Human Colon Tumor Cell Research Articles

Genotoxicity of 4-Hydroxy-2-Nonenal in Human Colon Tumor Cells Is Associated with Cellular Levels of Glutathione and the Modulation of Glutathione S-Transferase A4 Expression by Butyrate

The cellular production of 4-hydroxy-2-nonenal (HNE), a product of endogenous lipid peroxidation, constitutes a genotoxic risk factor for carcinogenesis. Our previous studies have shown that human HT29 colon cells developed resistance toward HNE injury after treatment with butyrate, a diet-associated gut fermentation product. This resistance was attributed to the induction of certain glutathione S-transferases (hGSTP1-1, hGSTM2-2, and hGSTA1-1) and also for the tripeptide glutathione (GSH) synthesizing enzymes. In the present study, we have investigated in HT29 cells whether hGSTA4-4, which has a high substrate specificity for HNE, was also inducible by butyrate and, thus, could contribute to the previously observed chemoresistance. In addition, we investigated if cellular depletion of GSH by L-buthionine-S,R-sulfoximine (BSO) enhances chemosensitivity to HNE injury in HT29 cells. Incubation of HT29 cells with butyrate (2-4 mM) significantly elicited a 1.8 to 3-fold upregulation of steady state hGSTA4 mRNA over 8-24 h after treatment. Moreover, 4 mM butyrate tended to increase hGSTA4-4 protein concentrations. Incubation with 100 microM BSO decreased cellular GSH levels by 77% without significant changes in cell viability. Associated with this was a 2-fold higher level of HNE-induced DNA damage as measured by the comet assay. Collectively, the results of this study and our previous work indicate that the genotoxicity of HNE is highly dependent on cellular GSH status and those GSTs that contribute toward HNE conjugation, including hGSTA4-4. Since HNE contributes to colon carcinogenesis, the favorable modulation of the GSH/GST system by butyrate may contribute to chemoprevention and reduction of the risks.

Suppression of tumor growth by oncolytic adenovirus-mediated delivery of an antiangiogenic gene, Soluble Flt-1

Armed oncolytic adenoviruses represent an appealing tumor treatment approach, as they can attack tumors at multiple levels. In this study, considering that angiogenesis plays a central role in tumor growth, we inserted an antiangiogenic gene, sflt-1(1-3) (the first three extracellular domains of FLT1, the hVEGF receptor-1), into an E1B-55-kDa-deleted oncolytic adenovirus (ZD55) to construct ZD55-sflt-1. Although soluble (s) Flt-1 did not affect tumor cell growth, ZD55-sflt-1 could specifically induce a cytopathic effect in tumor cells, like ONYX-015. The secretion of sFlt-1 from ZD55-sflt-1 was much higher than that from replication-deficient Ad-sflt-1 upon infection of SW620 human colon tumor cells, leading to a stronger inhibitory effect on VEGF-induced proliferation and tube formation ability of HUVECs. Moreover, marked reduction of tumor growth and long-term survival rates were observed in ZD55-sflt-1-treated nude mice with subcutaneous SW620 tumor. Its efficacy correlated with a decrease in microvessel density and an increase in apoptotic tumor cells. In addition, ZD55-sflt-1 showed a synergic effect with the chemotherapeutic agent 5-FU. These results indicate that ZD55-sflt-1, combining the advantages of oncolytic adenovirus and antiangiogenic gene therapy, is a powerful agent for human tumor treatment.

Tumor Necrosis Factor α-dependent Drug Resistance to Purine and Pyrimidine Analogues in Human Colon Tumor Cells Mediated through IKK

Development of drug resistance in cancer is one of the main challenges in chemotherapy, and many mechanisms are still unknown. In this study, we show that tumor necrosis factor alpha (TNFalpha) increases postdrug survival from 5-fluoro-2'-deoxyuridine (FdUrd) in two human colon tumor cell lines. This resulted in the development of drug-resistant cells in a TNFalpha-dependent manner. Interestingly, although the drug-resistant cells were selected using FdUrd, they are also resistant to a number of other antimetabolites in the DNA synthesis pathway in a TNFalpha-dependent manner. Only in the drug-resistant cells (p35-colo201) TNFalpha treatment resulted in G(0)-G(1) arrest but not in the parental colo201 and other cell types. Blocking TNFalpha-induced cell cycle arrest sensitized drug-resistant cells to FdUrd. TNFalpha-induced cell cycle arrest required IKK. IKK inhibition by a small molecule inhibitor or by the knockdown of IKKalpha, IKKbeta, or RelA/p65 using siRNA, but not the inhibition of JNK, MEK, p38, or caspase-8 pathways, blocked TNFalpha-induced G(0)-G(1) arrest and restored sensitivity to FdUrd of drug-resistant cells. TNFalpha reduced the transcripts and protein levels of phosphorylated retinoblastoma protein (Rb), Rb, E2F1, and Cdk4 only in drug-resistant p35-colo201 cells. This effect of TNFalpha was reversed by IKK inhibitor, suggesting that TNFalpha-induced cell cycle arrest is probably due to the reduction of Rb, E2F1, and Cdk4. Taken together, this study shows that, in vitro, TNFalpha-induced cell cycle arrest through IKK can provide a mechanism for the development of drug resistance to anti-cancer drugs, purine and pyrimidine analogues.

Antisense Inhibition of Methylenetetrahydrofolate Reductase Reduces Cancer Cell Survival In vitro and Tumor Growth In vivo

Many cancer lines are methionine dependent and decrease proliferation when methionine supply is limited. Methylenetetrahydrofolate reductase (MTHFR) generates the folate derivative for homocysteine remethylation to methionine. We investigated the effect of antisense-mediated inhibition of MTHFR on survival of human cancer cells. We examined the in vitro and in vivo anticancer effects of a combination of MTHFR antisense and standard cytotoxic drugs. Specific antisense against MTHFR (EX5) showed significant inhibitory effects on growth of human colon, lung, breast, prostate, and neuroblastoma tumor cells in vitro compared with that of the control oligonucleotide. Cytotoxic drugs (5-fluorouracil, cisplatin, or paclitaxel) potentiated the effect of EX5. In vivo, antisense alone or in combination with cytotoxic drugs inhibited the growth of human colon and lung carcinoma xenografts. In comparison with control oligonucleotide, treatment with EX5 inhibited growth of colon tumors and lung tumors by 60% and 45%, respectively. EX5 with 5-fluorouracil decreased growth of colon tumors by an additional 30% compared with EX5 alone, and EX5 with cisplatin decreased growth of lung tumors by an additional 40% compared with cisplatin alone. Growth inhibition by EX5 was associated with decreased amounts of MTHFR protein and with increased amounts of an apoptosis marker. Our results confirm that MTHFR inhibition decreases tumor growth and suggest that inhibition of MTHFR by antisense or small molecules may be a novel anticancer approach.

A Cytotoxic Carotenoid from the Marine Sponge Prianos osiros

Investigations of the marine sponge Prianos osiros, collected in Pohnpei, gave a new cytotoxic acetylenic carotenoid, (3R,3'R,5S)-3,3',5,19'-tetrahydroxy-7',8'-didehydro-gamma,epsilon-carotene-8-one. The absolute configuration of this carotenoid was solved by interpretation of IR, MS, and 2D NMR spectra and application of the modified Mosher's method. Compound 1 is cytotoxic toward cultured human colon tumor cells, HCT 116 (IC(50) 4.38 microg/mL).

Oligosaccharide Mimics Containing Galactose and Fucose Specifically Label Tumour Cell Surfaces and Inhibit Cell Adhesion to Fibronectin

With the aim of establishing a versatile and easy synthesis of branched saccharides for biological applications, we used molecular-dynamics simulations to model Lewis(y) to two classes of di- or triantennary saccharide mimetics. One set of mimetics was based on 1,3,5-tris(hydroxymethyl)cyclohexane (TMC) as the core, the other on furan, and both were derivatised with galactose and/or fucose. The TMC-based saccharides were biotinylated, while the furan disaccharides were treated with maleimide-activated biotin in a Diels-Alder fashion to yield oxazatricyclodecanes (OTDs). These were then assayed as cell-surface labels in human colon (SW480 and CaCo-2), liver (PLC), Glia (U333 CG 343) and ovary (SKOV-3) tumour cell lines. Discrete staining patterns were observed in all cells, usually at one or two poles of the cells, particularly with the asymmetric 3-beta-L-fucopyranosyloxymethyl-4-beta-D-galactopyranosyloxymethyl-OTD. Normal SV40-transformed fibroblasts (SV80) showed no staining. Adhesion of the highly metastatic mouse melanoma line B16 F10 to fibronectin was inhibited by 80 % by the TMC-digalactoside and by 30 % by 3,4-bis-(beta-D-galactopyranosyloxymethyl)furan. None of the saccharide mimetics inhibited the adhesion of the less metastatic B16 F1 line. Migration of B16 F10 cells through Matrigel was greatly inhibited by the TMC-digalactoside and weakly inhibited by the TMC-trigalactoside. The saccharide mimetics that had shown the best structural agreements with the terminal saccharides of Lewis(y) in the molecular dynamics simulation were also the most biologically potent compounds; this underlines the predictive nature of molecular dynamics simulations. The use of the non-saccharide cores enabled us to adapt spacer lengths and terminal saccharides to optimise the structures to bind more avidly to cell-surface lectins.

Structure-guided design of pyrazolo[1,5- a]pyrimidines as inhibitors of human cyclin-dependent kinase 2

The protein structure guided design of a series of pyrazolo[1,5- a]pyrimidines with high potency for human cyclin-dependent kinase 2 (CDK2) is described. Some examples were shown to inhibit the growth of human colon tumour cells, were equipotent for CDK1 and were selective against GSK-3β and other kinases.

In vitrogrowth inhibition of human colonic tumor cells by Verapamil

To investigate the effects and mechanisms of Verapamil on cultured human colonic tumor (HCT) cells. HCT cells were treated with different concentrations of Verapamil, and their proliferation was examined by MTT assay. The areas of sub-diploid peak were measured by flow cytometry, and the DNA ladder was found by agarose gel electrophoresis. The characteristic changes in morphology were observed under light microscopy. The cell nuclei (propidium iodide labeled, PI-labeled) and cellular distribution and concentration of calcium (Fluo-3-labeled) were studied by using laser confocal scanning microscope. The proliferation of HCT cells was inhibited by different concentrations of Verapamil. With the increase in concentration of Verapamil, the percent of G0-G1 phase cells in HCT cells increased and that of S phase cells decreased. After treating with different concentrations of Verapamil, flow cytometry showed that HCT cells were enlarged in areas of sub-diploid in a dose-dependent manner. Gel electrophoresis results displayed a typical DNA ladder. On staining with Wrights-Giemsa, the typical cellular apoptosis morphologic changes were also observed. PI-labeled cell nuclei were found markedly changed. In addition, we inspected that the 100 micromol/L Verapamil could increase the intracellular calcium ion concentration [Ca(2+)](i) in HCT cells. Verapamil can inhibit proliferation of HCT cells via inducing cell apoptosis.

DNA Topoisomerase I Inhibitor, Ergosterol Peroxide fromPenicillium oxalicum

Recently, we found that the MeOH extract of Penicillium oxalicum showed inhibitory activity towards DNA topoisomerase I. Subsequently, ergosterol peroxide, ergosterol, palmitoleic acid, and linoleic acid were isolated from the cultured mycelia of P. oxalicum. The structural determinations were based on physical and spectral analyses. Biological evaluation revealed that ergosterol peroxide inhibited the relaxation of supercoiled DNA (pBR322) induced by DNA topoisomerase I, and also showed marginal, selective cytotoxic activity against human colon tumor cells [COLO-205 (ED50=8.56 microg/mL].

Cytotoxic and Apoptotic Effects of Novel Heterodinucleoside Phosphates Consisting of 5‐Fluorodeoxyuridine and Ara‐C in Human Cancer Cell Lines

In search for possible alternatives in the treatment of human malignancies we investigated several new heterodinucleoside phosphates containing of 5‐Fluorodeoxyuridine (5‐FdUrd) and Arabinofuranosylcytosine (Ara‐C). We show that all dimers tested inhibited the number of colonies of CCL228, CCL227, 5‐FU resistant CCL227 and HT‐29 human colon tumor cells with IC50 values ranging from 0.65 to 1 nM. Dimer # 2 inhibited the number of sensitive and Ara‐C resistant H9 human lymphoma cells with IC50 values ranging from 200 to 230 nM. Since no significant difference in the cytotoxicity of the dimers could be observed between sensitive and resistant cells, these compounds might be used in the treatment of 5‐FU and Ara‐C resistant tumors.

Aurantosides G, H, and I: Three New Tetramic Acid Glycosides from a Papua New Guinea Theonella swinhoei

Aurantosides G-I (1-3) have been isolated from the lithistid sponge Theonella swinhoei from Papua New Guinea. Their structures were established by spectroscopic and chemical methods. Compounds 1-3 represent new monochloropentaenoyl tetramic acids with mono-, di-, and tri-N-saccharide substituents, respectively. Aurantosides G-I (1-3) failed to show any significant cytotoxicity against the human colon tumor cell line HCT-116.

Repetitive Treatments of Colon HT‐29 Cells with Diallyl Disulfide Induce a Prolonged Hyperacetylation of Histone H3 K14

Diallyl disulfide (DADS) is a sulfur compound derived from garlic. Several studies carried out in rodents have revealed protective effects of DADS against colon carcinogenesis. The antipromoting effects of DADS may be partly related to its ability to inhibit tumoral cell proliferation. In a previous study, we have shown that in two human colon tumor cell lines (HT-29 and Caco-2) seeded at a low density (0.2 x 10(6) cells/100-mm petri dish), DADS antiproliferative effects were associated with a transient increase of histone H3 K14 acetylation. Moreover, DADS could inhibit nuclear histone deacetylase activity. Therefore, in the present study, we examined the possible effects of different experimental conditions (HT-29 cells at high density, repetitive treatments with DADS) on the pattern of DADS-induced histone hyperacetylation. Using HT-29 cells seeded at a higher density (5 x 10(6) cells/100-mm petri dish), we found that DADS induced histone H3 K14 hyperacetylation rapidly (3 h). When administrated as single treatments, the DADS effect on histone H3 K14 remained transient. In contrast, repetitive treatment with DADS resulted in a prolonged hyperacetylation of histone H3 K14. Whatever the cell culture conditions were, DADS had no effect on histone H4 acetylation. Thus, in vitro, the cell density and pattern of DADS treatment influenced the HT-29 nuclear response to DADS. DADS belongs to food-borne molecules that may play a role in chromatin remodeling and contribute to the nutritional modulation of gene expression.

Tumor Cell Proliferation and Cyclooxygenase Enzyme Inhibitory Compounds in Amaranthus tricolor

Amaranthus tricolor is consumed as a vegetable in Asia. Bioassay-directed isolation of leaves and stems of A. tricolor yielded three galactosyl diacylglycerols (1-3) with potent cyclooxygenase and human tumor cell growth inhibitory activities. The purified compounds were characterized by spectroscopic methods. In addition, the fatty acid moieties in diacyl galactosyl glyerols were characterized by GC-MS analyses. The galactosyl diacylglycerols 1-3 inhibited the cyclooxygenase-1 (COX-1) enzyme by 78, 63, and 93% and the cyclooxygenase-2 (COX-2) enzyme by 87, 74, and 95%, respectively. These compounds were tested for antiproliferative activity using human AGS (gastric), CNS (central nervous system; SF-268), HCT-116 (colon), NCI-H460 (lung), and MCF-7 (breast) cancer cell lines. Compound 1 inhibited the growth of AGS, SF-268, HCT-116, NCI-H460, and MCF-7 tumor cell lines with IC50 values of 49.1, 71.8, 42.8, 62.5, and 39.2 mug/mL, respectively. For AGS, HCT-116, and MCF-7 tumor cell lines, the IC50 values of compounds 2 and 3 were 74.3, 71.3, and 58.7 microg/mL and 83.4, 73.1, and 85.4, respectively. This is the first report of the COX enzyme inhibitory activity for galactosyl glycerols and antiproliferative activities against human colon, breast, lung, stomach, and CNS tumor cell lines.

In vitro and in vivo antitumor activity of novel amphiphilic dimers consisting of 5-fluorodeoxyuridine and arabinofuranosylcytosine

Various heterodinucleoside phosphates of 5-fluorodeoxyuridine (5-FdUrd) and arabinofuranosylcytosine (Ara-C) have recently been synthesized as potent chemotherapeutic agents. 5-Fluorodeoxyuridine is being used in patients with colorectal carcinoma, whereas Ara-C is one of the most effective agents in the treatment of hematological malignancies. We now investigated the action of three novel amphiphilic dimers with different structures in various 5-fluorouracil (5-FU) sensitive and resistant human colon tumor cell lines (CCL228, CCL227, 5-FU resistant CCL227 and HT-29) as well as in L1210 murine leukemia cells. Activity of the heterodimers was determined by clonogenic and growth inhibition assays including the induction of programmed cell death. In addition, the in vivo effects were tested in L1210 leukemia bearing mice. We show that these compounds inhibited the number of colonies of 5-FU sensitive and resistant human colon tumor cell lines with IC50 values ranging from 0.65 to 1 nM. The investigated dimers induced dose-dependent apoptosis in HT-29 colon tumor cells as well as in L1210 leukemia cells. No significant difference in the cytotoxicity of these agents could be observed between 5-FU sensitive and resistant cells, indicating that these compounds might be used in the treatment of 5-FU resistant tumors. In L1210 leukemia bearing mice the survival of tumor-bearing animals was significantly increased in comparison with untreated control animals. We therefore conclude that these new heterodinucleoside phosphates of 5-FdUrd and Ara-C might be an additional option for the treatment of sensitive and 5-FU resistant colon cancer and hematological malignancies.

An uncontrolled phase II study evaluating anti-tumor efficacy and safety of ortataxel (BAY 59–8862) in patients with taxane-resistant non-small cell lung cancer

7136 Background: Ortataxel is a novel second-generation taxane, with potent growth inhibitory activity against human cell lines that express P-glycoprotein 170. Compared to paclitaxel and docetaxel, it is 20–30 times more potent as a growth inhibitory agent against human breast and colon tumor cell lines expressing P-gp 170. Ortataxel is also active in NSCL tumor xenografts. Based on these preclinical and additional phase I data, this phase II study was initiated to assess the safety and efficacy (response rate) of Ortataxel in patients (pts) with taxane-resistant NSCLC. Methods: Ortataxel was administered IV over 60 min every 3 wks at a dose of 75 mg/m2. Pts with taxane resistant NSCLC, with at least one measurable lesion, ECOG score 0, 1, or 2, adequate hematology and biochemistry, and no more than 2 prior anticancer chemotherapy regimens were eligible. Taxane resistance was defined as PD during treatment (Rx) or within 6 months of Rx with a prior taxane. The protocol was amended to include pts with brain metastases (BM). Results: 98 pts were enrolled. Clean data is available for the first 65 pts: 25 F, 40M; median age 60 (38 - 77); ECOG 0/1/2 - 19/37/9 respectively; prior chemo regimens: 0 (0 pt.), 1 (29 pts.), 2 (34 pts.), 3 (2 pts. received Iressa as 3rd regimen). Median number of cycles administered: 2 (1–11). 89% (58) pts received >90% planned dose intensity. Common drug related non-hematological toxicities included: fatigue 25(38%), nausea 19(29%), myalgia 18(27%), sensory neuropathy 17(26%), vomiting 15(23%), constipation 14(21%), diarrhea 11(17%), arthralgia 10(15%), alopecia 9(13%), stomatitis/pharyngitis 9(13%). Hematologic toxicities seen: G 3–4 neutropenia 49%, G3–4 anemia 7%. 4(6%) pts (without BM) had a PR, 25(38%) pts had SD, 31(48%) pts had PD. 5 pts were not evaluable (missing post baseline evaluations due to consent withdrawal in 2 pts and AE's in 3 pts). Conclusion: Four investigator-confirmed partial responses to Ortataxel were documented in this trial so far. An independent review is underway. The study continues to enroll only pts with BM in order to assess the drug in this subset of patients. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Bayer Healthcare; Pharma-Clinical Ltd.

An uncontrolled phase II study evaluating anti-tumor efficacy and safety of ortataxel (BAY 59–8862) in patients with taxane-resistant non-small cell lung cancer

7136 Background: Ortataxel is a novel second-generation taxane, with potent growth inhibitory activity against human cell lines that express P-glycoprotein 170. Compared to paclitaxel and docetaxel, it is 20–30 times more potent as a growth inhibitory agent against human breast and colon tumor cell lines expressing P-gp 170. Ortataxel is also active in NSCL tumor xenografts. Based on these preclinical and additional phase I data, this phase II study was initiated to assess the safety and efficacy (response rate) of Ortataxel in patients (pts) with taxane-resistant NSCLC. Methods: Ortataxel was administered IV over 60 min every 3 wks at a dose of 75 mg/m2. Pts with taxane resistant NSCLC, with at least one measurable lesion, ECOG score 0, 1, or 2, adequate hematology and biochemistry, and no more than 2 prior anticancer chemotherapy regimens were eligible. Taxane resistance was defined as PD during treatment (Rx) or within 6 months of Rx with a prior taxane. The protocol was amended to include pts with brain metastases (BM). Results: 98 pts were enrolled. Clean data is available for the first 65 pts: 25 F, 40M; median age 60 (38 - 77); ECOG 0/1/2 - 19/37/9 respectively; prior chemo regimens: 0 (0 pt.), 1 (29 pts.), 2 (34 pts.), 3 (2 pts. received Iressa as 3rd regimen). Median number of cycles administered: 2 (1–11). 89% (58) pts received >90% planned dose intensity. Common drug related non-hematological toxicities included: fatigue 25(38%), nausea 19(29%), myalgia 18(27%), sensory neuropathy 17(26%), vomiting 15(23%), constipation 14(21%), diarrhea 11(17%), arthralgia 10(15%), alopecia 9(13%), stomatitis/pharyngitis 9(13%). Hematologic toxicities seen: G 3–4 neutropenia 49%, G3–4 anemia 7%. 4(6%) pts (without BM) had a PR, 25(38%) pts had SD, 31(48%) pts had PD. 5 pts were not evaluable (missing post baseline evaluations due to consent withdrawal in 2 pts and AE's in 3 pts). Conclusion: Four investigator-confirmed partial responses to Ortataxel were documented in this trial so far. An independent review is underway. The study continues to enroll only pts with BM in order to assess the drug in this subset of patients. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Bayer Healthcare; Pharma-Clinical Ltd.

Discovery of the pyrrolo[2,1-f][1,2,4]triazine nucleus as a new kinase inhibitor template.

The pyrrolo[2,1-f][1,2,4]triazine nucleus was identified as a novel kinase inhibitor template which effectively mimics the well-known quinazoline kinase inhibitor scaffold. Attachment of a 4-((3-chloro-4-fluorophenyl)amino) substituent to the template provided potent biochemical inhibitors of the tyrosine kinase activity of EGFR, as well as inhibition of cellular proliferation of the human colon tumor cell line DiFi. Attachment of a 4-((3-hydroxy-4-methylphenyl)amino) substituent provided potent inhibitors of VEGFR-2 which also showed effects on the VEGF-dependent proliferation of human umbilical vein endothelial cells. Biological activity was maintained with substitution at positions 5 or 6, but not 7, suggesting that the former positions are promising sites for introducing side chains which modulate physicochemical properties. Preliminary inhibition studies with varying ATP concentrations suggest that, like the quinazoline-based kinase inhibitors, the pyrrolotriazine-based inhibitors bind in the ATP pocket.

Azoxymethane-induced pre-adipocyte factor 1 (Pref-1) functions as a differentiation inhibitor in colonic epithelial cells.

Inbred mice differ dramatically in their sensitivity to the colon carcinogen, azoxymethane (AOM). Identifying genes associated with this differential susceptibility in mice may ultimately reveal molecular mechanisms responsible for colon carcinogenesis. A cDNA array approach was taken to study gene expression changes induced by AOM in the colons of sensitive (A/J) and resistant (AKR) mice. Among the genes represented on the array, pre-adipocyte factor 1 (Pref-1), associated previously with suppression of adipocyte differentiation, was induced specifically by AOM in the distal colons of sensitive A/J mice (5.4-fold). Reverse transcription-PCR followed by sequence analysis revealed the presence of four alternative splice variants of Pref-1 mRNA in the colon. The potential significance of Pref-1 in colon tumorigenesis was explored in colon cancer cells infected with a retroviral construct containing the major splice variant. Over-expression of Pref-1A in HT-29 cells led to a marked resistance to butyrate-induced differentiation and growth inhibition. Our data indicate that Pref-1, a protein that suppresses differentiation and promotes colonocyte growth, may account in part for the sensitivity of A/J mice to AOM-induced carcinogenesis. In addition, detection of Pref-1 in a human colon tumor cell line suggests that it may also participate in human colon tumorigenesis.

Identification of 4-anilino-3-quinolinecarbonitrile inhibitors of mitogen-activated protein/extracellular signal-regulated kinase 1 kinase

A high-throughput screen for Ras-mitogen-activated protein kinase (MAPK) signaling inhibitors identified two series (class 1 and 2) of substituted 4-anilino-3-quinolinecarbonitriles as potent (IC(50)s <10 nmol/L) mitogen-activated protein/extracellular signal-regulated kinase 1 (MEK1) kinase inhibitors. These compounds had cyanoquinoline cores, but differed in their respective aniline groups [1a, 1b: 4-phenoxyphenylaniline; 2a, 2b: 3-chloro-4-(1-methylimidazol-2-sulfanyl)aniline]. These compounds were competitive inhibitors of ATP binding by MEK1 kinase, and they had minimal or no effect on Raf, epidermal growth factor receptor (EGFR), Akt, cyclin-dependent kinase 4 (CDK4), or MK2 kinases at concentrations >100-fold higher than those that inhibited MEK1 kinase. Both class 1 and 2 compounds inhibited in vitro growth of human tumor cell lines. A class 2 compound (2b) was the most potent inhibitor of human tumor cell growth in vitro, and this effect was linked to distinct suppression of MAPK phosphorylation in cells. Compound 2b did not affect phosphorylation status of other kinases, such as EGFR, Akt, and stress-activated protein (SAP)/c-jun-NH kinase (Jnk); nor did it affect overall tyrosine phosphorylation level in cells. However, compound 2b did inhibit MEK1 phosphorylation in cells. Inhibition of MEK1 phosphorylation by 2b was not due to a major effect on Raf kinase activity, because enzyme assays showed minimal Raf kinase inhibition. We believe compound 2b inhibits kinase activity upstream of Raf, and thereby affects MEK1 phosphorylation in cells. Even with the dual effect of 2b on MEK and MAPK phosphorylation, this compound was well tolerated and significantly inhibited growth of the human colon tumor cell line LoVo (at 50 and 100 mg/kg BID, i.p.) in a nude mouse xenograft model.

Dietary intervention with the probiotics Lactobacillus acidophilus 145 and Bifidobacterium longum 913 modulates the potential of human faecal water to induce damage in HT29clone19A cells.

Probiotics reduce the risk of colon cancer by inhibiting carcinogen-induced DNA damage in animals, but there are no analogous data in human subjects. To enhance knowledge of the effects of probiotics in human subjects, we have investigated the genotoxicity of faecal water after dietary intervention with standard yoghurt or with probiotic yoghurt, which included the strains Lactobacillus acidophilus 145 and Bifidobacterium longum 913. Faeces were collected from nine healthy volunteers after intervention with probiotic yoghurt or standard yoghurt. Faecal water was isolated and incubated with human colon tumour cells HT29clone19A. DNA strand breaks, oxidised DNA bases and damage after challenge with H2O2 were determined by micro-gel-electrophoresis. Faecal water was genotoxic in comparison with NaCl, but protected against H2O2-induced DNA strand breaks. The intervention with probiotic yoghurt significantly lowered faecal water genotoxicity compared with standard yoghurt. However, probiotic intervention also increased oxidative damage; this either reflected prooxidative activity or stimulation of endogenous defence systems. Altogether, the balance of effects favoured protection, since faecal water from the probiotic group reduced overall genetic damage. Thus, there was a reduction of strand break-inducing compounds in human faeces after dietary intervention with probiotic bacteria. This protection reflected results from previous studies in carcinogen-exposed animals where probiotics reduced DNA damage in colon cells.