Se-Lim Kim

SPION-loaded chitosan-linoleic acid nanoparticles to target hepatocytes.

The aim of this study was to develop a novel polymeric magnetic nanoprobe as an MRI contrast agent to target hepatocytes, as well as to evaluate the targeting ability of the nanoprobe with MRI in vivo. Superparamagnetic iron oxide nanocrystals (SPIONs) were synthesized by a thermal decomposition and seed growth method. An 1-ethyl-3-(3-(dimethylamino)-propyl) carbodiimide (EDC)-mediated reaction coupled water-soluble chitosan (WSC) to linoleic acid (LA). Twelve-nanometer-sized SPIONs were incorporated into the core of self-assembled WSC-LA nanoparticles. The morphology and size distribution of the SPION-loaded WSC-LA nanoparticles (SCLNs) were determined by transmittance electron microscopy (TEM) and dynamic light scattering (DLS), respectively. The encapsulation of SPIONs in the WSC-LA nanoparticles reduced the cytotoxicity of bare iron particles and enhanced their dispersion ability in water. The clustering of SPIONs into WSC-LA nanoparticles showed ultrasensitive magnetic behavior. After in vivo intravascular SCLN injection, MRI revealed relative signal enhancement in the liver. The localization of SCLN in hepatocytes was confirmed by Prussian blue staining and TEM analysis. We have successfully developed an ultrasensitive SCLN that effectively targets hepatocytes. The SCLN can be used as a contrast agent to aid in the diagnosis of hepatic diseases.

Superparamagnetic iron oxide nanoparticles-loaded chitosan-linoleic acid nanoparticles as an effective hepatocyte-targeted gene delivery system.

The goal of this study was to develop a gene delivery imaging system that targets hepatocytes to help diagnose and treat various liver diseases. To this end, we prepared superparamagnetic iron oxide nanoparticles (SPIO)-loaded with water-soluble chitosan (WSC)-linoleic acid (LA) nanoparticles (SCLNs) that formed gene complexes capable of localizing specifically to hepatocytes. We confirmed that (99m)Tc-labeled SCLNs delivered into mice via intravenous injection accumulated mainly in the liver using nuclear and magnetic resonance imaging. SCLN/enhanced green fluorescence protein (pEGFP) complexes were also successfully formed and were characterized with a gel retardation assay. SCLN/pEGFP complexes were transfected into primary hepatocytes, where GFP expression was observed in the cytoplasm. In addition, the injection of the gene complexes into mice resulted in significantly increased expression of GFP in hepatocytes in vivo. Furthermore, gene silencing was effectively achieved by administration of gene complexes loaded with specific siRNAs. In conclusion, our results indicate that the SCLNs have the potential to be useful for hepatocyte-targeted imaging and effective gene delivery into hepatocytes.

Folate receptor targeted imaging using poly (ethylene glycol)-folate: in vitro and in vivo studies.

The aim of this study was to ascertain the folate receptor (FR) targetability by an in vitro study and to acquire FR-targeted images in vivo models by using synthetic folate conjugates. PEG-folate was synthesized and labeled with 99mTc and fluorescein isothiocynate (FITC). Cell uptake studies were carried out in KB cells (FR-positive) and A549 cells (FR-negative) using FITC- and the 99mTc-labeled conjugates. The radiolabeled conjugate was intravenously injected to KB tumor xenografted mice. After it was injected, gamma images were recorded at 30 min, 1, 2, 3 and 4 hr. Cell uptake studies showed a difference between the KB cells and the A549 cells by flow cytometry analysis and gamma counting. On in vivo images, the tumor-to-normal muscle ratio was greater than 4. It ascertained that the PEG-folate conjugate specifically binds to the FR expressed on tumor cells in vitro. Moreover, it was possible to acquire the FR-targeted gamma images using PEG-folate conjugates in tumor models.

Synergistic antitumor effect of 5-fluorouracil in combination with parthenolide in human colorectal cancer

Parthenolide (PT), a NF-κB inhibitor, has recently been demonstrated as a promising anticancer agent that promotes apoptosis of cancer cells. 5-fluorouracil (5-FU) has been a drug of choice for treatment of colorectal cancer (CRC). Unfortunately, many of the therapies that use 5-FU alone or in combination with other agents are likely to become ineffective due to drug resistance. In the present study, we investigated the antitumor effect of PT combined with 5-FU on a human CRC cell line, SW620. The results demonstrated that combination of PT and 5-FU induced apoptosis which was determined using MTT, cell cycle analysis, annexin-V assay, and Hoechst 33258 staining. Apoptosis through the mitochondrial pathway was confirmed by detecting regulation of Bcl-2 family members, cytochrome C release, and activation of caspase 3 and 9. Moreover, intra-peritoneal injection of PT and 5-FU showed significant inhibition of tumor growth in the xenograft model. These results demonstrate that PT exhibits anticancer activity in human colorectal cancer in vitro and in vivo. These findings provide an efficacious strategy to overcome 5-FU resistance in certain CRC.

Parthenolide suppresses tumor growth in a xenograft model of colorectal cancer cells by inducing mitochondrial dysfunction and apoptosis

Parthenolide (PT), a principal active component in medicinal plants, has been used conventionally to treat migraine and inflammation. This component has recently been reported to induce apoptosis in cancer cells, through mitochondrial dysfunction. In the present study, we investigated PT-mediated cell death signaling pathway by focusing on the involvement of Bcl-2 family members in human colorectal cancer cells. We also investigated the inhibitory effect of PT on tumor growth in xenografts. Using the human colorectal cancer cell lines HT-29, SW620 and LS174T, we demonstrated that treatment of these cancer cells with PT induces apoptosis using MTT, Annexin V assay and Hoechst 33258 staining. Apoptosis through the mitochondrial pathway was confirmed by detecting regulation of Bcl-2 family members, cytochrome c release and caspase activation. Moreover, intraperitoneal injection of PT showed significant inhibition of tumor growth, angiogenesis in the xenograft model. These results demonstrate that PT exhibits anti-cancer activity in human colorectal cancer in vitro and in vivo. These findings may also provide a novel approach for the treatment of colorectal cancer.

Parthenolide exerts inhibitory effects on angiogenesis through the downregulation of VEGF/VEGFRs in colorectal cancer

Parthenolide (PT) is responsible for the bioactivities of feverfew (Tanacetum parthenium). Apart from its potent anti-inflammatory effects, this compound has been reported to induce apoptosis in various cancer cells. However, little is known about its role in the process of tumor angiogenesis. In the present study, we investigated the effects and potential mechanisms of action of PT on angiogenesis in human colorectal cancer (CRC). The anti-angiogenic effects of PT were evaluated in cultured human umbilical vein endothelial cells (HUVECs) and in the human CRC cell lines, HT-29, SW620 and HCT116. PT markedly inhibited vascular cell migration and capillary-like structure formation even at a dose which had not effects on cell viability. PT also suppressed the expression of angiogenic biomarker proteins [vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR)1 and VEGFR2] in both the HUVECs and CRC cells. Additionally, PT effectively inhibited tumor neovascularization in a HT-29 xenograft model. These results indicate that PT suppresses angiogenesis by reducing the expression of VEGF and its receptors and may be a viable drug candidate in anti-angiogenesis therapies for human CRC.

Parthenolide enhances sensitivity of colorectal cancer cells to TRAIL by inducing death receptor 5 and promotes TRAIL-induced apoptosis

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising cancer therapeutic agent. Recombinant human TRAIL has been evaluated in clinical trials, however, various malignant tumors are resistant to TRAIL. Parthenolide (PT) has recently been demonstrated as a highly effective anticancer agent and has been suggested to be used for combination therapy with other anticancer agents. In this study, we investigate the molecular mechanisms by which PT sensitizes colorectal cancer (CRC) cells to TRAIL-induced apoptosis. HT-29 (TRAIL-resistant) and HCT116 (TRAIL-sensitive) cells were treated with PT and/or TRAIL. The results demonstrated that combined treatment induced apoptosis which was determined using MTT, cell cycle analysis, Annexin V assay and Hoechst 33258 staining. Interestingly, we confirmed that HCT116 cells have much higher death receptor (DR) 5 than HT-29 cells and PT upregulates DR5 protein level and surface expression in both cell lines. Apoptosis through the mitochondrial pathway was confirmed by detecting regulation of Bcl-2 family members, p53 cytochrome C release, and caspase cascades. These results suggest that PT sensitizes TRAIL-induced apoptosis via upregulation of DR5 and mitochondria-dependent pathway. Combination treatment using PT and TRAIL may offer an effective strategy to overcome TRAIL resistance of certain CRC cells.

Synthesis and in vivo evaluation of 99m Tc-Transferrin conjugate for detection of inflamed site.

Transferrin is one of acute phase reactants in inflamed lesion. Expression of transferrin receptor is increased in activated macrophage during inflammation process. Conjugates of target ligand with novel water-soluble chitosan for fast excretion of background radioactivity have been shown to be selectively taken up via the target molecules. In this study, radiolabeled 99mTc–Transferrin conjugate was synthesized and evaluated its efficacy in vivo as a targeted agent for the rapid detection of inflamed lesion that expresses relatively high level of transferrin receptors. Transferrin was conjugated with HYNIC-chitosan and radiolabeled with 99mTc. The biodistribution and scintigraphic images with the 99mTc–HYNIC–chitosan–Transferrin conjugate (99mTc–Tfc) were studied in a murine infection model in which the infection was induced with Escherichia coli (2 × 106 colonies). The %ID/g was as follows: 1.612 ± 0.098, 2.473 ± 0.202 and 2.617 ± 0.646% at 30, 120 min and 6 h after injection, respectively. Gamma camera imaging rapidly visualized the infection/inflammation lesion, with the lesion-to-background ratio improving with time up to 5.68 ± 0.48. 99mTc–Tfc scintigraphy allows rapid and good imaging of an inflamed lesion.

Parthenolide Sensitizes Human Colorectal Cancer Cells to Tumor Necrosis Factor-related Apoptosis-inducing Ligand through Mitochondrial and Caspase Dependent Pathway

Background/Aims Combination therapy utilizing tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) in conjunction with other anticancer agents, is a promising strategy to overcome TRAIL resistance in malignant cells. Recently, parthenolide (PT) has proved to be a promising anticancer agent, and several studies have explored its use in combination therapy. Here, we investigated the molecular mechanisms by which PT sensitizes colorectal cancer (CRC) cells to TRAIL-induced apoptosis. Methods HT-29 cells (TRAIL-resistant) were treated with PT and/or TRAIL for 24 hours. The inhibitory effect on proliferation was detected using the 3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Annexin V staining, cell cycle analysis, and Hoechst 33258 staining were used to assess apoptotic cell death. Activation of an apoptotic pathway was confirmed by Western blot. Results Treatment with TRAIL alone inhibited the proliferation of HCT 116 cells in a dose-dependent manner, whereas proliferation was not affected in HT-29 cells. Combination PT and TRAIL treatment significantly inhibited cell growth and induced apoptosis of HT-29 cells. We observed that the synergistic effect was associated with misregulation of B-cell lymphoma 2 (Bcl-2) family members, release of cytochrome C to the cytosol, activation of caspases, and increased levels of p53. Conclusion Combination therapy using PT and TRAIL might offer an effetive strategy to overcome TRAIL resistance in certain CRC cells.

The effect of PPAR-γ agonist on 18F-FDG uptake in tumor and macrophages and tumor cells

PURPOSE The peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a member of the nuclear receptor superfamily of ligand-dependent transcription factors, and its role in adipogenesis and glucose metabolism has been well established. PPAR-gamma agonists have been shown to inhibit many cytokines and to have anti-inflammatory effects. In pathologic conditions, enhanced fluoro-2-deoxy-D-glucose (FDG) uptake is observed not only in malignant tumors but also in inflammatory lesions, and this uptake occurs through the glucose transporter in these cells. Thus, the present study was undertaken to investigate the potential of using PPAR-gammas glucose uptake ability as a diagnostic tool to differentiate between macrophage and tumor cells. MATERIALS AND METHODS Cellular uptake studies were carried out on macrophage and two tumor cell lines for comparison by using (18)F-FDG. Western blot analysis was performed to determine the expression levels of both the glucose transporter and hexokinase protein. To confirm the possibility of differentiation between tumor and inflammatory lesions using rosiglitazone based on in vitro studies, (18)F-FDG (3.7 x 10(6) Bq) uptake in A549 and RAW 264.7 xenograft mice was compared. RESULTS The cellular uptake study findings were quite different for macrophages and tumor cells. (18)F-FDG uptakes by macrophages decreased by about 60% but was increased twofold in tumor cells after rosiglitazone treatment. Moreover, the expressions of proteins related to glucose uptake correlated well with cellular glucose accumulation in both cell types. Higher tumor uptake was observed after the injection of rosiglitazone in A549 xenograft mice (1.58+/-0.55 to 4.66+/-1.16), but no significant change of (18)F-FDG uptake was shown in RAW 264.7 xenograft mice (4.04+/-1.16 to 4.00+/-0.14). CONCLUSION The present study demonstrates the roles of PPAR-gamma agonist on FDG uptake in macrophages and tumor cells in vitro and in vivo. Our findings suggest that rosiglitazone has the potential to increase the contrast between tumor and inflammatory lesions.