Resistance to the translation initiation inhibitor silvestrol is mediated by ABCB1/P-glycoprotein overexpression in acute lymphoblastic leukemia cells

Abstract

Protein synthesis is a powerful therapeutic target in leukemias and other cancers, but few pharmacologically viable agents are available that affect this process directly. The plant-derived agent silvestrol specifically inhibits translation initiation by interfering with eIF4A/mRNA assembly with eIF4F. Silvestrol has potent in vitro and in vivo activity in multiple cancer models including acute lymphoblastic leukemia (ALL) and is under pre-clinical development by the US National Cancer Institute, but no information is available about potential mechanisms of resistance. In a separate report, we showed that intraperitoneal silvestrol is approximately 100% bioavailable systemically, although oral doses were only 1% bioavailable despite an apparent lack of metabolism. To explore mechanisms of silvestrol resistance and the possible role of efflux transporters in silvestrol disposition, we characterized multi-drug resistance transporter expression and function in a silvestrol-resistant ALL cell line generated via culture of the 697 ALL cell line in gradually increasing silvestrol concentrations. This resistant cell line, 697-R, shows significant upregulation of ABCB1 mRNA and P-glycoprotein (Pgp) as well as cross-resistance to known Pgp substrates vincristine and romidepsin. Furthermore, 697-R cells readily efflux the fluorescent Pgp substrate rhodamine 123. This effect is prevented by Pgp inhibitors verapamil and cyclosporin A, as well as siRNA to ABCB1, with concomitant re-sensitization to silvestrol. Together, these data indicate that silvestrol is a substrate of Pgp, a potential obstacle that must be considered in the development of silvestrol for oral delivery or targeting to tumors protected by Pgp overexpression.

Fig. 1

Silvestrol resistance in 697-R cells is stable over time. 697-R cells exposed to 80 nM silvestrol every 2 weeks (697-R) or incubated without silvestrol for 14 weeks (697-R WS) were incubated with increasing concentrations of silvestrol for 72 h (N = 3). 697 parental cells were included for comparison. Viability was assessed by MTT assay, and results were calculated relative to time-matched untreated samples

Fig. 2

Pgp expression is elevated in 697-R cells. a Whole cell extracts from 697 and 697-R cells were immunoblotted for Pgp, using actin as a loading control. HL-60 cells without or with Pgp overexpression were included as negative and positive control, respectively. One of the three representative immunoblots is shown. b Live unfixed 697 and 697-R cells were incubated without antibody (black line), isotype control antibody (light gray line), or anti-Pgp-PE (medium gray line), washed, and examined by flow cytometry to determine surface Pgp expression. One of the three representative experiments is shown

Fig. 3

697-R cells show increased resistance to Pgp substrates. 697 and 697-R cells (N = 3) were incubated with increasing concentrations of vincristine, romidepsin, cycloheximide, and 2-F-ara-A. Viability was determined by MTT assay at 48 h, and results were calculated relative to time-matched untreated cells

Fig. 4

Pgp function is elevated in 697-R cells. 697 parental (left) and 697-R cells (right) were incubated with rhodamine 123 for 30 min, washed, and assessed by flow cytometry immediately and at several times after washout. Unstained cells were included as a negative control. One of the three representative experiments is shown

Fig. 5

Pgp inhibitors prevent rhodamine 123 efflux and resensitize 697-R cells to silvestrol. a 697 and 697-R were incubated with rhodamine 123 for 30 min, washed, and re-incubated in media with no inhibitor, verapamil (10 μM), or cyclosporin A (1 μM) for 2 h. Cells were then examined by flow cytometry. The mean fluorescent intensity (MFI) for each cell line is shown normalized to the MFI immediately following washout. b 697 and 697-R cells were treated with verapamil (10 μM), cyclosporin A (1 μM), silvestrol (80 nM), or combination. Viability was determined by MTT assay 48 h after treatment. Results are shown relative to time-matched untreated cells (N = 3)

Fig. 6

ABCB1 siRNA specifically inhibits Pgp and resensitizes 697-R cells to silvestrol. 697-R cells were mock-transfected (no siRNA) or transfected with scrambled, ABCB1 or GAPDH siRNA (N = 3). Cells were incubated for 48 h. a Cells were analyzed for rhodamine 123 efflux. b ABCB1 expression was determined by real-time RT-PCR. Fold change of ABCB1 mRNA after mock, scrambled, ABCB1 or GAPDH siRNA transfection is normalized to the ABCB1 expression of mock-treated samples. GAPDH mRNA expression after transfections was measured as a positive control. c Transfected cells were incubated with 80 nM silvestrol for an additional 72 h, and growth inhibition was determined by MTT assay. Results are presented relative to mock-transfected 697-R cells without treatment. 697 parental cells incubated with corresponding drugs were included as a positive control