Honokiol combined with curcumin sensitizes multidrug-resistant human lung adenocarcinoma A549/DDP cells to cisplatin

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.

Keywords: A549/DDP cells; P-glycoprotein; curcumin; honokiol; resistance.

Figure 1

Effect of HNK, CUR and DDP on the A549 and A549/DDP cells viability. The viabilities of A549 and A549/DDP cells treated with (A) HNK, (B) CUR and (C) DDP were assessed by MTS assay. The results were expressed as the mean ± standard deviation of three independent experiments and each was performed in triplicate. ^*P<0.05 and ^**P<0.01 vs. A549 cells. HNK, honokiol; CUR, curcumin; DDP, cisplatin; OD, optical density.

Figure 2

Effects of HNK or/and CUR on activity and proliferation of A549/DDP cells. The activity of (A) A549 and (B) A549/DDP cells administered HNK, CUR, DDP, HNK + CUR, HNK + DDP, CUR + DDP and HNK + CUR + DDP were examined by MTS assay. ^*P<0.05 and ^**P<0.01 vs. Control group, ^#P<0.05 vs. HNK + DDP group, ^$P<0.05 vs. CUR + DDP group. (C) The proliferation of A549 and A549/DDP cells administered with HNK, CUR, DDP, HNK + CUR, HNK + DDP, CUR + DDP and HNK + CUR + DDP were examined by colony formation assay. Quantification of (D) A549 and (E) A549/DDP cell colony formation. Magnification, x40. The results were expressed as the mean ± standard deviation of three independent experiments and each was performed in triplicate. ^*P<0.05 and ^**P<0.01 vs. control group, ^#P<0.05 vs. HNK + DDP group, ^$P<0.05 vs. CUR + DDP group. HNK, honokiol; CUR, curcumin; DDP, cisplatin; OD, optical density.

Figure 3

Effects of HNK or/and CUR on apoptosis of A549/DDP cells by flow cytometry. (A) A549/DDP cells treated with HNK, CUR, DDP, HNK + CUR, HNK + DDP, CUR + DDP and HNK + CUR + DDP for 48 h were harvested for flow cytometry analysis. (B) Annexin V/PI-stained cells were analyzed with the percentages of apoptosis cells. ^**P<0.01 vs. control group, ^##P<0.01 vs. HNK + DDP group or CUR + DDP group. HNK, honokiol; CUR, curcumin; DDP, cisplatin.

Figure 4

Effects of HNK or/and CUR on migration of A549/DDP cells by wound healing assay. (A) The migration abilities of A549/DDP cells treated with HNK, CUR, DDP, HNK + CUR, HNK + DDP, CUR + DDP and HNK + CUR + DDP for 24 or 48 h were examined by wound healing assay. Magnification, x200. (B) Wound healing percentage are shown in chart. ^*P<0.05 and ^**P<0.01 vs. control group, ^#P<0.05 vs. HNK + DDP group or CUR + DDP group. HNK, honokiol; CUR, curcumin; DDP, cisplatin.

Figure 5

Effects of HNK or/and CUR on invasion of A549/DDP cells by Transwell assay. (A) The invasion abilities of A549/DDP cells treated with HNK, CUR, DDP, HNK + CUR, HNK + DDP, CUR + DDP and HNK + CUR + DDP for 48 h were evaluated Transwell invasion assay and (B) analyzed. Magnification, x200. The results were expressed as the mean ± standard deviation of three independent experiments and each was performed in triplicate. ^**P<0.01 vs. control group, ^#P<0.05 vs. HNK + DDP group or CUR + DDP group. HNK, honokiol; CUR, curcumin; DDP, cisplatin.

Figure 6

Effects of HNK or/and CUR on P-gp protein nuclear volume of A549/DDP cells by immunofluorescence assay. (A) The P-gp protein nuclear volume of A549/DDP cells treated with HNK, CUR, DDP, HNK + CUR, HNK + DDP, CUR + DDP and HNK + CUR + DDP for 48 h were determined by immunofluorescence assay and (B) analyzed. Magnification, x200. ^**P<0.01 vs. control group, ^#P<0.05 vs. HNK + DDP group or CUR + DDP group. HNK, honokiol; CUR, curcumin; P-gp, P-glycoprotein; DDP, cisplatin.

Figure 7

Effects of HNK or/and CUR on the related mRNA expressions of AKT/Erk signal pathway by RT-qPCR. The mRNA expression of (A) P-gp, (B) P21, (C) MMP-2, (D) MMP-9, (E) Akt, (F) Erk1/2, (G) caspase-3, (H) caspase-9, (I) and PARP in A549/DDP cells treated with HNK, CUR, DDP, HNK + CUR, HNK + DDP, CUR + DDP and HNK + CUR + DDP for 48 h were evaluated by RT-qPCR assay. The results were expressed as the mean ± standard deviation of three independent experiments and each was performed in triplicate. ^**P<0.01 vs. control group, ^#P<0.05 vs. HNK + DDP group or CUR + DDP group. HNK, honokiol; CUR, curcumin; RT-qPCR, reverse transcription-quantitative PCR; P-gp, P-glycoprotein; MMP, matrix metalloproteinase; PARP, poly ADP-ribose polymerase; DDP, cisplatin; p, phosphorylated.

Figure 8

Effects of HNK or/and CUR on the related protein expression of the AKT/Erk signal pathway by western blot assay. (A) The protein expression of P-gp and (B) analysis. (C) The protein expression of P21, MMP-2 and MMP-9 and (D) analysis of in A549/DDP cells treated with HNK, CUR, DDP, HNK + CUR, HNK + DDP, CUR + DDP and HNK + CUR + DDP for 48 h were evaluated by western blot assay. (E) Protein levels of p-AKT, AKT, p-Erk1/2, Erk1/2, caspase-3, cleaved caspase-3, caspase-9, cleaved caspase-9, PARP and cleaved PARP and (F) densitometry analysis. The band intensity was quantified by ImageJ software. ^**P<0.01 vs. control group, ^#P<0.05 vs. HNK + DDP group or CUR + DDP group. HNK, honokiol; CUR, curcumin; MMP, matrix metalloproteinase; DDP, cisplatin.