Erinacine Facilitates the Opening of the Mitochondrial Permeability Transition Pore Through the Inhibition of the PI3K/ Akt/GSK-3β Signaling Pathway in Human Hepatocellular Carcinoma

Cell Physiol Biochem. 2018;50(3):851-867. doi: 10.1159/000494472. Epub 2018 Oct 24.

Abstract

Background/aims: Erinacine, which is extracted from the medicinal mushroom Hericium erinaceus, is known to play anticancer roles in human cancers. The following study aims to investigate the role of erinacine in the opening of the mitochondrial permeability transition pore (MPTP) in hepatocellular carcinoma (HCC) through the PI3K/Akt/GSK-3β signaling pathway and highlights the applicability of erinacine in HCC treatments.

Methods: HCC and paracancerous tissues were obtained from 85 HCC patients who've undergone surgical resection. Immunohistochemistry was adopted to detect positive expression of PI3K, Akt, and GSK-3β. Treatment of HepG-2 with LY294002 (an inhibitor of the PI3K/Akt/GSK-3β signaling pathway) and different concentration of erinacine was performed to determine the involvement of LY294002 in erinacine action. The expressions of PI3K, Akt, GSK-3β, CyclinD1, Vimentin, β-catenin, Bcl-2, E-cadherin, Bax, and caspase-9 were determined by RT-qPCR and Western blot analysis. Cell viability, colony formation rate, migration, invasion, cycle, and apoptosis were detected by MTT, colony formation, wound healing assay, Transwell assay, and flow cytometry, respectively. The size and weight of xenograft tumors were observed in nude mice. Mitochondrial membrane potential in HepG-2 was determined using laser scanning confocal microscopy following JC-1 staining. Mitochondrial Ca2+ indicator Rhod-2, AM was used to detect the changes of mitochondrial Ca2+, while western blot analysis was employed to detect the presence levels of cytochrome C (cyt-C).

Results: The results revealed that PI3K, Akt, and GSK-3β were up-regulated in HCC tissues. Erinacine or LY294002 led to a decrease in mitochondrial membrane potential, increase in intracellular mitochondrial Ca2+, and the release of cyt-C in mitochondria. In addition, Erinacine was found to decrease the mitochondrial membrane potential, expression of PI3K, Akt, GSK-3β, CyclinD1, Vimentin, β-catenin, and Bcl-2, cell proliferation, colony formation ability, migration, invasion, and xenograft tumor size, while E-cadherin, Bax, and caspase-9 expression, and cell apoptosis were elevated in a dose-dependent manner. Erinacine also stimulated the effects of LY294002 on the HCC. Following the addition of 500 μM Erinacine and MPTP opening inhibitor CsA, we found that the mitochondrial membrane potential level increased, while mitochondrial Ca2+ and Cyt-C decreased from the mitochondria.

Conclusion: The results from the study demonstrated that erinacine induced MPTP opening, facilitates the release of cyt-C, and inhibited cell proliferation, migration, and invasion, while it promotes apoptosis by inactivating the PI3K/Akt/GSK-3β signaling pathway, preventing the progression of HCC.

Keywords: Erinacine; Hepatocellular carcinoma; LY294002; Mitochondrial permeability transition pore; PI3K/Akt/GSK-3β signaling pathway.

MeSH terms

  • Animals
  • Apoptosis / drug effects
  • Carcinoma, Hepatocellular / metabolism
  • Carcinoma, Hepatocellular / pathology*
  • Cell Cycle Checkpoints / drug effects
  • Chromones / pharmacology
  • Diterpenes / pharmacology*
  • Female
  • Glycogen Synthase Kinase 3 beta / genetics
  • Glycogen Synthase Kinase 3 beta / metabolism
  • Hep G2 Cells
  • Humans
  • Liver Neoplasms / metabolism
  • Liver Neoplasms / pathology*
  • Male
  • Membrane Potential, Mitochondrial / drug effects
  • Mice, Inbred BALB C
  • Mice, Nude
  • Middle Aged
  • Mitochondrial Membrane Transport Proteins / drug effects*
  • Mitochondrial Membrane Transport Proteins / metabolism
  • Mitochondrial Permeability Transition Pore
  • Morpholines / pharmacology
  • Phosphatidylinositol 3-Kinases / genetics
  • Phosphatidylinositol 3-Kinases / metabolism
  • Proto-Oncogene Proteins c-akt / genetics
  • Proto-Oncogene Proteins c-akt / metabolism
  • Signal Transduction / drug effects*

Substances

  • Chromones
  • Diterpenes
  • Mitochondrial Membrane Transport Proteins
  • Mitochondrial Permeability Transition Pore
  • Morpholines
  • erinacine A
  • 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one
  • Glycogen Synthase Kinase 3 beta
  • Proto-Oncogene Proteins c-akt