Autophagy inhibition enhances the inhibitory effects of ursolic acid on lung cancer cells

Abstract

The aim of the present study was to identify natural compounds that bear significant anti‑tumor activity. Thus, the effects of 63 small molecules that were isolated from traditional Chinese medicinal herbs on A549 human non‑small cell lung cancer (NSCLC) and MCF‑7 breast cancer cells were examined. It was found that ursolic acid (UA), a natural pentacyclic triterpenoid, exerted significant inhibitory effect on these cells. Further experiments revealed that UA inhibited the proliferation of various lung cancer cells, including the NSCLC cells, H460, H1975, A549, H1299 and H520, the human small cell lung cancer (SCLC) cells, H82 and H446, and murine Lewis lung carcinoma (LLC) cells. UA induced the apoptosis and autophagy of NSCLC cells. The inhibition of the mammalian target of rapamycin (mTOR) signaling pathway, but not the activation of the extracellular signal‑regulated kinase 1/2 (ERK1/2) signaling pathway contributed to the UA‑induced autophagy of NSCLC cells. Moreover, the inhibition of autophagy by chloroquine (CQ) or siRNA for autophagy‑related gene 5 (ATG5) enhanced the UA‑induced inhibition of cell proliferation and promotion of apoptosis, indicating that UA‑induced autophagy is a pro‑survival mechanism in NSCLC cells. On the whole, these findings suggest that combination treatment with autophagy inhibitors may be a novel strategy with which enhance the antitumor activity of UA in lung cancer.

Figure 1

Identification of UA as a potent anticancer compound. (A and B) A549 and MCF-7 cells were treated with 63 natural compounds isolated from traditional Chinese medicinal herbs at 40 µM for 48 h. Cell viability was assessed by MTS assay and presented as relative viability normalized to control. (C) A total of 8 lung cancer cell lines were treated with various concentrations of UA for 48 h, cell viability was evaluated by MTS assay and shown as relative viability in comparison with control. (D) IC₅₀ values of UA for the indicated cell lines are expressed as the means ± SD. UA, ursolic acid.

Figure 2

UA inhibits proliferation and induces apoptosis of H460 and H1975 cells. (A and B) H460 and H1975 cells were treated with the indicated concentrations of UA for the indicated periods of time, and cell viability was determined by MTS assay. (C) Colony formation assay was performed in H460 and H1975 cells with indicated concentrations of UA. Representative images are shown. (D) Quantification of colonies. ^**P<0.01 and ^***P<0.001. (E) H460 and H1975 cells were treated with the indicated concentrations of UA for 48 h, and cell apoptosis was analyzed by flow cytometry using Annexin V-FITC/PI staining. Representative images are shown. (F) Quantification of flow cytometric analysis of apoptosis. ^***P<0.001. (G) H460 and H1975 cells were treated with the indicated concentrations of UA for 48 h, harvested, and subjected to western blot analysis using the indicated antibodies. Actin was used as a loading control. (H) Relative densitometric quantification of protein expression detected in (G). Data are presented as the means ± SD (n=3). ^*P<0.05, ^**P<0.01 and ^***P<0.001. UA, ursolic acid.

Figure 3

UA induces autophagy and increases the autophagic flux in NSCLC cells. (A) H460, H1975 and A549 cells were treated with UA at 10, 15 and 20 µM for 24 h, followed by obtaining images under an Olympus inverted phase-contrast microscope. (B) NSCLC cells were treated with the indicated concentrations of UA for 24 h, and the expression levels of LC3 were analyzed by western blot analysis (left panel). Relative densitometric quantification of LC3-II expression was performed using western blot bands and data are presented as the mean ± SD (n=3; right panel). ^aP<0.01 and ^bP<0.001. (C) H460 and H1975 cells were treated with 15 µM UA for 16 h, cells were then analyzed by immunofluorescence assay using LC3 antibody (green) and DAPI (blue). (D) H460 and H1975 cells were transfected with siNC or siATG5 for 36 h, then treated with UA at 15 µM for 24 and 48 h respectively, lysed, and whole cell lysates were subjected to western blot analysis using the indicated antibodies. Relative densitometric quantification of protein expression and data are presented as the means ± SD (n=3). ^aP<0.001 vs. siNC 0 µM UA, ^bP<0.001 vs. siNC 15 µM UA. (E) H460, H1975 and A549 cells were pre-treated without or with 5 µM CQ for 1 h, followed by UA treatment at 10 and 20 µM for 24 h, and the expression levels of LC3 were examined by western blot analysis. Relative densitometric quantification of the LC3-II expression and data are presented as the means ± SD (n=3). ^aP<0.01 vs. vehicle, ^bP<0.001 vs. vehicle, ^cP<0.001 vs. 5 µM CQ. UA, ursolic acid; CQ, chloroquine.

Figure 4

UA-induced autophagy is attributed to the inhibition of the PI3K/Akt/mTOR signaling pathway. (A) NSCLC cells were treated with the indicated concentrations of UA for 24 h, whole cell lysates were subjected to western blot analysis with the indicated antibodies. Actin was used as a loading control (left panels). Relative densitometric quantification of protein expression was performed using the western blot bands and data are presented as the means ± SD (n=3; right panels). ^aP<0.05 and ^bP<0.001. (B-D) NSCLC cells were pre-treated without or with indicated concentrations of LY294002, 3BDO or U0126 for 1 h, and then incubated with 15 or 20 µM UA for 24 h. Whole cell lysates were obtained and subjected to western blot analysis with the indicated antibodies. Relative densitometric quantification of protein expression was performed and data are presented as the means ± SD (n=3). ^aP<0.001 vs. vehicle, ^bP<0.001 vs. 20 µM UA, ^cP<0.001 vs. 15 µM UA. UA, ursolic acid; LY, LY294002.

Figure 5

Autophagy inhibition by CQ enhances the UA-induced inhibition of the proliferation and promotes the UA-induced apoptosis of NSCLC cells. (A) NSCLC cells were treated with the indicated concentrations of UA without or with 5 µM CQ for 48 h, cell viability was assessed by MTS assay and data are presented as relative viability to the control. ^***P<0.001. (B) H460 and H1975 cells were pre-treated without or with 5 µM CQ for 1 h, followed by treatment with UA at the indicated concentrations for 48 h, cell apoptosis was analyzed by flow cytometry using Annexin V-FITC/PI staining. Representative images are shown. (C) Quantification of flow cytometric analysis of apoptosis. ^***P<0.001. (D) H460 cells were pre-treated without or with 5 µM CQ for 1 h, then incubated with 10 µM UA for 48 h, lysed, and whole cell lysates were subjected to western blot analysis using the indicated antibodies. Actin was used as a loading control (left panel). Relative densitometric quantification of protein expression was performed and data are presented as the means ± SD (n=3; right panel). ^aP<0.001 vs. vehicle, ^bP<0.05 vs. 10 µM UA, ^cP<0.001 vs. 5 µM CQ. (E) H1975 cells were pre-treated without or with 5 µM CQ for 1 h, then incubated with UA at 15 µM for 48 h, whole cell lysates were subjected to western blot analysis with the indicated antibodies (left panel). Relative densitometric quantification of protein expression was performed and data are presented as the means ± SD (n=3; right panel). ^aP<0.001 vs. vehicle, ^bP<0.01 vs. vehicle, ^cP<0.001 vs. 15 µM UA, ^dP<0.001 vs. 5 µM CQ. (F) H1975 cells were pre-treated without or with 10 µM LY294002 for 1 h, followed by UA treatment at 20 µM for 24 h. The cells were harvested and subjected to western blot analysis using the indicated antibodies (left panel). Relative densitometric quantification of protein expression was performed and data are presented as the means ± SD (n=3; right panel). ^aP<0.001 vs. vehicle, ^bP<0.001 vs. 20 µM UA. UA, ursolic acid; CQ, chloroquine; LY, LY294002.

Figure 6

Autophagy inhibition by siRNA for ATG5 potentiates the UA-induced inhibition of the proliferation and promotes the UA-induced apoptosis of NSCLC cells. (A and B) H460 and H1975 cells were transfected with siNC or siATG5 for 36 h, treated with the indicated concentrations of UA for 48 h, and cell viability was determined by MTS assay. ^**P<0.01 and ^***P<0.001. The knockdown efficiency was determined by western blot analysis. Relative densitometric quantification of the ATG5 expression was performed and data are presented as the means ± SD (n=3). (C) H1975 cells were transfected with siNC or siATG5 for 36 h, treated with the indicated concentrations of UA for 48 h, and cell apoptosis was analyzed by flow cytometry using Annexin V-FITC/PI staining. Representative images are shown. (D) Quantification of flow cytometric analysis of apoptosis. ^***P<0.001. (E) H1975 cells were transfected with siNC or siATG5 for 36 h, then treated with 20 µM UA for 24 h. Whole cell lysates were subjected to western blot analysis using indicated antibodies. Actin was used as a loading control. (F) Relative densitometric quantification of protein expression was performed using bands in (E). Data are presented as the mean ± SD (n=3). ^aP<0.001 vs. siNC 0 µM UA, ^bP<0.001 vs. siNC 20 µM UA.