The human cyclin D1 gene generates two major isoforms, cyclin D1a and cyclin D1b, by alternative splicing. Although cyclin D1b mRNA is hardly expressed in normal human tissues, it is detected in approximately 60% of human bladder cancer tissues and cell lines. In the present study, to assess the therapeutic ability of cyclin D1b siRNA, we investigated the anti-oncogenic effects of cyclin D1b siRNA on human bladder cancer cell lines, SBT31A and T24, which express cyclin D1b mRNA. Knockdown of cyclin D1b by specific siRNA significantly suppressed cell proliferation, in vitro cell invasiveness and three-dimensional (3D) spheroid formation in these cell lines. Cell cycle analyses revealed that cyclin D1b siRNA inhibited G1-S transition in T24 cells. The increase in the sub-G1 fraction, morphological aberrant nuclei with nuclear fragmentation and caspase-3 activity in SBA31A cells treated with cyclin D1b siRNA showed that cyclin D1b siRNA induced apoptosis. In T24 cells, knockdown of cyclin D1b suppressed the expression of the stem cell marker CD44. Knockdown of cyclin D1b or CD44 suppressed the invasiveness under 3D spheroid culture conditions and expression of N-cadherin. Tumor growth of SBT31A cells in nude mice was significantly inhibited by cyclin D1b siRNA. Taken together, these results indicate that knockdown of cyclin D1b suppresses the malignant phenotypes of human bladder cancer cells via induction of apoptosis and suppression of cancer cell stemness and epithelial-mesenchymal transition. Applying cyclin D1b siRNA will be a novel therapy for cyclin D1b-expressing bladder cancers.

To clarify the evolutionary origin of the linkage of the MHC class III complement genes with the MHC class I and II genes, we isolated C4 cDNA from the banded hound shark (Triakis scyllium). Upon phylogenetic tree analysis, shark C4 formed a well-supported cluster with C4 of higher vertebrates, indicating that the C3/C4 gene duplication predated the divergence of cartilaginous fish from the main line of vertebrate evolution. The deduced amino acid sequence predicted the typical C4 three-subunits chain structure, but without the histidine residue catalytic for the thioester bond, suggesting the human C4A-like specificity. The linkage analysis of the complement genes, one C4 and two factor B (Bf) genes, to the shark MHC was performed using 56 siblings from two typing panels of T. scyllium and Ginglymostoma cirratum. The C4 and one of two Bf genes showed a perfect cosegregation with the class I and II genes, whereas two recombinants were identified for the other Bf gene. These results indicate that the linkage between the complement C4 and Bf genes, as well as the linkage between these complement genes and the MHC class I and II genes were established before the emergence of cartilaginous fish >460 million years ago.

Phosphorylation of pyruvate dehydrogenase by pyruvate dehydrogenase kinase 4 (PDK4) 4 inhibits its ability to induce a glycolytic shift. PDK4 expression is frequently upregulated in various cancer tissues, with its elevation being critical for the induction of the Warburg effect. PDK4 is an attractive target for cancer therapy given its effect on shifting glucose metabolism. Previous research has highlighted the necessity of identifying a potent compound to suppress PDK4 activity at the submicromolar concentrations. Here we identified natural diterpene quinones (KIS compounds) that inhibit PDK4 at low micromolar concentrations. KIS37 (cryptotanshinone) inhibited anchorage‐independent growth in three‐dimensional spheroid and soft agar colony formation assays of KRAS‐activated human pancreatic (MIAPaCa‐2 and Panc‐1) and colorectal (DLD‐1 and HCT116) cancer cell lines. KIS37 also suppressed KRAS protein expression in such cell lines. Furthermore, KIS37 suppressed phosphorylation of Rb protein and cyclin D1 protein expression via the PI3K‐Akt–mTOR signaling pathway under nonadherent culture conditions and suppressed the expression of cancer stem cell markers CD44, EpCAM, and ALDH1A1 in MIAPaCa‐2 cells. KIS37 also suppressed pancreatic cancer cell growth in both subcutaneous xenograft and orthotopic pancreatic tumor models in nude mice at 40 mg/kg (intraperitoneal dose) without any evident toxicity. Reduced ALDH1A1 expression was observed in KIS37‐treated pancreatic tumors, suggesting that cancer cell stemness was also suppressed in the orthotopic tumor model. The aforementioned results indicate that KIS37 administration is a novel therapeutic strategy for targeting PDK4 in KRAS‐activated intractable human pancreatic cancer.

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