Automated segmentation of 2D low-dose CT images of the psoas-major muscle using deep convolutional neural networks.

The psoas-major muscle has been reported as a predictive factor of sarcopenia. The cross-sectional area (CSA) of the psoas-major muscle in axial images has been indicated to correlate well with the whole-body skeletal muscle mass. In this study, we evaluated the segmentation accuracy of low-dose X-ray computed tomography (CT) images of the psoas-major muscle using the U-Net convolutional neural network, which is a deep-learning technique. Deep learning has been recently known to outperform conventional image-segmentation techniques. We used fivefold cross validation to validate the segmentation performance (n = 100) of the psoas-major muscle. For the intersection over union and CSA ratio, segmentation accuracies of 86.0 and 103.1%, respectively, were achieved. These results suggest that the U-Net network is competitive compared with the previous methods. Therefore, the proposed technique is useful for segmenting the psoas-major muscle even in low-dose CT images.

Novel Ridaifen-B Structure Analog Induces Apoptosis and Autophagy Depending on Pyrrolidine Side Chain.

Ridaifen (RID)-B is an analog derived from tamoxifen (TAM). TAM has an antitumor effect by acting as an antagonist to estrogen receptor (ER). However, TAM is known to also induces apoptosis in cancer cells that do not have ER. We clarified that RID-B induces cell death at a lower concentration than TAM, and causes ER-independent apoptosis and autophagy. Based on the results of previous studies, we assumed that RID-B had a unique target different from ER and examined structural activity correlation to determine what kinds of structural features are related to RID-B activity. As a result, we found there was activity even without one of phenyl groups (Ar3) in RID-B and revealed that two pyrrolidine side chains peculiar to RID-B are related to the action. Furthermore, analogs with shorter alkyl side chains induced autophagy, but analogs with certain length of alkyl side chains induced apoptosis. Also, although there is no doubt that RID-B induces apoptosis by causing mitochondrial injury, our results suggested that such injury induced mitochondria-selective autophagy. We revealed that RID-B induce mitophagy and that this mitophagy is a defense mechanism against RID-B. Our results suggest that autophagy was induced against apoptosis caused by mitochondrial dysfunction in RID-B, so the combination of autophagy inhibitor and anticancer-drug can be effective for cancer treatment.

Anti-proliferative effect of ridaifen-B on hepatoma cells.

Ridaifens (RIDs), a novel series of tamoxifen derivatives, exhibit a potent growth-inhibitory effect against numerous tumor cells regardless of the expression of estrogen receptors, and are thus promising candidates as novel anti-tumor drugs. RID-B is a first generation RIDs, and inhibits the proliferation of several tumor cell lines. However, the potentially growth inhibitory effect of RID-B against hepatoma cells, and the detailed mechanism underlying RID-B-mediated tumor cell death remain to be elucidated. The purpose of the current study was to evaluate the anti-proliferative effect of RID-B against hepatoma cells. The anti-proliferative effect of RID-B against human hepatoma Huh-7 cells was investigated by cell proliferation assay using WST-1 reagent, and caspase-3 activity was evaluated by using specific fluorescent substrate. In addition, DNA fragmentation in Huh-7 cells induced by RID-B was estimated by terminal deoxynucleotidyl transferase dUTP nick-end labelling assay, and binding of RID-B to double-stranded DNA was confirmed by mass spectrometry. RID-B (0.5, 1 and 2 µM) inhibited the growth of Huh-7 cells, seemingly dose-dependently, but did not inhibit the growth of normal primary rat hepatocytes in the same concentration range. Furthermore, the caspase-3 activity of Huh-7 cells was increased by RID-B (0.5 and 5 µM), and the anti-proliferative effect of RID-B (1 µM) on Huh-7 cells was partially suppressed by the addition of the caspase inhibitor, Z-VAD-FMK. Additionally, RID-B (10 µM) directly bound to double-stranded DNA, and the addition of DNA suppressed RID-B-mediated cell growth inhibition and DNA fragmentation in Huh-7 cells. From these data, it may be concluded that RID-B inhibited cell growth and induced apoptosis via activating caspase-3 and binding to DNA directly, leading to DNA fragmentation in hepatoma cells.

Ridaifen-F conjugated with cell-penetrating peptides inhibits intracellular proteasome activities and induces drug-resistant cell death.

Ridaifen-F (RID-F) potently inhibits proteolytic activities of the 20S proteasome but poorly inhibits those of the 26S proteasome. Here, we report preparation of several conjugates in which various peptides are connected to RID-F. Conjugates with peptides consisting of seven amino acid residues significantly inhibited the 26S proteasome. Particularly, RID-F conjugated to an octaarginine peptide (R8, a so-called cell-penetrating peptide) inhibited intracellular proteasome activities and induced cell death in drug-resistant KMS-11 myeloma cells. RID-F conjugated to hydrophobic peptides also inhibited the 26S proteasome but failed to induce cell death, suggesting poor penetration into cells. We infer that the R8 peptide has dual functions: (1) rapid penetration of conjugates into the cell increases intracellular drug concentrations sufficient for exhibition of its effect, and (2) recognition of the conjugates by the 26S proteasome stimulates drug entry into the catalytic chamber. In the presence of ATPγS, RID-F conjugates containing R8 inhibited the 26S proteasome more potently than in the presence of ATP, suggesting efficient entry of drugs into the catalytic chamber in a similar fashion to the substrate. Taken together with docking simulations of RID-F conjugate interactions with proteasome active sites, the second function of R8 peptide is plausible. Thus, the conjugation of nonpeptidic proteasome inhibitors to a cell-penetrating peptide could represent a viable strategy for overcoming the drug-resistance of tumor cells.

Ridaifen G, tamoxifen analog, is a potent anticancer drug working through a combinatorial association with multiple cellular factors.

Ridaifen-G (RID-G), a tamoxifen analog that we previously synthesized, has potent growth inhibitory activity against various cancer cell lines. Tamoxifen is an anticancer drug known to act on an estrogen receptor (ER) and other proteins. However, our previous studies interestingly suggested that the mechanism of action of RID-G was different from that of tamoxifen. In order to investigate the molecular mode of action of RID-G, we developed a novel chemical genetic approach that combined a phage display screen with a statistical analysis of drug potency and gene expression profiles in thirty-nine cancer cell lines. Application of this method to RID-G revealed that three proteins, calmodulin (CaM), heterogeneous nuclear ribonucleoproteins A2/B1 (hnRNP A2/B1), and zinc finger protein 638 (ZNF638) were the candidates of direct targets of RID-G. Moreover, cell lines susceptible to RID-G show similar expression profiles of RID-G target genes. These results suggest that RID-G involves CaM, hnRNP A2/B1, and ZNF638 in its growth inhibitory activity.