Induction of promyelocytic leukemia zinc finger protein by miR-200c-3p restores sensitivity to anti-androgen therapy in androgen-refractory prostate cancer and inhibits the cancer progression via down-regulation of integrin α3ß4.

PURPOSE: Androgen-refractory prostate cancer (ARPC) is one of the aggressive human cancers with metastatic capacity and resistance to androgen deprivation therapy (ADT). The present study investigated the genes responsible for ARPC progression and ADT resistance, and their regulatory mechanisms. METHODS: Transcriptome analysis, co-immunoprecipitation, confocal microscopy, and FACS analysis were performed to determine differentially-expressed genes, integrin α3ß4 heterodimer, and cancer stem cell (CSC) population. miRNA array, 3'-UTR reporter assay, ChIP assay, qPCR, and immunoblotting were used to determine differentially-expressed microRNAs, their binding to integrin transcripts, and gene expressions. A xenograft tumor model was used to assess tumor growth and metastasis. RESULTS: Metastatic ARPC cell lines (PC-3 and DU145) exhibiting significant downregulation of ZBTB16 and AR showed significantly upregulated ITGA3 and ITGB4. Silencing either one of the integrin α3ß4 heterodimer significantly suppressed ARPC survival and CSC population. miRNA array and 3'-UTR reporter assay revealed that miR-200c-3p, the most strongly downregulated miRNA in ARPCs, directly bound to 3'-UTR of ITGA3 and ITGB4 to inhibit the gene expression. Concurrently, miR-200c-3p also increased PLZF expression, which, in turn, inhibited integrin α3ß4 expression. Combination treatment with miR-200c-3p mimic and AR inhibitor enzalutamide showed synergistic inhibitory effects on ARPC cell survival in vitro and tumour growth and metastasis of ARPC xenografts in vivo, and the combination effect was greater than the mimic alone. CONCLUSION: This study demonstrated that miR-200c-3p treatment of ARPC is a promising therapeutic approach to restore the sensitivity to anti-androgen therapy and inhibit tumor growth and metastasis.

Megakaryocyte-Derived IL-8 Acts as a Paracrine Factor for Prostate Cancer Aggressiveness through CXCR2 Activation and Antagonistic AR Downregulation.

Prostate cancer is the fifth leading cause of cancer-related mortality in men, primarily because of treatment resistance, recurrence, and metastasis. In the present study, we investigated the role of paracrine interleukin-8 (IL-8) in the antagonistic expression of IL-8 and androgen receptor (AR), and the contribution of IL-8 to prostate cancer aggressiveness. In hormone-responsive LNCaP cells that do not express IL-8, recombinant IL-8 treatment significantly increased expressions of IL-8, CXC chemokine receptor 2 (CXCR2), matrix metalloproteinase (MMP)-2/9, Snail, and vimentin. IL-8 treatment significantly decreased AR and E-cadherin expression. IL-8-induced gene expression changes were suppressed by navarixin, a CXCR1/2 inhibitor, and gallein, a Gßγ inhibitor. In PC-3 androgen-refractory prostate cancer cells, IL-8 knockdown reduced expressions of CXCR2, MMP-2/9, Snail, and vimentin, and increased AR and E-cadherin expressions at the mRNA and protein levels. Co-culture with MEG-01 human megakaryocytic cells secreting high levels of IL-8 induced gene expression changes in both LNCaP and PC-3 cells, similar to those induced by IL-8 treatment. The altered gene expressions were accompanied by significant activation of transcription factor Snail in LNCaP and PC-3 cells. Treatment with the CXCR blocker navarixin inhibited the invasion of PC-3 cells but not LNCaP cells. However, invasion induced by MEG-01 was inhibited by navarixin in both LNCaP and PC-3 cells. The collective findings demonstrate that IL-8 enhances CXCR2 expression, which antagonistically regulates AR expression. More importantly, through changes in IL-8/CXCR2-regulated gene expression, IL-8 induces antiandrogen therapy resistance and epithelial-mesenchymal transition in prostate cancer.

Synthesis and anticancer evaluation of 6-azacyclonol-2,4,6-trimethylpyridin-3-ol derivatives: M3 muscarinic acetylcholine receptor-mediated anticancer activity of a cyclohexyl derivative in androgen-refractory prostate cancer.

We recently reported 2,4,5-trimethylpyridin-3-ol with C(6)-azacyclonol, whose code name is BJ-1207, showing a promising anticancer activity by inhibiting NOX-derived ROS in A549 human lung cancer cells. The present study was focused on structural modification of the azacyclonol moiety of BJ-1207 to find a compound with better anticancer activity. Ten new compounds (3A-3J) were prepared and evaluated their inhibitory actions against proliferation of eighteen cancer cell lines as a primary screening. Among the ten derivatives of BJ-1207, the effects of compounds 3A and 3J on DU145 and PC-3, androgen-refractory cancer cell lines (ARPC), were greater than the parent compound, and compound 3A showed better activity than 3J. Antitumor activity of compound 3A was also observed in DU145-xenografted chorioallantoic membrane (CAM) tumor model. In addition, the ligand-based target prediction and molecular docking study using DeepZema® server showed compound 3A was a ligand to M3 muscarinic acetylcholine receptor (M3R) which is overexpressed in ARPC. Carbachol, a muscarinic receptor agonist, concentration dependently increased proliferation of DU145 in the absence of serum, and it also activated NADPH oxidase (NOX). The carbachol-induced proliferation and NOX activity was significantly blocked by compounds 3A in a concentration-dependent manner. This finding might become a new milestone in the development of pyridinol-based anti-cancer agents against ARPC.

Inhibition of colitis by ring-modified analogues of 6-acetamido-2,4,5-trimethylpyridin-3-ol.

6-Aminopyridin-3-ol scaffold has shown an excellent anti-inflammatory bowel disease activity. Various analogues with the scaffold were synthesized in pursuit of the diversity of side chains tethering on the C(6)-position. Structure-activity relationship among the analogues was investigated to understand the effects of the side chains and their linkers on their anti-inflammatory activities. In this study, structural modification moved beyond side chains on the C(6)-position and reached to pyridine ring itself. It expedited us to synthesize diverse ring-modified analogues of a representative pyridine-3-ol, 6-acetamido-2,4,5-trimethylpyridin-3-ol (9). In the evaluation of compounds on their inhibitory actions against TNF-α-induced adhesion of monocytic cells to colonic epithelial cells, an in vitro model mimicking colon inflammation, the effects of compounds 9, 17, and 19 were greater than tofacitinib, an orally available anti-colitis drug, and compound 17 showed the greatest activity. In addition, TNF-α-induced angiogenesis, which permits more inflammatory cell migration into inflamed tissues, was significantly blocked by compounds 17 and 19 in a concentration-dependent manner. In the comparison of in vivo therapeutic effects of compounds 9, 17, and 19 on dextran sulfate sodium (DSS)-induced colitis in mice, compound 17 was the most potent and efficacious, and compound 19 was better than compound 9 which showed a similar degree of inhibitory effect to tofacitinib. Taken together, it seems that either the trimethyl system or the hydroxyl group on the pyridinol ring is essential to the activity. This finding might become a new milestone in the development of pyridinol-based anti-inflammatory bowel disease agents.

Potent Inhibitory Effect of BJ-3105, a 6-Alkoxypyridin-3-ol Derivative, on Murine Colitis Is Mediated by Activating AMPK and Inhibiting NOX.

Inflammatory bowel disease (IBD) is a chronic relapsing inflammation in the gastrointestinal tract. Biological therapeutics and orally available small molecules like tofacitinib (a JAK inhibitor) have been developed to treat IBD, but half of the patients treated with these drugs fail to achieve sustained remission. In the present study, we compared the therapeutic effects of BJ-3105 (a 6-alkoxypyridin-3-ol derivative) and tofacitinib in IBD. BJ-3105 induced activation of AMP-activated protein kinase (AMPK) in the kinase activity measurement and recovery from cytokine-induced AMPK deactivation in HT-29 human colonic epithelial cells. Similar to tofacitinib and D942 (an AMPK activator), BJ-3105 inhibited IL-6-induced JAK2/STAT3 phosphorylation and TNF-α-stimulated activation of IKK/NF-κB, and consequently, stimulus-induced upregulations of inflammatory cytokines and inflammasome components. In addition, unlike tofacitinib or D942, BJ-3105 inhibited NADPH oxidase (NOX) activation and consequent superoxide production induced by activators (mevalonate and geranylgeranyl pyrophosphate) of the NOX cytosolic component Rac. In mice, oral administration with BJ-3105 ameliorated dextran sulfate sodium (DSS)-induced colitis and azoxymethane/DSS-induced colitis-associated tumor formation (CAT) much more potently than that with tofacitinib. Moreover, BJ-3105 suppressed the more severe form of colitis and CAT formation in mice with AMPK knocked-out in macrophages (AMPKαfl/fl-Lyz2-Cre mice) with much greater efficacy than tofacitinib. Taken together, our findings suggest BJ-3105, which exerted a much better anti-colitis effect than tofacitinib through AMPK activation and NOX inhibition, is a promising candidate for the treatment of IBD.

Antitumor activity of BJ-1207, a 6-amino-2,4,5-trimethylpyridin-3-ol derivative, in human lung cancer.

Enhanced expression of NADPH oxidase (NOX) and the subsequent production of reactive oxygen species (ROS) are associated with lung cancer. In the present study, fifty 6-amino-2,4,5-trimethylpyridin-3-ol derivatives were screened for anticancer activity by targeting NOX2-derived ROS. The compounds suppressed ROS production and decreased cancer cell viability (R2 = 0.79). Among the derivatives, the compound coded BJ-1207, which contained a 4-(hydroxydiphenylmethyl)piperidine moiety, exhibited the most effective anticancer activity against A549 lung cancer cell line and eight other cancer cell lines, including H1299, MCF-7, MDA-MB-231, HT-29, SW620, Mia PaCa-2, PANC-1, and U937. BJ-1207 also showed significantly lower inhibitory effects on kinase insert domain receptor (KDR) and c-KIT tyrosine kinase but higher inhibitory activity on NOX than those of sunitinib, a multi-receptor tyrosine kinase (RTK) inhibitor. In addition, BJ-1207-induced inhibition of RTK-downstream signaling pathways, such as ROS production, and expression of target genes, such as stem cell factor and transforming growth factor-α, were similar to those induced by sunitinib. In the xenograft chick tumor model, BJ-1207 inhibited lung tumor growth to a similar or much greater extent than that of sunitinib or cisplatin, respectively. Overall, the present study showed that BJ-1207, a vitamin B6-derived 2,4,5-trimethylpyridin-3-ol compound with azacyclonol moiety at C (6)-position of the pyridine ring, inhibited NOX activity and that it is a promising lead compound for developing anticancer drugs against lung cancer.

Correction to: ß-Catenin gene promoter hypermethylation by reactive oxygen species correlates with the migratory and invasive potentials of colon cancer cells.

In the original version of above mentioned article an error occurred in Fig. 2. Panel g and panel h are included in the figure legend, but have not been published in the figure.

ß-Catenin gene promoter hypermethylation by reactive oxygen species correlates with the migratory and invasive potentials of colon cancer cells.

PURPOSE: Over half of the colon cancer patients suffer from cancer-related events, mainly metastasis. Loss of ß-catenin activity has previously been found to facilitate cancer cell dissociation and migration. Here, we aimed to investigate whether epigenetic silencing of ß-catenin induces human colon cancer cell migration and/or invasion. METHODS: HCT-116, Caco-2, HT-29 and SW620 cell migration and invasion capacities were assessed using scratch wound healing and Matrigel invasion assays, respectively. Confocal microscopy, qRT-PCR and Western blotting were performed to determine gene expression levels, whereas methylation-specific quantitative real-time PCR was used to assess the extent of ß-catenin gene (CTNNB1) promoter methylation after treatment of the cells with TPA, hydrogen peroxide, 5-aza-2'-deoxycytidine and/or VAS2870. RESULTS: We found that treatment of HT-29 and Caco-2 cells (differentiated and low metastatic) with 12-O-tetradecanoyl phorbol-13-acetate (TPA; a tumor promoter) suppressed E-cadherin and ß-catenin expression at both the mRNA and protein levels and, in addition, enhanced cell migration. Furthermore, we found that the CTNNB1 gene promoter methylation levels were higher in the more invasive HCT-116 and SW620 colon cancer cells than in HT-29 and CCD-841 (normal colon epithelial) cells. We also found that TPA or hydrogen peroxide induced CTNNB1 gene promoter methylation to a higher extent in HT-29 and CCD-841 cells than in HCT-116 and SW620 cells, and that the degree of CTNNB1 gene promoter methylation positively correlated with cell dissociation and migration. In addition, we found that co-treatment with 5-aza-2'-deoxycytidine (decitabine, a DNA methyl transferase inhibitor) and VAS2870 (a NADPH oxidase inhibitor) almost completely blocked the invasion of TPA-treated HT-29 and TPA-untreated HCT-116 and SW620 cells, and that these inhibitions surpassed those of the cells treated with decitabine or VAS2870 alone. CONCLUSIONS: From our data we conclude that the extent of CTNNB1 gene promoter methylation by reactive oxygen species correlates with the migratory and invasive abilities of colon cancer cells. Our results suggest that epigenetic regulation of CTNNB1 may serve as a novel avenue to block colon cancer cell migration and invasion.