Two new adenopeptins B and C inhibit sphere formation of pancreatic cancer cells.

Cancer remains one of the leading causes of death worldwide, particularly pancreatic cancer being lethal because of its aggressiveness and lack of early detection methods. A factor that contributes to malignancy are cancer stem cell-like characteristics promoted by the tumor-stromal interaction. Given that fibroblast conditioned medium (CM) promotes sphere formation of cancer cells, a cancer stem cell-like characteristic, its inhibitor could be a new anticancer agent. By exploring microbial cultures as a source, we found new compounds, namely, adenopeptins B (1) and C (2), from Acremonium sp. ESF00140. 1 and 2 selectively and potently inhibited the sphere formation of pancreatic cancer cells cultured in the fibroblast CM compared with the control medium. Oxygen consumption rate (OCR) assays showed that 1 and 2 inhibit OCR in pancreatic cancer cells. Studies of similar compounds suggested mitochondrial complex V inhibition. Therefore, results of measuring the activity of human mitochondrial complex V revealed that 1 and 2 inhibited its activity. Oligomycin A, an inhibitor of mitochondrial complex V, as well as 1 and 2, strongly inhibited the sphere formation of pancreatic cancer cells cultured in fibroblast CM. The addition of 1 and 2 to pancreatic cancer cells cultured in fibroblast CM increased reactive oxygen species (ROS) production compared with that in the control medium. In pancreatic cancer cells cultured in fibroblast CM, mitochondria significantly influence sphere formation, and targeting their function with 1 and 2 might provide a new therapeutic approach for pancreatic cancer.

Pancreatic Stromal Cell-derived Oncostatin M Confers Drug Resistance to a Multi-tyrosine Kinase Inhibitor in Pancreatic Cancer Cells.

BACKGROUND/AIM: Pancreatic cancer is known to have one of the worst prognoses of all cancers, and its tumor cells are highly resistant to chemotherapeutic drugs. Pancreatic cancer cells coexist with stromal cells; however, their involvement in anticancer drug resistance remains poorly understood. Thus, in this study, we analyzed drug sensitivity using an in vitro co-culture system containing pancreatic cancer cells and stromal cells treated with a compound library. MATERIALS AND METHODS: We examined the viability of the pancreatic cancer cell lines BxPC-3, Capan-1, and Panc-1 against compounds in an in vitro co-culture model containing pancreatic stromal cells (PSCs) and analyzed the protein expression for drug resistance by western blotting. RESULTS: We found that co-cultured pancreatic cancer cells were resistant to vandetanib, which is an inhibitor of multi-tyrosine kinases. The key factor involved in drug resistance in these pancreatic cancer cells was oncostatin M, which was secreted by stromal cells. The addition of oncostatin M increased the vandetanib resistance of pancreatic cancer cells, while it inhibited the suppression of insulin receptor substrate-1 (IRS1) and the phosphorylation of extracellular signal-regulated kinase (ERK) by vandetanib. CONCLUSION: Oncostatin M secreted by stromal cells derived from the pancreas activates the IRS1-ERK axis, causing resistance to vandetanib.

Quinofuracins F - I, new quinofuracin derivatives produced by Staphylotrichum boninense PF1444.

Four new quinofuracins F - I were isolated from the culture broth of Staphylotrichum boninense PF1444. The structures of quinofuracins F - I were elucidated by extensive spectroscopic analysis. These quinofuracins induced tumor suppressor protein p53-dependent cell death in human glioblastoma LNZTA3 cells.

Phenotypic screening system using three-dimensional (3D) culture models for natural product screening.

Recent progress in three-dimensional (3D) cell culture systems has attracted much attention in the fields of basic life science and drug development. Newly established methods include 3D co-culture, spheroid culture, and organoid culture; these methods enable more human tissue-like culture and have largely replaced traditional two-dimensional (2D) monolayer culture. By combining 3D culture methods with high-content imaging analysis, it is possible to obtain diverse and convincing data even during initial screening (which requires rapid and easy operating procedures). Until recently, 3D culture methods were considered expensive, time-consuming, complex, and unstable. However, by exploiting the self-assembling nature of cells and adding several technical improvements, we have developed several phenotypic screenings aimed at discovering anticancer compounds.

Quinofuracins A-E, produced by the fungus Staphylotrichum boninense PF1444, show p53-dependent growth suppression.

Quinofuracins A-E, novel anthraquinone derivatives containing ß-D-galactofuranose that were isolated from the fungus Staphylotrichum boninense PF1444, induced p53-dependent cell death in human tumor cells. The structures of quinofuracins A-E, including absolute configurations, were elucidated by extensive spectroscopic analysis and chemical transformation studies. Quinofuracins were classified into three groups according to the aglycone moieties. 5'-Oxoaverantin was present in quinofuracins A-C, whereas averantin and versicolorin B were identified in quinofuracins D and E, respectively. These quinofuracins induced p53-dependent growth suppression in human glioblastoma LNZTA3 cells.

A rare polymorphic variant of NBS1 reduces DNA repair activity and elevates chromosomal instability.

Failure to expeditiously repair DNA at sites of double-strand breaks (DSB) ultimately is an important etiologic factor in cancer development. NBS1 plays an important role in the cellular response to DSB damage. A rare polymorphic variant of NBS1 that resulted in an isoleucine to valine substitution at amino acid position 171 (I171V) was first identified in childhood acute lymphoblastic leukemia. This polymorphic variant is located in the N-terminal region that interacts with other DNA repair factors. In earlier work, we had identified a remarkable number of structural chromosomal aberrations in a patient with pediatric aplastic anemia with a homozygous polymorphic variant of NBS1-I171V; however, it was unclear whether this variant affected DSB repair activity or chromosomal instability. In this report, we demonstrate that NBS1-I171V reduces DSB repair activity through a loss of association with the DNA repair factor MDC1. Furthermore, we found that heterozygosity in this polymorphic variant was associated with breast cancer risk. Finally, we showed that this variant exerted a dominant-negative effect on wild-type NBS1, attenuating DSB repair efficiency and elevating chromosomal instability. Our findings offer evidence that the failure of DNA repair leading to chromosomal instability has a causal impact on the risk of breast cancer development.

In vivo imaging of proteasome inhibition using a proteasome-sensitive fluorescent reporter.

A proteasome degrades numerous regulatory proteins that are critical for tumor growth and is therefore recognized as a promising anticancer target. Determining proteasome activity in the tumors of mice bearing xenografts is essential for the development of novel proteasome inhibitors. We developed a system for in vivo imaging of proteasome inhibition in the tumors of living mice, using a proteasome-sensitive fluorescent reporter, ZsProSensor-1. This reporter consists of a green fluorescent protein, ZsGreen, fused to mouse ornithine decarboxylase, which is degraded by the proteasome without being ubiquitinated. In stably transfected cells expressing ZsProSensor-1, the fluorescent reporter was rapidly degraded under steady-state conditions, whereas it was stabilized in the presence of proteasome inhibitors. Subcutaneous inoculation of the transfected cells into nude mice resulted in tumor formation. When the proteasome inhibitor bortezomib was intravenously administered to mice bearing these tumors, the ZsProSensor-1 protein accumulated in the tumors and emitted a fluorescent signal in a dose-dependent manner. Robust fluorescence was sustained for 3 days and then gradually decreased to baseline levels within 15 days. Intravenous administration of bortezomib also showed potent antitumor activity. In contrast, oral administration of bortezomib did not result in fluorescent protein accumulation in tumors or exhibit any antitumor activity. These results indicate that in vivo imaging using the ZsProSensor-1 fluorescent protein can be used as an indicator of antitumor activity and will be a powerful tool for the development of novel proteasome inhibitors.

Mitochondrial inhibitors show preferential cytotoxicity to human pancreatic cancer PANC-1 cells under glucose-deprived conditions.

Large areas of tumor are nutrient-starved and hypoxic due to a disorganized vascular system. Therefore, we screened small molecules to identify cytotoxic agents that function preferentially in nutrient-starved conditions. We found that efrapeptin F had preferential cytotoxicity to nutrient-deprived cells compared with nutrient-sufficient cells. Because efrapeptin F acts as a mitochondrial complex V inhibitor, we examined whether inhibitors of complex I, II, III, and V function as cytotoxic agents preferentially in nutrient-deprived cells. Interestingly, these inhibitors showed preferential cytotoxicity to nutrient-deprived cells and caused cell death under glucose-limiting conditions, irrespective of the presence or absence of amino acids and/or serum. In addition, these inhibitors were preferentially cytotoxic to nutrient-deprived cells even under hypoxic conditions. Further, efrapeptin F showed antitumor activity in vivo. These data indicate that mitochondrial inhibitors show preferential cytotoxicity to cancer cells under glucose-limiting conditions, and these inhibitors offer a promising strategy for anticancer therapeutic.

The SYT-SSX fusion protein down-regulates the cell proliferation regulator COM1 in t(x;18) synovial sarcoma.

Chromosomal translocations are frequently associated with soft-tissue sarcomas. Fusion proteins generated by such translocations often play critical roles in tumorigenesis. Therefore, it is important to understand the function of the fusion protein to develop therapeutic interventions. The t(X;18)(p11.2;q11.2) translocation found in synovial sarcomas results in a fusion between the SYT gene on chromosome 18 and an SSX gene on the X chromosome. Although SYT-SSX fusion proteins appear to trigger synovial sarcoma development, little is known about the downstream targets of SYT-SSX. We found that the SYT-SSX fusion protein produces a dominant-negative function for SYT, which is a transcriptional coactivator. We then analyzed the gene expression profiles of SYT-SSX1-expressing HeLa cells using oligonucleotide microarrays and found that the SYT-SSX1 fusion protein directly down-regulated the expression of COM1, a regulator of cell proliferation. COM1 was found to be expressed at relatively low levels in synovial sarcoma tissues and cell lines. We then investigated the impact of conditional COM1 expression in the synovial sarcoma cell line. Increased COM1 expression resulted in induced apoptosis and in reduced cell growth and colony formation activity. Our results suggested that restoration of COM1 expression may be of therapeutic benefit in synovial sarcoma.