Lysine-Specific Histone Demethylase 1 Promotes Oncogenesis of the Esophageal Squamous Cell Carcinoma by Upregulating DUSP4.

Esophageal squamous cell carcinoma (ESCC) is a predominant subtype of esophageal cancer (EC) and has a poor prognosis due to its aggressive nature. Accordingly, it is necessary to find novel prognostic biomarkers and therapeutic targets for ESCC. Lysine-specific histone demethylase 1 (LSD1) plays a core role in the regulation of ESCC oncogenesis. However, the detailed mechanism of LSD1-regulated ESCC growth has not been elucidated. This study aims to explore molecular mechanism underlying the LSD1-regulated ESCC's oncogenesis. After LSD1 silencing, we detected differentially expressed genes (DEGs) in human ESCC cell line, TE-1, by transcriptome sequencing. Subsequently, we investigated expression pattern of the selected molecules in the ESCC tissues and cell lines by qRT-PCR and Western blotting. Furthermore, we explored the roles of selected molecules in ESCC using gene silencing and overexpression assays. Transcriptome sequencing showed that the expression of dual specificity phosphatase 4 (DUSP4) in TE-1 was significantly attenuated after the LSD1 silencing. In addition, the DUSP4 mRNA expression level was significantly higher in the ESCC tissues, especially in those derived from patients with invasion or metastasis. Moreover, the DUSP4 expression was positively associated with the LSD1 expression in the ESCC tissues. DUSP4 overexpression promoted proliferation, invasion, and migration of the ESCC cells, while DUSP4 silencing had an opposite effect. DUSP4 overexpression also enhanced tumorigenicity of the ESCC cells in vivo, while DUSP4 silencing inhibited tumor growth. Importantly, inhibition of cell proliferation, invasion, and migration by the LSD1 inhibitor (ZY0511) was reversed by DUSP4 overexpression. Conclusively, we found that LSD1 promotes ESCC's oncogenesis by upregulating DUSP4, the potential therapeutic and diagnostic target in ESCC.

Evaluation of exosomal miR-9 and miR-155 targeting PTEN and DUSP14 in highly metastatic breast cancer and their effect on low metastatic cells.

Breast cancer is one of the most prevalent cancers in women. Triple-negative breast cancer consists 15% to 20% of breast cancer cases and has a poor prognosis. Cancerous transformation has several causes one of which is dysregulation of microRNAs (miRNAs) expression. Exosomes can transfer miRNAs to neighboring and distant cells. Thus, exosomal miRNAs can transfer cancerous phenotype to distant cells. We used gene expression omnibus (GEO) datasets and miRNA target prediction tools to find overexpressed miRNA in breast cancer cells and their target genes, respectively. Exosomes were extracted from MDA-MB-231 and MCF-7 cells and characterized. Overexpression of the miRNAs of MDA-MB-231 cells and their exosomes were analyzed using quantitative Real-time PCR. The target genes expression was also evaluated in the cell lines. Luciferase assay was performed to confirm the miRNAs: mRNAs interactions. Finally, MCF-7 cells were treated with MDA-MB-231 cells' exosomes. The target genes expression was evaluated in the recipient cells. GSE60714 results indicated that miR-9 and miR-155 were among the overexpressed miRNAs in highly metastatic triple negative breast cancer cells and their exosomes. Bioinformatic studies showed that these two miRNAs target PTEN and DUSP14 tumor suppressor genes. Quantitative Real-time PCR confirmed the overexpression of the miRNAs and downregulation of their targets. Luciferase assay confirmed that the miRNAs target PTEN and DUSP14. Treatment of MCF-7 cells with MDA-MB-231 cells' exosomes resulted in target genes downregulation in MCF-7 cells. We found that miR-9 and miR-155 were enriched in metastatic breast cancer exosomes. Therefore, exosomal miRNAs can transfer from cancer cells to other cells and can suppress their target genes in the recipient cells.

MiR-122-5p inhibits cell migration and invasion in gastric cancer by down-regulating DUSP4.

OBJECTIVE: To explore the relationship between miR-122-5p and DUSP4 and their effects on gastric cancer (GC) cell mobility and invasiveness. METHODS: Abnormally expressed miRNAs and mRNAs were analyzed using microarrays. The miR-122-5p and DUSP4 mRNA expression levels in GC tissues and cells were determined by RT-qPCR. The target relationship between miR-122-5p and DUSP4 was validated by dual luciferase reporter assay. GC cell mobility and invasiveness were respectively observed by wound healing assay and transwell invasion assay. Western blot and immunohistochemistry were used for detection of the expressions of DUSP4 protein and MMP2 and MMP9 proteins related to cell invasion and migration. The migration and invasion abilities of gastric cancer cells in vivo were evaluated according to the number of lung metastatic nodules in mice. RESULTS: The expression of miR-122-5p in GC tissues and cells was significantly down-regulated, whereas DUSP4 expression was up-regulated. Bioinformatics prediction strategies and dual luciferase reporter assay verified the binding sites of miR-122-5p on 3'UTR of DUSP4 and the target relationship between miR-122-5p and DUSP4. Overexpression of miR-122-5p and knockdown of DUSP4 in BGC-823 cells observantly suppressed GC cell mobility and invasiveness, whereas downregulation of miR-122-5p expression promoted cell metastasis. MiR-122-5p inhibited GC cell mobility and invasiveness and pulmonary tumor metastasis via downregulation of DUSP4. CONCLUSION: MiR-122-5p restrained migration and invasion abilities of GC cells by repressing DUSP4.

Nimbolide suppresses non-small cell lung cancer cell invasion and migration via manipulation of DUSP4 expression and ERK1/2 signaling.

Nimbolide plays an important role in treating human diseases. In these years, the anticancer property of nimbolide has been paid more and more attention. However, the role of nimbolide in non-small cell lung cancer (NSCLC) remains unclear. In this study, we found that nimbolide treatment suppressed the invasion and migration of NSCLC cells, in a dose-dependent manner. Moreover, nimbolide treatment dose-dependently inhibited ERK1/2 activation, decreased Snail and MMP-3 expression, and increased E-cadherin expression. Further, we found that nimbolide treatment upregulated DUSP4 expression. DUSP4 knockdown attenuated nimbolide-mediated inhibition of cell invasion, migration and ERK1/2 activation. We also found that DUSP4 knockdown suppressed the effect of nimbolide on MMP-3, Snail and E-cadherin expression. Taken together, our study demonstrates that nimbolide treatment can upregulate the expression of DUSP4, thus inhibiting ERK1/2 activation. Inhibition of ERK1/2 pathway by nimbolide decreases MMP-3 and Snail expression, and increases E-cadherin expression, which finally inhibits NSCLC cell invasion and migration. Therefore, nimbolide may act as a novel drug to inhibit NSCLC invasion and metastasis through manipulation of ERK1/2 signaling and DUSP4 expression.

Dual-specificity phosphatase 26 (DUSP26) stimulates Aß42 generation by promoting amyloid precursor protein axonal transport during hypoxia.

Amyloid beta peptide (Aß) is a pathological hallmark of Alzheimer's disease (AD) and is generated through the sequential cleavage of amyloid precursor protein (APP) by ß- and γ-secretases. Hypoxia is a known risk factor for AD and stimulates Aß generation by γ-secretase; however, the underlying mechanisms remain unclear. In this study, we showed that dual-specificity phosphatase 26 (DUSP26) regulates Aß generation through changes in subcellular localization of the γ-secretase complex and its substrate C99 under hypoxic conditions. DUSP26 was identified as a novel γ-secretase regulator from a genome-wide functional screen using a cDNA expression library. The phosphatase activity of DUSP26 was required for the increase in Aß42 generation through γ-secretase, but this regulation did not affect the amount of the γ-secretase complex. Interestingly, DUSP26 induced the accumulation of C99 in the axons by stimulating anterograde transport of C99-positive vesicles. Additionally, DUSP26 induced c-Jun N-terminal kinase (JNK) activation for APP processing and axonal transport of C99. Under hypoxic conditions, DUSP26 expression levels were elevated together with JNK activation, and treatment with JNK inhibitor SP600125, or the DUSP26 inhibitor NSC-87877, reduced hypoxia-induced Aß generation by diminishing vesicle trafficking of C99 to the axons. Finally, we observed enhanced DUSP26 expression and JNK activation in the hippocampus of AD patients. Our results suggest that DUSP26 mediates hypoxia-induced Aß generation through JNK activation, revealing a new regulator of γ-secretase-mediated APP processing under hypoxic conditions. We propose the role of phosphatase dual-specificity phosphatase 26 (DUSP26) in the selective regulation of Aß42 production in neuronal cells under hypoxic stress. Induction of DUSP26 causes JNK-dependent shift in the subcellular localization of γ-secretase and C99 from the cell body to axons for Aß42 generation. These findings provide a new strategy for developing new therapeutic targets to arrest AD progression.

Decreased expression of dual specificity phosphatase 22 in colorectal cancer and its potential prognostic relevance for stage IV CRC patients.

Dual specificity phosphatase 22 (DUSP22) is a novel dual specificity phosphatase that has been demonstrated to be a cancer suppressor gene associated with numerous biological and pathological processes. However, little is known of DUSP22 expression profiling in colorectal cancer and its prognostic value. Our study aims to investigate the role of DUSP22 expression in the prognosis of colorectal cancer. We detected the mRNA expression in 92 paired primary colorectal cancer tissues and the corresponding adjacent normal tissues by using QuantiGenePlex assay. The Friedman test was used to determine the statistical difference of gene expression. Kaplan-Meier survival analysis was performed. Mann-Whitney test and Kruskal-Wallis test were used to conduct data analyses to determine the prognostic value. Statistical significance was set at P < 0.05. In 74 of 92 cases, DUSP22 mRNA was reduced in primary colorectal cancer tissues, compared to the adjacent normal tissues. The mRNA levels of DUSP22 were significantly lower in colorectal cancer tissues than in adjacent normal tissues (0.0290 vs. 0.0658; P < 0.001). Low expression of DUSP22 correlated significantly with large tumor size (P = 0.013). No association was observed between DUSP22 mRNA expression and differentiation, histopathological type, tumor invasion, lymph node metastases, metastases, TNM stage, and Duke's phase (all P > 0.05). Kaplan-Meier analysis indicated that DUSP22 expression had no significant relationship with overall survival in all patients (P > 0.05). Interestingly, low expression level of DUSP22 in stage IV patients had a poor survival measures with a marginal P value (P = 0.07). Reduced DUSP22 expression was found in colorectal cancer specimens. Low expression level of DUSP22 in stage IV patients had a poor survival outcome. Further study is required for the investigation of the role of DUSP22 in colorectal cancer.

Epigenetic silencing of DUSP9 induces the proliferation of human gastric cancer by activating JNK signaling.

Dual-specificity phosphatase 9 (DUSP9) is a strong negative regulator of transcription factor activating kinases (ERK, JNK and p38) in the mitogen-activated protein kinase (MAPK) pathways. The aim of this study was to examine the CpG island methylation status of DUSP9 using bisulfite sequencing PCR (BSP) in gastric cancer (GC). The investigation was conducted on 30 clinical GC samples and selected corresponding tumor-free normal gastric mucosa tissues, using BSP for the determination of the promoter methylation status. The methylation status of the tumor samples was compared to the corresponding tumor-free samples. DUSP9 was silenced by promoter region hypermethylation and G2/M phase arrest was induced by DUSP9 in the MKN-1 GC cell line. MKN-1 proliferation was suppressed by DUSP9 by inhibiting c-Jun, which was induced by JNK signaling. The expression levels of CCND1, c-Jun, CDK4 and CDK6 were upregulated while p21 was downregulated by DUSP9 in MKN-1 cells. However, DUSP9-induced resulted in the regulation of the levels of cycle-related molecules, whivh were inhibited when the JNK inhibitor SP600125 was added. In conclusion, DUSP9 was frequently methylated in human GC and the expression of DUSP9 is silenced by promoter region hypermethylation. The results of this study, combined with previous studies, suggested that therapeutic intervention to increase the expression or activity of DUSP9 may enable the activation of anti-proliferation signals in malignant cells.

Dual specificity phosphatase 4 gene expression in association with triple-negative breast cancer outcome.

Triple-negative breast cancer (TNBC) is an aggressive cancer with limited treatment options. Dual specificity phosphatase 4 (DUSP4) has recently been suggested as a potential marker of chemotherapy resistance for TNBC. DUSP4 gene expression levels were measured in breast cancer tissue from 469 TNBC patients aged 20-75 years who participated in the Shanghai Breast Cancer Survival Study, and their association with recurrence/breast cancer mortality and total mortality was evaluated. Information on breast cancer diagnosis, treatment, and disease progression was collected via medical chart review and multiple in-person follow-up surveys. A Cox regression model was applied in the data analyses. Over a median follow-up of 5.3 years (range: 0.7-8.9 years), 100 deaths and 92 recurrences/breast cancer deaths were documented. Expression levels of transcript variant 1 (NM_001394) and transcript variant 2 (NM_057158) of the DUSP4 gene were studied and were highly correlated (r = 0.76). Low DUSP4 expression levels, particularly of variant 1, were associated with both increased recurrence/breast cancer mortality and increased overall mortality. Hazard ratios with adjustment for age at diagnosis and TNM stage associated with below versus above the median expression level were 1.97 (95 % confidence interval (CI): 1.27-3.05) for recurrence/breast cancer mortality and 2.09 (95 % CI: 1.38-3.17) for overall mortality. Additional adjustment for expression levels of MKI67 and TP53, common treatment types, breast cancer subtype, and grade did not materially alter the observed associations. Low DUSP4 expression levels predict recurrence and mortality in TNBC patients independently from known clinical and molecular predictors.

Microarray phosphatome profiling of breast cancer patients unveils a complex phosphatase regulatory role of the MAPK and PI3K pathways in estrogen receptor-negative breast cancers.

Phosphatases are proteins with the ability to dephosphorylate different substrates and are involved in critical cellular processes such as proliferation, tumor suppression, motility and survival. Little is known about their role in the different breast cancer (BC) phenotypes. We carried out microarray phosphatome profiling in 41 estrogen receptor-negative (ER-) BC patients, as determined by immunohistochemistry (IHC), containing both ERBB2+ and ERBB2- in order to characterize the differences between these two groups. We characterized and confirmed the distinct phosphatome of the two main ER- BC subgroups (in two independent microarrays series) and that of ER+ BC (in three large independent series). Our findings point to the importance of the MAPK and PI3K pathways in ER- BCs as some of the most differentially expressed phosphatases (like DUSP4 and DUSP6) sharing ERK as substrate, or regulating the PI3K pathway (INPP4B, PTEN). It was possible to identify a selective group of phosphatases upregulated only in the ER- ERBB2+ subgroup and not in ER+ (like DUSP6, DUSP10 and PPAPDC1A among others), suggesting a role of these phosphatases in specific BC subtypes, unlike other differentially expressed phosphatases (DUSP4 and ENPP1) that seemed to have a role in multiple BC subtypes. Significant correlation was found at the protein level by IHC between the expression of DUSP6 and phospho-ERK (p=0.04) but not of phospho-ERK with DUSP4. To show the potential prognostic relevance of phosphatases as a functional group of genes, we derived and validated in two large independent BC microarray series a multiphosphatase signature enriched in differentially expressed phosphatases, to predict distant metastasis-free survival (DMFS). ER- ERBB2+, ER- ERBB2- and ER+ BC patients have a distinct pattern of phosphatase RNA expression with a potential prognostic relevance. Further studies of the most relevant phosphatases found in this study are warranted.

DUSP28 contributes to human hepatocellular carcinoma via regulation of the p38 MAPK signaling.

DUSP28, a member of the atypical dual­specificity phosphatase (DUSP) family, is a candidate tumor-related gene in hepatocellular carcinoma (HCC) selected by genome­wide approach, but its pathological role in HCC has not been elucidated. Here, we report for the first time that DUSP28 is involved in HCC progression. Quantitative real­time PCR and semi­quantitative RT-PCR showed notably elevated expression of DUSP28 in HCC specimens compared to that in corresponding adjacent non­tumor liver. DUSP28 overexpression promoted HCC cell proliferation, colony formation and soft agar colony formation in vitro while DUSP28 knockdown resulted in the opposite effects. Furthermore, the flow cytometric analysis indicated that DUSP28 could lead to an increased population of cancer cells in S phase, with a concomitant decrease of cells in G1 phase. Investigation of the mechanism revealed that DUSP28 could activate the p38 mitogen-activated protein kinase (MAPK) signaling pathway. Taken together, these data demonstrate that DUSP28 plays a significant role in HCC progression and may be a feasible molecular target for anti-cancer therapy.

Decreased expression and prognostic role of mitogen-activated protein kinase phosphatase 4 in hepatocellular carcinoma.

PURPOSE: This study aimed to investigate the potential role and prognostic significance of mitogen-activated protein kinase phosphatase 4 (MKP-4) in the pathology of hepatocellular carcinoma (HCC). METHODS: Western blot analysis and quantitative real-time polymerase chain reaction were performed to detect MKP-4 expression in HCC tissues, pericarcinomatous liver (PCL) tissues, and proliferating HCC cells. The detailed role of MKP-4 was further explored by MKP-4 downregulation in HepG2 cells using small interfering RNA (siRNA). Specimens of 134 HCC patients who had undergone hepatic resection were immunohistochemically evaluated for MKP-4 expression. RESULTS: MKP-4 protein and mRNA levels were significantly lower in HCC tissues than in PCL tissues. In vitro, its expression was gradually reduced following release of HepG2 cells from serum starvation. The cell counting kit-8 assay and Annexin-V-Fluos staining indicated that MKP-4 knockdown by siRNA in HCC cells enhanced cell survival and inhibited apoptosis. Univariate and multivariate analyses revealed that MKP-4 was a significant predictor for overall survival (OS) and time to recurrence (TTR). High MKP-4 expression was well correlated with prognosis independent of Edmondson grade and microvascular invasion (P < 0.001). CONCLUSIONS: MKP-4 expression was downregulated in HCC tissues and proliferating HCC cells and correlated with OS and TTR, which suggested that MKP-4 is a candidate prognostic marker for HCC.

MEK inhibition as a strategy for targeting residual breast cancer cells with low DUSP4 expression.

Lack of eradication of disseminated breast cancer by chemotherapy is a central clinical problem. Even tumors that show substantial shrinkage after drug treatment frequently relapse and eventually become refractory to all drugs available. The mechanisms underlying this lack of eradication are largely undefined and it is therefore difficult to develop curative strategies using systemic anti-cancer therapy. In a recent article low DUSP4 expression was reported to activate RAS-ERK signaling in residual breast cancer after neoadjuvant chemotherapy. This may be a druggable characteristic because MEK inhibition increases docetaxel sensitivity in a xenograft model.

The inhibitory effect of rapamycin on the oval cell response and development of preneoplastic foci in the rat.

Oval cell activation occurs under conditions of severe liver injury when normal hepatocyte proliferation is blocked. Recent studies have shown that a subset of hepatocellular carcinomas expresses oval cell markers, suggesting that these cells are targets of hepatocarcinogens. However, the signaling pathways that control oval cell activation and proliferation are not well characterized. Based on the role of the nutrient signaling kinase complex, mTORC1, in liver development, we investigated the role of this pathway in oval cell activation. Oval cell proliferation was induced in male Fisher rats by a modification of the traditional choline deficient plus ethionine model (CDE) or by 2-acetylaminoflourene treatment followed by 2/3 partial hepatectomy with or without initiation by diethylnitrosamine. To assess the role of mTOR in the oval cell response and development of preneoplastic foci, the effect of the mTORC1 inhibitor, rapamycin, was studied in all models. Rapamycin induced a significant suppression of the oval cell response in both models, an effect that coincided with a decrease in oval cell proliferation. Rapamycin administration did not affect the abundance of neutrophils or natural killer cells in CDE-treated liver or the expression of key cytokines. Gene expression studies revealed the fetal hepatocyte marker MKP-4 to be expressed in oval cells. In an experimental model of hepatic carcinogenesis, rapamycin decreased the size of preneoplastic foci and the rate of cell proliferation within the foci. mTORC1 signaling plays a key role in the oval cell response and in the development of preneoplastic foci. This pathway may be a target for the chemoprevention of hepatocellular carcinoma.

Glutathione peroxidase-1 deficiency augments proinflammatory cytokine-induced redox signaling and human endothelial cell activation.

Glutathione peroxidase-1 (GPx-1) is a crucial antioxidant enzyme, the deficiency of which promotes atherogenesis. Accordingly, we examined the mechanisms by which GPx-1 deficiency enhances endothelial cell activation and inflammation. In human microvascular endothelial cells, we found that GPx-1 deficiency augments intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression by redox-dependent mechanisms that involve NFκB. Suppression of GPx-1 enhanced TNF-α-induced ROS production and ICAM-1 expression, whereas overexpression of GPx-1 attenuated these TNF-α-mediated responses. GPx-1 deficiency prolonged TNF-α-induced IκBα degradation and activation of ERK1/2 and JNK. JNK or NFκB inhibition attenuated TNF-α induction of ICAM-1 and VCAM-1 expression in GPx-1-deficient and control cells, whereas ERK1/2 inhibition attenuated only VCAM-1 expression. To analyze further signaling pathways involved in GPx-1-mediated protection from TNF-α-induced ROS, we performed microarray analysis of human microvascular endothelial cells treated with TNF-α in the presence and absence of GPx-1. Among the genes whose expression changed significantly, dual specificity phosphatase 4 (DUSP4), encoding an antagonist of MAPK signaling, was down-regulated by GPx-1 suppression. Targeted DUSP4 knockdown enhanced TNF-α-mediated ERK1/2 pathway activation and resulted in increased adhesion molecule expression, indicating that GPx-1 deficiency may augment TNF-α-mediated events, in part, by regulating DUSP4.

Molecular pathways mediating the anti-inflammatory effects of calcitriol: implications for prostate cancer chemoprevention and treatment.

Calcitriol, the hormonally active form of vitamin D, exerts multiple anti-proliferative and pro-differentiating actions including cell cycle arrest and induction of apoptosis in many malignant cells, and the hormone is currently being evaluated in clinical trials as an anti-cancer agent. Recent research reveals that calcitriol also exhibits multiple anti-inflammatory effects. First, calcitriol inhibits the synthesis and biological actions of pro-inflammatory prostaglandins (PGs) by three mechanisms: i) suppression of the expression of cyclooxygenase-2, the enzyme that synthesizes PGs; ii) up-regulation of the expression of 15-hydroxyprostaglandin dehydrogenase, the enzyme that inactivates PGs; and iii) down-regulation of the expression of PG receptors that are essential for PG signaling. The combination of calcitriol and nonsteroidal anti-inflammatory drugs results in a synergistic inhibition of the growth of prostate cancer (PCa) cells and offers a potential therapeutic strategy for PCa. Second, calcitriol increases the expression of mitogen-activated protein kinase phosphatase 5 in prostate cells resulting in the subsequent inhibition of p38 stress kinase signaling and the attenuation of the production of pro-inflammatory cytokines. Third, calcitriol also exerts anti-inflammatory activity in PCa through the inhibition of nuclear factor-kappaB signaling that results in potent anti-inflammatory and anti-angiogenic effects. Other important direct effects of calcitriol as well as the consequences of its anti-inflammatory effects include the inhibition of tumor angiogenesis, invasion, and metastasis. We hypothesize that these anti-inflammatory actions, in addition to the other known anti-cancer effects of calcitriol, play an important role in its potential use as a therapeutic agent for PCa. Calcitriol or its analogs may have utility as chemopreventive agents and should be evaluated in clinical trials in PCa patients with early or precancerous disease.

Cdc25B dual-specificity phosphatase inhibitors identified in a high-throughput screen of the NIH compound library.

The University of Pittsburgh Molecular Library Screening Center (Pittsburgh, PA) conducted a screen with the National Institutes of Health compound library for inhibitors of in vitro cell division cycle 25 protein (Cdc25) B activity during the pilot phase of the Molecular Library Screening Center Network. Seventy-nine (0.12%) of the 65,239 compounds screened at 10 muM met the active criterion of > or =50% inhibition of Cdc25B activity, and 25 (31.6%) of these were confirmed as Cdc25B inhibitors with 50% inhibitory concentration (IC(50)) values <50 microM. Thirteen of the Cdc25B inhibitors were represented by singleton chemical structures, and 12 were divided among four clusters of related structures. Thirteen (52%) of the Cdc25B inhibitor hits were quinone-based structures. The Cdc25B inhibitors were further characterized in a series of in vitro secondary assays to confirm their activity, to determine their phosphatase selectivity against two other dual-specificity phosphatases, mitogen-activated protein kinase phosphatase (MKP)-1 and MKP-3, and to examine if the mechanism of Cdc25B inhibition involved oxidation and inactivation. Nine Cdc25B inhibitors did not appear to affect Cdc25B through a mechanism involving oxidation because they did not generate detectable amounts of H(2)O(2) in the presence of dithiothreitol, and their Cdc25B IC(50) values were not significantly affected by exchanging the dithiothreitol for beta-mercaptoethanol or reduced glutathione or by adding catalase to the assay. Six of the nonoxidative hits were selective for Cdc25B inhibition versus MKP-1 and MKP-3, but only the two bisfuran-containing hits, PubChem substance identifiers 4258795 and 4260465, significantly inhibited the growth of human MBA-MD-435 breast and PC-3 prostate cancer cell lines. To confirm the structure and biological activity of 4260465, the compound was resynthesized along with two analogs. Neither of the substitutions to the two analogs was tolerated, and only the resynthesized hit 26683752 inhibited Cdc25B activity in vitro (IC(50) = 13.83 +/- 1.0 microM) and significantly inhibited the growth of the MBA-MD-435 breast and PC-3 prostate cancer cell lines (IC(50) = 20.16 +/- 2.0 microM and 24.87 +/- 2.25 microM, respectively). The two bis-furan-containing hits identified in the screen represent novel nonoxidative Cdc25B inhibitor chemotypes that block tumor cell proliferation. The availability of non-redox active Cdc25B inhibitors should provide valuable tools to explore the inhibition of the Cdc25 phosphatases as potential mono- or combination therapies for cancer.