Downregulation Of CDC25A Research Articles

Oncogenic role of ALX3 in cervical cancer cells through KDM2B-mediated histone demethylation of CDC25A

BackgroundCell division cycle 25A (CDC25A) is a well-recognized regulator of cell cycle progression and is involved in cancer development. This work focused on the function of CDC25A in cervical cancer cell growth and the molecules involved.MethodsA GEO dataset GSE63514 comprising data of cervical squamous cell carcinoma (CSCC) tissues was used to screen the aberrantly expressed genes in cervical cancer. The CDC25A expression in cancer and normal tissues was predicted in the GEPIA database and that in CSCC and normal cells was determined by RT-qPCR and western blot assays. Downregulation of CDC25A was introduced in CSCC cells to explore its function in cell growth and the cell cycle progression. The potential regulators of CDC25A activity and the possible involved signaling were explored.ResultsCDC25A was predicted to be overexpressed in CSCC, and high expression of CDC25A was observed in CSCC cells. Downregulation of CDC25A in ME180 and C33A cells reduced cell proliferation and blocked cell cycle progression, and it increased cell apoptosis. ALX3 was a positive regulator of CDC25A through transcription promotion. It recruited a histone demethylase, lysine demethylase 2B (KDM2B), to the CDC25A promoter, which enhanced CDC25A expression through demethylation of H3k4me3. Overexpression of ALX3 in cells blocked the inhibitory effects of CDC25A silencing. CDC25A was found as a positive regulator of the PI3K/Akt signaling pathway.ConclusionThis study suggested that the ALX3 increased CDC25A expression through KDM2B-mediated demethylation of H3K4me3, which induced proliferation and cell cycle progression of cervical cancer cells.

Impact of Wnt/β-Catenin Inhibition on Cell Proliferation through CDC25A Downregulation in Soft Tissue Sarcomas.

Simple SummaryGrowing evidence suggests that Wnt signaling may be crucial for tumorigenesis and progression of soft tissue sarcomas (STS). Inhibitors of this pathway are currently in clinical trials or pre-clinical studies in order to validate its utility in different neoplasia. One of this inhibitors, PRI-724, is showing promising results for advanced pancreatic adenocarcinoma or ovarian cancer. We found that PRI-724 is able to suppress cell viability/proliferation and to increase cell death rates of soft tissue sarcomas cells in vitro. CDC25A, a target gene of Wnt signaling pathway, is essential for STS proliferation because its downregulation via siRNA was able to mimic the effect of PRT-724 on cell cycle arrest and evaluation of NCBI/GenBank data confirmed its overexpression in STS patients’ samples. Moreover, in vitro administration of PRI-724 along with standard STS chemotherapeutic drugs improved the efficacy of chemotherapy, suggesting that Wnt inhibition could be a promising new therapeutic strategy in STS.The Wnt signaling pathway is an important cellular mechanism for regulating differentiation processes as well as cell cycle events, and different inhibitors of this pathway, for example, PRI-724, are showing promising results in clinical trials for treatment of advanced pancreatic adenocarcinoma or ovarian cancer. Growing evidence suggests that Wnt signaling may also be crucial for tumorigenesis and progression of soft tissue sarcomas (STS), a malignant neoplasm with few therapeutic options at an advanced state. Our study with several STS cell lines and primary cultures shows that inhibition of Wnt/β-catenin signaling with PRI-724 is able to suppress cell viability/proliferation and to increase cell death rates. TCF/β-catenin-mediated transcriptional activity is decreased in treated cells, leading to downregulation of its target genes CCND1 and CDC25A. The latter was critical because its downregulation via siRNA was able to mimic the effect of PRI-724 on cell cycle arrest and cell death induction. An evaluation of NCBI/GenBank data confirmed that CDC25A mRNA is elevated in STS patients. Importantly, PRI-724 in combination with standard STS chemotherapeutics doxorubicin or trabectedin enhanced their antitumoral effect in a synergistic manner according to isobolographic analysis, suggesting that Wnt inhibition through PRI-724 could be a beneficial combination regime in patients with advanced STS.

Albendazole negatively regulates keratinocyte proliferation.

Increased keratinocyte proliferation occurs in the skin of psoriatic patients and is supposed to play a role in the pathogenesis of this disorder. Compounds interfering with keratinocyte proliferation could be useful in the management of psoriatic patients. To investigate whether albendazole, an anti-helmintic drug that regulates epithelial cell function in various systems, inhibits keratinocyte proliferation in models of psoriasis. Aldara-treated mice received daily topical application of albendazole. Keratinocyte proliferation and keratin (K) 6 and K16 expression were evaluated by immunohistochemistry and Western blotting and inflammatory cells/mediators were analysed by immunohistochemistry and real-time PCR. In human keratinocytes (HEKa and HaCaT) treated with albendazole, cell cycle and proliferation, keratins and cell cycle-associated factors were evaluated by flow cytometry, colorimetric assay and Western blotting respectively. Aldara-treated mice given albendazole exhibited reduced epidermal thickness, decreased number of proliferating keratinocytes and K6/K16 expression. Reduction of CD3- and Ly6G-positive cells in the skin of albendazole-treated mice associated with inhibition of IL-6, TNF-α, IL-1β, IL-17A, IL-36, CCL17, CXCL1, CXCL2 and CXCL5 expression. Treatment of keratinocytes with albendazole reduced K6/K16 expression and reversibly inhibited cell growth by promoting accumulation of cells in S-phase. This phenomenon was accompanied by down-regulation of CDC25A, a phosphatase regulating progression of cell cycle through S-phase, and PKR-dependent hyper-phosphorylation of eIF2α, an inhibitor of CDC25 translation. In Aldara-treated mice, albendazole activated PKR, enhanced eIF2α phosphorylation and reduced CDC25A expression. Data show that albendazole inhibits keratinocyte proliferation and exerts therapeutic effect in a murine model of psoriasis.

Exosome-derived miR-339-5p mediates radiosensitivity by targeting Cdc25A in locally advanced esophageal squamous cell carcinoma.

Cancer cells associated with radioresistance are likely to give rise to local recurrence and distant metastatic relapse. However, it remains unclear whether specific miRNAs have direct roles in radioresistance and/or prognosis. In this study, we find that miR-339-5p promotes radiosensitivity, and is downregulated in radioresistant subpopulations of esophageal cancer cells. Notably, miR-339-5p was selectively secreted into blood via exosomes, and that higher serum miR-339-5p levels were positively associated with radiotherapy sensitivity and good survival. Moreover, miR-339-5p expression was downregulated in the T3/T4 stage compared with T1/T2 stage in esophageal squamous cell carcinoma (ESCC) patients (P = 0.04), and low miR-339-5p expression in tissue was significantly associated with poor overall survival (P = 0.036) and disease-free survival (P = 0.037). Overexpression of miR-339-5p enhanced radiosensitivity in vitro and in vivo. Mechanistically, miR-339-5p enhances radiosensitivity by targeting Cdc25A, and is transcriptionally regulated by Runx3. Correlations were observed between miR-339-5p levels and Cdc25A/Runx3 levels in tissue samples. Intriguingly, combined analysis of miR-339-5p expression with Runx3 increased the separation of the survival curves obtained for either gene alone in the TCGA datasets (P = 0.009). Overall, exosome-derived miR-339-5p mediates radiosensitivity through downregulation of Cdc25A, and predicts pathological response to preoperative radiotherapy in locally advanced ESCC, suggesting it could be a promising non-invasive biomarker for facilitating personalized treatments.

Melatonin induces the apoptosis and inhibits the proliferation of human gastric cancer cells via blockade of the AKT/MDM2 pathway.

Globally, gastric cancer (GC) is one of the most common types of cancer and the third leading cause of cancer‑related death. In China, gastric and liver cancers have the highest mortality rates. Melatonin, also known as N-acetyl‑5-methoxytryptamine, is a hormone that is produced by the pineal gland in animals and regulates sleep and wakefulness. Melatonin has been shown to inhibit various carcinomas, including GC. There are many different hypotheses to explain the anticancer effects of melatonin, including stimulation of apoptosis, inhibition of cell growth, regulation of anticancer immunity, induction of free-radical scavenging, and the competitive inhibition of estrogen. However, the underlying mechanism by which these effects are elicited remains elusive. The aim of the present study was to investigate the effects of melatonin on human GC cells and determine the underlying molecular mechanism. We treated SGC-7901 GC cells with melatonin and analyzed the resulting protein changes using protein chip technology. Several proteins related to cell apoptosis and proliferation were identified and further tested in SGC-7901 GC cells. We found that melatonin induced cell cycle arrest and the downregulation of CDC25A, phospho-CDC25A (at Ser75), p21 (p21Cip1/p21Waf1) and phospho-p21 (at Thr145). Melatonin also induced upregulation of Bax, downregulation of Bcl-xL, an increase in cleaved caspase-9 level and activation of caspase-3, which confirmed the involvement of the mitochondria in melatonin‑induced apoptosis. Upstream regulators of the above proteins, MDM2, phospho-MDM2 (at Ser166) and AKT, phospho-AKT (at Thr308) were all attenuated by melatonin, which led to an increase in p53. The present study demonstrated that the oncostatic effects of melatonin on SGC-7901 GC cells are mediated via the blockade of the AKT/MDM2 intracellular pathway.

Sodium fluoride causes hepatocellular S-phase arrest by activating ATM-p53-p21 and ATR-Chk1-Cdc25A pathways in mice.

In this study, experimental pathology, flow cytometry (FCM), quantitative real-time polymerase chain reaction (qRT-PCR), and western blot (WB) were used to evaluate the effects of sodium fluoride (NaF) on hepatocellular cell cycle progression in mice. A total of 240 ICR mice were divided equally into four groups; the experimental groups received 12, 24, or 48 mg/kg NaF intragastrically for 42 days, while the control group received distilled water. Doses of NaF above 12 mg/kg increased the percentage of cells in S phase (S-phase arrest), reduced percentages of cells in G0/G1 or G2/M phase, and activated the ATM-p53-p21 and ATR-Chk1-Cdc25A pathways. Activation of these pathways was characterized by up-regulation of ATM, p53, p21, ATR, and Chk1 mRNA and protein expression, and down-regulation of Cdc25A, cyclin E, cyclin A, CDK2, CDK4, and proliferating cell nuclear antigen (PCNA) mRNA and protein expression. These results indicate that NaF caused S-phase arrest by activating the ATM-p53-p21 and ATR-Chk1-Cdc25A pathways.

Cdc25A Is a Critical Mediator of Ischemic Neuronal Death In Vitro and In Vivo.

Dysregulation of cell cycle machinery is implicated in a number of neuronal death contexts, including stroke. Increasing evidence suggests that cyclin-dependent kinases (Cdks) are inappropriately activated in mature neurons under ischemic stress conditions. We previously demonstrated a functional role for the cyclin D1/Cdk4/pRb (retinoblastoma tumor suppressor protein) pathway in delayed neuronal death induced by ischemia. However, the molecular signals leading to cyclin D/Cdk4/pRb activation following ischemic insult are presently not clear. Here, we investigate the cell division cycle 25 (Cdc25) dual-specificity phosphatases as potential upstream regulators of ischemic neuronal death and Cdk4 activation. We show that a pharmacologic inhibitor of Cdc25 family members (A, B, and C) protects mouse primary neurons from hypoxia-induced delayed death. The major contributor to the death process appears to be Cdc25A. shRNA-mediated knockdown of Cdc25A protects neurons in a delayed model of hypoxia-induced death in vitro Similar results were observed in vivo following global ischemia in the rat. In contrast, neurons singly or doubly deficient for Cdc25B/C were not significantly protective. We show that Cdc25A activity, but not level, is upregulated in vitro following hypoxia and global ischemic insult in vivo Finally, we show that shRNA targeting Cdc25A blocks Ser795 pRb phosphorylation. Overall, our results indicate a role for Cdc25A in delayed neuronal death mediated by ischemia.SIGNIFICANCE STATEMENT A major challenge in stroke is finding an effective neuroprotective strategy to treat cerebral ischemic injury. Cdc25 family member A (Cdc25A) is a phosphatase normally activated during cell division in proliferating cells. We found that Cdc25A is activated in neurons undergoing ischemic stress mediated by hypoxia in vitro and global cerebral ischemia in rats in vivo We show that pharmacologic or genetic inhibition of Cdc25A activity protects neurons from delayed death in vitro and in vivo Downregulation of Cdc25A led to reduction in retinoblastoma tumor suppressor protein (pRb) phosphorylation. An increase in pRb phosphorylation has been previously linked to ischemic neuronal death. Our results identify Cdc25A as a potential target for neuroprotectant strategy for the treatment of delayed ischemic neuronal death.

Pterostilbene Induces Cell Apoptosis and Cell Cycle Arrest in T-Cell Leukemia/Lymphoma by Suppressing the ERK1/2 Pathway.

Pterostilbene is a natural 3,5-dimethoxy analog of trans-resveratrol that has been reported to have antitumor, antioxidant, and anti-inflammatory effects. T-cell leukemia/lymphoma is one of the more aggressive yet uncommon non-Hodgkin lymphomas. Although there has been increasing research into T-cell leukemia/lymphoma, the molecular mechanisms of the antitumor effects of pterostilbene against this malignancy are still largely unknown. The aim of this study is to confirm the effects of pterostilbene in T-cell leukemia/lymphoma. Jurkat and Hut-78 cells treated with pterostilbene were evaluated for cell proliferation using Cell Counting Kit-8, and apoptosis, cell cycle progression, reactive oxygen species generation, and mitochondrial membrane potential were analyzed using flow cytometry. The level of protein expression was detected by western blot. The results demonstrated that pterostilbene significantly inhibited the growth of T-cell leukemia/lymphoma cell lines in vitro and induced apoptosis in a dose- and time-dependent manner. Moreover, pterostilbene treatment markedly induced S-phase cell cycle arrest, which was accompanied by downregulation of cdc25A, cyclin A2, and CDK2. Pterostilbene also induced the generation of reactive oxygen species and the loss of mitochondrial membrane potential and inhibited ERK1/2 phosphorylation. Taken together, our study demonstrated the potential of pterostilbene to be an effective treatment for T-cell leukemia/lymphoma.

The herbal medicine Cyperus amuricus inhibits proliferation of human hepatocellular carcinoma Hep3B cells by inducing apoptosis and arrest at the G0/G1 cell cycle phase.

Cyperus amuricus (C. amuricus) is one of the most common herbs in Oriental folk medicine for exerting astringent, diuretic, wound healing and other intestinal problems. However, little is known about the molecular mechanism of C.amuricus on anticancer activity. In the present study, the underlying mechanism of the anticancer effect of C. amuricus were elucidated. The methyl alcohol extract from the whole plant of C. amuricus exhibited cytotoxicity against Hep3B cells, but not against A549 and HaCaT cells. Consistent with an acceleration of the sub-G1 phase, downregulation of cdc25A, cyclinD1 and cyclinE, CDK4 and 2 as well as E2F-1, phospho-Rb, with concomitant of upregulation of p21CIP1/WAF1, p27KIPI and p16INK4a proteins, as evidenced by the appearance of cell cycle arrest, were detected in C. amuricus-treated Hep3B cells. Additionally, the sequential activation of various caspases (cleaved caspase-8, -9, -3, -7 and -6, and cleaved PARP) and the changed expression of other proteins related to the apoptosis pathway were observed after C. amuricus exposure. An increment in the pro-apoptotic proteins (Bim, tBid, Bax and Bak) and a reduction of anti-apoptotic protein (Bcl-2) regulate Hep3B cell death by controlling the permeability of mitochondrial membranes and the release of cytochromec from mitochondria into the cytosol with Apaf-1 after C. amuricus treatment. This is the first study indicating the potential of C.amuricus as a complementary agent for prevention and treatment of human liver cancer.

Dracorhodin perchlorate induces the apoptosis of glioma cells.

Dracorhodin perchlorate (Dp), a synthetic analogue of the antimicrobial anthocyanin red pigment, has recently been shown to induce apoptotic cell death in various types of cancer cells. Yet, the inhibitory effect of Dp on human glioma cells remains uninvestigated. Therefore, in the present study, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry were used to detect cell viability and cell cycle progression in glioma U87MG and T98G cells, respectively. Annexin V-FITC/propidium iodide double staining and JC-1 staining were separately applied to determine cellular apoptosis and mitochondrial membrane potential damage in the cells. The expression levels of associated proteins involved in cell cycle progression and apoptosis were measured by western blotting. The activities of caspase‑9/-3 were determined by Caspase-Glo-9/3 assay. The results indicated that Dp treatment significantly inhibited cell proliferation in a dose- and time-dependent manner, and blocked cell cycle progression at the G1/S phase in the U87MG and T98G cells via the upregulation of p53 and p21 protein expression, and simultaneous downregulation of Cdc25A, Cdc2 and P-Cdc2 protein expression. Additionally, Dp treatment led to the loss of cellular mitochondrial membrane potential, and the release of cytochrome c, and strongly induced the occurence of apoptosis. Increased expression levels of Bim and Bax protein and the downregulated expression of Bcl-2 protein were observed. Caspase-9/-3 were activated and their activities were elevated after Dp treatment. These findings indicate that Dp inhibits cell proliferation, induces cell cycle arrest and apoptosis in glioma cells, and is a possible candidate for glioma treatment.

The microRNA 424/503 cluster reduces CDC25A expression during cell cycle arrest imposed by transforming growth factor β in mammary epithelial cells.

Recently, we demonstrated that the microRNA 424(322)/503 [miR-424(322)/503] cluster is transcriptionally controlled by transforming growth factor β (TGF-β) in the mammary epithelium. Induction of this microRNA cluster impacts mammary epithelium fate by regulating apoptosis and insulin-like growth factor 1 (IGF1) signaling. Here, we expanded our finding to demonstrate that miR-424(322)/503 is an integral component of the cell cycle arrest mediated by TGF-β. Mechanistically, we showed that after TGF-β exposure, increased levels of miR-424(322)/503 reduce the expression of the cell cycle regulator CDC25A. miR-424(322)/503-dependent posttranscriptional downregulation of CDC25A cooperates with previously described transcriptional repression of the CDC25A promoter and proteasome-mediated degradation to reduce the levels of CDC25A expression and to induce cell cycle arrest. We also provide evidence that the TGF-β/miR-424(322)/503 axis is part of the mechanism that regulates the proliferation of hormone receptor-positive (HR(+)) mammary epithelial cells in vivo.

CDC25A Protein Stability Represents a Previously Unrecognized Target of HER2 Signaling in Human Breast Cancer: Implication for a Potential Clinical Relevance in Trastuzumab Treatment

The CDC25A-CDK2 pathway has been proposed as critical for the oncogenic action of human epidermal growth factor receptor 2 (HER2) in mammary epithelial cells. In particular, transgenic expression of CDC25A cooperates with HER2 in promoting mammary tumors, whereas CDC25A hemizygous loss attenuates the HER2-induced tumorigenesis penetrance. On the basis of this evidence of a synergism between HER2 and the cell cycle regulator CDC25A in a mouse model of mammary tumorigenesis, we investigated the role of CDC25A in human HER2-positive breast cancer and its possible implications in therapeutic response. HER2 status and CDC25A expression were assessed in 313 breast cancer patients and we found statistically significant correlation between HER2 and CDC25A (P = .007). Moreover, an HER2-positive breast cancer subgroup with high levels of CDC25A and very aggressive phenotype was identified (P = .005). Importantly, our in vitro studies on breast cancer cell lines showed that the HER2 inhibitor efficacy on cell growth and viability relied also on CDC25A expression and that such inhibition induces CDC25A down-regulation through phosphatidylinositol 3-kinase/protein kinase B pathway and DNA damage response activation. In line with this observation, we found a statistical significant association between CDC25A overexpression and trastuzumab-combined therapy response rate in two different HER2-positive cohorts of trastuzumab-treated patients in either metastatic or neoadjuvant setting (P = .018 for the metastatic cohort and P = .021 for the neoadjuvant cohort). Our findings highlight a link between HER2 and CDC25A that positively modulates HER2- targeted therapy response, suggesting that, in HER2-positive breast cancer patients, CDC25A overexpression affects trastuzumab sensitivity.

Compound K, a Ginsenoside Metabolite, Inhibits Colon Cancer Growth via Multiple Pathways Including p53-p21 Interactions

Compound K (20-O-beta-d-glucopyranosyl-20(S)-protopanaxadiol, CK), an intestinal bacterial metabolite of ginseng protopanaxadiol saponins, has been shown to inhibit cell growth in a variety of cancers. However, the mechanisms are not completely understood, especially in colorectal cancer (CRC). A xenograft tumor model was used first to examine the anti-CRC effect of CK in vivo. Then, multiple in vitro assays were applied to investigate the anticancer effects of CK including antiproliferation, apoptosis and cell cycle distribution. In addition, a qPCR array and western blot analysis were executed to screen and validate the molecules and pathways involved. We observed that CK significantly inhibited the growth of HCT-116 tumors in an athymic nude mouse xenograft model. CK significantly inhibited the proliferation of human CRC cell lines HCT-116, SW-480, and HT-29 in a dose- and time-dependent manner. We also observed that CK induced cell apoptosis and arrested the cell cycle in the G1 phase in HCT-116 cells. The processes were related to the upregulation of p53/p21, FoxO3a-p27/p15 and Smad3, and downregulation of cdc25A, CDK4/6 and cyclin D1/3. The major regulated targets of CK were cyclin dependent inhibitors, including p21, p27, and p15. These results indicate that CK inhibits transcriptional activation of multiple tumor-promoting pathways in CRC, suggesting that CK could be an active compound in the prevention or treatment of CRC.

Prevention of liver carcinogenesis by amarogentin through modulation of G 1 /S cell cycle check point and induction of apoptosis

Amarogentin, a secoiridoid glycoside, is an active component of the medicinal plant Swertia chirata. In this study, chemopreventive and chemotherapeutic actions of amarogentin were evaluated in a carbon tetrachloride (CCl(4))/N-nitrosodiethylamine (NDEA)-induced liver carcinogenesis mouse model system during continuous and posttreatment schedule. Better survival, no toxicity and increased body weight were noted in amarogentin-treated mice. Reduction in proliferation and increase in apoptosis frequency were evident in amarogentin-treated groups. In carcinogen control group moderate dysplasia, severe dysplasia and hepatocellular carcinoma were evident at 10th, 20th and 30th week, respectively. Amarogentin was found to prevent progression of liver carcinogenesis at mild dysplastic stage. Exposure to CCl(4)/NDEA resulted in upregulation of ppRb807/811, cyclinD1 and cdc25A at 10th week and additional activation of cMyc and mdm2 along with downregulation of LIMD1, p53 and p21 at 20th week. This was followed by activation of ppRb567 and downregulation of Rbsp3 at 30th week. Prevention of carcinogenesis by amarogentin in both groups might be due to cumulative upregulation of LIMD1, RBSP3, p16 and downregulation of cdc25A at 10th week along with activation of p53 and p21 and downregulation of ppRb807/811 and ppRb567 at 20th week, followed by downregulation of cyclinD1, cMyc and mdm2 at 30th week. During carcinogenesis reduction of apoptosis was evident since 20th week. However, amarogentin treatment could significantly induce apoptosis through upregulation of the Bax-Bcl2 ratio, activation of caspase-3 and poly ADP ribose polymerase cleavage. This is the first report of chemopreventive/therapeutic role of amarogentin during liver carcinogenesis through modulation of cell cycle and apoptosis.

Abstract 1685: Ginsenoside compound K inhibits colorectal tumorigenesis via multiple pathways including ATM/p53-p21

Abstract To date, colorectal cancer (CRC) remains one of the most prevalent malignancies in the United States. Health benefits derived from medicinal plants have been investigated for years. Ginseng has been shown to induce diverse pharmacological effects mediated predominantly by saponins, a type of ginsenoside contained in ginseng. Compound K (20-O-beta-D-glucopyranosyl-20(S)-protopanaxadiol, CK), an intestinal bacterial metabolite of ginseng protopanaxadiol saponins, has been shown to inhibit cell growth in a variety of tumors. However, the detailed mechanism, especially the effects of CK on cell cycle regulation in CRC cells, has not been examined. In addition, it is uncertain whether CK can inhibit colorectal tumor growth in vivo. In this study, we observed that CK significantly inhibited the growth of HCT-116 cells in athymic nude mouse xenograft models. Quantitative analysis revealed that CK significantly inhibited xenograft tumor growth from the 3rd week after CK administration (p<0.001); the higher dose (30mg/kg) of CK treatment exhibited a stronger effect than the lower dose (15mg/kg)(p<0.001). Using MTS assay, we showed that CK significantly inhibited the viability of human colorectal cancer cell lines HCT-116, HT-29 and SW480 in a dose- and time-dependent manner(with IC50 at around 10, 25, and 25uM respectively, p<0.01). In a flow cytometry assay, it was shown that CK could significantly induce HCT-116 cell apoptosis and arrest the cell cycle in the G1 phase. By screening with cell cycle and cell signal PCR arrays, some of the most relevant genes(such as CDKs/cyclins, BCL2, E2F4, p21CDKN1A, p15CDKN2B, TP53, etc.) and signal transduction pathways(such as p53, TGF-β, PI3K/AKT, pathways) were shown to be transcriptionally regulated after CK exposure. Western blots showed that the major regulated targets of CK are cyclin dependent inhibitors, including p21, p27, and p15. CK-induced colorectal cancer cell apoptosis and cell cycle arrest were related to the up-regulation of p53/p21, FoxO3a-p27/p15, Smad3, etc and down-regulation of cdc25A, CDK4/6 and cyclin D1/3 as well. Taken together, the results indicate that CK inhibits transcriptional activation of multiple tumor-promoting pathways in colorectal cancer, which suggest that CK could be useful for the prevention or treatment of CRC. Key words: Colorectal cancer, Ginsenoside, xenograft tumor, p53/p21, cell cycle arrest Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1685. doi:1538-7445.AM2012-1685

Abstract 3021: FLT3/ITD acute myeloid leukemia (AML) cells show C-MYC- and PIM-1-mediated CDC25A overexpression despite the presence of intrinsic DNA damage

Abstract Activating mutations of FLT3 occur in 30% of patients with AML, and are associated with poor prognosis. It has been reported that FLT3/ITD AML cells are sensitive to MDM2 inhibition-induced apoptosis (Blood 2010;116:71) and indeed, FLT3 inhibition synergizes with MDM2 inhibition in the induction of apoptosis in FLT3/ITD AML (Leukemia 2010;24:33). Considering the rarity of TP53 mutations in FLT3/ITD AML, use of MDM2 inhibitors in AML with mutant FLT3 may offer clinical benefits. We investigated the FLT3/ITD-specific p53 signaling profile that may be associated with high susceptibility of FLT3/ITD cells to MDM2 inhibition. [Methods] The MDM2 inhibitor Nutlin-3a was used. Murine pro-B cells stably transfected with expression vectors that encoded wild-type human FLT3 (Ba/F3-FLT3 cells) or human FLT3 containing an ITD mutation (Ba/F3-ITD cells) were provided by Dr Donald Small and AML samples were obtained from patients after informed consent. Apoptosis was quantified by Annexin binding assay and cells were transfected by the Amaxa Nucleofector I. Statistical analysis was performed using t test or Mann-Whitney U test. [Results] Ba/F3-ITD cells were more susceptible to Nutlin-induced apoptosis but not to AraC or doxorubicin than Ba/F3-FLT3 cells; high Nutlin-3a sensitivity was associated with efficient BAX activation (P < .001). Basal levels of total and phospho p53 were 7 times higher in Ba/F3-ITD than in Ba/F3-FLT3 cells; levels of γH2AX, a DNA damage marker, were 6 times higher in Ba/F3-ITD than in Ba/F3-FLT3 cells, and reactive oxygen species levels were also high in Ba/F3-ITD cells (26.2 ± 3.2% vs 6.1 ± 0.1%; P < .001). Although steady state p53 activation was not sufficient to inhibit cell growth or induce cell death in Ba/F3-ITD cells, Nutlin-3a potently activated p53 signaling and induced apoptosis. Interestingly, CDC25A, which is specifically degraded in response to DNA damage, was 30 times higher in Ba/F3-ITD cells compared to Ba/F3-FLT3 cells, despite the presence of DNA damage. Among patient AML samples (n=37), CDC25A levels were significantly higher in samples with mutant FLT3 (n=16) than those with wild-type FLT3 (n=21) (1.7 ± 1.2 vs 17.1 ± 6.7; P < .001). Knockdown experiments showed that CDC25A over-expression was partially induced by C-MYC and PIM-1. Nutlin-3a reduced CDC25A mRNA levels by 63.9 ± 0.4 % (P < 0.01). The proteasome inhibitor MG132 or the pan-caspase inhibitor Z-VAD-FMK did not rescue the CDC25A reduction. [Conclusion] CDC25A over-expression was closely associated with FLT3/ITD in AML. FLT3/ITD cells showed C-MYC- and PIM-1-mediated CDC25A over-expression despite the presence of intrinsic DNA damage. CDC25A over-expression has been reported in a variety of human cancers, and CDC25A has been considered a therapeutic target. CDC25A down-regulation may be potential mechanism by which MDM2 inhibition induces cell death in FLT3/ITD AML cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3021. doi:1538-7445.AM2012-3021

Activation of Cell Cycle Arrest and Apoptosis by the Proto-Oncogene Pim-2

Potent survival effects have been ascribed to the serine/threonine kinase proto-oncogene PIM-2. Elevated levels of PIM-2 are associated with various malignancies. In human cells, a single Pim-2 transcript gives rise mainly to two protein isoforms (34, 41 kDa) that share an identical catalytic site but differ at their N-terminus, due to in-frame alternative translation initiation sites. In this study we observed that the 34 kDa PIM-2 isoform has differential nuclear and cytoplasmic forms in all tested cell lines, suggesting a possible role for the balance between these forms for PIM-2's function. To further study the cellular role of the 34 kDa isoform of PIM-2, an N-terminally HA-tagged form of this isoform was transiently expressed in HeLa cells. Surprisingly, this resulted in increased level of G1 arrested cells, as well as of apoptotic cells. These effects could not be obtained by a Flag-tagged form of the 41 kDa isoform. The G1 arrest and apoptotic effects were associated with an increase in T14/Y15 phosphorylation of CDK2 and proteasom-dependent down-regulation of CDC25A, as well as with up-regulation of p57, E2F-1, and p73. No such effects were obtained upon over-expression of a kinase-dead form of the HA-tagged 34 kDa PIM-2. By either using a dominant negative form of p73, or by over-expressing the 34 kDa PIM-2 in p73-silenced cells, we demonstrated that these effects were p73-dependent. These results demonstrate that while PIM-2 can function as a potent survival factor, it can, under certain circumstances, exhibit pro-apoptotic effects as well.

An apolar extract of Critonia morifolia inhibits c-Myc, cyclin D1, Cdc25A, Cdc25B, Cdc25C and Akt and induces apoptosis

Investigating the bioactivity of traditional medical remedies under the controlled conditions of a laboratory is an option to find additional applications, novel formulations or lead structures for the development of new drugs. The present work analysed the anti‑neoplastic activity of increasing polar extracts of the rainforest plant Critonia morifolia (Asteraceae) that has been successfully used as traditional remedy to treat various inflammatory conditions in the long-lasting medical tradition of the Central American Maya, which was here also confirmed in vitro. The apolar petroleum ether extract exhibited the most potent anti‑proliferative and pro‑apoptotic effects in HL‑60 cells and triggered down-regulation of Cdc25C and cyclin D1 within 30 min followed by the inhibition of c-Myc expression and the onset of caspase-3 activation within 2 h. Subsequent to these very rapid molecular responses Chk2 and H2AX became phosphorylated (γ‑H2AX) after 4 h. Analysis of the cell cycle distribution showed an accumulation of cells in the G2-M phase within 8 h and after 24 h in S-phase. This was temporally paralleled by the down-regulation of Cdc25A, Cdc25B, Wee1 and Akt. Therefore, the attenuation of cell cycle progression in the G2-M phase was consistent with the known role of Chk2 for G2-M arrest and with the role of Cdc25B in S-phase progression. These findings suggest the presence of two distinct active principles in the petroleum ether extract of C. moriflia. These facilitated the strong apoptotic response evidenced by the rapid activation of caspase-3 that was later enforced by the inhibition of the survival kinase Akt. Importantly, the efficient down-regulation of Akt, which is successfully tested in current clinical trials, is a unique property of C. morifolia.

Involvement of Chk1–Cdc25A-cyclin A/CDk2 pathway in simvastatin induced S-phase cell cycle arrest and apoptosis in multiple myeloma cells

Statins have been demonstrated to effectively inhibit proliferation and induce apoptosis in cancer cells by inhibition of geranylgeranylation, however its novel molecular mechanism remains to be determined. Recently simvastatin has been found to result in the synergistic induction of apoptosis with 7-hydroxystaurosporine (UCN-01) (a Chk1 inhibitor) in myeloma cells. Therefore we hypothesized that Chk1 plays a role in the anti-myeloma effect of simvastatin. Interestingly, we found that simvastatin caused a dose-dependent increase in S phase cell cycle and induced significant apoptosis. The results of western blot showed that simvastatin-induced S-phase cell cycle arrest was associated with activation of Chk1, downregulation of Cdc25A, cyclin A and CDK2 expression. Additionally, simvastatin-induced apoptosis was accompanied by diminished Bcl-2 protein expression, increased cytosolic cytochrome c level, and activation of caspase 9 and caspase 3. Further investigation revealed that silence of Chk1 expression by Chk1 specific siRNA inhibited simvastatin-induced activation of Chk1, downregulation of Cdc25A, cyclin A and CDK2 expression, and diminished S phase cell cycle arrest. Additionally, inhibition of Chk1 expression enhanced simvastatin-induced downregulation of Bcl-2, caspase 9 cleavage and subsequent apoptosis. These results suggested that the Chk1-Cdc25A-cyclin A/CDk2 pathway was involved in simvastatin-induced S-phase cell cycle arrest and apoptosis in multiple myeloma cell lines.

High-Frequency Canonical Wnt Activation in Multiple Sarcoma Subtypes Drives Proliferation through a TCF/β-Catenin Target Gene, CDC25A

Wnt canonical signaling is critical for normal development as well as homeostasis of several epithelial tissues, and constitutive activation of this pathway is commonly observed in carcinomas. We show here that 50% of human sarcomas (n = 45) and 65% of sarcoma cell lines (n = 23) of diverse histological subtypes exhibit upregulated autocrine canonical Wnt signaling. Furthermore, in Wnt autocrine cell lines, we identify alterations including overexpression or gene amplification of Wnt ligands and/or LRP5/6 coreceptors and epigenetic silencing of different cell surface Wnt antagonists. Mutations in adenomatous polyposis coli (APC) gene were observed in two nonautocrine Wnt-positive sarcoma cell lines. Finally, downregulation of the activated Wnt pathway inhibited sarcoma cell proliferation both in vitro and in vivo by a mechanism involving the downregulation of CDC25A.