| Summary: | 碩士 === 中國醫藥大學 === 藥學系碩士班 === 101 === Chemotherapy is the most common treatment for cancer. However, the ability of cancer cells simultaneously becoming resistant to different drugs, a phenomenon called multidrug resistance (MDR), remains a big difficulty to successful chemotherapy and the drug efflux transporter, P-glycoprotein (P-gp), is reported to play an important role in this field. Although three generations of P-gp inhibitors have been developed to overcome this problem, the outcome of clinical trials among these agents has been negative. The discovering of “fourth generation inhibitors” aims at exploring potent and relative non-toxic substances from natural products. In the present study, we investigated the P-gp inhibitory effect and mechanism of ethanol extracts from Antrodia cinnamomea fruiting bodies (EEAC), bisdemethoxycurcumin (BDMC), demethoxycurcumin (DMC), and curcumin, the influence of common polymorphisms in ABCB1 gene was also evaluated.
HEK293 cell lines harbored some combinations of the common polymorphisms in human ABCB1 gene were established. Real-time RT-PCR was conducted to confirm the expression while rhodamine 123 accumulation and efflux assay and calcein-AM uptake assay were carried out to examine the function of P-gp. Influence of EEAC, BDMC, DMC and curcumin on P-gp expression and function was also evaluated by the above experiments. MDR1 shift assay and ATPase assay were conducted to figure out the interaction between P-gp and the tested substances.
The basal ABCB1 mRNA expression and P-gp efflux function of the established wild-type and variant-types P-gp expressing cell lines were confirmed. Results from rhodamine 123 efflux assay clearly demonstrated that EEAC, BDMC, DMC, and curcumin significantly inhibited wild-type P-gp efflux function with IC50 1.51μg/ml, 3.08μM, 1.56μM and 5.8μM, respectively. EEAC modulated wild-type P-gp through non-competitive inhibition while curcuminoids inhibited wild-type P-gp by uncompetitive kinetic. However, the P-gp mRNA expression was not changed after incubating the cells with the tested substances for 72 hours. Variant-type P-gp caused by ABCB1 gene mutations showed varied potency and distinct mechanism from wild-type P-gp inhibition. Moreover, results of ATPase assay revealed that these four substances influenced the P-gp ATPase function and may have interaction with other P-gp substrates.
This study successfully characterized EEAC, BDMC, DMC and curcumin, sourced from natural fungi and plants, as potential P-gp function modulators. The common polymorphism haplotypes in ABCB1 gene were demonstrated to affect the potency and mechanism of these candidate P-gp inhibitors. As the exploring of novel inhibitors from natural products is still in the early stages, the promising beginning and detailed mechanism investigation provided by this study will be helpful in the future in vivo studies and clinical practice.
|
|---|
