Prostate Carcinoma Cell Line PC-3 Research Articles

The IGF-Independent Role of IRS-2 in the Secretion of MMP-9 Enhances the Growth of Prostate Carcinoma Cell Line PC3.

Insulin receptor substrate-2 (IRS-2), a substrate of the insulin-like growth factor (IGF)-I receptor, is highly expressed in the prostate cancer cell line, PC3. We recently demonstrated that extracellular signal-regulated kinase (Erk1/2), a kinase downstream of IGF signaling, is activated in PC3 cells under serum starvation, and this activation can be inhibited by IRS-2 knockdown. Here, we observed that adding an IGF-I-neutralizing antibody to the culture medium inhibited the activation of Erk1/2. Suppression of Erk1/2 in IRS-2 knockdown cells was restored by the addition of a PC3 serum-free conditioned medium. In contrast, the IRS-2-silenced PC3 conditioned medium could not restore Erk1/2 activation, suggesting that IRS-2 promotes the secretion of proteins that activate the IGF signaling pathway. Furthermore, gelatin zymography analysis of the conditioned medium showed that matrix metalloproteinase-9 (MMP-9) was secreted extracellularly in an IRS-2 dependent manner when PC3 was cultured under serum starvation conditions. Moreover, MMP-9 knockdown suppressed Erk1/2 activation, DNA synthesis, and migratory activity. The IRS-2 levels were positively correlated with Gleason grade in human prostate cancer tissues. These data suggest that highly expressed IRS-2 activates IGF signaling by enabling the secretion of MMP-9, which is associated with hyperproliferation and malignancy of prostate cancer cell line, PC3.

Development of new TAK-285 derivatives as potent EGFR/HER2 inhibitors possessing antiproliferative effects against 22RV1 and PC3 prostate carcinoma cell lines

Epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) protein tyrosine kinases co-expressed in various cancers such as ovarian, breast, colon, and prostate subtypes. Herein, new TAK-285 derivatives (9a–h) were synthesised, characterised, and biologically evaluated as dual EGFR/HER2 inhibitors. Compound 9f exhibited IC50 values of 2.3 nM over EGFR and 234 nM over HER2, which is 38-fold of staurosporine and 10-fold of TAK-285 over EGFR. Compound 9f also showed high selectivity profile when tested over a small kinase panel. Compounds 9a–h showed IC50 values in the range of 1.0–7.3 nM and 0.8–2.8 nM against PC3 and 22RV1 prostate carcinoma cell lines, respectively. Cell cycle analysis, apoptotic induction, molecular docking, dynamics, and MM-GBSA studies confirmed the plausible mechanism(s) of compound 9f as a potent EGFR/HER2 dual inhibitor with an effective antiproliferative action against prostate carcinoma.

Single vesicle analysis of CD47 association with integrins and tetraspanins on extracellular vesicles released by T lymphoblast and prostate carcinoma cells.

CD47 regulates the trafficking of specific coding and noncoding RNAs into extracellular vesicles (EVs), and the RNA contents of CD47+ EVs differ from that of CD63+ EVs released by the same cells. Single particle interferometric reflectance imaging sensing combined with immunofluorescent imaging was used to analyse the colocalization of tetraspanins, integrins, and CD47 on EVs produced by wild type and CD47‐deficient Jurkat T lymphoblast and PC3 prostate carcinoma cell lines. On Jurkat cell‐derived EVs, β1 and α4 integrin subunits colocalized predominantly with CD47 and CD81 but not with CD63 and CD9, conserving the known lateral interactions between these proteins in the plasma membrane. Although PC3 cell‐derived EVs lacked detectable α4 integrin, specific association of CD81 with β1 and CD47 was preserved. Loss of CD47 expression in Jurkat cells significantly reduced β1 and α4 levels on EVs produced by these cells while elevating CD9+, CD63+, and CD81+ EVs. In contrast, loss of CD47 in PC3 cells decreased the abundance of CD63+ and CD81+ EVs. These data establish that CD47+ EVs are mostly distinct from EVs bearing the tetraspanins CD63 and CD9, but CD47 also indirectly regulates the abundance of EVs bearing these non‐interacting tetraspanins via mechanisms that remain to be determined.

Biological Synthesis of CdTe Quantum Dots and Their Anti-Proliferative Assessment Against Prostate Cancer Cell Line.

Quantum dots (QDs) are semiconducting materials which have a wide array of applications starting from semiconducting devices, in humidity and pressure sensors and in medical imaging including cancer therapy. In the present study, cadmium telluride (CdTe) QDs were synthesized by a biological method using yeast cells, Saccharomyces cerevisiae in modified Czapek's medium. QDs were characterized by transmission electron microscopy and X-ray diffraction. Cancer cells were treated with 2, 4, 8 and 16 μM concentrations of CdTe QDs for 24 h. The anti-proliferative activity was determined by using MTT assay, by evaluating the production of reactive oxygen species (ROS), and also by nuclear apoptosis and cell cycle analysis using a flow cytometer against human prostate carcinoma cell line PC-3. The size of the CdTe QDs was approximately 2 nm. In vitro anti-proliferative study showed that CdTe QDs induced cell death and nuclear apoptosis in a dosedependent manner. CdTe QDs induced significant increase in ROS level in PC-3 cells which was dose-dependent. Moreover, CdTe also arrested growth of PC-3 cells in the G2/M phase of the cell cycle. This study elucidates the apoptotic activity of CdTe QDs on prostate carcinoma which could provide useful insights to researchers for its clinical application.

Multilabel segmentation of cancer cell culture on vascular structures with deep neural networks.

.New increasingly complex in vitro cancer cell models are being developed. These new models seem to represent the cell behavior in vivo more accurately and have better physiological relevance than prior models. An efficient testing method for selecting the most optimal drug treatment does not exist to date. One proposed solution to the problem involves isolation of cancer cells from the patients’ cancer tissue, after which they are exposed to potential drugs alone or in combinations to find the most optimal medication. To achieve this goal, methods that can efficiently quantify and analyze changes in tested cell are needed. Our study aimed to detect and segment cells and structures from cancer cell cultures grown on vascular structures in phase-contrast microscope images using U-Net neural networks to enable future drug efficacy assessments. We cultivated prostate carcinoma cell lines PC3 and LNCaP on the top of a matrix containing vascular structures. The cells were imaged with a Cell-IQ phase-contrast microscope. Automatic analysis of microscope images could assess the efficacy of tested drugs. The dataset included 36 RGB images and ground-truth segmentations with mutually not exclusive classes. The used method could distinguish vascular structures, cells, spheroids, and cell matter around spheroids in the test images. Some invasive spikes were also detected, but the method could not distinguish the invasive cells in the test images.

Modelling the internalisation process of prostate cancer cells for PSMA-specific ligands

IntroductionIn prostate-specific membrane antigen (PSMA)-targeting radioligand therapy, small molecules are regularly internalised by the tumour cells. To determine the effectiveness of these ligands, the internalised fraction over time is derived from cell studies. Parameters, such as the ligand concentration and the number of cells, are experiment-specific and therefore a comparison between ligands is difficult. A more objective approach that allows better comparison is desirable. Therefore, the aim of this work was to develop a compartmental model that fully describes all relevant pharmacokinetic interactions of PSMA-specific ligands with prostate cancer cells. MethodsInternalisation studies were performed using the lymph node carcinoma of the prostate cell line LNCaP C4-2 and the prostatic carcinoma cell line PC-3. A new protocol was established for the determination of the PSMA-binding specificity by surface plasmon resonance (SPR). The experimental data in combination with parameters from literature were used for the modelling approach. ResultsA compartmental model which includes the relevant physiological mechanisms was developed. The basic model structure and some parameters originate from the literature. The PSMA-specific association and dissociation rates of Ga-PSMA-11 were measured using surface plasmon resonance technology. The ligand-induced internalisation and PSMA synthesis rates were estimated by fitting the developed model to experimental data obtained using LNCaP C4-2 cells. For all [68Ga]Ga-PSMA-11 concentrations and including four various incubation times, the ligand-induced internalisation was determined to be (3.6 ± 0.1) % min−1. ConclusionsThe presented approach is a prerequisite for better estimation and thus comparison of important ligand-cell interaction parameters, by combining SPR measurements, cell experiments and mathematical modelling. Advances in Knowledge and Implications for patientA compartmental model was developed for evaluation and comparison of PSMA-binding small molecules. A SPR protocol was established for the determination of PSMA-binding specificity.

Histone Deacetylase Inhibitor Modulates NKG2D Receptor Expression and Memory Phenotype of Human Gamma/Delta T Cells Upon Interaction With Tumor Cells.

The functional plasticity and anti-tumor potential of human γδ T cells have been widely studied. However, the epigenetic regulation of γδ T-cell/tumor cell interactions has been poorly investigated. In the present study, we show that treatment with the histone deacetylase inhibitor Valproic acid (VPA) significantly enhanced the expression and/or release of the NKG2D ligands MICA, MICB and ULBP-2, but not ULBP-1 in the pancreatic carcinoma cell line Panc89 and the prostate carcinoma cell line PC-3. Under in vitro tumor co-culture conditions, the expression of full length and the truncated form of the NKG2D receptor in γδ T cells was significantly downregulated. Furthermore, using a newly established flow cytometry-based method to analyze histone acetylation (H3K9ac) in γδ T cells, we showed constitutive H3K9aclow and inducible H3K9achigh expression in Vδ2 T cells. The detailed analysis of H3K9aclow Vδ2 T cells revealed a significant reversion of TEMRA to TEM phenotype during in vitro co-culture with pancreatic ductal adenocarcinoma cells. Our study uncovers novel mechanisms of how epigenetic modifiers modulate γδ T-cell differentiation during interaction with tumor cells. This information is important when considering combination therapy of VPA with the γδ T-cell-based immunotherapy for the treatment of certain types of cancer.

Tumor-Specific S/G2-Phase Cell Cycle Arrest of Cancer Cells by Methionine Restriction.

Cancer cells require elevated amounts of methionine (MET) and arrest their growth under conditions of MET restriction (MR). This phenomenon is termed MET dependence. Fluorescence-activated cell sorting (FACS) first indicated that the MET-dependent SV40-transformed cancer cells were arrested in the S and G2 phases of the cell cycle when under MR. This is in contrast to a G1-phase accumulation of cells, which occurs only in MET-supplemented medium at very high cell densities and which is similar to the G1 cell-cycle block which occurs in cultures of normal fibroblasts at high density. When the human PC-3 prostate carcinoma cell line was cultured in MET-free, homocysteine-containing (MET-HCY+) medium, there was an extreme increment in DNA content without cell division indicating that the cells were blocked in S phase. Recombinant methioninase (rMETase) treatment of cancer cells also selectively trapped cancer cells in S/G2: The cell cycle phase of the cancer cells was visualized with the fluorescence ubiquitination cell cycle indicator (FUCCI). At the time of rMETase-induced S/G2-phase trap, identified by the cancer cells' green fluorescence by FUCCI imaging, the cancer cells were administered S-phase-dependent chemotherapy drugs, which interact with DNA or block DNA synthesis such as doxorubicin, cisplatin, or 5-fluorouracil (5-FU) and which were highly effective in killing the cancer cells. In contrast, treatment of cancer cells with drugs in the presence of MET, only led to the majority of the cancer cell population being blocked in G0/G1 phase, identified by the cancer cells becoming red fluorescent in the FUCCI system. The G0/G1 blocked cells were resistant to the chemotherapy. MR has the potential for highly effective cell-cycle-based treatment strategy for cancer in the clinic.

The Role of microRNA-15A in the Development of Prostate Cancer – Effects on Cell Proliferation and Pro-Inflammatory Signalling

Abstract Worldwide prostate cancer is the second leading cause of cancer death among men after lung cancer. MicroRNAs are non-coding, endogenous RNAs and they play a role in tumorigenesis, RNA silencing and post-transcriptional regulation of gene expression. In this study we have investigated microRNA-15a impact on transcription factors cMYB and ETS1 in prostate-carcinoma cell line PC3. The PC3 cells were transfected with a synthetic analogue and inhibitor of microRNA-15a. The study was performed using reverse transcription polymerase chain reaction and flow cytometry methods for assessing the transcript and protein levels of cMYB and ETS1, NFκB stable reporter live cell line. Statistical analysis was performed using One–way ANOVA test. We found that cMYB and ETS1 are up-regulated by the synthetic analogue of microRNA-15a at the transcription and protein level. Transfection with microRNA-15a mimic resulted in NFκB transcription factor activation as found by using the live cell reporter system. There was some opportunistic activity exhibited by the synthetic inhibitor, but less pronounced. Our data suggest that microRNA-15a could participate in prostate cancer progression by modulating cell proliferation and pro-inflammatory signaling and paves a way for further in-depth investigation of the gene regulatory networks underneath.

Oxazoline derivatives of [17(20)E]-21-norpregnene – inhibitors of CYP17A1 activity and proliferation of prostate carcinoma cells

Nine new oxazolinyl derivatives of [17(20)E]-21-norpregnene, differing in the structure of steroid moiety, were investigated for their potency to inhibit the CYP17A1 catalytic activity, as well as growth and proliferation of LNCaP and PC-3 prostate carcinoma cell lines. The activity of one of the investigated compounds, 2´-{[(E)3β-hydroxyandrost-5-en-17-ylidene]methyl}-4´,5´-dihydro-1´,3´-oxazole (1), was found to be comparable with that of Abiraterone, a known inhibitor of CYP17A1 used for treatment of prostate cancer. A model of the interaction of oxazoline 1 with the active site of CYP17A1 was constructed by the molecular dynamics method. A correlation was found between the structure and the biological activity in the series of [17(20)E]-21-norpregnene oxazolinyl derivatives.

Induction of Oxidants Distinguishes Susceptibility of Prostate Carcinoma Cell Lines to p53 Gene Transfer Mediated by an Improved Adenoviral Vector.

Previously, the authors developed an adenoviral vector, Ad-PG, where transgene expression is regulated by a p53-responsive promoter. When used to transfer the p53 cDNA, a positive feedback mechanism is established. In the present study, a critical comparison is performed between Ad-PGp53 and AdRGD-PGp53, where the RGD motif was incorporated in the adenoviral fiber protein. AdRGD-PGp53 provided superior transgene expression levels and resulted in the killing of prostate carcinoma cell lines DU145 and PC3. In vitro, this effect was associated with increased production of cytoplasmic and mitochondrial oxidants, DNA damage as revealed by detection of phosphorylated H2AX, as well as cell death consistent with apoptosis. Differential gene expression of key mediators of reactive oxygen species pathways was also observed. Specifically, it was noted that induction of known p53-target genes Sestrin2 and PIG3, as well as a novel target, NOX1, occurred in PC3 cells only when transduced with the improved vector, AdRGD-PGp53. The participation of NOX1 was confirmed upon its inhibition using a specific peptide, resulting in reduced cell death. In situ gene therapy also resulted in significantly improved inhibition of tumor progression consistent with oxidant-induced DNA damage only when treated with the novel AdRGD-PGp53 vector. The study shows that the improved adenovirus overcomes limitations associated with other p53-expressing vectors and induces oxidant-mediating killing, thus supporting its further development for cancer gene therapy.

AB158. Atorvastatin induces autophagic cell death in prostate cancer cells in vitro

ObjectiveAlthough it is well known that apoptosis contributes to cancer cell death, the role of autophagy in cancer cell death has remained in dispute. Atorvastatin has been suggested to exhibit anti-cancer effects. The present study aimed to examine atorvastatin-induced autophagy-associated cell death and the autophagy-associated gene expression profile in the PC3 prostate carcinoma cell line.MethodsThe atorvastatin-induced process of autophagy in PC3 cells was determined via evaluation of the cellular expression levels of autophagosomal marker light-chain-3 (LC3)-II, using immunoblotting and counting of green fluorescent protein (GFP)-LC3-transfected autophagiccells. Apoptosis was examined by terminal deoxynucleotidyl transferase dUTP nick end labeling assay and an MTT assay was used to evaluate cell viability. Total RNA of PC3 cells was isolated for characterization of the gene expression profile following atorvastatin treatment.ResultsAtorvastatin treatment of PC3 cells for 24 h increased the expression of GFP-LC3-II by >25% and expression continued for >72 h, while apoptosis was not significantly induced within this time period. Four genes associated with the autophagy machinery were also significantly upregulated.ConclusionsIn the presence of atorvastatin, autophagy may be unable to abrogate cell damage and may therefore contribute to cellular dysfunction, leading to autophagic/type II programmed cell death. In response to atorvastatin treatment, the expression of genes involved in autophagic mediating pathways may have a role in tumor suppression.

Abstract 3304: Role of Vascular Endothelial Growth Factor C in promoting radioresistance of prostate cancer

Abstract Radiotherapy is an important treatment regimen for prostate cancer. We have shown that the lymphangiogenic growth factor, Vascular Endothelial Growth Factor C (VEGF-C), induces docetaxel resistance and autophagy in prostate cancer (Muders et al., Cancer Res 2009 and Stanton et al., Cancer Res 2013). In this study we have evaluated the role of VEGF-C and VEGF-C induced autophagy in radioresistance of prostate cancer using cell culture experiments and human tissue samples. The human prostate carcinoma cell lines PC-3, LNCaP and DU145 were used in these cell culture experiments. Clonogenic survival of prostate cancer cells, after RNA interference (RNAi) of VEGF-C, autophagy related gene 5 (ATG5), and after addition of recombinant human VEGF-C following irradiation therapy, was evaluated. Autophagic flux was tested after ionizing irradiation using a Light-Chain-3 II immunoblot. To confirm our in vitro findings, human tissues of patients who underwent radical prostatectomy and adjuvant radiotherapy at Mayo Clinic were evaluated for VEGF-C expression. The quantity and intensity of VEGF-C staining was evaluated by two pathologists independently and correlated with biochemical relapse free survival (BRFS). The follow-up time is up to 23 years after radiotherapy. Multivariate Cox analysis was performed. VEGF-C levels correlated with a significantly higher radioresistance in colony formation assays and human tissue samples by multivariate analysis. The risk of biochemical recurrence increased 2.8 fold (95% confidence interval: 1.109 to 7.237; p = 0.03) when VEGF-C was highly expressed in prostatectomy patients with adjuvant radiotherapy. In line with our studies on autophagy in VEGF-C mediated docetaxel resistance of prostate cancer, autophagic flux was reduced after VEGF-C depletion during radiation. Interestingly, RNAi for ATG5 showed no effect on radiosensitivity which suggests that the decrease in autophagic flux after VEGF-C depletion is not responsible for the increased radiosensitivity. We conclude that VEGF-C is an important mediator of radiotherapy resistance in prostate cancer. In contrast to our published results on chemotherapy resistance, VEGF-C induced autophagy appears to be less important for resistance against ionizing radiation. Other VEGF-C dependent mechanisms for therapy resistance are under investigation. Citation Format: Steffi Haberlau, Pia Hönscheid, Rafael E. Jimenez, Gustavo B. Baretton, Donald J. Tindall, Mechthild Krause, Kaustubh Datta, Michael H. Muders. Role of Vascular Endothelial Growth Factor C in promoting radioresistance of prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3304. doi:10.1158/1538-7445.AM2015-3304

Atorvastatin induces autophagic cell death in prostate cancer cells in vitro

Athough it is well known that apoptosis contributes to cancer cell death, the role of autophagy in cancer cell death has remained in dispute. Atorvastatin has been suggested to exhibit anti-cancer effects. The present study aimed to examine atorvastatin-induced autophagy-associated cell death and the autophagy‑associated gene expression profile in the PC3prostate carcinoma cell line. The atorvastatin‑induced process of autophagy in PC3cells was determined via evaluation of the cellular expression levels of autophagosomal marker light-chain-3 (LC3)‑II, using immunoblotting and counting of green fluorescent protein (GFP)‑LC3-transfected autophagic cells. Apoptosis was examined by terminal deoxynucleotidyl transferase dUTP nick end labeling assay and an MTT assay was used to evaluate cell viability. Total RNA of PC3cells was isolated for characterization of the gene expression profile following atorvastatin treatment. Atorvastatin treatment of PC3 cells for 24h increased the expression of green fluorescent protein‑LC3‑II by >25%, and expression continued for >72h, while apoptosis was not significantly induced within this time period. Four genes associated with the autophagy machinery were also significantly upregulated. In the presence of atorvastatin, autophagy may be unable to abrogate cell damage and may therefore contribute to cellular dysfunction, leading to autophagic/typeII programmed cell death. In response to atorvastatin treatment, the expression of genes involved in autophagic mediating pathways may have a role in tumor suppression.

Inhibitive effects of 131I labeling prostate stem cell antigen monoclonal antibody on prostate carcinoma cell line LNCap and PC3

Objective To investigate the inhibitive effects of 131Ⅰ labeling prostate stem cell antigen monoclonal antibody (131 Ⅰ-PSCA-McAb) on prostate carcinoma cell line LNCap and PC3 in vitro.Methods Prostate carcinoma LNCap cells and PC3 cells were cultured in vitro,and each cell line was divided into 4 groups:experimental group (adding 131Ⅰ-PSCA-McAb 2 g/L),monoclonal control group (adding PSCA-McAb 2 g/L),iodic control group (adding 131I 2 g/L),blank control group (adding saline solution 2 g/L).Cell inhibition of proliferation in each group was tested by methyl thiazol tetrazolium (MTF) assay; tumor apoptosis in each group was measured by flow cytometry; and invasiveness of cells was detected by Transwell invasion test.Results The inhibitory rates of LNCap experimental group and PC3 exerimental group reached (83.67 ± 2.52)％ and (80.33 ± 4.51)％,which obviously higher than monoclonal control groups [(73.33 ± 3.51)％ and (66.67 ± 5.03)％,P＜ 0.05] and iodic control groups [(53.33 ±4.51) ％ and (56.33 ± 2.52) ％,P ＜ 0.05].The apoptosis rates of LNCap experimental group and PC3 exerimental group reached (50.24 ±5.52)％ and (54.71 ±5.84)％ respectively,which obviously higher than monoclonal control groups [(42.23 ± 4.87)％ and (45.61 ± 5.60)％,P ＜0.05],iodic control groups [(26.75 ± 3.43) ％ and (22.26 ± 3.71) ％,P ＜ 0.05] and blank control groups [(14.18 ± 3.97) ％ and (15.14 ± 3.28) ％,P ＜ 0.05].The number of cells invading the membrane in LNCap experimental group and PC3 exerimental group was 33.34 ± 5.82 and 34.91 ± 6.10 respectively,which obviously less than monoclonal control groups (89.15 ± 7.96 and 92.04 ± 8.44,P ＜0.05),iodic control groups (97.83 ± 9.21 and 88.30 ± 8.12,P＜ 0.05) and blank control groups (119.04 ± 10.57 and 115.56 ± 11.08,P ＜ 0.05).Conclusion 131 Ⅰ-PSCA-mAb could effectively inhibit the growth and invasion ability of prostate carcinoma cell line LNCap and PC3,promote the apoptosis,and has the potential to become a new targeted therapy drug for the treatment of prostate carcinoma.131 Ⅰ-PSCA-McAb could effectively target PSCA and inhibit the growth of tumor,therefore was a promising radioimmunotherapy radiophamaceutical for prostate cancer. Key words: 131Ⅰ labeling prostate stem cell antigen monoclonal antibody; Cell proliferation; Cell apoptosis; Cell invasion; Radioimmunotherapy

The Synthesis and Biological Evaluation of Multifunctionalised Derivatives of Noscapine as Cytotoxic Agents

Noscapine, a phthalideisoquinoline alkaloid derived from Papaver somniferum, is a well-known antitussive drug that has a relatively safe in vitro toxicity profile. Noscapine is also known to possess weak anticancer efficacy, and since its discovery, efforts have been made to design derivatives with improved potency. Herein, the synthesis of a series of noscapine analogues, which have been modified in the 6', 9', 1 and 7-positions, is described. In a previous study, replacement of the naturally occurring N-methyl group in the 6'-position with an N-ethylaminocarbonyl was shown to promote cell-cycle arrest and cytotoxicity against three cancer cell lines. Here, this modification has been combined with other structural changes that have previously been shown to improve anticancer activity, namely halo substitution in the 9'-position, regioselective O-demethylation to reveal a free phenol in the 7-position, and reduction of the lactone to the corresponding cyclic ether in the 1-position. The incorporation of new aryl substituents in the 9'-position was also investigated. The study identified interesting new compounds able to induce G2/M cell-cycle arrest and that possess cytotoxic activity against the human prostate carcinoma cell line PC3, the human breast adenocarcinoma cell line MCF-7, and the human pancreatic epithelioid carcinoma cell line PANC-1. In particular, the ethyl urea cyclic ether noscapinoids and a compound containing a 6'-ethylaminocarbonyl along with 9'-chloro, 7-hydroxy and lactone moieties exhibited the most promising biological activities, with EC50 values in the low micromolar range against all three cancer cell lines, and these derivatives warrant further investigation.

Increased expression of p21Waf1/Cip1 and JNK with costimulation of prostate cancer cell activation by an siRNA Egr-1 inhibitor

The p21Waf1/Cip1 protein (hereafter, p21) and the c‑JunN-terminal kinase (JNK) are two well-characterized cell modulators that play a crucial role in cell differentiation, senescence and apoptosis. Here, we report that transcription of the p21Waf1/Cip1 and JNK-1 genes is affected by inhibition of the early growth response-1 (Egr-1) in response to a small interfering RNA [siRNA)-Egr-1] in LNCaP and PC-3 prostate carcinoma cell lines. The expression levels of protein were determined by western blotting, and apoptosis was measured by propidium iodide staining and flow cytometric analysis. Inhibition of Egr-1, p21 and JNK-1 was carried out by siRNAs. LNCaP and PC-3 cells exhibited readily detectable Egr-1, JNK and p21, even in low serum medium without the addition of other exogenous agents. The expression of Egr-1, p21 and JNK was strongly increased after treatment of the cells with TPA, tumor necrosis factor-α (TNF-α) or arsenite. Suppression of Egr-1 expression by siRNA abrogated the ability of TPA to induce Egr-1 and JNK-1 activities, moderately increasing the p21 activity and abrogating the anti-apoptotic effect of Egr-1 observed in the prostate cancer cell lines. Moreover, blockade of p21 and JNK was unable to decrease the activity of Egr-1, while siRNA against p21 abrogated the pro‑apoptotic effect of p21. The results demonstrated that Egr-1 acts as a key player in prostate tumor cell growth and survival, while p21 plays a key pro‑apoptotic role in LNCaP and PC-3 prostate carcinoma cell lines.

Time Evolution of Nanoparticle–Protein Corona in Human Plasma: Relevance for Targeted Drug Delivery

When nanoparticles (NPs) enter a biological fluid (e.g., human plasma (HP)), proteins and other biomolecules adsorb on the surface leading to formation of a rich protein shell, referred to as "protein corona". This corona is dynamic in nature and its composition varies over time due to continuous protein association and dissociation events. Understanding the time evolution of the protein corona on the time-scales of a particle's lifetime in blood is fundamental to predict its fate in vivo. In this study, we used lipid NPs, the cationic lipid 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl] (DC-Chol) and the zwitterionic lipid dioleoylphosphatidylethanolamine (DOPE), that are among the most promising nanocarriers both in vitro and in vivo. Here, we investigated the time evolution of DC-Chol-DOPE NPs upon exposure to HP. On time scales between 1 and 60 minutes, nanoliquid tandem mass spectrometry revealed that the protein corona of DC-Chol-DOPE NPs is mainly constituted of apolipoproteins (Apo A-I, Apo C-II, Apo D, and Apo E are the most enriched). Since the total apolipoprotein content is relevant, we exploited the protein corona to target PC3 prostate carcinoma cell line that expresses high levels of scavenger receptor class B type 1 receptor, which mediates the bidirectional lipid transfer between low-density lipoproteins, high-density lipoproteins, and cells. Combining laser scanning confocal microscopy experiments with flow cytometry we demonstrated that DC-Chol-DOPE/HP complexes enter PC3 cells by a receptor-mediated endocytosis mechanism.

Shedding of endogenous MHC class I‐related chain molecules A and B from different human tumor entities: Heterogeneous involvement of the “a disintegrin and metalloproteases” 10 and 17

The interaction of the MHC class I-related chain molecules A and B (MICA and MICB) with the corresponding natural killer group 2, member D (NKG2D) receptor triggers cytotoxic effector activity of natural killer cells and certain T-cell subsets and provides a costimulatory signal for cytokine production. Thus, the presence of MICA/B on transformed cells contributes to tumor immunosurveillance. Consequently, the proteolytic cleavage of MICA/B is regarded as an important immune escape mechanism of various cancer cells. To investigate the molecular machinery responsible for the shedding of endogenous MICA/B, we analyzed different human tumor entities including mammary, pancreatic and prostate carcinomas. Flow cytometry and enzyme-linked immunosorbent assay (ELISA) revealed that all tested tumor cells constitutively expressed MICA and MICB on the cell surface and also released NKG2D ligands into the supernatant. We demonstrate that the "a disintegrin and metalloproteases" (ADAMs) 10 and 17 are largely responsible for the generation of soluble MICA/B. Pharmacological inhibition of metalloproteases reduced the level of released MICA/B and increased cell surface expression. Studies using RNA interference not only revealed a prominent role of ADAM10 and ADAM17 in NKG2D ligand shedding but also a tumor cell-specific role of ADAM10 and/or ADAM17 in shedding of MICA or MICB. Moreover, we report that in the prostate carcinoma cell line PC-3, MICA was not shed at all but rather was secreted in exosomes. These data indicate that the release of NKG2D ligands from individual tumor entities is by far more complex than suggested in previously reported MICA/B transfection systems.

Type 10 Soluble Adenylyl Cyclase Is Overexpressed in Prostate Carcinoma and Controls Proliferation of Prostate Cancer Cells

cAMP signaling plays an essential role in modulating the proliferation of different cell types, including cancer cells. Until now, the regulation of this pathway was restricted to the transmembrane class of adenylyl cyclases. In this study, significant overexpression of soluble adenylyl cyclase (sAC), an alternative source of cAMP, was found in human prostate carcinoma, and therefore, the contribution of this cyclase was investigated in the prostate carcinoma cell lines LNCaP and PC3. Suppression of sAC activity by treatment with the sAC-specific inhibitor KH7 or by sAC-specific knockdown mediated by siRNA or shRNA transfection prevented the proliferation of prostate carcinoma cells, led to lactate dehydrogenase release, and induced apoptosis. Cell cycle analysis revealed a significant rise in the G(2) phase population 12 h after sAC inhibition, which was accompanied by the down-regulation of cyclin B(1) and CDK1. sAC-dependent regulation of proliferation involves the EPAC/Rap1/B-Raf signaling pathway. In contrast, protein kinase A does not play a role. In conclusion, this study suggests a novel sAC-dependent signaling pathway that controls the proliferation of prostate carcinoma cells.