Tzyh-Chyuan Hour

Resistance to Paclitaxel Is Proportional to Cellular Total Antioxidant Capacity

Paclitaxel, one of the most commonly prescribed chemotherapeutic agents, is active against a wide spectrum of human cancer. The mechanism of its cytotoxicity, however, remains controversial. Our results indicate that paclitaxel treatment increases levels of superoxide, hydrogen peroxide, nitric oxide (NO), oxidative DNA adducts, G2-M arrest, and cells with fragmented nuclei. Antioxidants pyruvate and selenium, the NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester, and the NO scavenger manganese (III) 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide all decreased paclitaxel-mediated DNA damage and sub-G1 cells. In contrast, the glutamylcysteine synthase inhibitor buthionine sulfoximine (BSO) and the superoxide dismutase (SOD) inhibitor 2-methoxyestradiol (2-ME) increased the sub-G1 fraction in paclitaxel-treated cells. These results suggest that reactive oxygen and nitrogen species are involved in paclitaxel cytotoxicity. This notion is further supported with the observation that concentrations of paclitaxel required to inhibit cell growth by 50% correlate with total antioxidant capacity. Moreover, agents such as arsenic trioxide (As2O3), BSO, 2-ME, PD98059, U0126 [mitogen-activated protein/extracellular signal-regulated kinase inhibitors], and LY294002 (phosphatidylinositol 3-kinase/Akt inhibitor), all of which decrease clonogenic survival, also decrease the total antioxidant capacity of paclitaxel-treated cells, regardless whether they are paclitaxel sensitive or paclitaxel resistant. These results suggest that paclitaxel chemosensitivity may be predicted by taking total antioxidant capacity measurements from clinical tumor samples. This, in turn, may then improve treatment outcomes by selecting out potentially responsive patients.

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.

Arsenic trioxide as a novel anticancer agent against human transitional carcinoma--characterizing its apoptotic pathway.

Arsenic trioxide (As2O3) has been shown to be an active agent against acute promyelocytic leukemia. Little is known about its therapeutic efficacy in human transitional carcinomas. In this study, the arsenic-mediated apoptotic pathway in transitional carcinoma cells was investigated. Three bladder transitional carcinoma cell lines were used, including a parental sensitive line and two resistant daughter lines (cisplatin and As2O3 resistant). The As2O3-mediated cytotoxicity to the three cell lines was studied in vitro in the presence or absence of buthionine sulfoximine (BSO), a chemotherapy modulator. In results, although a lesser extent of apoptosis was seen in cells treated with As2O3 alone, more significant apoptotic events were observed in the combined treatment of As2O3 and non-toxic concentrations of BSO (up to 10 μ M). These included the accumulation of sub-G1 fractions and internucleosomal DNA breakdown, which were preceded by production of reactive oxygen species, loss of mitochondrial membrane potential and activation of caspase-3. In conclusion, As2O3 in the presence of BSO may be an active agent against both chemonaive and cisplatin-resistant transitional carcinomas. The As2O3-mediated cytotoxicity appeared to go through the conventional apoptotic pathway. Our results have clinical implications and warrant further investigation.

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.

Curcumin induces apoptosis through an ornithine decarboxylase-dependent pathway in human promyelocytic leukemia HL-60 cells

Curcumin, a well-known dietary pigment derived from the food flavoring turmeric (Curcuma longa) exhibits anti-proliferative, anti-inflammatory, and anti-oxidative activities. Recently, studies have shown that a chemopreventive effect of curcumin could be due to the hyperproduction of reactive oxygen species (ROS) inducing apoptosis in tumor cells. In our previous studies, ornithine decarboxylase (ODC) overexpression prevented tumor necrosis factor alpha (TNF-alpha)- and methotrexate-induced apoptosis via reduction of ROS. Furthermore, ODC is the rate-limiting enzyme in polyamine biosynthesis and a target for chemoprevention. In this study, we found that enzyme activity and protein expression of ODC were reduced during curcumin treatment. Overexpression of ODC in human promyelocytic leukemia HL-60 parental cells could reduce curcumin-induced apoptosis, which leads to loss of mitochondrial membrane potential (Deltapsi(m)), through reducing intracellular ROS. Moreover, ODC overexpression prevented cytochrome c release and the activation of caspase-9 and caspase-3 following curcumin treatment. These results demonstrate that curcumin-induced apoptosis occurs through a mechanism of down-regulating ODC and along a ROS-dependent mitochondria-mediated pathway.

Genetic polymorphisms in oestrogen receptor-binding sites affect clinical outcomes in patients with prostate cancer receiving androgen-deprivation therapy

Abstract.  Huang C‐N, Huang S‐P, Pao J‐B, Hour T‐C, Chang T‐Y, Lan Y‐H, Lu T‐L, Lee H‐Z, Juang S‐H, Wu P‐P, Huang C‐Y, Hsieh C‐J, Bao B‐Y (Kaohsiung Medical University Hospital, Kaohsiung; Kaohsiung Medical University, Kaohsiung; Taipei City Hospital, Taipei; Kaohsiung Medical University, Kaohsiung; China Medical University, Taichung; National Taiwan University Hospital; Oriental Institute of Technology; National Taiwan University, Taipei; China Medical University Hospital, Taichung, Taiwan). Genetic polymorphisms in oestrogen receptor‐binding sites affect clinical outcomes in patients with prostate cancer receiving androgen‐deprivation therapy. J Intern Med 2012; 271: 499–509.

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.

Cytotoxicity of arsenic trioxide to transitional carcinoma cells

OBJECTIVES To explore the therapeutic efficacy of arsenic trioxide (As2O3) in human transitional cell carcinomas, we investigated the potential use of the compound as a chemotherapeutic agent and the possible cross-resistance with cisplatin in this malignancy. METHODS Three bladder transitional carcinoma cell lines, NTUB1, NTUB1/P (cisplatin-resistant), and NTUB1/As (As2O3-resistant), were used. The chemosensitivity of the three cell lines to cisplatin and As2O3 was determined by the microculture tetrazolium assay. The modulatory effect of buthionine sulfoximine (BSO) on As2O3 cytotoxicity was studied by combining the two agents simultaneously or sequentially and evaluated using the median-effect analysis. Cellular glutathione contents were determined using a biochemical method. RESULTS There was evident cross-resistance between cisplatin and As2O3 in the cell model used. BSO significantly enhanced As2O3 cytotoxicity in the three cell lines, indicating synergism in combination. In the presence of 3 microM BSO, the sensitivity of NTUB1, NTUB1/P, and NTUB1/As to As2O3 was increased 3, 7.4, and 8.4-fold, respectively. Among the three different combination schedules, greater cytotoxic effects were obtained by concurrent exposure to both agents. A significant dose-response relationship was found between the BSO concentrations and glutathione contents in NTUB1 (P = 0.007) and NTUB1/As (P = 0.05) but not NTUB1/P (P = 0.1) cells. CONCLUSIONS As2O3 in the presence of BSO may be an active agent against transitional cell carcinoma. Our results have clinical implications and warrant further investigation.

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.