Reversal of multidrug resistance by 5,5'-dimethoxylariciresinol-4-O-β-D-glucoside in doxorubicin-resistant human leukemia K562/DOX

Abstract

Objective: The objective of this study was to investigate the reversal effects of 5,5'-dimethoxylariciresinol-4'-O-β-D-glucoside (DMAG) extracted from traditional Chinese medicines Mahonia on multidrug resistance (MDR) of human leukemia cells to chemotherapeutic agents.

Materials and methods: MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed to determine the effect of DMAG on doxorubicin sensitivity to K562/DOX cells. Propidium iodide /Hoechst 33342 double staining assay was used to investigate the effect of DMAG on doxorubicin-induced cellular apoptosis. Intracellular accumulation of doxorubicin and rhodamine 123 assay were performed to evaluate the effect of DMAG on drugs efflux activity of P-glycoprotein.

Results: DMAG significantly enhanced the doxorubicin cytotoxicity to K562/DOX cells. In the presence of 1.0 μM of DMAG, the IC50 of doxorubicin decreased from 34.93 ± 1.37 μM to 12.51 ± 1.28 μM. DMAG of 1.0 μM significantly enhanced doxorubicin-induced cell apoptosis in K562/DOX cells and the enhancement was time-dependent. A significant increase in accumulation of doxorubicin in the presence of DMAG was observed. After treatment of the K562/DOX cells for 1 h with 15.0 μM doxorubicin alone, the fluorescence intensity was 33093.12. With the addition of 1.0 μM of DMAG, the fluorescence intensity of doxorubicin was 2.3-fold higher. A significant increase of accumulation of rhodamine 123 in the presence of DMAG was also observed. With the addition of 1.0 μM of DMAG, the fluorescence intensity was increased by 49.11% compared with rhodamine 123 alone.

Conclusion: DMAG was shown to effectively enhance chemosensitivity of resistant cells, which makes it might be a suitable candidate for potential MDR-reversing agents.

Keywords: 5,5’-dimethoxylariciresinol-4’-O-β-D-glucoside; doxorubicin; leukemia; multidrug resistance.

Figure 1

The effect of 5,5’-dimethoxylariciresinol-4’-O-β-D-glucoside (DMAG) and DOX on K562/DOX cell growth. (a) The effect of different concentrations DMAG on K562/DOX cell growth. (b) The effect of DOX alone, DOX in combination with 1.0 μM of DMAG or DOX in combination with 1.0 μM of verapamil on K562/DOX cell growth. Inhibitory rate of cells and survival rate of cells were assessed with MTT assay and expressed as mean ± standard deviations of three experiments

Figure 2

5,5’-dimethoxylariciresinol-4’-O-β-D-glucoside (DMAG)-mediated cell apoptosis in K562 and K562/DOX cells. (a) K562 cells were treated with 0.15 μM of doxorubicin in the presence or absence of 1.0 μM of DMAG or 1.0 μM of verapamil for 24 h and 48 h, and then mean fluorescence intensity (MFI) of propidium iodide (PI) in K562 cells were detected by Array Scan VTIHCS600 High-Contents. (b) K562/DOX cells were treated with 15.0 μM of doxorubicin in the presence or absence of 1.0 μM of DMAG or 1.0 μM of verapamil for 24 h and 48 h and then MFI of PI in K562/DOX cells were detected by Arrary Scan VTIHCS600 High-Contents. *Represents P < 0.01 compared with DOX group. *Represents P < 0.05 compared with DOX + verapamil group

Figure 3

Apoptosis images of K562 and K562/DOX cells determined by double staining of Heochst 33342 and propidium iodide with Arrary Scan VTIHCS600 High-Contents. A-1~A-4: K562 cells treated with 0.15 μM of doxorubicin in the presence or absence of 1.0 μM of 5,5’-dimethoxylariciresinol-4’-O-β-D-glucoside (DMAG) or 1.0 μM of verapamil for 24 h; A-5~A-8: K562 cells treated with 0.15 μM of doxorubicin in the presence or absence of 1.0 μM of DMAG or 1.0 μM of verapamil for 48 h. B-1~B-4: K562/DOX cells treated with 15.0 μM of doxorubicin in the presence of 1.0 μM of DMAG or 1.0 μM of verapamil for 24 h; B-5~B-8: K562/DOX cells treated with 15.0 μM of doxorubicin in the presence or absence of 1.0 μM of DMAG or 1.0 μM of verapamil for 48 h. A-1, A-5, B-1, B-5: negative control; A-2, A-6, B-2, B-6: DOX treatment group; A-3, A-7, B-3, B-7: DOX in combination with DMAG group; A-4, A-8, B-4, B-8: DOX in combination with verapamil group, ×40

Figure 4

5,5’-dimethoxylariciresinol-4’-O-β-D-glucoside (DMAG)- mediated intracellular accumulation of doxorubicin. The left showed intracellular doxorubicin fluorescence intensity. *Represents significant difference in MFI of doxorubicin between K562 cells and K562/DOX cells untreated and treated with verapamil (P < 0.01). **Represents significant difference in MFI of doxorubicin between K562/DOX cells treated with DMAG and those treated with verapamil (P < 0.01). The right shows VTIHCS600 High-Contents scanning images of intracellular doxorubicin accumulation. (a) K562 cells; (b) K562/DOX cells; (c) K562/ DOX cells treated with 1.0 μM of DMAG; (d) K562/DOX treated with 1.0 μM of verapamil, ×40

Figure 5

5,5’-dimethoxylariciresinol-4’-O-β-D-glucoside (DMAG)-mediated intracellular accumulation of rhodamine 123. Cells were pretreated with or without 1.0 μM of DMAG (verapamil as a positive control) for 1 h, then incubated with 5 mg/L of Hoechst 33342 for 10 min and 5 mg/L of Rh123 for another 1 h in the dark, (a) Fluorescence intensity of rhodamine 123 in the presence or absence of DMAG in K562 cells. (b) Fluorescence intensity of rhodamine 123 in the presence or absence of DMAG in K562/DOX cells.﹡Represents P < 0.05. (c) Image of fluorescence intensity of rhodamine 123. (c-1) K562 cells untreated. (c-2) K562 cells treated with 1.0 μM of 5,5’-dimethoxylariciresinol-4’-O-β-D-glucoside (DMAG). (c-3) K562 cells treated with 1.0 μM of verapamil. (c-4) K562/DOX cells untreated. (c-5) K562/DOX cells treated with 1.0 μM of DMAG. (c-6) K562/ DOX cells treated with 1.0 μM of verapamil.×40