Mitogenic and anti-apoptotic actions of adipocyte-derived hormone leptin in prostate cancer cells

OBJECTIVE

To investigate the proliferative and anti‐apoptotic effects of leptin on human prostate cancer cells, and the role of related signalling pathways in mediating these actions, as obesity is a possible risk factor for prostate cancer and leptin, an adipocyte‐derived hormone, has mitogenic action in various cell types.

MATERIALS AND METHODS

Two human prostate cancer cell lines, DU145 and PC‐3, were treated with leptin (5–100 ng/mL) for up to 48 h. Under serum‐free conditions, cell proliferation was measured using a colorimetric tetrazolium assay and apoptosis by an enzyme‐linked immunosorbent assay measuring cell death. Also, the phosphorylation of ERK1/2 and Akt was detected by Western blotting, and specific inhibitors of mitogen‐activated protein kinase (MAPK) (PD98059; 40 µm ) and phosphatidylinositol 3‐kinase (PI3‐K, LY294002; 40 µm ) were used to evaluate the role of these signalling pathways.

RESULTS

Leptin dose‐dependently increased the cell number in both cell lines for up to 48 h of incubation, the mean (sem ) percentage of the control being 189 (4.3)% for DU145 and 173 (7.5)% for PC‐3 (100 ng/mL leptin, 48 h; P < 0.01). Leptin also significantly reduced the number of apoptotic cells after 24 h of treatment, dose‐dependently caused ERK1/2 and Akt phosphorylation; pretreatment with inhibitors of MAPK and PI3‐K inhibited these responses.

CONCLUSION

These results show that chronic increases in leptin might enhance the growth of prostate cancer via the MAPK and PI3‐K pathways. Further studies are needed to investigate whether the ability of leptin to stimulate mitogenic/anti‐apoptotic signal transduction pathways could represent a target for anticancer drug discovery.     

p53 immunochemistry is an independent prognostic marker for outcome in conservatively treated prostate cancer

OBJECTIVE

To determine whether p53 is an independent biomarker of prostate cancer outcome against currently used biomarkers in a cohort of conservatively treated prostate cancers with long‐term follow‐up available.

PATIENTS AND METHODS

We examined p53 expression by immunohistochemistry in a cohort of 705 patients with clinically localized prostate cancer, who were treated conservatively. Patients were selected through UK Cancer Registries. End‐points included prostate cancer death and overall death rates. Standard biological variables, including diagnostic serum PSA, contemporary Gleason scoring, clinical staging and cancer extent were available. p53 expression was measured semi‐quantitatively on microscopic examination and compared with current clinical biomarkers.

RESULTS

p53 over expression was a significant predictor of cause‐specific survival (hazard ratio [HR] 2.95, 95% CI 2.05–4.25, P < 0.001) and overall survival (HR 2.37, 95% CI 1.84–3.05, P < 0.001). In multivariate analysis including competing biological variables p53 expression was still significantly linked to prostate cancer survival (HR 1.51, 95% CI 1.04–2.19, P = 0.03) and overall survival (HR 1.57, 95% CI 1.21–2.05, P = 0.001).

CONCLUSIONS

We conclude that p53 may have a role in the future assessment of newly diagnosed prostate cancer, as it significantly adds to the current prognostic model.     

Regulation of miR‐200c and miR‐141 by Methylation in Prostate Cancer

BACKGROUND

In prostate cancer (PCa), abnormal expression of several microRNAs (miRNAs) has been previously reported. Increasing evidence shows that aberrant epigenetic regulation of miRNAs is a contributing factor to their altered expression in cancer. In this study, we investigate whether expression of miR‐200c and miR‐141 in PCa is related to the DNA methylation status of their promoter.

METHODS

PCR analysis of miR‐200c and miR‐141, and CpG methylation analysis of their common promoter, was performed in PCa cell‐lines and in archived prostate biopsy specimens. The biological significance of miR‐200c and miR‐141 expression in prostate cancer cells was assessed by a series of in vitro bioassays and the effect on proposed targets DNMT3A and TET1/TET3 was investigated. The effect on promoter methylation status in cells treated with demethylating agents was also examined.

RESULTS

miR‐200c and miR‐141 are both highly elevated in LNCaP, 22RV1, and DU145 cells, but significantly reduced in PC3 cells. This correlates inversely with the methylation status of the miR‐200c/miR‐141 promoter, which is unmethylated in LNCaP, 22RV1, and DU145 cells, but hypermethylated in PC3. In PC3 cells, miR‐200c and miR‐141 expression is subsequently elevated by treatment with the demethylating drug decitabine (5‐aza‐2′deoxycytidine) and by knockdown of DNA methyltransferase 1 (DNMT1), suggesting their expression is regulated by methylation. Expression of miR‐200c and miR‐141 in prostate biopsy tissue was inversely correlated with methylation in promoter CpG sites closest to the miR‐200c/miR‐141 loci. In vitro, over‐expression of miR‐200c in PC3 cells inhibited growth and clonogenic potential, as well as inducing apoptosis. Expression of the genes DNMT3A and TET1/TET3 were down‐regulated by miR‐200c and miR‐141 respectively. Finally, treatment with the soy isoflavone genistein caused demethylation of the promoter CpG sites closest to the miR‐200c/miR‐141 loci resulting in increased miR‐200c expression.

CONCLUSIONS

Our findings provide evidence that miR‐200c and miR‐141 are under epigenetic regulation in PCa cells. We propose that profiling their expression and methylation status may have potential as a novel biomarker or focus of therapeutic intervention in the diagnosis and prognosis of PCa. Prostate 76:1146–1159, 2016. © 2016 The Authors. The Prostate published by Wiley Periodicals, Inc.     

Differential expression of angiopoietin-2 and vascular endothelial growth factor in androgen-independent prostate cancer models

OBJECTIVE

To investigate the relationship between microvessel density (MVD), blood vessel morphology and the expression of angiopoietin (Ang)‐1, Ang‐2, tyrosine kinase with immunoglobulin and epidermal growth factor homology domains (Tie)‐2, and vascular endothelial growth factor (VEGF) in androgen‐dependent (AD) and androgen‐independent (AI) prostate cancer models, to gain insight into the regulation of angiogenesis at different stages of prostate cancer.

MATERIALS AND METHODS

MVD and blood vessel morphology were evaluated by CD34 immunohistochemical staining. The mRNA and protein secretion of the Angs, Tie‐2 and VEGF were measured by real‐time polymerase chain reaction and enzyme‐linked immunosorbent assays, respectively, in LNCaP (AD) and LNCaP‐19, C4‐2, C4–2B₄ and PC‐3 (AI) prostate cancer xenografts in mice.

RESULTS

LNCaP, C4‐2 and C4–2B₄ xenografts had high expression of Ang‐2 and VEGF, similar MVD and blood vessel morphology_. However, the most angiogenic cell line LNCaP‐19 expressed low levels of both factors and had different vessel morphology. PC‐3 xenografts had a similar MVD to LNCaP, C4‐2 and C4–2B₄, but the Ang‐2 and VEGF expression as well as the vessel morphology were similar to LNCaP‐19.

CONCLUSION

The differences in MVD, blood vessel morphology and the expression of Ang‐2 and VEGF show that prostate cancer cells display angiogenic heterogeneity, which indicates different roles of these factors in the regulation of angiogenesis in different stages of prostate cancer.     

Elevated physiological levels of folic acid can increase in vitro growth and invasiveness of prostate cancer cells

What's known on the subject? and What does the study add? Evidence has emerged identifying folic acid supplementation as a potential risk factor for cancer development or progression. Long‐term folic acid supplementation has been shown to increase the risk of prostate cancer development by three‐fold. Sarcosine is a byproduct of folate metabolism and has been proposed as a biomarker for aggressive prostate cancer phenotypes. We looked at the effects of physiologically relevant levels of folic acid on in vitro prostate cancer cell growth and invasion, and demonstrated that higher levels can have the effect of increasing both of these biological processes. We also show that these changes toward a more aggressive phenotype are not linked to increased sarcosine levels, however other metabolic pathways may be involved.

OBJECTIVES

• ? To investigate the effects of different folic acid concentrations on the growth and invasiveness of prostate cancer cell lines.
• ? To determine if observed changes are correlated with changes in levels of the potential prostate cancer biomarker, sarcosine, a byproduct of folate metabolism.

MATERIALS AND METHODS

• ? The prostate cancer cell lines PC‐3, LNCaP and DU145 were cultured in media containing 4, 20 or 100 nm of folic acid and assayed for growth over 9 days by counting viable cells at 3‐day intervals, or for invasion by passage through a Matrigel‐coated transwell membrane.
• ? Cells grown in the different folic acid media were collected and subjected to metabolomic analysis by gas chromatography and mass spectrometry to measure levels of intracellular sarcosine.

RESULTS

• ? The results show that higher levels of folic acid can increase cell growth in PC‐3 and LNCaP prostate cancer cell lines, and may also increase the invasive capacity of PC‐3, LNCaP and DU145 cells.
• ? We did not observe a correlation between increased invasion from higher folic acid concentrations and levels of sarcosine, but there were significant changes in other metabolites in cells grown in higher levels of folic acid.

CONCLUSION

• ? These findings suggest that folic acid has an important and potentially negative role in prostate cancer progression.

     

Cryoablative response of prostate cancer cells is influenced by androgen receptor expression

OBJECTIVE

To investigate in prostate cancer cells the consequences of androgen‐insensitivity (AI) development on the cellular and molecular responses to freezing, as a challenge in prostate cancer treatment occurs when the androgen‐sensitive (AS) phenotype switches to an AI phenotype, the latter of which is often refractory to many therapies.

MATERIALS AND METHODS

PC‐3 (AI) and LNCaP (AS) were each genetically altered to express the opposite phenotype and subjected to an in vitro freezing model. Viability, caspase inhibitor and Western blot studies were used to determine the basis of the differential responses of AI and AS cells.

RESULTS

LNCaP high‐passage cells, formed by repeated passage of LNCaP (AS) cells, were AI and showed a phenotypic shift to freeze resistance matching the freeze response of PC‐3 cells (AI). While stably transfected androgen receptor (AR)‐transfected cells (PC‐3 AR) had a freezing sensitivity similar to that of the LNCaP (AS) cell line. Importantly, AI cell lines survived and recovered from freezing exposure to temperatures as low as ?40 °C whereas AS cell lines did not. Caspase inhibition studies and related fluorescent probes showed an elevated level of apoptotic involvement in both AS cell lines after freezing compared with their AI counterparts. Western blot analysis showed that AR expression was modified after exposure to freezing.

CONCLUSION

This study suggests that AS cancers may be far more sensitive to a freezing insult and this might be linked to elevated apoptosis and caspase activity. As such, cryoablation may prove most effective in cancer cells that have not yet progressed to a more resistant AI phenotype, but both generic variants can be fully ablated at sufficiently low temperatures.     

Function of mutant and wild‐type plexinB1 in prostate cancer cells

BACKGROUND

Semaphorins act as chemotactic cues for cell movement via their transmembrane receptors, plexins. Somatic missense mutations in the plexinB1 gene coupled with overexpression of the protein frequently occur in prostate tumors, indicating a role for plexinB1 in the pathogenesis of prostate cancer. However, the effect of semaphorin/plexin signaling is highly context dependent and whether plexinB1 acts as an inducer or inhibitor of prostate tumor progression in this context is not known.

METHODS

The response of prostate cancer cell lines to plexinB1 activation was assessed in migration, invasion, proliferation and protein phosphorylation assays. Expression was assessed by quantitative RTPCR and immunoblotting.

RESULTS

Different prostate cancer cell lines respond to Sema4D (the ligand for plexinB1) in diverse ways. Activation of endogenous plexinB1 enhances migration, invasion and anchorage‐independent growth of LNCaP prostate cancer cells via activation of ErbB2 and Akt. In contrast, Sema4D‐stimulation decreased the motility and proliferative capacity of PC3 cells. LNCaP has a missense mutation (Thr1697Ala) in the plexinB1 gene while LNCaP‐LN3, a derivative of LNCaP, expresses high levels of wild‐type plexinB1 only. Sema4D stimulation increases the motility and anchorage independent growth of both cell lines, showing that these responses are not dependent on the presence of the Thr1697Ala form of plexinB1. ErbB2 and plexinB1 are expressed in primary prostate epithelial cells.

CONCLUSIONS

PlexinB1 signals via ErbB2 to increase the invasive phenotype of prostate cancer cells. Both wild‐type and mutant forms of plexinB1 are potential targets for anti‐cancer therapy in prostate tumors that express ErbB2. Prostate 73:1326–1335, 2013. © 2013 The Authors. The Prostate published by Wiley Periodicals, Inc.     

Genistein combined polysaccharide enhances activity of docetaxel, bicalutamide and Src kinase inhibition in androgen-dependent and independent prostate cancer cell lines

OBJECTIVE

To determine the benefit of genistein combined polysaccharide (GCP) in combination with the androgen receptor antagonist bicalutamide, the antimicrotubule taxane docetaxel, and the Src kinase inhibitor pp2 as part of a treatment regimen for advanced prostate cancer (CaP).

MATERIALS AND METHODS

The growth inhibitory and apoptotic effects of GCP in combination with bicalutamide, docetaxel and pp2 were evaluated in both the androgen‐dependent LNCaP line, and three androgen‐independent lines: CWR22Rv1, PC‐3, and LNCaP‐R273H. The LNCaP‐R273H model is an LNCaP variant expressing a p53^GOF allele; like CWR22Rv1 and PC‐3, it is able to grow in a minimal androgen environment. The effects of GCP treatment in combination with the aforementioned drugs were measured using an MTT assay, Western blotting, flow cytometric analysis, and caspase activation assay. Altered schedules of drug administration were explored using combinations of GCP and docetaxel.

RESULTS

GCP potentiated the activity of docetaxel in all four cell lines, resulting in growth inhibition and increased apoptosis. The combination of GCP and bicalutamide had enhanced activity in both the LNCaP and LNCaP‐R273H lines, which may better represent patient tumour cells after progression to androgen independence. Administration of docetaxel followed by GCP resulted in a synergistic interaction in LNCaP cells, with increased apoptosis. By contrast, GCP administered first showed subadditivity, probably resulting from GCP‐mediated induction of G1 arrest interfering with docetaxel activity.

CONCLUSION

These data suggest that GCP, an isoflavone‐enriched compound with minimal side‐effects and far superior intestinal absorption rate of genistein, has significant clinical potential in combination with docetaxel, bicalutamide or targeted agents for the treatment of advanced CaP.     

Antiproliferative effects of c-myc antisense oligonucleotide in prostate cancer cells: A novel therapy in prostate cancer

Objectives

To explore the possibility of using antisense oligonucleotide therapy for prostate cancer, we investigated the effect of c-myc-antisense-oligonucleotide (c-myc-As-ODN) in human prostate cancer cell lines such as LNCaP, PC3, and DU 145.

Methods

LNCaP, PC3, and DU145 cells were incubated in the presence of c-myc-As-ODN. Dose (0 to 10 μM) and time dependent (1 to 6 days) effects on proliferation and viability were examined by [³H]thymidine incorporation and MTT assay, respectively. Flow cytometry analysis was carried out to analyze cell cycle status by determining the DNA content in LNCaP cells. Control cultures received either c-myc-sense-ODN or scrambled (nonsense) nucleotides.

Results

Time- and dose-dependent decreases in DNA synthesis and cell viability were noted for all three prostate cancer cell lines after c-myc-As-ODN treatment. Further studies using LNCaP cells indicated that these changes were accompanied by an increase in the percentage of cells with less than 2N DNA content after c-myc-As-ODN treatment. The results suggest that c-myc-As-ODN induces cell death. Comparison of a c-myc-As-ODN-treated group with a group subjected to isoleucine deprivation revealed that thymidine incorporation was almost the same in c-myc-As-ODN-treated LNCaP cells and in LNCaP cells at early S phase.

Conclusions

These results suggest that c-myc-As-ODN inhibits prostate cancer cell growth and proliferation mainly by decreasing cell viability.     

The combination of docetaxel and the somatostatin analogue lanreotide on androgen-independent docetaxel-resistant prostate cancer: experimental data

OBJECTIVE

To evaluate the effects of the association between docetaxel and the somatostatin analogue lanreotide on the androgen‐independent prostate cancer cell line PC3, either sensitive or made resistant to docetaxel (PC3R), as new drugs and new combinations have promising clinical activity in hormone‐refractory prostate cancer.

MATERIALS AND METHODS

We examined the effect of docetaxel and lanreotide on cell proliferation, with analysis of the mitogen‐activated protein kinase pathway and expression of cell‐cycle regulatory proteins.

RESULTS

Combined treatment with docetaxel and lanreotide inhibited PC3 cell growth in vitro through an enhanced induction of cell death, compared with treatment with either agent alone; this result was particularly evident on PC3R cells. The results suggested that lanreotide could act as a P glycoprotein inhibitor in PC3R cells.

CONCLUSION

The present results provide a promising therapeutic approach for using somatostatin analogues in hormone‐refractory prostate cancer, in which lanreotide could interact with docetaxel in PC3R cells, with possible explanatory mechanisms which involve P glycoprotein‐mediated docetaxel resistance.     

Differential involvement of the Fas receptor/ligand system in p53-dependent apoptosis in human prostate cancer cells

BACKGROUND: The objective of this study was to characterize the involvement of the Fas receptor/ligand system in p53-dependent apoptosis in human prostate cancer cells. METHODS: The effects of adenovirus-mediated p53 gene transfer (Ad5CMV-p53) into human prostate cancer LNCaP, DU145, and PC3 cells on their growth, apoptosis and Fas receptor/ligand expression were examined by the MTT assay, DNA fragmentation assay, and Northern blot analysis, respectively. The sensitivity of these cells to an agonistic anti-Fas receptor antibody (CH11) and the effects of an antagonistic anti-Fas ligand antibody (4H9) on Ad5CMV-p53-induced apoptosis were analyzed by the MTT assay and DNA fragmentation assay. RESULTS: Ad5CMV-p53 treatment resulted in substantial growth inhibition, induction of apoptosis and up-regulation of Fas receptor as well as Fas ligand mRNA expression in LNCaP, DU145 and PC3 cells. Despite the abundant expression of Fas receptor in all of these cells, CH11 induced apoptosis only in PC3 cells. Furthermore, 4H9 partially blocked the apoptosis induced by Ad5CMV-p53 in PC3 cells, but not in LNCaP and DU145 cells. CONCLUSIONS: The Fas receptor/ligand system is differentially involved in p53-dependent apoptosis in prostate cancer cells; therefore, reintroduction of wild-type p53 into prostate cancer cells may induce apoptosis through Fas receptor/ligand interaction as well as through an alternative pathway.     

Clusterin knockdown using the antisense oligonucleotide OGX-011 re-sensitizes docetaxel-refractory prostate cancer PC-3 cells to chemotherapy

OBJECTIVES

To characterize changes in secretory clusterin (sCLU) expression in prostate cancer cells after treatment with docetaxel and to determine whether sCLU knockdown can re‐introduce chemosensitivity in a docetaxel‐resistant, androgen‐independent human prostate cancer model.

PATIENTS AND METHODS

A tissue microarray was constructed for 84 radical prostatectomy (RP) specimens from a multicentre Phase II trial of neoadjuvant combined androgen ablation and docetaxel (CUOG‐P01a) and assessed for changes in the expression of the cytoprotective chaperone sCLU. The human prostate cancer cell line PC‐3 was repeatedly exposed to docetaxel chemotherapy in vitro, and a docetaxel‐resistant cell subline (PC‐3dR) was developed and analysed.

RESULTS

sCLU levels were significantly higher in RP specimens treated with neoadjuvant combined androgen ablation and docetaxel than in untreated specimens. Similarly, sCLU expression increased 2.5‐fold in the newly developed docetaxel‐refractory PC‐3dR cell line compared with parental PC‐3 cells. There was a dose‐dependent and sequence‐specific decrease in sCLU levels in PC‐3dR cells using OGX‐011, an antisense oligonucleotide against human sCLU. OGX‐011 and small‐interference RNA both chemosensitized PC‐3dR cells to docetaxel and mitoxantrone in vitro and apoptotic rates in PC‐3dR cells were significantly increased when OGX‐011 was combined with docetaxel. In vivo, growth of PC‐3dR xenografts in nude mice was synergistically inhibited by OGX‐011 combined with paclitaxel or mitoxantrone (by 76% and 44% compared with their mismatch controls, respectively).

CONCLUSION

The present findings indicate that targeted knockdown of sCLU enhances the effects of cytotoxic chemotherapy in docetaxel‐refractory cells, and provide preclinical proof of principle for clinical trials testing OGX‐011 in second‐line chemotherapy regimens for patients with docetaxel‐refractory prostate cancer.     

Cysteine-rich secretory protein 3 and β-microseminoprotein on prostate cancer needle biopsies do not have predictive value for subsequent prostatectomy outcome

What’s known on the subject? and What does the study add? Cysteine‐rich secretory protein 3 (CRISP‐3) and β‐microseminoprotein (β‐MSP) both have independent prognostic value for biochemical recurrence of prostate cancer after radical prostatectomy. The study investigates whether CRISP‐3 and β‐MSP have prognostic value on diagnostic prostate needle‐biopsies, which are more relevant for therapeutic decision‐making. On needle‐biopsies CRISP‐3 and β‐MSP do not have significant prognostic value.

OBJECTIVES

? To investigate whether cysteine‐rich secretory protein 3 (CRISP‐3) and/or β‐microseminoprotein (β‐MSP) expression in diagnostic prostate needle biopsies have predictive value for prostate cancer (PC) on radical prostatecomy (RP). ? To evaluate their potential clinical implementation in a preoperative setting.

PATIENTS AND METHODS

? In total, 174 participants from the European Randomized Study of Screening for Prostate Cancer, Rotterdam section, treated by RP for PC were included in the present study. ? CRISP‐3 and β‐MSP immunohistochemistry was performed on corresponding diagnostic needle biopsies. ? Outcome was correlated with clinicopathological parameters (prostate‐specific‐antigen, PSA; number of positive biopsies; Gleason score, GS; pT‐stage; surgical margins at RP) and significant PC at RP (pT3/4, or GS > 6, or tumour volume ≥0.5 mL) in the total cohort (n= 174) and in a subgroup with low‐risk features at biopsy (PSA ≤ 10 ng/ml, cT ≤ 2, PSA density <0.20 ng/mL/g, GS < 7 and ≤2 positive biopsy cores; n= 87).

RESULTS

? β‐MSP and CRISP‐3 expression in PC tissue was heterogeneous, with variable staining intensities occurring in the same tissue specimen. ? High expression of β‐MSP significantly correlated with GS < 7 at RP; it was not a predictor for significant PC at RP neither in the total group (n= 174; odds ratio, OR, 0.319; 95% confidence interval, CI, 0.060–1.695; P= 0.180), nor in the low‐risk group (n= 87; OR, 0.227; 95% CI, 0.040–1.274; P= 0.092). ? CRISP‐3 expression was not related to clinicopathological parameters, and did not predict significant PC at RP in the total group (n= 174; OR, 1.056; 95% CI, 0.438–2.545; P= 0.904) or the low‐risk group (n= 87; OR, 1.856; 95% CI, 0.626–5.506; P= 0.265).

CONCLUSIONS

? High β‐MSP expression correlated with low GS in subsequent RP specimens, supporting the view that β‐MSP exerts a tumour‐suppressive effect. ? No significant prognostic value of β‐MSP or CRISP‐3 in prostate needle biopsies for significant PC at RP was found. ? β‐MSP or CRISP‐3 do not have additional value in the therapeutic stratification of patients with PC.     

Lentivirus-mediated SMO RNA interference inhibits SMO expression and cell proliferation,and affects the cell cycle in LNCaP and PC3 cancer cell lines

Smoothened （SMO） is an important member of the Hedgehog signaling pathway. We constructed a specific recombinant lentiviral vector for RNA interference,targeting the SMO gene （NM_005631） to observe its effect on SMO expression,cell proliferation and the cell cycle in the human androgen-sensitive prostate cancer cell line,LNCaP,and in the androgen-independent prostate cancer cell line,PC3. Four siRNA sequences were designed and inserted into a lentiviral vector pGCSIL-GFP to construct four recombinant vectors. The vector with the highest interfering efficiency was co-transfected with packaging vectors （pHelper1.0 and pHelper2.0） in 293T cells to assemble lentivirus particles by liposome for infecting LNCaP and PC3 cell lines,respectively. The expression level of SMO mRNA,tumor cell proliferation and cell cycle were measured by quantitative realtime polymerase chain reaction （qRT-PCR）,3-（4,5）-dimethylthiahiazo （-z-yl）-3,5-di-phenytetrazoliumromide （MTT） assay and flow eytometry,respectively. Sequence results showed that recombinant lentiviral vectors were constructed successfully.pGCSIL-GFP-723 had the highest interfering efficiency,named Lv-SIL-SMO723 after co-transfection,with which LNCaP and PC3 cell lines were infected. Compared with the control groups,results showed significantly decreased （P〈0.05） SMO mRNA expressions of LNCaP and PC3,lower mean percentage of S-phase cells and higher mean percentage of G_2/M phase cells,as well as obviously slow proliferation （P〈0.01） of LNCaP in the infected group. Yet,the proliferation of PC3 was not altered （P〉0.05）. In conclusion,the recombinant lentivirus particles were able to suppress SMO expression,regulate the cell cycle in the LNCaP and PC3 cell lines and markedly inhibit proliferation of LNCaP cells but not PC3 cells.     

The value of EZH2, p27kip1, BMI‐1 and MIB‐1 on biopsy specimens with low‐risk prostate cancer in selecting men with significant prostate cancer at prostatectomy

OBJECTIVE

To assess the additional prognostic value of the molecular markers EZH2, MIB‐1, p27^kip1 and BMI‐1 on needle biopsies from men with low‐risk prostate cancer, as this disease in needle biopsies shows a heterogeneous clinical outcome, and while it is known that the expression of these tissue markers is predictive of the clinical outcome after radical prostatectomy (RP) their value in prostate biopsies is largely unknown.

PATIENTS AND METHODS

The study included men participating in a screening study, diagnosed with low‐risk prostate cancer and subsequently treated with RP. Immunohistochemical staining for EZH2, MIB‐1, p27^kip1 and BMI‐1 on the needle biopsies were (semi)quantitatively scored and expression levels were related to significant disease at RP. Clinical low‐risk prostate cancer was defined as a prostate‐specific antigen (PSA) level of ≤10 ng/mL, clinical T‐stage ≤2, biopsy Gleason score ≤6, a PSA density of <0.20 ng/mL/g and two or fewer positive cores. Significant PC at RP was defined as presence of any of extracapsular extension, Gleason pattern 4/5, or tumour volume ≥0.5 mL.

RESULTS

In all, 86 biopsy specimens were included; there was high EZH2 expression (>1.0%) in 42% and a low p27^kip expression (<90%) in 63%. Significant disease was present in 44 (51%) RP specimens. A high EZH2 (odds ratio 3.19, P = 0.043) and a low p27^kip1 (4.69, P = 0.036) were independent predictors for significant prostate cancer at RP.

CONCLUSIONS

The determination of EZH2 and p27^kip1 on diagnostic needle biopsies supports the selection of men with indolent prostate cancer at RP. Especially p27^kip1 could improve the pretreatment risk assessment of patients with low‐risk prostate cancer.     

Effect of extracellular ATP on the growth of hormone-refractory prostate cancer in vivo

OBJECTIVE

To investigate whether the antineoplastic action of ATP on hormone‐refractory prostate carcinoma (HRPC) cells in vitro also occurs in vivo, by examining the effect of ATP in vivo on tumours resulting from implanted HRPC cells in mice.

MATERIALS AND METHODS

HRPC tumour cells DU145 and PC‐3 were implanted into male nude athymic mice. The effect of daily intraperitoneal (i.p.) injections of ATP (25 mm ) on the growth of freshly implanted and established HRPC tumours was assessed. Histological examination using light and electron microscopy was used to confirm retention of the original ultrastructure of the implanted tumours.

RESULTS

Daily i.p. injections of ATP significantly reduced the growth of freshly implanted DU145 tumour by 57.8% (P = 0.003), and reduced the rate of growth of established DU145 tumour by 69.0% (P = 0.006). ATP also significantly reduced the growth of freshly implanted PC‐3 tumour by 68.9% (P < 0.001). ATP treatment had no adverse effects on the host mice.

CONCLUSION

Our results show, for the first time, that ATP effectively reduces the growth of advanced HRPC tumours in vivo. This may represent a step in establishing ATP as an effective agent for HRPC treatment.     

Combined p53 and MDM2 biomarker analysis shows a unique pattern of expression associated with poor prognosis in patients with renal cell carcinoma undergoing radical nephrectomy

What's known on the subject? and What does the study add? Unlike most other cancers mutations of the p53 gene (TP53), typically indicated by increased p53 expression, are rare in renal cell carcinomas (RCC) and there is no evidence that mutation of TP53 is associated with outcome or treatment response. However, whilst TP53 mutations are not linked with outcome, p53 expression is as we show here. Our study is the first to demonstrate simultaneously that patients with increased p53 expression (significantly associated with MDM2 expression), have reduced disease specific survival even though the expressed p53 is rarely mutated. We therefore identify increased expression of wild‐type p53 and MDM2 in RCC as targets for future therapeutic approaches.

OBJECTIVE

• ? To resolve much debated issues surrounding p53 function, expression and mutation in renal cell carcinoma (RCC), we performed the first study to simultaneously determine p53/MDM2 expression, TP53 mutational status (in p53‐positive patients) and outcome in RCC.

PATIENTS AND METHODS

• ? In total, 90 specimens obtained from patients with RCC, who were treated by radical nephrectomy, were analyzed by immunohistochemistry for p53 and MDM2 on a tissue microarray, and p53 was functionally and genetically analyzed in p53 positive samples.
• ? Outcome analysis was by the Kaplan–Meier method and univariate analysis was used to identify variables for subsequent multivariate analysis of correlations between clinical parameters and biomarker expression.

RESULTS

• ? Up‐regulation of p53 in RCC is strongly linked with MDM2 up‐regulation (P < 0.001).
• ? Increased coexpression of p53 and MDM2 identifies those patients with a significantly reduced disease‐specific survival by univariate (P= 0.036) and Cox multiple regression analysis (P= 0.027; relative risk, 3.20).
• ? Functional (i.e. functional analysis of separated alleles in yeast) and genetic analysis of tumours with increased p53 expression shows that most patients (86%) retain wild‐type p53.

CONCLUSIONS

• ? Coexpression of p53/MDM2 identifies a subset of patients with poor prognosis, despite all of them having organ‐confined disease.
• ? Up‐regulated p53 is typically wild‐type and thus provides a mechanistic explanation for the association between p53 and MDM2 expression: up‐regulated wild‐type p53 likely promotes the observed MDM2 coexpression.
• ? The results obtained in the present study suggest that the p53 pathway is altered in a tissue/disease‐specific manner and that therapeutic strategies targeting this pathway should be investigated to determine whether the tumour suppressive function of p53 can be rescued in RCC.

     

姜黄素对前列腺癌细胞核转录因子抑制蛋白表达的影响

目的观察姜黄素对前列腺癌细胞核转录因子抑制蛋白(IkBα)表达的影响,探讨姜黄素抑制前列腺癌细胞增殖的作用机制。方法分别用10、25、50、75和100μmol/L 浓度的姜黄素对雄激素依赖性及雄激素非依赖性前列腺癌细胞株 LNCaP 和 PC3进行干预,5、12和24 h 后采用噻唑蓝(MTT)比色法观察细胞增殖情况;采用流式细胞术测定24 h 后细胞周期变化;5 h 后 Western 印迹法检测细胞中 IkBα的表达。结果姜黄素显著抑制 LNCaP 及 PC3细胞的生长,呈剂量和时间依赖性;姜黄素将两种前列腺癌细胞阻滞于 G_2、M 期[LNCaP 与 PC3细胞,空白对照分别为(11.4±1.3)%与(17.3±1.7)%,100μmol/L 姜黄素作用后分别为(27.3±2.8)%与(33.4±4.0)%],从而诱导肿瘤细胞凋亡;姜黄素作用于 LNCaP 细胞后,细胞中 IkBα表达无变化(F=0.129,P>0.05);但作用于 PC3细胞后,细胞中 IkBα的表达明显增强,呈现出显著的剂量依赖性(F=31.618,P<0.05)。结论姜黄素通过活化 IkBα在 PC3细胞中的表达发挥抑制 PC3细胞增殖的作用。对于 LNCaP 细胞,姜黄素可能通过抗氧化、抑制细胞内代谢产物形成等方式抑制 LNCaP 细胞增殖。