BOS-93, a novel bromophenol derivative, induces apoptosis and autophagy in human A549 lung cancer cells via PI3K/Akt/mTOR and MAPK signaling pathway

Abstract

The novel bromophenol derivative, 3-(3-bromo-5-methoxy-4-(3-(piperidin-1-yl)propoxy)benzylidene)-N-(4-bromophenyl)-2-oxoindoline-5-sulfonamide (BOS-93), was synthesized in the CAS Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences (Qingdao, China). Experimental studies have demonstrated that it could induce apoptosis and autophagy in human A549 lung cancer cells, and it could also inhibit tumor growth in human A549 lung cancer xenograft models. In the present study, the molecular pathways underlying these effects were identified. The results demonstrated that BOS-93 could inhibit cell proliferation in A549 cells and block A549 cells at the G0/G1 phase. Furthermore, BOS-93 could induce apoptosis, activate caspase-3 and poly ADP ribose polymerase, and increase the B cell lymphoma (Bcl)-2 associated X protein/Bcl-2 ratio. Notably, BOS-93 could also induce autophagy in A549 cells. BOS-93-induced autophagy was confirmed by detecting light chain 3 (LC3)-I/LC3-II conversion and increasing expression of beclin1 and autophagy-related gene 14. Notably, BOS-93-induced autophagy could be inhibited by the autophagy inhibitor 3-MA. Flow cytometry, transmission electron microscopy (TEM) and western blot analysis indicated that BOS-93 induced apoptosis and autophagy activities by deactivating phosphoinositide 3-kinase/protein kinase B/mechanistic target of rapamycin and activating the mitogen-activated protein kinase signaling pathway. The present findings indicated that BOS-93 might be a novel anti-cancer agent for treatment of human lung cancer.

Keywords: apoptosis; autophagy; bromophenol; phosphoinositide 3-kinase/protein kinase B/mechanistic target of rapamycin signaling; reactive oxygen species.

Figure 1.

(A) Structure of BOS-93. (B) BOS-93 inhibits cell proliferation of A549 cells, 95D cells and NCI-H460 cells in a dose-dependent manner. Cell viability was evaluated by MTT assay. (C and D) BOS-93 inhibits colony formation of A549 cells. Data are expressed as mean + standard deviation (n=3). **P<0.01 vs. control group. BOS-93, 3-(3-bromo-5-methoxy-4-(3-(piperidin-1-yl)propoxy)benzylidene)-N-(4-bromophenyl)-2-oxoindoline-5-sulfonamide.

Figure 2.

BOS-93 induces G0/G1 arrest. (A and B) A549 cells were treated with BOS-93 for 48 h and then harvested for cell cycle analysis by flow cytometry. (C) A549 cells were treated with BOS-93 for 48 h and then cell cycle-associated proteins, including cyclin D1 and CDK4 were analyzed using western blotting. Data are expressed as mean + standard deviation (n=3). *P<0.05, **P<0.01 vs. control group. BOS-93, 3-(3-bromo-5-methoxy-4-(3-(piperidin-1-yl)propoxy)benzylidene)-N-(4-bromophenyl)-2-oxoindoline-5-sulfonamide; CDK, cyclin-dependent kinase.

Figure 3.

BOS-93 induces cell apoptosis. (A and B) A549 cells were treated with BOS-93 for 48 h, and then cells were stained with Annexin V/PI and analyzed by flow cytometry. (C) A549 cells were treated with BOS-93 for 48 h, and then cells were stained with Hoechst 33258 and photographed using fluorescence microscopy (scale bar, 50 µm). (D) A549 cells were treated with BOS-93 for 48 h and then apoptosis-associated proteins, including cleaved-PARP, Bcl-2, Bax and cleaved-caspase-3 were analyzed using western blotting. Data are expressed as mean + standard deviation (n=3). **P<0.01 vs. control group. BOS-93, 3-(3-bromo-5-methoxy-4-(3-(piperidin-1-yl)propoxy)benzylidene)-N-(4-bromophenyl)-2-oxoindoline-5-sulfonamide; PI, propidium iodide; PARP, poly ADP ribose polymerase; Bcl-2, B cell lymphoma-2; Bax, Bcl-2-associated X protein; CL, cleaved.

Figure 4.

BOS-93 induces autophagy in A549 cells. (A) Transmission electron microscopy examination of morphology changes of A549 cells with or without BOS-93 (10 µg/ml) for 48 h. (B) A549 cells were infected with recombinant adenovirus expressing green fluorescent protein-LC3 and then cells were treated with 10 µg/ml BOS-93 for 48 h. (C) Western blot analysis of LC3 in A549 cells following incubation with or without BOS-93. (D) A549 cells were pretreated for 1 h in the presence or absence of 3-MA (5 mM) prior to addition of BOS-93. Western blot analysis of LC3 was performed following addition of 10 µg/ml BOS-93 for 48 h. BOS-93, 3-(3-bromo-5-methoxy-4-(3-(piperidin-1-yl)propoxy)benzylidene)-N-(4-bromophenyl)-2-oxoindoline-5-sulfonamide; LC3, light chain 3; DMSO, dimethyl sulfoxide.

Figure 5.

(A) Effects of BOS-93 on the levels of beclin-1 and Atg14 proteins. A549 cells were treated with BOS-93 for 48 h and then autophagy-related proteins, including beclin-1 and Atg14 were analyzed using western blotting. (B) A549 cells were pretreated for 1 h in the presence or absence of 3-MA (5 mM) prior to addition of BOS-93. Western blot analysis of beclin-1 and Atg14 was performed following addition of 10 µg/ml BOS-93 for 48 h. (C) A549 cells were treated with BOS-93 (0, 2.5, 5 or 10 µg/ml) for 48 h. The expressions of PI3K, Akt, mTOR, p-PI3K, p-Akt and p-mTOR were assessed by western blot analysis. GAPDH was used to normalize protein content. All data were representative of three independent experiments. BOS-93, 3-(3-bromo-5-methoxy-4-(3-(piperidin-1-yl)propoxy)benzylidene)-N-(4-bromophenyl)-2-oxoindoline-5-sulfonamide; Atg14, autophagy-related gene 14; PI3K, phosphoinositide 3-kinase; Akt, protein kinase B; mTOR, mechanistic target of rapamycin; p, phosphorylated.

Figure 6.

BOS-93 induces ROS generation. (A and B) A549 cells were treated with various concentrations of BOS-93 for 48 h and then cells were stained with 2′,7′-dichlorofluorescein diacetate and analysis using flow cytometry. (C) A549 cells were treated with BOS-93 for 48 h and then the expressions of p38, ERK, p-p38 and p-ERK were analyzed using western blotting. Data are expressed as mean + standard deviation (n=3). **P<0.01 vs. control group. BOS-93, 3-(3-bromo-5-methoxy-4-(3-(piperidin-1-yl)propoxy)benzylidene)-N-(4-bromophenyl)-2-oxoindoline-5-sulfonamide; ROS, reactive oxygen species; ERK, extracellular signal-regulated kinase; p, phosphorylated.

Figure 7.

Effect of BOS-93 on tumor growth in A549-xenografted athymic mice. (A) BOS-93 was dissolved in 0.5% carboxymethyl cellulose-Na and administrated intraperitoneally once per day, tumor volume was recorded every 3 days until animals were sacrificed at day 21 (tumor images were taken on day 21). (B) The mean weights of treated and control mice were recorded every 3 days. (C) Immunohistochemistry for Ki 67 and LC3B were performed on paraffin-embedded tumor sections. (D) Organs from treated and control groups were stained with hematoxylin and eosin to evaluate the toxicity of BOS-93. Images were captured under ×100 magnification. BOS-93, 3-(3-bromo-5-methoxy-4-(3-(piperidin-1-yl)propoxy)benzylidene)-N-(4-bromophenyl)-2-oxoindoline-5-sulfonamide; LC3, light chain 3.

Figure 8.

The proposed molecular mechanisms of apoptosis and autophagy induced by BOS-93 in A549 cells. BOS-93, 3-(3-bromo-5-methoxy-4-(3-(piperidin-1-yl)propoxy)benzylidene)-N-(4-bromophenyl)-2-oxoindoline-5-sulfonamide; CDK, cyclin-dependent kinase; ROS, reactive oxygen species; ERK, extracellular signal-regulated kinase; PARP, poly ADP ribose polymerase; PI3K, phosphoinositide 3-kinase; Akt, protein kinase B; mTOR, mechanistic target of rapamycin; LC3, light chain 3.