Downregulation of AKT and MDM2, Melatonin Induces Apoptosis in AGS and MGC803 Cells
ABSTRACT
Melatonin, a neurohormone secreted by the pineal gland, has a variety of biological functions, such as circadian rhythms regulation, anti-oxidative activity, immunomodulatory effects, and anittumor, etc. At present, its antitumor effect has attracted people's attention due to its extensive tissue distribution, good tissue compatibility, and low toxic and side effects. In the gastrointestinal tract, there is high level of melatonin and many studies showed melatonin has effects of anti-gastric cancer. In this experiment, human gastric cancer cell lines AGS and MGC803 were used to investigate the intracellular molecular mechanism of melatonin against gastric cancer. After AGS and MGC803 have been treated with melatonin, the changes of cell morphology and cellular structure were observed under electron microscope. Flow cytometer and apoptosis detection kits were used to analyze the effect of apoptosis on AGS and MGC803. The alterations of apoptosis-related proteins Caspase 9, Caspase 3, and upstream regulators AKT, MDM2 including expression, phosphorylation, and activation were detected to analyze the intracellular molecular mechanism of melatonin inhibiting gastric cancer. In AGS and MGC803 cells with melatonin exposure, cleaved Caspase 9 was upregulated and Caspase 3 was activated; moreover, MDM2 and AKT expression and phosphorylation were downregulated. Melatonin promoted apoptosis of AGS and MGC803 cells by the downregulation of AKT and MDM2. Anat Rec, 302:1544–1551, 2019. © 2019 American Association for Anatomy
Gastric cancer (GC) was the third highest cause for Years of Life Losts (YLLs) of crude cancer since 2005 to 2015. In 2015, there were approximately 1.3 million incident cases of gastric cancer and about 819,000 deaths worldwide (Fitzmaurice et al., 2017). In China, the incidence of male GC is second only to lung cancer and that of female GC ranks third after breast cancer and lung cancer (Chen et al., 2016).
Melatonin, also known as N-acetyl-5-methoxy tryptamine, is a circadian rhythm monitoring hormone that is produced by the pineal gland. In addition to the circadian rhythm control, functions of melatonin also include the inhibition of sexually mature, immunomodulatory, antioxidant, anti-inflammatory, oncostatic activities, and so on (Karaaslan and Suzen, 2015; Talib, 2018). Presently, the anticancer effect of melatonin is more concerned by researchers. Melatonin could inhibit various carcinomas by inhibiting cell growth, promoting cellular apoptosis, scavenging free radicals, regulating anticancer immunity, and so on. Due to its competitively inhibiting estrogen, many researches concerned with melatonin against breast cancer. However, Melatonin is more closely related to the gastrointestinal tract because the concentration of melatonin in that is 10–100 times higher than in serum (Bubenik, 2008). Studies have shown that melatonin can enhance the immune system of the gastrointestinal tract, regulate fecal water, slow intestinal peristalsis, and protect the gastrointestinal tract from digestive enzymes, stomach acid, and drugs (Bubenik, 2002; Kolli et al., 2013). Our previous experimentation indicated melatonin could inhibit murine GC by downregulation Treg cells and accelerating the apoptosis of murine fore-gastric carcinoma cells (Liu et al., 2011). Proietti et al.’s (2011) breast cancer investigation exposed melatonin and D3 caused a reduction in Akt phosphorylation and MDM2 values leading to cell growth inhibition. Our research group used Cancer Signaling Phospho-Antibody Array and also found MDM2 modifications in the human GC (hGC) cell line SGC-803 treated with melatonin. In order to further verify the effect of melatonin on hGC cells and its molecular mechanism, this study selected hGC cell lines AGS and MGC803 to analyze the effect of melatonin on cell apoptosis and changes in apoptosis-related protein MDM2 and its upstream regulatory protein AKT to elucidate its intracellular molecular mechanism.
MATERIAL AND METHODS
Major Reagents and Cell Lines Culture
Cell lines of hGC AGS and MGC803 were purchased from Shanghai Institute for Biological Science, Chinese Academy of Sciences. AGS cells were cultured in Gibco DMEM/F12 (Thermo Fisher Scientific, Waltham, MA) and MGC803 in Roswell Park Memorial Institute (GE Healthcare Life Sciences,) medium supplemented both with 10% fetal bovine serum (GE Healthcare Life Sciences, Logan, UT). Melatonin was purchased from Sigma-Aldrich (St. Louis, MO). CellTiter 96® AQueous One Solution Cell Proliferation Assay and DeadEnd™ Fluorometric TUNEL (TdT-mediated dUTP Nick-End Labeling) System were from Promega (Madison, WI); FITC Annexin V Apoptosis Detection Kit I was from BD Pharmingen (Franklin Lakes, NJ); Antibodies: anti-MDM2 (ab137413), anti-phospho-MDM2 (at Ser166, ab170880), and goat anti-rabbit IgG (ab98505) were purchased from Abcam (Cambridge, UK); anti-AKT (4685S), anti-phospho-AKT (at Thr308, 4056S), anti-caspase-9 (9508S), and anti-GAPDH (2118S) were purchased from Cell Signaling Technology; and goat anti-mouse IgG (sc-2008) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Cell Observation
Melatonin was dissolved in ethanol prior to use. When melatonin was added into the cultures, the final concentration of ethanol in the culture medium never exceeded 1%. Based on previous studies, we selected 3 mmol/L melatonin and 24 hr of exposure for the follow-up experiments that these parameters reflect approximately a 50% inhibition of cell viability. AGS, MGC803 cells were seeded in 6-well plates at a density of 1 × 104 cells/mL. After 24 hr of culture, the cells were treated with 3 mmol/L melatonin for 24 hr and one group cells were added 1% ethanol as control. Cell morphology was observed with an electron microscope (EM208, FEI, Hillsboro, Oregon) after the melatonin treated.
Analysis of Apoptosis
We use the TUNEL assay to analyze cell apoptosis in situ. This assay measures nuclear DNA fragmentation of apoptotic cells tagged with fluorescein-12-dUTP, which can be visualized by fluorescence microscopy. AGS, MGC803 cells were cultured on coverslips and treated with either 3 mmol/L melatonin or 1% ethanol for 24 h. After three washes with PBS, the cells were fixed in 4% paraformaldehyde and incubated at 4°C for 25 min. Fixed cells were permeabilized with 0.2% Triton X-100 solution for 5 min. The cells were then incubated with a nucleotide mixture and rTdT buffer solution at 37°C for 60 min to allow the tailing reaction to occur in the dark. After terminating the reaction in 2X SSC, the cells were counterstained with PI for 15 min at room temperature. Positive apoptotic cells were identified by fluorescence microscopy (Observe. A1, Carl Zeiss Microscopy GmbH, Deutschland).
To analyze the rate of cellular apoptosis, we used the FITC Annexin V Apoptosis Detection Kit I following the manufacturer's instructions. Briefly, AGS, MGC803 cells were treated with 3 mmol/L melatonin for 24 hr and collected. In cells that have undergone apoptosis, phosphatidylserine, which is usually located in the inner leaflet of the plasma membrane, is translocated to the outer leaflet of the plasma membrane. Once on the outer surface of the membrane, PS is bound by FITC-labeled Annexin V and detected by flow cytometry.
Western Blotting
Melatonin-treated AGS, MGC803 cells, and control cells were lysed in cell lysis buffer (Beyotime Institute of Biotechnology, China) supplemented with a protease inhibitor cocktail and phosphatase inhibitors (Roche, Switzerland). Protein concentrations were measured using the Enhanced BCA Protein Assay kit (Beyotime Institute of Biotechnology). Protein extracts (40 μg) were subjected to 12% SDS-PAGE. The proteins in the gels were transferred to polyvinylidene difluoride membranes, which were then blocked in Tris-buffered saline containing 0.5% bovine serum albumin. Blocked membranes were incubated with primary antibodies: anti-MDM2 (1:1000 dilution), anti-phospho-MDM2 (at Ser166, 1:50000 dilution), anti-AKT (1:1000 dilution), anti-phospho-AKT (at Thr308, 1:1000 dilution), anti-caspase-9 (1:1000 dilution), and anti-GAPDH (1:1000 dilution). Proteins were detected by the addition of alkaline phosphatase-conjugated secondary antibody, goat anti-rabbit IgG (1:5000 dilution) or goat anti-mouse IgG (1:1000 dilution). Target proteins were visualized by the addition of CDP-Star reagents (Roche Diagnostics, Germany). The bands were detected using an ImageQuant LAS 4000 mini (GE Healthcare). Band intensities were quantified using ImageJ2x software (National Institutes of Health, Bethesda, Maryland) and the relative intensities to the internal GAPDH control were calculated.
Analysis of Caspase-3 Activity
AGS, MGC803 cells were seeded at a density of 1 × 104 cells/mL in 96-well plates. After 24 hr of culture, the cells were treated with 0 (1% ethanol as control) or 3 mmol/L melatonin for 24 hr. Melatonin- and vehicle-treated AGS, MGC803 cells were added 100 μl Caspase-Glo® 3/7 Reagent according to the manufacturer's instructions. This assay provides a luminogenic caspase-3/7 substrate that is released following caspase cleavage, and the subsequent production of light can be detected by a microplate luminometer (Orion Microplate Luminometer, Germany).
Data Analysis
The data represent the mean ± SD from at least three independent experiments. One-way ANOVA and the Student's paired t-test were used to determine statistical significance. P-value <0.05 was considered to indicate a statistically significant result. All analyses were performed using SPSS software (version 17.0).
RESULTS
The Observation under Electron Microscope
We use electron microscope for observing cell morphology of AGS and MGC803 after treatment with 3 mmol/L melatonin for 24 hr. Under the electron microscope, mitochondria of some cells exhibited vacuolation, and apoptotic bodies were appearance in the group of melatonin-treated cells (Fig. 1).
Melatonin Inducing Cellular Apoptosis in AGS and MGC803 Cells
TUNEL assay were used to assess apoptotic AGS, MGC803 cells in situ. As shown in Figure 2, apoptosis could be observed in the AGS and MGC803 cells treated with melatonin but not in the control cells under five random microscope views.
FITC Annexin Analysis Cellular Apoptosis
Then we used the FITC Annexin V Apoptosis Detection Kit I to determine the percentage of apoptotic cells using by flow cytometry. The percentages of melatonin-treated cells in the early stages of apoptosis were found to increase compared with the controls (Fig. 3). In the AGS, early apoptotic cells increased from 2.113 ± 0.241 to 9.460 ± 1.976 (P < 0.05); in the MGC803, early apoptotic cells increased from 0.577 ± 0.038 to 5.400 ± 0.748 (P < 0.01). All differences were statistically significant.
Molecular Mechanisms Underlying Melatonin's Effects
We checked changes in apoptosis-associated proteins caspase9 and caspase3 by Western blotting and CellTiter 96® AQueous One Solution Cell Proliferation Assay, respectively. The analysis demonstrated evidence of an increased level of cleaved caspase-9 (Fig. 4) and a significantly activated caspase3 (Table 1 and Fig. 5) in AGS, MGC803 cells after melatonin exposure compared with controls. To further understand the molecular mechanism underlying melatonin-induced cell apoptosis, we evaluated the expression and phosphorylation levels of upstream regulators MDM2 and AKT. Western blot analysis showed that both the expression levels of MDM2, AKT, and phosphorylation levels of phospho-MDM2 (at Ser166), phospho-AKT (at Thr308) decreased compared with the controls in AGS, MGC803 cells (Fig. 4).
)
| Cell line | Control | MLT | P |
|---|---|---|---|
| AGS | 7130.165 ± 308.029 | 18767.528 ± 608.932 | 0.005** |
| MGC803 | 4208.640 ± 191.267 | 17611.630 ± 1022.403 | 0.001** |
- ** P < 0.01 vs. blank control.
DISCUSSION
Melatonin is increasingly concerned on its oncostatic effects and is used in the auxiliary treatment of various tumors because of its good histocompatibility and low side effects. Melatonin exerts anticancer effects by inhibiting proliferation, promoting cell apoptosis, eliminating free radicals, regulating antitumor immunity, competitively inhibiting estrogen, and so on. However, Melatonin act on almost every tissue of human body and its function is diverse, and as soluble small molecules, it can play a role not only binding with membrane receptors, but also permeating directly through the cytoplasmic membrane, binding with a variety of receptors in the cytoplasm or nucleus. Therefore, the mechanism of melatonin oncostatic activity is relatively complex and still unclear. Our previous studies found that melatonin exhibited effective anticancer effects that were mediated by the stimulation of apoptosis, inhibition of cell growth, and reduction in the number of CD4+CD25+ regulatory T cells in mouse GC cells and in vivo (Liu et al., 2011; Xu et al., 2014). We also found that the nuclear receptor RORγ is involved in the effects of melatonin on hGC cells (Wang et al., 2016). A recent study demonstrated that melatonin induces AGS cell apoptosis via the activation of JNK and p38, and the suppression of NF-κB (Li et al., 2015). To further testify the mechanism of melatonin oncostatic activity, we examined the effect of melatonin on cell apoptosis and analyzed the apoptosis-associated proteins caspase9, caspase3 and upstream regulators MDM2 (murine double minute 2, also HDM2) and AKT in hGC cell lines AGS and MGC803. Once apoptosis initiates, the caspase family activates and phosphorylates downstream caspase, leading to target protein phosphorylation. Among them, caspase9 is the initial activated caspase in the mitochondrial apoptotic pathway, and caspase3 is executor which activates and phosphorylates the target protein and initiates apoptosis. The upregulation of cleaved caspase9 and activated caspase3 indicated that melatonin induced apoptosis via a mitochondrial apoptosis pathway.
MDM2was originally identified as an amplified oncogene on double-minute chromosomes in transformed mouse fibroblasts (Fakharzadeh et al., 1991). Numerous studies indicated that MDM2 was overexpressed in many human cancers, GC, Bladder, ovarian cancer, and so on (Ginath et al., 2001; Ohmiya et al., 2006; Onat et al., 2006; Nakajima et al., 2009; He et al., 2015). MDM2 is a negative regulator of the p53 tumor suppressor and also have p53-independent effects. MDM2 could influence the activities of other transcription factors besides of p53, such as p73 (Dobbelstein et al., 1999; Zeng et al., 1999; Gu et al., 2001; Watson et al., 2006), p65 (Cheney et al., 2008), Smad proteins (Sun et al., 1998; Yam et al., 1999), cyclin D1, c-Jun, c-Myc, and pRb (E2F1) (Xiao et al., 1995; Sdek et al., 2004). Moreover, MDM2 has been shown to influence chromatin modifications by interacting directly with chromatin (Thut et al., 1997; Minsky and Oren, 2004; Zhou et al., 2011). The phosphorylated form of MDM2 at Ser166 and at Ser186 is a substrate for AKT made it transfer into nuclear to act function as transcriptor. AKT need activation by its own phosphorylation. When AKT binds to PIP3 on the membrane, it's Ser473 and Thr308 residues are phosphorylated and activated. AKT and PIP3 is downstream signal molecule of G-protein coupled receptors which are the same type of membrane receptors of melatonin. In our study, melatonin exposure downregulate the expression and phosphorylation of AKT and MDM2, it is inferred that melatonin promoted apoptosis of human gastric cancer cells AGS, MGC803 by attenuating AKT activity, which leads to the inactivation of MDM2 (Fig. 6). However, the relationship between AKT, MDM2, and membrane receptors of melatonin should be confirmed by the more experiments. The conclusion partly confirms our precious study on melatonin against SGC-7901, another human gastric cancer cell line (Song et al., 2018). But, the change of p53 was inconsistent in these three cell lines, which inferred that there were other molecules involved in the downregulation of MDM2 besides of p53. We also need further experiments including a PDX (patient-derived tumor xenograft) model to find the downstream molecular network and confirm the conclusion in the future.
