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5, 7-Dimethoxyflavone sensitizes TRAIL-induced apoptosis through DR5 upregulation in hepatocellular carcinoma cells
Jian-Feng Yang1, Jian-Guo Cao, Li Tian
1Medical College, Hunan Normal University, Changsha 410013, China.
Purpose:
5, 7-dimethoxyflavone (DMF) has been reported to induce apoptosis in various cancer cells. The aim of this study was to examine whether DMF sensitizes human hepatocellular carcinoma (HCC) cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis and its mechanism.
Methods:
Human hepatocellular carcinoma cell lines Hep3B, Huh-7, and Hep G2 and human embryo liver L-02 cells were cultured in vitro. The cytotoxic activities were determined using MTT assay. The apoptotic cell death was examined using Flow cytometry using PI staining and DNA agarose gel electrophoresis. The activities of caspase-3, caspase-8, and caspase-9 were measured using ELISA. Intracellular ROS was measured by FCM using the fluorescent probe DCHF-DA, and the expression of DR4, DR5, CHOP, GPR78, and ATF4 proteins was analyzed using Western blot.
Results:
Our results demonstrated subtoxic concentrations of DMF sensitize HCC cells to TRAIL-induced apoptosis and induce the death receptor 5 (DR5) expression level, accompanying the generation of reactive oxygen species (ROS) and the upregulation of CHOP, GPR78, and ATF4 protein expression. Pretreatment with N-acetylcysteine (NAC) inhibited DMF-induced upregulation of DR5, CHOP, GPR78, and ATF4 protein expression and blocked the cotreatment-induced apoptosis. Furthermore, DMF-mediated sensitization of HCC cells to TRAIL was reduced by administration of a blocking antibody or small interfering RNAs for DR5, salubrinal, an inhibitor of ER stress, and the small interfering RNAs for CHOP. However, DMF could not induce the upregulation of DR5 expression, generation of ROS, and sensitization of TRAIL-induced apoptotic cell death in human embryo liver L-02 cells or normal human peripheral blood mononuclear cells (PBMCs).
Conclusion:
The present study demonstrates that DMF selectively enhances TRAIL-induced apoptosis by ROS-stimulated ER-stress triggering CHOP-mediated DR5 upregulation in HCC.
Insights
5,7-dimethoxyflavone (DMF) enhances TRAIL-induced apoptosis in liver cancer cells by increasing DR5 expression through ROS and ER stress. This compound selectively targets cancer cells, sparing normal cells.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- 5,7-dimethoxyflavone (DMF) is known to induce apoptosis in cancer cells.
- Hepatocellular carcinoma (HCC) is a significant global health concern.
- Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potential anti-cancer therapeutic.
Purpose of the Study:
- To investigate if DMF sensitizes HCC cells to TRAIL-mediated apoptosis.
- To elucidate the underlying molecular mechanisms of DMF's sensitizing effects.
Main Methods:
- Utilized human HCC cell lines (Hep3B, Huh-7, Hep G2) and normal liver cells (L-02).
- Assessed cytotoxicity via MTT assay and apoptosis using flow cytometry and DNA gel electrophoresis.
- Measured caspase activities, intracellular reactive oxygen species (ROS), and protein expression (DR4, DR5, CHOP, GPR78, ATF4) via ELISA and Western blot.
Main Results:
- DMF at subtoxic concentrations sensitized HCC cells to TRAIL-induced apoptosis.
- This sensitization was associated with increased DR5 expression, ROS generation, and upregulation of CHOP, GPR78, and ATF4.
- N-acetylcysteine (NAC) inhibited these effects, while DR5 or CHOP inhibition blocked DMF-mediated sensitization.
- DMF did not induce these effects in normal liver cells or peripheral blood mononuclear cells (PBMCs).
Conclusions:
- DMF selectively enhances TRAIL-induced apoptosis in HCC cells.
- The mechanism involves ROS-stimulated endoplasmic reticulum (ER) stress, leading to CHOP-mediated DR5 upregulation.
- DMF shows potential as a sensitizer for TRAIL-based cancer therapy in HCC.
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