Risk factors for hepatocellular carcinoma at baseline and 1 year after initiation of nucleos(t)ide analog therapy for chronic hepatitis B.

Nucleos(t)ide analogs (NAs) cannot completely suppress the risk of hepatocellular carcinoma (HCC) in patients with chronic hepatitis B (CHB). This study aimed to identify the risk factors for HCC development in naïve CHB patients treated with current NA. Patients receiving NA (n = 905) were recruited retrospectively from the 17 hospitals of the Japanese Red Cross Liver Study Group. All treatment-naïve patients had been receiving current NA continuously for more than 1 year until the end of the follow-up. We analyzed the accuracy of predictive risk score using the area under receiver operating characteristic curve. The albumin-bilirubin (ALBI) score was significantly improved by NA therapy (-0.171 ± 0.396; p < 0.001 at Week 48). A total of 72 (8.0%) patients developed HCC over a median follow-up of 6.2 (1.03-15.7) years. An independent predictive factor of HCC development was older age, cirrhosis, lower platelet counts at baseline and ALBI score, and alpha-fetoprotein (AFP) at 1 year after NA therapy according to multivariate analysis. The accuracy was assessed using the PAGE-B, mPAGE-B, aMAP, APA-B, and REAL-B scores that included these factors. Discrimination was generally acceptable for these models. aMAP and REAL-B demonstrated high discrimination with 0.866/0.862 and 0.833/0.859 for 3- and 5-year prediction from the status of 1 year after NA therapy, respectively. Baseline age and platelet count, as well as ALBI and AFP one year after NA, were useful for stratifying carcinogenesis risk. The aMAP and REAL-B scores were validated with high accuracy in Japanese CHB patients.

A Dual Anti-Inflammatory and Anti-Proliferative 3-Styrylchromone Derivative Synergistically Enhances the Anti-Cancer Effects of DNA-Damaging Agents on Colon Cancer Cells by Targeting HMGB1-RAGE-ERK1/2 Signaling.

The current anti-cancer treatments are not enough to eradicate tumors, and therefore, new modalities and strategies are still needed. Most tumors generate an inflammatory tumor microenvironment (TME) and maintain the niche for their development. Because of the critical role of inflammation via high-mobility group box 1 (HMGB1)-receptor for advanced glycation end-products (RAGE) signaling pathway in the TME, a novel compound possessing both anti-cancer and anti-inflammatory activities by suppressing the HMGB1-RAGE axis provides an effective strategy for cancer treatment. A recent work of our group found that some anti-cancer 3-styrylchromones have weak anti-inflammatory activities via the suppression of this axis. In this direction, we searched such anti-cancer molecules possessing potent anti-inflammatory activities and discovered 7-methoxy-3-hydroxy-styrylchromone (C6) having dual suppressive activities. Mechanism-of-action studies revealed that C6 inhibited the increased phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) under the stimulation of HMGB1-RAGE signaling and thereby suppressed cytokine production in macrophage-like RAW264.7 cells. On the other hand, in colorectal cancer HCT116 cells, C6 inhibited the activation of ERK1/2, cyclin-dependent kinase 1, and AKT, down-regulated the protein level of XIAP, and up-regulated pro-apoptotic Bax and caspase-3/7 expression. These alterations are suggested to be involved in the C6-induced suppression of cell cycle/proliferation and initiation of apoptosis in the cancer cells. More importantly, in cancer cells, the treatment of C6 potentiates the anti-cancer effects of DNA-damaging agents. Thus, C6 may be a promising lead for the generation of a novel class of cancer therapeutics.

A 3-styrylchromone converted from trimebutine 3D pharmacophore possesses dual suppressive effects on RAGE and TLR4 signaling pathways.

Receptor for advanced glycation end-products (RAGE) and Toll-like receptors (TLRs) are potential therapeutic targets in the treatment of acute and chronic inflammatory diseases. We previously reported that trimebutine, a spasmolytic drug, suppresses RAGE pro-inflammatory signaling pathway in macrophages. The aim of this study was to convert trimebutine to a new small molecule using in silico 3D pharmacophore similarity search, and dissect the mechanistic anti-inflammatory basis. Of note, a unique 3-styrylchromone (3SC), 7-methoxy-3-trimethoxy-SC (7M3TMSC), converted from trimebutine 3D pharmacophore potently suppressed both high mobility group box 1-RAGE and lipopolysaccharide-TLR4 signaling pathways in macrophage-like RAW264.7 cells. More importantly, 7M3TMSC inhibited the phosphorylation of extracellular signaling-regulated kinase 1 and 2 (ERK1/2) and downregulated the production of cytokines, such as interleukin-6. Furthermore, 3D pharmacophore-activity relationship analyses revealed that the hydrogen bond acceptors of the trimethoxy groups in a 3-styryl moiety and the 7-methoxy-group in a chromone moiety in this compound are significant in the dual anti-inflammatory activity. Thus, 7M3TMSC may provide an important scaffold for the development of a new type of anti-inflammatory dual effective drugs targeting RAGE/TLR4-ERK1/2 signaling.

Trimebutine suppresses Toll-like receptor 2/4/7/8/9 signaling pathways in macrophages.

Because of the critical roles of Toll-like receptors (TLRs) and receptor for advanced glycation end-products (RAGE) in the pathophysiology of various acute and chronic inflammatory diseases, continuous efforts have been made to discover novel therapeutic inhibitors of TLRs and RAGE to treat inflammatory disorders. A recent study by our group has demonstrated that trimebutine, a spasmolytic drug, suppresses the high mobility group box 1‒RAGE signaling that is associated with triggering proinflammatory signaling pathways in macrophages. Our present work showed that trimebutine suppresses interleukin-6 (IL-6) production in lipopolysaccharide (LPS, a stimulant of TLR4)-stimulated macrophages of RAGE-knockout mice. In addition, trimebutine suppresses the LPS-induced production of various proinflammatory cytokines and chemokines in mouse macrophage-like RAW264.7 cells. Importantly, trimebutine suppresses IL-6 production induced by TLR2-and TLR7/8/9 stimulants. Furthermore, trimebutine greatly reduces mortality in a mouse model of LPS-induced sepsis. Studies exploring the action mechanism of trimebutine revealed that it inhibits the LPS-induced activation of IL-1 receptor-associated kinase 1 (IRAK1), and the subsequent activations of extracellular signal-related kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), and nuclear factor-κB (NF-κB). These findings suggest that trimebutine exerts anti-inflammatory effects on TLR signaling by downregulating IRAK1‒ERK1/2‒JNK pathway and NF-κB activity, thereby indicating the therapeutic potential of trimebutine in inflammatory diseases. Therefore, trimebutine can be a novel anti-inflammatory drug-repositioning candidate and may provide an important scaffold for designing more effective dual anti-inflammatory drugs that target TLR/RAGE signaling.

Trimebutine attenuates high mobility group box 1-receptor for advanced glycation end-products inflammatory signaling pathways.

We previously identified papaverine as an inhibitor of receptor for advanced glycation end-products (RAGE) and showed its suppressive effect on high mobility group box 1 (HMGB1)-mediated responses to inflammation. Here, we found trimebutine to be a 3D pharmacophore mimetics of papaverine. Trimebutine was revealed to have more potent suppressive effects on HMGB1-induced production of pro-inflammatory cytokines, such as interleukin-6 and tumor necrosis factor-α in macrophage-like RAW264.7 cells and mouse bone marrow primarily differentiated macrophages than did papaverine. However, the inhibitory effect of trimebutine on the interaction of HMGB1 and RAGE was weaker than that of papaverine. Importantly, mechanism-of-action analyses revealed that trimebutine strongly inhibited the activation of RAGE downstream inflammatory signaling pathways, especially the activation of extracellular signal-regulated kinase 1 and 2 (ERK1/2), which are mediator/effector kinases recruited to the intracellular domain of RAGE. Consequently, the activation of Jun amino terminal kinase, which is an important effector kinase for the up-regulation of pro-inflammatory cytokines, was inhibited. Taken together, these results suggest that trimebutine may exert its suppressive effect on the HMGB1-RAGE inflammatory signal pathways by strongly blocking the recruitment of ERK1/2 to the intracellular tail domain of RAGE in addition to its weak inhibition of the extracellular interaction of HMGB1 with RAGE. Thus, trimebutine may provide a unique scaffold for the development of novel dual inhibitors of RAGE for inflammatory diseases.

Papaverine identified as an inhibitor of high mobility group box 1/receptor for advanced glycation end-products interaction suppresses high mobility group box 1-mediated inflammatory responses.

The interaction of high mobility group box 1 (HMGB1), which is secreted from immune and dying cells during cellular infection and injury, and receptor for advanced glycation end-products (RAGE) appears to be critical for acute and chronic inflammatory disorders. Here we designed a unique cyclic ß-hairpin peptide (Pepb2), which mimics the predicted RAGE-binding domain of HMGB1. Pepb2 competitively inhibited HMGB1/RAGE interaction. We then identified papaverine as a Pepb2 mimetic by in silico 3D-structural similarity screening from the DrugBank library. Papaverine was found to directly inhibit HMGB1/RAGE interaction. It also suppressed the HMGB1-mediated production of pro-inflammatory cytokines, IL-6 and TNF-α, in mouse macrophage-like RAW264.7 cells and bone marrow-derived macrophages. In addition, papaverine attenuated mortality in cecal ligation puncture-induced sepsis model mice. Taken together, these findings indicate that papaverine could become a useful therapeutic against HMGB1/RAGE-mediated sepsis and other inflammatory diseases.

Real-world efficacy and safety of sofosbuvir + ribavirin for hepatitis C genotype 2: A nationwide multicenter study by the Japanese Red Cross Liver Study Group.

AIM: This study aimed to describe the real-world efficacy and safety of sofosbuvir (SOF) + ribavirin (RBV) for chronic hepatitis C, genotype 2. METHODS: This was a retrospective analysis of a nationwide, multicenter registry including 914 hepatitis C genotype 2 Japanese patients treated with SOF + RBV for 12 weeks. The rate of sustained virologic response at 12 weeks after treatment (SVR12), incidence of adverse events, and changes in serological parameters were analyzed. RESULTS: Treatment was completed in 98.9% of patients. Ribavirin dose reduction was required in 29.7% of patients. The SVR12 rate was 96.8% in the intention-to-treat population and 97.6% in the per-protocol population. Factors associated with SVR12 were absence of advanced fibrosis (odds ratio, 5.76, P = 0.003) and interferon-treatment-naïve status (odds ratio, 4.79, P = 0.017). Dose reduction or total adherence of RBV was not associated with SVR. The resistance-associated substitution S282 T in NS5B was not detected in any patient at virologic failure. Serum albumin levels significantly increased, and the degree of increase was greater in patients with advanced fibrosis than in those without (0.21 ± 0.32 vs. 0.05 ± 0.29, P < 0.0001). Alpha-fetoprotein decreased significantly at end of treatment (P < 0.0001), and the degree of decrease was greater in patients with advanced fibrosis than in those without (21.7 ± 60.8 vs. 2.5 ± 15.5, P < 0.001). The most commonly reported adverse event was anemia (13.7%). CONCLUSIONS: Treatment with SOF + RBV was highly effective and safe in Japanese patients with HCV genotype 2 infection.

Factors predicting overall response and overall survival in hepatocellular carcinoma patients undergoing balloon-occluded transcatheter arterial chemoembolization: A retrospective cohort study.

AIM: This study aimed to investigate the factors predicting overall response and overall survival in hepatocellular carcinoma patients undergoing balloon-occluded transcatheter arterial chemoembolization (B-TACE). METHODS: Sixty-six patients treated with B-TACE at a Japanese tertiary referral hospital between January 2011 and August 2015 were included in this retrospective cohort study. RESULTS: The overall response was classified as complete response, partial response, stable disease, and progressive disease in 35 (53.0%), 7 (10.6%), 13 (19.7%), and 11 (16.7%) patients, respectively. The response rate was 63.6%, and the disease control rate was 83.3%. The number of tumors (hazard ratio [HR], 4.44; 95% confidence interval [CI], 1.26-15.7; P = 0.021) and α-fetoprotein level (HR, 11.40; 95% CI, 2.75-46.9; P < 0.001) were significantly associated with the tumor response in a multivariate analysis. The 1-, 2-, and 3-year survival rates were 76.8% (95% CI, 64.5-85.3%), 57.3% (95% CI, 42.3-69.7%), and 46.7% (95% CI, 30.7-61.2%), respectively. The median survival time was 902 days. Albumin (≥3.4 g/dL) (HR, 0.28; 95% CI, 0.12-0.63; P = 0.002) and overall response (complete response and partial response) (HR, 0.33; 95% CI, 0.16-0.71; P = 0.004) were factors significantly associated with overall survival in a multivariate analysis. No mortalities were observed, but biloma requiring percutaneous transhepatic biliary drainage occurred in one patient (1.5%). CONCLUSION: Balloon-occluded transcatheter arterial chemoembolization may exert a good antitumor effect and result in good overall survival in select hepatocellular carcinoma patients.

[Is Balloon-Occluded Transcatheter Arterial Chemoembolization Effective in Patients with Hepatocellular Carcinoma That Is Unresponsive to Conventional Transcatheter Arterial Chemoembolization ?]

We previously reported that the antitumor response of balloon-occluded transcatheter arterial chemoembolization(BTACE) is better than that of conventional transcatheter arterial chemoembolization(C-TACE). Thus far, little attention has been paid on the efficacy of B-TACE using the same antitumor agents for hepatocellular carcinoma(HCC)that is unresponsive to C-TACE, which is defined as C50% necrosis of the targeted nodules or the appearance of new lesions in the liver 1-3 months following one C-TACE procedure. Therefore, this study focused on the efficacy of B-TACE using the same antitumor agents for HCC that is unresponsive to C-TACE. Fourteen patients treated with B-TACE at our institution were retrospectively investigated between January 2011 and August 2015. The median age was 76(interquartile range[IQR]70-79)years, and 9 patients(64.3%)were men. A total of 9(64.3%)and 5(35.7%)patients had the Child-Pugh class A and B, respectively. The median maximum tumor diameter was 30(IQR 18-40)mm, and 4(28.6%), 3(21.4%), 0(0.0%), and 7(50.0%) patients had 1, 2, 3, andB4 tumors, respectively. The antitumor effects were CR, PR, SD, and PD in 6(42.9%), 1(7.1%), 3 (21.4%), and 7(28.6%)patients, respectively. The response and disease control rates were 50% and 71.4%, respectively. Our results suggest that B-TACE is an effective modality for the treatment of HCC that is unresponsive to C-TACE.

Structural insight into the active site of mushroom tyrosinase using phenylbenzoic acid derivatives.

So far, many inhibitors of tyrosinase have been discovered for cosmetic and clinical agents. However, the molecular mechanisms underlying the inhibition in the active site of tyrosinase have not been well understood. To explore this problem, we examined here the inhibitory effects of 4'-hydroxylation and methoxylation of phenylbenzoic acid (PBA) isomers, which have a unique scaffold to inhibit mushroom tyrosinase. The inhibitory effect of 3-PBA, which has the most potent inhibitory activity among the isomers, was slightly decreased by 4'-hydroxylation and further decreased by 4'-methoxylation against mushroom tyrosinase. Surprisingly, 4'-hydroxylation but not methoxylation of 2-PBA appeared inhibitory activity. On the other hand, both 4'-hydroxylation and methoxylation of 4-PBA increased the inhibitory activity against mushroom tyrosinase. In silico docking analyses using the crystallographic structure of mushroom tyrosinase indicated that the carboxylic acid or 4'-hydroxyl group of PBA derivatives could chelate with cupric ions in the active site of mushroom tyrosinase, and that the interactions of Asn260 and Phe264 in the active site with the adequate-angled biphenyl group are involved in the inhibitory activities of the modified PBAs, by parallel and T-shaped π-π interactions, respectively. Furthermore, Arg268 could fix the angle of the aromatic ring of Phe264, and Val248 is supposed to interact with the inhibitors as a hydrophobic manner. These results may enhance the structural insight into mushroom tyrosinase for the creation of novel tyrosinase inhibitors.

Discovery of a new type of scaffold for the creation of novel tyrosinase inhibitors.

Tyrosinase is known as the key enzyme for melanin biosynthesis, which is effective in preventing skin injury by ultra violet (UV). In past decades, tyrosinase has been well studied in the field of cosmetics, medicine, agriculture and environmental sciences, and a lot of tyrosinase inhibitors have been developed for their needs. Here, we searched for new types of tyrosinase inhibitors and found phenylbenzoic acid (PBA) as a unique scaffold. Among three isomers of PBA, 3-phenylbenzoic acid (3-PBA) was revealed to be the most potent inhibitor against mushroom tyrosinase (IC50=6.97µM, monophenolase activity; IC50=36.3µM, diphenolase activity). The kinetic studies suggested that the apparent inhibition modes for the monophenolase and diphenolase activities were noncompetitive and mixed type inhibition, respectively. Analyses by in silico docking studies using the crystallographic structure of mushroom tyrosinase indicated that the carboxylic acid group of the 3-PBA could adequately bind to two cupric ions in the tyrosinase. To prove this hypothesis, we examined the effect of modification of the carboxylic acid group of the 3-PBA on its inhibitory activity. As expected, the esterification abrogated the inhibitory activity. These observations suggest that 3-PBA is a useful lead compound for the generation of novel tyrosinase inhibitors and provides a new insight into the molecular basis of tyrosinase catalytic mechanisms.

Safety and efficacy of balloon-occluded transcatheter arterial chemoembolization using miriplatin for hepatocellular carcinoma.

AIM: Balloon-occluded transcatheter arterial chemoembolization (B-TACE) using a microballoon catheter was performed to administrate miriplatin, and the early therapeutic efficacy and safety of the procedure were evaluated. METHODS: Out of 158 patients who received miriplatin using B-TACE for hepatocellular carcinoma, 49 patients with a single lesion at either stage I or II (according to the Liver Cancer Study Group of Japan) were evaluated in comparison with 48 matched patients who received miriplatin using conventional TACE (C-TACE). RESULTS: The mean total dose and median dose of miriplatin in each group were 32.5 ± 31.7 mg and 20 mg (C-TACE) and 50.1 ± 31.3 mg and 40 mg (B-TACE), respectively (P < 0.01). The treatment effect (TE) on the target nodule classified as TE4, TE3, TE2 or TE1 was 39.6%, 33.3%, 25.0% and 2.1%, respectively, in the C-TACE group, and 55.1%, 38.8%, 4.1% and 2.0%, respectively, in the B-TACE group. Therefore, the TE was significantly higher in the B-TACE group (P < 0.05). Although abdominal blood tests revealed adverse, increased levels of serum alanine aminotransferase (ALT) in a significantly higher number of B-TACE-treated patients, serum ALT levels returned to baseline levels in all patients within 1 month. There were no significant differences in clinical symptoms between the two groups. CONCLUSION: Compared with C-TACE, B-TACE significantly improved cancer nodule control, and it was satisfactory in terms of safety. B-TACE is an effective procedure that enhances the effects of catheterization with miriplatin.

Structure-activity relationships of the thujaplicins for inhibition of human tyrosinase.

Tyrosinase inhibitors have become increasingly critical agents in cosmetic, agricultural, and medicinal products. Although a large number of tyrosinase inhibitors have been reported, almost all the inhibitors were unfortunately evaluated by using commercial available mushroom tyrosinase. Here, we examined the inhibitory effects of three isomers of thujaplicin (α, ß, and γ) on human tyrosinase and analyzed their binding modes using homology model and docking studies. As the results, γ-thujaplicin was found to strongly inhibit human tyrosinase with the IC50 of 1.15 µM, extremely superior to a well-known tyrosinase inhibitor kojic acid (IC50 = 571.17 µM). MM-GB/SA binding free energy decomposition analyses suggested that the potent inhibitory activity of γ-thujaplicin may be due to the interactions with His367, Ile368, and Val377 (hot spot amino acid residues) in human tyrosinase. Furthermore, the binding mode of α-thujaplicin indicated that Val377 and Ser380 may cause van der Waals clashes with the isopropyl group of α-thujaplicin. These results provide a novel structural insight into the hot spot of human tyrosinase for the specific binding of γ-thujaplicin and a way to optimize not only thujaplicins but also other lead compounds as specific inhibitors for human tyrosinase in a rational manner.

Supplementation of Nicotinic Acid and Its Derivatives Up-Regulates Cellular NAD+ Level Rather than Nicotinamide Derivatives in Cultured Normal Human Epidermal Keratinocytes.

Nicotinamide adenine dinucleotide (NAD+) plays a pivotal role in various physiological processes within mammalian cells, including energy metabolism, redox homeostasis, and genetic regulation. In the majority of mammalian cellular contexts, NAD+ biosynthesis primarily relies on vitamin B3, including nicotinamide (NAM) and nicotinic acid (NA). The concept of NAD+ augmentation therapy has recently emerged as a promising strategy to mitigate aging-associated phenomena, termed rejuvenation. Despite the involvement of diverse enzymatic cascades in NAD+ biosynthesis, certain cellular environments exhibit deficiencies in specific enzymes, suggesting cell type-dependent variability in optimal NAD+ precursor selection. However, the optimization of NAD+ precursors for topical formulations has received scant attention thus far. In the present investigation, we sought to delineate the most efficacious precursor for augmenting NAD+ levels in human skin keratinocytes. Remarkably, NA supplementation led to a significant 1.3-fold elevation in intracellular NAD+ levels, even in the presence of nicotinamide phosphoribosyltransferase inhibition by FK866. Additionally, NA mononucleotide demonstrated a 1.5-fold increase (but not significant) in NAD+ levels following 100 µM application. Conversely, NAM and its derivatives failed to elicit a NAD+ response in keratinocytes. Notably, NA supplementation elicited up-regulation of mitochondrial superoxide dismutase (SOD2) and sirtuin 3 (SIRT3), indicative of its beneficial impact on mitochondrial function. Furthermore, NA mitigated rotenone-induced mitochondrial reactive oxygen species (ROS) accumulation. Collectively, these findings advocate for the potential utility of NA in topical applications aimed at skin rejuvenation.

[Clinical evaluation of transcatheter arterial chemolipiodolization of miriplatin for multiple hepatocellular carcinoma].

The safety and efficacy of miriplatin-lipiodol suspension were investigated in 174 patients with hepatocellular carcinoma(HCC). We assessed 29 patients who underwent transarterial chemoembolization(TACE)for whole-liver multinodular HCC(mHCC), compared with 145 patients who underwent TACE for non-multinodular HCC(n-mHCC)as the controls. In the mHCC group, a treatment effect(TE)of 4 was obtained in 0%, TE3 in 38%, TE2 in 31%, and TE1 in 31%. In the n-mHCC group, TE4 was obtained in 24%, TE3 in 40%, TE2 in 32%, and TE1 in 4%. Efficacy was significantly higher in the mHCC group. In the mHCC group, Grade 3 adverse events(fever, elevated alanine aminotransferase, and thrombocytopenia)occurred in 4 patients(13. 7%). In the n-mHCC group, Grade 3 adverse events(ascites, elevated serum transaminase, and cytopenia)occurred in 33 patients(22. 7%). There was no significant difference in the change of Child-Pugh scores over time in 6 patients who underwent repetitive TACE for mHCC. In conclusion, TACE for whole-liver mHCC is generally safe, but its short-term therapeutic effects were not satisfactory. Variation in the TACE protocol using miriplatin, such as repetitive administration of miriplatin and a reduction in the treatment interval, can be alternative treatment choices for patients with whole-liver mHCC.

Respiratory frequency-tunable dynamic imaging for lung function: New exam method using chest X-ray cine imaging considering various respiratory diseases.

OBJECTIVES: A convenient way to conduct pulmonary function tests while preventing infectious diseases was proposed, together with countermeasures for severe coronavirus disease 2019 (COVID-19). The correlation between diagnosis result and diagnosis result was examined for patients with mild chronic obstructive pulmonary disease (COPD) of the most abounding as a subject of spirometry, and the possibility of using this method as an alternative to spirometry was examined. SETTING: This study was conducted in Kanagawa, Japan. PARTICIPANTS: Ten normal volunteers and 15 volunteers with mild COPD participated in this study. OUTCOME MEASURES: All images were taken by EXAVISTA (Hitachi, Japan) between October 2019 and February 2020. Continuous fluoroscopic images were taken in 12.5 frames per second for 10-20 s per subject. Images that do not adopt the automatic image processing of the equipment and only carry out the signal correction of each pixel were used for the analysis. RESULTS: The mean total dose for all volunteers was 0.2 mGy. There was no major discrepancy in the detection of lung field geometry, and no diagnostic problems were noted by the radiologist and physician. CONCLUSIONS: Existing X-ray cine imaging was used to extract frequency-tunable imaging. It is possible to identify abnormal regions on the images compared to spirometry, and it does not require maximum effort respiration; therefore, it is possible to perform a stable examination because the patient's physical condition and the ability of laboratory technicians on the day are less affected. This can also be used as a countermeasure in examining patients with infectious diseases. TRIAL REGISTRATION: UMIN UMIN000043868.

Hinokitiol-induced decreases of tyrosinase and microphthalmia-associated transcription factor are mediated by the endoplasmic reticulum-associated degradation pathway in human melanoma cells.

Tyrosinase (TYR) is a key enzyme for melanin production. We previously showed that hinokitiol, a naturally occurring seven-membered ring terpenoid, potently inhibits human TYR activity. Interestingly, hinokitiol was recently reported to decrease expression of TYR and microphthalmia-associated transcription factor (MITF), which is a main transcription factor of the TYR gene, in murine melanoma cells. However, the mechanisms by which hinokitiol decreases the intracellular levels of TYR and MITF have not been fully elucidated. Here, we investigated the underlying mechanisms of the decreases using cultured human melanoma cells. As a result, hinokitiol treatment decreased TYR protein level in a time- and dose-dependent manner in G361 human melanoma cells, while MITF protein level was decreased only at higher concentrations after 3 days treatment. Notably, the mRNA levels of TYR and MITF were slightly increased by hinokitiol treatment. Therefore, we focused on the degradation of TYR and MITF in endoplasmic reticulum (ER)-associated protein degradation (ERAD) pathway. Importantly, co-treatment of ERAD inhibitor with hinokitiol restored the protein levels of TYR and MITF to approximately 30% and 20% of total those in untreated control cells, respectively. Hinokitiol affected the ER homeostasis as well as degradation of TYR and MITF in two human melanoma cell lines, G361 and HT-144, but the changes of ER-stress markers under the hinokitiol treatment were different in the two human melanoma cell lines. Taken together, these observations indicate that hinokitiol may induce ER stress and trigger the degradation of unfolded newly synthesizing TYR and MITF via the ERAD pathway.

Bacterial production of recombinant human poly(ADP-ribose) glycohydrolase.

Poly(ADP-ribosyl)ation, which is mainly involved in DNA repair and replication, is catalyzed mainly by poly(ADP-ribose) polymerase-1 (PARP-1) and poly(ADP-ribose) glycohydrolase (PARG). Although recombinant human PARP-1 (hPARP-1) is commercially available, there are no reports on the preparation of recombinant human PARG (hPARG). Here, we report the efficient expression and purification of a recombinant hPARG-catalytic domain (hPARG-CD) from Escherichia coli (E. coli). hPARG-CD was expressed as a fusion protein with a glutathione S-transferase (GST) tag at the N-terminus and a hexahistidine (6His) tag at the C-terminus. Both high cell density and low temperature culture conditions were important for the maximum production of soluble recombinant hPARG-CD. After sequential affinity chromatography using immobilized metal affinity resin and glutathione-Sepharose (GSH-Sephasrose), more than 95% pure recombinant hPARG-CD was obtained with a yield of approximately 2mg per 1L of E. coli culture medium. The km and Vmax values of purified recombinant hPARG-CD were 9.0 µM and 35.6 µmol/min/mg protein, respectively. These kinetic values were similar to those of purified endogenous hPARG reported previously. Furthermore, the recombinant hPARG-CD was inhibited by known PARG inhibitors such as adenosine diphosphate (hydroxymethyl) pyrrolidinediol (ADP-HPD), eosin Y, and phloxine B. These results show that the recombinant hPARG-CD is useful to search for specific inhibitors and to elucidate the regulatory mechanisms of hPARG.

Respiratory effects of balloon occluded retrograde transvenous obliteration of gastric varices: a prospective controlled study.

BACKGROUND AND AIM: We evaluated the respiratory effects of balloon-occluded retrograde transvenous obliteration (BRTO) performed for the treatment of gastric varices complicating liver cirrhosis. METHODS: From 2005 to 2009, we performed BRTO in 20 patients with gastric fundal varices, by intravariceal injection of 5% ethanolamine oleate (EO) as the sclerosant. We studied the effect of BRTO on the respiratory gas exchange, chest X-ray findings, computed tomography (CT) findings, pulmonary function parameters, and (99m) Tc-MAA lung perfusion scintigraphy findings. Subjects undergoing balloon-occluded retrograde transvenous varicerography (BRTV) without injection of the sclerosant served as the controls. RESULTS: Arterial blood gas analysis revealed a decrease in the mean arterial partial oxygen tension (PaO(2)) (P < 0.01), and increase in the alveolar-arterial oxygen tension difference (AaDO(2)) after BRTO (P < 0.01), as compared with the results obtained before the BRTO, while breathing room air. No changes were observed after BRTV as compared with the previous findings. In addition, a significant correlation was observed between the change of the PaO(2) and the volume of the sclerosant injected (rs = 0.511, P = 0.011). Left-pleural effusion was noted on the chest CT in 20% of the patients. On pulmonary function testing, decrease of the vital capacity was noted in two of the 20 patients after BRTO. CONCLUSION: The aforementioned results suggest that BRTO performed using EO as the sclerosant induces pulmonary function disorders. The effect was found to depend on the total amount of EO injected. Therefore, careful respiratory monitoring seems necessary in patients undergoing BRTO, particularly those in whom large volumes of the sclerosant are used.

A Unique Anti-Cancer 3-Styrylchromone Suppresses Inflammatory Response via HMGB1-RAGE Signaling.

Background: High mobility group box 1 (HMGB1)-receptor for advanced glycation endo-products (RAGE) axis serves as a key player in linking inflammation and carcinogenesis. Recently, papaverine was revealed to suppress the HMGB1-RAGE inflammatory signaling pathway and cancer cell proliferation. Therefore, a dual suppressor targeting this axis is expected to become a new type of therapeutic agent to treat cancer. Methods: Papaverine 3D pharmacophore mimetic compounds were selected by the LigandScout software from our in-house, anti-cancer chemical library and assessed for their anti-inflammatory activities by a HMGB1-RAGE-mediated interleukin-6 production assay using macrophage-like RAW264.7 cells. Molecular-biological analyses, such as Western blotting, were performed to clarify the mechanism of action. Results: A unique 6-methoxy-3-hydroxy-styrylchromone was found to possess potent anti-inflammatory and anti-cancer activities via the suppression of the HMGB1-RAGE-extracellular signal-regulated kinase 1/2 signaling pathway. Furthermore, the 3D pharmacophore-activity relationship analyses revealed that the hydroxyl group at the C4' position of the benzene ring in a 3-styryl moiety was significant in its dual suppressive effects. Conclusions: These findings indicated that this compound may provide a valuable scaffold for the development of a new type of anti-cancer drug possessing anti-inflammatory activity and as a tool for understanding the link between inflammation and carcinogenesis.