The anticancer agent prodigiosin is not a multidrug resistance protein substrate

Abstract

The brilliant red pigments prodiginines are natural secondary metabolites that are produced by select species of Gram-negative and Gram-positive bacteria. These molecules have received significant attention due to their reported antibacterial, antifungal, immunosuppressive, and anticancer activities. In this study, a Serratia marcescens SER1 strain was isolated and verified using 16s rDNA. The prodigiosin was purified using silica chromatography and was analyzed by (1)H-NMR spectroscopy. The cell cytotoxic effects of the purified prodigiosin on multiple drug resistant cell lines that overexpress MDR1, BCRP, or MRP2 pumps were analyzed. Prodigiosin had nearly identical cytotoxic effects on the resistant cells in comparison to their parental lines. In agreement with the same prodigiosin cytotoxicity, FACS analysis of prodigiosin accumulation and efflux in MDR overexpressing cell lines also indicated that this pro-apoptotic agent operates independently of the presence of the MDR1, BCRP, or MRP transporter and may be a potential treatment for malignant cancer cells that overexpress multidrug resistance transporters.

FIG. 1.

Prodigiosin structure according to ¹H-NMR spectroscopy.

FIG. 2.

Growth rate of A2780 (A), EPG85-257 (B) cells and their resistant counterpart. Cells were seeded in 96-well plates at 1000 cells/well in RPMI-1640 culture medium. Cells were then counted using MTT assay during 7 days of seeding. Data are mean±SE of three independent experiments each in triplicate. The symbols () and () represent the mean absorbance difference between parental and resistant cells with p<0.001 and p<0.05, respectively.

FIG. 3.

Effects of prodigiosin on the survival of A2780 (A), EPG85-257 (B) cells and their resistant counterpart. Cells were cultured for 5 days with increasing doses of prodigiosin from 0 to 100 μM. Cell survival was measured by MTT assay. The values represent the means of three independent experiments performed in triplicate (Mean±SE). p>0.05 indicates that prodigiosin had nearly identical cytotoxicity on A2780 and EPG85-257 cells and their resistant counterpart.

FIG. 4.

FACS analysis of accumulation of daunorubicin, mitoxantrone, or prodigiosin in three individual experiments in a panel of parental and their resistant cell lines. Values are presented as the mean±SE. The symbols () and () represent the mean fluorescence difference between parental and resistant cells with p<0.001 and p<0.05, respectively. Dnr, daunorubicin; PG, prodigiosin; MX, mitoxantrone.

FIG. 5.

Efflux analysis of daunorubicin, mitoxantrone, and prodigiosin in EPG85-257 cells. The upper panel is EPG85-257 parent cells and the lower panel is EPG85-257RDB and EPG85-257RNOV resistant cells. No drug (autofluorescence; solid line), fluorescence levels after 60 min of efflux in the absence (dashed line) or presence (dotted line) of the inhibitors. Ver, verapamil; Novo, novobiocin.

FIG. 6.

Efflux analysis of daunorubicin and prodigiosin in A2780 cells. The upper panel is A2780 parent cells and the lower panel is A2780RCIS resistant cells. No drug (autofluorescence; solid line), fluorescence levels after 60 min of efflux in the absence (dashed line) or presence (dotted line) of the inhibitor. Indo, indomethacin.