Yun-Jeong Kang

Elevated Expression of Thymosin β4, Vascular Endothelial Growth Factor (VEGF), and Hypoxia Inducible Factor (HIF)-1α in Early-Stage Cervical Cancers

Recent studies have shown that thymosin β4 (TB-4) is highly related with tumor metastasis and angiogenesis. In addition, TB-4 induced the expression of VEGF in melanoma cells. We investigated the expression patterns of TB-4 and related angiogenic proteins, VEGF, and HIF-1α, at various stages of cervical cancers and also identified the expression pattern of these proteins in metastatic cervical cancers. Expression patterns of TB-4, VEGF, and HIF-1α were studied with tissue microarray containing 42 samples of cervical cancers. In addition, 15 cervical cancers and metastatic tumors in lymph nodes from patients who have metastatic tumors were also analyzed to confirm the role of TB-4, VEGF, and HIF -1α in cervical cancer metastasis. The expression levels of TB-4, VEGF, and HIF-1α were very weak at early cancer stages (stages 0 to 1A) but significantly increased at stage 1B. The numbers of blood vessels in tumors were also increased at stage 1B. The expression patterns of TB-4, VEGF, and HIF-1α were compared in tumors without lymph node metastasis, primary tumors with lymph node metastasis, and metastatic tumors in lymph nodes. The expression levels of TB-4, VEGF, and HIF-1α in primary tumors with lymph node metastasis and their metastatic tumors in lymph node were less than in tumors without lymph node metastasis. These data suggest that TB-4, VEGF, and HIF-1α triggered angiogensis and tumor invasiveness to surrounding tissues at early stage of cervical carcinoma but have a negative or no effect on the metastatic potential.

Trichinella spiralis infection induces angiogenic factor thymosin β4 expression.

Trichinella spiralis (T. spiralis) has been reported to up-regulate the expression of the angiogenic molecule vascular endothelial cell growth factor (VEGF) during nurse cell formation. In order to analyze the induction of angiogenesis by T. spiralis, the expression patterns of angiogenesis-related proteins were investigated by immunohistochemical analysis. VEGF expression was induced in the infected muscles at an early stage of infection (10 days after infection) and diminished after 3 weeks. Thymosin β4, a major factor which induces VEGF, showed increased expression in muscle fibers 10 days after infection, and the expression remained high in the nurse cells for 6 weeks, when the formation of the nurse cell complex was completed. The hypoxia inducible factor (HIF)-1α showed a diffuse expression pattern around the infected muscle fibers and was strongly expressed in inflammatory cells but was not related to the hypoxic condition caused by nurse cell formation. Localization of the hypoxic regions by the hypoxia marker, pimonidazole showed T. spiralis infection does not induce a hypoxic condition in nurse cells. These results suggest that the expression of VEGF and thymosin β4 induce angiogenesis and the expression of thymosin β4 remained elevated to potentially regulate nurse cell formation and maintenance.

Thymosin β4 expression in human tissues and in tumors using tissue microarrays.

Thymosin &bgr;4 has been reported to play the key roles in tumor growth, metastasis, and angiogenesis. Although the importance of thymosin &bgr;4 in angiogenesis and metastasis is known, few studies to show the expression patterns of thymosin &bgr;4 in human tissues including tumors have been conducted. The comparisons of the expression of thymosin &bgr;4 between the normal and tumor tissues are also needed to study the role of thymosin &bgr;4 in tumor formation. Using tissue microarray analysis, we compared the expression patterns of thymosin &bgr;4 in the normal human tissues and in the tumors to screen certain tumors and upregulated the expression of thymosin &bgr;4 by tumorigenesis. Thymosin &bgr;4 was highly expressed in the hepatic cells in the normal adult liver, duct, and acinar cells in pancreas, and muscle cells in the heart and also expressed highly in certain tumor cells, including osteosarcoma, colon adenocarcinoma, esophageal squamous cell carcinoma, kidney and urinary bladder transitional carcinoma, lung cancer, and liver cancer. Comparing the thymosin &bgr;4 expression between normal and tumors, thymosin &bgr;4 was upregulated specifically in osteosarcoma, colorectal carcinoma, and esophageal cancer. To confirm the over-expression of thymosin &bgr;4 in these tumors, we analyzed expression of thymosin &bgr;4 with each additional microarray of osteosarcoma, colorectal carcinoma, and esophageal cancer. The significant increased expression of thymosin &bgr;4 was observed in osteosarcoma and in colorectal cancer. These results suggest that the expression of thymosin &bgr;4 is highly related with tumorigenesis of certain tumors including the osteosarcoma and colorectal cancers.

DDX4 (DEAD box polypeptide 4) colocalizes with cancer stem cell marker CD133 in ovarian cancers.

DDX4 (DEAD box polypeptide 4), characterized by the conserved motif Asp-Glu-Ala-Asp (DEAD), is an RNA helicase which is implicated in various cellular processes involving the alteration of RNA secondary structure, such as translation initiation, nuclear and mitochondrial splicing, and ribosome and spliceosome assembly. DDX4 is known to be a germ cell-specific protein and is used as a sorting marker of germline stem cells for the production of oocytes. A recent report about DDX4 in ovarian cancer showed that DDX4 is overexpressed in epithelial ovarian cancer and disrupts a DNA damage-induced G2 checkpoint. We investigated the relationship between DDX4 and ovarian cancer stem cells by analyzing the expression patterns of DDX4 and the cancer stem cell marker CD133 in ovarian cancers via tissue microarray. Both DDX4 and CD133 were significantly increased in ovarian cancer compared to benign tumors, and showed similar patterns of expression. In addition, DDX4 and CD133 were mostly colocalized in various types of ovarian cancer tissues. Furthermore, almost all CD133 positive ovarian cancer cells also express DDX4 whereas CD133-negative cells did not possess DDX4, suggesting a strong possibility that DDX4 plays an important role in cancer stem cells, and/or can be used as an ovarian cancer stem cell marker.

Expression of Human Endogenous Retrovirus env Genes in the Blood of Breast Cancer Patients

Human endogenous retroviruses (HERV) env proteins have been recently reported to be significantly up-regulated in certain cancers. Specifically, mRNA and protein levels of HERV-K (HML-2) are up-regulated in the blood plasma or serum of breast cancer patients. Here, we collected blood samples of 49 breast cancer patients and analyzed mRNA expressions of various HERVs env genes including HERV-R, HERV-H, HERV-K, and HERV-P by real-time PCR. The expression of env genes were significantly increased in the blood of primary breast cancer patients but were decreased in patients undergoing chemotherapy to a similar level with benign patients. When we compared the group currently undergoing chemotherapy and those patients undergoing chemotherapy simultaneously with radiotherapy, HERVs env genes were reduced more in the chemotherapy only group, suggesting that chemotherapy is more effective in reducing HERV env gene expression than is radiotherapy. Among chemotherapy groups, HERV env gene expression was the lowest in the taxotere- or taxol-treated group, suggesting that taxotere and taxol can reduce HERVs env expression. These data suggest the potential to use HERVs env genes as a diagnosis marker for primary breast cancer, and further studies are needed to identify the mechanism and physiological significance of the reduction of HERV env gene expression during chemotherapy.

Expression Patterns of Thymosin β4 and Cancer Stem Cell Marker CD133 in Ovarian Cancers

Thymosin β4 (Tβ4), a small acidic actin binding peptide, is overexpressed in a side population of cancer stem cells and CD133-positive colorectal cancer stem cells. In order to understand the relationship between Tβ4 and CD133, we studied the expression patterns of Tβ4 and CD133 in ovarian cancers. The expression patterns of Tβ4 and CD133 were studied in normal ovaries, primary ovarian cancers, metastatic ovarian cancers, primary stomach cancers, and normal stomachs by Western blot and immunohistochemistry. Expression patterns and co-localization of Tβ4 and CD133 were examined by immunofluorescence and confocal laser-scanning microscopy. Tβ4 is overexpressed in primary ovarian cancers, but not in primary stomach cancers, when compared with normal controls. However, Tβ4 levels in metastatic stomach cancers to the ovary are significantly upregulated compared with levels in normal stomachs and primary stomach cancers. These results suggest that Tβ4 levels are related to tumorigenesis in ovarian cancers and metastasis in stomach cancers. The expression of Tβ4 in normal ovaries and normal stomachs was weak, but was co-localized with CD133 expression. Tβ4 expression was also co-localized with CD133 expression in primary ovarian carcinomas, metastatic ovarian cancers from stomach cancers and primary stomach cancers. These data suggest that Tβ4 expression is strongly related to CD133 expression and is a characteristic of stem cells or cancer stem cells.

Human ERV3-1 env protein expression in various human tissues and tumours

Human endogenous retrovirus group 3 member 1 (ERV3-1) has been implicated in the pathogenesis of several human diseases, including tumours. The gene expression profiles of ERV3-1 could provide us with important insights into the pathogenic relationship between ERV3-1 and cancer. Several studies have explored the mRNA expression profiles of ERV3-1 in normal and cancer tissues.1–6 Although the importance of ERV3-1 has been demonstrated in human tissues and cancers, few studies have investigated the expression pattern of ERV3-1 protein in human tissues and tumours. Here, we analysed the expression pattern of ERV3-1 viral envelope (env) protein in adult human organs and in tumours using a tissue microarray. We also compared the expression of ERV3-1 between normal and tumour tissues to study the relationship between ERV3-1 and tumour formation. Tissue microarray slides were purchased from SuperBioChips (SuperBioChips Laboratories, Seoul, Korea). Around 60 patients’ normal or tumour surgical specimens were put on individual tissue microarray slides. The normal tissue and tumours were obtained from patient surgical specimens and no clinical information except for the age and gender of each patient was available for the obtained tissues. For immunohistochemical analysis, tissue microarray slides were immunostained with rabbit polyclonal antibody to ERV3-1 (1:500 dilution; ab116723, Abcam, Cambridge, Massachusetts, USA) and stained using the Dako EnVision System (DAKO, Carpinteria, California, USA). Background staining was measured by the immunostaining of all slides without primary antibody or rabbit immunoglobulin G (IgG), and also confirmed antibody specificity by western blot analysis (figure 1). For protein expression assessment, staining intensity was scored as negative (−), weak (+), moderate (++), and strong (+++). The focal intensity of …

Trichinella spiralis infection reduces tumor growth and metastasis of B16-F10 melanoma cells

Recently, attempts have been made to use parasites as novel candidates for live vaccine vectors against solid tumors. In this study, we examined the effects of Trichinella spiralis (T. spiralis) infection on solid tumor growth and metastasis. After oral infection with T. spiralis larvae, B16-F10 cells were injected subcutaneously and intravenously into C57BL/6 mice to evaluate tumor growth and metastatic potential, respectively. Tumor growth and lung metastases in T. spiralis infected mice were significantly reduced compared with control mice. To elucidate the mechanism of tumor reduction by parasitic infection, we conducted cytokine arrays using mouse serum. CXCL9 and CXCL10 were increased in the infection group and decreased in the infection-tumor group. However, the expression level was not changed in the infection-metastasis group compared to the infection or control-metastasis groups. Although SDF-1 and IL-4 were increased in the infection group, there was no significant change in expression in the infection-tumor group or the infection-metastasis group. Additionally, IL-4 and KC were increased in the infection-tumor group compared to the control-tumor group, but there was no difference in expression between the control-metastasis group and the infection-metastasis group. CXCL13 was significantly increased in the infection-metastasis group only. These results suggest that T. spiralis infection reduced tumor growth and metastasis through a complex transition in cytokine regulation profiles including CXCL9, CXCL10, and CXCL13.

Thymosin β4 was upregulated in recurred colorectal cancers

Recurrent cancers usually metastasise to other organs and have a very poor prognosis. Recurrence and metastatic spread have been proposed to depend on cancer stem cells, which express CD133.1 The expression of the actin-binding protein, thymosin β4 (Tβ4), has been reported to be elevated in a side population of small cells known as cancer stem cells in the breast cancer cell lines MCF7 and MDA-MB231.2 A recent study has also shown that colorectal cancer cells (CR-CSCs) from different patients that were sorted by CD133 had higher Tβ4 levels than normal colorectal cancer cells. Additionally, a lentiviral strategy to downregulate Tβ4 expression in CR-CSCs lowered the capacity of the cells to grow and migrate in culture, and reduced the tumour size and aggressiveness of CR-CSCs in xenografted mice.3 We have previously reported that Tβ4 levels in metastatic stomach cancers to the ovary were significantly upregulated compared with levels in normal stomachs and primary stomach cancers. Furthermore, Tβ4 expression was colocalised with CD133 expression in primary ovarian carcinomas, metastatic ovarian cancers from stomach cancers, and primary stomach cancers, suggesting that Tβ4 expression is strongly related to CD133 expression and is a characteristic of stem cells or cancer stem cells.4 In the present study, we compared the expression patterns of Tβ4 and CD133 in primary and recurrent colorectal cancer cells from the same patient. We obtained the paraffin-embedded primary and recurrent colorectal cancers of 10 patients. Tβ4 and CD133 expression …

Trichinella spiralis infection induces β-actin co-localized with thymosin β4

Trichinella spiralis (T. spiralis) infection in muscle is characterized by the vascular network for the nurse cell-larva complex. We showed in a previous report that thymosin β4 was up-regulated during nurse cell formation by T. spiralis. As thymosin β4 (Tβ4) is the actin-sequestering protein that regulates actin polymerization, the expression pattern of β-actin during the nurse cell formation was analyzed. The protein level of β-actin in muscle fibers 10 days after infection was significantly increased, and its expression remained high in the nurse cells for six weeks. In order to investigate the functional relationship between Tβ4 and β-actin, localization of two proteins was analyzed. Immunofluorescence showed that Tβ4 and β-actin were co-localized in the T. spiralis-infected nurse cells from 10 days to six weeks. The expression patterns of other actin-binding proteins, including thymosin β10 (Tβ10), subunits of the Arp2/3 complex, subunits of Capping protein, profilin, and cofilin, were also analyzed at the mRNA level. Tβ10 expression was also increased during nurse cell formation. Expressions of the Arp2/3 complex was increased at 21 days after infection and Capping proteins was increased during nurse cell formation but shows different expression patterns, depending on the subunit. Profilin and cofilin were specifically increased in the muscle fibers from 14 days after infection. These data show that Tβ4 and β-actin are over-expressed during nurse cell formation upon T. spiralis infection and may be involved in nurse cell formation along with other actin-binding proteins.