Involvement of autophagy induction in penta-1,2,3,4,6-O-galloyl-β-D-glucose-induced senescence-like growth arrest in human cancer cells

Abstract

Growing evidence has demonstrated that autophagy plays important and paradoxical roles in carcinogenesis, while senescence is considered to be a crucial tumor-suppressor mechanism in cancer prevention and treatment. In the present study we demonstrated that both autophagy and senescence were induced in response to penta-1,2,3,4,6-O-galloyl-β-D-glucose (PGG), a chemopreventive polyphonolic compound, in multiple types of cancer cells. Analysis of these 2 events over the experimental time course indicated that autophagy and senescence occurred in parallel early in the process and dissociated later. The long-term culture study suggested that a subpopulation of senescent cells may have the capacity to reenter the cell cycle. Inhibition of autophagy by either a chemical inhibitor or RNA interference led to a significant reduction of PGG-induced senescence, followed by induction of apoptosis. These results suggested that autophagy promoted senescence induction by PGG and that PGG might exert its anticancer activity through autophagy-mediated senescence. For the first time, these findings uncovered the relationships among autophagy, senescence, and apoptosis induced by PGG. In addition, we identified that unfolded protein response signaling played a pivotal role in the autophagy-mediated senescence phenotype. Furthermore, our data showed that activation of MAPK8/9/10 (mitogen-activated protein kinase 8/9/10/c-Jun N-terminal kinases) was an essential upstream signal for PGG-induced autophagy. Finally, the key in vitro results were validated in vivo in a xenograft mouse model of human HepG2 liver cancer. Our findings provided novel insights into understanding the mechanisms and functions of PGG-induced autophagy and senescence in human cancer cells.

Keywords: MAPK8/9/10; PGG; UPR; apoptosis; autophagy; senescence.

Figure 1. PGG induces autophagy and senescence in HepG2, MCF-7, and A549 human cancer cells. (A) PGG caused a concentration-dependent increase of LC3-I to LC3-II conversion analyzed by western blotting in HepG2 (left), MCF-7 (middle), and A549 cells (right). (B) A punctate distribution of LC3 induced by PGG and detected by immunofluorescence staining. (C) Quantitative analysis of LC3-punctate cells for time-course experiments in PGG-treated and untreated cells. (D) Increase of autophagosome formation was involved in PGG-induced autophagy in HepG2 cells. The cells were treated with 25 μM PGG for 24 h in the presence or absence of 50 nM bafilomycin A₁ (added 2 h before cells harvest) and then LC3 was analyzed by western blotting. (E) PGG caused a concentration-dependent decrease of SQSTM1 analyzed by western blotting in HepG2 cells. (F and G) PGG-induced senescence-like phenotype in HepG2 cells. The cells were treated with 25 μM PGG for 24 h and SA-β-gal activity was measured with senescence-associated β-galactosidase staining. The stained cells were viewed and photographed under an inverted microscope (F); Time-course analysis of β-gal-positive cells (G). (H) Cell cycle distribution induced by PGG. HepG2 cells were treated with PGG for the indicated times and cell cycle was analyzed by flow cytometry (the data are representative of 3 experiments). (I and J) PGG stimulated IL6 secretion in HepG2 cells. The cells were treated with 25 μM PGG for the indicated times and the levels of IL6 mRNA and its secreted gene product were measured by Real-time PCR and an ELISA Kit respectively. **P < 0.01.

Figure 2. Autophagy is required for the senescent phenotype induced by PGG. (A) Effects of autophagy inhibitor 3-MA on PGG-induced autophagy in HepG2 cells. (B) Effects of autophagy inhibition by 3-MA on PGG-induced senescence phenotype (SA-β-gal activity) in HepG2 cells. The cells were treated with PGG in the presence or absence of 3-MA for 24 h and senescence phenotype was measured by senescence-associated β-galactosidase staining. (C) Quantitative analysis of β-gal-positive cells induced by PGG in the presence or absence of 3-MA. (D) Effects of autophagy inhibition by 3-MA on IL6 mRNA induction by PGG analyzed by real-time PCR. (E) Effects of autophagy inhibition by 3-MA on PGG-induced apoptosis. The cells were treated with PGG in the presence or absence of 3-MA for the indicated times and apoptosis was analyzed by ANXA5 staining. (F and G) Effects of autophagy inhibition by knockdown of ATG5 on PGG-induced senescence phenotype and apoptosis. The cells were transfected with 50 nmol/L of ATG5 siRNA using siPORT™ NeoFX™ Transfection Agent. After 24 h transfection, the cells were treated with 25 μM PGG for 24 or 48 h. ATG5, LC3-I to LC3-II conversion and PARP1 cleavage were analyzed using western blotting (F) and senescence phenotype was measured by senescence-associated β-galactosidase staining (G). (n = 3, **P < 0.01). (H) Effects of autophagy inhibition by knocking down ATG5 on cell cycle distribution induced by PGG. The cells were transfected with 50 nmol/L of ATG5 siRNA using siPORT™ NeoFX™ Transfection Agent. After 24 h transfection, the cells were treated with 25 μM PGG for 24 h and cell cycle was assessed by flow cytometry. (I–K) Effects of autophagy inhibition by knocking down ATG5 on senescence induction by PGG in a system where apoptosis was suppressed by knocking down CASP3. HepG2 cells were simultaneously transfected with 50 nmol/L of ATG5 siRNA and 60 nmol/L of CASP3 siRNA (I). After 24 h transfection, the cells were treated with 25 μM PGG for 48 h, and then cell death and senescence phenotype (SA-β-gal activity) were measured by a Cell Death ELISA Kit (J) and senescence-associated β-galactosidase staining (K) respectively (n = 3, **P < 0.01).

Figure 3. PGG induces the unfolded protein response (UPR) in HepG2, MCF-7, and A549 human cancer cells. The cells were treated with various concentrations of PGG for 24 h and ER stress associated markers were analyzed by western blotting. (A) HepG2 cells. (B) MCF-7 cells. (C) A549 cells.

Figure 4. Induction of autophagy contributed to PGG-induced activation of the UPR. (A) Time-course analysis of key parameters of autophagy and ER stress induced by PGG in HepG2 cells. The cells were treated with PGG for the indicated time and then LC3 and phosphorylation of ERN1 and EIF2S1 were assessed by western blotting. (B) Effects of autophagy inhibition by ATG5 knockdown on PGG-induced UPR. The cells were transfected ATG5 siRNA using siPORT™ NeoFX™ Transfection Agent. After 24 h transfection, the cells were treated with 25 μM PGG for 24 h and ERN1 and EIF2S1 phosphorylation were assessed by western blotting. (C) Effects of autophagy inhibition by its inhibitor on PGG-induced UPR. The cells were treated with 25 μM PGG in the presence or absence of 3-MA for 24 h and ER markers were assessed by western blotting. (D) Effects of ERN1 knockdown on PGG-induced autophagy. The cells were transfected ERN1 siRNA using siPORT™ NeoFX™ Transfection Agent. After 24 h transfection, the cells were treated with 25 μM PGG for 24 h and ERN1 and LC3 were assessed by western blotting. (E) Effects of ERN1 inhibitor on PGG-induced autophagy. The cells were treated with PGG in the presence or absence of 3-ethoxy-5,6-dibromosalicylaldehyde for 24 h and LC3 was assessed by western blotting.

Figure 5. Both the ERN1 and EIF2AK3 arms of UPR signaling pathways are involved in the PGG-induced senescent phenotype. (A and B) Effects of ERN1 or EIF2AK3 knockdown on PGG-induced senescence phenotype. HepG2 cells were transfected ERN1 or EIF2AK3 siRNA using siPORT™ NeoFX™ Transfection Agent. Twenty four h after transfection, the cells were treated with 25 μM PGG for 24 h and the senescence phenotype was measured by senescence-associated β-galactosidase staining. The stained cells were viewed and photographed under an inverted microscope (A); Quantitative analysis of β-gal-positive cells (B). (C) Effects of ERN1 or EIF2AK3 knockdown on PGG-induced apoptosis analyzed by ANXA5 staining after a 48 h treatment. (D) Effects of ERN1 inhibitor on the PGG-induced senescence phenotype. The cells were treated with PGG in the presence or absence of 3-ethoxy-5,6-dibromosalicylaldehyde for 24 h and the senescent phenotype was measured by senescence-associated β-galactosidase staining. (E) Effects of ERN1 inhibitor on PGG-induced apoptosis analyzed by ANXA5 staining after 24 and 48 h treatment.

Figure 6. MAPK8/9/10 activation lies upstream of PGG-induced autophagy-dependent senescence in HepG2 cells. (A) PGG induced a concentration-dependent MAPK8/9/10 phosphorylation (activation) analyzed by western blotting. (B and C) Effects of ERN1 or EIF2AK3 knockdown on PGG-induced MAPK8/9/10 phosphorylation. The cells were transfected with ERN1 (B) or EIF2AK3 (C) siRNA using siPORT™ NeoFX™ Transfection Agent. After 24 h transfection, the cells were treated with 25 μM PGG for 24 h and MAPK8/9/10 phosphorylation was assessed by western blotting. (D) Effects of MAPK8/9/10 inactivation by its inhibitor on PGG-induced autophagy and UPR. The cells were treated with 25 μM PGG in the presence or absence of SP600125 for 24 h. LC3 and phosphorylation of ERN1 and EIF2S1 were analyzed by western blotting. (E and F) Effects of MAPK8/9/10 inactivation by its inhibitor on the PGG-induced senescence phenotype. The cells were treated with PGG in the presence or absence of SP600125 for 24 h and the senescence phenotype was measured by senescence-associated β-galactosidase staining. (G) Effects of MAPK8/9/10 inactivation by its inhibitor on PGG-induced apoptosis. The cells were treated with PGG in the presence or absence of SP600125 for 48 h and apoptosis was assessed by ANXA5 staining.

Figure 7. PGG induces autophagy and senescence in vivo. PGG was given daily by i.p. injection (20 mg/kg body weight) starting 7 d after s.c. inoculation of cancer cells for 16 d. (A) Effects of PGG on tumor and bodyweight. (B) Western blotting analysis of the markers of autophagy and ER stress. (C) Senescence-associated β-galactosidase staining assay for detecting senescence phenotype.

Figure 8. Potential signaling pathways involved in PGG-induced autophagy and senescence in human cancer cells. PGG induced autophagy-dependent senescence through UPR (ERN1 and EIF2AK3) activation. MAPK8/9/10 activation was an essential upstream signal to trigger autophagy.