Penta-O-galloyl-beta-D-glucose induces G1 arrest and DNA replicative S-phase arrest independently of cyclin-dependent kinase inhibitor 1A, cyclin-dependent kinase inhibitor 1B and P53 in human breast cancer cells and is orally active against triple negative xenograft growth

Abstract

Introduction: Natural herbal compounds with novel actions different from existing breast cancer (BCa) treatment modalities are attractive for improving therapeutic efficacy and safety. We have recently shown that penta-1,2,3,4,6-O-galloyl-β-D-glucose (PGG) induced S-phase arrest in prostate cancer (PCa) cells through inhibiting DNA replicative synthesis and G(1) arrest, in addition to inducing cell death at higher levels of exposure. We and others have shown that PGG through intraperitoneal (i.p.) injection exerts a strong in vivo growth suppression of human PCa xenograft models in athymic nude mice. This study aims to test the hypothesis that the novel targeting actions of PGG are applicable to BCa cells, especially those lacking proven druggable targets.

Methods: Mono-layer cell culture models of p53-wild type estrogen receptor (ER)-dependent MCF-7 BCa cells and p53-mutant ER-/progesterone receptor (PR)- and Her2-regular (triple-negative) MDA-MB-231 BCa were exposed to PGG for a comprehensive investigation of cellular consequences and molecular targets/mediators. To test the in vivo efficacy, female athymic mice inoculated with MDA-MB-231 xenograft were treated with 20mg PGG/kg body weight by daily gavage starting 4 days after cancer cell inoculation.

Results: Exposure to PGG induced S-phase arrest in both cell lines as indicated by the lack of 5-bromo2'-deoxy-uridine (BrdU) incorporation into S-phase cells as well as G(1) arrest. Higher levels of PGG induced more caspase-mediated apoptosis in MCF-7, in strong association with induction of P53 Ser(15) phosphorylation, than in MDA-MB-231 cells. The cell cycle arrests were achieved without an induction of cyclin dependent kinase (CDK) inhibitory proteins P21(Cip1) and P27(Kip1). PGG treatment led to decreased cyclin D1 in both cell lines and over-expressing cyclin D1 attenuated G(1) arrest and hastened S arrest. In serum-starvation synchronized MCF-7 cells, down-regulation of cyclin D1 was associated with de-phosphorylation of retinoblastoma (Rb) protein by PGG shortly before G(1)-S transition. In vivo, oral administration of PGG led to a greater than 60% inhibition of MDA-MB231 xenograft growth without adverse effect on host body weight.

Conclusions: Our in vitro and in vivo data support PGG as a potential drug candidate for breast cancer with novel targeting actions, especially for a triple negative BCa xenograft model.

Figure 1

Growth inhibitory and cell death actions of PGG in MCF-7 and MDA-MB231 cells. (a) Overall inhibitory effects of PGG on MCF-7 and MDA-MB231 cell growth after 3 days of daily treatment with PGG in fresh medium. Values are mean ± standard error of the mean (n = 3 wells of 12-well plate). Statistical significance: ***P < 0.001; ****^,####P < 0.0001 versus untreated control. Results are representative of two independent experiments. (b) Immunoblot detection of apoptotic cPARP (cleaved poly-ADP-ribose polymerase), P53-Ser¹⁵ phosphorylation induced by PGG in MCF-7 or MDA-MB231 cells, and autophagy responses (pAMPK and LC3-II) in MDA-MB231 cells. Phase-contrast photomicrograph shows vaculolation typical of autophagy. The medium was not changed for PGG exposure of longer than 24 hours. DMSO, dimethyl sulfoxide; LC3, microtubule-associated protein 1 light chain 3; pAMPK, phospho-AMP kinase; PGG, penta-O-galloyl-β-D-glucose.

Figure 2

The effect of PGG on cell cycle distribution of MCF-7 (a) and MDA-MB231 (b) cells detected by propidium iodide/BrdU-bivariate flow cytometric analyses. Cells were exposed to increasing concentrations of PGG for 6, 24, and 48 hours. BrdU was added for the last 30 minutes to label S-phase cells active in DNA replication. Values are mean ± standard error of the mean (n = 4). Results are from two independent experiments with duplicate values at each concentration. The medium was not changed for PGG exposure of longer than 24 hours. Statistical significance: (a) BrdU incorporation at all three time points, one-way analysis of variance (ANOVA) P < 0.0001, with Dunnett's multiple comparison post test P value of less than 0.01 for 0 versus 12.5, 25, and 50 μM PGG. For G₁, 6 hours P < 0.05 for 0 versus 25 and 50 μM PGG; 24 hours P < 0.01 for 0 versus 12.5 or 50 μM PGG; 48 hours P < 0.01 for 0 versus 12.5 μM PGG and P < 0.05 for 0 versus 50 μM PGG. For S, 24 hours/48 hours P < 0.01 for 0 versus 12.5 and 25 μM PGG. (b) BrdU incorporation at all three time points, one-way ANOVA P < 0.0001, with Dunnett's multiple comparison post test P value <0.01 for 0 versus 12.5, 25, and 50 μM PGG. For G₁, 6 hours P < 0.05 for 0 versus 12.5, 25, and 50 μM PGG; 24 hours P < 0.01 for 0 versus 25 and 50 μM PGG; 48 hours P < 0.01 for 0 versus 50 μM PGG. For S, 24 hours/48 hours P < 0.01 for 0 versus 12.5 μM PGG. BrdU, 5-bromo-2'-deoxy-uridine; PGG, penta-O-galloyl-β-D-glucose.

Figure 3

Effect of PGG on cyclin D1, P21^Cip1, and P27^Kip1 and other select cell cycle proteins in MCF-7 and MDA-MB231 cells detected by Western blot analyses. (a) Cyclin D1, CDK4, P21^Cip1, and P27^Kip1 expression and P53-Ser¹⁵P in MCF-7 cells. β-Actin was re-probed as loading control. (b) P21^Cip1 and P27^Kip1 expression in MDA-MB231 cells. (c) Time course of cyclin D1 expression in MCF-7 and MDA-MB231 cells treated with PGG from 12 to 48 hours. Patterns are representative of two experiments. The medium was not changed for PGG exposure of longer than 24 hours. P21^Cip1, cyclin-dependent kinase inhibitor 1A; P27^Kip1, cyclin-dependent kinase inhibitor 1B; PGG, penta-O-galloyl-β-D-glucose.

Figure 4

The effect of PGG on cell cycle proteins in serum starvation-synchronized MCF-7 cells. PGG was included at time of serum stimulation (as time 0). Western blot was used to detect Cyclin D1 expression and phosphorylation of Retinoblastoma protein, activation of P53-Ser¹⁵P, and expressions of P21^Cip1 and estrogen receptor-alpha. cPARP, cleaved poly-ADP-ribose polymerase; ERα, estrogen receptor-alpha; FBS, fetal bovine serum; P21^Cip1, cyclin-dependent kinase inhibitor 1A; PGG, penta-O-galloyl-β-D-glucose.

Figure 5

Impact of overexpression of cyclin D1 on PGG-induced G₁ arrest in MCF-7 and MDA-MB231 cells. (a) Western blot verification of stable overexpression of cyclin D1 in MCF-7 and MDA-MB231 cells. (b) Cell cycle distribution of MCF-7 cells transfected with vector and cyclin D1 plasmid with or without PGG treatment for 24 hours. (c) Cell cycle distribution of MDA-MB231 cells transfected with vector and cyclin D1 plasmid with or without PGG treatment for 24 hours. Each bar reflects the average of two T25 flasks. The patterns are representative of two experiments. PGG, penta-O-galloyl-β-D-glucose.

Figure 6

Effect of PGG on G_0/1-S progression in synchronized MCF-7 cells. (a) The temporal kinetics of serum-stimulated progression of starvation-synchronized MCF-7 cells. Each time point was the average of duplicate flasks. *^,#P < 0.05; **^,##P < 0.01; ***^,###P < 0.001 versus 0 time. (b) Impact of delaying PGG treatment with reference to serum stimulation on G₁ arrest. Results are from two independent experiments with duplicate values at each time point. *^,#P < 0.05; **^,##P < 0.01 versus serum-free (SF) or PGG@0h-14 h. CM, complete medium; PGG, penta-O-galloyl-β-D-glucose.

Figure 7

PGG intake by oral gavage inhibits MDA-MB231 tumor growth in female athymic nude mice. Starting 4 days after cell inoculation, PGG (20 mg/kg) was gavaged with 2% Tween-80 as vehicle to these animals once a day. (a) Body weight. (b) Tumor volume. Values are mean ± standard deviation (n = 10 mice per group). Statistical significance: analysis of variance PGG effect on tumor size, P < 0.0001. PGG, penta-O-galloyl-β-D-glucose.