Customized small-sized clinostat using 3D printing and gas-permeable polydimethylsiloxane culture dish.

Over the past few decades, research on life in space has increased. Owing to the expensive nature of and the challenges associated with conducting experiments in real space, clinostats, which continuously randomize the gravity vector by using motors, have been used to generate simulated microgravity (SMG) on Earth. Herein, by using a 3D printing method, we develop a customized small-sized clinostat (CS clinostat) that is easy to manufacture, inexpensive, and robust. Moreover, we develop and fabricate a gas-permeable polydimethylsiloxane culture dish that fits inside the CS clinostat. To validate SMG generation, ovarian cancer cells (OV- 90, TOV-21G, and Caov-3) were applied to demonstrate a significant reduction in caveolin-1 expression, a biomarker of SMG, indicating SMG generation. The proposed CS clinostat system has good accessibility for SMG research, which makes it useful as a tool for biologists, who are unfamiliar with conventional clinostat equipment, to conduct preliminary studies in the space environment.

Lumbrokinase, a Fibrinolytic Enzyme, Prevents Intra-Abdominal Adhesion by Inhibiting the Migrative and Adhesive Activities of Fibroblast via Attenuation of the AP-1/ICAM-1 Signaling Pathway.

Intra-abdominal adhesion is a complication following abdominal surgery caused by the suppression of fibrinolytic activity and aggravated fibroblast invasion of the injured area, which may lead to chronic illnesses such as chronic pain, intestinal obstruction, and female infertility. This study hypothesized that lumbrokinase, a fibrinolytic enzyme extracted from the earthworm, supports the wound healing process. Therefore, we assessed the effect of lumbrokinase on intra-abdominal adhesion. Lumbrokinase treatment significantly decreased the severity and the area of intra-abdominal adhesion in vivo in a dose-dependent manner compared with the controls (untreated and hyaluronate-treated). Lumbrokinase-associated adverse effects were not observed. Immunohistochemical analysis of adhesion tissues revealed a loosened adhesive band between tissues, coupled with significantly decreased peritoneal thickening in the lumbrokinase-treated group versus the control group. Three-dimensional spheroid, MTT, and scratch wound migration assays using the IMR-90 human fibroblast cell line demonstrated that lumbrokinase significantly attenuated the migration and adhesive activity of fibroblasts without compromising cell proliferation. The luciferase assay and western blot analysis showed that lumbrokinase inhibited the AP-1/ICAM-1 cell adhesion signaling pathway. Therefore, lumbrokinase decreases intra-abdominal adhesion and peritoneal thickening by augmenting fibrinolytic action and inhibiting fibroblast migration and adhesive activity via attenuation of the AP-1/ICAM-1 signaling pathway. Lumbrokinase is thus a promising agent to prevent intra-abdominal adhesion.

DKK3, Downregulated in Invasive Epithelial Ovarian Cancer, Is Associated with Chemoresistance and Enhanced Paclitaxel Susceptibility via Inhibition of the ß-Catenin-P-Glycoprotein Signaling Pathway.

Dickkopf-3 (DKK3), a tumor suppressor, is frequently downregulated in various cancers. However, the role of DKK3 in ovarian cancer has not been evaluated. This study aimed to assess aberrant DKK3 expression and its role in epithelial ovarian carcinoma. DKK3 expression was assessed using immunohistochemistry with tissue blocks from 82 patients with invasive carcinoma, and 15 normal, 19 benign, and 10 borderline tumors as controls. Survival data were analyzed using Kaplan-Meier and Cox regression analysis. Paclitaxel-resistant cells were established using TOV-21G and OV-90 cell lines. Protein expression was assessed using Western blotting and immunofluorescence analysis. Cell viability was assessed using the MT assay and 3D-spheroid assay. Cell migration was determined using a migration assay. DKK3 was significantly downregulated in invasive carcinoma compared to that in normal, benign, and borderline tumors. DKK3 loss occurred in 56.1% invasive carcinomas and was significantly associated with disease-free survival and chemoresistance in serous adenocarcinoma. DKK3 was lost in paclitaxel-resistant cells, while ß-catenin and P-glycoprotein were upregulated. Exogenous secreted DKK3, incorporated by cells, enhanced anti-tumoral effect and paclitaxel susceptibility in paclitaxel-resistant cells, and reduced the levels of active ß-catenin and its downstream P-glycoprotein, suggesting that DKK3 can be used as a therapeutic for targeting paclitaxel-resistant cancer.

Synergistic Antiproliferative Effects of All-Trans Retinoic Acid and Paclitaxel on Autosomal Dominant Polycystic Kidney Disease Epithelial Cells.

Autosomal dominant polycystic kidney disease (ADPKD) is a genetic disorder characterized by uncontrollable epithelial cell growth, cyst formation, and kidney malfunction. In the present study, we investigated the antiproliferative effects of the treatment with the combination of paclitaxel (PAC) and all-trans retinoic acid (ATRA) on ADPKD epithelial cells. Our results show that the combined treatment with 1 nM PAC and 10 nM ATRA significantly suppressed ADPKD cell proliferation (20%), while the treatment with ATRA or PAC alone had no such effect. Treatment with PAC and ATRA induced cell cycle arrest at the G2/M phase and apoptosis by upregulating p53 and caspase-8 expression and increased the intracellular calcium (Ca2+) level possibly by enhancing Ca2+ uptake via plasma membrane channels. In addition, this treatment suppressed extracellular signal-regulated kinase signaling possibly through mitogen-activated protein kinase phosphatase-1 activation. Thus, the combination of PAC and ATRA can be explored as a potential treatment regimen for ADPKD.

ß-TrCP1-variant 4, a novel splice variant of ß-TrCP1, is a negative regulator of ß-TrCP1-variant 1 in ß-catenin degradation.

ß-transducin repeats-containing protein-1 (ß-TrCP1) serves as the substrate recognition subunit for SCFß-TrCP E3 ubiquitin ligases, which specifically ubiquitinate phosphorylated substrates. Three variants of ß-TrCP1 are known and act as homodimer or heterodimer complexes. Here, we identified a novel full-sequenced variant, ß-TrCP1-variant 4, which harbours exon II instead of exon III of variant 1, with no change in the open reading frame. The expression of ß-TrCP1-variant 4 is lower than that of variant 1 or 2 in ovarian cancer cell lines, whereas it is abundantly expressed in normal and cancerous ovarian tissues. Moreover, ß-TrCP1-variant 2 was aberrantly expressed more than variant 1 in ovarian cancer tissues whereas variant 1 was expressed more in normal tissues. Similar to variants 1 and 2, ß-TrCP1-variant 4 directly interacts with ß-catenin, one of the substrates of SCFß-TrCP E3 ubiquitin ligase and down-regulates the transcriptional activity and protein expression of ß-catenin with a significantly weaker effect than that by variants 1 and 2. However, the co-expression of ß-TrCP1-variant 4 with variant 1 in same proportion has no effect, whereas other combinations effectively down-regulate the activity of ß-catenin, indicating that the heterodimer of variants 1 and 4 has no function. Thus, ß-TrCP1-variant 4 could play a critical role in SCFß-TrCP E3 ligase-mediated ubiquitination by acting as a negative regulator of ß-TrCP1-variant 1.

Dickkopf-3 in Human Malignant Tumours: A Clinical Viewpoint.

Dickkopf-3 (DKK3), also known as REIC, is a secreted glycoprotein. DKK3 is aberrantly expressed in various types of human malignant tumours. Promoter methylation status, intracellular protein expression, and protein expression in tumour vessels are significantly correlated with clinical prognostic factors, including survival. In malignant cells, DKK3 is involved in the induction of apoptosis, inhibition of invasion, and remodelling of tumour vasculature. These activities are carried out via the regulation of the beta-catenin signalling and c-Jun N-terminal kinase-dependent cellular pathway, both of which are critical for carcinogenesis. This review explores the potential value of DKK3 as a clinical biomarker and a therapeutic candidate in human malignancies.