Renal Cell Cancer Cell Lines Research Articles

144 INHIBITORY EFFECT OF CATECHOL-O-METHYLTRANSFERASE-MEDIATED 4-HYDROXYESTRADIOL METABOLISM ON THE DEVELOPMENT OF HUMAN RENAL CELL CARCERS

You have accessJournal of UrologyKidney Cancer: Basic Research I1 Apr 2012144 INHIBITORY EFFECT OF CATECHOL-O-METHYLTRANSFERASE-MEDIATED 4-HYDROXYESTRADIOL METABOLISM ON THE DEVELOPMENT OF HUMAN RENAL CELL CARCERS Inik Chang, Jan Liu, Shahana Majid, Sharanjot Saini, Mohd Zaman, Soichiro Yamamura, Vary Shahryari, Takeshi Chiyomaru, Guoren Deng, Rajvir Dahiya, and Yuichiro Tanaka Inik ChangInik Chang San Francisco, CA More articles by this author , Jan LiuJan Liu San Francisco, CA More articles by this author , Shahana MajidShahana Majid San Francisco, CA More articles by this author , Sharanjot SainiSharanjot Saini San Francisco, CA More articles by this author , Mohd ZamanMohd Zaman San Francisco, CA More articles by this author , Soichiro YamamuraSoichiro Yamamura San Francisco, CA More articles by this author , Vary ShahryariVary Shahryari San Francisco, CA More articles by this author , Takeshi ChiyomaruTakeshi Chiyomaru San Francisco, CA More articles by this author , Guoren DengGuoren Deng San Francisco, CA More articles by this author , Rajvir DahiyaRajvir Dahiya San Francisco, CA More articles by this author , and Yuichiro TanakaYuichiro Tanaka San Francisco, CA More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.194AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Long-term exposure to estrogen and its metabolites may play an important role in renal cell carcinogenesis. Catechol-O-methyltransferase (COMT) participates in the estrogen metabolism pathway by neutralizing toxic substances. Although reduced COMT activity has been suggested to be a risk factor for estrogen-associated cancers, no studies have investigated the biological significance of COMT in the pathogenesis of human renal cell cancers (RCC). METHODS Gene expression was examined by quantitative RT-PCR, Western blotting and immunohistochemical staining. Cell proliferation was determined by MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)-based assay. Apoptosis was analyzed with an Annexin V-FITC/7-amino-actinomycin D staining system. Gene silencing was achieved by siRNA transfection. Gene expression profiling of selected apoptosis-related genes was investigated by PCR array. RESULTS We initially found that COMT levels are significantly decreased in human RCC tissues and cells suggesting a suppressive role of COMT in tumor development. However, transient over-expression of COMT has no effect on the proliferation, apoptosis and cell cycle progression of RCC cell lines. In contrast, when cells over-expressing COMT are treated with its substrate 4-hydroxyestradiol (4-OHE2), growth is inhibited by apoptotic cell death. We also found that COMT over-expression combined with 4-OHE2 induces up-regulation of growth arrest- and DNA damage-inducible protein α (GADD45α). We further show that down-regulation of GADD45α by a siRNA-mediated approach inhibits cell death, indicating the essential role of GADD45α in the underlying mechanism of COMT action in response to 4-OHE2. Finally, 4-methoxyestradiol fully reproduces the anti-proliferative function of COMT with 4-OHE2 by promoting GADD45α induction. CONCLUSIONS Our study shows that COMT in the presence of 4-OHE2 prevents RCC cell proliferation by enhancing apoptosis, and that GADD45α plays a critical role in the COMT-mediated inhibition of RCC. These findings will provide new direction for the biomarker detection and corresponding treatment in the prevention of human RCC. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e60 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Inik Chang San Francisco, CA More articles by this author Jan Liu San Francisco, CA More articles by this author Shahana Majid San Francisco, CA More articles by this author Sharanjot Saini San Francisco, CA More articles by this author Mohd Zaman San Francisco, CA More articles by this author Soichiro Yamamura San Francisco, CA More articles by this author Vary Shahryari San Francisco, CA More articles by this author Takeshi Chiyomaru San Francisco, CA More articles by this author Guoren Deng San Francisco, CA More articles by this author Rajvir Dahiya San Francisco, CA More articles by this author Yuichiro Tanaka San Francisco, CA More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Molecular characterization of temsirolimus-induced response in human renal and bladder cancer cell lines.

295 Background: Targeted therapies like mTOR inhibition is a clinically esthablished treatment modality for advanced renal cell cancer (RCC). We hypothesize that common elements of molecular signalling exists in RCC and transitional cell carcinoma of the bladder (TCC) that could provide a rational of the usage of this novel compound in human TCC. Therefore the goal of this investigation was to measure the in vivo and in vitro effect of temsirolimus/CCI-779 on human RCC and TCC cell lines on the molecular level. Methods: For in vivo experiments 3 RCC (786-O, A498, ACHN) cell lines and 7 TCC (T24, 5637, RT112, EJ-28, CLS-439, HB-CLS-1, HB-CLS-2) cell lines were compared. Effect of temsirolimus/CCI-779 was measured by real time impedance analysis (XCelligence, Roche). Following mRNA isolation microarray based mRNA expression analysis with 45.015 oligoprobes (G4112F, Agilent Technologies) was performed for molecular comparison of RCC and TCC cell lines. Expression patterns of 15 pathways were analyzed using the statistical software R (2.12.0) and the LIMMA package. Results: RCC and TCC cell lines demonstrated dose dependent inhibition of cellular growth with IC50 values of 10-20nM of temsirolimus/CCI-779 as measured by quantitative real time impedance analysis. Furthermore six out of 15 pathways including the mTOR and VEGF signalling were found with similar expression patterns following treatment with CCI-779 in both tumor entities. Conclusions: In vivo and in vitro analysis of temsirolimus mTOR inhitibtion on human bladder cancer cell lines support the hypothesis that a common molecular architectur exists in both tumor entities suggesting inhibition of mTOR in TCC as a possible target for further experimental therapeutic studies.

Catechol- O -methyltransferase-mediated metabolism of 4-hydroxyestradiol inhibits the growth of human renal cancer cells through the apoptotic pathway

Long-term exposure to estrogen and its metabolites may play an important role in renal cell carcinogenesis. Catechol-O-methyltransferase (COMT) participates in the estrogen metabolism pathway by neutralizing toxic substances. Although reduced COMT activity has been suggested to be a risk factor for estrogen-associated cancers, no studies have investigated the biological significance of COMT in the pathogenesis of human renal cell cancers (RCCs). We initially found that COMT levels are significantly decreased in human RCC tissues and cells suggesting it plays a suppressive role in tumor development. However, transient overexpression of COMT has no functional effect on RCC cell lines. In contrast, when cells overexpressing COMT are treated with its substrate 4-hydroxyestradiol (4-OHE(2)), growth is inhibited by apoptotic cell death. We also found that COMT overexpression combined with 4-OHE(2) induces upregulation of growth arrest- and DNA damage-inducible protein α (GADD45α). We further show that downregulation of GADD45α by a small interfering RNA-mediated approach inhibits cell death, indicating the essential role of GADD45α in the underlying mechanism of COMT action in response to 4-OHE(2). Finally, 4-methoxyestradiol fully reproduces the antiproliferative function of COMT with 4-OHE(2) by promoting GADD45α induction. Together, these findings show that COMT in the presence of 4-OHE(2) prevents RCC cell proliferation by enhancing apoptosis and that GADD45α plays a critical role in the COMT-mediated inhibition of RCC.

Evaluating rational non-cross-resistant combination therapy in advanced clear cell renal cell carcinoma: combined mTOR and AKT inhibitor therapy

Inhibition of the mammalian target of rapamycin (mTOR), a regulator of hypoxia inducible factor (HIF), is an established therapy for advanced renal cell cancer (RCC). Inhibition of mTOR results in compensatory AKT activation, a likely resistance mechanism. We evaluated whether addition of the Akt inhibitor perifosine to the mTOR inhibitor rapamycin would synergistically inhibit RCC. Select RCC cell lines were studied [786-O, A498 (VHL mutant), CAKI-1 (VHL wild type), and 769-P (VHL methylated)] with single agent and combination therapy. Growth inhibition was assessed by MTT and cell cycling by flow cytometry. Phospho-AKT (S473) and HIF-2α were assessed by Western blot. Total RNA was isolated from 786-O cells subjected to single agent and combination treatments. In these cells, genome-wide expression profiles were assessed, and real-time PCR was used to confirm a limited set of expression results. Three out of four cell lines (CAKI-1, 769-P, and 786-O) were sensitive to single-agent perifosine with 50% inhibitory concentrations ranging from 5 to 10μM. Perifosine blocked phosphorylation of AKT induced by rapamycin and inhibited HIF-2α expression in 786-O and CAKI-1. Combined treatment resulted in sub-additive growth inhibition. GeneChip analysis and pathway modeling revealed inhibition of the IL-8 pathway by these agents, concomitant with up-regulation of the KLF2 gene, a known suppressor of HIF1α. Perifosine is active in select RCC lines, abrogating the induction of AKT phosphorylation mediated by mTOR inhibition. Combined mTOR and AKT inhibition resulted in the modulation of pro-angiogenesis pathways, providing a basis for future investigations.

Abstract 136: hsa-miR-20a promotes tumorigenesis in ccRCC cancer cell lines

Abstract Objective & Background: We have previously reported specific microRNA profiles in renal cell cancer (RCC) cell lines in vitro (Kurisetty et al. AACR Annual Meeting 2010. A # 3045;). Among the miR signatures identified, miR-20a, a member of 17-92 Oncomir cluster, stood out as a highly upregulated miR in most RCC cell lines, regardless of their VHL status. The objectives of this study are to characterize the biological significance of miR 20A upregulation in RCC. Methods: ccRCC cell lines 786.O, ACHN and KV6 and HREC, were used for cell based biological/phenotypic assays. miRNA-gene target prediction analysis was performed using miRGEN and DIANA lab programs. miR-20a-Mimic (Mi) and miR-20a-Inhibitor (In) were transiently transfected into normal HREC cell line and RCC cell lines, respectively. Cell adhesion, proliferation, softagar colony formation and transwell invasion/migration assays were performed to investigate the influences of miR-20a-Mi/In transfected into normal and ccRCC cell lines. Conditioned medium from transfected cell lines was tested for its ability to induce in vitro angiogenesis in HUVECs. Western blotting was performed to identify the changes in E2F5 protein expression in the miR-20a-Mi/In transfected normal and ccRCC cell lines. Results: In normal HREC cells, upregulation of miR 20a (by miR-20a-Mi), significantly induced cell proliferation, adhesion, migration & invasion and soft agar colony formation. miR-20a knock down in RCC cells (by miR-20a-In) significantly reduced cell proliferation, adhesion, migration & invasion and colony formation to levels similar to non-malignant HREC cells. Prediction analyses through bioinformatics approaches identified E2F5, a transcription factor and a tumor suppressor of the E2F family of transcription factors, as a target of miR-20a. Compared to non-tumor HREC cells, E2F5 was significantly downregulated in RCC cell lines, and miR-20a inhibition enhanced E2F5 expression in cancer cells. miR-20a-Mi enhanced the ability of HREC conditioned medium to induce capillary formation in HUVECs. Conclusion: Our results suggest that miR-20a may promote the tumorigenic properties of RCC in vitro. Moreover, miR 20a may promote angiogenesis. We demonstrated that E2F5 is a target of miR 20a, is reduced in RCC cell lines, and its expression is enhanced by mir-20a inhibition, suggesting that E2F5 may mediate the tumor promoting effects of miR 20a. Molecular mechanisms of miR-20a function are currently being investigated further to understand its tumorigenic potential. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 136. doi:10.1158/1538-7445.AM2011-136

Abstract 4260: Sunitinib treatment induces Notch expression and activation in renal carcinoma cell lines

Abstract Introduction. Notch signaling is an evolutionary conserved pathway that plays a central role in stem cell biology, tumor formation, angiogenesis and cell fate decisions. Currently there is not any clear view as to whether Notch plays an oncogenic role in renal cell cancer (RCC). Notch-1 is activated after cytotoxic stimuli and may induce mTor pathway activation, leading to increased cell survival. We have investigated the role of Notch expression and activation in RCC cell lines in response to sunitinib treatment and the role of Notch in the activation of the mTor pathway. We elaborated three cell lines with a different VHL and PTEN mutation status: Caki-1 cells (VHL-wt, PTEN-wt), Caki-2 (VHL-mut, PTEN-wt) and 786-O (VHL-mut, PTEN-mut). Results: When Caki-1 and Caki-2 cells were exposed to sunitinib there was a marked upregulation in the expression of the Notch-1 mRNA and the target transcription factors RUNX1, RBPJ and HES1. The PI3K – mTor – S6K pathway was aslo upregulated. This upregulation was reversed when cells were pre-treated with a gamma-secretase inhibitor. However, both gamma secretase inhibitors and everolimus (an m-Tor inhibitor) had an antagonistic effect when combined with sunitinib (Combination Index >> 1.0). On the other hand, PTEN-mutant 786-O cells have a dysregulated mTor pathway and are more sensitive to inhinition of the m-Tor with everolimus. Sunitinib exposure did not result in further increase of the PI3K-mTor-S6K activation. Interestingly, combination of sunitinib with everolimus was synergistic in 786-O. Conclusions: VHL-wt and VHL-mutant RCC cells treated with sunitinib activate the Notch pathway. There are reports that Notch plays a tumor suppressor role in RCC and our results are on concert with that. Furthermore, Notch seems to regulate m-Tor pathway activation in cells with a wild-type PTEN. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4260. doi:10.1158/1538-7445.AM2011-4260

Noncytotoxic Differentiation Treatment of Renal Cell Cancer

Current drug therapy for metastatic renal cell cancer (RCC) results in temporary disease control but not cure, necessitating continued investigation into alternative mechanistic approaches. Drugs that inhibit chromatin-modifying enzymes involved in transcription repression (chromatin-relaxing drugs) could have a role, by inducing apoptosis and/or through differentiation pathways. At low doses, the cytosine analogue decitabine (DAC) can be used to deplete DNA methyl-transferase 1 (DNMT1), modify chromatin, and alter differentiation without causing apoptosis (cytotoxicity). Noncytotoxic regimens of DAC were evaluated for in vitro and in vivo efficacy against RCC cell lines, including a p53-mutated RCC cell line developed from a patient with treatment-refractory metastatic RCC. The cell division-permissive mechanism of action-absence of early apoptosis or DNA damage, increase in expression of HNF4α (hepatocyte nuclear factor 4α), a key driver associated with the mesenchymal to epithelial transition, decrease in mesenchymal marker expression, increase in epithelial marker expression, and late increase in cyclin-dependent kinase inhibitor CDKN1B (p27) protein-was consistent with differentiation-mediated cell-cycle exit. In vivo blood counts and animal weights were consistent with minimal toxicity of therapy. The distinctive mechanism of action of a dose and schedule of DAC designed for noncytotoxic depletion of DNMT1 suggests a potential role in treating RCC.

MDM-2 antagonists induce p53-dependent cell cycle arrest but not cell death in renal cancer cell lines

Renal cell cancers (RCC) are notoriously resistant to chemotherapy and radiotherapy. While mutations of the p53 tumor suppressor gene frequently contribute to therapy resistance in other epithelial cancers, p53 mutations are relatively rare in RCC. To date, there is conflicting evidence as to whether p53 signaling and function are otherwise proficient or defective in tumors with wild-type p53. In this study, we assayed p53 function in a series of RCC cell lines and normal proximal epithelial tubule cells using two different MDM-2 antagonists, Nutlin-3a and MI-219. Most cell lines with wild-type p53 responded to MDM-2 antagonists as evidenced by induction of p53 and its target gene p21. RCC cell lines treated with MDM-2 antagonists consistently accumulated in the G2/M phase of the cell cycle and this event was associated with inhibition of proliferation in RCC cell lines but not in normal proximal epithelial tubule cells. MDM-2 antagonists did not induce significant cell death in RCC cell lines, even with induction of p53-dependent pro-apoptotic genes. In contrast, MDM-2 antagonists caused significant cell death in LNCaP prostate adenocarcinoma cells. RCC cell lines with reduced p53, either by mutation or through ectopic expression of p53 shRNA, demonstrated enhanced sensitivity to cell death following sequential treatment with DNA damage and G2/M checkpoint abrogation. Our results suggest that wild-type p53 RCC cell lines are proficient in p53-dependent cell cycle arrest but defective in p53-dependent cell death.

Secreted frizzled‐related protein‐5 is epigenetically downregulated and functions as a tumor suppressor in kidney cancer

Secreted frizzled-related protein-5 (sFRP-5) has been identified as 1 of the secreted antagonists that bind Wnt protein. However, the functional significance of sFRP-5 in renal cell cancer (RCC) has not been reported. We hypothesized that sFRP-5 may be epigenetically downregulated through DNA methylation and histone modification and function as a tumor suppressor gene in RCC. Using tissue microarray and real-time RT-PCR, we found that sFRP-5 was significantly downregulated in kidney cancer tissues and cell lines, respectively. DNA bisulfite sequencing of the sFRP-5 promoter region in RCC cell lines showed it to be densely methylated, whereas there was few promoter methylation in normal kidney. The sFRP-5 expression was restored and the acetylation of H3 and H4 histones associated with the sFRP-5 promoter region were significantly increased after treatment with demethylation agent (5-Aza-dc) and histone deacetylase inhibitor (TSA). When RCC cells were transfected with the sFRP-5 gene, significant inhibition of anchorage independent colony formation and cell invasion were observed compared to controls. The sFRP-5 transfection also significantly induced apoptosis in RCC cells. In conclusion, this is the first report documenting that the sFRP-5 is downregulated by promoter methylation and histone acetylation and functions as a tumor suppressor gene by inducing apoptosis in RCC cells.

Sorafenib with doxorubicin augments cytotoxicity to renal cell cancer through PERK inhibition

Although cytokine therapy involving interleukin-2 or interferon-alpha has been employed for metastatic renal cell cancer (RCC) treatment, these therapies yielded limited response and benefit. Recently, several molecular-targeted agents have become available, and one newly developed anti-RCC agent, sorafenib (BAY 43-9006), is known to target multiple kinases. In this study, sorafenib was found to inhibit phosphorylation of the eukaryotic initiation factor-2alpha (eIF2alpha) and induce cell cycle arrest at G2/M phase and increase cell death. One of eIF2alpha kinases, PERK was responsible for eIF2alpha phosphorylation in RCC cells and PERK knockdown induced cell death similar to sorafenib treatment. The efficiency of sorafenib treatment correlated with phosphorylation level of eIF2alpha and nuclear Nrf2 expression level in eight RCC cell lines. Furthermore, sorafenib made Caki-1 and 786-O cells, but not ACHN cells sensitive to oxidative stress exerted by both hydrogen peroxide and doxorubicin. In addition, PERK knockdown sensitized Caki-1 and 786-O cells, but not ACHN cells to oxidative stress. In conclusion, levels of phospho-eIF2alpha and nuclear Nrf2 expression level in RCC might be a predictor of outcome in sorafenib treatment. In addition, PERK inhibition as well as sorafenib plus doxorubicin might be a promising therapeutic approach for RCC characterized by high levels of phosphorylated-eIF2alpha and nuclear Nrf2.

356 SECRETED FRIZZLED-RELATED PROTEIN-5 (SFRP-5) IS EPIGENETICALLY DOWNREGULATED AND FUNCTIONS AS A TUMOR SUPPRESSOR IN RENAL CELL CANCER

You have accessJournal of UrologyKidney Cancer: Basic Research III1 Apr 2010356 SECRETED FRIZZLED-RELATED PROTEIN-5 (SFRP-5) IS EPIGENETICALLY DOWNREGULATED AND FUNCTIONS AS A TUMOR SUPPRESSOR IN RENAL CELL CANCER Kazumori Kawakami, Soichiro Yamamura, Hiroshi Hirata, Koji Ueno, Sharanjot Saini, Shahana Majid, Yuichiro Tanaka, Ken Kawamoto, Hideki Enokida, Masayuki Nakagawa, and Rajvir Dahiya Kazumori KawakamiKazumori Kawakami San Francisco, CA More articles by this author , Soichiro YamamuraSoichiro Yamamura San Francisco, CA More articles by this author , Hiroshi HirataHiroshi Hirata San Francisco, CA More articles by this author , Koji UenoKoji Ueno San Francisco, CA More articles by this author , Sharanjot SainiSharanjot Saini San Francisco, CA More articles by this author , Shahana MajidShahana Majid San Francisco, CA More articles by this author , Yuichiro TanakaYuichiro Tanaka San Francisco, CA More articles by this author , Ken KawamotoKen Kawamoto Kagoshima, Japan More articles by this author , Hideki EnokidaHideki Enokida Kagoshima, Japan More articles by this author , Masayuki NakagawaMasayuki Nakagawa Kagoshima, Japan More articles by this author , and Rajvir DahiyaRajvir Dahiya San Francisco, CA More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.423AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Aberrant activation of the Wingless-type (Wnt) pathway plays a significant role in the carcinogenesis of several human cancers. Secreted frizzled-related protein-5 (sFRP-5) has been identified as one of the Wnt antagonists. The molecular function of sFRP-5 has never been examined in renal cell cancer (RCC). We hypothesize that sFRP-5 is epigenetically down-regulated and functions as a tumor suppressor in RCC. METHODS sFRP-5 expression levels were examined in 22 normal and 24 cancer regions of clinical samples using tissue microarray. sFRP-5 mRNA expression was also checked by TaqMan real time RT-PCR in RCC cell lines. We used bisulfite sequencing to check the methylation status of the sFRP-5 gene promoter. To examine the histone modification, chromatin immunoprecipitation (ChIP) assay was performed. We also established stable cell line (A498) transfected with either empty vector or the full-length sFRP-5 gene. Anchorage-independent colony formation assay, invasion assay and apoptosis analysis were carried out on the transfected A498 cell line. RESULTS Immunohistochemistry of tissue microarray and real time RT-PCR revealed that sFRP-5 was significantly down-regulated in RCC samples and cell lines, respectively. Bisulfite sequencing of the sFRP-5 promoter region showed it to be densely methylated. The sFRP-5 expression was restored and the acetylation of H3 and H4 histones associated with the sFRP-5 promoter region were significantly increased after treatment with demethylation agent (5-Aza-dc) and histone deacetylase inhibitor (TSA), respectively. Significant inhibition of anchorage-independent colony formation and cell invasion in sFRP-5 transfected cells compared to empty vector transfected cells were observed. In addition, the sFRP-5 transfection significantly induced apoptosis in RCC cells. CONCLUSIONS This is the first report documenting that the sFRP-5 is down-regulated by promoter methylation and histone acetylation and functions as a tumor suppressor gene by inducing apoptosis in RCC cells. © 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e141 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Kazumori Kawakami San Francisco, CA More articles by this author Soichiro Yamamura San Francisco, CA More articles by this author Hiroshi Hirata San Francisco, CA More articles by this author Koji Ueno San Francisco, CA More articles by this author Sharanjot Saini San Francisco, CA More articles by this author Shahana Majid San Francisco, CA More articles by this author Yuichiro Tanaka San Francisco, CA More articles by this author Ken Kawamoto Kagoshima, Japan More articles by this author Hideki Enokida Kagoshima, Japan More articles by this author Masayuki Nakagawa Kagoshima, Japan More articles by this author Rajvir Dahiya San Francisco, CA More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Vorinostat Enhances the Activity of Temsirolimus in Renal Cell Carcinoma Through Suppression of Survivin Levels

The mammalian target of rapamycin (mTOR) inhibitor temsirolimus has exhibited promising anticancer activity for the treatment of renal cell cancers (RCC). Survivin expression has been implicated in drug resistance and reducing its levels with the histone deacetylase (HDAC) inhibitor vorinostat may enhance the anticancer activity of temsirolimus. The sensitivity of RCC cell lines to the combination of temsirolimus and vorinostat was determined by measuring cell viability, clonogenic survival, and apoptosis. The effects of this combination on survivin levels were determined in vitro and in vivo. Survivin expression was silenced using small interfering RNA to evaluate its role in determining sensitivity to temsirolimus and vorinostat. The effect of the combination on angiogenesis was also determined in RCC xenograft models. Vorinostat synergistically improved the anticancer activity of temsirolimus in a panel of RCC cell lines in vitro and in two xenograft models in vivo. While each single agent led to a modest decrease in survivin levels, the combination dramatically reduced its expression, which correlated with an induction of apoptosis. Silencing survivin levels induced apoptosis and significantly improved the efficacy of temsirolimus and vorinostat. In addition, the temsirolimus/vorinostat combination led to a strong reduction in angiogenesis. Vorinostat augmented the anticancer activity of temsirolimus in both in vitro and in vivo models of RCC. The effectiveness of the combination was due to a decrease in survivin levels and corresponding induction of apoptosis, and enhanced inhibition of angiogenesis. Targeting survivin may be a promising therapeutic strategy to improve RCC therapy.

Immunotherapy for Human Renal Cell Carcinoma by Adoptive Transfer of Autologous Transforming Growth Factor β–Insensitive CD8+ T Cells

Transforming growth factor-beta (TGF-beta) is a potent immunosuppressor that has been associated with tumor evasion from the host immune surveillance and, thus, tumor progression. We tested a novel immunotherapy for human renal cell cancer (RCC) using a technique that involves the adoptive transfer of autologous tumor-reactive, TGF-beta-insensitive CD8(+) T cells into human RCC-challenged immunodeficient mice to identify its potent antitumor responses. The present study was conducted using a one-to-one adoptive transfer strategy to treat tumor-bearing severe combined immunodeficient (SCID/beige) mouse. The SCID/beige mice were humanized with peripheral blood mononuclear cells from patients with RCC (Hu-PBMC-SCID) before adoptive transfer. Autologous CD8(+) T cells were expanded ex vivo using autologous patient's dendritic cells pulsed with the tumor lysate and rendered TGF-beta insensitive by dominant-negative TGF-beta type II receptor. In addition, human RCC cell lines were generated using patients' tumor cells injected into SCID/beige mice. Using flow cytometry analysis, we confirmed the expression of the tumor-reactive, TGF-beta-insensitive CD8(+) T cells were the effector CD8(+) cells (CD27(-)CDRA(+)). Adoptive transfer of autologous TGF-beta-insensitive CD8(+) T cells into tumor-bearing Hu-PBMC-SCID mice induced robust tumor-specific CTL responses in vitro, were associated with tumor apoptosis, suppressed lung metastasis, and prolonged survival times in vivo. The one-to-one adoptive transfer strategy is an ideal in vivo murine model for studying the relationship between TGF-beta and immunosurveillance in RCC in vivo. Furthermore, this technique may offer the promise of a novel therapeutic option for the treatment of human patients with RCC.

Apoptotic and immunomodulatory effects of green tea extracts (GTE) on chemoresistant human tumor cells

e22101 Background: GTE and Epigallocatechin 3-gallate (EGCG), the most abundant polyphenol in GTE, have been shown to exert inhibitory effects on carcinogenesis; modulatory effects on tumor proliferation and differentiation; and immunomodulatory effects on tumor immunity in different pre-clinical models. These pluripotent effects suggest that GTE may have clinical activity that could be exploited for treatment of chemo-insensitive but immunologically responsive tumors. Thus, the present study investigated the effects of EGCG on the proliferation and immunologic sensitivity of human renal cell cancer (RCC) and malignant melanoma (MM) cell lines. Methods: Human RCC (769-P) and MM (A375) cell lines were tested following incubation in media ± 21.8μM EGCG, a pharmacologically-achievable concentration produced by 8, 200mg capsules GTE daily. Tumor proliferation was assessed by MTS assay; lytic sensitivity to IL2-activated human peripheral blood lymphocytes (IL2PBL) by 51Chromium release assay; and gene expression by quantitative RT-PCR assay. Results: EGCG produced significant inhibition of proliferation of both RCC and MM cells (61.5% and 67.3% of media control values respectively; p<0.01 by 2-tailed, paired t test). EGCG also caused significantly increased expression (≥ 2 times (×) media control values) of pro-apoptotic genes in both RCC: BCL2L1 (5.4×); BCL2L10 (7.9×); BIK (2.9×); Caspase 5 (6.3×); and Caspase 14 (6.3×) and MM cells: Caspase8 (2X) and Card6 (2.5X). Unfortunately, EGCG pre-treatment also decreased the sensitivity of RCC (43% of control) and MM (53% of control) to IL2PBL-mediated lysis (p<0.01 respectively). This occured in association with significant upregulation of Fas ligand mRNA in RCC (6.3×) and TRAF2 mRNA in MM (33 ×). Conclusions: The results of this pre-clinical study demonstrate that EGCG exerts significant antiproliferative effects against human RCC and MM cells in vitro in association with increased expression of different pro-apoptotic genes. Unfortunately, EGCG-treated cells also demonstrated reduced sensitivity to lysis by IL2-activated human lymphocytes. Taken together, the results suggest that GTE may have activity in vivo in patients against chemoresistant RCC or MM but should not be used in conjunction with IL2 therapy. No significant financial relationships to disclose.

The kinase inhibitors Sunitinib and Sorafenib differentially affect reactivity of NK cells against Renal Cell Cancer

Sunitinib and Sorafenib are multi‐kinase inhibitors licensed for the treatment of patients with advanced renal cell cancer (RCC) and target, among others, VEGF receptors, PDGF receptors, c‐kit and Flt3. Beyond that, Sorafenib inhibits c‐RAF and B‐RAF. Here we report that Sorafenib but not Sunitinib significantly (p<0.05, Mann‐Whitney U‐test) reduce cell surface CD107a expression (about 50%) on NK cells as a surrogate marker for degranulation in response to tumor targets. In line, pharmacological levels of Sorafenib but not Sunitinib significantly (p<0.05) diminish cytotoxicity of primary NK cells against the RCC cell lines A498, ACHN and CAKI‐2 (46% to 14%, 52% to 22% and 63% to 42%, respectively) in chromium release assays. While Sunitinib had no effect, Sorafenib, alike the specific c‐RAF inhibitor ZM336372 or the specific MEK inhibitor PD98059, markedly reduces ERK phosphorylation which regulates the movement of lytic granula towards target cells. Our results indicate that NK cell anti‐tumor reactivity is impaired by Sorafenib but not by Sunitinib due to the additional inhibitory effect of Sorafenib on the ERK pathway, which has been demonstrated to dictate NK cell cytotoxicity. We therefore conclude that Sunitinib is exquisitely suitable for combination with immunotherapeutic approaches in RCC patients.

The Kinase Inhibitors Sunitinib (Sutent®) and Sorafenib (Nexavar®) Differentially Affect Reactivity of NK Cells Against Renal Cell Cancer.

Sunitinib (Sutent®) and Sorafenib (Nexavar®) are multikinase inhibitors licensed for the treatment of patients with advanced renal cell cancer (RCC) because of their direct and indirect anti-tumor and anti-angiogenic effects. Sunitinib and Sorafenib target, among others, vascular endothelial growth factor receptors (VEGFR), platelet-derived growth factor receptors (PDGFR), the stem cell factor receptor (cKIT) and Fms-like tyrosine kinase-3 (Flt3). Beyond that, Sorafenib additionally inhibits c-RAF and B-RAF. NK cells play an important role in the immune surveillance of tumors. Their reactivity is governed by different surface receptors leading to activation of various kinases, phosphatases and transcription factors. Up to now, nothing is known regarding the influence of Sunitinib and Sorafenib on NK cells. Here we report that Sunitinib and Sorafenib differentially affect NK cell cytotoxicity. Analysis of peripheral blood mononuclear cells from 10 independent donors revealed that Sorafenib but not Sunitinib significantly (p<0.05, Mann-Whitney U-test) reduced cell surface CD107a expression (about 50%) on NK cells as a surrogate marker for degranulation in response to tumor targets. In line, pharmacological levels of Sorafenib significantly (p<0.05, Mann-Whitney U-test) diminished cytotoxicity of primary NK cells against the RCC cell lines A498, ACHN and CAKI-2 (from 46% to 14%, from 52% to 22% and from 63% to 42%, respectively) in chromium release assays. Again, no inhibition of cytotoxicity was observed in the presence of pharmacological concentrations of Sunitinib. Since activation of MAP kinases leading to ERK phosphorylation regulates the movement of lytic granula towards target cells, which is responsible for NK cell cytotoxicity we investigated the influence of Sunitinib and Sorafenib on ERK phosphorylation. While Sunitinib had no effect, Sorafenib, alike the specific c-RAF inhibitor ZM336372 or the specific MEK inhibitor PD98059, markedly reduced ERK phosphorylation as determined by Western Blot. Our results indicate that NK cell anti-tumor reactivity is impaired by Sorafenib but not by Sunitinib due to the additional inhibitory effect of Sorafenib on the ERK pathway, which has been demonstrated to dictate NK cell cytotoxicity. We therefore conclude that Sunitinib is exquisitely suitable for combination with immunotherapeutic approaches in RCC patients.

Indirubin-3‘-Monoxime, an Inhibitor of Cyclin Dependent Kinases, Induces Growth Inhibition and Apoptosis in Renal Cell Cancer in vitro

The compound indirubin is the active constituent of a traditionalChinese phytomedicine (Danggui Longhui Wan).This preparation has been used for years in the treatmentof chronic myelocytic leukemia. A derivate of the originalindirubin compound is indirubin-3'-monoxime which is aselective and potent inhibitor of cyclin-dependent kinases.In this study we investigated if indirubin-3'-monoximecan induce apoptosis and tumor cell death in 3 human(A498, CAKI-1, CAKI-2) and 1 murine (RENCA) renal cellcancer cell lines. The growth inhibitory and apoptosis inductionproperties were evaluated by EZ4U, a cytotoxicassay and by flow cytometry of annexin-V/PI stainingduring treatment with doses ranging from 5.0 to 15.0 µmindirubin-3'-monoxime over 72 h. To further evaluate theunderlying molecular actions of indirubin-3'-monoxime,survivin, a major antiaptotic protein, was additionallydetermined by intracellular flow cytometry. Our resultsshow that indirubin-3'-monoxime induces growth arrestand apoptosis in all RCC cell lines. All RCC lines expressedsurvivin. However, no clear correlation between apoptosisinduction and expression of survivin was found. As treatmentof metastatic RCC remains a challenge, the need for continuing assessment of novel agents in the treatment ofthis disease is mandatory. Indirubin-3'-monoxime seems acandidate for further evaluation.

Gene-modified T cells for adoptive immunotherapy of renal cell cancer maintain transgene-specific immune functions in vivo.

We have treated three patients with carboxy-anhydrase-IX (CAIX) positive metastatic renal cell cancer (RCC) by adoptive transfer of autologous T-cells that had been gene-transduced to express a single-chain antibody-G250 chimeric receptor [scFv(G250)], and encountered liver toxicity necessitating adaptation of the treatment protocol. Here, we investigate whether or not the in vivo activity of the infused scFv(G250)(+) T cells is reflected by changes of selected immune parameters measured in peripheral blood. ScFv(G250)-chimeric receptor-mediated functions of peripheral blood mononuclear cells (PBMC) obtained from three patients during and after treatment were compared to the same functions of scFv(G250)(+) T lymphocytes prior to infusion, and were correlated with plasma cytokine levels. Prior to infusion, scFv(G250)(+) T lymphocytes showed in vitro high levels of scFv(G250)-chimeric receptor-mediated functions such as killing of CAIX(+) RCC cell lines and cytokine production upon exposure to these cells. High levels of IFN-gamma were produced, whilst production of TNF-alpha, interleukin-4 (IL-4), IL-5 and IL-10 was variable and to lower levels, and that of IL-2 virtually absent. PBMC taken from patients during therapy showed lower levels of in vitro scFv(G250)-receptor-mediated functions as compared to pre-infusion, whilst IFN-gamma was the only detectable cytokine upon in vitro PBMC exposure to CAIX. During treatment, plasma levels of IFN-gamma increased only in the patient with the most prominent liver toxicity. IL-5 plasma levels increased transiently during treatment in all patients, which may have been triggered by the co-administration of IL-2. ScFv(G250)-receptor-mediated functions of the scFv(G250)(+) T lymphocytes are, by and large, preserved in vivo upon administration, and may be reflected by fluctuations in plasma IFN-gamma levels.

Von Hippel-Lindau Tumor Suppressor Protein Regulates the Assembly of Intercellular Junctions in Renal Cancer Cells through Hypoxia-Inducible Factor–Independent Mechanisms

Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene is responsible for the development of renal cell cancers (RCC), pheochromocytomas, and tumors in other organs. The best known function of VHL protein (VHL) is to target the hypoxia-inducible factor (HIF) for proteasome degradation. VHL is also required for the establishment of an epithelial-like cell shape in otherwise fibroblastic-like RCC cell lines. However, the underlying mechanisms and whether this is linked to HIF remain undetermined. Because the breakage of intercellular junctions induces a fibroblastic-like phenotype in multiple cancer cell models, we hypothesized that VHL may be required for the assembly of intercellular junctions in RCC cells. Our experiments showed that VHL in RCC cell lines is necessary for the normal organization of adherens and tight intercellular junctions, the maintenance of cell polarity, and control of paracellular permeability. Additionally, 786-O cells reconstituted with wild-type VHL and with a constitutively active form of HIF-2alpha did not reproduce any of the phenotypic alterations of VHL-negative cells. In summary, we show that VHL inactivation in RCC cells disrupts intercellular junctions and cell shape through HIF-independent events, supporting the concept that VHL has additional functions beside its role in the regulation of HIF.

A Third-Generation Bisphosphonate, Minodronic Acid (YM529), Augments the Interferon α/β-Mediated Inhibition of Renal Cell Cancer Cell Growth Both In vitro and In vivo

Abstract Purpose: Minodronic acid (YM529) is a third-generation nitrogen-containing bisphosphonate. Here, we have investigated the therapeutic efficacy of YM529 against renal cell cancer (RCC) alone or in combination with IFN both in vitro and in vivo. Experimental Design: One murine and eight human RCC cell lines were used for the in vitro studies and were subjected to a modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and Western blotting. Luciferase-labeled murine RCC cells (RENCALuc) were transplanted into the s.c. tissue or the renal subcapsule of syngeneic BALB/c mice. These mice were treated with YM529 and/or murine IFN and the growth of the cancer cells was monitored by an in vivo imaging system. Results: YM529 inhibited the growth of RCC cells in a dose- and time-dependent manner and enhanced the growth inhibitory potential of IFN in vitro. In the in vivo mouse models, YM529 did not markedly inhibit the RCC cell growth on its own but it augmented the anticancer effect of IFN (P &amp;lt; 0.05). The YM529-treated mice (with or without IFN) did not alter the γ/δ T-lymphocyte numbers. The various treatment regimens were also not associated with any adverse effects. However, YM529 combined with IFN reduced the serum vascular endothelial growth factor levels. Conclusions: Our study suggests that YM529 may be a potent anticancer agent for RCC. The efficacy and safety of IFN plus YM529 as a therapy for RCC should be verified by early-phase clinical trials.