Engineering and Preclinical Evaluation of Western Reserve Oncolytic Vaccinia Virus Expressing A167Y Mutant Herpes Simplex Virus Thymidine Kinase.

Viral replication of thymidine kinase deleted (tk-) vaccinia virus (VV) is attenuated in resting normal cells, enabling cancer selectivity, however, replication potency of VV-tk- appears to be diminished in cancer cells. Previously, we found that wild-type herpes simplex virus (HSV)-tk (HSV-tk) disappeared in most of the recombinant VV after multiple screenings, and only a few recombinant VV containing naturally mutated HSV-tk remained stable. In this study, VV-tk of western reserve (WR) VV was replaced by A167Y mutated HSV-tk (HSV-tk418m), to alter nucleoside selectivity from broad spectrum to purine exclusive selectivity. WOTS-418 remained stable after numerous passages. WOTS-418 replication was significantly attenuated in normal cells, but cytotoxicity was almost similar to that of wild type WR VV in cancer cells. WOTS-418 showed no lethality following a 5 × 108 PFU intranasal injection, contrasting WR VV, which showed 100% lethality at 1 × 105 PFU. Additionally, ganciclovir (GCV) but not BvdU inhibited WOTS-418 replication, confirming specificity to purine nucleoside analogs. The potency of WOTS-418 replication inhibition by GCV was > 10-fold higher than that of our previous truncated HSV-tk recombinant OTS-412. Overall, WOTS-418 demonstrated robust oncolytic efficacy and pharmacological safety which may delegate it as a candidate for future clinical use in OV therapy.

Preclinical safety evaluation of hepatic arterial infusion of oncolytic poxvirus.

PURPOSE: Oncolytic poxvirus has shown promise in treating various solid tumors, such as liver cancer, and administration of oncolytic poxvirus via the hepatic artery may provide more survival benefits than other routes of administration. However, there is a lack of safety information to guide the application of hepatic arterial infusion (HAI) of oncolytic poxvirus in human studies. To investigate the acute and chronic toxicity of HAI administration of oncolytic poxvirus in animals and provide safety information for future human studies. METHODS: VVtk-, a vaccinia poxvirus with inactivated thymidine kinase gene, was administered via HAI to rabbits with normal liver function under angiography (1×108 or 1×109 pfu), and rats with N-nitrosomorpholine-induced precancerous liver cirrhosis under open surgery (1×108 pfu). Body weights and survival were monitored and blood samples were collected for hematological and biochemical tests. Distribution of A56 (a specific marker for poxvirus infection) in rabbit organs was evaluated using immunofluorescence assays. RESULTS: HAI of high doses of VVtk- did not cause any acute or chronic changes in body weight, survival or in biochemical, hematological tests in the 2 animal models, and none of the changes showed dose dependency (in rabbit study), or were influenced by liver cirrhosis (in rat study). A56 was not detected in any of the major rabbit organs. CONCLUSION: HAI may provide a safe alternative route of oncolytic poxvirus administration for human studies.

Defensive mechanism in cholangiocarcinoma cells against oxidative stress induced by chlorin e6-based photodynamic therapy.

In this study, the effect of chlorin e6-based photodynamic therapy (Ce6-PDT) was investigated in human intrahepatic (HuCC-T1) and extrahepatic (SNU1196) cholangiocarcinoma (CCA) cells. The amount of intracellular Ce6 increased with increasing Ce6 concentration administered, or with incubation time, in both cell lines. The ability to take up Ce6 and generate reactive oxygen species after irradiation at 1.0 J/cm(2) did not significantly differ between the two CCA cell types. However, after irradiation, marked differences were observed for photodamage and apoptotic/necrotic signals. HuCC-T1 cells are more sensitive to Ce6-PDT than SNU1196 cells. Total glutathione (GSH) levels, glutathione peroxidase and glutathione reductase activities in SNU1196 cells were significantly higher than in HuCC-T1 cells. With inhibition of enzyme activity or addition of GSH, the phototoxic effect could be controlled in CCA cells. The intracellular level of GSH is the most important determining factor in the curative action of Ce6-PDT against tumor cells.

Effect of 5-aminolevulinic acid-encapsulate liposomes on photodynamic therapy in human cholangiocarcinoma cells.

Intracellular protoporphyrin IX (PpIX) generation following administration of 5-aminolevulinic acid (ALA) has been used in photodynamic therapy (PDT). Subsequent irradiation can lead to selective damage to photosensitizer-treated cells or tissues. In the present work, we describe the enhancement of ALA-induced PpIX accumulation using a liposome carrier. ALA-containing liposomes (Lipo-ALA) were prepared using dipalmitoyl-phosphatidyl choline and in vitro PDT effect was investigated against human cholangiocarcinoma HuCC-T1 cells. Lipo-ALA increased the uptake efficiency into tumor cells compared to ALA itself, which increased the phototoxic effect. A positive relationship was evident between small particle size, PpIX accumulation and cell death after Lipo-ALA based PDT.

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.

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/cm(2) when compared to ALA-PDT alone. Even though ROS levels increased with 0.25 J/cm(2) 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/cm(2) and 0.75 J/cm(2) 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.

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(ε-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.

Aminolevulinic acid derivatives-based photodynamic therapy in human intra- and extrahepatic cholangiocarcinoma cells.

Hexyl-aminolevulinic acid (HALA) was compared with aminolevulinic acid (ALA) in terms of improving ALA-based photodynamic therapy (PDT) for human intra- and extrahepatic cholangiocarcinoma (CCA) HuCC-T1 and SNU1196 cells. Because of the different uptake mechanisms of HALA, a relatively higher amount of protoporphyrin IX (PpIX) was induced in the both CCA cell types at low concentrations of HALA. Furthermore, higher expression of porphobilinogen deaminase, coproporphyrinogen III oxidase, and protoporphyrinogen oxidase, the key enzymes for synthesizing PpIX in the heme biosynthetic pathway, facilitated the exuberant generation of PpIX in HuCC-T1 cells. PpIX accumulation with ALA was markedly different between the two CCA cell types. Even at lower concentrations of ALA, SNU1196 cell successfully synthesized PpIX, due to the higher expression of the ALA transporter, mammalian H (+)/peptide co-transporter PEPT1. Considering the difference of PEPT1 or key enzyme expression, HALA could be a very effective substitute for ALA in doing PDT for cure of CCA.

Antitumor activity of sorafenib-incorporated nanoparticles of dextran/poly(dl-lactide-co-glycolide) block copolymer.

Sorafenib-incoporated nanoparticles were prepared using a block copolymer that is composed of dextran and poly(DL-lactide-co-glycolide) [DexbLG] for antitumor drug delivery. Sorafenib-incorporated nanoparticles were prepared by a nanoprecipitation-dialysis method. Sorafenib-incorporated DexbLG nanoparticles were uniformly distributed in an aqueous solution regardless of the content of sorafenib. Transmission electron microscopy of the sorafenib-incorporated DexbLG nanoparticles revealed a spherical shape with a diameter < 300 nm. Sorafenib-incorporated DexbLG nanoparticles at a polymer/drug weight ratio of 40:5 showed a relatively uniform size and morphology. Higher initial drug feeding was associated with increased drug content in nanoparticles and in nanoparticle size. A drug release study revealed a decreased drug release rate with increasing drug content. In an in vitro anti-proliferation assay using human cholangiocarcinoma cells, sorafenib-incorporated DexbLG nanoparticles showed a similar antitumor activity as sorafenib. Sorafenib-incorporated DexbLG nanoparticles are promising candidates as vehicles for antitumor drug targeting.

Effect of surfactant on 5-aminolevulinic acid uptake and PpIX generation in human cholangiocarcinoma cell.

Photodynamic therapy (PDT) is a palliative therapy and has been used to cure cholangiocarcinoma (CC), which has a poor prognosis and limited available curative therapy. PDT was shown to improve the median survival time of advanced-stage patients. Recently, 5-aminolevulinic acid (ALA) has been used as a pro-photosensitizer, which can be transferred to intercellular protoporphyrin IX (PpIX), which is a strong photosensitizer, via the heme pathway. The main limitation of using ALA in PDT is the hydrophilic properties of ALA, which results in low cellular uptake. In this study, non-ionic surfactants, pluronic F68 (PF68) and Tween 80 (TW80), were used to address this limitation. The human CC cell line, HuCC-T1, was cotreated with ALA and different concentrations of surfactants for 4h. The effect of surfactants was evaluated by monitoring the uptake of ALA, the fluorescence intensity of PpIX, and the cell survival rate after suitable light irradiation. Cotreatment with the surfactant resulted in an increased intracellular ALA level, PpIX formation, and phototoxicity.

Self-assembled nanoparticles of hyaluronic acid/poly(DL-lactide-co-glycolide) block copolymer.

We synthesized block copolymer composed of hyaluronic acid (HA) and poly(DL-lactide-co-glycolide) (PLGA) (HAbLG) for antitumor targeting. (1)H NMR was employed to confirm synthesis of block copolymer. At (1)H NMR study, HabLG nanoparticles showed HA intrinsic peaks only at D(2)O, indicating that they contained HA as a hydrophilic outer-shell and PLGA as a inner-core. Anti-tumor activity was studied using CD44-overexpressing HCT-116 human colon carcinoma cells. Addition of doxorubicin (DOX)-incorporated nanoparticles to tumor cells resulted in the expression of a strong red fluorescence color while they expressed very weak fluorescence when CD44 receptor was blocked with free HA. Flow cytometry data also showed similar results, indicating that the fluorescence intensity of tumor cells treated with nanoparticles was significantly decreased when CD44 receptor was blocked. These results indicate that HAbLG nanoparticles were able to target CD44-overexpressing tumor cells via receptor-mediated endocytosis.

Effect of 5-aminolevulinic acid-based photodynamic therapy via reactive oxygen species in human cholangiocarcinoma cells.

Cancer cells have been reported to exhibit an enhanced capacity for protoporphyrin IX (PpIX) synthesis facilitated by the administration of 5-aminolevulinic acid (ALA). We investigated the effect of ALA-based photodynamic therapy (PDT) on human cholangiocarcinoma cells (HuCC-T1). Since protoporphyrin IX (PpIX), a metabolite of ALA, can produce reactive oxygen species (ROS) under irradiation and then induce phototoxicity, ALA-based PDT is a promising candidate for the treatment of cholangiocarcinoma. When various concentrations of ALA (0.05-2 mM) were used to treat HuCC-T1 cells for 6 or 24 hours, the intracellular PpIX level increased according to the ALA concentration and treatment time. Furthermore, an increased amount of PpIX in HuCC-T1 cells induced increased production of ROS by irradiation, resulting in increased phototoxicity.

Doxorubicin-incorporated polymeric micelles composed of dextran-b-poly(DL-lactide-co-glycolide) copolymer.

BACKGROUND: Polymeric micelles using amphiphilic macromolecules are promising vehicles for antitumor targeting. In this study, we prepared anticancer agent-incorporated polymeric micelles using novel block copolymer. METHODS: We synthesized a block copolymer composed of dextran and poly (DL-lactide-co-glycolide) (DexbLG) for antitumor drug delivery. Doxorubicin was selected as the anticancer drug, and was incorporated into polymeric micelles by dialysis. Polymeric micelles were observed by transmission electron microscopy to be spherical and smaller than 100 nm, with a narrow size distribution. The particle size of doxorubicin-incorporated polymeric micelles increased with increasing drug content. Higher initial drug feeding also increased the drug content. RESULTS: During the drug-release study, an initial burst release of doxorubicin was observed for 10 hours, and doxorubicin was continuously released over 4 days. To investigate the in vitro anticancer effects of the polymeric micelles, doxorubicin-resistant HuCC-T1 cells were treated with a very high concentration of doxorubicin. In an antiproliferation study, the polymeric micelles showed higher cytotoxicity to doxorubicin-resistant HuCC-T1 cells than free doxorubicin, indicating that the polymeric micelles were effectively engulfed by tumor cells, while free doxorubicin hardly penetrated the tumor cell membrane. On confocal laser scanning microscopy, free doxorubicin expressed very weak fluorescence intensity, while the polymeric micelles expressed strong red fluorescence. Furthermore, in flow cytometric analysis, fluorescence intensity of polymeric micelles was almost twice as high than with free doxorubicin. CONCLUSION: DexbLG polymeric micelles incorporating doxorubicin are promising vehicles for antitumor drug targeting.

LRRK2 enhances oxidative stress-induced neurotoxicity via its kinase activity.

LRRK2 is an autosomal dominant gene whose mutations cause familial Parkinson's disease (PD). The LRRK2 protein contains a functional kinase and a GTPase domain. PD phenotypes caused by LRRK2 mutations are similar to those of idiopathic PD, implying that LRRK2 is an important participant in PD pathogenesis. Of LRRK2's PD-specific mutations, the G2019S is the most frequently observed one. Its over-expression is known to increase kinase activity and neurotoxicity compared to wild type (WT) LRRK2. Here, using a simple colorimetric cell viability assay, we analyzed LRRK2's neurotoxicity in dopaminergic SN4741 cells following treatment with hydrogen peroxide. When WT, G2019S, or empty vector was expressed in SN4741 cells, cell death was modestly and significantly increased in the order of G2019S>WT>vector. When these transfected cells were treated with hydrogen peroxide to mimic oxidative stress, cellular neurotoxicity was enhanced in the same order (i.e. G2019S>WT>vector). Moreover, incubation of SN4741 cells with conditioned medium from cells expressing G2019S and subjected to hydrogen peroxide treatment exhibited 10-15% more cell death than conditioned medium from cells transfected with vector or WT, suggesting that G2019S-expressing cells secrete a factor(s) affecting viability of neighboring cells. The kinase domain was mapped to be responsible for oxidative stress-induced neurotoxicity. In addition, over-expression of WT and G2019S LRRK2 lead to a weak, but significant, increase in intracellular reactive oxygen species (ROS) in the order of G2019S>WT as measured by DCFH-DA assay in both the presence and absence of H(2)O(2) treatment. Furthermore, in G2019S-expressing cells, co-expression of the anti-oxidant protein DJ-1 or ERK inhibitor treatment restored survival rate to a level similar to that of cells transfected with control vector under H(2)O(2) treatment. Taken together, our data suggest that the LRRK2 kinase domain increases the generation of ROS and causes enhanced neurotoxicity under H(2)O(2) treatment, which can be at least partially rescued by DJ-1 or the ERK inhibitor.