Inhibitory effects and molecular mechanisms of pentagalloyl glucose in combination with 5-FU on aggressive phenotypes of HepG2 cells

Abstract This study examined the inhibition and mechanism of natural product pentagalloyl glucose (PGG) against HepG2 cells and determined the effects of its combination with the clinical chemotherapeutic drug, 5-FU. PGG was found to inhibit the proliferation, migration and invasion of HepG2 cells, induced G1 arrest and apoptosis in both concentration- and time- dependent manners. The combination of PGG and 5-FU had synergistic effects on reversal the aggressive phenotypes of HepG2 cells, increasing the proportion of Bax/Bcl-2, promoting the activation of caspase-9 and caspase-3, and inducing apoptosis. This combination upregulated P27 and cyclin B1, and downregulated cyclin E1, leading to G1 phase arrest. The combination significantly downregulated MDR1 and LRP1, suggesting the potential to reverse the resistance to 5-FU. Graphical Abstract


Introduction
PGG is hydrolysable tannin found in a wide range of medicinal and edible plants. PGG-rich plants are used to treat malaria, inflammation, snake and scorpion bites, diabetes, chronic diarrhea, poisoning, and microbial infections. PGG has antioxidant and cancer cell-killing activities that are greater than the basic unit of hydrolysable tannins, gallic acid (Torres- Le on et al. 2017).
Combination therapy between a conventional chemotherapeutic drug and a natural product might increase chemotherapeutic sensitivity, and suppress the amount of toxic drugs (Housman et al. 2014). PGG sensitizes multiple myeloma cells to the cytotoxicity of the proteasome inhibitor MG132 (Tseeleesuren et al. 2018). Combination of 5-FU and protopanaxadiol inhibits the proliferation of HCT-116 cells, enhances the number of apoptotic cells, and reduces the tumor size in vivo (Wang et al. 2015). The synergistic effect of urushiol and its pechmann derivative combined with paclitaxel significantly inhibits HepG2 cell proliferation (Qi et al. 2018).
Hepatocellular carcinoma (HCC) is the third key cause of deaths from cancer (Torre et al. 2015). The chemotherapeutic drugs that are used to treat HCC have severe side effects, necessitating new drugs and strategies for patients with advanced liver cancer (Kalyan et al. 2015). In this study, we isolated PGG from Terminalia chebula fruit and found that PGG showed potent anti-proliferation effect on Hep G2 cells. We also measured the effects of PGG on cell cycle, apoptosis, migration, and invasion, even the combination of PGG and 5-FU.

Results and discussions
In the range of 5 $ 50 lM, PGG inhibited the proliferation of HepG2 cells in concentration-and time-dependent manners with IC 50 of 29.7 lM ( Figure S3). Treatment with PGG significantly increased the late apoptosis rate of HepG2 cells ( Figure S4). After PGG treatment, the expressions of Bax and AIF were up-regulated, the expression of Bcl-2, Bcl-x/l and XIAP were down regulated, and the Bcl-2/Bax ratio decreased ( Figure  S5), suggesting that the PGG induced HepG2 cell apoptosis might be mediated by mitochondrial signaling pathway.
In the scratch healing test, the cell mobility was significantly inhibited by PGG at 10.6, 21.2 and 31.8 lM ( Figure S6A); in a transwell chamber assay, PGG treatment significantly decreased the cell number of transmembrane invasion compared with the control group ( Figure S6B, P < 0.05).
Alone, 5-FU inhibited HepG2 cell proliferation in a concentration-dependent manner in the range of 1.5625 to 400 lM. In combination, 5-FU and PGG showed synergistic effects in a large concentration range. The Combination Index (CI) was calculated with Chour Talalay method and the lowest CI value (the greatest synergistic effects) was found when the ratio of 5-FU to PGG was 12.5 lM to 10 lM. This combination concentration was used in the following experiments.
After 24 h treatment with 5-FU and PGG combination, the apoptosis rate of HepG2 cells reached to 26.69% (P < 0.01 v.s. control cells, and P < 0.05 v.s. 5-FU or PGG treated cells), indicating that 5-FU and PGG combination could significantly induce the apoptosis of HepG2 cells ( Figure S8A, Table 1). The combination also significantly reduced the mitochondrial membrane potential of HepG2 cells as represented by the proportions of red and green fluorescences after treatment with JC-1 ( Figure S8B, Table 1).
As shown in Figure S8C and Table 2, combination of 5-FU and PGG almost completely arrested the cell cycle at G1 phase with the proportion of G1 phase cells being 81.31% (P < 0.01 v.s. PGG group, and P < 0.05 v.s. 5-FU group).
In scratch healing test, combination of 5-FU and PGG showed robust inhibition on the cell migration (P < 0.01 v.s. control and 5-FU group; P < 0.05 v.s. PGG group) (Figur S8D). In transwell chamber assay, the number of cells passing through the membrane was significantly reduced in 5-FU, PGG, and the combination groups (all P < 0.01 v.s. control, Figure S8E). Combination of 5-FU and PGG effectively inhibited the migration and invasion of HepG2 cells (Table 3).
PGG and 5-FU combination inhibited the expression of drug resistance proteins. Significantly higher expression of MDR1 (P-gp) was observed in cells treated with 5-FU alone than in the control cells. The expression of MDR1 in cells treated with PGG þ 5-FU was significantly lower than that treated with 5-FU alone. The expression of LRP1 in cells treated with PGG þ 5-FU was significantly lower than that in control cells and in cells treated with PGG or 5-FU alone ( Figure S9).
PGG and 5-FU changed the expression of apoptosis and cell cycle related proteins. Compared with control, PGG þ 5-FU treatment increased the expression of Bax, caspase-3, caspase-9, cyclinB1 and P27, decreased the expression of cyclin E1 and Akt, and Bcl-2 expression remained unchanged. Compared with the PGG group, the expression of Bax, caspase9, P27 and cyclinB1 protein increased in the combination group, and the expression of cyclinE1 and Akt protein decreased, and the difference was statistically significant ( Figure S9).

Conclusions
PGG and 5-FU synergistically inhibited the proliferation of HepG2 cells over a wide concentration range and in a concentration-dependent manner. This combination affected the expression of apoptosis-related and cyclin-related proteins, and induced apoptosis and G1 arrest in HepG2 cells. Also, the combination inhibited the invasion and migration of HepG2 cells, downregulated MDR1 and LRP1 that related to 5-FU resistance.

Disclosure statement
No potential conflict of interest was reported by the authors.