Rama P. Ramanujam

Doublecortin and CaM kinase-like-1 and leucine-rich-repeat-containing G-protein-coupled receptor mark quiescent and cycling intestinal stem cells, respectively.

It is thought that small intestinal epithelia (IE) undergo continuous self‐renewal primarily due to their population of undifferentiated stem cells. These stem cells give rise to transit amplifying (daughter/progenitor) cells, which can differentiate into all mature cell types required for normal gut function. Identification of stem cells in IE is paramount to fully understanding this renewal process. One major obstacle in gastrointestinal stem cell biology has been the lack of definitive markers that identify small intestinal stem cells (ISCs). Here we demonstrate that the novel putative ISC marker doublecortin and CaM kinase‐like‐1 (DCAMKL‐1) is predominantly expressed in quiescent cells in the lower two‐thirds of intestinal crypt epithelium and in occasional crypt‐based columnar cells (CBCs). In contrast, the novel putative stem cell marker leucine‐rich‐repeat‐containing G‐protein‐coupled receptor (LGR5) is observed in rapidly cycling CBCs and in occasional crypt epithelial cells. Furthermore, functionally quiescent DCAMKL‐1+ crypt epithelial cells retain bromo‐deoxyuridine in a modified label retention assay. Moreover, we demonstrate that DCAMKL‐1 is a cell surface expressing protein; DCAMKL‐1+ cells, isolated from the adult mouse small intestine by fluorescence activated cell sorting, self‐renew and ultimately form spheroids in suspension culture. These spheroids formed glandular epithelial structures in the flanks of athymic nude mice, which expressed multiple markers of gut epithelial lineage. Thus, DCAMKL‐1 is a marker of quiescent ISCs and can be distinguished from the cycling stem/progenitors (LGR5+). Moreover, DCAMKL‐1 can be used to isolate normal small intestinal stem cells and represents a novel research tool for regenerative medicine and cancer therapy. STEM CELLS 2009;27:2571–2579

DCAMKL-1 regulates epithelial-mesenchymal transition in human pancreatic cells through a miR-200a-dependent mechanism

Pancreatic cancer is an exceptionally aggressive disease in great need of more effective therapeutic options. Epithelial-mesenchymal transition (EMT) plays a key role in cancer invasion and metastasis, and there is a gain of stem cell properties during EMT. Here we report increased expression of the putative pancreatic stem cell marker DCAMKL-1 in an established KRAS transgenic mouse model of pancreatic cancer and in human pancreatic adenocarcinoma. Colocalization of DCAMKL-1 with vimentin, a marker of mesenchymal lineage, along with 14-3-3 σ was observed within premalignant PanIN lesions that arise in the mouse model. siRNA-mediated knockdown of DCAMKL-1 in human pancreatic cancer cells induced microRNA miR-200a, an EMT inhibitor, along with downregulation of EMT-associated transcription factors ZEB1, ZEB2, Snail, Slug, and Twist. Furthermore, DCAMKL-1 knockdown resulted in downregulation of c-Myc and KRAS through a let-7a microRNA-dependent mechanism, and downregulation of Notch-1 through a miR-144 microRNA-dependent mechanism. These findings illustrate direct regulatory links between DCAMKL-1, microRNAs, and EMT in pancreatic cancer. Moreover, they demonstrate a functional role for DCAMKL-1 in pancreatic cancer. Together, our results rationalize DCAMKL-1 as a therapeutic target for eradicating pancreatic cancers.

Diphenyl difluoroketone: a curcumin derivative with potent in vivo anticancer activity.

Diphenyl difluoroketone (EF24), a molecule having structural similarity to curcumin, was reported to inhibit proliferation of a variety of cancer cells in vitro. However, the efficacy and in vivo mechanism of action of EF24 in gastrointestinal cancer cells have not been investigated. Here, we assessed the in vivo therapeutic effects of EF24 on colon cancer cells. Using hexosaminidase assay, we determined that EF24 inhibits proliferation of HCT-116 and HT-29 colon and AGS gastric adenocarcinoma cells but not of mouse embryo fibroblasts. Furthermore, the cancer cells showed increased levels of activated caspase-3 and increased Bax to Bcl-2 and Bax to Bcl-xL ratios, suggesting that the cells were undergoing apoptosis. At the same time, cell cycle analysis showed that there was an increased number of cells in the G(2)-M phase. To determine the effects of EF24 in vivo, HCT-116 colon cancer xenografts were established in nude mice and EF24 was given i.p. EF24 significantly suppressed the growth of colon cancer tumor xenografts. Immunostaining for CD31 showed that there was a lower number of microvessels in the EF24-treated animals coupled with decreased cyclooxygenase-2, interleukin-8, and vascular endothelial growth factor mRNA and protein expression. Western blot analyses also showed decreased AKT and extracellular signal-regulated kinase activation in the tumors. Taken together, these data suggest that the novel curcumin-related compound EF24 is a potent antitumor agent that induces caspase-mediated apoptosis during mitosis and has significant therapeutic potential for gastrointestinal cancers.

Identification of a novel putative pancreatic stem/progenitor cell marker DCAMKL-1 in normal mouse pancreas

Stem cells are critical in maintaining adult homeostasis and have been proposed to be the origin of many solid tumors, including pancreatic cancer. Here we demonstrate the expression patterns of the putative intestinal stem cell marker DCAMKL-1 in the pancreas of uninjured C57BL/6 mice compared with other pancreatic stem/progenitor cell markers. We then determined the viability of isolated pancreatic stem/progenitor cells in isotransplantation assays following DCAMKL-1 antibody-based cell sorting. Sorted cells were grown in suspension culture and injected into the flanks of athymic nude mice. Here we report that DCAMKL-1 is expressed in the main pancreatic duct epithelia and islets, but not within acinar cells. Coexpression was observed with somatostatin, NGN3, and nestin, but not glucagon or insulin. Isolated DCAMKL-1+ cells formed spheroids in suspension culture and induced nodule formation in isotransplantation assays. Analysis of nodules demonstrated markers of early pancreatic development (PDX-1), glandular epithelium (cytokeratin-14 and Ep-CAM), and isletlike structures (somatostatin and secretin). These data taken together suggest that DCAMKL-1 is a novel putative stem/progenitor marker, can be used to isolate normal pancreatic stem/progenitors, and potentially regenerates pancreatic tissues. This may represent a novel tool for regenerative medicine and a target for anti-stem cell-based therapeutics in pancreatic cancer.

Activation of Apoptosis by 1-Hydroxy-5,7-Dimethoxy-2-Naphthalene-Carboxaldehyde, a Novel Compound from Aegle marmelos

We have identified a natural compound that activates apoptosis of epithelial cancer cells through activation of tumor necrosis factor-alpha (TNF-alpha), TNF receptor (TNFR)-associated death domain (TRADD), and caspases. The molecule 1-hydroxy-5,7-dimethoxy-2-naphthalene-carboxaldehyde (HDNC, marmelin) was isolated and characterized from ethyl acetate fraction of extracts of Aegle marmelos. HDNC treatment inhibited the growth of HCT-116 colon cancer tumor xenografts in vivo. Immunostaining for CD31 showed that there was a significant reduction in microvessels in the HDNC-treated animals, coupled with decreased cyclooxygenase-2, interleukin-8, and vascular endothelial growth factor mRNA. Using hexoseaminidase assay, we determined that HDNC inhibits proliferation of HCT-116 colon and HEp-2 alveolar epithelial carcinoma cells. Furthermore, the cancer cells showed increased levels of activated caspase-3 and induced G(1) cell cycle arrest, which was suppressed by caspase-3 inhibitors. HDNC induced TNF-alpha, TNFR1, and TRADD mRNA and protein expression. Moreover, caspase-8 and Bid activation, and cytochrome c release, were observed, suggesting the existence of a cross-talk between death receptor and the mitochondrial pathways. HDNC inhibited AKT and extracellular signal-regulated kinase phosphorylation both in cells in culture and in tumor xenografts. In addition, electrophoretic mobility shift assay and luciferase reporter assays showed that HDNC significantly suppressed TNF-alpha-mediated activation and translocation of nuclear factor-kappaB (NF-kappaB). This was further confirmed by Western blot analysis of nuclear extracts wherein levels of RelA, the p65 component of NF-kappaB, were significantly less in cells treated with HDNC. Together, the data suggest that the novel compound HDNC (marmelin) is a potent anticancer agent that induces apoptosis during G(1) phase of the cell cycle and could be a potential chemotherapeutic candidate.

Identification of the putative intestinal stem cell marker doublecortin and CaM kinase-like-1 in Barrett's esophagus and esophageal adenocarcinoma.

Background and Aim:  In Barretts esophagus (BE), the normal esophageal squamous epithelium is replaced with a specialized metaplastic columnar epithelium. BE is a premalignant lesion that can progress to esophageal adenocarcinoma (EAC). Currently, there are no early molecular indicators that would predict progression from BE to EAC. As the only permanent residents of the epithelium, stem cells have been implicated in this metaplastic progression. The aim of the present study was to determine the expression of doublecortin and CaM kinase‐like‐1 (DCAMKL‐1) and other putative gastrointestinal stem cell markers in normal esophageal mucosa (NEM), BE, and EAC.

Preparation of siRNA-Encapsulated PLGA Nanoparticles for Sustained Release of siRNA and Evaluation of Encapsulation Efficiency

Nanoparticles (NPs) formulated using poly (D,L-lactide-co-glycolide) (PLGA), a biodegradable, biocompatible, and clinically approved polymer, have been widely used for targeted drug delivery. Here we provide methods for preparing PLGA NPs that encapsulate small interfering RNA (siRNA). The siRNA NPs are formulated using a double-emulsion solvent evaporation technique with the addition of a small amount of the cationic polymer, polyethyleneimine, which significantly increases siRNA encapsulation.

Inhibition of angiogenesis- and inflammation-inducing factors in human colon cancer cells in vitro and in ovo by free and nanoparticle-encapsulated redox dye, DCPIP

BackgroundThe redox dye, DCPIP, has recently shown to exhibit anti-melanoma activity in vitro and in vivo. On the other hand, there is increasing evidence that synthetic nanoparticles can serve as highly efficient carriers of drugs and vaccines for treatment of various diseases. These nanoparticles have shown to serve as potent tools that can increase the bioavailability of the drug/vaccine by facilitating absorption or conferring sustained and improved release. Here, we describe results on the effects of free- and nanoparticle-enclosed DCPIP as anti-angiogenesis and anti-inflammation agents in a human colon cancer HCT116 cell line in vitro, and in induced angiogenesis in ovo.ResultsThe studies described in this report indicate that (a) DCPIP inhibits proliferation of HCT116 cells in vitro; (b) DCPIP can selectively downregulate expression of the pro-angiogenesis growth factor, VEGF; (c) DCPIP inhibits activation of the transcriptional nuclear factor, NF-κB; (d) DCPIP can attenuate or completely inhibit VEGF-induced angiogenesis in the chick chorioallantoic membrane; (e) DCPIP at concentrations higher than 6 μg/ml induces apoptosis in HCT116 cells as confirmed by detection of caspase-3 and PARP degradation; and (f) DCPIP encapsulated in nanoparticles is equally or more effective than free DCPIP in exhibiting the aforementioned properties (a-e) in addition to reducing the expression of COX-2, and pro-inflammatory proteins IL-6 and IL-8.ConclusionsWe propose that, DCPIP may serve as a potent tool to prevent or disrupt the processes of cell proliferation, tissue angiogenesis and inflammation by directly or indirectly targeting expression of specific cellular factors. We also propose that the activities of DCPIP may be long-lasting and/or enhanced if it is delivered enclosed in specific nanoparticles.

Identification of the putative intestinal stem cell marker DCAMKL-1 in Barrett's Esophagus and Esophageal Adenocarcinoma

Background and Aim:  In Barretts esophagus (BE), the normal esophageal squamous epithelium is replaced with a specialized metaplastic columnar epithelium. BE is a premalignant lesion that can progress to esophageal adenocarcinoma (EAC). Currently, there are no early molecular indicators that would predict progression from BE to EAC. As the only permanent residents of the epithelium, stem cells have been implicated in this metaplastic progression. The aim of the present study was to determine the expression of doublecortin and CaM kinase‐like‐1 (DCAMKL‐1) and other putative gastrointestinal stem cell markers in normal esophageal mucosa (NEM), BE, and EAC.

Identification of the putative intestinal stem cell marker doublecortin and CaM kinase-like-1 in Barrett's esophagus and esophageal adenocarcinoma: DCAMKL-1 in Barrett's and cancer

Background and Aim:  In Barretts esophagus (BE), the normal esophageal squamous epithelium is replaced with a specialized metaplastic columnar epithelium. BE is a premalignant lesion that can progress to esophageal adenocarcinoma (EAC). Currently, there are no early molecular indicators that would predict progression from BE to EAC. As the only permanent residents of the epithelium, stem cells have been implicated in this metaplastic progression. The aim of the present study was to determine the expression of doublecortin and CaM kinase‐like‐1 (DCAMKL‐1) and other putative gastrointestinal stem cell markers in normal esophageal mucosa (NEM), BE, and EAC.