Effects of Danhong injection, a traditional Chinese medicine, on nine cytochrome P450 isoforms in vitro.

Danhong injection (DHI) is made from Salvia miltiorrhiza Bunge. and Carthamus tinctorius L. extract and is widely used in the clinical treatment of cardiovascular and cerebrovascular diseases. This study aimed to evaluate the effect of DHI on cytochrome P450 (CYP450) enzymes in vitro to predict drug-drug interactions based on CYP450 as combination therapy. To assess the inhibitory effect of DHI on CYP450, we detected the IC50 value of DHI on CYP450 in vitro by liquid chromatography/tandem mass spectrometry (LC-MS/MS). Simultaneously, the induction effect of DHI on CYP450s was also evaluated. The relative induction ratios of DHI on CYP1A2, CYP2B6 and CYP3A4 activity were calculated by LC-MS/MS. The expression level of CYP3A4 mRNA was determined by reverse transcription PCR (RT-PCR). The LC-MS/MS data showed DHI intensively inhibit CYP2A6 activity and the intensity of inhibition was followed by CYP2C8, CYP3A4, CYP2C19, CYP2B6, CYP2D6, CYP1A2, CYP2E1 and CYP2C9 in vitro. The results of RT-PCR showed that there is a certain induction of DHI on CYP3A4 mRNA in human primary hepatocytes in vitro. The study suggested that drug-drug interactions might occur in clinical co-administration of drugs owing to the CYP2A6 inhibition and CYP3A4 induction.

Effects of diosmetin on nine cytochrome P450 isoforms, UGTs and three drug transporters in vitro.

Diosmetin (3', 5, 7-trihydroxy-4'-methoxyflavone), a natural flavonoid from traditional Chinese herbs, has been used in various medicinal products because of its anticancer, antimicrobial, antioxidant, estrogenic and anti-inflammatory activity. However, flavonoids could affect the metabolic enzymes and cause drug-drug interactions (DDI), reducing the efficacy of co-administered drugs and potentially resulting in serious adverse reactions. To evaluate its potential to interact with co-administered drugs, the IC50 value of phase I cytochrome P450 enzymes (CYPs), phase II UDP-glucuronyltransferases (UGTs) and hepatic uptake transporters (organic cation transporters (OCTs), organic anion transporter polypeptides (OATPs) and Na+-taurocholate cotransporting polypeptides (NTCPs)) were examined in vitro by LC-MS/MS. Diosmetin showed strong inhibition of CYP1A2 in a concentration-dependent manner. The intensity of the inhibitory effect was followed by CYP2C8, CYP2C9, CYP2C19 and CYP2E1. For CYP2A6, CYP2B6, CYP2D6 and CYP3A4, diosmetin was found to have no significant inhibitory effects, and the induction effect on CYPs was not significant. For UGTs, diosmetin had a minimal inhibitory effect. In addition, the inhibitory effects of diosmetin on OATP and OCT1 were weak, and it had little effect on NTCP. This finding indicated that drug-drug interactions induced by diosmetin may occur through co-administration of drugs metabolized by CYP1A2.

Colony-Stimulating Factor 1 Receptor Blockade Inhibits Tumor Growth by Altering the Polarization of Tumor-Associated Macrophages in Hepatocellular Carcinoma.

Colony-stimulating factor-1 (CSF-1) and its receptor, CSF-1R, regulate the differentiation and function of macrophages and play an important role in macrophage infiltration in the context of hepatocellular carcinoma. The therapeutic effects of CSF-1R blockade in hepatocellular carcinoma remain unclear. In this study, we found that CSF-1R blockade by PLX3397, a competitive inhibitor with high specificity for CSF-1R tyrosine kinase, significantly delayed tumor growth in mouse models. PLX3397 inhibited the proliferation of macrophages in vitro, but intratumoral macrophage infiltration was not decreased by PLX3397 in vivo Gene expression profiling of tumor-associated macrophages (TAM) showed that TAMs from the PLX3397-treated tumors were polarized toward an M1-like phenotype compared with those from vehicle-treated tumors. In addition, PLX3397 treatment increased CD8+ T-cell infiltration, whereas CD4+ T-cell infiltration was decreased. Further study revealed that tumor cell-derived CSF-2 protected TAMs from being depleted by PLX3397. In conclusion, CSF-1R blockade delayed tumor growth by shifting the polarization rather than the depletion of TAMs. CSF-1R blockade warrants further investigation in the treatment of hepatocellular carcinoma. Mol Cancer Ther; 16(8); 1544-54. ©2017 AACR.

An improved substrate cocktail for assessing direct inhibition and time-dependent inhibition of multiple cytochrome P450s.

AIM: The substrate cocktail is frequently used to evaluate cytochrome P450 (CYP) enzyme-mediated drug interactions and potential interactions among the probe substrates. Here, we re-optimized the substrate cocktail method to increase the reliability and accuracy of screening for candidate compounds and expanded the method from a direct CYP inhibition assay to a time-dependent inhibition (TDI) assay. METHODS: In the reaction mixtures containing human liver microsome (0.1 mg/mL), both the concentrations of a substrate cocktail (phenacetin for 1A2, coumarin for 2A6, bupropion for 2B6, diclofenac for 2C9, dextromethorphan for 2D6, and testosterone for 3A4) and the incubation time were optimized. Metabolites of the substrate probes were simultaneously analyzed by multiple-reaction monitoring (MRM) using a routine LC/MS/MS. Direct CYP inhibition was validated using 7 inhibitors (α-naphthoflavone, tranylcypromine, ticlopidine, fluconazole, quinidine, ketoconazole and 1-ABT). The time-dependent inhibition was partially validated with 5 inhibitors (ketoconazole, verapamil, quinidine, paroxetine and 1-ABT). RESULTS: The inhibition curve profiles and IC50 values of 7 CYP inhibitors were approximate when a single substrate and the substrate cocktail were tested, and were consistent with the previously reported values. Similar results were obtained in the IC50 shifts of 5 inhibitors when a single substrate and the substrate cocktail were tested in the TDI assay. CONCLUSION: The 6-in-1 substrate cocktail (for 1A2, 2A6, 2B6, 2C9, 2D6 and 3A) is reliable for assessing CYP inhibition and time-dependent inhibition of drug candidates.

The herbal compound Songyou Yin (SYY) inhibits hepatocellular carcinoma growth and improves survival in models of chronic fibrosis via paracrine inhibition of activated hepatic stellate cells.

Chronic fibrosis is a major risk factor for the development of hepatocellular carcinoma (HCC). The pathological progression of hepatic fibrosis has been linked to cellular processes that promote tumor growth and metastasis. Several recent studies have highlighted the cross-talk between tumor cells and activated hepatic stellate cells (aHSCs) in HCC. The herbal compound Songyou Yin (SYY) is known to attenuate hepatoma cell invasion and metastasis via down-regulation of cytokine secretion by aHSCs. However the underlying mechanism of SYY treatment in reversal of hepatic fibrosis and metastasis of liver cancers is not known. In the current study, a nude mouse model with liver fibrosis bearing orthotopic xenograft was established and we found that SYY could reduce associated fibrosis, inhibit tumor growth and improve survival. In the subcutaneous tumor model with fibrosis, we found that SYY could inhibit liver cancer. In vitro, hepatoma cells incubated with conditioned media (CM) from SYY treated aHSCs showed reduced proliferation, decrease in colony formation and invasive potential. SYY treated group showed altered gene expression, with 1205 genes up-regulated and 1323 genes down-regulated. Gene cluster analysis indicated that phosphatidylinositol-3-kinase (PI3K) was one of the key genes altered in the expression profiles. PI3K related markers were all significantly down-regulated. ELISA also indicated decreased secretion of cytokines which were regulated by PI3K/AKT signaling after SYY treatment in the hepatic stellate cell line, LX2. These data clearly demonstrate that SYY therapy inhibits HCC invasive and metastatic potential and improves survival in nude mice models with chronic fibrosis background via inhibition of cytokine secretion by activated hepatic stellate cells.

Arsenic trioxide induces differentiation of CD133+ hepatocellular carcinoma cells and prolongs posthepatectomy survival by targeting GLI1 expression in a mouse model.

BACKGROUND: Cancer stem cells (CSCs) play a key role in the posthepatectomy recurrence of hepatocellular carcinoma (HCC). CD133+ HCC cells exhibit liver CSC-like properties, and CSC differentiation-inducing therapy may lead these cells to lose their self-renewal ability and may induce terminal differentiation, which may in turn allow their malignant potential to be controlled. Because arsenic trioxide (As2O3) increases remission rates and prolongs survival among patients with acute promyelocytic leukemia by inducing differentiation and apoptosis of leukemic cells, we hypothesized that As2O3 might also inhibit HCC recurrence and prolong survival time after hepatectomy by inducing differentiation of HCC CSCs. METHODS: We evaluated the As2O3 induced differentiation of human HCC CSCs and its mechanism in vitro, and we investigated the effects of treatment with As2O3 on recurrence rates and median survival in a mouse xenograft model. RESULTS: We found that As2O3 induced HCC CSC differentiation by down-regulating the expression of CD133 and some stemness genes, thus inhibiting the cells' self-renewal ability and tumorigenic capacity without inhibiting their proliferation in vitro. In vivo experiments indicated that As2O3 decreased recurrence rates after radical resection and prolonged survival in a mouse model. As2O3, which shows no apparent toxicity, may induce HCC CSC differentiation by down-regulating the expression of GLI1. CONCLUSIONS: We found that As2O3 induced HCC CSC differentiation, inhibited recurrence, and prolonged survival after hepatectomy by targeting GLI1expression. Our results suggest that the clinical safety and utility of As2O3 should be further evaluated.

MicroRNA-26a inhibits angiogenesis by down-regulating VEGFA through the PIK3C2α/Akt/HIF-1α pathway in hepatocellular carcinoma.

BACKGROUND & AIMS: microRNAs (miRNAs) have been reported to regulate angiogenesis by down-regulating the expression of pro-angiogenic or anti-angiogenic factors. The aims of this study were to investigate whether miR-26a inhibited angiogenesis by down-regulating vascular endothelial growth factor A (VEGFA) and its clinical relevance in hepatocellular carcinoma (HCC). METHODS: The expression of miR-26a was modified in HepG2 and HCCLM3 cell lines respectively, and a panel of angiogenic factors was measured by real-time PCR in the cells. A luciferase reporter assay was used to validate the target gene of miR-26a. Specific inhibitors of signal transduction pathway and siRNA approaches were used to explore the regulatory mechanism of miR-26a. Migration and tube forming assays were conducted to show the changes of angiogenesis induced by miR-26a and its target genes. Finally animal studies were used to further validate those findings. RESULTS: Ectopic expression of miR-26a exhibited decreased levels of VEGFA in HepG2 cells. Migration and tube forming of human umbilical vein endothelial cells (HUVECs) were decreased in the conditioned medium from ectopic expression of miR-26a in HepG2 cells compared to control HepG2 cells. The pro-angiogenic effects of the conditioned medium of HepG2 cells on HUVECs were specifically decreased by LY294002, YC-1, and bevacizumab. Integrated analysis disclosed PIK3C2α as a downstream target gene of miR-26a. Ectopic expression of miR-26a suppressed ectopic and orthotopic tumor growth and vascularity in nude mice. The results in HCCLM3 were consistent with those in HepG2. miR-26a expression was inversely correlated with VEGFA expression in HCC patients. CONCLUSIONS: miR-26a modulated angiogenesis of HCC through the PIK3C2α/Akt/HIF-1α/VEGFA pathway. The expression of VEGFA was inversely correlated with miR-26a expression in HCC tumors.

Aspirin minimized the pro-metastasis effect of sorafenib and improved survival by up-regulating HTATIP2 in hepatocellular carcinoma.

BACKGROUND AIMS: We previously demonstrated the pro-metastasis effect of sorafenib in hepatocellular carcinoma (HCC), which is mediated by down-regulation of tumor suppressor HTATIP2. The aim of the present study was to determine whether aspirin minimizes this effect and improves survival. METHODS: The effects of sorafenib, aspirin, and combined sorafenib and aspirin were observed in HCCLM3 and HepG2 xenograft nude mice. Tumor growth, intrahepatic metastasis (IHM), lung metastasis, and survival were assessed. Polymerase chain reaction (PCR) array, real-time (RT)-PCR, and Western blotting were used to examine gene expression. The anti-invasion and anti-metastasis effects of aspirin were studied in HTATIP2-knockdown and HTATIP2-overexpressing HCC cell lines. The molecular mechanism of HTATIP2 regulation by aspirin was explored. RESULTS: Aspirin suppressed the pro-invasion and pro-metastasis effects of sorafenib in HCC and up-regulated HTATIP2 expression. Aspirin did not inhibit the proliferation of HCC cells, but it decreased the invasiveness of HCC with lower expression of HTATIP2 and increased expression of a set of markers, indicating a mesenchymal-to-epithelial transition in tumor cells. The up-regulation of HTATPI2 expression by aspirin is most likely mediated through inhibition of cyclooxygenase (COX) 2 expression. CONCLUSIONS: Aspirin minimized the pro-metastasis effect of sorafenib by up-regulating the tumor suppressor HTATIP2; this mechanism is mediated through inhibition of COX2.

Suppression of natural killer cells by sorafenib contributes to prometastatic effects in hepatocellular carcinoma.

Sorafenib, a multi-tyrosine kinase inhibitor, is a standard treatment for advanced hepatocellular carcinoma (HCC). The present study was undertaken to determine whether the growth and metastasis of HCC were influenced in mice receiving sorafenib prior to implantation with tumors, and to investigate the in-vivo and in-vitro effect of sorafenib on natural killer (NK) cells. In sorafenib-pretreated BALB/c nu/nu mice and C57BL/6 mice, tumor growth was accelerated, mouse survival was decreased, and lung metastasis was increased. However, the depletion of NK1.1(+) cells in C57BL/6 mice eliminated sorafenib-mediated pro-metastatic effects. Sorafenib significantly reduced the number of NK cells and inhibited reactivity of NK cells against tumor cells, in both tumor-bearing and tumor-free C57BL/6 mice. Sorafenib down-regulated the stimulatory receptor CD69 in NK cells of tumor-bearing mice, but not in tumor-free mice, and inhibited proliferation of NK92-MI cells, which is associated with the blocking of the PI3K/AKT pathway, and inhibited cytotoxicity of NK cells in response to tumor targets, which was due to impaired ERK phosphorylation. These results suggest immunotherapeutic approaches activating NK cells may enhance the therapeutic efficacy of sorafenib in HCC patients.

Fe-doped and ZnO-pillared titanates as visible-light-driven photocatalysts.

Fe-doped cesium titanate was obtained by a solid state reaction with a mixture of Cs(2)CO(3), TiO(2), and Fe(2)O(3). ZnO-pillared doped titanate nanocomposite was successfully fabricated by exfoliating doped titanate and restacking its nanosheets with ZnO nanoparticles. The resulting nanocomposite was characterized by powder X-ray diffraction, scanning electron microscope, X-ray photoelectron spectroscopy, N(2) adsorption-desorption measurement, thermogravimetric analysis and UV-vis spectroscopy. It was revealed that the present nanocomposite exhibits greatly increased specific surface area with mesoporous texture and that there exists an electronic coupling between the host sheets and the guest nanoparticles in the pillared system. The results of degradation of methylene blue under visible light radiation suggest that doping iron ions improves the material spectral response region and that hybridizing with ZnO nanopillars can suppress the recombination of photogenerated electron-hole pairs.

Overexpression of platelet-derived growth factor receptor alpha in endothelial cells of hepatocellular carcinoma associated with high metastatic potential.

PURPOSE: Little information is available on the heterogeneity of the vascular endothelium in hepatocellular carcinoma. The aim of this study was to identify the differentially expressed genes in tumor endothelial cells from highly metastatic hepatocellular carcinoma. EXPERIMENTAL DESIGN: Magnetic beads conjugated with anti-CD31 antibody were used to isolate vascular endothelial cells from hepatocellular carcinoma xenografts with different metastatic potentials in nude mice. Gene expression profiles for different endothelial cells were compared by use of cDNA microarray. The up-regulated gene was confirmed by reverse transcription-PCR, real-time PCR, and immunohistochemistry. RESULTS: cDNA microarray analysis revealed differential expression patterns in seven genes consistently presented in endothelial cells isolated from hepatocellular carcinoma with different metastatic potentials. Overexpression of platelet-derived growth factor receptor alpha was found only in the endothelium of highly metastatic hepatocellular carcinoma, which was confirmed by reverse transcription-PCR, real-time PCR, and immunohistochemistry. Oral administration of STI571 (imatinib mesylate or Glivec), a protein tyrosine kinase inhibitor of platelet-derived growth factor receptor, combined with s.c. injection of IFN-alpha not only effectively reduced tumor weight (by 81.8%) and microvessel density (by 70.2%) but also inhibited lung metastasis (by 100%). Furthermore, immunohistochemical analysis of platelet-derived growth factor receptor alpha in human hepatocellular carcinoma tissues revealed its correlation with postoperative recurrence, especially in patients without microvessel invasion. CONCLUSIONS: The gene expression of hepatocellular carcinoma vascular endothelium is different between tumors with different metastatic potential. Platelet-derived growth factor receptor alpha, which is overexpressed in endothelium of highly metastatic hepatocellular carcinoma, may serve as a biomarker for predicting metastasis and a therapeutic target for highly metastatic hepatocellular carcinoma.