The Clinical Significance Of Circulating Mir-21, Mir-142, Mir-143, And Mir-146a In Patients With Prostate Cancer

Prostate cancer (PCa) is the most common type of solid tissue cancer among men in western countries. To enlighten the underlying molecular mechanisms in the pathophysiology of PCa and to help the development of new diagnostics and treatment models, we planned to determine the levels of circulating miR-21, miR-142, miR-143, miR-146a, and RNU 44 levels as controls for the early diagnosis of PCa. The circulating miRNA levels in peripheral blood samples from 43 localized PCa patients, 12 metastatic PCa (MET) patients, and as a control group, 42 benign prostate hyperplasia (BPH) patients with a total of 97 volunteers were determined the by PCR method. No differences in the ΔCT values were found among the groups. In PCa and PCaMet groups the expression of miR21 and miR142 were higher compared to the BHP group. No other differences were observed among the other groups. miR21 expression in the PCa group was 6.29 folds upregulated whereas in the PCaMet group 10.84 folds upregulated. When the total expression of miR142 is evaluated, it showed a positive correlation with mir21 and mir 146 (both p<0.001). Also, the expression of miR146 shows a positive correlation with both miR21 and miR143 (both p<0.001). Expression of miRNAs was found to be an independent diagnostic factor in patients with Gleason score, PSA, and free PSA levels. In conclusion, our study showed that co-expression of miR-21, miR-142, miR-143, and miR-146a and the upregulation of miR-21 resulted in increased prostate carcinoma cell growth. In the PCaMet group, miR21 is the most upregulated of all miRNAs. These miRNA markers that are expressed in different levels may provide a novel diagnostic tool to help diagnose PCa with aggressive behavior.

Functional and mutational analysis after radiation and cetuximab treatment on prostate carcinoma cell line DU145

Background Epidermal Growth Factor Receptor is often overexpressed in advanced prostate carcinoma. In-vitro-studies in prostate carcinoma cell line DU145 have demonstrated increased sensibility to radiation after cetuximab treatment, but clinical data are not sufficient to date. Methods We analyzed effects of radiation and cetuximab in DU145 and A431 using proliferation, colony-forming-unit- and annexin-V-apoptosis-assays. Changes in protein expression of pEGFR and pERK1/2 after radiation and cetuximab treatment were analyzed. Using NGS we also investigated the impact of cetuximab long-term treatment. Results Cell counts in DU145 were reduced by 44% after 4 Gy (p = 0.006) and 55% after 4 Gy and cetuximab (p < 0.001). The surviving fraction (SF) was 0.69 after 2 Gy, 0.41 after 4 Gy and 0.15 after 6 Gy (each p < 0.001). Cetuximab treatment did not alter significantly growth reduction in 4 Gy radiated DU145 cells, p > 0.05 or SF, p > 0.05, but minor effects on apoptotic cell fraction in DU145 were detected. Using western blot, there were no detectable pEGFR and pERK1/2 protein signals after cetuximab treatment. No RAS mutation or HER2 amplification was detected, however a TP53 gen-mutation c.820G > T was found. Conclusions Radiation inhibits cell-proliferation and colony-growth and induces apoptosis in DU145. Despite blocking MAP-Kinase-pathway using cetuximab, no significant radiation-sensitizing-effect was detected. Cetuximab treatment did not induce resistance-mutations. Further research must clarify which combination of anti-EGFR treatment strategies can increase radiation-sensitizing-effects.

TPX2 Enhanced the Activation of the HGF/ETS-1 Pathway and Increased the Invasion of Endocrine-Independent Prostate Carcinoma Cells

The prognosis for endocrine-independent prostate carcinoma is still poor due to its highly metastatic feature. In the present work, TPX2 (the targeting protein for Xklp2), which is known as a micro-tubulin interacted protein, was identified as a novel coactivator of ETS-1, a transcription factor that plays a central role in mediating the metastasis of human malignancies. TPX2 enhanced the transcription factor activation of ETS-1 and increased the expression of ETS-1’s downstream metastasis-related genes, such as mmp3 or mmp9, induced by HGF (hepatocyte growth factor), a typical agonist of the HGF/c-MET/ETS-1 pathway. The protein-interaction between TPX2 and ETS-1 was examined using immunoprecipitation (IP). TPX2 enhanced the accumulation of ETS-1 in the nuclear and the recruitment of its binding element (EST binding site, EBS) located in the promoter region of its downstream gene, mmp9. Moreover, TPX2 enhanced the in vitro or in vivo invasion of a typical endocrine-independent prostate carcinoma cell line, PC-3. Therefore, TPX2 enhanced the activation of the HGF/ETS-1 pathway to enhance the invasion of endocrine-independent prostate carcinoma cells and thus it would be a promising target for prostate carcinoma treatment.

Radio-thermo-sensitivity Induced by Gold Magnetic Nanoparticles in the Monolayer Culture of Human Prostate Carcinoma Cell Line DU145

Background and Objective: Prostate cancer is the second cause of death in men worldwide. In this study, the cytotoxic effects of PLGA polymer-coated gold Magnetic Nanoparticles (MGNPs), as a novel treatment to enhance radiation and thermal sensitivity in the presence of hyperthermia (43°C) and electron beam, on DU145 prostate cancer cells were investigated. Methods: Nanoparticles were characterized using TEM, DLS, XRD and SAED methods. MGNPs entrance into the cells was determined using Prussian blue staining and TEM. Furthermore, the cytotoxic effects of combinatorial treatment modalities were assessed by applying colony and sphere formation assay. Results: Our results revealed that the decrease of colony and sphere numbers after combinatorial treatment of hyperthermia and radiation in the presence of nanoparticles was significantly higher than the other treatment groups (P<0.05). This treatment method proved that it has the capability of eliminating most of the DU145 cells (80-100%), and increased the value of the linear parameter (α) to 4.86 times. Conclusion: According to the study, magnetic gold nanoparticles, in addition to having a high atomic number, can effectively transmit heat produced inside them to the adjacent regions under hyperthermia, which increases the effects of radio-thermosensitivity, respectively.

Synergistic In-Vitro Effects through Radiation and Blocking the Epidermal Growth Factor Receptor (EGFR) with the Monoclonal Antibody Cetuximab in Prostate Carcinoma Cell Line DU145

Background: The EGF-receptor is often overexpressed in advanced prostate carcinoma. In-vitro studies in prostate carcinoma cell-line DU145 demonstrated increased sensibilities to radiation with Cetuximab; in-vivo effects were not detected. Methods: In vitro, we analyzed the effect of radiation and Cetuximab in cell-lines DU145 and A431 (reference), using a proliferation assay, colony-forming unit assay, and Annexin-V apoptosis assay. We analyzed changes in the protein expression of pEGFR and pERK1/2 post-radiation and Cetuximab. Additionally, we investigated the impact of Cetuximab long-term treatment on the development of secondary-resistance-mutations. Results: DU145 cell counts were reduced by 44% after 4Gy (p=0.006) and by 55% after 4Gy and Cetuximab (p<0.001). The surviving fraction was 0.69 after 2Gy; 0.41, 4Gy; and 0.15, 6Gy (p<0,001). The additional Cetuximab-treatment did not significantly alter the impact on growth reduction or on the surviving fraction. After radiation and Cetuximab-treatment minor effects on the apoptotic cell-fraction in DU145 were detected. Using western blot, there were no pEGFR and pERK1/2 protein signals after Cetuximab-treatment. While no mutations of RAS, BRAF, PI3KCA and no amplifications of HER2 were detected, there were a TP53 mutation before and after long-term treatment with Cetuximab. Conclusion: Radiation inhibits cell-proliferation and colony-growth and induces apoptosis in DU145. Despite blocking EGFR-MAP-Kinase pathway with Cetuximab, no significant radiation-sensitizing-effect was detected. Cetuximab-treatment did not cause typical resistance mutations in DU145. Further research must clarify whether a combination of anti-EGFR therapeutics and immune-oncological approaches can increase the radiation-sensitizing-effect.

Isoquinolinamine FX-9 Exhibits Anti-Mitotic Activity in Human and Canine Prostate Carcinoma Cell Lines

Current therapies are insufficient for metastatic prostate cancer (PCa) in men and dogs. As human castrate-resistant PCa shares several characteristics with the canine disease, comparative evaluation of novel therapeutic agents is of considerable value for both species. Novel isoquinolinamine FX-9 exhibits antiproliferative activity in acute lymphoblastic leukemia cell lines but has not been tested yet on any solid neoplasia type. In this study, FX-9′s mediated effects were characterized on two human (PC-3, LNCaP) and two canine (CT1258, 0846) PCa cell lines, as well as benign solid tissue cells. FX-9 significantly inhibited cell viability and induced apoptosis with concentrations in the low micromolar range. Mediated effects were highly comparable between the PCa cell lines of both species, but less pronounced on non-malignant chondrocytes and fibroblasts. Interestingly, FX-9 exposure also leads to the formation and survival of enlarged multinucleated cells through mitotic slippage. Based on the results, FX-9 acts as an anti-mitotic agent with reduced cytotoxic activity in benign cells. The characterization of FX-9-induced effects on PCa cells provides a basis for in vivo studies with the potential of valuable transferable findings to the benefit of men and dogs.