Cryptotanshinone suppresses tumorigenesis by inhibiting lipogenesis and promoting reactive oxygen species production in KRAS­activated pancreatic cancer cells.

Pyruvate dehydrogenase kinase 4 (PDK4) is an important regulator of energy metabolism. Previously, knockdown of PDK4 by specific small interfering RNAs (siRNAs) have been shown to suppress the expression of Κirsten rat sarcoma viral oncogene homolog (KRAS) and the growth of lung and colorectal cancer cells, indicating that PDK4 is an attractive target of cancer therapy by altering energy metabolism. The authors previously reported that a novel small molecule, cryptotanshinone (CPT), which inhibits PDK4 activity, suppresses the in vitro three­dimensional (3D)­spheroid formation and in vivo tumorigenesis of KRAS­activated human pancreatic and colorectal cancer cells. The present study investigated the molecular mechanism of CPT­induced tumor suppression via alteration of glutamine and lipid metabolism in human pancreatic and colon cancer cell lines with mutant and wild­type KRAS. The antitumor effect of CPT was more pronounced in the cancer cells containing mutant KRAS compared with those containing wild­type KRAS. CPT treatment decreased glutamine and lipid metabolism, affected redox regulation and increased reactive oxygen species (ROS) production in the pancreatic cancer cell line MIAPaCa­2 containing mutant KRAS. Suppression of activated KRAS by specific siRNAs decreased 3D­spheroid formation, the expression of acetyl­CoA carboxylase 1 and fatty acid synthase (FASN) and lipid synthesis. The suppression also reduced glutathione­SH/glutathione disulfide and increased the production of ROS. Knockdown of FASN suppressed lipid synthesis in MIAPaCa­2 cells, partially promoted ROS production and mildly suppressed 3D­spheroid formation. These results indicated that CPT reduced tumorigenesis by inhibiting lipid metabolism and promoting ROS production in a mutant KRAS­dependent manner. This PDK4 inhibitor could serve as a novel therapeutic drug for KRAS­driven intractable cancers via alteration of cell metabolism.

Cryptotanshinone, a novel PDK 4 inhibitor, suppresses bladder cancer cell invasiveness via the mTOR/ß­catenin/N­cadherin axis.

The phosphorylation of pyruvate dehydrogenase (PDH) by pyruvate dehydrogenase kinase (PDK) 4 inhibits its ability to induce a glycolytic shift. PDK4 expression is upregulated in various types of human cancer. Because PDK4 regulation is critical for metabolic changes in cancer cells, it is an attractive target for cancer therapy given its ability to shift glucose metabolism. It was previously shown that a novel PDK4 inhibitor, cryptotanshinone (CPT), suppressed the three­dimensional (3D)­spheroid formation of pancreatic and colorectal cancer cells. In the present study, the effects of CPT on the invasiveness of bladder cancer cells were investigated. CPT significantly suppressed the invasiveness and 3D­spheroid formation of T24 and J82 bladder cancer cells. CPT also suppressed the phosphorylation of PDH and ß­catenin, as well as the expression of N­cadherin, which are all critical for inducing epithelial­mesenchymal transition (EMT). The knockdown of ß­catenin or PDK4 using specific small interfering RNAs suppressed N­cadherin expression and invasiveness in T24 cells. An mTOR inhibitor also suppressed the phosphorylation of ß­catenin and N­cadherin expression. Furthermore, CPT injection significantly suppressed pancreatic tumor growth and peritoneal dissemination of highly metastatic SUIT­2 pancreatic cancer cells in a mouse orthotopic pancreatic cancer model, without evident toxicity. Moreover, immunohistochemistry analyses demonstrated decreased ß­catenin expression in CPT­treated pancreatic tumors compared with control tumors. Taken together, these results indicate that CPT reduced the invasiveness and metastasis of bladder cancer cells by suppressing EMT via the mTOR/ß­catenin/N­cadherin pathway.

Antitumor activity of potent pyruvate dehydrogenase kinase 4 inhibitors from plants in pancreatic cancer.

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.

Anti-oncogenic activities of cyclin D1b siRNA on human bladder cancer cells via induction of apoptosis and suppression of cancer cell stemness and invasiveness.

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.

Evolutionary analysis of two complement C4 genes: Ancient duplication and conservation during jawed vertebrate evolution.

The complement C4 is a thioester-containing protein, and a histidine (H) residue catalyzes the cleavage of the thioester to allow covalent binding to carbohydrates on target cells. Some mammalian and teleost species possess an additional isotype where the catalytic H is replaced by an aspartic acid (D), which binds preferentially to proteins. We found the two C4 isotypes in many other jawed vertebrates, including sharks and birds/reptiles. Phylogenetic analysis suggested that C4 gene duplication occurred in the early days of the jawed vertebrate evolution. The D-type C4 of bony fish except for mammals formed a cluster, termed D-lineage. The D-lineage genes were located in a syntenic region outside MHC, and evolved conservatively. Mammals lost the D-lineage before speciation, but D-type C4 was regenerated by recent gene duplication in some mammalian species or groups. Dual C4 molecules with different substrate specificities would have contributed to development of the antibody-dependent classical pathway.

Molecular mechanisms of ginsenoside Rp1-mediated growth arrest and apoptosis.

Ginsenoside Rp1, a semi-synthesized ginseng saponin, was shown to have chemopreventive action and anti-metastatic potential. However, the molecular mechanisms of Rp1 on cell growth and death are not fully understood. In this study, the antiproliferative effect of Rp1 on HeLa cells in vitro was investigated. Treatment with Rp1 at 40 microM inhibited the proliferation and partial accumulation of cells at the G1 phase. Rp1-mediated G1 arrest was accompanied by decreased expression of cyclin D1, E, and A and increased expression of p21 without any significant change in p53 or phospho-p53 (Ser15). On the other hand, prolonged incubation with Rp1 at 40 microM caused apoptosis and activation of caspase-3, -8, and -9. The participation of these three caspases in apoptosis was more clearly shown in experiments using inhibitors, which markedly prevented Rp1-induced apoptosis in the case of each caspase. Cleavage of the polyADP-ribose polymerase, often used as an apoptotic marker, was also found in Rp1-induced apoptosis. Among Bcl-2 family proteins (Bad, Bax, Bid, Bcl-2), Bax and Bid were activated by Rp1 treatment, which resulted in the release of cytochrome c from mitochondria, following activation of caspase-9. These observations indicate that multiple cell cycle regulatory proteins and apoptosis-inducing proteins are regulated by Rp1 and contribute to Rp1-induced growth arrest and apoptosis.

Molecular cloning of C4 gene and identification of the class III complement region in the shark MHC.

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.

Conservation of the modular structure of complement factor I through vertebrate evolution.

Mammalian complement factor I plays pivotal roles in the regulation of complement activation and generation of important biological activities from C3. The evolutionary origin of factor I has been unclear except with regard to the molecular cloning of factor I from amphibian Xenopus. Here, we report the identification and characterization of factor I cDNA from the liver of the banded houndshark. The deduced amino acid sequence of shark factor I showed a modular organization that was completely identical to that of mammalian factor I, suggesting the functional conservation of factor I throughout vertebrate evolution. Functionally important amino acid residues such as the basic residues at the processing site and the residues at the active site of the serine protease domain are conserved. Repeated sequences composed of 16 amino acids were inserted at a site between the leader peptide and the factor I/membrane attacking complex module in the shark factor I. This repeat is missing from mammalian and amphibian factor I, and the biological significance of the sequence, if any, is not clear at the moment. There was only one copy of the shark factor I gene, and Northern blotting analysis showed that the shark factor I gene was expressed only in the liver among several organs tested. While the lack of functional data does not exclude the possibility that factor I could have a different function, all these facts, together with the earlier reported data suggest the existence of a well developed complement system in cartilaginous fish.