Drp1-mediated mitochondrial fission contributes to baicalein-induced apoptosis and autophagy in lung cancer via activation of AMPK signaling pathway

Abstract

Baicalein (BA), a natural compound extracted from Scutellaria baicalensis Georgi, has been reported to exert antitumor effect in various cancers. However, the underlying mechanisms have not been well demonstrated. In the present study, we focused on the relationship between mitochondrial fission and BA-induced apoptosis and autophagy. We showed that BA inhibited cell viability and induced mitochondrial apoptosis in A549 and H1299 lung cancer cells. BA induced the loss of mitochondrial membrane potential (MMP) and the release of cytochrome c and apoptosis inducing factor (Aif) from mitochondria to cytoplasm. Meanwhile, BA induced autophagy and activated autophagic flux. Furthermore, we found that BA induced mitochondrial fission and mitochondrial impairment. Blocking mitochondrial fission by mdivi-1 attenuated BA-induced apoptosis and autophagy. Moreover, BA activated AMP-activated protein kinase (AMPK) pathway. Knockdown of AMPK with lentivirus encoded AMPKα also attenuated BA-induced mitochondrial fission, apoptosis and autophagy. Our in vivo data confirmed that BA inhibited tumor growth and induced apoptosis and autophagy in a Lewis lung carcinoma (LLC) xenograft model via activation of AMPK/mitochondrial fission pathway. Our study highlights the critical role of AMPK/mitochondrial fission pathway in the regulation of BA-induced apoptosis and autophagy. These results revealed the molecular mechanism of the anti-lung cancer property of BA and provided novel perspectives for the application of BA in the treatment of lung cancer.

Keywords: AMPK; apoptosis; autophagy; baicalein; mitochondrial fission.

Figure 1

BA inhibited viability and induced apoptosis in A549 and H1299 cells. (a) Chemical structure of BA. (b) A549 and H1299 cells were treated with BA at concentrations of 0 ~ 400 μM. WST-1 assay was performed to examine cell viability. (c) A549 and H1299 cells were treated with BA at concentrations of 80, 120, and 160 μM. Apoptosis analyses were performed by staining with Annexin V-FITC and PI and detected using flow cytometry. (d) The ratio of apoptosis was analyzed using FlowJo. (e) Nuclear condensation and fragmentation were performed using DAPI staining and detected by fluorescent microscopy (scale bar, 100 μm). (f) Positive cell ratio was analyzed using ImageJ. (g) The activity of caspase 3 was determined with using a caspase 3 detection kit. Data were from at least three independent experiments. *p<0.05, **p<0.01, ***p<0.001

Figure 2

BA activated mitochondrial apoptotic pathway in A549 and H1299 cells. (a) Mitochondrial membrane potentials (MMP) collapse in A549 and H1299 cells was monitored by TMRE staining and detected using flow cytometry. (b) MMP collapse was analyzed using FlowJo. (c) Intracellular ROS levels were determined using DCFH-DA staining and detected using flow cytometry. (d) ROS levels were analyzed using FlowJo. (e) The cleavage of PARP, caspase 3 and caspase 9 and protein expression levels of Bcl2 family members (Bcl2, Bclxl, Bax, Bak) were analyzed by western blotting. (f) Cytochrome c and Aif released from the mitochondria (Mito) to the cytoplasm (Cyto) were analyzed by western blotting. Cox IV was used as a loading control for mitochondrial gradient; β-actin was used as a loading control for cytosolic gradient. Data were from at least three independent experiments. *p<0.05, **p<0.01, ***p<0.001

Figure 3

BA induced Drp1-mediated mitochondrial fission and mitochondrial dysfunction in A549 and H1299 cells. (a) Cells were pretreated with mdivi-1 (15 μM) for 3 h, followed by treatment with BA (120 μM) for 48 h. Mitochondrial morphology was performed by MitoTracker red staining and detected by fluorescent microscopy (scale bar, 10 μm). (b) The expression levels of mitochondrial fission and fusion (Opa1, Mfn1) related proteins were determined by western blotting. (c) ATP production was determined using an ATP assay kit. (d) The expression levels of mitochondrial respiratory chain complexes were determined by western blotting. (e) Mitochondrial mass was determined by MitoTracker green staining and detected by flow cytometry. (f) Mean fluorescence intensity was analyzed using FlowJo. Data were from at least three independent experiments. **p<0.01, ***p<0.001

Figure 4

BA induced autophagy and autophagic flux in A549 and H1299 cells. (a) A549 and H1299 cells were pretreated with 3-MA (5 mM) for 3 h, followed by treatment with BA (120 μM) for 48 h. LC3 puncta was detected by staining with LC3 antibody and nucleus was detected by staining with DAPI through immunofluorescence using fluorescence microscopy (scale bar, 10 μm). (b) The expression levels of autophagy related proteins were determined by western blotting. (c) A549 and H1299 cells were pretreated with 3-MA (5 mM) for 3 h, followed by treatment with BA (120 μM) for 48 h. The expression levels of autophagy related proteins were determined by western blotting. (d) A549 and H1299 cells were pretreated with Baf-A1 (10 nM) for 3 h, followed by treatment with BA (120 μM) for 48 h. The expression levels of autophagy related proteins were determined by western blotting.

Figure 5

Mdivi-1 weakened BA-induced apoptosis and autophagy. A549 and H1299 cells were pretreated with mdivi-1 (15 μM) for 3 h, followed by treatment with BA (120 μM) for 48 h. (a) Cell viability was measured by WST-1 assay. (b) Apoptosis was performed by staining with Annexin V-FITC and PI and detected using flow cytometry. (c) Apoptosis ratio was analyzed using FlowJo. (d) The expression levels of mitochondrial apoptotic pathway related proteins were determined by western blotting. (e) LC3 puncta was detected by immunofluorescence using fluorescence microscopy (scale bar, 10 μm). (f) The expression levels of autophagy related proteins were determined by western blotting. Data were from at least three independent experiments. *p<0.05, ***p<0.001

Figure 6

BA induced Drp1-mediated mitochondrial fission via activation of AMPK pathway. (a) The expression levels of AMPKα and p-AMPKα (Thr172) were determined by western blotting. (b) A549 and H1299 cells were transfected by AMPKα lentivirus and control lentivirus and treated with or without BA (120 μM) for 48 h. The expression levels of AMPKα and p-AMPKα (Thr172) were determined by western blotting. (c) The expression levels of Drp1 and p-Drp1 (Ser616) were determined by western blotting. (d) Mitochondria morphology was determined MitoTracker red staining and detected by fluorescent microscopy (scale bar, 10 μm).

Figure 7

BA induced apoptosis and autophagy via activation of AMPK pathway. (a) Apoptosis analyses were performed by staining with Annexin V-APC and 7-AAD and detected using flow cytometry. (b) Apoptosis ratio was analyzed using FlowJo. (c) Cell viability was measured by WST-1 assay. (d) The expression levels of mitochondrial apoptotic pathway related proteins were determined by western blotting. (g) LC3 puncta was detected by immunofluorescence using fluorescence microscopy (scale bar, 10 μm). (f) The expression levels of autophagy related proteins were determined by western blotting. Data were from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001

Figure 8

BA inhibited tumor growth in vivo by apoptosis and autophagy via activation of the AMPK/mitochondrial fission pathway. (a) Tumor volumes in LLC xenograft mice were measured every two days in the control and BA (50 mg/kg and 100 mg/kg) groups (n = 10). (b) Gross images of tumors. (c) Weight of tumors (n = 6). (d) Changes of mice weight in each group over time (n = 10). The expression levels of mitochondrial apoptotic pathway related proteins (e), autophagy related proteins (f), and AMPK pathway related proteins (g) were determined by western blotting. (h) The morphology of mouse liver, heart and tumor was determined by H&E stanning (scale bar, 50 μm). (i) Apoptosis ratio was determined by TUNEL assay (scale bar, 50 μm). (j) The immunoreactivity of p-AMPKα, p-Drp1, and Drp1 were detected by IHC (scale bar, 50 μm). (k) A proposed model for BA-induced apoptosis and autophagy through AMPK-Drp1-mitochondrial fission pathway. *p<0.05, ***p<0.001