Juglone-induced apoptosis in human gastric cancer SGC-7901 cells via the mitochondrial pathway

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Abstract

This study was designed to investigate the effect of juglone on the apoptosis of human gastric cancer SGC-7901 cells. The cytotoxic activity of juglone on SGC-7901 cells was tested by the sulforhodamine B (SRB) assay. The morphological changes in the cells were observed by transmission electron microscopy (TEM). The apoptotic rate, the level of reactive oxygen species (ROS), mitochondrial transmembrane potential and the expression of cytochrome c protein were detected by flow cytometry (FCM). The expression of Bcl-2 and Bax proteins were examined by Western blot. Caspase 3 activity was determined with a microplate reader. Our results were as follows: the GI50 values for SGC-7901 cells were 36.51±1.05 μmol/L (24 h) and 25.37±1.19 μmol/L (48 h). After 24 h of exposure to juglone (5, 10, 15 and 20 μmol/L), the cells presented the typical morphological changes of apoptosis, and the rate of apoptosis was found to increase in a dose-dependent manner. After cells were treated with juglone at the same dose for 24 h, the level of ROS was significantly higher, the expression of Bcl-2 was significantly down-regulated and the expression of Bax was significantly up-regulated compared to the control. The mitochondrial transmembrane potential was significantly lower, and the expression of the cytochrome c protein was significantly higher relative to the control. Caspase 3 was activated in a concentration-dependent manner. In conclusion, juglone can induce apoptosis in SGC-7901 cells through a mitochondrial pathway that seems to be mediated by the generation of ROS and a reduction in the Bcl-2/Bax ratio.

Introduction

Gastric cancer is one of the most common malignancies in the world. The highest incidence rates occur in Eastern Asia (China, Japan, Republic of Korea, Democratic Republic of Korea and Mongolia), where the rates are 46 per 100,000 males and 21 per 100,000 females (Ferlay et al., 2004). Although the surgical treatment of gastric cancer is currently the main therapy, chemotherapy still plays an important role in comprehensive therapy. The therapeutic effect of chemotherapy drugs is limited, however, due to their adverse reactions and the resistance of tumor cells to chemotherapeutic agents. In the last few decades, natural products have become an increasingly important source of potential anticancer agents (Gatti and Perego, 2009; Johnson et al., 2008).

Juglone (5-hydroxy-1,4-naphtha-quinone) is a naturally-occurring naphthoquinone found in the roots, leaves, nut-hulls, bark and wood of Manchurian walnut (Juglands mandshurica), black walnut (Junglans nigra), walnut (Junglans regia) and butternut (Junglans cinerea) (Funt and Martin, 1993) trees. The bark, branches and exocarp of the immature green fruit of these medicinal plants have been used to treat gastric cancer, liver cancer, lung cancer and other types of cancer for a long time in China (Liu et al., 2004). Juglone has been reported to inhibit intestinal carcinogenesis induced by azoxymethane in rats and might be a promising chemopreventive agent in human intestinal neoplasia (Sugie et al., 1998). Juglone was also proven to be a potent cytotoxic agent in vitro in human tumor cell lines, including human colon carcinoma (HCT-15) cells, human leukemia (HL-60) cells and doxorubicin-resistant human leukemia (HL-60R) cells (Kamei et al., 1998; Segura-Aguilar et al., 1992). Our previous results suggested that juglone can inhibit the growth and induce apoptosis of sarcoma 180 cells in vivo. We observed the typical morphological changes that occur during apoptosis of sarcoma 180 cells by transmission electron microscopy (TEM) and detected the apoptosis rate by flow cytometry (FCM) to be 10.27±1.05% in a group treated with 8 μmol/kg of juglone (Ji et al., 2008b). The results from our in vivo work indicate that the mechanism of juglone's antitumor effect in human tumor cell lines is worth further study. In this work, we studied the growth inhibition and induction of apoptosis by juglone in human gastric cancer SGC-7901 cells and demonstrate that the antitumor effect is associated with the generation of reactive oxygen species (ROS), expression of Bcl-2 and Bax protein, mitochondrial depolarization, release of cytochrome c into the cytosol and activation of the caspase-3 cascade.

Section snippets

Cell lines and cell culture

Human gastric cancer SGC-7901 cells were purchased from the Institute for Cancer Research, Heilongjiang Cancer Hospital. SGC-7901 cells of a suitable concentration were inoculated in culture flasks containing RPMI-1640 solution with 10% of fetal bovine serum. The cultures were then incubated at 37 °C, with 5% CO2 and saturated humidity; culture transfer was performed once every 2–3 days.

Test drugs and chemical reagents

Juglone with a purity of 97% was purchased from Sigma-Aldrich Corporation; Hydroxycamptothecine Injection

The cytotoxic effect of juglone on SGC-7901 cells

Using the SRB assay, we examine the effect of juglone on the growth of SGC-7901 cells. Juglone was found to significantly inhibit cell growth in a dose-dependent manner. The GI50 value indicates the high sensitivity of this human gastric cancer line to juglone (Table 1). Juglone was found to potently inhibit the growth of SGC-7901 cells in vitro.

Effect of juglone on the ultrastructure of SGC-7901 cells

The results are shown in Fig. 1. In the control group, we observed the morphological characteristics of tumor cells, such as clear cellularity,

Discussion

Quinones are widely distributed in nature. Quinones have many biological functions and are applied as antibacterial agents, fungicides, antimalarials and anticancer drugs. The biological effects of quinones are largely mediated by the formation of reactive oxygen species through redox activation and the covalent modification of free thiols to form thioethers (Bolton et al., 2000). Juglone is 5-hydroxy-1,4-naphthoquinone and is a strong cytotoxic agent. Its cytotoxicity is based on its high

Conclusion

Juglone can potently inhibit the growth and induce apoptosis of SGC-7901 cells. The mechanism is mediated by the activation of the mitochondrial death pathway, which requires the generation of ROS, down-regulation of Bcl-2 protein expression and up-regulation of Bax protein expression.

Competing interests

The authors declare that they have no competing interests.

Acknowledgements

This work was supported by National Natural Science Foundation of China (no. 306008161) and the Research Fund for the Doctoral Program of Higher Education of China (no. 20060240001).

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