The Usefulness of Serum Interleukin-6 as a Predictor of Response to Atezolizumab plus Bevacizumab Combination Treatment in Hepatocellular Carcinoma.

INTRODUCTION: In atezolizumab plus bevacizumab (Atezo/Bev) combination treatment, both drugs act on the immune system. Previously, we reported that immunological changes after Atezo/Bev administration for unresectable hepatocellular carcinoma (uHCC) revealed significant alterations in interleukin (IL)-6, soluble IL-2 receptor, tumor necrosis factor-alpha, and programmed cell death-1 levels. Among these variable factors, serum levels of IL-6 can be easily measured on a commercial basis. Therefore, this study aimed to investigate the utility of serum IL-6 as a predictor of tumor response to Atezo/Bev treatment for uHCC. METHOD: The study included 44 patients with HCC treated with Atezo/Bev. Blood samples were collected before and 3 weeks after treatment, and tumor response was assessed using contrast-enhanced computed tomography 6 weeks after treatment. RESULTS: Significant changes in serum IL-6 levels were observed in patients treated with Atezo/Bev as first-line therapy but not in those treated with it as second-line or later-line therapy. In patients treated with Atezo/Bev as first-line therapy, serum IL-6 levels increased significantly after treatment in patients with a complete or partial response but not in patients with stable or progressive disease. Furthermore, compared to other tumor markers such as alpha-fetoprotein, Lens culinaris agglutinin-reactive fraction of alpha-fetoprotein, and des-gamma-carboxyprothrombin, serum IL-6 levels exhibited the highest sensitivity in predicting tumor response during the treatment period. CONCLUSION: In patients with uHCC treated with Atezo/Bev, serum IL-6 levels could serve as a potential predictor of tumor response. Elevated levels after treatment may indicate a favorable tumor response and prognosis.

Risk factors for lenvatinib-induced palmar-plantar erythrodysesthesia syndrome in patients with hepatocellular carcinoma: A retrospective study.

AIM: Lenvatinib mesylate (LEN) is an oral tyrosine kinase inhibitor used to treat various cancers, including hepatocellular carcinoma (HCC). HCC treatment with LEN is associated with a very high incidence of adverse events. This study was aimed at investigating the incidence of LEN-induced palmar-planter erythrodysesthesia syndrome (PPES) and its relationship with patient demographics by analyzing clinical laboratory data of LEN-treated patients with HCC. METHODS: This was a single-centre, retrospective study of patients with HCC who received LEN between April 19, 2018, and September 30, 2020. The observation period was from 1 week before the start of LEN administration to 1 month after the end of administration. RESULTS: Overall, 75 patients with HCC were enrolled. LEN-induced PPES was found in 48.0% (36/75 patients). In these patients, alkaline phosphatase (ALP), γ-Glutamyl transpeptidase (γ-GTP) and monocytes (MONO) were significantly high (ALP: p = 1.32 × 10-3, γ-GTP: p = 4.25 × 10-3 and MONO: p = 0.013). The cut off values of ALP, γ-GTP and MONO for LEN-induced PPES were estimated at 573 U/L, 89 U/L, and 310 counts/µL, respectively. In the multivariate analysis, γ-GTP and MONO were independent risk factors for LEN-induced PPES. CONCLUSIONS: High γ-GTP and high MONO were risk factors for LEN-induced PPES.

Second-line Treatment Strategy in Unresectable Hepatocellular Carcinoma After First-line Atezolizumab Plus Bevacizumab.

BACKGROUND/AIM: Atezolizumab plus beva-cizumab (AteBev) are an integral part of first-line therapy for unresectable hepatocellular carcinoma (uHCC), whereas no second-line regimen has been developed for these patients. This study evaluated the efficacy of second-line therapy for uHCC following AteBev treatment. PATIENTS AND METHODS: Sixty uHCC patients who were administered AteBev therapy were included in the study. Dynamic computed tomography was conducted after 6, 9, and 12 weeks, and blood tests were performed at baseline and after three weeks. RESULTS: After six weeks of AteBev therapy, 19 patients experienced partial response (PR), 12 had stable disease (SD), and 29 exhibited progressive disease (PD), with an overall response rate (ORR) of 31.7%. Of the 21 patients treated with lenvatinib as second-line treatment, one dropped out, nine experienced a compete response (CR) or PR, and 11 had SD or PD, resulting in an ORR of 45.0%. Serum levels of fibroblast growth factors (FGF)-19 increased substantially following lenvatinib therapy in the CR+PR group, although the levels decreased significantly in the SD+PD group. Soluble FGF-R4 levels did not differ significantly between the CR+PR group and the SD+PD group when assessed before and after lenvatinib treatment. CONCLUSION: Lenvatinib is useful as second-line treatment after Ate/Bev for uHCC patients who do not response to Ate/Bev treatment. Changes in serum FGF-19 levels after three weeks of AteBev therapy may serve as a biomarker for selecting lenvatinib as second-line therapy.

Remaining Issues Related to Serum Cytokines in Patients with Unresectable Hepatocellular Carcinoma Treated by Atezolizumab plus Bevacizumab Combination Treatment.

INTRODUCTION: Atezolizumab plus bevacizumab (AteBev) combination treatment is widely used as first-line systemic therapy for unresectable hepatocellular carcinoma (uHCC). We aimed to clarify therapeutic issues regarding serum cytokines and the immune reaction in patients with uHCC treated with AteBev. METHODS: We analyzed preserved serum from a previous prospective study on adult Japanese patients with chronic liver disease and uHCC who received AteBev treatment at our hospital. In that study, AteBev was administered intravenously every 3 weeks, and blood samples were collected before and after 3 weeks' treatment. Dynamic computed tomography was performed after 6 weeks of treatment to assess response. RESULTS: In the prospective study, 21 of the 59 patients showed partial response (PR) and 19 patients showed stable disease, but 19 patients showed progressive disease (PD). We found that serum levels of tumor necrosis factor-alpha, interleukin (IL)-6, and soluble IL-2 receptor (IL-2R) increased significantly in the PR group, but only soluble IL-2R increased significantly in the PD group. Regulatory T cells decreased significantly in the PD group, but there was no significant change in Th1 or Th2 cells from before to after treatment in any group. As regards soluble MHC-class I, pre-treatment levels were significantly lower in the PD group than in the PR group, and serum levels increased significantly with treatment in the PD group. CONCLUSION: These findings reveal a need to further improve T-cell priming and to further make T cells recognize tumor antigens in uHCC.

Importance of Atezolizumab Plus Bevacizumab Combination Treatment as First-line Therapy for Immunological Changes in Patients With Unresectable Hepatocellular Carcinoma.

BACKGROUND/AIM: The atezolizumab plus bevacizumab (AteBev) therapy is recommended as first-line treatment for unresectable hepatocellular carcinoma (uHCC). However, there remains a need to examine its efficacy with and without previous chemotherapy. Therefore, in patients with uHCC who underwent AteBev therapy, we aimed to clarify the effects of previous chemotherapy by examining serum immunological changes. PATIENTS AND METHODS: We retrospectively analyzed data of 29 patients with uHCC treated by AteBev therapy as part of a prospective study and divided participants into two groups depending on whether they had received prior chemotherapy. Dynamic computed tomography was performed after 6 weeks of treatment. Blood samples were collected at baseline and after 3 weeks of treatment. RESULTS: The group with prior treatment included 15 patients and the group without prior treatment included 14 patients. Objective response rates after six weeks of treatment were 13.3% and 28.6% in the groups with and without prior treatment, respectively. Serum levels of interleukin (IL)-6 and tumor necrosis factor-alpha showed no significant change in the group with prior treatment but increased significantly in the group without prior treatment. The percentage of regulatory T cells decreased significantly after treatment only in the group without prior treatment. CONCLUSION: In patients with uHCC, AteBev therapy can be expected to elicit an effective immune response in patients without prior treatment, but it may not do so in patients with prior treatment. Thus, AteBev appears to be more effective when used as first-line chemotherapy.

Risk Factors for Lenvatinib-Induced High-Grade Hypothyroidism in Patients with Hepatocellular Carcinoma: A Retrospective Study.

INTRODUCTION: Lenvatinib mesylate (LEN) is an orally administered tyrosine kinase inhibitor used for the treatment of various cancers, including hepatocellular carcinoma (HCC). HCC treatment with LEN is associated with a very high incidence of adverse events, especially hypothyroidism. This study investigated the incidence of hypothyroidism due to LEN and the relationships between hypothyroidism incidence and patient demographics by analyzing clinical laboratory data from HCC patients treated with LEN. METHODS: This was a single-center, retrospective study of HCC patients who received LEN between April 19, 2018, and September 30, 2020. The observation period was from 1 week before the start of LEN administration to 1 month after the end of administration. RESULTS: In total, 61 patients with HCC were enrolled. High-grade hypothyroidism (CTCAE Grade 2-3) was found in 36.1% (22/61 patients). In high-grade hypothyroidism, eosinophil (EOSINO) count was significantly low (p = 0.029). The cutoff value of EOSINO count was estimated to be approximately 150/µL. The adjusted odds ratios of high-grade hypothyroidism for current smoking and EOSINO count <150/µL were 0.237 (95% confidence interval: 0.063-0.893) and 4.219 (95% confidence interval: 1.119-15.92), respectively. CONCLUSION: The results showed that noncurrent smoking and EOSINO count <150/µL are risk factors for LEN-induced high-grade hypothyroidism.

Effects of Lenvatinib on Skeletal Muscle Volume and Cardiac Function in Patients with Hepatocellular Carcinoma.

INTRODUCTION: Previously, we reported that the tyrosine kinase inhibitor (TKI) sorafenib decreases serum levels of carnitine and reduces skeletal muscle volume. Moreover, others reported that TKIs might lead to cardiomyopathy or heart failure. Therefore, this study aimed to evaluate the effects of lenvatinib (LEN) on skeletal muscle volume and cardiac function in patients with hepatocellular carcinoma (HCC). METHODS: This retrospective study included 58 adult Japanese patients with chronic liver diseases and HCC treated with LEN. Blood samples were collected before and after 4 weeks of treatment, and serum carnitine fraction and myostatin levels were measured. Before and after 4-6 weeks of treatment, the skeletal muscle index (SMI) was evaluated from computed tomography images and cardiac function was assessed by ultrasound cardiography. RESULTS: After treatment, SMI, serum levels of total carnitine, and global longitudinal strain were significantly lower, but serum levels of myostatin were significantly higher. Left ventricular ejection fraction showed no significant change. CONCLUSION: In patients with HCC, LEN decreases serum levels of carnitine, skeletal muscle volume, and worsens cardiac function.

Lenvatinib Might Induce Activation of Host Immunity in Patients with Hepatocellular Carcinoma.

INTRODUCTION: Atezolizumab, an immune checkpoint inhibitor, plus bevacizumab, a monoclonal antibody that binds to vascular endothelial growth factor (VEGF), is an approved first-line systemic treatment for unresectable hepatocellular carcinoma (HCC). Immune checkpoint inhibitors are more effective in patients with HCC when administered with anti-VEGF drugs; however, these drugs affect host immunity. Lenvatinib is an anti-VEGF agent used to treat HCC; therefore, this study evaluated the effect of treatment of HCC with lenvatinib on host immunity in patients with chronic liver disease (CLD). METHODS: We studied adult Japanese patients with CLD and unresectable HCC treated with lenvatinib at our hospital. Lenvatinib was administered for 4 weeks (8 mg/day for bodyweight <60 kg; 12 mg/day for bodyweight >60 kg). Blood samples were collected at baseline and at 4 weeks of treatment and examined for immune-related changes. RESULTS: Forty-three patients were enrolled in this study. We found a significant increase in T helper (Th) 1 cells following 4 weeks of lenvatinib treatment, although there was no significant difference in Th2 cells and regulatory T cells. We also found a significant increase in serum levels of TNF-alpha, soluble TNF-alpha receptor I, and endothelial growth factor following 4 weeks of lenvatinib treatment. Furthermore, an increase in Th1 cells and serum levels of TNF-alpha was found in patients with partial response. CONCLUSION: Lenvatinib might induce Th1-dominant host immunity in patients with CLD and unresectable HCC treatment in patients who showed a partial response. These changes in host immunity may be a biomarker in HCC patients treated with lenvatinib.

Docosahexaenoic Acid Suppresses Oxidative Stress-Induced Autophagy and Cell Death via the AMPK-Dependent Signaling Pathway in Immortalized Fischer Rat Schwann Cells 1.

Autophagy is the process by which intracellular components are degraded by lysosomes. It is also activated by oxidative stress; hence, autophagy is thought to be closely related to oxidative stress, one of the major causes of diabetic neuropathy. We previously reported that docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) induced antioxidant enzymes and protected Schwann cells from oxidative stress. However, the relationship between autophagy and oxidative stress-induced cell death in diabetic neuropathy has not been elucidated. Treatment with tert-butyl hydroperoxide (tBHP) decreased the cell survival rate, as measured by an MTT assay in immortalized Fischer rat Schwann cells 1 (IFRS1). A DHA pretreatment significantly prevented tBHP-induced cytotoxicity. tBHP increased autophagy, which was revealed by the ratio of the initiation markers, AMP-activated protein kinase, and UNC51-like kinase phosphorylation. Conversely, the DHA pretreatment suppressed excessive tBHP-induced autophagy signaling. Autophagosomes induced by tBHP in IFRS1 cells were decreased to control levels by the DHA pretreatment whereas autolysosomes were only partially decreased. These results suggest that DHA attenuated excessive autophagy induced by oxidative stress in Schwann cells and may be useful to prevent or reduce cell death in vitro. However, its potentiality to treat diabetic neuropathy must be validated in in vivo studies.

Adaptation of lenvatinib treatment in patients with hepatocellular carcinoma and portal vein tumor thrombosis.

PURPOSE: The aim of this study was to clarify the adaptation of lenvatinib treatment in patients with hepatocellular carcinoma (HCC) and portal vein tumor thrombosis (PVTT). METHOD: Fifty-three patients with HCC were treated with lenvatinib. Before and after treatment blood sampling, patients were examined by computed tomography and ultrasonography. In patients with portal trunk invasion (Vp4), the analysis focused on the degree of occlusion due to the tumor in the portal trunk. In patients without major PVTT {ie, invasion of the primary branch of the portal vein [Vp3] or Vp4}, portal blood flow volume was measured by Doppler analysis; however, Doppler analysis is difficult to perform in patients with major PVTT, so the time from administration of the contrast agent to when it reached the primary branch of the portal vein (portal vein arrival time) was evaluated with the contrast agent Sonazoid. RESULTS: Patients with Vp4 had a significantly worse prognosis than patients with Vp3 and a significant increase in Child-Pugh score at 2 months. Patients with major PVTT had a poor prognosis if the degree of occlusion of the portal trunk was 70% or more. In patients without major PVTT, portal blood flow was significantly decreased after administration of lenvatinib; and in patients with major PVTT, the hepatic artery and portal vein arrival times were significantly increased. CONCLUSION: Lenvatinib treatment should be avoided in patients with Vp4 with a high degree of portal trunk occlusion because of concerns about decreased portal blood flow.

Importance of Compliance With Guidelines for the Prevention of Varicella-Zoster Virus Reactivation in Multiple Myeloma.

BACKGROUND/AIM: The importance of compliance with National Comprehensive Cancer Network (NCCN) guidelines for preventing varicella-zoster virus reactivation (VZVr) in multiple myeloma (MM) in a clinical setting has not been well investigated. PATIENTS AND METHODS: We retrospectively studied the clinical characteristics and outcomes of 118 patients with MM treated with proteasome inhibitors. RESULTS: Thirty-nine episodes of VZVr were observed in 37 patients (VZVr group). The proportion of prophylactic antiviral prescriptions and compliance with antiviral prophylaxis based on the NCCN Clinical Practice guidelines was 76% and 30% in the VZVr group, and 88% and 74% in the non-VZVr group, respectively. Multivariate analysis showed that compliance with the NCCN guidelines was the only independent risk factor for VZVr (p=0.0017). CONCLUSION: It is important that prophylactic antivirals are prescribed for an appropriate duration of time to prevent the reactivation of VZV in compliance with existing guidelines.

Sacraoxides A-G, Bioactive Cembranoids from Gum Resin of Boswellia sacra.

Seven undescribed cembranoids, sacraoxides A-G (1, 3-8) were isolated from the gum resin of Boswellia sacra. Their structures were elucidated by extensive physicochemical and spectroscopic analysis, as well as ECD calculation, modified Mosher's method and X-ray diffraction crystallography. Compounds 6 and 7 exhibited inhibitory activities on nitric oxide (NO) production induced by lipopolysaccharide in RAW264.7 cells with IC50 values of 24.9 ± 1.7 and 36.4 ± 2.9 µM.

Safety and Effectiveness of Ixazomib Dose-escalating Strategy in Ixazomib-Lenalidomide-Dexamethasone Treatment for Relapsed/Refractory Multiple Myeloma.

BACKGROUND/AIM: Gastrointestinal toxicity is common in patients receiving common therapy of ixazomib with lenalidomide and low-dose dexamethasone (IRd) for relapsed/refractory multiple myeloma. Here, we investigated the safety and effectiveness of ixazomib dosing schedules. PATIENTS AND METHODS: We retrospectively evaluated 17 consecutive patients treated with IRd (10 patients on ixazomib dose-escalation strategy (2.3 mg starting dose); seven patients on standard dose). RESULTS: The incidence of grade 3 or more haematological and grade 2 or more non-haematological adverse events was lower in the dose-escalation group than in the standard-dose group, and only that of diarrhoea was significantly lower. The median time to treatment interruption was significantly longer in the dose-escalation group than in the standard-dose group. There was no significant difference in the overall response rate (20% vs. 43%) and disease control rate (70% vs. 86%). CONCLUSION: A dose-escalation strategy to optimise ixazomib dosing may reduce treatment interruption due to adverse events without compromising its antitumor activity.

Tivantinib Decreases Hepatocyte Growth Factor-Induced BCRP Expression in Hepatocellular Carcinoma HepG2 Cells.

Tivantinib, a mesenchymal-epithelial transition factor (cMET) inhibitor, is a molecular targeting drug that kills hepatocellular carcinoma (HCC) cells. Tivantinib alone does not affect the overall survival of patients with HCC, and combination treatment with tivantinib and other therapies has not been evaluated. This study was conducted to clarify the effect of the tivantinib in regulating breast cancer therapy-resistant protein (BCRP), a key transporter of 5-fluorouracil (5-FU), and dihydropyridine dehydrogenase (DPYD), a major metabolic enzyme of 5-FU. To this end, cMET gene expression was determined by RT-PCR in HepG2 (human hepatoma) cells. The transcriptional start sites of BCRP were determined by 5'-rapid amplification of cDNA ends (5'-RACE). BCRP and DPYD mRNA levels were determined by real-time RT-PCR, and promoter activities were measured by dual-luciferase assays. Results show that hepatocyte growth factor (HGF) upregulated the mRNA level of BCRP, but not DPYD, in HepG2 cells. The upregulation of BCRP expression by HGF was down-regulated by tivantinib. We also identified two transcriptional start sites (E1α, E1ß) in BCRP by 5'-RACE. The transcriptional activity of the region -287 to E1α of BCRP was upregulated by HGF, which was decreased by tivantinib, whereas activity of the region -297 to E1ßo f BCRP was not affected by tivantinib. Therefore, tivantinib regulates BCRP expression upstream of exon 1α. Combination treatment of tivantinib and 5-FU should be further evaluated for HCC therapy.

Biphasic Increases of Cell Surface Calreticulin Following Treatment with Mitoxantrone.

Calreticulin (CRT) and calnexin (CNX), homologous major chaperones in the endoplasmic reticulum (ER), are known to translocate to the cell surface in response to chemotherapeutic agents, such as mitoxantrone (MIT), and cellular stresses, including apoptosis. Cell surface CRT (ecto-CRT) is relevant to the phagocytic uptake of cancer cells and dying cells, and pre-apoptotic exposure of CRT has been reported to result in enhanced immunogenicity of dying tumor cells, serving as a damage-associated molecular pattern (DAMP). In this study, HT-29 cells were treated with MIT to induce ER stress, and ecto-CRT and cell surface CNX were quantified by flow cytometry in the absence or presence of caspase inhibitors, a calpain inhibitor, or a scavenger of reactive oxygen species. The biphasic (early transient and late sustained) increase of ecto-CRT on HT-29 cells was observed after treatment with MIT. We confirmed that the early increase in ecto-CRT after 4 h of MIT treatment was not related to apoptosis, whereas the increase of ecto-CRT, as well as that of cell-surface CNX, during the later stage of treatment was caspase dependent and related to apoptosis. In addition, our results suggested that the early peak of ecto-CRT was mediated by activation of caspase 8 by ER stress. Thus, the physiological significance of the late increases in cell-surface CRT and/or CNX might be considered an "eat-me signal" for the removal of dead cells by phagocytosis, while the early increase in ecto-CRT caused by ER stress might enhance the immunogenicity of stressed tumor cells.

Noradrenaline-Induced Relaxation of Urinary Bladder Smooth Muscle Is Primarily Triggered through the ß3-Adrenoceptor in Rats.

ß-Adrenoceptors are subclassified into 3 subtypes (ß1-ß3). Among these, ß3-adrenoceptors are present in various types of smooth muscle and are believed to play a role in relaxation responses of these muscles. ß3-Adrenoceptors are also present in urinary bladder smooth muscle (UBSM), although their expression varies depending on the animal species. To date, there has been little information available about the endogenous ligand that stimulates ß3-adrenoceptors to produce relaxation responses in UBSM. In this study, to determine whether noradrenaline is a ligand of UBSM ß3-adrenoceptors, noradrenaline-induced relaxation was analyzed pharmacologically using rat UBSM. We also assessed whether noradrenaline metabolites were ligands in UBSM. In isolated rat urinary bladder tissues, mRNAs for ß1-, ß2-, and ß3-adrenoceptors were detected using RT-PCR. In UBSM preparations contracted with methacholine (3 × 10-5 M), noradrenaline-induced relaxation was not inhibited by the following antagonists: atenolol (10-6 M; selective ß1-adrenoceptor antagonist), ICI-118,551 (3 × 10-8 M; selective ß2-adrenoceptor antagonist), propranolol (10-7 M; non-selective ß-adrenoceptor antagonist), and bupranolol (10-7 M; non-selective ß-adrenoceptor antagonist). In the presence of propranolol (10-6 M), noradrenaline-induced relaxation was competitively inhibited by bupranolol (3 × 10-7-3 × 10-6 M) or SR59230A (10-7-10-6 M; selective ß3-adrenoceptor antagonist), with their pA2 values calculated to be 6.64 and 7.27, respectively. None of the six noradrenaline metabolites produced significant relaxation of methacholine-contracted UBSM. These findings suggest that noradrenaline, but not its metabolites, is a ligand for ß3-adrenoceptors to produce relaxation responses of UBSM in rats.

Influence of Sorafenib on Host Immunity in Patients with Liver Cirrhosis With Advanced Hepatocellular Carcinoma Stratified by Etiology.

AIM: We previously reported that sorafenib induces Th1 [interferon-γ (IFNγ)-positive interleukin 4 (IL4)-negative] dominance which prevents tumor cells from escaping the host immune system in patients with liver cirrhosis (LC) and advanced hepatocellular carcinoma (aHCC). However, in that study we did not assess the influence of sorafenib on host immunity according to the etiology of LC. Therefore, this study was retrospectively performed to evaluate the impact of sorafenib therapy for aHCC on host immunity in patients stratified according to the etiology of LC: Patients and Methods: A total of 116 adult Japanese patients with LC and aHCC received sorafenib therapy at our hospital. Blood samples were collected before and after treatment for 4 weeks. RESULTS: Twenty-two patients had hepatitis B virus (HBV)-related LC, 62 patients had hepatitis C virus (HCV)-related LC, 22 patients had alcoholic LC, and 10 patients had LC without these causative factors. In patients receiving sorafenib at a dose of 400 mg/day, patients in Child-Pugh class A, and patients with stage IVA aHCC, Th2 (IFNγ-negative/IL4-positive) cells decreased significantly after treatment, although there was no significant impact on the tumor response. In addition, Th2 cells decreased significantly in patients with HCV-related LC after treatment, while there were no significant changes in the other groups. CONCLUSION: Sorafenib might prevent tumor cells from escaping the host immune system in patients with aHCC and HCV-related LC, although it does not seem to do so in those with LC of other etiologies.

Comprehensive evaluation of antioxidant effects of Japanese Kampo medicines led to identification of Tsudosan formulation as a potent antioxidant agent.

Oxidative stress due to the overproduction of reactive oxygen species plays an important role in the pathogenesis of various diseases. In the present study, we comprehensively evaluated the antioxidant activities of 147 oral formulations of Japanese traditional herbal medicines (Kampo medicines), representing the entire panel of oral Kampo medicines listed in the Japanese National Health Insurance Drug List, using in vitro radical scavenging assays, including the 2,2-diphenyl-1-picrylhydrazyl free radical scavenging activity assay, the superoxide anion scavenging activity assay, and the oxygen radical absorption capacity assay. Three of the formulations tested, namely, Tsudosan, Daisaikoto, and Masiningan, showed the most potent in vitro antioxidant activities and were selected for further investigation of their intracellular and in vivo antioxidant effects. The results of the 2',7'-dichlorodihydrofluorescin diacetate assay demonstrated that all three Kampo medicines significantly inhibited hydrogen peroxide-induced oxidative stress in human hepatocellular liver carcinoma HepG2 cells. In addition, Tsudosan significantly increased the serum biological antioxidant potential values when orally administrated to mice, indicating that it also had in vivo antioxidant activity. The potent antioxidant activity of Tsudosan may be one of the mechanisms closely correlated to its clinical usage against blood stasis.

Globo-A Binds to the Recombinant Natural Cytotoxicity Receptor NKp44.

Natural killer (NK) cells play an important role in tumor immunity and infection control. The natural cytotoxicity receptors (NCRs) NKp46, NKp44 and NKp30 are involved in the control of the activation of NK cells. Few reports have investigated the ligands of NCRs. We previously reported the NCRs binding affinity to heparin and glycosaminoglycans. We also showed that multimeric sialyl Lewis X-expressing transferrin, secreted by human hepatoma HepG2 cells, binds to NKp46 and NKp44, but not to NKp30. In this study, we investigated the binding between NCRs and glycolipids. The possible binding of glycolipids to NCRs was screened by microarray, using the recombinant extracellular domain of NKp46, NKp44 and NKp30 tagged with 6×His (rNKp46, rNKp44 and rNKp30). We found that rNKp44 binds to Globo-A. However, we did not detect the interaction between rNKp46 or rNKp30 and any of the glycolipids investigated. Direct binding assays supported the results of the microarray screening. Therefore, we concluded that Globo-A is a novel ligand for NKp44 but not NKp46 and NKp30, and showed differences in the ligand selectivity of NCRs.

Pharmacological identification of ß-adrenoceptor subtypes mediating isoprenaline-induced relaxation of guinea pig colonic longitudinal smooth muscle.

Object We aimed to identify the ß-adrenoceptor (ß-AR) subtypes involved in isoprenaline-induced relaxation of guinea pig colonic longitudinal smooth muscle using pharmacological and biochemical approaches. Methods Longitudinal smooth muscle was prepared from the male guinea pig ascending colon and contracted with histamine prior to comparing the relaxant responses to three catecholamines (isoprenaline, adrenaline, and noradrenaline). The inhibitory effects of subtype-selective ß-AR antagonists on isoprenaline-induced relaxation were then investigated. Results The relaxant potencies of the catecholamines were ranked as: isoprenaline > noradrenaline ≈ adrenaline, whereas the rank order was isoprenaline > noradrenaline > adrenaline in the presence of propranolol (a non-selective ß-AR antagonist; 3 × 10-7 M). Atenolol (a selective ß1-AR antagonist; 3 × 10-7-10-6 M) acted as a competitive antagonist of isoprenaline-induced relaxation, and the pA2 value was calculated to be 6.49 (95% confidence interval: 6.34-6.83). The relaxation to isoprenaline was not affected by ICI-118,551 (a selective ß2-AR antagonist) at 10-9-10-8 M, but was competitively antagonized by 10-7-3 × 10-7 M, with a pA2 value of 7.41 (95% confidence interval: 7.18-8.02). In the presence of propranolol (3 × 10-7 M), the relaxant effect of isoprenaline was competitively antagonized by bupranolol (a non-selective ß-AR antagonist), with a pA2 value of 5.90 (95% confidence interval: 5.73-6.35). Conclusion These findings indicated that the ß-AR subtypes involved in isoprenaline-induced relaxation of colonic longitudinal guinea pig muscles are ß1-AR and ß3-AR.