ABSTRACT
High levels of intracellular reactive oxygen species (ROS) in cells is recognized as one of the major causes of cancer cell apoptosis and has been developed into a promising therapeutic strategy for cancer therapy. However, whether apoptosis associated biophysical properties of cancer cells are related to intracellular ROS functions is still unclear. Here, for the first time, we determined the changes of biophysical properties associated with the ROS-mediated oesophageal cancer KYSE-150 cell apoptosis using high resolution atomic force microscopy (AFM). Oridonin was proved to induce ROS-mediated KYSE-150 cell apoptosis in a dose dependent manner, which could be reversed by N-acetylcysteine (NAC) pretreatment. Based on AFM imaging, the morphological damage and ultrastructural changes of KYSE-150 cells were found to be closely associated with ROS-mediated oridonin-induced KYSE-150 cell apoptosis. The changes of cell stiffness determined by AFM force measurement also demonstrated ROS-dependent changes in oridonin induced KYSE-150 cell apoptosis. Our findings not only provided new insights into the anticancer effects of oridonin, but also highlighted the use of AFM as a qualitative and quantitative nanotool to detect ROS-mediated cancer cell apoptosis based on cell biophysical properties, providing novel information of the roles of ROS in cancer cell apoptosis at nanoscale.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Apoptosis/drug effects , Diterpenes, Kaurane/pharmacology , Epithelial Cells/drug effects , Reactive Oxygen Species/metabolism , Acetylcysteine/pharmacology , Antineoplastic Agents, Phytogenic/antagonists & inhibitors , Cell Line, Tumor , Diterpenes, Kaurane/antagonists & inhibitors , Dose-Response Relationship, Drug , Epithelial Cells/metabolism , Epithelial Cells/ultrastructure , Esophagus/drug effects , Esophagus/metabolism , Esophagus/ultrastructure , Free Radical Scavengers/pharmacology , Humans , Membrane Potential, Mitochondrial/drug effects , Microscopy, Atomic Force , Mitochondria/drug effects , Mitochondria/metabolism , Mitochondria/pathology , Oxidative Stress , Reactive Oxygen Species/agonists , Reactive Oxygen Species/antagonists & inhibitorsABSTRACT
Glaucocalyxin A (GLA) is a biologically active ent-kauranoid diterpenoid isolated from Rabdosia japonica var. glaucocalyx, a traditional Chinese medicinal herb, which has been shown to inhibit tumor cell proliferation. However, the mechanism underlying GLA-induced cytotoxicity remains unclear. In this study, we focused on the effect of GLA induction on apoptosis, the mitochondria-mediated death pathway and the accumulation of reactive oxygen species (ROS) in human leukemia cells (HL-60). GLA could induce a dose-dependent apoptosis in HL-60 cells as characterized by cell morphology, DNA fragmentation, activation of caspase-3, -9 and an increased expression ratio of Bax/Bcl-2. The mitochondrial membrane potential (Δψ(m)) loss and cytochrome c release from mitochondria to cytosol were observed during the induction. Moreover, GLA caused a time- and dose-dependent elevation of intracellular ROS level in HL-60 cells, and N-acetyl-l-cysteine (NAC, a well-known antioxidant) could block GLA-induced ROS generation and apoptosis. These data suggest that GLA induces apoptosis in HL-60 cells through ROS-dependent mitochondrial dysfunction pathway.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Apoptosis/drug effects , Drugs, Chinese Herbal/pharmacology , Leukemia/drug therapy , Mitochondria/drug effects , Antineoplastic Agents, Phytogenic/antagonists & inhibitors , Antioxidants/pharmacology , Biomarkers/metabolism , Caspase 3/metabolism , Caspase 9/metabolism , Cell Line, Tumor , Cell Survival/drug effects , DNA Fragmentation/drug effects , Diterpenes, Kaurane/antagonists & inhibitors , Diterpenes, Kaurane/pharmacology , G1 Phase/drug effects , HL-60 Cells , Humans , Inhibitory Concentration 50 , Leukemia/metabolism , Leukemia/pathology , Membrane Potential, Mitochondrial/drug effects , Mitochondria/enzymology , Mitochondria/metabolism , Reactive Oxygen Species/metabolism , Signal Transduction/drug effectsABSTRACT
Oridonin, isolated from Rabdosia rubescences, has been reported to exert cytotoxic effects on L929 cells. In this study, we investigated the mechanisms of FGF-2 protection of L929 cells from oridonin-induced apoptosis. Phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB) signal did not mediate this effect because the PI3K inhibitor wortmannin failed to reverse this protection and PKB activation was not observed in this process. In contrast, the extracellular signal-regulated kinase (ERK) was responsible for this rescue because its inhibition abolished the protective effect of fibroblast growth factor (FGF)-2. ERK had dual regulatory functions: mediating cell apoptosis or preventing cells from initiating the apoptotic response by phosphorylation or promoting expression of Bcl-2 in dependence of different stimuli. In L929 cells treated with oridonin alone, the activated ERK decreased the ratio of Bcl-2/Bax by mediating the phosphorylation of Bcl-2, resulting in apoptosis; the Ras inhibitor manumycin A and Raf inhibitor GW5074 failed to inhibit this apoptosis, indicating that there is a signal other than Ras/Raf pathway activated ERK. However, in the presence of FGF-2, Bcl-2 phosphorylation was blocked, and the Ras/Raf/ERK signal pathway was activated and protected against the oridonin-induced apoptosis by the alternative function of promoting of Bcl-2 expression.
