The Development of CDC25A-Derived Phosphoseryl Peptides That Bind 14-3-3ε with High Affinities.

Overexpression of the 14-3-3ε protein is associated with suppression of apoptosis in cutaneous squamous cell carcinoma (cSCC). This antiapoptotic activity of 14-3-3ε is dependent on its binding to CDC25A; thus, inhibiting 14-3-3ε - CDC25A interaction is an attractive therapeutic approach to promote apoptosis in cSCC. In this regard, designing peptide inhibitors of 14-3-3ε - CDC25A interactions is of great interest. This work reports the rational design of peptide analogs of pS, a CDC25A-derived peptide that has been shown to inhibit 14-3-3ε-CDC25A interaction and promote apoptosis in cSCC with micromolar IC50. We designed new peptide analogs in silico by shortening the parent pS peptide from 14 to 9 amino acid residues; then, based on binding motifs of 14-3-3 proteins, we introduced modifications in the pS(174-182) peptide. We studied the binding of the peptides using conventional molecular dynamics (MD) and steered MD simulations, as well as biophysical methods. Our results showed that shortening the pS peptide from 14 to 9 amino acids reduced the affinity of the peptide. However, substituting Gln176 with either Phe or Tyr amino acids rescued the binding of the peptide. The optimized peptides obtained in this work can be candidates for inhibition of 14-3-3ε - CDC25A interactions in cSCC.

A Comprehensive Overview of the Developments of Cdc25 Phosphatase Inhibitors.

Cdc25 phosphatases have been considered promising targets for anticancer development due to the correlation of their overexpression with a wide variety of cancers. In the last two decades, the interest in this subject has considerably increased and many publications have been launched concerning this issue. An overview is constructed based on data analysis of the results of the previous publications covering the years from 1992 to 2021. Thus, the main objective of the current review is to report the chemical structures of Cdc25s inhibitors and answer the question, how to design an inhibitor with better efficacy and lower toxicity?

Triterpenoids and lignans from Schisandra chinensis and their inhibition activities of Cdc25A/B phosphatases.

Three new highly modified lanostane triterpenoids schisanchinlactone A-C (1-3), together with six known compounds (4-9) were isolated from the stems and leaves of Schisandra chinensis. Their structures were established by extensive spectroscopic analyses. Compounds 7 and 8 showed significant inhibition of Cdc25A phosphatase with inhibitory rates of 85.5% and 98.1%, respectively, at the concentration of 100 µM.

Talaromynoids A-I, Highly Oxygenated Meroterpenoids from the Marine-Derived Fungus Talaromyces purpureogenus SCSIO 41517 and Their Lipid Accumulation Inhibitory Activities.

Nine new highly oxygenated 3,5-dimethylorsellinic acid-derived meroterpenoids, talaromynoids A-I (1-9), were isolated from the marine-derived fungus Talaromyces purpureogenus SCSIO 41517. Their structures including absolute configurations were elucidated by HRMS, NMR, single-crystal X-ray diffraction analysis, and electronic circular dichroism calculations. Compounds 1 and 7-9 possessed unprecedented 5/7/6/5/6/6, 6/7/6/6/6/5, 6/7/6/5/6/5/4, and 7/6/5/6/5/4 polycyclic systems, respectively. Biologically, compound 5 showed selective inhibitory activity against phosphatase CDC25B with an IC50 value of 13 µM. Moreover, 7-9 and 12 exhibited the activity of reducing triglyceride in 3T3-L1 adipocytes in a dosage-dependent manner.

Discovery of potent and selective Cdc25 phosphatase inhibitors via rapid assembly and in situ screening of Quinonoid-focused libraries.

Cell division cycle 25 (Cdc25) phosphatase is an attractive target for drug discovery. The rapid assembly and in situ screening of focused combinatorial fragment libraries using efficient modular reactions is a highly robust strategy for discovering bioactive molecules. In this study, we have utilized miniaturized synthesis to generate several quinonoid-focused libraries, by standard CuAAC reaction and HBTU-based amide coupling chemistry. Then the enzyme inhibition screening afforded some potent and selective Cdc25s inhibitors. Compound M5N36 (Cdc25A: IC50 = 0.15 ± 0.05 µM; Cdc25B: IC50 = 0.19 ± 0.06 µM; Cdc25C: IC50 = 0.06 ± 0.04 µM) exhibited higher inhibitory activity than the initial lead NSC663284 (Cdc25A: IC50 = 0.27 ± 0.02 µM; Cdc25B: IC50 = 0.42 ± 0.01 µM; Cdc25C: IC50 = 0.23 ± 0.01 µM). Moreover, M5N36 displayed about three-fold more potent against Cdc25C than Cdc25A and B, indicating that M5N36 could act as a relatively selective Cdc25C inhibitor. Cell viability evaluation, western blotting and molecular simulations provided a mechanistic understanding of the activity of M5N36. It showed promising anti-growth activity against the MDA-MB-231 cell line and desirable predicted physicochemical properties. Overall, M5N36 was proven to be a promising novel Cdc25C inhibitor.

Design, synthesis, and functional evaluation of triazine-based bivalent agents that simultaneously target the active site and hot spot of phosphatase Cdc25B.

Cdc25B phosphatase catalyzes the dephosphorylation and activation of cyclin-dependent kinases 2 (CDK2/CycA) and their overexpression has been reported in cancers. Although Cdc25B has received much attention as a drug target, its flat and featureless surface makes it challenging to develop new agents targeting this protein. In this study, we investigated the rational design of a series of bivalent triazine-based derivatives with the aim of simultaneously targeting the active site and the remote hotspot critical for the interaction with CDK2/CycA. Compounds 1e and 10, containing aromatic residues, were shown to inhibit Cdc25B activity selectively over Cdc25A at low micromolar concentration.

In Silico Identification of Small Molecules as New Cdc25 Inhibitors through the Correlation between Chemosensitivity and Protein Expression Pattern.

The cell division cycle 25 (Cdc25) protein family plays a crucial role in controlling cell proliferation, making it an excellent target for cancer therapy. In this work, a set of small molecules were identified as Cdc25 modulators by applying a mixed ligand-structure-based approach and taking advantage of the correlation between the chemosensitivity of selected structures and the protein expression pattern of the proposed target. In the first step of the in silico protocol, a set of molecules acting as Cdc25 inhibitors were identified through a new ligand-based protocol and the evaluation of a large database of molecular structures. Subsequently, induced-fit docking (IFD) studies allowed us to further reduce the number of compounds biologically screened. In vitro antiproliferative and enzymatic inhibition assays on the selected compounds led to the identification of new structurally heterogeneous inhibitors of Cdc25 proteins. Among them, J3955, the most active inhibitor, showed concentration-dependent antiproliferative activity against HepG2 cells, with GI50 in the low micromolar range. When J3955 was tested in cell-cycle perturbation experiments, it caused mitotic failure by G2/M-phase cell-cycle arrest. Finally, Western blotting analysis showed an increment of phosphorylated Cdk1 levels in cells exposed to J3955, indicating its specific influence in cellular pathways involving Cdc25 proteins.

Effective Pharmacophore for CDC25 Phosphatases Enzyme Inhibitors: Newly Synthesized Bromothiazolopyrimidine Derivatives.

BACKGROUND: Thiazolopyrimidine analogues are versatile synthetic scaffold possessing wide spectrum of biological interests involving potential anticancer activity. OBJECTIVE: To report the synthesis of novel bromothiazolopyrimidine derivatives and the study of both molecular modeling and in-vitro anticancer activity. METHODS: Novel bromothiazolopyrimidine derivatives 5-18 have been prepared from 2-bromo-3-(4- chlorophenyl)-1-(3,4-dimethylphenyl)-propenone 3 as a key starting compound. The anti-cancer activities of the new compounds were evaluated against HepG2, MCF-7, A549 and HCT116 cell lines. RESULTS: The compounds 16, 17 and 18 showed cytotoxic and growth inhibitory activities on both colon and lung cells. The cytotoxic activities of the novel synthetic compounds 8, 9, 11, 16, 17 and 18 were due to CDC25 phosphatases inhibition as shown by the enzymatic binding assay. Although compounds 8, 9 and 11 have only demonstrated CDC25B phosphatases inhibition. CONCLUSION: The novel bromothiazolopyrimidine derivatives showed promising in vitro anticancer activities against colon cancer HCT116 and lung cancer A549 cell lines comparable to the anticancer drug doxorubicin.

Epigallocatechin gallate induces chemopreventive effects on rats with diethylnitrosamine­induced liver cancer via inhibition of cell division cycle 25A.

Epigallocatechin gallate (EGCG), the most active monomer in green tea (GT), has demonstrated potential therapeutic and preventive effects on various tumors, including liver cancer. However, the anticancer mechanisms of EGCG in liver cancer remain to be elucidated. The abnormal expression of cell division cycle 25A (CDC25A) has been identified in liver cancer and is closely associated with malignancy and poor prognosis in patients with hepatocellular carcinoma (HCC). The present study used human hepatoma cell lines and rats with diethylnitrosamine (DEN)­induced HCC as models to investigate the association between the effect of EGCG on liver cancer and regulation of the p21waf1/Cip1/CDC25A axis. The results demonstrated that EGCG can inhibit the proliferation of HepG2 and Huh7 cells, reduce the expression of CDC25A and increase the expression of p21waf1/Cip1 in HepG2. In vivo, HCC was induced by DEN in Sprague­Dawley rats. EGCG significantly reduced tumor volume and improved the survival rates of rats with HCC. The expression levels of CDC25A mRNA and protein in liver tissues and the level of serum γ glutamyl transpeptidase in rats treated with EGCG were significantly decreased, while p21waf1/Cip1 mRNA and protein expression levels were increased compared with the HCC group, in the process of DEN­induced HCC. No significant difference in the chemopreventive effects on liver cancer was observed between GT extract and EGCG under an EGCG equivalence condition. Thus, EGCG can suppress human hepatoma cell proliferation and prolong the survival of rats with HCC, and the potential mechanism may be involved in EGCG­induced upregulation of p21waf1/Cip1 and downregulation of CDC25A.

Inhibition mechanism of naphthylphenylamine derivatives acting on the CDC25B dual phosphatase and analysis of the molecular processes involved in the high cytotoxicity exerted by one selected derivative in melanoma cells.

The dual phosphatases CDC25 are involved in cell cycle regulation and overexpressed in many tumours, including melanoma. CDC25 is a promising target for discovering anticancer drugs, and several studies focussed on characterisation of quinonoid CDC25 inhibitors, frequently causing undesired side toxic effects. Previous work described an optimisation of the inhibition properties by naphthylphenylamine (NPA) derivatives of NSC28620, a nonquinonoid CDC25 inhibitor. Now, the CDC25B•inhibitor interaction was investigated through fluorescence studies, shedding light on the different inhibition mechanism exerted by NPA derivatives. Among the molecular processes, mediating the specific and high cytotoxicity of one NPA derivative in melanoma cells, we observed decrease of phosphoAkt, increase of p53, reduction of CDC25 forms, cytochrome c cytosolic translocation and increase of caspase activity, that lead to the activation of an apoptotic programme. A basic knowledge on CDC25 inhibitors is relevant for discovering potent bioactive molecules, to be used as anticancer agents against the highly aggressive melanoma.

Medicinal chemistry insights into novel CDC25 inhibitors.

Cell division cycle 25 (CDC25) phosphatases, a kind of cell cycle regulators, have become an attractive target for drug discovery, as they have been found to be over-expressed in various human cancer cells. Several CDC25 inhibitors have achieved significant attention in clinical trials with possible mechanistic actions. Prompted by the significance of CDC25 inhibitors with medicinal chemistry prospect, it is an apt time to review the various drug discovery methods involved in CDC25 drug discovery including high throughput screening (HTS), virtual screening (VS), fragment-based drug design, substitution decorating approach, structural simplification approach and scaffold hopping method to seek trends and identify promising new avenues of CDC25 drug discovery.

Synthesis, anticancer activity, and molecular modeling of 1,4-naphthoquinones that inhibit MKK7 and Cdc25.

Cell division cycle 25 (Cdc25) and mitogen-activated protein kinase kinase 7 (MKK7) are enzymes involved in intracellular signaling but can also contribute to tumorigenesis. We synthesized and characterized the biological activity of 1,4-naphthoquinones structurally similar to reported Cdc25 and(or) MKK7 inhibitors with anticancer activity. Compound 7 (3-[(1,4-dioxonaphthalen-2-yl)sulfanyl]propanoic acid) exhibited high binding affinity for MKK7 (Kd = 230 nM), which was greater than the affinity of NSC 95397 (Kd = 1.1 µM). Although plumbagin had a lower binding affinity for MKK7, this compound and sulfur-containing derivatives 4 and 6-8 were potent inhibitors of Cdc25A and Cdc25B. Derivative 22e containing a phenylamino side chain was selective for MKK7 versus MKK4 and Cdc25 A/B, and its isomer 22f was a selective inhibitor of Cdc25 A/B. Docking studies performed on several naphthoquinones highlighted interesting aspects concerning the molecule orientation and hydrogen bonding interactions, which could help to explain the activity of the compounds toward MKK7 and Cdc25B. The most potent naphthoquinone-based inhibitors of MKK7 and/or Cdc25 A/B were also screened for their cytotoxicity against nine cancer cell lines and primary human mononuclear cells, and a correlation was found between Cdc25 A/B inhibitory activity and cytotoxicity of the compounds. Quantum chemical calculations using BP86 and ωB97X-D3 functionals were performed on 20 naphthoquinone derivatives to obtain a set of molecular electronic properties and to correlate these properties with cytotoxic activities. Systematic theoretical DFT calculations with subsequent correlation analysis indicated that energy of the lowest unoccupied molecular orbital E(LUMO), vertical electron affinity (VEA), and reactivity index ω of these molecules were important characteristics related to their cytotoxicity. The reactivity index ω was also a key characteristic related to Cdc25 A/B phosphatase inhibitory activity. Thus, 1,4-naphthoquinones displaying sulfur-containing and phenylamino side chains with additional polar groups could be successfully utilized for further development of efficacious Cdc25 A/B and MKK7 inhibitors with anticancer activity.

Identification of highly potent and selective Cdc25 protein phosphatases inhibitors from miniaturization click-chemistry-based combinatorial libraries.

Cell division cycle 25 (Cdc25) protein phosphatases play key roles in the transition between the cell cycle phases and their association with various cancers has been widely proven, which makes them ideal targets for anti-cancer treatment. Though several Cdc25 inhibitors have been developed, most of them displayed low activity and poor subtype selectivity. Therefore, it is extremely important to discover novel small molecule inhibitors with potent activities and significant selectivity for Cdc25 subtypes, not only served as drugs to treat cancer but also to probe its mechanism in transitions. In this study, miniaturized parallel click chemistry synthesis via CuAAC reaction followed by in situ biological screening were used to discover selective Cdc25 inhibitors. The bioassay results showed that compound M2N12 proved to be the most potent Cdc25 inhibitor, which also act as a highly selective Cdc25C inhibitor and was about 9-fold potent than that of NSC 663284. Moreover, M2N12 showed remarkable anti-growth activity against the KB-VIN cell line, equivalent to that of PXL and NSC 663284. An all-atom molecular dynamics (MD) simulation approach was further employed to probe the significant selectivity of M2N12 for Cdc25C relative to its structural homologs Cdc25A and Cdc25B. Overall, above results make M2N12 a promising lead compound for further investigation and structural modification.

Discovery of Novel Naphthylphenylketone and Naphthylphenylamine Derivatives as Cell Division Cycle 25B (CDC25B) Phosphatase Inhibitors: Design, Synthesis, Inhibition Mechanism, and in Vitro Efficacy against Melanoma Cell Lines.

CDC25 phosphatases play a critical role in the regulation of the cell cycle and thus represent attractive cancer therapeutic targets. We previously discovered the 4-(2-carboxybenzoyl)phthalic acid (NSC28620) as a new CDC25 inhibitor endowed with promising anticancer activity in breast, prostate, and leukemia cells. Herein, we report a structure-based optimization of NSC28620, leading to the identification of a series of novel naphthylphenylketone and naphthylphenylamine derivatives as CDC25B inhibitors. Compounds 7j, 7i, 6e, 7f, and 3 showed higher inhibitory activity than the initial lead, with Ki values in the low micromolar range. Kinetic analysis, intrinsic fluorescence studies, and induced fit docking simulations provided a mechanistic understanding of the activity of these derivatives. All compounds were tested in the highly aggressive human melanoma cell lines A2058 and A375. Compound 4a potently inhibited cell proliferation and colony formation, causing an increase of the G2/M phase and a reduction of the G0/G1 phase of the cell cycle in both cell lines.

Anti-inflammatory functions of the CDC25 phosphatase inhibitor BN82002 via targeting AKT2.

This study presents BN82002 as an anti-inflammatory drug candidate. It was found that BN82002 inhibited the production of nitric oxide (NO) and prostaglandin E2 (PGE2) in RAW 264.7 cells and peritoneal macrophages that were activated by toll-like receptor (TLR) 4 ligand, lipopolysaccharide (LPS). BN82002 dose-dependently down-regulated mRNA levels of nitric oxide synthase, tumor necrosis factor-α, and cyclooxygenase-2. The nuclear translocation of nuclear factor (NF)-κB (p65 and p50) was also blocked by BN82002 in RAW265.7 cells stimulated by LPS. According to reporter gene assay performed with NF-κB construct, BN82002 clearly reduced increased level of luciferase activity mediated by transcription factor NF-κB in LPS-treated RAW264.7 cells and in MyD88- and AKT2-overexpressing HEK293 cells. However, BN82002 did not inhibit NF-κB activity in AKT1- or IKKß-overexpressing HEK293 cells. NF-κB upstream signaling events specifically targeted AKT2 but had no effect on AKT1. The specific target of BN82002 was Tyr-178 in AKT2. BN82002 bound to Tyr-178 and interrupted the kinase activity of AKT2, according to a cellular thermal shift assay analysis of the interaction of BN82002 with AKT2 and an AKT2 mutant (Tyr-178 mutated to Ala; AKT2 Y178A). These results suggest that BN82002 could reduce inflammatory pathway by controlling NF-κB pathway and specifically targeting AKT2.

MiR-365 enhances the radiosensitivity of non-small cell lung cancer cells through targeting CDC25A.

Radioresistance is a major challenge in lung cancer radiotherapy (RT), and consequently, new radiosensitizers are urgently needed. MicroRNAs (miRNAs) have been demonstrated to participate in many important cellular processes including radiosensitization. MiR-365 is dysregulated in non-small cell lung cancer (NSCLC) and is able to restrain the development of NSCLC. However, the relationship between miR-365 and radiosensitivities of NSCLC cells remains largely unknown. Here we reveal that overexpression of miR-365 is able to enhance the radiosensitivity of NSCLC cells through targeting CDC25A. We found that the expression level of miR-365 was positively correlated with the radiosensitivity of NSCLC cell lines. Furthermore, our results showed that overexpression of miR-365 could sensitize A549 cells to the irradiation. However, knockdown of miR-365 in H460 cells could act the converse manner. Mechanically, miR-365 was able to directly target 3'UTR of cell division cycle 25A (CDC25A) mRNA and reduce the expression of CDC25A at the levels of mRNA and protein. And we confirmed that miR-365 could increase the radiosensitivity of NSCLC cells by targeting CDC25A using in vitro and in vivo assays. Taken together, restoration of miR-365 expression enhances the radiosensitivity of NSCLC cells by suppressing CDC25A, and miR-365 could be used as a radiosensitizer for NSCLC therapy.

Pharmacophore-guided discovery of CDC25 inhibitors causing cell cycle arrest and tumor regression.

CDC25 phosphatases play a key role in cell cycle transitions and are important targets for cancer therapy. Here, we set out to discover novel CDC25 inhibitors. Using a combination of computational methods, we defined a minimal common pharmacophore in established CDC25 inhibitors and performed virtual screening of a proprietary library. Based on the availability of crystal structures for CDC25A and CDC25B, we implemented a molecular docking strategy and carried out hit expansion/optimization. Enzymatic assays revealed that naphthoquinone scaffolds were the most promising CDC25 inhibitors among selected hits. At the molecular level, the compounds acted through a mixed-type mechanism of inhibition of phosphatase activity, involving reversible oxidation of cysteine residues. In 2D cell cultures, the compounds caused arrest of the cell cycle at the G1/S or at the G2/M transition. Mitotic markers analysis and time-lapse microscopy confirmed that CDK1 activity was impaired and that mitotic arrest was followed by death. Finally, the compounds induced differentiation, accompanied by decreased stemness properties, in intestinal crypt stem cell-derived Apc/K-Ras-mutant mouse organoids, and led to tumor regression and reduction of metastatic potential in zebrafish embryo xenografts used as in vivo model.

Silibinin Retards Colitis-associated Carcinogenesis by Repression of Cdc25C in Mouse Model.

PURPOSE: Silibinin possesses the efficacy of anticancer and anti-inflammation. We aimed to test whether silibinin could prevent colitis-associated carcinogenesis in mouse model. EXPERIMENTAL DESIGN: Azoxymethane (AOM) and dextran sulfate sodium (DSS) were used to induce colitis-associated tumorigenesis in C57BL mice. Six-to-eight-week-old male mice were gavaged with 350 or 750 mg/kg of silibinin for 10 weeks right after DSS administration. The mice were then sacrificed, and colon tissues were measured for tumor multiplicity and size. Molecular changes about proliferation, apoptosis and inflammation were tested. RESULTS: Silibinin feeding showed a dose-dependent inhibition on the size of tumor induced by AOM/DSS in mice. In addition, silibinin inhibited cell proliferation evidenced by a decrease (P < 0.05) in Ki-67 and proliferating cell nuclear antigen (PCNA). However, silibinin did not show any significant effect on inflammation, apoptosis, and the mRNA expression of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and vascular endothelial growth factor (VEGF). The experiments in vitro showed that silibinin induced cell cycle arrest at G2/M phase in CT-26 cells, a mouse colonic cancer cell line. Furthermore, silibinin reduced the expression of Cdc25C and blocked the dephosphorylation of CDK1 at multiple sites both in vitro and in vivo. CONCLUSIONS: Silibinin targets Cdc25C/CDK1 pathway and mitigates colitis-associated tumorigenesis in mice. Thus, our findings indicate the chemopreventive potential of silibinin for inflammation-associated colon cancer.

Shikonin inhibits cancer cell cycling by targeting Cdc25s.

BACKGROUND: Shikonin, a natural naphthoquinone, is abundant in Chinese herb medicine Zicao (purple gromwell) and has a wide range of biological activities, especially for cancer. Shikonin and its analogues have been reported to induce cell-cycle arrest, but target information is still unclear. We hypothesized that shikonin, with a structure similar to that of quinone-type compounds, which are inhibitors of cell division cycle 25 (Cdc25) phosphatases, will have similar effects on Cdc25s. To test this hypothesis, the effects of shikonin on Cdc25s and cell-cycle progression were determined in this paper. METHODS: The in vitro effects of shikonin and its analogues on Cdc25s were detected by fluorometric assay kit. The binding mode between shikonin and Cdc25B was modelled by molecular docking. The dephosphorylating level of cyclin-dependent kinase 1 (CDK1), a natural substrate of Cdc25B, was tested by Western blotting. The effect of shikonin on cell cycle progression was investigated by flow cytometry analysis. We also tested the anti-proliferation activity of shikonin on cancer cell lines by MTT assay. Moreover, in vivo anti-proliferation activity was tested in a mouse xenograft tumour model. RESULTS: Shikonin and its analogues inhibited recombinant human Cdc25 A, B, and C phosphatase with IC50 values ranging from 2.14 ± 0.21 to 13.45 ± 1.45 µM irreversibly. The molecular modelling results showed that shikonin bound to the inhibitor binding pocket of Cdc25B with a favourable binding mode through hydrophobic interactions and hydrogen bonds. In addition, an accumulation of the tyrosine 15-phosphorylated form of CDK1 was induced by shikonin in a concentration-dependent manner in vitro and in vivo. We also confirmed that shikonin showed an anti-proliferation effect on three cancer cell lines with IC50 values ranging from 6.15 ± 0.46 to 9.56 ± 1.03 µM. Furthermore, shikonin showed a promising anti-proliferation effect on a K562 mouse xenograph tumour model. CONCLUSION: In this study, we provide evidence for how shikonin induces cell cycle arrest and functions as a Cdc25s inhibitor. It shows an anti-proliferation effect both in vitro and in vivo by mediating Cdc25s.

CDC25A pathway toward tumorigenesis: Molecular targets of CDC25A in cell-cycle regulation.

The cell division cycle 25 (CDC25) phosphatases regulate key transitions between cell-cycle phases during normal cell division, and in the case of DNA damage, they are key targets of the checkpoint machinery that ensure genetic stability. Little is known about the mechanisms underlying dysregulation and downstream targets of CDC25. To understand these mechanisms, we silenced the CDC25A gene in breast cancer cell line MDA-MB-231 and studied downstream targets of CDC25A gene. MDA-MB-231 breast cancer cells were transfected and silenced by CDC25A small interfering RNA. Total messenger RNA (mRNA) was extracted and analyzed by quantitative real-time polymerase chain reaction. CDC25A phosphatase level was visualized by Western blot analysis and was analyzed by 2D electrophoresis and LC-ESI-MS/MS. After CDC25A silencing, cell proliferation reduced, and the expression of 12 proteins changed. These proteins are involved in cell-cycle regulation, programmed cell death, cell differentiation, regulation of gene expression, mRNA editing, protein folding, and cell signaling pathways. Five of these proteins, including ribosomal protein lateral stalk subunit P0, growth factor receptor bound protein 2, pyruvate kinase muscle 2, eukaryotic translation elongation factor 2, and calpain small subunit 1 increase the activity of cyclin D1. Our results suggest that CDC25A controls the cell proliferation and tumorigenesis by a change in expression of proteins involved in cyclin D1 regulation and G1/S transition.