Exploring in vitro, in vivo metabolism of mogroside V and distribution of its metabolites in rats by HPLC-ESI-IT-TOF-MS(n).

Mogroside V, a cucurbitane-type saponin, is not only the major bioactive constituent of traditional Chinese medicine Siraitiae Fructus, but also a widely used sweetener. To clarify its biotransformation process and identify its effective forms in vivo, we studied its metabolism in a human intestinal bacteria incubation system, a rat hepatic 9000g supernatant (S9) incubation system, and rats. Meanwhile, the distribution of mogroside V and its metabolites was also reported firstly. Seventy-seven new metabolites, including 52 oxidation products formed by mono- to tetra- hydroxylation/dehydrogenation, were identified with the aid of HPLC in tandem with ESI ion trap (IT) TOF multistage mass spectrometry (HPLC-ESI-IT-TOF-MS(n)). Specifically, 14 metabolites were identified in human intestinal bacteria incubation system, 4 in hepatic S9 incubation system, 58 in faeces, 29 in urine, 14 in plasma, 34 in heart, 33 in liver, 39 in spleen, 39 in lungs, 42 in kidneys, 45 in stomach, and 51 in small intestine. The metabolic pathways of mogroside V were proposed and the identified metabolic reactions were deglycosylation, hydroxylation, dehydrogenation, isomerization, glucosylation, and methylation. Mogroside V and its metabolites were distributed unevenly in the organs of treated rats. Seven bioactive metabolites of mogroside V were identified, among which mogroside IIE was abundant in heart, liver, spleen and lung, suggesting that it may contribute to the bioactivities of mogroside V. Mogroside V was mainly excreted in urine, whereas its metabolites were mainly excreted in faeces. To our knowledge, this is the first report that a plant constituent can be biotransformed into more than 65 metabolites in vivo. These findings will improve understanding of the in vivo metabolism, distribution, and effective forms of mogroside V and congeneric molecules.

Afatinib circumvents multidrug resistance via dually inhibiting ATP binding cassette subfamily G member 2 in vitro and in vivo.

Multidrug resistance (MDR) to chemotherapeutic drugs is a formidable barrier to the success of cancer chemotherapy. Expressions of ATP-binding cassette (ABC) transporters contribute to clinical MDR phenotype. In this study, we found that afatinib, a small molecule tyrosine kinase inhibitor (TKI) targeting EGFR, HER-2 and HER-4, reversed the chemoresistance mediated by ABCG2 in vitro, but had no effect on that mediated by multidrug resistance protein ABCB1 and ABCC1. In addition, afatinib, in combination with topotecan, significantly inhibited the growth of ABCG2- overexpressing cell xenograft tumors in vivo. Mechanistic investigations exhibited that afatinib significantly inhibited ATPase activity of ABCG2 and downregulated expression level of ABCG2, which resulted in the suppression of efflux activity of ABCG2 in parallel to the increase of intracellular accumulation of ABCG2 substrate anticancer agents. Taken together, our findings may provide a new and useful combinational therapeutic strategy of afatinib with chemotherapeutical drug for the patients with ABCG2 overexpressing cancer cells.

Lapatinib antagonizes multidrug resistance-associated protein 1-mediated multidrug resistance by inhibiting its transport function.

Lapatinib, a tyrosine kinase inhibitor, is used in the treatment of advanced or metastatic breast cancer overexpressing human epidermal receptor 2 (HER2). Lapatinib can modulate the function of ATP-binding cassette (ABC) transporters (ABCB1 and ABCG2), which are the major mechanism responsible for multidrug resistance (MDR) in cancer. In this study, we investigated the effect of lapatinib on multidrug resistance-associated protein 1 (MRP1 [ABCC1]), MRP2 (ABCC2), MRP4 (ABCC4) and lung relative resistance protein (LRP) drug efflux pumps. We demonstrated that lapatinib could enhance the efficacy of conventional chemotherapeutic agents in MRP1-overexpressing cells in vitro and in vivo, but no effect in MRP2-, MPR4- and LRP-overexpressing cells. Furthermore, lapatinib significantly increased the accumulation of rhodamine 123 (Rho123) and doxorubicin (DOX) in MRP1-overexpressing cells. However, lapatinib did not alter the protein or mRNA expression levels of MRP1. Further studies showed that the level of phosphorylation of AKT and extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) were not altered at the indicated concentrations of lapatinib. In conclusion, lapatinib enhanced the efficacy of conventional chemotherapeutic agents in MRP1-overexpressing cells by inhibiting MRP1 transport function without altering the level of AKT or ERK1/2 phosphorylation. These findings will encourage the clinical research of lapatinib combined with conventional chemotherapeutic drugs in MRP1-overexpressing cancer patients.

CEP-33779 antagonizes ATP-binding cassette subfamily B member 1 mediated multidrug resistance by inhibiting its transport function.

The overexpression of ATP-binding cassette (ABC) transporters often leads to the development of multidrug resistance (MDR), which is the major factor contributing to the failure of chemotherapy. The objective of this study was to investigate the enhancement of CEP-33779, a small-molecule inhibitor of Janus kinase 2 (JAK2), on the efficacy of conventional chemotherapeutic agents in MDR cells with overexpression of P-glycoprotein (ABCB1), multidrug resistance-associated protein 1 (ABCC1) and breast cancer resistance protein (ABCG2). Our results showed that CEP-33779, at nontoxic concentrations, significantly sensitized ABCB1 overexpressing MDR cells to its anticancer substrates. CEP-33779 significantly increased intracellular accumulation and decreased the efflux of doxorubicin by inhibiting the ABCB1 transport function. Furthermore, CEP-33779 did not alter the expression of ABCB1 both at protein and mRNA levels but did stimulate the activity of ABCB1 ATPase. CEP-33779 was predicted to bind within the large hydrophobic cavity of homology modeled ABCB1. In addition, the down-regulation of JAK2 by shRNA altered neither the expression of ABCB1 nor the cytotoxic effect of chemotherapeutic agents in ABCB1-overexpressing cells. Significantly, CEP-33779 enhanced the efficacy of vincristine against the ABCB1-overexpressing and drug resistant KBv200 cell xenograft in nude mice. In conclusion, we conclude that CEP-33779 enhances the efficacy of substrate drugs in ABCB1-overexpressing cells by directly inhibiting ABCB1 transport function. The findings encouraged to further study on the combination therapy of CEP-33779 with conventional chemotherapeutic agents in ABCB1 mediated-MDR cancer patients.

Afatinib enhances the efficacy of conventional chemotherapeutic agents by eradicating cancer stem-like cells.

Cancer stem cells (CSC) have garnered significant attention as a therapeutic focus, based on evidence that they may represent an etiologic root of treatment-resistant cells. Indeed, expression of the multidrug resistance protein ATP-binding cassette subfamily G member 2 (ABCG2) confers chemoresistance to CSCs, where it serves as a potential biomarker and therapeutic target. Here, we show that afatinib, a small-molecule inhibitor of the tyrosine kinases EGFR, HER2, and HER4, preferentially eliminated side population cells with CSC character, in both cell lines and patient-derived leukemia cells, by decreasing ABCG2 expression. In these cells, afatinib also acted in parallel to suppress self-renewal capacity and tumorigenicity. Combining afatinib with the DNA-damaging drug topotecan enhanced the antitumor effect of topotecan in vitro and in vivo. Mechanistic investigations suggested that ABCG2 suppression by afatinib did not proceed by proteolysis through the ubiquitin-dependent proteosome, lysosome, or calpain. Instead, we found that afatinib increased DNA methyltransferase activity, thereby leading to methylation of the ABCG2 promoter and to a decrease in ABCG2 message level. Taken together, our results advocate the use of afatinib in combination with conventional chemotherapeutic drugs to improve efficacy by improving CSC eradication.