Bu-Zhong-Yi-Qi Decoction, the Water Extract of Chinese Traditional Herbal Medicine, Enhances Cisplatin Cytotoxicity in A549/DDP Cells through Induction of Apoptosis and Autophagy

Figure 4

Combination treatment with BZYQD and cisplatin leads to autophagy induction in A549/DDP cells. (a) A549/DDP cells were pretreated with 100, 250, and 500 μg/ml BZYQD for 2 h, and then 40 μg/ml cisplatin was added for another 24 h. After drug cotreatment, cells were stained by indirect immunofluorescence to visualize LC3 puncta (green) by fluorescence microscopy. Cells were then stained with DAPI to visualize the nuclei (blue). (b) The formation of autophagosomes in treated cells was evaluated by TEM. Control cells seemed to be normal in both amount and ultrastructure morphology of mitochondria and lysosomes. Solo-cisplatin treated cells showed increasing numbers, swelling, and vacuolated mitochondria. BZYQD and cisplatin cotreated cells showed increasing the number of autophagosomes and secondary lysosomes, as well as autophagosomes formation. “Black arrow” indicates autophagosomes and “white arrow” indicates secondary lysosomes. (c) LC3-II and ATG7 were examined by western blot. GAPDH was a loading control. (d) A549/DDP cells were pretreated with 10 mM 3-MA for 4 h prior to cotreatment with BZYQD and cisplatin. After incubation, cell growth inhibition was determined by the Cell Counting Kit as described previously. Results are expressed as the mean ± SD of four independent experiments. (ANOVA followed by Bonferroni’s test.) BZYQD: Bu-Zhong-Yi-Qi decoction; LC3: microtubule-associated protein light chain-3; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; 3-MA: 3-methyladenine.

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