1 and ARG2 were enzymatically active and their decreased expression by siRNA resulted in reduced overall arginase activity and l-arginine metabolism. The decreased ARG1 and ARG2 expression also translated with diminished LNCaP cells cell growth and increased PBMC activation following exposure to LNCaP cells conditioned media. Finally, we found that interleukin-8 (IL-8) was also upregulated following androgen stimulation and that it directly increased the expression of ARG1 and ARG2 in the absence of androgens.

Conclusion/Significance

Our data provides the first detailed in vitro and in vivo account of an androgen-regulated immunosuppressive pathway in human PCa through the expression of ARG1, ARG2 and IL-8.     

Autoimmune pancreatocholangitis, non-autoimmune pancreatitis and primary sclerosing cholangitis: a comparative morphological and immunological analysis

Background

Autoimmune pancreatocholangitis (AIPC) is an emerging, not completely characterized disease. Aim of this study was the comprehensive evaluation of a series of AIPC patients, who were diagnosed and treated in a European institution between January 2003 and July 2006.

Methodology/Principal Findings

Thirty-three patients with histologically confirmed AIPC were analyzed and compared to 20 patients with non-autoimmune chronic pancreatitis (CP) and 14 patients with primary sclerosing cholangitis (PSC). Clinical features and conventional histopathology were taken into account. Immunohistochemistry and real-time quantitative PCR were used for the characterization of the inflammatory infiltrate and the stromal reaction. AIPC was localized in the pancreatic head in 94% of the patients. Intra- and/or extrapancreatic biliary tract involvement was present in 64% of the cases. The number of infiltrating T-lymphocytes, macrophages and total plasma cells was significantly higher in AIPC than in CP (3-, 4- and 8-fold increase, respectively). The absolute number of IgG4-positive plasma cells was higher in AIPC than in CP and PSC (7-fold and 35-fold increase, respectively), but significance was only reached in comparison with PSC. CXCR5- and CXCL13-positive cells were almost exclusively detected in AIPC.

Conclusions/Significance

AIPC is mainly a disease of the pancreatic head with possible extension into the periphery of the gland and/or into the biliary tract/gallbladder. The morphology of AIPC, as well as the immune- and stromal reaction is characteristic and comparable between cases with and without biliary tract involvement. Immunological markers (IgG4, CXCR5, CXCL13) can be of diagnostic relevance in specific settings.     

Prognostic Significance and Molecular Mechanism of ATP-Binding Cassette Subfamily C Member 4 in Resistance to Neoadjuvant Radiotherapy of Locally Advanced Rectal Carcinoma

Background

Mechanism of radioresistance in rectal carcinoma remains largely unknown. We aimed to evaluate the predictive role of ATP-binding cassette subfamily C member 4 (ABCC4) in locally advanced rectal carcinoma and explore possible molecular mechanisms by which ABCC4 confers the resistance to neoadjuvant radiotherapy.

Methods

The expression of ABCC4 and P53 mutant in biopsy tissue specimens from 121 locally advanced rectal carcinoma patients was examined using immunohistochemistry. The factors contributing to 3-year overall survival and disease-free survival were evaluated using the Kaplan-Meier method and Cox proportional hazard model. Lentivirus-mediated small hairpin RNA was applied to inhibit ABCC4 expression in colorectal carcinoma cell line RKO, and investigate the radiosensitivity in xenograft model. Intracellular cyclic adenosine monophosphate concentration and cell cycle distribution following irradiation were detected.

Results

High expression of ABCC4 and p53 mutant in pretreated tumors, poor pathological response, and high final tumor staging were significant factors independently predicted an unfavorable prognosis of locally advanced rectal carcinoma patients after neoadjuvant radiotherapy. Down-regulation of ABCC4 expression significantly enhanced irradiation-induced suppression of tumor growth in xenograft model. Furthermore, down-regulation of ABCC4 expression enhanced intracellular cyclic adenosine monophosphate production and noticeable deficiency of G1-S phase checkpoint in cell cycle following irradiation.

Conclusions

Our study suggests that ABCC4 serves as a novel predictive biomarker that is responsible for the radioresistance and predicts a poor prognosis for locally advanced rectal carcinoma after neoadjuvant radiotherapy.     

Induction of apoptosis coupled to endoplasmic reticulum stress in human prostate cancer cells by n-butylidenephthalide

Background

N-butylidenephthalide (BP) exhibits antitumor effect in a variety of cancer cell lines. The objective of this study was to obtain additional insights into the mechanisms involved in BP induced cell death in human prostate cancer cells.

Methods/Principal Findings

Two human prostate cancer cell lines, PC-3 and LNCaP, were treated with BP, and subsequently evaluated for their viability and cell cycle profiles. BP caused cell cycle arrest and cell death in both cell lines. The G0/G1 phase arrest was correlated with increase levels of CDK inhibitors (p16, p21 and p27) and decrease of the checkpoint proteins. To determine the mechanisms of BP-induced growth arrest and cell death in prostate cancer cell lines, we performed a microarray study to identify alterations in gene expression induced by BP in the LNCaP cells. Several BP-induced genes, including the GADD153/CHOP, an endoplasmic reticulum stress (ER stress)-regulated gene, were identified. BP-induced ER stress was evidenced by increased expression of the downstream molecules GRP78/BiP, IRE1-α and GADD153/CHOP in both cell lines. Blockage of IRE1-α or GADD153/CHOP expression by siRNA significantly reduced BP-induced cell death in LNCaP cells. Furthermore, blockage of JNK1/2 signaling by JNK siRNA resulted in decreased expression of IRE1-α and GADD153/CHOP genes, implicating that BP-induced ER stress may be elicited via JNK1/2 signaling in prostate cancer cells. BP also suppressed LNCaP xenograft tumor growth in NOD-SCID mice. It caused 68% reduction in tumor volume after 18 days of treatment.

Conclusions

Our results suggest that BP can cause G0/G1 phase arrest in prostate cancer cells and its cytotoxicity is mediated by ER stress induction. Thus, BP may serve as an anticancer agent by inducing ER stress in prostate cancer.     

Finasteride Inhibits Human Prostate Cancer Cell Invasion through MMP2 and MMP9 Downregulation

Introduction

The use of the 5-alpha reductase inhibitors (5-ARIs) finasteride and dutasteride for prostate cancer prevention is still under debate. The FDA recently concluded that the increased prevalence of high-grade tumors among 5-ARI-treated patients must not be neglected, and they decided to disallow the use of 5-ARIs for prostate cancer prevention. This study was conducted to verify the effects of finasteride on prostate cell migration and invasion and the related enzymes/proteins in normal human and tumoral prostatic cell lines.

Materials and Methods

RWPE-1, LNCaP, PC3 and DU145 cells were cultivated to 60% confluence and exposed for different periods to either 10 µM or 50 µM finasteride that was diluted in culture medium. The conditioned media were collected and concentrated, and MMP2 and MMP9 activities and TIMP-1 and TIMP-2 protein expression were determined. Cell viability, migration and invasion were analyzed, and the remaining cell extracts were submitted to androgen receptor (AR) detection by western blotting techniques. Experiments were carried out in triplicate.

Results

Cell viability was not significantly affected by finasteride exposure. Finasteride significantly downregulated MMP2 and MMP9 activities in RWPE-1 and PC3 cells and MMP2 in DU145 cells. TIMP-2 expression in RWPE-1 cells was upregulated after exposure. The cell invasion of all four tested cell lines was inhibited by exposure to 50 µM of finasteride, and migration inhibition only occurred for RWPE-1 and LNCaP cells. AR was expressed by LNCaP, RWPE-1 and PC3 cells.

Conclusions

Although the debate on the higher incidence of high-grade prostate cancer among 5-ARI-treated patients remains, our findings indicate that finasteride may attenuate tumor aggressiveness and invasion, which could vary depending on the androgen responsiveness of a patient’s prostate cells.     

Natural proteasome inhibitor celastrol suppresses androgen-independent prostate cancer progression by modulating apoptotic proteins and NF-kappaB

Background

Celastrol is a natural proteasome inhibitor that exhibits promising anti-tumor effects in human malignancies, especially the androgen-independent prostate cancer (AIPC) with constitutive NF-κB activation. Celastrol induces apoptosis by means of proteasome inhibition and suppresses prostate tumor growth. However, the detailed mechanism of action remains elusive. In the current study, we aim to test the hypothesis that celastrol suppresses AIPC progression via inhibiting the constitutive NF-κB activity as well as modulating the Bcl-2 family proteins.

Methodology/Principal Findings

We examined the efficacy of celastrol both in vitro and in vivo, and evaluated the role of NF-κB in celastrol-mediated AIPC regression. We found that celastrol inhibited cell proliferation in all three AIPC cell lines (PC-3, DU145 and CL1), with IC₅₀ in the range of 1–2 µM. Celastrol also suppressed cell migration and invasion. Celastrol significantly induced apoptosis as evidenced by increased sub-G1 population, caspase activation and PARP cleavage. Moreover, celastrol promoted cleavage of the anti-apoptotic protein Mcl-1 and activated the pro-apoptotic protein Noxa. In addition, celastrol rapidly blocked cytosolic IκBα degradation and nuclear translocation of RelA. Likewise, celastrol inhibited the expression of multiple NF-κB target genes that are involved in proliferation, invasion and anti-apoptosis. Celastrol suppressed AIPC tumor progression by inhibiting proliferation, increasing apoptosis and decreasing angiogenesis, in PC-3 xenograft model in nude mouse. Furthermore, increased cellular IκBα and inhibited expression of various NF-κB target genes were observed in tumor tissues.

Conclusions/Significance

Our data suggest that, via targeting the proteasome, celastrol suppresses proliferation, invasion and angiogenesis by inducing the apoptotic machinery and attenuating constitutive NF-κB activity in AIPC both in vitro and in vivo. Celastrol as an active ingredient of traditional herbal medicine could thus be developed as a new therapeutic agent for hormone-refractory prostate cancer.     

Ultrasonic Nanobubbles Carrying Anti-PSMA Nanobody: Construction and Application in Prostate Cancer-Targeted Imaging

To facilitate prostate cancer imaging using targeted molecules, we constructed ultrasonic nanobubbles coupled with specific anti-PSMA (prostate specific membrane antigen) nanobodies, and evaluated their in vitro binding capacity and in vivo imaging efficacy. The “targeted” nanobubbles, which were constructed via a biotin-streptavidin system, had an average diameter of 487.60 ± 33.55 nm and carried the anti-PSMA nanobody as demonstrated by immunofluorescence. Microscopy revealed targeted binding of nanobubbles in vitro to PSMA-positive cells. Additionally, ultrasonography indicators of nanobubble imaging (including arrival time, peak time, peak intensity and enhanced duration) were evaluated for the ultrasound imaging in three kinds of animal xenografts (LNCaP, C4-2 and MKN45), and showed that these four indicators of targeted nanobubbles exhibited significant differences from blank nanobubbles. Therefore, this study not only presents a novel approach to target prostate cancer ultrasonography, but also provides the basis and methods for constructing small-sized and high-efficient targeted ultrasound nanobubbles.     

Prostate Cancer Characteristics Associated with Response to Pre-Receptor Targeting of the Androgen Axis

Background

Factors influencing differential responses of prostate tumors to androgen receptor (AR) axis-directed therapeutics are poorly understood, and predictors of treatment efficacy are needed. We hypothesized that the efficacy of inhibiting DHT ligand synthesis would associate with intra-tumoral androgen ratios indicative of relative dependence on DHT-mediated growth.

Methods

We characterized two androgen-sensitive prostate cancer xenograft models after androgen suppression by castration in combination with the SRD5A inhibitor, dutasteride, as well as a panel of castration resistant metastases obtained via rapid autopsy.

Results

In LuCaP35 tumors (intra-tumoral T:DHT ratio 2∶1) dutasteride suppressed DHT to 0.02 ng/gm and prolonged survival vs. castration alone (337 vs.152 days, HR 2.8, p = 0.0015). In LuCaP96 tumors (T:DHT 10∶1), survival was not improved despite similar DHT reduction (0.02 ng/gm). LuCaP35 demonstrated higher expression of steroid biosynthetic enzymes maintaining DHT levels (5-fold higher SRD5A1, 41 fold higher, 99-fold higher RL-HSD, p<0.0001 for both), reconstitution of intra-tumoral DHT (to ∼30% of untreated tumors), and ∼2 fold increased expression of full length AR. In contrast, LuCaP96 demonstrated higher levels of steroid catabolizing enzymes (6.9-fold higher AKR1C2, 3000-fold higher UGT2B15, p = 0.002 and p<0.0001 respectively), persistent suppression of intra-tumoral DHT, and 6–8 fold induction of full length AR and the ligand independent V7 AR splice variant. Human metastases demonstrated bio-active androgen levels and AR full length and AR splice-variant expression consistent with the range observed in xenografts.

Conclusions

Intrinsic differences in basal steroidogenesis, as well as variable expression of full length and splice-variant AR, associate with response and resistance to pre-receptor AR ligand suppression. Expression of steroidogenic enzymes and AR isoforms may serve as potential biomarkers of sensitivity to potent AR-axis inhibition and should be validated in additional models.     

The Association between Polymorphisms in the MRPL4 and TNF-α Genes and Susceptibility to Allergic Rhinitis

Background

Allergic rhinitis (AR) is a chronic inflammatory disease of the nasal mucosa, involving a complex interaction between genetic and environmental factors. Evidence suggests that polymorphisms in the gene coding for mitochondrial ribosomal protein L4 (MRPL4), located in close proximity to intercellular adhesion molecule-1 (ICAM-1) gene on chromosome location 19p13.2, may influence the risk factor for the development of AR.

Objective

The aim of our study was to investigate any association between AR susceptibility and polymorphisms in ICAM-1 gene, as well as associations between AR risk and polymorphisms in MRPL4, nuclear factor-kappaB (NF-κB) and tumor necrosis factor alpha(TNF-α) genes, associated with ICAM-1 expression.

Methods

A cohort of 414 patients with AR and 293 healthy controls was enrolled from the Han Chinese population in Beijing, China. Blood was drawn for DNA extraction and total serum immunoglobulin E (IgE). A total of 14 single nucleotide polymorphisms (SNPs) in ICAM-1, NF-κB, TNF-α, and MRPL4 genes were selected using the CHB genotyping data from the International Haplotype Mapping (HapMap) and assessed for differences in frequencies of the alleles and genotypes between the AR patients and control subjects.

Results

TNF-α SNP rs1799964 and MRPL4 SNP rs11668618 were found to occur in significantly greater frequencies in the AR group compared to control group. There were no significant associations between SNPs in NF-κB, ICAM-1 and AR. The SNP-SNP interaction information analysis further indicated that there were no synergistic effects among the selected sets of polymorphisms.

Conclusions

Our results suggest a strong association between AR risk and polymorphisms of MRPL4 and TNF-α genes in Han Chinese population.     

Small interfering RNA against CD86 during allergen challenge blocks experimental allergic asthma

Background

CD86-CD28 interaction has been suggested as the principal costimulatory pathway for the activation and differentiation of naïve T cells in allergic inflammation. However, it remains uncertain whether this pathway also has an essential role in the effector phase. We sought to determine the contribution of CD86 on dendritic cells in the reactivation of allergen-specific Th2 cells.

Methods

We investigated the effects of the downregulation of CD86 by short interfering RNAs (siRNAs) on Th2 cytokine production in the effector phase in vitro and on asthma phenotypes in ovalbumin (OVA)-sensitized and -challenged mice.

Results

Treatment of bone marrow-derived dendritic cells (BMDCs) with CD86 siRNA attenuated LPS-induced upregulation of CD86. CD86 siRNA treatment impaired BMDCs’ ability to activate OVA-specific Th2 cells. Intratracheal administration of CD86 siRNA during OVA challenge downregulated CD86 expression in the airway mucosa. CD86 siRNA treatment ameliorated OVA-induced airway eosinophilia, airway hyperresponsiveness, and the elevations of OVA-specific IgE in the sera and IL-5, IL-13, and CCL17 in the bronchoalveolar lavage fluid, but not the goblet cell hyperplasia.

Conclusion

These results suggest that local administration of CD86 siRNA during the effector phase ameliorates lines of asthma phenotypes. Targeting airway dendritic cells with siRNA suppresses airway inflammation and hyperresponsiveness in an experimental model of allergic asthma.     

Association pattern of interleukin-1 receptor-associated kinase-4 gene polymorphisms with allergic rhinitis in a Han Chinese population

Objective

Interleukin-1 receptor-associated kinase-4 (IRAK-4) encodes a kinase that is essential for NF-kB activation in Toll-like receptor and T-cell receptor signaling pathways, indicating a possible crosstalk between innate and acquired immunities. We attempted to determine whether the polymorphisms in the Interleukin-1 receptor-associated kinase-4 (IRAK-4) gene are associated with allergic rhinitis (AR) in the Han Chinese population.

Methods

A population of 379 patients with AR and 333 healthy controls was studied. Blood was drawn for DNA extraction and total serum immunoglobulin E (IgE). A total of 11 single nucleotide polymorphisms (SNPs) in IRAK-4 were selected and individually genotyped.

Results

Significant allelic differences between cases and controls were obtained for the SNP of rs3794262 in the IRAK-4 gene. In the stratified analysis for gender, two SNPs (rs4251431 and rs6582484) in males appeared as significant associations. Subgroup analysis for the presence of different allergen sensitivities displayed associations only in the house dust mite-allergic cohorts (rs3794262, rs4251481). None of the selected SNPs in IRAK-4 was associated with total IgE level. The haplotype analyisis indicated GCCTGCGA was significantly associated with AR. The SNP-SNP interaction information analysis indicated that the selected sets of polymorphisms had no synergistic effect.

Conclusions

Our findings did not support the potential contribution of the IRAK-4 gene to serum IgE levels. However, the results demonstrated a gender- and allergen-dependant association pattern between polymorphisms in IRAK-4 and AR in Chinese population.     

High Efficiency Lipid-Based siRNA Transfection of Adipocytes in Suspension

Background

Fully differentiated adipocytes are considered to be refractory to introduction of siRNA via lipid-based transfection. However, large scale siRNA-based loss-of-function screening of adipocytes using either electroporation or virally-mediated transfection approaches can be prohibitively complex and expensive.

Methodology/Principal Findings

We present a method for introducing small interfering RNA (siRNA) into differentiated 3T3-L1 adipocytes and primary human adipocytes using an approach based on forming the siRNA/cell complex with the adipocytes in suspension rather than as an adherent monolayer, a variation of “reverse transfection”.

Conclusions/Significance

Transfection of adipocytes with siRNA by this method is economical, highly efficient, has a simple workflow, and allows standardization of the ratio of siRNA/cell number, making this approach well-suited for high-throughput screening of fully differentiated adipocytes.     

Amelioration of acute kidney injury in lipopolysaccharide-induced systemic inflammatory response syndrome by an aldose reductase inhibitor, fidarestat

Background

Systemic inflammatory response syndrome is a fatal disease because of multiple organ failure. Acute kidney injury is a serious complication of systemic inflammatory response syndrome and its genesis is still unclear posing a difficulty for an effective treatment. Aldose reductase (AR) inhibitor is recently found to suppress lipopolysaccharide (LPS)-induced cardiac failure and its lethality. We studied the effects of AR inhibitor on LPS-induced acute kidney injury and its mechanism.

Methods

Mice were injected with LPS and the effects of AR inhibitor (Fidarestat 32 mg/kg) before or after LPS injection were examined for the mortality, severity of renal failure and kidney pathology. Serum concentrations of cytokines (interleukin-1β, interleukin-6, monocyte chemotactic protein-1 and tumor necrosis factor-α) and their mRNA expressions in the lung, liver, spleen and kidney were measured. We also evaluated polyol metabolites in the kidney.

Results

Mortality rate within 72 hours was significantly less in LPS-injected mice treated with AR inhibitor both before (29%) and after LPS injection (40%) than untreated mice (90%). LPS-injected mice showed marked increases in blood urea nitrogen, creatinine and cytokines, and AR inhibitor treatment suppressed the changes. LPS-induced acute kidney injury was associated with vacuolar degeneration and apoptosis of renal tubular cells as well as infiltration of neutrophils and macrophages. With improvement of such pathological findings, AR inhibitor treatment suppressed the elevation of cytokine mRNA levels in multiple organs and renal sorbitol accumulation.

Conclusion

AR inhibitor treatment ameliorated LPS-induced acute kidney injury, resulting in the lowered mortality.