Nam Hee Won

Renal cell carcinoma does not express argininosuccinate synthetase and is highly sensitive to arginine deprivation via arginine deiminase.

Recently, pegylated arginine deiminase (ADI; EC 3.5.3.6) has been used to treat the patients with hepatocellular carcinoma or melanoma, in which the level of argininosuccinate synthetase (ASS) activity is low or undetectable. The efficacy of its antitumor activity largely depends on the level of intracellular ASS, which enables tumor cells to recycle citrulline to arginine. Thus, we examined the expression levels of ASS in various cancer cells and found that it is low in renal cell carcinoma (RCC) cells, rendering the cells highly sensitive to arginine deprivation by ADI treatment. Immunohistochemical analysis revealed that in biopsy specimens from RCC patients (n = 98), the expression of ASS is highly demonstrated in the epithelium of normal proximal tubule but not seen in tumor cells. Furthermore, RCC cells treated with ADI showed remarkable growth retardation in a dose dependent manner. ADI also exerted in vivo antiproliferative effect on the allografted renal cell carcinoma (RENCA) tumor cells and prolonged the survival of tumor‐bearing mice. Histological examination of the tumors revealed that tumor angiogenesis and vascular endothelial growth factor (VEGF) expression were significantly diminished by ADI administration. Therefore, these findings suggest that arginine deprivation by ADI could provide a beneficial strategy for the treatment of RCC in ways of inhibitions of arginine availability and neovascularization.

Inflammatory cytokines and lipopolysaccharide induce fas-mediated apoptosis in renal tubular cells

Background/Aims: Increased susceptibility of the kidney to acute renal failure (ARF) in the setting of sepsis even in the absence of systemic hypotension is well known. In the hypothesis that the proinflammatory cytokines and lipopolysaccharide (LPS) in gram-negative sepsis can directly cause renal tubular cell apoptosis via Fas- and caspase-mediated pathways, we examined apoptosis and Fas, Fas ligand, FADD expression, as well as PARP cleavage in cultured human proximal tubular cells under the cytokine and LPS-stimulated conditions. Methods: HK-2 cell, immortalized human proximal tubular cell lines, were treated with 5 and 30 ng/ml of tumor necrosis factor-α (TNF-α), 5 and 20 ng/ml of interleukin-1β (IL-1β) and 30 ng/ml LPS for 24 h. Fas expression was examined by RT-PCR and Fas ligand, Fas-associated protein with death domain (FADD) and poly ADP ribose polymerase (PARP) cleavage were examined by Western blot analysis. Apoptosis was assessed by flow cytometer using Annexin V-FITC and propidium iodide (PI) staining and also by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) methods. Results: Fas mRNA expression (ratio of Fas/L-19) increased in the TNF-α 5, 30 ng/ml and LPS treated group (p < 0.01, p < 0.01, p = 0.02), but there was no difference between the low- and high-dose TNF-α groups. Fas ligand protein expression did not increase in the low-dose TNF-α treated group, but it increased significantly in the high-dose TNF-α treated group (p< 0.01), IL-1β- and LPS-treated groups (p < 0.01, p = 0.01, p < 0.01, p = 0.02). The intracellular adaptor protein, FADD expression also increased significantly in the high-dose TNF-α- and IL-β-treated groups (p = 0.04, p = 0.04), but in the low-dose TNF-α and IL-β treated group, it did not show statistically significant differences. In the LPS group, FADD expression also showed an increased tendency, but it was not statistically significant (p = 0.09). Western blot for PARP, a DNA repair enzyme mainly cleaved by caspase 3, showed increased 89- and 24-kD PARP cleavage products in TNF-α, IL-1β and LPS treated cells. The degree of apoptosis examined by DNA fragmentation and translocation of membrane phosphatidyl serine significantly increased in cytokines and LPS treated groups. Conclusion: These results suggest that Fas- and caspase-mediated apoptosis of tubular cells by inflammatory cytokines and LPS can be one of the possible mechanisms of renal dysfunction in endotoxemia.

Prostaglandin E2 augments IL-10 signaling and function.

In inflamed joints of rheumatoid arthritis, PGE2 is highly expressed, and IL-10 and IL-6 are also abundant. PGE2 is a well-known activator of the cAMP signaling pathway, and there is functional cross-talk between cAMP signaling and the Jak-STAT signaling pathway. In this study, we evaluated the modulating effect of PGE2 on STAT signaling and its biological function induced by IL-10 and IL-6, and elucidated its mechanism in THP-1 cells. STAT phosphorylation was determined by Western blot, and gene expression was analyzed using real-time PCR. Pretreatment with PGE2 significantly augmented IL-10-induced STAT3 and STAT1 phosphorylation, as well as suppressors of cytokine signaling 3 (SOCS3) and IL-1R antagonist gene expression. In contrast, PGE2 suppressed IL-6-induced phosphorylation of STAT3 and STAT1. These PGE2-induced modulating effects were largely reversed by actinomycin D. Pretreatment with dibutyryl cAMP augmented IL-10-induced, but did not change IL-6-induced STAT3 phosphorylation. Misoprostol, an EP2/3/4 agonist, and butaprost, an EP2 agonist, augmented IL-10-induced STAT3 phosphorylation and SOCS3 gene expression, but sulprostone, an EP1/3 agonist, had no effect. H89, a protein kinase A inhibitor, and LY294002, a PI3K inhibitor, diminished PGE2-mediated augmentation of IL-10-induced STAT3 phosphorylation. In this study, we found that PGE2 selectively regulates cytokine signaling via increased intracellular cAMP levels and de novo gene expression, and these modulating effects may be mediated through EP2 or EP4 receptors. PGE2 may modulate immune responses by alteration of cytokine signaling in THP-1 cells.

Cancer cells induce metastasis-supporting neutrophil extracellular DNA traps

Treatment with DNase I–coated nanoparticles prevents metastasis by targeting neutrophil extracellular traps induced by cancer cells in a mouse model. Metastasis caught in a NET Neutrophil extracellular traps, or NETs, are DNA structures that are produced by neutrophils in response to infection and can promote the spread of cancer in the presence of infection. Park et al. discovered that even in the absence of infection, metastatic breast cancer cells can stimulate neutrophils to form NETs, which further support the spread of metastasis. The authors also demonstrated an approach to breaking this vicious cycle using nanoparticles coated with DNase I, an enzyme that breaks down DNA NETs. This treatment was effective in reducing lung metastases in mice, demonstrating the potential of NETs as a therapeutic target. Neutrophils, the most abundant type of leukocytes in blood, can form neutrophil extracellular traps (NETs). These are pathogen-trapping structures generated by expulsion of the neutrophil’s DNA with associated proteolytic enzymes. NETs produced by infection can promote cancer metastasis. We show that metastatic breast cancer cells can induce neutrophils to form metastasis-supporting NETs in the absence of infection. Using intravital imaging, we observed NET-like structures around metastatic 4T1 cancer cells that had reached the lungs of mice. We also found NETs in clinical samples of triple-negative human breast cancer. The formation of NETs stimulated the invasion and migration of breast cancer cells in vitro. Inhibiting NET formation or digesting NETs with deoxyribonuclease I (DNase I) blocked these processes. Treatment with NET-digesting, DNase I–coated nanoparticles markedly reduced lung metastases in mice. Our data suggest that induction of NETs by cancer cells is a previously unidentified metastasis-promoting tumor-host interaction and a potential therapeutic target.

Reduction of Renal Fibrosis as a Result of Liposome Encapsulated Clodronate Induced Macrophage Depletion after Unilateral Ureteral Obstruction in Rats

Background/Aim: Macrophages have been thought to play a role in renal tubulointerstitial fibrosis; recent reports have demonstrated an antifibrotic effect of macrophages in late-stage renal fibrosis. Liposome-encapsulated clodronate (LC) produces a selective and systemic depletion of phagocytic macrophages in vivo. To study the role of initial infiltrating macrophages in renal fibrosis, we compared the effects of pretreatment with LC and a liposome vehicle for control of the severity of renal fibrosis in a unilateral ureteral obstruction (UUO) rat model. Methods: One day after a single intravenous injection of LC or liposome vehicle, the rats underwent UUO. Following 1, 5, and 14 days, the kidneys were examined to evaluate macrophage infiltration and renal fibrosis. Results: LC depleted macrophages systemically and reduced renal fibrosis associated with UUO; this beneficial effect was accompanied by a decrease of transforming growth factor beta mRNA expression. The osteopontin expression was also reduced by pretreatment with LC. Conclusion: Initial interstitial infiltration of macrophages contributes to tubulointerstitial fibrosis in UUO.

BRAF mutation and AKAP9 expression in sporadic papillary thyroid carcinomas

Aim: We aimed to determine the BRAF mutation and AKAP9 expression in papillary thyroid carcinomas (PTCs). Methods and Results: In this study, we analysed 100 sporadic PTC specimens and we detected mutation in 62.2% of the conventional type PTCs (51/82), in 50% of the follicular variant type PTCs (3/6), in 50% of the diffuse sclerosing variant type PTCs (1/2), and in 30% of the microcarcinomas (3/10). All mutations involved a T→A transversion at the nucleotide 1796. The cases with BRAF mutation were significantly associated with extrathyroidal extension. We also evaluated the expression of AKAP9 protein by immunohistochemistry. The AKAP9 protein was seen as a single perinuclear dot in all the PTCs. Therefore, 58% of the specimens harboured the BRAF mutation and no case had AKAP9‐BRAF fusion in the sporadic PTCs. Conclusion: Our results suggest that the BRAF mutation can be a useful diagnostic and prognostic marker and a target for exploring novel cancer therapies to treat PTCs. AKAP9‐BRAF fusion may be a very rare event in sporadic PTCs.

Signaling pathway for 2,3,7,8-tetrachlorodibenzo- p-dioxin-induced TNF-alpha production in differentiated THP-1 human macrophages.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a prototypic halogenated aromatic hydrocarbon (HAH), is known as one of the most potent toxicants. At least a part of its toxic effects appears to be derived from its ability to induce TNF-α production. However, the signaling pathway of TCDD that leads to TNF-α expression has not been elucidated. In this study, we investigated the signaling mechanism of TCDD-induced TNF-α expression in PMA-differentiated THP-1 macrophages. TCDD induced both mRNA and protein expression of TNF-α in a dose- and time-dependent manner. Alpha-Naphthoflavone (NF), an aryl hydrocarbon receptor (AhR) inhibitor, prevented the TCDD-induced expression of TNF-α at both mRNA and protein levels. Genistein, a protein tyrosine kinase (PTK) inhibitor, and PD153035, an EGFR inhibitor, also blocked the increase of TNF-α expression by TCDD, indicating the role of EGFR in TCDD-induced TNF-α expression. On the other hand, PP2, a c-Src specific inhibitor, did not affect TCDD-induced TNF-α expression. EGFR phosphorylation was detected as early as 5 min after TCDD treatment. TCDD-induced EGFR activation was AhR-dependent since co-treatment with α-NF prevented it. ERK was found to be a downstream effector of EGFR activation in the signaling pathway leading to TNF-α production after TCDD stimulation. Activation of ERK was observed from 30 min after TCDD treatment. PD98059, an inhibitor of the MEK-ERK pathway, completely prevented the TNF-α mRNA and protein expression induced by TCDD, whereas inhibitors of JNK and p38 MAPK had no effect. PD153035, an EGFR inhibitor, as well as α-NF significantly reduced ERK phosphorylation, suggesting that ERK activation by TCDD was mediated by both EGFR and AhR. These results indicate that TNF-α production by TCDD in differentiated THP-1 macrophages is AhR-dependent and involves activation of EGFR and ERK, but not c-Src, JNK, nor p38 MAPK. A signaling pathway is proposed where TCDD induces sequential activation of AhR, EGFR and ERK, leading to the increased expression of TNF-α.

FINE NEEDLE ASPIRATION CYTOLOGY OF THE BREAST. EXPERIENCE AT AN OUTPATIENT BREAST CLINIC

OBJECTIVE To evaluate the accuracy of fine needle aspiration cytology (FNAC) of the breast at our institution and to perform quality assurance. STUDY DESIGN Two hundred forty-six cases with pathologic confirmation were selected and reviewed. A pathologist performed most of the aspirations at an outpatient breast clinic. We correlated cytologic and histologic findings and evaluated the influence of the size, location, grade, and pathologic subtypes and fibrosis in breast lesions on diagnostic results. RESULTS The likelihood ratios for malignant, suspicious, atypical, benign and unsatisfactory cytologic diagnoses were 98.71, 5.48, 1.09, 0.07 and 0.55, respectively. The absolute and complete sensitivities for malignant lesions were 64.5% and 90.3%, respectively. The specificity was 71.9%. False negative and positive rates were 4.3% and 0.7%, respectively. The predictive value for a malignant cytologic diagnosis was 98.4%. The rate of unsatisfactory samples was 9.3%. The rate of concordance between cytologic and histologic diagnosis was lower for large and diffusely growing lesions (benign and malignant), for malignancies with abundant fibrosis and of unusual types and for carcinomas of low grade. All axillary and recurrent chest wall lesions were diagnosed cytologically. Cell block sections were useful in a small number of cases. CONCLUSION Understanding the performance and limitations of FNAC can enhance its value as a diagnostic technique in the management of breast disease.

The effect of α-melanocyte-stimulating hormone on renal tubular cell apoptosis and tubulointerstitial fibrosis in cyclosporine A nephrotoxicity

Background. The pathogenesis of cyclosporine A (CsA)-induced nephrotoxicity has been known to be secondary to hemodynamic changes, but increasing evidence indicates that CsA has a direct toxicity to renal tubular cells, leading to their apoptosis and tubulointerstitial fibrosis. This study evaluated the mechanism for CsA-induced tubular cell apoptosis, tubulointerstitial fibrosis and its associated proteins, and the therapeutic effects of &agr;-melanocyte–stimulating hormone (MSH) on them. Methods. Male Sprague-Dawley rats fed with a low-sodium diet were divided into three treatment groups: group A (vehicle-injected group), group B (CsA 15 mg/kg-injected group), and group C(CsA+&agr;-MSH–injected group). After 42 days, creatinine clearance; blood CsA level; apoptosis; inflammation and tubulointerstitial fibrosis in renal tissue; and the expression of Bax, Bcl2, Fas, FasL, and transforming growth factor (TGF)-&bgr; protein were determined. Results. CsA-induced tubular cell apoptosis; cellular infiltration; and increase of Fas, Bax, TGF-&bgr; protein expression with significant tubulointerstitial fibrosis, and reduced Bcl2 protein expression. &agr;-MSH treatment prevented the Bax and TGF-&bgr; protein increase and induced Bcl2 protein increase, together with reduction of apoptosis, inflammation, and tubulointerstitial fibrosis. Conclusions. These findings suggest that chronic CsA nephrotoxicity is related to Bax and Bcl2-related apoptosis pathways, and that &agr;-MSH can attenuate the CsA-induced tubulointerstitial fibrosis as well as tubular cell apoptosis.

Fas (APO-1/CD95) ligand and Fas expression in renal cell carcinomas: correlation with the prognostic factors.

BACKGROUND AND OBJECTIVE Fas ligand (FasL, CD95L) is a type II transmembrane protein of the tumor necrosis factor family that induces cells to send an apoptotic signal to cells expressing Fas (CD95, APO-1). It has been shown that cancers have a dysregulated expression of Fas and FasL system, conferring a survival advantage. It is important to understand FasL and Fas expression in tumors, because the growth of cancer might be controlled by Fas-mediated apoptosis. METHODS The expressions of FasL and Fas were studied by immunohistochemical analyses in 51 cases of renal cell carcinomas and the adjacent normal renal tissues, respectively. In addition, their expressions were compared with prognostic factors, such as tumor size, nuclear grade, TNM stage, and histologic types. RESULTS In nonneoplastic renal tissues, FasL was expressed in all nephron segments, whereas Fas also expressed in all tubules, except for glomeruli. In renal cell carcinomas, FasL protein was detected in 50 (98.0%) of 51 cases, whereas Fas expressed in 38 (74.5%) of 51 cases. In fact, the immunostaining of Fas was less intense than that in the adjacent normal segments of all cases. The staining pattern showing both high expression of FasL and low expression of Fas was found in 36 (70.6%) (P = .04) of 51 cases, most of which were Fuhrman grade 2 or 3 tumors. However, the expression pattern did not correlate statistically with the tumor size, histologic type, or clinical stage. On the other hand, most grade 4 tumors displayed high expression of both FasL and Fas (P<.001). CONCLUSION These data indicate that high expression of FasL and low expression of Fas protein in renal cell carcinomas may play a role in evading surveillance of the immune system. In addition, the FasL and Fas expressions appear to have a therapeutic implication for high-grade tumors rather than a prognostic one.