Rhein ElicitsIn VitroCytotoxicity in Primary Human Liver HL-7702 Cells by Inducing Apoptosis through Mitochondria-Mediated Pathway

Abstract

Objective. To study rhein-induced apoptosis signaling pathway and to investigate its molecular mechanisms in primary human hepatic cells. Results. Cell viability of HL-7702 cells treated with rhein showed significant decrease in dose-dependent manner. Following rhein treatment (25 μM, 50 μM, and 100 μM) for 12 h, the detection of apoptotic cells was significantly analyzed by flow cytometry and nuclear morphological changes by Hoechst 33258, respectively. Fatty degeneration studies showed upregulation level of the relevant hepatic markers (P < 0.01). Caspase activities expressed significant upregulation of caspase-3, caspase-9, and caspase-8. Moreover, apoptotic cells by rhein were significantly inhibited by Z-LEHD-FMK and Z-DEVD-FMK, caspase-9 inhibitor, and caspase-3 inhibitor, respectively. Overproduction of reactive oxygen species, lipid peroxidation, and loss of mitochondrial membrane potential were detected by fluorometry. Additionally, NAC, a ROS scavenger, significantly attenuated rhein-induced oxidative damage in HL-7702 cells. Furthermore, real-time qPCR results showed significant upregulation of p53, PUMA, Apaf-1, and Casp-9 and Casp-3 mRNA, with no significant changes of Fas and Cytochrome-c. Immunoblotting revealed significant Cytochrome-c release from mitochondria into cytosol and no change in Fas expression. Conclusion. Taken together, these observations suggested that rhein could induce apoptosis in HL-7702 cells via mitochondria-mediated signal pathway with involvement of oxidative stress mechanism.

Apoptotic effects of rhein through the mitochondrial pathways, two death receptor pathways, and reducing autophagy in human liver L02 cells

“…Rhein, a component of traditional Chinese medicines such as P. multiflorum and R. palmatum L., may be one of the causes of liver damage caused by these Chinese medicines. It has been demonstrated to induce apoptosis activities in many cell lines, and caused hepatotoxicity in primary rat hepatocytes …”

“…It has been demonstrated to induce apoptosis activities in many cell lines, and caused hepatotoxicity in primary rat hepatocytes. [33][34][35] In this study, we conducted a series of experimental procedures of rhein and assessed rhein cytotoxicity on L02 cells. It has been suggested that more than one assay should be used to determine the cytotoxicity of chemicals for the reliability of the data.…”

Maspin suppresses growth, proliferation and invasion in cutaneous squamous cell carcinoma cells

“…When apoptosis occurs, phosphatidylserine (PS) located in the inner cytomembrane moved to the external cytomembrane, which could be specially combined by Annexin V, thus early apoptosis could be discovered by testing PS using flow cytometry (26). With apoptosis further proceeding, and through staining, condensed chromatin, nuclear fragmentation and apoptotic bodies in apoptotic cells exhibited intense fluorescence under a fluorescence microscope (27).…”

Glycitein induces reactive oxygen species‐dependent apoptosis and G0/G1 cell cycle arrest through the MAPK/STAT3/NF‐κB pathway in human gastric cancer cells

“…Data are given as the relative ratio of green to red fluorescence intensity, showing the level of depolarization of the mitochondrial membrane potential (Bounda, Zhou, Wang, & Feng, 2015). Harvested cells were treated with JC-1 (10 μg/mL) (Beyotime Institute of Biotechnology, Shanghai, China) at room temperature for 5 min to detect mitochondrial membrane potential (ΔΨm) depolarization.…”

“…States of ΔΨm depolarization were determined by flow cytometry and CytExpert software (version 1.2; Beckman Coulter, Inc., Brea, CA) was used to analyze the data. Data are given as the relative ratio of green to red fluorescence intensity, showing the level of depolarization of the mitochondrial membrane potential (Bounda, Zhou, Wang, & Feng, 2015).…”