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MP45-12 PROGNOSTIC IMPACT OF TUMOR ASSOCIATED MACROPHAGES IN PATIENTS WITH TRANSITIONAL CELL BLADDER CANCER
THE JOURNAL OF UROLOGYâ
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MP45-12 PROGNOSTIC IMPACT OF TUMOR ASSOCIATED MACROPHAGES IN PATIENTS WITH TRANSITIONAL CELL BLADDER CANCER Bo Wang, Shaoxu Wu, Zhuowei Liu, Wen Dong, Wang He, Xiaoliang Dong, Tianxin Lin, Jian Huang*, Guangzhou, China, People’s Republic of INTRODUCTION AND OBJECTIVES: Emerging evidence have shown that inflammation associated immune cells are involved in rin the molecular and cellular mechanisms of bacillus Calmette-Gue (BCG) immunotherapy of transitional cell bladder cancer (TCC). Macrophages (M&[phis]) is a critical mediator in the inflammatory response and can serve as a potential target for BCG. However, the clinical significance and related mechanisms of tumor associated M&[phis](TAM) during TCC progression are less clear. The aim of this study was to investigate the relationship between the type, density, and location of TAM within tumors and the clinical outcome of the TCC patients. METHODS: Immunohistochemistry and immunofluorescence were used to detect TAM phenotypes by using CD68 as a macrophage lineage marker and stabilin-1 as an M2 macrophage marker in situ. KaplaneMeier analysis and Cox proportional hazards regression models were applied to estimate overall survival (OS) and recurrencefree survival (RFS) in 302 TCC patients. FACS analysis and real-time quantitative PCR were used to detect M&[phis] phenotypes in vitro. RESULTS: In TCC tissues, M2 macrophage marker stabilin-1 is primarily expressed on the TAM, which is evident from the dual immunofluorescence staining for stabilin-1 and CD68. Both CD68þM& [phis] and stabilin-1þM&[phis] are distributed throughout the tissue, but often more prominent in stromal regions rather than intratumoral regions in TCC tissues (both P < 0.0001). After dichotomisation at the median cell density for CD68þM&[phis] and stabilin-1þM&[phis], only intratumoral stabilin-1þM&[phis] could represent an adverse prognostic predictor of both OS (P < 0.0001) and RFS (P ¼ 0.039). In addition, We used BCG to treat TAMs in vitro. The results showed that the expression of M1 markers were upregulated and the expression of M2 markers were downregulated on protein or mRNA levels. Notably, stabilin-1 was significantly downregulated on BCG-treated TAMs in vitro. CONCLUSIONS: Our findings suggest intratumoral stabilin1þM&[phis] could potentially be used as a poor prognostic marker for TCC patients. Tipping downregulating stabilin-1 expression may be one of the molecular mechanism of BCG immunotherapy.
Vol. 193, No. 4S, Supplement, Sunday, May 17, 2015
METHODS: Material consisted of tumor tissues from patients with low-grade (LGUC), high-grade (HGUC), normal urothelial tissues (NUC) and bladder cancer cell lines; T24 and UM-UC-3. Oxidative stress was induced by mETC inhibitors; rotenone and antimycin. Effect of autophagy induction and inhibition was evaluated by measuring cell viability, mitochondrial membrane potential (MMP), cytochrome-c release and caspase activation. RESULTS: N-Acetylcysteine (NAC), a ROS scavenger, blocked the autophagy induced by rotenone or antimycin (Fig1A). Inhibition of autophagy by wortmannin (Wm) or NAC led to increased apoptotic cell death in UCC (56-63%) as compared to rotenone (8%) and antimycin (9%) alone (Fig1B). Addition of NAC with rotenone or antimycin had no effect on MMP and cytochrome-c release, while Wm caused mitochondrial hyperpolarization and cytochrome-c release in UCC (Fig1CD). NAC treatment led to release of Smac, a mitochondrial protein, into cytosol which caused cleavage of XIAP, an anti-apoptotic protein, followed by increase in activation of caspase-9/-3 (Fig1E-I). Inhibition of autophagy by NAC treatment caused caspase-8 activation leading to Bid-mediated release of Smac (Fig1J). CONCLUSIONS: Autophagy is a cell survival mechanism during oxidative stress in urothelial carcinoma and its inhibition appears to be of potential clinical significance in improving therapeutic efficacy. To the best of our knowledge the present study is the first to document the functional role of Smac in mediating NAC-induced caspase-9 activation and apoptosis in UCC and targeting Smac could be used for exploring the therapeutic strategies in UCC.
Source of Funding: This study was funded by the National Natural Science Foundation of China (Grant No. U1301221, 81472384, 81372729, 81372883, 81272808, 81172431, 81101935, 81402106), and Guangdong Province Natural Scientific Foundation (Grant No. S2013020012671, 07117336, 10151008901000024), Specialized Research Fund for the Doctoral Program of Higher Education (for Tianxin Lin, 20130171110073), Sun Yat-Sen University Clinical Research 5010 Program (Grant No. 2007018), Elite Young Scholars Program of Sun Yat-Sen Memorial Hospital (for Tianxin Lin, J201401), and National Clinical Key Specialty Construcion Project for Department of Urology and Department of Oncology. Grant KLB09001 from the Key Laboratory of Malignant Tumor Gene Regulation and Target Therapy of Guangdong Higher Education Institutes, Sun-Yat-Sen University. Grant NO. 2014M562241 from China Postdoctoral Science Foundation.
MP45-13 N-ACETYLCYSTEINE-MEDIATED AUTOPHAGY INHIBITION LEADS TO CYTOCHROME-C INDEPENDENT CASPASE-9 ACTIVATION DURING OXIDATIVE STRESS IN UROTHELIAL CARCINOMA OF URINARY BLADDER Rani Ojha, Shrawan K. Singh*, Vivekanand Jha, Chandigarh, India INTRODUCTION AND OBJECTIVES: To investigate the effect of oxidative stress-induced autophagy on cell survival in different grades of urothelial carcinoma cells (UCC).
Source of Funding: Education and Research Cell, PGIMER, Chandigarh Indian Council of Medical Research (ICMR), New Delhi, India
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MP45-12 PROGNOSTIC IMPACT OF TUMOR ASSOCIATED MACROPHAGES IN PATIENTS WITH TRANSITIONAL CELL BLADDER CANCER Bo Wang, Shaoxu Wu, Zhuowei Liu, Wen Dong, Wang He, Xiaoliang Dong, Tianxin Lin, Jian Huang*, Guangzhou, China, People’s Republic of INTRODUCTION AND OBJECTIVES: Emerging evidence have shown that inflammation associated immune cells are involved in rin the molecular and cellular mechanisms of bacillus Calmette-Gue (BCG) immunotherapy of transitional cell bladder cancer (TCC). Macrophages (M&[phis]) is a critical mediator in the inflammatory response and can serve as a potential target for BCG. However, the clinical significance and related mechanisms of tumor associated M&[phis](TAM) during TCC progression are less clear. The aim of this study was to investigate the relationship between the type, density, and location of TAM within tumors and the clinical outcome of the TCC patients. METHODS: Immunohistochemistry and immunofluorescence were used to detect TAM phenotypes by using CD68 as a macrophage lineage marker and stabilin-1 as an M2 macrophage marker in situ. KaplaneMeier analysis and Cox proportional hazards regression models were applied to estimate overall survival (OS) and recurrencefree survival (RFS) in 302 TCC patients. FACS analysis and real-time quantitative PCR were used to detect M&[phis] phenotypes in vitro. RESULTS: In TCC tissues, M2 macrophage marker stabilin-1 is primarily expressed on the TAM, which is evident from the dual immunofluorescence staining for stabilin-1 and CD68. Both CD68þM& [phis] and stabilin-1þM&[phis] are distributed throughout the tissue, but often more prominent in stromal regions rather than intratumoral regions in TCC tissues (both P < 0.0001). After dichotomisation at the median cell density for CD68þM&[phis] and stabilin-1þM&[phis], only intratumoral stabilin-1þM&[phis] could represent an adverse prognostic predictor of both OS (P < 0.0001) and RFS (P ¼ 0.039). In addition, We used BCG to treat TAMs in vitro. The results showed that the expression of M1 markers were upregulated and the expression of M2 markers were downregulated on protein or mRNA levels. Notably, stabilin-1 was significantly downregulated on BCG-treated TAMs in vitro. CONCLUSIONS: Our findings suggest intratumoral stabilin1þM&[phis] could potentially be used as a poor prognostic marker for TCC patients. Tipping downregulating stabilin-1 expression may be one of the molecular mechanism of BCG immunotherapy.
Vol. 193, No. 4S, Supplement, Sunday, May 17, 2015
METHODS: Material consisted of tumor tissues from patients with low-grade (LGUC), high-grade (HGUC), normal urothelial tissues (NUC) and bladder cancer cell lines; T24 and UM-UC-3. Oxidative stress was induced by mETC inhibitors; rotenone and antimycin. Effect of autophagy induction and inhibition was evaluated by measuring cell viability, mitochondrial membrane potential (MMP), cytochrome-c release and caspase activation. RESULTS: N-Acetylcysteine (NAC), a ROS scavenger, blocked the autophagy induced by rotenone or antimycin (Fig1A). Inhibition of autophagy by wortmannin (Wm) or NAC led to increased apoptotic cell death in UCC (56-63%) as compared to rotenone (8%) and antimycin (9%) alone (Fig1B). Addition of NAC with rotenone or antimycin had no effect on MMP and cytochrome-c release, while Wm caused mitochondrial hyperpolarization and cytochrome-c release in UCC (Fig1CD). NAC treatment led to release of Smac, a mitochondrial protein, into cytosol which caused cleavage of XIAP, an anti-apoptotic protein, followed by increase in activation of caspase-9/-3 (Fig1E-I). Inhibition of autophagy by NAC treatment caused caspase-8 activation leading to Bid-mediated release of Smac (Fig1J). CONCLUSIONS: Autophagy is a cell survival mechanism during oxidative stress in urothelial carcinoma and its inhibition appears to be of potential clinical significance in improving therapeutic efficacy. To the best of our knowledge the present study is the first to document the functional role of Smac in mediating NAC-induced caspase-9 activation and apoptosis in UCC and targeting Smac could be used for exploring the therapeutic strategies in UCC.
Source of Funding: This study was funded by the National Natural Science Foundation of China (Grant No. U1301221, 81472384, 81372729, 81372883, 81272808, 81172431, 81101935, 81402106), and Guangdong Province Natural Scientific Foundation (Grant No. S2013020012671, 07117336, 10151008901000024), Specialized Research Fund for the Doctoral Program of Higher Education (for Tianxin Lin, 20130171110073), Sun Yat-Sen University Clinical Research 5010 Program (Grant No. 2007018), Elite Young Scholars Program of Sun Yat-Sen Memorial Hospital (for Tianxin Lin, J201401), and National Clinical Key Specialty Construcion Project for Department of Urology and Department of Oncology. Grant KLB09001 from the Key Laboratory of Malignant Tumor Gene Regulation and Target Therapy of Guangdong Higher Education Institutes, Sun-Yat-Sen University. Grant NO. 2014M562241 from China Postdoctoral Science Foundation.
MP45-13 N-ACETYLCYSTEINE-MEDIATED AUTOPHAGY INHIBITION LEADS TO CYTOCHROME-C INDEPENDENT CASPASE-9 ACTIVATION DURING OXIDATIVE STRESS IN UROTHELIAL CARCINOMA OF URINARY BLADDER Rani Ojha, Shrawan K. Singh*, Vivekanand Jha, Chandigarh, India INTRODUCTION AND OBJECTIVES: To investigate the effect of oxidative stress-induced autophagy on cell survival in different grades of urothelial carcinoma cells (UCC).
Source of Funding: Education and Research Cell, PGIMER, Chandigarh Indian Council of Medical Research (ICMR), New Delhi, India