Renal Cancer Cell Lines Caki-1 Research Articles

Novel thienopyrimidine-aminothiazole hybrids: Design, synthesis, antimicrobial screening, anticancer activity, effects on cell cycle profile, caspase-3 mediated apoptosis and VEGFR-2 inhibition

A series of novel hybrid compounds of hexahydrobenzo[4,5]thieno[2,3-d]pyrimidine with aminothiazole scaffolds were synthesized. The synthesized compounds were evaluated for their cytotoxic activity against the NCI-60 human tumor cell line panel. Compounds 7c, 7d and 7e exhibited significant antiproliferative activities at 10−5 M dose. Compound 7c exhibited excellent cytotoxic activity against CNS cancer cell lines including SNB-75 and SF-295 as well as renal cancer cell line CAKI-1 when compared with sorafenib as standard anticancer drug. In addition, compound 7d showed almost comparable anticancer activity to sorafenib against SNB-75 cell line and displayed moderate activity against SF-295 and CAKI-1 cell lines in comparison to sorafenib. Compound 7c inhibited the vascular endothelial growth factor receptor 2 (VEGFR-2) with IC50 of 62.48 ± 3.7 nM and decreased both total VEGFR-2 and phosphorylated VEGFR-2 in treated SNB-75 cells suggesting its ability to down regulate cell proliferation, growth, and survival.. The flow cytometric analysis showed that 7c displayed its cytotoxic activity through the reduction of the cellular proliferation and induction of cell cycle arrest at the G2/M phase. Compound 7c clearly boosted the level of the apoptotic caspase-3. All the synthesized compounds were also screened for their antibacterial and antifungal activity against four pathogenic strains of both Gram-positive and Gram-negative as well as Candida albicans. Only compound 7d exhibited antifungal activity against Candida albicans compared to nystatin as the standard antifungal compound.

Synthesis and antiproliferative evaluation of novel N-arylquinolones

Novel N-aryl-substituted quinolones were evaluated for antiproliferative activity. The chemical modifications were inspired by previously reported cytotoxic agents with structural similarities. Dimethylated anilines displayed the most potent effect in the renal cancer cell line Caki-1 and the breast cancer cell line MDA-MB-231, with GI50 values down to 24 µM. Further evaluation in the NCI60 cell lines revealed growth inhibition up to 50%, with the strongest effect observed in renal and lung cancer cell lines. In silico ADMET evaluation indicated favorable characteristics for both gastrointestinal and CNS uptake. The potential toxic electrophilic α,β-unsaturated carbonyl functionalities were shown to be inert to ethanethiol.

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.

RNA interference-mediated knockdown of astrocyte elevated gene-1 inhibits growth, induces apoptosis, and increases the chemosensitivity to 5-fluorouracil in renal cancer Caki-1 cells.

Astrocyte elevated gene-1 (AEG-1) is a recently discovered oncogene that has been reported to be highly expressed in various types of malignant tumors, including renal cell carcinoma. However, the precise role of AEG-1 in renal cancer cell proliferation and apoptosis has not been clarified. In this study, we transfected the renal cancer cell line Caki-1 with a plasmid expressing AEG-1 short hairpin RNA (shRNA) and obtained cell colonies with stable knockdown of AEG-1. We found that AEG-1 down-regulation inhibited cell proliferation and colony formation and arrested cell cycle progression at the sub-G1 and G0/G1 phase. Western blot analysis indicated that the expression of proliferating cell nuclear antigen (PCNA), cyclin D1 and cyclin E were significantly reduced following AEG-1 down-regulation. In addition, AEG-1 knockdown led to the appearance of apoptotic bodies in renal cancer cells, and the ratio of apoptotic cells significantly increased. Expression of the anti-apoptotic factor Bcl-2 was dramatically reduced, whereas the pro-apoptotic factors Bax, caspase-3 and poly (ADP-ribose) polymerase (PARP) were significantly activated. Finally, AEG-1 knockdown in Caki-1 cells remarkably suppressed cell proliferation and enhanced cell apoptosis in response to 5-fluorouracil (5-FU) treatment, suggesting that AEG-1 inhibition sensitizes Caki-1 cells to 5-FU. Taken together, our data suggest that AEG-1 plays an important role in renal cancer formation and development and may be a potential target for future gene therapy for renal cell carcinoma.

Synthesis, cytotoxic and antibacterial studies of p-benzyl-substituted NHC–silver(I) acetate compounds derived from 4,5-di-p-diisopropylphenyl- or 4,5-di-p-chlorophenyl-1H-imidazole

4,5-Di(p-isopropylphenyl)-1H-imidazole (2a) and 4,5-di(p-chlorophenyl)-1H-imidazole (2b) were prepared from the appropriately substituted benzoin precursors and then reacted with five p-substituted benzyl halide derivatives through N-dibenzylation to the symmetrically (3a–j) or through N-monobenzylation followed by N-methylation to the un-symmetrically substituted (5b) imidazolium halides. These halides were further used for the synthesis of the corresponding N-heterocyclic carbene–silver(I) (NHC–silver) acetate complexes 4a–j and 5c. The silver(I) compounds 4b, 4e and 4g were characterised by single crystal X-ray diffraction.Semiquantitative antibacterial studies against the Gram-negative bacteria Escherichia coli and the Gram-positive bacteria Staphylococcus aureus, using the Kirby–Bauer disc diffusion method, were carried out on the imidazolium halides 3a–j and NHC–silver(I) acetate complexes 4a–g and 4i–j. Imidazolium halide precursors 3a–j showed bacterial growth inhibition in the range of 1–5 mm (weak) while their NHC–silver(I) acetate derivatives 4a–g and 4i–j were found to inhibit bacterial growth in range 2–6 mm (weak to medium).The NHC–silver(I) acetate complexes 4a–g and 4i–j were found to be cytotoxic by producing IC50 values of 4.7 (±0.5), 2.1 (±0.8), 3.4 (±0.6), 15 (±2), 25 (±7), 24 (±3), 14 (±3), 21 (±5) and 50 (±2) μM against the breast cancer cell line MCF-7, respectively. The same NHC–silver(I) complexes 4a–g and 4i–j exhibited IC50 values of 8.7 (±1.5), 4.6 (±0.6), 5.5 (±0.6), 34 (±1), 42 (±4), 44 (±3), 29 (±1), 100 (±2) and 140 (±10) μM against the renal cancer cell-line Caki-1, respectively. The un-symmetrically substituted compound 5c has IC50 values of 24 (±3) μM against MCF-7 and 44 (±3) μM against Caki-1.

Abstract 4039: Harnessing the tumor adaptive response to hypoxia to identify novel combinations of the vascular disrupting agent BNC105 with targeted therapeutics.

Abstract BNC105 is a small molecule Vascular Disrupting Agent (VDA) that exerts anti-cancer activity through selective shut-down of tumor blood vessels. A single dose of BNC105 causes a very high degree of hypoxia resulting in >95% necrosis 24hrs post treatment in rodent tumor models. Tumor recovery from the hypoxic stress caused by BNC105 occurs by day 2 post-treatment. We have conducted immunohistochemical analysis to identify the molecular basis driving this tumor recovery. Using the mouse renal cancer orthotopic tumor model RENCA, we demonstrated that BNC105 causes significant vascular shutdown within the tumor mass and is followed by activation of a number of proteins that have been previously shown to be involved in tumor adaptive responses to hypoxia. These responses included: upregulation of GLUT1, a potential reflection of increased cancer cell dependency on glucose metabolism, increased expression of the angiogenic drivers HIFα and VEGFA and increased phosphorylation of mTOR and 4EBP1. Furthermore, there was upregulation of PERK and phosphorylation of eIF2α, reflecting activation of the Unfolded Protein Response (UPR). Similar observations were recorded when these proteins were studied in BNC105 treated mice bearing tumors of the human renal cancer cell lines Caki-1 (VHL wild type) and A-498 (VHL mutant), suggesting that these changes are likely to be generic to renal tumors being exposed to BNC105 induced hypoxia. These observations led us to investigate the potential therapeutic benefit of combining BNC105 with agents inhibiting the function of proteins being upregulated as a result of BNC105 induced hypoxia. Rapamycin was used to inhibit mTOR signalling. Treatment of mice bearing A498 tumors with BNC105, Rapamycin or the combination demonstrated greater tumor inhibition when the combination was used. Tumor growth inhibition with Rapamycin alone was 18% and BNC105 alone 12%. The Rapamycin + BNC105 combination treatment resulted in 47% tumor growth inhibition and was statistically significant compared to the inhibition seen with the monotherapies (p>0.05). The potential benefit of combining BNC105 with the proteasome inhibitor Bortezomib was also investigated. Upregulation of PERK has been shown to be associated with ER stress caused by accumulation of unfolded proteins. We hypothesised that concurrent inhibition of the proteasome with Bortezomib will increase cell stress resulting in increased tumor necrosis. Treatment of RENCA tumors with BNC105 + Bortezomib resulted in increased tumor necrosis compared to animals treated with Bortezomib or BNC105 alone. This suggests that the tumor is more susceptible to these agents when in combination. These data demonstrate that BNC105 can be combined with therapies targeting tumor adaptive responses to hypoxia to yield greater anti-tumor efficacy. Citation Format: Tina C. Lavranos, Daniel J. Inglis, Donna M. Beaumont, Annabell F. Leske, Chloe K. Brown, Gabriel Kremmidiotis. Harnessing the tumor adaptive response to hypoxia to identify novel combinations of the vascular disrupting agent BNC105 with targeted therapeutics. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4039. doi:10.1158/1538-7445.AM2013-4039

Novel symmetrically p-benzyl-substituted 4,5-diaryl-imidazole N-heterocyclic carbene-silver(I) acetate complexes – Synthesis and biological evaluation

Symmetrically substituted N-heterocyclic carbene (NHC) precursors 1a–e and 3a–e were synthesised by reacting 4,5-bisaryl-1H-imidazole with 2 equivalents of 4-benzyl bromide, 4-methylbenzyl bromide, 4-methoxybenzyl chloride, 4-methoxycarbonylbenzyl bromide or 4-cyanobenzyl bromide to give the 1,3-bis(p-benzyl substituted)-4,5-bisaryl-imidazolium halides. The NHC-silver(I) acetate complexes (1,3-bisbenzyl-4,5-bisphenyl-imidazole-2-ylidene) silver(I) acetate (2a), (1,3-bis(4-methylbenzyl)-4,5-bisphenyl-imidazole-2-ylidene) silver(I) acetate (2b), (1,3-bis(4-methoxybenzyl)-4,5-bisphenyl-imidazole-2-ylidene) silver(I) acetate (2c), (1,3-bis(4-methoxycarbonylbenzyl)-4,5-bisphenyl-imidazole-2-ylidene) silver(I) acetate (2d), (1,3-bis(4-cyanobenzyl)-4,5-bisphenyl-imidazole-2-ylidene) silver(I) acetate (2e), (1,3-bisbenzyl-4,5-bis(4-methoxyphenyl)-imidazole-2-ylidene) silver(I) acetate (4a), (1,3-bis(4-methylbenzyl)-4,5-bis(4-methoxyphenyl)-imidazole-2-ylidene) silver(I) acetate (4b), (1,3-bis(4-methoxybenzyl)-4,5-bis(4-methoxyphenyl)-imidazole-2-ylidene) silver(I) acetate (4c), (1,3-bis(4-methoxycarbonylbenzyl)-4,5-bis(4-methoxyphenyl)-imidazole-2-ylidene) silver(I) acetate (4d) and (1,3-bis(4-cyanobenzyl)-4,5-bis(4-methoxyphenyl)-imidazole-2-ylidene) silver(I) acetate (4e) were yielded by reacting these NHC precursors with silver(I) acetate. The silver(I) acetate complexes 2d, 2e and 4c were characterised by single crystal X-ray diffraction. Qualitative antibacterial studies against the Gram-negative bacteria Escherichia coli and the Gram-positive bacteria Staphylococcus aureus, using the Kirby–Bauer disc diffusion method were carried out on the ten NHC-silver(I) acetate complexes 2a–e and 4a–e. Also the IC50 values of these ten complexes were determined by an MTT-based assay against the human renal cancer cell line Caki-1 and the human breast cancer cell line MCF-7. The complexes 2a–e and 4a–e revealed the following IC50 values in μM against Caki-1: 14 (±1), 3.6 (±1.0), 4.2 (±0.5), 33 (±2), 59 (±4), 21 (±1), 21 (±2), 21 (±1), 34 (±2), 46 (±2) and against MCF-7: 5.8 (±0.6), 3.5 (±0.4), 5.4 (±0.3), 28 (±1), 25 (±2), 11 (±2), 5.0 (±0.3), 6.5 (±0.4), 17 (±1), 13 (±1); respectively.

Novel Nonsymmetrically p-Benzyl-Substituted (Benz)imidazole N-Heterocyclic Carbene-Silver(I) Acetate Complexes: Synthesis and Biological Evaluation

Nonsymmetrically substituted N-heterocyclic carbene (NHC) precursors 1a–d and 3a–d were synthesised by first reacting 1H-(benz)imidazole with p-cyanobenzyl bromide to give 4-(1H-imidazole-1-ylmethyl)benzonitrile (1) and 4-(1H-benzimidazole-1-ylmethyl)benzonitrile (3) and afterwards introducing benzyl bromide, 1-(bromomethyl)-4-methylbenzene, 1-(bromomethyl)-4-methoxybenzene, and methyl 4-(bromomethyl)benzoate. The NHC-silver(I) acetate complexes (1-benzyl-3-(4-cyanobenzyl)-2,3-dihydro-1H-imidazole-2-ylidene) silver(I) acetate (2a), (1-(4-cyanobenzyl)-3-(4-methylbenzyl)-2,3-dihydro-1H-imidazole-2-ylidene) silver(I) acetate (2b), (1-(4-cyanobenzyl)-3-[4-(methoxycarbonyl)benzyl]-2,3-dihydro-1H-imidazole-2-ylidene) silver(I) acetate (2c), (1-benzyl-3-(4-cyanobenzyl)-2,3-dihydro-1H-benzimidazole-2-ylidene) silver(I) acetate (4a), (1-(4-cyanobenzyl)-3-(4-methylbenzyl)-2,3-dihydro-1H-benzimidazole-2-ylidene) silver(I) acetate (4b), (1-(4-cyanobenzyl)-3-(4-methoxybenzyl)-2,3-dihydro-1H-benzimidazole-2-ylidene) silver(I) acetate (4c), and (1-(4-cyanobenzyl)-3-[4-(methoxycarbonyl)benzyl]-2,3-dihydro-1H-benzimidazole-2-ylidene) silver(I) acetate (4d) were yielded by reacting these NHC precursors with silver(I) acetate. The silver(I) acetate complex 4b was characterised by single crystal X-ray diffraction. Preliminary in vitro antibacterial studies against the Gram-positive bacteria Staphylococcus aureus and the Gram-negative bacteria Escherichia coli, using the Kirby-Bauer disc diffusion method, were carried out on the seven NHC-silver(I) acetate complexes 2a–c and 4a–d. Also the IC50 values of these seven complexes were determined by an MTT-based assay against the human renal cancer cell line Caki-1. The complexes 2a–c and 4a–c revealed the following IC50 values, respectively, 25 (±1), 15 (±2), 5.4 (±0.8), 16 (±2), 7.1 (±1), 20 (±4), and 14 (±1) μM.

281 Cancer: a less depressing outlook? Using antidepressants to induce autophagic programmed cell death in a resistant strain of Burkitt's lymphoma

STAT3, but not ERKs, mediates the IL-6–induced proliferation of renal cancer cells, ACHN and 769P.Although interleukin-6 (IL-6) has been suggested to function as an autocrine growth factor in renal cell carcinoma (RCC), the underlying mechanism responsible for the IL-6–induced proliferation of RCC has not been defined. The aim of this study was to characterize the signaling cascades mediating IL-6–induced proliferation and to investigate the use of effective novel interventions to block the IL-6–induced autocrine growth of renal cancer cells.IL-6–induced proliferation and intracellular signaling cascades were analyzed in four human renal cancer cell lines Caki-1, ACHN, 769P and A498. IL-6–induced activation of STAT3 (signal transducer and activator of transcription-1) and extracellular signal-regulated kinases (ERKs), and the effects of anti–IL-6 neutralizing antibody, Jak inhibitor AG 490, and MEK1 inhibitor PD 98059 were analyzed by Western blotting using phospho-specific antibodies. The DNA-binding activities of STATs were analyzed by EMSA. Apoptosis was determined by using nuclear staining and the TUNEL assay. Changes in the apoptosis-related proteins, bcl-2, bcl-xL, and bax were analyzed by Western blotting.IL-6 induced tyrosine phosphorylation and increased the DNA binding activity of STAT3 and, to a lesser extent, STAT1 in all cell lines except for Caki-1, which did not express the IL-6 receptor subunit gp130. ERKs were constitutively activated in all cell lines and the activation level was not up-regulated further by exogenously added IL-6 nor down-regulated by anti–IL-6 neutralizing antibody. IL-6–induced STAT3 tyrosine phosphorylation and DNA binding activity was inhibited by treatment with Jak specific inhibitor AG 490; however, it was not affected by the MEK1 inhibitor PD 98059. Moreover, treatment with AG 490 inhibited IL-6–induced proliferation of ACHN and 769P cells and induced apoptosis with the down-regulation of bcl-2 and the up-regulation of bax.This study identified STAT3, but not ERKs, to be a major mediator of IL-6–induced proliferation of renal cancer cells. Although ERKs were constitutively activated, ERKs were not found to be essential for the IL-6–induced proliferation and modulation of the STAT3 activity. Because the Jak specific inhibitor AG 490 effectively inhibited the IL-6–induced STAT3 activity and induced apoptosis, the blockade of the STAT3 signaling pathways is considered to be potentially useful as a novel therapeutic approach for RCC.

Decreased Acetylation of Histone H3 in Renal Cell Carcinoma: A Potential Target of Histone Deacetylase Inhibitors

Recent studies suggest that alterations in chromatin structure by histone acetylation may have an important role in the neoplastic process. We evaluated histone H3 acetylation in renal cell carcinoma and the effects of a histone deacetylase inhibitor on renal cancer cell lines. The expression of acetylated histone H3 (Lys9) in renal cell carcinoma and corresponding nonneoplastic tissue specimens was evaluated. We next assessed immunohistologically the relationships between acetylated histone H3 expression and clinicopathological parameters in 44 cases of renal cell carcinoma. Furthermore, we evaluated the effects of the histone deacetylase inhibitor depsipeptide on the renal cancer cell lines Caki-1, ACHN, 769P and 786O (ATCC). Acetylated histone H3 expression was decreased in 85.0% of renal cell carcinomas compared to levels in nonneoplastic tissue. Decreased expression was related to high nuclear tumor grade and advanced pathological tumor stage (p = 0.048 and 0.032, respectively). Depsipeptide inhibited cell line proliferation in a time and dose dependent manner. Depsipeptide induced apoptosis in Caki-1, ACHN and 786O, and induced G2 cell cycle arrest in 769P. Concomitantly depsipeptide increased acetylated histone H3 and p21(WAF/CIP1) expression, and induced Bcl-2 phosphorylation. These results suggest that the decreased acetylation of histone H3 is a common alteration in a malignant phenotype of renal cell carcinoma. Increasing the amount of acetylated histone H3 might be a therapeutic option for renal cell carcinoma.

Caspase-dependent and -independent induction of phosphatidylserine externalization during apoptosis in human renal carcinoma Cak1-1 and A-498 cells

Renal cell carcinoma is the most common neoplasm occurring in the kidney and is largely resistant to current chemotherapy. Understanding the mechanisms involved in renal carcinoma cell death may lead to novel and more effective therapies. In Caki-1 renal cancer cells, using phosphatidylserine externalization as a marker of apoptosis, the anti-cancer drugs 5-fluorouracil (5-FU), and its pro-drugs, doxifluridine (Dox) and floxuridine (Flox) proceeds via a caspase-dependent mechanism. In contrast, phosphatidylserine externalization produced by staurosporine in the renal cancer cell lines Caki-1 and A-498 proceeds via a caspase-independent mechanism. That is, the pan caspase inhibitor N-benzyloxycabonyl-Val-Ala-Asp-fluoromethylketone (ZVAD) did not ameliorate annexin V binding, cell shrinkage or changes in nuclear morphology. Subsequent experiments were conducted to determine mediators of phosphatidylserine externalization, using annexin V binding, when caspases were inhibited. Prior treatment of A-498 cells with cathepsin B (CA74 methyl ester), cathespsin D (pepstatin A) or calpain inhibitors (calpeptin, E64d) in the presence or absence of ZVAD did not ameliorate annexin V binding. The endonuclease inhibitor aurintricarboxylic acid (ATA), phospholipase A2 inhibitor bromoenol lactone (BEL), protein synthesis inhibitor cycloheximide (CH) and chloride channel blockers niflumic acid (NFA) and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) all had no effect on staurosporine-induced annexin V binding in A-498 cells either in the presence or absence of ZVAD. We also modulated sphingomyelin and the de novo pathways of ceramide synthesis and found no amelioration of staurosporine-induced annexin V binding in A-498 cells either in the presence or absence of ZVAD. These results indicate that 5-FU, Dox and Flox induce externalization of phosphatidylserine during apoptosis in Caki-1 renal cancer cells primarily through a caspase-dependent mechanism and that externalization of phosphatidylserine during apoptosis produced by staurosporine in the renal cancer cell line A-498 is independent of many of the common signaling pathways known to be involved in this process.

Leptin Promotes Invasiveness of Murine Renal Cancer Cells Via Extracellular Signal-Regulated Kinases and Rho Dependent Pathway

Obesity is a significant risk factor for renal cell carcinoma. The adipocyte derived cytokine leptin, which controls body weight homeostasis through food intake and energy expenditure, recently provided a potential link between obesity and cancer development. We examined whether leptin promotes the invasiveness of renal cancer cells and we investigated its underlying signaling pathway. Leptin receptor expression in the 6 human renal cancer cell lines Caki-1, ACHN, 769P, A498, SKRC44 and SKRC49, and in the murine renal cancer cell line Renca was examined by reverse transcriptase-polymerase chain reaction and Western blotting. The effect of leptin on renal cancer cell invasiveness was assessed by the invasion of cells through Matrigel coated Transwell inserts. Leptin induced intracellular signaling was examined by Western blotting. Leptin receptor was detected in all renal cancer cells examined at the mRNA and protein levels. Leptin increased Renca cell invasiveness at 1 ng/ml (p < 0.05). There was up to 3-fold invasiveness compared to untreated cells at 100 ng/ml with the activation of extracellular signal-regulated kinases and rho guanosine triphosphatase (p < 0.01). Leptin induced activation of rho guanosine triphosphatase was inhibited not only by the rho kinase inhibitor Y27632, but also by the MEK1 inhibitor PD98059, of which each inhibited leptin induced invasion of Renca cells (p < 0.01). Leptin promoted the invasiveness of murine renal cancer cells via extracellular signal-regulated kinases and rho guanosine triphosphatase dependent pathways. Rho guanosine triphosphatase was a downstream effector of extracellular signal-regulated kinases in leptin induced invasion. Leptin signaling could have a key role in renal cell carcinoma invasion.

Cell-surface matrix proteins and sialic acids in cell-crystal adhesion; the effect of crystal binding on the viability of human CAKI-1 renal epithelial cells.

To investigate the role of sialic acids and cellular matrix proteins as crystal-binding molecules in human calcium-oxalate nephrolithiasis. The well-defined human renal cancer cell line CAKI-1 was used a standard cell culture system. After enzymatic digestion of various cell surface molecules, the binding of alpha2,6 (Sambucus nigra, SN-) and alpha2,3 (Maackia amurensis, MA)-specific lectins to CAKI-1 cells was analysed. Simultaneously, the effect on adhesion and release of calcium oxalate monohydrate crystals was investigated (eight replicates). The effect of crystal adhesion on cell viability was assessed using Trypan blue exclusion (five replicates). Neuraminidase decreased MA-lectin binding of CAKI-1 cells by 39% (P < 0.05) but elevated SN-lectin binding by 812% (P < 0.05). Simultaneously, crystal binding to CAKI-1 cells was increased by 28% (P > 0.05). Pretreatment with collagenase type I, trypsin and dispase II reduced crystal-binding by 61-74% (P < 0.05) with no effect on sialic acid-specific lectin-binding. However, only collagenase type I and dispase (ratio 4 : 1) were also able to release crystals from their receptor-binding sites (P < 0.05). An increase in the number of cell surface-bound crystals correlated significantly with a decrease in cell viability (P < 0.05). alpha2,3-linked sialic acids protect cells from crystal-binding. Much greater SN-lectin binding associated with only moderately increased crystal binding argues against alpha2,6-linked sialic acids as a main target structure of crystals. In contrast, collagen type I, type IV and/or fibronectin seem to be potent crystal-binding molecules on human renal epithelial cells, with collagen type I involved in a potential second step of crystal-cell interaction.

STAT3, but not ERKs, mediates the IL-6–induced proliferation of renal cancer cells, ACHN and 769P

Although interleukin-6 (IL-6) has been suggested to function as an autocrine growth factor in renal cell carcinoma (RCC), the underlying mechanism responsible for the IL-6-induced proliferation of RCC has not been defined. The aim of this study was to characterize the signaling cascades mediating IL-6-induced proliferation and to investigate the use of effective novel interventions to block the IL-6-induced autocrine growth of renal cancer cells. IL-6-induced proliferation and intracellular signaling cascades were analyzed in four human renal cancer cell lines Caki-1, ACHN, 769P and A498. IL-6-induced activation of STAT3 (signal transducer and activator of transcription-1) and extracellular signal-regulated kinases (ERKs), and the effects of anti-IL-6 neutralizing antibody, Jak inhibitor AG 490, and MEK1 inhibitor PD 98059 were analyzed by Western blotting using phospho-specific antibodies. The DNA-binding activities of STATs were analyzed by EMSA. Apoptosis was determined by using nuclear staining and the TUNEL assay. Changes in the apoptosis-related proteins, bcl-2, bcl-xL, and bax were analyzed by Western blotting. IL-6 induced tyrosine phosphorylation and increased the DNA binding activity of STAT3 and, to a lesser extent, STAT1 in all cell lines except for Caki-1, which did not express the IL-6 receptor subunit gp130. ERKs were constitutively activated in all cell lines and the activation level was not up-regulated further by exogenously added IL-6 nor down-regulated by anti-IL-6 neutralizing antibody. IL-6-induced STAT3 tyrosine phosphorylation and DNA binding activity was inhibited by treatment with Jak specific inhibitor AG 490; however, it was not affected by the MEK1 inhibitor PD 98059. Moreover, treatment with AG 490 inhibited IL-6-induced proliferation of ACHN and 769P cells and induced apoptosis with the down-regulation of bcl-2 and the up-regulation of bax. This study identified STAT3, but not ERKs, to be a major mediator of IL-6-induced proliferation of renal cancer cells. Although ERKs were constitutively activated, ERKs were not found to be essential for the IL-6-induced proliferation and modulation of the STAT3 activity. Because the Jak specific inhibitor AG 490 effectively inhibited the IL-6-induced STAT3 activity and induced apoptosis, the blockade of the STAT3 signaling pathways is considered to be potentially useful as a novel therapeutic approach for RCC.