HT-29 Human Colon Carcinoma Cells Research Articles

CXCL12 Oligomerization Dictates Divergent Signaling and Migratory Responses in Intestinal Epithelial Cells

Chemotactic migration classically exhibits a bell-shaped response over a narrow concentration range. Previous studies have shown the chemokine, CXCL12, exists in a monomer-dimer equilibrium with heightened concentrations resulting in preferential accumulation of homodimeric chemokine. Little is understood how CXCL12 oligomerization induces distinct cellular effects through the same cognent receptor, CXCR4. The objective of these studies is to determine the effects of monomeric and dimeric CXCL12 on colorectal cancer cell migration. HCT116 and HT29 human colonic carcinoma cells were utilized to examine both chemotactic and restitutive migration respectively. Previously engineered preferential monomeric CXCL12(H25R), locked dimer CXCL12(2), and wild-type CXCL12(WT) were utilized in our analyses. Transwell inserts and aspiration wounded monolayers demonstrated that CXCL12(WT) and CXCL12(H25R) induced a strong chemotactic and restitutive migration response. In contrast, CXCL12(2) dimer was unable to elicit cellular migration. In agreement with those data, filamentous actin formation was induced by CXCL12(WT) and CXCL12(H25R) but not dimer. Intracellular calcium flux, a classical hallmark of G-protein coupled receptor signaling, was elicited equally by each oligomeric variant. This response was blocked with addition of CXCR4-specific inhibitors indicating each of the variants engaged the same cognate receptor. Similarly, CXCL12(2) dose-dependently inhibited CXCR4, but not CXCR1, mediated chemotaxis. The lack of migration was subsequently shown using nuclear magnetic resonance analyses to reflect distinct interactions of the monomeric and dimeric ligand for CXCR4 homotypic receptor dimers. These latter data support the notion that double occupancy of dimeric receptor limits migration while single occupancy of the same receptors evokes strong migration responses. Together these data underlie the importance of CXCL12 oligomerization in dictating epithelial migration and suggest the CXCL12 dimer may serve as a potential inhibitor of colorectal cancer metastasis.

Regulation of the Gene Encoding the Intestinal Bile Acid Transporter ASBT by CDX Transcription Factors

Chemotactic migration classically exhibits a bell-shaped response over a narrow concentration range. Previous studies have shown the chemokine, CXCL12, exists in a monomer-dimer equilibrium with heightened concentrations resulting in preferential accumulation of homodimeric chemokine. Little is understood how CXCL12 oligomerization induces distinct cellular effects through the same cognent receptor, CXCR4. The objective of these studies is to determine the effects of monomeric and dimeric CXCL12 on colorectal cancer cell migration. HCT116 and HT29 human colonic carcinoma cells were utilized to examine both chemotactic and restitutive migration respectively. Previously engineered preferential monomeric CXCL12(H25R), locked dimer CXCL12(2), and wild-type CXCL12(WT) were utilized in our analyses. Transwell inserts and aspiration wounded monolayers demonstrated that CXCL12(WT) and CXCL12(H25R) induced a strong chemotactic and restitutive migration response. In contrast, CXCL12(2) dimer was unable to elicit cellular migration. In agreement with those data, filamentous actin formation was induced by CXCL12(WT) and CXCL12(H25R) but not dimer. Intracellular calcium flux, a classical hallmark of G-protein coupled receptor signaling, was elicited equally by each oligomeric variant. This response was blocked with addition of CXCR4-specific inhibitors indicating each of the variants engaged the same cognate receptor. Similarly, CXCL12(2) dose-dependently inhibited CXCR4, but not CXCR1, mediated chemotaxis. The lack of migration was subsequently shown using nuclear magnetic resonance analyses to reflect distinct interactions of the monomeric and dimeric ligand for CXCR4 homotypic receptor dimers. These latter data support the notion that double occupancy of dimeric receptor limits migration while single occupancy of the same receptors evokes strong migration responses. Together these data underlie the importance of CXCL12 oligomerization in dictating epithelial migration and suggest the CXCL12 dimer may serve as a potential inhibitor of colorectal cancer metastasis.

English

Spica Prunellae (Prunella vulgaris fruiting spikes) has long been used as an important component in formulated prescriptions of Chinese traditional medicine to treat various kinds of cancer. However, the precise mechanism of the anti-cancer activity of Spica Prunellae remains to be elucidated. In this report, we investigated the cellular effects of the ethanol extract of Spica Prunellae (EESP) in the HT-29 human colon carcinoma cell line. We found that EESP inhibited the growth of HT-29 cells as evidenced by EESP-induced cell morphological changes and reduced cell viability in dose- and time-dependent manners. Furthermore, we demonstrated that the HT-29 cell growth inhibitory activity of EESP was due to apoptosis, as EESP treatment resulted in the loss of plasma membrane asymmetry (externalization of phosphatidylserine), collapse of mitochondrial membrane potential, activation of caspase-9 and caspase-3, and increase in the ratio of pro-apoptotic Bax to anti-apoptotic Bcl-2. Taken together, these results suggest that Spica Prunellae inhibits the growth of HT-29 colon cancer cells through mitochondrion-mediated apoptosis, which may, in part, explain its anti-cancer activity.   Key words: Apoptosis, anti-tumor, HT-29 cells, phytotherapy, mitochondria, Spica Prunellae.

Microarray analysis of differentially regulated genes in human neuronal and epithelial cell lines upon exposure to type A botulinum neurotoxin

Among the seven serotypes (A–G), type A botulinum neurotoxin (BoNT/A) is the most prevalent etiologic agent and the most potent serotype to cause foodborne botulism, characterized by flaccid muscle paralysis. Upon ingestion, BoNT/A crosses epithelial cell barriers to reach lymphatic and circulatory systems and blocks acetylcholine release at the pre-synaptic cholinergic nerve terminals of neuromuscular junctions (NMJs) resulting in paralysis. One of the unique features of BoNT/A intoxication is its neuroparalytic longevity due to its persistent catalytic activity. The persistent presence of the toxin inside the cell can induce host cell responses. To understand the pathophysiology and host response at the cellular level, gene expression changes upon exposure of human HT-29 colon carcinoma (epithelial) and SH-SY5Y neuroblastoma cell lines to BoNT/A complex were investigated using microarray analysis. In HT-29 cells, 167 genes were up-regulated while 60 genes were down-regulated, whereas in SH-SY5Y cells about 223 genes were up-regulated and 18 genes were down-regulated. Modulation of genes and pathways involved in neuroinflammatory, ubiquitin–proteasome degradation, phosphatidylinositol, calcium signaling in SH-SY5Y cells, and genes relevant to focal adhesion, cell adhesion molecules, adherens and gap junction related pathways in HT-29 cells suggest a massive host response to BoNT/A. A clear differential response in epithelial and neuronal cells indicates that the genes affected may play a distinct role in BoNTs cellular mode of action, involving these two types of host cells.

Anthocyanin‐rich extracts suppress the DNA‐damaging effects of topoisomerase poisons in human colon cancer cells

The effect of two anthocyanin-rich berry extracts (A, bilberry; B, red grape) on topoisomerases was investigated in a cell-free system and in human HT29 colon carcinoma cells. In parallel, their impact on DNA integrity was determined. The berry extracts suppressed the activity of topoisomerase I at concentrations ≥50 μg/mL. The activity of the topoisomerase II isoform was preferentially diminished (≥1 μg/mL). Within HT29 cells, the extracts were found to act as catalytic inhibitors without stabilizing the cleavable complex. Although topoisomerase activity was inhibited, none of the extracts induced DNA strand breaks up to 50 μg/mL. Moreover, pre- and coincubation of HT29 cells with A (≥1 μg/mL) significantly suppressed (p-value ≤0.001) the strand-breaking effects of camptothecin, whereas B was found to be less effective (1 μg/mL; p-value ≤0.05). Both extracts were found to significantly diminish doxorubicin-mediated DNA strand breaks at concentrations ≥1 μg/mL (p-value ≤0.001). Consistent with these results, the extracts suppressed doxorubicin-mediated enhancement of levels of topoisomerase II covalently linked to DNA in HT29 cells. These results raise the possibility that high intake of berry extracts may protect DNA and thus counteract the therapeutic effectiveness of orally applied topoisomerase poisons during chemotherapy.

Grape seed extract upregulates p21 (Cip1) through redox‐mediated activation of ERK1/2 and posttranscriptional regulation leading to cell cycle arrest in colon carcinoma HT29 cells

Abnormalities in cell cycle progression provide unlimited replicative potential to cancer cells, and therefore targeting of key cell cycle regulators could be a sound cancer chemopreventive strategy. Earlier, we found that grape seed extract (GSE) increases Cip/p21 protein level and inhibits growth and induces apoptosis in human colon carcinoma HT29 cells both in vitro and in vivo. However, the mechanism of GSE-induced p21 upregulation and its role in biological efficacy of GSE are not known, which were investigated here. GSE treatment of HT29 cells resulted in a strong dose- and time-dependent phosphorylation of extracellular signal regulated kinase 1/2 (ERK1/2), consistent with p21 induction. The inhibition of sustained ERK1/2 activation by GSE using pharmacological inhibitors abrogated GSE-induced p21 upregulation. Furthermore, pretreatment of cells with N-acetylcysteine inhibited GSE-induced ERK1/2 phosphorylation as well as p21 upregulation, suggesting the involvement of GSE-induced oxidative stress as an upstream event. Consistent with this, GSE also decreased intracellular level of reduced glutathione. Next, we determined whether GSE-induced signaling regulates p21 expression at transcriptional and/or translational levels. GSE was found to increase the stability of p21 message with resultant increase in p21 protein level, but it did not alter the protein stability to a great extent. Importantly, knock-down of p21 abrogated GSE-induced G(1) arrest suggesting that p21 induction by GSE is essential for its G(1) arrest effect. Together, our results for the first time identify a central role of p21 induction and associated mechanism in GSE-induced cell cycle arrest in HT29 cells.

The synthetic triterpenoid CDDO-Imidazolide suppresses experimental liver metastasis

Survival following diagnosis of liver metastasis remains poor and improved treatment strategies to combat liver metastases are needed. Synthetic triterpenoids, including 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Imidazolide or CDDO-Im), have been shown to inhibit primary tumor growth and lung metastasis in experimental models. Oral administration of CDDO-Im results in relatively high liver concentrations, suggesting that CDDO-Im may provide an approach to treatment of liver metastases. Here we assessed the effect of CDDO-Im on liver metastasis, using B16F1 (mouse melanoma) and HT-29 (human colon carcinoma) cells. In vitro, nanomolar concentrations of CDDO-Im arrested proliferation or induced cell death in both cell lines. In vivo, cells were injected via a surgically exposed mesenteric vein to target cells to the liver of mice. Mice were then treated with CDDO-Im (800 mg/kg diet) or vehicle control. Livers were removed at endpoint and metastatic burden was quantified by standard histology. In addition, a novel whole liver magnetic resonance imaging (MRI) technique was used to assess the effect of CDDO-Im on growing metastases as well as on non-dividing, solitary cancer cells present in the same livers. CDDO-Im treatment significantly decreased liver metastasis burden in both HT-29 (n = 8 treated, 10 control) and B16F1 (n = 15 treated, 16 control) injected mice (>60%, P < 0.05), but did not reduce the numbers of solitary B16F1 cancer cells (hypo-intensity) in the same livers (P = 0.9). This study demonstrates that CDDO-Im may be useful for the treatment metastatic liver disease as it successfully inhibits growth of actively proliferating liver metastases.

Peptides from Lactobacillus Hydrolysates of Bovine Milk Caseins Inhibit Prolyl-peptidases of Human Colon Cells

Prolyl-rich peptides derived from hydrolysates of bovine caseins have been previously shown to inhibit angiotensin converting enzyme (ACE) activity, suggesting that they may also be able to inhibit the enzymatic activities of prolyl-specific peptidases. This study shows that peptides derived from α(S1)-casein and β-casein inhibited the enzymatic activities of purified recombinant matrix metalloprotease (MMP)-2, MMP-7, and MMP-9. The inhibitory efficacy was sequence-dependent. These peptides also selectively inhibited the enzymatic activities of prolyl-amino-peptidases, prolyl-amino-dipeptidases, and prolyl-endopeptidases in extracts of HT-29 and SW480 human colon carcinoma cells, but not in intact cells. They were not cytotoxic or growth inhibitory for these cells. Thus, the prolyl-rich selected peptides were good and selective inhibitors of MMPs and post-proline-cleaving proteases, demonstrating their potential to control inadequate proteolytic activity in the human digestive tract, without inducing cytotoxic effects.

Effect of down-regulation of integrin-β1 expression on cell invasion and chemotherapy-resistance of human colon carcinoma HT-29 cell

Objective To investigate the effects of down-regulation of integrin-β1 expression on cell invasion and chemotherapy-resistance of human colon carcinoma cell line HT-29. Methods The down-regulation of integrin-β1 mRNA expression by antisense oligonucleotide technologies was determined with reverse transcription-polymerase chain reaction (RT-PCR). The invasive ability in vitro was evaluated by Transwell invasion chamber assay. Growth inhibition rates and apoptosis rates of HT-29 cells under different dosages of chemotherapeutic agents [5-fluorouracil (5-Fu)] were measured by methyl thiazol tetrazolium (MTT) colorimetric assay and flow cytometry (FCM). Results In contrast with control group,the expression of integrin-β1 mRNA of HT-29 cells were markedly down-regulated,and the number (59 ±12) of migrated HT-29 cells was significantly decreased in the experimental group (P ＜ 0. 05 ). Higher dosages ( 100 mg/L and 200 mg/L of 5-Fu) caused a greater increase of inhibition in experimental group than in control ( P ＜ 0. 05 ). Conclusion The results demonstrate that down-regulation of integrin-β1 expression can effectively decrease cell invasion and chemotherapy-resistance of human colon carcinoma cell line HT-29. Key words: Cell invasion; Chemotherapy-resistance; Colon carcinoma; Integrin-β

Growth Inhibition Effect of β-Catenin Small Interfering RNA–Mediated Gene Silencing on Human Colon Carcinoma HT-29 Cells

The β-catenin gene is a critical component of Wnt signaling pathway. Aberrant activation of Wnt/β-catenin signaling and subsequent upregulation of β-catenin is related to enhancing cell proliferation and developing colon polyps and colon cancer. In the present study, the effect of β-catenin knockdown on the growth and survival of the human colon cancer cell line HT-29 was investigated in vitro. The effect of knockdown of β-catenin on cell proliferation was investigated by MTT assay and colony formation. The cell cycle distribution was investigated by flow cytometry. Apoptosis was measured by nuclear staining and flow cytometry. The change of β-catenin and related proteins were determined by western blotting and immunofluorescence. The results showed that small interfering RNA directed against β-catenin markedly inhibited the expression and nuclear translocation of β-catenin and decreased the expression of known target genes such as cyclin D1 and c-myc; HT-29 cell proliferation was inhibited as indicated by growth reduction, cell cycle arrest in G0/G1 phase, and induction of apoptosis; and the inhibition of cell growth may be associated with switching off cyclin D1 and c-myc expression by small interfering RNA targeted against β-catenin in colon cancer HT-29 cells.

Hedyotis Diffusa Willd extract induces apoptosis via activation of the mitochondrion-dependent pathway in human colon carcinoma cells

Hedyotis Diffusa Willd has been used as a major component in several Chinese medicine formulations for the clinical treatment of colorectal cancer. However, the molecular mechanism of the anti-cancer activity of Hedyotis Diffusa Willd remains unclear. In the present study, we investigated the cellular effects of the ethanol extract of Hedyotis Diffusa Willd (EEHDW) in the HT-29 human colon carcinoma cell line. We found that EEHDW inhibited the growth of HT-29 cells demonstrating EEHDW-induced cell morphological changes and reduced cell viability in a dose- and time-dependent manner. Furthermore, we observed that EEHDW treatment resulted in DNA fragmentation, loss of plasma membrane asymmetry, collapse of mitochondrial membrane potential, activation of caspase-9 and caspase-3, and increase of the ratio of pro-apoptotic Bax to anti-apoptotic Bcl-2, suggesting that the HT-29 cell growth inhibitory activity of EEHDW was due to mitochondrion-mediated apoptosis, which may partly explain the anti-cancer activity of Hedyotis Diffusa Willd.

Regulation of p21, MMP-1, and MDR-1 Expression in Human Colon Carcinoma HT29 Cells by Tian Xian Liquid, a Chinese Medicinal Formula, In Vitro and In Vivo

Tian-Xian liquid (TXL), a commercially available Chinese medicine decoction, has been used as an anticancer dietary agent for more than 10 years without reported side effects. The safety and quality consistency of TXL and its mechanisms of action on antiproliferation, antimetastasis, and reversion of multidrug resistance (MDR) regimens were explored. In this study, an atomic absorption spectrophotometer and reversed phase high performance liquid chromatography with photodiode array detection (HPLC-DAD) were used to evaluate the main toxic elements and the quality consistency among different batches of TXL extracts, respectively. HT29 human colon cancer cell line and tumor-bearing nude mice were used. TXL was provided by China-Japan Feida Union Company Limited. The effect of TXL on in vitro proliferation of HT29 human colon cancer cell line was examined. The percentages of treated cells distributed in different phases of the cell cycles were analyzed by flow cytometry. Antiproliferative effect after treatment with TXL was assessed by determination of the protein levels of p21, cyclinD1, PCNA, and cdk-2, which are the key regulators for cell cycle progression. Meanwhile, the protein levels of MMP-1 and MDR-1 (multidrug resistance protein-1) were also determined to assess the effect of TXL on antimetastasis and reversion of MDR regimen, respectively. The contents of main toxic elements were lower in TXL extract compared with the standard set by the Department of Health of the Government of Hong Kong Special Administrative Region (SAR). Our HPLC results showed that the relative standard deviations of the amount of the 5 standards were less than 5% in different batches of TXL. Immunoblotting analysis revealed a dramatic induction of cyclin kinase inhibitor p21 as well as an inhibition of cyclinD1, PCNA, and cdk-2 in the TXL-treated in vitro models, thereby, impeding cell progression from G1/S phase. Results obtained from the in vivo study also demonstrated that TXL upregulated the protein level of p21 and downregulated the protein levels of MMP-1 and MDR-1. Results obtained from the present investigation not only demonstrate the safety and quality of TXL extract but also demonstrate that TXL possesses antiproliferative and antimetastatic activities and brings about reversion of MDR on HT29 cell and on xenografted tissue in tumor-implanted nude mice.

Cytotoxicity of new pyrazino[1,2-b]isoquinoline and 6,15-iminoisoquino[3,2-b]3-benzazocine compounds

Looking for optimised analogues of compound 2 that might be useful in colon cancer therapy, we here explore the in vitro cytotoxicity against MDA-MB 231 human breast carcinoma, A-549 human lung carcinoma and HT-29 human colon carcinoma cell lines of several analogues and derivatives. The effect of the R2-substituent and/or the introduction of an arylmethyl side-chain at C-3, as well as the presence of a double bond in the skeleton or a methoxy group at C-1 have been investigated. New 6,15-iminoisoquino[3,2-b]3-benzazocine compounds, related to the saframycin family, in which the C(7)–N(8)–C(9)-substructure contains a lactam function, a fused oxazolidine or an aminonitrile function were also studied, and many of them showed low micromolar GI50 values.

The ethyl acetate extract of Phellinus linteus grown on germinated brown rice induces G0/G1 cell cycle arrest and apoptosis in human colon carcinoma HT29 cells

It is well known that Phellinus linteus has a variety of biological functions, such as antitumor and immunomodulating activities. In our previous studies, we developed a P. linteus grown on germinated brown rice (PBR) and found that organic solvent extracts of PBR possessed immunomodulating activity to regulate a balance of cytokine network in mice. The components of PBR are ergosterol peroxide, gamma-aminobutyric acid (GABA) and Beta-glucan. In this study, we demonstrate that an organic solvent extract of P. linteus grown on PBR induced apoptotic cell death through the induction of G(0)/G(1) arrest of cell cycle and the apoptosis via DNA fragmentation in human colon carcinoma HT-29 cells. Cell death induced by the extract of P. linteus grown on PBR was shown to be associated with the upregulation of p21(CIP1/WAF1), the downregulation of cyclin D1, anti-apoptotic protein, Bcl-2, the release of cytochrome c, and the activation of caspase-9, caspase-3 and caspase-8. This study suggests that the ethyl acetate extract of P. linteus grown on PBR induces apoptosis accompanied by cell cycle arrest at G(0)/G(1) phase and regulates apoptosis-regulatory proteins, which may be applicable to anticancer therapy.

Abstract 5370: Potentiation of the novel topoisomerase I inhibitor indenoisoquinoline NSC743400 by Chk1/2 inhibitors in p53-deficient cells

Abstract Clincally available camptothecin derivatives, though highly effective against a broad range tumors, are limited by their chemical instability, solubility, ABCG2-mediated efflux and reversibility of Top1-DNA complexes. Indenoisoquinolines were identified as topoisomerase I (Top1) inhibitors by the COMPARE analysis of the NCI drug screen database. Although structurally similar to camptothecin, idenoisoquinolines are chemically stable, able to overcome ABCG2 efflux while forming more persistent Top1-DNA complexes and maintaining potent antitumor activity. The potential for indenoisoquinoline derivatives as possible clinical agents is further aided by the synergistic effect observed between camptothecin and Chk1/2 inhibitors. Chk1/2 kinases regulate cell cycle, DNA damage response via ATM, ATR and DNA-PK, which are activated by genomic instability. Previous studies have shown Chk1/2 inhibitors, such as UCN-01, can potentiate camptothecin-mediated cytotoxicity in p53 mutant cells by eliminating the DNA replication checkpoint. Further synthesis has led to the water-soluble indenoisoquinoline derivative, NSC743400 for clinical development. Current checkpoint kinase inhibitors, like PV1019 or AZD7762, offer a higher level of specificity than their predecessors that can be exploited by combination with indenoisoquinolines. Checkpoint inhibition showed synergistic antiproliferative activity with NSC743400 in human colon carcinoma HT-29 cells. Furthermore, AZD7762 was able to abrogate the late S-phase arrest induced by NSC743400 as early as 3h post treatment. Inhibition of checkpoint kinases also abrogated the DNA replication fork progression response to NSC743400. The effect of these inhibitors on the autophosphorylation of both Chk1 and Chk2 post NSC743400 was examined by Western blot. These results further substantiate the idea that checkpoint inhibition is a viable strategy in enhancing the antiproliferative effect of novel non-camptothecinn inhibitors such as the indenoisoquinolines. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5370.

Kimchi lactic acid bacteria and health benefits

Kimchi contains a high number of lactic acid bacteria (108~9/ml), organic acids (lactic, acetic, succinic, propionic acids, etc.), vitamin B complex, and modified functional phytochemicals formed during the fermentation process. Vitamin C, carotenoids, chlorophylls, tocopherol, isothiocyanates, indole‐3‐carbinol, flavonoids, β‐sitosterol, dietary fiber, capsaicinoids, unsaturated fatty acids, allyl sulfur compounds, gingerel, organic acid, lactic acid bacteria, se, etc are the nutraceuticals found in kimchi. The representative kimchi lactic acid bacteria (LAB) reported so far are Leu. mesenteroides, Leu. citreum, Leu. gasicomitatum, Leu. kimchii, Leu. inhae, Weissella Koreensis, Weissella cibaria, Lac. plantarum, Lac. sakei, Lac. delbrueckii, Lac. buchneri, Lac. brevis, Lac. fermentum, Ped. acidilactici and Ped. pentosaceus. LAB from kimchi revealed high preventive capability of the mutation induced by 4‐NQO, MeIQ and Trp‐p2 as much as (or higher than) Lac. acidophilus from dairy products, regardless of their viability. The cell wall fraction, rather than in the cytosol fraction was responsible for the antimutagenic activity of the lactic acid bacteria. Kimchi LAB exhibited antitumor, antimutagenic effect, increase immune function, decrease avian influenza virus, decrease atopy, antiallergy, production of GABA, etc. During the administration of kimchi in humans, the cell counts of Lactobacillus and Leuconostoc increased (p&lt;0.05). The enzymes of β‐glucosidase, β‐glucuronidase and nitroreductase in the colon decreased during the kimchi intake (p&lt;0.05). Kimchi LAB fermentation increased anticancer effect in HT‐29 human colon carcinoma cells. Optimally ripened (OpR) kimchi induced apoptosis as determined by DAPI staining and increased Bax, and decreased in the expression of Bcl‐2.

Human β-defensin-2 increases cholinergic response in colon epithelium

The human beta-defensin-2 (hBD-2) is expressed in epithelial cells of skin and respiratory and gastrointestinal tracts. Defensins are arginine-rich small cationic peptides with six intramolecular disulfide bonds and are antimicrobially active against a broad spectrum of pathogens. In addition, they have cytokine-like immunomodulatory properties. We hypothesized that hBD-2 also might influence epithelial cells themselves, thereby altering fluid composition in the gastrointestinal tract. We therefore tested its impact on electrogenic ion transport properties of distal colon in Ussing chamber experiments. Application of hBD-2 did not affect transepithelial voltage or resistance in cAMP-stimulated distal colon. However, it increased cholinergic Ca(2+)-dependent Cl(-) secretion. After 20 min of incubation with hBD-2, the effect of carbachol (CCh) on the equivalent short circuit current (I'(sc)) was enhanced twofold compared to vehicle-treated colon. Modulation of Ca(2+) signaling by hBD-2 was validated by Fura-2 measurements in human colon carcinoma HT29 cells. Twenty-minute incubation with hBD-2 increased the CCh-induced Ca(2+) transient by 20-30% compared to either vehicle-treated cells or cells treated with the defensins hBD-1, hBD-3, or HD-5. This effect was concentration-dependent, with an EC(50) of 0.043 microg/ml, and still present in the absence of extracellular Ca(2+). Also, the ionomycin-induced Ca(2+) transient was increased by hBD-2 treatment. We conclude that hBD-2 facilitates cholinergic Ca(2+)-regulated epithelial Cl(-) secretion. These findings contribute to the concept of a specific interaction of antimicrobial peptides with epithelial function.

Differential effects of resveratrol and novel resveratrol derivative, HS-1793, on endoplasmic reticulum stress-mediated apoptosis and Akt inactivation

Since resveratrol (3,4',5 tri-hydroxystilbene), which has been shown to inhibit multistage carcinogenesis, is not a potent cytotoxic compound, several studies were undertaken to obtain synthetic analogues of resveratrol with potent activity. We previously reported that a resveratrol derivative HS-1793 exhibits stronger antitumor effects than resveratrol in several cancer cell types. The present study was undertaken to reveal precise mechanism by which HS-1793 induces cell death. The induction of CCAAT/enhancer-binding protein-homologous protein (CHOP) and glucose-regulated protein (GRP)-78, and ER stress-specific XBP1 splicing were found in HT29 human colon carcinoma cells treated with resveratrol. Conversely, these manifestations were not observed in HT29 cells treated with HS-1793. Inhibition of caspase-4 activity by z-LEVD-fmk significantly reduced the induction of apoptosis by resveratrol but not by HS-1793. These findings suggest that HS-1793, contrary to resveratrol, does not induce ER stress-mediated apoptosis. Importantly, we observed that HS-1793 but not resveratrol decreased phosphorylated Akt level. We also demonstrated that HS-1793, compared to resveratrol, exerted more effective apoptosis inducing activity in Akt-activated cells. Taken together, the stronger antitumor activity of HS-1793 originates, at least in part, from its ability for Akt inactivation.

Antiproliferative Effect of Chitosan-Added Kimchi in HT-29 Human Colon Carcinoma Cells

The anticancer effects of chitosan-added kimchi were investigated by using an in vitro cellular system with HT-29 human colon carcinoma cells. Two different kinds of chitosan-soluble chitosan with a 90% degree of deacetylation and 3 cps viscosity and nonsoluble chitosan with a 95% degree of deacetylation and 22 cps viscosity-were used as sub-ingredients to increase anticancer effects of kimchi. The soluble chitosan-added kimchi (SK) and nonsoluble chitosan-added kimchi (NK) were stronger growth inhibitors in HT-29 cells than the control kimchi (CK) according to the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and the growth inhibition test. Treatment with SK and NK induced apoptosis, as determined by 4,6-diamidino-2-phenylindole staining, and resulted in the up-regulation of Bax expression and down-regulation of Bcl-2, cIAP-1, cellular inhibitor of apoptosis-2, cyclooxygenase-2, inhibitory nitric oxide synthase, and nuclear factor kappaB (NF-kappaB) expressions when compared to CK. The antiproliferative and anti-apoptotic effects appeared to be more pronounced in the cells treated with NK. The antiproliferative effects of the chitosan-added kimchi appeared to be associated with the induction of apoptosis through NF-kappaB or an NF-kappaB-dependent pathway. These results suggest that chitosan has potential to be a valuable active ingredient in functional kimchi products with anticancer effects.

Abstract B47: External Qi of Yan Xin Qigong induces cell cycle arrest and apoptosis in colon cancer cells through regulation of multiple signaling pathways

Abstract The concept external Qi of Qigong refers to the technology and ability of “Qi deployment” therapy and health preservation of traditional Chinese medicine (TCM). External Qi therapy of TCM has long been one of the medical practices in China. Long-term clinical observations and ongoing studies have shown positive effects of external Qi of Yan Xin Qigong (YXQ-EQ) on patients of various types of cancer. The molecular and cellular mechanisms underlying YXQ-EQ's antitumor effects have recently begun to be addressed. In the present study we investigated YXQ-EQ's effects on the growth and apoptosis of human colon carcinoma HT-29 cells. We show that YXQ-EQ treatment reduced the viability and inhibited clonogenic growth of HT-29 cells. In agreement, YXQ-EQ induced cell cycle G1 arrest and apoptosis in HT-29 cells. YXQ-EQ induced apoptosis is accompanied by DNA fragmentation and cleavage of procaspase-3 and -9 and poly(ribose-ADP) polymerase. YXQ-EQ inhibited phosphorylation of Akt and GSK3β, decreased the levels of cyclin D1 and hyper-phosphorylated Rb, and repressed the expression of antiapoptic proteins Bcl-XL, XIAP, and survivin. Concomitantly, YXQ-EQ increased the expression of cyclin-dependent kinase inhibitor p21CIP1/WAF1. These findings suggest that YXQ-EQ may induce cell cycle arrest and apoptosis in colon cancer cells through regulation of multiple signaling pathways that play important roles in cell proliferation, survival and apoptosis. Citation Information: Cancer Prev Res 2010;3(1 Suppl):B47.