Modulation of trabectedin (ET-743) hepatobiliary disposition by multidrug resistance-associated proteins (Mrps) may prevent hepatotoxicity

Abstract

Trabectedin is a promising anticancer agent, but dose-limiting hepatotoxicity was observed during phase I/II clinical trials. Dexamethasone (DEX) has been shown to significantly reduce trabectedin-mediated hepatotoxicity. The current study was designed to assess the capability of sandwich-cultured primary rat hepatocytes (SCRH) to predict the hepato-protective effect of DEX against trabectedin-mediated cytotoxicity. The role of multidrug resistance-associated protein 2 (Mrp2; Abcc2) in trabectedin hepatic disposition also was examined. In SCRH from wild-type Wistar rats, cytotoxicity was observed after 24-h continuous exposure to trabectedin. SCRH pretreated with additional DEX (1 microM) exhibited a 2- to 3-fold decrease in toxicity at 100 nM and 1000 nM trabectedin. Unexpectedly, toxicity in SCRH from Mrp2-deficient (TR(-)) compared to wild-type Wistar rats was markedly reduced. Depletion of glutathione from SCRH using buthionine sulfoximine (BSO) mitigated trabectedin toxicity associated with 100 nM and 1000 nM trabectedin. Western blot analysis demonstrated increased levels of CYP3A1/2 and Mrp2 in SCRH pretreated with DEX; interestingly, Mrp4 expression was increased in SCRH after BSO exposure. Trabectedin biliary recovery in isolated perfused livers from TR(-) rats was decreased by approximately 75% compared to wild-type livers. In conclusion, SCRH represent a useful in vitro model to predict the hepatotoxicity of trabectedin observed in vivo. The protection by DEX against trabectedin-mediated cytotoxicity may be attributed, in part, to enhanced Mrp2 biliary excretion and increased metabolism by CYP3A1/2. Decreased trabectedin toxicity in SCRH from TR(-) rats, and in SCRH pretreated with BSO, may be due to increased basolateral excretion of trabectedin by Mrp3 and/or Mrp4.

Fig. 1

Effect of DEX on trabectedin-mediated cytotoxicity in SCRH isolated from wild-type Wistar rats. Cytotoxicity was determined by measuring LDH release into the culture medium after 24 hr trabectedin (1 – 1000 nM) exposure. Each bar represents the mean ± S.E.M. (n=3) for samples from vehicle- (0.1 µM DEX; solid bars) or additional DEX (1 µM; white bars); *, p < 0.05.

Fig. 2

Trabectedin-mediated cytotoxicity in SCRH from wild-type and TR⁻ rats. Cytotoxicity was determined by measuring LDH release into the culture medium after 24 hr trabectedin (1 – 100 nM) exposure. Each bar represents the mean ± S.E.M. (n=3) for samples from wild-type (solid bars) or TR⁻ (white bars) SCRH; *, p < 0.05.

Fig. 3

Effect of BSO on trabectedin-mediated cytotoxicity. Cytotoxicity was determined by measuring LDH release into the culture medium after 24 hr trabectedin (50, 100 and 1000 nM) exposure. BSO (500 µM) was added to the culture medium 24 h prior to trabectedin exposure. Each bar represents the mean ± S.E.M. (n=3) for samples from vehicle- (solid bars) or BSO-pretreated (white bars) SCRH; *, p < 0.05.

Fig. 4

Representative Western blot from whole cell lysates for Mrp2, 3, 4 and CYP3A1/2 in SCRH pretreated with vehicle (0.1 µM DEX), additional DEX (1 µM) or BSO (500 µM) (25 µg protein/well; n=3).

Fig. 5

Trabectedin perfusate concentrations (Fig. 5.a) and cumulative biliary excretion (Fig. 5.b) in isolated perfused livers from wild-type (solid circle) and TR⁻ (white triangle) rats. Data are presented as mean ± S.D., n= 3/group.

Fig. 5

Trabectedin perfusate concentrations (Fig. 5.a) and cumulative biliary excretion (Fig. 5.b) in isolated perfused livers from wild-type (solid circle) and TR⁻ (white triangle) rats. Data are presented as mean ± S.D., n= 3/group.