PC-3 Human Prostate Cancer Research Articles

High hydrostatic pressure enhances the anti-proliferative properties of lotus bee pollen on the human prostate cancer PC-3 cells via increased metabolites

Ethnopharmacological relevanceThe beneficial effects of bee pollen on prostate diseases are well known. Clinicians confirm that, in nonbacterial prostate diseases, bee pollen improves the condition of patients effectively. However, there is insufficient evidence to rate effectiveness of bee pollen on prostate cancer. Aim of the studyHigh hydrostatic pressure (HHP), an effective non-thermal technique to improve the nutritional quality and bio-functionality of plant-based foods, was used to increase the anti-proliferative properties of Lotus (Nelumbo nucifera) bee pollen (LBP) in prostate cancer PC-3 cells via enhancement of bioactive compounds. Materials and methodsFreeze-dried lotus bee pollen produced from Fu Zhou city, Jiangxi province, China, was processed by high hydrostatic pressure (HHP). The anti-proliferative activities, apoptosis of ethanol and methanol extracts in prostate cancer PC-3 cells was evaluated using MTT method and Annexin-V/PI cell apoptosis assay kit, respectively. The changes of metabolites were determined using UPLC-Triple-TOF-MS analysis platform. ResultsHHP treatment enhanced anti-proliferative activities, cell apoptosis, cell cycle disruption, glutathione-depletion in prostate cancer PC-3 cells. The metabolomics analysis showed that some metabolites such as chaetoglobosin A, glutathione oxidized, cyanidin 3-rutinoside, brassicoside, sophoranone, curcumin II, soyasaponin II were significantly increased (p < 0.05) after the HHP treatment, PCA results shown that these bioactive components have quite correlation with anti-proliferative activities of lotus bee pollen on the PC-3 cells. The results indicated that HHP enhances the anti-prostate cancer activity of lotus bee pollen via increased metabolites.

Tumor suppressor REIC/Dkk-3 and its interacting protein SGTA inhibit glucocorticoid receptor to nuclear transport.

REIC/Dkk-3 is a tumor suppressor, and its expression is significantly downregulated in a variety of human cancer types. A previous study performed yeast two-hybrid screening and identified the small glutamine-rich tetratricopeptide repeat-containing protein α (SGTA), known as a negative modulator of cytoplasmic androgen receptor (AR) signaling, which is a novel interacting partner of REIC/Dkk-3. The previous study also indicated that the REIC/Dkk-3 protein interferes with the dimerization of SGTA and then upregulates the AR transport and signaling in human prostate cancer PC3 cells. Since the transport of some steroid receptors to nucleus is conducted similarly by dynein motor-dependent way, the current study aimed to investigate the role of SGTA and REIC/Dkk-3 in the transport of other glucocorticoid receptors (GR). In vitro reporter assays for the cytoplasmic GR transport were performed in human prostate cancer PC3 cells and 293T cells. As for the SGTA protein, a suppressive effect on the GR transport to the nucleus was observed in the cells. As for the REIC/Dkk-3 protein, an inhibitory effect was observed for the GR transport in PC3 cells. Under the depleted condition of SGTA by short-hairpin (sh)RNA, the downregulation of GR transport by REIC/Dkk-3 was significantly enhanced compared with the non-depleted condition in PC3 cells, suggesting a compensatory role of REIC/Dkk-3 in the SGTA mediated inhibition of GR transport. The current study therefore demonstrated that SGTA inhibited the cytoplasmic transport of GR in 293T and PC3 cells, and REIC/Dkk-3 also inhibited the cytoplasmic transport of GR in PC3 cells. These results may be used to gain novel insight into the GR transport and signaling in normal and cancer cells.

Synthesis and Anti-Proliferative Evaluations of New Heterocyclic Derivatives Using 5,6,8,9-Tetrahydropyrazolo[5,1-b]quinazolin-7(3H)-one Derivatives Derived from Cyclohexa-1,4-dione.

Recentlty, pyrazoloquinazoline derivatives acquired a special attention due to their wide range of pharmacological activities, especially therapeutic. Through the market, it was found that many pharmacological drugs containing the quinazoline nucleus were known. The aim of this work is to synthesize target molecules possessing not only anti-tumor activities but also kinase inhibitors. The target molecules were obtained through the synthesis of a series of 5,6,8,9- tetrahydropyrazolo[5,1-b]quinazolin-7(3H)-one derivatives 4a-i using the multi-component reactions of cyclohexane- 1,4-dione (1), the 5-amino-4-(2-arylhydrazono)-4H-pyrazol-3-ol derivatives 2a-c, the aromatic aldehydes 3a-c, respectively. The synthesized compounds were evaluated against c-Met kinase, PC-3 cell line, and different kinds of cancer cell lines together with normal cell line, tyrosine kinases, and Pim-1 kinase. Multi-component reactions were adopted using compound 1 to get different 5,6,8,9- tetrahydropyrazolo[5,1-b]quinazolin-7(3H)-one derivatives which underwent further heterocyclization reactions. The c-Met kinase activity of all compounds was evaluated using Homogeneous Time-Resolved Fluorescence (HTRF) assay, taking foretinib as the positive control. The anti-proliferative activity of all target compounds against the human prostatic cancer PC-3 cell line was measured using MTT assay using SGI-1776 as the reference drug. All the synthesized compounds were assessed for inhibitory activities against A549 (non-small cell lung cancer), H460 (human lung cancer), HT-29 (human colon cancer), and MKN-45 (human gastric cancer) cancer cell lines together with foretinib as the positive control by an MTT assay. Antiproliferative evaluations and c-Met kinase, Pim-1 kinase inhibitions were performed for the synthesized compounds, where the varieties of substituents through the aryl ring and the thiophene moiety afforded compounds with high activities. The compounds with high antiproliferative activity were tested towards c-Met and the results showed that compounds 4e, 4f, 4g, 4i, 6i, 6k, 6l, 8f, 8i, 10d, 10e, 10f, 10h, 12e, 12f, 12g, 12h, 12i, 14f, 14g, 14h, and 14i were the most potent compounds. A further selection of compounds for the Pim-1 kinase inhibition activity showed that compounds 4f, 6i, 6l, 8h, 8i, 8g, 10d, 12i, and 14f were the most active compounds to inhibit Pim-1.

Paper-Based Acoustofluidics for Separating Particles and Cells.

Paper is emerging as a versatile platform for automated fluid handling with a broad range of applications in medical diagnostics and analytical chemistry. However, selectively controlling analyte transport in paper to achieve concentration or selection has been a challenge for functional analysis. Here, by combining paper-based microfluidics with acoustics, we present a rapid and powerful method to size dependently control movement of microparticles and cells in paper using surface acoustic waves (SAW). We demonstrate the unique capability of the paper-based SAW approach to trap and concentrate microparticles in paper and release them when required, achieving collection efficiency of over 98%. Given the correlation between collection efficiency, size, and applied power, the paper-based SAW approach is applied to isolate a mixture of microparticles (1.1, 3.2, and 5 μm in diameter) into different regions and also to trap and concentrate human prostate cancer PC3 cells at a predetermined site. This paper-based SAW approach provides opportunities to develop powerful and low-cost selection and analysis tools, capable of processing complex multicomponent samples, with potential applications in medical diagnostics.

Endosulfan triggers epithelial-mesenchymal transition via PTP4A3-mediated TGF-β signaling pathway in prostate cancer cells

Endosulfan is a persistent organochlorine pesticide that bioaccumulates in human body through the food chain and thus represents a potential risk to public health. Despite epidemiological studies, the molecular mechanisms underlying the carcinogenic effects of endosulfan in the prostate remain poorly understood. In this study, we investigated the effect of endosulfan on epithelial-mesenchymal transition (EMT) in human prostate cancer PC3 and DU145 cells. Endosulfan induced alterations of EMT biomarkers, reflecting repression of E-cadherin expression and induction of fibronectin, snail2, ZEB2, Twist1 and Vimentin. The expression of Protein-tyrosine Phosphatase 4A3 (PTP4A3) at mRNA and protein levels was upregulated by endosulfan. PTP4A3 inhibitor reversed the changes of EMT biomarkers, PTP4A3 and p-Smad2/Smad2, but did not affect the upregulation of Cleaved-Notch1 and Jagged1 in endosulfan-exposed cells. Endosulfan promoted cell migration and invasion, which were rescued by specific inhibitors for PTP4A3, TGF-β signaling and Notch signaling, respectively. These findings suggest that endosulfan promoted cell migration and invasion with the induction of EMT through PTP4A3-mediated TGF-β signaling pathway in prostate cancer cells.

Suppressing PARylation by 2',5'-oligoadenylate synthetase 1 inhibits DNA damage-induced cell death.

High expression of 2',5'-oligoadenylate synthetase 1 (OAS1), which adds AMP residues in 2',5' linkage to a variety of substrates, is observed in many cancers as a part of the interferon-related DNA damage resistance signature (IRDS). Poly(ADP-ribose) (PAR) is rapidly synthesized from NAD+ at sites of DNA damage to facilitate repair, but excessive PAR synthesis due to extensive DNA damage results in cell death by energy depletion and/or activation of PAR-dependent programmed cell death pathways. We find that OAS1 adds AMP residues in 2',5' linkage to PAR, inhibiting its synthesis invitro and reducing its accumulation in cells. Increased OAS1 expression substantially improves cell viability following DNA-damaging treatments that stimulate PAR synthesis during DNA repair. We conclude that high expression of OAS1 in cancer cells promotes their ability to survive DNA damage by attenuating PAR synthesis and thus preventing cell death.

NOL6, a new founding oncogene in human prostate cancer and targeted by miR-590-3p.

We identified a new human prostate cancer oncogene, nucleolar protein 6 (NOL6), and screened for microRNAs that interfere with its expression in prostate cancer cells. A NOL6 shRNA plasmid was constructed and packaged into lentivirus to infect PC-3 cells. The ability of cell proliferation was evaluated by cell counting and colony formation. Cell cycle progression and apoptosis of PC-3 cells were detected by flow cytometry. A retrieval database was used to screen possible target microRNAs, and the effect of target miRNA overexpression on PC-3 cells was observed. The results showed that after NOL6 gene knockdown, PC-3 cell mitosis was blocked, proliferation was decreased, and the number of apoptotic cells were increased. The microRNA, hsa-miR-590-3p, that can regulate the NOL6 gene expression was identified. Overexpression of miR-590-3p in PC-3 cells by synthetic mimics resulted in abnormal mitosis, decreased cell proliferation, and an increase in apoptosis. In summary, we identified NOL6 as a novel oncogene in the human prostate cancer PC-3 cell line. The miRNA miR-590-3p interferes with NOL6 expression making it a potential treatment for prostate cancer.

Papain Mediated Synthesized Gold Nanoparticles Encore the Potency of Bioconjugated Flutamide.

Prostate cancer is the second most common cause of male cancer death after lung cancer in the US. Therefore, there is an urgent need for a highly effective therapeutic drug at substantially low doses. Anti-androgen drug flutamide was delivered to the prostate cancer cells using Papain Mediated Synthesized Gold Nanoparticles (PGNPs) as the drug delivery system. PGNPs and flutamide worked synergistically against cancer cells. Flutamide was used to bioconjugate with PGNPs to improve its efficacy against prostate cancer. The synthesis and bioconjugation of flutamide with PGNPs (F-PGNPs) were characterized by various characterization techniques such as UV-vis spectroscopy, Transmission Electron Microscopy (TEM), Dynamic Light Scattering (DLS), and zeta potential to ensure the synthesis, size, shape, size distribution, and stability. The drug loading efficiency of flutamide in F-PGNPs was confirmed and validated by UV-vis spectroscopy. Eventually, in vitro studies were performed to determine the potency of F-PGNPs, changes in nuclear morphology, and generation of Reactive Oxygen Species (ROS). The efficacy of F-PGNPs (IC50 is 46.54 μg/mL) was found to be improved significantly over pure flutamide (IC50 is 64.63 μg/mL) against human prostate cancer PC-3 cell line whereas F-PGNPs did not show any significant toxicity up to a fairly high concentration toward normal mouse macrophage J774A.1 cells. The apoptotic effects and ROS generation of F-PGNPs were analyzed by increased permeability of the cell membrane and condensed chromatin with deep blue and green fluorescent nucleus, respectively. The results clearly showed that F-PGNPs significantly improved the potency of flutamide by delivering it directly into the nucleus of cancer cells through caveolae-dependent endocytosis. Thus, the greater inhibitory effect of F-PGNPs over the pure drug would be of great advantage during prostate cancer treatment.

Checkpoint kinase inhibition in prostate cancer cells resistant to poly ADP-ribose polymerase inhibitors.

150 Background: Poly ADP-ribose polymerase inhibitors (PARPi) have shown promise in the treatment of metastatic castration-resistant prostate cancer patients with DNA damage response defects . The phase 3 PROfound trial showed olaparib delayed the time to radiographic progression or death as compared with abiraterone or enzalutamide. In addition to olaparib, three other PARPi are in Phase 3 trials in prostate cancer (PC): rucaparib, talazoparib, and niraparib. Despite responses, resistance is common and treatment options for PARPi-resistant patients are limited. In this study, we observed de novo activation of checkpoint kinases (CHEK) in talazoparib-resistant (TR) PC cells. Therefore, we hypothesized that targeting CHEK may mitigate resistance to PARPi in PC. Methods: We developed TR human prostate cancer PC3 (low BRCA2 protein due to heterozygous deletion of BRCA2) cells. We performed phosphoproteomic analysis to identify possible mechanisms of talazoparib resistance in PC3 cells and validated the results with Western blot. Results: TR-PC3 cells proliferated slower and had a significant increase in the phosphorylation of CHEK2 compared to parental (p) PC3. Treatment with a CHEK2-selective inhibitor, CCT241533, did not affect cell growth in TR-PC3 cells. Conversely, treatment with a CHEK 1/2 inhibitor, prexasertib, led to significant cell growth inhibition in TR-PC3 at a much lower IG 50% concentration compared to pPC3. RNAi-mediated knockdown validated the superior efficacy of combined CHEK1 and CHEK2 inhibition since this combination produced the greatest cell growth inhibition seen in both TR-PC3 and de novo PARPi-resistant p22RV1. Treatment of pPC-3 and p22RV1 with combinations of talazoparib and prexasertib showed greater cell growth inhibition compared to either drug alone. Conclusions: Resistance to PARPi in PC cells with deletion of BRCA2 may potentially be overcome with CHEK inhibition. Moreover, our preliminary data suggested that the effect of PARPi and CHEK inhibitors on PARPi/CHEK inhibitor-naïve PC cells was greatest when used together, indicating that patients with PC may experience greatest anti-tumor activity of the two drugs when they are used in combination.

Apigenin as an anticancer agent.

Apigenin is an edible plant-derived flavonoid that has been reported as an anticancer agent in several experimental and biological studies. It exhibits cell growth arrest and apoptosis in different types of tumors such as breast, lung, liver, skin, blood, colon, prostate, pancreatic, cervical, oral, and stomach, by modulating several signaling pathways. Apigenin induces apoptosis by the activation of extrinsic caspase-dependent pathway by upregulating the mRNA expressions of caspase-3, caspase-8, and TNF-α. It induces intrinsic apoptosis pathway as evidenced by the induction of cytochrome c, Bax, and caspase-3, while caspase-8, TNF-α, and B-cell lymphoma 2 levels remained unchanged in human prostate cancer PC-3 cells. Apigenin treatment leads to significant downregulation of matrix metallopeptidases-2, -9, Snail, and Slug, suppressing invasion. The expressions of NF-κB p105/p50, PI3K, Akt, and the phosphorylation of p-Akt decreases after treatment with apigenin. However, apigenin-mediated treatment significantly reduces pluripotency marker Oct3/4 protein expression which might be associated with the downregulation of PI3K/Akt/NF-κB signaling.

Pimozide Inhibits the Human Prostate Cancer Cells Through the Generation of Reactive Oxygen Species.

The United States Food and Drug Administration-approved antipsychotic drug, pimozide, has anticancer activities. However, the role of reactive oxygen species (ROS) in its effect on prostate cancer is not well-known. We examined cell proliferation, colony formation, migration, ROS production, and the expression of antioxidant-related genes after treatment of human prostate cancer PC3 and DU145 cells with pimozide. In addition, histopathology, ROS production, and superoxide dismutase (SOD) activity were analyzed after administering pimozide to TRAMP, a transgenic mouse with prostate cancer. Pimozide increased the generation of ROS in both cell lines and inhibited cell proliferation, migration, and colony formation. Oxidative stress induced by pimozide caused changes in the expression of antioxidant enzymes (SOD1, peroxiredoxin 6, and glutathione peroxidase 2) and CISD2. Co-treatment with glutathione, an antioxidant, reduced pimozide-induced ROS levels, and counteracted the inhibition of cell proliferation. Administration of pimozide to TRAMP mice reduced the progression of prostate cancer with increased ROS generation and decreased SOD activity. These results suggest that the antipsychotic drug, pimozide, has beneficial effects in prostate cancer in vivo and in vitro. The mechanism of pimozide may be related to augmenting ROS generation. We recommend pimozide as a promising anticancer agent.

Exosomes secreted by prostate cancer cells under hypoxia promote matrix metalloproteinases activity at pre-metastatic niches.

Approximately, 30 000 men die from prostate cancer (PCa) every year in the United States, mainly due to the metastasis. Thus, the key events associated with PCa metastasis are under rigorous investigation, with recent studies showing that preparation of pre-metastatic niches (PMN) in distant organs is an important step. However, the molecular basis for PMN preparation is still unclear. Hypoxia in primary tumors promotes aggressiveness; however, its precise role in metastasis is not clear. We recently reported that exosomes secreted by PCa cells under hypoxia promote stemness and invasiveness in naïve PCa cells; however, whether these extracellular vesicles also influence PMN remains unknown. In the present study, we isolated exosomes from human PCa PC3 cells under normoxic (21% O2 , exosomes secreted under normoxic condition [ExoNormoxic ]) and hypoxic (1% O2 , exosomes secreted under hypoxic condition [ExoHypoxic ]) conditions, and characterized their effect (10 µg exosomes, intraperitoneal (IP) treatment every 48 hours for 4 weeks) on key biomarkers associated with PMN in nude mice. Whole animal fluorescence imaging showed that ExoHypoxic treatment promotes matrix metalloproteinases (MMPs) activity in several putative metastatic sites. Histological studies confirmed that ExoHypoxic treatment enhanced the level of MMP2, MMP9, and extracellular matrix proteins (fibronectin and collagen) as well as increased the number of CD11b+ cells at selective PMN sites. Furthermore, proteomic profiling of exosomes by liquid chromatography/mass spectrometry identified cargo proteins in ExoNormoxic and ExoHypoxic as well as distinct canonical pathways targeted by them. These results suggest that exosomes secreted by PCa cells under hypoxia plausibly remodel distant PMN, and thus, could be a potential target to control metastatic PCa.

Epigallocatechin-3-Gallate Suppresses Vasculogenic Mimicry through Inhibiting the Twist/VE-Cadherin/AKT Pathway in Human Prostate Cancer PC-3 Cells.

Vasculogenic mimicry (VM) is the alternative process of forming vessel-like networks by aggressive tumor cells, and it has an important role in tumor survival, growth, and metastasis. Epigallocatechin-3-gallate (EGCG) is well known to have diverse bioactivities including anti-cancer effects. However, the efficacy of EGCG on VM is elusive. In this study, we explored whether and how EGCG affects VM in human prostate cancer (PCa) PC-3 cells. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Invasive and VM formation abilities were assessed by an invasion assay and a three-dimensional (3D) culture VM tube formation assay, respectively. Western blots were carried out. An immunofluorescence assay was performed to detect nuclear twist expression. EGCG effectively inhibited the invasive ability, as well as tubular channel formation, without affecting cell viability. EGCG significantly downregulated the expression of vascular endothelial cadherin (VE-cadherin) and its transcription factor, twist, N-cadherin, vimentin, phosphor-AKT, and AKT, but not phospho-erythropoietin-producing hepatocellular receptor A2 (EphA2) and EphA2. In addition, EGCG diminished the nuclear localization of twist. Treatment with SC79, an AKT activator, effectively rescued EGCG-inhibited VM formation. These results demonstrated for the first time that EGCG causes marked suppression of VM through inhibiting the twist/VE-cadherin/AKT pathway in human PCa PC-3 cells.

The cytotoxic activity of extracts of the brown alga Cystoseira tamariscifolia (Hudson) Papenfuss, against cancer cell lines changes seasonally

Brown seaweeds have been highlighted for their abundant production of bioactive substances that may provide novel drugs or drug scaffolds for a range of diseases, particularly cancer. Indeed, a number of promising compounds that can modulate growth arrest or apoptosis have already been isolated. As previous work has highlighted seasonal differences in concentrations of secondary metabolites, this study aimed to evaluate seasonal variation in the cytotoxic anticancer activity of Cystoseira tamariscifolia extracts. Primary and secondary metabolites were measured using colourimetric techniques and extracts were exposed to human leukaemia, HL60 and THP-1, and human prostate cancer PC3 cell lines in vitro, and cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The results confirm a large but inconsistent seasonal variation in the concentrations of biomolecules, although, generally, levels were higher in spring and summer. IC50 values for cytotoxicity also showed variability both seasonally and with extraction protocol, but again, this was inconsistent. These data suggest that when collecting brown algae in order to isolate novel bioactive compounds, repeated sampling across annual or biotic cycles of the seaweed and the use of multiple solvent extraction methods could give a greater possibility of discovering bioactive lead compounds.

Ethanol extracts of Hizikia fusiforme induce apoptosis in human prostate cancer PC3 cells via modulating a ROS-dependent pathway

Objective: To investigate whether ethanol extracts of Hizikia fusiforme could induce apoptosis in human prostate cancer PC3 cells. Methods: Cell viability was evaluated using 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide. Apoptosis and mitochondrial membrane potential (MMP) were measured using flow cytometry in PC3 cells. DNA damage was assessed by nuclear staining and DNA fragmentation assay. Expressions of apoptosis-associated proteins were determined by Western blotting assays. Activities of caspase-3, -8, and -9 were determined by colorimetric assay. Moreover, intracellular reactive oxygen species (ROS) generation was detected using a flow cytometer and fluorescence microscope. Results: Treatment of PC3 cells with ethanol extracts of Hizikia fusiforme inhibited proliferation, which was associated with induction of apoptosis, and accompanied by increased expression of Fas, Fas-ligand (FasL), Bax and tBid, and decreased expression of Bcl-2. In addition, ethanol extracts of Hizikia fusiforme reduced c-Flip expression and activated caspase-8, -9 and -3, resulting in an increase in poly (ADP-ribose) polymerase (PARP)cleavage. However, in the presence of a pan-caspase inhibitor, ethanol extracts of Hizikia fusiforme-mediated growth inhibition and apoptosis were significantly attenuated. Ethanol extracts of Hizikia fusiforme also destroyed the integrity of mitochondria due to the loss of MMP, leading to cytosolic release of cytochrome c. Moreover, the levels of ROS were markedly increased by treatment with ethanol extracts of Hizikia fusiforme, which was significantly suppressed by the ROS scavenger N-acetyl-L-cysteine. Further investigation of whether ethanol extracts of Hizikia fusiforme-induced apoptosis was related to the generation of ROS was conducted and the results showed that N-acetyl-L-cysteine fully blocked ethanol extracts of Hizikia fusiforme-induced apoptotic events including loss of MMP, activation of caspase-3, the cytosolic release of cytochrome c and cytotoxicity. Conclusions: Ethanol extracts of Hizikia fusiforme have chemopreventive potential via induction of ROS-dependent apoptosis. Therefore, ethanol extracts of Hizikia fusiforme may be useful for developing effective and selective natural sources to inhibit cancer cell proliferation.

Modulation of CXC-motif chemokine receptor 7, but not 4, expression is related to migration of the human prostate cancer cell LNCaP: regulation by androgen and inflammatory stimuli.

To elucidate the regulation, function of the chemokine CXC-motif ligand 12 (CXCL12) and its receptors (CXCR) 4 and 7 in prostate cancer tumor microenvironment. In-silico-analysis of expression in prostate cancer tissues. In-vitro comparison, testing of regulation in human prostate cancer cells LNCaP, DU145, and PC3. Dihydrotestosterone (DHT) treatments (0-10nM) were for 0-48h. The inflammatory agent Flagellin treatment (20ng/ml) was for 2h. Migration assays were performed for 24h using 10ng/ml CXCL12. Real-time PCR, western analysis, and migration assays were used to determine mRNA, protein, and functional changes, respectively. Malignant prostate cancer tissues exhibit higher CXCR4/7 mRNA ratio, and higher CXCR7 mRNA levels were detected in the androgen-responsive LNCaP cells. Putative androgen-responsive elements were identified in CXCR4, 7 gene, and exposure to DHT, flagellin increased CXCR4 mRNA but decreased CXCR7 mRNA levels in LNCaP cells. Androgen receptor siRNA significantly attenuated the effects of DHT on CXCR4, 7 mRNA in LNCaP cells. However, DHT and flagellin only decrease CXCR7 protein and additively increased migration of LNCaP cells towards CXCL12. Down regulation of CXCR7 protein by DHT and flagellin increased migration, supporting CXCR7 as decoy receptor counteracting CXCL12/CXCR4-mediated migration in prostate cancer cells.

Development of selective mono or dual PROTAC degrader probe of CDK isoforms

Cyclin-dependent kinase (CDK) family members are promising molecular targets in discovering potent inhibitors in disease settings, they function differentially. CDK2, CDK4 and CDK6, directly regulate the cell cycle, while CDK9 primarily modulates the transcription regulation. In discovering inhibitors of these CDKs, toxicity associated with off-target effect on other CDK homologs often posts as a clinical issue and hinders their further therapeutic development. To improve efficacy and reduce toxicity, here, using the Proteolysis Targeted Chimeras (PROTACs) approach, we design and further optimize small molecule degraders targeting multiple CDKs. We showed that heterobifunctional compound A9 selectively degraded CDK2. We also identified a dual-degrader, compound F3, which potently induced degradation of both CDK2 (DC50: 62 nM) and CDK9 (DC50: 33 nM). In human prostate cancer PC-3 cells, compound F3 potently inhibits cell proliferation by effectively blocking the cell cycle in S and G2/M phases. Our preliminary data suggests that PROTAC-oriented CDK2/9 degradation is potentially an effective therapeutic approach.

Integrated analysis of miRNA landscape and cellular networking pathways in stage-specific prostate cancer.

Dysregulation of miRNAs has been demonstrated in several human malignancies including prostate cancer. Due to tissue limitation and variable disease progression, stage-specific miRNAs changes in prostate cancer is unknown. Using chip-based microarray, we investigated global miRNA expression in human prostate cancer LNCaP, PC3, DU145 and 22Rv1 cells representing early-stage, advanced-stage and castration resistant prostate cancer in comparison with normal prostate epithelial cells. A total of 292 miRNAs were differentially expressed with 125 upregulated and 167 downregulated. These miRNAs were involved in pathways including drug resistance drug-efflux, adipogenesis, epithelial-to-mesenchymal transition, bone metamorphosis, and Th1/Th2 signaling. Regulation of miRNAs were interlinked with upstream regulators such as Argonaut 2 (AGO2), Double-Stranded RNA-Specific Endoribonuclease (DICER1), Sjogren syndrome antigen B (SSB), neurofibromatosis 2 (NF2), and peroxisome proliferator activated receptor alpha (PPARA), activated during stage-specific disease progression. Candidate target genes and pathways dysregulated in stage-specific prostate cancer were identified using CS-miRTar database and confirmed in clinical specimens. Integrative network analysis suggested some genes targeted by miRNAs include miR-17, let7g, miR-146, miR-204, miR-205, miR-221, miR-301 and miR-520 having a major effect on their dysregulation in prostate cancer. MiRNA-microarray analysis further identified miR-130a, miR-181, miR-328, miR146 and miR-200 as a panel of novel miRNAs associated with drug resistance drug-efflux and epithelial-to-mesenchymal transition in prostate cancer. Our findings provide evidence on miRNA dysregulation and its association with key functional components in stage-specific prostate cancer.

Effects of ADAM17 siRNA and doxorubicin on proliferation and apoptosis of prostate cancer PC-3 cells

Objective To investigate the effects of ADAM17 siRNA combined with adriamycin (ADM) on proliferation and apoptosis of human prostate cancer cell line PC-3. Methods PC-3 cells stably transfected with ADAM17 siRNA expression plasmid were treated with ADM. The proliferation rate was observed by MTT colorimetry, the apoptosis rate was detected by acridine orange staining and flow cytometry (FCM), and the activity of apoptotic protein caspase-3 was analyzed. Results Both ADAM17 siRNA and ADM alone and in combination could decrease the proliferation rate and increase the apoptosis rate of PC-3 cells in varying degrees. FCM data showed that the apoptosis rates of ADAM17 siRNA and ADM alone were(6.2±1.4)% and (17.2±1.9)% respectively, which were significantly different from (36.4±3.7)% of ADAM17 siRNA and ADM alone (P<0.01). Both of them also had a good synergistic effect on inducing caspase-3 activity. Conclusions ADAM17 siRNA and ADM have different effects on the proliferation and apoptosis of human prostate cancer PC-3, but the combination of ADAM17 siRNA and ADM is more effective. It is suggested that gene therapy combined with chemotherapy may be an important method to improve the curative effect and reduce the risk of recurrence and metastasis of prostate cancer. Key words: Prostatic Neoplasms; Cells, Cultured; RNA, Small Interfering; Doxorubicin; Cell Proliferation; Apoptosis

Inhibitory effects of 4-hydroperoxy-2-decenoic acid ethyl ester on phorbol ester- and TGF-β1-induced MMPs expression

Matrix metalloproteinases (MMPs), zinc-containing proteinases, play a critical role in tumour progression by degrading extracellular matrix components. MMP2 and MMP9 are secreted from tumour-associated macrophages as well as tumour cells and have been implicated in the formation of the tumour microenvironment. Therefore, the inhibition of these MMPs may suppress tumour progression and metastasis. 4-Hydroperoxy-2-decenoic acid ethyl ester (HPO-DAEE) is known to cause apoptosis in the human lung cancer cell line A549 by inducing endoplasmic reticulum (ER) stress. However, the effects of HPO-DAEE on tumour progression remain unclear. HPO-DAEE pre-treatment significantly suppressed phorbol 12-myristate 13-acetate (TPA)-triggered MMP activation in human monocytic THP-1 cells. It also enhanced the expression of haem oxygenase-1, an antioxidant enzyme, and suppressed the TPA-triggered intracellular accumulation of reactive oxygen species (ROS). Furthermore, HPO-DAEE suppressed transforming growth factor-β1-triggered human prostate cancer PC3 cell migration and this was accompanied by the inhibition of MMP expression and activities. The present results indicate that HPO-DAEE may exert inhibitory effects on tumour progression by suppressing MMP expression and activities.