Xiaokui Huo

Dioscin restores the activity of the anticancer agent adriamycin in multidrug-resistant human leukemia K562/adriamycin cells by down-regulating MDR1 via a mechanism involving NF-κB signaling inhibition.

The purpose of this study was to investigate the ameliorating effect of dioscin (1) on multidrug resistance (MDR) in adriamycin (ADR)-resistant erythroleukemic cells (K562/adriamycin, K562/ADR) and to clarify the molecular mechanisms involved. High levels of multidrug resistance 1 (MDR1) mRNA and protein and reduced ADR retention were found in K562/ADR cells compared with parental cells (K562). Dioscin (1), a constituent of plants in the genus Discorea, significantly inhibited MDR1 mRNA and protein expression and MDR1 promoter and nuclear factor κ-B (NF-κB) activity in K562/ADR cells. MDR1 mRNA and protein suppression resulted in the subsequent recovery of intracellular drug accumulation. Additionally, inhibitor κB-α (IκB-α) degradation was inhibited by 1. Dioscin (1) reversed ADR-induced MDR by down-regulating MDR1 expression by a mechanism that involves the inhibition of the NF-κB signaling pathway. These findings provide evidence to support the further investigation of the clinical application of dioscin (1) as a chemotherapy adjuvant.

Protostane Triterpenoids from the Rhizome of Alisma orientale Exhibit Inhibitory Effects on Human Carboxylesterase 2

Twelve new and 10 known protostane triterpenoids were isolated from the rhizome of Alisma orientale. Their structures were elucidated based on physical data analyses, including UV, HRESIMS, NMR experiments ((1)H, (13)C NMR, (1)H-(1)H COSY, HSQC, HMBC, and NOESY), and induced electronic circular dichroism. New compounds 1-12 were classified as protostanes (1-10), 29-norprotostane (11), and 24-norprotostane (12) by structure analyses. Furthermore, the inhibitory effects on human carboxylesterases (hCE-1, hCE-2) of compounds 1-22 were evaluated. Compounds 2, 6, 9, and 11 showed moderate inhibitory activities and were selective toward hCE-2 enzymes, with IC50 values of 8.68, 4.72, 4.58, and 2.02 μM, respectively. The inhibition kinetics of compound 11 toward hCE-2 were established, and the Ki value was determined as 1.76 μM using a mixed inhibition model. The interaction of bioactive compound 11 with hCE-2 was shown using molecular docking.

Alisol B 23-acetate protects against ANIT-induced hepatotoxity and cholestasis, due to FXR-mediated regulation of transporters and enzymes involved in bile acid homeostasis

Intrahepatic cholestasis is a clinical syndrome with systemic and intrahepatic accumulation of excessive toxic bile acids that ultimately cause hepatobiliary injury. Appropriate regulation of bile acids in hepatocytes is critically important for protection against liver injury. In the present study, we characterized the protective effect of alisol B 23-acetate (AB23A), a natural triterpenoid, on alpha-naphthylisothiocyanate (ANIT)-induced liver injury and intrahepatic cholestasis in mice and further elucidated the mechanisms in vivo and in vitro. AB23A treatment dose-dependently protected against liver injury induced by ANIT through reducing hepatic uptake and increasing efflux of bile acid via down-regulation of hepatic uptake transporters (Ntcp) and up-regulation of efflux transporter (Bsep, Mrp2 and Mdr2) expression. Furthermore, AB23A reduced bile acid synthesis through repressing Cyp7a1 and Cyp8b1, increased bile acid conjugation through inducing Bal, Baat and bile acid metabolism through an induction in gene expression of Sult2a1. We further demonstrate the involvement of farnesoid X receptor (FXR) in the hepatoprotective effect of AB23A. The changes in transporters and enzymes, as well as ameliorative liver histology in AB23A-treated mice were abrogated by FXR antagonist guggulsterone in vivo. In vitro evidences also directly demonstrated the effect of AB23A on FXR activation in a dose-dependent manner using luciferase reporter assay in HepG2 cells. In conclusion, AB23A produces protective effect against ANIT-induced hepatotoxity and cholestasis, due to FXR-mediated regulation of transporters and enzymes.

Enhancement effect of P-gp inhibitors on the intestinal absorption and antiproliferative activity of bestatin.

Bestatin is an immunomodulator with antitumor activity. This study was performed to investigate the effect of P-gp on the intestinal absorption and antiproliferative activity of bestatin. Our results showed that P-gp inhibitors significantly increased rat intestinal absorption of bestatin in vivo and in vitro. The net efflux ratio of bestatin was 2.2 across mock-/MDR1-MDCK cell monolayers and was decreased by P-gp inhibitors, indicating bestatin was a substrate of P-gp. Furthermore, the IC50 values of bestatin on U937 and K562 cells were decreased dramatically and the intracellular concentrations of bestatin were increased by incubation of cells with verapamil or Cyclosporin A. K562/ADR cells exhibited a higher IC50 value and a lower intracellular level of bestatin. The bestatin level in K562/ADR cells was partially restored by incubation with doxorubicin. However, P-gp and APN mRNA levels were not changed by bestatin. These results suggested that the intestinal absorption and accumulation in cancer cells for bestatin were limited by P-gp-mediated efflux. Additional attention should be paid to the alternative exposure of bestatin when bestatin was coadministered with drugs as P-gp substrates in clinic.

Involvement of Organic Anion-Transporting Polypeptides in the Hepatic Uptake of Dioscin in Rats and Humans

The objective of this study was to clarify the mechanism underlying hepatic uptake of dioscin (diosgenyl 2,4-di-O-a-L-rhamnopyranosyl-p-D-glucopyranoside), an herbal ingredient with antihepatitis activity, in rats and humans. The liver uptake index (LUI) in vivo, perfused rat liver in situ, rat liver slices, isolated rat hepatocytes, and human organic anion-transporting polypeptide (OATP)–transfected cells in vitro were used to evaluate hepatic uptake of dioscin. Values of 11.9% ± 1.6% and 15.0% ± 0.9% of dose for uptake of dioscin were observed by LUI in vivo and perfused rat livers in situ, respectively. The time course of dioscin uptake by rat liver slices was temperature-dependent. Uptake of dioscin by rat liver slices and isolated rat hepatocytes was inhibited significantly by Oatp modulators, such as ibuprofen (Oatp1a1 inhibitor), digoxin (Oatp1a4 substrate), and glycyrrhizic acid (Oatp1b2 inhibitor), but not by TEA or p-aminohippurate. Uptake of dioscin in rat hepatocytes and OATP1B3-human embryonic kidney (HEK) 293 cells indicated a saturable process with a Km of 3.75 ± 0.51 μM and 2.08 ± 0.27 μM, respectively. (–)-Epigallocatechin gallate, cyclosporin A, rifampicin, and telmisartan inhibited transport of dioscin in OATP1B3-HEK293 cells. However, transcellular transport of dioscin in OATP1B1- or OATP1B1/multidrug resistance-associated protein 2–Madin-Darby canine kidney strain II cells was not observed. These results indicate that hepatic uptake of dioscin is involved in OATP1B3 in humans, and multiple Oatps might participate in this process in rats.

Dasatinib reverses the multidrug resistance of breast cancer MCF-7 cells to doxorubicin by downregulating P-gp expression via inhibiting the activation of ERK signaling pathway.

Multidrug resistance (MDR) is one of the major obstacles to the efficiency of cancer chemotherapy, which often results from the overexpression of drug efflux transporters such as P-glycoprotein (P-gp). In the present study, we determined the effect of dasatinib which was approved for imatinib resistant chronic myelogenous leukemia (CML) and (Ph+) acute lymphoblastic leukemia (ALL) treatment on P-gp-mediated MDR. Our results showed that dasatinib significantly increased the sensitivity of P-gp-overexpressing MCF-7/Adr cells to doxorubicin in MTT assays; thus lead to an enhanced cytotoxicity of doxorubicin in MCF-7/Adr cells. Additionally, dasatinib increased the intracellular accumulation, inhibited the efflux of doxorubicin in MCF-7/Adr cells, and significantly enhanced doxorubicin-induced apoptosis in MCF-7/Adr cells. Further studies showed that dasatinib altered the expression levels of mRNA, protein levels of P-gp, and the phosphorylation of signal–regulated kinase (ERK) both in time-dependent (before 24 h) and dose-dependent manners at concentrations that produced MDR reversals. In conclusion, dasatinib reverses P-gp-mediated MDR by downregulating P-gp expression, which may be partly attributed to the inhibition of ERK pathway. Dasatinib may play an important role in circumventing MDR when combined with other conventional antineoplastic drugs.

Gamabufotalin, a bufadienolide compound from toad venom, suppresses COX-2 expression through targeting IKKβ/NF-κB signaling pathway in lung cancer cells

BackgroundGamabufotalin (CS-6), a major bufadienolide of Chansu, has been used for cancer therapy due to its desirable metabolic stability and less adverse effect. However, the underlying mechanism of CS-6 involved in anti-tumor activity remains poorly understood.MethodsThe biological functions of gamabufotalin (CS-6) were investigated by migration, colony formation and apoptosis assays in NSCLC cells. The nuclear localization and interaction between transcriptional co-activator p300 and NF-κB p50/p65 and their binding to COX-2 promoter were analyzed after treatment with CS-6. Molecular docking study was used to simulate the interaction of CS-6 with IKKβ. The in vivo anti-tumor efficacy of CS-6 was also analyzed in xenografts nude mice. Western blot was used to detect the protein expression level.ResultsGamabufotalin (CS-6) strongly suppressed COX-2 expression by inhibiting the phosphorylation of IKKβ via targeting the ATP-binding site, thereby abrogating NF-κB binding and p300 recruitment to COX-2 promoter. In addition, CS-6 induced apoptosis by activating the cytochrome c and caspase-dependent apoptotic pathway. Moreover, CS-6 markedly down-regulated the protein levels of COX-2 and phosphorylated p65 NF-κB in tumor tissues of the xenograft mice, and inhibited tumor weight and size.ConclusionsOur study provides pharmacological evidence that CS-6 exhibits potential use in the treatment of COX-2-mediated diseases such as lung cancer.

Biotransformation of 11-keto-β-boswellic acid by Cunninghamella blakesleana.

11-Keto-β-boswellic acid (KBA), as one of the active constituents in the gum resin of Boswellia serrata, possesses significant biological activities including anti-inflammatory activity. However, its extensive metabolism and low polarity has limited the systemic availability of KBA. The present research was aimed to obtain and explore the various possible derivatives of KBA through biotransformation by Cunninghamella blakesleana AS 3.970. A total of ten transformed compounds were isolated and purified, and their chemical structures were characterized as 7β-hydroxy-11-keto-β-boswellic acid; 7β, 15α-dihydroxy-11-keto-β-boswellic acid ; 7β, 16β-dihydroxy-11-keto-β-boswellic acid; 7β, 16α-dihydroxy-11-keto-β-boswellic acid; 7β, 22β-dihydroxy-11-keto-β-boswellic acid; 7β, 21β-dihydroxy-11-keto-β-boswellic acid; 7β, 20β-dihydroxy-11-keto-β-boswellic acid; 7β, 30-dihydroxy-11-keto-β-boswellic acid; 3α, 7β-dihydroxy-11-oxours-12-ene-24, 30-dioic acid and 3α, 7β-dihydroxy-30-(2-hydroxypropanoyloxy)-11-oxours-12-en-24-oic acid by various spectroscopic methods. The biotransformation processes include hydroxylation, oxidation and esterification. Primary structure-activity relationships (SAR) of inhibitory effects on NO production in RAW 264.7 macrophage cells are discussed.

Human transporters, PEPT1/2, facilitate melatonin transportation into mitochondria of cancer cells: an implication of the therapeutic potential

Melatonin is present in virtually all organisms from bacteria to mammals, and it exhibits a broad spectrum of biological functions, including synchronization of circadian rhythms and oncostatic activity. Several functions of melatonin are mediated by its membrane receptors, but others are receptor‐independent. For the latter, melatonin is required to penetrate membrane and enters intracellular compartments. However, the mechanism by which melatonin enters cells remains debatable. In this study, it was identified that melatonin and its sulfation metabolites were the substrates of oligopeptide transporter (PEPT) 1/2 and organic anion transporter (OAT) 3, respectively. The docking analysis showed that the binding of melatonin to PEPT1/2 was attributed to their low binding energy and suitable binding conformation in which melatonin was embedded in the active site of PEPT1/2 and fitted well with the cavity in three‐dimensional space. PEPT1/2 transporters play a pivotal role in melatonin uptake in cells. Melatonins membrane transportation via PEPT1/2 renders its oncostatic effect in malignant cells. For the first time, PEPT1/2 were identified to localize in the mitochondrial membrane of human cancer cell lines of PC3 and U118. PEPT1/2 facilitated the transportation of melatonin into mitochondria. Melatonin accumulation in mitochondria induced apoptosis of PC3 and U118 cells. Thus, PEPT1/2 can potentially be used as a cancer cell‐targeted melatonin delivery system to improve the therapeutic effects of melatonin in cancer treatment.

Protective Effects of Calycosin Against CCl4-Induced Liver Injury with Activation of FXR and STAT3 in Mice

PurposeInvestigating the hepatoprotective effect of calycosin against acute liver injury in association with FXR activation and STAT3 phosphorylation.MethodsThe acute liver injury model was established by intraperitoneal injection of CCl4 in C57BL/6 mice. Serum alanine aminotransferase, aspartate aminotransferase, HE staining and TUNEL assay were used to identify the amelioration of the liver histopathological changes and hepatocytes apoptosis after calycosin treatment. ELISA kit and 5-bromo-2-deoxyuridine immunohistochemistry were used to measure the liver bile acid concentration and hepatocyte mitotic rate in vivo. The relation between calycosin and activation of FXR and STAT3 was comfirmed using the Luciferase assay, Molecular docking, Real-time PCR and Western Blot in vitro.ResultsThe liver histopathological changes, hepatocytes apoptosis, liver bile acid overload and hepatocyte mitosis showed significant changes after calycosin treatment. Calycosin promoted the expression of FXR target genes such as FoxM1B and SHP but the effect was reversed by FXR suppressor guggulsterone. Molecular docking results indicated that calycosin could be embedded into the binding pocket of FXR, thereby increasing the expressions of STAT3 tyrosine phosphorylation and its target genes, Bcl-xl and SOCS3.ConclusionsCalycosin plays a critical role in hepatoprotection against liver injury in association with FXR activation and STAT3 phosphorylation.