nitrophenols and Colonic-Neoplasms

ABT-737, a small molecule BH-3 mimetic, is less effective against human colon cancers due to its resistance. Verticillin A is a natural compound, which was previously purified from verticillium-infected mushrooms. Hence, we aimed at overcoming the ABT737 resistance observed in CRC tumors by combining Verticillin A with ABT-737 and figuring out the potential mechanism. In this study, we observed that Verticillin A could sensitize colon cancer to ABT-737-induced cell death through induction of mitochondrial-dependent apoptosis. Verticillin A could significantly increase the BIMEL/MCL-1 ratio to overcome ABT737 resistance through the suppression of the MEK/ERK pathway. In addition, up-regulation of BIM protein levels to activate BAX translocation results in apoptosis induction. Altogether, our work suggested the potential application of Verticillin A as a MEK inhibitor in BH3-mimetic-based therapy.

Topics: Antineoplastic Agents; Bcl-2-Like Protein 11; Biphenyl Compounds; Cell Line, Tumor; Colonic Neoplasms; Drug Resistance, Neoplasm; Humans; Indoles; MAP Kinase Signaling System; Myeloid Cell Leukemia Sequence 1 Protein; Nitrophenols; Piperazines; Sulfonamides

There is increasing evidence that cancers are heterogeneous and contain a hierarchical organization consisting of cancer stem cells and their differentiated cell progeny. These cancer stem cells are at the core of the tumor as they represent the clonogenic cells within a tumor. Moreover, these cells are considered to contain selective therapy resistance, which suggests a pivotal role in therapy resistance and tumor relapse. Here we show that differentiated cells can re-acquire stemness through factors secreted from fibroblasts. This induced CSC state also coincides with re-acquisition of resistance to chemotherapy. Resistance induced in newly formed CSCs is mediated by the anti-apoptotic molecule BCLXL and inhibition of BCLXL with the BH3 mimetic ABT-737 sensitizes these cancer cells toward chemotherapy. These data point to an important interplay between tumor cells and their microenvironment in the regulation of stemness and therapy resistance.

Topics: AC133 Antigen; Antineoplastic Agents; bcl-X Protein; Biological Factors; Biomarkers; Biphenyl Compounds; Cell Communication; Cell Death; Cell Differentiation; Colonic Neoplasms; Culture Media, Conditioned; Drug Resistance, Neoplasm; Fibroblasts; Gene Expression Regulation, Neoplastic; Genes, Reporter; Green Fluorescent Proteins; Humans; Keratin-20; Mucin-2; Neoplastic Stem Cells; Nitrophenols; Piperazines; Primary Cell Culture; Receptors, G-Protein-Coupled; Signal Transduction; Spheroids, Cellular; Sulfonamides

The HMGB1 protein has multiple functions in tumor biology and can act both as a transcription factor and as a cytokine. HMGB1 is released during cell death, and in our previous studies we demonstrated that HMGB1 induces a distinct, necrosis-like cell death in glioblastoma. In epithelial malignant tumors such as colorectal cancer (CRC), the HMGB1-dependent effects show cross-talk with apoptotic signal transduction. Treatment of CRC cells with low concentrations of recombinant HMGB1 results in dose-dependent cytotoxicity which is morphologically characterized by the formation of giant mitochondria and does not share features of apoptosis. HMGB1-triggered cell death is associated with intracellular ROS release, and overexpression of Bcl-2 blocks both the increase of ROS as well as HMGB1-dependent cell death. Importantly, treatment with recombinant HMGB1 or overexpression of endogenous HMGB1 strongly sensitizes CRC cells to the cytotoxic activity of the pro-apoptotic death ligand TRAIL as well as the small molecule Bcl-2 family inhibitor ABT‑737. Moreover, treatment of CRC cells with TRAIL or ABT‑737 induces a release of endogenous HMGB1 into the extracellular space, and preincubation with glycyrrhizin, an HMGB1 inhibitor, significantly inhibits induction of cell death by TRAIL and ABT‑737, suggesting that HMGB1 functionally contributes to the execution of cell death triggered by pro-apoptotic agents. Finally, we investigated the expression of HMGB1 in human CRC tumor samples and found that loss of HMGB1 expression is associated with a more aggressive phenotype and a more advanced stage of disease in patients with CRC. Altogether, our findings demonstrate a functional link between cytotoxic signaling cascades triggered by HMGB1 and pro-apoptotic agents leading to an HMGB1-dependent sensitization to CRC cell death. Thus, a further evaluation of recombinant HMGB1 as part of an experimental combination treatment of CRC seems warranted.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Biphenyl Compounds; Colonic Neoplasms; Glycyrrhizic Acid; HCT116 Cells; HMGB1 Protein; Humans; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Recombinant Proteins; Sulfonamides; TNF-Related Apoptosis-Inducing Ligand

Colorectal cancer is characterized by high morbidity and mortality in developed countries. The lack of low-cost, easy-to-use screening diagnostic methods is one of the causes of late diagnosis of colorectal cancer. Beta-glucuronidase (GLU) is a lysosomal exoglycosidase involved in degradation of glycosaminoglycans of the cell membranes and extracellular matrix of normal and cancerous colon tissues. The aim of our research was to evaluate the activity of GLU in the serum of colorectal cancer and estimate its potential value in the diagnosis of colorectal cancer.. Blood samples were collected from 21 patients with colorectal adenocarcinoma and 17 healthy subjects. GLU activity was determined by the colorimetric method of Marciniak et al. by measuring the amount of p-nitrophenol released from 4-nitrophenyl-beta-D-glucuronide, at λ = 405 nm.. We found significantly greater activity of GLU (p<0.0001) in the serum of patients with colorectal cancer, as compared to the healthy subjects. The serum GLU activity significantly differentiates patients with colorectal cancer from healthy individuals.. Serum GLU activity has diagnostic value and may be used in the diagnosis of colon adenocarcinoma.

Topics: Adenocarcinoma; Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Colonic Neoplasms; Female; Glucuronidase; Humans; Male; Middle Aged; Nitrophenols

ABT-737 is a novel anti-apoptotic Bcl-2 family protein inhibitor with high affinity to Bcl-2, Bcl-xl and Bcl-w but relatively low affinity to Mcl-1/A1. Therefore, high level Mcl-1 usually confers human tumor cell resistance to ABT-737. At the present study, we observed that clitocine can induce apoptosis in six tested human colon cancer cell lines accompanied by suppression of Mcl-1. More interestingly, clitocine significantly enhances the ABT-737-mediated lethality by inducing apoptosis. At the molecular level we determined Mcl-1 is the potential target through which clitocine can sensitize human colon cancer cells to ABT-737 induced apoptosis. Knocking-down of Mcl-1 is sufficient to increase cancer cell susceptibility to ABT-737 while its over-expression can significantly reverse this susceptibility. We also determined that clitocine may activate Bak by disrupting the interaction between Mcl-1 and Bak to induce mitochondrial membrane permeabilization. Furthermore, silence of Bak with the specific siRNA effectively attenuates the apoptosis induction by co-treatment of clitocine and ABT-737. Finally, clitocine in combination with ABT-737 significantly suppress the xenograft growth in animal model. Collectively, our studies suggest clitocine can induce apoptosis and potentiate ABT-737 lethality in human colon cancer cells by disrupting the interaction of Mcl-1 and Bak to trigger apoptosis.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; Biphenyl Compounds; Caco-2 Cells; Cell Line, Tumor; Cell Membrane Permeability; Colonic Neoplasms; HCT116 Cells; Humans; Mice; Mice, Nude; Mitochondria; Myeloid Cell Leukemia Sequence 1 Protein; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Pyrimidine Nucleosides; Random Allocation; Sulfonamides; Xenograft Model Antitumor Assays

Anoikis, a Bax-dependent apoptosis triggered by detachment from the extracellular matrix, is often inhibited in metastatic cancer cells. Using a couple of isogenic human colon cancer cell lines derived either from the primary tumor (SW480) or from a lymph node metastasis (SW620), we found that only SW480 cells were sensitive to anoikis. Bim upregulation but not Mcl-1 degradation was determined to be a critical factor of anoikis initiation in SW480 cells. ERK-mediated phosphorylation targets Bim for ubiquitination and proteasomal degradation. A MEK inhibitor (PD0325901) was able to increase Bim expression in SW620 cells and to sensitize these cells to anoikis. Thus, in both cell lines anoikis is under the control of proteins of the Bcl-2 family. Most interestingly, the BH3-mimetic ABT-737 was found not only to increase the level of apoptosis in suspended SW480 cells but also to sensitize SW620 cells to anoikis. Accordingly, both cell lines cultured in suspension were found to be primed for death, as determined by the detection of Bcl-2:Bim and Bcl-xL:Bim complexes. In contrast, adherent SW480 and SW620 cells were resistant to ABT-737. This indicates that, whether or not they undergo anoikis, colon cancer cells that have detached from the extracellular matrix might go through a transient state, where they are sensitive to BH3 mimetics. This would confer to compounds such as Navitoclax or ABT-199 a therapeutic window where they could have anti-metastatic potential.

Topics: Anoikis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; Biomarkers, Tumor; Biphenyl Compounds; Cell Adhesion; Cell Line, Tumor; Colonic Neoplasms; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Epithelial-Mesenchymal Transition; Extracellular Signal-Regulated MAP Kinases; Humans; Membrane Proteins; Mitochondria; Nitrophenols; Piperazines; Protein Kinase Inhibitors; Proto-Oncogene Proteins; Sulfonamides

To investigate the metabolic enzymatic capacity of the colon mucosa to detoxify noxious carcinogenic compounds.. We investigated the activity of 2 conjugating enzymes-the microsomal uridine glucuronosyltransferase (UGT) and the cytosomal glutathione S-transferase (GST) in the uninvolved mucosa of the colon transversum and sigmoideum in patients with adenomatous polyps and colorectal cancer. Biopsies were taken from the mucosa during colonoscopies which were done for clinical (diagnostic) reasons. After storage, the biopsy material was homogenized and after differential centrifugation the enzyme assays were performed with 4-nitrophenol (UGT) and 1-chloro 2,4-dinitrobenzene (GST) as substrates.. About 48 patients were included of which 28 had adenomas and 20 had colorectal carcinomas confirmed by histopathology. Enzyme activities were expressed as nmol/mg per minute protein for the GST and as pmol/mg per minute protein for the UGT. Analysis of variance (F-test) indicated that both enzymes were more widely distributed in adenoma than in cancer patients. The means ± SD were smaller for cancer patients: GST for adenomas 268 ± 152 vs 241 ± 69 for carcinomas and UGT for adenomas 197 ± 200 vs 150 ± 86 for carcinomas.. Compared to patients with adenomatous colon polyps those with colorectal carcinoma exhibited a lower capacity of detoxifying enzyme metabolism and their activities clustered over a smaller range.

Topics: Adenoma; Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Biopsy; Carcinoma; Colonic Neoplasms; Dinitrochlorobenzene; Disease Progression; Female; Glucuronosyltransferase; Glutathione Transferase; Humans; Inactivation, Metabolic; Linear Models; Male; Middle Aged; Nitrophenols; Substrate Specificity

Several tumor immunotherapy approaches result in a low percentage of durable responses in selected cancers. We hypothesized that the insensitivity of cancer cells to immunotherapy may be related to an anti-apoptotic cancer cell milieu, which could be pharmacologically reverted through the inhibition of antiapoptotic Bcl-2 family proteins in cancer cells. ABT-737, a small molecule inhibitor of the antiapoptotic proteins Bcl-2, Bcl-w and Bcl-x(L), was tested for the ability to increase antitumor immune responses in two tumor immunotherapy animal models. The addition of systemic therapy with ABT-737 to the immunization of BALB/c mice with tumor antigen peptide-pulsed dendritic cells (DC) resulted in a significant delay in CT26 murine colon carcinoma tumor growth and improvement in survival. However, the addition of ABT-737 to either a vaccine strategy involving priming with TRP-2 melanoma antigen peptide-pulsed DC and boosting with recombinant Listeria monocytogenes expressing the same melanoma antigen, or the adoptive transfer of TCR transgenic cells, did not result in superior antitumor activity against B16 murine melanoma. In vitro studies failed to demonstrate increased cytotoxic lytic activity when testing the combination of ABT-737 with lymphokine activated killer (LAK) cells, or the death receptor agonists Fas, TRAIL-ligand or TNF-alpha against the CT26 and B16 cell lines. In conclusion, the Bcl-2 inhibitor ABT-737 sensitized cancer cells to the antitumor effect of antigen-specific immunotherapy in a vaccine model for the CT26 colon carcinoma in vivo but not in two immunotherapy strategies against B16 melanoma.

Topics: Animals; Antigens, Neoplasm; Apoptosis; Biphenyl Compounds; Cancer Vaccines; Cell Line, Tumor; Colonic Neoplasms; Cytotoxicity, Immunologic; Dendritic Cells; Drug Screening Assays, Antitumor; fas Receptor; Humans; Immunotherapy; Immunotherapy, Adoptive; Intramolecular Oxidoreductases; Killer Cells, Lymphokine-Activated; Listeria monocytogenes; Melanoma, Experimental; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Neoplasm Proteins; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Receptors, Death Domain; Recombinant Fusion Proteins; Sulfonamides; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha

UDP-glucuronosyltransferase (UGT) enzymes catalyze the glucuronidation and typically inactivation of endogenous and exogenous molecules including steroid hormones, bilirubin and many drugs. The UGT1A6 protein is expressed predominantly in liver and metabolizes small phenolic drugs including acetaminophen, salicylates and many beta-blockers. Interindividual variation in the capacity of humans to glucuronidate drugs has been observed.. We have identified a novel common single nucleotide polymorphism (SNP) in the human UGT1A6 gene resulting in a Ser7Ala change in encoded amino acid. We have further functionally characterized that polymorphism in the context of two previously reported polymorphisms, Thr181Ala and Arg184Ser. These non-synonymous cSNPs define four common haplotypes. Alleles appear with similar frequencies in Caucasian and African-American populations with distributions adhering to Hardy-Weinberg equilibrium. UGT1A6 genotype, rate of substrate glucuronidation and level of immunoreactive UGT1A6 protein was determined. A 25-fold variation in the rate of substrate glucuronidation and an 85-fold variation in level of immunoreactive protein were measured. Liver tissue samples that were homozygous for UGT1A6*2 exhibited a high rate of glucuronidation relative to tissues with other genotypes. Biochemical kinetic studies of recombinant UGT1A6 expressed in HEK293 cells indicated that the UGT1A6*2 allozyme, expressed homozygously, had almost two-fold greater activity toward p-nitrophenol than UGT1A6*1 and when expressed heterozygously (UGT1A6*1/*2) it was associated with low enzyme activity.. These data suggest that common genetic variation in human UGT1A6 confers functionally significant differences in biochemical phenotype as assessed in human tissue and cultured cells expressing recombinant allozymes. This genetic variation might impact clinical efficacy or toxicity of drugs metabolized by UGT1A6.

Topics: Black or African American; Carcinoma, Hepatocellular; Cells, Cultured; Colonic Neoplasms; Gene Frequency; Genetic Variation; Genotype; Glucuronates; Glucuronosyltransferase; Heterozygote; Homozygote; Humans; Isoenzymes; Liver Neoplasms; Microsomes, Liver; Nitrophenols; Pharmacogenetics; Polymorphism, Single Nucleotide; Recombinant Proteins; White People

COLO 201, human colon adenocarcinoma cells were incubated with artificial primers, p-nitrophenyl-glycoside derivatives at 1.0 mmol (mM) in the medium containing 10% fetal bovine serum to detect sugar chain elongation. However, when p-nitrophenyl-beta-N-acetylglucosamine (beta-GlcNAc-PNP) was added, the medium changed color to yellow and the cells were dead. To explain this finding, the cells were incubated with 1.0 mM each of beta-GlcNAc-PNP and 4-methylumbelliferyl-beta-N-acetylglucosamine, then the number of living cells was measured in a time course. In beta-GlcNAc-PNP, the living cells were decreased at 24 hours. The cells were survived with N-acetylglucosamine, whereas in the presence of p-nitrophenol (PNP) the living cells were decreased. It was suggested that PNP released from beta-GlcNAc-PNP induced the cell death. Activity of beta-D-N-acetylglucosaminidase was detected in fetal bovine serum. It was shown that PNP induced the cell death in time-and-dose dependent manner. Genomic DNA from COLO 201 analyzed by agarose gel electrophoresis was fragmentated. PNP analogues were tested for toxicity, and the results suggested that the phenolic OH-group linked to benzene ring and nitro-group linked to the structure in para-form (PNP) was the most effective.

Topics: Acetylglucosamine; Adenocarcinoma; Apoptosis; Cell Culture Techniques; Colonic Neoplasms; Dose-Response Relationship, Drug; Humans; Molecular Structure; Nitrophenols; Time Factors; Tumor Cells, Cultured

Irinotecan (CPT-11) is a topoisomerase I inhibitor commonly used in the treatment of colorectal tumors. It is a prodrug, converted to an active metabolite, SN-38, by carboxylesterases (CEs). CEs are ubiquitary enzymes that react with numerous substrates. A specific CPT-11 converting enzyme was isolated from rat serum, with different kinetic properties than other CEs. We determined kinetic properties of specific CPT-11 CE activity (CPT-CE) in human normal liver and colon tumors. Km were very similar (3.4 microM in liver and 3.8 microM in colon tumors), but Vmax was higher in liver (2.7 pmol/min/mg protein) than in colon tumor (1.7 pmol/min/mg protein). CPT-CE and total CE (using p-nitro-phenylacetate as substrate) were weakly correlated in colon tumors. The large interpatient variability observed in liver CPT-CE activity could play a potential role in the pharmacokinetic variability observed with irinotecan.

Topics: Animals; Antineoplastic Agents, Phytogenic; Camptothecin; Carboxylic Ester Hydrolases; Colon; Colonic Neoplasms; DNA Topoisomerases, Type I; Enzyme Activation; Enzyme Inhibitors; Humans; Irinotecan; Kinetics; Liver; Nitrophenols; Rats; Substrate Specificity; Topoisomerase I Inhibitors

Phenol sulfotransferase (PST) catalyzes the sulfate conjugation of phenolic and catechol drugs and neurotransmitters. Human platelets and brain contain at least two forms of PST. One form is relatively thermolabile (TL) and catalyzes the sulfate conjugation of monoamines such as dopamine. The other is thermostable (TS) and catalyzes the sulfation of "simple" phenols such as phenol and p-nitrophenol. We found that homogenates of human liver also contain two forms of PST that are similar to brain and platelet TL and TS PST with regard to substrate specificities, thermal stabilities and sensitivities to inhibitors. Optimal conditions were determined for the assay of these two activities in human liver homogenates. The apparent Km of liver homogenate TL PST for dopamine was 27 microM. The apparent Km of the TS form of the enzyme for p-nitrophenol was 0.94 microM. Human liver TS PST also catalyzed the sulfate conjugation of dopamine, but with an apparent Km of 5 mM, over two orders of magnitude higher than that of TL PST. Two different peaks of TS PST activity were separated from the TL activity by ion exchange chromatography of human liver preparations. Both peaks of TS PST activity were partially purified and characterized. Both had similar substrate specificities and inhibitor sensitivities. Km values of TS PST peak I for p-nitrophenol and for 3'-phosphoadenosine-5'-phosphosulfate were 0.91 and 0.86 microM, respectively, while the Km values of TS PST peak II for these two cosubstrates for the reaction were 0.43 and 0.64 microM, respectively. However, the TS PST activity in peak II was significantly more thermolabile than was the activity in peak I. These results are compatible with the conclusion that human liver homogenates contain at least two forms of PST, forms with properties similar to those of TS and TL PST in homogenates of human cerebral cortex and platelets. In addition, human liver contains two isozymes of TS PST.

Topics: Adult; Aged; Arylsulfotransferase; Cholelithiasis; Colonic Neoplasms; Dopamine; Enzyme Stability; Female; Hot Temperature; Humans; Isoenzymes; Kinetics; Liver; Liver Neoplasms; Male; Nitrophenols; Sulfurtransferases

K-m values of beta-N-acetylglucosaminidase (2-acetamido-2-deoxy-beta-D-glucoside acetamidodeoxyglucohydrolase EC 3.2.1.30), beta-N-acetylgalactosaminidase (EC 3.2.1.53), beta-galactosidase (beta-D-galactoside galactohydrolase EC 3.2.1.23) and alpha-L-fucosidase (alpha-L-fucoside fucohydrolase EC 3.2.1.51) of distal colonic tumours, induced in rats by 1,2-dimethylhydrazine, were found to be significantly different compared with the values for the enzymes of the colonic mucosa of the control and tumour-bearing animals and of the proximal colonic tumours. The inhibition kinetics data also showed a significant difference between the enzymes of the distal colon tumours and of other experimental tissues. The data on the effect of pH on enzyme kinetics (pK values) showed no significant difference in the catalytic groups of the active centres of enzymes from tumours and from the control colonic mucosa. Tumour beta-N-acetylglucosaminidase and beta-N-acetylgalactosaminidase compared with the enzymes from other experimental tissues were found to be different in their thermal inactivation kinetics. K-m values of 14 days old foetal intestinal beta-N-acetylglucosaminidase and beta-N-acetylgalactosaminidase were significantly different from the values obtained for the adult mucosal enzymes but were similar to those of the distal colonic tumour enzymes.

Topics: Acetylgalactosamine; Acetylglucosamine; alpha-L-Fucosidase; Animals; Colon; Colonic Neoplasms; Dimethylhydrazines; Fetus; Fucose; Galactose; Galactosidases; Gestational Age; Glycoside Hydrolases; Hexosaminidases; Hydrogen-Ion Concentration; Intestinal Mucosa; Kinetics; Lactones; Nitrophenols; Rats; Stereoisomerism

Topics: Adenocarcinoma; Animals; BCG Vaccine; Butyrates; Caprylates; Colonic Neoplasms; Erythrocytes; Esterases; Fatty Acids; Female; Histological Techniques; Hot Temperature; Humans; Isoelectric Focusing; Kidney; Liver; Lung; Mice; Naphthalenes; Nitrophenols; Species Specificity; Spleen; Urea

Topics: Animals; Antibodies; Arsphenamine; Carcinoembryonic Antigen; Carcinoma, Squamous Cell; Cell Line; Colonic Neoplasms; Cytotoxicity Tests, Immunologic; Glucose Oxidase; Goats; Haptens; HeLa Cells; Humans; Immunoglobulin G; Laryngeal Neoplasms; Nitrophenols; Peroxidases

Tumor cell lines exposed to immunoglobulins specific for cell surface antigens developed increased cellular incorporation of [(125)I]iododeoxyuridine and [(3)H]thymidine (up to 200-fold increases over cells treated with normal rabbit immunoglobulins). Antibody-stimulated cells multiplied more rapidly and lived longer than control cells in tissue culture. These observations were made both with cells substituted with 2,4,6-trinitrophenol and purified antibody against 2,4,6-trinitrophenol, and with several cell lines and their respective whole-cell antibodies. Antibodies that were stimulatory at low concentrations were cytotoxic at high concentrations. These observations may have significance in regard to enhancing effects of antibodies on tumor cell growth in vivo.

Topics: Animals; Antibodies, Neoplasm; Carcinoma; Cell Line; Colonic Neoplasms; Cross Reactions; DNA, Neoplasm; Epitopes; Female; HeLa Cells; Humans; Idoxuridine; Iodine Radioisotopes; Laryngeal Neoplasms; Mice; Neoplasms; Nitrophenols; Plasmacytoma; Rabbits; Thymidine; Tritium