Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “HT29 Cells”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 415 records · Page 23Linked to original sources
PubMed1999

Schedule-dependent cytotoxicity of SN-38 in p53 wild-type and mutant colon adenocarcinoma cell lines.

In this study the effects of SN-38 on colon adenocarcinoma cell lines expressing wild-type p53 (LS174T) or mutant non-functional p53 (HT29) have been investigated. On exposure to SN-38, HT29 cells rapidly progressed through G1 and S and arrested in G2/M. Release and concomitant increase in apoptosis after 48 h was concentration- and time-dependent (P < 0.001), being more rapid at higher concentrations, but reaching plateau at 10 ng ml(-1) with prolonged exposure. LS174T cells showed only a small increase in apoptosis, and only at high concentrations (50-100 ng ml(-1)). The main effect of SN-38 in LS174T cells was prolonged cell cycle arrest, which was independent of concentration. Arrest occurred in all phases of the cell cycle, with the distribution depending on concentration (P < 0.001) and not duration (P > 0.05). With increasing concentration, LS174T cells arrested in G2/M, S and G1. Cell cycle arrest was coincident with increased p53 expression in each phase of the cell cycle. Expression in G1 increased with time and concentration (P < 0.001, P = 0.01 respectively)whereas in S and G2/M p53 expression increased only with time (P< 0.001). Dose-dependent p53-associated G1 arrest, in the absence of DNA synthesis indicates an additional cytotoxic mechanism for SN-38, which requires higher concentrations than the S phase mechanism, and detection of which seems to involve p53. For incubations with the same ED (exposure x duration), apoptosis in HT29 cells was significantly higher for prolonged exposure to lower concentrations, whereas in LS174T cells there was a trend towards increased apoptosis with shorter exposures to higher concentrations, indicating a schedule effect of SN-38. Although expression of wild-type p53 leads to a more rapid induction of apoptosis, SN-38 cytotoxicity was generally greater in cells with mutant p53, as wild-type cells escaped apoptosis by p53 associated prolonged cell cycle arrest. Thus, pulsed schedules with higher doses may be more effective in cells expressing wild-type p53, whereas continued exposure with protracted schedules may be more active in cells expressing mutant p53.

Adenocarcinoma↗
PubMed1993

The role of the urokinase receptor in extracellular matrix degradation by HT29 human colon carcinoma cells.

Urokinase (u-PA) and the urokinase receptor (u-PAR), are thought to play a critical role in the invasive and metastatic properties of cancer cells. The HT29 human colon-carcinoma cell line was selected to evaluate these aspects. HT29 cells express u-PA receptors (100,000 sites/cell, KD = 1.5 nM), but no PA activity and therefore are unable to generate plasmin in the presence of plasminogen. These cells have been transfected with a human u-PA cDNA to investigate whether secreted u-PA would enhance in vitro extracellular matrix degradation, and whether the binding of u-PA to the cell surface is determinant. Five clones were selected for stable expression of high PA activity. These clones were capable of marked plasminogen-dependent degradation of R22 smooth-muscle-cell-derived extracellular matrix, whereas the parental cell line contributed to an insignificant breakdown only. Aprotinin, polyclonal anti-u-PA IgG, recombinant PAI-2, and co-culture with human PAI-I-producing mouse L cells significantly inhibited this degradation. Furthermore, a peptide displacing u-PA from its receptor as well as 2 different polyclonal anti-u-PA receptor IgGs decreased the breakdown after 24 hr by as much as 70% and 81%, respectively. These results show that the binding of u-PA to its receptor plays an important role in in vitro matrix breakdown by HT29 u-PA transfectants.

Carcinoma↗
PubMed1998

Use of replication deficient adenoviruses to investigate the role of G proteins in Ca2+ signalling in epithelial cells.

Here we report on the feasibility of using replication deficient adenoviruses to modify signal transduction systems in epithelia. We constructed two viruses, one expressing a dominant negative mutant of the alpha-subunit of Gq (Ad-EF1-dnG alpha q) and the other expressing the wild-type alpha-subunit of Gq (Ad-EF1-wtG alpha q). We used an adenovirus expressing green fluorescent protein (Ad-EF1-GFP20) to show that infection of cultured cells with an adenovirus results in at least 95% expression of the transgene in both HSG and HT29 cells. We also used an adenovirus that expresses no transgene (Ad-MX17) to demonstrate that adenoviral infection itself does not affect the resting concentration of cytosolic Ca2+ ([Ca2+]i) or the carbachol responses in these cells. We further show that Ad-EF1-dnG alpha q inhibits the increase in [Ca2+]i produced by muscarinic receptor activation in both the cell lines we studied. This inhibitory effect is not shared by Ad-EF1-wtG alpha q, which indicates that in both HSG and HT29 cells, the increase in [Ca2+]i produced by muscarinic receptor activation is largely mediated by activation of Gq. Neither virus affected the resting level of [Ca2+]i in these cells. Our findings confirm the feasibility of using replication deficient adenoviruses expressing dominant negative mutants to investigate the role of G proteins in signal transduction systems.

Adenoviridae↗
PubMed1998

Antiinflammatory actions of cat's claw: the role of NF-kappaB.

BACKGROUND: Uncaria tomentosa is a vine commonly known as cat's claw or 'uña de gato' (UG) and is used in traditional Peruvian medicine for the treatment of a wide range of health problems, particularly digestive complaints and arthritis. PURPOSE: The aim of this study was to determine the proposed anti-inflammatory properties of cat's claw. Specifically: (i) does a bark extract of cat's claw protect against oxidant-induced stress in vitro, and (ii) to determine if UG modifies transcriptionally regulated events. METHODS: Cell death was determined in two cell lines, RAW 264.7 and HT29 in response to peroxynitrite (PN, 300 microM). Gene expression of inducible nitric oxide synthase (iNOS) in HT29 cells, direct effects on nitric oxide and peroxynitrite levels, and activation of NF-kappaB in RAW 264.7 cells as influenced by UG were assessed. Chronic intestinal inflammation was induced in rats with indomethacin (7.5 mg/kg), with UG administered orally in the drinking water (5 mg/mL). RESULTS: The administration of UG (100 microg/mL) attenuated (P < 0.05) peroxynitrite-induced apoptosis in HT29 (epithelial) and RAW 264.7 cells (macrophage). Cat's claw inhibited lipopolysaccharide-induced iNOS gene expression, nitrite formation, cell death and inhibited the activation of NF-kappaB. Cat's claw markedly attenuated indomethacin-enteritis as evident by reduced myeloperoxidase activity, morphometric damage and liver metallothionein expression. CONCLUSIONS: Cat's claw protects cells against oxidative stress and negated the activation of NF-kappaB. These studies provide a mechanistic evidence for the widely held belief that cat's claw is an effective anti-inflammatory agent.

Animals↗
PubMed2006

Hypoxia-induced up-regulation of angiopoietin-2 in colorectal cancer.

Angiogenesis is a compensatory mechanism that enables malignant tumors to survive in an oxygen-deficient environment. To test our hypothesis that hypoxia stimulates the production of angiopoietin-2 (Ang-2) in colorectal cancer (CRC), we investigated the expression of Ang-2 in three cultured CRC cell lines, and in specimens from 11 CRC metastatic liver tumors. Hypoxia-induced Ang-2 mRNA expression was clearly evident in HCT116 cells that did not express Ang-2 under normoxic conditions. Ang-2 mRNA was detected only after 48 h in hypoxic serum-deprived cultures in a LoVo cell line, and under both normoxic and hypoxic conditions without any noticeable difference in the HT29 cells. There was a stepwise increase in Ang-2 expression from the periphery to the central part of the liver metastatic foci, whereas an inverse result was noted in tumor blood vessels, with a gradual decrease in CD31-positive ECs from the edge to the central region of the metastatic lesion. An expression pattern similar to Ang-2 was found in glucose transporter 1 (Glut-1), a known hypoxia-induced factor. These findings suggest that hypoxia plays an important role in inducing the expression of Ang-2 in CRC.

Angiopoietin-2↗
PubMed1994

pH regulation in HT29 colon carcinoma cells.

The pH regulation in HT29 colon carcinoma cells has been investigated using the pH-sensitive fluorescent indicator 2',7'-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF). Under control conditions, intracellular pH (pHi) was 7.21 +/- 0.07 (n = 22) in HCO3(-)-containing and 7.21 +/- 0.09 (n = 12) in HCO3(-)-free solution. HOE-694 (10 mumol/l), a potent inhibitor of the Na+/H+ exchanger, did not affect control pHi. As a means to acidify cells we used the NH4+/NH3 (20 mmol/l) prepulse technique. The mean peak acidification was 0.37 +/- 0.07 pH units (n = 6). In HCO3(-)-free solutions recovery from acid load was completely blocked by HOE-694 (1 mumol/l), whereas in HCO3(-)-containing solutions a combination of HOE-694 and 4,4'-diisothiocyanatostilbene-2,2'-disulphonate (DIDS, 0.5 mmol/l) was necessary to show the same effect. Recovery from acid load was Na(+)-dependent in HCO3(-)-containing and HCO3(-)-free solutions. Removal of external Cl- caused a rapid, DIDS-blockable alkalinization of 0.33 +/- 0.03 pH units (n = 15) and of 0.20 +/- 0.006 pH units (n = 5), when external Na+ was removed together with Cl-. This alkalinization was faster in HCO3(-)-containing than in HCO3(-)-free solutions. The present observations demonstrate three distinct mechanisms of pHi regulation in HT29 cells: (a) a Na+/H+ exchanger, (b) a HCO3-/Cl- exchanger and (c) a Na(+)-dependent HCO3- transporter, probably the Na(+)-HCO3-/Cl- antiporter.(ABSTRACT TRUNCATED AT 250 WORDS)

Acid-Base Equilibrium↗
PubMed2005

Potential anti-cancer effects of virgin olive oil phenols on colorectal carcinogenesis models in vitro.

The traditional Mediterranean diet is thought to represent a healthy lifestyle; especially given the incidence of several cancers including colorectal cancer is lower in Mediterranean countries compared to Northern Europe. Olive oil, a central component of the Mediterranean diet, is believed to beneficially affect numerous biological processes. We used phenols extracted from virgin olive oil on a series of in vitro systems that model important stages of colon carcinogenesis. The effect the extract on DNA damage induced by hydrogen peroxide was measured in HT29 cells using single cell microgel-electrophoresis. A significant anti-genotoxic linear trend (p=0.011) was observed when HT29 cells were pre-incubated with olive oil phenols (0, 5, 10, 25, 50, 75, 100 microg/ml) for 24 hr, then challenged with hydrogen peroxide. The olive oil phenols (50, 100 microg/ml) significantly (p=0.004, p=0.002) improved barrier function of CACO2 cells after 48 hr as measured by trans-epithelial resistance. Significant inhibition of HT115 invasion (p<0.01) was observed at olive oil phenols concentrations of 25, 50, 75, 100 microg/ml using the matrigel invasion assay. No effect was observed on HT115 viability over the concentration range 0, 25, 50 75, 100 microg/ml after 24 hr, although 75 and 100 microg/ml olive oil phenols significantly inhibited HT115 cell attachment (p=0.011, p=0.006). Olive oil phenols had no significant effect on metastasis-related gene expression in HT115 cells. We have demonstrated that phenols extracted from virgin olive oil are capable of inhibiting several stages in colon carcinogenesis in vitro.

Anticarcinogenic Agents↗
PubMed2004

Agonist-induced internalization of leukotriene B(4) receptor 1 requires G-protein-coupled receptor kinase 2 but not arrestins.

The leukotriene B(4) (LTB(4)) receptor (BLT1) becomes desensitized upon repeated agonist stimulation. Little is known, however, about BLT1 internalization, which follows desensitization in most G-protein-coupled receptors (GPCR). In the current study, transiently expressed BLT1 readily internalized, after LTB(4) stimulation, in RBL-2H3 cells that express high levels of endogenous GPCR kinase 2 (GRK2) but did not in COS-7 or human embryonic kidney (HEK) 293 cells, which do not overexpress GRK. The internalization of BLT1 could be blocked in RBL-2H3 cells by coexpressing dominant-negative (DN) GRK2 K220R and could be promoted in HEK293 cells by coexpressing wild-type (WT) GRK2. Coexpression of WT or DN nonvisual arrestins had no effect on BLT1 internalization. Moreover, upon stimulation with LTB(4), BLT1 did not induce arrestin-green fluorescence protein redistribution in either cell type, even in the presence of overexpressed GRK2. Coimmunoprecipitation experiments confirmed that BLT1 could associate with GRK2 but not with arrestins. A C-tail-truncated mutant of BLT1 lost the capacity to internalize and associate with GRK2 upon exposure to LTB(4), suggesting that the C-tail was required for receptor internalization and association with GRK2. Taken together, our results indicate that the C terminus of BLT1 plays a pivotal role in receptor internalization and GRK2 association. Moreover, ligand-induced BLT1 internalization is dependent on GRK2 but independent of arrestins. This may allow differential, cell-type-specific signaling in response to LTB(4), depending on GRK expression levels.

Animals↗
PubMed2003

Aberrant expression and activation of the thrombin receptor protease-activated receptor-1 induces cell proliferation and motility in human colon cancer cells.

The traditional view on the role of serine proteases in tumor biology has changed with the recent discovery of a family of protease-activated receptors (PARs). In this study we explored the expression and functional role of the thrombin receptor PAR-1 in human colon cancer cells. Reverse transcriptase-polymerase chain reaction analysis showed that PAR-1 mRNAs are present in 11 of 14 human colon cancer cell lines tested but not in normal human colonic epithelial cells. This is in line with the immunolocalization of PAR-1 in human colon tumors and its absence in normal human colonic mucosa. The functional significance of the aberrant expression of PAR-1 in colon cancer cells was then investigated. We found that 1) a prompt increase in intracellular calcium concentration was observed on thrombin (10 nmol/L) or PAR-1 agonist AP1 (100 micro mol/L) challenge of HT29 cells; 2) HT29 quiescent cells treated with thrombin (0.01 to 20 nmol/L) or AP1 (1 to 300 micro mol/L) exhibited dramatic mitogenic responses (3.5-fold increase in cell number). Proliferative effects of thrombin or AP1 were also observed in other colon cancer cell lines expressing PAR-1. This effect was reversed by the MEK inhibitor PD98059 in consonance with the ability of thrombin or AP1 to induce phosphorylation of p42/p44 extracellular-regulated protein kinases. 3) PAR-1 activation by thrombin or AP1 led to a two-fold increase in cell motility of wounded HT29-D4. Our results demonstrate for the first time the aberrant expression of the functional thrombin receptor PAR-1 in colon cancers and its important involvement in cell proliferation and motility. Thrombin should now be considered as a growth factor for human colon cancer.

Calcium↗
PubMed1995

Mapping of the vascular endothelial growth factor-producing hypoxic cells in multicellular tumor spheroids using a hypoxia-specific marker.

We have investigated the hypoxia inducibility of vascular endothelial growth factor (VEGF) in multicellular tumor spheroids of HT29 cells using a monoclonal antibody to a fluorinated bioreductive drug, EF5 [2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)aceta mide], a chemical probe for hypoxia. We have shown that VEGF expression is predominantly localized in interior spheroid cells that are sufficiently hypoxic to bioreductively activate the 2-nitroimidazole and produce immunologically detectable adducts of the EF5 compound. Northern blotting analyses demonstrated that VEGF165 is the predominant form of VEGF produced by HT29 cells and that the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate did not induce VEGF expression. This study demonstrates that VEGF expression is up-regulated in response to hypoxia and in the microenvironments found in human multicellular tumor spheroids. This investigation also illustrates the utility of the EF5 binding in multi-cellular tumor spheroids as a means of studying the expression and regulation of hypoxia-inducible genes.

Carcinoma↗
PubMed1996

A unique gene encodes spliceoforms of the B-cell adhesion molecule cell surface glycoprotein of epithelial cancer and of the Lutheran blood group glycoprotein.

Two new members of the Ig superfamily, the Lutheran (Lu) blood group glycoprotein and the B-cell adhesion molecule (B-CAM) epithelial cancer antigen, have been recently cloned from human placenta and colon cancer HT29 cell line, respectively. Although amino acid sequences deduced from cDNA analysis suggested that B-CAM should represent an abridged form of the Lu glycoprotein lacking the last 40 amino acids of the putative cytoplasmic tail, the relationship between the genes encoding these polypeptides has not been determined. In the present report, we showed by Southern blot analysis that the Lu and B-CAM cDNAs derived from a unique LU gene which exhibited an HindIII RFLP associated with the Lua/Lub blood group polymorphism. Accordingly, in situ hybridization of the Lu cDNA probe confirmed the localization of the Lutheran blood group locus to chromosome 19 q13.2-13.3, as previously shown for a B-CAM DNA probe. Sequence comparison between cDNA and genomic PCR fragments indicated that the Lu and B-CAM transcripts previously isolated are generated through the alternative use of internal splice donor and acceptor sites within an exon located at the 3' end of the LU gene. These spliceoforms corresponded to 2.5 kb and 4.0 kb mRNA species detectable by Northern blot in all tissues and cell lines in which the LU gene is expressed; their primary structures are consistent with the presence of both the Lu and B-CAM antigens on two glycoprotein isoforms. However, the 4.0 kb transcript was very poorly expressed as compared to the 2.5 kb species except in the colon carcinoma HT29 cell line, suggesting a differential regulation of the Lu/B-CAM messenger RNA in some tumor tissues.

Amino Acid Sequence↗
PubMed1994

Activation of AP-1 and of a nuclear redox factor, Ref-1, in the response of HT29 colon cancer cells to hypoxia.

Many solid tumors contain substantial fractions of hypoxic cells which are relatively resistant to both radiation therapy and certain cytotoxic drugs. We have previously shown that exposure of human HT29 cells to hypoxic conditions results in the overexpression of certain enzymes involved in the detoxication of xenobiotics, including NAD(P)H:(quinone acceptor) oxidoreductase (DT)-diaphorase, and gamma-glutamylcysteine synthetase, the rate-limiting enzyme in glutathione synthesis. This hypoxic effect on DT-diaphorase was shown to involve both transcriptional induction and altered message stability. We have investigated the effects of hypoxia on elements in the promoter region of DT-diaphorase. Electrophoretic mobility shift assays demonstrate the induction of a binding activity to the AP-1 response element of DT-diaphorase. Supershift assays suggest that this binding is due to AP-1 nuclear factors and that members of the jun family are induced to a greater degree than fos by hypoxia. Analysis of the kinetics of transcription factor expression indicates that the expression of c-jun and junD is induced during hypoxic exposure; mRNA levels fall during reoxygenation. Induction of fos on the other hand is not as florid during hypoxia (5-fold) and is most pronounced (17-fold) 24 h after the restoration of an oxic environment. Thus, the hypoxic response of DT-diaphorase expression is mediated in part through AP-1, initially by a jun-related mechanism and then by the involvement of fos. The affinity of transcription factors for the AP-1 binding site depends on the redox state of a cysteine residue located close to the DNA-binding region of both Fos and Jun. A nuclear protein, Ref-1, maintains the reduced state of Fos and Jun and promotes binding to AP-1. Nuclear extracts of HT29 cells exposed to hypoxia show markedly increased Ref-1 protein content. Elevation of ref-1 steady-state mRNA levels occurs as an early event following induction of hypoxia and persists when cells are restored to a normally oxygenated environment. Nuclear run-on analysis demonstrates that induction of transcription is the mechanism of ref-1 mRNA elevation. Electrophoretic mobility shift assays and immunodepletion assays were used to further define the interaction of Ref-1 with specific AP-1-binding proteins under hypoxic conditions. These data demonstrate that the induction of detoxicating enzyme expression in HT29 cells exposed to hypoxia results from the induction of both transactivating factors that bind to the AP-1 element and of redox proteins that enhance their affinity for this element.

Carbon-Oxygen Lyases↗
PubMedDate not supplied

Dendritic cell maturation induced by delivery of ultraviolet-mediated apoptotic colorectal cancer cell lines.

BACKGROUND: Dendritic cells (DCs) play a critical role in the immune response. The aim of this study was to investigate apoptotic tumor cells as an antigen source for DC maturation. MATERIALS AND METHODS: We compared the efficacy of ultraviolet (UV)-irradiation with that of gamma-irradiation in the induction of apoptosis of tumor cells. Phenotypic and functional changes of DCs were analyzed after co-incubation with UV-irradiated tumor cells. RESULTS: UV-irradiation (1.8 J/cm2) was a more reliable method of inducing apoptosis than gamma-irradiation (20,000 rad). The expression of costimulatory molecules on DCs was upregulated after co-incubation with UV-irradiated tumor cells. When we performed allogeneic mixed lymphocyte reaction assay, UV-irradiated tumor cells-pulsed DCs stimulated allogeneic T lymphocytes more efficiently than DCs pulsed with gamma-irradiated cells (HT29, p = 0.0419 and WiDr, p = 0.0076). CONCLUSION: UV-irradiation reliably induces apoptosis in cultured colorectal cancer cells. DCs mature in function and expression of costimulatory molecules after co-incubation with apoptotic tumor cells. The clinical implications warrant further study.

Antigens, CD↗
PubMedDate not supplied

Nitroxides as antioxidants: Tempol protects against EO9 cytotoxicity.

Nitroxide free radicals have been shown to be potent antioxidants in a variety of experimental models using diverse means of insults. Among other insults, nitroxides have been shown effective in inhibiting cytotoxicity of quinone-based drugs such as streptonigrin and mitomycin C. These drugs and other chemotherapeutic agents have the potential to undergo bioreductive activation by the normal reducing enzymes within a cell. In the present work we studied the effect of the nitroxide Tempol on the cytotoxicity induced by EO9, a mitomycin C analogue, in HT29 cells under aerobic and hypoxic conditions. The study was aimed to better understand the mechanism of EO9 cytotoxicity and the molecular level of the nitroxide's mode of protection. The reactions of Tempol with activated EO9, and the reactive species formed during EO9 activation were studied in a cell-free solution, using spin-trapping, and electron paramagnetic resonance (EPR) spectrometry. Our results indicate that EO9 induced similar cytotoxicity in HT29 cells under aerobic and hypoxic conditions while Tempol provided similar and almost complete protection to both aerobic and hypoxic cells. The results indicate that EO9 cytotoxicity is due to both 1- and 2-electron reductive activation processes, with aerobic toxicity caused by back-oxidation of the hydroquinone to the semiquinone, EO9.-. Tempol serves both as a useful tool in the study of the mechanisms of quinone-mediated cytotoxicity and as a potent antioxidant against the damaging effects of redox cycling quinones and semiquinones by scavenging of EO9.- or detoxification of O2.- and H2O2.

Aziridines↗
PubMed1999

Characterization of a membrane calcium pathway induced by rotavirus infection in cultured cells.

Some viruses induce changes in membrane permeability during infection. We have shown previously that the porcine strain of rotavirus, OSU, induced an increase in the permeability to Na+, K+, and Ca2+ during replication in MA104 cells. In this work, we have characterized the divalent cation entry pathway by measuring intracellular Ca2+ in fura-2-loaded MA104 and HT29 cells in suspension. The permeability to Ca2+ and other cations was evaluated by the change of the intracellular concentration following an extracellular cation pulse. Rotavirus infection induced an increase in permeability to Ca2+, Ba2+, Sr2+, Mn2+, and Co2+. The rate of cation entry decreased over time as the intracellular concentration increased during the first 20 s. This indicates that regulatory mechanisms, including channel inactivation, are triggered. La3+ did not enter the cell and blocked the entry of the divalent cations in a dose-dependent manner. Metoxyverapamil (D600), a blocker of L-type voltage-gated channels, partially inhibited the entry of Ca2+ in virus-infected MA104 and HT29 cells. The results suggest that rotavirus infection of cultured cells activates a cation channel rather than nonspecific permeation through the plasma membrane. This activation involves the synthesis of viral proteins through mechanisms yet unknown. The increase in intracellular Ca2+ induced by the activation of this channel may be related to the increase in cytoplasmic and endoplasmic reticulum Ca2+ pools required for virus maturation and cell death.

Animals↗
PubMed2004

Flavone inhibition of tumor growth via apoptosis in vitro and in vivo.

Colorectal carcinoma is a human malignant tumor, which is very resistant to currently available methods of treatment. Therefore, developing an effective agent with anti-colorectal carcinoma activity is important. In the present study, 8 structurally related flavones including flavone, 3-OH flavone, 5-OH flavone, 7-OH flavone, quercetin, kaempferol, quercetin, and morin were used to study their effects on colorectal carcinoma cells (HT29, COLO205, COLO320-HSR). Results of MTT assay indicated that flavone shows the most potent cytoxic effect among them on these three cell types. The cytotoxicity induced by flavone is mediated by inducing the occurrence of apoptosis characterized by the appearance of DNA ladders, apoptotic bodies and hypodiploid cells. Activation of caspase 3 protein procession and enzyme activity with inducing cleavage of caspase 3 substrates PARP was identified in flavone-treated cells, and an inhibitory peptide Ac-DEVD-FMK for caspase 3, but not Ac-YVAD-FMK for caspase 1, attenuates the cytotoxic effect of flavone in COLO205 and HT29 cells. Elevation of p21 but no p53 protein was observed in flavone-treated cells. Increasing intracellular peroxide level was detected in flavone-treated cells by DCHF-DA assay, and antioxidants such as tiron, catalase, SOD, PDTC, but not DPI, suppress flavone-induced cytotoxic effect. In vivo anti-tumor study indicates that flavone exhibits ability to inhibit tumor formation elicited by s.c. injection of COLO205 cells in nude mice, and apoptotic cells and an increase in p21, but not p53, protein were observed in tumor tissues derived from flavone-treated group. Additionally, flavone induced apoptosis in primary colon carcinoma cells COLO205-X with appearance of DNA ladders, caspase 3 protein procession, PARP protein cleavage, and an increase in p21 (not p53) protein. These data provide evidence to suggest that flavone is an effective agent to induce apoptosis in colorectal carcinoma cells in vitro and in vivo; activation of caspase 3, ROS production, and increasing p21 protein are involved.

Animals↗
PubMed1992

Expression of type I, but not type II insulin-like growth factor receptor on both undifferentiated and differentiated HT29 human colon carcinoma cell line.

The HT29 human colonic carcinoma cell line secretes insulin-like growth factor (IGF)-II. We have examined these cells for expression of IGF receptors. Competitive binding assays as affinity cross-linking experiments using 125I-IGF-II fail to reveal type II IGF receptors at the cell surface. In contrast, cross-linking studies with either 125I-IGF-I or 125I-IGF-II reveal an M(r) 135,000 protein that follows a peptide binding specificity characteristic of the alpha-subunit of the type I IGF receptor. However, 125I-IGF-II binding to this receptor is not inhibited at 4 C by alpha IR-3, a monoclonal antibody to the type I IGF receptor. Analysis of the competitive binding curves with each one of these radioligands suggests that HT29 cells express both a classical type I IGF receptor (about 6,000/cell; KdIGF-I = 0.48 nmol) and a variant one whose 125I-IGF-II binding is not blocked by alpha IR-3 (about 15,000/cell; KdIGF-II = 4.0 nmol). Endocytosis studies of specific cell-bound 125I-IGF-I or 125I-IGF-II suggest that ligand interaction with the classical, but not the variant, binding site is only able to induce receptor internalization. An identical IGF receptors pattern is observed with HT29-D4 clonal cells induced to differentiate by culture in a glucose-free medium.

Affinity Labels↗