Scoulerine is an isoquinoline alkaloid, which indicated promising suppression of cancer cells growth. However, the mode of action (MOA) remained unclear. Cytotoxic and antiproliferative properties were determined in this study. Scoulerine reduces the mitochondrial dehydrogenases activity of the evaluated leukemic cells with IC50 values ranging from 2.7 to 6.5 µM. The xCELLigence system revealed that scoulerine exerted potent antiproliferative activity in lung, ovarian and breast carcinoma cell lines. Jurkat and MOLT-4 leukemic cells treated with scoulerine were decreased in proliferation and viability. Scoulerine acted to inhibit proliferation through inducing G2 or M-phase cell cycle arrest, which correlates well with the observed breakdown of the microtubule network, increased Chk1 Ser345, Chk2 Thr68 and mitotic H3 Ser10 phosphorylation. Scoulerine was able to activate apoptosis, as determined by p53 upregulation, increase caspase activity, Annexin V and TUNEL labeling. Results highlight the potent antiproliferative and proapoptotic function of scoulerine in cancer cells caused by its ability to interfere with the microtubule elements of the cytoskeleton, checkpoint kinase signaling and p53 proteins. This is the first study of the mechanism of scoulerine at cellular and molecular level. Scoulerine is a potent antimitotic compound and that it merits further investigation as an anticancer drug.
- MeSH
- aktivace enzymů účinky léků MeSH
- antitumorózní látky chemie farmakologie MeSH
- apoptóza účinky léků MeSH
- berberinové alkaloidy chemie farmakologie MeSH
- estery chemie MeSH
- fosforylace účinky léků MeSH
- kaspasy metabolismus MeSH
- kontrolní body buněčného cyklu účinky léků MeSH
- kyseliny karboxylové chemie MeSH
- lidé MeSH
- membránový potenciál mitochondrií účinky léků MeSH
- mikrotubuly účinky léků metabolismus MeSH
- nádorové buněčné linie MeSH
- proliferace buněk účinky léků MeSH
- signální transdukce účinky léků MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- zlomy DNA účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Haemanthamine (HA) and sodium butyrate (NaB) are promising candidates for chemotherapy as a treatment for cancer. PURPOSE: We aimed to determine the anticancer potential of HA and NaB, alone and in combination, in A2780 ovarian cancer cells and concurrently investigated anticancer potential in contrast to non-cancer human MRC-5 fibroblasts. METHODS: Antiproliferative effects were determined by WST-1 assay and by Trypan blue exclusion staining. Cell cycle distributions were studied by flow cytometry and protein levels were determined by Western blotting. RESULTS: The combination of HA and NaB caused a significant decrease in the proliferation of A2780 cells compared to the stand-alone treatment of cells by HA or NaB. This effect was less pronounced in non-cancer MRC-5 fibroblasts. In the later intervals, the number of A2780 living cells was strongly decreased by treatment using a combination of NaB and HA. This simultaneous application had no considerable effect in MRC-5 fibroblasts. The combination of NaB and HA led to the suppression of cells in the G1 phase and caused an accumulation of cells in the S and G2 phase in comparison to those treated with NaB and HA alone. Treatment of cells with NaB alone led to the activation of proteins regulating the cell cycle. Notably, p21WAF1/Cip1 was upregulated in both A2780 and MRC-5 cells, while checkpoint kinases 1 and 2 were activated via phosphorylation only in A2780 cells. Unexpectedly, NaB in combination with HA suppressed the phosphorylation of Chk2 on threonine 68 and Chk1 on serine 345 in A2780 cells and downregulated p21WAF1/Cip1 in both tested cell lines. The sensitization of cells to HA and NaB treatment seems to be accompanied by increased histone acetylation. NaB-induced acetylation of histone H3 and H4 and histone acetylation increased markedly when a combination of NaB and HA was applied. Whereas the most prominent hyperacetylation after HA and NaB treatment was observed in A2780 cells, the acetylation of histones occurred in both cell lines. CONCLUSION: In summary, we have demonstrated the enhanced activity of HA and NaB against A2780 cancer cells, while eliciting no such effect in non-cancer MRC-5 cells.
- MeSH
- acetylace MeSH
- aktivace transkripce účinky léků MeSH
- alkaloidy amarylkovitých farmakologie MeSH
- buněčné dělení účinky léků MeSH
- buněčný cyklus účinky léků MeSH
- checkpoint kinasa 1 metabolismus MeSH
- checkpoint kinasa 2 metabolismus MeSH
- fenantridiny farmakologie MeSH
- fosforylace MeSH
- histony metabolismus MeSH
- inhibitor p21 cyklin-dependentní kinasy metabolismus MeSH
- kyselina máselná farmakologie MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- nádory vaječníků patologie MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
The aim of our work was to evaluate mechanisms leading to radiosensitization of MOLT-4 leukemia cells following valproic acid (VA) treatment. Cells were pretreated with 2 mM VA for 24 h followed by irradiation with a dose of 0.5 or 1 Gy. The effect of both noxae, alone and combined, was detected 1 and 24 hours after the irradiation. Induction of apoptosis was evaluated by a flow cytometry. The extent of DNA damage was further determined by phosphorylation of histone H2AX using confocal microscopy. Changes in protein expression were identified by SDS-PAGE/immunoblotting. Two-millimolar VA increased apoptosis induction after irradiation as well as phosphorylation of H2AX and provokes an increase in the level of p53 and its phosphorylation at Ser392 in 4 h post-irradiation. Likewise, p21 protein reached its maximal expression in 4 h after the irradiation of VA-treated cells. Twenty four hours later, only the p53 phosphorylated at Ser15 was detected. At the same time, the protein mdm2 (negative regulator of p53) was maximally activated. The 24-hour treatment of MOLT-4 leukemia cells with 2 mM VA results in radiosensitizing, increases apoptosis induction, H2AX phosphorylation, and also p53 and p21 activation.
- MeSH
- apoptóza účinky léků účinky záření MeSH
- fosforylace účinky léků účinky záření MeSH
- histony genetika metabolismus MeSH
- inhibitory enzymů farmakologie MeSH
- kyselina valproová farmakologie MeSH
- leukemie metabolismus MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- nádorový supresorový protein p53 genetika metabolismus MeSH
- oprava DNA účinky léků MeSH
- poškození DNA účinky léků účinky záření MeSH
- protoonkogenní proteiny c-mdm2 genetika metabolismus MeSH
- radiosenzibilizující látky farmakologie MeSH
- regulace genové exprese účinky léků účinky záření MeSH
- rho proteiny vázající GTP genetika metabolismus MeSH
- záření gama MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Ionizing radiation and somatostatin analogues are used for acromegaly treatment to achieve normalization or reduction of growth hormone hypersecretion and tumor shrinkage. In this study, we investigated a combination of somatostatin (SS14) with ionizing radiation of (60)Co and its effect on reparation of radiation-induced damage and cell death of somatomammotroph pituitary cells GH3. Doses of gamma-radiation 20-50 Gy were shown to inhibit proliferation and induce apoptosis in GH3 cells regardless of somatostatin presence. It has been found that the D(0) value for GH3 cells was 2.5 Gy. Somatostatin treatment increased radiosensitivity of GH3 cells, so that D(0) value decreased to 2.2 Gy. We detected quick phosphorylation of histone H2A.X upon irradiation by the dose 20 Gy and its colocalization with phosphorylated protein Nbs-1 in the site of double strand break of DNA (DSB). Number of DSB decreased significantly 24 h after irradiation, however, clearly distinguished foci persisted, indicating non repaired DSB, after irradiation alone or after combined treatment by irradiation and SS14. We found that SS14 alone triggers phosphorylation of Nbs1 (p-Nbs1), which correlates with antiproliferative effect of SS14. Irradiation also increased the presence of p-Nbs1. Most intensive phosphorylation of Nbs1 was detected after combined treatment of irradiation and SS14. The decrease of the number of the DSB foci 24 h after treatment shows a significant capacity of repair systems of GH3 cells. In spite of this, large number of unrepaired DSB persists for 24 h after the treatment. We conclude that SS14 does not have a radioprotective effect on somatomammotroph GH3 cells.
- MeSH
- adenom hypofýzy vylučující růstový hormon farmakoterapie chirurgie MeSH
- akromegalie farmakoterapie chirurgie MeSH
- apoptóza genetika účinky záření MeSH
- buněčný cyklus fyziologie účinky záření MeSH
- experimentální radiační poranění prevence a kontrola MeSH
- histony metabolismus účinky záření MeSH
- ionizující záření MeSH
- jaderné proteiny metabolismus účinky záření MeSH
- krysa rodu rattus MeSH
- modely nemocí na zvířatech MeSH
- nádorové buněčné linie MeSH
- nádory hypofýzy farmakoterapie chirurgie MeSH
- neparametrická statistika MeSH
- poškození DNA fyziologie účinky léků MeSH
- potkani Wistar MeSH
- proteiny buněčného cyklu metabolismus účinky záření MeSH
- radiochirurgie škodlivé účinky MeSH
- somatostatin fyziologie terapeutické užití MeSH
- somatotropní buňky metabolismus účinky léků účinky záření MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
