- Email: [email protected]
MP90-01 SERUM CHOLESTEROL LEVELS IMPACT TUMOR PROGRESSION IN A PTEN-DEFICIENT TRANSGENIC MOUSE MODEL OF PROSTATE CANCER
THE JOURNAL OF UROLOGYâ
e1144
Vol. 195, No. 4S, Supplement, Tuesday, May 10, 2016
MP89-19
MP89-20
ALTERED CAVEOLIN EXPRESSION IN DIABETIC PENIS: POSSIBLE ROLE IN ERECTILE DYSFUNCTION
TRACTION APPLIED TO PEYRONIE’S DISEASE CELLS REDUCES CELLULAR FIBROSIS
M. Raj Rajasekaran*, Johnny Fu, Adam Kassan, Alice Zemljic-Harpf, Valmik Bhargava, San Diego, CA; Karnam S Murthy, Richmond, VA; Hemal Patel, San Diego, CA
Jeffrey Campbell*, Ling DeYoung, Eric Chung, Gerald Brock, London, Canada
INTRODUCTION AND OBJECTIVES: Pathophysiology of increased severity of erectile dysfunction (ED) in diabetic men and their poor response to phosphodiesterase 5 (PDE5) inhibitors are unclear. A defective vascular endothelium and consequent impairment in formation and/or action of nitric oxide (NO) are implicated as possible causes. Endothelial NO synthase (eNOS) is localized to caveolae (plasma membrane lipid rafts). Caveolins (Cav), structural components of caveolae, are recognized to play a regulatory role in the vascular complications of diabetes. Our objectives are to: (i) evaluate changes in Cav-1 and Cav-3 expression in penile tissue of type 2 diabetes (T2DM) mice and (ii) measure impact of Cav-1 deletion on penile PDE5 activity. METHODS: We used wild type C57BL6, Cav-1 and Cav-3 KO male mice (n¼6 each). To create T2DM mouse model, C57BL6 (15-18 months) male mice were injected with a single dose streptozotocin (non-fasted, 75 mg/kg i.p.) and switched to high fat diet (HFD; 60% kcal of total calories). Two months after HFD, serum glucose and GTT (glucose tolerance test) were performed to confirm metabolic disorder. Animals were maintained on HFD for 4 months and euthanized to harvest penile tissues. Expression levels of Cav-1 and Cav-3 were determined by Western blot and PDE5 activity was measured using [3H]cGMP as the substrate. RESULTS: A significant increase in body weight and impairment in GTT were observed in HFD-fed mice (Fig 1). A significant decrease in both Cav-1 and Cav-3 levels were observed in T2DM mice (D1-3) compared to WT mice (C1-2; Fig 2A). Stimulation of PDE5 activity in response to NO donor, GSNO was significantly higher (~2 fold) in Cav1- KO (Fig 2B) compared to WT mice. CONCLUSIONS: Altered penile Cav-1 levels in T2DM mice and increased PDE5 activity after Cav-1 depletion suggests a regulatory role for Cav-1 in diabetes related ED. Targeting caveolins may be a novel approach to improve pharmacotherapeutic response in T2DM patients.
INTRODUCTION AND OBJECTIVES: Peyronie’s disease (PD) is a fibrotic condition of penile tunica albuginea (TA). Unfortunately, currently available medical management strategies for PD lack efficacy. Penile traction devices (PTD) have been gaining popularity as an early treatment option, however their exact mechanism remains poorly understood. The dense plaques identified in PD result from abnormal wound healing caused by an imbalance of fibrosis and fibrinolysis. Matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) play a central role in the regulation of this dynamic process. To investigate the pathophysiology underlying the reported success of PTDs we used a pressurized chamber system that allows for application of hydrostatic pressure with or without additional tension. We examined the concentrations of inflammatory cytokines expressed in cell culture media of human PD cells and control TA cells, and then applied tensile forces to simulate PTD for comparison. METHODS: Human tissue samples from nine PD plaques and six normal TA where collected intraoperatively. The third passage of the primary cell cultures of PD and controls were sub-cultured and their media was collected and analyzed. Sinusoidal tractional force was applied to the media for 24 hours using the Flexcellâ cyclic strain culture system. We measured concentration of cytokines involved in the pathophysiology of PD: Interleukin-6 (IL-6), MMP 1, 2, 3, 7, 8, 9, 10, 11, and 12; TIMP 1, 2, 3, and 4; and TGF b1, 2 and 3. We compared PD cell concentrations of these proteins before and after traction was applied. RESULTS: Significantly lower concentrations of MMP 2, 12 and 13 and significantly higher concentrations of TIMP 1 and 2 were measured in PD cell media compared to control TA cells. After traction was applied, there was a significant increase in MMP 2 and 13, and significant decrease in TIMP 2 concentrations of PD cells. Traction had no effect on control cells. There were no significant differences in TGF-b or IL-6 concentrations. CONCLUSIONS: A fine balance between the level of MMPs and TIMPs control the extent of local collagen deposition and plaque formation in PD. Tensile force applied to human PD cells increases the concentration of fibrinolytic MMPs and decreases the concentration of profibrotic TIMPs. In our unique study, the equalization of these important fibrosis mediators provides strong pathophysiologic evidence for the use of PTDs in PD plaque remodelling. Source of Funding: None
Prostate Cancer: Basic Research & Pathophysiology IV Moderated Poster Tuesday, May 10, 2016
10:30 AM-12:30 PM
MP90-01 SERUM CHOLESTEROL LEVELS IMPACT TUMOR PROGRESSION IN A PTEN-DEFICIENT TRANSGENIC MOUSE MODEL OF PROSTATE CANCER
Source of Funding: None
Emma Allott, Chapel Hill, NC; Elizabeth Masko, Durham, NC; Everardo Macias, Los Angeles, CA; Kristine Pelton, Keith Solomon, Boston, MA; Elahe Mostaghel, Seattle, WA; George Thomas, Portland, OR; Michael Freeman, Los Angeles, CA; Salvadore Pizzo, Durham, NC; Stephen Freedland*, Los Angeles, CA INTRODUCTION AND OBJECTIVES: Epidemiologic evidence supports a role for cholesterol, the precursor for steroid hormone
THE JOURNAL OF UROLOGYâ
Vol. 195, No. 4S, Supplement, Tuesday, May 10, 2016
synthesis, in prostate cancer progression. We determined the impact of modifying serum cholesterol levels on tumor progression in PTENdeficient transgenic mouse model of prostate cancer. METHODS: PTENloxP/loxP-Cre+ mice consuming ad libitum high fat, high cholesterol diets (40% fat, 1.25% cholesterol) were randomized after weaning to receive the cholesterol uptake inhibitor, ezetimibe (Zetia; 30 mg/kg diet), or no intervention, and sacrificed at 2, 3, or 4 months of age. At sacrifice, prostates were removed, weighed, and fixed in formalin. Serum cholesterol and testosterone levels were measured by ELISA and prostate androgens measured by mass spectrometry. Proliferation and apoptosis in tumor epithelium and stroma was assessed by Ki67 and TUNEL assay, respectively. RESULTS: Mice randomized to Zetia had lower serum cholesterol levels (p¼0.031). Cholesterol levels were positively correlated with prostate weight (p¼0.033) and tumor epithelial proliferation (p¼0.069), and negatively correlated with tumor epithelial apoptosis (p¼0.004). Tumor stromal cell proliferation was reduced in the Zetia group (p¼0.010). Increased cholesterol levels were associated with elevated intraprostatic DHEA, testosterone and androstendione (p¼0.043, p¼0.074, p¼0.031, respectively) but was unrelated to serum androgens (p¼0.08). Mice consuming Zetia diet had lower rates of intracystic adenocarcinoma at 2 and 3 months of age (0% vs. 14% and 30% vs. 44%, respectively). CONCLUSIONS: Using a PTEN-deficient transgenic mouse model of prostate cancer, we found that serum cholesterol reduction lowered intraprostatic androgen concentrations and slowed prostate cancer progression, supporting a potentially important role for cholesterol in prostate cancer. This study may provide new insight into the use of cholesterol-lowering interventions for prostate cancer prevention and treatment. Source of Funding: 1-R01-CA131235-01A1, 1K24CA160653
MP90-02 CARBOHYDRATE RESTRICTION OPPOSES TUMOR-PROMOTING EFFECTS OF OBESITY IN THE HI-MYC TRANSGENIC MOUSE MODEL OF PROSTATE CANCER. Emma Allott, Chapel Hill, NC; Everardo Macias, Los Angeles, CA; George Thomas, Portland, OR; Stephen Hursting, Chapel Hill, NC; Stephen Freedland*, Los Angeles, CA INTRODUCTION AND OBJECTIVES: Previously, we showed carbohydrate restriction slowed tumor growth in xenograft mouse prostate cancer models. Herein, we examined the impact of carbohydrate restriction on tumor progression within the context of dietinduced obesity in the Hi-Myc transgenic mouse model of prostate cancer. METHODS: At weaning, Hi-myc male transgenic mice were randomized to Western diet (WD; 40% fat, 42% carbohydrate; n¼39) or no carbohydrate ketogenic diet (NCKD; 82% fat, 1% carbohydrate; n¼44) for 3 or 6 months. At sacrifice, prostates were weighed and prostate histology, proliferation and apoptosis were evaluated by H&E, Ki67 and TUNEL, respectively. Body composition and bone mineral content were assessed by DEXA, serum cytokines measured using multiplex, and Akt/mTOR signaling assessed by Western blotting. RESULTS: Caloric intake was higher in the NCKD group, resulting in elevated body weights at 6 months of age (45.3g vs. 38.9g; p¼0.008). While mice randomized to NCKD had higher rates of adenocarcinoma at 6 months (100% vs. 80%, p¼0.04), they had significantly smaller prostates at both 3 and 6 months, following adjustment for body weight (p¼0.004 and p¼0.002, respectively). Despite elevated body weights, serum MCP-1 and IL-1a levels were lower in the NCKD group (p¼0.046 and p¼0.118, respectively) and relative Akt phosphorylation and phospho-S6 ribosomal protein levels were reduced in prostates of NCKD mice.
e1145
CONCLUSIONS: Despite higher caloric intake and elevated body weights, carbohydrate restriction reduced prostate weight, lowered MCP-1 levels, and downstream markers of mTOR/Akt activity. Together, these data suggest that carbohydrate restriction may reduce systemic inflammation and oppose the tumor-promoting effects of obesity, resulting in slower prostate cancer growth, possibility through reduced mTOR/Akt activity. Source of Funding: 1-R01-CA131235-01A1, 1K24CA160653
MP90-03 REACTIVE OXYGEN SPECIES INDUCTION BY CABAZITAXEL VIA INHIBITION OF THE SESTRIN FAMILY IN CASTRATIONRESISTANT PROSTATE CANCER Takeo Kosaka*, Hiroshi Hongo, Yasumasa Miyazaki, Eiji Kikuchi, Akira Miyajima, Mototsugu Oya, Tokyo, Japan INTRODUCTION AND OBJECTIVES: Cabazitaxel (CBZ) has become the most effective cytotoxic agent to demonstrate an improvement in survival in men with docetaxel-refractory CRPC. Accumulating evidence has suggested reactive oxygen species (ROS) production induced by taxanes in cancer cells may influence taxaneinduced cell death or drug resistance. However, ROS production by CBZ or its mechanistic action remains to be characterized in CRPC. In this study, we investigated PCa cell sensitivity to docetaxel (DTX) or CBZ by measurement of ROS production. METHODS: Three cell lines were used: LNCaP, a human PCa cell line that exhibits androgen-dependent proliferation; C4-2, a human PCa cell line that exhibits androgen-independent proliferation; and C42AT6, a CRPC cell line derived from C4-2. These cells were treated with increasing concentrations of docetaxel or CBZ in vitro. Gene expression profiles of CBZ- or DTX-treated cells were analyzed using a whole human genome microarray. FACS analysis was performed using a fluorogenic marker of ROS. Furthermore, we investigated the changes in expression of antioxidant enzymes induced by DTX and CBZ. RESULTS: Cabazitaxel showed significantly higher cytotoxicity than docetaxel in human CRPC cells. FACS analysis revealed that treatment with cabazitaxel significantly increased ROS generation than docetaxel in C4-2AT6 cells, accompanied by higher expression of phospho-p38. Microarray analysis revealed significant changes in ROS regulation-related genes by CBZ. qPCR analysis revealed CBZ induced C4-2AT6 cell death, accompanied by significantly decreased expression of Sestrin-3 (SESN3). To investigate whether CBZ-mediated cell death was caused by induction of ROS generation, C4-2AT6 cells were incubated with CBZ in the presence of the antioxidant N-acetylcysteine (NAC), which was observed to reduce cabazitaxel-induced cytotoxicity. Microarray analysis revealed dynamic changes in ROS regulation-related genes by CBZ, and qPCR analysis demonstrated CBZ reduced mRNA expression of SESN3. We observed that ROS reduction by SESN3 was significantly inhibited by cabazitaxel in a dose-dependent manner, but not by docetaxel. When C4-2AT6 cells were treated with cabazitaxel in the presence of siRNAs against SESN3, significant cytotoxicity was observed, accompanied by enhanced ROS production. CONCLUSIONS: Our results indicated that the higher sensitivity of human CRPC to CBZ, compared with DTX, involves ROS production via inhibition of SESN3 expression, an antioxidant enzyme. Source of Funding: This work was supported in part by a Grant-in-Aid for Young Scientists from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.
e1144
Vol. 195, No. 4S, Supplement, Tuesday, May 10, 2016
MP89-19
MP89-20
ALTERED CAVEOLIN EXPRESSION IN DIABETIC PENIS: POSSIBLE ROLE IN ERECTILE DYSFUNCTION
TRACTION APPLIED TO PEYRONIE’S DISEASE CELLS REDUCES CELLULAR FIBROSIS
M. Raj Rajasekaran*, Johnny Fu, Adam Kassan, Alice Zemljic-Harpf, Valmik Bhargava, San Diego, CA; Karnam S Murthy, Richmond, VA; Hemal Patel, San Diego, CA
Jeffrey Campbell*, Ling DeYoung, Eric Chung, Gerald Brock, London, Canada
INTRODUCTION AND OBJECTIVES: Pathophysiology of increased severity of erectile dysfunction (ED) in diabetic men and their poor response to phosphodiesterase 5 (PDE5) inhibitors are unclear. A defective vascular endothelium and consequent impairment in formation and/or action of nitric oxide (NO) are implicated as possible causes. Endothelial NO synthase (eNOS) is localized to caveolae (plasma membrane lipid rafts). Caveolins (Cav), structural components of caveolae, are recognized to play a regulatory role in the vascular complications of diabetes. Our objectives are to: (i) evaluate changes in Cav-1 and Cav-3 expression in penile tissue of type 2 diabetes (T2DM) mice and (ii) measure impact of Cav-1 deletion on penile PDE5 activity. METHODS: We used wild type C57BL6, Cav-1 and Cav-3 KO male mice (n¼6 each). To create T2DM mouse model, C57BL6 (15-18 months) male mice were injected with a single dose streptozotocin (non-fasted, 75 mg/kg i.p.) and switched to high fat diet (HFD; 60% kcal of total calories). Two months after HFD, serum glucose and GTT (glucose tolerance test) were performed to confirm metabolic disorder. Animals were maintained on HFD for 4 months and euthanized to harvest penile tissues. Expression levels of Cav-1 and Cav-3 were determined by Western blot and PDE5 activity was measured using [3H]cGMP as the substrate. RESULTS: A significant increase in body weight and impairment in GTT were observed in HFD-fed mice (Fig 1). A significant decrease in both Cav-1 and Cav-3 levels were observed in T2DM mice (D1-3) compared to WT mice (C1-2; Fig 2A). Stimulation of PDE5 activity in response to NO donor, GSNO was significantly higher (~2 fold) in Cav1- KO (Fig 2B) compared to WT mice. CONCLUSIONS: Altered penile Cav-1 levels in T2DM mice and increased PDE5 activity after Cav-1 depletion suggests a regulatory role for Cav-1 in diabetes related ED. Targeting caveolins may be a novel approach to improve pharmacotherapeutic response in T2DM patients.
INTRODUCTION AND OBJECTIVES: Peyronie’s disease (PD) is a fibrotic condition of penile tunica albuginea (TA). Unfortunately, currently available medical management strategies for PD lack efficacy. Penile traction devices (PTD) have been gaining popularity as an early treatment option, however their exact mechanism remains poorly understood. The dense plaques identified in PD result from abnormal wound healing caused by an imbalance of fibrosis and fibrinolysis. Matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) play a central role in the regulation of this dynamic process. To investigate the pathophysiology underlying the reported success of PTDs we used a pressurized chamber system that allows for application of hydrostatic pressure with or without additional tension. We examined the concentrations of inflammatory cytokines expressed in cell culture media of human PD cells and control TA cells, and then applied tensile forces to simulate PTD for comparison. METHODS: Human tissue samples from nine PD plaques and six normal TA where collected intraoperatively. The third passage of the primary cell cultures of PD and controls were sub-cultured and their media was collected and analyzed. Sinusoidal tractional force was applied to the media for 24 hours using the Flexcellâ cyclic strain culture system. We measured concentration of cytokines involved in the pathophysiology of PD: Interleukin-6 (IL-6), MMP 1, 2, 3, 7, 8, 9, 10, 11, and 12; TIMP 1, 2, 3, and 4; and TGF b1, 2 and 3. We compared PD cell concentrations of these proteins before and after traction was applied. RESULTS: Significantly lower concentrations of MMP 2, 12 and 13 and significantly higher concentrations of TIMP 1 and 2 were measured in PD cell media compared to control TA cells. After traction was applied, there was a significant increase in MMP 2 and 13, and significant decrease in TIMP 2 concentrations of PD cells. Traction had no effect on control cells. There were no significant differences in TGF-b or IL-6 concentrations. CONCLUSIONS: A fine balance between the level of MMPs and TIMPs control the extent of local collagen deposition and plaque formation in PD. Tensile force applied to human PD cells increases the concentration of fibrinolytic MMPs and decreases the concentration of profibrotic TIMPs. In our unique study, the equalization of these important fibrosis mediators provides strong pathophysiologic evidence for the use of PTDs in PD plaque remodelling. Source of Funding: None
Prostate Cancer: Basic Research & Pathophysiology IV Moderated Poster Tuesday, May 10, 2016
10:30 AM-12:30 PM
MP90-01 SERUM CHOLESTEROL LEVELS IMPACT TUMOR PROGRESSION IN A PTEN-DEFICIENT TRANSGENIC MOUSE MODEL OF PROSTATE CANCER
Source of Funding: None
Emma Allott, Chapel Hill, NC; Elizabeth Masko, Durham, NC; Everardo Macias, Los Angeles, CA; Kristine Pelton, Keith Solomon, Boston, MA; Elahe Mostaghel, Seattle, WA; George Thomas, Portland, OR; Michael Freeman, Los Angeles, CA; Salvadore Pizzo, Durham, NC; Stephen Freedland*, Los Angeles, CA INTRODUCTION AND OBJECTIVES: Epidemiologic evidence supports a role for cholesterol, the precursor for steroid hormone
THE JOURNAL OF UROLOGYâ
Vol. 195, No. 4S, Supplement, Tuesday, May 10, 2016
synthesis, in prostate cancer progression. We determined the impact of modifying serum cholesterol levels on tumor progression in PTENdeficient transgenic mouse model of prostate cancer. METHODS: PTENloxP/loxP-Cre+ mice consuming ad libitum high fat, high cholesterol diets (40% fat, 1.25% cholesterol) were randomized after weaning to receive the cholesterol uptake inhibitor, ezetimibe (Zetia; 30 mg/kg diet), or no intervention, and sacrificed at 2, 3, or 4 months of age. At sacrifice, prostates were removed, weighed, and fixed in formalin. Serum cholesterol and testosterone levels were measured by ELISA and prostate androgens measured by mass spectrometry. Proliferation and apoptosis in tumor epithelium and stroma was assessed by Ki67 and TUNEL assay, respectively. RESULTS: Mice randomized to Zetia had lower serum cholesterol levels (p¼0.031). Cholesterol levels were positively correlated with prostate weight (p¼0.033) and tumor epithelial proliferation (p¼0.069), and negatively correlated with tumor epithelial apoptosis (p¼0.004). Tumor stromal cell proliferation was reduced in the Zetia group (p¼0.010). Increased cholesterol levels were associated with elevated intraprostatic DHEA, testosterone and androstendione (p¼0.043, p¼0.074, p¼0.031, respectively) but was unrelated to serum androgens (p¼0.08). Mice consuming Zetia diet had lower rates of intracystic adenocarcinoma at 2 and 3 months of age (0% vs. 14% and 30% vs. 44%, respectively). CONCLUSIONS: Using a PTEN-deficient transgenic mouse model of prostate cancer, we found that serum cholesterol reduction lowered intraprostatic androgen concentrations and slowed prostate cancer progression, supporting a potentially important role for cholesterol in prostate cancer. This study may provide new insight into the use of cholesterol-lowering interventions for prostate cancer prevention and treatment. Source of Funding: 1-R01-CA131235-01A1, 1K24CA160653
MP90-02 CARBOHYDRATE RESTRICTION OPPOSES TUMOR-PROMOTING EFFECTS OF OBESITY IN THE HI-MYC TRANSGENIC MOUSE MODEL OF PROSTATE CANCER. Emma Allott, Chapel Hill, NC; Everardo Macias, Los Angeles, CA; George Thomas, Portland, OR; Stephen Hursting, Chapel Hill, NC; Stephen Freedland*, Los Angeles, CA INTRODUCTION AND OBJECTIVES: Previously, we showed carbohydrate restriction slowed tumor growth in xenograft mouse prostate cancer models. Herein, we examined the impact of carbohydrate restriction on tumor progression within the context of dietinduced obesity in the Hi-Myc transgenic mouse model of prostate cancer. METHODS: At weaning, Hi-myc male transgenic mice were randomized to Western diet (WD; 40% fat, 42% carbohydrate; n¼39) or no carbohydrate ketogenic diet (NCKD; 82% fat, 1% carbohydrate; n¼44) for 3 or 6 months. At sacrifice, prostates were weighed and prostate histology, proliferation and apoptosis were evaluated by H&E, Ki67 and TUNEL, respectively. Body composition and bone mineral content were assessed by DEXA, serum cytokines measured using multiplex, and Akt/mTOR signaling assessed by Western blotting. RESULTS: Caloric intake was higher in the NCKD group, resulting in elevated body weights at 6 months of age (45.3g vs. 38.9g; p¼0.008). While mice randomized to NCKD had higher rates of adenocarcinoma at 6 months (100% vs. 80%, p¼0.04), they had significantly smaller prostates at both 3 and 6 months, following adjustment for body weight (p¼0.004 and p¼0.002, respectively). Despite elevated body weights, serum MCP-1 and IL-1a levels were lower in the NCKD group (p¼0.046 and p¼0.118, respectively) and relative Akt phosphorylation and phospho-S6 ribosomal protein levels were reduced in prostates of NCKD mice.
e1145
CONCLUSIONS: Despite higher caloric intake and elevated body weights, carbohydrate restriction reduced prostate weight, lowered MCP-1 levels, and downstream markers of mTOR/Akt activity. Together, these data suggest that carbohydrate restriction may reduce systemic inflammation and oppose the tumor-promoting effects of obesity, resulting in slower prostate cancer growth, possibility through reduced mTOR/Akt activity. Source of Funding: 1-R01-CA131235-01A1, 1K24CA160653
MP90-03 REACTIVE OXYGEN SPECIES INDUCTION BY CABAZITAXEL VIA INHIBITION OF THE SESTRIN FAMILY IN CASTRATIONRESISTANT PROSTATE CANCER Takeo Kosaka*, Hiroshi Hongo, Yasumasa Miyazaki, Eiji Kikuchi, Akira Miyajima, Mototsugu Oya, Tokyo, Japan INTRODUCTION AND OBJECTIVES: Cabazitaxel (CBZ) has become the most effective cytotoxic agent to demonstrate an improvement in survival in men with docetaxel-refractory CRPC. Accumulating evidence has suggested reactive oxygen species (ROS) production induced by taxanes in cancer cells may influence taxaneinduced cell death or drug resistance. However, ROS production by CBZ or its mechanistic action remains to be characterized in CRPC. In this study, we investigated PCa cell sensitivity to docetaxel (DTX) or CBZ by measurement of ROS production. METHODS: Three cell lines were used: LNCaP, a human PCa cell line that exhibits androgen-dependent proliferation; C4-2, a human PCa cell line that exhibits androgen-independent proliferation; and C42AT6, a CRPC cell line derived from C4-2. These cells were treated with increasing concentrations of docetaxel or CBZ in vitro. Gene expression profiles of CBZ- or DTX-treated cells were analyzed using a whole human genome microarray. FACS analysis was performed using a fluorogenic marker of ROS. Furthermore, we investigated the changes in expression of antioxidant enzymes induced by DTX and CBZ. RESULTS: Cabazitaxel showed significantly higher cytotoxicity than docetaxel in human CRPC cells. FACS analysis revealed that treatment with cabazitaxel significantly increased ROS generation than docetaxel in C4-2AT6 cells, accompanied by higher expression of phospho-p38. Microarray analysis revealed significant changes in ROS regulation-related genes by CBZ. qPCR analysis revealed CBZ induced C4-2AT6 cell death, accompanied by significantly decreased expression of Sestrin-3 (SESN3). To investigate whether CBZ-mediated cell death was caused by induction of ROS generation, C4-2AT6 cells were incubated with CBZ in the presence of the antioxidant N-acetylcysteine (NAC), which was observed to reduce cabazitaxel-induced cytotoxicity. Microarray analysis revealed dynamic changes in ROS regulation-related genes by CBZ, and qPCR analysis demonstrated CBZ reduced mRNA expression of SESN3. We observed that ROS reduction by SESN3 was significantly inhibited by cabazitaxel in a dose-dependent manner, but not by docetaxel. When C4-2AT6 cells were treated with cabazitaxel in the presence of siRNAs against SESN3, significant cytotoxicity was observed, accompanied by enhanced ROS production. CONCLUSIONS: Our results indicated that the higher sensitivity of human CRPC to CBZ, compared with DTX, involves ROS production via inhibition of SESN3 expression, an antioxidant enzyme. Source of Funding: This work was supported in part by a Grant-in-Aid for Young Scientists from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.