An Ethanol Extract of Hawaiian Turmeric: Extensive In Vitro Anticancer Activity Against Human Colon Cancer Cells.

CONTEXT: Turmeric (Curcuma longa) is a food spice and colorant reported to be beneficial for human health. Curcumin (diferuloylmethane) is the major ingredient in turmeric, and existing data suggest that the spice, in combination with chemotherapy, provides a superior strategy for treatment of gastrointestinal cancer. However, despite its significant effects, curcumin suffers from poor bioavailability, due to poor absorption in the body. OBJECTIVE: The research team intended to evaluate a liquid extract of turmeric roots (TEx) that the team had formulated for its in vitro, anticancer activity against several human, colorectal cancer cell lines. DESIGN: The research team performed in vitro studies evaluating the anticancer efficacy via short and long-term assays and also evaluated invasion using Matrigel (Corning Life Sciences, Tewksbury, MA, USA). Further, in vitro anticancer activity of TEx was tested against 3-D cultures of HCT166 spheroids, which were subsequently analyzed by flow cytometry. SETTING: ADNA, Inc, Columbus, OH, USA; Foundation for Biomedical Research of the Academy of Athens, Athens, Greece; and Laboratory of Pharmacology, Faculty of Medicine, University of Thessaly, Larissa, Greece. INTERVENTION: The study used 4 human cell lines of colorectal cancer-HT29, HCT15, DLD1, and HCT116-and 2 breast cancer cell lines-SW480 and MDA-MB231. For a short-term assay, the extract was dissolved into culture mediums of HT29, HCT15, DLD1, HCT116, and SW480 at four 10-fold dilutions (100 to 0.1 µg/mL). For a long-term assay, TEx was added to the cultures of the same cell lines at 3 dilutions-20, 10, and 5 µg/mL. For an invasion assay, 100 µL per well of Matrigel was added and allowed to polymerize prior seeding of the MDA-MB231 cells. For cultures treated with the TEx, the TEx was mixed with the cell suspension prior to the seeding step. For the spheroid testing, the TEx was added to HCT116 cells either at the beginning of an experiment (ie, before the addition of the cancer cells), which was a chemopreventive approach, or 48 h later, on the addition of cells to the wells to allow the generation of spheroids, which was a chemotherapeutic approach. OUTCOME MEASURES: The in vitro activities of TEx were evaluated using a 48-h-incubation, short-term assay and a 2-wk, long-term (clonogenic) assay. To analyze the anti-invasive activity of the extract, images for the Matrigel invasion assay were taken with a camera at the 24-h time point. The in vitro, anticancer activity of TEx was also tested against 3-D cultures of HCT116 spheroids that were subsequently analyzed using flow cytometry. RESULTS: TEx had potently inhibited the growth of all human colon cancer cell lines tested in a dose- and time-dependent manner. TEx inhibited the formation of HCT116 spheroids when the cells were incubated with the extract. The extract also disrupted the formation of tubules formed by MDA-MB231 cells grown on Matrigel at concentrations that did not affect the overall viability of the cells, indicating a potent anti-invasive activity. CONCLUSIONS: These data suggest a potential therapeutic activity for TEx against human colon cancer, most likely due to the enhanced bioavailability of the turmeric.

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 Barrett's 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. METHODS: Human NEM, BE, EAC, and multitissue microarrays were analyzed for DCAMKL-1, and immunohistochemically scored based on staining intensity and tissue involvement, with epithelia and stroma scored separately. Total RNA isolated from BE and paired NEM was subjected to real-time reverse-transcription-polymerase chain reaction analysis for DCAMKL-1, leucine-rich repeat-containing G-protein-coupled receptor (LGR5), and Musashi-1 (Msi-1) mRNA expression. RESULTS: DCAMKL-1 was minimally expressed in squamous NEM, but increased in BE (with and without dysplasia) and EAC tissues. In EAC, we found increased stromal DCAMKL-1 staining compared to adjacent epithelia. Within the submucosa of dysplastic BE tissues, an increase in the endothelial cell expression of DCAMKL-1 was observed. Finally, an upregulation of DCAMKL-1, LGR5, and Msi-1 mRNA was seen in BE compared to squamous NEM. CONCLUSIONS: In the present study, we report the progressive increase of DCAMKL-1 expression in BE from dysplasia to EAC. Furthermore, there was an increase in putative stem cell markers DCAMKL-1, LGR5, and Msi-1 mRNA. Taken together, these data suggest that the regulation of resident stem cells might play an important role in the progression of BE to EAC.

Nanoparticle-based delivery of siDCAMKL-1 increases microRNA-144 and inhibits colorectal cancer tumor growth via a Notch-1 dependent mechanism.

BACKGROUND: The development of effective drug delivery systems capable of transporting small interfering RNA (siRNA) has been elusive. We have previously reported that colorectal cancer tumor xenograft growth was arrested following treatment with liposomal preparation of siDCAMKL-1. In this report, we have utilized Nanoparticle (NP) technology to deliver DCAMKL-1 specific siRNA to knockdown potential key cancer regulators. In this study, mRNA/miRNA were analyzed using real-time RT-PCR and protein by western blot/immunohistochemistry. siDCAMKL-1 was encapsulated in Poly(lactide-co-glycolide)-based NPs (NP-siDCAMKL-1); Tumor xenografts were generated in nude mice, treated with NP-siDCAMKL-1 and DAPT (γ-secretase inhibitor) alone and in combination. To measure let-7a and miR-144 expression in vitro, HCT116 cells were transfected with plasmids encoding the firefly luciferase gene with let-7a and miR-144 miRNA binding sites in the 3'UTR. RESULTS: Administration of NP-siDCAMKL-1 into HCT116 xenografts resulted in tumor growth arrest, downregulation of proto-oncogene c-Myc and Notch-1 via let-7a and miR-144 miRNA-dependent mechanisms, respectively. A corresponding reduction in let-7a and miR-144 specific luciferase activity was observed in vitro. Moreover, an upregulation of EMT inhibitor miR-200a and downregulation of the EMT-associated transcription factors ZEB1, ZEB2, Snail and Slug were observed in vivo. Lastly, DAPT-mediated inhibition of Notch-1 resulted in HCT116 tumor growth arrest and down regulation of Notch-1 via a miR-144 dependent mechanism. CONCLUSIONS: These findings demonstrate that nanoparticle-based delivery of siRNAs directed at critical targets such as DCAMKL-1 may provide a novel approach to treat cancer through the regulation of endogenous miRNAs.

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.

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.

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.

BACKGROUND: The 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. RESULTS: The 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-kappaB; (d) DCPIP can attenuate or completely inhibit VEGF-induced angiogenesis in the chick chorioallantoic membrane; (e) DCPIP at concentrations higher than 6 mug/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. CONCLUSIONS: We 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.

Curcumin and turmeric attenuate arsenic-induced angiogenesis in ovo.

Trivalent arsenic [As(III)] is currently approved by the FDA for the treatment of chronic and acute leukemias. However, As(III) has also demonstrated damaging effects on human health, including development of cardiovascular disease, diabetes, and cancer. Further, As(III) is a potent angiogenic agent. In this context, curcumin, an active ingredient in the dietary agent turmeric, has demonstrated potent antiproliferative, antiinflammatory, and antiangiogenic properties. In this report, we have shown that both curcumin and turmeric inhibit expression of vascular endothelial growth factor in HCT-116 human colon cancer cells exposed to As(III). Further, in the chicken chorioallantoic membrane assay model, treatment with low As(III) concentrations results in extensive increase in blood vessel density, which, however, is reduced in the presence of curcumin or turmeric. Collectively, the findings reported here strongly suggest that turmeric and curcumin can dramatically attenuate the process of angiogenesis induced by low As(III) concentrations.

14-3-3sigma-dependent resistance to cisplatin.

BACKGROUND: A major factor that impedes the clinical success of cisplatin-based chemotherapy for cancer is cisplatin resistance by cancer cells. MATERIALS AND METHODS: The sensitivity of parental HCT116 human colon cancer cell line and its isogenic gene-knockout sub-lines to cisplatin was determined by clonogenicity assay; furthermore, p53 activation, p21 expression, cell cycle arrest and senescence in these cells after cisplatin treatment were investigated. RESULTS: Parental cells were six times more resistant than 14-3-3sigma-knockout (sigma-KO) cells to cisplatin. Moreover, activation of p53, p53-dependent expression of p21 and p21-dependent senescence were observed in sigma-KO, but not parental cells after a treatment with a low cisplatin dose. CONCLUSION: A 14-3-3sigma-dependent mechanism inhibits p53 activation in parental cells treated with a low cisplatin dose, thereby blocking p21 expression that is essential for senescence and consequently conferring to the parental cells a significant degree of resistance to cisplatin.

Review: Chios mastic gum: a plant-produced resin exhibiting numerous diverse pharmaceutical and biomedical properties.

Chios mastic gum (CMG) is a resin produced by the plant Pistacia lentiscus var. chia. CMG is used to extract the mastic gum essential oil (MGO). CMG and MGO consist of nearly 70 constituents and have demonstrated numerous and diverse biomedical and pharmacological properties including (a) eradication of bacteria and fungi that may cause peptic ulcers, tooth plaque formation and malodor of the mouth and saliva; (b) amelioration or dramatic reduction of symptoms of autoimmune diseases by inhibiting production of pro-inflammatory substances by activated macrophages, production of cytokines by peripheral blood mononuclear cells in patients with active Crohn's disease, and suppression of production of inflammatory cytokines and chemokines in an asthma model in mice; (c) protection of the cardiovascular system by effectively lowering the levels of total serum cholesterol, low-density lipoprotein and triglycerides in rats, and protection of low-density lipoprotein from oxidation in humans; (d) induction of apoptosis in human cancer cells in vitro and extensive inhibition of growth of human tumors xenografted in immunodeficient mice; and (e) improvement of symptoms in patients with functional dyspepsia. Collectively taken, these numerous and diverse medical and pharmaceutical properties of CMG and MGO warrant further research in an effort to enhance specific properties and identify specific constituent(s) that might be associated with each property.

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.

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.

Urine and serum analysis of consumed curcuminoids using an IkappaB-luciferase surrogate marker assay.

BACKGROUND: curcumin metabolites are detectable in body fluids such as serum and urine. We have developed a novel assay that can detect metabolites in such body fluids by measuring their effect on the nuclear factor kappa B/inhibitor of kappa B (NF-κB/IκB) pathway. PATIENTS AND METHODS: fifteen healthy individuals were enrolled in the study and randomly assigned to two groups: control group (five) and curcumin group (ten). The test group ingested 8 g of the curcuminoids (C(3)-Complex) with 16 oz of bottled water. Blood and urine were collected at 0, 4, 8, and 24 h after ingestion. Degradation of the NF-κB/IκB complex was detected by the Genetic Expression and Measurement (GEM) assay using HCT116 cells stably transfected with PGL3-IκB firefly luciferase. RESULTS: using our novel GEM assay, the five controls who had not taken curcumin were identified. CONCLUSION: the GEM assay is a very sensitive and accurate non-invasive assay that could be utilized to detect metabolites in body fluids. It could also serve as a tool to determine participants' compliance during clinical research studies.

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.

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.