Novel Oral Derivative UD-017, a Highly Selective CDK7 Inhibitor, Exhibits Anticancer Activity by Inducing Cell-Cycle Arrest and Apoptosis in Human Colorectal Cancer

Objective: This study aimed to investigate the anticancer profile of a new cyclin-dependent kinase 7 (CDK7) inhibitor, UD-017, by examining its mechanism of action using HCT-116 colorectal cancer cells. Methods: The anticancer properties of UD-017 were assessed using several assays, including in vitro kinase, proliferation, and apoptosis assays, western blot analysis, and an in vivo xenograft mouse model. Results: UD-017 significantly inhibited CDK7 activity (IC50 = 16 nM) with high selectivity in an in vitro kinase assay testing a panel of over 300 proteins and lipid kinases. UD-017 also inhibited the growth of HCT-116 cells (GI50 = 19 nM) and inhibited the phosphorylation of various downstream mediators of CDK7 signaling. In cell cycle and apoptosis assays using HCT-116 cells, UD-017 increased the number of cells in both G1 and G2/M phases and induced apoptosis. In vivo, UD-017 inhibited tumor growth in an HCT-116 xenograft mouse model by 33%, 64%, and 88% at doses of 25, 50, and 100 mg/kg, respectively, with clear dose-dependency. Co-administration of 5-FU and 50 mg/kg UD-017 had a strong synergistic effect, as reflected in the complete inhibition of tumor growth. Conclusion: CDK7 may play a major role in colorectal cancer growth by regulating the cell cycle and apoptosis. UD-017 is a promising candidate therapeutic agent for the treatment of cancer involving CDK7 signaling

Identification of Physiologically Active Substances as Novel Ligands for MRGPRD

Mas-related G-protein coupled receptor member D (MRGPRD) is a G protein-coupled receptor (GPCR) which belongs to the Mas-related GPCRs expressed in the dorsal root ganglia (DRG). In this study, we investigated two novel ligands in addition to beta-alanine: (1) beta-aminoisobutyric acid, a physiologically active substance, with which possible relation to tumors has been seen together with beta-alanine; (2) diethylstilbestrol, a synthetic estrogen hormone. In addition to the novel ligands, we found that transfection of MRGPRD leads fibroblast cells to form spheroids, which would be related to oncogenicity. To understand the MRGPRD novel character, oncogenicity, a large chemical library was screened in order to obtain MRGPRD antagonists to utilize in exploring the character. The antagonist in turn inhibited the spheroid proliferation that is dependent on MRGPRD signaling as well as MRGPRD signals activated by beta-alanine. The antagonist, a small-molecule compound we found in this study, is a potential anticancer agent

MRGD, a MAS-related G-protein Coupled Receptor, Promotes Tumorigenisis and Is Highly Expressed in Lung Cancer

To elucidate the function of MAS-related GPCR, member D (MRGD) in cancers, we investigated the in vitro and in vivo oncogenic function of MRGD using murine fibroblast cell line NIH3T3 in which MRGD is stably expressed. The expression pattern of MRGD in clinical samples was also analyzed. We found that overexpression of MRGD in NIH3T3 induced focus formation and multi-cellular spheroid formation, and promoted tumors in nude mice. In other words, overexpression of MRGD in NIH3T3 induced the loss of contact inhibition, anchorage-independent growth and in vivo tumorigenesis. Furthermore, it was found that the ligand of MRGD, beta-alanine, enhanced spheroid formation in MRGD-expressing NIH3T3 cells. From investigation of clinical cancer tissues, we found high expression of MRGD in several lung cancers by immunohistochemistry as well as real time PCR. Based on these results, MRGD could be involved in tumorigenesis and could also be a novel anticancer drug target

Hydrolysis of sphingosylphosphocholine by neutral sphingomyelinases

AbstractSphingosylphosphocholine (SPC), the N-deacylated form of sphingomyelin (SM), is a naturally occurring lipid mediator. However, little is known about the metabolism of SPC. We here report an in vitro assay system for SPC-phospholipase C (PLC). Using this assay system, we demonstrated that nSMase1 and nSMase2, human neutral sphingomyelinases (SMases), are capable of hydrolyzing SPC efficiently under detergent-free conditions. Bacterial and plasmodial neutral SMases also showed SPC-PLC activity. The substrate specificity of neutral SMases that hydrolyze SM, SPC, and monoradyl glycerophosphocholine, but not diradyl glycerophosphocholine, suggested that a hydrogen-bond donor at the C-2 or sn-2 position in the substrate is required for recognition by the enzymes

Tumorigenesis of NIH3T3 cells stably expressing MRGD (NIH3T3-MRGD cells).

<p><b>A.</b> Representative pictures of focus formation in monolayer cultures of NIH3T3 cells stably expressing Mock (NIH3T3-Mock cells, left) or MRGD (NIH3T3-MRGD cells, right). Cells were stained with crystal violet after fixing with 4% paraformaldehyde. <b>B.</b> Representative pictures of NIH3T3-MRGD or NIH3T3-Mock spheroid on Days 1 and 7. <b>C.</b> Spheroid growth curves. The spheroid sizes of NIH3T3-MRGD (closed) or NIH3T3-Mock (open) at Days 2, 3, 5 and 7, are shown with their diameters (mean ± SD). * indicates p<0.005 (Mann-Whitney U test, 2 tails). <b>D.</b> Cell proliferation of spheroid cultures of NIH3T3-MRGD or NIH3T3-Mock. Luminescence of whole cell ATP contents (means ± SD) was measured at 6 days after plating. * indicates p<0.005 (Mann-Whitney U test, 2 tails).</p

IHC analysis of MRGD expression.

<p>Cell block samples were used to confirm the antibody specificity in immunostaining. HEK293/αvβ3 cells transfected with MRGD expression vector (<b>A</b>) or Mock vector (<b>B</b>) are shown. <b>C.</b> The representative staining of lung adenocarcinoma which is positive for MRGD.</p

MRGD ligand, beta-alanine, promoted cell proliferation of NIH3T3-MRGD cells.

<p>NIH3T3 cells transfected with MRGD or RASV12 were cultured in RPMI1640 containing 0.1% BSA and various concentration of beta-alanine for 7 days. Data were obtained from three independent cell cultures (means ± SD). * indicates p<0.05 (Mann-Whitney U test, 2 tails), compared with NIH3T3-MRGD without beta-alanine, and with NIH3T3-RASV12.</p

Tumor volumes of NIH3T3-MRGD or Mock bearing mice.

<p>Tumor volumes (mm³) were calculated according to the following equations: Tumor volume (mm³) = 1/2×(tumor length)×(tumor width)²</p