calpain and Carcinoma

Chlorpyrifos (CPF), a widely used organophosphorus pesticide has caused water pollution, threatening aquatic organisms. MicroRNAs (miRNAs) highly conserved noncoding RNAs, that regulate various cell death processes, including pyroptosis. To investigate the effect of CPF exposure on epithelioma papulosum cyprini (EPC) cell pyroptosis and the role of the miR-124-3p/CAPN1 axis, we established miR-124 overexpression and inhibition EPC cell models of CPF exposure. The target of the miR-124-3p/CAPN1 axis was primarily confirmed by the double luciferase reporter assay. Pyroptosis was demonstrated to occur in CPF-exposed EPC cells and was accompanied by mitochondrial membrane potential depletion, ROS level elevation and pyroptotic indicator expression upregulation. PD150606 was supplied as a CAPN1 inhibitor, alleviating CPF-induced mitochondrial dysfunction, and alleviating the increased expression of NLRP3, CASP1, IL1β and GSDMD. In conclusion, CPF induces pyroptosis by regulating the miR-124-3p/CAPN1 axis. This study enriches the cytotoxicity study of CPF, and provides new theoretical fundamentals for exploration of miRNA and its target protein response to water contaminants.

Topics: Calpain; Carcinoma; Chlorpyrifos; Humans; MicroRNAs; Organophosphorus Compounds; Pesticides; Pyroptosis

Long non-coding RNA MALAT1 (Metastasis-associated lung Adenocarcinoma transcript-1) has been demonstrated to play a critical role in the regulation of cancer progression and metastasis. However, little is known about MALAT1 in nasopharyngeal carcinoma (NPC) pathogenesis and progression.. Quantitative real-time PCR (qRT-PCR) was conducted to measure the expression of MALAT1, miR-124 and Capn4 mRNA in NPC cell lines. The protein level of Capn4 was examined by western blot analysis. Cell proliferation was detected by MTT assay, trypan blue exclusion method and colony formation analysis. Cell invasion was determined by transwell chamber assay. Expression of EMT-related proteins was detected by western blot. The potential targets of MALAT1 and miR-124 were verified by target prediction and luciferase reporter assay.. MALAT1 and Capn4 were upregulated while miR-124 expression was downregulated in NPC cell lines. MALAT1 knockdown inhibited proliferation, invasion and EMT of NPC cells. Moreover, MALAT1 improved Capn4 expression by sponging miR-124. MALAT1 upregulation abated miR-124-induced repression on NPC cell proliferation, invasion and EMT. Furthermore, Capn4 overexpression reversed the inhibitory effect of MALAT1 silencing on proliferation, invasion and EMT of NPC cells.. MALAT1 promoted proliferation, invasion and EMT of NPC cells through de-repressing Capn4 by sponging miR-124. The present study revealed a novel MALAT1/miR-124/Capn4 regulatory axis in NPC, contributing to a better understanding of the NPC pathogenesis and providing a promising therapeutic target for NPC therapy.

Topics: Calpain; Carcinoma; Cell Line, Tumor; Cell Movement; Cell Proliferation; Disease Progression; Down-Regulation; Epithelial-Mesenchymal Transition; Gene Expression Regulation, Neoplastic; Humans; MicroRNAs; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Neoplasm Invasiveness; RNA Interference; RNA, Long Noncoding; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Transfection; Up-Regulation

Impaired apoptosis is one of the hallmarks of cancer. Caspase-3 and -8 are key regulators of the apoptotic response and have been shown to interact with the calpain family, a group of cysteine proteases, during tumorigenesis. The current study sought to investigate the prognostic potential of caspase-3 and -8 in breast cancer, as well as the prognostic value of combinatorial caspase and calpain expression. A large cohort (n = 1902) of early stage invasive breast cancer patients was used to explore the expression of caspase-3 and -8. Protein expression was examined using standard immunohistochemistry on tissue microarrays. High caspase-3 expression, but not caspase-8, is significantly associated with adverse breast cancer-specific survival (P = 0.008 and P = 0.056, respectively). Multivariate analysis showed that caspase-3 remained an independent factor when confounding factors were included (hazard ratio (HR) 1.347, 95% confidence interval (CI) 1.086-1.670; P = 0.007). The analyses in individual subgroups demonstrated the significance of caspase-3 expression in clinical outcomes in receptor positive (ER, PR or HER2) subgroups (P = 0.001) and in non-basal like subgroup (P = 0.029). Calpain expression had been previously assessed. Significant association was also found between high caspase-3/high calpain-1 and breast cancer-specific survival in the total patient cohort (P = 0.005) and basal-like subgroup (P = 0.034), as indicated by Kaplan-Meier analysis. Caspase-3 expression is associated with adverse breast cancer-specific survival in breast cancer patients, and provides additional prognostic values in distinct phenotypes. Combinatorial caspase and calpain expression can predict worse prognosis, especially in basal-like phenotypes. The findings warrant further validation studies in independent multi-centre patient cohorts.

Topics: Adolescent; Adult; Aged; Breast Neoplasms; Calcium-Binding Proteins; Calpain; Carcinoma; Caspase 3; Caspase 8; Cell Line, Tumor; Estrogens; Female; Genes, erbB-2; Humans; Kaplan-Meier Estimate; Middle Aged; Neoplasm Proteins; Neoplasms, Hormone-Dependent; Progesterone; Prognosis; Tissue Array Analysis; Young Adult

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

Calpain small subunit 1 (Capn4) plays a key role in tumor migration or invasion. In this study, expression and function of Capn4 was investigated in human nasopharyngeal carcinoma (NPC). Here we report that both mRNA and protein levels of Capn4 were elevated in NPC tissues when compared to normal NP tissues. Similarly, Capn4 was also highly expressed in multiple NPC cell lines, compared to immortalized human nasopharyngeal epithelial cell line NP69. Moreover, expression of Capn4 was significantly correlated with Epstein-Barr virus infection, advanced stages, and lymph node or distant metastasis (P < 0.001). The patients with NPC displaying higher Capn4 had a significantly shorter overall survival (P = 0.002) and progression-free survival (P = 0.003). Furthermore, siRNA knockdown of Capn4 suppressed cell migration and invasion in vitro and in vivo. These events resulted from Capn4 downregulation were associated with reduced expression of matrix metalloproteinase 2 (MMP2), Snail, and Vimentin. Finally, we demonstrated that Capn4 upregulated MMP2 via nuclear factor-κB (NF-κB) activation, manifested by increased phosphorylation of p65, a subunit of NF-κB. Together, these findings argue a novel function of Capn4 in invasion and metastasis of NPC, and thereby suggest that Capn4 may represent an independent prognostic factor and a potential therapeutic target in NPC.

Topics: Animals; Biomarkers, Tumor; Cadherins; Calpain; Carcinoma; Cell Line, Tumor; Cell Movement; Disease-Free Survival; Enzyme Activation; Epstein-Barr Virus Infections; Female; Focal Adhesions; Gene Expression Regulation, Neoplastic; Humans; Male; Matrix Metalloproteinase 2; Mice; Mice, Inbred BALB C; Mice, Nude; Middle Aged; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Neoplasm Invasiveness; Neoplasm Metastasis; Phosphorylation; RNA Interference; RNA, Messenger; RNA, Small Interfering; Snail Family Transcription Factors; Transcription Factor RelA; Transcription Factors; Vimentin

The genesis and unique properties of the lymphovascular tumor embolus are poorly understood largely because of the absence of an experimental model that specifically reflects this important step of tumor progression. The lymphovascular tumor embolus is a blastocyst-like structure resistant to chemotherapy, efficient at metastasis and overexpressing E-cadherin (E-cad). Conventional dogma has regarded E-cad as a metastasis-suppressor gene involved in epithelial-mesenchymal transition. However, within the lymphovascular embolus, E-cad and its proteolytic processing by calpain and other proteases have a dominant oncogenic rather than suppressive role in metastasis formation and tumor cell survival. Studies using a human xenograft model of inflammatory breast cancer, MARY-X, demonstrated the equivalence of xenograft-generated spheroids with lymphovascular emboli in vivo with both structures demonstrating E-cad overexpression and specific proteolytic processing. Western blot revealed full-length (FL) E-cad (120 kDa) and four fragments: E-cad/NTF1 (100 kDa), E-cad/NTF2 (95 kDa), E-cad/NTF3 (85 kDa) and E-cad/NTF4 (80 kDa). Compared with MARY-X, only E-cad/NTF1 was present in the spheroids. E-cad/NTF1 was produced by calpain, E-cad/NTF2 by γ-secretase and E-cad/NTF3 by a matrix metalloproteinase (MMP). Spheroidgenesis and lymphovascular emboli formation are the direct result of calpain-mediated cleavage of E-cad and the generation of E-cad/NTF1 from membrane-associated E-cad rather than the de novo presence of either E-cad/NTF1 or E-cad/CTF1. E-cad/NTF1 retained the p120ctn-binding site but lost both the β-catenin and α-binding sites, facilitating its disassembly from traditional cadherin-based adherens junctions and its 360° distribution around the embolus. This calpain-mediated proteolysis of E-cad generates the formation of the lymphovascular embolus and is responsible for its unique properties of increased homotypic adhesion, apoptosis resistance and budding.

Topics: Amino Acid Sequence; Animals; Blood Vessels; Cadherins; Calpain; Carcinoma; Cell Adhesion; Cell Line, Tumor; Cell Survival; Embolism; Female; Humans; Inflammatory Breast Neoplasms; Lymphatic Vessels; Models, Biological; Molecular Sequence Data; Neoplasm Metastasis; Neoplasms; Neovascularization, Pathologic; Proteolysis; Transplantation, Heterologous

Pancreatic cancer, including cancer of the ampulla of Vater and bile duct, is very aggressive and has a poor five year survival rate; improved methods of patient stratification are required.. We assessed the expression of calpain-1, calpain-2 and calpastatin in two patient cohorts using immunohistochemistry on tissue microarrays. The first cohort was composed of 68 pancreatic adenocarcinomas and the second cohort was composed of 120 cancers of the bile duct and ampulla.. In bile duct and ampullary carcinomas an association was observed between cytoplasmic calpastatin expression and patient age (P = 0.036), and between nuclear calpastatin expression and increased tumour stage (P = 0.026) and the presence of vascular invasion (P = 0.043). In pancreatic cancer, high calpain-2 expression was significantly associated with improved overall survival (P = 0.036), which remained significant in multivariate Cox-regression analysis (hazard ratio = 0.342; 95% confidence interva l = 0.157-0.741; P = 0.007). In cancers of the bile duct and ampulla, low cytoplasmic expression of calpastatin was significantly associated with poor overall survival (P = 0.012), which remained significant in multivariate Cox-regression analysis (hazard ratio = 0.595; 95% confidence interval = 0.365-0.968; P = 0.037).. The results suggest that calpain-2 and calpastatin expression is important in pancreatic cancers, influencing disease progression. The findings of this study warrant a larger follow-up study.

Topics: Adult; Aged; Aged, 80 and over; Ampulla of Vater; Bile Duct Neoplasms; Biomarkers, Tumor; Calcium-Binding Proteins; Calpain; Carcinoma; Cohort Studies; Disease Progression; Female; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Male; Middle Aged; Pancreatic Neoplasms; Proportional Hazards Models; Tissue Array Analysis

Invasive cancer cells form dynamic adhesive structures associated with matrix degradation called invadopodia. Calpain 2 is a calcium-dependent intracellular protease that regulates adhesion turnover and disassembly through the targeting of specific substrates such as talin. Here, we describe a novel function for calpain 2 in the formation of invadopodia and in the invasive abilities of breast cancer cells through the modulation of endogenous c-Src activity. Calpain-deficient breast cancer cells show impaired invadopodia formation that is rescued by expression of a truncated fragment of protein tyrosine phosphatase 1B (PTP1B) corresponding to the calpain proteolytic fragment, which indicates that calpain modulates invadopodia through PTP1B. Moreover, PTP1B activity is required for efficient invadopodia formation and breast cancer invasion, which suggests that PTP1B may modulate breast cancer progression through its effects on invadopodia. Collectively, our experiments implicate a novel signaling pathway involving calpain 2, PTP1B, and Src in the regulation of invadopodia and breast cancer invasion.

Topics: Breast Neoplasms; Calpain; Carcinoma; Cell Adhesion; Cell Line; Cell Line, Tumor; Cell Movement; CSK Tyrosine-Protein Kinase; Down-Regulation; Extracellular Matrix; Female; Humans; Mutation; Neoplasm Invasiveness; Protein Tyrosine Phosphatase, Non-Receptor Type 1; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Pseudopodia; Signal Transduction; src-Family Kinases

Thapsigargin treatment of cultured cells leads to an increase in the intracellular calcium concentration, activation of calpain, and, in some cell types, apoptosis. Using a human prostate epithelial cell line that undergoes apoptosis in the presence of thapsigargin, we find decreased levels of IRS-1 protein levels during apoptosis. Inhibition of calpain prevents this decrease in IRS-1 protein; however, inhibitors of caspases or the proteasome are ineffective in maintaining IRS-1 levels. In terms of IGF-I-related second messenger proteins, the effect of thapsigargin is specific for IRS-1 since the protein levels of IGF-I receptor beta-subunit, Akt, Erk, and Shc are not affected. In addition to preventing the reduction in IRS-1, treatment of cells with calpain inhibitor II prevents apoptosis in response to thapsigargin. Finally, IRS-1 and calpain can be identified in protein complexes isolated using IRS-1-specific antibodies, indicating that calpain can associate with either IRS-1 or one of the proteins present in protein complexes that contain IRS-1. In total, these results suggest that IRS-1 may be targeted for degradation by calpain during apoptosis.

Topics: Apoptosis; Calcium; Calcium Signaling; Calpain; Carcinoma; Down-Regulation; Enzyme Inhibitors; Epithelial Cells; Gene Expression Regulation, Neoplastic; Humans; Insulin Receptor Substrate Proteins; Intracellular Fluid; Macromolecular Substances; Male; Phosphoproteins; Prostatic Neoplasms; Second Messenger Systems; Thapsigargin; Tumor Cells, Cultured

Animal, epidemiological and clinical studies have demonstrated the anti-tumor activity of pharmacological proteasome inhibitors and the cancer-preventive effects of green tea consumption. Previously, one of our laboratories reported that natural ester bond-containing green tea polyphenols (GTPs), such as (-)-epigallocatechin-3-gallate [(-)-EGCG] and (-)-gallocatechin-3-gallate [(-)-GCG], are potent and specific proteasome inhibitors. Another of our groups, for the first time, was able to enantioselectively synthesize (-)-EGCG as well as other analogs of this natural GTP. Our interest in designing and developing novel synthetic GTPs as proteasome inhibitors and potential cancer-preventive agents prompted our current study.. GTP analogs, (+)-EGCG, (+)-GCG, and a fully benzyl-protected (+)-EGCG [Bn-(+)-EGCG], were prepared by enantioselective synthesis. Inhibition of the proteasome or calpain (as a control) activities under cell-free conditions were measured by fluorogenic substrate assay. Inhibition of intact tumor cell proteasome activity was measured by accumulation of some proteasome target proteins (p27, I kappa B-alpha and Bax) using Western blot analysis. Inhibition of tumor cell proliferation and induction of apoptosis by synthetic GTPs were determined by G(1) arrest and caspase activation, respectively. Finally, inhibition of the transforming activity of human prostate cancer cells by synthetic GTPs was measured by a colony formation assay.. (+)-EGCG and (+)-GCG potently and specifically inhibit the chymotrypsin-like activity of purified 20S proteasome and the 26S proteasome in tumor cell lysates, while Bn-(+)-EGCG does not. Treatment of leukemic Jurkat T or prostate cancer LNCaP cells with either (+)-EGCG or (+)-GCG accumulated p27 and IkappaB-alpha proteins, associated with an increased G(1) population. (+)-EGCG treatment also accumulated the pro-apoptotic Bax protein and induced apoptosis in LNCaP cells expressing high basal levels of Bax, but not prostate cancer DU-145 cells with low Bax expression. Finally, synthetic GTPs significantly inhibited colony formation by LNCaP cancer cells.. Enantiomeric analogs of natural GTPs, (+)-EGCG and (+)-GCG, are able to potently and specifically inhibit the proteasome both, in vitro and in vivo, while protection of the hydroxyl groups on (+)-EGCG renders the compound completely inactive.

Topics: Adenocarcinoma; Apoptosis; bcl-2-Associated X Protein; Calpain; Carcinoma; Carrier Proteins; Caspases; Cell Line; Cysteine Endopeptidases; G1 Phase; Guanosine Triphosphate; Humans; Intracellular Signaling Peptides and Proteins; Jurkat Cells; Male; Microfilament Proteins; Multienzyme Complexes; Muscle Proteins; Phenols; Polymers; Prostatic Neoplasms; Proteasome Endopeptidase Complex; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Stereoisomerism; Tea; Transcriptional Elongation Factors

The p53 tumor suppressor protein is activated in cells in response to DNA damage and prevents the replication of cells sustaining genetic damage by inducing a cell cycle arrest or apoptosis. Activation of p53 is accompanied by stabilization of the protein, resulting in accumulation to high levels within the cell. p53 is normally degraded through the proteasome following ubiquitination, although the mechanisms which regulate this proteolysis in normal cells and how the p53 protein becomes stabilized following DNA damage are not well understood. We show here that p53 can also be a substrate for cleavage by the calcium-activated neutral protease, calpain, and that a preferential site for calpain cleavage exists within the N terminus of the p53 protein. Treatment of cells expressing wild-type p53 with an inhibitor of calpain resulted in the stabilization of the p53 protein. By contrast, in vitro or in vivo degradation mediated by human papillomavirus E6 protein was unaffected by the calpain inhibitor, indicating that the stabilization did not result from inhibition of the proteasome. These results suggest that calpain cleavage plays a role in regulating p53 stability.

Topics: Adenosine Triphosphate; Adenylyl Imidodiphosphate; Amino Acid Sequence; Breast Neoplasms; Calpain; Carcinoma; Chelating Agents; Cysteine Proteinase Inhibitors; Edetic Acid; Humans; Molecular Sequence Data; Mutation; Oncogene Proteins, Viral; Papillomaviridae; Repressor Proteins; Tumor Cells, Cultured; Tumor Suppressor Protein p53