ATC Cells Research Articles

Enhanced cytotoxic effect of camptothecin nanosponges in anaplastic thyroid cancer cells in vitro and in vivo on orthotopic xenograft tumors

Anaplastic carcinoma of the thyroid (ATC) is a lethal human malignant cancer with median survival of 6 months. To date, no treatment has substantially changed its course, which makes urgent need for the development of novel drugs or novel formulations for drug delivery. Nanomedicine has enormous potential to improve the accuracy of cancer therapy by enhancing availability and stability, decreasing effective doses and reducing side effects of drugs.Camptothecin (CPT) is an inhibitor of DNA topoisomerase-I with several anticancer properties but has poor solubility and a high degradation rate. Previously, we reported that CPT encapsulated in β-cyclodextrin-nanosponges (CN-CPT) increased solubility, was protected from degradation and inhibited the growth of prostate tumor cells both in vitro and in vivo. The aim of this study was to extend that work by assessing the CN-CPT effectiveness on ATC both in vitro and in vivo.Results showed that CN-CPT significantly inhibited viability, clonogenic capacity and cell-cycle progression of ATC cell lines showing a faster and enhanced effect compared to free CPT. Moreover, CN-CPT inhibited tumor cell adhesion to vascular endothelial cells, migration, secretion of pro-angiogenic factors (IL-8 and VEGF-α), expression of β-PIX, belonging to the Rho family activators, and phosphorylation of the Erk1/2 MAPK.Finally, CN-CPT significantly inhibited the growth, the metastatization and the vascularization of orthotopic ATC xenografts in SCID/beige mice without apparent toxic effects in vivo. This work extends the previous insight showing that β-cyclodextrin-nanosponges are a promising tool for the treatment of ATC.

Stage-Specific Embryonic Antigen-1 (SSEA-1) Expression in Thyroid Tissues.

Stage-specific embryonic antigen-1 (SSEA-1), also known as CD15, is a member of a cluster of differentiation antigens that have been identified in various normal tissues and in different types of cancers including papillary and medullary thyroid carcinoma. SSEA-1 may be expressed in normal stem cells and cancer stem-like cells. To evaluate the potential diagnostic and prognostic utility of SSEA-1 in thyroid tumors, we analyzed the expression of SSEA-1 in normal and neoplastic thyroid tissues by immunohistochemistry (IHC) using a tissue microarray with 158 different tissue cores. To evaluate the potential utility of SSEA-1 as a surface marker, we also assessed the expression of SSEA-1 in thyroid cell lines by flow cytometric analysis. SSEA-1 immunoreactivity was identified in malignant thyroid follicular epithelial cancers but not in the benign thyroid tissues. Anaplastic thyroid (ATC) (80%) and conventional papillary thyroid carcinoma (PTC) (60.7%) showed significantly higher percentage of cases that were SSEA-1 immunoreactive than follicular variant of papillary thyroid carcinoma (FVPTC) (20.6%) and follicular carcinoma (FCA) (32.1%). Flow cytometric analysis of cultured thyroid cell lines showed that a small subpopulation of ATC and PTC thyroid tumor cells had SSEA-1 immunoreactivity which may represent thyroid cancer stem-like cells. The ATC cells expressed more SSEA-1 immunoreactive cells than the PTC cell lines. Our findings suggest that expression of SSEA-1 immunoreactivity in thyroid neoplasms was associated with more aggressive thyroid carcinomas. SSEA-1 is a marker that detects malignant thyroid neoplasms in formalin-fixed paraffin-embedded thyroid tissue sections and may be a useful marker for thyroid cancer stem-like cells.

Recombinant human adenovirus p53 injection (rAd-p53) is a challenge for anaplastic thyroid cancer treatment.

e17561Background: to evaluate the activity and mechanism of rAd-p53 combined with chemotherapeutics in treatment of ATC in vitro and in vivoand clinical therapies. Methods: p53-mutant ATC cells (C6...

MP84-20 BIOINFORMATIC ANALYSIS UTILIZING THE ANDROGENIC SUPPRESSIVE EFFECT IDENTIFIED PROMISING CANDIDATE DRUGS FOR REPROGRAMMING DOCETAXEL-RESISTANT CASTRATION-RESISTANT PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology III1 Apr 2016MP84-20 BIOINFORMATIC ANALYSIS UTILIZING THE ANDROGENIC SUPPRESSIVE EFFECT IDENTIFIED PROMISING CANDIDATE DRUGS FOR REPROGRAMMING DOCETAXEL-RESISTANT CASTRATION-RESISTANT PROSTATE CANCER Hiroshi Hongo, Takeo Kosaka, Yota Yasumizu, Yasumasa Miyazaki, Eiji Kikuchi, Akira Miyajima, and Mototsugu Oya Hiroshi HongoHiroshi Hongo More articles by this author , Takeo KosakaTakeo Kosaka More articles by this author , Yota YasumizuYota Yasumizu More articles by this author , Yasumasa MiyazakiYasumasa Miyazaki More articles by this author , Eiji KikuchiEiji Kikuchi More articles by this author , Akira MiyajimaAkira Miyajima More articles by this author , and Mototsugu OyaMototsugu Oya More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.2244AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Recent evidence suggests CRPC cells may have an unknown regulation system to protect themselves from the androgenic suppressive effect. In this study, we investigated gene expression profiles of androgen-treated cells using a whole human genome microarray, and identified candidate drugs for reprogramming of docetaxel-resistant castration-resistant prostate cancer. METHODS Two cell lines were used: LNCaP and C4-2AT6, a castration-resistant prostate cancer cell line. Gene expression profiles of androgen-treated cells were analyzed and compared using CMAP to identify candidate drugs with the potential to revert an inverse gene signature pattern. RESULTS We investigated and compared the viability of prostate cancer cells treated with DHT at various concentrations. When treated with 0.1 nM DHT, LNCaP cells demonstrated significantly increased cell viability. On the other hand, C4-2AT6 cells showed significantly decreased cell viability when treated with 0.1 nM DHT. We next investigated and compared the gene signatures between LNCaP and C4-2AT6 cells treated with 0.1 nM DHT. CMAP analysis identified candidate drugs for reprogramming the gene expression changes. Based on the CMAP analysis data, candidate drugs were screened and tested for anti-tumor activity. The cytotoxic effect of docetaxel was first investigated by a cell viability assay in LNCaP and C4-2AT6 cells; relative cell viability when treated with 1nM docetaxel was 48.8 ± 0.1 % and 87.5 ± 1.9 %, respectively, suggesting increased docetaxel resistance in C4-2AT6 cells. Among the listed drugs, KOSH035 was identified as a candidate for reprogramming the castration resistance of docetaxel-resistant C4-2AT6 cells. We investigated the anti-tumor activity of KOSH035 on LNCaP and C4-2AT6 cells, and observed that relative cell viability was 72.2 ± 1.2 % and 72.8 ± 4.4 %, respectively. We further investigated the synergic effect of docetaxel and KOSH035. Relative cell viabilities of LNCaP and C4-2AT6 cells after treatment with both 1nM docetaxel and 10uM KOSH035 were 51.4 ± 0.5 % and 68.1 ± 2.0 %, respectively. The combination of docetaxel and KOSH035 showed a synergic effect on CRPC cells. CONCLUSIONS KOSH035 may be promising drug for reprogramming docetaxel-resistant PC. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e1097 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Hiroshi Hongo More articles by this author Takeo Kosaka More articles by this author Yota Yasumizu More articles by this author Yasumasa Miyazaki More articles by this author Eiji Kikuchi More articles by this author Akira Miyajima More articles by this author Mototsugu Oya More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

MP90-03 REACTIVE OXYGEN SPECIES INDUCTION BY CABAZITAXEL VIA INHIBITION OF THE SESTRIN FAMILY IN CASTRATION-RESISTANT PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology IV1 Apr 2016MP90-03 REACTIVE OXYGEN SPECIES INDUCTION BY CABAZITAXEL VIA INHIBITION OF THE SESTRIN FAMILY IN CASTRATION-RESISTANT PROSTATE CANCER Takeo Kosaka, Hiroshi Hongo, Yasumasa Miyazaki, Eiji Kikuchi, Akira Miyajima, and Mototsugu Oya Takeo KosakaTakeo Kosaka More articles by this author , Hiroshi HongoHiroshi Hongo More articles by this author , Yasumasa MiyazakiYasumasa Miyazaki More articles by this author , Eiji KikuchiEiji Kikuchi More articles by this author , Akira MiyajimaAkira Miyajima More articles by this author , and Mototsugu OyaMototsugu Oya More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.2547AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail 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 taxane-induced 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 C4-2AT6, 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. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e1145 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Takeo Kosaka More articles by this author Hiroshi Hongo More articles by this author Yasumasa Miyazaki More articles by this author Eiji Kikuchi More articles by this author Akira Miyajima More articles by this author Mototsugu Oya More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Characterization of thyroid cancer cell lines in murine orthotopic and intracardiac metastasis models.

Thyroid cancer incidence has been increasing over time, and it is estimated that ∼1950 advanced thyroid cancer patients will die of their disease in 2015. To combat this disease, an enhanced understanding of thyroid cancer development and progression as well as the development of efficacious, targeted therapies are needed. In vitro and in vivo studies utilizing thyroid cancer cell lines and animal models are critically important to these research efforts. In this report, we detail our studies with a panel of authenticated human anaplastic and papillary thyroid cancer (ATC and PTC) cell lines engineered to express firefly luciferase in two in vivo murine cancer models-an orthotopic thyroid cancer model as well as an intracardiac injection metastasis model. In these models, primary tumor growth in the orthotopic model and the establishment and growth of metastases in the intracardiac injection model are followed in vivo using an IVIS imaging system. In the orthotopic model, the ATC cell lines 8505C and T238 and the PTC cell lines K1/GLAG-66 and BCPAP had take rates >90 % with final tumor volumes ranging 84-214 mm(3) over 4-5 weeks. In the intracardiac model, metastasis establishment was successful in the ATC cell lines HTh74, HTh7, 8505C, THJ-16T, and Cal62 with take rates ≥70 %. Only one of the PTC cell lines tested (BCPAP) was successful in the intracardiac model with a take rate of 30 %. These data will be beneficial to inform the choice of cell line and model system for the design of future thyroid cancer studies.

Abstract 4367: Tumor suppressor miR-30a inhibits LOX expression and progression in anaplastic thyroid cancer

Abstract one of the most aggressive human malignancies with a median survival time of less than one year. A better understanding of the molecular events involved in ATC initiation and progression that could be targeted for therapy is gravely needed. There is limited data on the role of miRNAs in ATC. Screening of miRNA expression profile in thyroid cancer samples showed significant downregulation of miR-30a in ATC compared to differentiated thyroid cancer and normal tissue. Ectopic expression of miR-30a in vitro decreased invasion and migration of ATC cells with downregulation of epithelial-mesenchymal transition (EMT) markers and Lysyl Oxidase (LOX) expression, a predicted target of miR-30a. Using an in vivo mouse model of ATC metastases, miR-30a significantly decreased lung metastasis with decreased vimentin, CD44 and LOX expression in lung metastases tumor tissue. We also found that LOX and its downstream target TWIST-1 are highly expressed in ATC, and the expression of both were significantly, inversely correlated with miR-30a expression (p= 0.0026). In addition, the analysis of 454 primary thyroid cancer samples from The Cancer Genome Atlas dataset demonstrated upregulation of LOX and downregulation of miR-30a in high-risk thyroid cancer. These findings suggest that LOX and miR-30a are prognostic markers for aggressive thyroid cancer. To determine whether LOX is a direct effector of miR-30a, the 3′UTR region of LOX was cloned into a luciferase reporter plasmid. Transient transfection of 3′UTR-LOX along with miR-30a in ATC cell lines led to a decrease in luciferase activity when compared to the co-transfection of 3′UTR-LOX with negative control miRNA, demonstrating that LOX is a direct target of miR-30a. In vitro treatment of 4 ATC cell lines with β-aminoproprionitrile (BAPN), an irreversible inhibitor of LOX, had no effect on cell proliferation but inhibited cell invasion and migration and significantly decreased EMT markers and CD44 expression. Moreover, BAPN-treated mice with metastatic human ATC tumors had significantly fewer tumors and smaller size of lung metastases. Our data taken together support a critical role of miR-30a in ATC progression, which is mediated by LOX, and suggests that miR-30a and LOX is possible therapeutic target in ATC. Note: This abstract was not presented at the meeting. Citation Format: Myriem Boufraqech, Naris Nilubol, Lisa Zhang, Samira Sadowski, Sudheer Kumar Gara, Mei He, Sean Davis, Martha Quezado, Electron Kebebew. Tumor suppressor miR-30a inhibits LOX expression and progression in anaplastic thyroid cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4367. doi:10.1158/1538-7445.AM2014-4367

Limited in vitro efficacy of CYP17A1 inhibition on human castration resistant prostate cancer

Although accumulating evidence indicates high expression of CYP17A1(P45017A1) allows castration resistant prostate cancer (CRPC) to maintain high intratumoral androgen levels, the potential P45017A1 activity has not been characterized yet. The aim of this study was to examine the potential CYP17A1 activity including 17α-hydroxylase and 17,20-lyase activities in human CRPC and the effect of a CYP17A inhibitor. We used three human CRPC cell lines: C4-2 and C4-2AT6 which was established from C4-2 under androgen ablation conditions for 6months, and PC3. To ascertain the potential CYP17A1 activity, we cultured with the steroid precursors: 13C-[2,3,4]-progesterone (13C-Prog), and analyzed the sequential biosynthesis 13C-[2,3,4]-17-hydroxyprogesterone (13C-17OHP) and 13C-[2,3,4]-androstenedione(13C-Adione) by liquid chromatography/mass spectrometry (LC/MS/MS).The C4-2AT6 cells showed significantly higher CYP17A1 expression than C4-2 cells (p<0.001). LC/MS/MS analysis enabled us to detect the 13C-17-OHP and 13C-A-dione in these cell lines. The concentration ratio of 13C-Adione/13C-17OHP (Adione–17OHP ratio), which is thought to reflect the differences between 17-hydroxylase and 17,20-lyase activities, was then determined. The Adione–17OHP ratio in C4-2AT6 cells was significantly higher than that of C4-2 cells (p<0.001). Abiraterone were able to inhibit the CYP17A activities, although abiraterone did not have anti-proliferative effects on C4-2 and C4-2AT6 cells at clinically achievable concentrations of <1000nM in vitro. The present study clearly demonstrates CRPC have the dual activities of CYP17A1 mediated by 17-hydroxylase activity and 17,20-lyase activity. Abiraterone doesn’t have an in vitro anti-proliferative efficacy in CRPC cells, suggesting limited efficacy in vitro.

MP52-20 POST-TRANSLATIONAL MODIFICATION MEDIATED BY THE PROLYL ISOMERASE PIN1 ACCERELATE CANCER PROGRESSION IN CASTRATION RESISTANT PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Basic Research V1 Apr 2014MP52-20 POST-TRANSLATIONAL MODIFICATION MEDIATED BY THE PROLYL ISOMERASE PIN1 ACCERELATE CANCER PROGRESSION IN CASTRATION RESISTANT PROSTATE CANCER Yota Yasumizu, Takeo Kosaka, Yasumasa Miyazaki, Eiji Kikuchi, Akira Miyajima, and Mototsugu Oya Yota YasumizuYota Yasumizu More articles by this author , Takeo KosakaTakeo Kosaka More articles by this author , Yasumasa MiyazakiYasumasa Miyazaki More articles by this author , Eiji KikuchiEiji Kikuchi More articles by this author , Akira MiyajimaAkira Miyajima More articles by this author , and Mototsugu OyaMototsugu Oya More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.1630AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail Introduction and Objectives The prolyl isomerase Pin1 regulates phosphorylated status of proteins by catalyzing the cis-trans isomerization of phosphorylated Ser/Thr and Pro motifs, and plays an important role in cancer progression. The aim of this study was to establish a new treatment strategy for docetaxel resistant CRPC with a special focus on inhibition of Pin1. Methods Human CRPC cell lines: C4-2 and C4-2AT6 cell lines were used in this study. The C4-2AT6 cell line was established from C4-2 cells grown in androgen ablated serum for six months. The efficacy of Pin1 inhibitor: juglone was examined in CRPC cell lines. Human untreated prostate cancer tissues were obtained during radical prostatectomy at Keio University Hospital. Results We compared the cytotoxic effect of docetaxel (DTX) on C4-2 and C4-2AT6 cells by cell viability assay. In C4-2 and C4-2AT6 cells, the relative cell viability treated with 5nM DTX was 38.0% ± 5.2% and 59.1% ± 6.3%, respectively. These results indicated that C4-2AT6 cells showed significantly higher resistance to DTX than C4-2 cells. Next we evaluated the cytotoxic effect of juglone on C4-2AT6. The relative cell viability was 51.2 ± 3.2% with 30µM Juglone and 34.9 ± 3.8% with combined 5nM DTX and 30µM Juglone. Juglone significantly inhibited cell proliferation of C4-2AT6. When combined with DTX, Juglone showed significantly higher cytotoxicity than monotherapy (p<0.05). We previously demonstrated the efficacy of dual PI3K/mTOR inhibitor: NVP-BEZ235 against C4-2AT6. Pin1 regulates phosphorylated status of PI3K/Akt/mTOR pathway. Therefore, we examined the synergic effect of Juglone and NVP-BEZ235. The relative cell viability treated with 300nM NVP-BEZ235 was 49.3 ± 3.2% and 43.0 ± 3.8% with combined 300nM NVP-BEZ235 and 30µM Juglone. The combination of Juglone and NVP-BEZ235 shows little synergic effect on C4-2AT6 cells. In vivo, we examined a mouse xenogrftmodel of C4-2AT6 cells treated with DTX (2mg/kg) or NVP-BEZ235 (12.5mg/kg/day). In immunohistochemistry, tumors treated with DTX showed up-regulation of Pin1, while tumors treated with NVP-BEZ235 showed little expression of Pin1. These results suggest that Pin1 inhibitor shows the significant effect on tumors with up-regulation of Pin1. We confirmed the expression of Pin1 in human samples by immunohistochemistry. Specimens with a high Gleason score (>8) and a high pathological classification (pT3) showed significantly elevated Pin1 expression (p>0.05). Conclusions The combination of Juglone and DTX showed significant anti-tumor effects in CRPC. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e587 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Yota Yasumizu More articles by this author Takeo Kosaka More articles by this author Yasumasa Miyazaki More articles by this author Eiji Kikuchi More articles by this author Akira Miyajima More articles by this author Mototsugu Oya More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

MP24-14 METABOLIC SHIFT UNDER HYPOXIC ENVIRONMENT CAUSES RESISTANCE TO MTOR INHIBITOR IN HUMAN CASTRATION RESISTANT PROSTATE CANCER.

You have accessJournal of UrologyProstate Cancer: Basic Research I1 Apr 2014MP24-14 METABOLIC SHIFT UNDER HYPOXIC ENVIRONMENT CAUSES RESISTANCE TO MTOR INHIBITOR IN HUMAN CASTRATION RESISTANT PROSTATE CANCER. Yota Yasumizu, Takeo Kosaka, Yasumasa Miyazaki, Eiji Kikuchi, Akira Miyajima, and Mototsugu Oya Yota YasumizuYota Yasumizu More articles by this author , Takeo KosakaTakeo Kosaka More articles by this author , Yasumasa MiyazakiYasumasa Miyazaki More articles by this author , Eiji KikuchiEiji Kikuchi More articles by this author , Akira MiyajimaAkira Miyajima More articles by this author , and Mototsugu OyaMototsugu Oya More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.295AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Microenvironmental regulation of PI3K/Akt/mTOR signaling pathways and the efficacy of PI3K/mTOR inhibitors in hypoxic conditions have not been fully characterized yet. Recently we demonstrated that the activated PI3K/Akt/mTOR signaling pathway induced by androgen deprivation therapy or docetaxel explains in part the aggressiveness of castration resistant prostate cancer (CRPC). The aim of this study was to explore the efficacy of a dual PI3K and mTOR inhibitor and mTOR inhibitor for docetaxel resistant CRPC under hypoxia. METHODS Human CRPC cell lines: C4-2 and C4-2AT6 cell lines were used in this study. The C4-2AT6 cell line was established from C4-2 cells grown in androgen ablated serum for six months. We examined the changes in PI3K/Akt/mTOR pathway and hypoxia-related proteins under hypoxic conditions by using the dual PI3K and mTOR inhibitor NVP-BEZ235, and the mTOR inhibitor everolimus. RESULTS We investigated the expression of phosphorylated status in PI3K/Akt/mTOR pathway, HIF-1a, and pyruvate kinase isoform M2 (PKM2) in C4-2 and C4-2AT6 cells cultured under normoxia or hypoxia. PKM2 inactivates conversion of phosphoenolpyruvate to pyruvate and contributes to a metabolic shift that is required to cope with a hypoxic environment. Although under normoxia C4-2 and C4-2AT6 cells expressed elevated phosphorylated Akt (pAkt), phosphorylated S6 (pS6) and PKM2, a 5% hypoxic environment further induced expression of these proteins. These results indicate the PI3K/Akt/mTOR pathway is induced under hypoxic conditions. Next, we examined the cytotoxic effect of NVP-BEZ235 and everolimus under hypoxia by direct cell count. The relative cell number for treatment with 500nM NVP-BEZ235 and 100nM everolimus under 5% hypoxia compared to control was 0.46 ± 0.04 and 0.91 ± 0.11, respectively, while the relative cell number for treatment with the same doses of these drugs under normoxia was 0.47 ± 0.05 and 0.68 ± 0.08, respectively. NVP-BEZ235 showed a cytotoxic effect under hypoxia as well as normoxia (p<0.01). However, everolimus showed little cytotoxic effect under hypoxia. Western blot analysis revealed NVP-BEZ235 inhibited the expression of pAkt, pS6 and PKM2 in a dose-dependent manner. Everolimus did not inhibit the expression of pAkt or PKM2, although pS6 was fully inhibited. These results suggest that up-regulation of PKM2 is one possible mechanism of resistance to mTOR inhibitors in CRPC. CONCLUSIONS A hypoxic microenvironment modulates the sensitivity of mTOR inhibitors through PKM2 expression. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e264 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Yota Yasumizu More articles by this author Takeo Kosaka More articles by this author Yasumasa Miyazaki More articles by this author Eiji Kikuchi More articles by this author Akira Miyajima More articles by this author Mototsugu Oya More articles by this author Expand All Advertisement Advertisement PDF DownloadLoading ...

The novel histone deacetylase inhibitor thailandepsin A inhibits anaplastic thyroid cancer growth

BackgroundAnaplastic thyroid cancer (ATC) remains refractory to available surgical and medical interventions. Histone deacetylase (HDAC) inhibitors are an emerging targeted therapy with antiproliferative activity in a variety of thyroid cancer cell lines. Thailandepsin A (TDP-A) is a novel class I HDAC inhibitor whose efficacy remains largely unknown in ATC. Therefore, we aimed to characterize the effect of TDP-A on ATC. MethodsHuman-derived ATC cells were treated with TDP-A. IC50 was determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) rapid colorimetric assay, and cell proliferation was measured by viable cell count. Molecular mechanisms of cell growth inhibition were investigated by Western blot analysis of canonical apoptosis markers, intrinsic and extrinsic apoptosis regulators, and cell cycle regulatory proteins. Cell cycle staging was determined with propidium iodide flow cytometry. ResultsTDP-A dose- and time-dependently reduced cell proliferation. Increased cleavage of the apoptosis markers Caspase-9, Caspase-3, and poly adenosine diphosphate ribose polymerase were observed with TDP-A treatment. Levels of the intrinsic apoptosis pathway proteins BAD, Bcl-XL, and BAX remained unchanged. Importantly, the extrinsic apoptosis activator cleaved Caspase-8 increased dose-dependently, and the antiapoptotic proteins Survivin and Bcl-2 decreased. Among the cell cycle regulatory proteins, levels of CDK inhibitors p21/WAF1 and p27/KIP increased. Flow cytometry showed that ATC cells were arrested in G2/M phase with diminished S phase after TDP-A treatment. ConclusionsTDP-A induces a notable dose- and time-dependent antiproliferative effect on ATC, which is mainly attributed to extrinsic apoptosis with concomitant cell cycle arrest. TDP-A therefore warrants further preclinical and clinical investigations.

The Next Generation of Orthotopic Thyroid Cancer Models: Immunocompetent Orthotopic Mouse Models of BRAFV600E-Positive Papillary and Anaplastic Thyroid Carcinoma

While the development of new treatments for aggressive thyroid cancer has advanced in the last 10 years, progress has trailed headways made with other malignancies. A lack of reliable authenticated human cell lines and reproducible animal models is one major roadblock to preclinical testing of novel therapeutics. Existing xenograft and orthotopic mouse models of aggressive thyroid cancer rely on the implantation of highly passaged human thyroid carcinoma lines in immunodeficient mice. Genetically engineered models of papillary and undifferentiated (anaplastic) thyroid carcinoma (PTC and ATC) are immunocompetent; however, slow and stochastic tumor development hinders high-throughput testing. Novel models of PTC and ATC in which tumors arise rapidly and synchronously in immunocompetent mice would facilitate the investigation of novel therapeutics and approaches. We characterized and utilized mouse cell lines derived from PTC and ATC tumors arising in genetically engineered mice with thyroid-specific expression of endogenous Braf(V600E/WT) and deletion of either Trp53 (p53) or Pten. These murine thyroid cancer cells were transduced with luciferase- and GFP-expressing lentivirus and implanted into the thyroid glands of immunocompetent syngeneic B6129SF1/J mice in which the growth characteristics were assessed. Large locally aggressive thyroid tumors form within one week of implantation. Tumors recapitulate their histologic subtype, including well-differentiated PTC and ATC, and exhibit CD3+, CD8+, B220+, and CD163+ immune cell infiltration. Tumor progression can be followed in vivo using luciferase and ex vivo using GFP. Metastatic spread is not detected at early time points. We describe the development of the next generation of murine orthotopic thyroid cancer models. The implantation of genetically defined murine BRAF-mutated PTC and ATC cell lines into syngeneic mice results in rapid and synchronous tumor formation. This model allows for preclinical investigation of novel therapeutics and/or therapeutic combinations in the context of a functional immune system.

Nonthermal plasma induces apoptosis in ATC cells: involvement of JNK and p38 MAPK-dependent ROS.

PurposeTo determine the effects of nonthermal plasma (NTP) induced by helium (He) alone or He plus oxygen (O2) on the generation of reactive oxygen species (ROS) and cell death in anaplastic thyroid cancer cells.Materials and MethodsNTP was generated in He alone or He plus O2 blowing through a nozzle by applying a high alternating current voltage to the discharge electrodes. Optical emission spectroscopy was used to identify various excited plasma species. The apoptotic effect of NTP on the anaplastic thyroid cancer cell lines, such as HTH83, U-HTH 7, and SW1763, was verified with annexin V/propidium staining and TUNEL assay. ROS formation after NTP treatment was identified with fluorescence-activated cell sorting with DCFDA staining. The mitogen-activated protein kinase pathways and caspase cascade were investigated to evaluate the molecular mechanism involved and cellular targets of plasma.ResultsNTP induced significant apoptosis in all three cancer cell lines. The plasma using He and O2 generated more O2-related species, and increased apoptosis and intracellular ROS formation compared with the plasma using He alone. NTP treatment of SW1763 increased the expression of phosphor-JNK, phosphor-p38, and caspase-3, but not phosphor-ERK. Apoptosis of SW1763 as well as expressions of elevated phosphor-JNK, phosphor-p38, and caspase-3 induced by NTP were effectively inhibited by intracellular ROS scavengers.ConclusionNTP using He plus O2 induced significant apoptosis in anaplastic cancer cell lines through intracellular ROS formation. This may represent a new promising treatment modality for this highly lethal disease.

Curcumin Enhances Docetaxel-Induced Apoptosis of 8505C Anaplastic Thyroid Carcinoma Cells.

BackgroundAnaplastic thyroid cancer (ATC) is one of the most aggressive malignancies in humans, and its progression is poorly controlled by existing therapeutic methods. Curcumin has been shown to suppress inflammation and angiogenesis. In this study, we evaluated whether curcumin could augment docetaxel-induced apoptosis of ATC cells. We also analyzed changes in nuclear factor κB (NF-κB) and cyclooxygenase-2 (COX-2) expression levels to delineate possible mechanisms of their combined action.MethodsATC cells were cultured and treated with curcumin and docetaxel alone or in combination. The effects on cell viability were determined by MTS assay. Apoptosis was assessed by annexin V staining and confirmed by flow cytometric analysis. Caspase, COX-2, NF-κB levels were assayed by Western blotting.ResultsCurcumin combined with docetaxel led to lower cell viability than treatment with docetaxel or curcumin alone. Annexin V staining followed by flow cytometric analysis demonstrated that curcumin treatment enhanced the docetaxel-induced apoptosis of ATC cells. Additionally, curcumin inhibited docetaxel-induced p65 activation and COX-2 expression.ConclusionWe conclude that curcumin may enhance docetaxel's antitumor activity in ATC cells by interfering with NF-κB and COX-2. Our results suggest that curcumin may emerge as an attractive therapeutic candidate to enhance the antitumor activity of taxanes in ATC treatment.

Abstract A141: Dasatinib augments the antitumor effect of PLX4720 in a novel immunocompetent mouse model of BRAFV600E-positive anaplastic thyroid cancer.

Abstract Background: The MAPK and Src signaling pathways have emerged as critical drivers and mediators of papillary and anaplastic (PTC and ATC) thyroid cancer progression and the individual inhibition of either pathway has marked anti-tumor effects in mouse models. Unfortunately, neither drug induces apoptosis and drug resistance may develop when either is used. We postulated that the inhibition of the Src signaling pathway would further sensitize BRAFV600E-positive thyroid cancer cells to PLX4720 treatment. Methods: Proliferation and apoptosis were analyzed in four novel murine PTC and ATC cell lines derived from genetically engineered mice. Cells were treated with the BRAFV600E-inhibitor, PLX4720 (10μM), or the Src kinase inhibitor, dasatinib (50 nM), or a combination of the two drugs. Single and combinatorial dose response was determined and analyzed using Chou-Talalay. To determine the effect of combinatorial treatment in vivo, 105 murine ATC cells (TBP-3743 - BRAFV600E/WT; p53−/−) were orthotopically implanted in syngeneic immunocompetent mice. Mice were randomized into four groups and placed on treatment regimens 3 days following implantation, consisting of either control chow, PLX4720 chow (ab libitium), dasatinib gavage (50mg/kg) or a combination of PLX4720 chow and dasatinib gavage. Results: Basal pERK, pSrc(Y416) and pFAK(Y861) indicated active MAPK and Src signaling pathways in the thyroid cancer cell lines. PLX4720 treatment reduced pERK in the ATC lines, while dasatinib alone reduced both pSrc(Y416) and pFAK(Y861). The combination index (CI) of PLX4720 and dasatinib was calculated to be &amp;lt;1.0 for each line (TBP-3868 = 0.52, TBP-3743 = 0.45; TBPt-3403 = 0.56; TBPt-3610R = 0.07) indicating a synergistic effect on cellular proliferation. While neither PLX4720 nor dasatinib treatment induced significant apoptosis beyond baseline, combinatorial treatment induced apoptosis in the ATC lines TBP-3743 and TBPt-3403 as determined by caspase 3 and PARP cleavage in vitro. Orthotopic implantation of TBP-3743 resulted in the rapid formation of anaplastic tumors with CD3+, CD8+, B220+ and CD163+ immune cell infiltration. Relative to controls, which developed tumors two weeks post-implantation (231 mm3), treatment with PLX4720, dasatinib or a combination significantly reduced tumor volumes to 92 mm3, 123 mm3 and 62 mm3, respectively. Three weeks post-implantation, mean tumor volume significantly increased in mice treated with PLX4720 alone to 294 mm3 but not in mice treated with both PLX4720 and dasatinib which exhibited a modest increase to 118 mm3. Increased cleaved caspase 3 was detected in tumor sections from combination treated mice. Ultimately, the combination of PLX4720 and dasatinib resulted in increased apoptosis and reduced tumor volume relative to mice treated with PLX4720 alone. Conclusion: Combinatorial treatment of PTC and ATC cells with PLX4720 and dasatinib synergistically reduces proliferation and induces apoptosis in vitro and results in apoptosis and reduced tumor size relative to PLX4720 treatment alone in a pre-clinical mouse model of ATC. Together, these findings suggest that Src-inhibition enhances the anti-tumor effect of BRAFV600E-inhibition and the combination of these two FDA-approved drugs may have potential as a treatment paradigm for patients with ATC or refractory PTC. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A141. Citation Format: Pierre Vanden Borre, Viswanath Gunda, David McFadden, Peter Sadow, Sareh Parangi. Dasatinib augments the antitumor effect of PLX4720 in a novel immunocompetent mouse model of BRAFV600E-positive anaplastic thyroid cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A141.

The Role of Epithelial Mesenchymal Transition Markers in Thyroid Carcinoma Progression

Understanding the molecular mechanisms involved in thyroid cancer progression may provide targets for more effective treatment of aggressive thyroid cancers. Epithelial mesenchymal transition (EMT) is a major pathologic mechanism in tumor progression and is linked to the acquisition of stem-like properties of cancer cells. We examined expression of ZEB1 which activates EMT by binding to the E-box elements in the E-cadherin promoter, and expression of E-cadherin in normal and neoplastic thyroid tissues in a tissue microarray which included 127 neoplasms and 10 normal thyroid specimens. Thyroid follicular adenomas (n = 32), follicular thyroid carcinomas (n = 28), and papillary thyroid carcinomas (n = 57) all expressed E-cadherin and were mostly negative for ZEB1 while most anaplastic thyroid carcinomas (ATC, n = 10) were negative for E-cadherin, but positive for ZEB1. A validation set of 10 whole sections of ATCs showed 90% of cases positive for ZEB1 and all cases were negative for E-cadherin. Analysis of three cell lines (normal thyroid, NTHY-OR13-1; PTC, TPC-1, and ATC, THJ-21T) showed that the ATC cell line expressed the highest levels of ZEB1 while the normal thyroid cell line expressed the highest levels of E-Cadherin. Quantitative RT-PCR analyses showed that Smad7 mRNA was significantly higher in ATC than in any other group (p < 0.05). These results indicate that ATCs show evidence of EMT including decreased expression of E-cadherin and increased expression of ZEB1 compared to well-differentiated thyroid carcinomas and that increased expression of Smad7 may be associated with thyroid tumor progression.

Akt inhibition enhances the cytotoxic effect of apigenin in combination with PLX4032 in anaplastic thyroid carcinoma cells harboring BRAFV600E.

Aim of the present study was to evaluate the effect of apigenin in combination with BRAFV600E inhibitor PLX4032 on cell survival, and to investigate the influence of Akt inhibition on the combined effect of apigenin and PLX4032 in ATC cells harboring BRAFV600E. In 8505C and FRO cells harboring BRAFV600E, after treatment of apigenin and PLX4032, the cell viability decreased, and the percentage of dead cells increased in a time- and concentration-dependent manner, respectively. In apigenin- and PLX4032- treated cells, compared with apigenin alone-treated cells, the cell viability was lessened, and the percentage of dead cells was multiplied. In the addition of PLX4032 to apigenin, compared with the treatment of apigenin alone, the protein levels of cleaved PARP-1 and cleaved caspase-3 were elevated, and phospho-ERK protein levels were reduced, and the protein levels of total ERK, c-Myc, BRAF, phospho-Akt, phospho-p70S6K and phospho-4EBP1 were not varied. Compared with the treatment of PLX4032 alone, phosphop70S6K protein levels were reduced, and the other protein levels were not altered. Phospho-ERK protein levels were reduced only in 8505C cells. Under the co-treatment of apigenin and PLX4032, administration of the PI3K inhibitor wortmannin further decreased the cell viability, and increased the percentage of dead cells. In conclusion, our results suggest that PLX4032 augments apigenin-induced cytotoxicity in ATC cells harboring BRAFV600E. Moreover, Akt suppression potentiates the combined effect of apigenin and PLX4032 in ATC cells harboring BRAFV600E.

Dual PI3K/mTOR Inhibitor NVP-BEZ235 Sensitizes Docetaxel in Castration Resistant Prostate Cancer

Effective therapeutic strategies that can achieve long-term improvement in patients with castration resistant prostate cancer are urgently needed. We recently reported that the activated PI3K/Akt/mTOR signaling pathway induced by docetaxel explains resistance to docetaxel in castration resistant prostate cancer. In this study we explored the efficacy of NVP-BEZ235, a dual PI3K and mTORC1/2 inhibitor, for docetaxel resistant castration resistant prostate cancer. We used the 2 human castration resistant prostate cancer cell lines C4-2 and C4-2AT6. At our laboratory C4-2AT6 cells were established from C4-2 under androgen ablated treatment for 6 months. We investigated the efficacy of NVP-BEZ235 monotherapy and NVP-BEZ235 combined with docetaxel in vitro and in vivo. Increased phosphorylated Akt in C4-2AT6 cells was significantly inhibited by NVP-BEZ235 in a dose and time dependent manner. WST cell proliferation assay results in C4-2AT6 cells revealed that combined administration of NVP-BEZ235 and docetaxel had significant, synergistically greater cytotoxicity than NVP-BEZ235 or docetaxel monotherapy. Combined NVP-BEZ235 (40 mg/kg) and docetaxel (4 mg/kg) in vivo in a castrated mouse xenograft model inhibited C4-2AT6 tumor growth to a greater degree than in the monotherapy groups. Also, NVP-BEZ235 showed significant efficacy with docetaxel at a low concentration in vivo, suggesting that NVP-BEZ235 effectively decreased resistance to docetaxel. Results suggest that inhibition of the PI3K/Akt/mTOR signaling pathway by NVP-BEZ235 can overcome docetaxel resistance in human castration resistant prostate cancer. Our findings provide a molecular basis for the clinical use of combined administration of NVP-BEZ235 and docetaxel in patients with castration resistant prostate cancer.

506 INTRACRINE DYNAMICS OF CYP17A1 ACTIVITIES IN HUMAN CASTRATION RESISTANT PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Basic Research (III)1 Apr 2013506 INTRACRINE DYNAMICS OF CYP17A1 ACTIVITIES IN HUMAN CASTRATION RESISTANT PROSTATE CANCER Takeo Kosaka, Akira Miyajima, Takahiro Maeda, Hirohiko Nagata, Yota Yasumizu, Eiji Kikuchi, and Mototsugu Oya Takeo KosakaTakeo Kosaka Tokyo, Japan More articles by this author , Akira MiyajimaAkira Miyajima Tokyo, Japan More articles by this author , Takahiro MaedaTakahiro Maeda Tokyo, Japan More articles by this author , Hirohiko NagataHirohiko Nagata Tokyo, Japan More articles by this author , Yota YasumizuYota Yasumizu Tokyo, Japan More articles by this author , Eiji KikuchiEiji Kikuchi Tokyo, Japan More articles by this author , and Mototsugu OyaMototsugu Oya Tokyo, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.1900AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Although accumulating evidences indicates high expression of CYP17A1 allows castration resistant prostate cancer (CRPC) to maintain high intratumoral androgen levels, the potential CYP17A1 activity has not been characterized yet. The aim of this study was to examine the CYP17A1 expression and the potential CYP17A1 activity including 17α-hydroxylase and 17, 20-lyase activities in human CRPC. METHODS We used two human CRPC cell lines: C4-2 and C4-2AT6 which was established from C4-2 under androgen ablation conditions for six months. We quantified and compared transcripts and the protein expression of CYP17A1. To ascertain the potential CYP17A1 activity, we cultured with the steroid precursors: 13C-[2,3,4]-progesterone (13C-Prog), and analyzed the sequential biosynthesis 13C-[2,3,4]-17-hydroxyprogesterone (13C-17OHP) and 13C-[2,3,4]-androstenedione(13C-Adione). For the measurement of 13C-Adione and 13C-17OHP in cultured medium, an API-4000 triple stage quadrupole mass spectrometer connected to Agilent 1100, HTC-PAL, and an ESI ion source: liquid chromatography/mass spectrometry (LC/MS/MS) was employed. RESULTS Compared with C4-2 cells, quantitative PCR (qPCR) for C4-2AT6 cells showed significant increases of the expression of CYP17A1(p<0.001). Western blot analysis revealed significant increases of the expression of CYP17A1 at the protein level. LC/MS/MS analysis enabled us to detect the 13C-17-OHP and 13C-A-dione in these cell lines. The concentration ratio of 13C-Adione/13C-17-OHP (Adione-17-OHP ratio), which is thought to reflect the differences between 17-hydroxylase and 17, 20-lyase activities, was then determined. In C4-2 cells, the concentration of 13C-17-OHP and 13C-A-dione were 31.3±3.8 and 4.1±1.8 pg/mL; thus the Adione-17-OHP ratio was 0.135±0.07. On the other hand, in C4-2AT6 cells, the concentrations of 13C-17-OHP and 13C-Adione were 17.6±2.6 pg/mL and 6.9±0.5 pg/mL, and the Adione-17-OHP ratio was 0.402±0.09. Adione-17-OHP ratio was significantly higher in C4-2 than that of C4-2AT6 (p<0.001). These results indicated that both C4-2 and C4-2AT6 cells expressed direct CYP17A1 activities and that 17, 20-lyase activity was significantly higher in C4-2AT6 cells than in C4-2 cells. CONCLUSIONS These results demonstrated the direct evidence of intracrine dynamics of CYP17A1 activities mediated by 17α-hydroxylase activity and 17, 20-lyase activity in human CRPC. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e207-e208 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Takeo Kosaka Tokyo, Japan More articles by this author Akira Miyajima Tokyo, Japan More articles by this author Takahiro Maeda Tokyo, Japan More articles by this author Hirohiko Nagata Tokyo, Japan More articles by this author Yota Yasumizu Tokyo, Japan More articles by this author Eiji Kikuchi Tokyo, Japan More articles by this author Mototsugu Oya Tokyo, Japan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

973 A NEW STRATEGY FOR CASTRATION RESISTANT PROSTATE CANCER WITH TARGETING TO FEEDBACK MECHANISM OF PI3K/AKT/MTOR PATHWAY

You have accessJournal of UrologyProstate Cancer: Basic Research (V)1 Apr 2013973 A NEW STRATEGY FOR CASTRATION RESISTANT PROSTATE CANCER WITH TARGETING TO FEEDBACK MECHANISM OF PI3K/AKT/MTOR PATHWAY Yota Yasumizu, Akira Miyajima, Takeo Kosaka, Yasumasa Miyazaki, Suguru Shirotake, Eiji Kikuchi, and Mototsugu Oya Yota YasumizuYota Yasumizu Tokyo, Japan More articles by this author , Akira MiyajimaAkira Miyajima Tokyo, Japan More articles by this author , Takeo KosakaTakeo Kosaka Tokyo, Japan More articles by this author , Yasumasa MiyazakiYasumasa Miyazaki Tokyo, Japan More articles by this author , Suguru ShirotakeSuguru Shirotake Saitama, Japan More articles by this author , Eiji KikuchiEiji Kikuchi Tokyo, Japan More articles by this author , and Mototsugu OyaMototsugu Oya Tokyo, Japan More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.555AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The clinical trials of mTORC1 inhibiton in CRPC have failed to demonstarate anti-tumor effect despite the deficit of PTEN observed in about 70% of castration resistant prostate cancer (CRPC) with metastasis. The up-regulation of phosphorylated Akt (pAkt) through mTORC2 is reported as one factor of mTORC1 inhibitor resistance. In this study we aimed to establish a new strategy for docetaxel resistant CRPC, with a focus on regulatory system in PI3K/Akt/mTOR signaling pathway. METHODS The efficacy of PI3K/Akt/mTOR signaling pathway inhibition by the mTORC1 inhibitor: RAD001 and the PI3K and mTORC1/2 dual inhibitor: NVP-BEZ235 was examined in C4-2AT6 cells in vivo and in vitro. C4-2AT6 cells are a new CRPC cell line established by our laboratory under androgen ablation conditions and shows docetaxel resistance. RESULTS To examine the inhibitory effect of NVP-BEZ235 or RAD001, we performed the western blotting using C4-2AT6 cells. The expression of pAkt and phosphorylated S6 ribosomal protein (pS6) were significantly inhibited by 50nM NVP-BEZ235. On the other hand, the expression of pAKT was not inhibited by the treatment with RAD001, although the expression of pS6 was inhibited by 10nM RAD001. Next we tested the effect of NVP-BEZ235 or RAD001 to C4-2AT6 cells by WST assay. The mean cell viability at 24 h of treatment with 50nM NVP-BEZ235 and 10nM RAD001 was 59.2&undefined;} 6.4% and 61.2&undefined;} 7.2%, respectively. There was no significant difference between the two cell viability. The cell viability with 50nM NVP-BEZ235 and 5nM docetaxel was 41.2&undefined;} 5.2% and the cell viability with 10nM RAD001 and 5nM docetaxel was 50.1&undefined;} 4.8%. The combination of NVP-BEZ235 and docetaxel showed significantly higher anti-tumor effect than the combination of RAD001 and docetaxel (p<0.05). In vivo, we assigned castrated male nude mice to 4 groups; control, NVP-BEZ235 (12.5mg/kg/day), docetaxel (2mg/kg), and NVP-BEZ235 combined with docetaxel. We examined the time course changes in C4-2AT6 tumor growth with NVP-BEZ235 and docetaxel. The tumor volume of NVP-BEZ235 at day15 was 89.8&undefined;} 9.8%, and the tumor volume of docetaxel was 81.2&undefined;} 6.4%, compared to the control group. While the tumor volume of combined treatment was 6.1&undefined;} 5.5% of control. The combination therapy exhibited marked suppression of tumor growth compared to control, NVP-BEZ235 monotherapy, or docetaxel monotherapy (p<0.05). CONCLUSIONS The combination of NVP-BEZ235 and docetaxel showed significant anti-tumor effects in CRPC, suggesting the efficacy of PI3K/mTORC1/2 dual inhibitor for regulation of PI3K/Akt/mTOR pathway in CRPC. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e400 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Yota Yasumizu Tokyo, Japan More articles by this author Akira Miyajima Tokyo, Japan More articles by this author Takeo Kosaka Tokyo, Japan More articles by this author Yasumasa Miyazaki Tokyo, Japan More articles by this author Suguru Shirotake Saitama, Japan More articles by this author Eiji Kikuchi Tokyo, Japan More articles by this author Mototsugu Oya Tokyo, Japan More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...