Transient transcriptional silencing alters the cell cycle to promote germline stem cell differentiation in Drosophila.

Transcriptional silencing is a conserved process used by embryonic germ cells to repress somatic fate and maintain totipotency and immortality. In Drosophila, this transcriptional silencing is mediated by polar granule component (pgc). Here, we show that in the adult ovary, pgc is required for timely germline stem cell (GSC) differentiation. Pgc is expressed transiently in the immediate GSC daughter (pre-cystoblast), where it mediates a pulse of transcriptional silencing. This transcriptional silencing mediated by pgc indirectly promotes the accumulation of Cyclin B (CycB) and cell cycle progression into late-G2 phase, when the differentiation factor bag of marbles (bam) is expressed. Pgc mediated accumulation of CycB is also required for heterochromatin deposition, which protects the germ line genome against selfish DNA elements. Our results suggest that transient transcriptional silencing in the pre-cystoblast "re-programs" it away from self-renewal and toward the gamete differentiation program.

The effects of cordycepin on the cell proliferation, migration and apoptosis in human lung cancer cell lines A549 and NCI-H460.

OBJECTIVES: Our study aimed to evaluate the effect of cordycepin on human lung cancer cell lines A549 and NCI-H460. METHODS: Human lung cancer A549 cells and NCI-H460 cells were treated with different concentrations of cordycepin for different times. Cells incubated without cordycepin were defined as a control. The cell proliferation, migration and apoptosis were, respectively, determined by MTT assay, transwell migration assay and flow cytometry. Additionally, the expression levels of related proteins associated with cell cycle, epithelial-mesenchymal transition (EMT) and apoptosis were examined. KEY FINDINGS: The survival rate of A549 cells and NCI-H460 cells treated with cordycepin significantly decreased compared with untreated cells in a concentration-dependent manner, while the apoptosis rate increased. The migration number of cells treated with cordycepin significantly decreased as the increase in concentration. qRT-PCR and Western blot analysis showed that the aberrant expression of related molecules associated with cell cycle, migration and apoptosis was observed in the lung cancer cells, such as cyclin B, cyclin E, MMP-9, caspase-3 and Bcl-2. CONCLUSIONS: Cordycepin may exert inhibitory effects on the development of human lung cancer via inhibiting cell proliferation, suppressing migration and inducing apoptosis, suggesting that cordycepin may have therapeutic potential for the treatment of this disease.

NF-YA splice variants have different roles on muscle differentiation.

The heterotrimeric CCAAT-binding factor NF-Y controls the expression of a multitude of genes involved in cell cycle progression. NF-YA is present in two alternatively spliced isoforms, NF-YAs and NF-YAl, differing in 28 aminoacids in the N-terminal Q-rich activation domain. NF-YAs has been identified as a regulator of stemness and proliferation in mouse embryonic cells (mESCs) and human hematopoietic stem cells (hHSCs), whereas the role of NF-YAl is not clear. In the muscle system, NF-YA expression is observed in proliferating cells, but barely detectable in terminally differentiated cells in vitro and adult skeletal muscle in vivo. Here, we show that NF-YA inactivation in mouse myoblasts impairs both proliferation and differentiation. The overexpression of the two NF-YA isoforms differentially affects myoblasts fate: NF-YAs enhance cell proliferation, while NF-YAl boosts differentiation. The molecular mechanisms were investigated by expression profilings, detailing the opposite programs of the two isoforms. Bioinformatic analysis of the regulated promoters failed to detect a significant presence of CCAAT boxes in the regulated genes. NF-YAl activates directly Mef2D, Six genes, and p57kip2 (Cdkn1c), and indirectly the myogenic regulatory factors (MRFs). Specifically, Cdkn1c activation is induced by NF-Y binding to its CCAAT promoter and by reducing the expression of the lncRNA Kcnq1ot1, a negative regulator of Cdkn1c transcription. Overall, our results indicate that NF-YA alternative splicing is an influential muscle cell determinant, through direct regulation of selected cell cycle blocking genes, and, directly and indirectly, of muscle-specific transcription factors.

Silvestrol induces early autophagy and apoptosis in human melanoma cells.

BACKGROUND: Silvestrol is a cyclopenta[b]benzofuran that was isolated from the fruits and twigs of Aglaia foveolata, a plant indigenous to Borneo in Southeast Asia. The purpose of the current study was to determine if inhibition of protein synthesis caused by silvestrol triggers autophagy and apoptosis in cultured human cancer cells derived from solid tumors. METHODS: In vitro cell viability, flow cytometry, fluorescence microscopy, qPCR and immunoblot was used to study the mechanism of action of silvestrol in MDA-MB-435 melanoma cells. RESULTS: By 24 h, a decrease in cyclin B and cyclin D expression was observed in silvestrol-treated cells relative to control. In addition, silvestrol blocked progression through the cell cycle at the G2-phase. In silvestrol-treated cells, DAPI staining of nuclear chromatin displayed nucleosomal fragments. Annexin V staining demonstrated an increase in apoptotic cells after silvestrol treatment. Silvestrol induced caspase-3 activation and apoptotic cell death in a time- and dose-dependent manner. Furthermore, both silvestrol and SAHA enhanced autophagosome formation in MDA-MB-435 cells. MDA-MB-435 cells responded to silvestrol treatment with accumulation of LC3-II and time-dependent p62 degradation. Bafilomycin A, an autophagy inhibitor, resulted in the accumulation of LC3 in cells treated with silvestrol. Silvestrol-mediated cell death was attenuated in ATG7-null mouse embryonic fibroblasts (MEFs) lacking a functional autophagy protein. CONCLUSIONS: Silvestrol potently inhibits cell growth and induces cell death in human melanoma cells through induction of early autophagy and caspase-mediated apoptosis. Silvestrol represents a natural product scaffold that exhibits potent cytotoxic activity and could be used for the further study of autophagy and its relationship to apoptosis in cancer cells.

Amygdalin inhibits the growth of renal cell carcinoma cells in vitro.

Although amygdalin is used by many cancer patients as an antitumor agent, there is a lack of information on the efficacy and toxicity of this natural compound. In the present study, the inhibitory effect of amygdalin on the growth of renal cell carcinoma (RCC) cells was examined. Amygdalin (10 mg/ml) was applied to the RCC cell lines, Caki-1, KTC-26 and A498, for 24 h or 2 weeks. Untreated cells served as controls. Tumor cell growth and proliferation were determined using MTT and BrdU tests, and cell cycle phases were evaluated. Expression of the cell cycle activating proteins cdk1, cdk2, cdk4, cyclin A, cyclin B, cyclin D1 and D3 as well as of the cell cycle inhibiting proteins p19 and p27 was examined by western blot analysis. Surface expression of the differentiation markers E- and N-cadherin was also investigated. Functional blockade by siRNA was used to determine the impact of several proteins on tumor cell growth. Amygdalin treatment caused a significant reduction in RCC cell growth and proliferation. This effect was correlated with a reduced percentage of G2/M-phase RCC cells and an increased percentage of cells in the G0/1-phase (Caki-1 and A498) or cell cycle arrest in the S-phase (KTC-26). Furthermore, amygdalin induced a marked decrease in cell cycle activating proteins, in particular cdk1 and cyclin B. Functional blocking of cdk1 and cyclin B resulted in significantly diminished tumor cell growth in all three RCC cell lines. Aside from its inhibitory effects on growth, amygdalin also modulated the differentiation markers, E- and N-cadherin. Hence, exposing RCC cells to amygdalin inhibited cell cycle progression and tumor cell growth by impairing cdk1 and cyclin B expression. Moreover, we noted that amygdalin affected differentiation markers. Thus, we suggest that amygdalin exerted RCC antitumor effects in vitro.

PBK/TOPK mediates promyelocyte proliferation via Nrf2-regulated cell cycle progression and apoptosis.

Acute myeloid leukemia (AML) is a disorder involving hematopoietic stem cells, characterized by blockage of hematopoietic cell differentiation and an increase in clonal neoplastic proliferation. AML is associated with poor patient outcome. PBK/TOPK is a protein kinase derived from PDZ-binding kinase (PBK)/T-lymphokine-activated killer (T-LAK) cell-originated protein kinase (TOPK). Previous studies have shown that PBK/TOPK is expressed in hematologic tumors. In the present study, we aimed to investigate the role of PBK/TOPK in promyelocyte proliferation and to clarify the molecular mechanism. PBK/TOPK knockdown (KD) significantly decreased cell proliferation and viability in the NB4 and HL-60 promyelocytes. PBK/TOPK KD resulted in G2/M cell cycle arrest that attributed to a decrease in cdc2 and cyclin B expression. In addition, PBK/TOPK KD caused apoptosis, as evidenced by activation of the mitochondrial apoptotic pathway and an increase in TUNEL-positive cells. PBK/TOPK KD induced mitochondrial dysfunction and ROS generation, and inhibition of mitochondrial dysfunction and ROS production suppressed PBK/TOPK KD-induced cell cycle arrest and apoptosis. Moreover, PBK/TOPK KD decreased Nrf2 expression and ARE-binding activity. Overexpression of Nrf2 inhibited the PBK/TOPK KD-induced decrease in cdc2 and cyclin B expression and cell cycle arrest, and blocked ROS production and apoptosis. Based on literature and our results, it was demonstrated that Nrf2 may be a crucial regulator that mediates PBK/TOPK-exerted promotion of cell proliferation. PBK/TOPK stabilizes Nrf2, strictly regulates the ROS level, promotes cell cycle progression and inhibits apoptosis, contributing to the proliferation of promyelocytes. Our results provide new insights into the molecular mechanism of PBK/TOPK-mediated promyelocyte proliferation and shed light on the pathogenesis of AML.

Resistin is a survival factor for porcine ovarian follicular cells.

Previously, we demonstrated the expression of resistin in the porcine ovary, the regulation of its expression and its direct effect on ovarian steroidogenesis. The objective of this study was to examine the effect of resistin on cell proliferation and apoptosis in a co-culture model of porcine granulosa and theca cells. First, we analysed the effect of resistin at 1 and 10  ng/ml alone or in combination with FSH- and IGF1 on ovarian cell proliferation with an alamarBlue assay and protein expression of cyclins A and B using western blot. Next, the mRNA and protein expression of selected pro-apoptotic and pro-survival regulators of cell apoptosis, caspase-9, -8 and -3 activity and DNA fragmentation using real time PCR, western blot, fluorescent assay and an ELISA kit, respectively, were analysed after resistin treatment. Furthermore, we determined the effect of resistin on the protein expression of ERK1/2, Stat and Akt kinase. Using specific inhibitors of these kinases, we also checked caspase-3 activity and protein expression. We found that resistin, at both doses, has no effect on cell proliferation. The results showed that resistin decreased pro-apoptotic genes, which was confirmed on protein expression of selected factors. We demonstrate an inhibitory effect of resistin on caspase activity and DNA fragmentation. Finally, resistin stimulated phosphorylation of the ERK1/2, Stat and Akt and kinases inhibitors reversed resistin action on caspase-3 activity and protein expression to control. All of these results showed that resistin has an inhibitory effect on porcine ovarian cell apoptosis by activation of the MAPK/ERK, JAK/Stat and Akt/PI3 kinase signalling pathways.

Novel reversible selective inhibitor of CRM1 for targeted therapy in ovarian cancer.

BACKGROUND: Ovarian cancer represents the most fatal type of gynecological malignancies. Unfortunately, there are still no effective targeted treatment strategies for ovarian cancer. Overexpression of CRM1 has been correlated with poor prognosis of patients with ovarian cancer. AIM: In this study, we investigated the antitumor effects of a novel reversible inhibitor of CRM1 in ovarian cancer cells. METHODS: The effects of S109 on proliferation was detected by CCK-8, EdU, clonogenic assay. The protein expression were determined by Western blot. The subcellular localization of RanBP1 was analyzed by immunofluorescence microscopy assay. RESULTS: We demonstrated that S109 could induce nuclear accumulation of RanBP1, a canonical biomarker for CRM1 inhibition. This effect was clearly reversible in the majority of the cells, whereas the inhibitory effect of LMB could not be reversed. Our data reveal that treatment with S109 results in decrease in proliferation and colonogenic capacity of ovarian cancer cells by arresting cell cycle. Mechanistically, S109 treatment increase the expression of the cyclin-dependent kinase inhibitor p21, while it reduced the expression of cell cycle promoting proteins, Cyclin D1 and Cyclin B. CRM1 level itself was also down-regulated following S109 treatment. Furthermore, the nuclei of cells incubated with S109 accumulated tumor suppressor proteins (Foxo1, p27 and IκB-α). More importantly, Cys528 mutation of CRM1 abolished the ability of S109 to block proliferation of ovarian cancer cells. CONCLUSIONS: Together, our study identifies CRM1 as a valid target in ovarian cancer and provides a basis for the development of S109 in ovarian cancer.

Prognostic value of cyclin B in endometrial endometrioid adenocarcinoma.

Cyclins are a group of cell cycle regulatory proteins. Cyclin B acts as an activator to cyclin-dependent kinase 1 (CDK1), a protein kinase essential for G2/M phase transition. Deregulation of cyclins has been linked to a number of malignant neoplasms, but the impact on clinicopathological parameters seems to be cancer-specific. Overexpression of cyclin B has been shown to affect survival in some malignant tumors, including breast and esophageal cancer, but its impact on endometrial cancer has not been extensively studied. For this study, 211 endometrial endometrioid adenocarcinoma samples were obtained from patients surgically treated at the Oulu University Hospital. The samples were immunohistochemically stained and analyzed for cyclin B expression. The relationships between cyclin B expression and conventional prognostic factors were analyzed. A discrimination threshold for survival analyses was calculated by utilizing the receiver operator characteristic (ROC) method. Cyclin B expression correlated with grade and advanced stage. Survival analyses showed that cyclin B expression affects cancer-specific survival in univariate analysis. In multivariate analysis, the results were indicative that cyclin B may hold independent prognostic significance, but further studies are required to assess this.

Effects of suppressed autophagy on mitochondrial dynamics and cell cycle of N2a cells.

Autophagy dysregulation, mitochondrial dynamic abnormality and cell cycle re-entry are implicated in the vulnerable neurons of patients with Alzheimer's disease. This study was designed to testify the association among autophagy, mitochondrial dynamics and cell cycle in dividing neuroblastoma (N2a) cells. The N2a cells were cultured in vitro and treated with different concentrations of 3-methyladenine (3-MA). The cell viability was detected by methyl thiazolyl tetrazolium (MTT) assay. They were randomly divided into control group (cells cultured in normal culture medium) and 3-MA group (cells treated with 10 mmol/L 3-MA). The cell cycle was analyzed in the two groups 3, 6, 12, and 24 h after treatment by flow cytometry. Western blotting was used to evaluate the expression levels of mitofission 1 (Fis1), mitofusin 2 (Mfn2), microtubule-associated protein 1 light chain 3 (LC3), cell cycle-dependent kinase 4 (CDK4) and cdc2. The flow cytometry revealed that the proportion of cells in G(2)/M was significantly increased, and that in G0/G1 was significantly reduced in the 3-MA group as compared with the control group. Western blotting showed that the expression levels of Fis1, LC3, and CDK4 were significantly up-regulated in the 3-MA group at the four indicated time points as compared with the control group. Mfn2 was initially decreased in the 3-MA group, and then significantly increased at 6 h or 12 h. Cdc2 was significantly increased in the 3-MA group at 3 h and 6 h, and then dropped significantly at 12 h and 24 h. Our data indicated that 3-MA-induced suppressed autophagy may interfere with the cell cycle progression and mitochondrial dynamics, and cause cell death. There are interactions among cell cycle, mitochondrial dynamics and autophagy in neurons.

Combination treatment with paclitaxel and doxorubicin inhibits growth of human esophageal squamous cancer cells by inactivation of Akt.

Despite the fact that paclitaxel and doxorubicin are widely used as chemotherapy agents against several types of cancer, their combined effects on esophageal squamous cell carcinoma (ESCC) have never been fully elucidated. The present study was designed to investigate the biological effects of paclitaxel and doxorubicin in ESCC cells. Combination treatment with paclitaxel and doxorubicin significantly inhibited the proliferation of TE-12 cells in a dose-and time-dependent manner compared to treatment with paclitaxel or doxorubicin alone. FACS analysis showed that the percentage of cells in the G2/M phase was significantly increased at 12 h after treatment with the combination. Increased p-cdc2, p-Wee1 and p53 protein levels were observed, while Akt activation was suppressed by combination treatment with paclitaxel and doxorubicin. In addition, treatment with paclitaxel plus doxorubicin significantly increased apoptosis as indicated by increased cleaved poly(ADP-ribose) polymerase and cleaved caspase-7 and -9 levels. These results suggest that combination treatment with paclitaxel and doxorubicin induced G2/M cell cycle arrest and apoptosis in human ESCC cells by suppressing Akt activity. These findings highlight the potent apoptotic effect of combination therapy with paclitaxel and doxorubicin in ESCC cells and the potential clinical benefits of these two drugs in esophageal cancer.

The 3' untranslated region of the cyclin B mRNA is not sufficient to enhance the synthesis of cyclin B during a mitotic block in human cells.

Antimitotic agents are frequently used to treat solid tumors and hematologic malignancies. However, one major limitation of antimitotic approaches is mitotic slippage, which is driven by slow degradation of cyclin B during a mitotic block. The extent to which cyclin B levels decline is proposed to be governed by an equilibrium between cyclin B synthesis and degradation. It was recently shown that the 3' untranslated region (UTR) of the murine cyclin B mRNA contributes to the synthesis of cyclin B during mitosis in murine cells. Using a novel live-cell imaging-based technique allowing us to study synthesis and degradation of cyclin B simultaneously at the single cell level, we tested here the role of the human cyclin B 3'UTR in regulating cyclin B synthesis during mitosis in human cells. We observed that the cyclin B 3'UTR was not sufficient to enhance cyclin B synthesis in human U2Os, HeLa or hTERT RPE-1 cells. A better understanding of how the equilibrium of cyclin B is regulated in mitosis may contribute to the development of improved therapeutic approaches to prevent mitotic slippage in cancer cells treated with antimitotic agents.

Anticancer effects of O-desmethylangolensin are mediated through cell cycle arrest at the G2/M phase and mitochondrial-dependent apoptosis in Hep3B human hepatocellular carcinoma cells.

In the present study, in order to investigate the anticancer effects of O-desmethylangolensin (O-DMA) on human hepatocellular carcinoma Hep3B cells, we first examined the antiproliferative effect of O-DMA. When Hep3B cells were treated with O-DMA at various concentrations (5-200 µM) for 24, 48 or 72 h, cell proliferation decreased significantly in a dose- and time-dependent manner. Moreover, O-DMA exposure at the IC50 concentration for 72 h arrested cells at the G2/M phase, which was accompanied by a reduction in CDK1, and an increase in cyclin A and B. Under the same conditions, O-DMA significantly increased the number of sub-G1 phase cells. Additionally, an Annexin V assay revealed that exposure to O-DMA affected the rate of cell apoptosis. O-DMA caused the downregulation of Bcl-2 and upregulation of Bax, which led to cytochrome c release from the mitochondria and activation of caspase-3. Taken together, these data suggest that O-DMA exhibits anticancer activity by arresting the cell cycle at G2/M phase and causing mitochondrial-dependent apoptosis in Hep3B cells.

Role of hMLH1 in sterigmatocystin-induced G2 phase arrest in human esophageal epithelial Het-1A cells in vitro.

Sterigmatocystin (ST), a common environmental contaminant found across the world, is generally recognized as a potential carcinogen, mutagen and teratogen. Our previous epidemiological studies suggested that ST exposure might be a risk factor for esophageal cancer. However, the direct effects of ST on human esophageal epithelial cells are currently unknown. In the present study, we examined the effect of treating a human esophageal epithelial cell line (Het-1A) with ST on DNA damage, DNA repair mechanisms, and cell cycle distribution. We found that ST treatment could induce DNA damage and lead to a G2 phase arrest, associated with a marked up-regulation of G2/M regulatory proteins, including Cyclin B1, Cdc2/p-Cdc2, and Cdc25C/p-Cdc25C. Additionally, we found that the expression of two mismatch repair (MMR) proteins, hMLH1 and hMSH2, was up-regulated at both the mRNA and protein levels after ST treatment, suggesting that ST could induce the MMR system in Het-1A cells. Interestingly, ST-induced G2 phase arrest was mediated by hMLH1 up-regulation, but was independent of hMSH2. Treatment with hMLH1-siRNA prevented the up-regulation of Cyclin B1, Cdc2/p-Cdc2 and Cdc25C/p-Cdc25C in ST-treated cells, thereby inhibiting the subsequent G2 phase arrest of Het-1A cells. Moreover, we found that hMLH1 may act as a direct sensor of ST-mediated DNA damage. In conclusion, the study demonstrated that ST caused DNA damage and triggered G2 phase arrest in Het-1A cells, and hMLH1 participated in the ST-induced G2 phase arrest by up-regulating G2/M regulatory proteins.

High proliferation is associated with inferior outcome in male breast cancer patients.

Assessment of proliferation is important in female breast cancer and individual treatment decisions are based upon its results, especially in the luminal subgroups. Gene expression analyses fail to group male breast cancer into the intrinsic subgroups previously established in female breast cancer. Even though proliferation has been shown to divide male breast cancer into molecular subgroups with different prognoses, the clinical importance of proliferation markers has not yet been elucidated. Previous studies in male breast cancer have demonstrated contradictory results regarding the prognostic impact of histological grade and Ki-67, parameters strongly associated with proliferation. The aim of the present project was to study proliferation in male breast cancer by assessing other proliferation-related markers viz. cyclins A, B, D1 and mitotic count. A total of 197 male breast cancer cases with accessible paraffin-embedded material and outcome data were investigated. Immunohistochemical stainings were performed on tissue microarrays. Kaplan-Meier estimates and the Cox proportional regression models were used for survival analyses with breast cancer death as the event. The subset of patients with high expression of cyclin A (hazard ratio (HR) 3.7; P=0.001) and B (HR 2.7; P=0.02) demonstrated a poorer survival. Furthermore, high mitotic count was associated with an increased risk of breast cancer death (HR 2.5; P=0.01). In contrast, cyclin D1 overexpression was predictive of better breast cancer survival (HR 0.3; P=0.001). In conclusion, high levels of cyclin A and B expression and an elevated mitotic count result in a two to threefold higher risk for breast cancer death, whereas cyclin D1 overexpression halves the risk. The clinical utility of these proliferation markers needs further elucidation.

Effect of food restriction on reproductive-related genes and reproductive hormones in adult female rats.

OBJECTIVES: A number of factors involved in the control of energy balance and metabolism act as modulators of gonadal axis. Ghrelin, a peptide secreted from the stomach and hypothalamus, has emerged as an orexigenic food intake controlling signal acting upon hypothalamus. Recently, the potential reproductive role of ghrelin has received great attention. This study was designed to investigate the influence of food restriction and consequent metabolic hormone (ghrelin) on the level and gene expression of female reproductive hormones in adult rats. MATERIALS AND METHODS: To study the effect of chronic food restriction on ghrelin level in adult female rats and its relation to female reproductive hormones, 32 adult female Sprague Dawley rats divided into 4 groups: Group I (control group) comprised 8 rats fed ad libitum for 30 days, Group II, III and IV (food-restricted groups for 10, 20 and 30 days respectively) each consisted of 8 rats fed 50% of ad libitum intake determined by the amount of food consumed by the control group. RESULTS: Mean body weight of food restricted rats was observed to decrease during the period of the experiment. Food restriction produced significant increase of serum ghrelin with significant decrease of both gastric and hypothalamic ghrelin accompanied with significant increase in its gene expression in stomach and hypothalamus. Estradiol (E2), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels showed significant decrease correlated with down-regulation of gonadotropins, cyclin-dependent kinase (cdc2), cyclin B and kisspeptin (Kiss1) genes in food restricted rats compared with control group. CONCLUSIONS: Ghrelin could be one of the hormones responsible for the suppression of female reproductive axis in case of negative energy balance. Thus, ghrelin may operate as an autocrine/paracrine regulator of ovarian function. Overall, ghrelin may represent an additional link between body weight homeostasis and reproductive function.

Quinuclidinone derivative 6 induced apoptosis in human breast cancer cells via sphingomyelinase and JNK signaling.

Novel quinuclidinone derivatives have been previously reported by our laboratory. In this study, we investigated the impact of two novel quinuclidinone derivatives 4 and 6 on apoptotic signaling in breast cancer cells (MCF-7) and their normal counterparts (MCF-12a). Our data revealed that derivatives 4 and 6 reduced proliferation and induced apoptosis in breast cancer cells. However, derivative 6 was less cytotoxic to normal breast epithelial cells than breast cancer cells; therefore, we focused on derivative 6 for further investigation. Flow cytometric analysis showed that quinuclidinone derivative 6 reduced the percentage of MCF-7 cells in G(2)/M which is confirmed by increased expression levels of cyclin B, while it arrests MCF12a in G1 phase judging from increased p21. Quinuclidinone derivative 6 increased expression levels of p53 and Bax at both protein and mRNA levels and reduced expression level of Mdm2, Bcl2, Akt and Bcl-XL It also increased mitochondrial apoptotic pathways by activating release of cytochrome c which is consistent with activation of caspase-9 as confirmed by caspase-9 inhibitor LEHD-CHO. Finally, it increased sphingomyelinase signaling and ceramide formation as well as its downstream targets ERK1/2, p38, and JNK. Inhibition of ERK1/2 with PD98059 exerted little effect on the derivative 6-induced apoptosis and p38 inhibition with SB203580 slightly lessened apoptosis, whereas inhibition of JNK with SP600125 markedly suppressed derivative 6-induced apoptosis. These results indicate that derivative-6 induced the activation of sphingomyelinase signaling and that JNK played a pivotal role in induction of apoptosis in human breast cancer cells. In vivo studies and molecular docking experiments are now in progress for further anticancer investigations.

Chk1 knockdown confers radiosensitization in prostate cancer stem cells.

Radioresistance is responsible for treatment failure after radiotherapy in localized prostate cancer, while prostate cancer stem cells promote radioresistance by preferential activation of the DNA damage response. Chk1 inhibition has been shown to sensitize many tumor cells to radiation. However, whether Chk1 inhibition can potentiate the cytotoxic effects of radiation on prostate cancer stem cells remains to be elucidated. In this study, CD133+CD44+ cells were isolated using microbeads and were found to possess cancer stem cell properties. Using shRNA, Chk1 was knocked down in the sorted CD133+CD44+ cells. Our results demonstrated that Chk1 knockdown abrogated the radiation-induced G2/M arrest, inhibited DNA damage repair and promoted premature mitosis, leading to increased apoptosis in the radiated sorted CD133+CD44+ cells. Moreover, these effects were accompanied by caspase-2 activation and the inactivation of phosphorylated Cdc25C and Cdc2. Our results suggest that Chk1 knockdown increases the radiosensitivity of CD133+CD44+ prostate cancer stem cells. Chk1 knockdown in prostate cancer stem cells may be an effective therapeutic strategy against prostate cancer.

p53 Dimers associate with a head-to-tail response element to repress cyclin B transcription.

DNA damage induced by the topoisomerase I inhibitor SN38 activates cell cycle checkpoints which promote cell cycle arrest. This arrest can be abrogated in p53-defective cells by the Chk1 inhibitor 7-hydroxystaurosporine (UCN-01). Previously, we compared p53 wild-type MCF10A cells with derivatives whose p53 function was inhibited by over-expression of the tetramerization domain (MCF10A/OD) or expression of shRNA against p53 (MCF10A/Δp53). Treatment of SN38-arrested MCF10A/OD cells with UCN-01 abrogated S, but not G2 arrest, while the MCF10A/Δp53 cells abrogated both S and G2 arrest. The MCF10A/OD cells had reduced levels of cyclin B, suggesting that tetramerization of p53 is not required for repression of cyclin B gene expression. In the present study, we analyzed p53 oligomerization status using glutaraldehyde cross-linking. Following SN38 treatment, MCF10A cells contained oligomeric forms of p53 with molecular weights approximating monomers, dimers, trimers, and tetramers. However, MCF10A/OD cells possessed only monomers and dimers suggesting that these complexes may be involved in repression of cyclin B. While genes transcriptionally activated by p53 contain a consensus sequence with elements repeated in a head-to-head orientation, the cyclin B promoter contains similar elements oriented head-to-tail. Chromatin immunoprecipitation (ChIP) assays revealed that p53 associates with this head-to-tail element in both MCF10A and MCF10A/OD. Electrophoretic mobility shift assays (EMSA) using a biotin-labeled probe containing the head-to-tail element showed a shift in mobility consistent with the molecular weight of tetramers and dimers in MCF10A nuclear extract, but only the dimer in MCF10A/OD nuclear extract. Taken together, these results suggest a novel mechanism whereby p53 dimers associate with the head-to-tail element to repress cyclin B transcription.

Combined anti-tumor effects of IFN-α and sorafenib on hepatocellular carcinoma in vitro and in vivo.

Hepatocellular carcinoma (HCC) is among the most common and aggressive cancers worldwide, and novel therapeutic strategies are urgently required to improve clinical outcome. Interferon-alpha (IFN-α) and sorafenib are widely used as anti-tumor agents against various malignancies. In this study, we investigated the combined effects of IFN-α and sorafenib against HCC. We demonstrated that the combination therapy synergistically suppressed HCC cellular viability, arrested cell cycle propagation and induced apoptosis in HCC cells. Further research revealed that IFN-α and sorafenib collaboratively regulated the expression levels of cell cycle-related proteins Cyclin A and Cyclin B as well as the pro-survival Bcl-2 family proteins Mcl-1, Bcl-2 and Bcl-X(L). Moreover, sorafenib inhibited IFN-α induced oncogenic signaling of STAT3, AKT and ERK but not the activation of the tumor suppressor STAT1. Xenograft experiments also confirmed the combined effects of IFN-α and sorafenib on tumor growth inhibition and apoptosis induction in vivo. In conclusion, these results provide rationale for the clinical application of IFN-α and sorafenib combination therapy in HCC treatment.