Polyoxypregnane Aryl Esters Prepared from Metaplexis japonica (Thunb.) Makino and Their Role in Reversing Multidrug Resistance in HepG2/Dox Cells

<b>Objective</b>: The development of natural and new P-gp modulators to reverse tumor multidrug resistance (MDR). <b>Methods</b>: Test compounds were prepared from the plant <i>Metaplexis japonica</i>, and their ability to reverse P-glycoprotein (P-gp)-mediated MDR was investigated in HepG2/Dox cells. Their effects on P-gp expression and function and their interaction modes with P-gp were also investigated. <b>Results</b>: Natural product 3β,12β,14β, 17β,20(<i>S</i>)-pentahydroxy-5α-pregnan-12β-<i>O</i>-(<i>E</i>)-cinnamate (<b>1</b>) and its new semisynthetic derivative 3β12β,14β,17β,20(<i>S</i>)-pentahydroxy-5α-pregnan-3β-<i>O</i>-nicotinate-12β-<i>O</i>-(E)-cinnamate (<b>1a</b>) were obtained. At non-cytotoxic concentrations of 5 or 10 μM, they significantly reversed the resistance of HepG2/Dox cells to P-gp substrate drugs doxorubicin, paclitaxel, and vinblastine, with reversal folds of 7.1, 118.5, and 198.3 (<b>1</b>), and 18.8, 335.8, and 140.0 (<b>1a</b>), respectively, at 10 μM. Cell apoptosis and expression of caspase 9 were both triggered by the combination of 10 μM of compound <b>1</b> or <b>1a</b> and 500 nM of paclitaxel (<i>p</i> < 0.001). Compound <b>1</b> or <b>1a</b> did not affect P-gp expression, but it did significantly suppress the efflux of Rhodamine 123 out of HepG2/Dox cells (<i>p</i> < 0.001). On the Caco-2 cell monolayer, <b>1</b> and <b>1a</b> were shown to be non-substrates of P-gp, with efflux ratios of 0.83 and 0.89. Molecular docking revealed their strong binding energies (-8.2 and -8.4 kcal/mol) with P-gp, and their direct binding to P-gp was confirmed by their dissociation constants (5.53 µM for <b>1</b> and 3.72 µM for <b>1a</b>), determined using surface plasmon resonance. <b>Conclusions</b>: Compounds <b>1</b> and <b>1a</b> are potential P-gp modulators; they may reverse P-gp-MDR through interacting with P-gp to interfere with substrate binding and transporting, and have the potential to improve the efficacy of paclitaxel or vinblastine drugs for combating P-gp-mediated MDR in tumor cells.