calpain and Carcinoma--Squamous-Cell

Chemoresistance remains a challenge in the effective treatment of solid tumors, including oral squamous cell carcinoma (OSCC). Mitochondrial dynamics and autophagy have recently been implicated in the chemoresistance of cancer cells. The neutralization of ceramide is also associated with multidrug resistance, and ceramide synthase 6 (CerS6) is known to induce apoptosis. However, whether CerS6 regulates chemoresistance in OSCC is not clearly understood. Therefore, we investigated the role of CerS6 in the susceptibility of OSCC cells to cisplatin. In this study, we observed that cisplatin-resistant OSCC cells process lower levels of fission-state mitochondria and cell apoptosis than cisplatin-sensitive cells, and autophagy was activated in cisplatin-resistant OSCC cells. We found lower CerS6 expression in cisplatin-resistant OSCC cells. Overexpression of CerS6 with lentivirus-encoded CerS6 complementary DNA in cisplatin-resistant OSCC cells increased cisplatin sensitivity. Overexpression of CerS6 enhanced mitochondrial fission and apoptosis and attenuated cisplatin-induced autophagy in cisplatin-resistant OSCC cells. Further investigation indicated that CerS6 might function through altering calpain expression to enhance cisplatin sensitivity. Cisplatin-resistant OSCC cells xenografted onto a nude mouse model confirmed that CerS6 enhanced cisplatin chemotherapy sensitivity to reduce tumor volume. These data indicate that CerS6 could mediate an effective response to cisplatin in chemoresistant OSCC.

Topics: Animals; Apoptosis; Autophagy; Calpain; Carcinoma, Squamous Cell; Cell Line, Tumor; Cisplatin; Drug Resistance, Neoplasm; Enzyme Activation; Gene Expression Regulation, Neoplastic; Humans; Membrane Proteins; Mice, Nude; Mitochondrial Dynamics; Mouth Neoplasms; Sphingosine N-Acyltransferase

To identify the downstream regulated genes of GAEC1 oncogene in esophageal squamous cell carcinoma and their clinicopathological significance.. The anti-proliferative effect of knocking down the expression of GAEC1 oncogene was studied by using the RNA interference (RNAi) approach through transfecting the GAEC1-overexpressed esophageal carcinoma cell line KYSE150 with the pSilencer vector cloned with a GAEC1-targeted sequence, followed by MTS cell proliferation assay and cell cycle analysis using flow cytometry. RNA was then extracted from the parental, pSilencer-GAEC1-targeted sequence transfected and pSilencer negative control vector transfected KYSE150 cells for further analysis of different patterns in gene expression. Genes differentially expressed with suppressed GAEC1 expression were then determined using Human Genome U133 Plus 2.0 cDNA microarray analysis by comparing with the parental cells and normalized with the pSilencer negative control vector transfected cells. The most prominently regulated genes were then studied by immunohistochemical staining using tissue microarrays to determine their clinicopathological correlations in esophageal squamous cell carcinoma by statistical analyses.. The RNAi approach of knocking down gene expression showed the effective suppression of GAEC1 expression in esophageal squamous cell carcinoma cell line KYSE150 that resulted in the inhibition of cell proliferation and increase of apoptotic population. cDNA microarray analysis for identifying differentially expressed genes detected the greatest levels of downregulation of calpain 10 (CAPN10) and upregulation of trinucleotide repeat containing 6C (TNRC6C) transcripts when GAEC1 expression was suppressed. At the tissue level, the high level expression of calpain 10 protein was significantly associated with longer patient survival (month) of esophageal squamous cell carcinoma compared to the patients with low level of calpain 10 expression (37.73 ± 16.33 vs 12.62 ± 12.44, P = 0.032). No significant correction was observed among the TNRC6C protein expression level and the clinocopathologcial features of esophageal squamous cell carcinoma.. GAEC1 regulates the expression of CAPN10 and TNRC6C downstream. Calpain 10 expression is a potential prognostic marker in patients with esophageal squamous cell carcinoma.

Topics: Aged; Apoptosis; Biomarkers, Tumor; Calpain; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Proliferation; Esophageal Neoplasms; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Kaplan-Meier Estimate; Male; Middle Aged; Multivariate Analysis; Nuclear Proteins; Oligonucleotide Array Sequence Analysis; Prognosis; Proportional Hazards Models; RNA Interference; RNA-Binding Proteins; Time Factors; Tissue Array Analysis; Transfection

The transcription factors NF-kB, HIF-1 and vascular endothelial growth factors (VEGF) are known to play an important role in pathogenesis of squamous cell carcinoma of head and neck (SCCHN).. The aim of the study was to determine the NF-kB, HIF-1 and VEGF, expression their characteristics in squamous cell carcinoma of head and neck.. Transcription factors and VEGF expression were measured by ELISA kits. Proteasome and calpain activity were determined using specific fluorogenic substrate. Proteasome subunits composition was measured by Western blot analysis.. In the present study, we revealed the connection between SCCHN lymphogenous metastasis development and NF-kB p50 expression. An increase in total, 26S and 20S proteasome activities and calpain activity was observed in cancer tissues in comparison with agreed standard (non-transformed tissue). The dynamics of changes in proteasome activity and proteasome subunits content during lymph nodes metastasis development had a complex pattern. Nonparametric analysis of variance showed the connection between the extent of metastatic affection of regional lymph nodes, total proteasome activity and LMP2 expression. Proteasome and calpain systems corresponded and interacted with each other. We also revealed a positive correlation between the NF-kB p65 and p50 expression and proteasome activity.. Taken together, our results suggest that above mentioned transcription factors and intracellular proteolytic systems are involved in SCCHN progression and metastasis. Moreover, the opportunity of transcription factors regulation by proteasome takes place in oncogenesis of SCCHN. The results provide a basis for new prognostic tests and development of novel targeted therapy.

Topics: Blotting, Western; Calpain; Carcinoma, Squamous Cell; Female; Head and Neck Neoplasms; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Male; Middle Aged; NF-kappa B; Prognosis; Proteasome Endopeptidase Complex; Proteolysis; Vascular Endothelial Growth Factor A

Stimuli directed towards activation of apoptosis mechanisms are an attractive approach to eliminate evasion of apoptosis, a ubiquitous cancer hallmark. In these in vitro studies, kinetics and electric field thresholds for several apoptosis characteristics are defined in E4 squamous carcinoma cells (SCC) exposed to ten 300 ns pulses with increasing electric fields. Cell death was >95% at the highest electric field and coincident with phosphatidylserine externalization, caspase and calpain activation in the presence and absence of cytochrome c release, decreases in Bid and mitochondria membrane potential (Δψm) without apparent changes reactive oxygen species levels or in Bcl2 and Bclxl levels. Bid cleavage was caspase-dependent (55-60%) and calcium-dependent (40-45%). Intracellular calcium as an intrinsic mechanism and extracellular calcium as an extrinsic mechanism were responsible for about 30 and 70% of calcium dependence for Bid cleavage, respectively. The results reveal electric field-mediated cell death induction and progression, activating pro-apoptotic-like mechanisms and affecting plasma membrane and intracellular functions, primarily through extrinsic-like pathways with smaller contributions from intrinsic-like pathways. Nanosecond second pulsed electric fields trigger heterogeneous cell death mechanisms in E4 SCC populations to delete them, with caspase-associated cell death as a predominant, but not an unaccompanied event.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; bcl-X Protein; BH3 Interacting Domain Death Agonist Protein; Calpain; Carcinoma, Squamous Cell; Caspases; Cell Line, Tumor; Cell Membrane; Cytochromes c; Egtazic Acid; Electric Stimulation; Electricity; Enzyme Activation; Humans; Membrane Potential, Mitochondrial; Protease Inhibitors; Reactive Oxygen Species; Time Factors

Calpain, also named CAPN (for calcium-activated neutral protease), is a ubiquitous intracellular cytoplasmic non-lysosomal cysteine endopeptidase that requires calcium ions to exert its activity. Two major isoenzymes are known- micro -calpain (CAPN1) and m-calpain (CAPN2)-requiring micromolar and millimolar calcium concentrations for activation, respectively. Many known substrates of the different calpain isoenzymes, such as the transcription factors c-Fos and c-Jun, the tumour suppressor protein p53, protein kinase C, pp60src, or the adhesion molecule integrin, have been implicated in the pathogenesis of various malignancies including squamous (SCC) and basal (BCC) cell carcinomas of human skin, suggesting an important role of the calpain isoenzymes in malignant diseases. We have analysed the expression of CAP1 and CAPN2 protein and mRNA expression in BCCs and SCCs of human skin. Interestingly, CAPN1 immunoreactivity (streptavidin-peroxidase technique) was markedly reduced in BCCs compared to normal human skin or SCCs, while in contrast CAPN1 mRNA levels (determined by real-time PCR) were markedly elevated in BCCs and SCCs compared to normal human skin. No differences were found analysing CAPN2 protein and mRNA expression in normal human skin, BCCs and SCCs. In conclusion, we have demonstrated for the first time alterations in calpain mRNA expression and protein content in malignant skin tumours that may be of importance for the tumorigenesis and growth characteristics of BCCs and SCCs. However, our results do not allow conclusions on the function of CAPN1 and CAPN2 in BCCs and SCCs. It is not known if the CAPN genes in BCCs or SCCs exhibit functionally inactivating mutations or whether decreased CAPN1 protein expression in BCCs and elevated CAPN1 mRNA in BCCs and SCCs reflect a feedback loop coupled with increased degradation or proteolysis of CAPN1 protein.

Topics: Blotting, Northern; Calpain; Carcinoma, Basal Cell; Carcinoma, Squamous Cell; Gene Expression; Humans; Immunoenzyme Techniques; Neoplasm Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Neoplasm; Skin; Skin Neoplasms

We examined the activation of mu-calpain in human epidermoid carcinoma KB cells following a rise in intracellular calcium concentration using antibodies specifically recognizing different activation states of mu-calpain. KB cells possess calpastatin activity in large excess of calpain activity as analyzed by ion exchange HPLC. Stimulation of the cells with a calcium ionophore, ionomycin, caused production of the autolytic intermediate form (M(r) = 78 k) of mu-calpain derived from the preautolysis form (80 k), while the fully autolyzed postautolysis form (76 k) remained below detectable levels at all times. The appearance of the autolytic intermediate paralleled limited proteolysis of the membrane-associated calpastatin fractions (110 k and 106 k); the resulting fragments (68 k and 45 k) were released into the cytosol. Both the production of the autolytic mu-calpain intermediate and the limited proteolysis of calpastatin in cell lysates in the presence of calcium were inhibited by a synthetic calpastatin peptide, indicating that proteolysis of calpastatin was indeed catalyzed by calpain and that the autolytic intermediate may have exerted the proteolytic activity. Furthermore, mu-calpain autolysis and calpastatin degradation, upon ionomycin treatment, were both augmented by epidermal growth factor (EGF). These results suggest that calpastatin serves not only as an inhibitor but also as a substrate for calpain at cell membranes and that intracellular conditions associated with the cell cycle may affect the activation of mu-calpain.

Topics: Amino Acid Sequence; Antibody Specificity; Autolysis; Binding Sites; Calcium; Calcium-Binding Proteins; Calpain; Carcinoma, Squamous Cell; Cysteine Proteinase Inhibitors; Enzyme Activation; Humans; Molecular Sequence Data; Tumor Cells, Cultured

Little is known about the relative intracellular localizations of the calcium-dependent proteases, calpains, and their naturally occurring inhibitor, calpastatin. In the present study, the intracellular localization of mu-calpain, m-calpain, and calpastatin was studied at the light microscopic level in proliferating A431 cells. Highly specific antibodies against the three antigens revealed distinct staining patterns in interphase and mitotic cells. Most notably, calpastatin in interphase cells was localized near the nucleus in tube-like, or large granular structures, while the calpains were more uniformly distributed through the cytoplasm in either a fibrillar form (mu-calpain) or a diffuse or fine granular form (m-calpain). The distribution patterns of the two calpain isozymes were distinctly different during mitosis. m-Calpain was concentrated at the mitotic spindle poles and midbody, while mu-calpain appeared to accumulate at the cell membrane and the spindles. Four other human cell lines as well as normal human monocytes were examined to determine if the calpains-calpastatin segregation patterns are common to other cells or are unique to the A431 line. With the exception of abundant nuclear mu-calpain in the C-33A cervical carcinoma, the segregation of the proteins was similar to that of A431. These studies indicate that calpains may be localized at regions which are relatively poor in calpastatin content. Proteins at these sites may be susceptible to calpain-catalyzed cleavage.

Topics: Antibodies, Monoclonal; Blotting, Western; Calcium-Binding Proteins; Calpain; Carcinoma, Squamous Cell; Cell Cycle; Cell Division; Cytoskeleton; Fluorescent Antibody Technique; Humans; Immunohistochemistry; Interphase; Subcellular Fractions; Tumor Cells, Cultured

Antisera were prepared against three synthetic peptides with amino acid sequences identical to those surrounding the three major autophosphorylation sites of the epidermal growth factor (EGF) receptor. The affinity-purified antibodies reacted strongly in an enzyme-linked immunosorbent assay against the immunizing peptide but showed little cross-reaction with the other two phosphorylation site peptides. EGF receptors labelled by autophosphorylation could be specifically precipitated by each of the phosphorylation site antibodies. The antibodies recognised EGF receptors labelled at each of the autophosphorylation sites, indicating that they could bind to the immunizing sequences irrespective of their states of phosphorylation. The antibodies were able to inhibit EGF receptor autophosphorylation without affecting EGF-stimulated tyrosine kinase activity towards exogenous peptide substrates, suggesting that the kinase and autophosphorylation sites were in distinct domains. Immunofluorescent staining of A431 cells showed that the autophosphorylation site sequences resided inside the cell. The autophosphorylation sites were shown to be within a domain of 20 000 mol. wt. which could be cleaved from the receptor through limited proteolysis by the calcium-dependent protease, calpain. The position of cleavage of the EGF receptor by the protease was mapped to lie between residues 996 and 1059. These results are discussed in the context of a model for the structure and function of the human EGF receptor.

Topics: Amino Acid Sequence; Antibodies; Calpain; Carcinoma, Squamous Cell; Cell Line; Epidermal Growth Factor; Epitopes; ErbB Receptors; Fluorescent Antibody Technique; Humans; Methionine; Peptide Fragments; Phosphorylation; Protein-Tyrosine Kinases; Receptors, Cell Surface; Sulfur Radioisotopes

Limited proteolysis converts the native (Mr 170 000) epidermal growth factor (EGF) receptor to the Mr 150 000 form of the receptor. Calcium-activated, neutral protease (purified to homogeneity from beef lung), chymotrypsin, and elastase were all similarly effective in generating the 150-kilodalton (150-kDa) form of the receptor in detergent-solubilized, membrane vesicles shed from A-431 cells. The rate of autophosphorylation with [gamma-32P]ATP of the 150-kDa form was only 10% of the rate with the native receptor. This decreased rate was not due to loss of kinase activity, since the phosphorylation of angiotensin was virtually unchanged after limited proteolysis of the native receptor kinase. However, maps of elastase-produced peptides from 170-kDa forms and elastase-generated 150-kDa forms of the EGF receptor showed that the major autophosphorylation sites in these two forms were totally different. Confirming this difference in autophosphorylation sites was the finding that the 32P label in the autophosphorylated native receptor could not be recovered in the 150-kDa form following proteolysis. This label was quantitatively recovered in 30-15-kDa peptide fragments generated simultaneously with the 150-kDa form of the receptor. Therefore, the decreased autophosphorylation of the 150-kDa form results from the loss of preferred autophosphorylation sites on the native receptor. Only 1-3% of the phosphate incorporated in the native receptor during autophosphorylation could be found on the 150-kDa autophosphorylation sites. Hence, autophosphorylation of the tyrosine sites in the 150-kDa form of the EGF receptor is markedly enhanced by removing the major sites autophosphorylated on the native form of the receptor.

Topics: Animals; Calpain; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Epidermal Growth Factor; ErbB Receptors; Kinetics; Mice; Molecular Weight; Phosphorylation; Protein-Tyrosine Kinases; Receptors, Cell Surface