HT-29 Human Colon Adenocarcinoma Cells Research Articles

Abstract B46: Dietary acrylamide differentially modulates azoxymethane-induced F344 rat colon tumor formation and ectopic growth of human colon tumor cells in nude (nu/nu) mice

Abstract Acrylamide, a known rodent and a possible human carcinogen is ubiquitously formed in foods at high temperature. We earlier reported that dietary acrylamide at doses known to cause rodent tumors, did not increase the formation of azoxymethane (AOM)-induced colon aberrant crypt foci (ACF), putative precancerous lesions. On the contrary, we observed a significant decrease in ACF in rats receiving acrylamide at 10 and 50 mg/kg diet and a trend to increase ACF at the lowest dose (5mg acrylamide/kg diet) tested. In this study using two rodent models, we assessed if dietary acrylamide, at levels reflecting those in popular human foods, modulated colon tumor formation and progression. In Experiment-1, 4 groups of weanling male F344 rats received AIN-93G diets with acrylamide at 0.5, 1.0 or 2.0 mg/kg diet or without (0 mg/kg diet, control). After two weeks, rats in each diet group received either two weekly s.c. injections of AOM or saline. All rats were on the experimental diets ad libitum for 20 weeks post AOM/saline injections after which they were killed and their colons assessed for tumors. In Experiment-2, athymic nude (nu/nu) mice were s.c. injected with HT-29 human colon adenocarcinoma cells and tumors were allowed to grow for 3 weeks after which they received one of the 4 experimental diets; tumor growth was monitored bi-weekly and mice were killed 4 weeks after. None of the saline-injected rats had tumors in their colon. 23 rats that received acrylamide had blood in their stool at 19 weeks post-AOM injections, and hence the study was stopped in a timely manner at 20 weeks post-AOM injections. The colon tumor incidence was 54.2% in the control group and 66.67% in the highest acrylamide-treated group (2 mg/kg diet). Tumor multiplicity was similar across all diet groups. However, tumor size and burden were higher in the group that received 2 mg/kg acrylamide by comparison to the control. These results suggest acrylamide by itself is not a “carcinogen” at upper exposure levels commonly seen in popular foods, but acts as a “co-carcinogen” at the highest tested dose by exacerbating the risk of developing colon tumors by a known colon-specific carcinogen AOM. The nude mice study indicated no differences in ectopic human colon tumor growth between acrylamide-treated and control mice suggesting that acrylamide at the levels tested does not aid in the progression of established human tumors in this experimental model. Our data provides characterization of acrylamide as a possible colon cancer promoter in association with a known and possibly other environmental tumor initiators/promoters. This additional information will be useful in defining the health risks of acrylamide when exposed through the diet, and will assist regulators in developing a better understanding of the relevance to humans. Citation Information: Cancer Prev Res 2011;4(10 Suppl):B46.

Microanalytical method development for Fe, Cu and Zn determination in colorectal cancer cells

Microanalytical methods suitable for the determination of Fe, Cu in HT-29 (human colon adenocarcinoma) cells treated with different iron compounds (Fe(II) sulfate, Fe(III) chloride, Fe(III) citrate and Fe(III) transferrin) and cultured in medium supplemented or not with 10% (v/v) fetal calf serum (FCS) by total reflection X-ray fluorescence spectrometry (TXRF) and simultaneous graphite furnace atomic absorption spectrometry (GF-AAS) were developed. The developed TXRF method was also suitable for Zn determination in the samples. The main advantage of the proposed methods is the execution of all sample preparation steps following incubation and prior to the elemental analysis in the same Eppendorf tubes. Sample preparation was performed at microscale (115μL sample volume) with 65% nitric acid and 30% hydrogen peroxide. According to scanning electron microscopic measurements, the organic matrix of the cell samples could be eliminated to the extent that accurate results were obtained for Cu and Fe by analyzing the same samples by TXRF and GF-AAS. Concerning the iron uptake, HT-29 cells incubated in FCS-free medium contained Fe in cca. 5–50 times higher amounts compared to cells cultured in FCS supplemented medium. Pronounced differences in the iron uptake compared to the iron supply (inorganic vs. organic chelated as well as iron(II) vs. iron(III)) were observed in the case of cell lines incubated in FCS-free medium.

Novel irreversible EGFR tyrosine kinase inhibitor 324674 sensitizes human colon carcinoma HT29 and SW480 cells to apoptosis by blocking the EGFR pathway

BackgroundEpidermal growth factor receptor (EGFR) is widely expressed in multiple solid tumors including colorectal cancer by promoting cancer cell growth and proliferation. Therefore, the inhibition of EGFR activity may establish a clinical strategy of cancer therapy. MethodsIn this study, using human colon adenocarcinoma HT29 and SW480 cells as research models, we compared the efficacy of four EGFR inhibitors in of EGFR-mediated pathways, including the novel irreversible inhibitor 324674, conventional reversible inhibitor AG1478, dual EGFR/HER2 inhibitor GW583340 and the pan-EGFR/ErbB2/ErbB4 inhibitor. Cell proliferation was assessed by MTT analysis, and apoptosis was evaluated by the Annexin-V binding assay. EGFR and its downstream signaling effectors were examined by western blotting analysis. ResultsAmong the four inhibitors, the irreversible EGFR inhibitor 324674 was more potent at inhibiting HT29 and SW480 cell proliferation and was able to efficiently induce apoptosis at lower concentrations. Western blotting analysis revealed that AG1478, GW583340 and pan-EGFR/ErbB2/ErbB4 inhibitors failed to suppress EGFR activation as well as the downstream mitogen-activated protein kinase (MAPK) and PI3K/AKT/mTOR (AKT) pathways. In contrast, 324674 inhibited EGFR activation and the downstream AKT signaling pathway in a dose-dependent manner. ConclusionOur studies indicated that the novel irreversible EGFR inhibitor 324674 may have a therapeutic application in colon cancer therapy.

Preparation and Photodynamic Activities of Silicon(IV) Phthalocyanines Substituted with Permethylated β‐Cyclodextrins

A series of silicon(IV) phthalocyanines linked to two permethylated β-cyclodextrin moieties through different spacers at the axial positions have been synthesized and characterized. The effects of these spacers on the photophysical properties and in vitro photodynamic activities have also been examined. Having two bulky hydrophilic substituents, all of these compounds are soluble and essentially nonaggregated in DMF and even in aqueous media. The fluorescence and singlet oxygen quantum yields in DMF are lower for the analogue with the shortest separation between the amino group in the spacer and the phthalocyanine ring. The fluorescence quantum yield of this compound increases in water probably due to protonation of the amino group, which inhibits the reductive quenching process. This series of compounds also exhibit photocytotoxicity toward HT29 human colon adenocarcinoma and HepG2 human hepatocarcinoma cells with IC(50) values in the range of 0.04-1.32 μM. The analogue with an α,ω-aminohydroxypentyl linker shows the highest potency, which can be ascribed to its high cellular uptake and high efficiency in generating intracellular reactive oxygen species. This compound also shows preferential localization in the lysosome, induces cell death mainly through apoptosis, and inhibits the growth of tumor in vivo. The results suggest that it is a promising photosensitizer for photodynamic therapy.

Evaluation of the Effects of the Flavonoid Apigenin on Apoptotic Pathway Gene Expression on the Colon Cancer Cell Line (HT29)

Apigenin (4',5,7-trihydroxyflavone) is one of the leading components supporting targeted treatment options. We explored the cytotoxic and apoptotic effects of various doses of apigenin administered alone and together with 5-fluorouracil (5-FU)-a chemotherapeutic agent with high cytotoxicity-for different incubation periods, on morphologic, DNA, RNA (messenger RNA [mRNA]), and protein levels on the p53 mutant HT29 human colon adenocarcinoma cell line. Treatment with apigenin alone for a 72-hour incubation at 90-μM dose resulted in an apoptotic percentage of 24.92% (P=.001). A higher percentage (29.13%) was observed after treatment with the same dose of apigenin plus 5-FU for the same incubation period (P=.001). These results were confirmed as mRNA and protein expression levels of caspase-3 increased 2.567-fold and mRNA expression levels of caspase-8 increased 3.689-fold compared with the control group. On the other hand, mRNA expression levels of mammalian target of rapamycin (mTOR) and cyclin D1 (CCND1) decreased by 0.423-fold and 0.231-fold, respectively. To our knowledge this is the first study showing that treatment with apigenin alone results in cell cycle arrest through activation of caspase cascade and stimulation of apoptosis in HT29 cells. It also shows that use of apigenin plus 5-FU further increases this effect. This study draws attention to the probable clinical effectiveness of apigenin plus a chemotherapeutic agent with high cytotoxicity. It also highlights the induction of desirable apoptotic effects by targeting the caspase cascade pathway through administration of reduced doses for shorter incubation periods.

Effects of adrenaline in human colon adenocarcinoma HT-29 cells

Aims Stress has been implicated in the development of cancers. Adrenaline levels are increased in response to stress. The effects of adrenaline on colon cancer are largely unknown. The aims of the study are to determine the effects of adrenaline in human colon adenocarcinoma HT-29 cells and the possible underlying mechanisms involved. Main methods The effect of adrenaline on HT-29 cell proliferation was determined by [ 3H] thymidine incorporation assay. Expression of cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF) were detected by Western blot. Matrix metalloproteinase-9 (MMP-9) activity and prostaglandin E 2 (PGE 2) release were determined by zymography and enzyme immunoassay, respectively. Key findings Adrenaline stimulated HT-29 cell proliferation. This was accompanied by the enhanced expression of COX-2 and VEGF in HT-29 cells. Adrenaline also upregulated MMP-9 activity and PGE 2 release. Adrenaline stimulated HT-29 cell proliferation which was reversed by COX-2 inhibitor sc-236. COX-2 inhibitor also reverted the action of adrenaline on VEGF expression and MMP-9 activity. Further study was performed to determine the involvement of β-adrenoceptors. The stimulatory action of adrenaline on colon cancer growth was blocked by atenolol and ICI 118,551, a β 1- and β 2-selective antagonist, respectively. This signified the role of β-adrenoceptors in this process. In addition, both antagonists also abrogated the stimulating actions of adrenaline on COX-2, VEGF expression, MMP-9 activity and PGE 2 release in HT-29 cells. Significance These results suggest that adrenaline stimulates cell proliferation of HT-29 cells via both β 1- and β 2-adrenoceptors by a COX-2 dependent pathway.

Lithocholic Acid Is an Eph-ephrin Ligand Interfering with Eph-kinase Activation

Eph-ephrin system plays a central role in a large variety of human cancers. In fact, alterated expression and/or de-regulated function of Eph-ephrin system promotes tumorigenesis and development of a more aggressive and metastatic tumour phenotype. In particular EphA2 upregulation is correlated with tumour stage and progression and the expression of EphA2 in non-trasformed cells induces malignant transformation and confers tumorigenic potential. Based on these evidences our aim was to identify small molecules able to modulate EphA2-ephrinA1 activity through an ELISA-based binding screening. We identified lithocholic acid (LCA) as a competitive and reversible ligand inhibiting EphA2-ephrinA1 interaction (Ki = 49 µM). Since each ephrin binds many Eph receptors, also LCA does not discriminate between different Eph-ephrin binding suggesting an interaction with a highly conserved region of Eph receptor family. Structurally related bile acids neither inhibited Eph-ephrin binding nor affected Eph phosphorylation. Conversely, LCA inhibited EphA2 phosphorylation induced by ephrinA1-Fc in PC3 and HT29 human prostate and colon adenocarcinoma cell lines (IC50 = 48 and 66 µM, respectively) without affecting cell viability or other receptor tyrosine-kinase (EGFR, VEGFR, IGFR1β, IRKβ) activity. LCA did not inhibit the enzymatic kinase activity of EphA2 at 100 µM (LANCE method) confirming to target the Eph-ephrin protein-protein interaction. Finally, LCA inhibited cell rounding and retraction induced by EphA2 activation in PC3 cells. In conclusion, our findings identified a hit compound useful for the development of molecules targeting ephrin system. Moreover, as ephrin signalling is a key player in the intestinal cell renewal, our work could provide an interesting starting point for further investigations about the role of LCA in the intestinal homeostasis.

Semisynthesis of triptolide analogues: Effect of γ-lactone and C-14 substituents on cytotoxic activities

Triptolide γ-lactone and C-14 analogues were prepared and evaluated cytotoxity against human lung adenocarcinoma epithelial A549 cells and human colon adenocarcinoma HT-29 cells. γ-Lactone substructure and C-14 substituents affected the biological activities significantly.

Target‐Selective Drug Delivery through Liposomes Labeled with Oligobranched Neurotensin Peptides

The structure and the in vitro behavior of liposomes filled with the cytotoxic drug doxorubicin (Doxo) and functionalized on the external surface with a branched moiety containing four copies of the 8-13 neurotensin (NT) peptide is reported. The new functionalized liposomes, DOPC-NT₄Lys(C₁₈)₂, are obtained by co-aggregation of the DOPC phospholipid with a new synthetic amphiphilic molecule, NT₄ Lys(C₁₈)₂, which contains a lysine scaffold derivatized with a lipophilic moiety and a tetrabranched hydrophilic peptide, NT8-13, a neurotensin peptide fragment well known for its ability to mimic the neurotensin peptide in receptor binding ability. Dynamic light scattering measurements indicate a value for the hydrodynamic radius (RH) of 88.3±4.4 nm. The selective internalization and cytotoxicity of DOPC-NT₄ Lys(C₁₈)₂ liposomes containing Doxo, as compared to pure DOPC liposomes, were tested in HT29 human colon adenocarcinoma and TE671 human rhabdomyosarcoma cells, both of which express neurotensin receptors. Peptide-functionalized liposomes show a clear advantage in comparison to pure DOPC liposomes with regard to drug internalization in both HT29 and TE671 tumor cells: FACS analysis indicates an increase in fluorescence signal of the NT₄-liposomes, compared to the DOPC pure analogues, in both cell lines; cytotoxicity of DOPC-NT₄ Lys(C₁₈)₂-Doxo liposomes is increased four-fold with respect to DOPC-Doxo liposomes in both HT29 and TE671 cell lines. These effects could to be ascribed to the higher rate of internalization for DOPC-NT₄ Lys(C₁₈)₂-Doxo liposomes, due to stronger binding driven by a lower dissociation constant of the NT₄-liposomes that bind the membrane onto a specific protein, in contrast to DOPC liposomes, which approach the plasma membrane unselectively.

Fermented Nondigestible Fraction from Common Bean (Phaseolus vulgaris L.) Cultivar Negro 8025 Modulates HT‐29 Cell Behavior

The aim of the study was to evaluate the effect of a fermented nondigestible fraction (FNDF) of cooked bean (Phaseolus vulgaris L.) cultivar Negro 8025 on human colon adenocarcinoma HT-29 cell survival. Negro 8025 was chosen for in vitro fermentation based on comparison of chemical composition with 2 other cultivars: Azufrado Higuera and Pinto Durango. Negro 8025 had 58% total dietary fiber, 27% resistant starch, and 20 mg of (+)-catechin equivalents per gram of sample. Short-chain fatty acids (SCFAs) production and pH of the medium were measured after fermentation as indicators of colon protection through induced arrest on cell culture and apoptosis. Butyrate and pH of FNDF of Negro 8025 were higher than the control fermented raffinose extract. The FNDF inhibited HT-29 cell survival in a time- and concentration-dependent manner. The lethal concentration 50 (LC(50)) was 13.63% FNDF (equivalent to 7.36, 0.33, and 3.31 mmol of acetic, propionic, and butyric acids, respectively). DNA fragmentation, an apoptosis indicator, was detected by the TdT-mediated dUTP nick end labeling method in cells treated with the LC(50)-FNDF and a synthetic mixture of SCFAs mimicking LC(50)-FNDF. Our results suggest that common bean is a reliable source of fermentable substrates in colon, producing compounds with potential chemoprotective effect on HT-29 colon adenocarcinoma cells, so it may present an effective alternative to mitigate colon cancer development.

Thermodynamics of Solution, Interaction with Calf Thymus DNA and Anticancer Activity of Phenylhydrazone Derivatives

The solubilities in water (W) and in 3 mol⋅L−1 aqueous acetonitrile at 298.15 K of thiophene-2-, furan-2- and pyrrole-2-carboxaldehyde phenylhydrazone along with their nitro and 2,4-dinitro derivatives, referred to as PCT, PCF, PCP, NPCT, NPCF, NPCP, DPCT, DPCF and DPCP, respectively, are reported. The standard Gibbs energy of transfer from W to solvent mixtures (\(\Delta G^{\mathrm{transf}}_{\mathrm{W-mix}}\)) was estimated for each solute. The results indicate that the transfer process for all systems is a spontaneous process. Calculations under the scaled-particle theory suggest that the work of cavity creation (ΔΔGc), which is associated with the transfer of the phenylhydrazone from W to mix, dominates the magnitude of \(\mathrm {\Delta} G^{\mathrm{transf}}_{\mathrm{W-mix}}\). UV spectroscopic measurements suggest that these compounds bind calf thymus(CT)-DNA via intercalation mode in a buffer–3 mol⋅L−1 acetonitrile solution. The binding constant (Kb) depends on the nitro-substitution on the phenyl moiety and the electronegativity of the heteroatom in the heterocyclic ring. The corresponding Gibbs energy of binding (\(\Delta G_{\mathrm{DNA-B}}^{0}\)) of phenylhydrazone derivatives to CT-DNA shows a marked dependence with ΔΔGc. The anticancer activity on human breast cancer cell lines MDA-231, MDA-435 and HT-29 human colon adenocarcinoma cell line was evaluated for the compounds NPCT, NPCF, DPCT and PCT.

Phthalocyanine−Polyamine Conjugates as Highly Efficient Photosensitizers for Photodynamic Therapy

A series of silicon(IV) phthalocyanines substituted axially with different polyamine moieties have been prepared. Their fluorescence quantum yields (Φ(F) = 0.03-0.08) in N,N-dimethylformamide are low because of reductive quenching by the amino moieties. The values are significantly increased in aqueous media (Φ(F) = 0.12-0.21) as a result of protonation of the amino substituents. All the compounds are highly photocytotoxic against human colon adenocarcinoma HT29 cells and Chinese hamster ovary cells with IC(50) values as low as 1.1 nM. Flow cytometric studies of two selected compounds (2 and 5) against HT29 cells have shown that they induce apoptosis extensively. As shown by confocal microscopy, these two compounds also show high affinity toward the lysosomes, but not the mitochondria, of the cells. Their in vivo photodynamic activity has also been investigated using HT29 tumor bearing nude mice. Both of them can effectively inhibit the growth of the tumor without causing apparent injury to the liver of the mice.

Nem-szteroid gyulladásgátlók alkalmazása az 5-fluorouracil hatékonyságának növelésére kísérletes rendszerekben

The elevated cyclooxygenase-2 (COX-2) expression has been shown to affect the carcinogenesis and tumor progression processes, including cell proliferation, motility and angiogenesis. COX-2 is overexpressed in approximately 80% of sporadic colorectal carcinomas and COX-2 enzyme is the best defined target of non-steroidal anti-inflammatory drugs (NSAIDs). In the chemotherapy of colorectal carcinomas 5-fluorouracil (5-FU) has been the most important of the basic drugs for more than 40 years. In order to improve the effectiveness of 5-FU therapy different biological modifiers i.e. inhibitors of its catabolism or activators of anabolism have been studied recently. The rate-limiting enzyme of 5-FU catabolism is dihydropyrimidine dehydrogenase (DPD) since more than 80% of the administered 5-FU is catabolized by DPD. Tumoral DPD has become of clinical interest because elevated intratumoral DPD can decrease the tumor response to 5-FU therapy. The main purpose of our experiments was to investigate the effect of COX inhibitors on the efficacy of 5-FU on high and low COX-2 expressing HCA-7 and HT-29 human colon adenocarcinoma cell lines, respectively, and also on xenografts derived from HT-29 cells. The cytotoxic and antitumor effects of 5-FU in the presence of low doses of indomethacin (non-selective COX-2 inhibitor) and that of NS-398 (highly selective COX-2 inhibitor) on HT-29 and HCA-7 cells and also on the HT-29 xenograft were investigated. In addition, our intention was to understand the mechanism(s) by which NSAIDs could enhance the cytotoxic effect of 5-FU. Our data indicated that the elevated COX-2 expression of HCA-7, the collagen-induced HT-29-C cells and of the HT-29 xenograft were associated with reduced 5-FU sensitivity. Based on the fact that at the same time DPD activity was also increased it might be conceivable that a possible explanation for the decrease of 5-FU sensitivity is the co-existence of high COX-2 and DPD activity. Indomethacin or NS-398 enhanced in a simultaneous and significant manner the sensitivity and cytotoxic effect of 5-FU on high COX-2 expressing cells and xenografts through the modulation of DPD - decrease of its mRNA expression and/or enzyme activity. Based on our results it could be presumable that 5-FU efficacy is limited by the COX-2 associated high DPD expression and activity in patients with colorectal cancer as well, therefore further clinical studies are warranted to decide if NSAIDs in the therapeutic protocol might improve the antitumor potency of 5-FU. Réti A. Application of non-steroidal anti-inflammatory drugs to enhance 5-fluorouracil efficacy in experimental systems.

Abstract 2562: Role of Pyruvate Kinase isozyme type M2 in response and resistance to oxaliplatin in HT29 human colon adenocarcinoma cell lines

Abstract BACKGROUND: Resistance to platinum-based chemotherapy is a multifactorial and complex process resulting in a major obstacle for effective treatment. In a previous work we identified low levels of PKM2 (mRNA and protein) as a putative oxaliplatin-resistance marker by using an in vitro model of resistance and a comparative proteomic approach. Our results were also validated in advanced colorectal cancer patients treated with oxaliplatin (Martinez-Balibrea, Molecular cancer therapeutics, 2009). PKM2 has been reported to translocate to the nucleus and activate apoptosis after H2O2, UV or a somatostatin analog treatment. The aim of this work was to study the role of PKM2 in the molecular mechanisms involved in response and resistance to oxaliplatin and identify proteins related to PKM2 expression levels. EXPERIMENTAL DESIGN: We silenced PKM2 expression by using siRNA techniques in the human adenocarcinoma cell line HT29. Validation of knockdown was done by Western Blotting (WB) and RTQPCR. To study the effect of PKM2 silencing in the resistance phenotype an MTT assay was performed to compare the viability between PKM2-silenced cells and those transfected with an unspecific siRNA (NTC). Cells were treated 24 hours with oxaliplatin at 15 µM. To verify the PKM2 nuclear translocation and its possible role in activating apoptosis after H2O2 and oxaliplatin treatment, HT29 and HCT116 cells were treated with H2O2 and oxaliplatin at equivalent doses and harvested at 0, 2 and 4h after treatment. Sub-cellular fractionation and WB as well as immunoflorescenece techniques were performed to visualize nuclear translocation of PKM2. To study possible changes in the proteome of HT29 cells as a consequence of PKM2 silencing, 2D- DIGE and Mass Spectrometry techniques were performed. RESULTS: Forty-eight hours after transfection we obtained a 95% of PKM2 knockdown by RTQPCR (mRNA level) and also by WB (protein level). A slightly increase of viability was observed in PKM2-silenced cells in comparison to NTC cells after oxaliplatin exposition (50% vs. 35%). Preliminary results showed that both oxaliplatin and H2O2 induced PKM2 nuclear translocation in HT29 and HCT116 cells 4 hours after the exposition to these agents. By 2D-DIGE we determined changes in the proteome of HT29 cells as a consequence of PKM2 silencing. The identification and validation of those proteins showing up- or down-regulation are in process and will be presented during the annual meeting. CONCLUSIONS: PKM2 knockdown alters sensitivity to oxaliplatin and protein expression patterns in HT29 cells. Oxaliplatin induces nuclear translocation of PKM2. Further studies are guaranteed to evaluate the role of PKM2 in activating apoptosis after oxaliplatin exposure. 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 2562.

Antiproliferative Activity of Greek Propolis

The butanolic extract and the isolated chemical constituents, mainly diterpenes and flavonoids, from Greek propolis have been tested for their cytostatic activities against human malignant and normal cell strains. The extract and the diterpenes were found to be the most active against HT-29 human colon adenocarcinoma cells, without affecting normal human cells. Manool, a diterpene isolated for the first time from Greek propolis, was the most active compound, arresting the cancer cells at the G(2)/M phase of the cell cycle.

Synthesis, molecular modeling and biological evaluation of novel tadalafil analogues as phosphodiesterase 5 and colon tumor cell growth inhibitors, new stereochemical perspective

The synthesis of novel tadalafil analogues in which the benzodioxole moiety is replaced by 2-bromophenyl; the chiral carbons swing from R, R to R, S, S, R and S, S; the piperazinedione ring is maintained or reduced to the 5-membered imidazolidinedione or thioxoimidazolinone is described. The prepared analogues were evaluated for their capacity to inhibit the cyclic guanosine monophosphate (cGMP) selective phosphodiesterase 5 (PDE5) isozyme and the growth of human HT-29 colon adenocarcinoma cells. The R absolute configuration of C-5 in the β-carboline-hydantoin and C-6 in the β-carboline-piperazinedione derivatives was found to be essential for the PDE5 inhibition. In addition, tadalafil analogues that were synthesized from l-tryptophan were more active than those derived from d-tryptophan, which is of economic value and expands the horizon for the discovery of new carbolines as PDE5 inhibitors. While some analogues displayed potent tumor cell growth inhibitory activity, there was no apparent correlation with their PDE5 inhibitory activity, which leads us to conclude that other PDE isozymes or PDE5 splice variants may be involved.

Fish fatty acids alter markers of apoptosis in colorectal adenoma and adenocarcinoma cell lines but fish consumption has no impact on apoptosis-induction ex vivo

Previous studies suggest that the n-3 polyunsaturated fatty acids (PUFAs) eicosapenteinoic acid (EPA) and docosahexaenoic acid (DHA), constituents of fish oil, exert chemopreventive activity in colon cancer. One of the mechanisms involved is the facilitation of apoptosis. While a pro-apoptotic potential of n-3 PUFAs has been suggested, it is still unclear whether additional consumption of fish will also lead to comparable results. The aim of this study was to assess EPA- and DHA-mediated effects on endpoints of apoptosis and to use a novel biomarker-approach to measure modulation of apoptosis by consumption of fish. LT97 human colon adenoma and HT29 human colon adenocarcinoma cells were used to investigate modulation of apoptosis by EPA, DHA or linoleic acid (LA) using a set of endpoints, namely phosphatidylserine staining with Annexin-V (flow cytometry), Bcl-2 expression (Real-time RT-PCR), and Bid, caspase 3, 8 and 9 expression as well as PARP cleavage (Western Blot). Furthermore, faecal water (FW) of volunteers (n = 89) from a human trial intervening with fish was used to investigate changes in apoptosis by flow cytometry. DHA was more effective at inducing apoptosis than EPA. LT97 cells were more prone to DHA and EPA induced apoptosis than HT29 cells. Treatment of LT97 cells with FW from volunteers consuming fish did not result in any changes in apoptosis. Taken together, our results show that adenoma cells are highly susceptible to n-3 PUFA-induced apoptosis. By using a biomarker-approach (FW) to measure apoptosis-induction ex vivo no change in apoptosis after additional fish consumption was detectable.

Synthesis and photobiological properties of novel silicon(IV) phthalocyanines axially modified by paracetamol and 4-hydroxyphenylacetamide

Two novel axial-disubstituted silicon(IV) phthalocyanines (compounds 1 and 2) have been prepared by introducing paracetamol (a common antipyretic analgesic) or its isomer 4-hydroxyphenylacetamide at the axial positions of silicon(IV) phthalocyanine, respectively. Their photophysical and biological properties have been examined. Both compounds are highly soluble and exhibit very similar absorption spectra in N, N-dimethylformamide, which is typical for non-aggregated phthalocyanines. Both compounds are photocytotoxic against HT29 human colon adenocarcinoma cells. Compound 2 shows a very high in vitro photodynamic activity, with the IC50 value down to 15 nM. In contrast, compound 1 exhibits a much lower in vitro photodynamic activity toward HT29 cells, which can be attributed to its higher aggregating trend in the biological medium and lower singlet oxygen quantum yield.

Bowman-Birk inhibitors in lentil: Heterologous expression, functional characterisation and anti-proliferative properties in human colon cancer cells

A full-length cDNA, encoding a Bowman-Birk protease inhibitor (BBI), was isolated from lentil immature seeds. The deduced amino acid sequence was longer than that of the BBI extracted from lentil seeds and contained two binding sites; the first inhibitory site inhibits trypsin whereas the second one inhibits chymotrypsin. In order to characterize this lentil BBI, a longer (complete) and its C-terminally processed (mature) form were heterologously expressed in the yeast Pichia pastoris. The recombinant BBI proteins proved to be active against trypsin and chymotrypsin, showing Ki values at nanomolar levels. Mass spectrometry analysis revealed that complete BBI was composed of an array of molecular masses, whereas mature BBI showed the presence of a major peak of the expected size. The effects of mature BBI on the growth of human colon adenocarcinoma HT29 and colonic fibroblast CCD-18Co cells were evaluated. Lentil BBI was able to inhibit the growth of such cells at concentrations higher than 19 μM, in a concentration-dependent manner; by contrast, the CCD18-Co cells were unaffected. These data broaden our knowledge of the beneficial biological activities of naturally-occurring BBI proteins and address the need for systematic evaluation of natural variants in order to design novel strategies in preventive medicine.

Widdrol induces cell cycle arrest, associated with MCM down-regulation, in human colon adenocarcinoma cells

Widdrol, an odorous compound extracted from Juniperus chinensis, has been shown to inhibit the in vitro growth of in human cancer cells. This study was conducted on cultured human colon adenocarcinoma HT29 cells to elucidate the possible mechanisms by which widdrol exerts its anti-proliferative activity, which until now has remained poorly understood. It was found that widdrol induces accumulation of sub-G1 phase and arrests in the G1 phase of the cell cycle. Induction of G1 arrest by widdrol was correlated with induction of Chk2, p53 phosphorylation and CDK inhibitor p21 expression as well as inhibition of cyclin E, cyclin-dependent kinase (CDK2) and retinoblastoma protein (pRB). Moreover, mini-chromosome maintenance (MCM) proteins were markedly down-regulated in HT29 cells treated with widdrol. Altogether, these results show widdrol possesses potential anti-cancer activity against colon adenocarcinoma cells by inhibiting their proliferation and inducing cell cycle G1 arrest.