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Enhanced Sensitivity to Sunitinib by Inhibition of Akt1 Expression in Human Castration- resistant Prostate Cancer PC3 Cells Both In Vitro and In Vivo 

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Presentation on theme: "Enhanced Sensitivity to Sunitinib by Inhibition of Akt1 Expression in Human Castration- resistant Prostate Cancer PC3 Cells Both In Vitro and In Vivo "— Presentation transcript:

1 Enhanced Sensitivity to Sunitinib by Inhibition of Akt1 Expression in Human Castration- resistant Prostate Cancer PC3 Cells Both In Vitro and In Vivo  Masatomo Nishikawa, Hideaki Miyake, Masato Fujisawa  Urology  Volume 85, Issue 5, Pages 1215.e e7 (May 2015) DOI: /j.urology Copyright © 2015 Elsevier Inc. Terms and Conditions

2 Figure 1 (A) Expression levels of Akt1 in PC3 sublines (PC3/P, parental cell line; PC3/C, control vector–only transfected cell line; PC3/sh-Akt1#1 to #4, Akt1 shRNA–transfected cell lines). Protein was extracted from each cell line, and Western blotting was performed to analyze the expression levels of Akt1 and β-actin in PC3 sublines. (B) In vitro cell growth of PC3 sublines. The in vitro proliferation of PC3/C and PC3/sh-Akt1#2 was measured daily in triplicate using the MTT assay. Bars, standard deviation. (C) Effects of the treatment with sunitinib on the in vitro cell growth of PC3 sublines. PC3/C and PC3/sh-Akt1#2 were treated with the indicated doses of sunitinib. After 48 hours of incubation, cell growth was determined in triplicate using the MTT assay in 3 independent experiments. *Significantly different from PC3/C (P <.05). Bars, standard deviation. (D) Effects of the treatment with sunitinib on the in vitro migration ability of the PC3 sublines. PC3/C and PC3/sh-Akt1#2 were treated with sunitinib. After 12 hours of incubation, migration ability was determined in triplicate by using the gap closure assay in 3 independent experiments. Bars, standard deviation. *Significantly different from PC3/C (P <.05). (E) Effects of the treatment with sunitinib on the in vitro invasive ability of the PC3 sublines. PC3/C and PC3/sh-Akt1#2 were treated with sunitinib. After 48 hours of incubation, invasive ability was determined in triplicate using the in vitro tumor cell invasion assay in 3 independent experiments. Bars, standard deviation. Urology  , 1215.e e7DOI: ( /j.urology ) Copyright © 2015 Elsevier Inc. Terms and Conditions

3 Figure 2 (A) Changes in the expression patterns of key molecules involved in signal transduction in PC3 sublines after the treatment with sunitinib. Expression levels of p-Akt1, Akt2, Akt3 total and p-mammalian target of rapamycin, total and p-p44/42 mitogen-activated protein kinase, and hypoxia inducible factor 1α (HIF-1α) in PC3 sublines before and after the treatment with sunitinib were analyzed by Western blotting. (B) Changes in the expression patterns of key molecules involved in apoptosis in PC3 sublines after the treatment with sunitinib. Expression levels of Bcl-2, Bcl-xL, Bax, clusterin, and β-actin in PC3 sublines before and after the treatment with sunitinib were analyzed by Western blotting. HIF-1α, hypoxia inducible factor 1α; MAPK, mitogen-activated protein kinase; mTOR, mammalian target of rapamycin. Urology  , 1215.e e7DOI: ( /j.urology ) Copyright © 2015 Elsevier Inc. Terms and Conditions

4 Figure 3 Effects of the treatment with sunitinib on the in vivo growth of PC3 sublines. Twenty nude mice were subcutaneously given 5 × 106 cells of each PC3 subline, then randomly selected for treatment with either 40 mg/kg of sunitinib or vehicle 4 times per week for 4 weeks. The subcutaneous tumor volume was measured using calipers. Bars, standard deviation. ** and *significantly different from PC3/C (P <.01 and P <.05, respectively). Urology  , 1215.e e7DOI: ( /j.urology ) Copyright © 2015 Elsevier Inc. Terms and Conditions

5 Figure 4 Histopathologic study of PC3 tumors after treatment with sunitinib. In vivo subcutaneous tumors were harvested from nude mice undergoing the treatment with sunitinib or vehicle for 8 weeks according to the schedule described in Figure 3. Sections from each tumor tissue were examined by TUNEL staining and immunohistochemical staining with antibodies against Ki-67, CD31, p-p44/42 mitogen-activated protein kinase, Bcl-2, and Bax. MAPK, mitogen-activated protein kinase; MTT, Thiazolyl Blue Tetrazolium Bromide; TUNEL, TdT-mediated dUTP nick end labeling. Urology  , 1215.e e7DOI: ( /j.urology ) Copyright © 2015 Elsevier Inc. Terms and Conditions

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