nitrophenols and Carcinoma--Hepatocellular

Hepatocellular carcinoma (HCC) represents a global health challenge with limited therapeutic options. Anti-angiogenic immune checkpoint inhibitor-based combination therapy has been introduced for progressed HCC, but improves survival only in a subset of HCC patients. Tyrosine-kinase inhibitors (TKI) such as sorafenib represent an alternative treatment option but have only modest efficacy. Using different HCC cell lines and HCC tissues from various patients reflecting HCC heterogeneity, we investigated whether the sorafenib response could be enhanced by combination with pro-apoptotic agents, such as TNF-related apoptosis-inducing ligand (TRAIL) or the BH3-mimetic ABT-737, which target the death receptor and mitochondrial pathway of apoptosis, respectively. We found that both agents could enhance sorafenib-induced cell death which was, however, dependent on specific BH3-only proteins. TRAIL augmented sorafenib-induced cell death only in NOXA-expressing HCC cells, whereas ABT-737 enhanced the sorafenib response also in NOXA-deficient cells. ABT-737, however, failed to augment sorafenib cytotoxicity in the absence of BIM, even when NOXA was strongly expressed. In the presence of NOXA, BIM-deficient HCC cells could be in turn strongly sensitized for cell death induction by the combination of sorafenib with TRAIL. Accordingly, HCC tissues sensitive to apoptosis induction by sorafenib and TRAIL revealed enhanced NOXA expression compared to HCC tissues resistant to this treatment combination. Thus, our results suggest that BH3-only protein expression determines the treatment response of HCC to different sorafenib-based drug combinations. Individual profiling of BH3-only protein expression might therefore assist patient stratification to certain TKI-based HCC therapies.

Topics: Apoptosis; Bcl-2-Like Protein 11; Biphenyl Compounds; Carcinoma, Hepatocellular; Caspases; Cell Death; Cell Line, Tumor; Cell Proliferation; Cell Survival; Enzyme Activation; Humans; Ki-67 Antigen; Liver Neoplasms; Mitochondrial Membrane Transport Proteins; Myeloid Cell Leukemia Sequence 1 Protein; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Sorafenib; Sulfonamides; TNF-Related Apoptosis-Inducing Ligand

Diacid metabolite as the stable form of norcantharidin (DM-NCTD) derived from Chinese blister beetle (Mylabris spp.). The previous studies reported that DM-NCTD could enhance ABT-737-triggered cell viability inhibition and apoptosis in hepatocellular carcinoma (HCC) cell lines. To translate this synergistic therapy into in vivo anticancer treatment, a folate receptor-targeted lipid bilayer-supported chlorodimethyloctadecylsilane-modified mesoporous silica nanoparticle (FA-LB-CHMSN) with DM-NCTD loaded in CHMSN and ABT-737 in lipid bilayer was prepared, which could promote the cancer cell uptake of the drugs through folate receptor-mediated endocytosis. The structure and the properties of the nanoparticle were evaluated. FA-LB-CHMSN with DM-NCTD/ABT-737 loaded induced apparent tumor cell apoptosis and showed remarkably tumor inhibition in H22 tumor-bearing mice model, with significant cellular apoptosis in the tumor and no obvious toxicity to the tissues. We expect that this nanoparticle could be of interest in both biomaterial investigations for HCC treatment and the combination of chemotherapeutic drugs for synergistic therapies.

Topics: Animals; Antineoplastic Agents; Biphenyl Compounds; Bridged Bicyclo Compounds, Heterocyclic; Carcinoma, Hepatocellular; Folic Acid; Lipid Bilayers; Liver; Liver Neoplasms; Mice; Nanoparticles; Nitrophenols; Piperazines; Silicon Dioxide; Sulfonamides

ABT-737, a B-cell lymphoma 2 homology 3 mimetic, not only induces cell apoptosis by inhibiting the interaction of B-cell lymphoma 2 and Bax but also induces cell autophagy by interrupting the interaction of B-cell lymphoma 2 and Beclin1. Several recent studies have reported that ABT-737 has antitumor efficacy in diverse cancers. However, another study showed that hepatocellular carcinoma cells with high B-cell lymphoma 2 expression were resistant to ABT-737 compared to hepatocellular carcinoma cells with low B-cell lymphoma 2 expression. It was also found that ABT-737-induced autophagy is crucial for drug resistance. Here, we observed that of B-cell lymphoma 2 expression in Adriamycin-resistant human hepatocellular carcinoma HepG2/ADM cells is higher than that in human hepatocellular carcinoma HepG2 cells. Therefore, we further confirmed the mechanism and effect of autophagy induced by ABT-737 on apoptosis in HepG2/ADM cells with high B-cell lymphoma 2 expression. Our results showed that ABT-737 induced apoptosis and autophagy in time- and dose-dependent manner in HepG2/ADM cells, and this ABT-737-induced autophagy was Beclin1-dependent. In addition, we demonstrated that ABT-737 induced reactive oxygen species-mediated autophagy, and the reactive oxygen species-inhibitor N-acetyl-l-cysteine suppressed the reactive oxygen species-induced autophagy and ABT-737-induced increase in HepG2/ADM cell apoptosis. Furthermore, autophagy inhibitors increased HepG2/ADM cell apoptosis. In conclusion, our study further confirms that Beclin1- and reactive oxygen species-dependent autophagy induced by ABT-737 also plays a protective function in HepG2/ADM cells, which show B-cell lymphoma 2 expression higher than that in HepG2 cells.

Topics: Apoptosis; Autophagy; Beclin-1; Biphenyl Compounds; Carcinoma, Hepatocellular; Doxorubicin; Drug Resistance, Neoplasm; Hep G2 Cells; Humans; Liver Neoplasms; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Sulfonamides

At present, the therapeutic treatment strategies for patients with hepatocellular carcinoma (HCC) remain unsatisfactory, and novel methods are urgently required to treat this disease. Members of the B cell lymphoma (Bcl)-2 family are anti‑apoptotic proteins, which are commonly expressed at high levels in certain HCC tissues and positively correlate with the treatment resistance of patients with HCC. ABT-737, an inhibitor of Bcl-2 anti-apoptotic proteins, has been demonstrated to exhibit potent antitumor effects in several types of tumor, including HCC. However, treatment with ABT-737 alone also activates certain pro-survival signaling pathways, which attenuate the antitumor validity of ABT-737. Curcumin, which is obtained from Curcuma longa, is also an antitumor potentiator in multiple types of cancer. In the present study, the synergistic effect of curcumin and ABT-737 on HCC cells was investigated for the first time, to the best of our knowledge. It was found that curcumin markedly enhanced the antitumor effects of ABT-737 on HepG2 cells, which was partially dependent on the induction of apoptosis, according to western blot analysis and flow cytometric apoptosis analysis. In addition, the sustained activation of the ROS-ASK1-c-Jun N-terminal kinase pathway may be an important mediator of the synergistic effect of curcumin and ABT-737. Collectively, these results indicated that the combination of curcumin and ABT-737 can efficaciously induce the death of HCC cells, and may offer a potential treatment strategy for patients with HCC.

Topics: Apoptosis; Biphenyl Compounds; Carcinoma, Hepatocellular; Cell Proliferation; Curcumin; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Liver Neoplasms; MAP Kinase Kinase 4; MAP Kinase Kinase Kinase 5; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Sulfonamides

To study the therapeutic effect of norcantharidin (NCTD) combined with ABT-737 on hepatocellular carcinoma cells and the molecular mechanism.. Two hepatocellular carcinoma (HCC) cell lines, HepG2 and SMMC-7721, were selected. ABT-737 and NCTD were allocated into groups to be used alone or in combination. HepG2 and SMMC-7721 cells were cultured in vitro. Liver cancer cells in the logarithmic phase of growth were vaccinated and cultured to the cell wall stage; these cells were treated for 48 h with different concentrations of NCTD, or ABT-737, or NCTD combined with ABT-737. The cell proliferation inhibition rate was detected by methyl thiazolyl tetrazolium. The expression of Mcl in HCC cells was detected by Western Blotting, and the cells in each group after treatment had apoptosis detected by flow cytometry. The proliferation inhibition rate, the expression of Mcl-1 in cells and the apoptosis inducing effect of treatment were observed in each group, and the effect of NCTD on ABT-737 in the treatment of HCC and its mechanism of action were analyzed.. As the concentration of NCTD increased, the cell proliferation inhibition rate gradually decreased; and the treatment effect of ABT-737 1-3 μm combined with NCTD on cell proliferation inhibition was stronger than that of ABT-737 alone. The difference was statistically significant (P < 0.05). In observing the expression of Mcl-1 in cells after the treatment of different concentrations of NCTD, this was partially inhibited after treatment with NCTD 15 μm, and the expression of Mcl-1 was almost undetectable after treatment with NCTD 30 μm and 60 μm. The effect on inducing apoptosis with the treatment of ABT-737 or NCTD alone for 48 h was lower than that of the control group. The difference was not statistically significant (P > 0.05). The effect on inducing apoptosis in HepG2 and SMMC-7721 cells with the treatment of ABT-737 combined with NCTD for 48 h was greater than that of ABT-737 or NCTD alone. The difference was statistically significant (P < 0.05).. NCTD combined with ABT-737 has a positive role in the treatment of HCC, and it has great value in clinical research.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Biphenyl Compounds; Bridged Bicyclo Compounds, Heterocyclic; Carcinoma, Hepatocellular; Cell Proliferation; Cytochromes c; Dose-Response Relationship, Drug; Hep G2 Cells; Humans; Liver Neoplasms; Myeloid Cell Leukemia Sequence 1 Protein; Nitrophenols; Piperazines; Sulfonamides; Time Factors

The Bcl-2 inhibitor ABT-737 has shown promising antitumor efficacy in vivo and in vitro. However, some reports have demonstrated that HCC cells are resistant to ABT-737, and the corresponding molecular mechanisms of this resistance are not well known. In this study, we found that HCC cells with high levels of Bcl-2 were markedly resistant to ABT-737 compared to HCC cells with low levels of Bcl-2. In HCC cells with high levels of Bcl-2 (such as HepG2 cells), ABT-737 induced protective autophagy via the sequential triggering of reactive oxygen species (ROS) accumulation, short-term activation of JNK, enhanced phosphorylation of Bcl-2, and dissociation of Beclin 1 from the Bcl-2/Beclin 1 complex. Moreover, autophagy suppressed the overactivation of the ROS-JNK pathway and protected against apoptosis. In HCC cells with low levels of Bcl-2 (i.e., Huh7 cells), ABT-737 induced apoptosis via the sequential stimulation of ROS, sustained activation of JNK, enhanced translocation of Bax from the cytosol to the mitochondria, and release of cytochrome c. In sum, this study indicated that the activation of the ROS-JNK-autophagy pathway may be an important mechanism by which HCC cells with high levels of Bcl-2 are resistant to ABT-737.

Topics: Apoptosis; Autophagy; Biphenyl Compounds; Carcinoma, Hepatocellular; Gene Expression Regulation, Neoplastic; Hep G2 Cells; Humans; Liver Neoplasms; MAP Kinase Signaling System; Nitrophenols; Piperazines; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Sulfonamides

Advanced stages of tumour and development of metastases are the two major problems in treating liver tumours such as hepatoblastoma (HB) and hepatocellular carcinoma (HCC), in paediatric patients. Modulation of apoptosis in HB cells enhances the sensitivity of these cells towards various drugs and has been discussed to enforce treatment. We analysed the effect of apoptosis modulators, BH3 mimetics, on mechanisms of dissemination such as adhesion or migration of HB and HCC cells. BH3 mimetics such as ABT-737 and obatoclax can reduce cell migration in a scratch assay as well as adhesion of HB and HCC cells to matrigel. Immunofluorescence staining of F-actin demonstrated that development of lamellipodia, which are important for migration, decreased. BH3 mimetics increase the level of activated caspases 3 and 7 in HUH6 cells. This results in the degradation of GTPase Cdc42, which can be determined by western blot analysis. A pan-caspase inhibitor can block the migration and degradation of Rho-GTPase. In summary, our study showed that BH3 mimetics not only enhance drug sensitivity but also may prevent metastasis by inhibiting HB and HCC cell motility.

Topics: Actins; Antineoplastic Agents; Apoptosis; Biphenyl Compounds; Blotting, Western; Carcinoma, Hepatocellular; Caspase 3; Caspase 7; Caspase Inhibitors; cdc42 GTP-Binding Protein; Cell Adhesion; Cell Movement; Cytoskeleton; Enzyme Activation; Fluorescent Antibody Technique; Hepatoblastoma; Humans; Indoles; Neoplasm Metastasis; Nitrophenols; Oligopeptides; Piperazines; Proteolysis; Pseudopodia; Pyrroles; Sulfonamides

Small-molecule cell-permeable Bcl-2/Bcl-xL antagonist ABT-737 has recently emerged as a novel cancer therapeutic agent because it potently induces apoptosis in certain cancer cells. However, since ABT-737 binds to Mcl-1 with low affinity, ABT-737-mediated apoptosis signaling is inhibited in hepatocellular carcinoma (HCC) cells and other solid cancer cells due to the elevated expression of Mcl-1. Accordingly, strategies that target Mcl-1 are explored for overcoming ABT-737-resistance. In this study, we reported that Norcantharidin (NCTD), a small-molecule anticancer drug derived from Chinese traditional medicine blister beetle (Mylabris), induced transcriptional repression of Mcl-1 and considerably enhanced ABT-737-triggered cell viability inhibition and apoptosis in multiple HCC cell lines. Moreover, we observed that the enhancement of ABT-737-mediated apoptosis by NCTD was associated with activation of mitochondrial apoptosis signaling pathway, which involved cytosolic release of cytochrome c, cleavage of caspase-9 and caspase-3. Additionally, knockdown of Bax/Bak, the key effectors permeabilizing mitochondrial outer membrane significantly attenuated the enhancement, indicating mitochondrial apoptosis pathway played an essential role in the execution of the apoptosis. Finally, knockdown of Mcl-1 substantially potentiated ABT-737-mediated apoptotic cell death, confirming the potency of Mcl-1 repression by NCTD in enhancing ABT-737-induced apoptosis. These results therefore suggest that combination treatment with NCTD can overcome ABT-737 resistance and enhance ABT-737 therapeutic efficacy in treating human HCC.

Topics: Antineoplastic Agents; Apoptosis; Biphenyl Compounds; Bridged Bicyclo Compounds, Heterocyclic; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Humans; Liver Neoplasms; Myeloid Cell Leukemia Sequence 1 Protein; Nitrophenols; Piperazines; Sulfonamides; Transcription, Genetic

The human hepatocellular carcinoma (HCC) represents biologically aggressive and chemo-resistant cancers. Owing to the low affinity with the apoptotic factor Mcl-1, the BH3 mimetic drug ABT-737 failed to exert potent cancer-killing activities in variety of cancer models including HCC. The current study demonstrated that combining ABT-737 and Celastrol synergistically suppressed HCC cell proliferation, and induced apoptosis which was accompanied with the activation of caspase cascade and release of cytochrome c from mitochondria. Further study revealed that the enhanced Noxa caused by Celastrol was the key factor for the synergy, since small interfering RNA-mediated knockdown of Noxa expression in HCC cells resulted in decreased apoptosis and attenuated anti-proliferative effects of the combination. In addition, our study unraveled that, upon Celastrol exposure, the activation of endoplasmic reticulum (ER) stress, specifically, the eIF2α-ATF4 pathway played indispensable roles in the activation of Noxa, which was validated by the observation that depletion of ATF4 significantly abrogated the Noxa elevation by Celastrol. Our findings highlight a novel signaling pathway through which Celastrol increase Noxa expression, and suggest the potential use of ATF4-mediated regulation of Noxa as a promising strategy to improve the anti-cancer activities of ABT-737.

Topics: Apoptosis; Biphenyl Compounds; Blotting, Western; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Survival; Drug Synergism; Endoplasmic Reticulum Stress; Humans; Immunoprecipitation; Membrane Potential, Mitochondrial; Nitrophenols; Pentacyclic Triterpenes; Piperazines; Proto-Oncogene Proteins c-bcl-2; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Sulfonamides; Triterpenes

The activity of the α-L-fucosidase (AFU) enzyme represents an excellent test for diagnosis of hepatocellular carcinoma (HCC) and fucosidosis recognized in inborn disorder of metabolism and increases the sensitivity of detection to 95.5% in patients with HCC. Therefore, the determination of the activity of AFU enzyme is very important and can be used as a screening tool for the early diagnosis of tumors for HCC patients. A simple, accurate, and sensitive potentiometric method was developed for measuring the activity of AFU. The method was based upon measuring the concentration of 2-chloro-4-nitrophenol (2-chloro-4-NP) using a 2-chloro-4-NP-rhodamine B ion pair in a PVC membrane sensor. The electrode shows a linear, reproducible, and stable potentiometric response with an anionic Nernstian slope of -51.13 ± 0.6 mV/decade over a wide range of concentrations 10(-5)-10(-2) M and a detection limit of 1.0 × 10(-6) M of 2-chloro-4-NP. The membrane exhibits a fast response time of 30 s, over a pH range of 4.0-6.5. The selectivity coefficients indicate excellent selectivity for 2-chloro-4-NP over a number of interfering species, e.g., chloride, nitrate, sulfate, chromate urea, albumin, glucose, uric acid, and total protein. The prepared sensor has been used successfully for the determination of 2-chloro-4-NP produced from the hydrolysis of 2-chloro-4-NP-α-L-fucopyranoside substrate. It was also applied for the determination α-L-fucosidase enzyme of 33 serum samples of healthy subjects and patients. The average recoveries ± RSD for the healthy subjects, cirrhosis of chronic hepatitis C and B, and HCC serum samples were 102.6 ± 1.01%, 101.5 ± 0.95%, and 100.1 ± 1.1%, respectively. The results obtained are in good agreement with those obtained by standard methods.

Topics: Adult; Aged; alpha-L-Fucosidase; Biosensing Techniques; Carcinoma, Hepatocellular; Female; Hepatitis C, Chronic; Humans; Limit of Detection; Male; Membranes, Artificial; Middle Aged; Nitrophenols; Polyvinyl Chloride; Potentiometry; Rhodamines; Young Adult

Proteins of the BCL2 family are key regulators of apoptosis. Their expression levels are frequently altered in cancers, enabling tumor cells to survive. To gain insight into the pathogenesis of hepatocellular carcinoma (HCC), we performed a comprehensive survey of the expression of the members of the BCL2 family in samples obtained from surgically resected HCCs. Here, we report the occurrence of a new molecular anomaly, consisting of a strong reduction in the expression of the proapoptotic protein BAD in HCC compared with surrounding nontumoral tissue. We investigate the function of BAD in a panel of HCC cell lines. Using gene overexpression and RNA interference, we show that BAD is involved in the cytotoxic effects of sorafenib, a multikinase blocker, which is currently the sole therapeutic drug effective for the treatment of HCC. Finally, we report that ABT-737, a compound that interacts with proteins of the BCL2 family and exhibits a BAD-like reactivity, sensitizes HCC cells toward sorafenib-induced apoptosis. Collectively, our findings indicate that BAD is a key regulator of apoptosis in HCC and an important determinant of HCC cell response to sorafenib.

Topics: Antineoplastic Agents; Apoptosis; Benzenesulfonates; Biomarkers, Tumor; Biphenyl Compounds; Blotting, Western; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Drug Synergism; Humans; Liver; Liver Neoplasms; Niacinamide; Nitrophenols; Phenylurea Compounds; Piperazines; Proto-Oncogene Proteins c-bcl-2; Pyridines; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sorafenib; Sulfonamides

Tumor cells are characterized by uncontrolled proliferation, often driven by activation of oncogenes, and apoptosis resistance. The oncogenic kinase inhibitor sorafenib can significantly prolong median survival of patients with advanced hepatocellular carcinoma (HCC), although the response is disease-stabilizing and cytostatic rather than one of tumor regression. Bcl-xL (B cell lymphoma extra large), an antiapoptotic member of the B cell lymphoma-2 (Bcl-2) family, is frequently overexpressed in HCC. Here, we present in vivo evidence that Bcl-xL overexpression is directly linked to the rapid growth of solid tumors. We also examined whether ABT-737, a small molecule that specifically inhibits Bcl-xL but not myeloid cell leukemia-1 (Mcl-1), could control HCC progression, especially when used with sorafenib. Administration of ABT-737, even at an in vivo effective dose, failed to suppress Huh7 xenograft tumors in mice. ABT-737 caused the levels of Mcl-1 expression to rapidly increase by protein stabilization. This appeared to be related to resistance to ABT-737, because decreasing Mcl-1 expression levels to the baseline by a small interfering RNA-mediated strategy made hepatoma cells sensitive to this agent. Importantly, administration of ABT-737 to Mcl-1 knockout mice induced severe liver apoptosis, suggesting that tumor-specific inhibition of Mcl-1 is required for therapeutic purposes. Sorafenib transcriptionally down-regulated Mcl-1 expression specifically in tumor cells and abolished Mcl-1 up-regulation induced by ABT-737. Sorafenib, not alone but in combination with ABT-737, efficiently induced apoptosis in hepatoma cells. This combination also led to stronger suppression of xenograft tumors than sorafenib alone.. Bcl-xL inactivation by ABT-737 in combination with sorafenib was found to be safe and effective for anti-HCC therapy in preclinical models. Direct activation of the apoptosis machinery seems to unlock the antitumor potential of oncogenic kinase inhibitors and may produce durable clinical responses against HCC.

Topics: Animals; Antineoplastic Agents; Apoptosis; bcl-X Protein; Benzenesulfonates; Biphenyl Compounds; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Down-Regulation; Humans; Liver Neoplasms; Mice; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Transplantation; Niacinamide; Nitrophenols; Phenylurea Compounds; Piperazines; Proto-Oncogene Proteins c-bcl-2; Pyridines; RNA, Small Interfering; Sorafenib; Sulfonamides

1: The metabolism by HepG2 cell from two sources (M1, M2) of 12 substrates is reported: ethoxyresorufin, ethoxycoumarin, testosterone, tolbutamide, chlorzoxazone, dextromethorphan, phenacetin, midazolam, acetaminophen, hydroxycoumarin, p-nitrophenol and 1-chloro-2,4-dinitrobenzene (CDNB), and a pharmaceutical compound, EMD68843. 2: Activities varied markedly. Some were present in M1 (CYP1A, CYP2C9, CYP2E1) but absent in M2. M1 had a more complete set of Phase I enzymes than M2. CYP1A2, CYP2C9, CYP2D6, CYP2E1 and CYP3A activities were present at levels similar to human hepatocytes. Phase II metabolism differed between M1 and M2. M1 conjugated hydroxycoumarin and p-nitrophenol to glucuronides only, whereas M2 produced sulfates. Glutathione conjugation of CDNB metabolism was 10-fold higher in M1 than in M2, but was still much lower than in human hepatocytes. CYP2E, CYP2C, CYP2B6 and CYP3A (but not CYP1A, glucuronyl S-transferase or S-transferase) were inducible in M1. Metabolites of EMD68843, produced by induced (but not uninduced) M1 were the same as those produced in human hepatocytes. 3: In conclusion, HepG2 cells have both Phase I and II enzymes, which activities and at what levels depend on the source and culture conditions. Therefore, HepG2 cells routinely used in in vitro assays should be characterized for their drug-metabolizing capabilities before any results can be fully interpreted.

Topics: Aryl Hydrocarbon Hydroxylases; Benzofurans; Carcinoma, Hepatocellular; Cell Culture Techniques; Cell Line, Tumor; Coumarins; Dinitrochlorobenzene; Enzyme Activation; Humans; Indoles; Kinetics; Liver Neoplasms; Nitrophenols; Oxazines; Piperazines; Substrate Specificity; Umbelliferones; Vilazodone Hydrochloride

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

The expression of high- and low-molecular weight acid phosphatase (HMr- and LMr-AP) and zinc ion-dependent acid phosphatase (HMr-ZnAP and LMr-ZnAP) was compared in normal human liver and in Hep G2 human hepatocarcinoma cell line extracts. The investigation was carried out using Sephadex G-100 chromatography, molecular weight determination, and analysis of some distinctive biochemical characteristics and immunochemical properties. Normal human liver and Hep G2 cell lines expressed both HMr- and LMr-AP enzymes although in different proportions. HMr-ZnAP was detected only in human liver extract, while LMr-ZnAP was present only in hepatoma cell extract, indicating that they were differentially expressed in normal and transformed human liver cells.

Topics: Acid Phosphatase; Animals; Blotting, Western; Carcinoma, Hepatocellular; Cell Fractionation; Chlorides; Dextrans; Enzyme Activation; Gels; Humans; Hydrogen-Ion Concentration; Indicators and Reagents; Liver; Liver Neoplasms, Experimental; Nitrophenols; Organophosphorus Compounds; Tumor Cells, Cultured; Zinc Compounds

1. The objective of this study was to examine the usefulness of the hepatoma cell line Hep G2 as a model for human sulphoconjugation of drugs, in particular stereoselective conjugation. 2. Using the substrates p-nitrophenol and dopamine, we found sulphation activities consistent with the presence of both the phenol (P) and the monoamine (M) form of the human phenolsulphotransferases in these cells. 3. The Kmapp was 3.0 microM for the sulphation of p-nitrophenol. This activity was inhibited selectively by 2,6-dichloro-4-nitrophenol, IC50 6 microM. The Kmapp was 39 microM for the sulphation of dopamine. This activity was selectively inhibited by elevated temperature. 4. The chiral adrenergic drugs (+/-)-terbutaline and (+/-)-4-hydroxypropranolol were both sulphated stereoselectively with Kmapp and Vmaxapp values for each enantiomer virtually identical to previous observations with human liver cytosol. 5. In a direct comparison, the estimated activity of the P form of phenolsulphotransferase in the Hep G2 cell line was 30% of that in human liver, whereas, surprisingly, the activity of the M form of phenolsulphotransferase was 4.5 times higher in the Hep G2 cells than in the liver.

Topics: Arylsulfotransferase; Carcinoma, Hepatocellular; Dopamine; Humans; Liver; Liver Neoplasms; Nitrophenols; Propranolol; Sulfates; Temperature; Terbutaline; Tumor Cells, Cultured

Occurrence of estrone, estradiol, and testosterone glucuronyltranferase activities was tested in a well-differentiated hepatoma, Reuber H35. Transferase activities for estrone and estradiol were found in the hepatoma. The Michaelis-Menten kinetics of these two microsomal glucuronyltransferase activities were similar in hepatoma and in liver preparations, except for a somewhat higher apparent Km for estradiol in the hepatoma preparations. Under the same experimental conditions, only trace amounts of testosterone glucuronyltransferase activity could be detected in the hepatoma preparations. By contrast, in liver microsomal preparations, testosterone glucuronyltransferase activity was the highest among the steroid glucuronyltransferase activities tested.

Topics: Animals; Carcinoma, Hepatocellular; Estradiol; Estrone; Glucuronates; Glucuronosyltransferase; Hexosyltransferases; In Vitro Techniques; Kinetics; Liver Neoplasms; Male; Microsomes, Liver; Neoplasms, Experimental; Nitrophenols; Rats; Rats, Inbred ACI; Testosterone; Uridine Diphosphate Glucuronic Acid

Beta-Xylosides stimulate 2- to 6-fold the synthesis of glycosaminoglycans by three types of nonconnective tissue cells (RG-C6, NB41A, and rat hepatoma cells, and normal and simian virus 40 (SV40)-transformed normal human skin fibroblasts. The effect, which is specific for the anomeric linkage and the glycone, is observed in the presence and absence of puromycin. Beta-Xylosides may substitute for xylosylated core protein as initiators of synthesis of chondroitin sulfate chains. No stimulation of synthesis of heparan sulfate was observed. With the use of a fluorogenic xyloside, 4-methylumbelliferyl-beta-D-xyloside, it was demonstrated that the free chondroitin sulfate chains secreted into the medium bear the xyloside at the reducing end, and have an average molecular weight of 16,500.

Topics: Animals; Carcinoma, Hepatocellular; Cells, Cultured; Chondroitin; Chromatography, Gel; Dermatan Sulfate; Fibroblasts; Galactose; Glycosaminoglycans; Glycosides; Heparitin Sulfate; Humans; Hyaluronic Acid; Hymecromone; Liver Neoplasms; Mice; Neuroblastoma; Neuroglia; Nitrophenols; Rats; Simian virus 40; Skin; Spectrometry, Fluorescence; Sulfur Radioisotopes; Sulfuric Acids; Tritium; Xylose

Topics: Amines; Animals; Bilirubin; Carcinoma, Hepatocellular; Cattle; Clone Cells; Depression, Chemical; Glucuronates; Glucuronidase; Glucuronosyltransferase; Liver Neoplasms; Methods; Neoplasms, Experimental; Nitrophenols; Phenols; Rats; Serum Albumin, Bovine; Time Factors

Topics: Animals; Carcinoma 256, Walker; Carcinoma, Hepatocellular; Glucuronates; Glucuronosyltransferase; Kinetics; Liver; Liver Neoplasms; Neoplasms, Experimental; Nitrophenols; Rats; Rats, Inbred ACI; Sulfuric Acids; Sulfurtransferases

Topics: Aniline Compounds; Animals; Bilirubin; Binding Sites; Carcinoma, Hepatocellular; Cell Line; Clone Cells; Glucuronates; In Vitro Techniques; Liver Neoplasms; Male; Nitrophenols; Phenols; Proadifen; Rats

Topics: Animals; Carcinoma, Hepatocellular; Clone Cells; Coumarins; Digitonin; Edetic Acid; Female; Glucosamine; Glucuronates; Glucuronosyltransferase; Hexosyltransferases; Liver Neoplasms; Male; Microsomes, Liver; Nitrophenols; Nitrosamines; Orotic Acid; Phenols; Proteins; Rats; Stimulation, Chemical; Time Factors

Topics: Alkaline Phosphatase; Amino Acids; Ascitic Fluid; Binding, Competitive; Carcinoma, Hepatocellular; Chromatography, DEAE-Cellulose; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Glycerophosphates; Humans; Kinetics; Leucine; Liver Neoplasms; Mathematics; Nitrophenols; Norleucine; Phenols; Phenylalanine; Phosphates; Tryptophan

Topics: Animals; Carbon Isotopes; Carcinoma, Hepatocellular; Chlorpromazine; Clone Cells; Culture Techniques; Depression, Chemical; Glucuronidase; Liver Neoplasms; Nitrophenols; Phenols; Rats

Topics: Aflatoxins; Aminopyrine; Animals; Benzopyrenes; Carcinogens; Carcinoma, Hepatocellular; Cell Transformation, Neoplastic; Diet; Hyperplasia; Lipotropic Agents; Liver; Liver Neoplasms; Male; Mixed Function Oxygenases; Neoplasms, Experimental; Nitrophenols; Oxidoreductases; Precancerous Conditions; Rats; Thymidine; Transferases; Tritium

Topics: Acid Phosphatase; Alkaline Phosphatase; Animals; Calcium; Carcinogens; Carcinoma, Hepatocellular; Castration; Cell Membrane; Cholesterol; Edetic Acid; Estrone; Female; Glycerophosphates; Liver; Liver Neoplasms; Magnesium; Male; Neoplasms, Experimental; Nitrophenols; Phenobarbital; Potassium; Rats; Sex Factors; Testosterone; Zinc

Topics: Animals; Carcinoma, Hepatocellular; Electron Transport Complex IV; Liver; Liver Neoplasms; Malate Dehydrogenase; Microscopy, Electron; Mitochondria, Liver; Monoamine Oxidase; Nitrophenols; Phosphotransferases; Rats; Respiration; Staining and Labeling

Topics: Animals; Carcinoma, Hepatocellular; Cell Line; Culture Techniques; Female; Glucosyltransferases; Glucuronidase; Liver Neoplasms; Male; Microsomes, Liver; Neoplasm Transplantation; Neoplasms, Experimental; Nitrophenols; Rats

Topics: 2,4-Dinitrophenol; Ascites; Carcinoma, Hepatocellular; Dinitrophenols; Liver; Liver Neoplasms; Metabolism; Neoplasms; Neoplasms, Experimental; Nitrophenols