1,2,3,4,6-Penta-O-galloyl-β-D-glucose, an active substance derived from natural foods, can block the cell cycle, induce apoptosis, and exert anti-tumor effects. Studies have shown that β-PGG has a powerful cancer cell-killing effect and its effect is superior to that of gallic acid27. Another study found that PGG blocked the cell cycle of human multiple myeloma cells RPMI8226 in the G1 phase and induced apoptosis in vitro28. PGG can also downregulate the expression of Cyclin D1. A previous study revealed that low concentrations of PGG blocked the cycle of ER + breast cancer T-47D and BT-474 cells in S phase, whereas high concentrations of PGG blocked the cycle of cells in G1 phase14. Moreover, PGG was found to downregulate the expression of HURP and BCL-2, and increase the expression of BAX to induce apoptosis as an anti-ER breast cancer14. In colorectal cancer cells, PGG induced endogenous apoptosis by upregulating the expression of P53, P21, and cleaved CASP328. In addition, in vivo experiments found that PGG cured cancer cachexia by inhibiting IR and IGF1R in pancreatic cancer cells, thereby reducing glycolytic enzymes, hepatic gluconeogenesis, skeletal muscle protein hydrolysis, and fat lipolysis in tumor grafts. Dong et al.29 reported that PGG exerted its anti-cancerous effects in vivo by activating MAPK8/9/10, ERN1, and EIF2S1 signaling pathways through autophagy mediated senescence to exert its anti-hepatocellular carcinoma activity29. However, although previous studies have shown that PGG can inhibit cancer activity, the effect of PGG on liver cancer and its potential mechanism have not yet been evaluated. This study aimed at clarifying the effect of PGG on the proliferation and apoptosis of liver cancer cells, elucidating the mechanism of action of network pharmacology, and exploring the relationship between its mechanism and the p53 signaling pathway.
Network pharmacology has the potential to expand the druggable space of proteins involved in complex diseases by mapping unexplored targets of natural products, thereby identifying new therapeutic approaches for diseases30. In this study, 363 targets of PGG against liver cancer were identified using PharmMapper, SwissTargetPrediction, and GeneCards databases. GO and KEGG enrichment analyses revealed that PGG treatment of liver cancer was mainly associated with the p53 signaling pathway. It is worth noting that P53 was identified as a tumor suppressor gene in 50% of human cancers in the late 1980s and 1990s. Specifically, genes with P53 mutations were found in 50% of all human cancers31,32. One study revealed that P53 is activated by various stresses to halt cancer progression by causing transient or permanent growth arrest, DNA repair, or advancing the cell death program33.
To further validate the results of network pharmacology and explore whether PGG promotes apoptosis in hepatocellular carcinoma pairs through activation of the p53 signaling pathway, an in vitro hepatocellular carcinoma model was established using HepG2 cells. Results obtained after performing the CCK-8 assay showed that PGG could inhibit proliferation of hepatocellular carcinoma HepG2 cells in a time-dependent manner. Flow cytometry analysis showed that PGG blocked cell growth in the S phase and increased drug concentrations blocked cells in the G0/G1 phase. P21, the first identified CDK inhibitor, binds to cell cycle protein complexes, such as A/CDK2, E/CDK2, D1/CDK4, and D2/CDK4, thereby inhibiting phosphorylation of pRB proteins34. It has been reported that P53 induces P21 to inhibit the cell cycle protein E/CDK2 in response to DNA damage, thereby inhibiting the G1/S transition35. Western blot analysis showed that PGG can lead to accumulation and activation of P21.
Apoptosis occurs when internal or external factors activate the programmed cell death process. Notably, dysregulated apoptosis is a common feature of malignant tumors. In this study, flow cytometry results showed that PGG could cause apoptosis, and the apoptotic state gradually shifted towards early apoptosis as the PGG concentration increased. After combining this result with changes in mitochondrial membrane potential and Ca2+ concentration, we hypothesized that apoptosis was mainly endogenous. To further investigate the relationship between β-PGG inhibition of apoptosis and the p53 signaling pathway, the changes of related genes and proteins on the p53 signaling pathway were first analyzed by qRT-PCR. Results showed that the mRNA expressions of P21, PUMA, IGF-BP3, CASP3, and Cytochrome C genes in the cells were increased with highly significant differences (p < 0.01). In addition, the mRNA expression of CASP9 and PERP was significantly increased (p < 0.05), whereas the mRNA expression of BAX and P53 showed no significant difference. Notably, the expression of BCL-2 gene was significantly decreased (p < 0.05). Second, western blot analysis was performed to detect the increased expression of P53, P21, Cleaved CASP3, CASP9, Cytochrome C and BAX proteins, and the decreased expression of BCL-2 protein (p < 0.05). Activated P53 regulated the expression of downstream P21 protein, thereby resulting in an increase in P21 protein levels in HepG2 cells with a significant difference (p < 0.01). Currently, it is increasingly becoming apparent that the p53 signaling pathway plays an important role in apoptosis26, and its activation can lead to cellular angiogenesis, inhibition of apoptosis, and DNA repair, ultimately resulting in cancer development and progression.
In summary, this study has demonstrated that β-PGG achieves its anti-tumor effects in vitro mainly through two aspects. On one hand, it affects the cell cycle by upregulating the expression of P21 gene and protein; whereas on the other hand, it induces apoptosis in HepG2 cells by increasing the expression of P53, PUMA, and CASP9 proteins, thereby causing CASP3 to shear and its shedder content to increase the ratio of BAX to BCL-2 and promote Cytochrome C release. It is worth mentioning that the mechanisms of hepatocarcinogenesis and development are complex and thus further in vivo experiments are needed. Overall, this study provides more possibilities for the treatment of hepatocellular carcinoma with the help of network pharmacology and provides a reference for the development of related health food products.