Comparative analysis of prostatic acid phosphatase and prostate-specific antigen mRNA levels in hyperplastic prostate stimulated with steroid hormones and growth factors

Prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) are the markers of human prostatic gland. However, it is still not completely understood if and how, steroid hormones and growth factors affect their expression and metabolism in the respect to the major pathologies of the gland. Appropriate studies were carried out on histopathologically diagnosed benign prostatic hyperplasia - BPH (n = 42) using tissue slices and cells derived from them. They were incubated with steroid hormones: 5-α-dihydrotestosterone (DHT), estradiol (E) and growth factors: epidermal growth factor (EGF), basic fibroblastic growth factor (bFGF) under culture conditions for up to 24 hours. 32P-labelled specific oligonucleotide probes were used to analyze total RNA isolated from each sample for the presence of PAP and PSA mRNAs. DHT, E, bFGF, EGF or both DHT + bFGF and DHT + EGF increased PAP and PSA mRNA levels in a time- and dose-dependent manner. The highest and statistically significant increase (P <0.001) for PAP mRNA was observed when DHT + bFGF were present in the medium while for PSA mRNA if DHT + EGF were added to the medium. Slow but constant decrease of PAP and PSA mRNA levels was observed in the absence of each of these factors in the incubation medium. The results suggest that early expression of PSA and PAP genes and/or their mRNA stability strongly depend on DHT while differ in their response to EGF and bFGF

Increased Akt signaling resulting from the loss of androgen responsiveness in prostate cancer

The mechanisms responsible for the switch of prostate cancer from androgen-sensitive (AS) to androgen-insensitive (AI) form are not well understood. Regulation of androgen receptor (AR), through which androgens control the expression of genes involved in prostate cells proliferation, migration and death also involves its cross-talk with the other signaling pathways, transcription factors and coregulatory proteins, such as β-catenin. With the aim to determine their possible contribution in triggering the switch from AS to AI form, which occurs upon androgen deprivation therapy - AR, Akt and β-catenin expression were knocked-down with respective siRNAs. Treatment of LNCaP prostate cells with siRNA for AR significantly reduced their proliferation (45-70%), expression of nuclear β- catenin, cyclin-D1, cyclin-G1, c-Myc as well as activity of metalloproteinases (MMPs) -2,-7,-9 and cell migration. Surprisingly, after longer (over 72 hrs) silencing of AR in LNCaP cells, elevated levels of p-Akt were detected and enhanced proliferation as well as expression of nuclear β-catenin, cyclin-D1, c-Myc and activity of MMPs were observed. Such effects were not observed in either PC-3 or DU145 AI cells. However, silencing of Akt and /or β-catenin in those as well as in LNCaP cells led to their decreased proliferation and migration. Our findings suggest that in prostate cancer cells, either AR or Akt signaling prevails, depending on their initial androgen sensitivity and its availability. In AI prostate cancer cells, Akt takes over the role of AR and more effectively contributes through the same signaling molecule, β-catenin, to AI cancer progression

Could the kinetin riboside be used to inhibit human prostate cell epithelial-mesenchymal transition?

The epithelial-mesenchymal transition (EMT) is a molecular process connected to higher expression of vimentin and increased activity of transcription factors (Snail, Twist) which restrains E-cadherin. EMT has been linked to prostate cancer metastatic potential, therapy resistance, and poor outcomes. Kinetin riboside (9-(b-dribofuranosyl)-6-furfurylaminopurine, KR) is a naturally occurring cytokinin, which induces apoptosis and shows strong antiproliferative activity against various human cancer cell lines. To establish the effect of KR on human prostate cell lines, expression of, e.g. AR, E-, N-cadherins, Vimentin, Snail, Twist, and MMPs, was analysed at mRNA and protein levels using Western Blot and RT-PCR and/or RQPCR techniques. KR inhibited the growth of human prostate cancer cells, but also, to a small extent, of normal cells. This effect depended on the type of the cells and their androgen sensitivity. KR also decreased the level of p-Akt, which takes part in androgen signalling modulation. The antiapoptotic Bcl-2 protein was down-regulated in cancer cell lines, while that of Bax is up-regulated upon KR exposure. KR contributed to re-expression of the E-cadherin as well as to significant changes in cell migration. Taken together, our results indicate for the first time that KR can be proposed as a factor for signalling pathways regulation that participates in the inhibition of development of aggressive forms of prostate cancer, and may alter the approach to therapeutic interventions. We propose KR as a potent inhibitor of EMT in human prostate cells

Integrin-linked kinase regulates cadherin switch in bladder cancer

Cadherin switch is specific of epithelial-mesenchymal transition (EMT) and is closely related to tumor cell invasion. However, the molecular mechanism that promotes the phenotypic changes remains unclear and elusive. We found that integrin-linked kinase (ILK) is a key factor involved in cadherin switch. The expression and activity of ILK are elevated in a variety of cancers but its mechanisms are not exactly understood. In this report, we studied the role and mechanism of ILK in EMT of human bladder cancer. We showed that silencing of ILK expression by small interfering RNA (siRNA) significantly abolished the nuclear translocation or the presence of markers associated with EMT like Snail, Twist, Zeb, and beta-catenin. ILK knockdown by siRNA suppressed N-cadherin expression and increased re-expression of E-cadherin in bladder cancer cells. We suggest that ILK is a major signaling factor involved in EMT. It is essential to understand the molecular mechanism of EMT in aim to possibly use it in search for new therapeutic targets. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s13277-016-5354-x) contains supplementary material, which is available to authorized users

Szybka metoda oceny stężenia witaminy A, witaminy E oraz kotyniny w surowicy krwi kobiet z śródnabłonkową neoplazją (CIN)i rakiem szyjki macicy

Abstract Objective: The aim of this study was to elaborate on the analytical method for quantitative determination of retinol and α-tocopherol in serum of women diagnosed with CIN and cervical cancer. The basic problem in the analysis of the vitamins content in biological material is their low physiological concentration level and instability. Liquid chromatography with diode array detector (DAD) was applied. Material and methods: The material consisted of serum and urine collected from 12 women diagnosed with cervical intraepithelial neoplasia (CIN) and 16 diagnosed with cervical cancer. The method was evaluated for the following parameters: linearity, recovery, sensitivity, precision, accuracy, selectivity, stability, limit of quantification (LOQ) and limit of detection (LOD). Results: Results showed good linearity (r2 ≥0,99) in the range 0,1μg/ml-10mg/ml for retinol and 0,25μg/ml-15μg/ml for α-tocopherol. The Lower Limit of Detection was 0,15μg/ml for vitamin E and 0,05μg/ml for vitamin A. The within-run R.S.Ds were below 5,2% at all concentration levels and the between-run R.S.Ds were below 10,0% at all concentration levels. Conclusions: The advantage of this method is that it measures both compounds in a more rapid, reproducible and accurate manner when compared to the previous HPLC studies. The compounds (vitamin A and E and internal standards) are measured in the same sample at the same time. Quantitative determination of cotinine may reveal active smokers and subjects exposed to environmental tobacco smoke, which is independent measurable carcinogenetic co-factor. The following study is a part of a project determining non-viral causative agents in cervical carcinogenesis.Streszczenie Cel pracy: Celem niniejszej pracy jest ocena przydatności analitycznej metody oznaczania ilościowego retinolu i alfa-tokoferolu w surowicy kobiet z rozpoznaną śródnabłonkową neoplazją (CIN) i rakiem szyjki macicy. Głównym problemem w analizowaniu zawartości witamin w materiale biologicznym jest ich niskie fizjologiczne stężenie i niestabilność. W niniejszym badaniu zastosowano metodę ciekłej chromatografii z detektorem diodowym (DAD). Materiał i metodyka: Materiał badawczy stanowiło osocze i mocz, 12 kobiet z rozpoznaną śródnabłonkową neoplazją szyjki macicy (CIN) i 16 z rakiem szyjki macicy. Metodę oceniono pod kątem następujących parametrów: liniowość, powtarzalność, czułość, precyzja, dokładność, selektywność, stabilność, granica kwantyfikacji (limit of quantification - LOQ) i detekcji (limit of detection - LOD). Wyniki: Uzyskano wysoka liniowość (r2 ≥0,99) w zasięgu 0,1μg/ml-10mg/ml dla retinolu i 0,25μg/ml-15μg/ml dla alfa-tokoferolu. Dolna granica wykrywalności wyniosła 0,15μg/ml dla witaminy E i 0,05μg/ml dla witaminy A. Zasięg metody R.S.Ds wyniósł 5,2% we wszystkich poziomach stężeń a interwałowy zasięg R.S.Ds poniżej 10,0% we wszystkich poziomach stężeń. Wnioski: Zaleta zbadanej metody jest fakt, iż można przy jej pomocy uzyskać wyniki w szybszy, bardziej powtarzalny i dokładniejszy sposób, niż w opisywanych w literaturze badaniach z użyciem HPLC. Dodatkową zaleta stosowanej metody jest możliwość pomiaru z tej samej próbki witamin A i E. Ilościowa ocena stężenia kotyniny może wskazać pacjentki, będące aktywnymi palaczkami lub narażonymi na palenie bierne. W aspekcie karcinogenezy w obrębie szyjki macicy może być to cenna wiadomość dotycząca istotnego a często zatajanego kofaktora nowotworzenia. Niniejsze badanie jest częścią projektu oceniającego pozawirusowe czynniki onkogenne w procesie karcinogenezy w obr´bie szyjki macicy

Aberrant promoter methylation may be responsible for the control of CD146 (MCAM) gene expression during breast cancer progression

The CD146 (also known as MCAM, MUC-18, Mel-CAM) was initially reported on in 1987, as a protein crucial for melanoma invasion. Recently, it has been confirmed that CD146 is involved in progression and poor overall survival of many other cancers, including breast cancer. Importantly, in independent studies, CD146 was reported to be a trigger of epithelial to mesenchymal transition in breast cancer cells. The goal of our current study was to verify possible involvement of an epigenetic mechanism behind regulation of the CD146 expression in breast cancer cells, as it has been previously reported for prostate cancer. First, we analysed the response of breast cancer cells, varying in the initial CD146 mRNA and protein content, to an epigenetic modifier, 5-aza-2-deoxycytidine, and subsequently the methylation status of CD146 gene promoter was investigated, using direct bisulfite sequencing. We observed that treatment with a demethylating agent led to induction of CD146 expression in all analysed breast cancer cell lines, both at the mRNA and protein levels, which was accompanied by an elevated expression of selected mesenchymal markers. Importantly, CD146 gene promoter analysis showed aberrant CpG island methylation in 2 out of 3 studied breast cancer cells lines, indicating epigenetic regulation of the CD146 gene expression. In conclusion, our study revealed for the first time that aberrant methylation may be involved in expression control of CD146, a very potent EMT inducer in breast cancer cells. Altogether, the data obtained may provide basis for novel therapies, as well as diagnostic approaches enabling sensitive and very accurate detection of breast cancer cells.

Dysregulation of transcription factor activity during formation of cancer-associated fibroblasts

The reciprocal interactions between cancer cells and the quiescent fibroblasts leading to the activation of cancer-associated fibroblasts (CAFs) serve an important role in cancer progression. Here, we investigated the activation of transcription factors (TFs) in prostate fibroblasts (WPMY cell line) co-cultured with normal prostate or tumorous cells (RWPE1 and RWPE2 cell lines, respectively). After indirect co-cultures, we performed mRNA-seq and predicted TF activity using mRNA expression profiles with the Systems EPigenomics Inference of Regulatory Activity (SEPIRA) package and the GTEx and mRNA-seq data of 483 cultured fibroblasts. The initial differential expression analysis between time points and experimental conditions showed that co-culture with normal epithelial cells mainly promotes an inflammatory response in fibroblasts, whereas with the cancerous epithelial, it stimulates transformation by changing the expression of the genes associated with microfilaments. TF activity analysis revealed only one positively regulated TF in the RWPE1 co-culture alone, while we observed dysregulation of 45 TFs (7 decreased activity and 38 increased activity) uniquely in co-culture with RWPE2. Pathway analysis showed that these 45 dysregulated TFs in fibroblasts co-cultured with RWPE2 cells may be associated with the RUNX1 and PTEN pathways. Moreover, we showed that observed dysregulation could be associated with FER1L4 expression. We conclude that phenotypic changes in fibroblast responses to co-culturing with cancer epithelium result from orchestrated dysregulation of signaling pathways that favor their transformation and motility rather than proinflammatory status. This dysregulation can be observed both at the TF and transcriptome levels