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MP21-09 PLECTIN-1 DEPLETION IN MUSCLE INVASIVE BLADDER CANCER CELL LINES PROMOTES PHOSPHORYLATION OF FER TYROSINE KINASE AND ENHANCES INVASION POTENTIAL
THE JOURNAL OF UROLOGYâ
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MP21-07 HIGH-DOSE CHEMOTHERAPEUTICS USED IN INTRAVESICAL THERAPY INDUCE MITOCHONDRIAL DYSFUNCTION FOLLOWED BY NECROSIS-LIKE CELL DEATH IN BLADDER CANCER CELLS Takahiro Yoshida*, Suita, Osaka, Japan; Hiroaki Okuyama, Masahi Nakayama, Kazuo Nishimura, Masahiro Inoue, Osaka, Osaka, Japan; Norio Nonomura, Suita, Osaka, Japan INTRODUCTION AND OBJECTIVES: Non-muscle invasive bladder cancer is often treated by intravesical chemotherapy following transurethral resection. Mechanism of the effect of intravesical chemotherapy has yet to be elucidated. In this study, we investigated cell death mechanism induced by high-dose chemotherapeutics using a newly-developed culture method for primary cancer cells. METHODS: We have developed a novel culture method which enables preparation and culture of primary cancer cells as multicellular spheroids which are termed cancer tissue-originated spheroids (CTOSs) (Okuyama 2013 J Urol). First we generated a bladder cancer patient-derived xenograft. We prepared CTOSs from the xenograft, and exposed to high-dose (1mg/ml) of epirubicin (e-ADM) or mitomycin C (MMC) for 2 hours, or low-dose (0.01mg/ml) of those chemotherapeutics. We investigated absorption of chemotherapeutics into CTOSs and assessed the mode of cell death. Next, we prepared CTOSs directly from surgical specimens of bladder cancer and tested the individual responses after exposure to high-dose e-ADM and MMC. RESULTS: At high dose, e-ADM was promptly and homogenously delivered into cancer cells in the CTOSs. High-dose e-ADM and MMC decreased ATP levels in CTOSs within an hour accompanied with mitochondrial swelling and reduction of mitochondrial membrane potential, while plasma membrane integrity was maintained. Some of the molecules in mitochondrial apoptotic pathway were subsequently activated, while at later time point the cells lost integrity of cell membrane without DNA laddering. In contrast, CTOSs exposed to low-dose of eADM or MMC exhibited typical DNA laddering. Intriguingly, the decrease of ATP levels was varied among CTOSs derived from 20 surgical specimens. CONCLUSIONS: High-dose chemotherapeutics used in intravesical therapy may induce necrosis-like cell death, different from typical apoptosis caused by chemotherapeutics at the dose of systemic chemotherapy. Each bladder cancer may have a different response to high-dose chemotherapeutics used in intravesical therapy, indicating necessity of chemosensitivity test aiming for personalized medicine.
Vol. 191, No. 4S, Supplement, Sunday, May 18, 2014
METHODS: YTS-1 is a BCa cell line established from muscle invasive BCa and highly positive for PDPN and C2GnT. KK-47 is a BCa cell line established from non-muscle invasive BCa and expresses a faint level of PDPN and C2GnT. YTS-1 cells were transfected with PDPN-siRNA or C2GnT-siRNA to silence of PDPN or C2GnT expression. KK47 cells were transfected with pcDNA3.1-PDPN and/or pcDNA3.1-C2GnT to make KK47-PDPN, KK47-C2GnT and KK47C2PDPN which are highly positive for PDPN, C2GnT or both of them. These cells were subjected to growth factor assays and invasion assay. Co-culture system of BCa cells and platelet was used to evaluate growth kinetics of the BCa cells and production of growth factors. In vitro invasion assay was used to evaluate invasion potential of the BCa cells. RESULTS: In the co-culture system of BCa cells and platelet, BCa cells culturing with platelet had higher growth activity than without platelet (p<0.05). YTS-1 produced significantly higher amounts of bFGF, EGF, NGF beta, PDGF-BB, TGF-beta and VEGF when they are co-cultured with platelet than they are cultured without platelet (p<0.05). O-glycosylation of PDPN on BCa cells significantly increased the production of growth factors (p<0.05). When PDPN is knocked-down, invasive potential of YTS-1 was markedly reduced. (p¼0.005). CONCLUSIONS: These results suggest that expression of PDPN and its O-glycosylation enhance growth factors production, proliferation activity, and invasion potential of bladder cancer cells through interaction with platelet.
Source of Funding: None. Source of Funding: The Japan Society for the Promotion of Science
MP21-08 GLYCAN MODIFICATION OF PODOPLANIN ENHANCES GROWTH FACTOR PRODUCTION THROUGH STABILIZATION OF PLATELET AGGREGATION IN BLADDER CANCER CELL Yuki Tobisawa*, Shingo Hatakeyama, Tohru Yoneyama, Teppei Okamoto, Hayato Yamamoto, Kazuyuki Mori, Akiko Okamoto, Atsushi Imai, Takahiro Yoneyama, Yasuhiro Hashimoto, Takuya Koie, Chikara Ohyama, Hirosaki, Japan INTRODUCTION AND OBJECTIVES: Platelet aggregation together with cancer cell is one of the key factors for cancer progression and metastasis. Podoplanin (PDPN), which is known as a lymphatic endothelial marker, expressed on cancer cell surface has a potential of platelet aggregation through interaction with CLEC-2 expressed on Platelet. Core2 b-1,6-N-acetylglucosaminyltransferase (C2GnT) is a key enzyme for O-glycosylation of membrane proteins. Although PDPN has a core2 O-glycosylation site on their mucin domain, its roll in cancer progression is still unclear. Here, we examine the roles of PDPN and their O-glycosylation in bladder cancer (BCa) progression and invasion.
MP21-09 PLECTIN-1 DEPLETION IN MUSCLE INVASIVE BLADDER CANCER CELL LINES PROMOTES PHOSPHORYLATION OF FER TYROSINE KINASE AND ENHANCES INVASION POTENTIAL Hiromichi Iwamura*, Tohru Yoneyama, Yuki Tobisawa, Shingo Hatakeyama, Takuya Koie, Chikara Ohyama, Hirosaki, Japan INTRODUCTION AND OBJECTIVES: Plectin-1 which acts as a cytoskeletal crosslinker protein affects mechanical as well as dynamic properties of cytoskeleton. It was reported that Plectin-1 directly bind to Fer tyrosine kinase (Fer) and suppress phosphorylation of Fer and downstream signaling in mouse fibroblasts [Lunter et al., BBRC: 296, 904-910, 2002]. Plectin-1 is often impaired in muscle invasive bladder cancer [Okusa et al., J Electrophoresis: 52, 19-23, 2008], yet roles and underlying mechanisms of plectin-1 and Fer in bladder cancer
THE JOURNAL OF UROLOGYâ
Vol. 191, No. 4S, Supplement, Sunday, May 18, 2014
progression remain unclear. Here, we investigated the role of plectin-1 and Fer in bladder cancer cell lines. METHODS: We used the 2 bladder cancer cell lines YTS-1 (muscle invasive) and RT4 (superficial). Expression of plectin-1 and Fer in bladder cancer cells was analyzed by immunofluorescence (IF) staining and immunoblotting (IB). Interaction of plectin-1 and Fer in bladder cancer cells was analyzed by immunoprecipitation (IP). Bladder cancer cells were tested for invasion potential against basal membrane extract (BME) gel or bladder smooth muscle cell (BdSMC) monolayer. We also examined the role of Fer in invasion capacity using Fer knockdown YTS-1 cells. Furthermore, we examined expression of phospho-Fer in bladder cancer tissues. RESULTS: IB and IF staining showed that expression of plectin-1 was much higher in RT4 compared with YTS-1. Although the expression of Fer was decreased in YTS-1, expression of phosphor-Fer was much higher in YTS-1 compared with RT4. Phosphorylation of ERK or AKT which is a downstream signaling molecules of Fer was also upregulated in YTS-1. IP showed that plectin-1 bound to Fer in RT4 but not bound to Fer in YTS-1. In YTS-1, Fer was bound to vimentin. Invasion capacity against BME gel and BdSMC in YTS-1 was much higher than those of RT4. Immunohistochemical analyses of bladder cancer tissues showed that the upregulation of phospho-Fer correlated with poor prognosis of bladder cancer. CONCLUSIONS: These results indicate that plectin-1 depletion in muscle invasive bladder cancer cells promotes phosphorylation of Fer and enhances invasion capacity.
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Unfortunately, bladder cancer has the high recurrence rate after treatment, even when superficial tumors are completely removed by a laser or electrical force. We hypothesize that the aberrant activation between stromal cells and bladder cancer cells is probably involved in the high recurrence rate of bladder cancer. The development of efficient tumor models that mimic human disease would benefit to investigate new cancer therapeutic agents. Here we have developed a new model of bladder tumors by recombining rat mesenchymal cells with T24 human bladder cancer cells. METHODS: Rat mesenchymal cells were prepared from 18-day SD rat fetuses. Briefly, bladder, urogenital sinuses, and stomach were dissected from the fetuses and separated into epithelial and mesenchymal components. Recombinants were prepared by mixing 5 x 105 T24 cells and 1 x 105 rat mesenchymal cells in suspension. Pelleted cells were resuspended in neutralized type I rat tail collagen gels, and then grafted beneath the renal capsule of adult male nude mice. RESULTS: As generally known, T24 tumors without rat mesenchymal cells were not well grown in vivo. In contrast, T24 tumors that formed with rat mesenchymal cells derived from urogenital sinus (UGM) and bladder (BLM) but not stomach (STM) showed accelerated cell proliferation and angiogenesis. In in vitro cell culture experiments, several growth factors such as EGF, TGF-alpha, and IL-6 stimulated cell proliferation of T24 cells. Only IL-6 treatment activated cellular PI3/ Akt pathway in T24 cells, whereas IL-6 treatment reduced VEGF secretion from T24 cells. Cell motility of T24 cells was significantly increased by EGF and TGF-alpha treatment. CONCLUSIONS: In this study, we demonstrate that our recombination model using mesenchymal cells may be useful to understand the tumor-stromal interactions in bladder cancer. Identifying mechanisms underlying the aberrant activation between stromal cells and bladder cancer cells is an initial step toward developing new cancer therapeutic agents in bladder cancer. Source of Funding: none
MP21-11 UP-REGULATED NFkB ACTIVATION IS A POTENT THERAPEUTIC TARGET IN ACQUIRED PLATINUM-RESISTANT BLADDER CANCER: THE EFFICACY OF NFkB BLOCKADE BY DEHYDROXYMETHYL DERIVATIVE OF EPOXYQUINOMICIN (DHMEQ) Source of Funding: none
MP21-10 DEVELOPMENT OF AN EFFICIENT BLADDER TUMOR MODEL BY RECOMBINING WITH MESENCHYMAL CELLS Kenichiro Ishii*, Takeshi Sasaki, Manabu Kato, Tsu, Japan; Kenjiro Ito, Motomu Sakuragi, Nobuyuki Oda, Tsukuba, Japan; Hideki Kanda, Yasushi Yamada, Kiminobu Arima, Taizo Shiraishi, Yoshiki Sugimura, Tsu, Japan INTRODUCTION AND OBJECTIVES: Aberrant activation of tumor-stromal interactions is considered to play a critical role in the promotion of tumorigenesis. Many solid tumors are composed of carcinoma cells and surrounding stromal cells named as carcinomaassociated fibroblasts (CAFs). They secrete a number of growth factors such as EGF family, FGFs, HGF, and IGFs, which are also produced by rat mesenchymal cells (Ishii et al., Endocr Relate Cancer, 2009). Bladder cancer is the second most common genitourinary malignancy, with transitional cell carcinoma representing 90% of the cases.
Yujiro Ito*, Eiji Kikuchi, Nobuyuki Tanaka, Takeo Kosaka, Ryuichi Mizuno, Akira Miyajima, Kazuo Umezawa, Mototsugu Oya, Tokyo, Japan INTRODUCTION AND OBJECTIVES: No study has addressed the efficacy of NFkB blockade when bladder tumors developed acquired resistance toward CDDP treatments. We investigated the changes in NFkB expression and therapeutic impact of an NFkB inhibitor in acquired platinum-resistant, T24PR cells. METHODS: T24PR cells were newly established as acquired platinum-resistant sublines of T24 by culturing in CDDP-containing medium for 6 months (Br J Cancer, 2011). Expression of p65 protein in two fractionated cell lines was determined by Western blotting analysis. DNA-binding activity of NFkB was detected by electrophoretic mobility shift assay. Using a novel NFkB inhibitor, DHMEQ, the cytotoxic effects and induction of apoptosis were analyzed in vitro and in vivo. RESULTS: Nuclear activation and protein expression of p65 were enhanced in T24PR cells compared with T24 cells. DHMEQ inhibited cell growth in a dose-dependent manner in both cell lines, however, the half maximal inhibitory concentration of T24PR cells (6.9mg/ml) was significantly lower than that of T24 cells (17.3mg/ml). Using the TUNEL assay, the apoptotic index induced by DHMEQ (10mg/ml) at 48 hours was 23.64.8% in T24 cells and 46.18.5% in
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MP21-07 HIGH-DOSE CHEMOTHERAPEUTICS USED IN INTRAVESICAL THERAPY INDUCE MITOCHONDRIAL DYSFUNCTION FOLLOWED BY NECROSIS-LIKE CELL DEATH IN BLADDER CANCER CELLS Takahiro Yoshida*, Suita, Osaka, Japan; Hiroaki Okuyama, Masahi Nakayama, Kazuo Nishimura, Masahiro Inoue, Osaka, Osaka, Japan; Norio Nonomura, Suita, Osaka, Japan INTRODUCTION AND OBJECTIVES: Non-muscle invasive bladder cancer is often treated by intravesical chemotherapy following transurethral resection. Mechanism of the effect of intravesical chemotherapy has yet to be elucidated. In this study, we investigated cell death mechanism induced by high-dose chemotherapeutics using a newly-developed culture method for primary cancer cells. METHODS: We have developed a novel culture method which enables preparation and culture of primary cancer cells as multicellular spheroids which are termed cancer tissue-originated spheroids (CTOSs) (Okuyama 2013 J Urol). First we generated a bladder cancer patient-derived xenograft. We prepared CTOSs from the xenograft, and exposed to high-dose (1mg/ml) of epirubicin (e-ADM) or mitomycin C (MMC) for 2 hours, or low-dose (0.01mg/ml) of those chemotherapeutics. We investigated absorption of chemotherapeutics into CTOSs and assessed the mode of cell death. Next, we prepared CTOSs directly from surgical specimens of bladder cancer and tested the individual responses after exposure to high-dose e-ADM and MMC. RESULTS: At high dose, e-ADM was promptly and homogenously delivered into cancer cells in the CTOSs. High-dose e-ADM and MMC decreased ATP levels in CTOSs within an hour accompanied with mitochondrial swelling and reduction of mitochondrial membrane potential, while plasma membrane integrity was maintained. Some of the molecules in mitochondrial apoptotic pathway were subsequently activated, while at later time point the cells lost integrity of cell membrane without DNA laddering. In contrast, CTOSs exposed to low-dose of eADM or MMC exhibited typical DNA laddering. Intriguingly, the decrease of ATP levels was varied among CTOSs derived from 20 surgical specimens. CONCLUSIONS: High-dose chemotherapeutics used in intravesical therapy may induce necrosis-like cell death, different from typical apoptosis caused by chemotherapeutics at the dose of systemic chemotherapy. Each bladder cancer may have a different response to high-dose chemotherapeutics used in intravesical therapy, indicating necessity of chemosensitivity test aiming for personalized medicine.
Vol. 191, No. 4S, Supplement, Sunday, May 18, 2014
METHODS: YTS-1 is a BCa cell line established from muscle invasive BCa and highly positive for PDPN and C2GnT. KK-47 is a BCa cell line established from non-muscle invasive BCa and expresses a faint level of PDPN and C2GnT. YTS-1 cells were transfected with PDPN-siRNA or C2GnT-siRNA to silence of PDPN or C2GnT expression. KK47 cells were transfected with pcDNA3.1-PDPN and/or pcDNA3.1-C2GnT to make KK47-PDPN, KK47-C2GnT and KK47C2PDPN which are highly positive for PDPN, C2GnT or both of them. These cells were subjected to growth factor assays and invasion assay. Co-culture system of BCa cells and platelet was used to evaluate growth kinetics of the BCa cells and production of growth factors. In vitro invasion assay was used to evaluate invasion potential of the BCa cells. RESULTS: In the co-culture system of BCa cells and platelet, BCa cells culturing with platelet had higher growth activity than without platelet (p<0.05). YTS-1 produced significantly higher amounts of bFGF, EGF, NGF beta, PDGF-BB, TGF-beta and VEGF when they are co-cultured with platelet than they are cultured without platelet (p<0.05). O-glycosylation of PDPN on BCa cells significantly increased the production of growth factors (p<0.05). When PDPN is knocked-down, invasive potential of YTS-1 was markedly reduced. (p¼0.005). CONCLUSIONS: These results suggest that expression of PDPN and its O-glycosylation enhance growth factors production, proliferation activity, and invasion potential of bladder cancer cells through interaction with platelet.
Source of Funding: None. Source of Funding: The Japan Society for the Promotion of Science
MP21-08 GLYCAN MODIFICATION OF PODOPLANIN ENHANCES GROWTH FACTOR PRODUCTION THROUGH STABILIZATION OF PLATELET AGGREGATION IN BLADDER CANCER CELL Yuki Tobisawa*, Shingo Hatakeyama, Tohru Yoneyama, Teppei Okamoto, Hayato Yamamoto, Kazuyuki Mori, Akiko Okamoto, Atsushi Imai, Takahiro Yoneyama, Yasuhiro Hashimoto, Takuya Koie, Chikara Ohyama, Hirosaki, Japan INTRODUCTION AND OBJECTIVES: Platelet aggregation together with cancer cell is one of the key factors for cancer progression and metastasis. Podoplanin (PDPN), which is known as a lymphatic endothelial marker, expressed on cancer cell surface has a potential of platelet aggregation through interaction with CLEC-2 expressed on Platelet. Core2 b-1,6-N-acetylglucosaminyltransferase (C2GnT) is a key enzyme for O-glycosylation of membrane proteins. Although PDPN has a core2 O-glycosylation site on their mucin domain, its roll in cancer progression is still unclear. Here, we examine the roles of PDPN and their O-glycosylation in bladder cancer (BCa) progression and invasion.
MP21-09 PLECTIN-1 DEPLETION IN MUSCLE INVASIVE BLADDER CANCER CELL LINES PROMOTES PHOSPHORYLATION OF FER TYROSINE KINASE AND ENHANCES INVASION POTENTIAL Hiromichi Iwamura*, Tohru Yoneyama, Yuki Tobisawa, Shingo Hatakeyama, Takuya Koie, Chikara Ohyama, Hirosaki, Japan INTRODUCTION AND OBJECTIVES: Plectin-1 which acts as a cytoskeletal crosslinker protein affects mechanical as well as dynamic properties of cytoskeleton. It was reported that Plectin-1 directly bind to Fer tyrosine kinase (Fer) and suppress phosphorylation of Fer and downstream signaling in mouse fibroblasts [Lunter et al., BBRC: 296, 904-910, 2002]. Plectin-1 is often impaired in muscle invasive bladder cancer [Okusa et al., J Electrophoresis: 52, 19-23, 2008], yet roles and underlying mechanisms of plectin-1 and Fer in bladder cancer
THE JOURNAL OF UROLOGYâ
Vol. 191, No. 4S, Supplement, Sunday, May 18, 2014
progression remain unclear. Here, we investigated the role of plectin-1 and Fer in bladder cancer cell lines. METHODS: We used the 2 bladder cancer cell lines YTS-1 (muscle invasive) and RT4 (superficial). Expression of plectin-1 and Fer in bladder cancer cells was analyzed by immunofluorescence (IF) staining and immunoblotting (IB). Interaction of plectin-1 and Fer in bladder cancer cells was analyzed by immunoprecipitation (IP). Bladder cancer cells were tested for invasion potential against basal membrane extract (BME) gel or bladder smooth muscle cell (BdSMC) monolayer. We also examined the role of Fer in invasion capacity using Fer knockdown YTS-1 cells. Furthermore, we examined expression of phospho-Fer in bladder cancer tissues. RESULTS: IB and IF staining showed that expression of plectin-1 was much higher in RT4 compared with YTS-1. Although the expression of Fer was decreased in YTS-1, expression of phosphor-Fer was much higher in YTS-1 compared with RT4. Phosphorylation of ERK or AKT which is a downstream signaling molecules of Fer was also upregulated in YTS-1. IP showed that plectin-1 bound to Fer in RT4 but not bound to Fer in YTS-1. In YTS-1, Fer was bound to vimentin. Invasion capacity against BME gel and BdSMC in YTS-1 was much higher than those of RT4. Immunohistochemical analyses of bladder cancer tissues showed that the upregulation of phospho-Fer correlated with poor prognosis of bladder cancer. CONCLUSIONS: These results indicate that plectin-1 depletion in muscle invasive bladder cancer cells promotes phosphorylation of Fer and enhances invasion capacity.
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Unfortunately, bladder cancer has the high recurrence rate after treatment, even when superficial tumors are completely removed by a laser or electrical force. We hypothesize that the aberrant activation between stromal cells and bladder cancer cells is probably involved in the high recurrence rate of bladder cancer. The development of efficient tumor models that mimic human disease would benefit to investigate new cancer therapeutic agents. Here we have developed a new model of bladder tumors by recombining rat mesenchymal cells with T24 human bladder cancer cells. METHODS: Rat mesenchymal cells were prepared from 18-day SD rat fetuses. Briefly, bladder, urogenital sinuses, and stomach were dissected from the fetuses and separated into epithelial and mesenchymal components. Recombinants were prepared by mixing 5 x 105 T24 cells and 1 x 105 rat mesenchymal cells in suspension. Pelleted cells were resuspended in neutralized type I rat tail collagen gels, and then grafted beneath the renal capsule of adult male nude mice. RESULTS: As generally known, T24 tumors without rat mesenchymal cells were not well grown in vivo. In contrast, T24 tumors that formed with rat mesenchymal cells derived from urogenital sinus (UGM) and bladder (BLM) but not stomach (STM) showed accelerated cell proliferation and angiogenesis. In in vitro cell culture experiments, several growth factors such as EGF, TGF-alpha, and IL-6 stimulated cell proliferation of T24 cells. Only IL-6 treatment activated cellular PI3/ Akt pathway in T24 cells, whereas IL-6 treatment reduced VEGF secretion from T24 cells. Cell motility of T24 cells was significantly increased by EGF and TGF-alpha treatment. CONCLUSIONS: In this study, we demonstrate that our recombination model using mesenchymal cells may be useful to understand the tumor-stromal interactions in bladder cancer. Identifying mechanisms underlying the aberrant activation between stromal cells and bladder cancer cells is an initial step toward developing new cancer therapeutic agents in bladder cancer. Source of Funding: none
MP21-11 UP-REGULATED NFkB ACTIVATION IS A POTENT THERAPEUTIC TARGET IN ACQUIRED PLATINUM-RESISTANT BLADDER CANCER: THE EFFICACY OF NFkB BLOCKADE BY DEHYDROXYMETHYL DERIVATIVE OF EPOXYQUINOMICIN (DHMEQ) Source of Funding: none
MP21-10 DEVELOPMENT OF AN EFFICIENT BLADDER TUMOR MODEL BY RECOMBINING WITH MESENCHYMAL CELLS Kenichiro Ishii*, Takeshi Sasaki, Manabu Kato, Tsu, Japan; Kenjiro Ito, Motomu Sakuragi, Nobuyuki Oda, Tsukuba, Japan; Hideki Kanda, Yasushi Yamada, Kiminobu Arima, Taizo Shiraishi, Yoshiki Sugimura, Tsu, Japan INTRODUCTION AND OBJECTIVES: Aberrant activation of tumor-stromal interactions is considered to play a critical role in the promotion of tumorigenesis. Many solid tumors are composed of carcinoma cells and surrounding stromal cells named as carcinomaassociated fibroblasts (CAFs). They secrete a number of growth factors such as EGF family, FGFs, HGF, and IGFs, which are also produced by rat mesenchymal cells (Ishii et al., Endocr Relate Cancer, 2009). Bladder cancer is the second most common genitourinary malignancy, with transitional cell carcinoma representing 90% of the cases.
Yujiro Ito*, Eiji Kikuchi, Nobuyuki Tanaka, Takeo Kosaka, Ryuichi Mizuno, Akira Miyajima, Kazuo Umezawa, Mototsugu Oya, Tokyo, Japan INTRODUCTION AND OBJECTIVES: No study has addressed the efficacy of NFkB blockade when bladder tumors developed acquired resistance toward CDDP treatments. We investigated the changes in NFkB expression and therapeutic impact of an NFkB inhibitor in acquired platinum-resistant, T24PR cells. METHODS: T24PR cells were newly established as acquired platinum-resistant sublines of T24 by culturing in CDDP-containing medium for 6 months (Br J Cancer, 2011). Expression of p65 protein in two fractionated cell lines was determined by Western blotting analysis. DNA-binding activity of NFkB was detected by electrophoretic mobility shift assay. Using a novel NFkB inhibitor, DHMEQ, the cytotoxic effects and induction of apoptosis were analyzed in vitro and in vivo. RESULTS: Nuclear activation and protein expression of p65 were enhanced in T24PR cells compared with T24 cells. DHMEQ inhibited cell growth in a dose-dependent manner in both cell lines, however, the half maximal inhibitory concentration of T24PR cells (6.9mg/ml) was significantly lower than that of T24 cells (17.3mg/ml). Using the TUNEL assay, the apoptotic index induced by DHMEQ (10mg/ml) at 48 hours was 23.64.8% in T24 cells and 46.18.5% in