Effect of Inhibition of the Lysophosphatidic Acid Receptor 1 on Metastasis and Metastatic Dormancy in Breast Cancer

Background Previous studies identified the human nonmetastatic gene 23 (NME1, hereafter Nm23-H1) as the first metastasis suppressor gene. An inverse relationship between Nm23-H1 and expression of lysophosphatidic acid receptor 1 gene (LPAR1, also known as EDG2 or hereafter LPA1) has also been reported. However, the effects of LPA1 inhibition on primary tumor size, metastasis, and metastatic dormancy have not been investigated. Methods The LPA1 inhibitor Debio-0719 or LPA1 short hairpinned RNA (shRNA) was used. Primary tumor size and metastasis were investigated using the 4T1 spontaneous metastasis mouse model and the MDA-MB-231T experimental metastasis mouse model (n = 13 mice per group). Proliferation and p38 intracellular signaling in tumors and cell lines were determined by immunohistochemistry and western blot to investigate the effects of LPA1 inhibition on metastatic dormancy. An analysis of variance-based two-tailed t test was used to determine a statistically significant difference between treatment groups. Results In the 4T1 spontaneous metastasis mouse model, Debio-0719 inhibited the metastasis of 4T1 cells to the liver (mean = 25.2 liver metastases per histologic section for vehicle-treated mice vs 6.8 for Debio-0719-treated mice, 73.0% reduction, P < .001) and lungs (mean = 6.37 lesions per histologic section for vehicle-treated mice vs 0.73 for Debio-0719-treated mice, 88.5% reduction, P < .001), with no effect on primary tumor size. Similar results were observed using the MDA-MB-231T experimental pulmonary metastasis mouse model. LPA1 shRNA also inhibited metastasis but did not affect primary tumor size. In 4T1 metastases, but not primary tumors, expression of the proliferative markers Ki67 and pErk was reduced by Debio-0719, and phosphorylation of the p38 stress kinase was increased, indicative of metastatic dormancy. Conclusion The data identify Debio-0719 as a drug candidate with metastasis suppressor activity, inducing dormancy at secondary tumor site

Zebrafish-Based Screening Models for the Identification of Anti-Metastatic Drugs

Metastasis, a leading contributor to the morbidity of cancer patients, occurs through a multi-step process: invasion, intravasation, extravasation, colonization, and metastatic tumor formation. Each process is not only promoted by cancer cells themselves but is also affected by their microenvironment. Given this complexity, drug discovery for anti-metastatic drugs must consider the interaction between cancer cells and their microenvironments. The zebrafish is a suitable vertebrate animal model for in vivo high-throughput screening studies with physiological relevance to humans. This review covers the zebrafish model used to identify anti-metastatic drugs

Development of an in vivo tissue-engineered, autologous heart valve (the biovalve) : preparation of a prototype model

Objective: This study aimed to develop an autologous heart valve without using traditional in vitro tissue-engineering methods, which necessitate complicated cell management protocols under exceptionally clean laboratory facilities. Methods: An autologous heart valve construct composed of trileaflets was prepared using a specially designed mold. The mold was prepared by covering a silicone rod with a crown-shaped tubular polyurethane scaffold containing 3 horns. The mold was implanted in the dorsal subcutaneous space in Japan White rabbits for 4 weeks. After harvesting, the implanted trileaflet valve-shaped structure with an internal diameter of either 5 or 20 mm was obtained by trimming the membranous tissue formed between the horns located around the silicone rod. The valve substitute was examined both macroscopically and histologically. The tensile strength of the leaflets was measured to rupture. The degree of regurgitation in valve function was evaluated using a flow circuit by calculating the ratio of the regurgitation volume to the forward flow volume. Results: After implantation, the mold was completely covered with connective tissue consisting mostly of collagen and fibroblasts. Harvesting of the mold was straightforward, because there was little adhesion between the formed tissue and the native skin tissue. The trileaflet heart valve construct was obtained after withdrawing the inserted rods and trimming the membranous tissues formed between the horns of the scaffold. It was firmly attached to the scaffold, the interstices and surface of which revealed connective tissues composed of components similar to those of the leaflet tissue. Although the mechanical properties of the leaflet tissue were less efficient than those of the native porcine aortic valve leaflets, satisfactory valvular functions were demonstrated under pulsatile conditions using a flow circuit. No regurgitation was observed under retrograde hydrostatic pressures of up to 60 mm Hg, the physiologic pressure acting on the aortic valves during retrograde aortic flow. Conclusions: The biovalve, an autologous, in vivo tissue-engineered, trileaflet, valve-shaped construct, was developed using our novel in-body tissue architecture technology. The biovalve has the potential to be an ideal prosthetic heart valve, with excellent biocompatibility to the growth of the recipient’s heart

Characteristics of Nodulation and Nitrogen Fixation in the Improved Supernodulating Soybean (Glycine max L. Merr.) Cultivar ‘Sakukei 4’

Supernodulating soybean lines have more than several times as many nodules as normal cultivars. They are expected to have high nitrogen-fixing ability and enhanced productivity, but their yields have been inferior to those of normal genotypes. We have recently developed a new supernodulating cultivar,‘Sakukei 4’(formerly ‘En-b0-1-2’, presently‘Kanto 100’), with improved growth and yield. The objective of the present study was to identify the characteristics of the nodulation and nitrogen-fixing ability of Sakukei 4. In pot trials, the nodule number of Sakukei 4 was 8.3 times that of a normal cultivar,‘Enrei’, and the nodule weight per plant was 2.3 to 2.8 times the value for Enrei. The acetylene reduction activity per plant in Sakukei 4 was higher than that in Enrei and conventional supernodulating genotypes, especially during the late growth stage. Compared with conventional supernodulating lines, the improved vegetative growth in shoots and roots of Sakukei 4, especially after flowering, probably enhanced its nitrogen-fixing ability per plant. We consider that its high nitrogen-fixing ability at the seed-filling stage, would help increase its yield in fields with low nitrogen fertility

Characteristics of Growth and Yield Formation in the Improved Genotype of Supernodulating Soybean (Glycine maxL. Merr.)

Most of the supernodulating soybean lines were agronomically inferior to conventional genotypes. Our previous tests with ‘En6500’, a supernodulating mutant derived from a variety ‘Enrei’, revealed that its low growth and yield were not necessarily due to supernodulation. In our attempts to improve the yield of En6500 through crossing with Enrei, we recently succeeded in selecting a supernodulating line showing vigorous growth. Field experiments with this new supernodulating line ‘En-bO-1-2’ for three years revealed that it yielded much more than En6500. When the overall yield level was low, it even tended to yield more than Enrei. En-bO-1-2 thus showed a remarkably higher productivity than other supernodulating lines reported so far. Its improved yield was largely due to : (a) better seed filling, (b) vigorous vegetative growth during flowering period, and (c) high leaf area index and leaf N content that enabled production of more photosynthates to enhance N fixation and dry matter accumulation during the period of pod and seed development