An Accelerated Wound-Healing Surgical Suture Engineered with an Extracellular Matrix.

A suture is a ubiquitous medical device to hold wounded tissues together and support the healing process after surgery. Surgical sutures, having incomplete biocompatibility, often cause unwanted infections or serious secondary trauma to soft or fragile tissue. In this research, UV/ozone (UVO) irradiation or polystyrene sulfonate acid (PSS) dip-coating is used to achieve a fibronectin (FN)-coated absorbable suture system, in which the negatively charged moieties produced on the suture cause fibronectin to change from a soluble plasma form into a fibrous form, mimicking the actions of cellular fibronectin upon binding. The fibrous fibronectin coated on the suture can be exploited as an engineered interface to improve cellular migration and adhesion in the region around the wounded tissue while preventing the binding of infectious bacteria, thereby facilitating wound healing. Furthermore, the FN-coated suture is found to be associated with a lower friction between the suture and the wounded tissue, thus minimizing the occurrence of secondary wounds during surgery. It is believed that this surface modification can be universally applied to most kinds of sutures currently in use, implying that it may be a novel way to develop a highly effective and safer suture system for clinical applications.

SFMBT2-Mediated Infiltration of Preadipocytes and TAMs in Prostate Cancer.

Infiltration of diverse cell types into tumor microenvironment plays a critical role in cancer progression including metastasis. We previously reported that SFMBT2 (Scm-like with four mbt domains 2) regulates the expression of matrix metalloproteinases (MMPs) and migration and invasion of cancer cells in prostate cancer. Here we investigated whether the down-regulation of SFMBT2 regulates the infiltration of preadipocytes and tumor-associated macrophages (TAMs) in prostate cancer. We found that the down-regulation of SFMBT2 promotes the infiltration of preadipocytes and TAMs through up-regulation of CXCL8, CCL2, CXCL10, and CCL20 expression in prostate cancer. Expression of CXCL8, CCL2, CXCL10, and CCL20 was also elevated in prostate cancer patients having a higher Gleason score (≥8), which had substantially lower SFMBT2 expression. We also found that the up-regulation of CXCL8, CCL2, CXCL10, and CCL20 expression is dependent on NF-κB activation in prostate cancer cells expressing a low level of SFMBT2. Moreover, increased IL-6 from infiltrated preadipocytes and TAMs promoted migration and invasion of prostate cancer cells expressing a low level of SFMBT2. Our study may suggest that SFMBT2 a critical regulator for the infiltration of preadipocytes and TAMs into the prostate tumor microenvironment. Thus, the regulation of SFMBT2 may provide a new therapeutic strategy to inhibit prostate cancer metastasis, and SFMBT2 could be used as a potential biomarker in prostate cancer metastasis.

Protopine isolated from Nandina domestica induces apoptosis and autophagy in colon cancer cells by stabilizing p53.

The tumor suppressor p53 plays essential roles in cellular protection mechanisms against a variety of stress stimuli and its activation induces apoptosis or autophagy in certain cancer cells. Here, we identified protopine, an isoquinoline alkaloid isolated from Nandina domestica, as an activator of the p53 pathway from cell-based natural compound screening based on p53-responsive transcription. Protopine increased the p53-mediated transcriptional activity and promoted p53 phosphorylation at the Ser15 residue, resulting in stabilization of p53 protein. Moreover, protopine up-regulated the expression of p21WAF1/CIP1 and BAX, downstream genes of p53, and inhibited the proliferation of HCT116 colon cancer cells. Apoptosis was elicited by protopine as indicated by caspase-3/7 activation, poly ADP ribose polymerase cleavage, and increased population of Annexin V-FITC-positive cells. Furthermore, protopine induced the formation of microtubule-associated protein 1 light chain 3 (LC3) puncta and LC3-II turnover, typical biochemical markers of autophagy, in HCT116 cells. Our findings suggest that protopine exerts its antiproliferative activity by stimulating the p53 pathway and may have potential as a chemopreventive agent for human colon cancer.

CCAAT/enhancer-binding protein-ß functions as a negative regulator of Wnt/ß-catenin signaling through activation of AXIN1 gene expression.

Axin1, a concentration-limiting component of the ß-catenin destruction complex, negatively regulates the Wnt/ß-catenin pathway. Axin1 concentration is reported to be regulated by proteasomal degradation; however, its transcriptional regulation has not yet been reported. Here, we demonstrated that CCAAT/enhancer-binding protein-ß (C/EBP-ß) activates axis inhibition protein 1 (AXIN1) gene expression, thereby attenuating Wnt/ß-catenin signaling. C/EBP-ß interacted with cis-regulatory element for C/EBP-ß in the 5'-upstream sequences of the AXIN1 gene and increased AXIN1 promoter activity. Functional analysis using Drosophila and zebrafish models established that C/EBP-ß negatively regulates the Wnt/ß-catenin pathway. Small-molecule-based up-regulation of C/EBP-ß induces AXIN1 gene expression and down-regulates the intracellular ß-catenin level, thereby inhibiting hepatoma cell growth. Thus, our findings provide a unique mechanistic insight into the regulation of Axin homeostasis and present a novel strategy for the development of anticancer therapeutics targeting Wnt/ß-catenin signaling.

Wnt signaling promotes androgen-independent prostate cancer cell proliferation through up-regulation of the hippo pathway effector YAP.

Aberrant up-regulation of Wnt/ß-catenin signaling is associated with the development and progression of prostate cancer, but the underlying mechanism is unclear. Here we show that in the absence of androgens, the Wnt/ß-catenin pathway activates AR-mediated transcription through up-regulation of the Hippo pathway effector Yes-associated protein (YAP). Wnt3a-conditioned medium (Wnt3a-CM) promotes the growth of LNCaP cells and increases AR and YAP protein levels. Moreover, Wnt3a-CM induces the nuclear translocation of YAP and the AR, but not ß-catenin, thereby activating the expression of AR- and YAP-dependent genes, in an androgen-independent manner. In addition, depletion of YAP with small interfering RNA (siRNA) prevented Wnt3a-CM-mediated up-regulation of AR-dependent gene expression. Thus, our findings provide mechanistic insight into the proposed cross-talk between the Wnt/ß-catenin and Hippo pathways in androgen-independent prostate cancer development.

SFMBT2 (Scm-like with four mbt domains 2) negatively regulates cell migration and invasion in prostate cancer cells.

Metastatic prostate cancer is the leading cause of morbidity and mortality in men. In this study, we found that expression level of SFMBT2 is altered during prostate cancer progression and has been associated with the migration and invasion of prostate cancer cells. The expression level of SFMBT2 is high in poorly metastatic prostate cancer cells compared to highly metastatic prostate cancer cells. We also found that SFMBT2 knockdown elevates MMP-2, MMP-3, MMP-9, and MMP-26 expression, leading to increased cell migration and invasion in LNCaP and VCaP cells. SFMBT2 interacts with YY1, RNF2, N-CoR and HDAC1/3, as well as repressive histone marks such as H3K9me2, H4K20me2, and H2AK119Ub which are associated with transcriptional repression. In addition, SFMBT2 knockdown decreased KAI1 gene expression through up-regulation of N-CoR gene expression. Expression of SFMBT2 in prostate cancer was strongly associated with clinicopathological features. Patients having higher Gleason score (≥ 8) had substantially lower SFMBT2 expression than patients with lower Gleason score. Moreover, tail vein or intraprostatic injection of SFMBT2 knockdown LNCaP cells induced metastasis. Taken together, our findings suggest that regulation of SFMBT2 may provide a new therapeutic strategy to control prostate cancer metastasis as well as being a potential biomarker of metastatic prostate cancer.

Involvement of transcription repressor Snail in the regulation of human telomerase reverse transcriptase (hTERT) by transforming growth factor-ß.

Human telomerase reverse transcriptase (hTERT), a catalytic subunit of telomerase, is the primary determinant for telomerase enzyme activity, which has been associated with cellular immortality. Expression of the hTERT gene is regulated by various extracellular (external) stimuli and is aberrantly up-regulated in more than 90% of cancers. Here we show that hTERT gene expression was repressed in response to transforming growth factor-ß (TGF-ß) by a mechanism dependent on transcription factors Snail and c-Myc. TGF-ß activated Snail and down-regulated c-Myc gene expression. In addition, ectopic expression of Snail strongly inhibited hTERT promoter activity, although co-expression of c-Myc abrogated this effect. Chromatin immunoprecipitation (ChIP) analysis revealed that TGF-ß decreased c-Myc occupancy and dramatically increased recruitment of Snail to the E-box motifs of the hTERT promoter, thereby repressing hTERT expression. Our findings suggest a dynamic alteration in hTERT promoter occupancy by Snail and c-Myc is the mechanistic basis for TGF-ß-mediated regulation of hTERT.

Green tea polyphenol EGCG suppresses Wnt/ß-catenin signaling by promoting GSK-3ß- and PP2A-independent ß-catenin phosphorylation/degradation.

(-)-Epigallocatechin-3-gallate (EGCG), the major polyphenol in green tea, has been reported to inhibit the Wnt/ß-catenin pathway, which is aberrantly up-regulated in colorectal cancers, but its precise mechanism of action remains unclear. Here, we used a sensitive cell-based system to demonstrate that EGCG suppresses ß-catenin response transcription (CRT), activated by Wnt3a-conditioned medium (Wnt3a-CM), by promoting the degradation of intracellular ß-catenin. EGCG induced ß-catenin N-terminal phosphorylation at the Ser33/37 residues and subsequently promoted its degradation; however, this effect was not observed for oncogenic forms of ß-catenin. Pharmacological inhibition or depletion of glycogen synthase kinase-3ß (GSK-3ß) did not abrogate the EGCG-mediated ß-catenin degradation. EGCG did not affect the activity and expression of protein phosphatase 2A (PP2A). Consistently, the phosphorylation and degradation of ß-catenin was found in adenomatous polyposis coli (APC) mutated colon cancer cells after EGCG treatment. EGCG repressed the expression of cyclin D1 and c-myc, which are ß-catenin/T-cell factor-dependent genes, and inhibited the proliferation of colon cancer cells. Our findings suggest that EGCG exerts its cancer-preventive or anticancer activity against colon cancer cells by promoting the phosphorylation and proteasomal degradation of ß-catenin through a mechanism independent of the GSK-3ß and PP2A.

Cardamonin suppresses the proliferation of colon cancer cells by promoting ß-catenin degradation.

Aberrant accumulation of intracellular ß-catenin and subsequent activation of ß-catenin response transcription (CRT) in intestinal epithelial cells is a frequent early event during the development of colon cancer. Here we show that cardamonin, a chalcone isolated from Aplinia katsumadai Hayata, inhibited CRT in SW480 colon cancer cells that carry inactivating mutation in the adenomatous polyposis coli (APC) gene. Cardamonin also down-regulated intracellular ß-catenin levels in SW480 cells without affecting its mRNA levels. Interestingly, pharmacological inhibition of the proteasome prevented the cardamonin-induced down-regulation of ß-catenin. In addition, cardamonin suppressed the expression of cyclin D1 and c-myc, which are known ß-catenin/T cell factor (TCF)-dependent genes. Moreover, cardamonin inhibited the growth of various colon cancer cells and induced G2/M cell cycle arrest in SW480 colon cancer cells. These findings indicate that cardamonin is a potential chemotherapeutic agent against colon cancer.

Small molecule-based promotion of PKCα-mediated ß-catenin degradation suppresses the proliferation of CRT-positive cancer cells.

Aberrant accumulation of intracellular ß-catenin is a well recognized characteristic of several cancers, including prostate, colon, and liver cancers, and is a potential target for development of anticancer therapeutics. Here, we used cell-based small molecule screening to identify CGK062 as an inhibitor of Wnt/ß-catenin signaling. CGK062 promoted protein kinase Cα (PKCα)-mediated phosphorylation of ß-catenin at Ser33/Ser37, marking it for proteasomal degradation. This reduced intracellular ß-catenin levels and consequently antagonized ß-catenin response transcription (CRT). Pharmacological inhibition or depletion of PKCα abrogated CGK062-mediated phosphorylation and degradation of ß-catenin. In addition, CGK062 repressed the expression of the genes encoding cyclin D1, c-myc, and axin-2, ß-catenin target genes, and thus inhibited the growth of CRT-positive cancer cells. Furthermore, treatment of nude mice bearing PC3 xenograft tumors with CGK062 at doses of 50 mg/kg and 100 mg/kg (i.p.) significantly suppressed tumor growth. Our findings suggest that CGK062 exerts its anticancer activity by promoting PKCα-mediated ß-catenin phosphorylation/degradation. Therefore, CGK062 has significant therapeutic potential for the treatment of CRT-positive cancers.

Small molecule-based disruption of the Axin/ß-catenin protein complex regulates mesenchymal stem cell differentiation.

The Wnt/ß-catenin pathway plays important roles in the differentiation of multiple cell types, including mesenchymal stem cells. Using a cell-based chemical screening assay with a synthetic chemical library of 270 000 compounds, we identified the compound SKL2001 as a novel agonist of the Wnt/ß-catenin pathway and uncovered its molecular mechanism of action. SKL2001 upregulated ß-catenin responsive transcription by increasing the intracellular ß-catenin protein level and inhibited the phosphorylation of ß-catenin at residues Ser33/37/Thr41 and Ser45, which would mark it for proteasomal degradation, without affecting CK1 and GSK-3ß enzyme activities. Biochemical analysis revealed that SKL2001 disrupted the Axin/ß-catenin interaction, which is a critical step for CK1- and GSK-3ß-mediated phosphorylation of ß-catenin at Ser33/37/Thr41 and Ser45. The treatment of mesenchymal stem cells with SKL2001 promoted osteoblastogenesis and suppressed adipocyte differentiation, both of which were accompanied by the activation of Wnt/ß-catenin pathway. Our findings provide a new strategy to regulate mesenchymal stem cell differentiation by modulation of the Wnt/ß-catenin pathway.

Induction of apoptosis in colon cancer cells by a novel topoisomerase I inhibitor TopIn.

The tumor suppressor p53 plays an important role in cellular emergency mechanisms through regulating the genes involved in cell cycle arrest and apoptosis. To identify small molecules that can activate p53-responsive transcription, we performed chemical screening using genetically engineered HCT116 reporter cells. We found that TopIn (7-phenyl-6H-[1,2,5]oxadiazolo[3,4-e]indole 3-oxide) efficiently activated p53-mediated transcriptional activity and induced phosphorylation of p53 at Ser15, thereby stabilizing the p53 protein. Furthermore, TopIn upregulated the expression of p21(WAF1/CIP1), a downstream target of p53, and suppressed cellular proliferation in various colon cancer cells. Additionally, TopIn induced DNA fragmentation, caspase-3/7 activation and poly ADP ribose polymerase cleavage, typical biochemical markers of apoptosis, in p53 wild-type and mutated colon cancer cells. Finally, we found that TopIn inhibited topoisomerase I activity, but not topoisomerase II, in vitro and induced the formation of the topoisomerase I-DNA complex in HCT116 colon cancer cells. Unlike camptothecin (CPT) and its derivative SN38, TopIn did not affect the activity of the ATP-binding cassette transporter breast cancer resistance protein (BCRP) or multidrug-resistant protein-1 (MDR-1). These results suggest that TopIn may present a promising new topoisomerase I-targeting anti-tumor therapeutics.

Galangin suppresses the proliferation of ß-catenin response transcription-positive cancer cells by promoting adenomatous polyposis coli/Axin/glycogen synthase kinase-3ß-independent ß-catenin degradation.

Galangin is a naturally occurring bioflavonoid with anticancer activity against certain human cancers, yet little is known about its mechanism of action. Here, we used a chemical biology approach to reveal that galangin suppresses ß-catenin response transcription (CRT), which is aberrantly up-regulated in colorectal and liver cancers, by promoting the degradation of intracellular ß-catenin. Inhibition of glycogen synthase kinase-3ß (GSK-3ß) activity or mutation of the GSK-3ß-targeted sequence from ß-catenin was unable to abrogate the galangin-mediated degradation of ß-catenin. In addition, galangin down-regulated the intracellular ß-catenin levels in cancer cells with inactivating mutations of adenomatous polyposis coli (APC) or Axin, which are components of the ß-catenin destruction complex. Galangin repressed the expression of ß-catenin/T-cell factor-dependent genes, such as cyclin D1 and c-myc, and thus inhibited the proliferation of CRT-positive cancer cells. Structure-activity data indicated that the major structural requirements for galangin-mediated ß-catenin degradation are hydroxyl groups at positions 3, 5, and 7. Our findings suggest that galangin exerts its anticancer activity by promoting APC/Axin/GSK-3ß-independent proteasomal degradation of ß-catenin.

Oligodeoxyribozymes that cleave beta-catenin messenger RNA inhibit growth of colon cancer cells via reduction of beta-catenin response transcription.

Abnormal regulation of Wnt/beta-catenin signaling followed by increased levels of the beta-catenin protein have been identified in enhanced cellular proliferation and development of colon polyps and cancers. To inhibit beta-catenin gene expression in colon cancer cells, RNA-cleaving oligodeoxyribozyme (DNAzyme) was employed to destroy the beta-catenin mRNA. We designed a strategy to identify the cleavage sites in beta-catenin RNA with a pool of random sequences from a DNAzyme library and identified four potential DNAzyme-working sites. DNAzymes were constructed for the selected target sites and were tested for the ability to cleave beta-catenin RNA. When introduced into the cells, the selected DNAzymes decreased the expression of beta-catenin significantly as well as its downstream gene, cyclin D1. Additionally, we designed short hairpin RNA that targets the same cleavage site for the selected DNAzyme. The designed short hairpin RNA also inhibited beta-catenin gene expression in colon cancer cells. Our studies show that RNA-cleaving DNAzymes and RNA interference targeted to beta-catenin significantly reduced beta-catenin-dependent gene expression, resulting in inhibition of colon cancer cell growth. These results indicate that the functional antisense oligonucleotides directed against beta-catenin might have potential as a therapeutic intervention to treat colon cancer.

Murrayafoline A attenuates the Wnt/beta-catenin pathway by promoting the degradation of intracellular beta-catenin proteins.

Molecular lesions in Wnt/beta-catenin signaling and subsequent up-regulation of beta-catenin response transcription (CRT) occur frequently during the development of colon cancer. To identify small molecules that suppress CRT, we screened natural compounds in a cell-based assay for detection of TOPFalsh reporter activity. Murrayafoline A, a carbazole alkaloid isolated from Glycosmis stenocarpa, antagonized CRT that was stimulated by Wnt3a-conditioned medium (Wnt3a-CM) or LiCl, an inhibitor of glycogen synthase kinase-3beta (GSK-3beta), and promoted the degradation of intracellular beta-catenin without altering its N-terminal phosphorylation at the Ser33/37 residues, marking it for proteasomal degradation, or the expression of Siah-1, an E3 ubiquitin ligase. Murrayafoline A repressed the expression of cyclin D1 and c-myc, which is known beta-catenin/T cell factor (TCF)-dependent genes and thus inhibited the proliferation of various colon cancer cells. These findings indicate that murrayafoline A may be a potential chemotherapeutic agent for use in the treatment of colon cancer.

Isoreserpine promotes beta-catenin degradation via Siah-1 up-regulation in HCT116 colon cancer cells.

Aberrant accumulation of intracellular beta-catenin in intestinal epithelial cells is a frequent early event during the development of colon cancer. To identify small molecules that decrease the level of intracellular beta-catenin, we performed cell-based chemical screening using genetically engineered HEK293 reporter cells to detect compounds that inhibit TOPFlash reporter activity, which was stimulated by Wnt3a-conditioned medium. We found that isoreserpine promoted the degradation of intracellular beta-catenin by up-regulation of Siah-1 in HEK293 and HCT116 colon cancer cells. Moreover, isoreserpine repressed the expression of beta-catenin/T-cell factor (TCF)-dependent genes, such as cyclin D1 and c-myc, resulting in the suppression of HCT116 cell proliferation. Our findings suggest that isoreserpine can potentially be used as a chemotherapeutic agent against colon cancer.

Stimulation of protein kinase C-alpha suppresses colon cancer cell proliferation by down-regulation of beta-catenin.

We reported previously that protein kinase C-alpha (PKC-alpha) negatively regulates Wnt/beta-catenin signalling pathway. The current study explores the role of PKC-alpha in the regulation of proliferation of colon cancer cells, which contain aberrant up-regulation of intracellular beta-catenin. In colon tissue and cells, an inverse correlation was observed between the expression levels of PKC-alpha and intracellular beta-catenin. Activation of PKC-alpha inhibited beta-catenin response transcription by down-regulation of intracellular beta-catenin and induced phosphorylation of the N-terminal serine and threonine residues (Ser33/Ser37/Thr41) of beta-catenin, marking it for proteasomal degradation, in colon cancer cells. Pharmacological inhibition or depletion of PKC-alpha-abrogated PKC-alpha-mediated beta-catenin down-regulation and phosphorylation in colon cancer cells. Notably, the Ser45 residue of beta-catenin was essential for PKC-alpha-induced beta-catenin down-regulation in colon cancer cells. Moreover, PKC-alpha activation repressed the expression of cyclin D1 and c-myc, which are known beta-catenin target genes, and thus inhibited the growth of colon cancer cells. These findings suggest that PKC-alpha negatively regulates colon cancer cell proliferation viabeta-catenin phosphorylation/down-regulation and may facilitate the development of new strategies to treatment of colon cancer.

Protein-kinase-C-mediated beta-catenin phosphorylation negatively regulates the Wnt/beta-catenin pathway.

Normally, the Wnt/beta-catenin pathway controls developmental processes and homeostasis, but abnormal activation of this pathway is a frequent event during the development of cancer. The key mechanism in regulation of the Wnt/beta-catenin pathway is the amino-terminal phosphorylation of beta-catenin, marking it for proteasomal degradation. Here we present small-molecule-based identification of protein kinase C (PKC)-mediated beta-catenin phosphorylation as a novel mechanism regulating the Wnt/beta-catenin pathway. We used a cell-based chemical screen to identify A23187, which inhibits the Wnt/beta-catenin pathway. PKC was activated by A23187 treatment and subsequently phosphorylated N-terminal serine (Ser) residues of beta-catenin, which promoted beta-catenin degradation. Moreover, the depletion of PKCalpha inhibited the phosphorylation and degradation of beta-catenin. Therefore, our findings suggest that the PKC pathway negatively regulates the beta-catenin level outside of the Wnt/beta-catenin pathway.

Hexachlorophene inhibits Wnt/beta-catenin pathway by promoting Siah-mediated beta-catenin degradation.

Aberrant activation of Wnt/beta-catenin signaling and subsequent up-regulation of beta-catenin response transcription (CRT) is a critical event in the development of human colon cancer. Thus, Wnt/beta-catenin signaling is an attractive target for the development of anticancer therapeutics. In this study, we identified hexachlorophene as an inhibitor of Wnt/beta-catenin signaling from cell-based small-molecule screening. Hexachlorophene antagonized CRT that was stimulated by Wnt3a-conditioned medium by promoting the degradation of beta-catenin. This degradation pathway is Siah-1 and adenomatous polyposis colidependent, but glycogen synthase kinase-3beta and F-box beta-transducin repeat-containing protein-independent. In addition, hexachlorophene represses the expression of cyclin D1, which is a known beta-catenin target gene, and inhibits the growth of colon cancer cells. Our findings suggest that hexachlorophene attenuates Wnt/beta-catenin signaling through the Siah-1-mediated beta-catenin degradation.