RNA-Integrity and 8-Isoprostane Levels Are Stable in Prostate Tissue Samples Upon Long-Term Storage at -80°C.

Sampling of prostate tissue (n = 97) was performed in conjunction with planned radical prostatectomies, in collaboration with Biobank1®. The tissue used in this study was collected during the period 2003-2016, quickly frozen, and kept at -80°C until assayed in 2018. RNA extraction was performed with two different protocols (miRNeasy and mirVana™), and RNA quality was determined by measuring the RNA Integrity Number (RIN). The level of isoprostanes is widely recognized as a specific indicator of lipid peroxidation both in vitro and in vivo. The level of 8-isoprostane was measured because it is the main oxidation product of arachidonic acid, the most abundant phospholipid fatty acid. The level of 8-isoprostane was measured using enzyme immunoassay. There was no statistically significant difference in yield between the samples isolated with the mirVana protocol compared to the miRNeasy protocol. Average RIN was 2.8 units higher with the mirVana extraction protocol compared to the miRNeasy protocol (p < 0.001). For miRNeasy extractions, RINs were 7.1 for prostatectomies in 2005-2007 and 6.2 for those in 2018 (p < 0.001). For mirVana extractions, the difference in RIN score between the two groups regarding years of collection was not statistically significant. There was no significant increase in the levels of 8-isoprostane between the 2005-2007 samples and the 2018. The conclusion is that there is no oxidation of phospholipids with increasing storage time up to 15 years.

Increased levels of serum miR-148a-3p are associated with prostate cancer.

Prostate cancer (PCa) is one of the most common types of cancer and the fifth leading cause of death among men worldwide. The tools for diagnosing PCa have limited value, and to improve correct diagnosis there is a need for markers that can contribute to a more precise diagnosis, which would lead to proper treatment of only those patients who need it. Micro RNA (miRNA) plays a key role in the development of cancer and is therefore a potential marker for PCa. Next-generation sequencing was used to discover differences in miRNA expression between serum samples from PCa patients and healthy controls, and the results were validated by quantitative real-time polymerase chain reaction. Detection of the miRNA of interest was attempted in prostate tissue by in situ hybridization. All samples were collected in collaboration with Biobank1® . By miRNA sequencing of serum samples, significant expression of some miRNAs in patients with PCa and healthy controls was detected. This study showed that miR-148a-3p is upregulated in men with PCa, and the miRNA is differentially expressed in PCa patients compared to healthy controls. The results also showed that miR-148a-3p is located in prostate tissue.

Gene expressional changes in prostate fibroblasts from cancerous tissue.

Prostate cancer is the most common type of cancer in men. It is assumed that the tumor microenvironment of the prostate contributes to invasion and metastasis. Stroma-epithelial crosstalk has shown to change with progression of prostate cancer, and thereby the stromal compartment might be an attractive target in diagnostic and therapeutic approaches to prostate cancer. The purpose of this project was to study the reciprocal influence between fibroblasts and cancer cells in prostate cancer. Prostate fibroblast primary cultures from areas with cancer and hyperplasia were cocultivated with cells of the PC-3 lineage. Gene expression profiles of both cell types were studied to reveal possible associations to cancer invasion and metastasis. There were 383 differentially expressed genes between fibroblasts from cancerous areas and fibroblasts from areas with hyperplasia before cocultivation with PC-3 cells. Several of the differentially expressed gene classes are associated with cancer development and metastasis. After cocultivation, there were 26 differentially expressed genes between cancerous and hyperplastic fibroblasts. There were only three differentially expressed genes between PC-3 cells that had been cocultivated with cancerous fibroblasts and PC-3 cells that had been cocultivated with hyperplastic fibroblasts. The fibroblasts from cancer areas showed a different expression pattern from the characteristics reported as reactive stroma in previous studies. We found tenascin C to be downregulated, which is contrary to previous findings. TGF-ß3 and TGF-ßR3 were also downregulated, which has been associated with disturbance of TGF-ß signaling during prostate cancer progression. Cocultivation with PC-3 cells seems to make the cancerous and hyperplastic fibroblasts more alike each other, as the number of differentially expressed genes decreases. It is desirable to find out if the reduction in differential gene expression is attributable to that hyperplastic fibroblasts become more alike the cancerous fibroblasts or vice versa. Also, we think that the lower expression levels of c-Jun and c-Fos in cancerous fibroblasts without coculture may cause loss of normal fibroblast differentiation, proliferation and inflammatory response, and hence, favor the proliferation and invasion of cancer cells.

A new method to provide a fresh frozen prostate slice suitable for gene expression study and MR spectroscopy.

BACKGROUND: Fresh frozen tissue from radical prostatectomy specimens is highly valuable material for research on gene expression and cellular metabolites. The purpose of this study was to develop a standardized method to provide a representative high quality research sample from radical prostatectomy specimens without interfering with the routine histopathological procedure. METHODS: A complete transversal slice is collected and snap-frozen before formalin fixation and routine processing of the remaining gland. The freezing preserves the original geometric shape, thus allowing subsampling of specific cell populations without thawing. RNA was extracted from 53 cylindrical subsamples (diameter 3 mm, thickness 2 mm) from 16 consecutive frozen slices. The histological pattern was evaluated by microscopy of a cryosection from sample before further analysis. RESULTS: Using this novel harvesting method close to 400 slices have been collected. Whenever tumor was present in both adjacent surrounding hematoxylin-eosin sections, we found cancer in 88% of the frozen slices. The extracted RNA showed very high quality with a mean RNA integrity number of 9.16 (SD 0.53). The MR spectra showed metabolic profiles containing several resonances, which deserve further evaluation as possible biomarkers for prostate cancer. After MR analysis the RNA was still highly intact with a mean RNA integrity number of 8.40 (SD 1.53), which makes it possible to correlate transcriptomic and metabolomic profiles of the extracted samples. CONCLUSION: We present a safe and standardized method for procurement of a high quality fresh frozen prostate slice, suitable for gene expression analysis and MR spectroscopy.

[Epithelial-to-mesenchymal transition and prostate cancer]. / Epitelial-mesenkymal transisjon og prostatakreft.

BACKGROUND: Prostate cancer is highly prevalent and is a frequent cause of death in men. As for most other cancers the prognosis is largely determined by the occurrence of metastases. Future treatment of prostate cancer should focus on inhibition of the cancer cells' ability to invade surrounding tissues--and to metastasise. In order to develop such therapies, it is important to unveil the mechanisms that lead to an invasive phenotype. Development of invasive tumours resemble processes involved in embryonic development, e.g. during formation of the mesoderm. The latter is characterised by a sequence of events whereby epithelial ectodermic cells acquire a migratory phenotype, which directly parallels the formation of invasive behaviour in carcinomas. MATERIAL AND METHODS: The present review is based on articles published in well-recognized journals of high international ranking. Some of the considerations also draw on the authors' personal experience in clinical work and basic research. RESULTS: Gastrulation is the process in which the three types of tissue stem cells move to different areas in the embryo (morphogenetic movement) and form the basis for various tissues and organs. This overview calls attention to the fact that events during carcinoma development, are strikingly similar to cellular changes during gastrulation. INTERPRETATION: A thorough understanding of gastrulation may provide a fruitful framework for new insight into cancer cell invasion and metastasis. This knowledge may in turn be exploited to develop drugs with anti-invasive properties which could revolutionise the treatment of carcinoma of the prostate and other sites.

Transcription levels of invasion-related genes in prostate cancer cells are modified by inhibitors of tyrosine kinase.

Previous reports have shown that genistein and tyrphostin AG-1478, two tyrosine kinase inhibitors (TKIs), exert multiple cellular effects in prostate carcinoma cells, e.g. a reduction in the production of urokinase plasminogen activator (uPA) and its receptor uPAR, and a decrease in the cells' ability to invade an artificial basement membrane. Microarray technology was used to measure alterations in mRNA levels caused by TKI treatment in two prostatic carcinoma cell lines, PC-3 and DU-145. Genistein treatment led to a reduction of at least 50% in 78 genes in PC-3, while 82 were twofold upregulated. In DU-145, the same treatment resulted in a 50% decreased transcript level in 120 genes, and increased expression in 25 genes. Tyrphostin AG-1478 produced a 50% reduction in mRNA levels in 58 genes in DU-145, whereas no alterations were demonstrated using the tyrphostin in PC-3 cells. Among the effects of TKIs, a lowered uPA and uPAR transcription was demonstrated in genistein-treated cells, while a few metalloproteinases (MMPs) were affected. Transcription of various integrin subunits was also downregulated overall. Several alterations in gene transcription were demonstrated in PC-3 and DU-145 after TKI treatment. This knowledge could be of importance in the search for new therapeutic strategies in prostate cancer treatment, and the interplay between the various effects needs to be investigated further.

Urokinase plasminogen activator receptor (uPAR) expression is reduced by tyrosine kinase inhibitors.

Previously we reported that tyrosine kinase inhibitors (TKI) produced a reduction in uPA expression in prostatic cancer cells, and that TKI-treated cells were less invasive compared to untreated cells. Nevertheless, no change in cell migration was observed when TKI-treated cells were supplied with external uPA, thus indicating more complex mechanisms leading to decreased cell invasion. uPAR expression was measured with an enzyme-linked immunosorbent assay (ELISA) in PC-3 and DU-145 prostate carcinoma cells treated with the two TKI genistein and AG-1478. uPAR mRNA levels were measured with real-time reverse transcriptase-polymerase chain reaction (RT-PCR). uPAR immunocytochemistry was used to examine the receptor distribution in cells grown on a reconstituted basal lamina. Immunocytochemistry showed an intense uPAR immunostaining in invading cells, particularly in the leading edge membrane. Treatment with genistein and AG-1478 led to a decreased expression of uPAR in DU-145, but not in PC-3. Furthermore, a reduction of uPAR mRNA was found in TKI-treated DU-145 cells, while PC-3 was not affected. Our results indicate a possible role of TKI as cancer suppressors by acting as a regulator of uPAR expression.

Tyrosine kinase inhibitors alter adhesivity of prostatic cancer cells to extracellular matrix components.

Tyrosine kinase inhibitors (TKIs) are thought to have potential as a new generation of anti-cancer drugs. Since invasiveness, the main characteristic of malignant behaviour, is believed to depend on altered cell-matrix interactions, we investigated the effect of two potent TKIs, genistein and tyrphostin AG-1478, on the interaction of prostate cancer cells with extracellular matrix components. PC-3 and DU-145 cells were treated with various concentrations of genistein and tyrphostin AG-1478. Adhesion to extracellular matrix was assayed using fluorescence-labelled cells seeded on collagen type I, collagen type IV, fibronectin, laminin and vitronectin. The expression levels of integrin beta1, alpha2, alpha3 and alpha5 subunits were measured using flow cytometry of cells labelled with monoclonal murine antibodies. Genistein treatment reduced the ability of both cell lines to adhere to the matrix proteins tested. This effect was more pronounced for PC-3 cells than for DU-145 cells. Genistein treatment decreased the expression of beta1 integrins by 40% in PC-3 cells and 22% in DU-145. AG-1478 treatment slightly reduced the ability of DU-145 cells to adhere, but did not decrease PC-3 cell adhesion. Nevertheless, expression levels were reduced for most integrins tested, except the expression of alpha-5, for which no significant effect was measured. Our results point to a possible role of TKIs as suppressors of prostate carcinoma cell adhesion to extracellular matrix components, by acting as inhibitors of integrin expression.

The invasive behaviour of prostatic cancer cells is suppressed by inhibitors of tyrosine kinase.

Proteolytic enzymes, and especially urokinase plasminogen activator (uPA), play an important role in tumour invasion and metastasis. Previously we demonstrated that the production of urokinase plasminogen activator (uPA) was decreased by several tyrosine kinase inhibitors (TKI) in two prostatic carcinoma cell lines. The effect of the two TKI genistein and tyrphostin AG-1478 was investigated in the prostate carcinoma cell lines PC-3 and DU-145. A reconstituted basal lamina (Matrigel) was used as a migration barrier. The production of matrix metalloproteinases (MMP) was also measured. Roles of plasminogen and uPA were examined. Cell invasion was increased by plasminogen, but this enhanced cell migration was counteracted by TKI treatment. The increased cell invasion induced by plasminogen was decreased by at least 60% in both cell lines when alpha-2 anti-plasmin was added to the assay. Cells in the absence of plasminogen were not affected by TKI. External uPA failed to regenerate the decreased cell invasion caused by TKI. The production of MMP was inhibited by both TKI. Our results indicate a possible role of TKI as inhibitors of cancer cell invasion by inhibiting uPA and MMP production.

Inhibitors of tyrosine kinase inhibit the production of urokinase plasminogen activator in human prostatic cancer cells.

Urokinase-type plasminogen activator (uPA) seems to be an important protease in prostate cancer invasion, and tyrosine phosphorylation is thought to play a role in the regulation of its production. The amount of uPA was measured with a synthetic peptide substrate after treatment with various concentrations of tyrosine kinase inhibitors (TKI). The effect on proliferation and apoptosis was also assayed. Non-toxic levels of genistein or the tyrphostin AG 490 produced up to 50% reduction of the uPA production in PC-3 and DU-145. The tyrphostins AG 1296 and AG 1478 inhibited uPA production in PC-3 cells, whereas DU-145 showed a slight increase of uPA production. TKI neither induced any detectable apoptosis, nor was there any reduction in proliferation rate. TKI can profoundly modify the production of uPA in prostatic cancer cells, thus indicating their possible use as suppressors of the invasive phenotype. The therapeutic potential of TKI warrants further investigation.