Anticancer effects of Poncirus fructus on hepatocellular carcinoma through regulation of apoptosis, migration, and invasion

Abstract

Poncirus fructus (PF) is a phytochemical compound extracted from the dry, immature fruits of Poncirus trifoliate . PF is traditionally used to treat gastrointestinal disorders, allergies, and inflammatory disease. In East Asia, PF is also known for its anticancer properties. There are numerous reports on the anticancer and anti-inflammatory effects of PF in a wide range of cancers and gastrointestinal diseases, respectively. However, the role of PF in inducing apoptosis and suppressing the invasiveness of hepatocellular carcinoma (HCC) remains unclear. This study investigated the ability of PF to induce apoptosis and inhibit the invasiveness and migratory ability of HCC cell lines (Hep3B and Huh7). Wound healing, Transwell migration and invasion, and colony-formation assays, as well as flow cytometry, were used to analyze cell proliferation, migration, invasion, and apoptosis. Epithelial-mesenchymal transition (EMT)-related and apoptotic proteins were assessed by western blotting. The mitochondrial membrane potential of the Hep3B and Huh7 cells was observed with tetramethylrhodamine ethyl ester. The reactive oxygen species (ROS) level was determined by dihydroethidium (DHE) staining. PF treatment significantly decreased the proliferation of Hep3B and Huh7 cells in a dose-dependent manner, reduced the mitochondrial membrane potential, increased ROS levels, decreased the protein levels of Bcl-2, and increased the protein levels of Bax and cleaved caspase-3 and 9, suggesting that PF mediated HCC apoptosis via a mitochondrial pathway. Our findings showed that PF prevented HCC cell migration and invasion by inhibiting the EMT process and downregulating MMP-2 and MMP-9 activities. The results suggest the potential anticancer effects of PF by inhibiting proliferation, inducing apoptosis, and reducing the invasion and migration of HCC cells.

3,3′-Diindolylmethane Suppresses the Growth of Hepatocellular Carcinoma by Regulating Its Invasion, Migration, and ER Stress-Mediated Mitochondrial Apoptosis

“…Previously, numerous studies have suggested that efflux of Ca 2+ from ER to mitochondria can result in mitochondrial dysfunction, causing overproduction of ROS, which in turn results in cell death [ 75 , 76 ]. Several studies have reported that overproduction of mitochondrial ROS can result in mitochondrial membrane depolarization, causing a loss in mitochondrial membrane potential which initiates mitochondrial-dependent apoptotic pathway [ 4 , 77 ]. Although many studies have investigated the relationship among ROS, Ca 2+ , and apoptosis in several cancer cell lines [ 78 , 79 ], little is known about the role of mitochondria, ROS, and Ca 2+ in the induction of apoptosis caused by DIM.…”

“…Mitochondrial membrane potentials (ΨΔm) of Hep3B and Huh7 cells were determined using tetramethylrhodamine ethyl ester perchlorate (TMRE) dye as described previously [ 4 ]. Briefly, Hep3B and Huh7 cells were treated with DIM (0, 40, 60, and 80 µM) for 24 h and stained with TMRE (400 nmol/L) for 20 min.…”

“…Intracellular ROS levels in Hep3B and Huh7 cells were determined using a fluorescent dihydroethidium (DHE) probe and an OxiSelect In Vitro ROS/RNS Assay Kit (Cell BioLabs, San Diego, CA, USA). To analyze ROS using the DHE probe, cells were stained with 5 µM of DHE, labeled with 4′,6-diamidono-2-phenylindole, dihydrochloride (DAPI), and examined under a fluorescence microscope using a ×63 oil immersion objective lens as described previously [ 4 ]. To determine the amount of ROS produced using the OxiSelect In vitro ROS/RNS Assay Kit following the manufacturer’s instruction, Hep3B and Huh7 cells were harvested after DIM treatment.…”

“…Changes in protein expression level in response to DIM treatment were determined by Western blot analysis. Methods of protein extraction, quantification, and Western blotting were mentioned previously [ 4 , 46 ]. Proteins were transferred from gels onto PVDF membranes and incubated with primary antibodies overnight.…”

“…It is associated with an extremely poor prognosis [ 1 , 2 ]. Gradual increases of chronic liver disease and cirrhosis due to viral hepatitis, metabolic disorders, and alcohol intake are important risk factors for the development of HCC worldwide [ 3 , 4 ]. Due to its strong invasion ability and the complexity of the metastatic process, treatment options for HCC patients are very limited and its prognosis is dire [ 5 , 6 ].…”