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The recombinant kringle domain of urokinase-type plasminogen activator (UK1) has been shown to inhibit angiogenesis in vitro and in vivo and suppress brain tumor in vivo. To avoid limitations in application due to mass production of recombinant proteins, we dissected UK1 sequences to seven peptides based on structure and amino acid characteristics, and examined the anti-angiogenic activities for the constructed peptides. UP-7 peptide derived from ß-sheets region of UK1 was the most potent inhibitor of the proliferation and migration of endothelial cells (ECs) in vitro, and it also inhibited in vivo angiogenesis in the mouse matrigel plug assay. Such anti-angiogenic activities were not exerted by the scrambled peptide. At molecular level, UP-7 inhibited the VEGF or bFGF-induced phosphorylation of FAK and ERK1/2 and suppressed formation of stress fibers and focal adhesions. It also inhibited the attachment and spreading of ECs onto immobilized fibronectin. In a lung cancer animal model xenografted with UP-7-non-sensitive NCI H460, systemic treatment of UP-7 effectively suppressed tumor growth through inhibition of angiogenesis. Interestingly, breast cancer cells such as MDA-MB231 were sensitive to UP-7 in proliferation differently from other cancer cells. It inhibited the migration and invasion of LM-2-MDA-MD231 and suppressed the phosphorylation of FAK. Accordingly, UP-7 potently inhibited lung metastatic growth of LM2-MDA-MB-231 in experimental metastasis model. Taken together, these results suggest that novel UK1-derived peptide, UP-7 can be effectively used for treatment of the metastatic growth of breast cancer. (NRF-2012R1A1A2007175, NRF-2012R1A5A2051476) Citation Format: Young Ae Joe, Hyun-Kyung Kim, Purevjargal Naidansuren, Seung Woo Lee, Rae-Kwon Kim, Su-Jae Lee, Suk Keun Lee, Yong-Kil Hong. Urokinase-derived peptide UP-7 effectively inhibits metastatic growth of breast cancer through suppression of FAK activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4901. doi:10.1158/1538-7445.AM2017-4901

The increasing number of endoscopic endonasal transsphenoidal approaches (EETSA) has resulted in several sinonasal complications, including voice changes. Here, we compared preoperative and postoperative voice changes according to age.We retrospectively reviewed the medical records. Patients were divided into the following three groups according to age: ≤ 30 years, 31 to 60 years, and ≥ 60 years. Patients underwent preoperative voice evaluation using acoustic analysis, a nasometer, and the voice handicap index (VHI). A nasometer was used to measure the nasalance scores. The subjects read or repeated three speech stimuli. For each stimulus, mean nasalance scores were obtained and age dependence was analyzed. Repeat testing was performed 6 months postoperatively.One hundred and seventy patients who underwent binostril four-hand EETSA with a bilateral modified rescue flap were included in this study. Mean nasalance scores for the oral passage, oronasal passage, and nasal passage--as well as mean jitter scores, shimmer scores, and VHI--were significantly increased in all of the EETSA patients (P 0.05 for all). Mean nasalance scores for the nasal sentences were significantly increased in the ≤ 30 age group (all, P 0.05). Scores for the oronasal passage and nasal passage were significantly increased in the 31 to 60 age group (all, P 0.05). Scores for the oronasal passage, nasal passage, jitter, and shimmer were significantly increased in the 60 age group (all, P 0.05).Endoscopic endonasal transsphenoidal approaches might contribute to voice changes. In addition, older age might produce hypernasality and roughness after EETSA. Patients who plan to undergo EETSA should be informed that their voice may change after the operation.4.

Immunotherapy emerged as a promising therapeutic approach to highly incurable malignant gliomas due to tumor-specific cytotoxicity, minimal side effect, and a durable antitumor effect by memory T cells. But, antitumor activities of endogenously activated T cells induced by immunotherapy such as vaccination are not sufficient to control tumors because tumor-specific antigens may be self-antigens and tumors have immune evasion mechanisms to avoid immune surveillance system of host. Although recent clinical results from vaccine strategy for malignant gliomas are encouraging, these trials have some limitations, particularly their failure to expand tumor antigen-specific T cells reproducibly and effectively. An alternative strategy to overcome these limitations is adoptive T cell transfer therapy, in which tumor-specific T cells are expandedex vivorapidly and then transferred to patients. Moreover, enhanced biologic functions of T cells generated by genetic engineering and modified immunosuppressive microenvironment of host by homeostatic T cell expansion and/or elimination of immunosuppressive cells and molecules can induce more potent antitumor T cell responses and make this strategy hold promise in promoting a patient response for malignant glioma treatment. Here we will review the past and current progresses and discuss a new hope in adoptive T cell therapy for malignant gliomas.

Cancer stem cells (CSCs) have been reported to be critical in the initiation, maintenance, and progression of cancers. The expression of stem cell markers, such as podoplanin (PDPN), CD133, and nestin, may have been correlated with malignant progression. However, the effects of CSCs and stem cell markers on clinical outcomes in cancer patients remain unclear. In this study, we assessed the prognostic roles of glioma CSCs (gCSCs) isolation and stem cell markers in patients with primary glioblastoma (pGBM). A cohort of 39 patients with pGBM was separated into two groups, those positive or negative for gCSCs, and the correlation between gCSC and patient survival was evaluated. We observed significantly different cumulative survival (P=0.045) when comparing patients positive for gCSCs patients and negative for gCSC. Among the patients positive for gCSCs, we observed no significant differences in survival between those whose gCSCs were each positive or negative for PDPN, CD133, or nestin. This study strongly supports the prognostic value of gCSCs isolation on the survival of patients with pGBM.

Human malignant gliomas are highly vascularized and aggressive tumors. Angiogenesis inhibitors have been shown to induce regression of a variety of primary and metastatic tumors in vivo. However, their usefulness in treating brain tumors is not well understood. Angiostatin, a multiple kringle (1–4 of 5)–containing fragment of plasminogen, is one of the highly effective natural cryptic angiogenesis inhibitors. In our study, the therapeutic efficacy of non-glycosylated and small molecular size recombinant kringles 1–3 (rPK1–3) was examined in the treatment of brain tumors generated by stereotactic intracerebral implantation of U-87 human glioma cells in nude mice. Mice bearing tumors 7 days post-implant were treated daily with rPK1–3 (100 mg/kg) s.c. for 21 days. Treated animals showed suppressed brain tumor growth by greater than 71.2% along with a 3-fold increase of apoptotic index and suppressed vascularization by 78.9%, without any observable signs of toxicity. Analysis of bFGF and VEGF expression in the tumors of treated animals using immuno-histochemical methods showed near complete absence of growth factors. Our results indicate that the non-glycosylated, small molecular size rPK1–3 is an efficient tumoristatic agent for the treatment of intracranial human glioma xenografts in mice and might provide new strategies for the treatment of brain tumors. Int. J. Cancer 82:694–699, 1999. © 1999 Wiley-Liss, Inc.

The results are reported of a retrospective review of the presentation and outcome of 43 pineal region tumors treated from 1982 to 1996, including 20 identified tumors: 5 germinomas, 8 teratomas, 2 embryonal carcinomas, 1 endodermal sinus tumor, 2 pineocytomas and 2 pineoblastomas. Of the 43 tumors reviewed, 36 were located in the pineal region, 5 in the suprasellar, and 2 in both the pineal and suprasellar regions. Twenty patients underwent surgical resection: total in 6 and partial in 10, while only a biopsy was taken in 4 cases. Fifteen patients were managed on the basis of serum CSF tumor markers and radiation response. Twenty-three patients with germinomas received radiotherapy (RT) and had a 5-year survival rate of 87%. Fifteen patients with non-germinomatous germ cell tumors received RT and chemotherapy following direct surgery, and 5 died (mortality rate of 33.3%). The overall survival rate of the 43 patients with pineal tumors was 79.1% (34/43) and the death rate was 20.9% (9/43). It is now recognized that the wide variety of tumor types found in the pineal region necessitates different modes of treatment, and improved microsurgical and stereotactic surgical techniques have made mortality and morbidity rates acceptably low. Because the radiation response and CSF cytology are not enough to determine optimum treatment, a tissue diagnosis should be obtained in all patients.

To retrospectively compare the utility of perfusion magnetic resonance imaging (MRI) in distinguishing treatment-related changes from recurrent disease in glioma patients.Thirty-one patients with histologically diagnosed gliomas and increased enhancement after or during concurrent (chemo-) radiation therapy were enrolled. They underwent dynamic contrast-enhanced (DCE) permeability MRI followed by dynamic susceptibility contrast (DSC) perfusion MRI. The vascular transfer constant (rK(trans)) and initial areas under the concentration curve (riAUC) were obtained from DCE MRI, and cerebral blood volume (rCBV) was obtained from DSC MRI. Patients were classified as having treatment-related changes or recurrent tumours based on clinicoradiological results or pathological results from surgery.Nineteen patients were diagnosed as having recurrences and 12 patients as having treatment-related changes. The rK(trans), riAUC, and rCBV values in the recurrent group were significantly higher than the values in the group with treatment-related changes (p0.05). For all 31 patients, there was no significant difference between DSC MRI and DCE MRI for the differentiating power between recurrence and treatment-related changes (p = 0.7227). However, when including only the 24 patients with concordant values of rK(trans) and riAUC, DCE MRI showed a significant AUC value of 0.786 in the receiver operating characteristic (ROC) curve analysis (p = 0.003), whereas DSC MRI did not (AUC = 0.643, p = 0.229).MRI perfusion images appear to show promise in distinguishing treatment-related changes from recurrent tumours. When both rK(trans) and riAUC show concordant values, DCE MRI seems to be more powerful than DSC MRI in the differentiation of recurrence from treatment-related changes.

A nasoseptal flap is used to reconstruct defects in the skull base when cerebrospinal fluid (CSF) leaks after the endoscopic endonasal transsphenoidal approach (EETSA). We evaluated the usefulness of elevating bilateral nasoseptal flaps with the EETSA. Sixty-seven patients (71 procedures, including 4 revisions) underwent the EETSA with bilateral nasoseptal flap elevation. We retrospectively reviewed patients' medical records, including demographic data, surgical procedures, outcomes, and complications. The entire sellar floor was exposed after elevating bilateral nasoseptal flaps. We reconstructed the defect using a right nasoseptal flap in 14 cases with intraoperative CSF leakage. The denuded sphenoidal sinus was covered with a left nasoseptal flap in 13 cases with excessive loss of sphenoidal sinus mucosa. Unused flaps (57 right flaps and 58 left flaps) were repositioned in the original sites. No postoperative CSF leak occurred. All sphenoidal sinuses covered with the left nasoseptal flap healed well without excessive crust. Two patients experienced immediate postoperative bleeding. Septal perforation occurred in 1 patient who underwent a revision operation. Bilateral nasoseptal flap elevation provided good exposure of the sellar floor with the EETSA. The nasoseptal flap could be used to reconstruct the defect after the EETSA and to cover the denuded sphenoidal sinus. The unused flaps could be repositioned in their original sites to minimize the septal defect and could be reused in revision surgery. We suggest that elevating bilateral nasoseptal flaps is a useful surgical technique in a variety of settings with the EETSA.

The presence of glioma stromal mesenchymal stem‑like cells (GS-MSLCs) in tumors from glioma patients has been previously reported. The mechanisms through which these cells function as a part of the glioma microenvironment, however, remain incompletely understood. We investigated the biological effects of GS-MSLCs on glioma cancer stem cells (gCSCs), testing the hypothesis that GS-MSLCs alter the biological characteristics of gCSCs. GS-MSLCs and gCSCs were isolated from different glioblastoma (GBM) specimens obtained from patients. In in vitro experiments, gCSCs were cultured alone or co-cultured with GS-MSLCs, and gCSCs cell counts were compared between the two groups. In addition, two groups of orthotopic GBM xenografts in mice were created, one using gCSCs from the monoculture group and one using gCSCs isolated from the co-culture group, and tumor volume and survival were analyzed. Furthermore, in vivo proliferation, apoptosis and vessel formation were examined using immunohistochemical analyses. In vitro cell counts for gCSCs co-cultured with GS-MSLCs increased 3-fold compared to gCSCs cultured alone. In orthotopic xenograft experiments, mice injected with gCSCs isolated from the co-culture group had significantly larger tumor volume, measured on day 40 after injection, and their survival times were shorter. Immunohistochemical analysis showed increased tumor expression of CD31, indicative of enhanced microvessel formation in mice injected with gCSCs co-cultured with GS-MSLCs compared to mice injected with gCSCs cultured alone. However, proliferation (PCNA) and apoptosis (TUNEL) markers showed no significant difference between the two groups. In conclusion, GS-MSLCs may influence the biological properties of gCSCs, shifting them towards a more aggressive status; moreover, increased angiogenesis may be a critical component of this mechanism.

It was presented that mesenchymal stem cells (MSCs) can be isolated from western glioma specimens. However, whether MSCs exist in glioma specimens of different ethnicities is unknown. To verify the existence of MSCs in an independent cohort, we undertook studies to isolate MSCs from a group of Korean patients. We hypothesized that cells resembling MSCs that were deemed mesenchymal stemlike cells (MSLCs) exist in an independent cohort of Korean gliomas.We cultured fresh glioma specimens using the protocols used for culturing MSCs. The cultured cells were analyzed with fluorescence-activated cell sorting (FACS) for surface markers associated with MSCs. Cultured cells were exposed to mesenchymal differentiation conditions. To presume possible locations of MSLCs in the glioma, sections of glioma were analyzed by immunofluorescent labeling for CD105, CD31, and NG2.From nine of 31 glioma specimens, we isolated cells resembling MSCs, which were deemed Korean glioma stroma MSLCs (KGS-MSLCs). KGS-MSLCs were spindle shaped and adherent to plastic. KGS-MSLCs had similar surface markers to MSCs (CD105(+), CD90(+), CD73(+), and CD45(-)). KGS-MSLCs were capable of mesenchymal differentiation and might be located around endothelial cells, pericytes, and in a disorganized perivascular area inside glioma stroma.We found that cells resembling MSCs indeed exist in an independent cohort of glioma patients, as presented in western populations. We could presume that the possible location of KGS-MSLCs was in perivascular area or in glioma stroma that was a disorganized vascular niche. It might be possible that KGS-MSLCs could be one of constituent of stroma of glioma microenvironment.

The existence of cancer stem cells (CSCs) in glioblastoma has been proposed. However, the unknown knowledge that is yet to be revealed is the presence of glioma CSCs (gCSCs) in correlation to each WHO grades of glioma. We approached this study with a hypothesis that specimens from high-grade gliomas would have higher isolation rate of gCSCs in comparison to those of lower-grade gliomas. The glioma specimens were obtained from patients and underwent gliomasphere assay. The gliomaspheres were chosen to be analyzed with immunocytochemisty for surface markers. Then the selected gliomaspheres were exposed to neural differentiation conditions. Lastly, we made mouse orthotopic glioma models to examine the capacity of gliomagenesis. The gliomaspheres were formed in WHO grade IV (13 of 21) and III (two of nine) gliomas. Among them, WHO grade IV (11 of 13) and III (two of two) gliomaspheres showed similar surface markers to gCSCs and were capable of neural differentiation. Lastly, among the chosen cells, 10 of 11 WHO grade IV and two of two WHO grade III gliomaspheres were capable of gliomagenesis. Thus, overall, the rates of existence of gCSCs were more prominent in high-grade gliomas: 47.6 % (10 of 21) in WHO grade IV gliomas and 22.2 % (two of nine) in WHO grade III gliomas, whereas WHO grade II and I gliomas showed virtually no gCSCs. This trend of stage-by-stage increase of gCSCs in gliomas showed statistical significance by chi-square test linear-by-linear association. We prove that the rates of existence of gCSCs increase proportionally as the WHO grades of gliomas rise.

Currently, the interaction between the niche and glioma cancer stem cells (gCSCs) is gaining attention. However, there are few studies concerned with the effects of repeated exposure to a new microenvironment on gCSCs characteristics. In this study, serial in vivo subtransplantation was performed to create a new microenvironment. We evaluated and compared the biological characteristics of gCSCs after serial in vivo subtransplantation.We cultured gCSCs from human glioma specimens according to cultured gliomasphere methods. The isolated gCSCs were termed zero-generation gCSCs (G0-gCSCs). By subsequent serial subtransplantation, we obtained first-generation gCSCs (G1-gCSCs) and second-generation gCSCs (G2-gCSCs). We evaluated and compared the biological characteristics of G0-gCSCs, G1-gCSCs, and G2-gCSCs. The in vitro characteristics included the morphology, surface marker profiles, and neural differentiation capacity and the in vivo characteristics was the survival of mice xenografts. Additionally, brain sections were analyzed using PCNA, TUNEL, and CD31 staining.We observed no significant differences in the in vitro characteristics of G0-gCSCs, G1-gCSCs, and G2-gCSCs. However, the survival time of mice glioma xenografts was significantly decreased upon serial subtransplantation. In addition, immunohistochemical analyses showed that the number of TUNEL(+) cells was significantly decreased while the number of CD31(+) cells was significantly increased with serial in vivo subtransplantation.There were significant in vivo biological changes in gCSCs upon serial in vivo subtransplantation, which were shorter xenograft survival, increased angiogenesis, and decreased apoptosis. This study suggests that the repeated exposure to new microenvironments may affect the biological changes in gCSCs in vivo.

Animal models are important for defining paradigms of tumor immunology and for evaluating therapeutic efficacy of immunotherapy. Many animal models have been used for evaluating in vivo characteristics of malignant gliomas and their responses to therapy. No animal model, however, is perfect because malignant glioma has a very heterogeneous biological behavior. There are so many parallels between mouse and human immunology, but there are significant discrepancies in immune system. Animal models for vaccine therapy can be classified as transplantable tumor models and models of spontaneous tumor in genetically engineered animals. Although transplantable tumor models have been used to test immunotherapeutic efficacy and remain a mainstay in study of brain tumor immunology, a lot of tumor vaccines that look promising in experimental animals have turned out to be ineffective clinically. Recent advances of laboratory techniques and understanding of genetic and molecular characteristics of gliomas allows for animal models of gliomas with similar biologic characteristics. Well-designed glioma models that accurately reflect the biology, pathology and clinical behaviors of human gliomas can provide more useful preclinical informations to predict clinical efficacy of novel immunotherapies and cancer vaccines.

Objective This study was performed to determine the safety and outcome of concurrent chemoradiotherapy (CCRT) and adjuvant chemotherapy with temozolomide for Korean patients with a newly diagnosed glioblastoma. Methods Patients were recruited from four institutions between 2004 and 2007. The patients received fractionated focal irradiation in daily fractions of 2 Gy given 5 days per week for 6 weeks and daily temozolomide, followed by 6 cycles of adjuvant temozolomide. The primary endpoint was overall survival (OS) and the secondary endpoints were progression-free survival (PFS), response, and safety. Results A total of 103 patients were enrolled in this study. Ninety-six patients (93%) completed the CCRT and 54 patients (52%) received 6 cycles of adjuvant temozolomide. The response rate was 73% (53/73) and the tumor control rate was 92% (67/73). Of the 96 patients who completed the CCRT, the median OS was 18.0 months and the 1- and 2-year OS rates were 74 and 38%, respectively. The median PFS was 10.0 months and the 1- and 2-year PFS rates were 33 and 16%, respectively. The only significant prognostic factor of survival was the extent of surgical resection (p

Animal models are important for defining paradigms of tumor immunology and for evaluating therapeutic efficacy of immunotherapy. Many animal models have been used for evaluating in vivo characteristics of malignant gliomas and their responses to therapy. No animal model, however, is perfect because malignant glioma has a very heterogeneous biological behavior. There are so many parallels between mouse and human immunology, but there are significant discrepancies in immune system. Animal models for vaccine therapy can be classified as transplantable tumor models and models of spontaneous tumor in genetically engineered animals. Although transplantable tumor models have been used to test immunotherapeutic efficacy and remain a mainstay in study of brain tumor immunology, a lot of tumor vaccines that look promising in experimental animals have turned out to be ineffective clinically. Recent advances of laboratory techniques and understanding of genetic and molecular characteristics of gliomas allows for animal models of gliomas with similar biologic characteristics. Well-designed glioma models that accurately reflect the biology, pathology and clinical behaviors of human gliomas can provide more useful preclinical informations to predict clinical efficacy of novel immunotherapies and cancer vaccines.

The Onodi cell is the posterior-most ethmoid air cell and an important anatomical variant because of the intimate spatial relationship with the optic nerve, internal carotid artery, and sellar floor during sphenoid sinus surgery. The authors evaluated the incidence of Onodi cells, their clinical importance, and the association between preoperative radiological findings and surgical findings.Case series with chart review.Tertiary care medical center.The authors retrospectively reviewed the medical records of 162 cases, including preoperative paranasal sinus computed tomography (PNS CT) findings and the findings with the endoscopic endonasal transsphenoidal approach (EETSA). They evaluated the prevalence of Onodi cells and the clinical manifestations in the patients with these cells. They also examined the clinical significance of these cells during EETSA.Onodi cells were identified in the preoperative PNS CT of 53 patients, whereas Onodi cells were observed in 54 (33.3%) of the 162 patients at EETSA. The Onodi cells were bilateral in 23 patients and unilateral in 31. In all cases, the Onodi cells limited the exposure of the sellar floor. Only after removing these cells was the entire sellar floor exposed so that the tumors could be removed completely.Onodi cells were observed more frequently than in previous studies, and 98.1% of them were identified on preoperative PNS CT. When reviewing PNS CT images preoperatively, one needs to identify the presence of Onodi cells. The Onodi cells must be removed to completely resect tumors located in the sellar region during EETSA.

We have determined O(6)-methylguanine-DNA-methyltransferase (MGMT) promoter methylation status by methylation-specific polymerase chain reaction (MSP) in 22 paraffin-embedded specimens of glioblastoma multiforme. A MGMT methylation-specific high resolution melting (HRM) assay was performed to compare the methylation levels of the tumorous and non-tumorous portions of each sample, which were selectively collected using a microdissection technique. MGMT methylation was detected in 10 patients using MSP, while 8 patients had both methylated and unmethylated MGMT promoters. HRM assays showed that there was no difference in the level of methylation between tumorous and non-tumorous portions of each sample. In patients with MSP-positive tumors, the overall survival (median, 22 months) was longer as compared to those with MSP-negative tumors (median, 14 months). A correlation between the methylation status of the MGMT promoter and MGMT protein expression was observed in 12 samples. This study demonstrates that MGMT methylation is not restricted to glioblastoma cells. Additionally, methylation-specific HRM is a feasible approach that can be readily applied to the methylation analysis of MGMT. A further study will be needed to determine the dynamic change of MGMT methylation in the tumor environment.

Objective The incidence of leptomeningeal dissemination from malignant glioma is rare, so the clinical features of this are not well documented yet. We attempted to determine the clinical features of leptomeningeal dissemination from malignant gliomas. Methods We retrospectively analyzed 11 cases of leptomeningeal dissemination of malignant glioma, who were treated at our institution between 2006 and 2009. We investigated the clinical features of these patients by considering the following factors : tumor locations, the events of ventricular opening during surgery and the cerebrospinal fluid (CSF) profiles, including the cytology. Results The group was composed of 9 males and 2 females. The histological diagnosis of their initial intracranial tumors were 4 primary glioblastoma, 3 anaplastic astrocytoma, 1 anaplastic oligoastrocytoma, 2 ganglioglioma and 1 pleomorphic xanthoastrocyotma with anaplastic features. The mean age of the patients at the time of the initial presentation was 42.8±10.3 years. The mean time between surgery and the diagnosis of spinal dissemination was 12.3±7.9 (3-28) months. The mean overall survival after dissemination was 2.7±1.3 months. All our patients revealed a history of surgical opening of the ventricles. Elevated protein in the CSF was reported for eight patients who had their CSF profiles checked. Conclusion We propose that in the malignant gliomas, the surgical opening of ventricles can cause the spinal leptomeningeal dissemination and the elevated protein content of CSF may be a candidate marker of leptomeningeal dissemination.

The alkylating agent temozolomide (TMZ) is an effective drug used for the treatment of malignant gliomas. However, tumor relapse combined with the development of drug resistance remains a significant problem. To clarify the mechanism of the resistance of glioma cells to TMZ chemotherapy, TMZ-resistant glioma cell lines (TR cells) were generated using U373 and U251 human glioma cells, and TMZ-resistance was confirmed via viability and apoptosis assays. The TMZ-resistance of TR cells was not associated with the TMZ-resistance molecule O(6)-methylguanine-DNA-methyltransferase. Notably, the expression level of signal transducers and activators of transcription 3 (STAT3) and serine 727-phosphorylated STAT3 (pSTAT3-Ser727) was highly increased in TR cells, while that of 705-phosphorylated STAT3 (pSTAT3-Tyr705) was decreased. The inhibition of STAT3 expression by small interfering RNA enhanced TR cell TMZ sensitivity. These results suggest that STAT3 contributes to TMZ-resistance in gliomas and is a potential target for the reversal of TMZ-resistance in patients with a recurrent glioma.

In this study, we investigated the potential of combined treatment with temozolomide (TMZ) chemotherapy and tumor antigen-pulsed dendritic cells (DCs) and the underlying immunological factors of TMZ chemoimmunotherapy with an intracranial GL26 glioma animal model. The combined treatment enhanced the tumor-specific immune responses and prolonged the survival more effectively than either single therapy in GL26 tumor-bearing animals. Apoptosis was induced in the tumors of the animals by the treatment with TMZ. Calreticulin (CRT) surface exposure was detected by immunofluorescence staining of TMZ-treated GL26 cells. TMZ chemotherapy increased tumor antigen cross-priming from tumor cells, leading to cross-priming of tumor antigen-specific CD4+T cells and CD8+T cells. This chemotherapy appeared to suppress the frequency of CD4+CD25+regulatory T cells (Treg). Moreover, this combined therapy resulted in an increase in the tumor infiltration of CD4+and CD8+T cells. Collectively, the findings of this study provide evidence that the combination of TMZ chemotherapy and treatment with DC-based vaccines leads to the enhancement of antitumor immunity through increased tumor-specific immune responses via the cross-priming of apoptotic tumor cell death mediated by CRT exposure and, in part, the suppression of Treg. Therefore, CRT exposure, regulatory T cells, and cross-priming by TMZ chemotherapy may be immunological factors related to the enhancement of the antitumor effects of chemoimmunotherapy in an experimental brain tumor model.

Topoisomerase II alpha (Top2alpha) is an attractive candidate to be used as a tumor antigen for cancer immunotherapy, because it is abundantly expressed in various tumors and serves as a target for a number of chemotherapeutic agents. In this study, we demonstrated the immunogenicity of Top2alpha, using dendritic cells (DC) electroporated with RNA encoding the Top2alpha C-terminus (Top2alphaCRNA/DC). Top2alphaCRNA/DC were able to demonstrate in vitro stimulation of T cells from mice that were previously vaccinated with Top2alpha-expressing tumor lysate-pulsed DC. Vaccination with Top2alphaCRNA/DC induced Top2alpha-specific T cell responses in vivo as well as antitumor effects in various murine tumor models including MC-38, B16F10, and GL26. DC pulsed with p1327 (DSDEDFSGL), defined as an epitope presented by H-2K(b), also induced Top2alpha-specific immune responses and antitumor effects. Based on these data, Top2alpha is suggested to be a universal target for cancer immunotherapy.

The kringle domain of urokinase-type plasminogen activator (UK1) has anti-angiogenic and anti-tumor effects. Celecoxib, an inhibitor of cyclooxygenase type 2, also suppresses angiogenesis and tumor growth. To look for potential additive effects in their activities, we examined the anti-angiogenic and anti-tumor effects of the combination of UK1 and celecoxib for malignant gliomas. In vitro , the combination of UK1 and celecoxib enhanced inhibition of proliferation, migration, and tube formation of endothelial cells, although showing no enhancement of inhibition of U87 cell growth. However, in vivo models, combination treatment of intracerebral U87 malignant glioma xenografts in nude mice with UK1 (10 mg/kg/day) and celecoxib (10 mg/kg/day) at lower doses resulted in even more potent inhibition of tumor growth than each monotherapy (by 81% compared to untreated tumors), with drastic decrease of the expression of angiogenesis-related factors and increase of apoptosis in the tumor tissues. Interestingly, UK1 inhibited VEGF or bFGF-induced phosphorylation of ERK1/2 in ECs, whereas celecoxib showed no such effects. However, celecoxib inhibited U87 cell growth and directly suppressed their VEGF production. Therefore, our data suggest that combined use at low doses of UK1 and celecoxib with different anti-angiogenic mechanisms provides a desirable strategy for anti-glioma therapy.

Objective : Interferon-β‚ (IFN-β) has been used in the treatment of cancers. Inhibition of the enzyme cyclooxygenase (COX) with celecoxib had a significantly suppressive effect on tumor growth, angiogenesis, and metastasis in a variety of tumors. The aim of this study was to elucidate the antiglioma effect of combined treatment with IFN- β and celecoxib in U87 glioma model. Methods : The in vitro effects of IFN-β (50-1,000 IU/mL) and celecoxib (50-250 µM) alone or combination of both on the proliferation and apoptosis of U87 cells were tested using MTT assay, FACS analysis and DNA condensation. To determine the in vivo effect, nude mice bearing intracerebral U87 xenograft inoculation were treated with IFN- β intraperitoneally (2×10 5 IU/day for 15 days), celecoxib orally (5, 10 mg/kg) or their combination. Results : IFN- β or celecoxib showed an inhibitory effect on the proliferation of U87 cells. When U87 cells were treated with IFN-β and celecoxib combination, it seemed that IFN-β interrupted the antiproliferative and apoptotic activity of celecoxib. No additive effect was observed on the survival of the tumor bearing mice by the combination of IFN-β and celecoxib. Conclusion : These results suggest that IFN-β seems to inhibit the antiglioma effect of celecoxib, therefore combination of IFN-β and celecoxib may be undesirable in the treatment of glioma.

The recombinant kringle domain (UK1) of urokinase-type plasminogen activator (uPA) has been shown to possess anti-angiogenic activity in vitro and in vivo. It has also been found to inhibit in vivo malignant glioma growth. In contrast, direct interaction of the kringle domain of uPA and integrin alphavbeta3 has been reported to be involved in plasminogen and leukocyte activation by uPA. Since integrin alphavbeta3 is involved in tumor angiogenesis, we investigated whether integrin alphavbeta3 is involved in the inhibitory function of UK1 in angiogenesis, by examining its anti-migratory activity. In a modified Boyden chamber assay, the Pichia-expressed UK1 dose-dependently inhibited the VEGF-induced migration of human umbilical vein endothelial cells (HUVECs). However, in the absence of growth factor stimulation, soluble UK1 alone did not induce or inhibit HUVEC migration. In cell adhesion, immobilized UK1 promoted HUVEC adhesion and spreading which were compared to BSA. Pretreatment of the anti-alphavbeta3 integrin antibody, significantly inhibited HUVEC binding to immobilized UK1, whereas neither anti-alpha2beta1 nor anti-alpha5beta1 integrin antibody had any effect, although pre-treatment of the soluble UK1 showed no marked alteration of the binding level of anti-alphavbeta3 antibody to HUVECs in FACS analysis. In a modified Boyden chamber assay, the function blocking antibodies against integrins alphavbeta3, alpha2beta1 and alpha5beta1 did not completely prevent the inhibitory effect of UK1 in HUVEC migration. These results suggest that UK1 interacts with integrin alphavbeta3, but its anti-migratory activity on endothelial cells is not significantly mediated by integrin alphavbeta3.

Survivin (SVV), a member of the inhibitors of apoptosis, has been considered as an ideal tumor-associated antigen due to its broad expression pattern in many types of human malignancies. Here, SVV-specific immune responses were evaluated with dendritic cells (DCs) loaded with a recombinant tatPTD fused SVV (tatSVV) protein both in vitro and in vivo system. The tatPTD fusion allowed for highly efficient and reproducible protein delivery into the DCs even in the presence of a phagocytosis inhibitor. The level of SVV-specific cytotoxic T-cell response appeared to be significantly higher for the T-cell line that was primed with the tatSVV-pulsed DCs, as compared to the T-cell line produced with naked (n)-SVV-pulsed DCs in vitro. Mice that received a xenogeneic tatSVV-pulsed DC vaccine developed a higher immune response than mice vaccinated with the nSVV-pulsed DCs and exhibited prolonged mean survival rate, though mice injected with the DCs alone or the nSVV-pulsed DC also showed anti-tumor effects in the subcutaneous GL26 glioma model. These results suggest that vaccination with DCs pulsed with tatSVV is an effective way for the SVV-targeting cancer vaccine to induce an effective anti-tumor responses.

We analyzed the clinical efficacy and toxicity of concurrent therapy as a first line modality for malignant glioma patients. From 1998 to 2004, 39 patients, 22 with glioblastoma (GM), nine with anaplastic astrocytoma (AA), 7 with anaplastic oligodendroglioma (AO) and 1 with anaplastic oligodendro-astrocytoma (AOA) were enrolled in this study. The median age was 46.2 years (range 8-67). Both external involved field radiotherapy and chemotherapy, composed of CCNU (75-110 mg/m(2)), procarbazine (60 mg/m(2)) and vincristine (1.4 mg/m(2)), were started simultaneously two weeks after surgery. The median progression-free survival time for the GM, AA, and AO patients was 6, 26, and 31 months, respectively. The median survival of the patients with GM and AA was 27 and 41 months. The two-year survival rate of the GM and AA patients was 50.4 and 66.7%, respectively. Grade III/IV hematological toxicity was reduced from 25.6 to 13% after reduction of the dose of CCNU (75 mg/m(2)). Radiation necrosis was confirmed by pathologic examination in four patients (10.3%). The median interval from the completion of radiotherapy to the diagnosis of necrosis was 19 weeks. Modified concurrent chemoradiotherapy may be a feasible option for treating malignant glioma with acceptable toxicity.

In a previous report, the recombinant kringle domain (UK1) of the urokinase type plasminogen activator (uPA) showed antiangiogenic activity. Here, we investigated in vivo antitumor effects of the UK1 of human uPA employing a brain tumor model. The systemic administration of UK1 purified from pichia expression (10 and 50 mg/kg/day intraperitoneally for 25 days) led to suppress the growth of a U87 human glioma xenograft, implanted into the brains of male BALB/cSlc nude mice, by 35% and 80%, respectively. In the immunohistochemical analysis, the tumors treated with UK1 showed decreased vascularity and expression of angiogenesis-related factors including vascular endothelial growth factor (VEGF), angiogenin, alpha-smooth muscle actin, von Willebrand's factor, and CD31 (PECAM-1 [Platelet endothelial cell adhesion molecule-1]), and increased apoptosis. UKl inhibited the in vitro proliferation and tube formation of VEGF-stimulated endothelial cells but not the proliferation of glioma cells. These results suggest that UK1 inhibits the malignant glioma growth by suppression of angiogenesis.

We analyzed clinical data of 376 patients with histologically confirmed anaplastic oligodendroglioma or anaplastic oligoastrocytoma (AOA) from 2000 to 2010. Post-operative therapy was performed in 354 patients (94.1 %), of which 133 received radiotherapy (RT) alone and 189 received both RT and chemotherapy. The use of procarbazine, lomustine, and vincristine (PCV) decreased (57.6 % in 2000-2001 vs. 28.6 %in 2010) and the use of temozolomide (TMZ) increased (0 % in 2000-2001 vs. 61.9 % in 2010) over the study period. A combination of chemotherapy and RT was used more often than RT alone in young patients and patients with a good performance status. The 1p/19q co-deletion status and O-6-methyguanine-DNA methyltransferase methylation were analyzed since 2004. Among the patients who received chemotherapy, TMZ was used more often in patients with AOA and PCV was used more often in patients with the 1p/19q codeletion. Median progression-free survival (PFS) was 2.9 years and overall survival (OS) was 8.7 years. (5-year and 10-year OS rates were 58% and 45%, 5-year and 10-year PFS rates were 38% and 24%). Younger age, frontal lobe location of the tumor, gross total removal, 1p/19q codeletion, and initial RT were associated with longer PFS and OS rates. A clear improvement in progression-free and overall survival was observed for RT and combined CT/RT in compared with CT only. Postoperative RT appears to improve survival for entire group thus total removal and 1p/19q codeletion may not be sufficient criteria to omit RT as a treatment option. These results suggest that RT should continue to be offered as the standard treatment option. Taken together, treatment pattern for oligodendroglial tumors changed significantly across the study period in Korea. In particular, TMZ has replaced PCV, and the use of molecular markers as well as RT alone has increased, but a optimal therapeutic option remains to be defined though the prospective randomized studies.

OBJECTIVES: High-dose methotrexate chemotherapy (HD-MTX) is the mainstay in treatment of primary CNS lymphoma (PCNSL) and has been known to have acute or late toxicity on skin, gastrointestinal mucosa, liver, kidneys, and bone marrow. However, hyperacute adverse events (HADE), which occurred within one hour of HD-MTX administration, are rarely known in PCNSL patients. We report our HADE experiences with 9 consecutive cases. METHODS: Of 51 PCNSL patients, 38 immunocompetent adult PCNSL patients treated with HD-MTX at Seoul St. Mary's Hospital, The Catholic University of Korea between 2004 and 2015 were investigated. Our HD-MTX monotherapy regimen consists of induction period (8 g/m2 on day 1, 10, and 20 for 3 cycles) and maintenance period (3.5 g/m2, every 4 weeks for 3 cycles and then every 3 months for 3 cycles). Medical records were reviewed for each of the HADE events, retrospectively. RESULTS: Of 38 patients who received HD-MTX of 247 cumulative cycles, 9 patients (23.7%; 4 men and 5 women) presented 13 HADE. Median age of the patients was 58 years (range 47 to 64 years) at the time of the event. HADE developed during the maintenance period in 5 patients, at the early induction period in 1 patient and at the rechallenging period for recurrence in 3 patients. HADE symptoms were variable; most commonly skin rashes in 12 events (92.3%), abdominal pain in 2 events (15.4%), and most severely hypotension in 1 event (7.7%). HD-MTX monotherapy was discontinued in 4 patients, continued with corticosteroid premedication and symptomatic application of antihistamine with or without MTX dose modification in 5 patients, and completed in 2 patients. CONCLUSION: Although HD-MTX appears to be a relatively safe medication, greater awareness of possible HADE in HD-MTX chemotherapy is needed for its prevention, early diagnosis and management. Studies to clarify its mechanism should be warranted.

This study investigated the treatment of anaplastic oligodendroglial tumors across nine Korean institutions. We reviewed the medical records from 381 patients with histologically confirmed anaplastic oligodendroglioma or anaplastic oligoastrocytoma (AOA) from 2000 to 2010. Clinical factors and treatment patterns were analyzed for each year. Post-operative therapy was performed in 354 patients (94.1 %), of which 133 received radiotherapy (RT) alone and 189 received both RT and chemotherapy. RT alone was the preferred treatment toward the end of the study period (29.4 % in 2000-2001 vs. 56.3 % in 2010, P = 0.005). The use of procarbazine, lomustine, and vincristine (PCV) decreased (57.6 % in 2000-2001 vs. 28.6 % in 2010, P = 0.001) and the use of temozolomide (TMZ) increased (0 % in 2000-2001 vs. 61.9 % in 2010, P < 0.001) over the study period. A combination of chemotherapy and RT was used more often than RT alone in young patients (P = 0.036) and patients with a good performance status (P = 0.023). The 1p/19q co-deletion status and O-6-methyguanine-DNA methyltransferase methylation were analyzed since 2004 but were not significant factors for determining whether to administer chemotherapy. Among the patients who received chemotherapy, TMZ was used more often in patients with AOA (P = 0.007) and PCV was used more often in patients with either multiple lesions (P = 0.027) or the 1p/19q co-deletion (P = 0.026). Our results demonstrate that the treatment pattern for oligodendroglial tumors changed significantly across the study period. In particular, TMZ has replaced PCV, and the use of molecular markers as well as RT alone has increased, but a unified protocol remains to be established.

Objective Complete sellar floor reconstruction is critical to avoid postoperative cerebrospinal fluid (CSF) leakage during transsphenoidal surgery. Recently, the pedicled nasoseptal flap has undergone many modifications and eventually proved to be valuable and efficient. However, using these nasoseptal flaps in all patients who undergo transsphenoidal surgery, including those who had none or only minor CSF leakage, appears to be overly invasive and time-consuming. Methods Patients undergoing endoscopic endonasal transsphenoidal tumor surgery within a 5 year-period were reviewed. Since 2009, we classified the intraoperative CSF leakage into grades from 0 to 3. Sellar floor reconstruction was tailored to each leak grade. We did not use any tissue grafts such as abdominal fat and did not include any procedures of CSF diversions such as lumbar drainage. Results Among 200 cases in 188 patients (147 pituitary adenoma and 41 other pathologies), intraoperative CSF leakage was observed in 27.4% of 197 cases : 14.7% Grade 1, 4.6% Grade 2a, 3.0% Grade 2b, and 5.1% Grade 3. Postoperative CSF leakage was observed in none of the cases. Septal bone buttress was used for Grade 1 to 3 leakages instead of any other foreign materials. Pedicled nasoseptal flap was used for Grades 2b and 3 leakages. Unused septal bones and nasoseptal flaps were repositioned. Conclusion Modified classification of intraoperative CSF leaks and tailored repair technique in a multilayered fashion using an en-bloc harvested septal bone and vascularized nasoseptal flaps is an effective and reliable method for the prevention of postoperative CSF leaks.

Background Optimal treatment for recurrent primary central nervous system lymphomas (PCNSLs) has not been defined yet and there is no general consensus about the salvage chemotherapy after high-dose methotrexate (HD-MTX)-based chemotherapy. The purpose of the present study was to evaluate the efficacy and safety of procarbazine, lomustine, and vincristine (PCV) chemotherapy for recurrent PCNSLs. Methods We reviewed eight immunocompetent patients (five males/three females, mean age: 56 years) who received salvage PCV chemotherapy (procarbazine 60 mg/m(2), days 8 through 21: CCNU 110 mg/m(2), day 1: vincristine 2 mg, days 8 and 28) for recurrent PCNSL and two patients switched to PCV chemotherapy due to severe adverse effects of HD-MTX chemotherapy. Radiologic responses, survival, and adverse effects were analyzed. Results Of the eight recurrent PCNSLs, three patients (37.5%) showed radiologic complete response, one patient (12.5%) showed partial response, and four patients (50%) showed progressive disease after PCV chemotherapy. Median progression free survival (PFS) from the first administration of PCV to relapse or last follow-up was 7 months (range 5-32 months) and median overall survival was 8 months (range 2-41 months). The two patients who switched to PCV chemotherapy showed PFS of 9 and 5 months from the beginning of PCV to relapse. The common side effects were thrombocytopenia, neutropenia, and peripheral neuropathy. There were 4 grade III or IV myelo-suppression, but no fatal complications, including severe hemorrhage or infection, were observed. Conclusion Salvage PCV chemotherapy has a moderate anti-lymphoma activity for recurrent PCNSLs after the HD-MTX-based chemotherapy with tolerable toxicity.

Temozolomide (TMZ) is a DNA methylating agent that has shown promising antitumor activity against high grade glioma. Interferon-beta (IFN-beta) is known to have antiproliferative and antiangiogenic activities. The aim of this study was to elucidate whether an antiglioma effect could be potentiated by the combination of TMZ and IFN-beta. In vitro, the combination of these drugs suppressed the proliferative and migratory activities, as well as enhance of the apoptosis and cell cycle (S phase) arrest of U-87 cells more efficiently than TMZ or IFN-beta alone. IFN-beta exerted a potent inhibitory effect on the proliferation of human umbilical vein endothelial cells (HUVEC); however, no additive or synergistic effect was observed with the addition of TMZ. To determine in vivo effect, nude mice bearing intracerebral U-87 xenograft inoculation were treated with intraperitoneal administration of PBS, TMZ (15 mg/kg for 3 days), IFN-beta (2x10(5) IU for 15 days), and a TMZ + IFN-beta combination. The combined treatment (median 62.0+/-8.6 days, P=0.0005) was observed to significantly increase the survival of the animals compared to treatment with PBS (median 30.0+/-2.5 days), TMZ (median 41.0+/-3.5 days) or IFN-beta (mean 36.0+/-2.5 days). These results suggest that antiglioma activity can be enhanced by the combination of TMZ and IFN-beta, providing the possibility for a new strategy development in the management of malignant glioma.

Temozolomide (TMZ) is a DNA methylating agent that has shown promising antitumor activity against high grade glioma. Interferon-beta (IFN-beta) is known to have antiproliferative and antiangiogenic activities. The aim of this study was to elucidate whether an antiglioma effect could be potentiated by the combination of TMZ and IFN-beta. In vitro, the combination of these drugs suppressed the proliferative and migratory activities, as well as enhance of the apoptosis and cell cycle (S phase) arrest of U-87 cells more efficiently than TMZ or IFN-beta alone. IFN-beta exerted a potent inhibitory effect on the proliferation of human umbilical vein endothelial cells (HUVEC); however, no additive or synergistic effect was observed with the addition of TMZ. To determine in vivo effect, nude mice bearing intracerebral U-87 xenograft inoculation were treated with intraperitoneal administration of PBS, TMZ (15 mg/kg for 3 days), IFN-beta (2x10(5) IU for 15 days), and a TMZ + IFN-beta combination. The combined treatment (median 62.0+/-8.6 days, P=0.0005) was observed to significantly increase the survival of the animals compared to treatment with PBS (median 30.0+/-2.5 days), TMZ (median 41.0+/-3.5 days) or IFN-beta (mean 36.0+/-2.5 days). These results suggest that antiglioma activity can be enhanced by the combination of TMZ and IFN-beta, providing the possibility for a new strategy development in the management of malignant glioma.

Intracranial cavernous angiomas are benign vascular malformations and can be divided into intra-axial and extra-axial lesions. Extra-axial cavernous angiomas are relatively rare and usually arise in relation to the dura mater and mimick meningiomas. We report a case of cavernous angioma that occurred in the falx cerebri of a 22-yr-old female patient with the special focus on neuroradiologic findings. This is the fourth case of cavernous angioma in the falx cerebri reported in the literature to our knowledge.

Although chemotherapy remains among the best treatment options for most cancers, adjuvant therapies such as dendritic cell (DC)-based immunotherapy have been added to treatment protocols to destroy residual tumor cells. IFN-gamma secreting T cells specific for survivin was found after temozolomide (TMZ) treatment in C57BL/6 mice intracranial (i.c.) inoculated with GL26 cells. Furthermore, combination treatment with low-dose TMZ (2.5mg/kg/day, i.p.) chemotherapy followed by vaccination with survivin RNA-transfected DCs (1 x 10(6)cells/mouse, s.c.) enhanced T cells responses specific for survivin and improved survival rate compared with DC vaccination alone or TMZ treatment alone in tumor inoculated mice. However, these enhancements of T cells responses by TMZ treatment were not observed in mice without tumor inoculation. These results suggested that cross-priming by TMZ may enhance antitumor immunity of DC vaccination in murine brain tumor model.

Baculovirus pseudotyped with vesicular stomatitis virus G protein (Bac-VSV-G) was found to efficiently transduce and express transgenes on mammalian cells. In this study, this recombinant virus was used for induction of anti-tumor immunity against murine telomerase reverse transcriptase (mTERT) and was compared with RNA-electroporated dendritic cells (DCs) in a murine glioma model. Splenocytes from the mice vaccinated with Bac-VSV-G expressing mTERT (Bac-VSVGmTERT) showed significantly increased numbers of mTERT-specific IFN-c-secreting T cells using an ELISPOT technique, and also showed increased NK cell activity. In addition, the TERT-specific T cells activated by Bac-VSVG-mTERT and mTERT RNA-electroporated DCs were predominantly CD4 + T cells and CD8 + T cells, respectively. The protective anti-tumor effect of Bac-VSVG-mTERT was similar to that of mTERT RNA-electroporated DCs. These results suggest that the pseudotype baculovirus expressing TERT may be a good candidate for a genetic vaccine for use in the treatment of malignant gliomas. � 2006 Elsevier Ireland Ltd. All rights reserved.

The two-kringle domain of tissue-type plasminogen activator (TK1-2) has been identified as a novel angiogenesis inhibitor. In the previous study, purified Pichia-derived TK1-2 has been shown to suppress in vivo growth of human lung and colon cancer cells. Here, we demonstrate that E. coli-derived non-glycosylated TK1-2 suppresses tumor growth more potently than Pichia-derived TK1-2 and prolongs the survival of tumor bearing mice. The recombinant TK1-2 prepared through E. coli expression, His-tag affinity chromatography and in vitro refolding was injected intraperitoneally once daily into nude mice 7 days after subcutaneous implantation with PC14 lung cancer cells (n=10). Measurement of tumor volumes indicated that low-dose TK1-2 treatment (10 mg/kg) suppressed tumor growth by approximately 85.2% (p0.01), while high-dose TK1-2 treatment (50 mg/kg) even more potently inhibited tumor growth (93.8%) (p0.005). Treatment of TK1-2 also prolonged the survival of tumor-bearing mice in a dose-dependent fashion. In an independent HCT116 xenograft model, E. coli-derived TK1-2 was more effective in suppressing tumor growth than Pichia-derived TK1-2. Immunohistochemical analysis of tumor tissue also revealed that the expression of VEGF, SMA-alpha, TNF-alpha and angiogenin was less positive in the E. coli-derived TK1-2-treated group than in the Pichia-derived TK1-2-treated group. These results suggest that E. coli-derived refolded, non-glycosylated TK1-2 can be used more effectively as an anti-cancer agent.