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1.  Group choreography: mechanisms orchestrating the collective movement of border cells 
Cell movements are essential for animal development and homeostasis but also contribute to disease. Moving cells typically extend protrusions towards a chemoattractant, adhere to the substrate, contract and detach at the rear. It is less clear how cells that migrate in interconnected groups in vivo coordinate their behaviour and navigate through natural environments. The border cells of the Drosophila melanogaster ovary have emerged as an excellent model for the study of collective cell movement, aided by innovative genetic, live imaging, and photomanipulation techniques. Here we provide an overview of the molecular choreography of border cells and its more general implications.
PMCID: PMC4099007  PMID: 23000794
2.  PMA synergistically enhances apicularen A-induced cytotoxicity by disrupting microtubule networks in HeLa cells 
BMC Cancer  2014;14:36.
Combination therapy is key to improving cancer treatment efficacy. Phorbol 12-myristate 13-acetate (PMA), a well-known PKC activator, increases the cytotoxicity of several anticancer drugs. Apicularen A induces cytotoxicity in tumor cells through disrupting microtubule networks by tubulin down-regulation. In this study, we examined whether PMA increases apicularen A-induced cytotoxicity in HeLa cells.
Cell viability was examined by thiazolyl blue tetrazolium (MTT) assays. To investigate apoptotic potential of apicularen A, DNA fragmentation assays were performed followed by extracting genomic DNA, and caspase-3 activity assays were performed by fluorescence assays using fluorogenic substrate. The cell cycle distribution induced by combination with PMA and apicularen A was examined by flow cytometry after staining with propidium iodide (PI). The expression levels of target proteins were measured by Western blotting analysis using specific antibodies, and α-tubulin mRNA levels were assessed by reverse transcription polymerase chain reaction (RT-PCR). To examine the effect of combination of PMA and apicularen A on the microtubule architecture, α-tubulin protein and nuclei were visualized by immunofluorescence staining using an anti-α-tubulin antibody and PI, respectively.
We found that apicularen A induced caspase-dependent apoptosis in HeLa cells. PMA synergistically increased cytotoxicity and apoptotic sub-G1 population induced by apicularen A. These effects were completely blocked by the PKC inhibitors Ro31-8220 and Go6983, while caspase inhibition by Z-VAD-fmk did not prevent cytotoxicity. RNA interference using siRNA against PKCα, but not PKCβ and PKCγ, inhibited cytotoxicity induced by combination PMA and apicularen A. PMA increased the apicularen A-induced disruption of microtubule networks by further decreasing α- and β-tubulin protein levels in a PKC-dependent manner.
These results suggest that the synergy between PMA and apicularen A is involved by PKCα activation and microtubule disruption, and that may inform the development of novel approaches to treat cancer.
PMCID: PMC3901760  PMID: 24447339
PMA; Apicularen A; PKCα; Cell death; Microtubule disruption
3.  Docosahexaenoic acid induces autophagy through p53/AMPK/mTOR signaling and promotes apoptosis in human cancer cells harboring wild-type p53 
Autophagy  2011;7(11):1348-1358.
Docosahexaenoic acid (DHA) has been reported to induce tumor cell death by apoptosis. However, little is known about the effects of DHA on autophagy, another complex well-programmed process characterized by the sequestration of cytoplasmic material within autophagosomes. Here we show that DHA increased both the level of microtubule-associated protein 1 light chain 3 and the number of autophagic vacuoles without impairing autophagic vesicle turnover, indicating that DHA induces not only apoptosis but also autophagy. We also observed that DHA-induced autophagy was accompanied by p53 loss. Inhibition of p53 increased DHA-induced autophagy and prevention of p53 degradation significantly led to the attenuation of DHA-induced autophagy, suggesting that DHA-induced autophagy is mediated by p53. Further experiments showed that the mechanism of DHA-induced autophagy associated with p53 attenuation involved an increase in the active form of AMP-activated protein kinase and a decrease in the activity of mammalian target of rapamycin. In addition, compelling evidence for the interplay between autophagy and apoptosis induced by DHA is supported by the findings that autophagy inhibition suppressed apoptosis and further autophagy induction enhanced apoptosis in response to DHA treatment. Overall, our results demonstrate that autophagy contributes to the cytotoxicity of DHA in cancer cells harboring wild-type p53.
PMCID: PMC3242799  PMID: 21811093
DHA; autophagy; apoptosis; p53; cancer; mTOR; AMPK; p27
5.  Protein-bound polysaccharide from Phellinus linteus inhibits tumor growth, invasion, and angiogenesis and alters Wnt/β-catenin in SW480 human colon cancer cells 
BMC Cancer  2011;11:307.
Polysaccharides extracted from the Phellinus linteus (PL) mushroom are known to possess anti-tumor effects. However, the molecular mechanisms responsible for the anti-tumor properties of PL remain to be explored. Experiments were carried out to unravel the anticancer effects of PL.
The anti-cancer effects of PL were examined in SW480 colon cancer cells by evaluating cell proliferation, invasion and matrix metallo-proteinase (MMP) activity. The anti-angiogenic effects of PL were examined by assessing human umbilical vein endothelial cell (HUVEC) proliferation and capillary tube formation. The in vivo effect of PL was evaluated in an athymic nude mouse SW480 tumor engraft model.
PL (125-1000 μg/mL) significantly inhibited cell proliferation and decreased β-catenin expression in SW480 cells. Expression of cyclin D1, one of the downstream-regulated genes of β-catenin, and T-cell factor/lymphocyte enhancer binding factor (TCF/LEF) transcription activity were also significantly reduced by PL treatment. PL inhibited in vitro invasion and motility as well as the activity of MMP-9. In addition, PL treatment inhibited HUVEC proliferation and capillary tube formation. Tumor growth of SW480 cells implanted into nude mice was significantly decreased as a consequence of PL treatment, and tumor tissues from treated animals showed an increase in the apoptotic index and a decrease in β-catenin expression. Moreover, the proliferation index and microvessel density were significantly decreased.
These data suggest that PL suppresses tumor growth, invasion, and angiogenesis through the inhibition of Wnt/β-catenin signaling in certain colon cancer cells.
PMCID: PMC3154178  PMID: 21781302
6.  Identification of Novel Phosphorylation Motifs Through an Integrative Computational and Experimental Analysis of the Human Phosphoproteome 
Protein phosphorylation occurs in certain sequence/structural contexts that are still incompletely understood. The amino acids surrounding the phosphorylated residues are important in determining the binding of the kinase to the protein sequence. Upon phosphorylation these sequences also determine the binding of certain domains that specifically bind to phosphorylated sequences. Thus far, such ‘motifs’ have been identified through alignment of a limited number of well identified kinase substrates.
Experimentally determined phosphorylation sites from Human Protein Reference Database were used to identify 1,167 novel serine/threonine or tyrosine phosphorylation motifs using a computational approach. We were able to statistically validate a number of these novel motifs based on their enrichment in known phosphopeptides datasets over phosphoserine/threonine/tyrosine peptides in the human proteome. There were 299 novel serine/threonine or tyrosine phosphorylation motifs that were found to be statistically significant. Several of the novel motifs that we identified computationally have subsequently appeared in large datasets of experimentally determined phosphorylation sites since we initiated our analysis. Using a peptide microarray platform, we have experimentally evaluated the ability of casein kinase I to phosphorylate a subset of the novel motifs discovered in this study. Our results demonstrate that it is feasible to identify novel phosphorylation motifs through large phosphorylation datasets. Our study also establishes peptide microarrays as a novel platform for high throughput kinase assays and for the validation of consensus motifs. Finally, this extended catalog of phosphorylation motifs should assist in a systematic study of phosphorylation networks in signal transduction pathways.
PMCID: PMC3124146  PMID: 21720494
Phosphorylation; Motifs; Peptide array
7.  A serum-stable branched dimeric anti-VEGF peptide blocks tumor growth via anti-angiogenic activity 
Experimental & Molecular Medicine  2010;42(7):514-523.
Angiogenesis is critical and indispensable for tumor progression. Since VEGF is known to play a central role in angiogenesis, the disruption of VEGF-VEGF receptor system is a promising target for anti-cancer therapy. Previously, we reported that a hexapeptide (RRKRRR, RK6) blocked the growth and metastasis of tumor by inhibiting VEGF binding to its receptors. In addition, dRK6, the D-form derivative of RK6, retained its biological activity with improved serum stability. In the present study, we developed a serum-stable branched dimeric peptide (MAP2-dRK6) with enhanced anti-VEGF and anti-tumor activity. MAP2-dRK6 is more effective than dRK6 in many respects: inhibition of VEGF binding to its receptors, VEGF- and tumor conditioned medium-induced proliferation and ERK signaling of endothelial cells, and VEGF-induced migration and tube formation of endothelial cells. Moreover, MAP2-dRK6 blocks in vivo growth of VEGF-secreting colorectal cancer cells by the suppression of angiogenesis and the subsequent induction of tumor cell apoptosis. Our observations suggest that MAP2-dRK6 can be a prospective therapeutic molecule or lead compound for the development of drugs for various VEGF-related angiogenic diseases.
PMCID: PMC2912478  PMID: 20543548
angiogenesis inhibitors; colorectal neoplasms; peptides; receptors, vascular endothelial growth factor; vascular endothelial growth factors
8.  Transduction of artificial transcriptional regulatory proteins into human cells 
Nucleic Acids Research  2008;36(16):e103.
Protein transduction (PT) is a method for delivering proteins into mammalian cells. PT is accomplished by linking a small peptide tag—called a PT domain (PTD)—to a protein of interest, which generates a functional fusion protein that can penetrate efficiently into mammalian cells. In order to study the functions of a transcription factor (TF) of interest, expression plasmids that encode the TF often are transfected into mammalian cells. However, the efficiency of DNA transfection is highly variable among different cell types and is usually very low in primary cells, stem cells and tumor cells. Zinc-finger transcription factors (ZF-TFs) can be tailor-made to target almost any gene in the human genome. However, the extremely low efficiency of DNA transfection into cancer cells, both in vivo and in vitro, limits the utility of ZF-TFs. Here, we report on an artificial ZF-TF that has been fused to a well-characterized PTD from the human immunodeficiency virus-1 (HIV-1) transcriptional activator protein, Tat. This ZF-TF targeted the endogenous promoter of the human VEGF-A gene. The PTD-attached ZF-TF was delivered efficiently into human cells in vitro. In addition, the VEGF-A-specific transcriptional repressor retarded the growth rate of tumor cells in a mouse xenograft experiment.
PMCID: PMC2532713  PMID: 18644841
9.  Activity and expression of urokinase-type plasminogen activator and matrix metalloproteinases in human colorectal cancer 
BMC Cancer  2006;6:211.
Matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9), and urokinase-type plasminogen activator (uPA) are involved in colorectal cancer invasion and metastasis. There is still debate whether the activity of MMP-2 and MMP-9 differs between tumors located in the colon and rectum. We designed this study to determine any differences in the expression of MMP-2, MMP-9 and uPA system between colon and rectal cancer tissues.
Cancer tissue samples were obtained from colon carcinoma (n = 12) and rectal carcinomas (n = 10). MMP-2 and MMP-9 levels were examined using gelatin zymography and Western blotting; their endogenous inhibitors, tissue inhibitor of metalloproteinase-2 (TIMP-2) and tissue inhibitor of metalloproteinase-1 (TIMP-1), were assessed by Western blotting. uPA, uPAR and PAI-1 were examined using enzyme-linked immunosorbent assay (ELISA). The activity of uPA was assessed by casein-plasminogen zymography.
In both colon and rectal tumors, MMP-2, MMP-9 and TIMP-1 protein levels were higher than in corresponding paired normal mucosa, while TIMP-2 level in tumors was significantly lower than in normal mucosa. The enzyme activities or protein levels of MMP-2, MMP-9 and their endogenous inhibitors did not reach a statistically significant difference between colon and rectal cancer compared with their normal mucosa. In rectal tumors, there was an increased activity of uPA compared with the activity in colon tumors (P = 0.0266), however urokinase-type plasminogen activator receptor (uPAR) and plasminogen activator inhibitor-1 (PAI-1) showed no significant difference between colon and rectal cancer tissues.
These findings suggest that uPA may be expressed differentially in colon and rectal cancers, however, the activities or protein levels of MMP-2, MMP-9, TIMP-1, TIMP-2, PAI-1 and uPAR are not affected by tumor location in the colon or the rectum.
PMCID: PMC1563482  PMID: 16916471
10.  Comparison of the Efficacy of Oral Capecitabine versus Bolus 5-FU in Preoperative Radiotherapy of Locally Advanced Rectal Cancer 
The effects of treatment with oral capecitabine vs. bolus 5-FU, administered concurrently with preoperative radiotherapy, were compared in the treatment of locally advanced rectal cancer (LARC). One hundred and twenty-seven patients with LARC received concurrent preoperative chemoradiation using two cycles bolus 5-FU (500 mg/m2/day) plus leucovorin (LV, 20 mg/m2/day) (Group I). Another LARC group received concurrent chemoradiation using two cycles 1,650 mg/m2/day of oral capecitabine and 20 mg/m2/day of LV (Group II, 97 patients). Radiation was delivered to the primary tumor at 50.4 Gy in both groups. Definitive surgery was performed 6 weeks after the completion of chemoradiation. A pathologic complete remission was achieved in 11.4% of patients in Group I and in 22.2% of patients in Group II (p=0.042). The down-staging rates of the primary tumor and lymph nodes were 39.0/68.7% in Group I and 61.1/87.5% in Group II (p=0.002/0.005). Sphincter-preserving surgery was possible in 42.1% of patients in Group I and 66.7% of those in Group II (p=0.021). Grade 3 or 4 leucopenia, diarrhea, and radiation dermatitis were statistically more prevalent in Group I than in Group II, while the opposite was true for grade 3 hand-foot syndrome. Preoperative chemoradiation using oral capecitabine was better tolerated than bolus 5-FU and was more effective in the promotion of both down-staging and sphincter preservation in patients with LARC.
PMCID: PMC2733979  PMID: 16479065
Fluorouracil; capecitabine; Radiotherapy; drug therapy; Rectal Neoplasms

Results 1-10 (10)