Cell division cycle 20 promotes cell proliferation and invasion and inhibits apoptosis in osteosarcoma cells

Cdc20 (cell division cycle 20 homologue) has been reported to exhibit an oncogenic role in human tumorigenesis. However, the function of Cdc20 in osteosarcoma (OS) has not been investigated. In the current study, we aim to explore the role of Cdc20 in human OS cells. Multiple approaches were used to measure cell growth, apoptosis, cell cycle, migration and invasion in OS cells after depletion of Cdc20 or overexpression of Cdc20. We found that down-regulation of Cdc20 inhibited cell growth, induced apoptosis and triggered cell cycle arrest in OS cells. Moreover, Cdc20 down-regulation let to inhibition of cell migration and invasion in OS cells. Consistently, overexpression of Cdc20 in OS cells promoted cell growth, inhibited apoptosis, enhanced cell migration and invasion. Mechanistically, our Western blotting results showed that overexpression of Cdc20 reduced the expression of Bim and p21, whereas depletion of Cdc20 upregulated Bim and p21 levels in OS cells. Altogether, our findings demonstrated that Cdc20 exerts its oncogenic role partly due to regulation of Bim and p21 in OS cells, suggesting that targeting Cdc20 could be useful for the treatment of OS.     

S-phase kinase-associated protein 2 promotes cell growth and motility in osteosarcoma cells

Skp2 (S-phase kinase-associated protein 2) plays an oncogenic role in a variety of human cancers. However, the function of Skp2 in osteosarcoma (OS) is elusive. Therefore, in the current study, we explore whether Skp2 exerts its oncogenic function in OS. The cell growth, apoptosis, invasion and cell cycle were measured in OS cells after Skp2 overexpression. We found that overexpression of Skp2 enhanced cell growth, and inhibited cell apoptosis in OS cells. Moreover, we observed that upregulation of Skp2 accelerated cell cycle progression in OS cells. Furthermore, the ability of migration and invasion was enhanced in Skp2 overexpressing OS cells. Mechanically, our Western blotting data suggested that Skp2 decreased the expression of E-cadherin, Foxo1, p21, and p57, but increased MMP-9 in OS cells. In conclusion, our study demonstrated that Skp2 exhibited an oncogenic function in OS cells, suggesting that inhibition of Skp2 may be a novel approach for the treatment of OS.     

GANT61 plays antitumor effects by inducing oxidative stress through the miRNA-1286/RAB31 axis in osteosarcoma

Osteosarcoma (OS) is a rare malignancy of bone associated with poor clinical outcomes. The antitumor effects of GANT61 on OS is unclear. To investigate antitumor effects and mechanism of GANT61 in OS cells and xenograft model. Effects of GANT61 on cell viability, clone formation, cell cycle, apoptosis, migration, and invasion ability of OS cells were assessed. Reactive oxygen species (ROS) levels measured by dichlorofluorescein fluorescence were used to evaluate oxidative stress. The Xenograft model was constructed to investigate the antitumor effects of GANT61 in vivo. The microRNA (miRNA)-1286 was downregulated, while RAB31 upregulated in OS tissues and cells. GANT61 inhibited viability, migration, and invasion ability of OS cells (SaOS-2 and U2OS), and induced apoptosis and the ROS production, along with miRNA-1286 upregulation and RAB13 downregulation. After knockdown of miRNA-1286, GANT6-induced cell inhibition was attenuated, along with RAB31 upregulation. Inversely, miRNA-1286 overexpression downregulated RAB31. Dual-luciferase reporter assay verified that miR-1286 negatively targeted RAB13. Moreover, the knockdown of RAB31 stimulated apoptosis and ROS production while inhibited viability, migration, and invasion of GANT61-treated cells. In vivo experiments further confirmed that GANT61 inhibited tumor growth and RAB13 expression, but enhanced miRNA-1286. The study demonstrated that GANT61 inhibited cell aggressive phenotype and tumor growth by inducing oxidative stress through the miRNA-1286/RAB31 axis. Our findings provided a potential antitumor agent for the OS clinical treatment.     

Geldanamycin induces cell cycle arrest in K562 erythroleukemic cells

Geldanamycin (GA), a benzoquinone ansamycin, is one of the specific inhibitors of 90-kDa heat shock protein and induces growth inhibition and apoptosis in certain cancer cell lines. We have investigated the mechanism of GA-induced growth inhibition in K562 erythroleukemic cells. DNA flow-cytometric analysis indicated that GA-induced growth arrest was associated with G2/M phase arrest of the cell cycle. GA treatment down-regulated the expression of cyclin B1 and inhibited phosphorylation of Cdc2 protein, both key regulatory proteins at the G2/M boundary. GA also markedly inhibited the Cdc2 kinase activity, which may be in part a result of up-regulation of p27KIP1 by GA. The present results suggest a novel mechanism that p27KIP1 could be involved in the regulation of G2 to M phase transition.     

Arsenic trioxide inhibits cell growth and motility via up-regulation of let-7a in breast cancer cells

Arsenic trioxide (ATO) has been reported to exert its anti-cancer activities in human cancers. However, the molecular mechanism of ATO-triggered anti-tumor activity has not been fully elucidated. Recently, multiple studies demonstrated that ATO could regulate miRNAs in human cancers. Therefore, in this study, we investigated whether ATO regulated let-7a in breast cancer cells. We found that ATO upregulated let-7a level in breast cancer cells. We also found that up-regulation of let-7a inhibited cell growth and induced apoptosis and retarded cell migration and invasion. We also observed that up-regulation of let-7a enhanced cell growth inhibition and invasion suppression induced by ATO treatment. Our findings suggest that ATO suppressed cell growth, stimulated apoptosis, and retarded cell invasion partly via upregulation of let-7a in breast cancer cells. Our study provides a new anti-tumor mechanism of ATO treatment in breast cancer.     

Phosphatidylinositol 3-kinase and frabin mediate Cryptosporidium parvum cellular invasion via activation of Cdc42

Cryptosporidium parvum invades target epithelia via a mechanism that involves host cell actin reorganization. We previously demonstrated that C. parvum activates the Cdc42/neural Wiskott-Aldrich syndrome protein network in host cells resulting in actin remodeling at the host cell-parasite interface, thus facilitating C. parvum cellular invasion. Here, we tested the role of phosphatidylinositol 3-kinase (PI3K) and frabin, a guanine nucleotide exchange factor specific for Cdc42 in the activation of Cdc42 during C. parvum infection of biliary epithelial cells. We found that C. parvum infection of cultured human biliary epithelial cells induced the accumulation of PI3K at the host cell-parasite interface and resulted in the activation of PI3K in infected cells. Frabin also was recruited to the host cell-parasite interface, a process inhibited by two PI3K inhibitors, wortmannin and LY294002. The cellular expression of either a dominant negative mutant of PI3K (PI3K-Deltap85) or functionally deficient mutants of frabin inhibited C. parvum-induced Cdc42 accumulation at the host cell-parasite interface. Moreover, LY294002 abolished C. parvum-induced Cdc42 activation in infected cells. Inhibition of PI3K by cellular overexpression of PI3K-Deltap85 or by wortmannin or LY294002, as well as inhibition of frabin by various functionally deficient mutants, decreased C. parvum-induced actin accumulation and inhibited C. parvum cellular invasion. In contrast, the overexpression of the p85 subunit of PI3K promoted C. parvum invasion. Our data suggest that an important component of the complex process of C. parvum invasion of target epithelia results from the ability of the organism to trigger host cell PI3K/frabin signaling to activate the Cdc42 pathway, resulting in host cell actin remodeling at the host cell-parasite interface.     

Diosgenin inhibits the migration of human breast cancer MDA-MB-231 cells by suppressing Vav2 activity

Diosgenin, a naturally occurring steroidal saponin, possess tumor therapeutic potential. However, the effect of diosgenin on cancer metastasis remains poorly understood. In this study, we performed in vitro experiments to investigate the inhibitory activity of diosgenin on human breast cancer MDA-MB-231 cell migration, and reveal the possible mechanism. Diosgenin caused a marked inhibition of cell migration in MDA-MB-231 cell by transwell assay. In addition, diosgenin significantly impacted MDA-MB-231 cell migratory behavior under real-time observation. We also found diosgenin significantly inhibited actin polymerization, Vav2 phosphorylation and Cdc42 activation, which might be, at least in part, attributed to the anti-metastatic potential of diosgenin. These findings reveal a new therapeutic potential of diosgenin for human breast cancer metastasis therapy.     

Dasatinib Modulates Invasive and Migratory Properties of Canine Osteosarcoma and has Therapeutic Potential in Affected Dogs

BACKGROUND: This investigation sought to elucidate the relationship between hepatocyte growth factor (HGF)–induced metastatic behavior and the tyrosine kinase inhibitors (TKIs) crizotinib and dasatinib in canine osteosarcoma (OS). Preliminary evidence of an apparent clinical benefit from adjuvant therapy with dasatinib in four dogs is described. METHODS: The inhibitors were assessed for their ability to block phosphorylation of MET; reduce HGF-induced production of matrix metalloproteinase (MMP); and prevent invasion, migration, and cell viability in canine OS cell lines. Oral dasatinib (0.75 mg/kg) was tested as an adjuvant therapy in four dogs with OS. RESULTS: Constitutive phosphorylation of MET was detected in two cell lines, and this was unaffected by 20-nM incubation with either dasatinib or crizotinib. Incubation of cell lines with HGF (MET ligand) increased cell migration and invasion in both cell lines and increased MMP-9 activity in one. Dasatinib suppressed OS cell viability and HGF-induced invasion and migration, whereas crizotinib reduced migration and MMP-9 production but did not inhibit invasion or viability. CONCLUSIONS: Invasion, migration, and viability of canine OS cell lines are increased by exogenous HGF. HGF induces secretion of different forms of MMP in different cell lines. The HGF-driven increase in viability and metastatic behaviors we observed are more uniformly inhibited by dasatinib. These observations suggest a potential clinical benefit of adjuvant dasatinib treatment for dogs with OS.     

SD-208, a Novel Protein Kinase D Inhibitor,Blocks Prostate Cancer Cell Proliferation and Tumor Growth In Vivo by Inducing G2/M Cell Cycle Arrest

Protein kinase D (PKD) has been implicated in many aspects of tumorigenesis and progression, and is an emerging molecular target for the development of anticancer therapy. Despite recent advancement in the development of potent and selective PKD small molecule inhibitors, the availability of in vivo active PKD inhibitors remains sparse. In this study, we describe the discovery of a novel PKD small molecule inhibitor, SD-208, from a targeted kinase inhibitor library screen, and the synthesis of a series of analogs to probe the structure-activity relationship (SAR) vs. PKD1. SD-208 displayed a narrow SAR profile, was an ATP-competitive pan-PKD inhibitor with low nanomolar potency and was cell active. Targeted inhibition of PKD by SD-208 resulted in potent inhibition of cell proliferation, an effect that could be reversed by overexpressed PKD1 or PKD3. SD-208 also blocked prostate cancer cell survival and invasion, and arrested cells in the G2/M phase of the cell cycle. Mechanistically, SD-208-induced G2/M arrest was accompanied by an increase in levels of p21 in DU145 and PC3 cells as well as elevated phosphorylation of Cdc2 and Cdc25C in DU145 cells. Most importantly, SD-208 given orally for 24 days significantly abrogated the growth of PC3 subcutaneous tumor xenografts in nude mice, which was accompanied by reduced proliferation and increased apoptosis and decreased expression of PKD biomarkers including survivin and Bcl-xL. Our study has identified SD-208 as a novel efficacious PKD small molecule inhibitor, demonstrating the therapeutic potential of targeted inhibition of PKD for prostate cancer treatment.     

A Cdc25A antagonizing K vitamin inhibits hepatocyte DNA synthesis in vitro and in vivo

Thioalkyl containing K vitamin analogs have been shown to be potent inhibitors of hepatoma cell growth and antagonizers of protein tyrosine phosphatase activity. We now show that they inhibit the activity of specific protein tyrosine phosphatases (PTP) in cell-free conditions in vitro, particularly the dual specificity phosphatase Cdc25A. Using primary cultures of adult rat hepatocytes that are in G0/G1 phase until stimulated into DNA synthesis by epidermal growth factor, we found that 2-(2-mercaptoethanol)-3-methyl-1,4-naphthoquinone or Compound 5 (Cpd 5) inhibited hepatocyte DNA synthesis and PTP activity in cell culture and in vivo after a two-thirds partial hepatectomy. We found a selective inhibition of Cdc25A activity in vitro, using both synthetic substrates and authentic cellular substrate, immunoprecipitated phospho-Cdk4. Intact Cpd 5-treated cells had decreased cellular Cdc25A activity and increased tyrosine phosphorylation of Cdk4, resulting in decreased phosphorylation of retinoblastoma (Rb). Loss of Cdk4 activity was confirmed using Cdk4 immunoprecipitates from either Cpd 5-treated or untreated cells and measuring its kinase activity using GST-Rb as target. We found a similar order of activity for inhibition of growth and Cdc25A activity using several thiol-containing analogs. Cdc25A inhibitors may thus be useful for defining biochemical pathways involving protein tyrosine phosphorylation that mediate cell growth inhibition.     

Involvement of HDAC1 in E-cadherin expression in prostate cancer cells; its implication for cell motility and invasion

In this study, we investigate the molecular mechanism by which histone deacetylase (HDAC) inhibitors exert anti-invasiveness effect against prostate cancer cells. We first evaluate the growth inhibition effect of HDAC inhibitors in prostate cancer cells, which is accompanied by induction of p21^WAF1 expression and accumulation of acetylated histones. And we found that the migration and invasion of prostate cancer cells is strongly inhibited by treatment with HDAC inhibitors. In parallel, E-cadherin level is highly up-regulated in HDAC inhibitor-treated prostate cancer cells. And siRNA knockdown of E-cadherin significantly diminishes the anti-invasion effect of HDAC inhibitors, indicating that E-cadherin overexpression is one of possible mechanism for anti-invasion effect of HDAC inhibitors. Furthermore, specific downregulation of HDAC1, but not HDAC2, causes E-cadherin expression and subsequent inhibition of cell motility and invasion. Collectively, our data demonstrate that HDAC1 is a major repressive enzyme for E-cadherin expression as well as HDAC inhibitor-mediated anti-invasiveness.     

Diosgenin, a naturally occurring steroid, suppresses fatty acid synthase expression in HER2-overexpressing breast cancer cells through modulating Akt, mTOR and JNK phosphorylation

Fatty acid synthase (FAS) expression is markedly elevated in HER2-overexpressing breast cancer cells. In this study, diosgenin, a plant-derived steroid, was found to be effective in suppressing FAS expression in HER2-overexpressing breast cancer cells. Diosgenin preferentially inhibited proliferation and induced apoptosis in HER2-overexpressing cancer cells. Furthermore, diosgenin inhibited the phosphorylation of Akt and mTOR, and enhanced phosphorylation of JNK. The use of pharmacological inhibitors revealed that the modulation of Akt, mTOR and JNK phosphorylation was required for diosgenin-induced FAS suppression. Finally, we showed that diosgenin could enhance paclitaxel-induced cytotoxicity in HER2-overexpressing cancer cells. These results suggested that diosgenin has the potential to advance as chemopreventive or chemotherapeutic agent for cancers that overexpress HER2.     

Paeoniflorin inhibits cell growth and induces cell cycle arrest through inhibition of FoxM1 in colorectal cancer cells

Paeoniflorin (PF) exhibits tumor suppressive functions in a variety of human cancers. However, the function of PF and molecular mechanism in colorectal cancer are elusive. In the present study, we investigated whether PF could exert its antiproliferative activity, anti-migration, and anti-invasive function in colorectal cancer cells. We found that PF inhibited cell growth and induced apoptosis and blocked cell cycle progression in the G0/G1 phase in colorectal cancer cells. Moreover, we found that PF suppressed cell migration and invasion in colorectal cancer cells. FoxM1 has been reported to play an important oncogenic role in human cancers. We also determine whether PF inhibited the expression of FoxM1, leading to its anti-cancer activity. We found that PF treatment in colorectal cancer cells resulted in down-regulation of FoxM1. The rescue experiments showed that overexpression of FoxM1 abrogated the tumor suppressive function induced by PF treatment. Notably, depletion of FoxM1 promoted the anti-tumor activity of PF in colorectal cancer cells. Therefore, inhibition of FoxM1 could participate in the anti-tumor activity of PF in colorectal cancer cells.     

Upregulation of Cdc2 and cyclin A during apoptosis of endothelial cells induced by cleaved high-molecular-weight kininogen

We (8) reported that the cleaved high-molecular-weight kininogen (HKa) and its domain 5 (D5) inhibited angiogenesis. Further studies (15) revealed that D5 could inhibit cell proliferation and induce apoptosis of proliferating endothelial cells, which together may represent a critical part of antiangiogenic activity of HKa and D5. In the present study, we further examined the effect of HKa on cell cycle progression and cell viability. We report that HKa induced a significant upregulation of Cdc2 and cyclin A in proliferating endothelial cells, concurrent with a marked increase of Cdc2 activity. The increased expression of Cdc2 and cyclin A by HKa was not associated with an apparent change in cell cycle profiles of basic fibroblast growth factor-stimulated proliferating cells, but closely correlated with a marked increase of apoptosis, suggesting that the elevated Cdc2 activity is involved in HKa-induced apoptosis of proliferating endothelial cells. Our results support an emerging hypothesis that Cdc2 and cyclin A are important regulators for cell cycle as well as for apoptosis.     

Pds1p, an inhibitor of anaphase in budding yeast, plays a critical role in the APC and checkpoint pathway(s)

We report the isolation and characterization of pds1 mutants in Saccharomyces cerevisiae. The initial pds1-1 allele was identified by its inviability after transient exposure to microtubule inhibitors and its precocious dissociation of sister chromatids in the presence of these microtubule inhibitors. These findings suggest that pds1 mutants might be defective in anaphase arrest that normally is imposed by a spindle-damage checkpoint. To further examine a role for Pds1p in anaphase arrest, we compared the cell cycle arrest of pds1 mutants and PDS1 cells after: (a) the inactivation of Cdc16p or Cdc23p, two proteins that are required for the degradation of mitotic cyclins and are putative components of the yeast anaphase promoting complex (APC); (b) the inactivation of Cdc20p, another protein implicated in the degradation of mitotic cyclins; and (c) the inactivation of Cdc13 protein or gamma irradiation, two circumstances that induce a DNA- damage checkpoint. Under all these conditions, anaphase is inhibited in PDS1 cells but not in pds1 mutants. From these results we suggest that Pds1 protein is an anaphase inhibitor in PDS1 cells but not in pds1 mutants. From these results we suggest that Pds1 protein is an anaphase inhibitor that plays a critical role in the control of anaphase by both APC and checkpoints. We also show that pds1 mutants exit mitosis and initiate new rounds of cell division after gamma irradiation and Cdc13p inactivation but no after nocodazole-treatment or inactivation of Cdc16p, Cdc20p or Cdc23p function. Therefore, in the DNA-damage checkpoint, Pds1p is required for the inhibition of cytokinesis and DNA replication as well as anaphase. The role of Pds1 protein in anaphase inhibition and general cell cycle regulation is discussed.     

IpaC induces actin polymerization and filopodia formation during Shigella entry into epithelial cells.

Shigella proteins that are targeted to host cells by a type III secretion apparatus are essential for reorganization of the cytoskeleton during cell invasion. We have developed a semi-permeabilized cell assay that tests the effects of bacterial proteins on the actin cytoskeleton. The Shigella IpaC protein was found to induce the formation of filopodial and lamellipodial extensions in these semi-permeabilized cells. Microinjection of IpaC into cells, or cellular expression of IpaC also led to the formation of filopodial structures. Monoclonal antibodies (mAbs) directed against the C-terminus of IpaC inhibited the IpaC-induced extensions, whereas an anti-N-terminal IpaC mAb stimulated extensive lamellae formation. Shigella induced foci of actin polymerization in the permeabilized cells and these were inhibited by anti-C-terminal IpaC mAbs. Consistent with a role for IpaC in Shigella-induced cytoskeletal rearrangements during entry, stable transfectants expressing IpaC challenged with Shigella showed increased bacterial internalization. IpaC-induced extensions were inhibited by a dominant-interfering form of Cdc42 or the Cdc42-binding domain of WASP, whereas a dominant-interfering form of Rac resulted in inhibition of lamellae formation. We conclude that IpaC leads to activation of Cdc42 which in turn, causes activation of Rac, both GTPases being required for Shigella entry.     

Suppression of Autophagy Enhanced Growth Inhibition and Apoptosis of Interferon-β in Human Glioma Cells

Interferon-beta (IFN-β) is a cytokine with anti-viral, anti-proliferative, and immunomodulatory effects. In this study, we investigated the effects of IFN-β on the induction of autophagy and the relationships among autophagy, growth inhibition, and apoptosis induced by IFN-β in human glioma cells. We found that IFN-β induced autophagosome formation and conversion of microtubule associated protein 1 light chain 3 (LC3) protein, whereas it inhibited cell growth through caspase-dependent cell apoptosis. The Akt/mTOR signaling pathway was involved in autophagy induced by IFN-β. A dose- and time-dependent increase of p-ERK 1/2 expression was also observed in human glioma cells treated with IFN-β. Autophagy induced by IFN-β was suppressed when p-ERK1/2 was impaired by treatment with U0126. We also demonstrated that suppression of autophagy significantly enhanced growth inhibition and cell apoptosis induced by IFN-β, whereas inhibition of caspase-dependent cell apoptosis impaired autophagy induced by IFN-β. Collectively, these findings indicated that autophagy induced by IFN-β was associated with the Akt/mTOR and ERK 1/2 signaling pathways, and inhibition of autophagy could enhance the growth inhibitory effects of IFN-β and increase apoptosis in human glioma cells. Together, these findings support the possibility that autophagy inhibitors may improve IFN-β therapy for gliomas.