Tae Won Kwak

Dextran-b-poly(L-histidine) copolymer nanoparticles for ph-responsive drug delivery to tumor cells

Purpose Nanoparticles based on stimuli-sensitive drug delivery have been extensively investigated for tumor targeting. Among them, pH-responsive drug targeting using pH-sensitive polymers has attracted attention because solid tumors have an acidic environment. A dextran-b-poly(L-histidine) (DexPHS) copolymer was synthesized and pH-responsive nanoparticles were fabricated for drug targeting. Methods and results A DexPHS block copolymer was synthesized by attaching the reductive end of dextran to the amine groups of poly(L-histidine). pH-responsive nanoparticles incorporating doxorubicin were fabricated and studied in HuCC-T1 cholangiocarcinoma cells. Synthesis of DexPHS was confirmed by 1H nuclear magnetic resonance spectroscopy, with specific peaks of dextran and PHS observed at 2–5 ppm and 7.4–9.0 ppm, respectively. DexPHS nanoparticles showed changes in particle size with pH sensitivity, ie, the size of the nanoparticles increased at an acidic pH and decreased at a basic pH. DexPHS block copolymer nanoparticles incorporating doxorubicin were prepared using the nanoprecipitation dialysis method. The doxorubicin release rate was increased at acidic pH compared with basic pH, indicating that DexPHS nanoparticles have pH-sensitive properties and that drug release can be controlled by variations in pH. The antitumor activity of DexPHS nanoparticles incorporating doxorubicin were studied using HuCC-T1 cholangiocarcinoma cells. Viability was decreased in cells treated with nanoparticles at acidic pH, whereas cell viability in response to treatment with doxorubicin did not vary according to changes of pH. Conclusion Our results indicated that DexPHS polymeric micelles are promising candidates for antitumor drug targeting.

Synergistic Anticancer Effects of Vorinostat and Epigallocatechin-3-Gallate against HuCC-T1 Human Cholangiocarcinoma Cells

The aim of this study was to investigate the effect of the combination of vorinostat and epigallocatechin-3-gallate against HuCC-T1 human cholangiocarcinoma cells. A novel chemotherapy strategy is required as cholangiocarcinomas rarely respond to conventional chemotherapeutic agents. Both vorinostat and EGCG induce apoptosis and suppress invasion, migration, and angiogenesis of tumor cells. The combination of vorinostat and EGCG showed synergistic growth inhibitory effects and induced apoptosis in tumor cells. The Bax/Bcl-2 expression ratio and caspase-3 and -7 activity increased, but poly (ADP-ribose) polymerase expression decreased when compared to treatment with each agent alone. Furthermore, invasion, matrix metalloproteinase (MMP) expression, and migration of tumor cells decreased following treatment with the vorinostat and EGCG combination compared to those of vorinostat or EGCG alone. Tube length and junction number of human umbilical vein endothelial cells (HUVECs) decreased as well as vascular endothelial growth factor expression following vorinostat and EGCG combined treatment. These results indicate that the combination of vorinostat and EGCG had a synergistic effect on inhibiting tumor cell angiogenesis potential. We suggest that the combination of vorinostat and EGCG is a novel option for cholangiocarcinoma chemotherapy.

Ursodeoxycholic acid-conjugated chitosan for photodynamic treatment of HuCC-T1 human cholangiocarcinoma cells.

Chitosan was hydrophobically modified with ursodeoxycholic acid (UDCA) to fabricate nano-photosensitizer for photodynamic therapy (PDT) of HuCC-T1 cholangiocarcinoma cells. Synthesis of UDCA-conjugated chitosan (ChitoUDCA) was confirmed using (1)H NMR spectra. Chlorin E6 (Ce6) was used as a photosensitizer and incorporated into ChitoUDCA nanoparticles through formation of ion complexes. Morphology of Ce6-incorporated ChitoUDCA nanoparticles was observed using TEM and their shapes were spherical with sizes around 200-400 nm. The PDT potential of Ce6-incorporated ChitoUDCA nanoparticles were studied with HuCC-T1 human cholangiocarcinoma cells. The results showed that ChitoUDCA nanoparticles enhances of Ce6 uptake into tumor cells, phototoxicity, and ROS generation compared to Ce6 itself. Furthermore, Ce6-incorporated ChitoUDCA nanoparticles showed quenching in aqueous solution and sensing at tumor cells. We suggest that Ce6-incorporated ChitoUDCA nanoparticles are promising candidates for PDT of cholangiocarcinoma cells.

Preclinical evaluation of sorafenib-eluting stent for suppression of human cholangiocarcinoma cells

Background Cholangiocarcinoma is a malignant tumor arising from the epithelium of the bile ducts. In this study, we prepared sorafenib-loaded biliary stents for potential application as drug-delivery systems for localized treatment of extrahepatic cholangiocarcinoma. Methods A sorafenib-coated metal stent was prepared using an electrospray system with the aid of poly(ɛ-caprolactone) (PCL), and then its anticancer activity was investigated using human cholangiocellular carcinoma (HuCC)-T1 cells in vitro and a mouse tumor xenograft model in vivo. Anticancer activity of sorafenib against HuCC-T1 cells was evaluated by the proliferation test, matrix metalloproteinase (MMP) activity, cancer cell invasion, and angiogenesis assay in vitro and in vivo. Results The drug-release study showed that the increased drug content on the PCL film induced a faster drug-release rate. The growth of cancer cells on the sorafenib-loaded PCL film surfaces decreased in a dose-dependent manner. MMP-2 expression of HuCC-T1 cells gradually decreased according to sorafenib concentration. Furthermore, cancer cell invasion and tube formation of human umbilical vein endothelial cells significantly decreased at sorafenib concentrations higher than 10 mM. In the mouse tumor xenograft model with HuCC-T1 cells, sorafenib-eluting PCL films significantly inhibited the growth of tumor mass and induced apoptosis of tumor cells. Various molecular signals, such as B-cell lymphoma (Bcl)-2, Bcl-2-associated death promoter, Bcl-x, caspase-3, cleaved caspase-3, Fas, signal transducer and activator of transcription 5, extracellular signal-regulated kinases, MMP-9 and pan-janus kinase/stress-activated protein kinase 1, indicated that apoptosis, inhibition of growth and invasion was cleared on sorafenib-eluting PCL films. Conclusion These sorafenib-loaded PCL films are effective in inhibiting angiogenesis, proliferation and invasion of cancer cells. We suggest that sorafenib-loaded PCL film is a promising candidate for the local treatment of cholangiocarcinoma.

Synergistic effects of 5-aminolevulinic acid based photodynamic therapy and celecoxib via oxidative stress in human cholangiocarcinoma cells.

5-Aminolevulinic acid (ALA)-based photodynamic therapy (PDT) has the potential to kill cancer cells via apoptotic or necrotic signals that are dependent on the generation of intracellular reactive oxygen species (ROS). Celecoxib is an anti-inflammatory drug that induces intracellular ROS generation. We investigated whether the combined application of celecoxib and ALA-PDT improved the efficacy of PDT in human cholangiocarcinoma cells and in tumor bearing mice. In vitro, combined treatment of celecoxib and ALA-PDT increased phototoxicity and intracellular ROS levels after irradiation with 0.75 J/cm2 when compared to ALA-PDT alone. Even though ROS levels increased with 0.25 J/cm2 of irradiation, it did not influence phototoxicity. When heme oxygenase-1, a defensive protein induced by oxidative stress, was inhibited in the combined treatment group, phototoxicity was increased at both 0.25 J/cm2 and 0.75 J/cm2 of irradiation. We identified the combined effect of ALA-PDT and celecoxib through the increase of oxidative stress such as ROS. In vivo, about 40% tumor growth inhibition was observed with combined application of ALA-PDT and celecoxib when compared to ALA-PDT alone. The combined application of ALA-PDT and celecoxib could be an effective therapy for human cholangiocarcinoma. Moreover, use of a heme oxygenase-1 inhibitor with PDT could play an important role for management of various tumors involving oxidative stress.

Anticancer activities of epigallocatechin-3-gallate against cholangiocarcinoma cells

Purpose Epigallocatechin-3-gallate (EGCG) is an antioxidant agent derived from green tea. Because it has chemopreventive and anti-invasive effect against various cancer cells, EGCG can be used to inhibit proliferation and invasion of cholangiocarcinoma (CCA) cells. Methods The anticancer effects of EGCG were studied using human CCA cells (HuCC-T1). Apoptosis was analyzed by Western blotting. Invasion and migration of cancer cells were assessed with Matrigel® and wound healing assays. An animal tumor xenograft model of HuCC-T1 was used to study the in vivo antitumor activities of EGCG. Results EGCG effectively inhibited the growth of HuCC-T1 cells with no adverse effects on the viability of 293T cells. EGCG induced apoptotic cell death at 5 µg/mL concentration. It inhibited the expression of mutant p53 and induced apoptotic molecular signals such as Bax/Bcl-2, Caspase, and cytochrome C. Furthermore, EGCG dose-dependently inhibited the activity of matrix metalloproteinase (MMP)-2/9, invasion, and migration. In the animal tumor xenograft model of HuCC-T1 cells, EGCG was subcutaneously administered beside the tumor for local treatment. EGCG efficiently inhibited growth of the tumor and suppressed carcinogenic molecular signals such as Notch1, MMP-2/9, and proliferating cell nuclear antigen. Conclusion EGCG induced apoptosis of cancer cells without adverse effects on normal cells. EGCG inhibited growth, invasion, and migration of HuCC-T1 cells. We suggest EGCG as a promising candidate for local treatment of CCA.

Redox-Responsive nanophotosensitizer composed of chlorin e6-conjugated dextran for photodynamic treatment of colon cancer cells

We synthesized dextran-chlorin e6 conjugates having disulfide linkage for specific targeting of colonic region and cancer cells. Reductive end group of dextran was treated with sodium borocyanohydride and conjugated with cystamine. Cystamine end group was conjugated with carboxylic acid of chlorin e6 (DEX6ss). DEX6ss conjugates were formed as spherical nanoparticles with small sizes less than 100 nm. Chlorin e6 (Ce6) was specifically released from DEX6ss nanoparticles in the presence of dextranase or glutathione (GSH), indicating that DEX6ss nanoparticles have responsiveness against dextranase and redox-environment. In darktoxicity test using normal cells and cancer cells, Ce6 and DEX6ss nanoparticles were practically nontoxic. Intracellular delivery of DEX6ss nanoparticles was significantly improved compared to Ce6 itself. DEX6ss nanoparticles achieved significantly higher ROS production and phototoxicity against HCT116 colon cancer cells than Ce6 itself. Furthermore, DEX6ss nanoparticles showed enhanced tumor targeting efficiency and longer retention in the tumor tissues at in vivo animal study with HCT116 tumor-bearing mice. Furthermore, DEX6ss nanoparticles have responsiveness against colonic enzyme, dextranase, indicating that they have potential of colon-specific delivery and dextranase-specific drug delivery capacity. We fabricated colon-specific and tumor-targetable nanophotosensitizer using DEX6ss conjugates. They showed improved cellular uptake ratio, phototoxicity, and colon-specificity. We suggest that DEX6ss nanoparticles can be considered as a promising candidate for PDT of colon cancer.

Anticancer activity of streptochlorin, a novel antineoplastic agent, in cholangiocarcinoma

Background The aim of this study is to investigate the anticancer activity of streptochlorin, a novel antineoplastic agent, in cholangiocarcinoma. Methods The anticancer activity of streptochlorin was evaluated in vitro in various cholangiocarcinoma cell lines for apoptosis, proliferation, invasiveness, and expression of various protein levels. A liver metastasis model was prepared by splenic injection of HuCC-T1 cholangiocarcinoma cells using a BALB/c nude mouse model to study the systemic antimetastatic efficacy of streptochlorin 5 mg/kg at 8 weeks. The antitumor efficacy of subcutaneously injected streptochlorin was also assessed using a solid tumor xenograft model of SNU478 cells for 22 days in the BALB/c nude mouse. Results Streptochlorin inhibited growth and secretion of vascular endothelial growth factor by cholangiocarcinoma cells in a dose-dependent manner and induced apoptosis in vitro. In addition, streptochlorin effectively inhibited invasion and migration of cholangiocarcinoma cells. Secretion of vascular endothelial growth factor and activity of matrix metalloproteinase-9 in cholangiocarcinoma cells were also suppressed by treatment with streptochlorin. Streptochlorin effectively regulated metastasis of HuCC-T1 cells in a mouse model of liver metastasis. In a tumor xenograft study using SNU478 cells, streptochlorin significantly inhibited tumor growth without changes in body weight when compared with the control. Conclusion These results reveal that streptochlorin is a promising chemotherapeutic agent to the treatment of cholangiocarcinoma.

Simple nanophotosensitizer fabrication using water-soluble chitosan for photodynamic therapy in gastrointestinal cancer cells

The polysaccharide chitosan has abundant cationic amine groups, and can form ion-complexes with anionic molecules such as the strong photosensitizer chlorin e6 (Ce6). In this study, water-soluble chitosan (WSC) was used to fabricate Ce6-incorporated nanophotosensitizers (Abbreviated as ChitoCe6 nanophotosensitizer) via a self-assembling process. This was accomplished by dissolving WSC in pure water and then directly mixing the solution with solid Ce6 causing ion complex formation between WSC and Ce6. The resulting nanophotosensitizer was spherical in shape and had a particle size of less than 300nm. The photodynamic effect of ChitoCe6 nanophotosensitizer was evaluated using gastrointestinal (GI) cancer cells. At in vitro study using SNU478 cholangiocarcinoma cells, ChitoCe6 nanophotosensitizer showed improved Ce6 uptake by tumor cells, reactive oxygen species production, and cellular phototoxicity. An in vivo study using SNU478-bearing nude mice showed that the ChitoCe6 nanophotosensitizer efficiently accumulated in the tumor tissue and inhibited tumor growth more than treatment with Ce6 alone. Furthermore, ChitoCe6 nanophotosensitizer was also efficiently absorbed through tissue layers in an ex vivo study using porcine bile duct explants. ChitoCe6 nanophotosensitizer showed enhanced photosensitivity and photodynamic effects against cancer cells in vitro and in vivo. We present ChitoCe6 nanophotosensitizer as a promising candidate for photodynamic therapy of GI cancer.

Vorinostat-eluting poly(DL-lactide-co-glycolide) nanofiber-coated stent for inhibition of cholangiocarcinoma cells

Purpose The aim of this study was to fabricate a vorinostat (Zolinza™)-eluting nanofiber membrane-coated gastrointestinal (GI) stent and to study its antitumor activity against cholangiocarcinoma (CCA) cells in vitro and in vivo. Methods Vorinostat and poly(DL-lactide-co-glycolide) dissolved in an organic solvent was sprayed onto a GI stent to make a nanofiber-coated stent using an electro-spinning machine. Intact vorinostat and vorinostat released from nanofibers was used to assess anticancer activity in vitro against various CCA cells. The antitumor activity of the vorinostat-eluting nanofiber membrane-coated stent was evaluated using HuCC-T1 bearing mice. Results A vorinostat-incorporated polymer nanofiber membrane was formed on the surface of the GI stent. Vorinostat was continuously released from the nanofiber membrane over 10 days, and its release rate was higher in cell culture media than in phosphate-buffered saline. Released vorinostat showed similar anticancer activity against various CCA cells in vitro compared to that of vorinostat. Like vorinostat, vorinostat released from nanofibers induced acetylation of histone H4 and inhibited histone deacetylases 1⋅3⋅4/5/7 expression in vitro and in vivo. Furthermore, vorinostat nanofibers showed a higher tumor growth inhibition rate in HuCC-T1 bearing mice than vorinostat injections. Conclusion Vorinostat-eluting nanofiber membranes showed significant antitumor activity against CCA cells in vitro and in vivo. We suggest the vorinostat nanofiber-coated stent may be a promising candidate for CCA treatment.