MicroRNA-5572 Is a Novel MicroRNA-Regulating SLC30A3 in Sporadic Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive degenerative disease caused by the loss of motor neurons. Although the pathogenesis of sporadic ALS (sALS) remains unclear, it has recently been suggested that disorders of microRNA (miRNA) may be involved in neurodegenerative conditions. The purpose of this study was to investigate miRNA levels in sALS and the target genes of miRNA. Microarray and real-time RT-PCR analyses revealed significantly-decreased levels of miR-139-5p and significantly increased levels of miR-5572 in the spinal cords of sALS patients compared with those in controls. We then focused on miR-5572, which has not been reported in ALS, and determined its target gene. By using TargetScan, we predicted SLC30A3 as the candidate target gene of miR-5572. In a previous study, we found decreased SLC30A3 levels in the spinal cords of sALS patients. We revealed that SLC30A3 was regulated by miR-5572. Taken together, these results demonstrate that the level of novel miRNA miR-5572 is increased in sALS and that SLC30A3 is one of the target genes regulated by miR-5572.

Dexamethasone exacerbates cisplatin-induced muscle atrophy.

Dexamethasone for antiemetic therapy is typically administered with anticancer drugs such as cisplatin. We previously reported that cisplatin upregulates the muscle-specific E3 ubiquitin ligase genes, namely muscle ring-finger protein 1 (MuRF1) and atrophy gene-1 (atrogin-1), and promotes muscle atrophy in mice. It is well known that dexamethasone causes upregulation of MuRF1 and Atrogin-1 expression in skeletal muscles. Although it is speculated that a combination of dexamethasone and cisplatin worsens muscle atrophy, there are no reports based on research. We thereby investigated the effects of cisplatin and dexamethasone, alone or in combination, on the expression of MuRF1 and Atrogin-1 in murine skeletal muscles and C2C12 myotubes. Mice were intraperitoneally injected with cisplatin or the vehicle control once daily for 4 days. Dexamethasone or the vehicle control was subcutaneously administered 30 minutes prior to the administration of cisplatin. Dexamethasone enhanced MuRF1 and Atrogin-1 gene expression upregulated by cisplatin in murine quadriceps muscles and C2C12 myotubes. Cisplatin-caused upregulation of myostatin and downregulation of IGF-1 gene expression were also enhanced by co-administration of dexamethasone in murine quadriceps muscles and C2C12 myotubes. This study shows that the combination treatment of cisplatin and dexamethasone exacerbated muscle atrophy in mice. Therefore, this treatment regimen might exacerbate muscle atrophy in cancer patients.

[Investigation of Colorectal Cancer Cases among Patients Aged over 75 Years Who Received Postoperative Adjuvant Chemotherapy].

BACKGROUND: As the aging of the Japanese population progresses, the administration of postoperative adjuvant chemotherapy( AC)to the elderly is also expected to increase. OBJECTIVE: To examine the characteristics of AC in cases of colorectal cancer among elderly people aged over 75 years. PATIENTS: Forty-eight cases of colorectal cancer patients who received AC. METHODS: The clinicopathological factors, including 14 patient-related factors, 6 operation-related factors, and 2 AC-related factors, as well as the long-term outcomes, were compared between the elderly group of patients aged over 75 years(group O, 12 cases)and the non-elderly group(group Y, 36 cases). RESULTS: Significant differences were observed between groups in neutrophil count(p=0.044), operation time(p=0.044), AC regimen(p=0.006), and administration completion status (p=0.046). Compared to group Y, a higher proportion of oral drug alone(92% vs 39%)and completion rate of the initial setting dose(75% vs 39%)were observed in group O. There was no significant difference in the 2-year disease-free survival rate. CONCLUSION: Oral preparations of AC may be useful from the viewpoint of tolerability in the elderly.

[A Case of Advanced Breast Cancer in an Elderly Patient That Was Well Controlled by Mohs' Paste, Endocrine Therapy, and Denosumab].

We report a case of advanced left breast cancer with continuous bleeding from the primary tumor surface in an 81-year-old woman. Pathological findings showed invasive carcinoma with positive expression of hormone receptors. CT and bone scintigraphy showed primary tumor invasion of the greater pectoral muscle and multiple bone metastases. She received Mohs' chemosurgery, endocrine therapy, and denosumab. The bleeding was maintained by weekly treatment. Most of the bulging surface of the tumor decreased and dried up 2 months later. The tumor disappeared almost completely, and the bone metastases achieved stable status 3 months later. Until currently, the primary tumor and metastasis have been controlled for 1.5 years. Mohs' paste was effective for controlling bleeding and exudates of unresectable cancer bulging from the skin surface and played an important role in cancer treatment with systemic therapy.

ELR+ chemokine-mediated neutrophil recruitment is involved in 2,4,6-trinitrochlorobenzene-induced contact hypersensitivity.

Contact dermatitis is a form of delayed-type hypersensitivity characterized by localized thickening, papules, redness and vesicles of the skin. A model of contact dermatitis involving repeated challenge of a hapten is adapted to assess dermatitis as characterized by skin thickening. Recently, it was reported that neutrophils have crucial roles in contact hypersensitivity. We thus examined the involvement of CXC chemokines bearing the glutamic acid-leucine-arginine (ELR) motif ("ELR+ chemokines") and neutrophils in the ear swelling induced by 2,4,6-trinitrochlorobenzene (TNCB) challenges in the present study. Mice were sensitized by application of TNCB on their abdominal skin. They were then challenged thrice with TNCB to the ear. The CXCR2 antagonist SB225002 (9 mg/kg, i.p.) was administered before each TNCB challenge. Gene expressions and protein levels of the ELR+ chemokines CXCL1, 2 and 5 was increased markedly in mouse ear after the final TNCB challenge. In addition, we indicated that gene expression of CXCL1 was enhanced in the epidermis and dermis upon TNCB challenge. Expression of the CXCL2 gene was enhanced in the epidermis, and that of the CXCL5 gene was enhanced in the dermis. The swelling induced by TNCB challenges was significantly attenuated by SB225002. Furthermore, the increases in myeloperoxidase activity, and expression of myeloperoxidase and neutrophil elastase induced by TNCB challenge in mouse ear were inhibited by SB225002. These data suggest that ear swelling resulting from TNCB challenges might be concerned by upregulated ELR+ chemokine-induced neutrophil recruitment.

Effect of acute treadmill exercise on cisplatin-induced muscle atrophy in the mouse.

Cisplatin, a platinum-based anti-cancer drug, is one of the most effective broad-spectrum anti-cancer agents used against various cancers. It has been recently suggested that low skeletal muscle mass is predictive of mortality in patients with cancer. Although several molecules produced by the actual tumor itself contribute to skeletal muscle impairment, we recently suggested that the administration of cisplatin could increase levels of muscle RING finger-1 (MuRF1) and atrogin-1, possibly leading to muscle atrophy in the mouse. Exercise is an important factor that induces muscle protein synthesis and muscle hypertrophy by enhancing the positive effects of the Akt/mTOR/p70S6 kinase pathway. In the present study, we therefore investigated the effect of treadmill exercise on cisplatin-induced muscle atrophy. C57BL/6J mice were treated with cisplatin (3 mg/kg, i.p.) or saline for four consecutive days. On day 4, the quadriceps and gastrocnemius muscles were isolated from the mice. The animals in the treadmill exercise groups were forced to run on a motorized treadmill for 20 min once a day for 9 days. In addition to muscle mass, the decrease in myofiber diameter associated with cisplatin administration was significantly restored by treadmill exercise. This exercise also significantly attenuated cisplatin-induced upregulation of MuRF1 and atrogin-1 in quadriceps and gastrocnemius muscle. The decreased Akt, p70S6 kinase, and Foxo3a phosphorylation observed with cisplatin treatment was significantly recovered by treadmill exercise in both the muscles. In the present study, myostatin (Mstn) gene expression, upregulated by cisplatin administration, was also attenuated by treadmill exercise. These findings suggest that treadmill exercise could attenuate cisplatin-induced muscle atrophy, at least partially, and could improve prognosis.

Active Ingredients of Hange-shashin-to, Baicalelin and 6-Gingerol, Inhibit 5-Fluorouracil-Induced Upregulation of CXCL1 in the Colon to Attenuate Diarrhea Development.

5-Fluorouracil (5-FU) is widely used as an anti cancer drug and is known to cause severe diarrhea. Recently we suggested that levels of chemokine (C-X-C motif) ligand 1 (CXCL1) and neutrophil recruitment in the colonic mucosa were drastically increased by the 5-FU administration in mice. Hange-shashin-to (HST) is prescribed in Japan for treat gastritis, stomatitis, and inflammatory diarrhea. We therefore examined the effects of HST and its active ingredients on 5-FU-induced CXCL1 upregulation in cultured colon tissue, and also examined the effects of HST on 5-FU-induced diarrhea development in the mouse. The distal colon isolated from the mouse was incubated with 5-FU and HST. Mice were given 5-FU (50 mg/kg, intraperitoneally (i.p.)) daily for four days. HST (300 mg/kg, per os (p.o.)) was administered 30 min before mice received 5-FU. mRNA levels of CXCL1 in the colon were examined using quantitative RT-PCR. 5-FU enhanced CXCL1 mRNA in the colon but the effect by 5-FU was markedly suppressed by application of HST and its active ingredients, baicalein and 6-gingerol. Nuclear factor kappa B (NF-κB) was activated by 5-FU treatment in cultured colon tissue, which was also suppressed by HST and the combination of baicalein and 6-gingerol. Furthermore, HST reduced 5-FU-induced diarrhea development. Under such experimental condition, CXCL1 gene, protein levels of neutrophil elastase and myeloperoxidase upregulation induced by 5-FU in the colon was attenuated by HST. These findings suggest that HST, especially baicalein and 6-gingerol, prevent the development of neutrophil recruitment and diarrhea by the inhibition of NF-κB activity.

Interference of Skin Scratching Attenuates Accumulation of Neutrophils in Murine Allergic Contact Dermatitis Model.

We recently reported that swelling resulting from 2,4,6-trinitrochlorobenzene (TNCB) challenge might be associated with recruitment of neutrophils. However, it is not known whether neutrophil recruitment is affected by scratching at inflamed sites or not. Therefore, the effects of an Elizabethan collar on the TNCB-induced upregulation of ELR-positive chemokines (CXCL1, CXCL2, and CXCL5) and neutrophil recruitment were investigated. Mice were sensitized by the application of TNCB on abdominal skin. Then, the mice were challenged three times with TNCB to auricle of the ear. To prevent scratching at inflamed sites, an Elizabethan collar was placed on the mice from just before the first challenge until the end of the experiment. The effects of the Elizabethan collar on the TNCB-induced upregulation of CXCLs chemokines and recruitment of neutrophil were investigated. The increase of ear swelling by TNCB challenge was inhibited by the Elizabethan collar. TNCB-challenge-induced upregulation of TNF-α, IL-1ß, IL-6, ELR+ chemokines, MPO, and ELA2 was also attenuated by the Elizabethan collar. The gene expression of CXCL1, CXCL2, and CXCL5 human homolog IL-8 was enhanced by TNF-α and IL-1ß in human dermal fibroblasts and epidermal keratinocytes. We here suggest that scratching the site of inflammation leads to neutrophil accumulation mediated by TNF-α and IL-1ß/ELR+ chemokines in TNCB-challenge-induced contact dermatitis in mice.

Mutational analyses of Plasmodium falciparum and human S-adenosylhomocysteine hydrolases.

S-adenosylhomocysteine hydrolase is a prospective target for developing new anti-malarial drugs. Inhibition of the hydrolase results in an anti-cellular effect due to the suppression of adenosylmethionine-dependent transmethylations. Based on the crystal structure of Plasmodium falciparum S-adenosylhomocysteine hydrolase which we have determined recently, we performed mutational analyses on P. falciparum and human enzymes. Cys59 and Ala84 of the parasite enzyme, and the equivalent residues on the human enzyme, Thr60 and Gln85, were examined. Mutations of Cys59 and Thr60 caused dramatic impact on inhibition by 2-fluoronoraristeromycin without significant effect both on its kinetic parameters and on inhibition constant against noraristeromycin. In addition, the impact was independent from the electronegativity of the side chain of the substituting residue. These results showed that steric hindrance between a functional group at the 2-position of an adenine nucleoside inhibitor and Thr60 of the human enzyme, not an electrostatic effect, contributed to inhibitor selectivity.

Crystal structure of S-adenosyl-L-homocysteine hydrolase from the human malaria parasite Plasmodium falciparum.

The human malaria parasite Plasmodium falciparum is responsible for the death of more than a million people each year. The emergence of strains of malarial parasite resistant to conventional drug therapy has stimulated searches for antimalarials with novel modes of action. S-Adenosyl-L-homocysteine hydrolase (SAHH) is a regulator of biological methylations. Inhibitors of SAHH affect the methylation status of nucleic acids, proteins, and small molecules. P.falciparum SAHH (PfSAHH) inhibitors are expected to provide a new type of chemotherapeutic agent against malaria. Despite the pressing need to develop selective PfSAHH inhibitors as therapeutic drugs, only the mammalian SAHH structures are currently available. Here, we report the crystal structure of PfSAHH complexed with the reaction product adenosine (Ado). Knowledge of the structure of the Ado complex in combination with a structural comparison with Homo sapiens SAHH (HsSAHH) revealed that a single substitution between the PfSAHH (Cys59) and HsSAHH (Thr60) accounts for the differential interactions with nucleoside inhibitors. To examine roles of the Cys59 in the interactions with nucleoside inhibitors, a mutant PfSAHH was prepared. A replacement of Cys59 by Thr results in mutant PfSAHH, which shows HsSAHH-like nucleoside inhibitor sensitivity. The present structure should provide opportunities to design potent and selective PfSAHH inhibitors.

Synthesis of 5'-methylenearisteromycin and its 2-fluoro derivative with potent antimalarial activity due to inhibition of the parasite S-adenosylhomocysteine hydrolase.

5'-methylenearisteromycin 5 and its 2-fluoro derivative 6, which were designed as antimalarial agents because of their AdoHcy hydrolase inhibition, were synthesized from D-ribose, using a stereoselective intramolecular radical cyclization as the key step to construct the carbocyclic structure. These compounds were evaluated as AdoHcy hydrolase inhibitors with the recombinant human and malarial parasite enzymes. Although 5 and 6 were both potent inhibitors of the malarial parasite AdoHcy hydrolase, the 2-fluoro derivative 6 proved to be superior due to its lower inhibitory effect on the human enzyme. In addition, 6 was identified as a potent antimalarial agent using an in vitro assay system with Plasmodium falciparum.

Three-dimensional structure of S-adenosyl-L-homocysteine hydrolase from Plasmodium falciparum.

Structural information of Plasmodium falciparum S-adenosyl-L-homocysteine hydrolase (PfSAHH) has been expected to provide new-type chemotherapeutic agents against malaria. Here we report the crystal structure of PfSAHH. The present structure should provide opportunities to design potent and selective PfSAHH inhibitors.

Synthesis of carbocyclic and acyclic nucleosides possessing 2-fluoroadenine derivatives and their inhibitory activities against Plasmodium falciparum SAH hydrolase.

Carbocyclic and acyclic nucleosides possessing 2-fluoroadenine, such as 2-fluoronoraristeromycin (6) and 2-fluoro-9-[(2S,3R)-2,3,4-trihydroxy-butyl-1-yl]adenine (8), were synthesized and their inhibitory activities against human and Plasmodium falciparum recombinant SAH hydrolase were investigated.