Polyphenols Isolated from Allium cepa L. Induces Apoptosis by Induction of p53 and Suppression of Bcl-2 through Inhibiting PI3K/Akt Signaling Pathway in AGS Human Cancer Cells.

Background: The extract of Allium cepa Linn is commonly used as adjuvant food for cancer therapy. We assumed that it includes a potential source of anti-cancer properties. Methods: We investigated anti-cancer effects of polyphenols extracted from lyophilized A. cepa Linn (PEAL) in AGS human cancer cells. Results: PEAL inhibited cell growth in a dose-dependent manner. It was related to caspase-dependent apoptosis. We confirmed this finding with annexin V staining. PEAL up-regulated p53 expression, and subsequent Bax induction, down regulated Bcl-2 protein, anti-apoptotic protein. In addition, PEAL suppressed Akt activity and PEAL-induced apoptosis were significantly accentuated with Akt inhibitor (LY294002). Conclusions: Our data suggested that PEAL induce caspase-dependent apoptosis through mitochondrial pathway by up-regulating p53 protein, and subsequent Bax protein as well as by modulating Bcl-2 protein, and that PEAL induces caspase-dependent apoptosis at least in part through the inhibition of phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. This study provides evidence that PEAL might be useful for the treatment of cancer.


INTRODUCTION
Allium cepa Linn (onion, A. cepa), a member of the family Liliaceae, has been used as a supplementary folk remedy for cancer treatment. A. cepa has plentiful flavonoids and organosulfur compounds, 1 which have exhibited anticancer properties. A French epidemiological study showed that high intake of A. cepa was correlated with lower risk of cancer. 2 A. cepa extract has been demonstrated to have inhibitory effects on carcinogenesis. In addition, A. cepa and its constituents have anti-cancer potential. 3 However, the molecular mechanisms of the anti-cancer effects of polyphenols extracted from lyophilized A. cepa Linn (PEAL) are poorly understood in human cancer cells.
As advances in medical science, our lifespan has been extended. The elderly cancer patients and their cancerrelated mortality are expected to increase because the elderly have a high risk for cancer development. Therefore, it is essential to find out less toxic agents in controlling cancer for this population. To meet the demands for finding out less toxic agents, dietary agents has arisen as one of the candidates for the possibility of controlling cancer with minimal toxicity because these dietary agents are known to have or enhance anti-inflammatory and/or anti-cancer activity without showing any toxicities. 4 Moreover, many studies suggested that the most dominant mechanism of anti-cancer effects of the dietary agents be apoptosis triggered by modulating numerous molecular targets.
Apoptosis is a type I programmed cell death requiring active-energy consuming process. It features cytoplasmic shrinkage, blebbing of the plasma membrane, chromatin condensation, and DNA degradation. 5 Apoptosis usually occurs through intrinsic pathway, which is mediated by mitochondria.
In this study, we investigated the mechanisms of anticancer effects of PEAL on human AGS cells.

Cell viability
For the cell viability assay, the cells were seeded onto 24-well plates at a concentration of

Statistical analysis
Data represent means±standard deviations. Statistical significance was determined using the one-way analysis of variance (ANOVA) with post-test Neuman-Keuls for more than two groups and Student's t test for two groups. P＜ 0.05 was accepted as statistically significant.

PEAL induces apoptosis in human AGS cancer cells
To investigate the anti-cancer activity of PEAL, AGS cells were treated with various concentrations of PEAL for 48 h.
The cell growth was assessed by MTT assay. The MTT assay revealed that the growth of AGS cells were inhibited by PEAL treatment in a dose-dependent manner, and the 50% inhibition of cell growth (IC50) was less than 50 μg/mL Finally we also measured the early apoptotic cells (Annexin V ＋ /PI − ) by flow cytometry. The early apoptotic cells were increased in a dose-dependent manner (Fig. 1D).

PEAL-induced apoptosis is caspase-dependent in human AGS cancer cells
We then assessed the effects of PEAL at a concentration of 50 μg/mL on caspases and their substrates (PARP). PEAL decreased the expression levels of procaspase-3 in a timedependent manner. With the decrease of procaspases, the cleavages of PARP were increased in a time-dependent manner ( Fig. 2A). This finding suggests that PEAL induce caspase-dependent apoptosis.  (Fig. 2D).

PEAL down-regulates
These findings suggest that activation of p53 protein might be the important mechanisms of PEAL-induced apoptosis in AGS cells.

PEAL induces apoptosis at least in part by inhibiting
Akt activity PI3K/Akt pathway plays an important role in regulating apoptosis and cell death. In addition, the expression of anti-apoptotic Bcl-2 protein is regulated by Akt. Hence, we investigated the effects of PEAL on Akt in AGS cells.
Since the activity of Akt is regulated by phosphorylation, augmented the effect of PEAL on cell viability and apoptosis ( Fig. 3B-D). We also confirmed the findings by measuring mitochondrial membrane potential (MMP, ΔΨm).
Western blot revealed that the addition of LY294002 to PEAL significantly augmented Bax Induction with near complete inhibition of Bcl-2. PEAL also induced ERK phosphorylation (Fig. 3A), but an ERK inhibitor augmented PEAL-induced cytotoxicity in AGS cells (Fig. 3H), indicating ERK activation was associated with survival pathway to escape PEAL-induced apoptosis. Taken together, these findings suggest that PEAL induce apoptosis at least in part by inhibiting phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway.

DISCUSSION
This study was designed to determine whether PEAL has anti-cancer properties in human cancer cells, and to further investigate the underlying mechanisms of the down-regulated Bcl-2 expression which plays a crucial role in apoptosis (Fig. 4B). 7 The major component of PEAL was quercetin. 8,9 It has been reported that quercetin induces caspase-dependent apoptosis through down-regulation of Bcl-2, 10 and mitochondrial-mediated apoptosis pathway, 11 which is consistent with our results. In addition, quercetin enhances anti-cancer activity by up-regulating p53 protein. 12 Quercetin-induced apoptosis was associated with Akt pathway. 10 These findings support our findings, and we confirmed with an Akt inhibitor (LY294002).
However, in a certain cell line, PEAL-induced apoptosis was associated with anti-apoptotic factor c-FLIP, Bcl-xL, and cIAP-1 rather than Bcl-2. 13  which are easily absorbed from the intestine. 17 PEAL also contains isorhamnetin glucosides, which also have the same merit in the bioavailability. Isorhamnetin is a phytochemical present in many plants and is also a metabolite of quercetin. isorhamnetin is reported to have anti-cancer effects. 18,19 In summary, this study demonstrated that PEAL induced caspase-dependent apoptosis in AGS human gastric cancer cells. The apoptosis was triggered through mitochondrial pathway by up-regulating p53, and subsequent Bax induction as well as by modulating Bcl-2 protein (Fig. 4). In addition, PEAL induced caspase-dependent apoptosis at least in part through the inhibition of phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway indicating that PI3K/Akt is the upstream signaling that regulate the apoptotic effect of PEAL. This study provides evidence that PEAL might have anticancer property on human gastric cancer cells.

CONFLICT OF INTEREST
The authors declare no conflict of interest.