Eun Kyoung Yang

Resveratrol derivatives potently induce apoptosis in human promyelocytic leukemia cells.

Resveratrol has been shown to possess antioxidant and anticancer activities, but little is known on the effect of resveratrol derivatives. Recently we have isolated resveratrol and its dimers and trimers from peony (Paeonia lactiflora) seeds, and reported their strong antioxidant and cytotoxic activity. In the present study, we have evaluated cellular effects of resveratrol derivatives; viniferin, gnetin H, and suffruticosol B on the proliferation and apoptosis in HL-60 cells in vitro. All resveratrol and its derivatives reduced viability of HL-60 cells in a dose-dependent manner with their IC50 values of 20-90 µM. Ascending orders of IC50 values were suffruticosol B, gnetin H, viniferin and resveratrol respectively. HL-60 cells treated with the four stilbenes exhibited the distinct morphological changes characteristics of cell apoptosis such as chromatin condensation, apoptotic bodies, and DNA fragmentations. A time-dependent histogram of the cellular DNA analyzed by flow cytometry revealed a rapid increase in subdiploid cells and a concomitant decrease in diploid cells exposed to 100 µM resveratrol for 0-24 h. Cells treated with 25 µM of resveratrol, viniferin, gnetin H, and suffruticosol B for 24 h resulted in increment of sub-G1 population by 51, 5, 11 and 59%, respectively. Treatment of cells with 0-20 µM resveratrol for 5 h produced a concentration-dependent decrease in cytochrome P450 (CYP) 1B1 mRNA levels. Suffruticosol B also suppressed CYP1B1 gene expression. These results demonstrated that resveratrol oligomers also strongly suppressed HL-60 cell proliferation, and induced DNA damage. In addition, CYP1B1 gene supression may suggest an involvement in the resveratrol-induced apoptosis in HL-60 cells.

Genetic association of angiogenesis- and hypoxia-related gene polymorphisms with osteonecrosis of the femoral head

Multiple factors have been implicated in the development of osteonecrosis of the femoral head (ONFH). In particular, non-traumatic ONFH is directly or indirectly related to injury of the vascular supply to the femoral head. Thus, hypoxia in the femoral head caused by impaired blood flow may be an important risk factor for ONFH. In this study, we investigated whether genetic variations of angiogenesis- and hypoxia-related genes contribute to an increased risk for the development of ONFH. Candidate genes were selected based on known hypoxia and angiogenesis pathways. An association study was performed using an Affymetrix Targeted Genotyping 3K Chip array with 460 ONFH patients and 300 control subjects. We showed that single nucleotide polymorphisms (SNPs) in the genes TF, VEGFC, IGFBP3, and ACE were associated with an increased risk of ONFH. On the other hand, SNPs in the KDR and NRP1 genes were associated with protection against ONFH. The most important finding was that one SNP (rs2453839) in the IGFBP3 gene was significantly associated with a higher risk of ONFH (P = 0.0061, OR 7.74). In subgroup analysis, most candidate gene variations that were associated with ONFH occurred in the idiopathic subgroup. Among other SNPs, ACE SNPs were associated with steroid-induced ONFH (P = 0.0018-0.0037, OR > 3). Collectively, our findings suggest that genetic variations in angiogenesis- and hypoxia-related genes may help to identify susceptibility factors for the development of ONFH in the Korean population.

Generation of anti-tumour immune response using dendritic cells pulsed with carbonic anhydrase IX-Acinetobacter baumannii outer membrane protein A fusion proteins against renal cell carcinoma

Carbonic anhydrase IX (CA9), a specific molecular marker for renal cell carcinoma (RCC), serves as a potential target for RCC‐specific immunotherapy using dendritic cells (DCs). However, pulsing of DCs with CA9 alone is not sufficient for generation of a therapeutic anti‐tumour immune response against RCC. In this study, in order to generate a potent anti‐tumour immune response against RCC, we produced recombinant CA9‐Acinetobacter baumannii outer membrane protein A (AbOmpA) fusion proteins, designated CA9‐AbOmpA, and investigated the ability of DCs pulsed with CA9‐AbOmpA fusion proteins in a murine renal cell carcinoma (RENCA) model. A recombinant CA9‐AbOmpA fusion protein was composed of a unique proteoglycan‐related region of CA9 (1–120 amino acids) fused at the C‐terminus with transmembrane domain of AbOmpA (1–200 amino acids). This fusion protein was capable of inducing DC maturation and interleukin (IL)‐12 production in DCs. Interaction of DCs pulsed with CA9‐AbOmpA fusion proteins with naive T cells stimulated secretion of IL‐2, interferon (IFN)‐γ and tumour necrosis factor (TNF)‐α in T cells. Lymphocytes harvested from mice immunized with DCs pulsed with CA9‐AbOmpA fusion proteins secreted IFN‐γ and showed a specific cytotoxic activity against CA9‐expressing RENCA (RENCA‐CA9) cells. Administration of CA9‐AbOmpA‐pulsed DC vaccine suppressed growth of RENCA‐CA9 cells in mice with an established tumour burden. These results suggest that DCs pulsed with CA9‐AbOmpA fusion proteins generate a specific anti‐tumour immune response against RCC, which can be utilized in immunotherapy of RCC.

Histone deacetylase inhibitor, CG200745, attenuates cardiac hypertrophy and fibrosis in DOCA-induced hypertensive rats

CG200745 is a novel inhibitor of histone deacetylases (HDACs), initially developed for treatment of various hematological and solid cancers. Because it is water-soluble, it can be administered orally. We hypothesized that the HDAC inhibitor, CG200745, attenuates cardiac hypertrophy and fibrosis in deoxycorticosterone acetate (DOCA)-induced hypertensive rats. For establishment of hypertension, 40 mg/kg of DOCA was subcutaneously injected four times weekly into Sprague-Dawley rats. All the rats used in this study including those in the sham group had been unilaterally nephrectomized and allowed free access to drinking water containing 1% NaCl. Systolic blood pressure was measured by the tail-cuff method. Blood chemistry including sodium, potassium, glucose, triglyceride, and cholesterol levels was analyzed. Sections of the heart were visualized after trichrome and hematoxylin and eosin stain. The expression of hypertrophic genes such as atrial natriuretic peptide A (Nppa) and atrial natriuretic peptide B (Nppb) in addition to fibrotic genes such as Collagen-1, Collagen-3, connective tissue growth factor (Ctgf), and Fibronectin were measured by quantitative real-time PCR (qRT-PCR). Injection of DOCA increased systolic blood pressure, heart weight, and cardiac fibrosis, which was attenuated by CG200745. Neither DOCA nor CG200745 affected body weight, vascular contraction and relaxation responses, and blood chemistry. Injection of DOCA increased expression of both hypertrophic and fibrotic genes, which was abrogated by CG200745. These results indicate that CG200745 attenuates cardiac hypertrophy and fibrosis in DOCA-induced hypertensive rats.

17Beta-Estradiol Inhibits Calcium-Activated Potassium Channel Expressions in Rat Whole Bladder

Purpose: To investigate the effect of estrogen on the expression of calcium-activated potassium (KCa) channels in an overactive bladder rat model. To this end, mRNA and protein levels of KCa channel subtypes in the bladder of ovariectomized rats were measured by reverse transcription polymerase chain reaction and western blotting, respectively. Methods: Ten-week-old female Sprague-Dawley rats were divided randomly into 3 groups: sham-operated control group (n=11), ovariectomy group (n=11), and the group treated with estrogen after ovariectomy (n=12). Rats in the last group were subcutaneously injected with 17β-estradiol (50 μg/kg) every other day for 2 weeks, whereas rats in the other 2 groups received vehicle (soybean oil) alone. Two weeks after treatment, the whole bladder was excised for mRNA and protein measurements. Results: Protein levels of the large-conductance KCa (BK) channels in the ovariectomy group were 1.5 folds higher than those in the sham-operated control group. However, the protein levels of the other KCa channel subtypes did not change significantly upon bilateral ovariectomy. Treatment with 17β-estradiol after ovariectomy restored BK channel protein levels to the control value. In contrast, BK channel mRNA levels were not significantly affected by either ovariectomy alone or 17β-estradiol treatment. The small-conductance KCa type 3 channel (SK3) mRNA and protein levels decreased to 75% of control levels upon 17β-estradiol treatment. Conclusions: These results suggest that 17β-estradiol may influence urinary bladder function by modulating BK and SK3 channel expression.

Immunostimulatory Activity of Dendritic cells pulsed with carbonic anhydrase IX and Acinetobacter baumannii outer membrane protein A

Dendritic cell (DC)-based immunotherapy is a potent therapeutic modality for treating renal cell carcinoma (RCC), but development of antigens specific for tumor-targeting and anti-tumor immunity is of great interest for clinical trials. The present study investigated the ability of DCs pulsed with a combination of carbonic anhydrase IX (CA9) as an RCC-specific biomarker and Acinetobacter baumannii outer membrane protein A (AbOmpA) as an immunoadjuvant to induce anti-tumor immunity against murine renal cell carcinoma (RENCA) in a murine model. Murine bone-marrow-derived DCs pulsed with a combination of RENCA lysates and AbOmpA were tested for their capacity to induce DC maturation and T cell responses in vitro. A combination of RENCA lysates and AbOmpA up-regulated the surface expression of co-stimulatory molecules, CD80 and CD86, and the antigen presenting molecules, major histocompatibility (MHC) class I and class II, in DCs. A combination of RENCA lysates and AbOmpA also induced interleukin-12 (IL-12) production in DCs. Next, the immunostimulatory activity of DCs pulsed with a combination of CA9 and AbOmpA was determined. A combination of CA9 and AbOmpA up-regulated the surface expression of co-stimulatory molecules and antigen presenting molecules in DCs. DCs pulsed with a combination of CA9 and AbOmpA effectively secreted IL-12 but not IL-10. These cells interacted with T cells and formed clusters. DCs pulsed with CA9 and AbOmpA elicited the secretion of interferon-γ and IL-2 in T cells. In conclusion, a combination of CA9 and AbOmpA enhanced the immunostimulatory activity of DCs, which may effectively induce anti-tumor immunity against human RCC.