Therapeutic efficacy of 211At-radiolabeled 2,6-diisopropylphenyl azide in mouse models of human lung cancer.

Targeted α-particle therapy (TAT) is an attractive alternative to conventional therapy for cancer treatment. Among the available radionuclides considered for TAT, astatine-211 (211At) attached to a cancer-targeting molecule appears very promising. Previously, we demonstrated that aryl azide derivatives could react selectively with the endogenous acrolein generated by cancer cells to give a diazo compound, which subsequently forms a covalent bond with the organelle of cancer cells in vivo. Herein, we synthesized 211At-radiolabeled 2,6-diisopropylphenyl azide (ADIPA), an α-emitting molecule that can selectively target the acrolein of cancer cells, and investigated its antitumor effect. Our results demonstrate that a single intratumor or intravenous administration of this simple α-emitting molecule to the A549 (human lung cancer) cell-bearing xenograft mouse model, at a low dose (70 kBq), could suppress tumor growth without inducing adverse effects. Furthermore, because acrolein is generally overproduced by most cancer cells, we believe ADIPA is a simple TAT compound that deserves further investigation for application in animal models and humans with various cancer types and stages.

Identification and characterization of sperm motility-initiating substance-2 gene in internally fertilizing Cynops species.

Sperm motility-initiating substance (SMIS) is an oviductal protein critical for internal fertilization in urodeles. It contributes to the establishment of various reproductive modes in amphibians and is thus a unique research model for the gene evolution of gamete-recognizing ligands that have diversified among animal species. In this study, a paralogous SMIS gene, smis2, was identified via the RNA sequencing of the oviduct of the newt, Cynops pyrrhogaster. The base sequence of the smis2 gene was homologous (˃90%) to that of the original smis gene (smis1), and deduced amino acid sequences of both genes conserved six cysteine residues essential for the cysteine knot motif. Furthermore, smis2 complementary DNA was identified in the oviduct of Cynops ensicauda, and the base substitution patterns also suggested that the smis gene was duplicated in the Salamandridae. Nonsynonymous/synonymous substitution ratios of smis1 and smis2 genes were 0.79 and 2.6, respectively, suggesting that smis2 gene evolution was independently driven by positive selection. Amino acid substitutions were concentrated in the cysteine knot motif of SMIS2. The smis2 gene was expressed in some organs in addition to the oviduct; in contrast, SMIS1 was only expressed in the oviduct. The SMIS2 protein was suggested to be produced and secreted at least in the oviduct and redundantly act in sperm. These results suggest that smis1 plays the original role in the oviduct, whereas smis2 may undergo neofunctionalization, which rarely occurs in gene evolution.

Tumor Targeting of 211At-Labeled Antibody under Sodium Ascorbate Protection against Radiolysis.

Astatine-211 (211At) is an alpha emitter applicable to radioimmunotherapy (RIT), a cancer treatment that utilizes radioactive antibodies to target tumors. In the preparation of 211At-labeled monoclonal antibodies (211At-mAbs), the possibility of radionuclide-induced antibody denaturation (radiolysis) is of concern. Our previous study showed that this 211At-induced radiochemical reaction disrupts the cellular binding activity of an astatinated mAb, resulting in attenuation of in vivo antitumor effects, whereas sodium ascorbate (SA), a free radical scavenger, prevents antibody denaturation, contributing to the maintenance of binding and antitumor activity. However, the influence of antibody denaturation on the pharmacokinetics of 211At-mAbs relating to tumor accumulation, blood circulation time, and distribution to normal organs remains unclear. In this study, we use a radioactive anti-human epidermal growth factor receptor 2 (anti-HER2) mAb to demonstrate that an 211At-induced radiochemical reaction disrupts active targeting via an antigen-antibody interaction, whereas SA helps to maintain targeting. In contrast, there was no difference in blood circulation time as well as distribution to normal organs between the stabilized and denatured immunoconjugates, indicating that antibody denaturation may not affect tumor accumulation via passive targeting based on the enhanced permeability and retention effect. In a high-HER2-expressing xenograft model treated with 1 MBq of 211At-anti-HER2 mAbs, SA-dependent maintenance of active targeting contributed to a significantly better response. In treatment with 0.5 or 0.2 MBq, the stabilized radioactive mAb significantly reduced tumor growth compared to the denatured immunoconjugate. Additionally, through a comparison between a stabilized 211At-anti-HER2 mAb and radioactive nontargeted control mAb, we demonstrate that active targeting significantly enhances tumor accumulation of radioactivity and in vivo antitumor effect. In RIT with 211At, active targeting contributes to efficient tumor accumulation of radioactivity, resulting in a potent antitumor effect. SA-dependent protection that successfully maintains tumor targeting will facilitate the clinical application of alpha-RIT.

Romosozumab successfully regulated progressive osteoporosis in a patient with autosomal dominant polycystic kidney disease undergoing hemodialysis.

Osteoporosis is a crucial complication in patients with chronic kidney disease (CKD), similar to that in the general population. Although romosozumab, a monoclonal antibody targeting sclerostin, has been administered for patients with CKD, its clinical effectiveness in these patients, especially in patients on hemodialysis (HD), remains to be studied. Herein, we report the case of a 42-year-old man on HD who developed severe osteoporosis. Serum calcium levels were extremely high, bone metabolic markers were abnormal, and the patient had pathological fractures. The bone biopsy indicated a bone metabolism disorder and high bone turnover. We administered romosozumab once a month as an intervention for bone alteration. Through the 10-month usage, bone metabolic markers improved, and the decrease in bone mineral density was ameliorated. We hypothesized that romosozumab could be a therapeutic option for osteoporosis in patients undergoing HD, especially in those with bone mineralization disorders.

Evaluation of CBSX Proteins as Regulators of the Chloroplast Thioredoxin System.

The chloroplast-localized cystathionine ß-synthase X (CBSX) proteins CBSX1 and CBSX2 have been proposed as modulators of thioredoxins (Trxs). In this study, the contribution of CBSX proteins to the redox regulation of thiol enzymes in the chloroplast Trx system was evaluated both in vitro and in vivo. The in vitro biochemical studies evaluated whether CBSX proteins alter the specificities of classical chloroplastic Trx f and Trx m for their target proteins. However, addition of CBSX proteins did not alter the specificities of Trx f and Trx m for disulfide bond reduction of the photosynthesis-related major thiol enzymes, FBPase, SBPase, and NADP-MDH. In vivo analysis showed that CBSX-deficient mutants grew similarly to wild type plants under continuous normal light conditions and that CBSX deficiency did not affect photo-reduction of photosynthesis-related thiol enzymes by Trx system at several light intensities. Although CBSX proteins have been suggested as modulators in the chloroplast Trx system, our results did not support this model, at least in the cases of FBPase, SBPase, and NADP-MDH in leaves. However, fresh weights of the cbsx2 mutants were decreased under short day. Since Trxs regulate many proteins participating in various metabolic reactions in the chloroplast, CBSX proteins may function to regulate other chloroplast Trx target proteins, or serve as modulators in non-photosynthetic plastids of flowers. As a next stage, further investigations are required to understand the modulation of Trx-dependent redox regulation by plastidal CBSX proteins.

Critical Role of NdhA in the Incorporation of the Peripheral Arm into the Membrane-Embedded Part of the Chloroplast NADH Dehydrogenase-Like Complex.

The chloroplast NADH dehydrogenase-like (NDH) complex mediates ferredoxin-dependent plastoquinone reduction in the thylakoid membrane. In angiosperms, chloroplast NDH is composed of five subcomplexes and further forms a supercomplex with photosystem I (PSI). Subcomplex A (SubA) mediates the electron transport and consists of eight subunits encoded by both plastid and nuclear genomes. The assembly of SubA in the stroma has been extensively studied, but it is unclear how SubA is incorporated into the membrane-embedded part of the NDH complex. Here, we isolated a novel Arabidopsis mutant chlororespiratory reduction 16 (crr16) defective in NDH activity. CRR16 encodes a chloroplast-localized P-class pentatricopeptide repeat protein conserved in angiosperms. Transcript analysis of plastid-encoded ndh genes indicated that CRR16 was responsible for the efficient splicing of the group II intron in the ndhA transcript, which encodes a membrane-embedded subunit localized to the connecting site between SubA and the membrane subcomplex (SubM). To analyze the roles of NdhA in the assembly and stability of the NDH complex, the homoplastomic knockout plant of ndhA (ΔndhA) was generated in tobacco (Nicotiana tabacum). Biochemical analyses of crr16 and ΔndhA plants indicated that NdhA was essential for stabilizing SubA and SubE but not for the accumulation of the other three subcomplexes. Furthermore, the crr16 mutant accumulated the SubA assembly intermediates in the stroma more than that in the wild type. These results suggest that NdhA biosynthesis is essential for the incorporation of SubA into the membrane-embedded part of the NDH complex at the final assembly step of the NDH-PSI supercomplex.

211At-labeled immunoconjugate via a one-pot three-component double click strategy: practical access to α-emission cancer radiotherapeutics.

α-Emission radiotherapeutics has potential to be one of most effective cancer therapeutics. Herein, we report a facile synthesis of an 211At-labeled immunoconjugate for use as an α-emission molecular targeting therapy. We synthesized a tetrazine probe modified with closo-decaborate(2-), a prosthetic group that forms a bioavailable stable complex with 211At. Our one-pot three-component double-click labeling method was used to attach decaborate to trastuzumab (anti-HER2 antibody) using decaborate-tetrazine and TCO-aldehyde probes without reducing the antibody binding affinity. Labeling the decaborate-attached trastuzumab with 211At produced in the cyclotron at the RIKEN Nishina Center, at which highly radioactive 211At can be produced, readily furnished the 211At-labeled trastuzumab with a maximum specific activity of 15 MBq µg-1 and retention of the native binding affinity. Intratumor injection of the 211At-labeled trastuzumab in BALB/c nude mice implanted with HER2-expressing epidermoid cancer cells yielded efficient accumulation at the targeted tumor site as well as effective suppression of tumor growth.

Cancer stem-like properties of hormonal therapy-resistant breast cancer cells.

BACKGROUND: Presently, hormonal therapy targeting estrogen receptors is the most effective treatment available for luminal breast cancer. However, many patients relapse after the therapy. It has been suggested that cancer stem-like cells are involved with hormonal therapy resistance; in the present study, we evaluated this hypothesis. METHODS: In the present study, we used our previously established hormonal therapy-resistant cell lines, including aromatase inhibitor (AI)-resistant cells (Type 1 and Type 2) and fulvestrant-resistant cells (MFR). RESULTS: AI-resistant cell lines expressing ER (Type 1 V1 and V2) showed high cancer stemness in terms of their CD44/CD24 expression and side populations, which were stimulated by the addition of estrogen and inhibited by fulvestrant. However, ALDH activity was lower than in the ER-negative resistant cells, suggesting that the stemness of luminal cells is distinct from that of basal-like breast cancer cells. The migration and invasion activity of the ER-positive Type 1 V1 and V2 cells were higher than in the ER-negative cell lines, Type 2 and MFR. CONCLUSIONS: Fractionation of parental cells based on CD44/CD24 expression and colony formation assay indicated that CD44+/CD24+ cells might be the origin of hormonal therapy-resistant cells. This population reconstituted various other subpopulations under estrogen deprivation. These results indicate that hormonal therapy resistance is closely related to the cancer stem cell-like properties of luminal breast cancer.

A case of Ménétrier's disease seemingly caused by hilar cholangiocarcinoma.

A 54-year-old man presented to our department with abdominal discomfort and anorexia and was diagnosed as having Ménétrier's disease (MD) with hilar cholangiocarcinoma. Based on his clinical examination, there was no evidence of Helicobacter pylori or cytomegalovirus (CMV) infection. Although we administered proton pump inhibitor and high-calorie enteral nutrition, hypoproteinemia did not improve, and the refractory protein-losing enteropathy persisted. However, interestingly, MD improved immediately after resection of the hilar cholangiocarcinoma. Generally, the etiology of MD is unknown, but H. pylori and CMV infections have been implicated. To our knowledge, there has been no previous report indicating that a malignant tumor could be involved in the etiology of MD. Thus, we report an extremely rare case of MD which is seemingly caused by malignancy.

CTRP6 is an endogenous complement regulator that can effectively treat induced arthritis.

The complement system is important for the host defence against infection as well as for the development of inflammatory diseases. Here we show that C1q/TNF-related protein 6 (CTRP6; gene symbol C1qtnf6) expression is elevated in mouse rheumatoid arthritis (RA) models. C1qtnf6(-/-) mice are highly susceptible to induced arthritis due to enhanced complement activation, whereas C1qtnf6-transgenic mice are refractory. The Arthus reaction and the development of experimental autoimmune encephalomyelitis are also enhanced in C1qtnf6(-/-) mice and C1qtnf6(-/-) embryos are semi-lethal. We find that CTRP6 specifically suppresses the alternative pathway of the complement system by competing with factor B for C3(H2O) binding. Furthermore, treatment of arthritis-induced mice with intra-articular injection of recombinant human CTRP6 cures the arthritis. CTRP6 is expressed in human synoviocytes, and CTRP6 levels are increased in RA patients. These results indicate that CTRP6 is an endogenous complement regulator and could be used for the treatment of complement-mediated diseases.

Thrombomodulin in the management of acute cholangitis-induced disseminated intravascular coagulation.

AIM: To evaluate the need for thrombomodulin (rTM) therapy for disseminated intravascular coagulation (DIC) in patients with acute cholangitis (AC)-induced DIC. METHODS: Sixty-six patients who were diagnosed with AC-induced DIC and who were treated at our hospital were enrolled in this study. The diagnoses of AC and DIC were made based on the 2013 Tokyo Guidelines and the DIC diagnostic criteria as defined by the Japanese Association for Acute Medicine, respectively. Thirty consecutive patients who were treated with rTM between April 2010 and September 2013 (rTM group) were compared to 36 patients who were treated without rTM (before the introduction of rTM therapy at our hospital) between January 2005 and January 2010 (control group). The two groups were compared in terms of patient characteristics at the time of DIC diagnosis (including age, sex, primary disease, severity of cholangitis, DIC score, biliary drainage, and anti-DIC drugs), the DIC resolution rate, DIC score, the systemic inflammatory response syndrome (SIRS) score, hematological values, and outcomes. Using logistic regression analysis based on multivariate analyses, we also examined factors that contributed to persistent DIC. RESULTS: There were no differences between the rTM group and the control group in terms of the patients' backgrounds other than administration. DIC resolution rates on day 9 were higher in the rTM group than in the control group (83.3% vs 52.8%, P < 0.01). The mean DIC scores on day 7 were lower in the rTM group than in the control group (2.1 ± 2.1 vs 3.5 ± 2.3, P = 0.02). The mean SIRS scores on day 3 were significantly lower in the rTM group than in the control group (1.1 ± 1.1 vs 1.8 ± 1.1, P = 0.03). Mortality on day 28 was 13.3% in the rTM group and 27.8% in the control group; these rates were not significantly different (P = 0.26). Multivariate analysis identified only the absence of biliary drainage as significantly associated with persistent DIC (P < 0.01, OR = 12, 95%CI: 2.3-60). Although the difference did not reach statistical significance, primary diseases (malignancies) (P = 0.055, OR = 3.9, 95%CI: 0.97-16) and the non-use of rTM had a tendency to be associated with persistent DIC (P = 0.08, OR = 4.3, 95%CI: 0.84-22). CONCLUSION: The add-on effects of rTM are anticipated in the treatment of AC-induced DIC, although biliary drainage for AC remains crucial.

Differences in stemness properties associated with the heterogeneity of luminal-type breast cancer.

BACKGROUND: Luminal-type breast cancers are the most abundant subtype. Endocrine therapies targeting estrogen receptor (ER) or estradiol (E2) synthesis have achieved marked improvement in disease-free and overall survival of ER-positive cancers. However, approximately one-third of these cancers are poorly responsive to endocrine therapies, suggesting nonuniform tumor cell characteristics of this subtype. Recently, the tumorigenesis theory which states that CSCs are capable of self-renewal, tumorigenicity, and therapeutic resistance, became widely accepted. We investigated the relationship between the heterogeneity of luminal-type breast cancer and stemness properties. MATERIALS AND METHODS: CSC surface markers and expression of stemness-related genes, including Octamer-binding transcription factor 4 (OCT4), Nanog homeobox (NANOG), and Kruppel-like factor 4 (KLF4), were analyzed in clinical samples. ER activities were analyzed using the adenovirus vector carrying the ERE-green fluorescent protein (GFP). We separated the luminal-type breast cancers into 2 groups according to stemness-related gene expression patterns in mammospheres. RESULTS: The group that predominantly expressed NANOG mRNA showed a high percentage of the cells that were positive for CD44 and negative for CD24 and Hoechst (possessing high-stemness properties), younger patient age, higher p53 expression, and tended to show higher histological grade and higher topoisomerase IIα expression. The ERE-GFP assay revealed that the luminal-type breast cancer mammospheres were heterogeneous. Mammospheres from several specimens lacked ER activity and responsiveness to E2 but some retained ER activities. CONCLUSION: ERE activity differences might be associated with endocrine therapy effectiveness. Mammosphere stemness properties could be a useful and novel criterion for subclassification of luminal-type breast cancers.

Retear of anterior cruciate ligament grafts in female basketball players: a case series.

BACKGROUND: Incidence of anterior cruciate ligament (ACL) injuries in young female basketball players is higher than that in male basketball players. Graft retears are more frequent with the increasing number of ACL reconstructions. The present study aimed to examine the incidence of retears in competitive female basketball players. METHODS: Sixty-four female basketball players (aged 12 to 29 years) who underwent primary anatomic double-bundle ACL reconstruction using hamstring grafts participated in the study. We investigated incidence, mechanism, and patient characteristics of ACL graft retears. Mann-Whitney U test was used for statistical analysis, and the level of significance was determined at P < 0.05. RESULTS: Six patients suffered from ACL graft retear (9.4%). Mean duration between primary ACL reconstruction and incidence of retears was 11.7 months. However, there were no other postoperative graft ruptures after 24 months. Primary injury and retear mechanisms varied by patient. At six months after the primary ACL reconstruction surgery, mean quadriceps and hamstring strengths were 81% and 87%, respectively, indicating favorable recovery of muscle strength. However, preoperative quadriceps and hamstring strength in the retear group were 65% and 71%, respectively. In particular, preoperative quadriceps strength in the retear group demonstrated a lower value than that in the uninjured group (P < 0.05). CONCLUSIONS: We observed a high incidence of ACL graft retears in competitive female basketball players, as previously reported. Considering the timing of graft retear occurrences, an early return to playing basketball should be avoided following ACL reconstruction. Closer attention should be paid to player preoperative condition, as well as muscle strength and postoperative status.