Qianying Chen

Chronic inflammation up-regulates P-gp in peripheral mononuclear blood cells via the STAT3/Nf-κb pathway in 2,4,6-trinitrobenzene sulfonic acid-induced colitis mice

Patients with inflammatory bowel diseases, including Crohn’s disease and ulcerative colitis, often suffer drug intolerance. This resistance can be divided into intrinsic resistance and acquired resistance. Although there is agreement on acquired resistance, studies regarding intrinsic resistance have demonstrated inconsistencies, especially for Crohn’s disease. For this reason, an animal model of Crohn’s disease was induced with 2,4,6-trinitrobenzene sulfonic acid solution (TNBS), and intrinsic resistance was analyzed by measuring the function and expression of P-glycoprotein (P-gp) in peripheral mononuclear blood cells (PMBC), followed by mechanistic studies. The results revealed reduced retention of cyclosporine A in PMBC over-expressing P-gp in a TNBS-treated group and enhanced secretion of the cytokines IL-1β, IL-6, IL-17, and TNF-α as well as LPS in plasma. These cytokines and LPS can induce P-gp expression through the STAT3/Nf-κb pathway, contributing to a decrease of cyclosporine A retention, which can be reversed by the application of a P-gp inhibitor. Our results demonstrated that the sustained chronic inflammation could induce the intrinsic resistance presented as P-gp over-expression in PBMC in Crohn’s disease through STAT3/Nf-κb pathway and this resistance might be reversed by combinational usage of P-gp inhibitors.

Alternation of adriamycin penetration kinetics in MCF-7 cells from 2D to 3D culture based on P-gp expression through the Chk2/p53/NF-κB pathway

Monolayer cells are largely different from tumor masses, and might misguide drug screenings. 3D in vitro cell culture models simulate the characteristics of tumor masses in vivo and have recently been used in many studies of anti-cancer drugs. Among various 3D cell culture models, multi-cellular layer (MCL) models allow for the direct quantitative assessment of the penetration of chemotherapeutic agents through solid tissue environments without requiring the use of fluorescently labeled drugs or imaging molecules. Therefore, in our present study, a 3D-no base and embedded MCF-7 MCL model was successfully developed for a 14-day culture. Over time, its thickness and cell layers increased and exhibited highly proliferative properties and drug resistance to adriamycin (ADR) with markedly elevated IC50 values. Meanwhile, G2/M stage cycle arrest was also observed, which likely up-regulated P-gp expression through the Chk2/p53/NF-κB pathway. The elevated P-gp expression altered the ADR penetration kinetics in MCF-7 MCLs in vitro by accelerating the apparent penetration of ADR through the intercellular spaces of the MCLs. Additionally, a decreased ADR retention within tumor cells was observed, but could be significantly reversed by a P-gp inhibitor. The attenuated ADR retention in the deeper cells of tumor masses was confirmed in xenografted mice in vivo. This phenomenon could be elucidated by the mathematical modeling of penetration kinetics parameters. Our study provided a new model that evaluated and improved the quantification of the drug penetration kinetics, revealed the relationship between P-gp and drug penetration through tumor masses, and suggested the potential molecular mechanisms.

Non-antibiotic agent ginsenoside 20(S)-Rh2 enhanced the antibacterial effects of ciprofloxacin in vitro and in vivo as a potential NorA inhibitor

The aim of this study is to explore the potential enhancing effect of ginsenoside 20(S)-Rh2 (Rh2) towards ciprofloxacin (CIP) against Staphylococcus aureus (S. aureus) infection in vitro and in vivo, and analyze the possible mechanisms through NorA inhibition from a target cellular pharmacokinetic view. In combination with non-toxic dosage of Rh2, the susceptibilities of S. aureus strains to CIP were significantly augmented, and the antibacterial kinetics of CIP in the S. aureus strains were markedly promoted. This enhancing effect of Rh2 towards CIP was also observed in S. aureus infected peritonitis mice, with elevated survival rate and reduced bacteria counts in blood. However, Rh2 did not influence the plasma concentrations of CIP. Further analysis indicated that Rh2 significantly promoted the accumulations of CIP in S. aureus, and inhibited the NorA mediated efflux of pyronin Y. The expressions of NorA gene on S. aureus were positively correlated with the enhancing effect of Rh2 with CIP. This is the first report of the enhancing effect of Rh2 with CIP for S. aureus infection in vitro and in vivo, of which it is probably that Rh2 inhibited NorA-mediated efflux and promoted the accumulation of CIP in the bacteria.

Glycyrrhetic acid, but not glycyrrhizic acid, strengthened entecavir activity by promoting its subcellular distribution in the liver via efflux inhibition

Abstract Entecavir (ETV) is a superior nucleoside analogue used to treat hepatitis B virus (HBV) infection. Although its advantages over other agents include low viral resistance and the elicitation of a sharp decrease in HBV DNA, adverse effects such as hepatic steatosis, hepatic damage and lactic acidosis have also been reported. Glycyrrhizin has long been used as hepato‐protective medicine. The clinical combination of ETV plus glycyrrhizin in China displays better therapeutic effects and lower rates of liver damage. However, there is little evidence explaining the probable synergistic mechanism that exists between these two drugs from a pharmacokinetics view. Here, alterations in the plasma pharmacokinetics, tissue distribution, subcellular distribution, and in vitro and in vivo antiviral activity of ETV after combination with glycyrrhizic acid (GL) were analysed to determine the synergistic mechanisms of these two drugs. Specific efflux transporter membrane vesicles were also used to elucidate their interactions. The primary active GL metabolite, glycyrrhetic acid (GA), did not affect the plasma pharmacokinetics of ETV but promoted its accumulation in hepatocytes, increasing its distribution in the cytoplasm and nucleus and augmenting the antiviral efficiency of ETV. These synergistic actions were primarily due to the inhibitory effect of GA on MRP4 and BCRP, which transport ETV out of hepatocytes. In conclusion, GA interacted with ETV at cellular and subcellular levels in the liver through MRP4 and BCRP inhibition, which enhanced the antiviral activity of ETV. Our results partially explain the synergistic mechanism of ETV and GL from a pharmacokinetics view, providing more data to support the use of these compounds together in clinical HBV treatment. Graphical abstract Figure. No Caption available.

Magnesium Isoglycyrrhizinate attenuates lipopolysaccharide-induced depressive-like behavior in mice.

Magnesium Isoglycyrrhizinate (MI) is a magnesium salt of 18α-GA stereoisomer which has been reported to exert hepatoprotective activity. The aim of the present study was to ascertain the underlying mechanisms behind the action of Magnesium Isoglycyrrhizinate on neuroinflammatation and oxidative stress in LPS-stimulated mice. Mice were pretreated with Magnesium Isoglycyrrhizinate (MI, 25, 50mg/kg) as well as fluoxetine (Flu, positive control, 20mg/kg) once daily for one week before intraperitoneal injection of LPS (0.83mg/kg). Pretreatments with MI and Flu significantly improved immobility time in tail suspension test (TST) and forced swim test (FST) as well as locomotor activity in open-field test (OFT). In addition, the levels of pro-inflammatory cytokines and oxidative stress in serum and hippocampus were also suppressed effectively by MI and Flu administrations. Western blot analysis showed the up-regulated levels of p-Jak3, p-STAT3, p-NF-κBp65, and p-IκBα in mice exposed to LPS, while different degrees of down-regulation in these expression were observed in MI (25, 50mg/kg) and Flu (20mg/kg) groups respectively. Taken together, our obtained results demonstrated that Magnesium Isoglycyrrhizinate (MI) exhibited an antidepressant-like effect in LPS-induced mice, which might be mediated by JAK/STAT/NF-κB signaling pathway.

Shenmai injection enhances the cytotoxicity of chemotherapeutic drugs against colorectal cancers via improving their subcellular distribution

Shenmai injection (SMI) is a Chinese patent-protected injection, which was mainly made of Red Ginseng and Radix Ophiopogonis and widely used for treating coronary heart disease and tumors by boosting Qi and nourishing Yin. In this study we examined whether SMI could produce direct synergetic effects on the cytoxicity of adriamycin (ADR) and paclitaxel (PTX) in colorectal cancers in vivo and in vitro, and explored the underlying pharmacokinetic mechanisms. BALB/c nude mice with LoVo colon cancer xenografts were intraperitoneally injected with ADR (2 mg·kg−1·3d−1) or PTX (7.5 mg·kg−1·3d−1) with or without SMI (0.01 mL·g−1·d−1) for 13 d. Co-administration of SMI significantly enhanced the chemotherapeutic efficacy of ADR and PTX, whereas administration of SMI alone at the given dosage did not produce visible anti-cancer effects, The chemosensitizing action of SMI was associated with increased concentrations of ADR and PTX in the plasma and tumors. In Caco-2 and LoVo cells in vitro, co-treatment with SMI (2 μL/mL) significantly enhanced the cytotoxicity of ADR and PTX, and resulted in some favorable pharmacokinetic changes in the subcellular distribution of ADR and PTX. In addition, SMI-induced intracellular accumulation of ADR was closely correlated with the increased expression levels of P-glycoprotein in 4 colon cancer cell lines (r2=+0.8558). SMI enhances the anti-cancer effects of ADR and PTX in colon cancers in vivo and in vitro by improving the subcellular distributions of ADR and PTX.

Simultaneous determination of tenofovir alafenamide and its active metabolites tenofovir and tenofovir diphosphate in HBV-infected hepatocyte with a sensitive LC–MS/MS method

HIGHLIGHTSA LC–MS/MS method was established for simultaneous determination of tenofovir prodrug tenofovir alafenamide (TAF) and its metabolites tenofovir (TFV) and TFV diphosphate (TFV‐DP).Samples were collected by direct lyzing with 50% methanol, followed by further protein precipitation using pure methanol.Intracellular pharmacokinetics of TFV‐DP, TFV and TAF were investigated in HepG2.2.15 cells. Tenofovir was generated quickly along with rapid elimination of TAF, and immediately further phosphorylated into active form TFV‐DP largely. ABSTRACT Tenofovir (TFV), a first‐line anti‐viral agent, has been prepared as various forms of prodrugs for better bioavailability, lower systemic exposure and higher target cells loading of TFV to enhance efficacy and reduce toxicity. TFV undergoes intracellular phosphorylation to form TFV diphosphate (TFV‐DP) in target cell to inhibit viral DNA replication. Hence, TFV‐DP is the key active metabolite that exhibits anti‐virus activity, its intracellular exposure and half‐life determine the final activity. Therefore, simultaneous monitoring prodrug, TFV and TFV‐DP in target cells will comprehensively evaluate TFV prodrugs, both considering the stability of ester prodrug, and the intracellular exposure of TFV‐DP. Thus we intended to develop a convenient general analytical method, taking tenofovir alafenamide (TAF) as a representative of TFV prodrugs. A sensitive LC–MS/MS method was developed, and TAF, TFV and TFV‐DP were separated on a XSelect HSS T3 column (4.6 mm × 150 mm, 3.5 &mgr;m, Waters) with gradient elution after protein precipitation. The method provided good linearity for all the compounds (2–500 nM for TFV and TAF; 20–5000 nM for TFV‐DP) with the correlation coefficients (r) greater than 0.999. Intra‐ and inter‐day accuracies (in terms of relative error, RE < 10.4%) and precisions (in terms of coefficient of variation, CV < 14.1%) satisfied the standard of validation. The matrix effect, recovery and stability were also within acceptable criteria. Finally, we investigated the intracellular pharmacokinetics of TAF and its active metabolites in HepG2.2.15 cells with this method.

A Promising Microtubule Inhibitor Deoxypodophyllotoxin Exhibits Better Efficacy to Multidrug-Resistant Breast Cancer than Paclitaxel via Avoiding Efflux Transport

Multidrug resistance (MDR) is a common limitation for the clinical use of microtubule-targeting chemotherapeutic agents, and it is the main factor for poor prognoses in cancer therapy. Here, we report on deoxypodophyllotoxin (DPT), a promising microtubule inhibitor in phase 1, as a promising candidate to circumvent this obstacle. DPT remarkably suppressed tumor growth in xenograft mice bearing either paclitaxel (PTX)-sensitive MCF-7/S or acquired resistance MCF-7/Adr (MCF-7/A) cells. Also, DPT exhibited similar accumulation in both tumors, whereas PTX displayed much a lower accumulation in the resistant tumors. In vitro, DPT exhibited a much lower resistance index (0.552) than those of PTX (754.5) or etoposide (38.94) in both MCF-7/S and MCF-7/A cells. Flow cytometry analysis revealed that DPT (5 and 10 nM) caused arrest of the G2/M phase in the two cell lines, whereas PTX (up to 10 nM) had no effect on cell-cycle progression of the MCF-7/A cells. Microtubule dynamics assays revealed that DPT destabilized microtubule assembly in a different mode. Cellular pharmacokinetic assays indicated comparable intracellular and subcellular accumulations of DPT in the two cell lines but a much lower retention of PTX in the MCF-7/A cells. Additionally, transport assays revealed that DPT was not the substrate of P-glycoprotein, breast cancer resistance protein, or MDR-associated protein 2, indicating a lower occurrence rate of MDR. DPT might be a promising microtubule inhibitor for breast cancer therapy, especially for treatment of drug-resistant tumors.

LC–MS/MS method for the simultaneous determination of Lys-MCC-DM1, MCC-DM1 and DM1 as potential intracellular catabolites of the antibody-drug conjugate trastuzumab emtansine (T-DM1)

&NA; Lysine‐MCC‐DM1, MCC‐DM1 and DM1 are potential catabolites of trastuzumab emtansine (T‐DM1). A convenient liquid chromatography‐tandem mass spectrometry (LC–MS/MS) method was developed and validated to detect these catabolites simultaneously in in vitro investigations for the first time. Protein precipitation was utilized to prepare the samples. Chromatographic separation was achieved on a Phenomenex Kinetex C18 column (100 × 2.1 mm, 2.6 &mgr;m) with mobile‐phase gradient elution. The calibration curves of each analyte ranging from 1 to 100 nM showed good linearity (r2 > 0.995). The method was validated successfully and applied to the intracellular catabolism and regulation of T‐DM1. HighlightsLC–MS/MS method for simultaneous determination of Lys‐MCC‐DM1, MCC‐DM1 and DM1 was developed.Immediate protein precipitation was used instead of DM1 derivatization for sample preparation.The method was validated and applied to intracellular catabolism investigations of T‐DM1.

Bevacizumab-enhanced antitumor effect of 5-fluorouracil via upregulation of thymidine phosphorylase through vascular endothelial growth factor A/vascular endothelial growth factor receptor 2-specificity protein 1 pathway

Bevacizumab (Bv) can be used synergistically with fluoropyrimidine‐based chemotherapy to treat colorectal cancer. Whether and how it affects the delivery of fluoropyrimidine drugs is unknown. The present study aimed to explore the effect of Bv on the delivery of 5‐fluorouracil (5‐FU) to tumors and the underlying mechanism from metabolic perspective. Bv enhanced the anti‐tumor effects of 5‐FU in LoVo colon cancer xenograft mice and increased the 5‐FU concentration in tumors without affecting hepatic 5‐FU metabolism. Interestingly, Bv remarkably upregulated thymidine phosphorylase (TP) in tumors, which mediated the metabolic activation of 5‐FU. Although TP is reported to promote angiogenesis and resistance, the combination of Bv and 5‐FU resulted in anti‐angiogenesis and vessel normalization in tumors, indicating that the elevated TP mainly contributed to the enhanced response to 5‐FU. Bv also induced TP upregulation in LoVo cancer cells. Treatment with vascular endothelial growth factor receptor 2 (VEGFR2) antagonist apatinib and VEGFR2 silencing further confirmed TP upregulation. Bv and apatinib both enhanced the cytotoxicity of 5‐FU in LoVo cells, but there was no synergism with adriamycin and paclitaxel. We further demonstrated that the effect of Bv was dependent on VEGFR2 blockade and specificity protein 1 activation via MDM2 inhibition. In summary, Bv enhanced the accumulation of 5‐FU in tumors and the cytotoxicity of 5‐FU via TP upregulation. We provide data to better understand how Bv synergizes with 5‐FU from metabolic perspective, and it may give clues to the superiority of Bv in combination with fluoropyrimidine drugs compared to other chemotherapeutic drugs in colon cancer.