Molecular and pharmacological effects of schisandrol A and gomisin A on multidrug resistant cancer cells

Abstract

Multidrug resistance (MDR) is defined as the chemotherapeutic resistant of cancer cells to a broad spectrum of structurally unrelated drugs. Overexpression of membrane transporter P-glycoprotein (P-gp) is the most extensively studied MDR pathway. P-gp modulator screening program in our laboratory has shown that Fructus Schisandrae (the dried fruit of Schisandra chinensis (Turcz.) Baill.) modulates P-gp-mediated MDR. Through bioassay guided fractionation two dibenzocyclooctadiene lignans schisandrol A (SCH) and gomisin A (GOM) were identified and we studied their modulatory mechanisms on P-gp-mediated MDR. In P-gp overexpressing subline HepG2-DR cells SCH or GOM was relatively non-toxic (IC50 > 150M after 72 h incubation) but without altering P-gp expression they restored the cytotoxic actions of anticancer drugs such as vinblastine and doxorubicin that are P-gp substrates. The toxicity of SCH or GOM itself was not altered by P-gp competitive modulator verapamil suggesting that they are P-gp modulators rather than substrates. Although SCH and GOM share similar chemical structure their P-gp modulatory mechanisms are not exactly the same. They showed differential effects on the P-gp-associated ATPase activity. SCH activated while GOM inhibited the P-gp-associated ATPase activity suggesting that SCH contained some substrate-like properties but GOM acted as pure P-gp inhibitor. Consistent with this observation SCH showed a mixed-type inhibition while GOM showed an uncompetitive inhibition on progesterone- or verapamil-stimulated P-gp-associated ATPase activity. In HepG2-DR cells both SCH and GOM were able to increase cellular retention of the P-gp substrate rhodamine 123 (Rh-123) and GOM showed more effective than SCH. The combined effect of verapamil with SCH or GOM was basically additive, implying that SCH or GOM might simultaneously bind on P-gp with verapamil. Moreover, binding of transport substrates with P-gp would result in a P-gp conformational change favoring UIC-2 antibody reactivity but SCH or GOM impeded UIC-2 binding, suggesting that binding of SCH or GOM altered P-gp conformation in a manner distinct from P-gp-substrate binding. These results suggested that SCH or GOM modulates the P-gp-dependent MDR in HepG2-DR cells possibly by simultaneously binding with substrates onto P-gp and altering the P-gp-substrate interaction. We also investigated the effect of SCH or GOM on the drug metabolizing activity of CYP3A4, which usually show cross substrate/inhibitor specificity with P-gp. SCH or GOM inhibited CYP3A4 activity and GOM (IC50 = 1.39M) showed higher potency than SCH (IC50 = 32.02M). Therefore the pharmacokinetic interaction of SCH or GOM with chemotherapeutic agents should be carefully considered when combined use in vivo. Apart from their modulation on P-gp-mediated MDR SCH or GOM showed vincristine sensitizing effect on both 2008 ovarian cancer cells and its MRP1-transfected subline. Addition of GSH could not reverse this effect suggesting that it was unrelated to the modulation of MRP1-mediated MDR. No sensitizing effect was observed when SCH or GOM was combined with doxorubicin or taxol indicating that the action mechanism was P-gp-independent. We studied the mechanism for this vincristine sensitizing effect. SCH or GOM enhanced vincristine-induced mitosis arrest, apoptosis and Cdc2 dephosphorylation/activation. Combined drug treatments regulated Wee1 and Cdc25C through phosphorylation/dephosphorylation and nuclear/cytoplasmic translocation in favor of Cdc2 activation. Olomoucine inhibited phosphorylation of Wee1 and Cdc25C suggesting the presence of a Cdc2-mediated positive feedback loop. The enhanced apoptosis was associated with increases of caspases 8 activation. The actions of SCH or GOM were independent from p53 as shown firstly that SCH or GOM suppressed vincristine-stimulated p53 up-regulation. Direct inhibitory interaction of p53/Cdc2 was not detected and SCH or GOM did not affect the expression levels of p53 transactivation targets. Lastly transient transfection and expression of wild-type and dominant negative p53 had no effect on SCH- or GOM-mediated vincristine sensitizing effect. Taken together, our results suggested that besides their modulatory effect on P-gp-mediated MDR, SCH or GOM is able to potentiate vincristine-mediated mitotic arrest, Cdc2 activation and apoptosis in a P-gp- and p53-independet manner.

Date of Award	15 Feb 2007
Original language	English
Awarding Institution	City University of Hong Kong
Supervisor	M YANG (Supervisor) & Wang Fun FONG (Supervisor)