Nam GH et al. (2019), In vitro and in vivo Induction of p53-Dependent...

ECB-ART-47254

Front Oncol 2019 May 22;9:406. doi: 10.3389/fonc.2019.00406.

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In vitro and in vivo Induction of p53-Dependent Apoptosis by Extract of Euryale ferox Salisb in A549 Human Caucasian Lung Carcinoma Cancer Cells Is Mediated Through Akt Signaling Pathway.

Nam GH , Jo KJ , Park YS , Kawk HW , Kim SY , Kim YM .

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Lung cancer is one of the leading causes of death, and mortality rates have steadily been increasing. Recently, several studies were conducted to develop novel, physiologically active compounds from medicinal plant extracts. Several plant-derived extracts and molecules regulate and inhibit signaling molecules associated with the growth and proliferation of cancer cells. Euryale ferox salisb is a medicinal plant that is effective against different types of cancers. In this study, we investigated the apoptotic effects of E. ferox salisb extract (ESE) in A549 lung cancer cells, exerted by the inhibition of the Akt protein and activation of the p53 protein. Our results show that ESE induces apoptosis via the regulation of mitochondrial outer membrane potential and generation of reactive oxygen species (ROS). We demonstrate that apoptosis is induced in a p53-dependent manner when cells are treated with pifithrin-α (a p53 inhibitor) and LY294002 (an Akt inhibitor). The apoptotic effects from ESE were observed in vivo in Balb/c-nu mice bearing A549 xenografts. Altogether, these results suggest that E. ferox salisb extracts exert anti-cancer effects in a p53-dependent manner.

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Species referenced: Echinodermata
Genes referenced: bax ehfl LOC100893907 LOC105441782 LOC115919910 LOC582192 LOC583082 LOC590297 ros1

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	Figure 1. The identification of active compounds in ESE. (A) HPLC profiles of ESE (total extract) (B) HPLC profiles of standard resveratrol (33.489). (C) HPLC profiles of standard alliin (5.595). (D) HPLC profiles of standard gallic acid (10.583). X-axis represents retention time (min), Y-axis represents absorption units (AU). Detector was set at 240 nm.
	Figure 2. ESE suppresses A549 Human Caucasian lung carcinoma cancer cells proliferation Cell proliferation rate was measured by MTT assay. And LDH assay was performed for assessing cell deaths. Cytotoxicity was induced by ESE. (A) Various cancer cell lines were treated with the indicated concentrations of ESE (150 μg/ml) for 24 h. (B) Various cancer cell lines were treated with the indicated concentrations of ESE (150 μg/ml) for 6–48 h. The most effective anti-cancer effect in 24 h. Anti-cancer effect in 48 h also worked well, but the difference was not significant compared to 24 h. (C) MRC-5 Human lung Fibroblast cells were treated with various concentrations of ESE for 24 h through MTT assay. (D) MRC-5 Human lung Fibroblast cells were treated with various concentrations of ESE for 24 h through LDH assay. (E) A549 Human Caucasian lung carcinoma cancer cell proliferation rate was measured by MTT assay (F) A549 Human Caucasian lung carcinoma cancer cell viability rate was measured by LDH assay. N represents untreated cells and delivery is a control for treatment without ESE. The statistical analysis of the data was carried out by use of ANOVA test. ***P < 0.001 compared to N (untreated groups). N.S; not significant (each experiment, n = 3). The error bars represent the standard error.
	Figure 3. ESE induces apoptosis in A549 Human Caucasian lung carcinoma cancer cells. (A) Cells were treated with the indicated concentrations of ESE for 24 h. Cells stained with Muse™ Annexin V and Dead Cell Assay kit and analyzed by Muse™ Cell Analyzer. Data shows four cell populations—Live, Dead, Late Apoptosis/Dead, Early Apoptosis. Y-axis is labeled for viability [stained by 7-AAD (a dead cell marker)] and X-axis is labeled for Annexin V [binding with phosphatidylserine(PS)]. (B) Cell apoptosis observed using Hoechst 33342 staining and image-based monitoring. Cells were treated with the indicated concentrations of ESE for 24 h. Florescence detected by confocal microscope and Image-based monitoring detected by optical microscope. N represents untreated cells. (C) Nuclear abnormalities rate measured. The number of nuclear counted from the randomly selected fields. N represents untreated cells. The statistical analysis of the data was carried out by use of ANOVA test. ***P < 0.001 compared to N (untreated groups). N.S; not significant (each experiment, n = 3). The error bars represent the standard error.
	Figure 4. Observation of various apoptosis inducers in cancer cells. (A) Cells were treated with the indicated concentrations of ESE (50–150 μg/ml) for 24 h. Cells stained with Muse™ Cell Cycle Kit and analyzed by Muse™ Cell Analyzer. X-axis is labeled for propidium iodide (PI) and Y-axis is numbers of cells. propidium iodide (PI) discriminates cells at different stages of the cell cycle, based on differential DNA content. (B) Representative histograms from Muse™ Cell Analyzer in the A549 Human Caucasian lung carcinoma cancer cells treated with various concentration of ESE. Assays were performed in triplicate. (C) Cells stained with Muse Oxidative Stress Kit and analyzed by Muse™ Cell Analyzer. X-axis is labeled for ROS positive cells. It is based on dihydroethidium (DHE), a well-characterized reagent that has been extensively used to detect ROS. The reagent is cell permeable and it has long been postulated that DHE upon reaction with superoxide anions undergoes oxidation to form the DNA-binding fluorophore ethidium bromide or a structurally similar product which intercalates with DNA resulting in red fluorescence. (D) Cells stained with Muse™ MitoPotential Kit and analyzed by Muse™ Cell Analyzer. This parameter is displayed in the X-axis. A dead cell marker (7-AAD) is also used as an indicator of cell membrane structural integrity and cell death. It is excluded from live, healthy cells, as well as early apoptotic cells. Dead cells thus show increased fluorescence in the Y-axis. (E) Activation of Caspase-3/7 was analyzed by Apo-ONE Homogeneous Caspase 3/7 Assay Kit. Cells were treated with ESE (50–150 μg/ml) for 24 h. N represents untreated cells. The statistical analysis of the data was carried out by use of ANOVA test. P < 0.05, **P < 0.001 compared to N (untreated groups). N.S.; not significant (each experiment, n = 3). The error bars represent the standard error.
	Figure 5. ESE induce apoptosis through Akt signaling pathway suppression and p53-dependent manner. (A) The expression of (cleaved) PARP, (p)Aktser473, (p)MDM2ser166, p53, COX-2, pro-caspase 3, Bcl-2, Bak, Bax, and β-actin were analyzed by Western blot analysis. The β-actin probe served as protein-loading control. The figures show non-adjacent bands from the same blot. (B) The expression of (cleaved) PARP, (p)Aktser473, (p)MDM2ser166, p53, COX-2, pro-caspase 3, Bcl-2, Bak, Bax, and β-actin were analyzed by Western blot analysis. Cells were pre-treated with 20 μM LY294002 or 40 μM Pifithrin-α for 60 min and co-treated with 100 μg/ml ESE 24 h. The β-actin probe served as protein-loading control. The figures show non-adjacent bands from the same blot. (C) Cell proliferation rate was measured by MTT assay. Cells were pre-treated with 20 μM LY294002 or 40 μM Pifithrin-α for 60 min and co-treated with 100 μg/ml ESE 24 h. (D) Activation of Caspase-3/7 was analyzed by Apo-ONE Homogeneous Caspase 3/7 Assay Kit. Cells were pre-treated with 20 μM LY294002 or 40 μM Pifithrin-α for 60 min and co-treated with 100 μg/ml ESE 24 h. N represents untreated cells. The statistical analysis of the data was carried out by use of ANOVA test. P < 0.05, **P < 0.001 compared to N (untreated groups). ###P < 0.001 compared between ‘ESE’ with ‘P+ESE’. N.S.; not significant (each experiment, n = 3). The error bars represent the standard error.

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