The C-Circle Biomarker Is Secreted by Alternative-Lengthening-of-Telomeres Positive Cancer Cells inside Exosomes and Provides a Blood-Based Diagnostic for ALT Activity.

C-Circles, self-primed telomeric C-strand templates for rolling circle amplification, are the only known alternative-lengthening-of-telomeres (ALT)-specific molecule. However, little is known about the biology of C-Circles and if they may be clinically useful. Here we show that C-Circles are secreted by ALT+ cancer cells inside exosomes, and that a blood-based C-Circle Assay (CCA) can provide an accurate diagnostic for ALT activity. Extracellular vesicles were isolated by differential centrifugation from the growth media of lung adenocarcinoma, glioblastoma, neuroblastoma, osteosarcoma, and soft tissue sarcoma cell lines, and C-Circles were detected in the exosome fraction from all eleven ALT+ cancer cell lines and not in any extracellular fraction from the eight matching telomerase positive cancer cell lines or the normal fibroblast strain. The existence of C-Circles in ALT+ exosomes was confirmed with exosomes isolated by iodixanol gradient separation and CD81-immunoprecipitation, and C-Circles in the exosomes were protected from nucleases. On average, 0.4% of the total ALT+ intracellular C-Circles were secreted in the exosomes every 24 h. Comparing the serum-based and tumor-based CCAs in 35 high risk neuroblastoma patients divided randomly into ALT+ threshold derivation and validation groups, we found the serum-based CCA to have 100% sensitivity (6/6), 70% specificity (7/10), and 81% concordance (13/16). We conclude that the secretion of C-Circles by ALT+ cancer cells in the exosomes provides a stable blood-based biomarker and a potential clinical diagnostic for ALT activity.

3'-hydroxy-4'-methoxy-ß-methyl-ß-nitrostyrene inhibits tumorigenesis in colorectal cancer cells through ROS-mediated DNA damage and mitochondrial dysfunction.

The ß-nitrostyrene family has been shown to suppress cell proliferation and induce apoptosis in types of various cancers. However, the mechanisms underlying the anticancer effects of ß-nitrostyrenes in colorectal cancer remain poorly understood. In this study, we synthesized a ß-nitrostyrene derivative, CYT-Rx20 (3'-hydroxy-4'-methoxy-ß-methyl-ß-nitrostyrene), and investigated its anticancer activities in human colorectal cancer cells both in vitro and in vivo. Our findings showed that treatment with CYT-Rx20 reduced cell viability and induced DNA damage in colorectal cancer cells. In addition, CYT-Rx20 induced cell cycle arrest of colorectal cancer cells at the G2/M phase and upregulated the protein expression of phospho-ERK, cyclin B1, phospho-cdc2 (Tyr15), aurora A, and aurora B, while it downregulated the expression of cdc25A and cdc25C. Furthermore, we found that CYT-Rx20 caused accumulation of intracellular reactive oxygen species (ROS) and reduction of mitochondrial membrane potential. The effects of CYT-Rx20 on cell viability, DNA damage, and mitochondrial membrane potential were reversed by pretreatment with the thiol antioxidant N-acetyl-L-cysteine (NAC), suggesting that ROS-mediated DNA damage and mitochondrial dysregulation play a critical role in these events. Finally, the nude mice xenograft study showed that CYT-Rx20 significantly reduced tumor growth of implanted colorectal cancer cells accompanied by elevated protein expression of aurora A, aurora B, γH2AX, phosphor-ERK, and MDA in the tumor tissues. Taken together, these results suggest that CYT-Rx20 may potentially be developed as a novel ß-nitrostyrene-based anticancer agent for colorectal cancer.

Extracellular Visfatin-Promoted Malignant Behavior in Breast Cancer Is Mediated Through c-Abl and STAT3 Activation.

PURPOSE: Visfatin is an adipocytokine involved in cellular metabolism, inflammation, and cancer. This study investigated the roles of extracellular visfatin in breast cancer, and explored underlying mechanisms in clinical and experimental settings. EXPERIMENTAL DESIGN: Associations of serum visfatin with clinicopathologic characteristics and patient survival were assessed with Cox regression models and Kaplan-Meier analyses. Effects of extracellular visfatin on cultured breast cancer cells were examined, followed by in vivo investigation of tumor growth and metastasis in xenograft animal models. Imatinib and Stattic were used to inhibit c-Abl and STAT3 activation, respectively. RESULTS: Breast cancer patients with high serum visfatin levels were associated with advanced tumor stage, increased tumor size and lymph node metastasis, and poor survival. Elevated phosphorylation of c-Abl and STAT3 in breast tumor tissues were correlated with high serum visfatin levels in patients. Visfatin-promoted in vitro cell viability and metastatic capability were suppressed by imatinib (c-Abl inhibitor) and Stattic (STAT3 inhibitor). Increased in vivo cell invasiveness was observed in zebrafish xenografted with visfatin-pretreated breast cancer cells. Tumor growth and lung metastasis occurred in visfatin-administered mice xenografted with breast cancer cells. Tail vein-injected mice with visfatin-pretreated breast cancer cells showed increased lung metastasis, which was suppressed by imatinib. CONCLUSIONS: Serum visfatin levels in breast cancer patients reveal potential prognostic values, and our findings that visfatin promoted breast cancer through activation of c-Abl and STAT3 may provide an important molecular basis for future design of targeted therapies that take into account different serum visfatin levels in breast cancer. Clin Cancer Res; 22(17); 4478-90. ©2016 AACR.

Different metal-ion-induced dimeric self-assembling cavities based on thiacalix[4]benzocrown-4 isomers.

To construct the self-assembly of metal-ion-induced well-ordered architectures based on calixcrowns, isomeric thiacalix[4]benzocrowns-4 1 and 2 with rigid and small crown units were employed as the new scaffolds. They all show remarkable selectivity for Ag(+) and their complexation ability towards Ag(+) results in two novel dimeric aggregates of calixcrowns, which were first evidenced by ESI-MS, (1)H and DOSY-NMR spectra. Ultimately, X-ray diffraction experiments confirmed unambiguously the existence of the two metal-ion-induced dimers in lower rim/lower rim mode, and showed that dimerization of calixcrown 1 or 2 in the presence of Ag(+) could form dimeric supramolecular cavity with a small inner room. Moreover, the positional isomerism of their crown units (o-benzocrown unit for ligand 1 and m-benzocrown unit for 2) led to a dramatic change in the configuration of the two dimeric cavities 1·Ag(+) and 2·Ag(+). For dimeric cavity 1·Ag(+), two silver centers seamed two thiacalix[4]crown molecules together and resided at the edge of the dimeric self-assembling cavity; for dimeric cavity 2·Ag(+), one Ag(+) stitched two thiacalixcrowns together and was encapsulated in the center of the self-assembling cavity, while the other Ag(+) is tied down to one end of the dimer. Consequently, as a result of the different construction of dimeric cavities 1·Ag(+) and 2·Ag(+) the extended structures of the complexes are also different. The neighbour self-assembling cavities 1·Ag(+) are mutually oriented side-by-side and form a 1-D rectilineal polymeric chain. While, the neighbour self-assembling cavities 2·Ag(+) arrange themselves in a typical head-to-tail fashion to form a zig-zag polymeric chain.

A novel indole-3-carbinol derivative inhibits the growth of human oral squamous cell carcinoma in vitro.

Indole-3-carbinol (I3C), a naturally occurring phytochemical found in cruciferous vegetables, has received much attention due to its translational potential in cancer prevention and therapy. In this study, we investigated the antitumor effects of OSU-A9, a structurally optimized I3C derivative, in a panel of oral squamous cell carcinoma cell lines, SCC4, SCC15, and SCC2095. The antiproliferative effect of OSU-A9 was approximately two-orders-of-magnitude higher than that of I3C. Importantly, normal human oral keratinocytes were less sensitive to OSU-A9 than oral cancer cells. This antiproliferative effect of OSU-A9 was attributable to the induction of mitochondrial-dependent apoptosis as evidenced by sub-G1 accumulation of cells, poly ADP-ribose polymerase cleavage, and cytochrome c release from the mitochondria. OSU-A9 down regulates Akt and NF-κB signaling pathways, leading to changes in many downstream effectors involved in regulating cell cycle and apoptosis. Moreover, the observed down regulation of IKKα and IKKß expression by OSU-A9 is not reported for I3C. OSU-A9 also induces both the production of reactive oxygen species and the endoplasmic reticulum stress. Taken together, these results suggest the translational value of OSU-A9 in oral squamous cell cancer therapy in the future.