Induction of apoptosis by hinokitiol, a potent iron chelator, in teratocarcinoma F9 cells is mediated through the activation of caspase‐3

Abstract

Hinokitiol, a potent iron chelator, has been reported to induce differentiation in teratocarcinoma F9 cells with a reduction of viable cells. In this study, we examined the steps leading to eventual cell death by hinokitiol during differentiation. Hinokitiol induced DNA fragmentation of F9 cells in a concentration- and time-dependent manner. This effect was also observed in a cell-free system using the nuclei from intact cells and the cytosols from hinokitiol-treated cells. In contrast, hinokitiol methyl ether and hinokitiol-Fe (III) complex, which are deficient in iron-chelating activity, showed no DNA fragmentation activity in both cell culture and cell-free systems. These results suggest that iron deprivation by hinokitiol may be involved in the induction of apoptosis of F9 cells. Caspase-3, one of the key enzymes in the apoptotic cascade, was specifically activated by hinokitiol treatment, but not by the other two derivatives. In addition, its specific inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, strongly blocked hinokitiol-induced DNA fragmentation. These results indicate that iron deprivation by hinokitiol can induce apoptosis of F9 cells through the activation of caspase-3.

Novel di-2-pyridyl–derived iron chelators with marked and selective antitumor activity: in vitro and in vivo assessment

“…7,9 Moreover, we have shown that the closely related group of PKIH chelators binds Fe(II) and mediates Fenton chemistry, resulting in DNA degradation. 73 In this investigation, an increase in ROS was detected after 2-hour incubation with Dp44mT, coinciding with h-cytc release ( Figure 7A). Hence, as with the doxorubicin-Fe complex that redox cycles to generate radicals, 83 the Fe complex of Dp44mT is redox active, and this could play a role in its cytotoxic effects.…”

“…74 Hence, based on the close structural similarities between the PKIH and DpT chelators (Figure 1), these latter ligands will be tridentate and will have a high affinity for Fe. Considering the formation constants for the related PCIH 74 and the solution and electrochemistry of the PKIH analogs, 73 it can be predicted that the DpT analogs will bind Fe(II) with high affinity. As with all Fe chelators, these ligands will, to some extent, also bind Zn(II) and Cu(II).…”

Comparison of Cytotoxicity of Cysteine and Homocysteine for Renal Epithelial Cells

“…The cell pellets were dissolved in 0.5% SDS solution for protein assay. Caspase-3 activity was colorimetrically measured using Ac-DEVD-MCA [Ac-Asp-Glu-Val-Asp-α-4(-methyl-coumaryl-7-amide)] as substrate [16]The assay was performed in 96-well microtiter plates by incubating 20 µl of cell lysates with 140 µl of reaction buffer (100 m M Hepes-KOH pH 7.5, 10% sucrose, 0.1% CHAPS 10 m M DTT, 0.1 mg/ml ovalbumin, and 1 m M EDTA) and 40 µl of 1 m M substrate at 37°C for 30 min. The caspase-3 activity was determined in the fluorescence spectrophotometer at 349–460 nm.…”

Hinokitiol inhibits vasculogenic mimicry activity of breast cancer stem/progenitor cells through proteasome-mediated degradation of epidermal growth factor receptor

“…Hinokitiol may induce G1 arrest in malignant melanoma cells through increased cyclin-dependent kinase inhibitor 1B protein expression (15). Hinokitiol was also indicated to cause caspase-dependent apoptosis (16) and differentiation (17) in teratocarcinoma F9 cells. A recent study indicated that hinokitiol induced autophagy in murine breast and colorectal cancer cells via downregulation of the v-akt murine thymoma viral oncogene homolog 1/mechanistic target of rapamycin (serine/threonine kinase) signaling pathway, which led to cell death (18).…”