calpain and Kidney-Neoplasms

We analyzed the dynamics of the expression of transcription factors, VEGF and its receptor VEGFR2, serine-threonine protein kinase mTOR and activity of proteasome and calpain in patients with metastatic renal cancer during therapy with tyrosine kinase inhibitor Votrient and mTOR blocker Afinitor. The expression of hypoxic nuclear factor HIF-1α in the tumor tissue decreased during therapy with the target preparations. The decrease of VEGF and its receptor VEGFR2 was observed only in patients treated with mTOR inhibitor. The increase in calpain activity in the tumor tissue was observed in both groups. These findings extend our understanding of the mechanism of action of target anticancer preparations as allow considering the studied markers as predictors in choosing optimal therapy.

Topics: Antineoplastic Combined Chemotherapy Protocols; Calpain; Carcinoma, Renal Cell; Everolimus; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Indazoles; Kidney Neoplasms; Middle Aged; Molecular Targeted Therapy; Nephrectomy; NF-kappa B; Pyrimidines; Sulfonamides; TOR Serine-Threonine Kinases; Treatment Outcome; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-2

Runt-related transcription factor 2 (RUNX2), a specific transcription factor of osteocytes, has been confirmed to be involved in the malignant biological behavior of various tumor cells, including renal cell carcinoma. However, the mechanism of action of RUNX2 in renal cell carcinoma cells is not yet fully understood. In this study, RUNX2-negative A498 cells and strongly positive ACHN cells were selected as the study subjects. An invasion chamber assay was used to detect the invasive ability of the cells. The expression of each protein was detected by Western blotting or immunofluorescence assays. The invasive ability of A498 cells was enhanced after the expression of RUNX2 protein was upregulated, whereas ACHN cells decreased after the expression of RUNX2 protein was silenced. The expression of calcium-activated neutral protease 2 (Calpain2) and fibronectin (FN) proteins was upregulated in A498 cells overexpressing RUNX2 protein, whereas it was downregulated after the downregulation of RUNX2 protein expression in ACHN cells. It was found that Calpain2 small interfering RNA (siRNA) or calpain inhibitor calpeptin could inhibit the expression of FN in ACHN and A498 cells overexpressing RUNX2. Calpain2 siRNA or calpeptin inhibited the invasion of A498 cells overexpressing RUNX2. Similarly, in ACHN cells, Calpain2 siRNA or calpeptin inhibited cell invasion. RUNX2 upregulates FN protein expression via Calpain2, thereby mediating renal cell carcinoma invasion.

Topics: Calpain; Carcinoma, Renal Cell; Cell Line, Tumor; Cell Proliferation; Core Binding Factor Alpha 1 Subunit; Humans; Kidney Neoplasms; RNA, Small Interfering

Calpain small subunit 1 (Capns1) has shown its correlation with the metastasis and invasion of hepatocellular carcinoma and intrahepatic cholangiocarcinoma. However, the expression and function of Capns1 in human renal cell carcinoma (RCC) have not been clarified. This study aimed to examine the expression of Capns1 in RCC tissues and cell lines and to assess its role performed in RCC.. Capns1 expression was evaluated in 75 pairs of RCC and matched adjacent non-tumor tissues by immunohistochemistry. The prognostic value of Capns1 in RCC was assessed by Kaplan-Meier and Cox regression analyses. The action of Capns1 in the proliferation, adhesion, migration, and invasion of RCC cells and the effects on matrix metalloproteinase (MMP) 2 and 9 expression were evaluated after Capns1 silence.. Capns1 expression was significantly higher in RCC tissues compared with the adjacent non-tumor tissues. Multivariate analysis showed that Capns1 overexpression was an independent poor prognostic marker in RCC. The silencing of Capns1 prohibited cell adhesion and impaired the migration and invasion ability of 786-O cells in vitro. Furthermore, Capns1 silence reduced MMP2 and MMP9 expression.. Capns1 overexpression predicts poor prognosis and correlates with tumor progression in RCC. Capns1 expression might serve a prognostic marker and therapeutic target for RCC.

Topics: Calpain; Carcinoma, Renal Cell; Cell Line, Tumor; Correlation of Data; Disease Progression; Female; Gene Expression Regulation, Neoplastic; Humans; Kidney Neoplasms; Male; Middle Aged; Prognosis

Calpain small subunit 1 (Capn4) has been shown to correlate with the metastasis/invasion of clear cell renal cell carcinoma (ccRCC). This study aimed to further elucidate the molecular mechanisms underlying Capn4-mediated ccRCC progression. The mRNA expression levels in ccRCC cells were measured by quantitative real-time PCR. The effects of Capn4 on cell adhesion, invasion, and migration were examined by cell adhesion assay, cell invasion assay, and wound-healing assay, respectively. The protein levels were detected by western blot analysis. The effect of Capn4 on cancer metastasis in vivo was assessed in a nude mice xenograft model. It was found that Capn4 was up-regulated in the ccRCC cells, and Capn4 overexpression suppressed cell adhesion activity and increased cell invasion and migration in 786-O cells, while Capn4 silencing increased cell adhesion activity and impaired the invasion and migration ability of Caki-1 cells. Capn4 overexpression also increased the protein level of cleaved talin in 786-O cells, while Capn4 silencing decreased the protein level of cleaved talin in Caki-1 cells. The focal adhesion kinase (FAK)/AKT/MAPK signaling was activated by Capn4 overexpression in 786-O cells, and was inhibited by Capn4 down-regulation in Caki-1 cells. Capn4 overexpression increased the protein levels of matrix metalloproteinase 2 (MMP-2), vimentin, N-cadherin, and down-regulated E-cadherin in 786-O cells, while Capn4 silencing decreased the protein levels of MMP-2, vimentin, N-cadherin, and up-regulated E-cadherin in Caki-1 cells. Capn4 also promoted cancer metastasis in the in vivo nude mice xenograft model. Our results implicate the functional role of Capn4 in ccRCC invasion and migration, which may contribute to cancer metastasis in ccRCC.

Topics: Animals; Calpain; Carcinoma, Renal Cell; Cell Line; Cell Line, Tumor; Focal Adhesion Protein-Tyrosine Kinases; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; Kidney Neoplasms; Mice, Nude; Neoplasm Invasiveness; Neoplasm Metastasis; Signal Transduction; Talin; Transplantation, Heterologous

Renal medullary carcinoma (RMC) is a rare and highly aggressive neoplasm that most often occurs in the setting of sickle cell trait or sickle cell disease (SCD). Most patients present with metastatic disease resistant to conventional chemotherapy, and therefore there is an urgent need for molecular insight to propose new therapies.. To determine the molecular alterations and oncogenic pathways that drive RMC development.. A series of five frozen samples of patients with RMC was investigated by means of gene expression profiling, array comparative genomic hybridization, and RNA and whole exome sequencing (WES).. RNA and DNA sequencing read data were analyzed to detect gene fusions and somatic mutations. Gene fusions mutations were validated by real-time polymerase chain reaction and fluorescence in situ hybridization. Gene expression profiling was analyzed by unsupervised hierarchical clustering and Gene Set Enrichment Analysis (Broad Institute, Cambridge, MA, USA).. We observed inactivation of the tumor suppressor gene SMARCB1 in all tumors. In all four cases developed in patients with SCD, we identified an original mechanism of interchromosomal balanced translocations that disrupt the SMARCB1 sequence and thus contribute to its inactivation. Gene expression profiling revealed that RMC shares common oncogenic pathways with pediatric malignant rhabdoid tumors, another tumor subtype characterized by SMARCB1 deficiency.. RMCs are characterized by an original mechanism of interchromosomal balanced translocations that disrupt the SMARCB1 sequence. WES reveals that RMCs show no other recurrent genetic alteration and an overall stable genome, underscoring the oncogenic potency of SMARCB1 inactivation.. Our comprehensive molecular study supports a pivotal role of the tumor suppressor gene SMARCB1 in the development of renal medullary carcinoma. The use of therapeutic strategies based on the biologic effects of its inactivation should now open new perspectives for this typically lethal malignancy.

Topics: Adolescent; Adult; Anemia, Sickle Cell; Calpain; Carcinogenesis; Carcinoma; Child; Comparative Genomic Hybridization; DNA-Binding Proteins; Exome Sequencing; Gene Expression Profiling; Gene Fusion; Humans; Kidney Neoplasms; Nuclear Proteins; Nuclear Receptor Subfamily 1, Group F, Member 1; RNA, Long Noncoding; Sequence Analysis, RNA; SMARCB1 Protein; Trans-Activators; Transcription Factors; Translocation, Genetic

To investigate the mRNA and protein levels of calpain small subunit-1 (Capn4) in human clear cell renal cell carcinoma (ccRCC), to analyse the relationship between Capn4 mRNA level and pathological stage of ccRCC, and to examine the potential of Capn4 as a prognostic factor in ccRCC.. mRNA and protein levels of Capn4 were measured in pairs of tumour tissues and matched adjacent nontumour tissue obtained from patients with ccRCC by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blotting, respectively. Associations of the mRNA level of Capn4 with pathological tumour stage and the overall survival of ccRCC patients were also analysed.. Capn4 mRNA and protein level were significantly higher in ccRCC tumour tissues compared with adjacent nontumour tissues as assessed by qRT-PCR and Western blotting, respectively. Higher Capn4 mRNA levels were observed in patients with more advanced pathological stage of ccRCC and were also associated with decreased overall survival of patients with ccRCC.. The findings from this study indicate that Capn4 has the potential to be an independent prognostic indicator for ccRCC.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Calpain; Carcinoma, Renal Cell; Disease Progression; Female; Gene Expression Regulation, Neoplastic; Humans; Kidney; Kidney Neoplasms; Male; Middle Aged; RNA, Messenger; Treatment Outcome

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 Cak(i)-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 Cak(i)-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 A(2) 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 Cak(i)-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.

Topics: Amino Acid Chloromethyl Ketones; Aniline Compounds; Antimetabolites, Antineoplastic; Apoptosis; Benzoic Acid; Benzylidene Compounds; Calpain; Caspases; Cathepsin B; Cathepsin D; Cell Line, Tumor; Cell Size; Ceramides; Cisplatin; Exocytosis; Fumonisins; Humans; Kidney Neoplasms; Naphthalenes; Niflumic Acid; Nitrobenzoates; Phosphatidylserines; Pyrones; Sphingomyelin Phosphodiesterase; Staurosporine; Triterpenes

Calpain, also named CANP (for calcium-activated neutral protease), is an intracellular cytoplasmatic non-lysosomal cysteine endopeptidase that requires calcium ions for activity. Many substrates of the calpain isoenzymes, such as the transcription factors c-Fos and c-Jun, the tumor supressor protein p53, protein kinase C, pp60c-src and the adhesion molecule integrin, have been implicated in the pathogenesis of different human tumors, suggesting an important role of the calpains in malignant diseases. We now report differential expression of the calpain I gene (CL I) in a variety of tumors, extending our study to a larger series of renal cell carcinomas. Using Northern-blot analysis, we studied calpain I expression in 30 renal cell carcinomas as compared with matched healthy tissues. Tumor samples were classified according to their histological type: 21 clear cell carcinomas, 4 chromophobe carcinomas, 3 papillary carcinomas and 2 oncocytomas. In renal tumor samples, calpain I gene mRNA was expressed at highly variable levels, significantly depending on the different histological types. Moreover, there was a correlation of higher calpain I expression with increased malignancy: within the clear cell carcinoma subset, tumor samples with advanced nodal status (N1 and N2) showed a significantly higher calpain I expression than tumors without metastasis to regional lymph nodes. Our data suggest an important role of calpain isoenzymes in carcinogenesis and tumor progression.

Topics: Biomarkers, Tumor; Blotting, Northern; Calpain; Carcinoma, Renal Cell; Gene Expression; Humans; Kidney Neoplasms; Lymphatic Metastasis; RNA, Messenger

mu-Calpain is a calcium-dependent neutral thiol protease activated by micromolar concentrations of calcium. mu-Calpain is implicated in various cellular functions regulated by calcium including exocytosis, cell fusion, apoptosis and control of cell proliferation. We studied the effect of 1,25-(OH)2D3 on mu-calpain levels in the human renal cell carcinoma line SK-RC-29 using monoclonal antibodies to the 80 kDa subunit of mu-calpain. Exposure of low density cultures (15000 cells/cm2) to 1,25-(OH)2D3 (100nM) for 48 hours resulted in 1.5-3 fold increase of mu-calpain cell content. The effect was not observed in higher density cultures (40000 cells/cm2). mu-Calpain content of high density cultures was higher than that of low density cultures and similar to that in low density cultures treated by 1,25-(OH)2D3. The cellular content of two other calcium binding proteins, annexin II and annexin VI was not affected by the hormone. 1,25-(OH)2D3 did not affect cell number or viability therefore its effect on mu-calpain is not secondary to changes in cell density. The effect of 1,25-(OH)2D3 was dose-dependent apparent already at 1nM and was not observed with 24,25-(OH)2D3. Increase in mu-calpain content may underlie some of the actions of 1,25-(OH)2D3 on classical and non classical target cells.

Topics: Calcitriol; Calpain; Carcinoma, Renal Cell; Cell Count; Densitometry; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Humans; Immunoblotting; Kidney Neoplasms; Tumor Cells, Cultured