Tetrandrine Interaction with ABCB1 Reverses Multidrug Resistance in Cancer Cells Through Competition with Anti-Cancer Drugs Followed by Downregulation of ABCB1 Expression

Abstract

The overexpression of ABC transporters induced by anticancer drugs has been found to be the main cause of multidrug resistance. It is actually also a strategy by which cancer cells escape being killed. Tetrandrine is a natural product extracted from the stem of Tinospora crispa. In this study, tetrandrine showed synergistic cytotoxic activity in combinational use with chemotherapeutic drugs, such as Doxorubicin, Vincristine, and Paclitaxel, in both drug-induced and MDR1 gene-transfected cancer cells that over-expressed ABCB1/P-glycoprotein. Tetrandrine stimulated P-glycoprotein ATPase activity, decreased the efflux of [³H]-Paclitaxel and increased the intracellular accumulation of [³H]-Paclitaxel in KB-C2 cells. Furthermore, SW620/Ad300 and KB-C2 cells pretreated with 1 μM tetrandrine for 72 h decreased P-glycoprotein expression without changing its cellular localization. This was demonstrated through Western blotting and immunofluorescence analysis. Interestingly, down-regulation of P-glycoprotein expression was not correlated with gene transcription, as the MDR1 mRNA level exhibited a slight fluctuation in SW620/Ad300 and KB-C2 cells at 0, 24, 48, and 72 h treatment time points. In addition, molecular docking analysis predicted that tetrandrine had inhibitory potential with the ABCB1 transporter. Our results suggested that tetrandrine can antagonize MDR in both drug-selected and MDR1 gene-transfected cancer cells by down regulating the expression of the ABCB1 transporter, followed by increasing the intracellular concentration of chemotherapeutic agents. The combinational therapy using tetrandrine and other anticancer drugs could promote the treatment efficiency of drugs that are substrates of ABCB1.

Keywords: ABC transporter; ABCB1/P-gp; multidrug resistance (MDR); tetrandrine; transgenic cancer cell.

Figure 1

Cytotoxicity of tetrandrine in the parental and resistant cell lines. (A) Chemical structure of tetrandrine. MTT assay on the effect of tetrandrine in cells: (B) SW620 and SW620/Ad300; (C) KB-3-1 and KB-C2; (D) HEK293/pcDNA3.1 and HEK293/ABCB1.

Figure 2

Cell viability assay in three pairs of cells treated with different chemical substrates singly or in combination with 1 μM or 3 μM tetrandrine. A(1)–(6): Doxorubicin with and without 1 μM or 3 μM tetrandrine (Dox + Tet); B(1)–(6): Vincristine with and without 1 μM or 3 μM tetrandrine (Vin + Tet); C(1)–(6): Paclitaxel with and without 1 μM or 3 μM tetrandrine (Pac + Tet); D(1)–(6): control Cisplatin with or without 1 μM or 3 μM tetrandrine (Cis + Tet).

Figure 3

Tetrandrine combined with different anti-cancer drugs reverses the ABCB1-mediated drug resistance in ABCB1 overexpressing cell lines. (A–D): Sw620 and Sw620/Ad300; (E–H): KB-3-1 and KB-C2; (I–L): HEK293/pcDNA3.1 and HEK293/ABCB1. * p < 0.05, ^# p < 0.01 versus the no tetrandrine group.

Figure 4

(A) Intracellular [³H]-paclitaxel in parental cells KB-3-1 and ABCB1-overexpressing cells KB-C2 pretreated with or without tetrandrine (3 μM and 5 μM) and Verapamil (3 μM). (B) Intracellular [³H]-paclitaxel in parental KB-3-1 cells at different time points. (C) Intracellular [³H]-paclitaxel in ABCB1-overexpression KB-C2 cells at different time points. Verapamil, an inhibitor of ABCB1, was used as a positive control. * p < 0.05, versus the controls.

Figure 5

Variation of orthovanadate sensitive ABCB1 ATPase activity with increasing concentration of tetrandrine from 0 to 40 μM.

Figure 6

(A) The expression of ABCB1 and GAPDH in SW620/Ad300 cells after treatment with 1 µM tetrandrine at 0, 24, 48, and 72 h time points. (B) representation of ABCB1/GAPDH relative ratio of A; (C) The expression of ABCB1 and GAPDH in KB-C2 cells after treatment with 1 µM tetrandrine at 0, 24, 48, and 72 h time points. (D) representation of ABCB1/GAPDH relative ratio of C; (E) Immunofluorescent staining on the influence of tetrandrine on the subcellular localization of ABCB1 in SW620/Ad300 cells at 0, 24, 48, and 72 h time points. The nuclei were stained by PI (Propidium Iodide) and shown in red.

Figure 7

Relative ABCB1/GAPDH mRNA expression of gene MDR1 in MDR cells after treatment with 1 μM tetrandrine for 0, 24, 48, or 72 h. (A) SW620/Ad300 cells; (B) KB-C2 cells. The difference was not statistically significant (p > 0.05).

Figure 8

(A) Comparison between binding positions of tetrandrine (blue) and verapamil (red) as ball-and-stick models. The human homology ABCB1 is depicted as yellow ribbons. (B) Induced-fit docking (IFD) predicted docked conformation of tetrandrine as ball and stick model is shown within the drug-binding site of ABCB1, with the atoms colored as carbon–green, hydrogen–white, oxygen–red, nitrogen–blue. Important amino acids are depicted as sticks with the same color scheme as above except that carbon atoms are represented in grey. Only polar hydrogens were shown. Dotted blue line indicates π-π stacking interaction, while dotted dark green line indicates cation-π interaction. Values of the relevant distances are given in Å.

Figure 8

(A) Comparison between binding positions of tetrandrine (blue) and verapamil (red) as ball-and-stick models. The human homology ABCB1 is depicted as yellow ribbons. (B) Induced-fit docking (IFD) predicted docked conformation of tetrandrine as ball and stick model is shown within the drug-binding site of ABCB1, with the atoms colored as carbon–green, hydrogen–white, oxygen–red, nitrogen–blue. Important amino acids are depicted as sticks with the same color scheme as above except that carbon atoms are represented in grey. Only polar hydrogens were shown. Dotted blue line indicates π-π stacking interaction, while dotted dark green line indicates cation-π interaction. Values of the relevant distances are given in Å.