formazans and Colonic-Neoplasms

Cetuximab is widely used in patients with metastatic colon cancer expressing wildtype KRAS. However, acquired drug resistance limits its clinical efficacy. Exosomes are nanosized vesicles secreted by various cell types. Tumor cell-derived exosomes participate in many biological processes, including tumor invasion, metastasis, and drug resistance. In this study, exosomes derived from cetuximab-resistant RKO colon cancer cells induced cetuximab resistance in cetuximab-sensitive Caco-2 cells. Meanwhile, exosomes from RKO and Caco-2 cells showed different levels of phosphatase and tensin homolog (PTEN) and phosphor-Akt. Furthermore, reduced PTEN and increased phosphorylated Akt levels were found in Caco-2 cells after exposure to RKO cell-derived exosomes. Moreover, an Akt inhibitor prevented RKO cell-derived exosome-induced drug resistance in Caco-2 cells. These findings provide novel evidence that exosomes derived from cetuximab-resistant cells could induce cetuximab resistance in cetuximab-sensitive cells, by downregulating PTEN and increasing phosphorylated Akt levels.

Topics: Analysis of Variance; Antineoplastic Agents, Immunological; Blotting, Western; Caco-2 Cells; Cell Line, Tumor; Cell Survival; Cetuximab; Colonic Neoplasms; Drug Resistance; Exosomes; Formazans; Humans; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Tetrazolium Salts; Time Factors

Fifty-four strains of lactic acid bacteria obtained from fermented dairy milks were investigated for possible use as probiotics and for colon cancer biological products. Five of these strains inhibited growth of eight food-borne pathogens including Helicobacter pylori, Escherichia coli, and Salmonella typhimurium. Three of these strains survived at pH 2.5 and in 0.3% bile salts. Additionally they produced no haemolysis, were resistant to kanamycin and adhered to Caco-2 cells. 16S rRNA gene sequences of probiotic strains indicated that RM11 and RM28 were Enterococcus faecium and Lactobacillus fermentum, respectively. Both the cultured medium and live whole cells from probiotic strains were tested for antiproliferation of colon cancer cells through MTT and Trypan Blue exclusion assays. The probiotic strains of E. faecium RM11 and L. fermentum RM28 also triggered antiproliferation of colon cancer cells at the rates of 21-29%, and 22-29%, respectively. This suggested that both strains could be used as potential probiotics in functional food or for colon cancer biological products.

Topics: Animals; Bile Acids and Salts; Caco-2 Cells; Cell Survival; Colonic Neoplasms; DNA, Bacterial; DNA, Ribosomal; Enterococcus faecium; Escherichia coli; Formazans; Growth Inhibitors; Helicobacter pylori; Hemolysis; Humans; Hydrogen-Ion Concentration; Limosilactobacillus fermentum; Milk; Probiotics; RNA, Ribosomal, 16S; Salmonella typhimurium; Tetrazolium Salts; Trypan Blue

The effects of the alpha-diketone derivatives 2,3- and 3,4-hexanediones were investigated in three non-neuronal cell lines (MCF7, HepG(2) and CaCo-2) as well as in the neuroblastoma line, SH-SY5Y. The MTT reduction assay was employed to determine the necrotic effects of the alpha-diketones and the neurotoxin 2,5-hexanedione over 4, 24 and 48 hr exposures. Flow cytometry was also used to study the effects of the three isomers on the cell cycle of the SH-SY5Y line only. With 2,5-hexanedione, the mean MTT IC(50) decreased more than 10-fold from 4 to 48 hr. The toxicities of both alpha-diketones were similar, with a more than 18-fold increase in sensitivity of the SH-SY5Y at 24 hr compared to that of 4 hr. With flow cytometry at 48 hr, SH-SY5Y apoptosis with 2,5-hexanedione rose throughout the concentration range evaluated (0-30 mM) while 2,3- and 3,4-hexanediones showed apoptosis over the concentration range 1-1.6 mM, with 3,4-hexanedione being the more potent compared to the 2,3-isomer. At 1.6 mM nearly all the cells had entered apoptosis in the presence of the 3,4-isomer, (94.9 +/- 1.4%) but only 57.5 +/- 4.1% of the 2,3-isomer-treated cells had reached that stage. The 2,3- and 3,4-isomers in diets alone may not pose a serious threat to human health. Further studies may be necessary to evaluate the effects of other dietary components on their toxicity. These alpha-diketones also display a degree of toxic selectivity towards neuroblastoma cells, which may have therapeutic implications.

Topics: Adenocarcinoma; Apoptosis; Breast Neoplasms; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Survival; Colonic Neoplasms; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Female; Flow Cytometry; Formazans; Hexanones; Humans; Isomerism; Neuroblastoma; Neurons; Tetrazolium Salts

4-Methyl-2,7-diamino-5,10-diphenyl-4,9-diazapyrenium hydrogensulfate (ADAP) is a potential antitumor compound because of its DNA and RNA intercalating ability. In this study, cellular uptake, intracellular distribution as well as mechanism of action, antitumor activity in vitro and toxicity in vivo of ADAP were investigated.. Based on the fluorescence properties of ADAP, its entry and distribution into live cells were analyzed by fluorescence microscopy. The in vitro antiproliferative activity was determined using MTT test. For screening of topoisomerase II-targeted effects of ADAP, the cell-free assay and immunoband depletion assay were used. Expression of the genes c-mos, c-N-ras, c-Ki-ras, c-H-ras, p53 and caspase 3 in Caco-2 cells treated with ADAP was examined by RT-PCR. Toxicity in vivo was determined using C3HHf/Bu Zgr/Hr mice treated by single or multiple doses of ADAP at a concentration of 25 mg/kg.. ADAP in microM concentrations entered into MIAPaCa-2 cell's cytoplasm in 5 min and into nuclei in 60 min after administration. Intracellular distribution of ADAP depended on the period of treatment time. ADAP (0.1-100 microM) strongly inhibited the growth of both mouse (FsaR, SCCVII) and human tumor cells (HeLa, Caco-2, HT-29, MIAPaCa-2, HBL, HEp-2, SW620, MCF-7) compared to its weak cytotoxicity on controls and normal cells (WI38). Results of both topoisomerase II assays showed that ADAP is not a topoisomerase II poison. Expression of investigated genes was dependent on the incubation time, except for p53 and c-H-ras. Morphological changes in tissues and organs of mice were not observed. Results of patohistological analysis have been confirmed by hematological and clinical-chemical analysis of blood of treated and non-treated animals.. ADAP is a strongly bioactive compound with antitumor potential in vitro. The antitumor potential in vivo remains to be identified.

Topics: Adenocarcinoma; Aminoquinolines; Animals; Antineoplastic Agents; Caco-2 Cells; Cell Line, Tumor; Cell Survival; Colonic Neoplasms; DNA Topoisomerases, Type II; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Female; Formazans; Gene Expression; Humans; Intercalating Agents; Male; Mice; Mice, Inbred C3H; Quinolinium Compounds; RNA, Messenger; Tetrazolium Salts

Ecteinascidin 743 (Et743; NSC 648766) is a potent antitumor agent presently in clinical trials. Et743 selectively alkylates guanine N2 from the minor groove of duplex DNA and bends the DNA toward the major groove. This differentiates Et743 from other DNA-alkylating agents presently in the clinic. To date, the cellular effects of Et743 have not been elucidated. Recently, Et743 DNA adducts have been found to suppress gene expression selectively and to induce topoisomerase I (top1) cleavage complexes in vitro and top1-DNA complexes in cell culture. In the present study, we characterized the DNA damage and the cell cycle response induced by Et743 in human colon carcinoma HCT116 cells. Alkaline elution experiments demonstrated that micromolar concentrations of Et743 produced comparable frequencies of DNA-protein cross-links and DNA single-strand breaks. The single-strand breaks were protein-cross-linked and were not associated with detectable DNA double-strand breaks. By contrast with camptothecin, these lesions persisted for several hours after drug removal and were not formed at 4 degrees C. Et743 treatment induced transient p53 elevation, dose-dependent cell cycle accumulation in G2-M and in G1- and S-phase, and inhibition of DNA synthesis. The sensitivity of camptothecin-resistant mouse leukemia P388/ CPT45 cells, which fail to express detectable top1, was similar to the sensitivity of wild-type P388 cells, suggesting that top1 is not a critical target for the antiproliferative activity of Et743.

Topics: Antineoplastic Agents, Alkylating; Cell Cycle; Cell Division; Cell Survival; Colonic Neoplasms; Dioxoles; DNA Damage; DNA Topoisomerases, Type I; DNA, Neoplasm; Dose-Response Relationship, Drug; Flow Cytometry; Formazans; Humans; Immunoenzyme Techniques; Isoquinolines; Proliferating Cell Nuclear Antigen; Tetrahydroisoquinolines; Tetrazolium Salts; Trabectedin; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Tumoral thymidine phosphorylase (TP) appears to play a dual role by being involved in neoangiogenesis and by activating 5FU prodrugs at the tumoral target site. The aim of the study was to investigate more thoroughly these potential physiological and pharmacological roles of TP. A rat carcinoma cell line (PROb) was transfected with TP/PD-ECGF in order to study the effect of the overexpression of this enzyme (1) on the sensitivity of cells to 5'DFUR and 5FU in vitro and (2) on tumour growth in vivo by using a syngenic tumour model in the BDIX rat (hepatic tumours, sub-cutaneous tumours). Cytotoxic effects of 5'DFUR, and to a lesser extent those of 5FU, were enhanced in TP clones as compared to control cells: there was a highly significant correlation between TP activity and in vitro sensitivity to 5'DFUR (r2= 0.91, P = 0.0002, n = 8) and, to a lesser extent, to 5FU (r2= 0.49, P = 0.053, n = 8). The impact of TP transfection on tumour growth was relatively modest and concerned only the initial stages of tumour expansion. Staining of TP tumours for endothelial (factor VIII) cells was always higher than controls. The staining ratio (TP/controls) tended to be reduced as tumours increased in size. The stability of TP expression was checked both in vitro (TP activity measurement) and in vivo (RT-PCR determinations) and there was no loss of TP expression over time which could be advanced to explain the progressive weakening of the impact of TP overexpression on both tumour growth and neoangiogenesis.

Topics: Animals; Antimetabolites, Antineoplastic; Colonic Neoplasms; Drug Resistance, Neoplasm; Floxuridine; Fluorouracil; Formazans; Neoplasm Transplantation; Neovascularization, Pathologic; Pentosyltransferases; Pyrimidine Phosphorylases; Rats; Tetrazolium Salts; Thymidine Phosphorylase; Transfection; Tumor Cells, Cultured

Topotecan is a topoisomerase I inhibitor with demonstrated anticancer activity in preclinical and clinical studies. The purpose of the present study was to evaluate drug-drug interactions in therapeutic regimens that would combine topotecan with microtubule-interfering agents, such as Taxol and vinblastine.. The cytotoxic activities of various drug combinations and schedules of administration were measured in a colon cancer cell line using the MTT assay. Western blot and flow cytometry were performed to determine the effects of Taxol and vinblastine on topoisomerase I and Bcl-xL protein levels and cell cycle distribution.. Brief incubation of colon cancer cells with low concentrations of either Taxol or vinblastine increased the efficacy of a subsequent treatment with topotecan. Preincubation of cells with vinblastine or Taxol reduced by 10- to 40-fold the concentration of topotecan necessary to induce a 50% decrease in cell survival. The effects were maximal when the cells were treated for 5 h with microtubule-interfering agents and then incubated for 19 h in drug-free medium before the addition of topotecan. Under these conditions, both Taxol and vinblastine caused an increase in topoisomerase I protein levels, fraction of S phase cells, and extent of Bcl-xL phosphorylation immediately prior to the addition of topotecan. All these factors may contribute to the increased efficacy of topotecan observed with sequential therapy.. Combinations of topotecan and microtubule-interfering agents result in synergistic anticancer activity when the drugs are administered sequentially. The promising preclinical data presented here encourage clinical testing of these drug combinations using a sequential schedule of administration.

Topics: Antineoplastic Agents; Antineoplastic Agents, Phytogenic; bcl-X Protein; Blotting, Western; Cell Cycle; Colonic Neoplasms; DNA Topoisomerases, Type I; Dose-Response Relationship, Drug; Drug Interactions; Drug Synergism; Enzyme Inhibitors; Formazans; Humans; Microtubules; Paclitaxel; Proto-Oncogene Proteins c-bcl-2; Tetrazolium Salts; Topoisomerase I Inhibitors; Topotecan; Tumor Cells, Cultured; Vinblastine

Epidemiological studies suggest that beta-carotene is able to modulate the risk of cancer. A number of in vitro studies reported that beta-carotene inhibits the growth of cancer cells; however, so far little is known about the molecular mechanisms of the antiproliferative effect of beta-carotene. Here we have investigated the effects of two beta-carotene preparations, (i) beta-carotene dissolved in tetrahydrofuran (final concentration in cell culture medium: 0.5%) and (ii) beta-carotene incorporated in a water dispersible bead form, on cultured human colon carcinoma cells HT29. The treatment of cells with beta-carotene up to 30 microM for 72 h led to a significant increase in the cellular beta-carotene concentration and formation of retinol. Beta-Carotene showed only low cytotoxicity for confluent cells tested up to 30 microM, but at dietary relevant concentrations for the intestinal tract (10, 30 microM) beta-carotene was strongly cytotoxic for growing cells and induced apoptosis in HT29 cells as assessed by the Annexin-V assay (the maximal effect was observed 15 h after treatment with beta-carotene). Exposure of cells to retinol at concentrations yielding cellular retinol levels similar to those observed by beta-carotene treatment had no antiproliferative or cytotoxic effect. Furthermore, beta-carotene did not affect the activation of the extracellular signal-regulated kinases (ERK1 and ERK2) that are essential for cellular growth. In summary, beta-carotene can inhibit growth of human colon carcinoma cells in vitro by induction of apoptosis in proliferating cells.

Topics: Apoptosis; beta Carotene; Cell Division; Colonic Neoplasms; Flow Cytometry; Formazans; Growth Inhibitors; HT29 Cells; Humans; Mitogen-Activated Protein Kinases; Phosphorylation; Tetrazolium Salts; Vitamin A