A-Mei Huang

Loss of CCAAT/enhancer binding protein δ promotes chromosomal instability

The transcription factor CCAAT/enhancer binding protein δ (Cebpd, also known as C/EBPδ, CRP3, CELF, NF-IL6β) is implicated in diverse cellular functions such as the acute phase response, adipocyte differentiation, learning and memory, and mammary epithelial cell growth control. Here, we report that lack of Cebpd causes genomic instability and centrosome amplifications in primary embryonic fibroblasts derived from 129S1 mice. Upon spontaneous immortalization, Cebpd-deficient fibroblasts acquire transformed features such as impaired contact inhibition and reduced serum dependence. These data identify a novel role for Cebpd in the maintenance of chromosomal stability and suggest a potential tumor suppressor function in vivo.

Ursolic acid derivatives induce cell cycle arrest and apoptosis in NTUB1 cells associated with reactive oxygen species.

Twenty-three ursolic acid (1) derivatives 2-24 including nine new 1 derivatives 5, 7-11, 20-22 were synthesized and evaluated for cytotoxicities against NTUB1 cells (human bladder cancer cell line). Compounds 5 and 17 with an isopropyl ester moiety at C-17-COOH and a succinyl moiety at C-3-OH showed potent inhibitory effect on growth of NTUB1 cells. Compounds 23 and 24 with seco-structures prepared from 1 also showed the increase of the cytotoxicity against NTUB1 cells. Exposure of NTUB1 to 5 (40 microM) and 23 (20 and 50 microM) for 24h significantly increased the production of reactive oxygen species (ROS) while exposure of NTUB1 to 5 (20 and 40 microM) and 23 (20 and 50 microM) for 48 h also significantly increased the production of ROS while exposure of cells to 17 did not increase the amount of ROS. Flow cytometric analysis exhibited that treatment of NTUB1 with 5 or 17 or 23 led to the cell cycle arrest accompanied by an increase in apoptotic cell death after 24 or 48 h. These data suggest that the presentation of G1 phase arrest and apoptosis in 5- and 23-treated NTUB1 for 24 h mediated through increased amount of ROS in cells exposed with 5 and 23, respectively, while the presence of G2/M arrest before accumulation of cells in sub-G1 phase in 5-treated cells for 48 h also due to increased amount of ROS in cells exposed with 5. The inhibition of tubulin polymerization and cell cycle arrest at G2/M following by apoptosis presented in the cell cycle of 23 also mediates through the increase amount of ROS induced by treating NTUB1 with 23 for 48 h.

Loss of CCAATsenhancer binding protein δ promotes chromosomal instability

The transcription factor CCAAT/enhancer binding protein δ (Cebpd, also known as C/EBPδ, CRP3, CELF, NF-IL6β) is implicated in diverse cellular functions such as the acute phase response, adipocyte differentiation, learning and memory, and mammary epithelial cell growth control. Here, we report that lack of Cebpd causes genomic instability and centrosome amplifications in primary embryonic fibroblasts derived from 129S1 mice. Upon spontaneous immortalization, Cebpd-deficient fibroblasts acquire transformed features such as impaired contact inhibition and reduced serum dependence. These data identify a novel role for Cebpd in the maintenance of chromosomal stability and suggest a potential tumor suppressor function in vivo.

CCAAT/enhancer binding protein delta (CEBPD) elevating PTX3 expression inhibits macrophage-mediated phagocytosis of dying neuron cells

The CCAAT/enhancer binding protein delta (CEBPD, C/EBPδ, NF-IL6β) is induced in many inflammation-related diseases, suggesting that CEBPD and its downstream targets may play central roles in these conditions. Neuropathological studies show that a neuroinflammatory response parallels the early stages of Alzheimers disease (AD). However, the precise mechanistic correlation between inflammation and AD pathogenesis remains unclear. CEBPD is upregulated in the astrocytes of AD patients. Therefore, we asked if activation of astrocytic CEBPD could contribute to AD pathogenesis. In this report, a novel role of CEBPD in attenuating macrophage-mediated phagocytosis of damaged neuron cells was found. By global gene expression profiling, we identified the inflammatory marker pentraxin-3 (PTX3, TNFAIP5, TSG-14) as a CEBPD target in astrocytes. Furthermore, we demonstrate that PTX3 participates in the attenuation of macrophage-mediated phagocytosis of damaged neuron cells. This study provides the first demonstration of a role for astrocytic CEBPD and the CEBPD-regulated molecule PTX3 in the accumulation of damaged neurons, which is a hallmark of AD pathogenesis.

Transcriptional up-regulation of SOD1 by CEBPD: A potential target for cisplatin resistant human urothelial carcinoma cells

Bladder cancer is the fourth most common type of cancer in men (ninth in women) in the United States. Cisplatin is an effective agent against the most common subtype, urothelial carcinoma. However, the development of chemotherapy resistance is a severe clinical problem for the successful treatment of this and other cancers. A better understanding of the cellular and molecular events in response to cisplatin treatment and the development of resistance are critical to improve the therapeutic options for patients. Here, we report that expression of the CCAAT/enhancer binding protein delta (CEBPD, C/EBPdelta, NF-IL6beta) is induced by cisplatin in the human bladder urothelial carcinoma NTUB1 cell line and is specifically elevated in a cisplatin resistant subline. Expression of CEBPD reduced cisplatin-induced reactive oxygen species (ROS) and apoptosis in NTUB1 cells by inducing the expression of Cu/Zn-superoxide dismutase (SOD1) via direct promoter transactivation. Several reports have implicated CEBPD as a tumor suppressor gene. This study reveals a novel role for CEBPD in conferring drug resistance, suggesting that it can also be pro-oncogenic. Furthermore, our data suggest that SOD inhibitors, which are already used as anti-angiogenic agents, may be suitable for combinatorial chemotherapy to prevent or treat cisplatin resistance in bladder and possibly other cancers.

Ling-Zhi Polysaccharides Potentiate Cytotoxic Effects of Anticancer Drugs against Drug-Resistant Urothelial Carcinoma Cells

The combined effects of ling-zhi polysaccharide fraction 3 (LZP-F3) and anticancer drugs (cisplatin and arsenic trioxide) were examined in three human urothelial carcinoma (UC) cells (parental, NTUB1; cisplatin-resistant, N/P(14); and arsenic-resistant, N/As(0.5)). MTT assay and median-effect analysis revealed that LZP-F3 could profoundly reverse the chemosensitivity of N/P(14) and N/As(0.5) to cisplatin and arsenic, respectively, in a dose-dependent manner, which involved activation of p38 and down-regulation of Akt and XPA. A dose of 10 mug/mL of LZP-F3 induced significant G1 arrest in N/P(14) and N/As(0.5) cells by flow cytometry, which may be mediated by the induction of p21(WAF1/CIP1). The combination of LZP-F3 and arsenic trioxide produced a significant synergistic growth inhibition of NTUB1 and N/As(0.5) cells. Similar results were also found in N/P(14) cells. These molecular events of combined effects involved significant and earlier induction of Fas, caspase 3 and 8 activation, Bax and Bad up-regulation, Bcl-2 and Bcl-x(L) down-regulatuion, and cytochrome c release.

Hepatocyte growth factor stimulates the growth and activates mitogen-activated protein kinase in human hepatoma cells.

Hepatocyte growth factor (HGF) is a potent mitogen for hepatocytes and various epithelial cells. Unexpectedly, it has been reported to inhibit the growth of hepatoma cells in vitro. To clarify this phenomenon, we examined the effects of recombinant baculovirus-expressed HGF on the growth of 6 human hepatoma cell lines. The growth of Hep3B and HepG2 cells was markedly stimulated to 1.8- and 1.7-fold, respectively, PLC/PRF/5 to 1.4-fold, and SK-Hep-1 to 1.2-fold in a dose-dependent manner under HGF concentrations below 20 ng/ml. Neither HuH-7 nor HCC36 were affected. None of these cells were inhibited. All these cells expressed c-Met, the membrane receptor for HGF, and their c-Met would be activated to be phosphorylated upon addition of HGF. They also contained the ERK2 subgroup of mitogen-activated protein kinases (MAPKs). When HGF was added, their ERK2 would also be phosphorylated. The extent of ERK2 phosphorylation was partially correlated to their growth response to HGF. In conclusion, HGF could stimulate the growth of certain human hepatoma cells, probably through activation of c-Met and MAPKs.

Antioxidant xanthone derivatives induce cell cycle arrest and apoptosis and enhance cell death induced by cisplatin in NTUB1 cells associated with ROS

In an effort to develop novel antioxidant as anticancer agents, a series of xanthones were prepared. In vitro screening, the synthetic xanthones revealed significant inhibitory effects on xanthine oxidase and ABTS radical-cation scavenging activity. The selective compounds 2 and 8 induced an accumulation of NTUB1 cells in the G(1) phase arrest and cellular apoptosis by the increase of ROS level. The combination of cisplatin and 2 significantly enhanced the cell death in NTUB1 cells. Compounds 2 and 8 did not show cytotoxic activity in selected concentrations against SV-HUC1 cells. The present results suggested that antioxidants 2 and 8 may be used as anticancer agent for enhancing the therapeutic efficacy of anticancer agents and to reduce their side effect.

Characterization of membranous and cytoplasmic EGFR expression in human normal renal cortex and renal cell carcinoma

Metastatic renal cell carcinoma (RCC) is highly resistant to conventional systemic treatments, including chemotherapy, radiotherapy and hormonal therapies. Previous studies have shown over-expression of EGFR is associated with high grade tumors and a worse prognosis. Recent studies suggest anticancer therapies targeting the EGFR pathway have shown promising results in clinical trials of RCC patients. Therefore, characterization of the level and localization of EGFR expression in RCC is important for target-dependent therapy. In this study, we investigated the clinical significance of cellular localization of EGFR in human normal renal cortex and RCC. RCC and adjacent normal kidney tissues of 63 patients were obtained for characterization of EGFR expression. EGFR protein expression was assessed by immunohistochemistry on a scale from 0 to 300 (percentage of positive cells × staining intensity) and Western blotting. EGFR membranous staining was significantly stronger in RCC tumors than in normal tissues (P < 0.001). In contrast, EGFR cytoplasmic staining was significantly higher in normal than in tumor tissues (P < 0.001). The levels of membranous or cytoplasmic EGFR expression in RCC tissues were not correlated with sex, tumor grade, TNM stage or overall survival (P > 0.05). These results showed abundant expression of membranous EGFR in RCC, and abundant expression of cytoplasmic EGFR in normal tissues. EGFR expression in RCC was mostly located in the cell membrane, whereas the EGFR expression in normal renal tissues was chiefly seen in cytoplasm. Our results suggest different locations of EGFR expression may be associated with human renal tumorigenesis.

18β-Glycyrrhetinic acid derivatives induced mitochondrial-mediated apoptosis through reactive oxygen species-mediated p53 activation in NTUB1 cells

Twenty six 18β-glycyrrhetinic acid (GA) (1) derivatives 2-27 including twelve new GA derivatives 10, 11, 13-17, 21-25 were synthesized and evaluated for cytotoxicities against NTUB1 cells (human bladder cancer cell lines). seco-Compounds 9, 25, and 27 are the most potent compounds of this series, inhibiting cell growth of human NTUB1 cells with an IC(50) values of 2.34 ± 0.28, 4.76 ± 1.15, and 3.31 ± 0.61 μM, respectively. Exposure of NTUB1 to 25 for 24h significantly increased the production of reactive oxygen species (ROS). Flow cytometric analysis exhibited that treatment of NTUB1 with 25 did not induce cell cycle arrest but accompanied by an increase of apoptotic cell death in a dose-dependant manner after 24h. Mitochondrial membrane potential (MMP) decreased significantly in a dose-dependant manner when the NTUB1 cells were exposed to 25 for 24h. Marked collapse of the MMP suggested that dysfunction of the mitochondria may be involved in the oxidative burst and apoptosis induced by 25. Western blot analysis shows that NTUB1 cells treated with 25 increased the level of p-p53 in a dose-dependant manner. Further, NAC treatment prevented p53 phosphorylation stimulated by 25. These results suggested that 25 induced a mitochondrial-mediated apoptosis in NTUB1 cells through activation of p53, which are mainly mediated ROS generated by 25.