Radical Retropubic Prostatectomy for Localized Prostate Cancer in Renal Transplant Patients

OBJECTIVE To investigate the feasibility of radical retropubic prostatectomy (RRP) in renal transplant recipients with clinically localized prostate cancer. METHODS A prospective protocol was established between August 2004 and November 2007. In that period, 8 patients diagnosed with localized prostate cancer were submitted to RRP, and their clinicopathologic data were reviewed. RESULTS The mean age (standard deviation) at surgery was 59.6 +/- 6.7 years (range, 49-67 years). All patients had TIC tumors, except for 1 with a T2A tumor. The mean preoperative prostate-specific antigen value was 4.5 +/- 1.8 ng/mL (range, 1.6-7.0 ng/mL). The mean interval between renal transplantation and RRP was 89.9 +/- 65.1 months (range, 40-209 months). The procedure was well tolerated without major complications, and all patients were discharged on the fifth postoperative day. There was no impairment to bladder descent caused by the presence of the allograft or the ureteroneocystostomy. Urethrovesical anastomosis was easily performed in all cases in the standard manner. Blood transfusion was needed in 2 patients (1 received 2 U and another 5 U of blood). The mean operative duration was 183 +/- 29.7minutes (range, 150-240 minutes), the mean estimated blood loss was 656 +/- 576 mL (range, 100-2000 mL), and no deterioration of graft function was observed. All patients were followed, and the mean follow-up was 10.5 months (range, 2-30 months). Prostate-specific antigen was undetectable in all cases during this time frame. CONCLUSIONS Radical retropubic prostatectomy in renal transplant patients is safe, effective, and can be easily performed in the same manner as described by Walsh, regardless of the presence of the allograft. The only necessary technical modification is the avoidance of ipsilateral lymphadenectomy to prevent damage to the transplanted organ. UROLOGY 72: 1362-1365, 2008. (C) 2008 Elsevier Inc.

Polymorphisms of the matrix metalloproteinases associated with prostate cancer

Prostate cancer (PCa) is the most common type of malignant tumor in Brazilian males. Single nucleotide polymorphisms (SNPs) have been demonstrated to be present in the promoter region of matrix metalloproteinase (MMP) genes and have been associated with the development and progression of some cancers. In this study, our aim was to investigate the association between the polymorphisms of MMP1, 2, 7, and 9 and susceptibility, and their correlation with the classic prognostic parameters of PCa. For genes MMP1, 2 and 9, the frequencies of the polymorphic homozygote genotypes were higher in the control group than in the PCa group (P<0.0001). We conclude that the MMP1, 2 and 9 polymorphisms are more common in the control group than in patients with PCa, and may have a protective effect in the development of this neoplasia.

Loss of samples in the tissue microarray technique: comparison between slides using adhesive tape and silanized slides

The tissue microarray (TMA) technique allows multiple tissue samples in a single block. Commercial adhesive tape is used to avoid the loss of tissue samples during the immunostaining process. Few reports exist in the literature comparing the use of these adhesive tapes to other adhesive techniques. The objective of this study was to compare loss of sections adhered to slides using commercial adhesive tapes versus using silanized only slides. TMA was constructed with varying tissues using a fixed-base device (Beecher Instruments), placing 108 cylinders of 1 mm diameter in duplicate, spaced 1.2 mm apart. Section of 4 mu m were cut from the TMA block and adhered to 30 silanized slides and 30 commercial glass slides using adhesive tape, according to manufacturer`s recommendations. Vimentin immunoexpression was evaluated by immunohistochemistry. Antigenic recovery was realized in citrate buffer using a microwave oven. Cylinder loss in the immunohistochemical process was quantified and expressed as: total (>80%), almost complete (75-79%), or partial (50-74%). The commercial adhesive tape group presented lesser total loss (1.1 versus 6.4%), almost complete loss (2.2 versus 3.5%), and partial loss (2.1 versus 3.8%) than the silanized slide group (ANOVA, P < 0.05). The sum of total and almost complete losses in the silanized slide group was 9.9%, greater than the losses in slides using commercial adhesive tapes (3.3%) and less than reported and considered acceptable in the literature (10-30%). In conclusion, the use of silanized only slides presents very satisfactory results, requires less training, and reduces costs significantly, thus justifying their use in research.

Cytotoxic effects of butanolic extract from Pfaffia paniculata (Brazilian Ginseng) on cultured human breast cancer cell line MCF-7

Roots of Pfaffia paniculata have been well documented for multifarious therapeutic values and have also been used for cancer therapy in folk medicine. This study has been performed in a human breast tumor cell line, the MCF-7 cells. These are the most commonly used model of estrogen-positive breast cancer, and it has been originally established in 1973 at the Michigan Cancer Foundation from a pleural effusion taken from a woman with metastatic breast cancer. Butanolic extract of the roots of P. paniculata showed cytotoxic effect MCF-7 cell line. as determined with crystal violet assay, cellular death with acridine orange/ethidium bromide staining, and cell proliferation with immunocytochemistry of bromodeoxyuridine (BrdU). Subcellular alterations were evaluated by electron microscopy. Cells treated With butanolic extract showed degeneration of cytoplasmic components and profound morphological and nuclear alterations. The results show that this butanolic extract indeed presents cytotoxic substances, and its fractions merit further investigations. (C) 2008 Elsevier GmbH. All rights reserved.

Oral squamous cell carcinoma: status of tight junction claudins in the different histopathological patterns and relationship with clinical parameters. A tissue-microarray-based study of 136 cases

Aims Claudins are integral transmembrane proteins of the tight junctions, critical for maintaining cell adhesion and polarity. Alterations in the expression of individual claudins have been detected in carcinomas and appear to correlate with tumour progression. Methods In this study, a panel of anti-claudin antibodies (anti-claudins 1, 2, 3, 4, 5 and 7) was employed to map claudin expression in 136 cases of oral squamous cell carcinoma (OSCC) organised in a tissue microarray. Results Claudins were expressed in a reticular pattern up to the prickle layer in normal mucosal epithelium. In OSCC, claudins were strongly present in well-differentiated tumours, they presented mild and low expression in moderately differentiated OSCC, and were negative in poorly differentiated OSCC; the absences of claudin 1 (p = 0.002) and claudin 4 (p<0.001) were associated with moderately/poorly differentiated tumours. Strong expression of claudin 4 was associated with decreased perineural infiltration (p = 0.024). Claudins 5 and 7 were mostly negative or weakly expressed in all cases studied. Expression of claudin 7 was associated with the early clinical stages of the disease, whereas loss of claudin 7 tended to be more frequent in advanced stages of OSCC (p = 0.054). Absence of claudin 7 was also associated with absent vascular infiltration (p = 0.045) and with presence of recurrence (p = 0.052). Conclusions Claudin expression patterns showed a strong correlation with histological type of OSCC; claudin expression was decreased in areas of invasion, and negative in poorly differentiated tumours. This pattern may be related to evolution and prognosis of these tumours, especially in the case of claudin 7, which seems to be associated with a poor prognosis in OSCC.

Nerve growth factor stimulates proliferation and survival of human breast cancer cells through two distinct signaling pathways

We show here that the neurotrophin nerve growth factor (NGF), which has been shown to be a mitogen for breast cancer cells, also stimulates cell survival through a distinct signaling pathway. Breast cancer cell lines (MCF-7, T47-D, BT-20, and MDA-MB-231) were found to express both types of NGF receptors: p140(trkA) and p75(NTR). The two other tyrosine kinase receptors for neurotrophins, TrkB and TrkC, were not expressed. The mitogenic effect of NGF on breast cancer cells required the tyrosine kinase activity of p140(trkA) as well as the mitogen-activated protein kinase (MAPK) cascade, but was independent of p75(NTR). I, contrast, the anti-apoptotic effect of NGF (studied using the ceramide analogue C2) required p75(NTR) as well as the activation of the transcription factor NF-kB, but neither p140(trkA) nor MAPK was necessary. Other neurotrophins (BDNF, NT-3, NT-4/5) also induced cell survival, although not proliferation, emphasizing the importance of p75(NTR) in NGF-mediated survival. Both the pharmacological NF-KB inhibitor SN50, and cell transfection with IkBm, resulted in a diminution of NGF anti-apoptotic effect. These data show that two distinct signaling pathways are required for NGF activity and confirm the roles played by p75(NTR) and NF-kappaB in the activation of the survival pathway in breast cancer cells.

Proteomic analysis reveals that 14-3-3 sigma in downregulated in human breast cancer cells

The class of molecular chaperones known as 14-3-3 is involved in the control of cellular growth by virtue of its apparent regulation of various signaling pathways, including the Raf/mitogen-activated protein kinase pathway. In breast cancer cells, the sigma form of 14-3-3 has been shown to interact with cyclin-dependent kinases and to control the rate of entry into mitosis. To test for a direct role for 14-3-3 in breast epithelial cell neoplasia, me have quantitated 14-3-3 protein levels using a proteomic approach based on two-dimensional electrophoresis and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF). We show here that 14-3-3 sigma protein is strongly down-regulated in the prototypic breast cancer cell lines MCF-7 and MDA-MB-231 and in primary breast carcinomas as compared with normal breast epithelial cells. In contrast, levels of the alpha, beta, delta, or zeta isoforms of 14-3-3 mere the same in both normal and transformed cells. The data support the idea that 14-3-3 sigma is involved in the neoplastic transition of breast epithelial cells by virtue of its role as a tumor suppressor; as such, it may constitute a robust marker with clinical efficacy for this pathology.

Enhanced effector and memory CTL responses generated by incorporation of receptor activator of NF-kappa B(RANK)/RANK ligand costimulatory molecules into dendritic cell immunogens expressing a human tumor-specific antigen

The outcome of dendritic cell (DC) presentation of Ag to T cells via the TCR/MHC synapse is determined by second signaling through CD80/86 and, importantly, by ligation of costimulatory ligands and receptors located at the DC and T cell surfaces. Downstream signaling triggered by costimulatory molecule ligation results in reciprocal DC and T cell activation and survival, which predisposes to enhanced T cell-mediated immune responses. In this study, we used adenoviral vectors to express a model tumor Ag (the E7 oncoprotein of human papillomavirus 16) with or without coexpression of receptor activator of NF-kappaB (RANK)/RANK ligand (RANKL) or CD40/CD40L costimulatory molecules, and used these transgenic DCs to immunize mice for the generation of E7-directed CD8(+) T cell responses. We show that coexpression of RANK/RANKL, but not CD40/CD40L, in E7-expressing DCs augmented E7-specific IFN-gamma-secreting effector and memory T cells and E7-specific CTLs. These responses were also augmented by coexpression of T cell costimulatory molecules (RANKL and CD40L) or DC costimulatory molecules (RANK and CD40) in the E7-expressing DC immunogens. Augmentation of CTL responses correlated with up-regulation of CD80 and CD86 expression in DCs transduced with costimulatory molecules, suggesting a mechanism for enhanced T cell activation/survival. These results have generic implications for improved tumor Ag-expressing DC vaccines, and specific implications for a DC-based vaccine approach for human papillomavirus 16-associated cervical carcinoma.

Differential expression of CDC25 phosphatases splice variants in human breast cancer cells

Background: CDC25 phosphatases control cell cycle progression by activating cyclin dependent kinases. The three CDC25 isoforms encoding genes are submitted to alternative splicing events which generate at least two variants for CDC25A and five for both CDC25B and CDC25C. An over-expression of CDC25 was reported in several types of cancer, including breast cancer, and is often associated with a poor prognosis. Nevertheless, most of the previous studies did not address the expression of CDC25 splice variants. Here, we evaluated CDC25 spliced transcripts expression in anti-cancerous drug-sensitive and resistant breast cancer cell lines in order to identify potential breast cancer biomarkers. Methods: CDC25 splice variants mRNA levels were evaluated by semi-quantitative RT-PCR and by an original real-time RT-PCR assay. Results: CDC25 spliced transcripts are differentially expres-sed in the breast cancer cell lines studied. An up-regulation of CDC25A2 variant and an increase of the CDC25C5/C1 ratio are associated to the multidrug-resistance in VCREMS and DOXOR breast cancer cells, compared to their sensitive counterpart cell line MCF-7. Additionally, CDC25B2 tran-script is exclusively over-expressed in VCREMS resistant cells and could therefore be involved in the development of certain type of drug resistance. Conclusions: CDC25 splice variants could represent interesting potential breast cancer prognostic biomarkers.

Regulation of histone H2A.Z expression is mediated by sirtuin 1 in prostate cancer

Histone variants seem to play a major role in gene expression regulation. In prostate cancer, H2A.Z and its acetylated form are implicated in oncogenes’ upregulation. SIRT1, which may act either as tumor suppressor or oncogene, reduces H2A.Z levels in cardiomyocytes, via proteasome-mediated degradation, and this mechanism might be impaired in prostate cancer cells due to sirtuin 1 downregulation. Thus, we aimed to characterize the mechanisms underlying H2A.Z and SIRT1 deregulation in prostate carcinogenesis and how they interact. We found that H2AFZ and SIRT1 were up- and downregulated, respectively, at transcript level in primary prostate cancer and high-grade prostatic intraepithelial neoplasia compared to normal prostatic tissues. Induced SIRT1 overexpression in prostate cancer cell lines resulted in almost complete absence of H2A.Z. Inhibition of mTOR had a modest effect on H2A.Z levels, but proteasome inhibition prevented the marked reduction of H2A.Z due to sirtuin 1 overexpression. Prostate cancer cells exposed to epigenetic modifying drugs trichostatin A, alone or combined with 5-aza-2’-deoxycytidine, increased H2AFZ transcript, although with a concomitant decrease in protein levels. Conversely, SIRT1 transcript and protein levels increased after exposure. ChIP revealed an increase of activation marks within the TSS region for both genes. Remarkably, inhibition of sirtuin 1 with nicotinamide, increased H2A.Z levels, whereas activation of sirtuin 1 by resveratrol led to an abrupt decrease in H2A.Z. Finally, protein-ligation assay showed that exposure to epigenetic modifying drugs fostered the interaction between sirtuin 1 and H2A.Z. We concluded that sirtuin 1 and H2A.Z deregulation in prostate cancer are reciprocally related. Epigenetic mechanisms, mostly histone post-translational modifications, are likely involved and impair sirtuin 1-mediated downregulation of H2A.Z via proteasome-mediated degradation. Epigenetic modifying drugs in conjunction with enzymatic modulators are able to restore the normal functions of sirtuin 1 and might constitute relevant tools for targeted therapy of prostate cancer patients

SMYD3 contributes to a more aggressive phenotype of prostate cancer and targets Cyclin D2 through H4K20me3

Prostate cancer (PCa) is one of the most incident cancers worldwide but clinical and pathological parameters have limited ability to discriminate between clinically significant and indolent PCa. Altered expression of histone methyltransferases and histone methylation patterns are involved in prostate carcinogenesis. SMYD3 transcript levels have prognostic value and discriminate among PCa with different clinical aggressiveness, so we decided to investigate its putative oncogenic role on PCa.We silenced SMYD3 and assess its impact through in vitro (cell viability, cell cycle, apoptosis, migration, invasion assays) and in vivo (tumor formation, angiogenesis). We evaluated SET domain's impact in PCa cells' phenotype. Histone marks deposition on SMYD3 putative target genes was assessed by ChIP analysis.Knockdown of SMYD3 attenuated malignant phenotype of LNCaP and PC3 cell lines. Deletions affecting the SET domain showed phenotypic impact similar to SMYD3 silencing, suggesting that tumorigenic effect is mediated through its histone methyltransferase activity. Moreover, CCND2 was identified as a putative target gene for SMYD3 transcriptional regulation, through trimethylation of H4K20.Our results support a proto-oncogenic role for SMYD3 in prostate carcinogenesis, mainly due to its methyltransferase enzymatic activity. Thus, SMYD3 overexpression is a potential biomarker for clinically aggressive disease and an attractive therapeutic target in PCa.

Effects of Environmental Organochlorine Pesticides on Human Breast Cancer: Putative Involvement on Invasive Cell Ability

Human exposure to persistent organic pollutants (POPs) is a certainty, even to long banned pesticides like o,p′-dichlorodiphenyltrichloroethane (o,p′-DDT), and its metabolites p,p′-dichlorodiphenyldichloroethylene (p,p′-DDE), and p,p′-dichlorodiphenyldichloroethane (p,p′-DDD). POPs are known to be particularly toxic and have been associated with endocrine-disrupting effects in several mammals, including humans even at very low doses. As environmental estrogens, they could play a critical role in carcinogenesis, such as in breast cancer. With the purpose of evaluating their effect on breast cancer biology, o,p′-DDT, p,p′-DDE, and p,p′-DDD (50–1000 nM) were tested on two human breast adenocarcinoma cell lines: MCF-7 expressing estrogen receptor (ER) α and MDA-MB-231 negative for ERα, regarding cell proliferation and viability in addition to their invasive potential. Cell proliferation and viability were not equally affected by these compounds. In MCF-7 cells, the compounds were able to decrease cell proliferation and viability. On the other hand, no evident response was observed in treated MDA-MB-231 cells. Concerning the invasive potential, the less invasive cell line, MCF-7, had its invasion potential significantly induced, while the more invasive cell line MDA-MB-231, had its invasion potential dramatically reduced in the presence of the tested compounds. Altogether, the results showed that these compounds were able to modulate several cancer-related processes, namely in breast cancer cell lines, and underline the relevance of POP exposure to the risk of cancer development and progression, unraveling distinct pathways of action of these compounds on tumor cell biology.

Post-Treated Prostate Cancer: Normal Findings and Signs of Local Relapse on Multiparametric Magnetic Resonance Imaging

The use of multiparametric magnetic resonance imaging (mp-MRI) for prostate cancer has increased over recent years, mainly for detection, staging, and active surveillance. However, suspicion of recurrence in the set of biochemical failure is becoming a significant reason for clinicians to request mp-MRI. Radiologists should be able to recognize the normal post-treatment MRI findings. Fibrosis and atrophic remnant seminal vesicles after prostatectomy are often found and must be differentiated from local relapse. Moreover, brachytherapy, external beam radiotherapy, cryosurgery, and hormonal therapy tend to diffusely decrease the signal intensity of the peripheral zone on T2-weighted images (T2WI) due to the loss of water content, consequently mimicking tumor and hemorrhage. The combination of T2WI and functional studies like diffusion-weighted imaging and dynamic contrast-enhanced improves the identification of local relapse. Tumor recurrence tends to restrict on diffusion images and avidly enhances after contrast administration either within or outside the gland. The authors provide a pictorial review of the normal findings and the signs of local tumor relapse after radical prostatectomy, external beam radiotherapy, brachytherapy, cryosurgery, and hormonal therapy.

Improving the early diagnostic of prostate cancer by multiple biomarker detection with new biosensing devices

Prostate cancer (PCa) is the most common form of cancer in men, in Europe (World Health Organization data). The most recent statistics, in Portuguese territory, confirm this scenario, which states that about 50% of Portuguese men may suffer from prostate cancer and 15% of these will die from this condition. Its early detection is therefore fundamental. This is currently being done by Prostate Specific Antigen (PSA) screening in urine but false positive and negative results are quite often obtained and many patients are sent to unnecessary biopsy procedures. This early detection protocol may be improved, by the development of point-of-care cancer detection devices, not only to PSA but also to other biomarkers recently identified. Thus, the present work aims to screen several biomarkers in cultured human prostate cell lines, serum and urine samples, developing low cost sensors based on new synthetic biomaterials. Biomarkers considered in this study are the following: prostate specific antigen (PSA), annexin A3 (ANXA3), microseminoprotein-beta (MSMB) and sarcosine (SAR). The biomarker recognition may occurs by means of molecularly imprinted polymers (MIP), which are a kind of plastic antibodies, and enzymatic approaches. The growth of a rigid polymer, chemically stable, using the biomarker as a template allows the synthesis of the plastic antibody. MIPs show high sensitivity/selectivity and present much longer stability and much lower price than natural antibodies. This nanostructured material was prepared on a carbon solid. The interaction between the biomarker and the sensing-material produces electrical signals generating quantitative or semi-quantitative data. These devices allow inexpensive and portable detection in point-of-care testing.

Localization of MCT2 at peroxisomes is associated with malignant transformation in prostate cancer

Previous studies on monocarboxylate transporters expression in prostate cancer (PCa) have shown that monocarboxylate transporter 2 (MCT2) was clearly overexpressed in prostate malignant glands, pointing it out as a putative biomarker for PCa. However, its localization and possible role in PCa cells remained unclear. In this study, we demonstrate that MCT2 localizes mainly at peroxisomes in PCa cells and is able to take advantage of the peroxisomal transport machinery by interacting with Pex19. We have also shown an increase in MCT2 expression from non-malignant to malignant cells that was directly correlated with its peroxisomal localization. Upon analysis of the expression of several peroxisomal ß-oxidation proteins in PIN lesions and PCa cells from a large variety of human prostate samples, we suggest that MCT2 presence at peroxisomes is related to an increase in ß -oxidation levels which may be crucial for malignant transformation. Our results present novel evidence that may not only contribute to the study of PCa development mechanisms but also pinpoint novel targets for cancer therapy.