Cell Lines Caki-1 Research Articles

PO-032 The knock-down of ferritin heavy subunit induces xenobiotic-resistance in K562 cells through the activation of NF-kB pathway

IntroductionRenal Cell Carcinoma (RCC) is the most common kidney with still unclear pathophysiology. The most common types of RCC are clear cel (ccRCC) and papillary (pRCC). Recently, it have been proposed that renin-angiotensin system (RAS), mainly including angiotensin II, is involved in development and progression of RCC. Angiotensin-(1–7) [Ang-(1–7)] is a RAS molecule, acting through Mas receptor (MasR), that has recently gained more attention because its ability to inhibit proliferation and migration of lung, breast and prostate cancer cells. Because high levels of Ang-(1–7) are observed in the kidney, we have decided to investigate role of Ang-(1–7) on RCC cells.Material and methodsThe study was performed on Caki-1 and Caki-2 cell lines that represent ccRCC and pRCC phenotype respectively. Cell proliferation was assessed by AlamarBlue assay and wound healing assay was used for quantification of cell migration. Cell were treated with different combinations of 0,001–10 uM of Ang-(1–7) and/or MasR antagonist A-779 for 6–72 hour depending on the experiment.Results and discussionsWe found that Ang-(1-7) increase cell migratory abilities of both Caki 1 and Caki 2 cells after 6, 12, 18 and 24 hour (p<0.01). The effect is inhibited by blockade of Mas receptor by A-779, that have no effect on migration when administrated alone. In the proliferation assay, 0,1 uM Ang-(1–7) caused 20% increase of cell proliferation, while 1 uM A-779 inhibited proliferation up to 30%, however, both results were not statistically significant (p>0.05)ConclusionThis study demonstrates potential role of Ang-(1–7) and Mas receptor in pathogenesis of RCC, mostly by promoting cell migration and increasing metastatic potential. Results may help to better understand molecular mechanism underlying progression of this tumour and find new potential targets for therapy. Discrepancy between pro-migratory effect and no impact on cell proliferation, role of Ang-(1–7) and MasR requires further studies and confirmation in animal model of RCC.

Abstract 898: Clear cell renal cell carcinoma resistance to RTKs inhibitors is mediated by c-Met receptor and MCPIP1

Abstract Introduction Clear cell renal cell carcinoma (ccRCC) treatment with small molecules that inhibit multiple receptor tyrosine kinases (RTKs),showed in approximately 38% of patients significant tumor control. However, despite the efficacy of treatment, ccRCC often develops resistance to targeted drugs and the majority of patients who receive such treatment exhibit progressive disease after 1 year. Several hypotheses have been proposed regarding the mechanisms underlying resistance to RTKs inhibitors, but the precise pathways have not yet been fully elucidated. Aim The main objective of our study was to determine the role of c-Met and anti-inflammatory protein MCPIP1 in the acquisition of resistance to RTKs inhibitors in ccRCC. Materials and methods ccRCC cell lines (Caki-1 and Caki-2) as well as normal epithelial cell lines (Hek293, RPTEC/TERT1) were treated with sunitinib, sorafenib or SU11274 constantly for 24h, 1 and 3 weeks. The level and localization of key proteins were examined using western blots and IF staining. ccRCC patient samples were divided into four histological grades, and levels of genes or proteins were investigated using microarray analysis, western blots, protein arrays, mass spectrometry and real time PCR. Results Our study shows that short term (24h) and long term (3 weeks) stimulation with sunitinib or sorafenib induced phosphorylation of c-Met receptor, STAT3 and Src kinase. Furthermore, sunitinib treatment resulted in the acquisition of cancer stem cells features and the formation of clones. In addition, after sunitinib and sorafenib treatment we observed a decrease in the protein level of MCPIP1, postulated tumor suppressor. Interestingly, after MCPIP1 overexpression, we observed c-Met downregulation, regulated by RNase activity of MCPIP1. Moreover, our results show that in patient samples, together with MCPIP1 downregulation, level of phosphorylated and total c-Met receptor increase. Conclusions We showed that acquisition of therapy resistance in ccRCC may be affected by MCPIP1 decrease, together with phopsphorylation of c-Met receptor and it can be partially reversed by overexpressing the MCPIP1 protein, which may act as a potent tumor suppressor. Proposed research may help in understanding the mechanisms responsible for tumor resistance to targeted therapy. Obtained results may contribute to increased understanding of the biology of clear cell renal cell carcinoma, which in the future may help in identifying new, more effective therapeutic purposes or improving existing ones. Acknowledgment This study was supported by research grant from the National Science Centre 2013/09/D/NZ/00249, 2017/25/N/NZ5/03014 and grant from Jagiellonian University BMN 16/2017. Citation Format: Paulina Marona, Judyta Górka, Jolanta Jura, Katarzyna Miękus. Clear cell renal cell carcinoma resistance to RTKs inhibitors is mediated by c-Met receptor and MCPIP1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 898.

Abstract 2840: Characterization of pazopanib resistance in renal cell carcinoma cell lines

Abstract BACKGROUND: Together with Sunitinib (Sun), Pazopanib (Paz) is considered as a gold standard in metastatic renal cell carcinoma treatment (RCC). Similarly to other tyrosine kinase inhibitors (TKI), development of resistance constitutes the main problem of Paz-based therapy. This warrants the in vivo investigations of the processes related to Paz resistance development. METHODS: Parental human RCC cell lines, 786-O/wt and CAKI-2/wt, were continuously treated with Paz at IC50 concentration and Paz-resistant 786-O and CAKI-2 cell subpopulations (786-O/res and CAKI-2/res) were developed. Parental and resistant cells were compared in terms of the malignant phenotype by WST-1 proliferation, cross-resistance to another TKI, migration wound healing assays and signaling pathways analysis by Western blot. MiRNA expression was analyzed using microarrays (Agilent Technologies). RESULTS: The in vitro analysis of RCC cells growth under Paz treatment revealed that this drug inhibited proliferation of parental 786-O and CAKI-2 cells with IC50 (inhibitory concentration of 50%) of 14 and 11 µmol/L, respectively. The continuous treatment with this drug resulted in the gradual resistance development in both tested cell lines. At the end of the period of continuous treatment (32 passages, 3 months) the resistance level of 786-O/res and CAKI-2/res cells was increased 2-fold compared with 786-O/wt and CAKI-2/wt. The analysis of cell motility revealed that the short-termed exposure to Paz retarded the migration of both parental and Paz-resistant 786-O and CAKI-2 cells. However, no differences in the motility rates of parental and resistant cells were observed. The analysis of the signaling pathways that might be involved in the resistance development revealed no changes in the expression of epithelial-to-mesenchymal transition (EMT) markers N-cadherin, vimentin and β-catenin between parental and Paz-resistant subpopulations of both cell lines. Cell response to Paz treatment differs between the tested parental RCC cell lines CAKI-2 and 786-O in terms of the expression of phosphorylated forms of the growth-promoting molecules of Akt-mTOR and MAPK-ERK pathways. A significant change in miRNA expression was found in both cell lines at different time points of treatment compared to parenteral cells. CONCLUSIONS: Our data suggest that the process of Paz resistance development is cell line specific. Akt-mTOR and MAPK-ERK signaling pathways seem to be related to Paz resistance development in RCC. Development of resistance is associated with specific miRNA alterations. Further studies have to analyze the functional effects of miRNAs on resistance and possible signaling pathways. Citation Format: Bozhena Vynnytska-Myronovska, Joana Heinzelmann, Sebastian Hölters, Michael Stoeckle, Kerstin Junker. Characterization of pazopanib resistance in renal cell carcinoma cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2840.

Abstract 2021: The CXCL12/CXCR4 axis drives epithelial-mesenchymal transition in renal cell carcinoma

Abstract Introduction. Renal cell carcinoma (RCC) is the one of the most common cancers diagnosed in adult men with more than 40000 new cases annually. Recurrence or metastasis of the disease occurs in more than 30% of patients and death from disease occurs in most of these patients due to limited therapeutic options. Clearly, a better understanding of factors contributing to RCC metastasis might improve therapeutic development and patient outcomes. One of the steps required for tumor cell progression from localized disease to metastatic disease is the acquisition of invasive abilities through epithelial to mesenchymal transition (EMT). EMT promotes loss of epithelial markers by downregulation of E-cadherin, gain of mesenchymal markers like Vimentin, and N-cadherin, and cytoskeletal rearrangement that enables cells to leave the primary site and intravasate into the blood or lymphatic systems and thereby colonize distant sites. Metastasis and colonization are highly directed mechanisms in which cancer cells migrate toward specific preferred organs that express specific protein receptors. One of these receptors is CXCR4, which is upregulated in advanced cancer including RCC and is correlated with poor survival. In this study we explored the hypothesis that CXCR4/CXCL12 signaling promotes migratory ability by inducing EMT in vitro in the RCC cell lines Caki-2 and 786-0. Methods. To pursue these studies we utilized in vitro mammalian cell culture, cell proliferation (WST) assays, cell migration (scratch) assays, and protein analysis methods including immunoblot and immunofluorescence. Results. These studies showed that RCC cell lines express CXCR4 that is activated by CXCL12 to induce cellular proliferation and promote a wound healing migratory response dependent on EMT cytoskeletal rearrangement by Rho GTPases. Moreover, CXCR4/CXCL12 induces down-regulation of E-cadherin, up-regulation of vimentin, N-cadherin and the upregulation of EMT transcription factors SLUG/SNAIL, consistent with EMT signaling activation. Lastly, CXCR4/CXCL12 activation promotes phosphorylation of AKT and ERK, suggesting that these signaling pathways are coupled to CXCR4/CXCL12-mediated RCC progression. Conclusions. Our findings suggest that CXCR4/CXCL12 activation in RCC has a pivotal role in EMT induction by the acquisition of a migratory and invasive phenotype. Understanding the underlying signaling pathways employed by the CXCL12/CXCR4 axis may shed new light on the migration/ invasion mechanisms that allow RCC cells to spread to distant organs and assist with the design and testing of new therapeutic strategies to treat advanced metastatic RCC. Citation Format: Alisa Zhilin-Roth, Jill A. Macoska. The CXCL12/CXCR4 axis drives epithelial-mesenchymal transition in renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2021.

Klotho inhibits EGF-induced cell migration in Caki-1 cells through inactivation of EGFR and p38 MAPK signaling pathways.

Klotho is a single-pass transmembrane protein with documented anti-cancer properties. Recent reports have implicated Klotho as an inhibitor of transforming growth factor β1 induced cell migration in renal fibrosis. Overexpression of epidermal growth factor receptor (EGFR) is known to promote tumor initiation and progression in clear-cell renal cell carcinoma (cRCC). We tested our hypothesis that Klotho inhibits EGF-mediated cell migration in cRCC by interfering with the EGFR signaling complex and mitogen-activated protein kinase (MAPK) pathways. We performed cell adhesion, migration, and biochemical studies in vitro using Caki-1 cell line. In addition, we validated the cell culture studies with expression analysis of six de-identified FFPE tissues from primary and metastatic cRCC patients. Our studies show that Klotho inhibited EGF-induced Caki-1 de-adhesion and decreased spreading on collagen type 1. Klotho also inhibited EGF-induced α2β1 integrin-dependent cell migration on collagen type 1. To test the involvement of MAPK pathways in EGF-induced Caki-1 cell motility, the cells were pretreated with either SB203580, a specific p38 MAPK inhibitor, or Klotho. SB203580 blocked the EGF-induced Caki-1 cell migration. Klotho had a comparable inhibitory effect. Our FFPE clinical specimens revealed decreased Klotho mRNA expression compared to a control, non-cancer kidney tissue. The decrease in Klotho mRNA levels correlated with increased c-Src expression, while E-Cadherin was relatively reduced in metastatic FFPE specimens where Klotho was least expressed. Taken together, these results suggest that secreted Klotho inhibits EGF-induced pro-migratory cell morphological changes and migration in Caki-1 cells. Our data additionally suggest that decreased Klotho expression may be involved in cRCC metastasis.

Vitamin D receptor suppresses proliferation and metastasis in renal cell carcinoma cell lines via regulating the expression of the epithelial Ca2+ channel TRPV5.

We previously demonstrated that transient receptor potential vanilloid subfamily 5 (TRPV5) expression was decreased in renal cell carcinoma (RCC) compared with that in normal kidney tissues, a finding that was correlated with vitamin D receptor (VDR) expression, but further investigations is warranted. The aim of this study was to elucidate whether VDR could regulate the expression of TRPV5 and affect proliferation and metastasis in RCC. In this study, we used lentivirus to conduct the model of VDR overexpression and knockdown caki-1 and 786-O RCC cell lines in vitro. The results demonstrated that VDR overexpression significantly inhibited RCC cells proliferation, migration and invasion, and promoted apoptosis by the MTT, transwell cell migration/invasion and flow cytometry assays, respectively. However, VDR knockdown in RCC cells had the opposite effect. The RNA-sequence assay, which was assessed in caki-1 cells after VDR overexpression and knockdown, also indicated that significantly differentially expressed genes were associated with cell apoptotic, differentiation, proliferation and migration. RT-PCR and western blot analysis showed that VDR knockdown increased TRPV5 expression and VDR overexpression decreased TRPV5 expression in caki-1 cells. Furthermore, knockdown of TRPV5 expression suppressed the VDR knockdown-induced change in the proliferation, migration and invasion in caki-1 cells. Taken together, these findings confirmed that VDR functions as a tumour suppressor in RCC cells and suppresses the proliferation, migration and invasion of RCC through regulating the expression of TRPV5.

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.

A heterometallic ruthenium-gold complex displays antiproliferative, antimigratory, and antiangiogenic properties and inhibits metastasis and angiogenesis-associated proteases in renal cancer.

Heterobimetallic compounds are designed to harness chemotherapeutic traits of distinct metal species into a single molecule. The ruthenium-gold (Ru-Au) family of compounds based on Au-N-heterocyclic carbene (NHC) fragments [Cl2(p-cymene)Ru(μ-dppm)Au(NHC)]ClO4 was conceived to combine the known antiproliferative and cytotoxic properties of Au-NHC-based compounds and the antimigratory, antimetastatic, and antiangiogenic characteristic of specific Ru-based compounds. Following recent studies of the anticancer efficacies of these Ru-Au-NHC complexes with promising potential as chemotherapeutics against colorectal, and renal cancers in vitro, we report here on the mechanism of a selected compound, [Cl2(p-cymene)Ru(μ-dppm)Au(IMes)]ClO4 (RANCE-1, 1). The studies were carried out in vitro using a human clear cell renal carcinoma cell line (Caki-1). These studies indicate that bimetallic compound RANCE-1 (1) is significantly more cytotoxic than the Ru (2) or Au (3) monometallic derivatives. RANCE-1 significantly inhibits migration, invasion, and angiogenesis, which are essential for metastasis. RANCE-1 was found to disturb pericellular proteolysis by inhibiting cathepsins, and the metalloproteases MMP and ADAM which play key roles in the etiopathogenesis of cancer. RANCE-1 also inhibits the mitochondrial protein TrxR that is often overexpressed in cancer cells and facilitates apoptosis evasion. We found that while auranofin perturbed migration and invasion to similar degrees as RANCE-1 (1) in Caki-1 renal cancer cells, RANCE-1 (1) inhibited antiangiogenic formation and VEGF expression. We found that auranofin and RANCE-1 (1) have distinct proteolytic profiles. In summary, RANCE-1 constitutes a very promising candidate for further preclinical evaluations in renal cancer.

Down-regulation of miR-210-3p encourages chemotherapy resistance of renal cell carcinoma via modulating ABCC1

BackgroundATP-binding cassette transporter super-family including ABCC1 and MDR-1 were involved in multi-drug resistance (MDR) of renal cell carcinoma (RCC) patients. Several miRNAs were confirmed to promote the MDR and the survival of tumor cells.MethodsThe RCC cell lines Caki-2 with vinblastine-resistant (Caki-2/VBL) or doxorubicin-resistant (Caki-2/DOX) were constructed, respectively. The expressions of miR-210-3p, ABCC1 and MDR-1 protein were determined by qRT-PCR and Western blot assays. The viability of RCC cells was assessed by MTT assay. The regulatory relationship between miR-210-3p and ABCC1 was analyzed by Dual Luciferase assay. The effect of miR-210-3p in vivo was investigated with a tumor xenograft model in mice.ResultsMiR-210-3p expression was observed to significantly decrease in Caki-2/VBL and Caki-2/DOX cells. Meanwhile, ABCC1 and MDR-1 were significantly increased in Caki-2/VBL and Caki-2/DOX cells. ABCC1 was a novel target of miR-210-3p and negatively regulated by miR-210-3p. And miR-210-3p improved drug-sensitivity of RCC cells. Down-regulation of ABCC1 could reverse the effect of miR-210-3p knockdown on the drug-resistance and the level of MDR-1 in drug-sensitive RCC cells.ConclusionWe confirmed that down-regulation of miR-210-3p increased ABCC1 expression, thereby enhancing the MRP-1-mediated multidrug resistance of RCC cells.

Synthesis and Evaluation of Antioxidant and Cytotoxicity of the N-Mannich Base of Berberine Bearing Benzothiazole Moieties.

Berberine, a quaternary ammonium salt from the protoberberine group of benzylisoquinoline alkaloids has drawn high attention for its several biological potencies. To furnish new rationalized derivatives based on berberine core which can deliver promising antioxidant and cytotoxic activities. The N-Mannich base of an isoquinoline alkaloid, berberine, bearing substituted benzothiazole moieties was obtained. Novel synthesized analogues were in vitro screened for antioxidant efficacy toward 2,2-diphenyl- 1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) free radicals and in vitro cytotoxicity towards cervical cancer cell lines (HeLa and CaSki), an ovarian cancer cell line (SK-OV-3) and human renal cancer cell line (Caki-2). Cytotoxicity of the compounds toward normal cell lines was examined using the Madin-Darby canine kidney (MDCK) non-cancer cell line. Analogues bearing a methoxy functional group (5e), acid functionality (5c), and a cyano group (5m) showed remarkable radical scavenging potential in DPPH and ABTS bioassays. Potent cytotoxicity exhibited by berberine against the HeLa cell line was attributable to the presence of a 2-aminobenzothaizole moiety (5a) and its 6-chloro congener (5g) on the berberine core, and the 6-cyano group (5m) on the benzothiazole ring revealed strong sensitivity for the CaSki cell line, whereas subjected scaffolds demonstrated diminished activity against the SK-OV-3 cell line. In addition, the compound with a 2-aminobenzothaizole moiety (5a), compound with methoxy functional group (5e) and compound with cyano group appeared with the most significant cytotoxicity effect in Caki-2 cell line. Their structures have been elucidated by FT-IR, 1H NMR, 13C NMR, and elemental analyses (CHN) essential research. N-Mannich bases of berberine were efficiently generated utilizing pharmacologically diverse substituted 2-aminobenzothiazole entities and final compounds were found remarkably active in antioxidant and cytotoxic assay. Hence, such types of compounds can be further studied or rationalized in future drug discovery studies.

CIP2A Promotes Proliferation, Invasion and Chemoresistance to Cisplatin in Renal Cell Carcinoma.

CIP2A is a well-known oncoprotein whose expression is elevated in multiple human solid tumor types. However, its role in renal cell carcinoma (RCC) development is poorly understood. Thus, in our present study, we used the renal cancer cell lines 786-O, A498 and CAKI-1 and the renal epithelial cell line HK-2 to clarify the function of CIP2A in RCC. We found that CIP2A expression is much higher in the RCC cells than in the normal renal epithelial cell. Lentivirus covered coding region CIP2A cDNA sequence and CIP2A siRNA were used to up and down regulate CIP2A expression in vitro. We found that overexpression of CIP2A promoted G1/S transition and cell proliferation. In addition, up-regulation of CIP2A significantly enhanced the invasion and migration capabilities of the cells. Furthermore, CIP2A promoted epithelial-mesenchymal transformation (EMT) and chemoresistance to cisplatin in RCC cells. Taken together, our findings demonstrate that CIP2A plays an important role in proliferation, invasion and chemoresistance to cisplatin in RCC cells. CIP2A may serve as an ideal molecular target for RCC therapeutics.

Three-Dimensional Cell Culture Model Utilization in Renal Carcinoma Cancer Stem Cell Research.

Specific 3D conditions of cancer cell lines have been optimized over last years, with growing significance of serum-free and xeno-free culture variants. The choice of proper culture media enables cancer stem cells proliferation in primary and stable cell lines. To obtain renal cell cancer stem-like phenotype, we employed media dedicated for mesenchymal cells and adult stem cells. Developed RCC cell line 3D culture system enables effective drug testing, including tyrosine kinase inhibitor anti-cancer cell toxicity. To induce formation of 3D spheroids by RCC cell lines, StemXvivo and NutriStem media must be used. Usage of laminin- or poly-D-lysine coated plates enhances also the formation of spheroids in 3D-promoting media. Seeding is optimal with Caki-1 or ACHN cell lines as well as 786-O or HKCSC cells. Our bio-mimic 3D RCC cell culture model promotes cell viability and stem-related gene expression including E-cadherin, N-cadherin, HIF1, HIF2, VEGF, Sox2, Pax2, and Nestin. 3D spheroid formation ability and spheroid volume increase are disturbed upon drug treatment. Untreated 3D structures reach ~100μm in diameter at the end of 14-day long experiment. Sorter-based cell cycle analysis and Ki-67 staining should be conducted to verify specific toxicity. We suggest that due to the more complex architecture 3D RCC culture is more relevant to investigate the in vivo-like tumor drug response.

Integrin α5 triggers the metastatic potential in renal cell carcinoma.

The therapy of advanced renal cell carcinoma (RCC) is still a major challenge. To intervene therapeutically a deeper comprehension of the particular steps of metastasis is necessary. In this context membrane bound receptors like integrins play a decisive role. We analyzed the integrin α5 expression in 141 clear cell RCC patients by Western blot. Patients with RCC expressed a significant higher level of integrin α5 in tumor than in normal tissue. The integrin α5 expression correlated with tumor grade, the development of distant metastases within five years after tumor nephrectomy and reduced survival. The RCC cell lines Caki-1 and CCF-RC1, which highly express integrin α5, were treated with fibronectin in combination with or without an inhibiting anti-integrin α5 antibody. Afterwards the migration, adhesion, viability and prominent signaling molecules were analyzed. Both cell lines showed a significant reduced migration potential as well as a decreased adhesion potential to fibronectin after treatment with an integrin α5 blocking antibody. A contribution of the AKT and ERK1/2 signaling pathways could be demonstrated. The results indicate integrin α5 as a potent marker to discriminate patients’ tumor prognosis. Consequently the integrin subunit α5 can be considered as a target for individual therapy of advanced RCC.

Potential biological process of X-linked inhibitor of apoptosis protein in renal cell carcinoma based upon differential protein expression analysis.

The X-linked inhibitor of apoptosis protein (XIAP) is the best characterized member of the IAP family and is a potent inhibitor of the caspase/apoptosis pathway. It has also been revealed that XIAP has additional biological functions that rely on its direct inhibition of apoptosis. In the present study, stably transfected Caki-1 cells with XIAP-knockdown were generated, and an isobaric tag for relative and absolute quantitation-based proteomics approach was employed to investigate the regulatory mechanism of XIAP in renal cell carcinoma (RCC). The results demonstrate that the sensitivity of the RCC cell line to apoptotic stimulation increased markedly with XIAP-knockdown. A number of differentially expressed proteins were detected between the original Caki-1 cell line and the XIAP-knockdown Caki-1 cell line; 87 at 0 h (prior to etoposide treatment), 178 at 0.5 h and 169 at 3 h, while no differentially expressed proteins were detected (ratio >1.5 or <0.5; P<0.05) at 12 h after etoposide treatment. Through analysis of the differentially expressed proteins, it was revealed that XIAP may participate in the tumor protein p53 pathway, the Wnt signaling pathway, glucose metabolism, endoplasmic reticulum stress, cytoskeletal regulation and DNA repair. These results indicate that XIAP may have a number of biological functions and may provide an insight into the biomedical significance of XIAP overexpression in RCC.

In Vitro Antitumor Effects of AHR Ligands Aminoflavone (AFP 464) and Benzothiazole (5F 203) in Human Renal Carcinoma Cells.

We investigated activity and mechanism of action of two AhR ligand antitumor agents, AFP 464 and 5F 203 on human renal cancer cells, specifically examining their effects on cell cycle progression, apoptosis, and migration. TK-10, SN12C, Caki-1, and ACHN human renal cancer cell lines were treated with AFP 464 and 5F 203. We evaluated cytotoxicity by MTS assays, cell cycle arrest, and apoptosis by flow cytometry and corroborated a mechanism of action involving AhR signal transduction activation. Changes in migration properties by wound healing assays were investigated: 5F 203-sensitive cells show decreased migration after treatment, therefore, we measured c-Met phosphorylation by Western blot in these cells. A 5F 203 induced a decrease in cell viability which was more marked than AFP 464. This cytotoxicity was reduced after treatment with the AhR inhibitor α-NF for both compounds indicating AhR signaling activation plays a role in the mechanism of action. A 5F 203 is sequestered by TK-10 cells and induces CYP1A1 expression; 5F 203 potently inhibited migration of TK-10, Caki-1, and SN12C cells, and inhibited c-Met receptor phosphorylation in TK-10 cells. AhR ligand antitumor agents AFP 464 and 5F 203 represent potential new candidates for the treatment of renal cancer. A 5F 203 only inhibited migration of sensitive cells and c-Met receptor phosphorylation in TK-10 cells. c-Met receptor signal transduction is important in migration and metastasis. Therefore, we consider that 5F 203 offers potential for the treatment of metastatic renal carcinoma. J. Cell. Biochem. 118: 4526-4535, 2017. © 2017 Wiley Periodicals, Inc.

An increase in long non-coding RNA PANDAR is associated with poor prognosis in clear cell renal cell carcinoma

BackgroundNearly 30% of clear cell renal cell carcinoma (ccRCC) patients present with metastasis at the time of diagnosis, and the prognosis for these patients is poor. Therefore, novel potential prognostic biomarkers and therapeutic targets for ccRCC could be helpful. Emerging evidence indicates that lncRNAs play important roles in cancer tumorigenesis and could be used as potential biomarkers or therapeutic targets. PANDAR (promoter of CDKN1A antisense DNA damage activated RNA) is a relatively novel lncRNA that plays an important role in the development of multiple cancers. However, the clinical significance and molecular mechanism of PANDAR in ccRCC are still elusive. In the present study, we attempted to elucidate the role of PANDAR in ccRCC.MethodsThe relative expression level of lncRNA PANDAR was quantified by real-time qPCR in 62 paired ccRCC tissues and in renal cancer cell lines, and its association with overall survival was assessed by statistical analysis. The biological functions of lncRNA PANDAR on ccRCC cells were determined both in vitro and in vivo.ResultsPANDAR expression was significantly upregulated in tumor tissues and cell lines compared with normal counterparts. Moreover, PANDAR served as an independent predictor of overall survival, and increased PANDAR expression was positively correlated with an advanced TNM stage. Further experiments demonstrated that PANDAR silencing can significantly inhibit cell proliferation and invasion, induce cell cycle arrest in the G1 phase and significantly promote apoptosis in 7860 and Caki-1 cell lines. In addition, in vivo experiments confirmed that downregulation of PANDAR inhibited the tumorigenic ability of 7860 cells in nude mice. Silencing of PANDAR also inhibited the expression of Bcl-2 and Mcl-1 and upregulated the expression of Bax in vivo.ConclusionsOur results suggest that PANDAR is involved in ccRCC progression and may serve as a potential prognostic biomarker and therapeutic target.

Efficacy of a single-dose injection of CLR 131 (1-131-CLR1404) in a Caki-2 athymic nude mouse model.

e14087 Background: CLR 131 is a novel radioiodinated therapeutic that exploits the selective uptake and retention of phospholipid ethers (PLEs) by malignant cells. Study was to evaluate the therapeutic effect of CLR 131 when administered as a single dose intravenous injection in Caki-2 tumor bearing mice. Caki-2 cells are a human clear cell renal cell carcinoma (CCRCC). Methods: The Caki-2 cell line (human clear cell carcinoma) was purchased from American Type Culture Collection (ATCC, Rockville, MD) and maintained in McCoy’s 5a media supplemented with 10% fetal bovine serum. Female athymic nude mice (Hsd: Athymic Nude-Foxn1nu); 4-5 weeks of age, 16-18 g (Harlan, Indianapolis, IN) were injected subcutaneously with 1x106 viable cells (in 100 µL Dulbecco’s PBS) into the right flank. The study was initiated when tumor size had reached a pre-determined size (100-250 mm3). The mice were given potassium iodide at a concentration of 0.1% in their drinking water to block possible free iodide in the drug formulation. A single dose of ~110µCi of CLR 131 was given at Day 0 (N = 6 per group). A control dose of I-127-CLR1404 was given at ~110 µCi dose and was injected via tail vein on Day 0. Results: Tumor growth of the treatment group was significantly inhibited. The control group showed exponential growth after day 20 post-injection while the treatment group maintained the initial tumor volume up to day 75 post injection. By day 65, the control group increased 10.75-fold compared to the treatment group in average tumor volume. CLR 131 provides survival benefit for Caki-2 bearing mice. Kaplan Meier survival showed significant survival benefit with this model. Conclusions: The results of the study indicate that a single dose of CLR 131 on Caki-2 tumor bearing model showed a significant inhibition of tumor growth as well as significant survival benefit.

Notch signaling plays a crucial role in cancer stem-like cells maintaining stemness and mediating chemotaxis in renal cell carcinoma

BackgroundCancer stem cells (CSCs) are correlated with the initiation, chemoresistance and relapse of tumors. Notch pathway has been reported to function in CSCs maintenance, but whether it is involved in renal cell carcinoma (RCC) CSCs maintaining stemness remain unclear. This study aims to explore the effect of Notch pathway on stemness of CSCs in RCC and the underlying mechanisms.MethodsThe CD133+/CD24+ cells were isolated from RCC ACHN and Caki-1 cell line using Magnetic-activated cell sorting and identified by Flow cytometry analysis. RT-PCR and immunoblot analyses were used for determining the stemness maker expression. The effect of Notch pathway on function of CSCs was assessed by self-renewal ability, chemosensitivity, invasive and migratory ability tumorigenicity in vivo using soft agar colony formation assay, sphere-forming assay, MTT assay, Transwell assay.ResultsHere, we found that the sorted CD133+/CD24+cells possessed elevated stemness maker CTR2, BCL-2, MDR1, OCT-4, KLF4, compared with parental cells, as well as enhanced self-renewal ability, stronger resistance to cisplatin and sorafenib, increased invasion and migration, and higher tumorigenesis in vivo, suggesting the CD133+/CD24+ cells have the stem-like characteristics of CSCs and thus identified as RCC CSCs. Then the enhanced notch1, notch2, Jagged1, Jagged2, DLL1 and DLL4 expression were detected in RCC CSCs and blockage of Notch1 or notch2 using pharmacological inhibitor MRK-003 or its endogenous inhibitor Numb resulted in loss of its stemness features: self-renewal, chemoresistance, invasive and migratory potential, and tumorigenesis in vivo. Moreover, it is confirmed that overexpression of notch1 up-regulated CXCR4 inRCC CSCs and augmented SDF-1-induced chemotaxis in RCC CSCs in vitro, which could be rescued when treatment of CXCR4 inhibitor, suggesting that notch signaling promotes the chemotaxis of RCC CSCs by SDF-1/CXCR4 axis.ConclusionsOur results provide a new mechanism of RCC CSCs maintaining stemness via notch pathway as well as a potential therapeutic target in human RCC.

The Endocannabinoid, Anandamide, Induces Cannabinoid Receptor-Independent Cell Death in Renal Proximal Tubule Cells

Background: The endocannabinoid (EC) system is well characterized in the central nervous system but scarcely studied in peripheral organs. In this paper, we newly identify the effect of the EC anandamide (AEA) upon renal proximal tubule cells. Methods: Measurement of lactate dehydrogenase (LDH) release after treatment of primary renal proximal tubule cells (RPTEC) and renal carcinoma cell line (Caki-1) with AEA, arachidonic acid (AA), ethanolamide (EtAm), EC receptor CB1 antagonist (AM251), CB2 receptor antagonist (SR144528), TRPV1 receptor antagonist (capsazepine), degradation enzyme fatty acid amide hydrolase (FAAH) antagonist (URB597), antioxidants GSH-EE; Trolox, GSH depletor BSO, membrane cholesterol depletor (MCD), apoptosis inhibitor zVAD, necroptosis inhibitor Nec-1 or ferroptosis inhibitor Fer-1. Western blot and qRT-PCR analysis plus determination of reactive oxygen species (ROS) via H2-DCFDA were performed. Histology for EC enzymes, N-acetylphosphatidylethanolamine-hydrolyzing phospholipase D (NAPE-PLD) and FAAH, as well as the determination of physiological levels of ECs in human and rat renal tissue via liquid chromatography were conducted. Results: AEA both dose- and time-dependently induces cell death in RPTEC and Caki-1 within hours, characterized by cell blebbing, not influenced by blocking the described EC receptors by AM251, SR144528, capsazepine or FAAH by URB597 or MCD. Cell death is mediated via ROS. There is no difference found in the histology of the enzymes FAAH and NAPE-PLD in human renal tissue with interstitial nephritis. Blocking of apoptotic, necroptotic or ferroptotic cell death does not lead to a reduction in LDH release in vitro. Conclusion: The endocannabinoid anandamide induces cell death in renal proximal tubule cell in a time- and dose-dependent manner. This pathway is mediated via ROS and is independent of cannabinoid receptors, membrane cholesterol or FAAH activity.

CRISPR/Cas9-editing-based modeling of hypoxia in renal cancer cells

Uncontrolled growth in the cell mass of malignant tumors induces intensive angiogenesis. However, the demands of the cancer cells for nutrients and oxygen remain only partially met. Hypoxia is a process that accompanies malignant transformation and evokes changes in the DNA methylation profile in solid tumors. To a certain extent, these changes, including the hypermethylation of tumor suppressor gene promoters, are related to the decrease in the activity of Tet proteins under the conditions of oxygen and free radical deficit. Stabilization, accumulation, and nuclear translocation of the transcription factor HIF1α are the key molecular events in hypoxia. We modified the clear-cell renal cancer cell line Caki1 to stabilize the HIF1α protein and characterized a model cell line that will enable the studies of the mechanisms of changes of the DNA methylation level at a constant activity of Tet proteins and a gene transcription profile characteristic of hypoxia. The CRISPR/Cas9 DNA editing system was used to edit the VHL gene. The mutant VHL protein contained a disrupted alpha-helix at the C-terminus and could not participate in the molecular pathway of proteasomal degradation of the HIF1α factor; therefore, the latter accumulated in the nucleus and activated the specific target genes. An analysis of gene transcription revealed the induction of hypoxia-associated genes in the modified cell line. The developed Сaki-1/VHLmut model can be used to discriminate between the effects evoked by oxygen-suppressed hydroxylases of the Tet family and other hypoxia-associated mechanisms of DNA methylation/demethylation.