NTUB1 Cells Research Articles

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/EBPδ, NF-IL6β) 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.

Synthesis and biological evaluation of 2′,5′-dimethoxychalcone derivatives as microtubule-targeted anticancer agents

A series of novel 2′,5′-dimethoxylchalcone derivatives including 18 new compounds were synthesized and evaluated for cytotoxicities against two human cancer cell lines, NTUB1 (human bladder cancer cell line) and PC3 (human prostate cancer cell line). All these derivatives except for 21 exhibited significant cytotoxic effect against NTUB1 and PC3 cell lines. Compounds 13 and 17 with 4-carbamoyl moiety showed potent inhibitory effect on growth of NTUB1 and PC3 cells. Flow cytometric analysis demonstrated that treatment of NTUB1 cells with 1 μM 13 and 17 induced G1 phase arrest accompanied by an increase in apoptotic cell death of NTUB1 cells after 24 h. Treatment of PC3 cells with 1 μM and 3 μM 13, and 1 μM and 3 μM 17 induced S and G1, and G1 and G2/M phase arrests, respectively, accompanied by an increase in apoptotic cell death. These data suggested that 13 and 17 with different 4-carbamoyl moiety displayed same cell cycle arrest in NTUB1 cells while different doses of 13 and 17 revealed different cell cycle arrest in PC3 cells. Cell morphological study of 17 indicated that more cells rounding up or dead associated with tubulin polymerization. Compound 17 showed an increased α-tubulin level in polymerized microtubule fraction in a dose-dependent manner while 500 nM paclitaxel also showed similar effect in NTUB1 cells by Western blot analysis. The result suggested that 17 may be used as microtubule-targeted agents.

Abstract A97: Combination of a bifunctional alkylating agent and arsenic trioxide is effectively against the drug resistant tumor cells

Abstract Inherited or acquired resistance to therapeutic agents is a crucial factor causing the failure of cancer chemotherapy. In this study, we explored the combination effect of a synthetic bifunctional alkylating agent, BO-1012, and arsenic trioxide (ATO) on killing H460 cells, derived from human large cell lung carcinoma and more resistant to BO-1012 and ATO as compared to several other cancer cell lines used, such as H1299, PC3, U87, MCF7, and OECM1 cells. We observed a synergistic enhancement of cell killing effects by treatment of H460 cells with BO-1012 for 1 h and followed with ATO for 72 h as compared to individual agents. By aid of modified comet assay, the repair of BO-1012-induced DNA interstrand crosslinks was significantly inhibited by ATO. Consequently, the protein levels as well as the nuclei foci of H2AX, a marker for DNA double strand breaks, was remarkably increased in H460 cells treated with in combination of BO-1012 and ATO. Accompanied with increased DNA damage, combined treatment resulted in severe G2/M accumulation as well as apoptosis. We further demonstrated that combination treatment with BO-1012 and ATO synergistically reduced tumor volumes of H460 cells subcutaneously inoculated into nude mice, indicating the effectiveness of combined treatment with BO-1012 and ATO against the inherited resistant H460 cells in xenograft mice model. Similar findings were demonstrated by using NTUB1/P cells, cisplatin resistant human bladder carcinoma cells derived from NTUB1 cells. These present results suggest that combination of bifunctional alkylating agents and ATO may be a rationale strategy for treatment of inherited or acquired drug-resistant cancers. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A97.

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 μM) and 23 (20 and 50 μM) for 24 h significantly increased the production of reactive oxygen species (ROS) while exposure of NTUB1 to 5 (20 and 40 μM) and 23 (20 and 50 μM) 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.

Comet assay with nuclear extract incubation

Alkaline comet assay is a simple sensitive method for detecting DNA strand breaks. However, at the time of cell lysis, only a fraction of the entire DNA damage appears as DNA strand breaks, while some DNA strand breaks may have been rejoined and some DNA lesions may still remain unexcised. We showed that nuclear extract (NE) prepared from human cells could excise the DNA adducts induced by UVC, X-ray, and methyl methanesulfonate (MMS). Thus, the comet assay with NE incubation allows a closer estimation of total DNA damage. Among the human urothelial carcinoma cell lines we tested, the NE of NTUB1 cells showed higher activity in excising the DNA adducts induced by UVC, but with a lower activity in excising the DNA adducts induced by MMS than the NE of BFTC905 cells. Moreover, under the same dose of X-ray irradiation, a larger difference in total DNA damage between two cell lines was revealed in comet assay incubated with NE than without NE. Therefore, the comet assay with NE incubation may be useful in the research of cancer risk, drug resistance, and DNA repair proteins.

Megestrol acetate antagonizes cisplatin cytotoxicity.

Megestrol acetate (MGA) is being widely used for the improvement of appetite and performance status in patients receiving chemotherapy, especially cisplatin-containing therapy. However, little is known about whether MGA has an effect on cisplatin cytotoxicity. We have investigated this using two transitional carcinoma cell lines, i.e. the cisplatin-sensitive parental line NTUB1 and the resistant daughter line NTUB1/P. Combined effects of MGA and cisplatin were assayed with a microculture chemosensitivity method. We explored the level changes of several cisplatin detoxification mechanisms, including metallothionein (MT), glutathione S-transferase-pi (GST-pi) and glutathione (GSH) levels in cells treated with or without MGA. After treatment with 10 microns MGA for 24 h, the cisplatin IC50s of NTUB1 and NTUB1/P increased 1.4- (p = 0.03) and 1.6- (p = 0.02) fold, respectively. By median effect analysis, the combinations of MGA and cisplatin in the two cells appeared to produce an antagonistic interaction. By Northern analysis, MT transcript levels in both cells were significantly upregulated after treatment with MGA, as compared to those without treatment. Exposure to MGA in either sensitive or resistant cells did not alter GST-pi levels as shown by immunoblotting analysis. Cellular GSH content was increased only in NTUB1/P (p = 0.0036) but remained unchanged in NTUB1 cells (p = 0.29) after MGA exposure. In conclusion, MGA may antagonize cisplatin cytotoxicity by upregulating cellular MT and GSH levels. Use of MGA in cisplatin-containing chemotherapy may impair tumor response by antagonizing cisplatin antitumor activity.