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Updated: May 27, 2026
Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro
Published on: July 17, 2018
Casticin-induced apoptosis involves death receptor 5 upregulation in hepatocellular carcinoma cells
1Department of Pathology, The Third Xiangya Hospital of Central South University, Changsha 410013, Hunan Province, China.
Aim:
To investigate the apoptotic activities of casticin in hepatocellular carcinoma (HCC) cells and its molecular mechanisms.
Methods:
PLC/PRF/5 and Hep G2 cell lines were cultured in vitro and the inhibitory effect of casticin on the growth of cells was detected by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolim bromide (MTT) assay. The apoptotic cell death was examined using the cell apoptosis enzyme linked immunosorbent assay (ELISA) detection kit, flow cytometry (FCM) after propidium iodide (PI) staining and DNA agarose gel electrophoresis. The caspase activities were measured using ELISA. Reactive oxygen species (ROS) production was evaluated by FCM after dichlorodihydrofluorescein diacetate (DCFH-DA) probe labeling. Intracellular glutathione (GSH) content was measured using a glutathione assay kit. The expression of death receptor (DR)4 and DR5 proteins was analyzed by Western blotting and FCM.
Results:
Casticin significantly inhibited the growth of human HCC (PLC/PRF/5 and Hep G2) cells in a dose-dependent manner (P < 0.05). Casticin increased the percentage of the sub-G1 population in HCC cells in a concentration-dependent manner. The potency of casticin to PLC/PRF/5 cells was higher than that of 5-flurouracil (26.8% ± 4.8% vs 17.4% ± 5.1%) at 10 μmol/L for 24 h. Casticin increased the levels of Histone/DNA fragmentation and the levels of active caspase-3, -8 and -9 in a concentration-dependent manner (P < 0.05). Treatment with 30 μmol/L casticin for 24 h resulted in the formation of a DNA ladder. Casticin reduced the GSH content (P < 0.05), but did not affect the level of intracellular ROS in PLC/PRF/5 and Hep G2 cells. The thiol antioxidants, acetylcysteine (NAC) and GSH restored GSH content and attenuated casticin-induced apoptosis. In contrast, the nonthiol antioxidants, butylated hydroxyanisole and mannitol failed to do so. In the HCC cells treated with casticin for 24 h, DR5 protein level was increased. The expression of DR5 protein induced by casticin was inhibited by NAC. Pretreatment with DR5/Fc chimera protein, a blocking antibody, effectively attenuated the induction of apoptosis by casticin.
Conclusion:
Casticin-induced apoptosis of HCC cells is involved in GSH depletion and DR5 upregulation.
Insights
Casticin effectively inhibits hepatocellular carcinoma (HCC) cell growth by inducing apoptosis. This process involves glutathione (GSH) depletion and upregulation of death receptor 5 (DR5).
Area of Science:
- Oncology
- Molecular Biology
- Pharmacology
Background:
- Hepatocellular carcinoma (HCC) is a significant global health concern with limited effective treatments.
- Casticin, a natural compound, has shown potential anti-cancer properties that warrant further investigation.
- Understanding the molecular mechanisms of casticin's action is crucial for developing novel therapeutic strategies.
Purpose of the Study:
- To investigate the apoptotic activities of casticin in hepatocellular carcinoma (HCC) cells.
- To elucidate the molecular mechanisms underlying casticin-induced apoptosis in HCC.
- To evaluate casticin's efficacy compared to a standard chemotherapeutic agent.
Main Methods:
- In vitro culture of PLC/PRF/5 and Hep G2 HCC cell lines.
- Cell viability assessed by MTT assay.
- Apoptosis evaluated by ELISA, flow cytometry (FCM) with propidium iodide (PI) staining, and DNA agarose gel electrophoresis.
- Caspase activities, reactive oxygen species (ROS), and intracellular glutathione (GSH) levels measured.
- Expression of death receptor (DR)4 and DR5 proteins analyzed by Western blotting and FCM.
- Effect of antioxidants (acetylcysteine, GSH, butylated hydroxyanisole, mannitol) and DR5 blocking antibody investigated.
Main Results:
- Casticin significantly inhibited HCC cell growth in a dose-dependent manner.
- Casticin induced apoptosis, evidenced by increased sub-G1 population, DNA fragmentation, and elevated caspase-3, -8, and -9 activities.
- Casticin reduced intracellular GSH levels but did not affect ROS.
- Thiol antioxidants restored GSH and attenuated casticin-induced apoptosis, while non-thiol antioxidants did not.
- Casticin upregulated DR5 protein expression, which was inhibited by acetylcysteine and attenuated by a DR5 blocking antibody.
- Casticin demonstrated higher potency than 5-fluorouracil in inhibiting PLC/PRF/5 cells.
Conclusions:
- Casticin effectively induces apoptosis in hepatocellular carcinoma cells.
- The mechanism involves significant depletion of intracellular glutathione (GSH).
- Upregulation of death receptor 5 (DR5) plays a critical role in casticin-mediated apoptosis.
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