The Extracts from Allium hookeri induces p53-independent Apoptosis through Mitochondrial Intrinsic Pathways in AGS Human Gastric Carcinoma Cells

Copyright: © 2018 Nam GH, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The Extracts from Allium hookeri induces p53-independent Apoptosis through Mitochondrial Intrinsic Pathways in AGS Human Gastric Carcinoma Cells


Introduction
Cancer is one of the biggest causes of death worldwide, and the death rate from cancer continues to increase in the developed countries and other countries [1]. Today, every third person suffers from cancer [2]. Among various cancer types, stomach cancer is known to be the most frequent cancer regardless of gender. Recent advances in cancer diagnosis and treatment have emphasized the importance of early screening to increase the survival rate of stomach cancer patients [3]. If cancer is diagnosed early, it will be treated with gastric mucosectomy or surgery using a laparoscope. However, in the case of metastasis to other parts of the body, the patient will be treated with anticancer chemotherapy without becoming an indication for the operation. As a result, stomach cancer patients may experience post-removal dumping syndrome, nutritional deficiency, and anemia during the treatment process, and anticancer chemotherapy may cause misdiagnosis, vomiting, constipation, diarrhea, hypostosis, and bone marrow functions.
In previous research, it has been pointed out that this causes patients with stomach cancer to experience physical and psychological pain, leading thus to reduced quality of life [4]. For that reason, many studies have recently been conducted to develop new physiologically active substances based on the efficacy of various natural substances that are harmless to humans, as the natural extracts can inhibit and regulate the signaling molecules associated with the growth and proliferation of cancer cells [5]. Among such natural products, Allium hookeri, which has a variety of physiological activities, such as anti-inflammatory property, is a vegetable mainly consumed in the Himalayas where it is used for various inflammation-related and cancer diseases. Now that Allium hookeri is brought Korea, there is a growing interest in its ingredients and efficacy [6,7]. It was reported that the anti-inflammatory effect of Allium hookeri prevents cell growth and mutagenesis and induces apoptosis (death of a cell) in cancer cells [8]. Therefore, seeking for a new anticancer substance using such natural materials, research is currently underway on which natural extracts induce apoptosis and thus have anticancer effects [9]. Apoptosis is a very important process, because it shrinks cells, condenses nuclei, and forms cell membrane bubbles due to the programmed cell death, and if the cells do not die properly, mutations of some cells will be promoted with cancer [10].
Apoptosis's pathways are divided into extrinsic pathways and intrinsic pathways, especially in mitochondria, which play an important role in the intrinsic pathways of apoptosis. The method is influenced by the relative ratios of Bcl-2, an anti-apoptotic protein, and Bax and Bak proteins of pro-apoptotic protein [11]. Akt (serine/threonine kinase), one of the regulating proteins of apoptosis intrinsic pathways, regulates cell proliferation, differentiation, and growth, and inhibits Bax and Bak [12]. The mTOR activated by Akt is involved in tumor formation and is regulated in cell growth and proliferation [13,14]. In addition, mTOR induces ubiquitation of p53 by phosphorylating MDM2 (Murine Double Minute 2) and affects cell growth [15]. The affected p53 adjusts gene expression involved in cell death with a transcription factor [16]. Therefore, caspase cascade is induced under the influence of the apoptosis intrinsic pathway, and apoptosis is induced through the activity of caspase-3 [17]. Therefore, in the present study, we sought to determine the expression of the anticancer activity mechanism in the AGS human gastric cancer cells, and whether or not the suppression of the proliferation of the protein would be caused by intrinsic apoptosis.

Cell culture
AGS Human Gastric Carcinoma Cells were obtained from the American Type Culture Collection (ATCC, USA). The cells were grown in RPMI-1640 medium (Hyclone, USA) containing 10% fetal bovine serum (Hyclone, USA) and 1% antibiotics (100 mg/L streptomycin with 100 U/mL penicillin) in a 5% CO 2 at 37°C. The cells were suspended by Trypsin-EDTA (Hyclone, USA) and separated at 1 × 10 6 cells/mL per plate, every 48-72 hours.

MTT assay
Cells seeded on 96-well micro plates at 4000 cells/well were incubated with the test compounds for indicated time period. Respectively medium was removed and then incubated with 40 μl of MTT solution (2 mg/mL MTT in PBS) for 1 h. MTT is converted to a blue formazan. Absorbance was determined using an auto reader.

Determination of apoptosis by Annexin V staining
The cells were seeded at 1 × 10 6 cells/mL in plate and incubated for 24 h. The cells were treated with the AHE for 24 h in a 5% CO 2 atmosphere at 37°C. The inhibitor was pre-treated for 30-120 min before treating AHE. Total cells were harvested, collected by centrifugation, washed with PBS. Cells were stained with Annexin V for 20 min. apoptosis was analyzed using a Muse TM Cell analyzer (Merck, Germany).

Western blotting
The cells were seeded at 1 × 10 6 cells/mL plate. Then, the cells were treated with the AHE for 24 h in a 5% CO 2 atmosphere at 37°C. The inhibitor was pre-treated for 30-120 min before treating AHE. The cells were scraped with RIPA lysis buffer. The primary antibody was then added following by overnight at 4°C, following the addition of the secondary antibody was added following by 30-120 min.

Statistical analysis
MTT assay was statistically analyzed using t-test (SPSS Inc.; Chicago, USA). A value of P<0.05 was indicated to a statistically significant difference.

Impact of AHE on AGS human gastric cancer cell proliferation
MMT assay after processing AHE (15,35,35,70,105,140, and 170 μg/mL) was applied to AGS human gastric cancer cells to confirm the effect of AHE on AGS and gastric cancer cell growth rate. The results show that, when the AHE was treated for 24 hours, the cell proliferation was suppressed in a dose-dependent manner ( Figure 1A). The results of measuring the cytotoxicity of AHE to fibroblast cells of normal cells confirm that the proliferation rate of fibroblast cells was maintained at least 90% ( Figure 1B). When apoptosis was induced, the cells were broken up, the cells were condensed in chromatin, the bubbles formed in the plasma membrane, and the cells were divided into cells. Lamellipodia and philopodia, which play important roles in cell migration, were also lost and the cells lost adhesion. Therefore, we observed that such morphological changes in apoptosis and cell the upstream regulators of apoptosis intrinsic pathway, but also affected downstream regulators. As shown in Figure 3, the reduction of p-Akt, p-mTOR and Bcl-2, Bcl-XL and pro caspase-3, which are anti-apoptotic proteins involved in cell proliferation, was confirmed. An increase in Bax, Bak, and cleaved-PARP of p53 and pro-apoptotic protein of the cancer suppressor gene was also confirmed. PARP, a protein decomposed by caspase-3, maintains the stability of DNA repair and genes. If a fragment is cut, it will lose its function. Therefore, it is possible to know that apoptosis by caspase activity is induced by the expression of the cleared-PARP [22,23]. In conclusion, we could learn that AHE induces apoptosis through an intrinsic pathway in AGS human gastric cancer cells.

The effects of p53 and Akt and mTOR inhibitors on AGS human gastric cancer cells growth
In order to confirm the relationship between AHE-induced cytotoxicity and apoptotic pathways, after the treatment of LY294002 (Akt inhibitor) or rapamycin (mTOR inhibitor), and the co-treatment of pifitrin-α (p53 inhibitor) by AHE were performed. We found that the AHE treatment group, as well as the LY294002 and the rapamycin treatment group showed a similar decrease as compared to the control. Moreover, the pifitrin-α treatment group did not show a significant difference with the control group. However, if pifitrin-α and AHE were co-treated, Despite the treatment of Pifitrin-α, the number decreased in the same way as in the AHE treatment group (Figure 4). These results mean that AHE induces cytotoxicity and apoptosis through Akt, mTOR dephosphorylation, and p53 independent pathway downregulation.

Confirmation of apoptosis effect and apoptosis intrinsic pathway-related protein expression by p53 and Akt and mTOR inhibitor
In order to investigate the apoptosis effect of AGS human gastric cancer cells through intrinsic pathway, Anexin-V staining analysis was conducted using flow cytometry ( Figure 5). Furthermore, as shown in Figure 6, we confirmed the control relation between the upstream regulators (p-Akt, p-mTOR and p53) and downstream regulators proliferation were inhibited when AHE was treated with AGS human gastric cancer cells. As a result, as shown in Figure 1C, we were able to see that the cells in the control group, which did not process anything, were relatively dense, uniform, and normally attached. It was confirmed that the higher the concentration of AHE, the lower the density of the cells, and the more cells become irregular [18].

Determination of the Application of AGS human gastric cancer cells by AHE
Early apoptosis occurs in the translocation of phosphatidylserine, an early sign of programmed cell death and the cell membrane remains undamaged in the process of translocation [19,20]. To distinguish whether the death of AGS stomach cancer cells under AHE processing is caused by apoptosis or necrosis, we measured the level of intracellular phosphatidylserine (PS) expression, which is considered to be the early stage of apoptosis development using Annexin V ( Figure 2). As shown in Figure 1B, the ratio of Annexin V-positive cells was low in the control (5.9%). However, as the concentration of AHE increased, the percent of annexin V-positive cells increased as well: increasing the concentration of 23.17% (70 μg/mL), 26.20% (105 μg/mL), 31.90% (140 μg/mL). Throughout this process, we observed that the treatment of AHE and the inhibition of the growth of AGS human gastric cancer cells were caused by apoptosis.

Confirmation of apoptosis intrinsic pathway-related protein expression by AHE
Mitochondria play an important role in apoptosis. Intrinsic pathway and Bcl-2 family of proteins also play an important role in maintaining its membrane potential [21]. Bcl-2 and Bcl-XL, which are anti-apoptotic proteins, are moved to maintain the stability of the mitochondria membrane and the pro-apoptotic proteins Bax and Bak move in the mitochondrial membrane to play a role of increasing membrane permeability. The caspase cade-inducing caspase-3 is activated to decompose the PARP protein. Therefore, in the present study, Western blotting was performed in order to confirm the degree of expression of not only Akt, mTOR, and p53 proteins, which are   (Bcl-2, Bax, Bak, and pro caspase-3) by Western blot analysis after cotreatment or single-treatment with LY294002 and rapamycin, Pifitrinα and AHE. Similar to the MTT assay, the Anexin-V/ staining analysis results were verified by apoptosis. When AHE was single-treated, ca. 31.62% apoptosis was induced, When LY294002 was single-treated, ca. 28.02% apoptosis was induced. Furthermore, when rapamycin and pifithrin-α were single-treated, ca. 23.36% and 3.28% of apoptosis was observed, respectively. Even though the p53 was decreased by pifithrin-α, the group that co-treated pifithrin-α and AHE was similar to single-treated AHE. Based on the results of Western blotting, it was confirmed that the expression of p-Akt, p-mTOR and p53 proteins was reduced by the LY294002 and rapamycin and pifithrin-α treatments ( Figure 6). Therefore, the expression of the pro-caspase-3, Bcl-2 as the downstream regulators of the signal pathway was reduced, and the expression of the Bax, Bak, and cleaved PARP increased. When comparing the AHE treatment with apoptosis-related protein expression, we observed that the protein regulation of these apoptosis intrinsic pathway signaling paths that occurred was not different from when the LY294002 and rapamycin processing was performed. Previous studies have reported that the treatment of LY294002 and rapamycin affects the downstream regulators and induces apoptosis [24]. The pifithrin-α treating group decreased the expression of p53. Since the p53 does not affect Bax and Bak proteins whose activity increased, our results confirmed that AHE affects AGS human gastric cancer cells through the p53-independent pathway. As a result, apoptosis induced by AHE processing in the present study was controlled by the intrinsic pathway. This effect was confirmed to occur through the reduction of Akt and mTOR activity and the p53-independent pathway. Based on our results, it can be concluded that active ingredient analysis and various additional experiments should be conducted in the future to confirm the possibility of actual use of AHE as a therapeutic agent for stomach cancer.

Discussion and Conclusion
Stomach cancer is the most common cancer worldwide, including Korea [25]. Therefore, treatment methods that induce apoptosis are urgently needed to treat stomach cancer. In the present study, we investigated the apoptosis effect of extract from Allium hookeri, a plant species that proved to be effective in cancer treatment in the previous literature [26]. Akt signaling pathway is frequently activated in cancers including glioblastoma multiforme (GBM), Ras signaling pathway where this kinase network regulates survival [27,28]. We showed that AHE induces apoptosis by inhibiting Akt signaling pathway. Akt(serine/ threonine kinase) mediates of PI3K-initiated signaling pathway by phosphorylating and inhibiting death-inducing proteins and blocks phosphorylates mTOR, MDM2 [29,30]. Phosphorylated-mTOR has emerged as a major regulator of cell proliferation and protein synthesis [31,32]. Previous studies have shown that natural substances inhibit Akt, mTOR induce apoptosis through the intrinsic pathway [33,34]. Furthermore, Phosphorylated-MDM2 inhibits the transcriptional activity of p53 and more importantly, promotes its apoptosis by intrinsic pathway [30,35,36] we confirmed that AHE can induce apoptosis through its downregulation of Akt and mTOR whereby proteins associated with intrinsic pathway work by p53-independent pathway [37]. First, we demonstrated the cytotoxicity effect of AHE through the MTT assay. As a result, AHE significantly reduced viability of cells in the dose-dependent manner. In addition, we detected apoptotic cells using Annexin V staining and found that treatment of AHE on AGS human gastric cancer cells changes the number of apoptotic cells in the dose-dependent manner. Furthermore, in order to investigate if apoptosis goes through the intrinsic pathway, we used Western blotting to confirm the degree of expression of not only Akt, mTOR, and p53 proteins, which are the upstream regulators of apoptosis, Intrinsic pathway, but also affected downstream regulators [38,39]. Then, we were able to observe that the reduction of p-Akt, p-mTOR and Bcl-2, Bcl-XL and pro caspase-3, which are anti-apoptotic proteins involved in cell proliferation. An increase in Bax, Bak, and cleaved-PARP of p53 and pro-apoptotic protein of the cancer suppressor gene were also confirmed. Additionally, to confirm the effect of Akt and mTOR and p53 in AHE-induced apoptosis, we induced Akt, mTOR and p53 inhibitors. The LY294002 treated group, rapamycin-treated group, and AHE single-treated group were induced in similar number of cell toxicity and apoptosis. Even when co-treated with AHE and pifithrin-α, the groups had reduced apoptosis (pifithrin-α blocks the expression of p53 and reduces apoptosis [40]). It was not inhibited and apoptosis was normally induced. These results mean that AHE induces cytotoxicity and apoptosis through Akt, mTOR dephosphorylation, and p53-independent pathway downregulation.