Growth Inhibition Of HT-29 Cells Research Articles

Identification of functional compounds in baru (Dipteryx alata Vog.) nuts: Nutritional value, volatile and phenolic composition, antioxidant activity and antiproliferative effect

This work aimed to contribute to the nutritional and functional characterization of roasted baru nuts, a seed widely consumed and produced in Brazil. Baru nut was characterized in terms of its nutritional value and volatile composition (SPME-GC–MS analysis). The ultrasound assisted extraction was used to extract free and bound phenolic compounds that were identified by LC-DAD-ESI-MS/MS method. Bioactivity assays were carried out to evaluate the antioxidant activity (ORAC and HOSC assay) and anticancer effect (inhibition of HT29 cell growth and targeting of cancer stemness) of baru nut extracts and phenolic compounds. Results showed that baru is a good source of protein and monounsaturated fatty acids, specifically oleic acid (47.20 g/100 g). The predominant volatile compounds are hexanal (71.18%) and 2,5-dimethyl-pyrazine (9.43%). The main phenolic compounds identified were gallic acid and its derivatives, such as gallic acid esters and gallotannins. Among all, gallic acid and methyl gallate seemed to be the main compounds responsible for the high antioxidant activity. The antiproliferative effect evaluated of baru extracts in HT29 cell line showed ability to impair cell growth in both monolayer and spheroid cultures and to reduce ALDH+ population. These results supply new information about the functional compounds presents in baru nut, which are important sources of natural antioxidants and antiproliferative compounds.

Wpływ ekstraktów z liści kaliny koralowej (Viburnum opulus L.) na wzrost ludzkich komórek jelita

Introduction. Cranberrybush (Viburnum opulus L.) is a plant rich in phenols. Chlorogenic acid is a main compound responsible for its antioxidant activity. This plant also has antibacterial and anti-inflammatory properties, for this reason it is used for prevention, as well as for the treatment of many diseases, mainly those of the genitourinary tract, but also of the heart or lungs. So far, there are few studies that suggest V. opulus has anti-cancer activity and could become a supplement in such therapies. Aim. The aim of the study was to assess and compare the effect of both leaf extract (ELK) and phenol-rich leaf extract from cranberrybush (FELK) on the growth of colon cancer and normal colon cells. Material and methods. The HPLC method allowed to estimate the chemical composition of ELK and FELK. In addition, two colon cancer cell lines HT29 and SW480 were used, as well as normal epithelial cells CCD841CoN. The cells were treated with various concentrations of V. opulus extracts. The cell viability was assessed by MTT test after 24 h, 48 h and 72 h, respectively. Results. ELK and FELK extracts had moderate effect on the inhibition of HT29 cell growth. The cell viability of SW480 was much more pronounced after ELK and FELK treatment. The FELK extract inhibited the growth of HT29 and SW480 more significantly compared to ELK. The growth of CCD841CoN cells was higher after FELK treatment than ELK. Conclusions. The purified leaf extract of V. opulus (FELK) was richer in phenolic compounds than the unpurified ELK, more significantly inhibited the growth of HT29 and SW480 colon cancer cells as well as had a more gentle influence on the growth of normal epithelial CCD841CoN colon cells.

A natural flavonoid lawsonaringenin induces cell cycle arrest and apoptosis in HT-29 colorectal cancer cells by targeting multiple signalling pathways.

Colorectal cancer is the third most common malignancy in the world having a high mortality rate. Flavonoids possess many biological activities including anti-cancer activity. lawsonaringenin (LSG) is a flavonoid isolated from leaves of Lawsonia alba Lam. The objective of this study was to demonstrate the anti-cancer potential of LSG in colorectal cancer for the first time. The HT-29 cells were treated with LSG or 5-fluoruracil, as a positive control, to determine its effect on cell cytotoxicity by a MTT cell proliferation assay, and cell cycle progression and apoptosis using flowcytometry. We also determined the mechanisms underlying LSG-mediated growth inhibition of HT-29 cells by by investigating the expression of key oncogenes and apoptosis genes using q-RT PCR and immunocytochemical analysis. The cell cytotoxicity data showed that the IC50 value of LSG was significantly less than the IC50 value of 5-FU (50µM). The anti-proliferative effect of LSG was mediated by arresting cells in the S phase of the cell cycle which then led to the induction of apoptosis the q-RT PCR and immunocytochemical analysis showed that LSG reduced the expression of ß-catenin (non-phosphorylated) and its downstream signalling target c-Myc, whereas it increased the phosphorylation of ß-catenin. Furthermore, LSG also downregulated the expression of oncogene K-Ras and anti-apoptotic proteins, Bcl-2, and Bcl-xL. In conclusion, our data demonstrates that LSG exerted its anti-tumor activity by arresting the cell cycle in S phase, and by downregulating the expression of oncogenes including ß-catenin, c-Myc, K-Ras and anti-apoptosis proteins Bcl-2 and Bcl-xL. This study suggests a potential use of natural flavonoid, lawsonaringenin, to attenuate colorectal cancer growth; however, further pre-clinical/clinical studies are required to establish its role as a therapeutic agent.

Treatment mechanism of matrine in combination with irinotecan for colon cancer.

The inhibitory effect of matrine (MA) was studied in combination with irinotecan (CPT-11) on proliferation of human colon carcinoma cell line HT29. We also explored the mechanism of cell apoptosis induction in HT29. HT29 cells were treated with different concentrations of MA and CPT-11 alone and in combination. The growth inhibition in HT29 cells was evaluated using MTT assay. Apoptosis was detected using AV-PI double staining flow cytometry. Transmission electron microscopy was used to detect structural changes in cells. Topoisomerase (TOPO) I, Bax and Caspase-3 expression levels were evaluated using western blot analysis. MA and CPT-11 alone and in combination, inhibited the proliferation of HT29 cells, whereas the combination treatment exhibited higher inhibitory effect (P<0.01). This suggests the existence of synergistic cytotoxicity. Compared with each treatment alone, the combination treatment caused more significant damage to cell structure, and caused a significantly higher apoptosis rate (P<0.01). Additionally, the combination treatment increased TOPO I, Bax and Caspase-3 expression levels (P<0.01). In conclusion, MA in combination with CPT-11 synergistically inhibited HT29 cell proliferation and induced apoptosis in these cells. The mechanism may be related to upregulation of the TOPO I, Bax and Caspase-3 protein expression.

Molecular mechanisms of apoptosis and cell selectivity of zinc dithiocarbamates functionalized with hydroxyethyl substituents

In the solid state each of three binuclear zinc dithiocarbamates bearing hydroxyethyl groups, {Zn[S2CN(R)CH2CH2OH]2}2 for R=iPr (1), CH2CH2OH (2), and Me (3), and an all alkyl species, [Zn(S2CNEt2)2]2 (4), features a centrosymmetric {ZnSCS}2 core with a step topology; both 1 and 3 were isolated as monohydrates. All compounds were broadly cytotoxic, specifically against human cancer cell lines compared with normal cells, with greater potency than cisplatin. Notably, some selectivity were indicated with 2 being the most potent against human ovarian carcinoma cells (cisA2780), and 4 being more cytotoxic toward multidrug resistant human breast carcinoma cells (MCF-7R), human colon adenocarcinoma cells (HT-29), and human lung adenocarcinoma epithelial cells (A549). Based on human apoptosis PCR-array analysis, caspase activities, DNA fragmentation, cell apoptotic assays, intracellular reactive oxygen species (ROS) measurements and human topoisomerase I inhibition, induction of apoptosis in HT-29 cells is demonstrated via both extrinsic and intrinsic pathways. Compounds 2–4 activate the p53 gene while 1 activates both p53 and p73. Cell cycle arrest at the S and G2/M phases correlates with inhibition of HT-29 cell growth. Cell invasion is also inhibited by 1–4 which is correlated with down-regulation of NF-κB.

Research on Biological Materials with Oxazinone Derivatives Induce Apoptosis in HT-29 Cells

Objective: To monitor the induction of apoptosis in HT-29 cells by three compounds of oxazinone derivatives (A-C) with similar structures and research the mechanism of those oxazinone derivatives in induction of apoptosis in HT-29 cells. Methods: HT-29 cells were used for the determination of cytotoxicity elicited by oxazinone compounds. Cytotoxic effects of these compounds in HT-29 cells are monitored by a Real-Time Cell Analyser system. Results: All the oxazinone derivatives exhibited growth inhibition in HT-29 cells in a concentration-dependent manner. Conclusion: All the three compounds of oxazinone derivatives (A-C) could exhibit growth inhibition in HT-29 cells in a dose-dependent manner.

Beta-catenin mediates the apoptosis induction effect of celastrol in HT29 cells

AimWe evaluated the apoptosis induction effects of celastrol in human colorectal cancer cell line HT29 in WNT/beta-catenin pathway. Main methodsHT29 cells were treated with various concentrations (10–100μM) for 24h, MTT assay was performed to examine the effect of celastrol on growth inhibition of HT29 cells. Beta-catenin siRNA was used for transfection of cells. Cell apoptosis was detected through both DNA laddering analysis and Tdt-mediated dUTP nick end labeling (TUNEL) assay. Western blot analysis and real-time reverse transcription polymerase chain reaction technologies were applied to assess the expression level of c-Myc, Bax, and Bcl-2 in HT29 cells. Key findingsTreatment of HT29 cells with celastrol resulted in a growth inhibition effect, and the IC50 value was 56μM. Celastrol induced HT29 cells apoptosis, and increased the nuclear translocation of beta-catenin. Apoptosis induction effects of celastrol were significantly attenuated by beta-catenin siRNA transfection. Beta-catenin siRNA markedly increased mRNA and protein levels of c-Myc compared with control siRNA. Beta-catenin siRNA significantly inhibited the expression of Bax and Bcl-2 in celastrol-treated HT29 cells. SignificanceBeta-catenin mediates the apoptosis induction effects of celastrol in HT29 cells.

Acetylbritannilactone suppresses growth via upregulation of kr�ppel-like transcription factor 4 expression in HT-29 colorectal cancer cells

Acetylbritannilactone (ABL) is a new active compound isolated from Inula Britannica L, a traditional Chinese medicinal herb. It has been reported that ABL can inhibit the proliferation of vascular smooth muscle cells (VSMCs) and neointima formation after balloon injury in rats. ABL also shows chemopreventive properties by inducing cell apoptosis in breast and ovarian cancers, but the antitumor activity and the molecular targets of ABL in colon cancer cells have not been determined. In this study, we showed that ABL inhibits the growth in dose- and time-dependent manners by inducing cell cycle arrest in G0/G1 phase of HT-29 human colon cancer cells. This suppression was accompanied by a strong decrease of cyclin E and CDK4 protein levels, and an increase in p21 protein expression in HT-29 cells. We also show that ABL-induced growth inhibition is associated with the upregulation of KLF4 expression. The overexpression of KLF4 by infection with pAd-KLF4 resulted in growth inhibition, with decrease in the protein levels of cyclin E and CDK4, and increase in the expression of p21, similarly to the effects of ABL. Conversely, knockdown of KLF4 using a specific siRNA impaired the ABL-induced growth inhibition in HT-29 cells. These results suggest that KLF4 as an important cellular target of ABL mediates the growth inhibition of HT-29 cells induced by ABL via upregulation of p21 expression.

Ghrelin inhibits 5‐fluorouracil‐induced apoptosis in colonic cancer cells

The aim of the present study was to investigate if ghrelin inhibits apoptosis in colonic cancer cells. Cell viability in HT-29 cells was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptosis was measured using 4',6-diamidino-2-phenylindole staining and flow cytometry. The protein expression of Bcl-2, Bax, and caspase-3 activation was examined using Western blotting. Ghrelin dose dependently decreased the growth inhibition of HT-29 cells induced by 5-fluorouracil (5-FU). Cells treated with 5-FU displayed chromatin condensation and nuclear fragmentation, which are typical changes of apoptosis. However, co-treatment with ghrelin reduced these changes. Flow cytometry after staining with Annexin V and propidium iodide showed that ghrelin decreased the apoptotic rate of HT-29 cells induced by 5-FU. Caspase-3 activation was significantly lower in the co-treated group than in the group treated with 5-FU alone. In addition, ghrelin reversed the 5-FU-induced Bcl-2/Bax protein ratio. Ghrelin inhibits 5-FU-induced apoptosis in colon cancer cells through the regulation of the Bcl-2/Bax protein ratio.

Lubiprostone Suppresses Growth of Colon Cancer Cells in vitro and in vivo

Purpose: Colon and other tumors show low levels of expression of 15-hydroxy-prostaglandin dehydrogenase [NAD+] (HPGD), also known as 15-PGDH, a prostone synthetase, which converts prostaglandins to their metabolites, prostones. 15-PGDH, a tumor suppressor, is low in HT-29 colon cancer cells. Deficiency in 15-PGDH results in high prostaglandin levels (and perhaps low prostone levels), which in turn may lead to stimulation of tumor growth. Lubiprostone is a prostone derivative that activates ClC-2 Cl- channels and is used for the treatment of chronic idiopathic constipation in adults and irritable bowel syndrome with constipation in adult women. Cellular effects of lubiprostone include activation of ion channels, subsequent membrane hyperpolarization, reduced intracellular free calcium ([Ca2+]i), and no effect on [cAMP]i. In contrast, PGE1 and PGE2 cause membrane depolarization and increased [Ca2+]i and [cAMP]i by binding to EP receptors (Prostaglandin and Other Lipid Mediators 86: 56-60, 2008). The effect of lubiprostone on tumor growth rate was investigated using wild-type, mock-transfected and 15-PGDH-transfected HT-29 colon cancer cells and HT-29-derived tumors orthotopically implanted on the cecum mucosa of nude mice. Methods: For in vitro studies, HT-29 cells were plated in 60-mm Petri dishes in triplicate and counted in a 1 mm2 area using a microscope grid in a randomly chosen field. Lubiprostone or vehicle (0.1% DMSO) was added after 48 h of plating and cells were then counted 24, 48, 72 and 96 h later. For the animal study, HT-29 tumors were surgically implanted on the cecum mucosa in nude mice and tumor sizes were measured once/week. Lubiprostone and vehicle were given daily orally at day 15 after tumor implantation and monitored at day 22 and day 29 after implantation. Results: Growth of HT-29 cells was significantly inhibited even at 24 h by 100 nM lubiprostone in vitro and inhibited up to about 73% by 72 h. Lubiprostone inhibition of HT-29 cell growth was dose dependent with IC50=1.8±0.3 nM. In PGDH-transfected-HT-29 cells, the inhibitory effect of lubiprostone was lost compared to mock-transfected or non-transfected cells. Lubiprostone (1, 3, 5 mg/kg) also significantly suppressed growth of HT-29 orthotopically implanted tumors by 40-50%. Conclusion: Lubiprostone suppresses growth of HT-29 colon cancer cells in vitro and HT-29 tumor growth in vivo. The mechanism might involve interference with the growth effects caused by high prostaglandin levels in tumor cells with low 15-PGDH (low prostone synthetase activity). These results suggest that lubiprostone might be useful for treatment and/or prevention of colon and potentially other cancers. Supported by Sucampo Pharmaceuticals, Inc. Disclosure: John Cuppoletti is a consultant to, holds stock options in, and has a research grant from Sucampo Pharmaceuticals Inc. Danuta H. Malinowska and Jayati Chakrabarti are supported by the grant from Sucampo Pharmaceuticals Inc. Takashi Sekida is an employee of Sucampo Pharmaceuticals, Inc. Ryuji Ueno is CEO and Chief Scientific Officer, Chairman of the Board and Founder of Sucampo Pharmaceuticals, Inc. This research was supported by an industry grant from Sucampo Pharmaceuticals Inc.

Rosiglitazone enhances the radiosensitivity of p53-mutant HT-29 human colorectal cancer cells

Combined-modality treatment has improved the outcome in cases of various solid tumors, and radiosensitizers are used to enhance the radiotherapeutic efficiency. Rosiglitazone, a synthetic ligand of peroxisome proliferator-activated receptors γ used in the treatment of type-2 diabetes, has been shown to reduce tumor growth and metastasis in human cancer cells, and may have the potential to be used as a radiosensitizer in radiotherapy for human colorectal cancer cells. In this study, rosiglitazone treatment significantly reduced the cell viability of p53-wild type HCT116 cells but not p53-mutant HT-29 cells. Interestingly, rosiglitazone pretreatment enhanced radiosensitivity in p53-mutant HT-29 cells but not HCT116 cells, and prolonged radiation-induced G 2/M arrest and enhanced radiation-induced cell growth inhibition in HT-29 cells. Pretreatment with rosiglitazone also suppressed radiation-induced H2AX phosphorylation in response to DNA damage and AKT activation for cell survival; on the contrary, rosiglitazone pretreatment enhanced radiation-induced caspase-8, -9, and -3 activation and PARP cleavage in HT-29 cells. In addition, pretreatment with a pan-caspase inhibitor, zVAD-fmk, attenuated the levels of caspase-3 activation and PARP cleavage in radiation-exposed cancer cells in combination with rosiglitazone pretreatment. Our results provide proof for the first time that rosiglitazone suppresses radiation-induced survival signals and DNA damage response, and enhances the radiation-induced apoptosis signaling cascade. These findings can assist in the development of rosiglitazone as a novel radiosensitizer.

건조 마늘, 홍삼 및 이들 혼합물의 생리활성

Water extracts were extracted on water bath for 3 hrs at 90℃ after 5 times water was added to hot-air dried garlic (DG) and red ginseng (RG), respectively. Its extracts were dried on rotary evaporator, and then the mixture samples were prepared from RG extracts added to DG extracts at 5, 10, and 15%. To test biological activities such as DPPH, nitrite scavenging, tyrosinase, α-glucosidase, human gastric (AGS), and human colon cancer cell (HT-29) growth, dryness of DG, RG, and mixture samples were diluted 500～10,000 ㎍/mL adding deionized water, respectively. DPPH scavenging activities of mixture sample were slightly higher than DG and RG sample alone. Nitrite scavenging activity was the highest in DG, and the other samples were below 45%. Inhibition activity of tyrosinase was below 35.0% in tested all samples. Inhibition activity of α-glucosidase was lower in DG or RG alone, while its activity of mixture sample was increased in proportion to concentration of RG extracts. Inhibition of AGS cell growth was more effective in mixture samples than DG and RG alone, while inhibition of HT-29 cell growth was more effective in DG or RG alone than mixture samples.

A novel zinc-chelating small molecule exhibits cancer cell growth inhibition in vitro and in vivo by induction of tumor suppressors early growth response 1 (Egr-1) and Krüppel-Like Factor 4 (KLF4)

14084 Background: Lead compound LT-253 was selected from a group of 2-indolyl imidazol [4,5-d] phenanthroline derivatives with anticancer activity. It shows potent and selective anti-proliferative activity against several human cancer types in vitro, and in vivo in xenograft mouse models of human colon carcinoma (HT-29) and non-small cell lung carcinoma (H460). Methods: The mechanism of cell growth inhibition of LT-253 was investigated in HT-29 colon cancer cells using the XTT cell proliferation assay, flow cytometry and apoptosis assays. In vitro and in vivo zinc chelation was determined by competition assays using fluorescent and chromophoric chelators. Gene expression studies were performed by human genome microarray analysis and confirmed by quantitative real- time PCR. The transcription factor activity profile of LT-253-treated cells was determined by a multiplex transcription factor array and electrophoretic mobility shift assays. The functional role of specific genes was evaluated by siRNA gene knock-down. Results: LT-253 functions as chelator of zinc in vitro, and of intracellular labile zinc in HT-29 cells. Moreover, LT-253-mediated HT-29 cell growth inhibition was reversed by zinc supplementation. Gene expression profiling confirmed sustained changes in zinc-sensitive genes such as metallothionine and several zinc transporters, but not copper-sensitive or iron-sensitive genes. LT-253 induces cancer cell growth inhibition primarily through G1/S phase cell cycle arrest. Gene expression and transcription factor activities of both Egr-1 and KLF4 are induced within 4 hr post LT-253 treatment. Moreover, increased expression of both Egr-1 and KLF4 is observed in LT-253-sensitive cancer cell lines of various origins. Importantly, Egr-1 and KLF4 gene knock-down by siRNA reversed the LT-253-mediated cell growth inhibition of HT-29 cells. Conclusion: Selective chelation of intracellular labile zinc pool by LT-253 triggers immediate induction of stress-responsive tumor suppressor Egr-1 and sustained induction of zinc-responsive tumor suppressor KLF4, leading to G1/S phase cell cycle arrest and inhibition of tumor growth. No significant financial relationships to disclose.

Trans-10,cis-12, not cis-9,trans-11, conjugated linoleic acid decreases ErbB3 expression in HT-29 human colon cancer cells.

To examine whether trans-10,cis-12 CLA (t10c12) or cis-9,trans-11 CLA (c9t11) inhibits heregulin (HRG)-beta-stimulated cell growth and HRG-beta-ErbB3 signaling in HT-29 cells. We cultured HT-29 cells in the absence or presence of the CLA isomers and/or the ErbB3 ligand HRG-beta. MTT assay, [3H]thymidine incorporation, Annexin V staining, RT-PCR, Western blotting, immunoprecipitation, and in vitro kinase assay were performed. HRG-beta increased cell growth, but did not prevent t10c12-induced growth inhibition. T10c12 inhibited DNA synthesis and induced apoptosis of HT-29 cells, whereas c9t11 had no effect. T10c12 decreased the levels of ErbB1, ErbB2, and ErbB3 proteins and transcripts in a dose-dependent manner, whereas c9t11 had no effect. Immunoprecipitation/Western blot studies revealed that t10c12 inhibited HRG-beta-stimulated phosphorylation of ErbB3, recruitment of the p85 subunit of phosphoinositide 3-kinase (PI3K) to ErbB3, ErbB3-associated PI3K activities, and phosphorylation of Akt. However, c9t11 had no effect on phospho Akt levels. Neither t10c12 nor c9t11 had any effect on HRG-beta-induced phosphorylation of ERK-1/2. These results indicate that the inhibition of HT-29 cell growth by t10c12 may be induced via its modulation of ErbB3 signaling leading to inhibition of Akt activation.

Trans-10, cis-12 conjugated linoleic acid reduces insulin-like growth factor-II secretion in HT-29 human colon cancer cells.

We previously demonstrated that a mixture of conjugated linoleic acid (CLA) isomers decreases colon cancer incidence in rats treated with 1,2-dimethylhydrazine. Our in vitro studies have also shown that CLA inhibits the growth of HT-29 cells, a human colon cancer cell line. When we compared the individual potencies of the two main isomers found in the mixture of CLA isomers (e.g., cis-9, trans-11 [c9t11] and trans-10, cis-12 [t10c12]), t10c12 CLA decreased viable cell numbers in a dose-dependent manner. By contrast, c9t11 CLA had no effect. Therefore, the present study examined whether the decreased cell growth is related to changes in secretion of insulin-like growth factor (IGF)-II and/or IGF-binding proteins (IGFBPs) that have been shown to regulate HT-29 cell proliferation. Cells were incubated in serum-free medium with various concentrations of the individual CLA isomers, and immunoblot analysis of 24-hour, serum-free, conditioned media using a monoclonal anti-IGF-II antibody was performed. HT-29 cells secreted both mature 7,500 apparent molecular weight (M(r)) and higher-M(r) forms of IGF-II. t10c12 CLA decreased the levels of the higher-M(r) and the mature form of IGF-II in a dose-dependent manner, whereas c9t11 CLA had no effect. Ligand blot analysis of conditioned medium using (125)I-IGF-II revealed that the production of IGFBP-2 and IGFBP-4 was also decreased by t10c12 CLA, whereas c9t11 CLA had no effect. Exogenous IGF-II abrogated the growth inhibition induced by t10c12 CLA. These results indicate that inhibition of HT-29 cell growth by t10c12 CLA may be mediated by decreasing IGF-II secretion in these cells.

Ceramide involvement in homocamptothecin- and camptothecin-induced cytotoxicity and apoptosis in colon HT29 cells

Topoisomerase I inhibitors of the camptothecin (CPT) family have emerged as potent clinical chemotherapeutic agents in first-line treatment of solid colorectal cancer and in second-line for 5-fluorouracil resistant patients. CPT and homocamptothecin (hCPT), derivative with enhanced lactone stability, induced growth inhibition in HT29 cells via p53-independent apoptosis. hCPT- and CPT-induced apoptosis was dependent on caspase-3 but not caspase-1. We report here substantial evidence that ceramide, resulting from de novo pathway or catabolism modulation, acted as a second messenger of these antitumor drugs in HT29 cells and leads to the activation of caspase-3. In addition, hCPT and CPT may favor ceramide signaling by disturbing sites of synthesis (Golgi) and trafficking of glucosylceramide from Golgi to lipid droplets. This work contributes to the understanding of the mechanism of action of CPTs, and suggests that inhibitors of glycosylation or activators of de novo metabolism could be of clinical interest in enhancing the effects of CPTs.

Genistein induces apoptosis and topoisomerase II-mediated DNA breakage in colon cancer cells

The present study was undertaken to determine if (a) genistein induces topo II-mediated DNA damage in HT-29 colon cancer cells; and (b) if this damage is required to induce apoptosis. DNA damage was evaluated using the comet assay. Apoptosis was determined by the ethidium bromide/acridine orange staining technique. DNA breakage was noted within 1 h of treatment. Apoptosis was only induced with high concentrations (⩾60 μM) of genistein. Marked inhibition of HT-29 cell growth was evident at concentrations ranging from 60 to 150 μM. This was associated with a cell cycle arrest at G 2/M. Similar findings were obtained in SW-620 and SW-1116 colon cancer cell lines. Aclarubicin, a topo II antagonist, reduced genistein-induced DNA breaks but did not reduce apoptosis. These data suggest that, in colon cancer cells, topo II serves as the enzymatic target of genistein. Furthermore, topo II-mediated DNA cleavage is not required for the induction of apoptosis.

Extracellular adenosine deprivation induces epithelial differentiation of HT29 cells: evidence for a concomitant adenosine A 1/A 2 receptor balance regulation

HT29 cells display an undifferentiated phenotype in culture. However, numerous treatments are able to induce both epithelial differentiation and cell growth inhibition. We have previously demonstrated that adenosine and its analogues act through specific adenosine receptors to modulate cell proliferation in HT29 and other human colon adenocarcinoma cell lines. Among the treatments tested, the most potent inhibition of HT29 cell growth was induced by deprivation of extracellular adenosine using adenosine deaminase. Here, we investigated the capacity of adenosine deaminase to initiate epithelial differentiation. After 1 month of daily addition of 10 U/ml adenosine deaminase to the culture medium, HT29 cells were cloned by limited dilution. Among the clones obtained, we focused our attention on clone 13. Microscopic visualization and proliferation studies indicated that cells from this clone grew very slowly and in a pseudo-monolayer, in marked contrast with the situation observed in the mother HT29 cell line. In addition, clone 13 cells displayed epithelial features that mimic the enterocytic differentiation of Caco-2 cells. These modifications were accompanied by dramatic changes in the activity of adenosine receptors, as demonstrated by pharmacological studies. In contrast to the original HT29 cells, clone 13 as well as Caco-2 cells displayed (i) a very low number of adenosine A 1 receptors, and (ii) increases in intracellular cAMP levels when challenged with adenosine analogues. It is hypothesized that a loss of adenosine A 1 receptors, with no change or a concomitant increase in adenosine A 2 receptors, results in the emergence of adenosine A 2 receptor-mediated differentiation and inhibition of proliferation, through a cAMP-dependent pathway.

Cellular uptake, cytotoxicity and DNA-binding studies of the novel imidazoacridinone antineoplastic agent C1311.

C1311 is a novel therapeutic agent with potent activity against experimental colorectal cancer that has been selected for entry into clinical trial. The compound has previously been shown to have DNA-binding properties and to inhibit the catalytic activity of topoisomerase II. In this study, cellular uptake and mechanisms by which C1311 interacts with DNA and exerts cytotoxic effects in intact colon carcinoma cells were investigated. The HT29 colon cancer cell line was chosen to follow cellular distribution of C1311 over a time course of 24 h at drug concentrations that just inhibited cell proliferation by 50% or 100%. Nuclear uptake of C1311 and co-localization with lysosomal or mitochondrial dyes was examined by fluorescence microscopy and effects on these cellular compartments were determined by measurement of acid phosphatase levels, rhodamine 123 release or DNA-binding behaviour. The strength and mode of DNA binding was established by thermal melting stabilization, direct titration and viscometric studies of host duplex length. The onset of apoptosis was followed using a TUNEL assay and DNA-fragmentation to determine a causal relationship of cell death. Growth inhibition of HT29 cells by C1311 was concomitant with rapid drug accumulation in nuclei and in this context we showed that the compound binds to duplex DNA by intercalation, with likely A/T sequence-preferential binding. Drug uptake was also seen in lysosomes, leading to lysosomal rupture and a marked increase of acid phosphatase activity 8 h after exposure to C1311 concentrations that effect total growth inhibition. Moreover, at these concentrations lysosomal swelling and breakdown preceded apoptosis, which was not evident up to 24 h after exposure to drug. Thus, the lysosomotropic effect of C1311 appears to be a novel feature of this anticancer agent. As it is unlikely that C1311-induced DNA damage alone would be sufficient for cytotoxic activity, lysosomal rupture may be a critical component for therapeutic efficacy. © 1999 Cancer Research Campaign

Induction of retinoic acid receptor β mediates growth inhibition in retinoid resistant human colon carcinoma cells

BACKGROUNDThe molecular mechanisms underlying the differential sensitivity of human colon carcinoma cells to retinoid mediated growth inhibition are poorly understood.AIMTo identify the intracellular mechanisms responsible for resistance against retinoid mediated...