Isoquercitrin Suppresses Colon Cancer Cell Growth in Vitro by Targeting the Wnt/β-Catenin Signaling Pathway

Flavonoids are plant-derived polyphenolic molecules that have potential biological effects including anti-oxidative, anti-inflammatory, anti-viral, and anti-tumoral effects. These effects are related to the ability of flavonoids to modulate signaling pathways, such as the canonical Wnt signaling pathway. This pathway controls many aspects of embryonic development and tissue maintenance and has been found to be deregulated in a range of human cancers. We performed several in vivo assays in Xenopus embryos, a functional model of canonical Wnt signaling studies, and also used in vitro models, to investigate whether isoquercitrin affects Wnt/β-catenin signaling. Our data provide strong support for an inhibitory effect of isoquercitrin on Wnt/β-catenin, where the flavonoid acts downstream of β-catenin translocation to the nuclei. Isoquercitrin affects Xenopus axis establishment, reverses double axes and the LiCl hyperdorsalization phenotype, and reduces Xnr3 expression. In addition, this flavonoid shows anti-tumoral effects on colon cancer cells (SW480, DLD-1, and HCT116), whereas exerting no significant effect on non-tumor colon cell (IEC-18), suggesting a specific effect in tumor cells in vitro. Taken together, our data indicate that isoquercitrin is an inhibitor of Wnt/β-catenin and should be further investigated as a potential novel anti-tumoral agent.     

Ectopic Expression of eIF-4E in Human Colon Cancer Cells Promotes the Stimulation of Adhesion Molecules by Transforming Growth Factorβ

Transforming growth factor β1 (TGFβ) inhibits cellular proliferation, promotes differentiation, and stimulates the expression and secretion of the extracellular matrix adhesion molecules fibronectin and laminin and the colon-associated intercellular adhesion molecule carcinoembryonic antigen. This is collectively called the TGFβ-mediated adhesion response and occurs in the human colon cancer cell line Moser while the cell line KM12SM is relatively unresponsive to TGFβ. We have previously shown that TGFβ rapidly stimulates protein kinase C (PKC) phosphotransferase activity in the Moser cells and that the induction of the adhesion response (but not antiproliferation) by TGFβ is dependent on PKC. Because resistance to growth factors may be due to translational suppression and the translation initiation factor eIF-4E may alleviate translational suppression, we determined the effect of eIF-4E expression on the responses of Moser and KM12SM cells to TGFβ. Ectopic expression of eIF-4E in the TGFβ-responsive Moser cells enhanced the activation of PKC by TGFβ and the induction of the adhesion response, especially the secretion of adhesion molecules, but not the antiproliferative response. Ectopic expression of eIF-4E in the TGFβ-resistant KM12SM cells increased TGFβ stimulation of PKC and the TGFβ-mediated adhesion response (but not antiproliferation). The secretion of adhesion molecules was significantly increased by TGFβ. These results showed in these cells that eIF-4E promotes TGFβ-regulated adhesion but not antiproliferation in a PKC-dependent manner.     

UDP-Glucuronosyltransferase 1A Determinates Intracellular Accumulation and Anti-Cancer Effect of β-Lapachone in Human Colon Cancer Cells

β-lapachone (β-lap), an NAD(P)H:quinone oxidoreductase 1 (NQO1) targeting antitumor drug candidate in phase II clinical trials, is metabolically eliminated via NQO1 mediated quinone reduction and subsequent UDP-glucuronosyltransferases (UGTs) catalyzed glucuronidation. This study intends to explore the inner link between the cellular glucuronidation and pharmacokinetics of β-lap and its apoptotic effect in human colon cancer cells. HT29 cells S9 fractions exhibited high glucuronidation activity towards β-lap, which can be inhibited by UGT1A9 competitive inhibitor propofol. UGT1A siRNA treated HT29 cells S9 fractions displayed an apparent low glucuronidation activity. Intracellular accumulation of β-lap in HCT116 cells was much higher than that in HT29 cells, correlated with the absence of UGT1A in HCT116 cells. The cytotoxic and apoptotic effect of β-lap in HT29 cells were much lower than that in HCT116 cells; moreover, β-lap triggered activation of SIRT1-FOXO1 apoptotic pathway was observed in HCT116 cells but not in HT29 cells. Pretreatment of HT29 cells with UGT1A siRNA or propofol significantly decreased β-lap’s cytotoxic and apoptotic effects, due to the repression of glucuronidation and the resultant intracellular accumulation. In conclusion, UGT1A is an important determinant, via switching NQO1-triggered redox cycle to metabolic elimination, in the intracellular accumulation of β-lap and thereafter its cytotoxicity in human colon cancer cells. Together with our previous works, we propose that UGTs determined cellular pharmacokinetics is an important determinant in the apoptotic effects of NQO1 targeting substrates serving as chemotherapeutic drugs.     

Curcumin Enhances the Effect of Chemotherapy against Colorectal Cancer Cells by Inhibition of NF-κB and Src Protein Kinase Signaling Pathways

Objective

Development of treatment resistance and adverse toxicity associated with classical chemotherapeutic agents highlights the need for safer and effective therapeutic approaches. Herein, we examined the effectiveness of a combination treatment regimen of 5-fluorouracil (5-FU) and curcumin in colorectal cancer (CRC) cells.

Methods

Wild type HCT116 cells and HCT116+ch3 cells (complemented with chromosome 3) were treated with curcumin and 5-FU in a time- and dose-dependent manner and evaluated by cell proliferation assays, DAPI staining, transmission electron microscopy, cell cycle analysis and immunoblotting for key signaling proteins.

Results

The individual IC₅₀ of curcumin and 5-FU were approximately 20 µM and 5 µM in HCT116 cells and 5 µM and 1 µM in HCT116+ch3 cells, respectively (p<0.05). Pretreatment with curcumin significantly reduced survival in both cells; HCT116+ch3 cells were considerably more sensitive to treatment with curcumin and/or 5-FU than wild-type HCT116 cells. The IC₅₀ values for combination treatment were approximately 5 µM and 1 µM in HCT116 and 5 µM and 0.1 µM in HCT116+ch3, respectively (p<0.05). Curcumin induced apoptosis in both cells by inducing mitochondrial degeneration and cytochrome c release. Cell cycle analysis revealed that the anti-proliferative effect of curcumin and/or 5-FU was preceded by accumulation of CRC cells in the S cell cycle phase and induction of apoptosis. Curcumin potentiated 5-FU-induced expression or cleavage of pro-apoptotic proteins (caspase-8, -9, -3, PARP and Bax), and down-regulated anti-apoptotic (Bcl-xL) and proliferative (cyclin D1) proteins. Although 5-FU activated NF-κB/PI-3K/Src pathway in CRC cells, this was down-regulated by curcumin treatment through inhibition of IκBα kinase activation and IκBα phosphorylation.

Conclusions

Combining curcumin with conventional chemotherapeutic agents such as 5-FU could provide more effective treatment strategies against chemoresistant colon cancer cells. The mechanisms involved may be mediated via NF-κB/PI-3K/Src pathways and NF-κB regulated gene products.     

Honokiol Inhibits Non-Small Cell Lung Cancer Cell Migration by Targeting PGE2-Mediated Activation of β-Catenin Signaling

Lung cancer remains a leading cause of death due to its metastasis to distant organs. We have examined the effect of honokiol, a bioactive constituent from the Magnolia plant, on human non-small cell lung cancer (NSCLC) cell migration and the molecular mechanisms underlying this effect. Using an in vitro cell migration assay, we found that treatment of A549, H1299, H460 and H226 NSCLC cells with honokiol resulted in inhibition of migration of these cells in a dose-dependent manner, which was associated with a reduction in the levels of cyclooxygenase-2 (COX-2) and prostaglandin E₂ (PGE₂). Celecoxib, a COX-2 inhibitor, also inhibited cell migration. Honokiol inhibited PGE₂-enhanced migration of NSCLC cells, inhibited the activation of NF-κB/p65, an upstream regulator of COX-2, in A549 and H1299 cells, and treatment of cells with caffeic acid phenethyl ester, an inhibitor of NF-κB, also inhibited migration of NSCLC cells. PGE₂ has been shown to activate β-catenin signaling, which contributes to cancer cell migration. Therefore, we checked the effect of honokiol on β-catenin signaling. It was observed that treatment of NSCLC cells with honokiol degraded cytosolic β-catenin, reduced nuclear accumulation of β-catenin and down-regulated matrix metalloproteinase (MMP)-2 and MMP-9, which are the down-stream targets of β-catenin and play a crucial role in cancer cell metastasis. Honokiol enhanced: (i) the levels of casein kinase-1α, glycogen synthase kinase-3β, and (ii) phosphorylation of β-catenin on critical residues Ser⁴⁵, Ser^33/37 and Thr⁴¹. These events play important roles in degradation or inactivation of β-catenin. Treatment of celecoxib also reduced nuclear accumulation of β-catenin in NSCLC cells. FH535, an inhibitor of Wnt/β-catenin pathway, inhibited PGE₂-enhanced cell migration of A549 and H1299 cells. These results indicate that honokiol inhibits non-small cell lung cancer cells migration by targeting PGE₂-mediated activation of β-catenin signaling.     

Mechanisms of Epithelial Cell–Cell Adhesion and Cell Compaction Revealed by High-resolution Tracking of E-Cadherin–?Green Fluorescent Protein

Cadherin-mediated adhesion initiates cell reorganization into tissues, but the mechanisms and dynamics of such adhesion are poorly understood. Using time-lapse imaging and photobleach recovery analyses of a fully functional E-cadherin/GFP fusion protein, we define three sequential stages in cell–cell adhesion and provide evidence for mechanisms involving E-cadherin and the actin cytoskeleton in transitions between these stages. In the first stage, membrane contacts between two cells initiate coalescence of a highly mobile, diffuse pool of cell surface E-cadherin into immobile punctate aggregates along contacting membranes. These E-cadherin aggregates are spatially coincident with membrane attachment sites for actin filaments branching off from circumferential actin cables that circumscribe each cell. In the second stage, circumferential actin cables near cell–cell contact sites separate, and the resulting two ends of the cable swing outwards to the perimeter of the contact. Concomitantly, subsets of E-cadherin puncta are also swept to the margins of the contact where they coalesce into large E-cadherin plaques. This reorganization results in the formation of a circumferential actin cable that circumscribes both cells, and is embedded into each E-cadherin plaque at the contact margin. At this stage, the two cells achieve maximum contact, a process referred to as compaction. These changes in E-cadherin and actin distributions are repeated when additional single cells adhere to large groups of cells. The third stage of adhesion occurs as additional cells are added to groups of >3 cells; circumferential actin cables linked to E-cadherin plaques on adjacent cells appear to constrict in a purse-string action, resulting in the further coalescence of individual plaques into the vertices of multicell contacts. The reorganization of E-cadherin and actin results in the condensation of cells into colonies. We propose a model to explain how, through strengthening and compaction, E-cadherin and actin cables coordinate to remodel initial cell–cell contacts to the final condensation of cells into colonies.     

miRNA-29c Suppresses Lung Cancer Cell Adhesion to Extracellular Matrix and Metastasis by Targeting Integrin β1 and Matrix Metalloproteinase2 (MMP2)

Our pilot study using miRNA arrays found that miRNA-29c (miR-29c) is differentially expressed in the paired low-metastatic lung cancer cell line 95C compared to the high-metastatic lung cancer cell line 95D. Bioinformatics analysis shows that integrin β1 and matrix metalloproteinase 2 (MMP2) could be important target genes of miR-29c. Therefore, we hypothesized that miR-29c suppresses lung cancer cell adhesion to extracellular matrix (ECM) and metastasis by targeting integrin β1 and MMP2. The gain-of-function studies that raised miR-29c expression in 95D cells by using its mimics showed reductions in cell proliferation, adhesion to ECM, invasion and migration. In contrasts, loss-of-function studies that reduced miR-29c by using its inhibitor in 95C cells promoted proliferation, adhesion to ECM, invasion and migration. Furthermore, the dual-luciferase reporter assay demonstrated that miR-29c inhibited the expression of the luciferase gene containing the 3′-UTRs of integrin β1 and MMP2 mRNA. Western blotting indicated that miR-29c downregulated the expression of integrin β1 and MMP2 at the protein level. Gelatin zymography analysis further confirmed that miR-29c decreased MMP2 enzyme activity. Nude mice with xenograft models of lung cancer cells confirmed that miR-29c inhibited lung cancer metastasis in vivo, including bone and liver metastasis. Taken together, our results demonstrate that miR-29c serves as a tumor metastasis suppressor, which suppresses lung cancer cell adhesion to ECM and metastasis by directly inhibiting integrin β1 and MMP2 expression and by further reducing MMP2 enzyme activity. The results show that miR-29c may be a novel therapeutic candidate target to slow lung cancer metastasis.     

Targeted Disruption of β-Arrestin 2-Mediated Signaling Pathways by Aptamer Chimeras Leads to Inhibition of Leukemic Cell Growth

β-arrestins, ubiquitous cellular scaffolding proteins that act as signaling mediators of numerous critical cellular pathways, are attractive therapeutic targets because they promote tumorigenesis in several tumor models. However, targeting scaffolding proteins with traditional small molecule drugs has been challenging. Inhibition of β-arrestin 2 with a novel aptamer impedes multiple oncogenic signaling pathways simultaneously. Additionally, delivery of the β-arrestin 2-targeting aptamer into leukemia cells through coupling to a recently described cancer cell-specific delivery aptamer, inhibits multiple β-arrestin-mediated signaling pathways known to be required for chronic myelogenous leukemia (CML) disease progression, and impairs tumorigenic growth in CML patient samples. The ability to target scaffolding proteins such as β-arrestin 2 with RNA aptamers may prove beneficial as a therapeutic strategy.

Highlights

• An RNA aptamer inhibits β-arrestin 2 activity.
• Inhibiting β-arrestin 2 impedes multiple tumorigenic pathways simultaneously.
• The therapeutic aptamer is delivered to cancer cells using a cell-specific DNA aptamer.
• Targeting β-arrestin 2 inhibits tumor progression in CML models and patient samples.

     

Enhancement of Metastatic and Invasive Capacity of Gastric Cancer Cells by Transforming Growth Factor-β1

Transforming growth factor-β (TGF-β),a multifunctional cytokine,exerts contradictory rolesin different kinds of cells.A number of studies have revealed its involvement in the progression of many typesof tumors.To investigate the effect of TGF-β on gastric carcinoma,SGC7901,BGC823 and MKN28 (aTGF-β-resistant cell line) adenocarcinoma clones were used.After pretreatment in serum-free medium withor without 10 ng/ml TGF-β1,their experimental metastatic potential,chemotaxis,and invasive and adhesiveability were measured.Furthermore,zymography for gelatinase was processed.Liver colonies were alsomeasured 4 weeks after inoculation of SGC7901,BGC823 and MKN28 in Balb/c nude mice,and an increasein the number of surface liver metastases was seen in SGC7901 (from 11.0±3.0 to 53.3±3.3) and BGC823(from 9.3±2.5 to 60.0±2.8) groups,whereas there was no difference between MKN28 groups (from 35.2±3.8 to 38.5±2.7).In vitro experiments showed that TGF-β1 increased the adhesion capacity of SGC7901and BGC823 cells to immobilized reconstituted basement membrane/fibronectin matrices and promoted theirpenetration through reconstituted basement membrane barriers.Zymography demonstrated that enhancedinvasive potential was partly due to the increased type Ⅳ collagenolytic (gelatinolytic) activity,but there wasno difference in type Ⅳ collagenolytic activity and other biological behaviors between MKN28 groups.Theseresults suggested that TGF-β1 might modulate the metastatic potential of gastric cancer cells by promotingtheir ability to break down and penetrate basement membrane barriers and their adhesive and motile activities.We speculated that TGF-β1 might act as a progression-enhancing factor in gastric cancer.Therefore blockageof TGF-β or TGF-β signaling might prevent gastric cancer cells from invading and metastasizing.     

Design and Synthesis of a Novel Antimicrobial Peptide Targeting β-catenin in Human Breast Cancer Cell lines

International Journal of Peptide Research and Therapeutics - Antimicrobial peptides which play a vital role in an innate immune defense mechanism of various organisms can be regarded as novel...     

PTB-Associated Splicing Factor (PSF) Is a PPARγ-Binding Protein and Growth Regulator of Colon Cancer Cells

Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear receptor that plays an essential role in cell proliferation, apoptosis, and inflammation. It is over-expressed in many types of cancer, including colon, stomach, breast, and lung cancer, suggesting that regulation of PPARγ might affect cancer pathogenesis. Here, using a proteomic approach, we identify PTB-associated splicing factor (PSF) as a novel PPARγ-interacting protein and demonstrate that PSF is involved in several important regulatory steps of colon cancer cell proliferation. To investigate the relationship between PSF and PPARγ in colon cancer, we evaluated the effects of PSF expression in DLD-1 and HT-29 colon cancer cell lines, which express low and high levels of PPARγ, respectively PSF affected the ability of PPARγ to bind, and expression of PSF siRNA significantly suppressed the proliferation of colon cancer cells. Furthermore, PSF knockdown induced apoptosis via activation of caspase-3. Interestingly, DLD-1 cells were more susceptible to PSF knockdown-induced cell death than HT-29 cells. Our data suggest that PSF is an important regulator of cell death that plays critical roles in the survival and growth of colon cancer cells. The PSF-PPARγ axis may play a role in the control of colorectal carcinogenesis. Taken together, this study is the first to describe the effects of PSF on cell proliferation, tumor growth, and cell signaling associated with PPARγ.     

Tyrosine Phosphorylation of β-Catenin and Plakoglobin Enhanced by Hepatocyte Growth Factor and Epidermal Growth Factor in Human Carcinoma Cells

The effect of hepatocyte growth factor /scatter factor (HGF/SF) and epidermal growth factor (EGF) on cadherin-mediated adhesion of human carcinoma cells was studied. HGF/SF induced scattering of colonic adenocarcinoma HT29 and gastric adenocarcinomas MKN7 and MKN74 cells. Likewise, EGF induced scattering of HT29 and MKN7 cells. These cells expressed E-cadherin, which was concentrated at cell-cell contact sites. When the scattering of these cells was induced by HGF/SF or EGF, the E-cadherin concentration at cell-cell boundaries tended to decrease. Irnmunoblotting analyses, however, demonstrated that these growth factor treatments did not alter the expression of E-cadherin and E-cadherin-associated proteins, α- and β-catenin and plakoglobin. β-Catenin, plakoglobin and an unidentified 115-kDa molecule associated with E-cadherin were found to be phosphorylated at tyrosine residues, and these phosphorylations were enhanced by the growth factor treatments. These results suggest that HGF/SF and EGF may modulate the function of the cadherin-catenin system via tyrosine phosphorylation of cadherin-associated proteins.     

Responses of Chrysanthemum Cells to Mechanical Stimulation Require Intact Microtubules and Plasma Membrane–Cell Wall Adhesion

Plant cells are highly susceptible and receptive to physical factors, both in nature and under experimental conditions. Exposure to mechanical forces dramatically results in morphological and microstructural alterations in their growth. In the present study, cells from chrysanthemum (Dendranthema morifolium) were subjected to constant pressure from an agarose matrix, which surrounded and immobilized the cells to form a cell-gel block. Cells in the mechanically loaded blocks elongated and divided, with an axis preferentially perpendicular to the direction of principal stress vectors. After a sucrose-induced plasmolysis, application of peptides containing an RGD motif, which interferes with plasma membrane-cell wall adhesion, reduced the oriented growth under stress conditions. Moreover, colchicines, but not cytochalasin B, abolished the effects of mechanical stress on cell morphology. Cellulose staining revealed that mechanical force reinforces the architecture of cell walls and application of mechanical force, and RGD peptides caused aggregative staining on the surface of plasmolyzed protoplasts. These results provide evidence that the oriented cell growth in response to compressive stress requires the maintenance of plasmalemma-cell wall adhesion and intact microtubules. Stress-triggered wall development in individual plant cells was also demonstrated.     

Selective Induction of Necrotic Cell Death in Cancer Cells by β-Lapachone through Activation of DNA Damage Response Pathway

Most efforts thus far have been devoted to develop apoptosis inducers for cancer treatment. However, apoptotic pathway deficiencies are a hallmark of cancer cells. We propose that one way to bypass defective apoptotic pathways in cancer cells is to induce necrotic cell death. Here we show that selective induction of necrotic cell death can be achieved by activation of the DNA damage response pathways. While β-lapachone induces apoptosis through E2F1 checkpoint pathways, necrotic cell death can be selectively induced by β-lapachone in a variety of cancer cells. We found that β-lapachone, unlike DNA damaging chemotherapeutic agents, transiently activates PARP1, a main regulator of the DNA damage response pathway, both in vitro and in vivo. This occurs within minutes of exposure to β-lapachone, resulting in selective necrotic cell death. Inhibition of PAR blocked β-lapachone-induced necrosis. Furthermore, necrotic cell death induced by β-lapachone was significantly reduced in PARP1 knockout cell lines. Our data suggest that selective necrotic cell death can be induced through activation of DNA damage response pathways, supporting the idea of selective necrotic cell death as a therapeutic strategy