Betulinaldehyde exhibits effective anti-tumor effects in A549 cells by regulating intracellular autophagy

Abstract

It is of great significance to find new effective drugs for an adjuvant therapy targeting lung cancer to improve the survival rate and prognosis of patients with the disease. Previous studies have confirmed that certain Chinese herbal extracts have clear anti-tumor effects, and in our preliminary study, betulinaldehyde was screened for its potential anti-tumor effects. The current study thus aimed to confirm the anti-tumor effect of betulinaldehyde, using in vitro experiments to explore its underlying molecular mechanism. It was found that betulinaldehyde treatment significantly inhibited the viability, proliferation, and migration of A549 cells in a dose-dependent manner. In addition, betulinaldehyde inhibited the activation of Akt, MAPK, and STAT3 signaling pathways in A549 cells in a time-dependent manner. More importantly, betulinaldehyde also decreased the expression level of SQSTM1 protein, increased the expression level of LC3 II, and increased the autophagy flux in A549 cells. The pretreatment of A549 cells with the autophagy inhibitor, 3-methyladenine, could partially negate the anti-tumor effects of betulinaldehyde. These findings suggest that betulinaldehyde could significantly inhibit the oncological activity of A549 cells by regulating the intracellular autophagy level, making it a potentially effective option for the adjuvant therapy used to treat lung cancer in the future.

Figure 1

Betulinaldehyde inhibited the viability and colony formation of A549 cells. (A) The chemical structure of betulinaldehyde. (B) A549 cells were treated with different concentrations of betulinaldehyde for 24 h and the cell viability was detected via CCK8. (C,D) The colony formation ability was detected via crystal violet staining. All values are expressed as the mean ± SD (n = 3). Statistical significance was evaluated by one-way analysis of variance followed by LSD for post hoc test. **P < 0.01 versus 0 μM group.

Figure 2

The effect of betulinaldehyde on the intracellular signaling pathways of A549 cells. (A) A549 cells were treated with 20-μM betulinaldehyde at different time points before the cell extracts were analyzed using the western blotting technique. The statistical analyses of (B) p-Akt, (C) p-ERK1/2, and (D) p-STAT3 are included. Cropped images are displayed, uncropped blots are displayed in Supplementary Fig. 1. The images were collected from different parts of the same gel with same loading amounts. The blot gels shown are representatives of three different experiments (n = 3). All values are expressed as the mean ± SD (n = 3). Statistical significance was evaluated by one-way analysis of variance followed by LSD for post hoc test. *P < 0.05, **P < 0.01 versus 5 min group.

Figure 3

The effect of betulinaldehyde on the SQSTM1 and LC3-II levels of A549 cells. (A) A549 cells were treated with different concentrations of betulinaldehyde for 24 h before the cell extracts were analyzed using the western blotting technique. The statistical analyses of the (B) SQSTM1 levels and (C) LC3-II levels are included. Cropped images are displayed, uncropped blots are displayed in Supplementary Fig. 2. The images were collected from different parts of the same gel with same loading amounts. The blot gels shown are representatives of three different experiments (n = 3). All values are expressed as the mean ± SD (n = 3). Statistical significance was evaluated by one-way analysis of variance followed by LSD for post hoc test. *P < 0.05, **P < 0.01 versus 0 μM group.

Figure 4

Betulinaldehyde promoted the autophagy flux in A549 cells. (A) A549 cells were treated with different concentrations of betulinaldehyde for 24 h before the autophagy flux was analyzed via a confocal laser scanning microscope using mCherry-GFP-LC3B. (B) 3-MA was also used to determine the effects of 20-μM betulinaldehyde on the autophagy flux in A549 cells. (C,D) Quantification of LC3B positive autolysomomes or autophagosomes in A549/mCherry-GFP-LC3B cells. (E) Expressions of SQSTM1 and LC3-II were tested at the protein level by western blotting. The statistical analyses of the (F) SQSTM1 levels and LC3-II levels are included. Cropped images are displayed, uncropped blots are displayed in Supplementary Fig. 3. The images were collected from different parts of the same gel with same loading amounts. The blot gels shown are representatives of three different experiments (n = 3). All values are expressed as the mean ± SD (n = 3). Comparisons among multiple groups were performed using one-way analysis of variance. The differences between control and other groups were analyzed using LSD multiple comparison test. *P < 0.05, **P < 0.01 versus control group, ^#P < 0.05, ^##P < 0.01 versus Bet group (Bet = betulinaldehyde, 3-MA = 3-methyladenine). Scale bar, 20 μm.

Figure 5

Autophagy mediated the effect of betulinaldehyde on viability and proliferation of A549 cells. (A) A549 cells were treated with 20-μM betulinaldehyde for 24 h with or without 3-MA before the cell viability was determined via CCK8. (B) The cell proliferation was measured via EdU staining. (C) The statistical analyses of the proliferation ability. (D,E) Colony formation of the A549 cells analyzed via crystal violet staining. All values are expressed as the mean ± SD (n = 3). Comparisons among multiple groups were performed using one-way analysis of variance. The differences between control and other groups were analyzed using LSD multiple comparison test. **P < 0.01 versus control group, #P < 0.05, ##P < 0.01 versus Bet group (Bet = betulinaldehyde, 3-MA = 3-methyladenine). Scale bar, 100 μm.

Figure 6

Autophagy mediated the effect of betulinaldehyde on migration of A549 cells. (A) A549 cells were treated with betulinaldehyde (20 μM) for 24 h, with or without 3-MA, and the intercellular spacing was observed at different time points. (B) The statistical analyses of the migration ability. All values are expressed as the mean ± SD (n = 3). Statistical significance was evaluated by one-way analysis of variance followed by LSD for post hoc test. *P < 0.05, **P < 0.01 versus control group, ^#P < 0.05, ^##P < 0.01 versus Bet group (Bet = betulinaldehyde, 3-MA = 3-methyladenine). Scale bar, 100 μm.

Figure 7

Betulinaldehyde inhibited the growth of A549 tumor xenografts in nude mice in vivo. After adaptive feeding for one week, A549 cells were implanted into nude mice via subcutaneous injection under the axilla, and the mice were treated with different concentrations of betulinaldehyde when the tumor grew to a suitable size. (A) The xenograft tumors were separated when the animals were killed. (B) The tumor growth was measured every 4 days. Results were presented as mean ± SD (n = 7). (C) The total body weight was monitored for 21 days. (D–F) The expressions of SQSTM1 and LC3-II were tested at the protein level and the data were quantitated to be represented as mean ± SD (n = 3). Cropped images are displayed, uncropped blots are displayed in Supplementary Fig. 4. The images were collected from different parts of the same gel with same loading amounts. The blot gels shown are representatives of three different experiments (n = 3). Comparisons among multiple groups were performed using one-way analysis of variance. The differences between control and treatment groups were analyzed using LSD multiple comparison test. *P < 0.05, **P < 0.01 versus control group, ^#P < 0.05, ^##P < 0.01 versus Bet group (Bet-L = betulinaldehyde [50 mg/kg]; Bet-M = betulinaldehyde [100 mg/kg]; Bet-H = betulinaldehyde [200 mg/kg].

Figure 8

The effect of betulinaldehyde on the expression of proliferation and apoptosis markers in tumor tissues. (A) Representative IHC images of VEGF and Ki67 of the tumors. (B,C) Quantification analysis of immunoreactive cells for VEGF and Ki67. Data are presented as mean ± SD (n = 4). (D) TUNEL analysis image at 3 weeks after transplantation. (E) Quantified TUNEL positive apoptotic cells count. All values are expressed as the mean ± SD (n = 4). Comparisons among multiple groups were performed using one-way analysis of variance. The differences between control and treatment groups were analyzed using LSD multiple comparison test. *P < 0.05, **P < 0.01 versus control group, ^#P < 0.05, ^##P < 0.01 versus Bet group (Bet-L = betulinaldehyde [50 mg/kg]; Bet-M = betulinaldehyde [100 mg/kg]; Bet-H = betulinaldehyde [200 mg/kg]. Scale bar, 50 μm.