Cdc25C Protein Research Articles

Abstract 1310: Increased cytoplasmic CDC25A phosphatase in cutaneous squamous cell carcinoma leads to 14-3-3 relocalization and suppression of apoptosis

Abstract Non-melanoma skin cancer, the most common cancer in the United States, frequently results from chronic exposure to ultraviolet (UV) irradiation. UV-induced DNA damage activates cell cycle arrest checkpoints through degradation of the cyclin-dependent kinase activators, the Cell Division Cycle 25 (CDC25) phosphatases, which are over-expressed and, thus, deregulate cell cycle control in many human cancers. To investigate a role for CDC25 in skin cancer, CDC25 levels were examined in both UV-induced mouse tumors and human skin cancer. CDC25A and CDC25C protein and transcripts were increased in benign and malignant skin tumors from UV-exposed v-rasHa transgenic Tg.AC mice. CDC25B protein but not transcripts was increased in mouse skin tumors compared to normal skin. Similarly, CDC25A and CDC25C but not CDC25B were elevated in human squamous cell carcinoma (SCC) and in the premalignant precursors actinic keratosis and Bowen's disease when compared to skin. While CDC25A was localized primarily to nuclei in normal epidermis, both the premalignant precursors and SCC exhibited strong cytoplasmic CDC25A localization. To determine whether increased CDC25A stimulates proliferation in skin SCC, CDC25A levels were modulated by siRNA-targeting or forced expression in cultured SCC cells, but no difference in proliferation resulted. Because cytoplasmic CDC25A can bind to the apoptotic regulator 14-3-3, we hypothesized that the increase in cytoplasmic CDC25A may suppress apoptosis. Forced over-expression of CDC25A in SCC cells suppressed cell death, whereas CDC25A silencing increased apoptotic cell death. Phosphorylation of CDC25A at serine178, a docking site for 14-3-3 proteins, was elevated in the cytoplasmic compartment of SCCs, and cytoplasmic 14-3-3ϵ was increased in cutaneous SCC.Mutation of the nuclear export sequence on CDC25A relocalized both CDC25A and 14-3-3ϵ to the nucleus, while mutation of 14-3-3 binding sites on CDC25A led to a decrease in 14-3-3ϵ levels. Furthermore, silencing of 14-3-3ϵ increased cell death by 60%. Taken together, these data suggest that increased cytoplasmic CDC25A in cutaneous SCC may suppress apoptotic cell death through its interaction with 14-3-3ϵ. Citation Format: Jenan Almatouq, Kristen Ho, Laura A. Hansen. Increased cytoplasmic CDC25A phosphatase in cutaneous squamous cell carcinoma leads to 14-3-3 relocalization and suppression of apoptosis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1310. doi:10.1158/1538-7445.AM2014-1310

Activation of JNK contributes to evodiamine-induced apoptosis and G2/M arrest in human colorectal carcinoma cells: a structure-activity study of evodiamine.

Evodiamine (EVO; 8,13,13b,14-tetrahydro-14-methylindolo[2′3′-3,4]pyrido[2,1-b]quinazolin-5-[7H]-one derived from the traditional herbal medicine Evodia rutaecarpa was reported to possess anticancer activity; however, the anticancer mechanism is still unclear. In this study, we investigated the anticancer effects of EVO on human colon COLO205 and HT-29 cells and their potential mechanisms. MTT and lactate dehydrogenase (LDH) release assays showed that the viability of COLOL205 and HT-29 cells was inhibited by EVO at various concentrations in accordance with increases in the percentage of apoptotic cells and cleavage of caspase-3 and poly(ADP ribose) polymerase (PARP) proteins. Disruption of the mitochondrial membrane potential by EVO was accompanied by increased Bax, caspase-9 protein cleavage, and cytochrome (Cyt) c protein translocation in COLO205 and HT-29 cells. Application of the antioxidant N-acetyl-L-cysteine (NAC) inhibited H2O2-induced reactive oxygen species (ROS) production and apoptosis, but did not affect EVO-induced apoptosis of COLO205 or HT-29 cells. Significant increases in the G2/M ratio and cyclinB1/cdc25c protein expression by EVO were respectively identified in colon carcinoma cells via a flow cytometric analysis and Western blotting. Induction of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) protein phosphorylation was detected in EVO-treated cells, and the JNK inhibitor, SP600125, but not the ERK inhibitor, U0126, inhibited EVO-induced phosphorylated JNK protein expression, apoptosis, and G2/M arrest of colon carcinoma cells. Data of the structure-activity analysis showed that EVO-related chemicals containing an alkyl group at position 14 were able to induce apoptosis, G2/M arrest associated with increased DNA ladder formation, cleavage of caspase-3 and PARP, and elevated cycB1 and cdc25c protein expressions in COLO205 and HT-29 cells. Evidence supporting JNK activation leading to EVO-induced apoptosis and G2/M arrest in colon carcinoma cells is provided, and alkylation at position 14 of EVO is a critical substitution for treatment of colonic cancer.

Inhibitory effects of α-pinene on hepatoma carcinoma cell proliferation.

Pine needle oil from crude extract of pine needles has anti-tumor effects, but the effective component is not known. In the present study, compounds from a steam distillation extract of pine needles were isolated and characterized. Alpha-pinene was identified as an active anti-proliferative compound on hepatoma carcinoma BEL-7402 cells using the MTT assay. Further experiments showed that α-pinene inhibited BEL-7402 cells by arresting cell growth in the G2/M phase of the cell cycle, downregulating Cdc25C mRNA and protein expression, and reducing cycle dependence on kinase 1(CDK1) activity. Taken together, these findings indicate that α-pinene may be useful as a potential anti-tumor drug.

Anti-oxidative and Anti-cancer Activities by Cell Cycle Regulation of Salsola collina Extract

Salsola collina, also known as Russian thistle, is widely distributed in and around waste facilities, roadsides, and drought and semi-drought areas, and is used as a traditional folk remedy in Chinese medicine for the treatment of hypertension. In this study, we have evaluated the anti-oxidative and anti-cancer activities of the ethanol extract of S. collina Pall. (EESC), and the molecular mechanisms of its anti-cancer effects on human colon carcinoma HT29 cells. EESC exhibited anti-oxidative activity through DPPH radical scavenging capacity and showed cytotoxic activity in a dose-dependent manner in HT29 cells. After EESC treatment, HT29 cells altered their morphology, becoming smaller and irregular in shape. EESC also induced cell accumulation in the G2/M phase in a dose-dependent manner, accompanied by a decrease of cell population in the G1 phase. The G2/M arrest by EESC was associated with the increased expression of cyclin-dependent kinase (CDK) inhibitor p21 and Wee1 kinase, which phosphorylates, or inactivates, Cdc2. EESC treatment induced the phosphorylation of Cdc2 and Cdc25C, and inhibited cyclin A and Cdc25C protein expression. In addition, S arrest was induced by the highest concentration of EESC treatment, associated with a decrease of cyclin A and Cdk2 expression. These findings suggest that EESC may possess remarkable anti-oxidative activity and exert an anti-cancer effect in HT29 cells by cell cycle regulation.

Chk1, but not Chk2, is responsible for G2/M phase arrest induced by diallyl disulfide in human gastric cancer BGC823 cells

Diallyl disulfide (DADS) has been shown to cause G2/M phase cell cycle arrest in several human cancers. Here we demonstrate a mechanism by which DADS induces G2/M phase arrest in BGC823 human gastric cancer cells via Chk1. From cell cycle gene array results, we next confirmed that cyclin B1 expression was decreased by DADS, while the expression of p21, GADD45α and p53 were increased. Despite the lack of change in Chk1 gene expression in response to DADS according to the array analysis, intriguingly overexpression of Chk1, but not Chk2, exhibited increased accumulation in G2/M phase. Moreover, overexpression of Chk1 promoted the effect of DADS-induced G2/M arrest. Augmented phosphorylation of Chk1 by DADS was observed in Chk1-transfected cells, followed by downregulation of Cdc25C and cyclin B1 proteins. In contrast, phosphorylated Chk2 showed no obvious change in Chk2-transfected cells after DADS treatment. Furthermore, knockdown of Chk1 by siRNA partially abrogated DADS-induced downregulation of Cdc25C and cyclin B1 proteins and G2/M arrest. In contrast, knockdown of Chk2 did not show these effects. Therefore, these data indicate that DADS may specifically modulate Chk1 phosphorylation, and DADS-induced G2/M phase arrest in BGC823 cells could result in part from Chk1-mediated inhibition of the Cdc25C/cyclin B1 pathway.

Antitumor activity of di-n-butyl-(2,6-difluorobenzohydroxamato)tin(IV) against human gastric carcinoma SGC-7901 cells via G2/M cell cycle arrest and cell apoptosis.

Di-n-butyl-(2,6-difluorobenzohydroxamato)Tin(IV) (DBDFT), a potential antitumor agent against malignancies, exhibited high activities both in vitro and in vivo. Flow cytometric analysis showed that treatment with DBDFT against Human Gastric Carcinoma (SGC-7901) cells induced a concentration and time-dependent cell accumulation in the G2/M phase of the cell cycle with a parallel depletion of the percentage of cells in G0/G1, the cell apoptosis was observed by characteristic morphological changes and AnnexinV/PI dual-immunofluorescence staining. Fluorescence quantitative FQ- PCR and western blot results showed that G2/M-phase arrest was correlated with up-regulation of cyclin dependent kinase inhibitor p21, Chk2 and CyclinB1, whereas the expressions of other G2/M regulatory check-point protein, Cdc2, and feedback loop protein Cdc25C were obviously down-regulated in a p53-independent manner after the SGC-7901 cells were treated with DBDFT (2.5, 5.0, 7.5 µmol·L−1) compared with the control. Furthermore, the up-regulation of Bax and down-regulation of Bcl-2 as well as the activation of caspase-3 were observed, which indicated that DBDFT treatment triggered the mitochondrial apoptotic pathway with an increase of Bax/Bcl-2 ratios, resulting in mitochondrial membrane potential loss and caspase-9 activation in DBDFT treated SGC-7901 cells. In summary, the results illustrated the involvement of multiple signaling pathways targeted by DBDFT in mediating G2/M cell cycle arrest and apoptosis in SGC-7901 cells, which suggested that DBDFT might have therapeutic potential against gastric carcinoma as an effective compound.

Tributyltin induces a G2/M cell cycle arrest in human amniotic cells via PP2A inhibition-mediated inactivation of the ERK1/2 cascades

The molecular mechanisms underlying the cell cycle alterations induced by tributyltin (TBT), a highly toxic environmental contaminant, remain elusive. In this study, cell cycle progression and some key regulators in G2/M phase were investigated in human amniotic cells treated with TBT. Furthermore, protein phosphatase (PP) 2A and the ERK cascades were examined. The results showed that TBT caused a G2/M cell cycle arrest that was accompanied by a decrease in the total cdc25C protein level and an increase in the p-cdc2 level in the nucleus. TBT caused a decrease in PP2A activity and inhibited the ERK cascade by inactivating Raf-1, resulting in the dephosphorylation of MEK1/2, ERK1/2, and c-Myc. Taken together, TBT leads to a G2/M cell cycle arrest in FL cells, an increase in p-cdc2 and a decrease in the levels of total cdc25C protein, which may be caused by the PP2A inhibition-mediated inactivation of the ERK1/2 cascades.

Kaempferol Induces Cell Cycle Arrest in HT-29 Human Colon Cancer Cells.

Background:A greater intake of vegetables and fruits has been linked to a reduced incidence of colon cancer. Flavonoids are polyphenolic compounds which are broadly distributed in fruits and vegetables and display a remarkable spectrum of physiological activities, including anti-carcinogenic effects. The objective of this study was to determine the mechanisms by which kaempferol, a flavonol present in tea, apples, strawberries, and beans, inhibits the growth of HT-29 human colon cancer cells.Methods:To examine the effects of kaempferol on cell cycle progression in HT-29 cells, cells were treated with various concentrations (0–60 μmol/L) of kaempferol. Cell proliferation and DNA synthesis were evaluated by MTT assay and [3H]thymidine incorporation assay, respectively. Fluorescence-activated cell sorting analyses were conducted to calculate cell cycle phase distribution. Western blot analyses and in vitro kinase assays were used to estimate the expression of proteins involved in the regulation of cell cycle progression and the activity of cyclin-dependent kinase (CDK)s, respectively.Results:Kaempferol decreased viable cell numbers and [3H]thymidine incorporation into DNA of HT-29 cells in a dose-dependent manner. Kaempferol induced G1 cell cycle arrest within 6 h and G2/M arrest at 12 h. Kaempferol inhibited the activity of CDK2 and CDK4 as well as the protein expression of CDK2, CDK4, cyclins D1, cyclin E, and cyclin A, and suppressed the phosphorylation of retinoblastoma protein. Additionally, kaempferol decreased the levels of Cdc25C, Cdc2, and cyclin B1 proteins, as well as the activity of Cdc2.Conclusions:The present results indicate that kaempferol induces G1 and G2/M cell cycle arrest by inhibiting the activity of CDK2, CDK4, and Cdc2. The induction of cell cycle arrest may be one of the mechanisms by which kaempferol exerts anti-carcinogenic effects in colon cancer cells.

The Telomere Deprotection Response Is Functionally Distinct from the Genomic DNA Damage Response

Loss of chromosome end protection through telomere erosion is a hallmark of aging and senescence. Here we developed an experimental system that mimics physiological telomere deprotection in human cells and discovered that the telomere deprotection response is functionally distinct from the genomic DNA damage response. We found that, unlike genomic breaks, deprotected telomeres that are recognized as DNA damage but remain in the fusion-resistant intermediate state activate differential ataxia telangiectasia mutated (ATM) signaling where CHK2 is not phosphorylated. Also unlike genomic breaks, we found that deprotected telomeres do not contribute to the G2/M checkpoint and are instead passed through cell division to induce p53-dependent G1 arrest in the daughter cells. Telomere deprotection is therefore an epigenetic signal passed between cell generations to ensure that replication-associated telomere-dependent growth arrest occurs in stable diploid G1 phase cells before genome instability can occur.

Berberine inhibits the growth of human colorectal adenocarcinoma in vitro and in vivo

Berberine is an alkaloid isolated from the Chinese herbal medicine Huanglian, and has long been used as an antibiotic. Its antineoplastic properties were subsequently discovered in vitro. The purpose of this study was to investigate the effects of berberine on the growth of human colorectal carcinoma cells in vitro and in vivo. The results showed that berberine inhibited human colorectal adenocarcinoma (LoVo) cell growth in a time- and dose-dependent manner. A WST-1 assay showed that the IC50 value after 72 h was 40.79 ± 4.11 μM. Cell cycle analysis of 40 μM berberine-treated LoVo cells by flow cytometry showed accumulation of cells in the G2/M phase. The inhibition of LoVo cell growth by berberine was associated with the suppression of cyclin B1, cdc2, and cdc25c proteins. Berberine at a dose of 50 mg kg(-1) day(-1) showed inhibitory rates of 45.3% in a human colorectal adenocarcinoma xenograft in nude mice. The combination of berberine and 5-fluorouracil (5-FU) had a higher inhibitory rate (59.8%) than the berberine group (36.4%, P = 0.01), but no significant difference was observed between the 5-FU group (43.0%, P = 0.06) and the combination group. These results support the possibility that berberine may be useful as an alternative therapy for colorectal carcinoma.

Androgens Upregulate Cdc25C Protein by Inhibiting Its Proteasomal and Lysosomal Degradation Pathways

Cdc25C is a cell cycle protein of the dual specificity phosphatase family essential for activating the cdk1/Cyclin B1 complex in cells entering into mitosis. Since altered cell cycle is a hallmark of human cancers, we investigated androgen regulation of Cdc25C protein in human prostate cancer (PCa) cells, including androgen-sensitive (AS) LNCaP C-33 cells and androgen-independent (AI) LNCaP C-81 as well as PC-3 cells. In the regular culture condition containing fetal bovine serum (FBS), Cdc25C protein levels were similar in these PCa cells. In a steroid-reduced condition, Cdc25C protein was greatly decreased in AS C-33 cells but not AI C-81 or PC-3 cells. In androgen-treated C-33 cells, the Cdc25C protein level was greatly elevated, following a dose- and a time-dependent manner, correlating with increased cell proliferation. This androgen effect was blocked by Casodex, an androgen receptor blocker. Nevertheless, epidermal growth factor (EGF), a growth stimulator of PCa cells, could only increase Cdc25C protein level by about 1.5-fold. Altered expression of Cdc25C in C-33 cells and PC-3 cells by cDNA and/or shRNA transfection is associated with the corresponding changes of cell growth and Cyclin B1 protein level. Actinomycin D and cycloheximide could only partially block androgen-induced Cdc25C protein level. Treatments with both proteasomal and lysosomal inhibitors resulted in elevated Cdc25C protein levels. Immunoprecipitation revealed that androgens reduced the ubiquitination of Cdc25C proteins. These results show for the first time that Cdc25C protein plays a role in regulating PCa cell growth, and androgen treatments, but not EGF, greatly increase Cdc25C protein levels in AS PCa cells, which is in part by decreasing its degradation. These results can lead to advanced PCa therapy via up-regulating the degradation pathways of Cdc25C protein.

Purification and biochemical analysis of catalytically active human cdc25C dual specificity phosphatase

We describe a reliable and efficient method for the purification of catalytically active and mutant inactive full-length forms of the human dual specificity phosphatase cdc25C from bacteria. The protocol involves isolating insoluble cdc25C protein in inclusion bodies, solubilization in guanidine HCL, and renaturation through rapid dilution into low salt buffer. After binding renatured proteins to an ion exchange resin, cdc25C elutes in two peaks at 350 and 450 mM NaCl. Analysis by gel exclusion chromatography and enzymatic assays reveals the highest phosphatase activity is associated with the 350 mM NaCl with little or no activity present in the 450 mM peak. Furthermore, active cdc25C has a native molecular mass of 220 kDa consistent with a potential tetrameric complex of the 55-kDa cdc25C protein. Assaying phosphatase activity against artificial substrates pNPP and 3-OMFP reveals a 220 kDa form of the phosphatase is active in a non-phosphorylated state. The protein effectively activates cdk1/cyclin B prokinase complexes in vitro in the absence of cdk1 kinase activity in an orthovanadate sensitive manner but is inactivated by A-kinase phosphorylation. In vitro phosphorylation of purified cdc25C by cdk1/cyclin B1, cdk2/cyclin A2 and cdk2/cyclin E shows that distinct TP/SP mitotic phosphorylation sites on cdc25C are differentially phosphorylated by these 3 cdk/cyclin complexes associated with different levels of cdc25C activation. Finally, we show that endogenous native cdc25C from human cells is present in high molecular weight complexes with other proteins and resolves mostly above 200-kDa. These data show that untagged cdc25C can be purified with a simple protocol as an active dual specificity phosphatase with a native molecular mass consistent with a homo-tetrameric configuration.

Decitabine, a DNA methyltransferases inhibitor, induces cell cycle arrest at G2/M phase through p53-independent pathway in human cancer cells

Decitabine (5-aza-2′-deoxycytidine), an inhibitor of DNA methyltransferases, has a wide range of anti-metabolic and anti-cancer activities. Decitabine also induces cell cycle arrest at G2/M phase and apoptosis in human cancer cells. However, the cellular and molecular mechanisms of this cell cycle arrest are poorly understood. In the present study, we investigated the roles of the tumor suppressor p53 and the cyclin-dependent kinase (Cdk) inhibitor p21 following decitabine-induced G2/M arrest in human cancer cells. DNA flow cytometric analyses indicated that decitabine induced a G2/M arrest in AGS gastric and A549 lung carcinoma cell lines, which have wild type p53. Western blot analyses using whole cell lysates from AGS cells demonstrated that decitabine treatment did not change the steady-state level of Cdks and Cdk inhibitor p27, but it partially inhibited expression of cyclin A, cyclin B1, and Cdc25C proteins. However, similar results were found using the A549 cell line, where decitabine induced a dramatic up-regulation of both p53 and p21 expression, and the increased levels of p21 were associated with increased binding of p21 with Cdks, cyclin A, and cyclin B1. Knockdown of p53 by small interfering RNA (siRNA) markedly abolished p53 induction by decitabine in AGS cells, yet p53 siRNA had no attenuating effect on p21 induction. In addition, depletion of p21 expression with siRNA, but not p53, significantly attenuated decitabine-induced G2/M arrest. We also observed that decitabine strongly induced G2/M arrest associated with p21 induction in both p53 allele-null (–/–) HCT116 and wild type p53 (+/+) HCT116 cell lines. Therefore, our data indicated that p21 plays a crucial role in decitabine-induced G2/M arrest and operates in a p53-independent manner.

Histone deacetylase induces accelerated maturation in Xenopus laevis oocytes

In oocyte maturation in Xenopus laevis, nuclear material induces rapid maturation and is required for entry into meiosis II. Nuclear material contains a large number of RNAs and proteins, including histone deacetylase (HDAC); however, it is not known which materials induce accelerated maturation. The HDAC activity modifies transcription rate and is required for normal meiosis; however, its function in oocyte maturation is still unclear. We investigated the function of HDAC activity, which is localized in the nuclear material, in the regulation of the speed of oocyte maturation. Inhibition of HDAC activity with trichostatin A (TSA) induced hyperacetylation of histone H3 and prolonged oocyte maturation. In contrast, increase in HDAC activity with an injection of FLAG-tagged maternal histone deacetylase (HDACm-FLAG) mRNA induced deacetylation of histone H3 and reduced the duration of oocyte maturation. Cdc2 kinase, Cdc25C or mitogen-activated protein kinase (MAPK), which are key regulators of the meiosis, were activated coincidently with maturation progression. In oocytes, the mRNA level of Cdc25C, an activator of Cdc2, was increased by HDACm-FLAG mRNA-injection; in contrast, the mRNA level of Cdc2 inhibitor Wee1 was increased by TSA treatment. These results suggest that HDAC activity is involved in the control of maturation speed through the regulation of mRNA levels of cell cycle regulators. Thus, HDACm is a candidate for the nuclear material component that induces rapid maturation in Xenopus oocytes.

Butyrate induces reactive oxygen species production and affects cell cycle progression in human gingival fibroblasts

Short-chain fatty acids, such as butyric acid and propionic acid, are metabolic by-products generated by periodontal microflora such as Porphyromonas gingivalis, and contribute to the pathogenesis of periodontitis. However, the effects of butyrate on the biological activities of gingival fibroblasts (GFs) are not well elucidated. Human GFs were exposed to various concentrations of butyrate (0.5-16 mm) for 24 h. Viable cells that excluded trypan blue were counted. Cell cycle distribution of GFs was analyzed by propidium iodide-staining flow cytometry. Cellular reactive oxygen species (ROS) production was measured by flow cytometry using 2',7'-dichlorofluorescein (DCF). Total RNA and protein lysates were isolated and subjected to RT-PCR using specific primers or to western blotting using specific antibodies, respectively. Butyrate inhibited the growth of GFs, as indicated by a decrease in the number of viable cells. This event was associated with an induction of G0/G1 and G2/M cell cycle arrest by butyrate (4-16 mm) in GFs. However, no marked apoptosis of GFs was noted in this experimental condition. Butyrate (> 2 mm) inhibited the expression of cdc2, cdc25C and cyclinB1 mRNAs and reduced the levels of Cdc2, Cdc25C and cyclinB1 proteins in GFs, as determined using RT-PCR and western blotting, respectively. This toxic effect of butyrate was associated with the production of ROS. These results suggest that butyrate generated by periodontal pathogens may be involved in the pathogenesis of periodontal diseases via the induction of ROS production and the impairment of cell growth, cell cycle progression and expression of cell cycle-related genes in GFs. These events are important in the initiation and prolongation of inflammatory processes in periodontal diseases.

Cell division cycle 25 homolog c effects on low-dose hyper-radiosensitivity and induced radioresistance at elevated dosage in A549 cells

The underlying mechanisms behind both low-dose hyper-radiosensitivity (HRS) and induced radioresistance (IRR), generally occurring at elevated radiation levels, remain unclear; however, elucidation of the relationship between cell cycle division 25 homolog c (Cdc25c) phosphatase and HRS/IRR may provide important insights into this process. Two cell lines with disparate HRS status, A549 and SiHa cells, were selected as cell models for comparison of dose-dependent Cdc25c phosphatase expression subsequent to low-dose irradiation. Knockdown of Cdc25c in A549 cells was mediated by transfection with a pGCsi-RAN-U6neo vector containing hairpin siRNA sequences. S216-phosphorylated Cdc25c protein [p-Cdc25c (Ser216)], cell survival and mitotic ratio were measured by western blot, colony-forming assay and histone H3 phosphorylation analysis. Variant p-Cdc25c (Ser216) expression was observed in the two cell lines after irradiation. The p-Cdc25c (Ser216) expression noted in SiHa cells after administration of 0–1 Gy radiation was similar to the radioresistance model; however, in A549 cells, the dose response for the phosphorylation of the Cdc25c Ser216 residue overlapped the level required to overcome the HRS response. Furthermore, Cdc25c repression prior to low-dose radiation induced more distinct HRS and prevented the development of IRR. The dose required to overcome the HRS response coincided with the effect of early G2-phase checkpoint arrest in A549 cells (approximately 0.3 Gy), and Cdc25c knockdown in A549 cells (approximately 0.5 Gy) corresponded to the phosphorylation of the Cdc25c Ser216 residue. Resultant data confirmed that dose-dependent Cdc25c phosphatase does effectively act as an early G2-phase checkpoint, thus indicating mechanistic importance in the HRS to IRR transition in A549 cells.

Inactivation of PI3K/Akt signaling mediates proliferation inhibition and G2/M phase arrest induced by andrographolide in human glioblastoma cells

AimsAndrographolide, a principal diterpenoid lactone isolated from the traditional herbal medicine Andrographis paniculata, has been reported to show anti-tumor activity. Since the high lipid solubility of andrographolide permits it to penetrate the blood–brain barrier and concentrate in the brain, we hypothesized that andrographolide may be a potential chemotherapeutic agent for the treatment of glioblastomas. To clarify this point, we investigated the growth inhibitory effect and mechanisms of actions of andrographolide on human glioblastoma U251 and U87 cells. Main methodsMTT assay and trypan blue exclusion assay were used to investigate the proliferation inhibitory and cytotoxic effects of andrographolide, respectively. Cell cycle distribution was analyzed using flow cytometry. Apoptosis analysis proceeded by detecting the cleavage of caspase-3. The levels of proteins were probed by Western blotting. Key findingsThe results showed that non-toxic concentrations of andrographolide inhibited the proliferation of human glioblastoma cells through induction of G2/M arrest, which was accompanied by down-regulating Cdk1 and Cdc25C proteins. Additionally, andrographolide decreased the activity of PI3K/Akt signaling, as demonstrated by down-regulation of the expression of phos-PI3K, phos-Akt, phos-mTOR and phos-p70s6k in U251 and U87 cells. Furthermore, additive effects on the proliferation inhibition, G2/M arrest and down-regulation of G2/M phase-related proteins were observed, when a combined treatment of andrographolide with PI3K inhibitor LY294002 was used in U251 and U87 cells. SignificanceWe prove that andrographolide inhibits the proliferation of human glioblastoma cells via inducing G2/M arrest, which is mediated by inhibiting the activity of PI3K/Akt signaling.

Abstract 1614: Grape seed proanthocyanidins inhibit pancreatic cancer cell growth in vitro and in vivo through induction of apoptosis and inhibition of PI3K/Akt pathway

Abstract The American Cancer Society reported that 35,420 Americans died in 2009 because of pancreatic cancer. It is an aggressive malignancy that is frequently diagnosed at an advanced stage with poor prognosis. Phosphoinositide 3-kinase (PI3K)/Akt is a potent survival pathway that may mediate resistance to chemotherapeutic drugs and conventional therapies in pancreatic cancer patients. Thus, PI3K/Akt is considered as a promising therapeutic target for this cancer. Dietary phytochemicals offer promising options for the development of effective strategies for the prevention of cancer, and thus can be utilized as complementary and alternative medicine. Therefore, to develop newer and more effective chemotherapeutic agent for pancreatic cancer, we have examined the effect of grape seed proanthocyanidins (GSPs) on the growth of pancreatic cancer cell lines and the molecular mechanisms underlying this effect using PANC-1, AsPC-1 and Miapaca-2 cell lines as a model. The effect of GSPs on pancreatic cancer cell lines was studied on cell proliferation, cell cycle regulation, apoptosis and PI3K/Akt pathway using MTT assay, western blotting and FACS analysis. In vitro treatment of pancreatic cancer cells (Miapaca-2, PANC-1, and AsPC-1) with GSPs resulted in: (i) growth inhibition, which was associated with G2/M-phase arrest, and includes the inhibition of the expressions of cyclin B1, Cdc25B and Cdc25c proteins. (ii) Induction of apoptosis of pancreatic cancer cells by GSPs was associated with the enhanced expression of Bax, and caspase-3 activation while reduced levels of anti-apoptotic proteins (Bcl2, Bcl-xl). (iii) Blocking of PI3K/Akt pathway. Exposure of Miapaca-2 and PANC-1 cancer cells to PI3K inhibitor (wortmannin) also resulted in a dose-dependent induction of apoptosis in these cells. The in vitro data were further verified using in vivo tumor xenograft model. Administration of GSPs (0.2% and 0.5%, w/w) as a supplement of an AIN76A control diet resulted in a dose-dependent inhibition of the growth of Miapaca-2 tumor xenografts in athymic nude mice (35-65%; P<0.05-0.01) compared to non-GSPs-treated controls. The growth inhibitory effect of dietary GSPs on the pancreatic xenograft tumors was associated with the induction of apoptotic cell death of tumor cells, and induction of Bax expression and activation of caspase-3 while decreased the expression of anti-apoptotic proteins. Further, the levels of phospho-Akt and the proteins of PI3K family (PI3K 110 and PI3K 85) were markedly decreased in the xenograft tumors treated with GSPs compared with controls. Together, this preclinical study provides evidence that the chemotherapeutic effect of GSPs on the growth of pancreatic cancer cells in vitro and in vivo is mediated, at least in part, through the induction of apoptosis, G2/M phase arrest and blocking of PI3K/Akt cell survival pathway. 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 1614. doi:1538-7445.AM2012-1614

Rapid alterations of cell cycle control proteins in human T lymphocytes in microgravity

In our study we aimed to identify rapidly reacting gravity-responsive mechanisms in mammalian cells in order to understand if and how altered gravity is translated into a cellular response. In a combination of experiments using "functional weightlessness" provided by 2D-clinostats and real microgravity provided by several parabolic flight campaigns and compared to in-flight-1g-controls, we identified rapid gravity-responsive reactions inside the cell cycle regulatory machinery of human T lymphocytes. In response to 2D clinorotation, we detected an enhanced expression of p21 Waf1/Cip1 protein within minutes, less cdc25C protein expression and enhanced Ser147-phosphorylation of cyclinB1 after CD3/CD28 stimulation. Additionally, during 2D clinorotation, Tyr-15-phosphorylation occurred later and was shorter than in the 1 g controls. In CD3/CD28-stimulated primary human T cells, mRNA expression of the cell cycle arrest protein p21 increased 4.1-fold after 20s real microgravity in primary CD4+ T cells and 2.9-fold in Jurkat T cells, compared to 1 g in-flight controls after CD3/CD28 stimulation. The histone acetyltransferase (HAT) inhibitor curcumin was able to abrogate microgravity-induced p21 mRNA expression, whereas expression was enhanced by a histone deacetylase (HDAC) inhibitor. Therefore, we suppose that cell cycle progression in human T lymphocytes requires Earth gravity and that the disturbed expression of cell cycle regulatory proteins could contribute to the breakdown of the human immune system in space.

Molecular mechanism of Aurora kinase inhibitor PHA739358 in inhibited proliferation and induced apoptosis of breast cancer cells

To explore the molecular mechanisms of Aurora kinase inhibitor PHA739358 in inhibited proliferation and in vitro induced apoptosis of breast cancer cells. The in vitro cultured T47D cells in logarithmic growth phase were used. The effects of PHA739358 on cell proliferation were examined by MTT (methyl thiazolyl tetrazolium) assay. The variety of nuclear and spindle morphologies was examined by immunofluorescence. And G2/M arrest was determined by flow cytometry. The morphological changes of apoptotic cells were observed by fluorescent microscope. The levels of Aurora kinase relative protein expression phosphonate-AuroraA (p-AuroraA), AuroraA, phosphonate-histone H3 (p-histone H3), histone H3, cell cycle-specific protein expression CyclinB1, cell cycle regulative protein expression Cdc2, Cdc25c, phosphonate-Cdc2 (p-Cdc2), phosphonate-Cdc25c (p-Cdc25c) and apoptosis relative protein expression PARP, Bcl-2 and Bax were detected by Western blot. The apoptotic rate was examined by flow cytometer. PHA739358 obviously inhibited the proliferation of T47D cells after a 24 h or 48 h treatment in a dose-dependent and time-dependent manner. Their IC50 values were (3.44 ± 0.54) and (0.21 ± 0.67) µmol/L respectively. Flow cytometry showed that G2/M arrested in a dose-dependent manner. PHA739358 dose-dependently and time-dependently promoted the dissection of PARP (poly (ADP-ribose) polymerase); down-regulated the expressions of Bcl-2, p-AuroraA, p-histone H3, CyclinB1 and up-regulated the levels of Bax, p-Cdc25c, p-Cdc2, P21 and P53 protein. PHA739358 had no significant effects on the expressions of AuroraA and histone H3. Flow cytometry and fluorescence microscope showed that PHA739358 significantly induced apoptosis. Flow cytometry showed the rate of apoptosis significantly increased from 0.31% ± 0.03% to 40.6% ± 0.81%. The proliferation-inhibiting and apoptosis-inducing effects of PHA739358 may provide new therapeutic approaches of breast cancer.