Ribociclib Inhibits P-gp-Mediated Multidrug Resistance in Human Epidermoid Carcinoma Cells

Abstract

The efficacy of cancer chemotherapy can be attenuated or abrogated by multidrug resistance (MDR) in cancer cells. In this study, we determined the effect of the CDK4/6 inhibitor, ribociclib (or LEE011), on P-glycoprotein (P-gp)-mediated MDR in the human epidermoid carcinoma MDR cell line, KB-C2, which is widely used for studying P-gp-mediated MDR in cancers. The incubation of KB-C2 cells with ribociclib (3-9 µM) increased the efficacy of colchicine, a substrate for P-gp. The cell expression of P-gp was down-regulated at both translation and transcription levels. Furthermore, ribociclib produced a 3.5-fold increase in the basal activity of P-gp ATPase, and the concentration required to increase basal activity by 50% (EC₅₀) was 0.04 μM. Docking studies indicated that ribociclib interacted with the drug-substrate binding site of P-gp. The short-term and long-term intracellular accumulation of doxorubicin greatly increased in the KB-C2 cells co-cultured with ribociclib, indicating ribociclib inhibited the drug efflux activity of P-gp. The results of our study indicate that LEE011 may be a potential agent for combined therapy of the cancers with P-gp mediated MDR.

Keywords: CDKs 4 and 6; P-glycoprotein (ABCB1 protein); cancer; cyclin dependent kinases; multidrug resistance (MDR); ribociclib-LEE011.

FIGURE 1

Reversal effect of ribociclib on MDR in KB-C2 cell line. Ribociclib reverses MDR in KB-C2 cells but does not significantly affect the efficacy of colchicine in the parental KB-3-1 cells. Ribociclib at 9 µM significantly decreased the IC₅₀ of colchicine in KB-C2 cancer cells (p < 0.05).

FIGURE 2

ABCB1 expression deviation between the robociclib/LEE011 treated KB-C2 cells and the robociclib non-treated KB-C2 cells. (A) Western blot indicated that P-gp expression was significantly downregulated by incubating the cells with 9 µM of ribociclib for 48 h (B) ABCB1 mRNA level was analyzed by RT-q-PCR, using GAPDH mRNA as the inner reference. The cells without robociclib treatment were set as control.

FIGURE 3

Structural basis for the interaction of ribociclib with P-gp. Docking analysis of the 3-dimentional structure of the ribociclib-P-gp complex were performed using HEX 8.0 software. (A) Ribociclib interacted with the NBD domain near the interface at the inner side. (B) The magnified region showing the amino groups that interact with ribociclib (C, D) Spatial structure and charge distributions of the site that binds ribociclib.

FIGURE 4

A 3-D structural model showing the electrostatic interaction between ribociclib and P-gp. Under physiological conditions, the N,N-dimethylamide cluster is positively charged and may bind to a cavity containing E273 and E1129 with negative charges due to the dissociation of hydrogen protons from the carboxyl group at neutral or higher pH values.

FIGURE 5

The interaction between ribociclib and the P-gp transporter alters the ATPase activity of P-gp and drug efflux activity in KB-C2 cells. (A) ATPase activity of P-gp following incubation with ribociclib. (B) A comparison of the drug accumulation in MDR KB-C2 cells over-expressing P-gp and the drug sensitive KB-3-1 cells in the presence or absence of ribociclib. The cells were co-cultured with ribociclib (9 µM) and Dox (0.2 µM) for 2 h. The level of Dox fluorescence was measured at 550 nm. (C) A Scheme showing that ribociclib alters ATPase activity and drug efflux by interacting with the P-gp transporter.

FIGURE 6

Ribociclib significantly increases the accumulation of doxorubicin (Dox) in KB-C2 cells after 72 h of incubation, producing apoptosis or necrosis. KB-C2 and parental KB-3-1 cells were co-cultured with doxorubicin (1 µM for KB-C2 and 0.1 µM for KB-3-1) and ribociclib 1 (9 µM) for 72 h. The cells incubated with vehicle were used as controls. The fluorescence of Dox was imaged using fluorescent microscopy (set to the red fluorescent channel). The schematic figure shows that ribociclib increases the accumulation of Dox by 1) decreasing its efflux, which increases apoptosis and 2) downregulating the levels of P-gp protein expression which also increases the intracellular levels of Dox.