Antiproliferative activity of Humulus lupulus extracts on human hepatoma (Hep3B), colon (HT-29) cancer cells and proteases, tyrosinase, ß-lactamase enzyme inhibition studies.

The aims of this study were to examine the antiproliferation of Humulus lupulus extracts on human hepatoma carcinoma (Hep3B) and human colon carcinoma (HT-29) cell lines along with enzyme inhibitory effects of the crude extracts. Potential cell cytotoxicity of six different H. lupulus extracts were assayed on various cancer cells using MTT assay at 24, 48 and 72 h intervals. Methanol-1 extract has inhibited the cell proliferation with doses of 0.6-1 mg/mL in a time dependent (48 and 72 hours) manner in Hep3B cells with 70% inhibition, while inhibitory effect was not seen in colon cancer cells. Acetone extract has increased the cell proliferation at low doses of 0.1 mg/mL for 72 h in Hep3B cells and 0.1-0.2 mg/mL for 48 and 72 h in HT29 cells. The inhibitory effects of the extracts were compared by relative maximum activity values (V(max)) using proteases such as α-chymotrypsin, trypsin and papain, tyrosinase and ß-lactamase (penicillinase).

Hypoxic Regulation of the KLK4 Gene in two Different Prostate Cancer Cells Treated with TGF- ß.

The human kallikrein-related peptidase (KLK) family which consists of 15 members is associated with prostate cancer and other cancers. It has been reported that overexpression of KLK4 in prostate cancer correlates with bone metastasis or advanced stage. Hypoxia occurs in the early stages of prostate cancer due to the accumulation of acidic metabolites or reactive oxygen species (ROS). In our study, KLK4 gene expression in hypoxic conditions in PC-3 and LNCaP cells which are treated with TGF-ß was evaluated with mRNA, protein, and promoter activity levels. A chemical hypoxia model was created and confirmed at mRNA and protein level. No statistically significant cytotoxic effect of CoCl2 and TGF-ß was observed in PC-3 and LNCaP cells with the MTT test. Four different truncated KLK4 gene promoter constructs were cloned in pmetLuc expression vector and basal activities of all promoter fragments were analyzed. The activities of P1 (-447/ + 657), P2 (-103/ + 657), and P3 (-267/ + 657) promoter fragments increased in hypoxic conditions except P4 (+555/ + 657), which does not contain the SMAD and HRE region. KLK4 mRNA levels in both PC-3 and LNCaP cells increased in the hypoxia and hypoxia/TGF groups compared to the non-treated groups. The stimulating effect of TGF-ß is correlated with the increase in SMAD2/3 mRNA levels. KLK4 expression is up-regulated by TGF-ß, especially under hypoxic conditions, and its interaction with the SMAD pathway is determined with different inhibitor experiments. HIF-1α and SMAD transcription factors bind to the KLK4 promoter showing the direct interaction of HIF-1α (-80/-52) and SMAD (+163/+194) regions with EMSA.

Mutation of active site residues Asn67 to Ile, Gln92 to Val and Leu204 to Ser in human carbonic anhydrase II: influences on the catalytic activity and affinity for inhibitors.

Site-directed mutagenesis has been used to change three amino acid residues involved in the binding of inhibitors (Asn67Ile; Gln92Val and Leu204Ser) within the active site of human carbonic anhydrase (CA, EC 4.2.1.1) II (hCA II). Residues 67, 92 and 204 were changed from hydrophobic to hydrophilic ones, and vice versa. The Asn67Ile and Leu204Ser mutants showed similar k(cat)/K(M) values compared to the wild type (wt) enzyme, whereas the Gln92Val mutant was around 30% less active as a catalyst for CO(2) hydration to bicarbonate compared to the wt protein. Affinity for sulfonamides/sulfamates was decreased in all three mutants compared to wt hCA II. The effect was stronger for the Asn67Ile mutant (the closest residue to the zinc ion), followed by the Gln92Val mutant (residue situated in the middle of the active site) and weakest for the Leu204Ser mutant, an amino acid situated far away from the catalytic metal ion, at the entrance of the cavity. This study shows that small perturbations within the active site architecture have influences on the catalytic efficiency but dramatically change affinity for inhibitors among the CA enzymes, especially when the mutated amino acid residues are nearby the catalytic metal ion.

Evaluation of in vitro effects of some analgesic drugs on erythrocyte and recombinant carbonic anhydrase I and II.

The in vitro effects of the injectable form of analgesic drugs, dexketoprofen trometamol, dexamethasone sodium phosphate, metamizole sodium, diclofenac sodium, thiocolchicoside, on the activity of purified human carbonic anhydrase I and II were evaluated. The effect of these drugs on erythrocyte hCA I and hCA II was compared to recombinant hCA I and hCA II expressed in Ecoli. IC(50) values of the drugs that caused inhibition were determined by means of activity percentage diagrams. The IC(50) concentrations of dexketoprofen trometamol and dexamethasone sodium phosphate on hCA I were 683 µM and 4250 µM and for hCA II 950 µM and 6200 µM respectively. Conversely, the enzyme activity was increased by diflofenac sodium. In addition, thiocolchicoside has not any affect on hCA I and hCA II. The effect of these drugs on erythrocyte hCA I and hCA II were consistent with the inhibition of recombinant enzymes.

Essential oil chemical composition, antimicrobial, anticancer, and antioxidant effects of Thymus convolutus Klokov in Turkey.

In this study, the chemical composition, antimicrobial, antioxidant, and anticancer effects of Thymus convolutus Klokov oil and its main compound camphor were investigated. The oil was isolated from T. convolutus using hydrodistillation method, analyzed by gas chromatography/mass spectrometry (GC-MS), and 66 compounds were identified. The main component was determined as camphor at 16.6%. The antioxidant properties were identified with the DPPH (2,2'-diphenyl-1-picrylhydrazyl) radical-scavenging method and, 33.39 ± 0.25% DPPH was scavenging in 1000 µg/mL of essential oil. The strong antimicrobial activity was observed against Escherichia coli, Enterobacter aerogenes, Proteus vulgaris, and Pseudomonas aeruginosa with MIC values of 125 µg/mL. Aspergillus flavus was more sensitive (28%) against T. convolutus essential oil than other fungi. The cytotoxic effect of oil was analyzed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) method. Camphor was effective on human hepatoma cells (Hep3B) at concentrations of 1 mg/mL, 500, 250, and 125 µg/mL, while essential oil of T. convolutus was found to be effective at concentrations of 250 and 125 µg/mL. A reduction in cell proliferation was observed in colon carcinoma cells (HT-29) treated with 500 µg/mL camphor for 48 h. No statistically significant effect was found in Umbilical Vein Endothelial Cells (HUVEC) treated with essential oil and camphor.

Mutation of Phe91 to Asn in human carbonic anhydrase I unexpectedly enhanced both catalytic activity and affinity for sulfonamide inhibitors.

Site-directed mutagenesis has been used to change one amino acid residue considered non essential (Phe91Asn) to catalysis in carbonic anhydrase (CA, EC 4.2.1.1) isozyme I (hCA I), but which is near the substrate binding pocket of the enzyme. This change led to a steady increase of 16% of the catalytic activity of the mutant hCA I over the wild type enzyme, which is a gain of 50% catalytic efficiency if one compares hCA I and hCA II as catalysts for CO(2) hydration. This effect may be due to the bigger hydrophobic pocket in the mutant enzyme compared to the wild type one, which probably leads to the reorganization of the solvent molecules present in the cavity and to a diverse proton transfer pathway in the mutant over the non mutated enzyme. To our surprise, the mutant CA I was not only a better catalyst for the physiologic reaction, but in many cases also showed higher affinity (2.6-15.9 times) for sulfonamide/sulfamate inhibitors compared to the wild type enzyme. As the residue in position 91 is highly variable among the 13 catalytically active CA isoforms, this study may shed a better understanding of catalysis/inhibition by this superfamily of enzymes.

Upregulation of PSMD4 gene by hypoxia in prostate cancer cells.

Ubiquitin-proteasome pathways have a crucial role in tumor progression. PSMD4 (Rpn10, 26S proteasome non-ATPase subunit 4), which is a subunit of the regulatory particle, is a major ubiquitin (Ub) receptor of 26S proteasome. PSMD4 overexpression has been observed in colon carcinoma, hepatocellular carcinoma, and breast cancer. In this work, we elucidated the effect of hypoxia on PSMD4 gene expression in prostate cancer cells (PC3). Chemically mimicked hypoxia drastically upregulated PSMD4 gene expression at both mRNA and protein levels. Transient transfection experiments indicated that all promoter fragments were active in PC3 cells. Hypoxia increased transcriptional activity of all PSMD4 promoter constructs. EMSA analysis shows that HIF-1a transcription factor binds to the hypoxia response element (HRE) present within the -98/+52 region of PSMD4 promoter. We also used human umbilical vein endothelial cell (HUVEC) as a different cell model, in which increased PSMD4 expression was seen only at 24 h. The increased expression of the PSMD4 level in the PC3 cell line was not parallel to the expression in hypoxic HUVEC.

Carbonic anhydrase III is a new target of HIF1α in prostate cancer model.

Carbonic Anhydrase III (CAIII) belongs to a member of the alpha Carbonic Anhydrase (CA) family. Although some CA members are strongly up-regulated by HIF1-α, it is not known about the transcriptional regulation of CAIII in prostate cancer cells, PCa. Therefore, we aimed to identify regulatory regions important for the regulation of CAIII gene under hypoxic conditions in human prostate cancer cells (PC3). The present study, for the first time, demonstrated that the chemically mimicked hypoxic condition led to the induced CAIII mRNA and protein expression in prostate cancer cells. Transcriptional regulation of CAIII was investigated by transient transfection assay that indicates that the most active promoter activity was in the region of P2 -699/+86. Hypoxic condition also upregulates the basal activity of for P1;-941/+86 and P2;-699/+86 constructs containing putative Hypoxia Response Element (HRE) region located in -268/-252. EMSA analysis of HRE located in -268/-252 bases, showed one DNA-protein binding complexes. Competition assays indicated this complex is resulted from HIF1α interactions. In addition, site-directed mutagenesis of potential HIF1α binding sites diminished a DNA-protein complex. These findings suggest that CAIII is a hypoxia-regulated gene and valuable for targeting of prostate cancer tumors in hypoxic condition.

SP1 and USF differentially regulate ADAMTS1 gene expression under normoxic and hypoxic conditions in hepatoma cells.

ADAM metallopeptidase with thrombospondin type I motif, 1 (ADAMTS1) that has both antiangiogenic and aggrecanase activity was dysregulated in many pathophysiologic circumstances. However, there is limited information available on the transcriptional regulation of ADAMTS1 gene. Therefore, this study mainly aimed to identify regulatory regions important for the regulation of ADAMTS1 gene under normoxic and hypoxic conditions in human hepatoma cells (HEP3B). Cultured HEP3B cells were exposed to normal oxygen condition, and Cobalt chloride (CoCl2) induced the hypoxic condition, which is an HIF-1 inducer. The cocl2-induced hypoxic condition led to the induced ADAMTS1 mRNA and protein expression in Hepatoma cells. Differential regulation of SP1 and USF transcription factors on ADAMTS1 gene expression was determined by transcriptional activity, mRNA and protein level of ADAMTS1 gene. Ectopic expression of SP1 and USF transcription factors resulted in the decrease in ADAMTS1 transcriptional activity of all promoter constructs consistent with mRNA and protein level in normoxic condition. However, overexpression of SP1 and USF led to the increase of ADAMTS1 gene expressions at mRNA and protein level in hypoxic condition. On the other hand, C/EBPα transcription factor didn't show any statistically significant effect on ADAMTS1 gene expression at mRNA, protein and transcriptional level under normoxic and hypoxic condition.