Fumie Shiraishi

Anti-angiogenic effects of differentiation-inducing factor-1 involving VEGFR-2 expression inhibition independent of the Wnt/β-catenin signaling pathway.

BackgroundDifferentiation-inducing factor-1 (DIF-1) is a putative morphogen that induces cell differentiation in Dictyostelium discoideum. DIF-1 inhibits proliferation of various mammalian tumor cells by suppressing the canonical Wnt/β-catenin signaling pathway. To assess the potential of a novel cancer chemotherapy based on the pharmacological effect of DIF-1, we investigated whether DIF-1 exhibits anti-angiogenic effects in vitro and in vivo.ResultsDIF-1 not only inhibited the proliferation of human umbilical vein endothelial cells (HUVECs) by restricting cell cycle in the G0/G1 phase and degrading cyclin D1, but also inhibited the ability of HUVECs to form capillaries and migrate. Moreover, DIF-1 suppressed VEGF- and cancer cell-induced neovascularization in Matrigel plugs injected subcutaneously to murine flank. Subsequently, we attempted to identify the mechanism behind the anti-angiogenic effects of DIF-1. We showed that DIF-1 strongly decreased vascular endothelial growth factor receptor-2 (VEGFR-2) expression in HUVECs by inhibiting the promoter activity of human VEGFR-2 gene, though it was not caused by inhibition of the Wnt/β-catenin signaling pathway.ConclusionThese results suggested that DIF-1 inhibits angiogenesis both in vitro and in vivo, and reduction of VEGFR-2 expression is involved in the mechanism. A novel anti-cancer drug that inhibits neovascularization and tumor growth may be developed by successful elucidation of the target molecules for DIF-1 in the future.

DIF-1 inhibits the Wnt/β-catenin signaling pathway by inhibiting TCF7L2 expression in colon cancer cell lines.

We previously reported that differentiation-inducing factor-1 (DIF-1), a morphogen in Dictyostelium discoideum, inhibits the proliferation of human cancer cell lines by inducing β-catenin degradation and suppressing the Wnt/β-catenin signaling pathway. To determine whether β-catenin degradation is essential for the effect of DIF-1, we examined the effect of DIF-1 on human colon cancer cell lines (HCT-116, SW-620 and DLD-1), in which the Wnt/β-catenin signaling pathway is constitutively active. DIF-1 strongly inhibited cell proliferation and arrested the cell cycle in the G(0)/G(1) phase via the suppression of cyclin D1 expression at mRNA and protein levels without reducing β-catenin protein. TCF-dependent transcriptional activity and cyclin D1 promoter activity were revealed to be inhibited via suppression of transcription factor 7-like 2 (TCF7L2) expression. Luciferase reporter assays and EMSAs using the TCF7L2 promoter fragments indicated that the binding site for the transcription factor early growth response-1 (Egr-1), which is located in the -609 to -601 bp region relative to the start codon in the TCF7L2 promoter, was involved in DIF-1 activity. Moreover, RNAi-mediated depletion of endogenous TCF7L2 resulted in reduced cyclin D1 promoter activity and protein expression, and the overexpression of TCF7L2 overrode the inhibition of the TCF-dependent transcriptional activity and cyclin D1 promoter activity induced by DIF-1. Therefore, DIF-1 seemed to inhibit the Wnt/β-catenin signaling pathway by suppressing TCF7L2 expression via reduced Egr-1-dependent transcriptional activity in these colon cancer cell lines. Our results provide a novel insight into the mechanisms by which DIF-1 inhibits the Wnt/β-catenin signaling pathway.

Differential effects of organic nitrates on arterial diameter among healthy Japanese participants with different mitochondrial aldehyde dehydrogenase 2 genotypes: randomised crossover trial

Objectives To determine whether polymorphisms at codon 487 (*1, GAA=Glu; *2, AAA=Lys) of mitochondrial aldehyde dehydrogenase 2 (ALDH2) influence nitroglycerine (glyceryl trinitrate (GTN))-induced vasodilation, and whether GTN or isosorbide dinitrate (ISDN) is a more effective antianginal agent in each ALDH2 genotype. Design A randomised, open-label, crossover trial with 117 healthy Japanese (20–39 years) whose genotypes were determined (*1/*1, n=47; *1/*2, n=48; *2/*2, n=22) was performed at Kyushu University Hospital, Fukuoka, Japan. Participants were randomly assigned to treatment: sublingual spray of GTN (0.3 mg) or ISDN (1.25 mg). After ≥1 week, measurements were repeated using the other drug. The main outcome measures were the maximal rate of increase in the brachial artery diameter determined by ultrasonography, the time required to attain maximal dilation (Tmax) and the time required to attain 90% maximal dilation (T0.9). Results The maximal artery diameter increase in response to GTN or ISDN did not differ among genotypes. However, GTN Tmax was significantly longer for *2/*2 (299.7 s, 269.0–330.4) than *1/*1 (254.7 s, 238.6–273.4; p=0.0190). GTN T0.9 was significantly longer in the *1/*2 (206.1 s, 191.7–219.3) and *2/*2 (231.4 s, 211.8–251.0) genotypes than *1/*1 (174.9 s, 161.5–188.3; p=0.0068, p<0.0001, respectively). In contrast, the time-course of ISDN-induced vasodilation did not differ among genotypes. GTN Tmax and T0.9 among *1 allele carriers (*1/*1 and *1/*2) were significantly shorter than those of ISDN, whereas the time course of GTN and ISDN vasodilation did not differ among participants carrying *2/*2. Conclusions The amplitude of GTN-induced vasodilation was not influenced by the ALDH2 genotype, but the response was significantly delayed in *2 allele carriers, especially *2/*2. GTN dilated the artery more quickly than ISDN in *1/*1 and *1/*2, but not in *2/*2. Trial registration number UMIN000001492 (UMIN-CTR database).

The effect of troponin C substitution on the Ca2+-sensitive ATPase activity of vertebrate and invertebrate myofibrils by troponin Cs with various numbers of Ca2+-binding sites

The effect of four different classes of troponin C with different numbers of Ca(2+)-binding sites was investigated on the Ca(2+)-activation profiles of the ATPase of troponin C-depleted myofibrils prepared from vertebrate fast skeletal (rabbit), vertebrate cardiac (bovine) and invertebrate crustacean tail striated (crayfish, lobster) muscles. Troponin C from vertebrate sources [fast skeletal (rabbit, chicken) with four Ca(2+)-binding sites, and cardiac (bovine, chicken) with three Ca(2+)-binding sites] activated the Ca(2+)-ATPase of troponin C-depleted myofibrils from the vertebrate skeletal or cardiac muscles, but did not activate the invertebrate troponin C-depleted crustacean myofibrils. On the other hand, two kinds of crustacean (crayfish, lobster) troponin C with two Ca(2+)-binding sites activated only crustacean troponin C-depleted myofibrils. One invertebrate molluscan (Akazara scallop) troponin C with one Ca(2+)-binding site did not activate the Ca(2+)-ATPase of the troponin C-depleted myofibrils from either vertebrate or crustacean striated muscles. The results obtained from the four kinds of combinations of troponin C and troponin C-depleted myofibrils from vertebrate skeletal and cardiac muscles demonstrated that the characteristic cooperativity of the Ca(2+)-activation profiles of both vertebrate skeletal and cardiac myofibrils was determined by the skeletal or cardiac origin of troponin C molecules, irrespective of the animal species, and the Ca(2+)-affinity of the myofibrillar ATPase was related to the skeletal or cardiac origin of both the troponin C and myofibrils. These findings indicated that each of the four classes of troponin C has its own characteristic Ca(2+)-activation profile for each kind of myofibril examined in the present study.

The exchange of Ca2+-receptive protein complex (troponin) in the myofibrils of fast and slow skeletal muscles

In order to compare the role of the Ca2+-receptive protein (troponin), in the characteristic myofibrillar contractile response of chicken fast and slow skeletal muscles, the troponin in both kinds of myofibrils were partially exchanged, under slightly acidic conditions. The Ca2+- or Sr2+-activation of the ATPase of fast (or slow) skeletal myofibrils hybridized with slow (or fast) skeletal troponin profiles were also investigated. The results indicated that the Ca2+- or Sr2+-affinity of the myofibrillar ATPase activity were related to the species of troponin. This procedure for replacing troponin in myofibrils under physiological conditions in thus considered to be useful for the study of the Ca2+-regulatory mechanism in myofibrillar contraction.