N-alkylated isatins evade P-gp mediated efflux and retain potency in MDR cancer cell lines

Abstract

The search for novel anticancer therapeutics with the ability to overcome multi-drug resistance (MDR) mechanisms is of high priority. A class of molecules that show potential in overcoming MDR are the N-alkylated isatins. In particular 5,7-dibromo-N-alkylisatins are potent microtubule destabilizing agents that act to depolymerize microtubules, induce apoptosis and inhibit primary tumor growth in vivo. In this study we evaluated the ability of four dibrominated N-alkylisatin derivatives and the parent compound, 5,7-dibromoisatin, to circumvent MDR. All of the isatin-based compounds examined retained potency against the MDR cell lines; U937VbR and MES-SA/Dx5 and displayed bioequivalent dose-dependent cytotoxicity to that of the parental control cell lines. We show that one mechanism by which the isatin-based compounds overcome MDR is by circumventing P-glycoprotein (P-gp) mediated drug efflux. Thus, as the isatin-based compounds are not susceptible to extrusion from P-gp overexpressing tumor cells, they represent a promising alternative strategy as a stand-alone or combination therapy for treating MDR cancer.

Keywords: Cancer treatment; Cell biology; Medicinal chemistry.

Fig. 1

Chemical structures of 5,7-dibromoisatin (1) and the N-alkylisatin derivatives 5,7-dibromo-N-(p-hydroxymethylbenzyl)isatin (2); 5,7-dibromo-N-(p-phenylbenzyl)isatin (3); 5,7-dibromo-N-(p-cinnamyl)isatin (4) and 5,7-dibromo-N-(napthalen-1-ylmethyl)isatin (5) and the commercial anticancer agents paclitaxel, doxorubicin, vinblastine and colchicine.

Fig. 2

U937VbR and MES-SA/Dx5 overexpress ABCB1 mRNA and its protein product P-gp. (A) Real-time quantitative PCR (RT-qPCR) of the ABCB1 mRNA transcript level in MES-SA, MES-SA/Dx5, U937 and U937VbR cell lines. Raw expression values were normalized to the internal reference gene, β-actin and expressed as a fold increase to respective cells lines. (B) Fold-increase in P-gp. Immunofluorescent staining of P-gp on the surface of parental and resistant cell lines as determined by flow cytometry. Values are the mean (± SD) of triplicates.

Fig. 3

U937VbR and MES-SA/Dx5 cell lines exhibit cross resistance to the anticancer drugs vinblastine, colchicine, paclitaxel and doxorubicin. Cell viability of (A) U937 (black bars) or U937VbR (grey bars) and (B) MES-SA (black bars) or MES-SA/Dx5 (grey bars) after 48 h treatment with 0.1 μM of the commercial anticancer agents vinblastine, colchicine, paclitaxel and doxorubicin. Values are the mean (± SEM) of triplicates.

Fig. 4

Dose-dependent cytotoxicity of the isatin derivatives 1-5 is maintained in the vinblastine resistant U937 (U937VbR) subline. Cells were incubated with increasing concentrations of either (A) 5,7-dibromoisatin (1), (B) 5,7-dibromo-N-(p-hydroxymethylbenzyl)isatin (2), (C) 5,7-dibromo-N-(p-phenylbenzyl)isatin (3), (D) 5,7-dibromo-N-(p-cinnamyl)isatin (4), (E) 5,7-dibromo-N-(naphthalen-1-ylmethyl)isatin (5) or (F) vinblastine (as a control). The relative sensitivity of the U937 (black) and U937VbR (red) cell lines to the drugs was determined by MTS assay after 48 h incubation. Values are the mean (± SEM) of triplicates.

Fig. 5

Dose-dependent cytotoxicity of the isatin derivatives 1-5 is maintained in doxorubicin resistant MES-SA (MES-SA/Dx5) subline. Cells were incubated with increasing concentrations of either (A) 5,7-dibromoisatin (1), (B) 5,7-dibromo-N-(p-hydroxymethylbenzyl)isatin (2), (C) 5,7-dibromo-N-(p-phenylbenzyl)isatin (3), (D) 5,7-dibromo-N-(p-cinnamyl)isatin (4), (E) 5,7-dibromo-N-(napthalen-1-ylmethyl)isatin (5) or (F) colchicine (as a control). The relative sensitivity of the MES-SA (black) and MES-SA/Dx5 (red) cell lines to the drugs was determined by MTS assay after 48 h incubation. Values are the mean (± SEM) of triplicates.

Fig. 6

N-alkylisatins retain their mode of action against the vinblastine resistant U937VbR subline. Morphological effects of commercial and N-alkylisatin based microtubule destabilizers on U937 and U937VbR cells. Cells were treated with either (A/B) DMSO vehicle control, (C/D) 60 nM vinblastine, (E/F) 10 nM colchicine or 2.5 μM of the N-alkylisatins (G/H) 5,7-dibromo-N-(p-hydroxymethylbenzyl)isatin (2), (I/J) 5,7-dibromo-N-(p-cinnamyl)isatin (4) and (K/L) 5,7-dibromo-N-(naphthalen-1-ylmethyl)isatin (5) for 24 h. Images were obtained by brightfield microscopy on an inverted light microscope at 40x magnification.

Fig. 7

N-Alkylisatins are not substrates or inhibitors of P-gp. (A) Relative effect of the isatins on Pgp-mediated extrusion of Calcein AM. Cells (MES-SA/Dx5) were incubated with compounds 1-5 (40 μM), CSA (40 μM) or vehicle control (2% v/v) for 40 min. Data is normalized to CSA treated cells (100% inhibition) and presented as the mean ± SD (n = 12) determined using live cell imaging (IncuCyte ZOOM). ***P < 0.001; ns = not significant. (B) Effect of test compounds on P-gp ATPase activity. P-gp membranes in the presence of compounds 1-5 (in 1% DMSO) or verapamil (positive control) at either 40 μM or 200 μM were treated with MgATP as per the Pgp-Glo^TM assay protocol. Change in luminescence in relative light units (RLU) was calculated by subtracting the RLU of each test compound from the RLU of sodium orthovanadate (selective inhibitor of P-gp ATPase) to correct for P-gp independent ATP consumption. Change in luminescence is inversely proportional to ATP levels, which are negatively correlated with the activity of P-gp ATPase and therefore P-gp-mediated transport. Red line indicates basal activity. Data are presented as the mean ± SD (n = 3). **P < 0.01; *P < 0.05; ns = not significant.