ANT2 shRNA downregulates miR-19a and miR-96 through the PI3K/Akt pathway and suppresses tumor growth in hepatocellular carcinoma cells

MicroRNAs (miRNAs) are negative regulators of gene expression, and miRNA deregulation is found in various tumors. We previously reported that suppression of adenine nucleotide translocase 2 (ANT2) by short hairpin RNA (shRNA) inhibits hepatocellular carcinoma (HCC) development by rescuing miR-636 expression. However, the tumor-suppressive mechanisms of ANT2 shRNA are still poorly understood in HCC. Here, we hypothesized that miRNAs that are specifically downregulated by ANT2 shRNA might function as oncomiRs, and we investigated the roles of ANT2 shRNA-regulated miRNAs in the pathogenesis of HCC. Our data show that miR-19a and miR-96, whose expression is regulated by ANT2 suppression, were markedly upregulated in HCC cell lines and clinical samples. Ectopic expression of miR-19a and miR-96 dramatically induced the proliferation and colony formation of hepatoma cells in vitro, whereas inhibition of miR-19a and miR-96 reduced these effects. To investigate the in vivo function, we implanted miR-96-overexpressing HepG2 cells in a xenograft model and demonstrated that the increase in miR-96 promoted tumor growth. We also found that miR-19a and miR-96 inhibited expression of tissue inhibitor of metalloproteinase-2. Taken together, our results suggest that ANT2-regulated miR-19a and miR-96 play an important role in promoting the proliferation of human HCC cells, and the knockdown of ANT2 directly downregulates miR-19a and miR-96, ultimately resulting in the suppression of tumor growth.

Introduction of the CIITA gene into tumor cells produces exosomes with enhanced anti-tumor effects

Exosomes are small membrane vesicles secreted from various types of cells. Tumor-derived exosomes contain MHC class I molecules and tumor-specific antigens, receiving attention as a potential cancer vaccine. For induction of efficient anti-tumor immunity, CD4+ helper T cells are required, which recognize appropriate MHC class II-peptide complexes. In this study, we have established an MHC class II molecule-expressing B16F1 murine melanoma cell line (B16F1-CIITA) by transduction of the CIITA (Class II transactivator) gene. Exosomes from B16-CII cells (CIITA-Exo) contained a high amount of MHC class II as well as a tumor antigen TRP2. When loaded on dendritic cells (DCs), CIITA-Exo induced the increased expression of MHC class II molecules and CD86 than the exosomes from the parental cells (Exo). In vitro assays using co-culture of immunized splenocytes and exosome-loaded DCs demonstrated that CIITA-Exo enhanced the splenocyte proliferation and IL-2 secretion. Consistently, compared to B16-Exo, CIITA-Exo induced the increased mRNA levels of inflammatory cytokines such as TNF-alpha, chemokine receptor CCR7 and the production of Th1-polarizing cytokine IL-12. A tumor preventive model showed that CIITA-Exo significantly inhibited tumor growth in a dose-dependent manner. Ex vivo assays using immunized mice demonstrated that CIITA-Exo induced a higher amount of Th1-polarized immune responses such as Th1-type IgG2a antibodies and IFN-gamma cytokine as well as TRP2-specific CD8+ T cells. A tumor therapeutic model delayed effects of tumor growth by CIITA-Exo. These findings indicate that CIITA-Exo are more efficient as compared to parental Exo to induce anti-tumor immune responses, suggesting a potential role of MHC class II-containing tumor exosomes as an efficient cancer vaccine.

Plasma Thioredoxin Level and Its Correlation to Myocardial Damage in Patients with Acute Myocardial Infarction who Underwent Successful Primary Angioplasty

BACKGROUND AND OBJECTIVES: Oxidative stress is thought to play important role in cardiovascular disease. Thioredoxin is an important biomarker for determining the degree of oxidative stress. However, the relationship between the plasma thioredoxin levels and myocardial damage has not been investigated. SUBJECTS AND METHODS: We measured the plasma thioredoxin levels in the patients suffering with acute myocardial infarction and who also underwent successful primary angioplasty. We then compared the plasma thioredoxin levels and the clinical parameters in acute myocardial infarction patients (n=37) in order to examine the relationship between oxidative stress and myocardial damage. RESULTS: The plasma thioredoxin level was significantly related with the initial WBC count (r=0.349, p<0.05) and the myocardial damage, the peak CK level (r=0.489, p<0.01), the CK increment (r=0.452, p<0.05), the peak MB level (r=0.417, p<0.05), and the MB increment (r=0.364, p<0.05). We divided the patients into two groups according to the plasma thioredoxin levels. There was a significant difference in myocardial damage between the low and high plasma thoiredoxin levels at the initial WBC count (10174.2+/-3380.4/uL vs 13500+/-3740.7/uL, respectively; p<0.01) and the cardiac enzyme, the peak CK level (2565.2+/-1389.9 IU/L vs 4045.9+/-1978.9 IU/L, respectively; p=0.02), the CK increment (2309.6+/-1351.8 IU/L vs 3762.8+/-2079.7 IU/L, respectively; p=0.03), the peak MB level (208.7+/-127.5 IU/L vs 322.7+/-146.3 IU/L, respectively; p=0.02), and the MB increment (173.8+/-128.4 IU/L vs 277.7+/-158.9 IU/L, respectively; p=0.05). CONCLUSION: High thioredoxin levels were associated with the degree of oxidative stress and the extent of myocardial damage. Thioredoxin levels may be used as a new surrogate biomarker for the severity of oxidative stress and the extent of myocardial damage in the patients suffering with acute myocardial infarction.

Perivascular Delivery of Paclitaxel with F-127 Pluronic Gel Inhibits Neointimal Hyperplasia in a Rat Carotid Artery Injury Model

BACKGROUND AND OBJECTIVES: The local delivery of drugs to the arterial wall represents a strategy for the treatment of fibroproliferative vascular disease. Paclitaxel has been shown to inhibit vascular smooth muscle cell proliferation and migration, which contribute to neointimal formation. This study tested whether the perivascular delivery of paclitaxel can prevent neointimal formation in a rat carotid artery injury model. MATERIALS AND METHODS: The ability of locally-administered paclitaxel to prevent the neointimal hyperplastic response was tested by incorporating 10 microgram paclitaxel into 40% F-127 pluronic gel, which was then applied to the adventitial surface of the rat carotid artery immediately following balloon injury. Fourteen days after angioplasty, the neointimal growth was compared between paclitaxel- (n=12) and pluronic gel only treated (control group, n=11) rats. RESULTS: The paclitaxel-treated group showed significant neointimal formation reductions compared to the control group (0.10+/-0.05 versus 0.21+/-0.05 mm2, p<0.05). The perivascular application of paclitaxel produced a highly localized pattern of neointimal growth inhibition in the arterial cross-section. Although 10 microgram paclitaxel showed no significant cytotoxicity, 20 microgram paclitaxel (n=3) demonstrated cytotoxicity, with medial cell drop out in the region of application. CONCLUSION: We have demonstrated that the local extravascular application of 40% F-127 pluronic gel containing paclitaxel provides an effective mechanism for inhibiting the proliferative response to vascular injury in the rat. The cellular response to paclitaxel is highly focal. Locally sustained delivery of paclitaxel, as little as 10 microgram, was effective in preventing neointimal growth, without destroying medial wall smooth muscle cells.