[논문]Role of Intracellular <tex>$Ca^{2+}$</tex> Signal in the Ascorbate-Induced Apoptosis in a Human Hepatoma Cell Line

Lee , Yong-Soo

doi:10.1007/bf02975889

Role of Intracellular $Ca^{2+}$ Signal in the Ascorbate-Induced Apoptosis in a Human Hepatoma Cell Line 원문보기

Archives of pharmacal research : a publication of the Pharmaceutical Society of Korea, v.27 no.12, 2004년, pp.1245 - 1252

Lee , Yong-Soo (College of Pharmacy, Duksung Women's University)

Abstract ▼ AI-Helper

Although ascorbate (vitamin C) has been shown to have anti-cancer actions, its effect on human hepatoma cells has not yet been investigated, and thus, the exact mechanism of this action is not fully understood. In this study, the mechanism by which ascorbate induces apoptosis using HepG2 human hepatoblastoma cells is investigated. Ascorbate induced apoptotic cell death in a dose-dependent manner in the cells, was assessed through flow cytometric analysis. Contrary to expectation, ascorbate did not alter the cellular redox status, and treatment with antioxidants (N-acetyl cysteine and N,N-diphenyl-p-phenylenediamine) had no influence on the ascorbate-induced apoptosis. However, ascorbate induced a rapid and sustained increase in intracellular $Ca^{2+}$ concentration. EGTA, an extracellular $Ca^{2+}$ chelator did not significantly alter the ascorbate-induced intracellular $Ca^{2+}$ increase and apoptosis, whereas dantrolene, an intracellular $Ca^{2+}$ release blocker, completely blocked these actions of ascorbate. In addition, phospholipase C (PLC) inhibitors (U-73122 and manoalide) significantly suppressed the intracellular $Ca^{2+}$ release and apoptosis induced by ascorbate. Collectively, these results suggest that ascorbate induced apoptosis without changes in the cellular redox status in HepG2 cells, and that the PLC-coupled intracellular $Ca^{2+}$ release mechanism may mediate ascorbate-induced apoptosis.

주제어

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문제 정의

Thus, the purpose of this study was to elucidate the mechanism by which ascorbate induces apoptosis in tumor cells. Specifically, the roles of ROS and intracellular Ca2+ were investigated in the mechanism of ascorbateinduced apoptosis using HepG2 human hepatoblastoma cells as a model cellular system.

대상 데이터

(Eugene, OR). Fetal bovine serum (FBS) and antibiotics (penicillin and streptomycin mixture) were purchased from GIBCO (Grand isiand, NY). DCFH-DA, BAPTA/AM, and Fura-2/AM were prepared as stock solutions in dimethyl sulfoxide (DMSO), and then, they were diluted with aqueous medium to the desired concentrations.
For loading DCFH-DA into the cells, cells were incubated with the dye for 2 hrs at a final concentration of 5 |니VI at 37℃. Fluorescence (excitation wavelength set at 485 nm and the emission wavelength at 530 nm) was monitored in a well-stirred cuvette.
The HepG2 human hepatoblastoma cell line was purchased from American Type Culture Collection (Rockville, MA). Powdered Eagle's minimum essential medium (MEM) and Earle's basal salt soution (EBSS), trypsin solution, sodium pyruvate, sodium ascorbate, N-acetyl cysteine (NAC), /N, /N-diphenyl-p-phenylenediamine dantrolene (DPPD), manoalide, 1 -(6-(17β-3-methoxyestra-1, 3, 5(10)-trien-17-yl)amino)hexyl)-1 H-pyrrole-2, 5, -dione (U-73122), ethylene glycol-bis-(aminoethylether)-ethane-N, N,N,N-tetraacetic acid (EGTA), propidium iodide (PI), ribonuclease A, and all salt powders were obtained from Sigma Chemical CO.

데이터처리

All experiments were performed four times. The data were expressed as means ± standard error of the mean (SEM), and the results were analyzed using one way analysis of variance (ANOVA) and the Student-Newman-Keul's test for individual comparisons. P values less than 0.

이론/모형

No involvement of the cellular redox status in the ascorbate-fnduced apoptosis in HepG2 human hepatoblastoma cells. Data (A) show changes in ROS levels as a function of time, which was measured using the DCF fluorescenGe method. The arrow shows the time point for the addition of ascorbate (5 mM).

성능/효과

and Orrenius, 1997). Consistent to this implication, in this study, it was also shown that ascorbate appeared to induce apoptosis through the rise of intracellular Ca²⁺, since ascorbate induced a rapid and sustained increase in intracellular Ca2+ concentration (Fig 3A), and intracellular Ca²⁺ chelation with BAPTA/AM effectively prevented ascorbate-induced apoptosis (Fig. 3B). In addition to apoptosis, cell proliferation and differentiation have been linked to the stimulation of the intracellular Ca2+ signal (Munaron et al.
In conclusion, ascorbate induced apoptosis in HepG2 human hepatoblastoma cells and the activation of PLC, and the subsequent release of intracellular Ca²⁺ from IP3-sensitive stores may be essentially involved in the mechanism of this action. The results from this study further suggest that ascorbate may be valuable for the therapeutic intervention of human hepatomas.
, 1998). The results of this study also suggest that the rise in intracellular Ca²⁺ and apoptotic effects of ascorbate may occur apart from ascorbate incorporation because ascorbate rapidly increased the intracellular Ca²⁺ level, and the major target of ascorbate appeared to be the PLC residing in the plasma membrane. The PLC family is further classified into three subgroups: β, γ, and β.

후속연구

Although speculated, ascorbate may act on the putative membrane receptor coupled to the activation of PLC via stimulation of certain types of G proteins. This possibility should be determined in a future study.

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