Purpose: Pancreatic cancer is characterized by intratumoral hypoxia, early and aggressive local invasion, and metastatic potential.
Hypoxia-inducible factor-1 (HIF-1) is the major transcriptional ...activator of hypoxia-responsive genes and intratumoral hypoxia
is associated with increased risk of metastasis. However, the behavior of the cells having HIF-1 activity during the malignant
progression in pancreatic cancer has not been tested.
Experimental Design: We orthotopically transplanted pancreatic cancer cells stably transfected with a HIF-1–dependent luciferase reporter gene
and monitored HIF-1 activity in vivo in control and POP33-treated mice. POP33 is a novel prodrug, which has potential to increase caspase-3 activity and induce
apoptosis in HIF-1–active/hypoxic cells.
Results: In vivo optical imaging showed that HIF-1 activity proceeded along with local invasion, the peritoneal dissemination, and the liver
metastasis. HIF-1–active hypoxic cells were selectively eradicated by POP33. Moreover, selective killing of HIF-1–active hypoxic
cells significantly suppressed malignant progression, resulting in a significant improvement in survival rate.
Conclusions: These results show that HIF-1–active cells constitute a large proportion of invading and metastatic cells and suggest that
eradication of these cells may improve the outcome in advanced pancreatic cancer, a condition for which no effective therapy
currently exists.
HSC fate decisions are regulated by cell-intrinsic and cell-extrinsic cues. The latter cues are derived from the BM niche. Membrane-type 1 matrix metalloproteinase (MT1-MMP), which is best known for ...its proteolytic role in pericellular matrix remodeling, is highly expressed in HSCs and stromal/niche cells. We found that, in MT1-MMP−/− mice, in addition to a stem cell defect, the transcription and release of kit ligand (KitL), stromal cell–derived factor-1 (SDF-1/CXCL12), erythropoietin (Epo), and IL-7 was impaired, resulting in a trilineage hematopoietic differentiation block, while addition of exogenous KitL and SDF-1 restored hematopoiesis. Further mechanistic studies revealed that MT1-MMP activates the hypoxia-inducible factor-1 (HIF-1) pathway via factor inhibiting HIF-1 (FIH-1) within niche cells, thereby inducing the transcription of HIF-responsive genes, which induce terminal hematopoietic differentiation. Thus, MT1-MMP in niche cells regulates postnatal hematopoiesis, by modulating hematopoietic HIF-dependent niche factors that are critical for terminal differentiation and migration.
The small number of high-migratory cancer cells in a cell population make studies on high-migratory cancer cells difficult. For the development of migration assays for such cancer cells, several ...microfluidic devices have been developed. However, they measure migration that is influenced by microstructures and they collect not only high-migratory cells, but also surrounding cells. In order to find high-migratory cells in cell populations while suppressing artifacts and to collect these cells while minimizing damages, we developed a microfluidic high-migratory cell collector with the ability to sort cancer cells according to cellular migration and mechanical detachment. High-migratory cancer cells travel further from the starting line when all of the cells are seeded on the same starting line. The high-migratory cells are detached using a stretch of cell adhesive surface using a water-driven balloon actuator. Using this cell collector, we selected high-migratory HeLa cells that migrated about 100m in 12 h and collected the cells.
This paper proposes a microfluidic device for screening molecules such as aptamers, antibodies, proteins, etc. for target cell-specific binding molecules. The discovery of cancer cell-specific ...binding molecules was the goal of this study. Its functions include filtering non-target cell-binding molecules, trapping molecules on the surface of target cells, washing away unbound molecules, and collecting target cell-specific binding molecules from target cells. These functions were effectively implemented by using our previously developed micro pillar arrays for cell homogeneous dispersion and pneumatic microvalves for tall microchannels. The device was also equipped with serially connected filter chambers in which non-target cells were cultured to reduce the molecules binding to non-target cells as much as possible. We evaluated the performance of the device using cancer cell lines (N87 cells as target cells and HeLa cells as non-target cells) and two fluorescent dye-labeled antibodies: Anti-human epidermal growth factor receptor 2 (anti-HER2) antibody that binds to target cells and anti-integrin antibody that binds to non-target cells. The results showed that the device could reduce anti-integrin antibodies to the detection limit of fluorescent measurement and collect anti-HER2 antibodies from the target cells.
Hypoxia-inducible factor (HIF) functions as a master transcriptional regulator for adaptation to hypoxia by inducing adaptive changes in gene expression for regulation of proliferation, angiogenesis, ...apoptosis and energy metabolism. Cancers with high expression of the alpha subunit of HIF (HIFα) are often malignant and treatment-resistant. Therefore, the development of a molecular probe that can detect HIF activity has great potential value for monitoring tumor hypoxia. HIF prolyl hydroxylases (HPHDs) act as oxygen sensors that regulate the fate of HIFα protein through its oxygen-dependent degradation (ODD) domain. We constructed a recombinant protein PTD-ODD-HaloTag (POH) that is under the same ODD regulation as HIFα and contains protein transduction domain (PTD) and an interchangeable labeling system. Administration of near-infrared fluorescently labeled POH (POH-N) to mouse models of cancers allowed successful monitoring of HIF-active regions. Immunohistochemical analysis for intratumoral localization of POH probe revealed its specificity to HIF-active cells. Furthermore, lack of the PTD domain or a point mutation in the ODD domain abrogated the specificity of POH-N to HIF-active cells. Overall results indicate that POH is a practical probe specific to HIF-active cell in cancers and suggest its large potential for imaging and targeting of HIF-related diseases.
Small proteins that have a high affinity for cancer cell surface markers can be promising cheap alternatives to antibodies (antibody mimetics). Various types of antibody mimetics have thus been ...extensively developed. We recently found that a target-binding peptide binds to its target molecule more strongly when it is structurally constrained. To apply this finding to the development of chemically synthesizable small antibody mimetics, we have established an efficient method of creating such proteins, named fluctuation-regulated affinity proteins (FLAPs). To identify desirable scaffolds, first, 13 human proteins (46-104 aa) were selected from the Protein Data Bank. Then, thirteen graft acceptor (GA) sites that efficiently immobilize the grafted peptide structure were identified from six small protein scaffolds using molecular dynamics simulation. To assess the designed antibody mimetics in vitro, human epidermal growth factor receptor 2 (HER2)-binding peptides were selected from the anti-HER2 antibody drugs trastuzumab and pertuzumab by calculating the binding energy, and these were then grafted into the GA sites of scaffolds to create 65 FLAP candidates. The FLAP candidates were expressed in bacteria as fusion proteins with Renilla luciferase (Rluc), and their relative binding affinity to HER2 was easily determined by measuring the Rluc bioluminescence intensity without protein purification. Finally, four out of the 65 showed specific binding to HER2 with a dissociation constant (KD) of 24-65 nM, and these were used for the detection of HER2-expressing cancer cells. Our design strategy will promote the development of antibody mimetics for the effective treatment of cancers and other diseases.
Cyclooxygenase (COX)-2, a rate-limiting enzyme of prostaglandin (PG) production, is overexpressed in colorectal adenomas and adenocarcinomas, and its inhibition by nonsteroidal antiinflammatory drugs ...protects against colorectal cancer. Mechanisms of cancer promotion by COX-2 are not fully understood, but signaling through prostaglandin (PG)E2receptors is a contributing factor. The major PGE2receptors on epithelial cells, EP2and EP4, increase cAMP production, which promotes growth and inhibits apoptosis in some cell types. Here, we show that cAMP agonists, including PGE2, cholera toxin, and a membrane-permeant cAMP analog, protect normal and transformed intestinal epithelial cells from apoptosis induced by diverse stimuli. This protection is associated with cAMP-mediated, rapid induction of cellular inhibitor of apoptosis protein (c-IAP)-2 and delayed induction of LIVIN, but not of six other members of the IAP family. Concurrently and characteristic of IAP functions, the activity, but not generation, of the cleaved form of the central executioner caspase 3 is inhibited. Induction of c-IAP2 expression by cAMP agonists is accompanied by phosphorylation of cAMP response element binding protein and cAMP response element-dependent activation of transcriptional reporters. Furthermore, inhibition of COX-2 in cells overexpressing the enzyme decreases c-IAP2 expression and promotes apoptosis, both of which are reversible by PGE2addition, suggesting that COX-2-promoted antiapoptosis is mediated by release of PGE2and subsequent cAMP-dependent c-IAP2 induction. These results help to explain the cancer chemoprotective effects of nonsteroidal antiinflammatory drugs by defining a mechanism through which cAMP signaling can promote the development of colorectal and possibly other epithelial cancers by means of disruption of normal apoptotic processes.
Solid tumors containing more hypoxic regions show a more malignant phenotype by increasing the expression of genes encoding angiogenic and metastatic factors. Hypoxia-inducible factor-1 (HIF-1) is a ...master transcriptional activator of such genes, and thus, imaging and targeting hypoxic tumor cells where HIF-1 is active are important in cancer therapy. In the present study, HIF-1 activity was monitored via an optical in vivo imaging system by using a luciferase reporter gene under the regulation of an artificial HIF-1-dependent promoter, 5HRE. To monitor tumor hypoxia, we isolated a stable reporter-transfectant, HeLa/5HRE-Luc, which expressed more than 100-fold luciferase in response to hypoxic stress, and observed bioluminescence from its xenografts. Immunohistochemical analysis of the xenografts with a hypoxia marker, pimonidazole, confirmed that the luciferase-expressing cells were hypoxic. Evaluation of the efficacy of a hypoxia-targeting prodrug, TOP3, using this optical imaging system revealed that hypoxic cells were significantly diminished by TOP3 treatment. Immunohistochemical analysis of the TOP3-treated xenografts confirmed that hypoxic cells underwent apoptosis and were removed after TOP3 treatment. These results demonstrate that this model system using the 5HRE-luciferase reporter construct provides qualitative information (hypoxic status) of solid tumors and enables one to conveniently evaluate the efficacy of cancer therapy on hypoxia in malignant solid tumors.
PDF
