Pancreatic cancer is an aggressive malignancy that is generally refractory to chemotherapy, thus posing experimental and clinical challenges. In this study, the antiproliferative effect of the triterpenoid compound cucurbitacin B was tested in vitro and in vivo against human pancreatic cancer cells. Dose-response studies showed that the drug inhibited 50% growth of seven pancreatic cancer cell lines at 10 À7 mol/L, whereas clonogenic growth was significantly inhibited at 5 Â 10 À8 mol/L. Cucurbitacin B caused dose-and timedependent G 2 -M-phase arrest and apoptosis of pancreatic cancer cells. This was associated with inhibition of activated JAK2, STAT3, and STAT5, increased level of p21 WAF1 even in cells with nonfunctional p53, and decrease of expression of cyclin A, cyclin B1, and Bcl-XL with subsequent activation of the caspase cascade. Interestingly, the combination of cucurbitacin B and gemcitabine synergistically potentiated the antiproliferative effects of gemcitabine on pancreatic cancer cells. Moreover, cucurbitacin B decreased the volume of pancreatic tumor xenografts in athymic nude mice by 69.2% (P < 0.01) compared with controls without noticeable drug toxicities. In vivo activation of JAK2/STAT3 was inhibited and expression of Bcl-XL was decreased, whereas caspase-3 and caspase-9 were up-regulated in tumors of drug-treated mice. In conclusion, we showed for the first time that cucurbitacin B has profound in vitro and in vivo antiproliferative effects against human pancreatic cancer cells, and the compound may potentate the antiproliferative effect of the chemotherapeutic agent gemcitabine. Further clinical studies are necessary to confirm our findings in patients with pancreatic cancer.

Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide) is an ingredient of chili peppers with inhibitory effects against cancer cells of different origin. We examined the activity of capsaicin on breast cancer cells in vitro and in vivo. The drug potently inhibited growth of ER-positive (MCF-7, T47D, BT-474) and ER-negative (SKBR-3, MDA-MB231) breast cancer cell lines, which was associated with G 0 /G 1 cell-cycle arrest, increased levels of apoptosis and reduced protein expression of human epidermal growth factor receptor (EGFR), HER-2, activated extracellular-regulated kinase (ERK) and cyclin D1. In contrast, cell-cycle regulator p27 KIP1, caspase activity as well as poly-ADP ribose polymerase (PARP) cleavage were increased. Notably, capsaicin blocked breast cancer cell migration in vitro and decreased by 50% the size of MDA-MB231 breast cancer tumors growing orthotopically in immunodeficient mice without noticeable drug side effects. in vivo activation of ERK was clearly decreased, as well as expression of HER-2 and cyclin D1, whereas caspase activity and PARP cleavage products were increased in tumors of drug-treated mice. Besides, capsaicin potently inhibited the development of preneoplastic breast lesions by up to 80% without evidence of toxicity. Our data indicate that capsaicin is a novel modulator of the EGFR/HER-2 pathway in both ERpositive and -negative breast cancer cells with a potential role in the treatment and prevention of human breast cancer.

Approximately 20-25% of traumatic brain injury (TBI) subjects develop acute lung injury (ALI), but the pathomechanisms of TBI-induced ALI remain poorly defined. Our previous work has shown that the inflammasome plays a critical role in TBI-induced secondary pathophysiology and that inflammasome proteins are released in extracellular vesicles (EV) after TBI. Here we investigated whether EV-mediated inflammasome signaling contributed to the etiology of TBI-induced ALI. C57/BL6 male mice were subjected to controlled cortical impact (CCI), and the brains and lungs were examined for inflammasome activation and ALI at 4 and 24 h after TBI. We show that TBI releases EV containing inflammasome proteins into serum that target the lung to cause ALI, supporting activation of a neural-respiratory-inflammasome axis. Administration of a low-molecular-weight heparin (enoxaparin, a blocker of EV uptake) or treatment with a monoclonal antibody against apoptosis speck-like staining protein containing a caspase recruitment domain (anti-ASC) after adoptive transfer of EV isolated from TBI-injured mice significantly inhibited inflammasome activation in the lungs of recipient mice resulting in improved ALI scores.This axis constitutes an important arm of the innate inflammatory response in lung pathology after TBI and targeting this axis represents a novel therapeutic treatment for TBI-induced ALI.

We have recently shown that a 150-bp Col10a1 distal promoter (−4296 to −4147 bp) is sufficient to direct hypertrophic chondrocyte-specific reporter (LacZ) expression in vivo. More recently, through detailed sequence analysis we identified two putative tandem-repeat Runx2 binding sites within the 3′-end of this 150-bp region (TGTGGG-TGTGGC, −4187 to −4176 bp). Candidate electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation, and transfection studies demonstrate that these putative Runx2 sites bind Runx2 and mediate upregulated Col10a1/reporter activity in vitro. Transgenic studies using the 5′-sequence without Runx2 sites were not able to drive the cell-specific LacZ reporter activity, suggesting the in vivo requirement of the Runx2 sites located in the 3′-end in mediating Col10a1/reporter expression. Indeed, mutating the Runx2 sites in the context of the 150-bp promoter abolishes its capacity to drive hypertrophic chondrocyte-specific reporter expression in transgenic mice. We have also generated multiple transgenic mouse lines using only the 3′-sequence containing the Runx2 sites to drive the LacZ gene. Interestingly, no hypertrophic chondrocyte-specific blue staining was observed in these transgenic mice. Together, our data support that Runx2 directly interacts with murine Col10a1 cis-enhancer. This interaction is required but not sufficient for cell-specific Col10a1 promoter activity in vivo. Additional cooperative/repressive elements within the 5′- or 3′-sequences of this 150-bp promoter are needed to work with Runx2 together to mediate cell-specific Col10a1 expression. Further delineation of these elements/factors has the potential to identify novel therapeutic targets for multiple skeletal disorders, including osteoarthritis, that show abnormal Col10a1 expression and altered chondrocyte maturation. © 2011 American Society for Bone and Mineral Research

The inflammasome is a key contributor to the inflammatory innate immune response after stroke. We have previously shown that inflammasome proteins are released in extracellular vesicles (EV) after brain and spinal cord injury. In addition, we have shown that inflammasome proteins offer great promise as biomarkers of central nervous system (CNS) injury following brain trauma. In the present study, we used a Simple Plex Assay (Protein Simple), a novel multi-analyte automated microfluidic immunoassay platform, to analyze serum and serum-derived EV samples from stroke patients and control subjects for inflammasome protein levels of caspase-1, apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC), Interleukins (IL)-1β, and (IL)-18. Receiver operator characteristic (ROC) curves with associated confidence intervals obtained from the analysis of serum samples revealed that the area under the curve (AUC) for ASC was 0.99 with a confidence interval between 0.9914 and 1.004, whereas the AUC for caspase-1, IL-1β, and IL-18 were 0.75, 0.61, and 0.67, respectively. Thus, these data indicate that ASC is a potential biomarker of stroke and highlight the role of the inflammasome in the inflammatory response after brain ischemia.