Aloe-emodin-induced apoptosis in human gastric carcinoma cells

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Abstract​

The purpose of this study was to investigate the anticancer effect of aloe-emodin, an anthraquinonecompound present in the leaves of Aloe vera, on two distinct human gastric carcinoma cell lines, AGS and NCI-N87. We demonstrate that aloe-emodin induced cell death in a dose- and time-dependent manner. Noteworthy is that the AGS cells were generally more sensitive than the NCI-N87 cells. Aloe-emodin caused the release of apoptosis-inducing factor and cytochrome c from mitochondria, followed by the activation of caspase-3, leading to nuclear shrinkage and apoptosis. In addition, exposure to aloe-emodin suppressed the casein kinase II activity in a time-dependent manner and was accompanied by a reduced phosphorylation of Bid, a downstream substrate of casein kinase II and a pro-apoptotic molecule. These preclinical studiessuggest that aloe-emodin represents a suitable and novel chemotherapeutic drug candidate for the treatment of human gastric carcinoma.

 

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Introduction​

Aloe-emodin is a natural active compound present in the leaves of Aloe vera (Reynolds, 1985). Some studies have indicated that aloe-emodin has a number of biological properties, including antiviral, antimicrobial, and hepatoprotective activities (Eshun and He, 2004). Aloe-emodin has also been reported to exhibit an anticancer activity on neuroectodermal tumors, lung squamous cell carcinoma, and hepatoma cells (Pecere et al., 2000, Lee et al., 2001, Kuo et al., 2002). In addition, aloe-emodin has been shown to inhibit S-phase progression in a transformed glia cell line and in a human glioma cell line, sensitize HeLa cells to As2O3 via the generation of reactive oxygen species, and affect the anticancer activity of cisplatin through blocking the activation of extracellular signal-regulated kinase (Acevedo-Duncan et al., 2004, Yi et al., 2004, Mijatovic et al., 2005). However, the effect of aloe-emodin on human gastric cancer cells has not yet been studied.

Apoptosis is an actively regulated process of cell death and the intrinsic pathway of apoptosis involves mitochondria (Penninger and Kroemer, 2003). Mitochondrial outer membrane permeabilization in response to cell death triggers (e.g., DNA damage) is an important early step which is regulated by Bcl-2 and controls the release of proteins, such as cytochrome c, from the mitochondria to the cytoplasm where they initiate apoptosis, ultimately leading to cell death (Kim et al., 2006). Apoptosis-inducing factor, another mitochondrial protein that is released into the cytosol and nucleus, induces chromatin condensation and DNA fragmentation (Susin et al., 1999). Most likely, members of caspase superfamily will be produced and activated that will hasten the cell death process involving the caspase-dependent apoptotic pathway (Kim et al., 2006).

Casein kinase II is a conserved and ubiquitous protein serine/threonine kinase engaged in various functions, including normal and abnormal cell proliferation (Kikkawa et al., 1992). Remarkable, casein kinase II is localized in both the cytoplasm and the nuclear compartment of healthy cells, but is predominantly present in the nuclear compartment of cancer cells and basically deregulated in all carcinoma studied to date (Ahmad et al., 2005). Recent evidence links casein kinase II to apoptosis (Ahmed et al., 2002). A role of casein kinase II in the modulation of caspase susceptibility has been observed with Bid, a proapoptotic member of the Bcl-2 family. It was shown that casein kinase II could phosphorylate Bid, close to the caspase-8 cleavage site, thereby preventing cells from undergoing apoptosis (Desagher et al., 2001). Thus, casein kinase II is an important target for the treatment of cancer as disruption of the casein kinase II activity prevents the phosphorylation of Bid thereby allowing the cleavage of Bid by caspases followed by apoptosis (Ahmad et al., 2005, Unger et al., 2004). Interestingly, an extensive downregulation of casein kinase II by antisense casein kinase II in prostate cancer xenografts eventually led to a complete disappearance of the tumor (Ahmad et al., 2005).

 

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Here, we employed two distinct human gastric carcinoma cell lines, AGS and NCI-N87, representative for the diffuse type and intestinal type of gastric carcinoma, respectively, to evaluate the anticancer effect of aloe-emodin. Observation of nuclei and cell morphologies, and examination of releases of apoptosis-inducing factor and cytochrome c demonstrated the apoptotic activity of aloe-emodin. The role of casein kinase II in aloe-emodin-induced apoptosis was also examined. We report for the first time that the natural compound aloe-emodin can induce apoptosis in human gastric carcinoma cells.

Section snippets​

Aloe-emodin​

Aloe-emodin (1,8-dihydroxy-3-[hydroxymethyl]-anthraquinone, CAS registry Number 481-72-1, EU number 2075717, purity ⩾ 95%) was purchased from Sigma–Aldrich Co. (St. Louis, MO, USA). It was dissolved in 1% dimethylsulfoxide (DMSO) to a concentration of 7.4 mM and stored at −20 °C until used.

Cell culture and treatments​

Human gastric carcinoma cell lines (AGS and NCI-N87) were obtained from the Food Industry Research and Development Institute (Hsinchu, Taiwan). Cells were cultured in RPMI-1640 supplemented with 10%

Aloe-emodin induces cell death in a dose- and time-dependent manner​

The effect of aloe-emodin on the viability of AGS and NCI-N87 gastric carcinoma cells was examined using the XTT viability assay. Exposure of AGS cells to various concentrations of aloe-emodin (i.e., 0.07, 0.11, 0.15, and 0.19 mM) resulted in a dose- and time-dependent decrease in cell viability relative to control cell cultures (Fig. 1a). The IC50 value for 72 h of exposure to aloe-emodin was below 0.07 mM. A similar effect of aloe-emodin was observed after 48 or 72 h incubation using the NCI-N87

 

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Discussion​

In the present study, we demonstrated that aloe-emodin has an anticancer activity on AGS and NCI-N87 gastric carcinoma cells and that the cytotoxic mechanism involves the induction of apoptosis. The latter was characterized by a release of apoptosis-inducing factor and cytochrome c from the mitochondria to the cytosol, activation of caspase 3, and inhibition of the activity of casein kinase II, and thus of downstream phosphorylation of Bid. Using the human lung squamous carcinoma cell line

Conflict of interest statement​

None declared.

Acknowledgments​

This work was supported by the National Science Council, Republic of China (to K.-Y. Lin and S.-H. Chen) and Taipei Medical University (to C.-C. Chang and C.-P. Lin).

 

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activation of caspase 3,