A498 Renal Cell Carcinoma Cells Research Articles

Co-Expression of Multiple PAX Genes in Renal Cell Carcinoma (RCC) and Correlation of High PAX Expression with Favorable Clinical Outcome in RCC Patients.

Renal cell carcinoma (RCC) is the most common form of kidney cancer, consisting of multiple distinct subtypes. RCC has the highest mortality rate amongst the urogenital cancers, with kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), and kidney chromophobe carcinoma (KICH) being the most common subtypes. The Paired-box (PAX) gene family encodes transcription factors, which orchestrate multiple processes in cell lineage determination during embryonic development and organogenesis. Several PAX genes have been shown to be expressed in RCC following its onset and progression. Here, we performed real-time quantitative polymerase chain reaction (RT-qPCR) analysis on a series of human RCC cell lines, revealing significant co-expression of PAX2, PAX6, and PAX8. Knockdown of PAX2 or PAX8 mRNA expression using RNA interference (RNAi) in the A498 RCC cell line resulted in inhibition of cell proliferation, which aligns with our previous research, although no reduction in cell proliferation was observed using a PAX2 small interfering RNA (siRNA). We downloaded publicly available RNA-sequencing data and clinical histories of RCC patients from The Cancer Genome Atlas (TCGA) database. Based on the expression levels of PAX2, PAX6, and PAX8, RCC patients were categorized into two PAX expression subtypes, PAXClusterA and PAXClusterB, exhibiting significant differences in clinical characteristics. We found that the PAXClusterA expression subgroup was associated with favorable clinical outcomes and better overall survival. These findings provide novel insights into the association between PAX gene expression levels and clinical outcomes in RCC patients, potentially contributing to improved treatment strategies for RCC.

Shikonin Inhibits Cell Growth of Sunitinib-Resistant Renal Cell Carcinoma by Activating the Necrosome Complex and Inhibiting the AKT/mTOR Signaling Pathway.

Simple SummaryAdvanced renal cell carcinoma (RCC) remains an incurable disease, despite the establishment of new therapeutic options during the last decades. Unfortunately, drug resistance inevitably evolves, making better treatment strategies essential. Shikonin (SHI) from traditional Chinese medicine has shown promising antitumor properties with other tumor entities. Thus, in the current investigation, we evaluated the impact of SHI on therapy-sensitive and therapy-resistant RCC cells. SHI led to significant inhibition of progressive tumor cell growth in both therapy-sensitive and therapy-resistant RCC cells. This was accompanied by cell cycle arrest and reduction in cell cycle activating proteins, which results in blockage of cell division. SHI also activated necrosome complex proteins, leading to necroptosis, a programmed cell death. Furthermore, SHI inhibited the AKT/mTOR pathway, pivotal for cell survival and growth. Thus, SHI may hold promise in improving the treatment of RCC, even in its advanced form.Therapy resistance remains a major challenge in treating advanced renal cell carcinoma (RCC), making more effective treatment strategies crucial. Shikonin (SHI) from traditional Chinese medicine has exhibited antitumor properties in several tumor entities. We, therefore, currently investigated SHI’s impact on progressive growth and metastatic behavior in therapy-sensitive (parental) and therapy-resistant Caki-1, 786-O, KTCTL-26, and A498 RCC cells. Tumor cell growth, proliferation, clonogenic capacity, cell cycle phase distribution, induction of cell death (apoptosis and necroptosis), and the expression and activity of regulating and signaling proteins were evaluated. Moreover, the adhesion and chemotactic activity of the RCC cells after exposure to SHI were investigated. SHI significantly inhibited the growth, proliferation, and clone formation in parental and sunitinib-resistant RCC cells by G2/M phase arrest through down-regulation of cell cycle activating proteins. Furthermore, SHI induced apoptosis and necroptosis by activating necrosome complex proteins. Concomitantly, SHI impaired the AKT/mTOR pathway. Adhesion and motility were cell line specifically affected by SHI. Thus, SHI may hold promise as an additive option in treating patients with advanced and therapy-resistant RCC.

Abstract 2618: Tumor multicomponent targeting nanoparticle for synergistic killing of drug resistant renal cell carcinoma

Abstract Targeted therapy in renal cell carcinoma (RCC) has limited clinical response due to the development of drug resistance. Drug resistance can be acquired through multiple pathways including, impairment of apoptosis, overexpression of hypoxia promoting carbonic anhydrase 9 (CA9), and infiltration of folate receptor (FR) positive tumorigenic macrophages. Our hypothesis was to overcome the drug resistance by developing a tumor stimuli-responsive nanoparticle (NP) that can carry combination drugs, such as Cabozantinib (CB) and CFM 4.16 in RCC. The rationale of choosing the CB in combination with CFM4.16 was to induce synergistic anti-RCC effect by modulating the multiple tumor survival pathways, such as caspase 3, Akt/p-Akt, MET. One of the major challenges of the CB and CFM4.16 combination therapy is their poor water solubility and limited targetability to cancer. Thus, we came up with a smart strategy of developing dual folate receptor (FR) and carbonic anhydrase-9 (CA9) biomarker directed nanoparticles that can target the tumor stroma and hypoxia of RCC, respectively. In this proof of concept study, we synthesized SMA-TPGS and SMA polymer (PL) comprising of FR and CA9 dual targeting ligand, namely, FR-CA9-OL using reagent free ‘click' chemistry. FR-CA9-PL was encapsulated either with CFM4.16 or with CB to obtain nanoparticles, called FR-CA9-C4.16 and FR-CA9-CB respectively. The nanoparticles were prepared by using a co-solvent dialysis method that resulted in small-particle size with a range of 80-100 nm diameter, narrow polydispersity index of ∼ 0.15 and high drug loading of 30% (wt./wt.). The homogenous and high drug loading characteristics of nanoparticles are optimal for penetrating hypoxia of RCC. The IC50 of FR-CA9-C4.16 was improved by 60-fold as compared to CFM4.16 treatment and IC50 of FR-CA9-CB is improved by 16-fold compared to CB treatment in aggressive Everolimus-resistant (Evr-res) A498 RCC cells. The combination of FR-CA9-C4.16 with FR-CA9-CB demonstrated synergistic killing of Evr-res-A498 with CI value 0.1-0.5. The signature characteristic and superior anticancer effect of nanoparticles in Evr-resistant RCC provide a worthwhile strategy for overcoming drug resistance in RCC. Citation Format: Kushal Vanamala, Samaresh Sau, Shivani Naik, Arun K. Rishi, Arun Iyer. Tumor multicomponent targeting nanoparticle for synergistic killing of drug resistant renal cell carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2618.

Holliday junction‑recognition protein modulates apoptosis, cell cycle arrest and reactive oxygen species stress in human renal cell carcinoma.

Holliday junction recognition protein (HJURP) is involved in the regulation of mortality in various cell types, including renal cell carcinoma (RCC) cells. The specific mechanisms by which HJURP regulates RCC cell apoptosis and the cell cycle have not been previously investigated, to the best of our knowledge. In the present study, the expression of HJURP in RCC tissues and adjacent paracancerous renal tissue, as well as in RCC cell lines, was analyzed using reverse transcription‑quantitative PCR and western blot analysis. The A498 RCC cells were transfected with an HJURP overexpression vector, which resulted in reduced proliferation, as demonstrated using immunofluorescence staining, a Cell Counting Kit‑8 assay and a colony formation assay. Flow cytometry and terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labelling assays were used to determine the effect of HJURP on the cell cycle and apoptosis of RCC cells. Proteins associated with the reactive oxygen species (ROS) status were analyzed using western blot analysis. The expression of HJURP was lower in RCC tissues and cells compared with that in the adjacent paracancerous renal tissues and control cells. Furthermore, overexpression of HJURP resulted in a decrease in cell viability and proliferation invitro. Overexpression of HJURP resulted in cell cycle arrest at the G0/G1 phase, cell apoptosis and an increase in ROS stress. In addition, the phosphorylated/total sirtuin 1 (SIRT1) protein ratio was decreased, whereas the expression of peroxisome proliferator‑activated receptor (PPAR)γ was increased in the HJURP‑overexpressing RCC cells. In clinical practice, decreased HJURP expression may be associated with poor prognosis in patients with RCC. These results suggest that HJURP may regulate cell apoptosis and proliferation in RCC cells and this may be mediated by PPARγ/SIRT1. Thus, HJURP may be used as a predictor of prognosis in patients with RCC.

Classic SRY-box protein SOX7 functions as a tumor suppressor regulating WNT signaling and is methylated in renal cell carcinoma.

SOX7 (SRY-related high mobility group box 7), a high mobility group protein, is reported to be down-regulated in several cancer types, which indicates an important role in tumorigenesis; however, its biologic role in renal cell carcinoma (RCC) pathogenesis remains unknown. We studied the alterations and functions of SOX7 in RCC. We detected its broad expression in multiple human normal tissues, including kidney, but frequent down-regulation in RCC cell lines and primary tumors. Promoter CpG methylation seems to directly mediate SOX7 silencing in RCC cells, which could be reversed by demethylation treatment. SOX7 methylation was detected in primary RCC tumors, but rarely in normal kidney tissues. Restoration of SOX7 in silenced 786-O and A498 RCC cell lines inhibited their cell growth by inducing G0/G1 arrest, whereas SOX7 knockdown promoted RCC cell proliferation. We also found that SOX7 silencing resulted in the activation of WNT signaling and the induction of epithelial to mesenchymal transition. In conclusion, the current study demonstrates that SOX7 is frequently inactivated by promoter CpG methylation in RCC and functions as a tumor suppressor by regulating WNT signaling.-Wang, L., Fan, Y., Zhang, L., Li, L., Kuang, G., Luo, C., Li, C., Xiang, T., Tao, Q., Zhang, Q., Ying, J. Classic SRY-box protein SOX7 functions as a tumor suppressor regulating WNT signaling and is methylated in renal cell carcinoma.

Acquired resistance to temsirolimus is associated with integrin α7 driven chemotactic activity of renal cell carcinoma in vitro.

The mechanistic target of the rapamycin (mTOR) inhibitor, temsirolimus, has significantly improved the outcome of patients with renal cell carcinoma (RCC). However, development of temsirolimus-resistance limits its effect and metastatic progression subsequently recurs. Since integrin α7 (ITGA7) is speculated to promote metastasis, this investigation was designed to investigate whether temsirolimus-resistance is associated with altered ITGA7 expression in RCC cell lines and modified tumor cell adhesion and invasion. Caki-1, KTCTL-26, and A498 RCC cell lines were driven to temsirolimus-resistance by exposing them to temsirolimus over a period of 12 months. Subsequently, adhesion to human umbilical vein endothelial cells, to immobilized fibronectin, or collagen was investigated. Chemotaxis was evaluated with a modified Boyden chamber assay and ITGA7 expression by flow cytometry and western blotting. Chemotaxis significantly decreased in temsirolimus-sensitive cell lines upon exposure to low-dosed temsirolimus, but increased in temsirolimus-resistant tumor cells upon reexposure to the same temsirolimus dose. The increase in chemotaxis was accompanied by elevated ITGA7 at the cell surface membrane with simultaneous reduction of intracellular ITGA7. ITGA7 knock-down significantly diminished motility of temsirolimous-sensitive cells but elevated chemotactic activity of temsirolimus-resistant Caki-1 and KTCTL-26 cells. Therefore, ITGA7 appears closely linked to adhesion and migration regulation in RCC cells. It is postulated that temsirolimus-resistance is associated with translocation of ITGA7 from inside the cell to the outer surface. This switch forces RCC migration forward. Whether ITGA7 can serve as an important target in combatting RCC requires further investigation.

Axitinib induces DNA damage response leading to senescence, mitotic catastrophe, and increased NK cell recognition in human renal carcinoma cells.

Tyrosine kinase inhibitors (TKIs) including axitinib have been introduced in the treatment of renal cell carcinoma (RCC) because of their anti-angiogenic properties. However, no evidence are presently available on a direct cytotoxic anti-tumor activity of axitinib in RCC.Herein we reported by western blot analysis that axitinib treatment induces a DNA damage response (DDR) initially characterized by γ-H2AX phosphorylation and Chk1 kinase activation and at later time points by p21 overexpression in A-498 and Caki-2 RCC cells although with a different potency. Analysis by immunocytochemistry for the presence of 8-oxo-7,8-dihydro-2'-deoxyguanosine in cellular DNA and flow cytometry using the redox-sensitive fluorescent dye DCFDA, demonstrated that DDR response is accompanied by the presence of oxidative DNA damage and reactive oxygen species (ROS) generation. This response leads to G2/M cell cycle arrest and induces a senescent-like phenotype accompanied by enlargement of cells and increased senescence-associated β-galactosidase activity, which are abrogated by N-acetyl cysteine (NAC) pre-treatment. In addition, axitinib-treated cells undergo to cell death through mitotic catastrophe characterized by micronucleation and abnormal microtubule assembly as assessed by fluorescence microscopy.On the other hand, axitinib, through the DDR induction, is also able to increase the surface NKG2D ligand expression. Accordingly, drug treatment promotes NK cell recognition and degranulation in A-498 RCC cells in a ROS-dependent manner.Collectively, our results indicate that both cytotoxic and immunomodulatory effects on RCC cells can contribute to axitinib anti-tumor activity.

Aberrant Methylation of the 1p36 Tumor Suppressor Gene RIZ1 in Renal Cell Carcinoma.

Retinoblastoma protein-interacting zinc finger gene 1(RIZ1) functions as a tumor suppressor. Hypermethylation-mediated RIZ1 silencing has been reported in several cancers, but not in renal cell carcinoma (RCC) yet. We examined the RIZ1 expression and methylation in a panel of RCC cell lines and 50 primary tumors using semiquantitative/quantitative polymerase chain reaction (PCR), methylation specific PCR, and bisulfite sequencing genomic. We also explored the relationship between methylation status of RIZ1 and clinicopathological features in RCC patients. RIZ1 expression was down-regulated or lost in OS-RC-2, 769-P, Caki-1, 786-O and A498 RCC cell lines. Restored expression of RIZ1 was detected after addition of 5-aza-2'-deoxycytidine with/without trichostatin A, suggesting that DNA methylation directly mediates its silencing. The RIZ1 expression was significantly reduced in RCCs compared to adjacent non-malignant renal samples (P<0.001). Aberrant methylation was detected in 15 of 50 (30%) RCCs and in 2 of 28 (7%) adjacent non- malignant renal samples (P=0.02). No statistically significant correlation between methylated and unmethylated cases with regard to age, gender, pathological stage and grade was observed. RIZ1 expression is down-regulated in human RCC, and this down-regulation is associated with methylation. RIZ1 methylation may play a role in renal carcinogenesis.

Induction of cell cycle arrest, DNA damage, and apoptosis by nimbolide in human renal cell carcinoma cells.

Nimbolide is a tetranortriterpenoid isolated from the leaves and flowers of Azadirachta indica which has been shown to exhibit anticancer, antioxidant, anti-inflammatory, and anti-invasive properties in a variety of cancer cells. However, the anti-tumor effect on human renal cell carcinoma (RCC) cells is unknown. In this study, we found that nimbolide treatment had a cytotoxic effect on 786-O and A-498 RCC cells in a dose-dependent manner. According to flow cytometric analysis, nimbolide treatment resulted in G2/M arrest in 786-O and A-498 cells accompanied with an increase in the phosphorylation status of p53, cdc2, cdc25c, and decreased expressions of cyclin A, cyclin B, cdc2, and cdc25c. Nimbolide also caused DNA damage in a dose-dependent manner as determined by comet assay and measurement of γ-H2AX. In addition, apoptotic cells were observed in an Annexin V-FITC/propidium iodide double-stained assay. The activities of caspase-3, -9, and poly ADP-ribose polymerase (PARP) were increased, and the expression of pro-caspase-8 was decreased in nimbolide-treated 786-O and A-498 cells. Western blot analysis revealed that the levels of intrinsic-related apoptotic proteins Bax and extrinsic-related proteins (DR5, CHOP) were significantly increased in nimbolide-treated 786-O and A-498 cells. In addition, the expressions of Bcl-2 and Mcl-1 were decreased in 786-O and A-498 cells after nimbolide treatment. We conclude that nimbolide can inhibit the growth of human RCC cells by inducing G2/M phase arrest by modulating cell cycle-related proteins and cell apoptosis by regulating intrinsic and extrinsic caspase signaling pathways. Nimbolide may be a promising therapeutic strategy for the treatment of RCC.

MP23-14 FUNCTIONAL SIGNIFICANCE OF TUMOR SUPPRESSIVE MICRORNA-143/145 CLUSTER IN RENAL CELL CARCINOMA

You have accessJournal of UrologyKidney Cancer: Basic Research I1 Apr 2014MP23-14 FUNCTIONAL SIGNIFICANCE OF TUMOR SUPPRESSIVE MICRORNA-143/145 CLUSTER IN RENAL CELL CARCINOMA Hideki Enokida, Hirofumi Yoshino, Takeshi Chiyomaru, Toshihiko Itesako, Naohiko Seki, and Masayuki Nakagawa Hideki EnokidaHideki Enokida More articles by this author , Hirofumi YoshinoHirofumi Yoshino More articles by this author , Takeshi ChiyomaruTakeshi Chiyomaru More articles by this author , Toshihiko ItesakoToshihiko Itesako More articles by this author , Naohiko SekiNaohiko Seki More articles by this author , and Masayuki NakagawaMasayuki Nakagawa More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.882AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Our recent studies of microRNA (miRNA) expression signatures have demonstrated that the miRNA-143/145 cluster is significantly downregulated in several types of cancer and represents a putative tumor-suppressive miRNA in human cancers. The aim of this study was to investigate the functional significance of the miR-143/145 cluster in cancer cells and to identify novel molecular targets of the miR-143/145 cluster in renal cell carcinoma (RCC). METHODS We evaluated miR-143 and miR-145 expressions in 18 pair of clear cell RCC and adjacent noncancerous tissues by stem-loop RT-PCR. We performed gain-of-function studies (cell migration and invasion assays) by mature miRNAs transfection into RCC cell lines (786-O and A498). To identify the biological processes or pathways potentially regulated by the miR-143/145 cluster, we applied genome-wide gene expression analysis and in silico study; TargetScan database and GENECODIS software, which assigned a number of the putative miRNA targets to known pathways in KEGG. Furthermore, we performed gene expression analyses of all candidate genes involved in each of the pathways by using microarray expression data containing 53 clear cell RCCs and 23 noncancerous kidney tisuues, and constructed heat map diagrams. RESULTS The expression levels of miR-143 and miR-145 were significantly downregulated in RCC tissues compared with adjacent noncancerous tissues. A significant positive correlation was recognized between miR-143 and miR-145 expression. Restoration of mature miR-143 or miR-145 in 786-O and A498 RCC cells revealed that both mature miRNAs significantly inhibited cancer cell proliferation and invasion, suggesting that the miR-143/145 cluster functioned as a tumor suppressor in RCC. Gene expression data and in silico database analysis implied several promising target genes of the cluster, including hexokinase-2 (HK2) gene, which encodes a glycolytic enzyme crucial for the Warburg effect in cancer cells. Luciferase reporter assays showed that both miR-143 and miR-145 directly regulated HK2. In clinical RCC specimens, the expression of HK2 was significantly higher in cancer tissues than in noncancerous tissues. Silencing HK2 RCC enhanced cell proliferation and invasion, suggesting that HK2 has oncogenic functions in RCC. CONCLUSIONS Thus, our data demonstrated that loss of the tumor-suppressive miR-143/145 cluster enhanced RCC cell proliferation and invasion through targeting HK2 that is crucial for the Warburg effect. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e248 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Hideki Enokida More articles by this author Hirofumi Yoshino More articles by this author Takeshi Chiyomaru More articles by this author Toshihiko Itesako More articles by this author Naohiko Seki More articles by this author Masayuki Nakagawa More articles by this author Expand All Advertisement Advertisement PDF DownloadLoading ...

Tumor‐suppressive microRNA‐143/145 cluster targets hexokinase‐2 in renal cell carcinoma

Our recent studies of microRNA (miRNA) expression signatures have indicated that the miR-143/145 cluster is significantly downregulated in several types of cancer and represents a putative tumor-suppressive miRNA in human cancers. The aim of this study was to investigate the functional significance of the miR-143/145 cluster in cancer cells and to identify novel molecular targets of the miR-143/145 cluster in renal cell carcinoma (RCC). The expression levels of miR-143 and miR-145 were significantly downregulated in RCC tissues compared with adjacent non-cancerous tissues. A significant positive correlation was recognized between miR-143 and miR-145 expression. Restoration of mature miR-143 or miR-145 in 786-O and A498 RCC cells revealed that both mature miRNAs significantly inhibited cancer cell proliferation and invasion, suggesting that the miR-143/145 cluster functioned as a tumor suppressor in RCC. Gene expression data and in silico database analysis showed that the hexokinase-2 (HK2) gene, which encodes a glycolytic enzyme crucial for the Warburg effect in cancer cells, was a candidate target of the miR-143/145 cluster. Luciferase reporter assays showed that both miR-143 and miR-145 directly regulated HK2. In RCC clinical specimens, the expression of HK2 was significantly higher in cancer tissues than in non-cancerous tissues. Silencing HK2 suppressed RCC cell proliferation and invasion, suggesting that HK2 has oncogenic functions in RCC. Thus, our data showed that loss of the tumor-suppressive miR-143/145 cluster enhanced RCC cell proliferation and invasion through targeting HK2.

In Vitro Assessment of the Efficacy of Thermal Therapy in Human Renal Cell Carcinoma

To define the temperature-time points that result in cell death in a human renal cell carcinoma cell (RCC) line in vitro. Cellular viability and clonogenic cell survival were determined for human A498 RCC cells after thermal treatment. Various temperature (45 degrees C to 70 degrees C) and time (1 to 30 minutes) combinations were used. Cell viability was assessed by vital dye uptake and clonogenic cell survival. Mathematical Arrhenius modeling was performed to construct a graphic display of A498 cell thermal sensitivity. Temperature-time points at which 99% or greater cell death occurred according to the vital dye assay were 55 degrees C for 30 minutes, 60 degrees C for 10 minutes, and 65 degrees C for 8 minutes. Clonogenic survival studies confirmed that cells treated at these temperature-time points failed to grow even after 10 days. These in vitro results show that short exposure to temperatures higher than 70 degrees C is lethal in the A498 RCC cell line. Lower temperatures in the 60 degrees C range require more prolonged heating to cause cell death. Knowledge of these temperatures will be useful to better plan and monitor complex radiofrequency ablations.

Comparative analysis of COX‐2, vascular endothelial growth factor and microvessel density in human renal cell carcinomas

Cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF) are frequently up-regulated in malignant tumours and play a role in proliferation, apoptosis, angiogenesis and tumour invasion. In the present study, the expression of COX-2 and VEGF in renal cell carcinoma (RCC) was analysed and correlated with the microvessel density (MVD). COX-2 and VEGF were analysed by realtime reverse transcriptase-polymerase chain reaction and immunohistochemistry. The MVD was assessed by CD31 immunohistochemistry. The expression of COX-2 and VEGF was determined in the RCC cell lines A498 and Caki-1 under short-term hypoxia and in multicellular tumour cell aggregates. COX-2 was expressed in RCC by tumour epithelia, endothelia and macrophages in areas of cystic tumour regression and tumour necrosis. COX-2 protein in RCC was not altered in comparison with normal renal tissue. VEGF mRNA was up-regulated in RCC and positively correlated with MVD. RCC with high up-regulation of VEGF mRNA showed weak intracytoplasmic expression of VEGF in tumour cells. Intracytoplasmic VEGF protein expression was negatively correlated with MVD. In RCC with necrosis the MVD was reduced in comparison with RCC without necrosis. A498 RCC cells down-regulated COX-2 and up-regulated VEGF under conditions of hypoxia. In Caki-1 cells COX-2 expression remained stable, whereas VEGF was significantly up-regulated. In multicellular A498 cell aggregates COX-2 and VEGF were up-regulated centrally, whereas no gradient was found in Caki-1 cells. COX-2 and VEGF are potential therapeutic targets because COX-2 and VEGF are expressed in RCC and associated cell populations such as endothelia and monocytes/macrophages.

Tubulogenesis by microvascular endothelial cells is mediated by vascular endothelial growth factor (VEGF) in renal cell carcinoma.

To investigate the role of vascular endothelial growth factor (VEGF) in tumour angiogenesis in renal cell carcinoma (RCC). The expression of VEGF was examined in tissue samples from 25 patients with RCC using the reverse-transcriptase polymerase chain reaction (RT-PCR). Cellular localization of VEGF was studied in normal kidney and RCC tissues. Tube formation by human omental microvascular endothelial (HOME) cells co-cultured with A498 RCC cells was quantified in a three-dimensional collagen gel using computer-image analysis. RT-PCR detected VEGF m-RNA in tissue from 20 of 25 patients with RCC. An immunohistochemical study revealed that VEGF was primarily localized in the cytosol of normal renal tubule cells and RCC cells. Tube formation by HOME cells was increased in the presence of A498 cells overexpressing VEGF mRNA, induced by exogenous VEGF in a dose-dependent manner and completely inhibited by an anti-VEGF antibody. VEGF, which is produced and released from RCC cells, may elicit tumour angiogenesis by inducing microvessel tubulogenesis in patients with RCC. The co-culture system may be useful for screening inhibitors of tumour angiogenesis in RCC.