Multifactorial anticancer effects of digalloyl-resveratrol encompass apoptosis, cell-cycle arrest, and inhibition of lymphendothelial gap formation in vitro - PubMed (original) (raw)

. 2010 Apr 27;102(9):1361-70.

doi: 10.1038/sj.bjc.6605656.

P Saiko, C Vonach, K Viola, N Huttary, N Stark, R Popescu, M Gridling, N T-P Vo, I Herbacek, A Davidovits, B Giessrigl, S Venkateswarlu, S Geleff, W Jäger, M Grusch, D Kerjaschki, W Mikulits, T Golakoti, M Fritzer-Szekeres, T Szekeres, G Krupitza

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Multifactorial anticancer effects of digalloyl-resveratrol encompass apoptosis, cell-cycle arrest, and inhibition of lymphendothelial gap formation in vitro

S Madlener et al. Br J Cancer. 2010.

Abstract

Background: Digalloyl-resveratrol (di-GA) is a synthetic compound aimed to combine the biological effects of the plant polyhydroxy phenols gallic acid and resveratrol, which are both radical scavengers and cyclooxygenase inhibitors exhibiting anticancer activity. Their broad spectrum of activities may probably be due to adjacent free hydroxyl groups.

Methods: Protein activation and expression were analysed by western blotting, deoxyribonucleoside triphosphate levels by HPLC, ribonucleotide reductase activity by (14)C-cytidine incorporation into nascent DNA and cell-cycle distribution by FACS. Apoptosis was measured by Hoechst 33258/propidium iodide double staining of nuclear chromatin and the formation of gaps into the lymphendothelial barrier in a three-dimensional co-culture model consisting of MCF-7 tumour cell spheroids and human lymphendothelial monolayers.

Results: In HL-60 leukaemia cells, di-GA activated caspase 3 and dose-dependently induced apoptosis. It further inhibited cell-cycle progression in the G1 phase by four different mechanisms: rapid downregulation of cyclin D1, induction of Chk2 with simultaneous downregulation of Cdc25A, induction of the Cdk-inhibitor p21(Cip/Waf) and inhibition of ribonucleotide reductase activity resulting in reduced dCTP and dTTP levels. Furthermore, di-GA inhibited the generation of lymphendothelial gaps by cancer cell spheroid-secreted lipoxygenase metabolites. Lymphendothelial gaps, adjacent to tumour bulks, can be considered as gates facilitating metastatic spread.

Conclusion: These data show that di-GA exhibits three distinct anticancer activities: induction of apoptosis, cell-cycle arrest and disruption of cancer cell-induced lymphendothelial disintegration.

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Figures

Figure 1

Figure 1

Chemical structures of (A) gallic acid (GA) and (B) digalloyl-resveratrol (di-GA).

Figure 2

Figure 2

Induction of apoptosis by (A) GA and (B) di-GA in HL-60 cells. Cells were incubated with increasing concentrations of drugs for 24 h, and then double stained with Hoechst 33258 and propidium iodide. Afterwards cells were examined under the microscope with UV light connected to a DAPI filter. Nuclei with a morphological phenotype indicating apoptosis were counted and percentages of apoptotic cells were calculated. Experiments were conducted in triplicate. Error bars indicate s.e.m., asterisks significance (P<0.05). (C) Activation of caspase 3 and cleavage of PARP on treatment with di-GA. Logarithmically growing HL-60 cells were incubated with 10 μ

M

di-GA for 0.5, 2, 4, 8 and 24 h. Afterwards cells were lysed and protein expression was analysed by western blotting. The anti-caspase 3 antibody recognises only the cleaved peptide indicating its activation. Anti-PARP antibody recognises the full-length form (116 kDa) and the signature-type cleaved product (85 kDa) that is generated by active caspase 3. The antibody against _β_-actin was used to monitor equal sample loading.

Figure 3

Figure 3

Effect of di-GA on the cell cycle of HL-60 cells. (A) Logarithmically growing HL-60 cells were incubated with increasing concentrations of di-GA for 24 h and then subjected to FACS analysis. Experiments were conducted in triplicate. Error bars indicate s.e.m., asterisks significance (P<0.05). HL-60 cells were incubated with 10 μ

M

di-GA for 0.5, 2, 4, 8 and 24 h, lysed, and the (B) expression of p21Cip/Waf, the phosphorylation of threonine202/tyrosine204-Erk1/2 (p-Erk1/2) and serine217/221-MEK1/2 (p-MEK), and (C) phosphorylation of threonine68-Chk2 (p-Chk2), serine17-Cdc25A (p-Cdc25A), tyrosine15-Cdc2 (p-Cdc2), and the protein levels of cyclin D1, E were analysed by western blotting. _β_-Actin served as loading control.

Figure 4

Figure 4

(A) Measurement of the in situ effect of di-GA on ribonucleotide reductase (RR) activity. HL-60 cells were incubated with 1, 2.5 and 5 μ

M

di-GA for 24 h at 37 °C in a humidified atmosphere containing 5% CO2 to assess changes in RR in situ activity. Then, cells were pulsed with 14C-cytidine (Sigma-Aldrich; 3 _μ_l in a 5 ml cell suspension) for 30 min at 37 °C. Afterwards the cells were collected and the radioactivity that became incorporated into genomic DNA was measured. (B) Effect of di-GA on intracellular dNTP pools in HL-60 cells. HL-60 cells were incubated with 5, 10 and 40 μ

M

di-GA for 24 h. Then the cells were prepared for HPLC analysis and the dNTP levels were determined according to the protocol described in the ‘Materials and methods’ section. Experiments were conducted in triplicate. Error bars indicate s.e.m., asterisks significance (P<0.05).

Figure 5

Figure 5

Effect of di-GA on MCF-7 spheroid-induced gap formation in lymphendothelial cell monolayers. (A) LEC monolayers that were exposed to MCF-7 spheroid (left side panels), MCF-7 spheroid treated with 40 μ

M

di-GA (middle panels) and HLF spheroid (right side panels). Upper panels are phase-contrast micrographs showing the respective spheroids, the panels below show the identical power fields using FITC filter and exhibit green stained LECs underneath the respective spheroids. Bars in the lower right corners of upper panels indicate 100 μ

M

. (B) MCF-7 tumor spheroids were preincubated with solvent (control), or 5 and 50 μ

M

berberine; 5, 10, 25, 40 and 80 μ

M

di-GA; 80 μ

M

GA; 50 μ

M

RV; 50 μ

M

NDGA; 100 μ

M

baicalein; 200 μ

M

aspirin; 25 μ

M

mannitol; 600 U ml−1 catalase; 200 μ

M

carboxy-PTIO and 100 μ

M

probucol, and then placed on top of cytotracker stained LEC monolayers that were also treated with respective agents for 4 h. Then, the size of the gaps that were formed in the LEC monolayers by MCF-7 spheroids (through repulsion of LECs) was measured using an inverted microscope connected to an FITC filter and equipped with Axiovision 4.5 software (Carl Zeiss). As negative controls normal human lung fibroblast (HLF) spheroids were used. Rho/rac (small GTPases regulating cell migration), LOX (lipoxygenase), COX (cyclooxygenases) and ROS (reactive oxygen species) indicate which mechanisms and phenomena are inhibited by the respective agents. Experiments were conducted in triplicate, and the underneath areas of at least 12 spheroids were analysed. Error bars indicate s.e.m., asterisks significance (P<0.05).

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