Accumulation of polyhydroxybutyrate (PHB) seems to be a common metabolic strategy adopted by many bacteria to cope with cold environments. This work aimed at evaluating and understanding the cryoprotective effect of PHB. At first a monomer of PHB, 3-hydroxybutyrate, was identified as a potent cryoprotectant capable of protecting model enzyme (lipase), yeast (Saccharomyces cerevisiae) and bacterial cells (Cupriavidus necator) against the adverse effects of freezing-thawing cycles. Further, the viability of the frozen-thawed PHB accumulating strain of C. necator was compared to that of the PHB non-accumulating mutant. The presence of PHB granules in cells was revealed to be a significant advantage during freezing. This might be attributed to the higher intracellular level of 3-hydroxybutyrate in PHB accumulating cells (due to the action of parallel PHB synthesis and degradation, the so-called PHB cycle), but the cryoprotective effect of PHB granules seems to be more complex. Since intracellular PHB granules retain highly flexible properties even at extremely low temperatures (observed by cryo-SEM), it can be expected that PHB granules protect cells against injury from extracellular ice. Finally, thermal analysis indicates that PHB-containing cells exhibit a higher rate of transmembrane water transport, which protects cells against the formation of intracellular ice which usually has fatal consequences.
BACKGROUND: Dysregulation of the balance between cell growth and death in the colonic epithelium is associated with cancer promotion. Understanding how cell death in this self-renewing tissue is regulated and how it is influenced by interaction of specific dietary components, especially fat and fibre, could lead to improved treatment and prevention strategies for cancer. AIM OF THE STUDY: The effects of two types of polyunsaturated fatty acids (PUFAs)--arachidonic (AA, 20:4, n-6) or docosahexaenoic (DHA, 22:6, n-3)--on the response of human colon adenocarcinoma HT-29 cells to sodium butyrate (NaBt) were investigated. METHODS: The parameters reflecting cell proliferation and cell death were studied together with oxidative response, mitochondrial membrane potential (MMP) and changes of selected regulatory molecules associated with cell cycle (p27(Kip1) and p21(Cip1/WAF1)) and apoptosis (caspase-3, caspase-9, poly (ADP-ribose) polymerase--PARP, Bcl-2, Bax, Bak,Mcl-1). RESULTS: We demonstrated that pre-treatment with either AA or DHA attenuated cell cycle arrest caused by NaBt which is associated with modulation of p27(Kip1), but not p21(Cip1/WAF1) protein expression. On the other hand, PUFAs sensitised HT-29 cells to NaBt-induced apoptosis. An increased amount of floating cells and cells in the subG(0)/G(1) population was associated with increased reactive oxygen species production, lipid peroxidation, decrease of MMP, activation of caspase-3 and -9, PARP cleavage, and decrease in the expression of antiapoptotic Mcl-1 protein. The observed effects were modulated by the addition of a protein synthesis inhibitor, cycloheximide, and partially reversed by the antioxidant Trolox. CONCLUSIONS: PUFAs may have beneficial effects in the colon enhancing apoptosis induced by NaBt. Alteration of cell membrane lipid composition and potentiation of oxidative processes accompanied by changes in mitochondria followed by stimulation of apoptotic cascade components play a role in these effects.
- MeSH
- Adenocarcinoma * metabolism MeSH
- Apoptosis drug effects MeSH
- Cell Cycle drug effects MeSH
- HT29 Cells MeSH
- Butyrates * metabolism MeSH
- Microscopy, Fluorescence MeSH
- Caspase 3 MeSH
- Caspase 9 MeSH
- Caspases metabolism drug effects MeSH
- Arachidonic Acids administration & dosage MeSH
- Docosahexaenoic Acids administration & dosage MeSH
- Humans MeSH
- Membrane Potentials drug effects MeSH
- Colonic Neoplasms * metabolism MeSH
- Fatty Acids, Unsaturated * metabolism MeSH
- Lipid Peroxidation drug effects MeSH
- Poly(ADP-ribose) Polymerases metabolism drug effects MeSH
- Proto-Oncogene Proteins metabolism drug effects MeSH
- Flow Cytometry MeSH
- Reactive Oxygen Species metabolism MeSH
- Dose-Response Relationship, Drug MeSH
- Blotting, Western MeSH
- Check Tag
- Humans MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
- Comparative Study MeSH
OBJECTIVES: The resistance of tumor cells to cisplatin remains a major cause of treatment failure in cancer patients. In this study, the ability of Pt(IV) complex with adamantylamine-LA-12 and its reduced counterpart with lower oxidation state Pt(II)-LA-9 to overcome intrinsic cisplatin resistance was investigated. METHODS: The ovarian adenocarcinoma SK-OV-3 cells were exposed to cisplatin, LA-9, or LA-12 for 72 h and the effects of drug concentrations that caused 10% or 50% inhibition of cell proliferation were determined. After 24-72 h of sustained exposure viability, apoptosis and inhibition of proliferation were analyzed. DNA synthesis and cell cycle analysis were performed simultaneously in order to determine the modulation of cell cycle after platinum complexes treatment. RESULTS: Lung Resistance-related Protein (LRP/MVP) was detected in SK-OV-3 cells but not in the other two ovarian cancer lines with different sensitivity to cisplatin. LRP/MVP overexpression may be an important factor contributing to intrinsic cisplatin resistance. Interestingly, Pt(IV) complex-LA-12 had approximately 2.7-fold lower IC(50) concentration than LA-9 or cisplatin in SK-OV-3 cells. Moreover, LA-12 caused persistent accumulation of cells in S-phase of the cell cycle while LA-9 and cisplatin treatment-induced S-phase arrest was transient and shifted to G(2)/M-phase at later intervals. Apoptosis seemed to be not the dominant type of cell death caused by such the derivatives, but it was the most intensive after LA-12 treatment. CONCLUSIONS: We found strong differences between effects of Pt(IV) complex-LA-12 and Pt(II) derivatives-LA-9 and cisplatin on cytokinetic parameters. Overall, LA-12 but not its reduced Pt(II) counterpart LA-9 is the compound effective in p53 null human ovarian cancer cells and it is able to overcome intrinsic cisplatin resistance in these cells.
- MeSH
- Adenocarcinoma drug therapy metabolism pathology MeSH
- Amantadine analogs & derivatives administration & dosage MeSH
- Cell Cycle drug effects MeSH
- Cell Growth Processes drug effects MeSH
- Drug Resistance, Neoplasm MeSH
- Cisplatin administration & dosage MeSH
- DNA, Neoplasm biosynthesis MeSH
- Financing, Organized MeSH
- Humans MeSH
- Cell Line, Tumor MeSH
- Neoplasm Proteins biosynthesis MeSH
- Ovarian Neoplasms drug therapy metabolism pathology MeSH
- Organoplatinum Compounds administration & dosage pharmacology MeSH
- Poly(ADP-ribose) Polymerases metabolism MeSH
- Antineoplastic Combined Chemotherapy Protocols pharmacology MeSH
- Vault Ribonucleoprotein Particles antagonists & inhibitors MeSH
- Blotting, Western MeSH
- Check Tag
- Humans MeSH
- Female MeSH
BACKGROUND: Fatty acid-induced apoptosis and ER stress of pancreatic β-cells contribute to the development of type 2 diabetes, however, the molecular mechanisms involved are unclear. AIMS: In this study we have tested the role of caspase-2 and suggested ER stress mediator JNK in saturated fatty acid-induced apoptosis of the human pancreatic β-cells NES2Y. RESULTS: We found that stearic acid at apoptosis-inducing concentration activated ER stress signaling pathways, i.e. IRE1α, PERK and ATF6 pathways, in NES2Y cells. During stearic acid-induced apoptosis, JNK inhibition did not decrease the rate of apoptosis nor the activation of caspase-8, -9, -7 and -2 and PARP cleavage. In addition, inhibition of JNK activity did not affect CHOP expression although it did decrease the induction of BiP expression after stearic acid treatment. Caspase-2 silencing had no effect on PARP as well as caspase-8, -9 and -7 cleavage and the induction of CHOP expression, however, it also decreased the induction of BiP expression. Surprisingly, caspase-2 silencing was accompanied by increased phosphorylation of c-Jun. CONCLUSIONS: We have demonstrated that caspase-2 as well as JNK are not key players in apoptosis induction by saturated fatty acids in human pancreatic β-cells NES2Y. However, they appear to be involved in the modulation of saturated fatty acid-induced ER stress signaling, probably by a mechanism independent of c-Jun phosphorylation.
- MeSH
- Apoptosis drug effects MeSH
- Insulin-Secreting Cells cytology metabolism MeSH
- DNA-Binding Proteins genetics metabolism MeSH
- Phosphorylation MeSH
- JNK Mitogen-Activated Protein Kinases antagonists & inhibitors metabolism MeSH
- Caspase 2 chemistry genetics metabolism MeSH
- Caspase 7 metabolism MeSH
- Caspase 8 metabolism MeSH
- Caspase 9 metabolism MeSH
- Stearic Acids pharmacology MeSH
- Humans MeSH
- RNA, Small Interfering metabolism MeSH
- Poly(ADP-ribose) Polymerases metabolism MeSH
- Heat-Shock Proteins metabolism MeSH
- RNA Interference MeSH
- Signal Transduction drug effects MeSH
- Endoplasmic Reticulum Stress drug effects MeSH
- Activating Transcription Factor 6 metabolism MeSH
- Transcription Factor CHOP metabolism MeSH
- Transcription Factors genetics metabolism MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The homeostasis of the gut epithelium relies upon continuous renewal and proliferation of crypt-resident intestinal epithelial stem cells (IESCs). Wnt/β-catenin signaling is required for IESC maintenance, however, it remains unclear how this pathway selectively governs the identity and proliferative decisions of IESCs. Here, we took advantage of knock-in mice harboring transgenic β-catenin alleles with mutations that specifically impair the recruitment of N- or C-terminal transcriptional co-factors. We show that C-terminally-recruited transcriptional co-factors of β-catenin act as all-or-nothing regulators of Wnt-target gene expression. Blocking their interactions with β-catenin rapidly induces loss of IESCs and intestinal homeostasis. Conversely, N-terminally recruited co-factors fine-tune β-catenin's transcriptional output to ensure proper self-renewal and proliferative behaviour of IESCs. Impairment of N-terminal interactions triggers transient hyperproliferation of IESCs, eventually resulting in exhaustion of the self-renewing stem cell pool. IESC mis-differentiation, accompanied by unfolded protein response stress and immune infiltration, results in a process resembling aberrant "villisation" of intestinal crypts. Our data suggest that IESC-specific Wnt/β-catenin output requires selective modulation of gene expression by transcriptional co-factors.
- MeSH
- Algorithms MeSH
- beta Catenin chemistry metabolism MeSH
- Cell Differentiation MeSH
- Chromatin metabolism MeSH
- Phenotype MeSH
- Transcription, Genetic * MeSH
- Homeostasis MeSH
- Hyperplasia MeSH
- JNK Mitogen-Activated Protein Kinases metabolism MeSH
- Stem Cells metabolism MeSH
- RNA, Messenger genetics metabolism MeSH
- Mutation genetics MeSH
- Mutant Proteins metabolism MeSH
- Mice MeSH
- Organoids metabolism MeSH
- Cell Proliferation MeSH
- Chromatin Assembly and Disassembly MeSH
- Base Sequence MeSH
- Signal Transduction MeSH
- Intestinal Mucosa cytology MeSH
- Transcription Factors metabolism MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Antifungal drug ketoconazole causes severe drug-drug interactions by influencing gene expression and catalytic activity of major drug-metabolizing enzyme cytochrome P450 CYP3A4. Ketoconazole is administered in the form of racemic mixture of two cis-enantiomers, i.e. (+)-ketoconazole and (-)-ketoconazole. Many enantiopure drugs were introduced to human pharmacotherapy in last two decades. In the current paper, we have examined the effects of ketoconazole cis-enantiomers on the expression of CYP3A4 in human hepatocytes and HepG2 cells and on catalytic activity of CYP3A4 in human liver microsomes. We show that both ketoconazole enantiomers induce CYP3A4 mRNA and protein in human hepatocytes and HepG2 cells. Gene reporter assays revealed partial agonist activity of ketoconazole enantiomers towards pregnane X receptor PXR. Catalytic activity of CYP3A4/5 towards two prototypic substrates of CYP3A enzymes, testosterone and midazolam, was determined in presence of both (+)-ketoconazole and (-)-ketoconazole in human liver microsomes. Overall, both ketoconazole cis-enantiomers induced CYP3A4 in human cells and inhibited CYP3A4 in human liver microsomes. While interaction of ketoconazole with PXR and induction of CYP3A4 did not display enantiospecific pattern, inhibition of CYP3A4 catalytic activity by ketoconazole differed for ketoconazole cis-enantiomers ((+)-ketoconazole IC₅₀ 1.69 µM, Ki 0.92 µM for testosterone, IC₅₀ 1.46 µM, Ki 2.52 µM for midazolam; (-)-ketoconazole IC₅₀ 0.90 µM, Ki 0.17 µM for testosterone, IC₅₀ 1.04 µM, Ki 1.51 µM for midazolam).
- MeSH
- Biocatalysis drug effects MeSH
- Hep G2 Cells MeSH
- Cytochrome P-450 CYP3A genetics metabolism MeSH
- Transcription, Genetic drug effects MeSH
- Hepatocytes drug effects enzymology MeSH
- Microsomes, Liver drug effects metabolism MeSH
- Ketoconazole chemistry pharmacology MeSH
- Kinetics MeSH
- Humans MeSH
- RNA, Messenger genetics metabolism MeSH
- Gene Expression Regulation, Enzymologic drug effects MeSH
- Genes, Reporter MeSH
- Stereoisomerism MeSH
- Receptors, Steroid genetics metabolism MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The expression of the transcription factor encoded by the Wilms tumor gene 1 (WT1) is associated with a variety of human cancers. WT1 protein has been reported to serve as a target antigen for tumor-specific immune responses. We observed that the immunization of mice with peptide vaccines derived from WT1 in a mixture with the CpG adjuvant (ODN 1826) by tattoo administration was superior to subcutaneous delivery of the peptides in combination with CpG formulated with the mineral oil adjuvant or a DNA vaccine or a recombinant vaccinia virus vaccine expressing the truncated WT1 protein. Tattooing with the WT1122-140 and WT1126-134 peptide elicited the response of WT1-specific interferon-γ-producing T cells. Peptide vaccine administered with a tattoo device had an antitumor effect on the growth of the prostate tumor cell line TRAMP-C2, provided that the transforming growth factor-β produced by tumor cells was neutralized by anti-TGFβ monoclonal antibody. The treatment of the tumor-bearing mice with 5-azadeoxycytidine or poly IC did not work in synergy with the peptide vaccine.
- MeSH
- Adjuvants, Immunologic MeSH
- Azacitidine analogs & derivatives pharmacology therapeutic use MeSH
- Injections, Intradermal MeSH
- Antibodies, Monoclonal immunology MeSH
- Mice, Inbred BALB C MeSH
- Mice MeSH
- Cell Line, Tumor MeSH
- Prostatic Neoplasms therapy MeSH
- Poly I-C pharmacology therapeutic use MeSH
- WT1 Proteins administration & dosage immunology MeSH
- Antineoplastic Agents pharmacology therapeutic use MeSH
- Cancer Vaccines administration & dosage MeSH
- Vaccines, Subunit administration & dosage MeSH
- Tattooing MeSH
- Transforming Growth Factor beta antagonists & inhibitors immunology MeSH
- Animals MeSH
- Check Tag
- Male MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Inhibitors of cyclin-dependent kinases 9 have been developed as potential anticancer drugs for the treatment of multiple myeloma. We have previously prepared a library of arylazo-3,5-diaminopyrazole inhibitors of CDKs. Here, we describe a novel member, AAP1742 (CDK9 inhibition with IC(50) = 0.28 μm), that reduces the viability of multiple myeloma cell lines in low micromolar concentrations. Consistent with inhibition of CDK9, AAP1742 decreases the phosphorylation of RNA polymerase II and inhibits mRNA synthesis of anti-apoptotic proteins Mcl-1, Bcl-2, and XIAP, followed by apoptosis in the RPMI-8226 cell line in a dose- and a time-dependent manner. These results are consistent with the biochemical profile of AAP1742 and further suggest cellular inhibition of CDK9 as a possible target for anticancer drugs.
- MeSH
- Enzyme Activation drug effects MeSH
- Apoptosis drug effects MeSH
- Azo Compounds chemistry pharmacology MeSH
- Cyclin-Dependent Kinase 9 antagonists & inhibitors metabolism MeSH
- Down-Regulation drug effects MeSH
- Phosphorylation drug effects MeSH
- Humans MeSH
- RNA, Messenger metabolism MeSH
- Mitochondria metabolism MeSH
- Multiple Myeloma metabolism pathology MeSH
- Cell Line, Tumor MeSH
- Myeloid Cell Leukemia Sequence 1 Protein genetics metabolism MeSH
- Antineoplastic Agents chemistry pharmacology MeSH
- Proto-Oncogene Proteins c-bcl-2 genetics metabolism MeSH
- Pyrazoles chemistry pharmacology MeSH
- RNA Polymerase II metabolism MeSH
- X-Linked Inhibitor of Apoptosis Protein genetics metabolism MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The aim of this study was to characterize an in vitro modulating effect of three commensal Lactobacillus strains on cellular differentiation of non-transformed crypt-like rat small intestinal cell line IEC-18. IEC-18 was grown on extracellular matrix, with or without presence of Lactobacillus strains. Gene expression of IEC-18 bacterial detection system - such as Toll-like receptors TLR-2, TLR-4, signal adapter MyD88, cytoplasmic NOD2 receptor, inflammatory cytokines IL-18, IL-1beta, chemokine IL-8 and enzyme caspase-1 - was evaluated using real-time PCR. Expression and localization of TLR-2, TLR-4, IL-18 and caspase-1 proteins was demonstrated by Western blotting and immunofluorescent staining. Secretion of IL-18 to apical and basolateral surfaces was assayed by ELISA. Our results suggested that L. casei LOCK0919 accelerated differentiation of IEC-18 by stimulating TLR-2, TLR-4, MyD88, IL-18, caspase-1 mRNAs and proteins. L. casei LOCK0919 increased expression and transfer of villin and beta-catenin from cytoplasm to cell membrane. Presence of L. rhamnosus LOCK0900 resulted in detachment of IEC-18 layer from extracellular matrix leading to induction of IL-1beta, of TLR-2 and IL-8 mRNAs and stimulation of MyD88, caspase-1 and cytosolic receptor NOD2 mRNAs. L. rhamnosus LOCK0908 was not recognized by TLR-2 or TLR-4 receptors. Lactobacilli-IEC-18 crosstalk enhanced immune and barrier mucosal functions.
- MeSH
- beta Catenin biosynthesis MeSH
- Cell Differentiation drug effects MeSH
- Cytokines biosynthesis MeSH
- Epithelial Cells drug effects MeSH
- Interleukin-18 biosynthesis MeSH
- Caspase 1 biosynthesis MeSH
- Rats MeSH
- Lacticaseibacillus rhamnosus * MeSH
- Lacticaseibacillus casei * MeSH
- RNA, Messenger biosynthesis MeSH
- Microfilament Proteins biosynthesis MeSH
- Probiotics pharmacology MeSH
- Gene Expression Regulation drug effects MeSH
- Intestinal Mucosa cytology drug effects MeSH
- Subcellular Fractions metabolism MeSH
- Toll-Like Receptors biosynthesis drug effects MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
