Endoplasmic reticulum stress-mediated autophagy protects against β,β-dimethylacrylshikonin-induced apoptosis in lung adenocarcinoma cells

Abstract

β,β-Dimethylacrylshikonin (DMAS) is an anti-cancer compound extracted from the roots of Lithospermum erythrorhizon. The present study aims to investigate the effects of DMAS on human lung adenocarcinoma cells in vitro and explore the mechanisms of its anti-cancer action. We showed that DMAS markedly inhibited cell viability in a dose- and time-dependent way, and induced apoptosis as well as autophagy in human lung adenocarcinoma cells. Furthermore, we found that DMAS stimulated endoplasmic reticulum stress and mediated autophagy through the PERK-eIF2α-ATF4-CHOP and IRE1-TRAF2-JNK axes of the unfolded protein response in human lung adenocarcinoma cells. We also showed that the autophagy induced by DMAS played a prosurvival role in human lung adenocarcinoma cells and attenuated the apoptotic cascade. Collectively, combined treatment of DMAS and pharmacological autophagy inhibitors could offer an effective therapeutic strategy for lung adenocarcinoma treatment.

Keywords: apoptosis; autophagy; endoplasmic reticulum stress; lung adenocarcinoma; β,β-dimethylacrylshikonin.

Figure 1

β,β‐Dimethylacrylshikonin (DMAS) inhibits cell viability and induces apoptosis in human lung adenocarcinoma cells. A, Chemical structure of DMAS. B, PC‐9 cells were treated with DMAS for 24 and 48 h, and then cell viability was evaluated by MTT assay. C, PC‐9 cells were treated with DMAS (15 μmol/L) for 24 h and then stained with DAPI solution. Morphological and nuclear changes were assessed by fluorescence microscopy. White arrows indicate condensed nuclei. D, After treatment with DMAS for 24 h, PC‐9 cells were stained with Annexin V‐FITC/propidium iodide (PI) and detected by flow cytometry analysis. E, PC‐9 cells were treated with DMAS for 24 h. Expression levels of indicated proteins were detected by western blot assay. F, PC‐9 cells were incubated with DMAS for 24 h with or without pretreatment with Z‐VAD‐FMK (10 μmol/L). Expression levels of indicated proteins were studied by western blot assay. All data are expressed as mean ± SD. *P < .05 compared to the control group. PARP, poly ADP ribose polymerase

Figure 2

β,β‐Dimethylacrylshikonin (DMAS) induces autophagy in human lung adenocarcinoma cells. A, PC‐9 and A549 cells were treated with DMAS (15 μmol/L) for 24 h and then stained with MDC. Acidic vesicles were observed under a fluorescence microscope. B, PC‐9 and A549 cells were treated with DMAS (15 μmol/L) for 24 h, then transmission electron microscopy (TEM) was carried out. Black arrows show membrane‐bound vacuoles characteristic of autophagosomes. C, PC‐9 and A549 cells were treated with DMAS (15 μmol/L) for the indicated times. Expression levels of indicated proteins were analyzed by western blot assay. All data are expressed as mean ± SD. *P < .05 compared to the control group. D, PC‐9 and A549 cells were exposed to DMAS for 24 h. Expression levels of indicated proteins were detected by western blot assay. E, PC‐9 and A549 cells were transfected with lentivirus Stub‐RFP‐Sens‐GFP‐LC3 and subsequently treated with DMAS (15 μmol/L) for 24 h. Fluorescent images were obtained by fluorescence microscopy and typical images are presented. White arrows indicate autophagosomes

Figure 3

β,β‐Dimethylacrylshikonin (DMAS) induces autophagy flux in human lung adenocarcinoma cells. A, PC‐9 and A549 cells were transfected with control siRNA or Atg5 siRNA and subsequently exposed to DMAS (15 μmol/L) for 24 h. Expression levels of indicated proteins were examined by western blot assay. B, PC‐9 and A549 cells were pretreated with 3‐methyladenine (3‐MA; 5 mmol/L) for 1 h, and then coincubated with DMAS (15 μmol/L) for another 24 h. Expression levels of indicated proteins were detected by western blot assay. C, PC‐9 and A549 cells were pretreated with Bafilomycin A1 (20 nmol/L) for 1 h, and then cotreated with DMAS (15 μmol/L) for another 24 h. Expression levels of indicated proteins were analyzed by western blot assay. D, PC‐9 and A549 cells were pretreated with chloroquine (CQ; 3 μmol/L) for 1 h, and then coincubated with DMAS (15 μmol/L) for another 24 h. Expression levels of indicated proteins were studied by western blot assay. All data are expressed as mean ± SD. *P < .05 compared to the control group

Figure 4

β,β‐Dimethylacrylshikonin (DMAS) induces endoplasmic reticulum (ER) stress in human lung adenocarcinoma cells. PC‐9 and A549 cells were incubated with DMAS for 24 h. Expression levels of indicated proteins were evaluated by western blot assay. All data are expressed as mean ± SD. *P < .05 compared to the control group

Figure 5

β,β‐Dimethylacrylshikonin (DMAS) induces autophagy by endoplasmic reticulum (ER) stress‐evoked PERK‐eIF2α‐ATF4‐CHOP and IRE1‐TRAF2‐JNK upregulation. A, PC‐9 and A549 cells were transfected with control siRNA or CHOP siRNA for 48 h, and then exposed to DMAS (15 μmol/L) for 24 h. Expression levels of indicated proteins were detected by western blot assay. B, PC‐9 and A549 cells were transfected with control siRNA or ATF4 siRNA for 48 h, and then incubated with DMAS (15 μmol/L) for 24 h. Expression levels of indicated proteins were analyzed by western blot assay. C, PC‐9 and A549 cells were pretreated with SP600125 (50 μmol/L) for 1 h, and then cotreated with DMAS (15 μmol/L) for another 24 h. Expression levels of indicated proteins were examined by western blot assay. D, PC‐9 and A549 cells were pretreated with 4‐phenylbutyrate (4‐PBA; 5 mmol/L) for 30 min, and then cotreated with DMAS (15 μmol/L) for another 24 h. Expression levels of indicated proteins were studied by western blot assay. All data are expressed as mean ± SD. *P < .05 compared to the control group

Figure 6

Autophagy plays a protective role in β,β‐dimethylacrylshikonin (DMAS)‐treated human lung adenocarcinoma cells. (A,B) PC‐9 and A549 cells were transfected with control siRNA or Atg5 siRNA for 48 h, and then treated with DMAS (15 μmol/L) for 24 h. Expression levels of indicated proteins were measured by western blot assay. Apoptotic cells were studied through the Annexin V‐FITC/propidium iodide (PI) staining assay. (C,D) PC‐9 and A549 cells were pretreated with 3‐methyladenine 3‐MA; 5 mmol/L) for 1 h, and then coincubated with DMAS (15 μmol/L) for another 24 h. Expression levels of indicated proteins were evaluated by western blot assay. Apoptotic cells were detected through the Annexin V‐FITC/PI staining assay. (E,F) PC‐9 and A549 cells were pretreated with Bafilomycin A1 (20 nmol/L) for 1 h, and then cotreated with DMAS (15 μmol/L) for another 24 h. Expression levels of indicated proteins were analyzed by western blot assay. Apoptotic cells were detected by Annexin V‐FITC/PI staining assay. (G,H) PC‐9 and A549 cells were pretreated with chloroquine (CQ; 3 μmol/L) for 1 h, and then coincubated with DMAS (15 μmol/L) for another 24 h. Expression levels of indicated proteins were studied by western blot assay. Apoptotic cells were examined by Annexin V‐FITC/PI staining assay. All data are expressed as mean ± SD. *P < .05 compared to the control group. PARP, poly ADP ribose polymerase

Figure 7

Endoplasmic reticulum (ER) stress inhibition confers sensitivity to β,β‐dimethylacrylshikonin (DMAS) in human lung adenocarcinoma cells. A, PC‐9 and A549 cells were transfected with control siRNA or CHOP siRNA for 48 h, and then incubated with DMAS (15 μmol/L) for another 24 h. Expression levels of indicated proteins were studied by western blot assay. B, PC‐9 and A549 cells were transfected with control siRNA or ATF4 siRNA for 48 h, and then exposed to DMAS (15 μmol/L) for 24 h. Expression levels of indicated proteins were measured by western blot analysis. C, PC‐9 and A549 cells were pretreated with SP600125 (50 μmol/L) for 1 h, and then coincubated with DMAS (15 μmol/L) for 24 h. Expression levels of indicated proteins were examined by western blot assay. D, PC‐9 and A549 cells were pretreated with 4‐phenylbutyrate (4‐PBA; 5 mmol/L) for 30 min, and then cotreated with DMAS (15 μmol/L) for 24 h. Expression levels of indicated proteins were detected by western blot assay. All data are expressed as mean ± SD. *P < .05 compared to the control group. ns, no significant difference. PARP, poly ADP ribose polymerase