The roots of Platycodon grandiflorus are widely used as a crude drug. The active components include a variety of triterpenoid saponins. Recent studies have revealed that Cyt P450 monooxygenases (P450s) function as triterpene oxidases in triterpenoid saponin biosynthesis in many plant species. However, there have been no reports regarding triterpene oxidases in P. grandiflorus. In this study, we performed transcriptome analysis of three different P. grandiflorus tissues (roots, leaves and petals) using RNA sequencing (RNA-Seq) technology. We cloned six P450 genes that were highly expressed in roots, and classified them as belonging to the CYP716A, CYP716D and CYP72A subfamilies. We heterologously expressed these P450s in an engineered yeast strain that produces β-amyrin, one of the most common triterpenes in plants. Two of the CYP716A subfamily P450s catalyzed oxidation reactions of the β-amyrin skeleton. One of these P450s, CYP716A140v2, catalyzed a three-step oxidation reaction at C-28 on β-amyrin to produce oleanolic acid, a reaction performed by CYP716A subfamily P450s in a variety of plant species. The other P450, CYP716A141, catalyzed the hydroxylation of β-amyrin at C-16β. This reaction is unique among triterpene oxidases isolated to date. These results enhance our knowledge of functional variation among CYP716A subfamily enzymes involved in triterpenoid biosynthesis, and provide novel molecular tools for use in synthetic biology to produce triterpenoid saponins with pre-defined structures.

BackgroundHepatocellular carcinoma (HCC) develops in some patients who achieve sustained virological response (SVR) against hepatitis C virus (HCV) infection via anti-HCV therapy. To examine the pathogenesis of HCC development after HCV eradication, histopathological changes and clinical markers were evaluated in SVR patients.MethodsOf 654 SVR patients treated with interferon (IFN)-based therapies, 34 patients who had undergone liver biopsy before initiating IFN therapy and after SVR achievement were enrolled: 11 patients with HCC and 23 patients without HCC (male/female, 9/2 and 8/15, respectively: age, 58 ± 5 and 54 ± 11 years, respectively). We compared the clinical and histopathological factors between the two groups. Immunohistochemistry for Cytoglobin (CYGB) and α smooth muscle actin (α-SMA) was also performed.ResultsAt baseline, prior to initiating the IFN-based therapy, there were significant differences between the SVR-non-HCC and SVR-HCC groups in the male gender, HBc antibody positivity, prothrombin activity, and histological inflammatory grade. Histopathological evaluation, using the new Inuyama classification system, revealed an improvement in the inflammatory grade, from 2.1 ± 0.6 to 1.0 ± 0.6 (p < 0.0001), whereas the fibrosis stage remained unchanged, from 2.3 ± 0.9 to 2.0 ± 1.2 (p = 0.2749), during the 97 ± 72-month observation period in the SVR-HCC group. Both the grade and stage scores were significantly improved in the SVR-non-HCC group. The area of collagen deposition, evaluated using Sirius red staining, showed a marked decrease, from 18.6 ± 7.6% to 7.7 ± 4.6%, in the SVR-non-HCC group, with no change in the SVR-HCC group. CYGB- and α-SMA-positive hepatic stellate cells (HSCs), indicative of the HSC activated phenotype, remained in the fibrotic tissue of livers among patients in the SVR-HCC group.ConclusionStagnation of fibrosis regression is associated with a high risk for HCC after SVR. HSC activation may inhibit improvement in fibrosis after SVR and potentially contribute to hepatocarcinogenesis.

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Radiofrequency ablation (RFA) with artificial pleural effusion and/or artificial ascites has recently been recognized as a useful device for the treatment of tumors located on the liver surface and in the hepatic dome. Sixty patients (including 32 naive patients) with hepatocellular carcinoma (HCC) underwent RFA with artificial pleural effusion and/or ascites. We decided the precise indication for the use of artificial infusion according to the location of tumor. The total local recurrence rates at 1 and 2 years were 4% and 22%, respectively. The estimated survival rates of 32 naive patients at 1 and 3 years were 90% and 78%, respectively. The local recurrence rates of a tumor size of <3 cm and > = 3 cm at 2 years were 22% and 17%, respectively.

Oxygen (O 2 ) is required for cytochrome P450 (CYP)-dependent drug metabolism. Cytoglobin (CYGB) is a unique globin expressed exclusively in hepatic stellate cells (HSCs). However, its role in O 2 -dependent metabolism in neighboring hepatocytes remains unknown. This study provides evidence that CYGB in HSCs is involved in acetaminophen (N-acetyl-p-aminophenol; APAP)-induced hepatotoxicity. Serum alanine aminotransferase levels were higher in wild-type mice than in Cygb-null mice. Wild-type mice exhibited more severe hepatocyte necrosis around the central vein area compared with Cygb-null mice, thus indicating that CYGB deficiency protects against APAP-induced liver damage. Although no difference in the hepatic expression of CYP2E1, a key enzyme involved in APAP toxicity, was observed between wild-type and Cygb-null mice, the serum levels of the APAP metabolites cysteinyl-APAP and N-acetyl-cysteinyl-APAP were decreased in Cygb-null mice, suggesting reduced APAP metabolism in the livers of Cygb-null mice. In primary cultures, APAP-induced hepatocyte damage was increased by co-culturing with wild-type HSCs but not with Cygb-null HSCs. In addition, cell damage was markedly alleviated under low O 2 condition (5% O 2 ), suggesting the requirement of O 2 for APAP toxicity. Carbon tetrachloride-induced liver injury (CYP2E1-dependent), but not lipopolysaccharide/ D-galactosamine-induced injury (CYP2E1-independent), was similarly alleviated in Cygb-null mice. Considering the function of CYGB as O 2 carrier, these results strongly support the hypothesis that HSCs are involved in the CYP2E1-mediated xenobiotic activation by augmenting O 2 supply to hepatocytes. In conclusion, CYGB in HSCs contributes to the CYP-mediated metabolism of xenobiotics in hepatocytes by supplying O 2 for enzymatic oxidation. The metabolism of xenobiotics, including clinical drugs, occurs primarily in the liver. Most xenobiotic detoxification metabolism involves cytochrome P450 (CYP) enzymes, which are predominantly expressed in the liver, 1 and these processes occasionally result in acute hepatocyte damage. For instance, acetaminophen (N-acetyl-p-aminophenol; APAP) and carbon tetrachloride (CCl 4 ) are metabolized by CYP to generate toxic intermediates.APAP is commonly used as an antipyretic and analgesic drug. CYP-mediated APAP metabolism is known to generate N-acetyl-p-benzoquinone imine (NAPQI), which binds to cellular macromolecules and initiates hepatocyte damage if present at high concentrations under glutathione (GSH) depletion. 2 CYP1A2, CYP2A6, CYP2E1, and CYP3A have been identified as enzymes that generate NAPQI from APAP. 3 Among these CYPs, CYP2E1 has the lowest K m for APAP, and the CYP2E1-dependent metabolism of APAP can result in hepatotoxicity. 4 CYP2E1 is a monooxygenase that requires molecular oxygen (O 2 ) for its enzymatic activity. A recent study reported that the APAP-induced damage to primary mouse hepatocytes was reduced under a low O 2 concentration (5% O 2 ) compared with normoxic conditions (20% O 2 ). 5 Thus, APAP-indu...

Inosine triphosphatase polymorphism appeared to correlate with anemia incidence and RBV dose reduction during SOF/RBV therapy, but not the clinical outcome. Careful monitoring of Hb concentrations and prompt adjustment of RBV doses are required for successful treatment, particularly in patients harboring the ITPA CC genotype or age ≥ 65 years.

The response rate and overall survival after sorafenib administration in patients with advanced hepatocellular carcinoma are unsatisfactory. We herein present the case of a 65-year-old man with multiple lung metastases of hepatocellular carcinoma. Because the patient had liver cirrhosis of Child-Pugh B accompanied by pancytopenia, sorafenib administration was initiated at a dose of 400 mg daily. Although he received sorafenib for only 21 days, the patient exhibited complete regression of the tumors. There was no clinical evidence of recurrence without the administration of anticancer treatment. It is unique that short-term sorafenib treatment achieved a complete response.