In vitro study on the anticancer effects of oxalipalladium against PC3 human prostate carcinoma cells.

Prostate cancer is a common type of cancer in men with high incidence and mortality. Our aim was to investigate the effects of oxalipalladium (ox-Pd) on metastatic human prostate cancer PC3 cells and compare them with the effects of oxaliplatin (ox-Pt) (as an approved cancer drug). We synthesized ox-Pd through a new chemical method and used FT-IR, 1H NMR, 13C NMR, and MS analyzes to characterize it. The effects of ox-Pd on PC3 cells viability, apoptosis, cell cycle, migration, and gene expression were examined. Inhibition of topoisomerase IIα activity was investigated by pHOT1 plasmid relaxation and kDNA decatenation assays. Chemical tests showed ox-Pd with the correct composition and structure. For the first time, the exact fragmentation pathway of ox-Pd and its difference with ox-Pt was obtained by MS analysis. Ox-Pd significantly decreased PC3 cell viability with less/no toxicity effect on MHFB-1 normal skin fibroblasts. Wound scratch assay confirmed the strong anti-migratory activity of ox-Pd. According to flow cytometry analysis, this drug increased the number of PC3 cells in late apoptosis and decreased DNA replication and mitosis. Furthermore, pHOT1 plasmid relaxation and kDNA decatenation assays showed that ox-Pd strongly inhibited the catalytic activity of topoisomerase IIα. The expression of topoisomerase IIα, Bcl-2, P21, and survivin was decreased while the expression of Bax and p53 was increased under ox-Pd treatment. We provide the first evidence that ox-Pd exhibits more selective anticancer effects on PC3 cells compared to ox-Pt. Taken together, these data strongly suggest a therapeutic window for ox-Pd in cancer.

Effects of Red Sorghum-Derived Deoxyanthocyanidins and Their O-ß-D-Glucosides on E-Cadherin Promoter Activity in PC-3 Prostate Cancer Cells.

Although much less common than anthocyanins, 3-Deoxyanthocyanidins (3-DAs) and their glucosides can be found in cereals such as red sorghum. It is speculated that their bioavailability is higher than that of anthocyanins. Thus far, little is known regarding the therapeutic effects of 3-DAs and their O-ß-D-glucosides on cancer, including prostate cancer. Thus, we evaluated their potential to decrease cell viability, to modulate the activity of transcription factors such as NFκB, CREB, and SOX, and to regulate the expression of the gene CDH1, encoding E-Cadherin. We found that 4',7-dihydroxyflavylium chloride (P7) and the natural apigeninidin can reduce cell viability, whereas 4',7-dihydroxyflavylium chloride (P7) and 4'-hydroxy-7-O-ß-D-glucopyranosyloxyflavylium chloride (P3) increase the activities of NFkB, CREB, and SOX transcription factors, leading to the upregulation of CDH1 promoter activity in PC-3 prostate cancer cells. Thus, these compounds may contribute to the inhibition of the epithelial-to-mesenchymal transition in cancer cells and prevent the metastatic activity of more aggressive forms of androgen-resistant prostate cancer.

Methanolic extract of Teucrium persicum up-regulates and induces the membrane restoration of E-cadherin protein in PC-3 cells.

Teucrium persicum Boiss. an Iranian endemic plant is used in Iranian traditional medicine. E-cadherin transmembrane protein participates in adherens junctions and is the main partner for ß-catenin protein. The GC-MS analysis was used to detect the chemical constituents of the methanolic extract. Its effects on the transcription of the E-cadherin encoding gene, cellular levels, and localization of E-cadherin protein in PC-3 cells were investigated. About 70 chemical constituents were identified. Indirect immunofluorescence microscopy and western blotting results revealed the restoration of E-cadherin protein at cell adhesion contact sites in cells treated with T. persicum extract. Gene expression studies revealed that the extract increased the transcription of the E-cadherin encoding gene in PC-3 cells. These results suggest that T. persicum extract may contain potent compounds that provide further support for the anticancer properties of T. persicum. Surely, detailed molecular investigations are needed to find the mechanism(s) behind these effects.

Anticancer Activity of the Marine Triterpene Glycoside Cucumarioside A2-2 in Human Prostate Cancer Cells.

Despite recent advances in the treatment of metastatic castration-resistant prostate cancer (CRPC), treatment is inevitably hampered by the development of drug resistance. Thus, new drugs are urgently needed. We investigated the efficacy, toxicity, and mechanism of action of the marine triterpene glycoside cucumarioside A2-2 (CA2-2) using an in vitro CRPC model. CA2-2 induced a G2/M-phase cell cycle arrest in human prostate cancer PC-3 cells and caspase-dependent apoptosis executed via an intrinsic pathway. Additionally, the drug inhibited the formation and growth of CRPC cell colonies at low micromolar concentrations. A global proteome analysis performed using the 2D-PAGE technique, followed by MALDI-MS and bioinformatical evaluation, revealed alterations in the proteins involved in cellular processes such as metastatic potential, invasion, and apoptosis. Among others, the regulation of keratin 81, CrkII, IL-1ß, and cathepsin B could be identified by our proteomics approach. The effects were validated on the protein level by a 2D Western blotting analysis. Our results demonstrate the promising anticancer activity of CA2-2 in a prostate cancer model and provide insights on the underlying mode of action.

Sesquiterpenoids from the Resina Commiphora Promoting the Apoptotic Activity of PC-3 Prostate Cancer Cells.

Four new germacrane-type sesquiterpenes commiphoranes M1-M4 (1-4) together with eighteen sesquiterpenes were isolated from the Resina Commiphora. The structures and relative configurations of new substances were determined by using spectroscopic methods. Biological activity investigation revealed that nine compounds including 7, 9, 14, 16, (+)-17, (-)-17, 18, 19, and 20 could induce the apoptosis of prostate cancer originated PC-3 cells, through classic apoptosis signaling pathway, even using flow cytometry showed that the compound (+)-17 caused apoptosis of PC-3 cells more than 40 %, suggesting their potential therapeutic application in the development of novel drugs against prostate cancer.

SNAT2 is responsible for hyperosmotic induced sarcosine and glycine uptake in human prostate PC-3 cells.

Solute carriers (SLC) are important membrane transport proteins in normal and pathophysiological cells. The aim was to identify amino acid SLC(s) responsible for uptake of sarcosine and glycine in prostate cancer cells and investigate the impact hereon of hyperosmotic stress. Uptake of 14C-sarcosine and 3H-glycine was measured in human prostate cancer (PC-3) cells cultured under isosmotic (300 mOsm/kg) and hyperosmotic (500 mOsm/kg) conditions for 24 h. Hyperosmotic culture medium was obtained by supplementing the medium with 200 mM of the trisaccharide raffinose. Amino acid SLC expression was studied using RT-PCR, real-time PCR, and western blotting. siRNA knockdown of SNAT2 was performed. Experiments were conducted in at least 3 independent cell passages. The uptake of Sar and Gly was increased approximately 8-ninefold in PC-3 cells after 24 h hyperosmotic culture. PAT1 mRNA and protein could not be detected, while SNAT2 was upregulated at the mRNA and protein level. Transfection with SNAT2-specific siRNA reduced Vmax of Sar uptake from 2653 ± 38 to 513 ± 38 nmol mg protein-1 min-1, without altering the Km value (3.19 ± 0.13 vs. 3.42 ± 0.71 mM), indicating that SNAT2 is responsible for at least 80% of Sar uptake in hyperosmotic cultured PC-3 cells. SNAT2 is upregulated in hyperosmotic stressed prostate cancer cells and SNAT2 is responsible for cellular sarcosine and glycine uptake in hyperosmotic cultured PC-3 cells. Sar is identified as a substrate for SNAT2, and this has physiological implications for understanding cellular solute transport in prostate cancer cells.

Nrf2 antioxidant pathway and apoptosis induction and inhibition of NF-κB-mediated inflammatory response in human prostate cancer PC3 cells by Brassica oleracea var. acephala: An in vitro study.

BACKGROUND: Brassica oleracea var. acephala is a good source of health-promoting biologically active compounds like phenolics, vitamins, and glucosinolates. METHODS AND RESULTS: This in vitro research was conducted to evaluate the apoptotic, antioxidant, anti-inflammatory, and antiproliferative properties of ethanolic extract of Brassica oleracea var. acephala (EEBO) in PC3 prostate cancer cells. The LC-MS/MS technique was applied to identify the biomolecules of EEBO. The MTT assay was used to evaluate the cytotoxic effects of EEBO on PC3 cells. Moreover, qRT-PCR was used to examine the expression levels of Nrf2, NQO1, HO-1, NF-κB, TNF-α, IL-6, BAX, and BCL-2 in PC3 cell line. MMP was predicted by Rhodamine 123 staining, and release of cytochrome c was detected by an ELISA kit. Further, apoptosis was quantified by DNA fragmentation assay. The Western blotting method was used to detect the protein expression levels, and The DPPH assay was applied to determine the antioxidant effect of EEBO. The formula and structure of 19 biomolecules were predicted by LC-MS/MS. EEBO exhibited scavenging activity for DPPH. The MTT test showed EEBO reduced the viability of PC3 cells. The mRNA and protein levels of NRF2 pathway genes and BAX were increased, but those of the NF-κB pathway genes and BCL-2 were decreased in the EEBO-treated cells. Moreover, EEBO led to the diminution of MMP and enhanced the release of cytochrome c and DNA fragmentation, which resulted in apoptosis. CONCLUSIONS: Molecular changes due to the anticancer impact of EEBO on PC3 were involved in the induction of Nrf2 antioxidant pathway and apoptosis and inhibition of inflammation.

Inhibition of cancer cell-derived exosomal microRNA-183 suppresses cell growth and metastasis in prostate cancer by upregulating TPM1.

BACKGROUND: Emerging evidence continues to highlight the significant role of microRNAs (miRNAs) in the regulation of cancer growth and metastasis. Herein, the current study aimed to elucidate the role of exosomal miR-183 in prostate cancer development. METHODS: Initially, public microarray-based gene expression profiling of prostate cancer was employed to identify differentially expressed miRNAs. The putative target gene TPM1 of miR-183 was subsequently predicted, followed by the application of a luciferase reporter assay and examination of the expression patterns in prostate cancer patients and cell lines. The effects of miR-183 and TPM1 on processes such as cell proliferation, invasion and migration were evaluated using in vitro gain- and loss-of-function experiments. The effect of PC3 cells-derived exosomal miR-183 was validated in LNCaP cells. In vivo experiments were also performed to examine the effect of miR-183 on prostate tumor growth. RESULTS: High expression of miR-183 accompanied with low expression of TPM1 was detected in prostate cancer. Our data indicated that miR-183 could target and downregulate TPM1, with the overexpression of miR-183 and exosomal miR-183 found to promote cell proliferation, migration, and invasion in prostate cancer. Furthermore, the tumor-promoting effect of exosome-mediated delivery of miR-183 was subsequently confirmed in a tumor xenograft model. CONCLUSIONS: Taken together, the key findings of our study demonstrate that prostate cancer cell-derived exosomal miR-183 enhance prostate cancer cell proliferation, invasion and migration via the downregulation of TPM1, highlighting a promising therapeutic target against prostate cancer.

Quinazolin-4(3H)-one based agents bearing thiadiazole-urea: Synthesis and evaluation of anti-proliferative and antiangiogenic activity.

A series of quinazolin-4(3H)-one based agents containing thiadiazole-urea were designed, synthesized, and biologically evaluated. The proliferation rate of PC3 cells was moderately reduced by compound 9f (IC50 = 17.7 µM)which was comparable with sorafenib (IC50 = 17.3 µM). There was also a significant reduction in the number of HUVEC cells, when they were exposed to compound 9y (IC50 = 6.1 µM). To test the potential of compounds in inducing apoptosis, Annexin V-FITC/propidium iodide double staining assay was used. After the treatment of HUVEC cells with 9f, they underwent apoptotic effects. A substantial effort was dedicated to gathering comprehensive data across CAM assay. These data showed that 9f moderately inhibits the growth of corresponding blood vessels. Finally, the outcomes of Western blotting proposed a mechanism of action, by which the phosphorylation of VEGFR-2 is inhibited by compounds 9f and 9y.

Design, synthesis and antitumour evaluation of novel anthraquinone derivatives.

We report the design, synthesis, and biological evaluation of 13 new and 1 known anthraquinone derivatives which exerted cytotoxicity against PC3, A549 and NTUB1 cell lines. The results indicate that, among these 14, compounds-1 and 14 showed the highest growth inhibitory effect on NTUB1 and PC3 cells, respectively. Compound-1 at lower doses targets DNA, induces DNA damage and subsequently triggers G2/M arrest and apoptotic cell death at 24 h. Previously we reported that 14 induced PC3 cell autophagy and in treated PC3 cells, cleaved caspase-3 and cleaved PARP, and survivin did not increase and increase, respectively. The autophagic and necrotic cell deaths mediated by 14-triggered ROS generation. Our study is the first to investigate the biological mechanism of 14 action in detail. We find that when 14 was co-administrated with Bafilomycin A1 (BAF) in PC3 cells, rapid necrotic cell death occurred with no cleaved caspase-3 and cleaved PARP activation and increasing the expression of survivin. We further show that necrotic signaling in these cells coincided with production of reactive oxygen species. In the present study, we developed methods to synthesize five new 14 analogues for studing the structure-activity relationships. This study could provide valuable sight to find new antitumor agents for cancer therapy.

Immunocytochemical Analysis of Endogenous Frizzled-(Co-)Receptor Interactions and Rapid Wnt Pathway Activation in Mammalian Cells.

The differential activation of Wnt pathways (canonical: Wnt/ß-catenin; non-canonical: planar cell polarity (PCP), Wnt/Ca2+) depends on the cell-specific availability and regulation of Wnt receptors, called Frizzled (FZD). FZDs selectively recruit co-receptors to activate various downstream effectors. We established a proximity ligation assay (PLA) for the detection of endogenous FZD-co-receptor interactions and analyzed time-dependent Wnt pathway activation in cultured cells. Prostate cancer cells (PC-3) stimulated by Wnt ligands (Wnt5A, Wnt10B) were analyzed by Cy3-PLA for the co-localization of FZD6 and co-receptors (canonical: LRP6, non-canonical: ROR1) at the single-cell level. Downstream effector activation was assayed by immunocytochemistry. PLA allowed the specific (siRNA-verified) detection of FZD6-LRP6 and FZD6-ROR1 complexes as highly fluorescent spots. Incubation with Wnt10B led to increased FZD6-LRP6 interactions after 2 to 4 min and resulted in nuclear accumulation of ß-catenin within 5 min. Wnt5A stimulation resulted in a higher number of FZD6-ROR1 complexes after 2 min. Elevated levels of phosphorylated myosin phosphatase target 1 suggested subsequent Wnt/PCP activation in PC-3. This is the first study demonstrating time-dependent interactions of endogenous Wnt (co-)receptors followed by rapid Wnt/ß-catenin and Wnt/PCP activation in PC-3. In conclusion, the PLA could uncover novel signatures of Wnt receptor activation in mammalian cells and may provide new insights into involved signaling routes.

Estrogen Receptor Signaling Pathways Involved in Invasion and Colony Formation of Androgen-Independent Prostate Cancer Cells PC-3.

Castration-resistant prostate cancer (CRPC) is an advanced and androgen-independent form of prostate cancer. Recent studies of rapid actions mediated by estrogen in the prostate and its relationship with CRPC are emerging. We have previously shown that estrogen receptor (ER) promotes migration and invasion of the androgen-independent prostate cancer cells PC-3, but the signaling pathways involved in these events remain to be elucidated. Therefore, this study aimed to analyze the role of ERα and ERß in the activation of SRC, and the involvement of SRC and PI3K/AKT on invasion and colony formation of the PC-3 cells. Our results showed that the activation of ERα (using ERα-selective agonist PPT) and ERß (using ERß-selective agonist DPN) increased phosphorylation of SRC in PC-3 cells. In the presence of the selective inhibitor for SRC-family kinases PP2, the effects of DPN and PPT on transmigration and soft agar colony formation assays were decreased. Furthermore, SRC is involved in the expression of the non-phosphorylated ß-catenin. Finally, using PI3K specific inhibitor Wortmannin and AKT inhibitor MK2206, we showed that PI3K/AKT are also required for invasion and colony formation of PC-3 cells simulated by ER. This study provides novel insights into molecular mechanisms of ER in PC-3 cells by demonstrating that ER, located outside the cell nucleus, activates rapid responses molecules, including SRC and PI3K/AKT, which enhance the tumorigenic potential of prostate cancer cells, increasing cell proliferation, migration, invasion, and tumor formation.

The MTT Assay: Utility, Limitations, Pitfalls, and Interpretation in Bulk and Single-Cell Analysis.

The MTT assay for cellular metabolic activity is almost ubiquitous to studies of cell toxicity; however, it is commonly applied and interpreted erroneously. We investigated the applicability and limitations of the MTT assay in representing treatment toxicity, cell viability, and metabolic activity. We evaluated the effect of potential confounding variables on the MTT assay measurements on a prostate cancer cell line (PC-3) including cell seeding number, MTT concentration, MTT incubation time, serum starvation, cell culture media composition, released intracellular contents (cell lysate and secretome), and extrusion of formazan to the extracellular space. We also assessed the confounding effect of polyethylene glycol (PEG)-coated gold nanoparticles (Au-NPs) as a tested treatment in PC-3 cells on the assay measurements. We additionally evaluated the applicability of microscopic image cytometry as a tool for measuring intracellular MTT reduction at the single-cell level. Our findings show that the assay measurements are a result of a complicated process dependant on many of the above-mentioned factors, and therefore, optimization of the assay and rational interpretation of the data is necessary to prevent misleading conclusions on variables such as cell viability, treatment toxicity, and/or cell metabolism. We conclude, with recommendations on how to apply the assay and a perspective on where the utility of the assay is a powerful tool, but likewise where it has limitations.

Stimulation of neuroendocrine differentiation in prostate cancer cells by GHRH and its blockade by GHRH antagonists.

Prostate cancer is the second leading cause of cancer-related deaths among men in developed countries. Neuroendocrine prostate cancer, in particular, is associated with an aggressive phenotype and a poor prognosis. Neuroendocrine cells produce and secrete peptide hormones and growth factors in a paracrine/autocrine manner which promote the progression of the disease. Recent studies have demonstrated that extracellular vesicles or exosomes are released by prostate cancer cells, supporting the spread of prostate cancer. Hence, the aim of this study was to investigate the effect of growth hormone-releasing hormone (GHRH) on neuroendocrine differentiation (NED) in the androgen-dependent prostate cancer cell line LNCaP and the molecular mechanisms underlying these effects. GHRH induced an increase in the percentage of neurite-bearing cells and in the protein levels of Neuron-Specific Enolase. Both effects were blocked by the GHRH receptor antagonist MIA-690. In addition, pretreatment of these cells with the calcium chelator BAPTA, the EGFR inhibitor AG-1478 or the HER2 inhibitor AG-825 reduced the effect of GHRH, suggesting that the GHRH-induced stimulation of NED involves calcium channel activation and EGFR/HER2 transactivation. Finally, PC3-derived exosomes led to an increase in NED, cell proliferation and cell adhesion. Altogether, these findings suggest that GHRH antagonists should be considered for in the management of neuroendocrine prostate cancer.

Thioredoxin interacting protein (TXNIP) acts as a tumor suppressor in human prostate cancer.

Prostate cancer (PCa) is one of the most common malignant tumors in the world. Thioredoxin interacting protein (TXNIP) is downregulated in a variety of human tumors and plays an important role in tumor suppression. However, the expression level and biological functions of TXNIP in PCa have not been identified yet. Therefore, this study aims to investigate the expression and biological functions of TXNIP in PCa. We reported that the expression of TXNIP was significantly decreased in PCa and associated with clinicopathological features. Overexpression of TXNIP could significantly inhibited PC-3 cells proliferation, migration, invasion, and glucose uptake. Additionally, overexpression of TXNIP could remarkably block cell cycle in the G0/G1 phase and promoted cell apoptosis. Furthermore, TXNIP expression correlated inversely with GLUT1 expression in PCa. Taken together, our results for the first time revealed that TXNIP was decreased in PCa. Moreover, TXNIP might act as a tumor suppressor of PCa and correlated with tumor occurrence and development. Our findings cast a new light on better understanding the occurrence and development of PCa and indicated that TXNIP might be favorable for PCa molecular target therapy.

Curcumin stimulates angiogenesis through VEGF and expression of HLA-G in first-trimester human placental trophoblasts.

Curcumin has a protective role in placental diseases like preeclampsia and preterm birth. Very little is known about its functional effects on growth, angiogenesis, and epigenetic activities of human first trimester placenta. HTR8/SVneo trophoblasts cells were used as model for human first trimester placenta. Effects of curcumin (≥80%) in these cells were investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), radioactive thymidine uptake, quantitative real-time polymerase chain reaction (qRT-PCR), promoter DNA methylation, qRT-PCR array, tube formation, wound healing, and immunoblot assays. PC3 (prostate cancer), JEG-3 (trophoblast), and HMEC-1 (endothelial) cells were used as control in various experiments. Unlike in PC3 cells, curcumin stimulated growth, proliferation, and viability in HTR8/SVneo cells. Curcumin increased tube formation, and messenger RNA (mRNA) expression of angiogenic factors such as vascular endothelial growth factor A (VEGFA) and protein expression of proangiogenic factor VEGF receptor-2 and fatty acid-binding protein-4 (FABP4) in these cells. Curcumin-stimulated tube formation was associated with an increased expression of VEGFR2 and FABP4. The stimulatory effects of curcumin were inhibited by VEGFR2 (SU5416) and FABP4 (BMS309403) inhibitors. Curcumin also significantly increased both mRNA and protein expression of HLA-G in HTR8/SVneo cells. Curcumin increased mRNA expression of DNMT3A and NOTCH signaling system whereas down-regulated mRNA expression of HSD11ß2. Curcumin enhanced hypomethylation of gene promoters against oxidative stress and DNA damage pathway mediators. Curcumin promotes cell growth, migration, and thus angiogenic potential of these cells. Increased expression of HLA-G by curcumin, hitherto unknown, is a novel finding since HLA-G not only favors the immune environment for invasive trophoblasts but also positively modulates angiogenesis.

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.

Silencing of carbonic anhydrase I enhances the malignant potential of exosomes secreted by prostatic tumour cells.

We report results showing that the silencing of carbonic anhydrase I (siCA1) in prostatic (PC3) tumour cells has a significant impact on exosome formation. An increased diameter, concentration and diversity of the produced exosomes were noticed as a consequence of this knock-down. The protein composition of the exosomes' cargo was also altered. Liquid chromatography and mass spectrometry analyses identified 42 proteins significantly altered in PC3 siCA1 exosomes compared with controls. The affected proteins are mainly involved in metabolic processes, biogenesis, cell component organization and defense/immunity. Interestingly, almost all of them have been described as 'enhancers' of tumour development through the promotion of cell proliferation, migration and invasion. Thus, our results indicate that the reduced expression of the CA1 protein enhances the malignant potential of PC3 cells.

PROSTAGLANDIN E2 stimulates cancer-related phenotypes in prostate cancer PC3 cells through cyclooxygenase-2.

Cyclooxygenase (COX)-derived prostaglandin E2 (PGE2 ) affects many mechanisms that have been shown to play roles in carcinogenesis. Recently, we found that, in androgen-independent prostate cancer PC3 cells, PGE2 acts through an intracrine mechanism by which its uptake by the prostaglandin transporter (PGT) results in increased intracellular PGE2 (iPGE2 ), leading to enhanced cell proliferation, migration, invasion, angiogenesis, and loss of cell adhesion to collagen I. These iPGE2 -mediated effects were dependent on hypoxia-inducible factor 1-α (HIF-1α), whose expression increased upon epidermal growth factor receptor (EGFR) transactivation by a subset of intracellular PGE2 receptors. Here, we aimed to study the role of COX in PGE2 protumoral effects in PC3 cells and found that the effects were prevented by inhibition of COX-2, which highlights its crucial role amplifying the levels of iPGE2 . Treatment with exogenous PGE2 determined a transcriptional increase in COX-2 expression, which was abolished by genetic or pharmacologic inhibition of PGT. PGE2 -induced increase in COX-2 expression and, thereby, in transcriptional increase in HIF-1α expression was due to EGFR activation, leading to the activation of Phosphoinositide 3-kinase/Akt, Extracellular signal -regulated kinases 1/2, p38 and Mitogen- and stress-activated protein kinase-1 (PI3K/Akt, Erk1/2, p38 and MSK-1). Collectively, the data suggest that EGFR-dependent COX-2 upregulation by a novel positive feedback loop triggered by iPGE2 underlies the intracrine pro-tumoral effects of PGE2 in PC3 cells. Therefore, this feedback loop may be relevant in prostate cancer for the maintenance of PGE2 -dependent cancer cell growth through amplifying the activity of the COX-2 pathway.

Evaluation of the anticancer and anti-metastasis effects of novel synthetic sodium channel blockers in prostate cancer cells in vitro and in vivo.

BACKGROUND: Voltage-gated sodium channels (VGSCs) are involved in several cellular processes related to cancer cell growth and metastasis, including adhesion, proliferation, apoptosis, migration, and invasion. We here in investigated the effects of S0154 and S0161, two novel synthetic sodium channel blockers (SCBs), on human prostate cancer cells (PC3, DU145, and LnCaP) and a prostate cancer xenograft model. METHODS: The MTT assay was used to assess the anticancer effects of SCBs in PC3, DU145, and LnCaP cells. Sodium indicator and glucose uptake assays were used to determine the effects of S0154 and S0161 in PC3 cells. The impact of these SCBs on the proliferation, cell cycle, apoptosis, migration, and invasion of PC3 cells were determined using a CFDA-SE cell proliferation assay, cell cycle assay, annexin V-FITC apoptosis assay, transwell cell invasion assay, and wound-healing assay, respectively. The protein expression levels of Nav1.6, Nav1.7, CDK1, cyclin B1, MMP2, MMP9 in PC3 cells were analysis by Western blotting. The in vivo anticancer activity was evaluated using a PC3 xenograft model in nude mice. RESULTS: S0154 and S0161 both showed anticancer and anti-metastatic effects against prostate cancer cells and significantly inhibited cell viability, with IC50 values in the range of 10.51-26.60 µmol/L (S0154) and 5.07-11.92 µmol/L (S0161). Both compounds also increased the intracellular level of sodium, inhibited the protein expression of two α subunits of VGSCs (Nav1.6 and Nav1.7), and caused G2/M phase cell cycle arrest, with no or minor effects on cell apoptosis. Concentrations of 5 and 10 µmol/L of S0154 and S0161 significantly decreased the glucose uptake of PC3 cells. The compounds also inhibited the proliferation of PC3 cells and decreased their invasion in transwell assays. Furthermore, S0161 exerted antitumor activity in an in vivo PC3 xenograft model in nude mice, inhibiting the growth of the tumors by about 51% compared to the control group. CONCLUSIONS: These results suggest that S0154 and S0161 have anticancer and anti-metastasis effects in prostate cancer cells both in vitro and in vivo, supporting their further development as potential therapeutic agents for prostate cancer.