Both Ser361 phosphorylation and the C-arrestin domain of thioredoxin interacting protein are important for cell cycle blockade at the G1/S checkpoint.

Thioredoxin interacting protein (TXNIP) is a novel tumor suppressor that is down-regulated in several cancer tissues and tumor cell lines. Overexpression of TXNIP causes cell cycle arrest at the G1/S checkpoint in the hepatocellular carcinoma cell line HuH-7. TXNIP contains putative phosphorylation sites, but the effects of its phosphorylation have not been fully characterized. TXNIP also contains two α-arrestin domains (N-arrestin and C-arrestin) whose functions are not fully understood. Here, we reveal an association between TXNIP and cell cycle regulatory proteins (p27kip1, Jun activation domain-binding protein 1 (JAB1), Cdk2, and cyclin E), suggesting its participation in cell cycle regulation. We observed phosphorylation of TXNIP and used both in vivo and in vitro kinase assays to demonstrate that TXNIP can be phosphorylated by p38 mitogen-activated protein kinase. Furthermore, we also identified Ser361 in TXNIP as one of the major phosphorylation sites. Cell cycle analysis showed that Ser361 phosphorylation participates in TXNIP-mediated cell cycle arrest. In addition, the C-arrestin domain may also play an important role in cell cycle arrest. We also showed that phosphorylation at Ser361 may be important for the association of TXNIP with JAB1 and that the C-arrestin domain is necessary for the nuclear localization of this molecule. Collectively, these studies reveal that TXNIP participates in cell cycle regulation through association with regulatory proteins, especially JAB1, and that C-arrestin-dependent nuclear localization is important for this function. This work may facilitate the development of a new cancer therapy strategy that targets TXNIP as a key molecule inhibiting cancer cell growth via cell cycle blockade at the G1/S checkpoint.

Additive antitumour effect of D­allose in combination with cisplatin in non-small cell lung cancer cells.

D­allose is a rare sugar which has been shown to have growth inhibitory effects in several kinds of malignancies. However, the effect of D­allose on lung cancer progression has not been previously studied. To investigate the antitumour effect of D­allose in lung cancer cells and its mechanism, human non-small cell lung cancer (NSCLC) cell lines (squamous cell carcinomas: EBC1 and VMRC­LCD; adenocarcinomas: A549, HI1017, RERF­LC­A1 and NCI-H1975) were treated with D­allose (50 mM) with or without cisplatin (5 µM). D­allose inhibited cell growth, particularly in EBC1 and VMRC­LCD cells. In combination with cisplatin, D­allose had a synergistic growth inhibitory effect. D­allose increased the expression of thioredoxin interacting protein (TXNIP) at mRNA and protein levels. D­allose decreased the proportion of cells in G1 phase and increased those in S and G2/M phases. For in vivo experiments, EBC1 cells were inoculated into BALB/c-nu mice. After tumourigenesis, D­allose and cisplatin were injected. In this mouse xenograft model, additional treatment with D­allose showed a significantly greater tumour inhibitory effect compared with cisplatin alone, accompanied by lower Ki­67 and higher TXNIP expression. In conclusion, D­allose inhibited NSCLC cell proliferation in vitro and tumour progression in vivo. In combination with cisplatin, D­allose had an additional antitumour effect. Specifically, increased TXNIP expression and subsequent G2/M arrest play a role in D­allose-mediated antitumour effects in NSCLC.

Characterization of moss ent-kaurene oxidase (CYP701B1) using a highly purified preparation.

CYP701B1 of the moss, Physcomitrella patents, might be a unique cytochrome P450 having the ent-kaurene oxidase (KO) activity occurring in nonvascular plant. Phylogenetic analysis suggested that the gene encoding CYP701B1 was diverged from a common ancestral gene encoding KO of vascular plants. CYP701B1 expressed in Phichia yeast microsomes was purified and characterized. The purified CYP701B1 catalyzed the oxidation of ent-kaurene to ent-kaurenoic acid through three successive monooxygenations, and the rate-limiting step of this oxidation might be the initial step that forms ent-kaurenol. CYP701B1 was a typical ferric low-spin cytochrome P450 and was completely moved to high-spin state upon binding with ent-kaurene, and apparent Kd of ent-kaurene estimated by the spectral change caused by this spin-state shift was 2.5 µM. The potent KO inhibitor uniconazole, an azole compound with molecular size similar to ent-kaurene, bound CYP701B1 with high affinity. However, ketoconazole, an azole compound whose molecular size is larger than ent-kaurene could not bind to CYP701B, though it binds strongly with CYP51, lanosterol 14-demethylase. The results indicated that the active site of CYP701B1 is fitted for the molecular size of ent-kaurene. The P450 monooxygenase adapted for ent-kaurene oxidation might appear in land plants before evolutionary divergence into vascular and nonvascular plants.

Inhibitory effect of isoamericanol A from Jatropha curcas seeds on the growth of MCF-7 human breast cancer cell line by G2/M cell cycle arrest.

Although various parts of J. curcas (Jatropha curcas L., Euphorbiaceae) have long been used as traditional folk medicines for their antiviral, analgesic, and/or antidotal efficacies, we are the first to investigate the role of anti-carcinogenicity of isoamericanol A (IAA) from the seed extract. Our results showed that IAA is capable of inhibiting cell proliferation in a dose-dependent manner on the human cancer cell lines of MCF-7, MDA-MB231, HuH-7, and HeLa. Flow cytometry analysis showed IAA significantly induces cell cycle arrest at G2/M on MCF-7 cells. At both protein and mRNA levels examined by western blot and real-time PCR, the results revealed increased expression of BTG2 (B-cell translocation gene 2), p21 (p21(WAF1/CIPI) ), and GADD45A (growth arrest and DNA-damage-inducible, alpha) after IAA treatment, but inversed expression in CDK1 (cyclin-dependent kinase 1) and cyclins B1 and B2. All these effects contribute to G2/M cell cycle arrest. Furthermore, these results coincide with the changes in molecular expressions determined by DNA-microarray analysis. Our findings indicate that IAA has an inhibitory effect on cell proliferation of MCF-7 through cell cycle arrest, giving it great potential as a future therapeutic reagent for cancers.

D-Allose Inhibits Cancer Cell Growth by Reducing GLUT1 Expression.

Glucose is a major energy source for mammalian cells and is transported into cells via cell-specific expression of various glucose transporters (GLUTs). Especially, cancer cells require massive amounts of glucose as an energy source for their dysregulated growth and thus over-express GLUTs. d-allose, a C-3 epimer of d-glucose, is one of rare sugars that exist in small quantities in nature. We have shown that d-allose induces the tumor suppressor gene coding for thioredoxin interacting protein (TXNIP) and inhibits cancer cell growth by G1 cell cycle arrest. It has also been reported that GLUTs including GLUT1 are over-expressed in many cancer cell lines, which may contribute to larger glucose utilization. Since d-allose suppresses the growth of cancer cells through the upregulation of TXNIP expression, our present study focused on whether d-allose down-regulates GLUT1 expression via TXNIP expression and thus suppresses cancer cell growth. Western blot and real-time PCR analyses revealed that d-allose significantly induced TXNIP expression and inhibited GLUT1 expression in a dose-dependent manner in three human cancer cell lines: hepatocellular carcinoma (HuH-7), Caucasian breast adenocarcinoma (MDA-MB-231), and neuroblastoma (SH-SY5Y). In these cell lines, d-allose treatment inhibited cell growth. Importantly, d-allose treatment decreased glucose uptake, as measured by the uptake of 2-deoxy d-glucose. Moreover, the reporter assays showed that d-allose decreased the expression of luciferase through the hypoxia response element present in the tested promoter region. These results suggest that d-allose may cause the inhibition of cancer growth by reducing both GLUT1 expression and glucose uptake.

Beneficial effect of D-allose for isolated islet culture prior to islet transplantation.

BACKGROUND: Pretransplant restoration of islets damaged during isolation remains to be solved. In this study, we examined the effect of D-allose on islets isolated from rat pancreata prior to islet transplantation. METHODS: Rat islets isolated from fresh pancreata were cultured overnight in Roswell Park Memorial Institute 1640 solution in the absence (group 1) or presence (group 2) of D-allose. Then we assessed stimulation index of insulin, and cure rate after islet transplantation to diabetic nude mice. We also measured malondialdehyde level and caspase 3 activity of islets after the overnight culture for assessment of the oxidative stress and the apoptosis. RESULTS: D-allose significantly improved insulin secretion of islets. The stimulation index in group 2 was significantly higher than in group 1. Cure rate after transplantation in group 2 was higher than in group 1 especially in the first week. The malondialdehyde level in group 2 was significantly lower than in group 1. But the caspase 3 activities in both groups did not differ. CONCLUSIONS: D-allose treatment of isolated islet culture prior to transplantation restored islet function and increased successful transplant rate. The results of this study suggested that D-allose improved function of damaged islets through its anti-oxidative activity.

Rare sugar D-psicose prevents progression and development of diabetes in T2DM model Otsuka Long-Evans Tokushima Fatty rats.

BACKGROUND: The fundamental cause of overweight and obesity is consumption of calorie-dense foods. We have introduced a zero-calorie sweet sugar, d-psicose (d-allulose), a rare sugar that has been proven to have strong antihyperglycemic and antihyperlipidemic effects, and could be used as a replacement of natural sugar for the obese and diabetic subjects. AIM: Above mentioned efficacy of d-psicose (d-allulose) has been confirmed in our previous studies on type 2 diabetes mellitus (T2DM) model Otsuka Long-Evans Tokushima Fatty (OLETF) rats with short-term treatment. In this study we investigated the long-term effect of d-psicose in preventing the commencement and progression of T2DM with the mechanism of preservation of pancreatic ß-cells in OLETF rats. METHODS: Treated OLETF rats were fed 5% d-psicose dissolved in water and control rats only water. Nondiabetic control rats, Long-Evans Tokushima Otsuka (LETO), were taken as healthy control and fed water. To follow the progression of diabetes, periodic measurements of blood glucose, plasma insulin, and body weight changes were continued till sacrifice at 60 weeks. Periodic in vivo body fat mass was measured. On sacrifice, pancreas, liver, and abdominal adipose tissues were collected for various staining tests. RESULTS: d-Psicose prevented the commencement and progression of T2DM till 60 weeks through the maintenance of blood glucose levels, decrease in body weight gain, and the control of postprandial hyperglycemia, with decreased levels of HbA1c in comparison to nontreated control rats. This improvement in glycemic control was accompanied by the maintenance of plasma insulin levels and the preservation of pancreatic ß-cells with the significant reduction in inflammatory markers. Body fat accumulation was significantly lower in the treatment group, with decreased infiltration of macrophages in the abdominal adipose tissue. CONCLUSION: Our findings suggest that the rare sugar d-psicose could be beneficial for the prevention and control of obesity and hyperglycemia with the preservation of ß-cells in the progression of T2DM.

Rare sugar D-allose strongly induces thioredoxin-interacting protein and inhibits osteoclast differentiation in Raw264 cells.

Oxidative stress modulates the osteoclast differentiation via redox systems, and thioredoxin 1 (Trx) promotes the osteoclast formation by regulating the activity of transcription factors. The function of Trx is known to be regulated by its binding partner, thioredoxin-interacting protein (TXNIP). We previously reported that the expression of TXNIP gene is strongly induced by a rare sugar D-allose. In this study, we tested the hypothesis that D-allose could inhibit the osteoclast differentiation by regulating the Trx function. We used a murine Raw264 cell line that differentiates to the osteoclast by the receptor activator of nuclear factor-κB ligand (RANKL) treatment. The effect of sugars was evaluated by tartrate-resistant acid phosphatase staining. The expression and localization of TXNIP and Trx protein were examined by Western blotting and immunohistochemisty. The activity of the nuclear factor-κB, nuclear factor of activated T cells, and activator protein 1 transcription factors was measured by the luciferase reporter assay. The addition of D-allose (25 mmol/L) inhibited the osteoclast differentiation down to 9.53% ± 1.27% of a receptor activator of nuclear factor-κB ligand-only treatment. During the osteoclast differentiation, a significant increase of TNXIP was observed by D-allose treatment. The immunohistochemical analysis showed that both Trx and TXNIP existed in the nucleus in preosteoclasts and osteoclasts. Overexpression of TXNIP by plasmid transfection also inhibited the osteoclast formation, indicating the functional importance of TXNIP for the osteoclast differentiation. Transcriptional activity of the activator protein 1, nuclear factor-κB, and nuclear factor of activated T cells, known to be modulated by Trx, were inhibited by D-allose. In conclusion, our data indicate that D-allose is a strong inhibitor of the osteoclast differentiation, and this effect could be caused by TXNIP induction and a resulting inhibition of the Trx function.

Inhibitory effects of isoliquiritigenin and licorice extract on voltage-dependent K(+) currents in H9c2 cells.

The effect of isoliquiritigenin (ISL), a component of licorice, on the voltage-dependent, ultra-rapidly activating delayed-rectifier K(+) current (IKur) was examined in H9c2 cells, a cell-line derived from rat cardiac myoblasts. IKur was recorded using the whole-cell patch clamp method with a pipette solution containing 140 mM K(+). Depolarizing voltage pulses of 200-ms duration were given with 10-mV steps every 10 s from -40 mV holding potential. ISL inhibited IKur in a concentration-dependent manner. The median inhibitory concentration (IC(50)) of ISL was approximately 0.11 microM and the Hill coefficient was 0.71. Using CHO cells expressing Kv1.5 IKur channels, ISL also inhibited Kv1.5 IKur, but less potently than the IKur current in H9c2 cells. Furthermore, in H9c2 cells, the licorice extract itself inhibited IKur in a manner similar to ISL. We conclude that ISL, one component of licorice, is a potent inhibitor of K(+) channels, which specifically in H9c2 cells could be Kv2.1, and that this inhibition may be involved in various pharmacological effects of licorice.

[Anesthetic management of two cases with placenta percreta that caused massive hemorrhage during cesarean section].

We report the anesthetic management of two cases with placenta percreta that caused massive hemorrhage during cesarean section. These pregnant women, with a past history of cesarean section underwent elective operation after being diagnosed with total placenta previa and suspected adhesion of the placenta. The placenta percreta became evident after laparotomy and the patients underwent total hysterectomy after infant expulsion. They went into serious hemorrhagic shock, and recovered after the application of intensive cardiovascular support and blood transfusion. In recent years, the incidence of adhesion of the placenta has increased, but definitive, preoperative diagnosis is difficult; especially for the severe type: placenta percreta. Therefore, intensive management is necessary for the anesthesia of pregnant women with suspected adhesion of the placenta, including adequate preparation of transfused blood, since it might be difficult to save the mother's life after the onset of massive hemorrhage.