Geon-Tae Park

Advanced new strategies for metastatic cancer treatment by therapeutic stem cells and oncolytic virotherapy

The field of therapeutic stem cell and oncolytic virotherapy for cancer treatment has rapidly expanded over the past decade. Oncolytic viruses constitute a promising new class of anticancer agent because of their ability to selectively infect and destroy tumor cells. Engineering of viruses to express anticancer genes and specific cancer targeting molecules has led to the use of these systems as a novel platform of metastatic cancer therapy. In addition, stem cells have a cancer specific migratory capacity, which is available for metastatic cancer targeting. Prodrug activating enzyme or anticancer cytokine expressing stem cells successfully inhibited the proliferation of cancer cells. Preclinical models have clearly demonstrated anticancer activity of these two platforms against a number of different cancer types and metastatic cancer. Several systems using therapeutic stem cells or oncolytic virus have entered clinical trials, and promising results have led to late stage clinical development. Consequently, metastatic cancer therapies using stem cells and oncolytic viruses are extremely promising. The following review will focus on the metastatic cancer targeting mechanism of therapeutic stem cells and oncolytic viruses, and potential challenges ahead for advancing the field.

Anti-proliferative Effect of Engineered Neural Stem Cells Expressing Cytosine Deaminase and Interferon-β Against Lymph Node-derived Metastatic Colorectal Adenocarcinoma in Cellular and Xenograft Mouse Models.

Purpose Genetically engineered stem cells may be advantageous for gene therapy against various human cancers due to their inherent tumor-tropic properties. In this study, genetically engineered human neural stem cells (HB1.F3) expressing Escherichia coli cytosine deaminase (CD) (HB1.F3.CD) and human interferon-β (IFN-β) (HB1.F3.CD.IFN-β) were employed against lymph node–derived metastatic colorectal adenocarcinoma. Materials and Methods CD can convert a prodrug, 5-fluorocytosine (5-FC), to active 5-fluorouracil, which inhibits tumor growth through the inhibition of DNA synthesis,while IFN-β also strongly inhibits tumor growth by inducing the apoptotic process. In reverse transcription polymerase chain reaction analysis, we confirmed that HB1.F3.CD cells expressed the CD gene and HB1.F3.CD.IFN-β cells expressed both CD and IFN-β genes. Results In results of a modified trans-well migration assay, HB1.F3.CD and HB1.F3.CD.IFN-β cells selectively migrated toward SW-620, human lymph node–derived metastatic colorectal adenocarcinoma cells. The viability of SW-620 cells was significantly reduced when co-cultured with HB1.F3.CD or HB1.F3.CD.IFN-β cells in the presence of 5-FC. In addition, it was found that the tumor-tropic properties of these engineered human neural stem cells (hNSCs) were attributed to chemoattractant molecules including stromal cell-derived factor 1, c-Kit, urokinase receptor, urokinase-type plasminogen activator, and C-C chemokine receptor type 2 secreted by SW-620 cells. In a xenograft mouse model, treatment with hNSC resulted in significantly inhibited growth of the tumor mass without virulent effects on the animals. Conclusion The current results indicate that engineered hNSCs and a prodrug treatment inhibited the growth of SW-620 cells. Therefore, hNSC therapy may be a clinically effective tool for the treatment of lymph node metastatic colorectal cancer.

Effects of microalgal polyunsaturated fatty acid oil on body weight and lipid accumulation in the liver of C57BL/6 mice fed a high fat diet

Abstract Dietary polyunsaturated fatty acids (PUFAs), which are abundant in marine fish oils, have recently received global attention for their prominent anti-obesogenic effects. Among PUFAs, eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3), which are n-3 long-chain PUFAs widely referred to as omega-3 oils, were reported to prevent the development of obesity in rodents and humans. In the present study, we evaluated the anti-obesity effects of microalgal oil on high-fat induced obese C57BL/6 mice, compared with commercial omega-3 fish oil and vegetable corn oil. Microalgal oil is an inherent mixture of several PUFAs, including EPA, DHA and other fatty acids produced from a marine microalgal strain of Thraustochytriidae sp. derived mutant. It was found to contain more PUFAs (>80%) and more omega-3 oils than commercial omega-3 fish oil (PUFAs >31%) and corn oil (PUFAs 59%). All three types of oils induced weight loss in high-fat-induced obese mice, with the loss induced by microalgal oil being most significant at 9 weeks (10% reduction). However, the oils tested did not improve blood lipid levels, although microalgal oil showed an apparent inhibitory effect on lipid accumulation in the liver. These findings may be attributed to the higher PUFA content, including omega-3 oils of microalgal oil than other oils. Collectively, these findings suggest that microalgal oil, derived from Thraustochytriidae sp. derived mutant, is a prominent candidate for replacement of omega-3 fish oils based on its apparent anti-obesity effect in vivo.

Potential Anti-proliferative and Immunomodulatory Effects of Marine Microalgal Exopolysaccharide on Various Human Cancer Cells and Lymphocytes In Vitro

Marine microalgal exopolysaccharides (EPSs) have drawn great attention due to their biotechnological potentials such as anti-viral, anti-oxidant, anti-lipidemic, anti-proliferative, and immunomodulatory activities, etc. In the present study, the EPS derived from microalgae Thraustochytriidae sp.-derived mutant GA was investigated for its anti-proliferation and immunomodulation. Anti-cancer efficacy of the microalgal EPS was examined for the alterations in cell proliferation and cell cycle-related gene expression that occur in three types of human cancer cell lines, BG-1 ovarian, MCF-7 breast, and SW-620 colon cancer cell lines, by its treatment. Alterations in immunoreactivity by the microalgal EPS were examined by measuring its influence on the growth of T and B lymphocytes and cytokine production of T cells. In cell viability assay, the microalgal EPS inhibited cancer cell growth at the lowest concentration of 10−11 dilution and in a dose-responsive manner within the range of dilution of 10−11~10−3. In addition, the protein expression of cell cycle progression genes such as cyclin D1 and E in these cancer cell lines was significantly reduced by the microalgal EPS in a dose- and a time-dependant manner. In cell proliferation assay using T and B cells, the microalgal EPS induced B cell proliferation even at the lowest dilution of 10−11, but not T cells. In cytokine assay, the microalgal EPS decreased the formation of IL-6 and INF-γ at 10−3 dilution compared to the control and had no significant effects on TNF-α. Collectively, these findings suggest that the EPS derived from microalgae Thraustochytriidae sp. GA has an anti-proliferative activity against cancer cells and an immunomodulatory effect by having an influence on B cell proliferation and cytokine secretion of T cells.

Application of SV40 T-transformed human corneal epithelial cells to evaluate potential irritant chemicals for in vitro alternative eye toxicity

Assessment of eye irritation potential is important to human safety, and it is necessary for various cosmetics and chemicals that may contact the human eye. Until recently, the Draize test was considered the standard method for estimating eye irritation, despite its disadvantages such as the need to sacrifice many rabbits for subjective scoring. Thus, we investigated the cytotoxicity and inflammatory response to standard eye irritants using SV40 T-transformed human corneal epithelial (SHCE) cells as a step toward development of an animal-free alternative eye irritation test. MTT and NRU assays of cell viability were performed to investigate the optimal experimental conditions for SHCE cell viability when cells were exposed to sodium dodecyl sulfate (SDS) as a standard eye irritant at 6.25×10(-3) to 1×10(-1)%. Additionally, cell viability of SHCE cells was examined in response to six potential eye irritants, benzalkonium chloride, dimethyl sulfoxide, isopropanol, SDS, Triton X-100 and Tween 20 at 5×10(-3) to 1×10(-1)%. Finally, we estimated the secretion level of cytokines in response to stimulation by eye irritants in SHCE cells. SHCE cells showed a good response to potential eye irritants when the cells were exposed to potential irritants for 10min at room temperature (RT), and cytokine production increased in a concentration-dependent manner, indicating that cytotoxicity and cytokine secretion from SHCE cells may be well correlated with the concentrations of irritants. Taken together, these results suggest that SHCE cells could be an excellent alternative in vitro model to replace in vivo animal models for eye irritation tests.

The growth of K562 human leukemia cells was inhibited by therapeutic neural stem cells in cellular and xenograft mouse models

To confirm the anti-tumor effect of engineered neural stem cells (NSCs) expressing cytosine deaminase (CD) and interferon-β (IFN-β) with prodrug 5-fluorocytosine (FC), K562 chronic myeloid leukemia (CML) cells were co-cultured with the neural stem cell lines HB1.F3.CD and HB1.F3.CD.IFN-β in 5-FC containing media. A significant decrease in the viability of K562 cells was observed by the treatment of the NSC lines, HB1.F3.CD and HB1.F3.CD.IFN-β, compared with the control. A modified trans-well assay showed that engineered human NSCs significantly migrated toward K562 CML cells more than human normal lung cells. In addition, the important chemoattractant factors involved in the specific migration ability of stem cells were found to be expressed in K562 CML cells. In a xenograft mouse model, NSC treatments via subcutaneous and intravenous injections resulted in significant inhibitions of tumor mass growth and extended survival dates of the mice. Taken together, these results suggest that gene therapy using genetically engineered stem cells expressing CD and IFN-β may be effective for treating CML in these mouse models.

Abstract B14: Injection of therapeutic stem cells expressing cytosine deaminase and interferon-beta resulted in the inhibition of K562 human chronic myeloid leukemia cells in a xenograft model

Gene-directed enzyme/prodrug therapies have been found to be more advantageous compared to conventional cancer treatment method. One of these, a cytosine deaminase (CD)/5-fluorocytosine (5-FC) system, is known to induce apoptosis of cancer cells by converting 5-FC, a prodrug, to its metabolically active form, 5-fluorouracil. In this study, human neural stem cells (hNSCs) derived enzyme/prodrug therapy was used to treat leukemia. The parental hNSCs, HB1.F3, were engineered to express E. coli CD and/or human interferon-β. To manufacture animal models xenografted with leukemia, K-562 cells (1×106) were mixed with Matrigel (BD Biosciences, Bedford, MA, USA) at 1:1 volume ratio of Matrigel to PBS in 100 μl and injected subcutaneously (s.c.) into the back of athymic nude mice. This animal study was performed for 24 days after hNSCs injections. When tumor volume reached 500 mm3, CM-Dil pre-labeled hNSCs (4 x 106 cells per mouse) were injected subcutaneously adjacent to the tumor mass. Another group, CM-dil pre-labeled hNSCs (4 x 106 cells per mouse) was injected intravenously. hNSCs were injected on the first day of each week. Two days after the injection of hNSCs, all mice received i.p. injections of 5-FC (500 mg/kg/day in 100 μl PBS) every day for 24 days. At 24 h after the last 5-FC treatment, the mice were euthanized and tumor masses were harvested for molecular analysis. In a xenografted mouse model administered with hNSCs intravenously or subcutaneously, hNSC significantly inhibits the growth of tumor mass and extends survival date in the presence of a prodrug. In addition, HB1.F3.CD.IFN-β treatment group showed more antitumor effect compared with HB1.F3.CD treatment group, indicating that IFN- β may have a synergistic effect for directly killing leukemia tumors. The present results represent that engineered hNSCs and prodrug treatment inhibited the proliferation of leukemia. These results suggest that gene therapy employing genetically engineered stem cells expressing CD and IFN-β may be effective for treating leukemia. Note: This abstract was not presented at the conference. Citation Format: Geon-Tae Park, Kyung-Chul Choi. Injection of therapeutic stem cells expressing cytosine deaminase and interferon-beta resulted in the inhibition of K562 human chronic myeloid leukemia cells in a xenograft model [abstract]. In: Proceedings of the AACR International Conference: New Frontiers in Cancer Research; 2017 Jan 18-22; Cape Town, South Africa. Philadelphia (PA): AACR; Cancer Res 2017;77(22 Suppl):Abstract nr B14.