A series of triterpenoid pyrones was synthesized and subsequently modified to introduce phthalimide or phthalate moieties into the triterpenoid skeleton. These compounds underwent in vitro cytotoxicity screening, revealing that a subset of six compounds exhibited potent activity, with IC50 values in the low micromolar range. Further biological evaluations, including Annexin V and propidium iodide staining experiment revealed, that all compounds induce selective apoptosis in cancer cells. Measurements of mitochondrial potential, cell cycle analysis, and the expression of pro- and anti-apoptotic proteins confirmed, that apoptosis was mediated via the mitochondrial pathway. These findings were further supported by cell cycle modulation and DNA/RNA synthesis studies, which indicated a significant increase in cell accumulation in the G0/G1 phase and a marked reduction in S-phase cells, alongside a substantial inhibition of DNA synthesis. The activation of caspase-3 and the cleavage of PARP, coupled with a decrease in the expression of Bcl-2 and Bcl-XL proteins, underscored the induction of apoptosis through the mitochondrial pathway. Given their high activity and pronounced effect on mitochondria function, trifluoromethyl pyrones 1f and 2f, and dihydrophthalimide 2h have been selected for further development.
- MeSH
- Apoptosis MeSH
- DNA metabolism MeSH
- Phthalimides pharmacology MeSH
- Phthalic Acids * MeSH
- Membrane Potential, Mitochondrial MeSH
- Mitochondria metabolism MeSH
- Cell Line, Tumor MeSH
- Neoplasms * drug therapy MeSH
- Antineoplastic Agents * therapeutic use MeSH
- Pyrones pharmacology MeSH
- Triterpenes * pharmacology MeSH
- Publication type
- Journal Article MeSH
Kojic acid is an industrially important secondary metabolite produced by Aspergillus oryzae. The construction of genetic materials for kojic acid related genes is important for understanding the mechanism of kojic acid synthesis in A. oryzae. However, multigene simultaneous knockout mutants for kojic acid synthesis genes remain limited because A. oryzae is multinuclear and good selectable markers are scarce. Here, we firstly successfully obtained single mutants of kojA, kojR, and kojT by our previously constructed CRISPR/Cas9 system in A. oryzae, which demonstrated the feasibility of the targeting sgRNAs for kojA, kojR, and kojT. Then, the AMA1-based genome-editing system for multiplex gene editing was developed in A. oryzae. In the multiplex gene-editing system, two guide RNA expression cassettes were ligated in tandem and driven by two U6 promoters in the AMA1-based autonomously replicating plasmid with the Cas9-expression cassette. Moreover, the multiplex gene-editing technique was applied to target the kojic acid synthesis genes kojA, kojR, and kojT, and the double and triple mutants within kojA, kojR, and kojT were obtained successfully. Additionally, the selectable marker pyrG was knocked out in the single and triple mutants of kojA, kojR, and kojT to obtain the auxotrophic strains, which can facilitate to introduce a target gene into the single and triple mutants of kojA, kojR, and kojT for investigating their relationship. The multiplex gene-editing system and release of these materials provide a foundation for further kojic acid research and utilization.
- MeSH
- Aspergillus oryzae * genetics metabolism MeSH
- CRISPR-Cas Systems MeSH
- Fungal Proteins genetics metabolism MeSH
- Pyrones metabolism MeSH
- Publication type
- Journal Article MeSH
The aim of the present study is to evaluate the role of ATM (KU55933) and DNA-PK (NU7441) inhibitors in the repair of double-strand breaks and downstream signaling of DNA damage introduced by ionizing radiation. The irradiation of MCF-7 cells alone increased the proportion of cells in the G1 phase in comparison with mock-treated cells. After ATM inhibitor pretreatment, the cells were more accumulated in the G2 phase, whereas DNA-PK inhibitor application increased the percentage of cells in the G1 phase. ATM and DNA-PK inhibitor application alone increased the sensitivity of MCF-7 cells to ionizing radiation; however, combining both inhibitors together resulted in a further enhancement of cell death. Unexpectedly, combining both inhibitors decreased the percentage of senescent cells and increased G2 cell cycle arrest 3 days after treatment. After irradiation, the p21 protein was increased and Chk1 and Chk2 were activated. These proteins were not increased in cells pretreated with the ATM inhibitor prior to ionizing radiation exposure, albeit DNA-PK inhibitor application did not affect the amount of proteins detected. Formation of γH2AX was found to be ATM and DNA-PK dependent, application of the ATM inhibitor suppressed incidence of γH2AX, whereas DNA-PK caused persistence of γH2AX. Our results suggest that the further investigation of the ATM inhibitor in combination with the DNA-PK inhibitor as sensitizers preventing cell senescence and promoting cell death in breast carcinoma MCF-7 cells is warranted.
- MeSH
- Ataxia Telangiectasia Mutated Proteins antagonists & inhibitors metabolism MeSH
- Cell Death drug effects MeSH
- Checkpoint Kinase 2 metabolism MeSH
- Chromones pharmacology MeSH
- DNA-Binding Proteins antagonists & inhibitors MeSH
- G1 Phase drug effects MeSH
- G2 Phase drug effects MeSH
- Histones MeSH
- Cyclin-Dependent Kinase Inhibitor p21 metabolism MeSH
- Radiation, Ionizing MeSH
- Cell Cycle Checkpoints drug effects MeSH
- Humans MeSH
- MCF-7 Cells MeSH
- Morpholines pharmacology MeSH
- Cell Line, Tumor MeSH
- DNA Repair drug effects MeSH
- DNA Damage drug effects MeSH
- DNA-Activated Protein Kinase antagonists & inhibitors metabolism MeSH
- Protein Kinases metabolism MeSH
- Antineoplastic Combined Chemotherapy Protocols pharmacology MeSH
- Pyrones pharmacology MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
We compared the effects of inhibitors of kinases ATM (KU55933) and ATR (VE-821) (incubated for 30 min before irradiation) on the radiosensitization of human promyelocyte leukaemia cells (HL-60), lacking functional protein p53. VE-821 reduces phosphorylation of check-point kinase 1 at serine 345, and KU55933 reduces phosphorylation of check-point kinase 2 on threonine 68 as assayed 4 h after irradiation by the dose of 6 Gy. Within 24 h after gamma-irradiation with a dose of 3 Gy, the cells accumulated in the G2 phase (67 %) and the number of cells in S phase decreased. KU55933 (10 μM) did not affect the accumulation of cells in G2 phase and did not affect the decrease in the number of cells in S phase after irradiation. VE-821 (2 and 10 μM) reduced the number of irradiated cells in the G2 phase to the level of non-irradiated cells and increased the number of irradiated cells in S phase, compared to irradiated cells not treated with inhibitors. In the 144 h interval after irradiation with 3 Gy, there was a considerable induction of apoptosis in the VE-821 group (10 μM). The repair of the radiation damage, as observed 72 h after irradiation, was more rapid in the group exposed solely to irradiation and in the group treated with KU55933 (80 and 77 % of cells, respectively, were free of DSBs), whereas in the group incubated with 10 μM VE-821, there were only 61 % of cells free of DSBs. The inhibition of kinase ATR with its specific inhibitor VE-821 resulted in a more pronounced radiosensitizing effect in HL-60 cells as compared to the inhibition of kinase ATM with the inhibitor KU55933. In contrast to KU55933, the VE-821 treatment prevented HL-60 cells from undergoing G2 cell cycle arrest. Taken together, we conclude that the ATR kinase inhibition offers a new possibility of radiosensitization of tumour cells lacking functional protein p53.
- MeSH
- Leukemia, Promyelocytic, Acute pathology MeSH
- Apoptosis drug effects MeSH
- Ataxia Telangiectasia Mutated Proteins antagonists & inhibitors MeSH
- HL-60 Cells MeSH
- Protein Kinase Inhibitors pharmacology MeSH
- G2 Phase Cell Cycle Checkpoints drug effects MeSH
- Humans MeSH
- Morpholines pharmacology MeSH
- DNA Repair drug effects MeSH
- Pyrazines pharmacology MeSH
- Pyrones pharmacology MeSH
- Sulfones pharmacology MeSH
- Radiation Tolerance drug effects MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
DNA lesions trigger the DNA damage response (DDR) machinery, which protects genomic integrity and sustains cellular survival. Increasing data underline the significance of the integrity of the DDR pathway in chemotherapy response. According to a recent work, persistent exposure of A549 lung carcinoma cells to doxorubicin induces an initial DDR-dependent checkpoint response, followed by a later DDR-independent, but p27(Kip1)-dependent one. Prompted by the above report and to better understand the involvement of the DDR signaling after chemotherapeutic stress, we examined the potential role of the canonical DDR pathway in A549 cells treated with doxorubicin. Exposure of A549 cells, prior to doxorubicin treatment, to ATM, ATR and DNA-PKcs inhibitors either alone or in various combinations, revealed that the earlier documented two-step response was DDR-dependent in both steps. Notably, inhibition of both ATM and ATR or selective inhibition of ATM or DNA-PKcs resulted in cell-cycle re-entry despite the increased levels of p27(Kip1) at all time points analyzed. We further investigated the regulation of p27(Kip1) protein levels in the particular setting. Our results showed that the protein status of p27(Kip1) is mainly determined by p38-MAPK, whereas the role of SKP2 is less significant in the doxoroubicin-treated A549 cells. Cumulatively, we provide evidence that the DNA damage signaling is responsible for the prolonged cell cycle arrest observed after persistent chemotherapy-induced genotoxic stress. In conclusion, precise identification of the molecular mechanisms that are activated during the chemotherapeutic cycles could potentially increase the sensitization to the therapy applied.
- MeSH
- Ataxia Telangiectasia Mutated Proteins antagonists & inhibitors MeSH
- A549 Cells MeSH
- Chromones pharmacology MeSH
- Doxorubicin pharmacology MeSH
- Cyclin-Dependent Kinase Inhibitor p27 physiology MeSH
- Caffeine pharmacology MeSH
- G2 Phase Cell Cycle Checkpoints drug effects MeSH
- Humans MeSH
- p38 Mitogen-Activated Protein Kinases metabolism MeSH
- Morpholines pharmacology MeSH
- DNA Damage MeSH
- DNA-Activated Protein Kinase antagonists & inhibitors MeSH
- S-Phase Kinase-Associated Proteins metabolism MeSH
- Antineoplastic Agents pharmacology MeSH
- Pyrones pharmacology MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
The effect of protein kinase C (PKC) inhibitors on porcine oocyte activation by calcium ionophore A23187 was studied. Calcium ionophore applied in a 50 microM concentration for 10 min induced activation in 74% of oocytes matured in vitro. When the ionophore-treated oocytes were exposed to the effect of bisindolylmaleimide I, which inhibits calcium-dependent PKC isotypes (PKC-alpha, -beta(I), -beta(II), -gamma,) and calcium-independent PKC isotypes (PKC-delta, -epsilon), the portion of activated oocytes decreased (at a concentration of 100 nM, 2% of the oocytes were activated). Go6976, the inhibitor of calcium-dependent PKC isotypes PKC-alpha, -beta(I) did not prevent the action of the oocytes treated with calcium ionophore in concentrations from 1 to 100 microM. The inhibitor of PKC-beta(I) and beta(II) isotypes, hispidin, in a concentration of 2 microM-2 mM, was not effective either. The inhibitor of PKC-delta isotype, rottlerin, suppressed activation of the oocytes by calcium ionophore (no oocyte was activated at 10 microM concentration). The PKC-delta isotype in matured porcine oocytes, studied by Western blot analysis, appeared as non-truncated PKC-delta of 77.5 kDa molecular weight, on the one hand, and as truncated PKC-delta, which was present in the form of a doublet of approximately 62.5 and 68 kDa molecular weight, on the other hand. On the basis of these results, it can be supposed that PKC participates in the regulation of processes associated with oocyte activation. Calcium-dependent PKC-alpha, -beta isotypes do not seem to play any significant role in calcium activation. The activation seems to depend on the activity of the calcium-independent PKC-delta isoform.
- MeSH
- Acetophenones pharmacology MeSH
- Benzopyrans pharmacology MeSH
- Calcimycin pharmacology MeSH
- Indoles pharmacology MeSH
- Protein Kinase Inhibitors pharmacology MeSH
- Ionophores pharmacology MeSH
- Isoenzymes antagonists & inhibitors physiology classification MeSH
- Carbazoles pharmacology MeSH
- Maleimides pharmacology MeSH
- Oocytes physiology drug effects MeSH
- Swine physiology MeSH
- Protein Kinase C antagonists & inhibitors physiology classification MeSH
- Pyrones pharmacology MeSH
- Calcium physiology MeSH
- Blotting, Western MeSH
- Animals MeSH
- Check Tag
- Female MeSH
- Animals MeSH
- Publication type
- Case Reports MeSH
- MeSH
- Antifungal Agents pharmacology chemistry MeSH
- Chelating Agents pharmacology metabolism MeSH
- Fermentation MeSH
- Research Support as Topic MeSH
- Antineoplastic Agents pharmacology metabolism MeSH
- Pyrones pharmacology metabolism MeSH
- Relative Biological Effectiveness MeSH
- Publication type
- Review MeSH
Kava-kava je uklidňující a posilující nápoj připravovaný z kořene pepřovníku druhu Piper methysticum Forst. Tento druh roste v celém Jižním Pacifiku, kde je domorodci využíván k přípravě nápoje již déle než 3000 let. Kava-kava působí relaxačně, zlepšuje mentální funkce a zvyšuje odolnost proti stresu a úzkosti. Droga tlumí bolest a může být užívána také jako prostředek pro snížení nervozity a při nespavosti. Farmakologické vlastnosti kava-kava jsou podmíněny přítomností několika látek, známých jako kavapyron. Jedná se o směs látek odvozených od pyron-2-onu.
Kava-kava is the cordial natural beverage that is made from the root of Piper methysticum Forst, a species of pepper. It is cultivated throughout the South Pacific and it has been used by Pacific Islanders for over 3,000 years. Kava-kava has the ability to relax the body while promoting mental clarity, what makes it a natural choice for the relief of stress and anxiety. Kava's analgesic properties can help to manage pain. The drug may also be used for nervousness and insomnia. Pharmacological properties of kava-kava are determined by the family of compounds known as kavapyron. There is a mixture of compounds derived from pyron-2-one.
- MeSH
- Anti-Anxiety Agents MeSH
- Phytotherapy MeSH
- Kava MeSH
- Plant Roots MeSH
- Beverages MeSH
- Neuromuscular Agents MeSH
- Pyrones pharmacology chemistry metabolism MeSH
- Publication type
- Review MeSH
- MeSH
- DNA biosynthesis MeSH
- Phosphorylation MeSH
- Rats MeSH
- Leukemia L1210 drug therapy MeSH
- Humans MeSH
- Tumor Cells, Cultured MeSH
- Antineoplastic Agents pharmacology chemical synthesis chemistry MeSH
- Pyrones pharmacology chemical synthesis chemistry MeSH
- Thiocyanates pharmacology chemical synthesis chemistry MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Humans MeSH
- Animals MeSH
