Puerarin Promotes Parthanatos in Hepatocellular Carcinoma Cells by Blocking the Phosphorylation of the PI3K/AKT/mTOR Pathway
WANG Yingyi1, JIANG Jingwen2, WANG Liangyu1*
This study aims to investigate the effect of puerarin on parthanatos in HCC (hepatocellular carcinoma) cells. Human HCC cell lines Hep3B, HepG2, Huh7, and SK-HEP-1 were cultured and divided into a blank group (0 μmoL/L), low-concentration puerarin group (10 μmoL/L), medium-concentration puerarin group (50 μmoL/L), and high-concentration puerarin group (100 μmoL/L). CCK-8 and EdU (5-ethynyl-2’-deoxyuridine) assays were used to detect the effect of puerarin on cell proliferation. Western blot was employed to detect the effects of low/medium/high doses of puerarin on the expression of PARP-1 [poly (ADP-ribose) polymerase 1]/AIF (apoptosis-inducing factor) knockdown/PTEN (phosphatase and tensin homolog deleted on chromosome ten) knockdown, PAR [poly (ADP-ribose)], as well as proteins in the PI3K (phosphatidylinositol 3-kinase)/AKT (protein kinase B)/mTOR (mammalian target of rapamycin) pathway. The following indicators were detected using Western blot: the effects of low, medium, and high doses of puerarin on the expression of PARP-1, AIF, PTEN, PAR, and proteins in the PI3K/AKT/mTOR pathway; and the effects of puerarin on the expression of PTEN, PARP-1, AIF, and proteins in the PI3K/AKT/mTOR pathway after the addition of a PARP-1 inhibitor (Olaparib), PTEN knock-down, addition of an AKT agonist (PTP1B-IN-24), or addition of a PI3K agonist (740Y-P). A nude mouse xenograft model was established to observe the effects of puerarin feeding on tumor volume and mass. TUNEL staining was used to detect apoptosis; immunofluorescence staining was used to detect AKT expression, and Western blot was used to detect the expression levels of PARP-1 and AIF proteins. The results showed that, compared with the Control group, puerarin at low/medium/high doses dose-dependently inhibited the proliferative activity of HCC cells (P<0.05), significantly reduced the phosphorylation levels of the PI3K/AKT/mTOR pathway (P<0.05), increased the protein expression levels of PAR, PARP-1, and AIF (P<0.05), and promoted the nuclear translocation of AIF protein. Compared with the Olaparib group/PTEN-KD group/PTP1B-IN-24 group, there was no significant change in cell proliferation ability after the addition of puerarin (P>0.05). Among the low/medium/high doses of puerarin, compared with the Control group, the expression of PAR, PARP-1, and AIF in the 740Y-P group was significantly decreased (P<0.05), while the expression of PI3K/AKT/mTOR pathway related proteins was significantly increased (P<0.05). In vivo experiments showed that puerarin feeding significantly inhibited the growth of HCC tumor volume and weight (P<0.001), increased the expression levels of PARP-1 and AIF proteins (P<0.01), and promoted AKT expression. Puerarin promotes parthanatos in HCC cells by blocking the phosphorylation of the PI3K/AKT/mTOR pathway.
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