555 NANOPARTICLE BCG HAS IDENTICAL DIRECT ANTITUMOR EFFECT AS LIVE BCG, BUT HAS A DIFFERENT ANTITUMOR MECHANISM

Vol. 187, No. 4S, Supplement, Sunday, May 20, 2012

mechanism for erlotinib resistance and invasive potential. The relationship between the miRNAs involved in erlotinib resistance and proteins known to be involved in EMT will be explored. Source of Funding: None

554 A KEY ROLE FOR ARF IN DRUG RESISTANCE IN INVASIVE BLADDER CANCER Takashi Kobayashi*, Joan Delto, Cory Abate-Shen, New York, NY INTRODUCTION AND OBJECTIVES: Although most superficial bladder cancers can be removed transurethrally with excellent prognosis, clinical outcome is much more problematic for patients with muscle-invasive disease. Indeed, invasive bladder cancer is a major clinical challenge since it is highly associated with postoperative recurrence and metastasis. Current treatments for those lethal forms of bladder cancer include systemic chemotherapy and molecular targeted therapy; however, survival is poor since most patients develop resistance to the drugs within a short timeframe. Clearly, there is a need to identify novel therapeutic options for invasive bladder cancer as well as a greater understanding of the molecular mechanisms of drug resistance. METHODS: We have been investigating mechanisms of drug resistance using genetically-engineered mouse models of invasive bladder cancer based on the combinatorial deletion of p53 and Pten in bladder epithelium. p53 and PTEN are frequently inactivated in human bladder cancers, particularly those with poor prognosis. Indeed, allograft tumors established from these mice responded initially to the treatment with cisplatin, docetaxel, or rapamycin but eventually developed resistance. RESULTS: We have observed that these p53; PTEN deficient tumors express robust levels of p19Arf, while targeted deletion of Arf retards the aquisition of resistance following drug treatment. The significance of this expression was further suggested by analysis of a published gene expression profile of human bladder cancers which revealed that high Arf expression is an independent predictor of poor survival of invasive bladder cancer patients. Furthermore, following drug treatment in the mouse model, the Arf-positive, compared to the Arf-negative, tumors were significantly enriched in extracellular matrix/ integrin signaling gene signature consistent with activation of downstream PI3K-mTOR pathway, suggesting that a model for drug resistance is via PI3K pathway activation. We have observed similar results in human bladder cancer cell lines either following knock-down of p14Arf in J82 cells that express endogenous p14Arf or following forced expression of p14Arf in ARF-negative UMUC3 human bladder cancer cells, and in both cases it was coincident with deregulated activation of PI3K-mTOR pathway. CONCLUSIONS: Thus, we propose that Arf confers drug resistance on bladder cancer by activating PI3K-mTOR pathway, highlighting a potential therapeutic target for advanced invasive bladder cancer patients. Source of Funding: AUA Foundation, American Association for Cancer Research

555 NANOPARTICLE BCG HAS IDENTICAL DIRECT ANTITUMOR EFFECT AS LIVE BCG, BUT HAS A DIFFERENT ANTITUMOR MECHANISM Kazuyuki Mori*, Tohru Yoneyama, Yuki Tobisawa, Yusuke Ishibashi, Hiromi Murasawa, Hayato Yamamoto, Hirofumi Ishimura, Shingo Hatakeyama, Shigemasa Kudo, Takahiro Yoneyama, Takuya Koie, Noritaka Kamimura, Chikara Ohyama, Hirosaki, Japan INTRODUCTION AND OBJECTIVES: In our previous report on AUA2007, we generated nanoparticle BCG, which exerts a direct antitumor effect identical to that by live BCG against J82 and KK47

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bladder cancer cells, however, we did not analyze the mechanism. In this study, we performed cell cycle analysis and detection of apoptosis. METHODS: The Tokyo 172 BCG sub-strain suspension was disrupted by a French press, and the particle distribution was determined in a particle analyzer. After removing the non-disrupted bacteria by centrifugation at 6800 ⫻g, the supernatant was centrifuged at 18,000 ⫻g. The supernatant (CM) and the precipitate (CW) were lyophilized to obtain nanoparticle BCG. The bladder cancer cell lines J82 and KK47 were maintained in RPMI1640 medium. Live BCG, CW, CM, and CW⫹CM (mix) were added to the culture medium at a final concentration of 0.1 mg/mL and cultured for 5 days, and the viable cells were counted using trypan blue staining. Cells and culture sup were collected and stained with propidium iodide, and cell cycle analysis and detection of apoptosis using flow cytometry was performed. RESULTS: After 5 days of culturing, the viability of cells treated with BCG and nanoparticle BCG significantly decreased to 72.4% and 73.4% (J82 cells) and 68.2% and 67.0% (KK47 cells) of control cells, respectively. The percentage of apoptosis in control cells, cells treated with BCG, and cells treated with nanoparticle BCG was 3.61%, 7.61%, and 4.02% in J82 cells, and 1.53%, 7.14%, and 0.07% in KK47 cells, respectively. BCG induced apoptosis, but nanoparticle BCG did not. The percentage of total S phase in control cells, cells treated with BCG, and cells treated with nanoparticle BCG was 26.19%, 32.39%, and 27.48% in J82 cells, and 27.88%, 26.78%, and 27.48% in KK47 cells, respectively. In J82 cells, BCG significantly increased the percentage of total S phase cells, but nanoparticle BCG did not. CONCLUSIONS: Nanoparticle BCG had identical direct antitumor effect as live BCG; however, it did not induce apoptosis. These results suggest that nanoparticle BCG can be used to develop new therapeutic drugs through the different antitumor mechanism against live BCG-failure cases, invasive bladder cancer, or other tissue cancer. Source of Funding: None

556 NOVEL NF-KAPPAB INHIBITOR DHMEQ: THE NEW STRATEGY FOR ACQUIRED CHEMO-RESISTANT BLADDER CANCER Yujiro Ito*, Eiji Kikuchi, Nobuyuki Tanaka, Hiroshi Shirakawa, Akira Miyajima, Yutaka Horiguchi, Jun Nakashima, Ken Nakagawa, Kazuo Umezawa, Mototsugu Oya, Tokyo, Japan INTRODUCTION AND OBJECTIVES: The gold standard treatment for metastatic bladder cancer is CDDP-based combined chemotherapy. However many bladder tumors finally progress after acquiring resistance toward chemotherapeutic agents, and no effective 2nd line is currently available. We have synthesized a dehydroxymethyl derivative of epoxyquinomicin, abbreviated DHMEQ, from a natural product, and we previously reported the efficacy of DHMEQ as a potent modality in urogenital cancers. In the study, we investigated the therapeutic impact of DHMEQ in acquired chemo-resistant bladder cancer. METHODS: Four invasive human bladder cancer cell lines, T24, T24PR (Platinum-Resistant), T24GR (Gemcitabine-Resistant), and T24TR (Taxan-Resistant) were used in vitro, while in vivo, T24 and T24PR cells were used. T24PR, T24GR, and T24TR were newly established at our institution as acquired chemo-resistant sublines by culturing in each chemotherapeutic agent (CDDP, gemcitabine, paclitaxel) containing medium for 6 months. RESULTS: First we evaluated the acquired resistance of T24PR, T24GR, and T24TR cells toward each chemotherapeutic agent both in vitro and in vivo. DHMEQ inhibited the DNA-binding activity of NF-kappaB by blocking its nuclear translocation, and cell viability was significantly suppressed by DHMEQ even in these chemo-resistant cells. Moreover, IC50 of DHMEQ was significantly lower than that of parent cells (T24PR 17.4␮g/ml, T24GR 18.1␮g/ml, T24TR 17.4␮g/ml and T24 55.6␮g/ml). Using, apoptosis were strongly induced in these chemo-resistant cells compared with parent cells (T24PR 46.1%, T24GR 49.3%, T24TR 43.8% and T24 23.6%) as shown in Figure 1. EMSA assay showed that the status of NF-kappaB was strongly