Abstract
The aim of the present study was to discuss the effects and underlying mechanisms of honokiol (HNK) and/or curcumin (CUR) in sensitization of multidrug-resistant human lung adenocarcinoma A549/DDP cells to cisplatin (DDP). An MTS assay was performed to detect the cytotoxicity of HNK, CUR and DDP in A549 and A549/DDP cells and compare their sensitivity. The A549/DDP cells were then divided into 8 groups: Control, HNK, CUR, DDP, HNK + CUR, HNK + DDP, CUR + DDP and HNK + CUR + DDP. Cell proliferation was measured by MTS assay and colony formation assay, cell apoptosis was detected by flow cytometry, cell invasion was evaluated by Transwell assay and cell migration was determined by a wound healing assay. In order to investigate the possible mechanisms, P-glycoprotein (P-gp) protein expression was measured by western blotting and immunofluorescence assays. The mRNA expression levels of AKT, Erk1/2, cyclin-dependent kinase inhibitor 1 (P21), caspase 3, cleaved caspase 3, caspase 9, cleaved caspase 9, poly (ADP-ribose) polymerase (PARP), cleaved PARP, matrix metalloproteinase (MMP)-2 and MMP-9 were examined by reverse transcription-quantitative (RT-q) PCR assay, and the protein expression levels of phosphorylated (p)-AKT, p-Erk1/2, P21, caspase 3, cleaved caspase 3, caspase 9, cleaved caspase 9, PARP, cleaved PARP, MMP-2 and MMP-9 proteins expression by western blot assay. The MTS assay demonstrated that HNK (5 µg/ml), CUR (10 µg/ml) and DDP (5 µg/ml) had no obvious toxicity to A549/DDP cells, and HNK, CUR and DDP were more sensitive in A549 cells compared with A549/DDP cells. The optimal concentrations of HNK (5 µg/ml), CUR (10 µg/ml) and DDP (5 µg/ml) were chosen to carry out the further experiments. Compared with the control group, no significant change was observed in cell proliferation, apoptosis, migration, invasion and related mRNA and protein expression in HNK, CUR, DDP and HNK + CUR groups. The cell proliferation rate in the HNK + DDP and CUR + DDP groups was significantly suppressed with cell apoptosis significantly increased, respectively. The invasion cell number and wound healing rate of HNK + DDP and CUR + DDP groups were significantly depressed compared with the control group, respectively. Immunofluorescence demonstrated that the nuclear volume of P-gp in HNK + DDP and CUR + DDP groups were significantly downregulated compared with the control group, respectively. The RT-qPCR assay demonstrated that the AKT, Erk1/2 and P21 mRNA expression levels were significantly decreased and cleaved caspase 3, cleaved caspase 9 and cleaved PARP were increased in HNK + DDP and CUR + DDP groups compared with the control group. The western blotting results were consistent with the RT-qPCR results. NK + CUR + DDP had improved effects on A549/DDP compared with HNK + DDP or CUR + DDP group, respectively. HNK and/or CUR could improve the sensitivity of DDP to A549/DDP cell by the regulation of P-gp, inducing apoptosis, and inhibiting migration and invasion via AKT/ERK signal pathway in an in vitro study.