calpain and Lung-Neoplasms

We investigated the association between genetic variants in the histone modification regions and the prognosis of lung adenocarcinoma after curative surgery. Potentially functional SNPs were selected using integrated analysis of ChIP-seq and RNA-seq. The SNPs were analyzed in a discovery set (n = 166) and a validation set (n = 238). The associations of the SNPs with overall survival (OS) and disease-free survival (DFS) were analyzed. A total of 279 SNPs were selected for genotyping. Among these, CAPN1 rs17583C>T was significantly associated with better OS and DFS (P = 0.001 and P = 0.007, respectively), and LINC00959 rs4751162A>G was significantly associated with worse DFS (P = 0.008). Luciferase assays showed a significantly lower promoter activity of CAPN1 in the rs17583 T allele than C allele (P = 0.008), and consistently the CT + TT genotypes had significantly lower CAPN1 expression than CC genotype (P = 0.01) in clinical samples. The rs4751162 G allele had higher promoter activity of GLRX3 than A allele (P = 0.05). The motif analyses and ChIP-qPCR confirmed that the variants are located in the active promoter/enhancer regions where transcription factor binding occurs. This study showed that genetic variants in the histone modification regions could predict the prognosis of lung adenocarcinoma after surgery.

Topics: Adenocarcinoma of Lung; Biomarkers, Tumor; Calpain; Carrier Proteins; Female; Gene Expression Regulation, Neoplastic; Histones; Humans; Lung Neoplasms; Male; Middle Aged; Polymorphism, Single Nucleotide; Prognosis; RNA, Long Noncoding; Survival Rate

The main cause of death in non-small-cell lung cancer (NSCLC) is tumor progression, in which metastasis and invasion play an important role. The metastatic cascade is marked by a change in morphological, biological, biochemical and functional characteristics, including the acquisition of cellular mobility. The migration activity of tumor cells determines the work of actin-binding proteins that cause their functional partners CAP1 and cofilin. Of interest is the study of the regulation of working tandem CAP1/cofilin in NSCLC. The mechanism that regulates the level of proteins in cells is proteolysis, carried out by proteasomes and calpains. Therefore, the aim of this study was to estimate the expression of CAP1/CFL1 mRNA and their protein level in NSCLC tissues, and to analyze the possible mechanisms of their regulation by the proteasome and calpain systems. Samples of NSCLC and histological unchanged lung tissue were used (n = 42). The CAP1 and CFL1 mRNA expressions were determined by real-time PCR, the contents of proteins encoded by them were determined by Western blotting, and the activity of proteasomes and calpains by the fluorimetric method. There was an increase in the expression of mRNA and protein levels of CAP1 and cofilin in the tumor tissue compared with the unchanged lung tissue. The expression of mRNA and the level of CAP1 in tumor tissue increased during growth of the primary tumor. The cofilin level in the tumor tissue decreases against the background of increased expression of its mRNA. At the same time, during tumor growth, the activity of proteasomes and calpains increased. A negative regression relationships between the activity of proteasomes and the levels of CAP1 and cofilin, as well as the activity of calpains and the level of cofilin, were found. It can be assumed that proteasomes and calpains are involved in the degradation of CAP1 and cofilin. The data obtained suggest the importance of CAP1, cofilin and proteolytic systems in the tumor transformation and lymphogenous metastasis.

Topics: Calpain; Carcinoma, Non-Small-Cell Lung; Cell Cycle Proteins; Cell Movement; Cofilin 1; Cytoskeletal Proteins; Female; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Lymphatic Metastasis; Male; Middle Aged; Proteolysis; RNA, Messenger; Signal Transduction

Lung cancer is diagnosed at an advanced stage due to its unrecognized symptoms, resulting in high mortality. In recent decades, research into the development of an early diagnostic method for lung cancer has expanded in order to overcome the high mortality rate. Calpain 2 (CAPN2) has been suggested as a tumor marker linked to angiogenesis, cell proliferation, and migration in non-small cell lung cancer. In this study, CAPN2 enzyme-activatable near-infrared peptide sensor linked to human serum albumin (HSA-CAPN2) was developed. Intracellular localization and strong recovered fluorescence signals of HSA-CAPN2 were observed in

Topics: A549 Cells; Animals; Biomarkers, Tumor; Biosensing Techniques; Calpain; Carcinoma, Non-Small-Cell Lung; Cell Transformation, Neoplastic; Early Detection of Cancer; Humans; Lung Neoplasms; Mice; Nanotechnology; Optical Imaging; Serum Albumin, Human

Calpain 1 (CAPN1) has been found to be a promoter of cancer progression. PTPN1 as a physiological target molecule of CAPN1 plays a dephosphorylated role on multiple receptor tyrosine kinases. This study aimed to reveal the effects of CAPN1/PTPN1 on malignant phenotype and EGFR-TKI resistance of lung adenocarcinoma (LUAD) cells.. A total of 84 primary LUAD tissues and paired paracancerous normal tissues were collected. Quantitative real-time PCR (qRT-PCR) and immunohistochemical (IHC) methods were used to measure the expression of CAPN1 and PTPN1 in tissues. qRT-PCR and western blot were used to detect the expressions of CAPN1, PTPN1, c-Met and PIK3R2 in cell lines. Cell counting kit-8 (CCK-8), colony formation and transwell assay were carried out to evaluate cell erlotinib resistance, proliferation, migration and invasion. Co-IP assay was used to verify the interaction between proteins. Cycloheximide (CHX) was applied to block protein synthesis.. CAPN1, c-Met and PIK3R2 were significantly upregulated and the correlation was positive in LUAD, while PTPN1 was decreased. EGFR-sensitive mutation was related to CAPN1/PTPN1. in vitro studies showed that PTPN1 can mediate dephosphorylation of c-Met and PIK3R2 by binding with both, thereby weakening cell proliferation, metastasis and erlotinib resistance, while CAPN1 could enhance the degradation of PTPN1 protein as a cancer promoter.. CAPN1 enhances the malignant behavior and erlotinib resistance of LUAD cells via degrading PTPN1 and then activating c-Met/PIK3R2, which suggests CAPN1/PTPN1 may serve as tumor markers or potential targets for diagnosis and treatment of LUAD.. Significant findings of the study Superior CAPN1 and inferior PTPN1 were related to activation of c-Met/PIK3R2 in lung adenocarcinoma. Moreover, regulations of CAPN1 and PTPN1 induced the changes of malignant behavior and erlotinib resistance. What this study adds Our findings confirmed that CAPN1/PTPN1 play crucial roles on proliferation, metastasis and erlotinib resistance of LUAD cells as c-Met/PIK3R2 regulators, and validated the regulatory mechanism of CAPN1 on PTPN1 in tumor model for the first time.

Topics: Adenocarcinoma of Lung; Antineoplastic Agents; Apoptosis; Biomarkers, Tumor; Calpain; Case-Control Studies; Cell Movement; Cell Proliferation; Drug Resistance, Neoplasm; Erlotinib Hydrochloride; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Phosphatidylinositol 3-Kinases; Prognosis; Protein Tyrosine Phosphatase, Non-Receptor Type 1; Proto-Oncogene Proteins c-met; Survival Rate; Tumor Cells, Cultured

Topics: Animals; Apoptosis; Calpain; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Movement; Cell Proliferation; Drug Resistance, Neoplasm; ErbB Receptors; Female; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Lung Neoplasms; Male; Mice, Nude; Paclitaxel; Tissue Array Analysis

Calpain 1 is a proinflammatory calcium-activated cysteine protease, which can be partly externalized. Extracellular calpains limit inflammatory processes and promote tissue repair, through cell proliferation and migration. Toll like receptor (TLR) 2 has been identified as a target of extracellular calpains in lymphocytes. The aim was to investigate the externalization of calpain 1 and the release of soluble TLR2 during tumor progression of pulmonary lepidic predominant adenocarcinoma (LPA). Extracellular calpain 1, soluble fragment of TLR2 and cytokines were analyzed by ELISA in bronchoalveolar lavage fluid (BALF) supernatants from patients with LPA (n=68). Source of calpain was analyzed by immunohistochemistry and soluble TLR2 by flow cytometry on polymorphonuclear neutrophils (PMN) and human lung cancer cell lines. Extracellular calpain 1, secreted by tumor cells, was associated to tumor progression, neutrophilic inflammation, with a poor prognostic factor on survival (P=0.003). TLR2 was expressed on PMN and tumor cells and decreased after calpain exposure. Soluble fragment of TLR2 in BALF supernatants was correlated to the extracellular calpain 1 concentration (r=0.624; P<0.001), and its high level was associated with tumor progression and a pro-inflammatory environment. Extracellular calpain 1 secreted by tumor cells, could participate in inflammatory microenvironment and tumor progression through TLR2 in LPA.

Topics: Adenocarcinoma; Aged; Bronchoalveolar Lavage Fluid; Calpain; Cell Line, Tumor; Disease Progression; Female; Humans; Inflammation; Lung Neoplasms; Male; Neoplasm Proteins; Neutrophils; Prognosis; Toll-Like Receptor 2

Our previous study suggested the anti-tumor activity of sepia ink oligopeptide (SIO). Here we sought to investigate the underlying molecular mechanism.. Cell proliferation was evaluated by cell counting kit-8 (CCK-8) assay. Cell apoptosis was determined by Annexin V/Propidium Iodide (PI) staining. The mitochondria pathway was characterized by quantification of Bcl-2, Bax, Caspase-9 and Cyto-C. The death receptor pathway was analyzed by determinement of Fas, Caspase-8 and NIK. The endoplasmic reticulum (ER)-dependent pathway was determined by measurement the expression of CHOP, Caspase-12, GRP78 and Calpain. The associated gene expression was quantified by RT-PCR and protein level was determined by immunoblotting.. We demonstrated treatment with structurally modified SIO (CSIO, 5 µM) significantly inhibited cell proliferation and induced apoptosis in lung cancer cell line A549. The mitochondrial pathway, death receptor pathway and ER stress induced apoptosis were stimulated upon CSIO treatment. The administration with respective inhibitors including midiv-1 (50 µM for 2 h), PDTC (20 µM PDTC for 30 min) and ALLN (20 mM ALLN for 5 h) readily reversed the apoptosis inducing effect of CSIO.. Our data demonstrates that CSIO is capable of induction apoptosis in lung cancer cell line, which is mediated by all three classical apoptotic pathways. Our results warrant further in vivo investigations of the anti-tumor potential of CSIO.

Topics: A549 Cells; Animals; Apoptosis; bcl-2-Associated X Protein; Calpain; Caspase 12; Caspase 8; Caspase 9; Cell Proliferation; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; fas Receptor; Humans; Ink; Leupeptins; Lung Neoplasms; Mitochondria; Oligopeptides; Proline; Proto-Oncogene Proteins c-bcl-2; Sepia; Thiocarbamates; Transcription Factor CHOP

Gefitinib, an epidermal growth factor receptor (EGFR)‑specific drug, is effective for ~1 year, after which resistance is inevitable. Calpain 2 (CAPN2) is known to serve a role in the drug response and resistance in certain cancer therapies. However, the full function of CAPN2, particularly in non‑small cell lung cancer, has not yet been elucidated. In the present study, CAPN2 expression in gefitinib‑resistant lung adenocarcinoma cells was investigated. CAPN2 function in these cells was further evaluated using gene knockdown both in vitro and in vivo. The results demonstrated that CAPN2 was strongly associated with gefitinib‑resistance, and CAPN2 mRNA and protein expression levels were significantly increased in gefitinib‑resistant cell lines. Furthermore, CAPN2 knockdown inhibited gefitinib‑resistant cell proliferation in vitro and in vivo. CAPN2 conferred gefitinib‑resistance by inhibiting cell apoptosis and arresting the cell cycle. CAPN2 knockdown also induced caspase activation and mitochondrial dysfunction, and its function in gefitinib resistance appeared to be largely mediated by EGFR/protein kinase B/survivin signaling pathway activation. These results suggest that CAPN2 is responsible for EGFR‑tyrosine kinase inhibitor resistance, and CAPN2 inhibition may be used to provide therapeutic benefits in the treatment of gefitinib resistance.

Topics: Animals; Apoptosis; Biomarkers, Tumor; Calpain; Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cell Movement; Cell Proliferation; Drug Resistance, Neoplasm; ErbB Receptors; Gefitinib; Humans; Inhibitor of Apoptosis Proteins; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Mice, Nude; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Quinazolines; Signal Transduction; Survivin; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

In patients with breast cancer and lung cancer, chymotrypsin-like and caspase-like activities of proteasomes and total activity of calpains in the primary tumor nodes and lymphogenic metastasis are elevated in comparison with the corresponding normal tissues. The development of lymphogenic metastases of breast cancer and lung cancer was associated with opposite change in caspase-like activity of proteasomes. These results can be useful for the development of methods for evaluation of aggressiveness of breast and lung cancer.

Topics: Adult; Breast Neoplasms; Calpain; Female; Humans; Lung Neoplasms; Middle Aged; Neoplasm Metastasis; Proteasome Endopeptidase Complex

Triple-negative breast cancers (TNBC) often exhibit an aggressive phenotype. Disulfiram (DSF) is an approved drug for the treatment of alcohol dependence, but has also been shown to kill TNBC cells in a copper (Cu)-dependent manner. Exactly how this occurs has not been clearly elucidated. We sought to investigate the mechanisms responsible for DSF/Cu-dependent induction of apoptosis and suppression of lung colonization by TNBC cells. DSF/Cu induced anoikis and significantly suppressed cell migration and invasion with negative effects on focal adhesions, coinciding with vimentin breakdown and calpain activation in TNBC cells. In a xenograft tumor model, DSF suppressed tumor growth and lung nodule growth, which was also associated with calpain activation. These findings warrant further investigation of disulfiram as a potential treatment for metastatic TNBC.

Topics: Animals; Anoikis; Antineoplastic Agents; Calpain; Cell Line, Tumor; Cell Movement; Cell Proliferation; Copper; Cytoskeleton; Disulfiram; Dose-Response Relationship, Drug; Enzyme Activation; Female; Focal Adhesions; Humans; Lung Neoplasms; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Proteolysis; Signal Transduction; Time Factors; Triple Negative Breast Neoplasms; Tumor Burden; Vimentin; Xenograft Model Antitumor Assays

The receptor tyrosine kinase MET and its ligand, the hepatocyte growth factor, are essential to embryonic development, whereas deregulation of MET signaling is associated with tumorigenesis leading to various cancers, including lung carcinoma. Mutations in the MET kinase domain lead to constitutive kinase activity and are associated with tumorigenesis. In lung cancer, however, some mutations are found in the juxtamembrane domain, and their functional consequences are unknown. Because the juxtamembrane domain of MET is targeted by several proteolytic cleavages, involved in its degradation during cell death or under steady-state conditions, we evaluated the influence of these mutations on the MET proteolytic cleavages. In stably transfected epithelial cells expressing MET, the juxtamembrane mutations R970C, P991S, and T992I were found not to modify the known caspase or presenilin-dependent regulated intramembrane proteolysis. Yet when overexpressed, the R970C variant caused generation of an as yet undescribed 45-kDa fragment (p45 MET). This fragment was found in the confluent lung cancer cell line NCI-H1437 carrying the R970C mutation and at a lesser extent in cell lines expressing WT MET, suggesting that R970C mutation favors this cleavage. Generation of p45 MET required the activity of the calpain proteases, confirming the involvement of proteolysis. Ectopic expression of reconstituted p45 MET in epithelial cell lines favored cell scattering and invasion indicating active role of this fragment in HGF/SF induced responses. Hence, although the juxtamembrane mutations of MET do not affect its known proteolytic cleavages, the R970C MET variant favors calpain dependent proteolytic cleavage in lung cancer cells.

Topics: Blotting, Western; Calpain; Cell Line, Tumor; Epithelial Cells; Fluorescent Antibody Technique; Gene Knockdown Techniques; Humans; Lung Neoplasms; Mutation; Protein Domains; Proto-Oncogene Proteins c-met; Signal Transduction

Small cell lung cancer (SCLC) is characterized by excellent initial response to chemotherapy and radiation therapy with a majority of the patients showing tumor shrinkage and even remission. However, the challenge with SCLC therapy is that patients inevitably relapse and subsequently do not respond to the first line treatment. Recent clinical studies have investigated the possibility of camptothecin-based combination therapy as first line treatment for SCLC patients. Conventionally, camptothecin is used for recurrent SCLC and has poor survival outcomes. Therefore, drugs which can improve the therapeutic index of camptothecin should be valuable for SCLC therapy. Extensive evidence shows that nutritional compounds like capsaicin (the spicy compound of chili peppers) can improve the anti-cancer activity of chemotherapeutic drugs in both cell lines and animal models. Statistical analysis shows that capsaicin synergizes with camptothecin to enhance apoptosis of human SCLC cells. The synergistic activity of camptothecin and capsaicin is observed in both classical and variant SCLC cell lines and, in vivo, in human SCLC tumors xenotransplanted on chicken chorioallantoic membrane (CAM) models. The synergistic activity of capsaicin and camptothecin are mediated by elevation of intracellular calcium and the calpain pathway. Our data foster hope for novel nutrition based combination therapies in SCLC.

Topics: Animals; Apoptosis; Calpain; Camptothecin; Capsaicin; Carcinoma, Small Cell; Cell Line, Tumor; Chickens; Drug Synergism; Humans; Lung Neoplasms

Macrophages highly populate tumour microenvironment and are referred to as tumor-associated macrophages (TAMs). The inflammasome is a multiprotein complex responsible of IL-1 like cytokines release, which biology has been widely studied by using bone-marrow-derived macrophages to mimic a physiological and/or host defense condition. To understand the role of this complex in lung tumor-associated macrophages (TAMs), we isolated and cultured broncho-alveolar lavage (BAL)-derived cells of lung tumor-bearing mice. The stimulation of lung TAMs with LPS+ATP increased the release of IL-1β. The inhibition of NLRP3 by means of glybenclamide significantly reduced IL-1β release. Similarly, C3H-derived, caspase-1 ko and caspase-11 ko TAMs released significantly reduced levels of IL-1β. Moreover, the stimulation of lung TAMs with the sole LPS induced a significant release of IL-1α, which was significantly reduced after caspase-1 pharmacological inhibition, and in TAMs genetically lacking caspase-1 and caspase-11. The inhibition of calpain I/II by means of MDL28170 did not alter IL-1α release after LPS treatment of lung TAMs. To note, the inoculation of LPS-treated bone marrow-derived macrophages into carcinogen-exposed mice increased lung tumor formation. In contrast, the depletion of TAMs by means of clodronate liposomes reduced lung tumorigenesis, associated to lower in vivo release of IL-1α and IL-1β.In conclusion, our data imply lung tumor lesions are populated by macrophages which pro-tumor activity is regulated by the activation of the NLRP3 inflammasome that leads to the release of IL-1α and IL-1β in a caspase-11/caspase-1-dependent manner.

Topics: Adenosine Triphosphate; Animals; Bronchoalveolar Lavage Fluid; Calpain; Carcinogenesis; Caspase 1; Caspase Inhibitors; Caspases; Caspases, Initiator; Cells, Cultured; Clodronic Acid; Dipeptides; Female; Glyburide; Inflammasomes; Interleukin-1alpha; Interleukin-1beta; Lipopolysaccharides; Lung Neoplasms; Macrophages, Alveolar; Methylnitrosourea; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Knockout; Neoplasms, Experimental; NLR Family, Pyrin Domain-Containing 3 Protein; Signal Transduction; Specific Pathogen-Free Organisms; Toll-Like Receptor 4

The expression of calpain small subunit 1 (Capn4) is correlated with the invasion of several types of tumors. However, the roles of Capn4 in non-small cell lung cancer (NSCLC) remain unclear. In this study, we found that the expression of Capn4 in NSCLC tissues was much higher than that in nontumorous samples. High levels of Capn4 expression were associated with lymph node metastasis and large tumor size in NSCLC patients. The 5-year overall survival rate in the Capn4(high) group was significantly lower than that in the Capn4(low) group. In multivariate analysis, Capn4 was identified as an independent prognostic factor for overall survival. Moreover, in an in vitro analysis, downregulation of Capn4 expression by siRNA suppressed the invasive potential of lung cancer cells. Finally, we demonstrated that Capn4 enhanced the invasion ability of lung cancer cells by upregulating the expression of matrix metalloproteinase 2. Our findings indicated that Capn4 may represent a potential therapeutic target and a novel prognostic marker of NSCLC.

Topics: Aged; Calpain; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Female; Gene Knockdown Techniques; Humans; Lung Neoplasms; Lymphatic Metastasis; Male; Matrix Metalloproteinase 2; Middle Aged; Prognosis; Up-Regulation

Upon activation by its ligand hepatocyte growth factor/scatter factor, the receptor tyrosine kinase Met promotes survival, proliferation, and migration of epithelial cells during embryogenesis. Deregulated Met signaling can also promote cancer progression and metastasis. Met belongs to the functional family of dependence receptors whose activity switches from pro-survival to pro-apoptotic during apoptosis upon caspase cleavage. Although apoptosis resistance is a hallmark of cancer cells, some remain sensitive to other cell death processes, including necrosis induced by calcium stress. The role and fate of Met during necrotic cell death are unknown. Following treatment with calcium ionophores, cell lines and primary cells undergo necrosis, and the full-length Met receptor is efficiently degraded. This degradation is achieved by double cleavage of Met in its extracellular domain by a metalloprotease of the A disintegrin and metalloproteinase (ADAM) family and in its intracellular domain by calpains (calcium-dependent proteases). These cleavages separate the Met extracellular region from its kinase domain, thus preventing Met activity and its potential pro-survival activity. Although the intracellular fragment is very similar to the fragment generated by caspases, it displays no pro-apoptotic property, likely because of the presence of the last few amino acids of Met, known to inhibit this pro-apoptotic function. The fragments identified here are observed in lung tumors overexpressing the Met receptor, along with fragments previously identified, suggesting that proteolytic cleavages of Met are involved in its degradation in tumor tissues. Thus, Met is a modulator of necrosis, able to protect cells when activated by its ligand but efficiently degraded by proteolysis when this process is engaged.

Topics: ADAM Proteins; Animals; Apoptosis; Calcium; Calpain; Caspases; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Enzyme Activation; Epithelial Cells; HEK293 Cells; Hepatocyte Growth Factor; Humans; Ionomycin; Lung Neoplasms; Mice; Necrosis; Neoplasm Metastasis; Proto-Oncogene Proteins c-met; RNA Interference; RNA, Small Interfering; Signal Transduction

Small cell lung cancer (SCLC) has an annual mortality approaching that of breast and prostate cancer. Although sensitive to initial chemotherapy, SCLC rapidly develops resistance, leading to less effective second-line therapies. SCLC cells often overexpress Bcl-2, which protects cells from apoptosis both by sequestering pro-apoptotic family members and by modulating inositol 1,4,5-trisphosphate receptor (IP3R)-mediated calcium signaling. BH3-mimetic agents such as ABT-263 disrupt the former activity but have limited activity in SCLC patients. Here we report for the first time that Bcl-2-IP3 receptor disruptor-2 (BIRD-2), a decoy peptide that binds to the BH4 domain of Bcl-2 and prevents Bcl-2 interaction with IP3Rs, induces cell death in a wide range of SCLC lines, including ABT-263-resistant lines. BIRD-2-induced death of SCLC cells appears to be a form of caspase-independent apoptosis mediated by calpain activation. By targeting different regions of the Bcl-2 protein and different mechanisms of action, BIRD-2 and ABT-263 induce cell death synergistically. Based on these findings, we propose that targeting the Bcl-2-IP3R interaction be pursued as a novel therapeutic strategy for SCLC, either by developing BIRD-2 itself as a therapeutic agent or by developing small-molecule inhibitors that mimic BIRD-2.

Topics: Aniline Compounds; Apoptosis; Calcium; Calpain; Caspases; Cell Line, Tumor; Drug Synergism; Enzyme Activation; Humans; Lung Neoplasms; Models, Biological; Peptides; Small Cell Lung Carcinoma; Sulfonamides

Capsaicin, the pungent ingredient of chili peppers, displays potent anti-neoplastic activity in a wide array of human cancer cells. The present manuscript examines the signaling pathways underlying the apoptotic activity of capsaicin in human small cell lung cancer (SCLC) in vitro and in vivo. Studies in neuronal cells show that capsaicin exerts its biological activity via the transient receptor potential vanilloid (TRPV) superfamily of cation-channel receptors. The TRPV family is comprised of six members (TRPV1-6). Capsaicin is a known agonist of the TRPV1 receptor. We observed that capsaicin-induced apoptosis in human SCLC cells was mediated via the TRPV receptor family; however it was independent of TRPV1. Surprisingly, the apoptotic activity of capsaicin required the TRPV6 receptor. Depletion of TRPV6 receptor by siRNA methodology abolished the apoptotic activity of capsaicin in SCLC cells. Immunostaining and ELISA showed that TRPV6 receptor was robustly expressed on human SCLC tissues (from patients) and SCLC cell lines but almost absent in normal lung tissues. This correlates with our results that capsaicin induced very little apoptosis in normal lung epithelial cells. The pro-apoptotic activity of capsaicin was mediated by the intracellular calcium and calpain pathway. The treatment of human SCLC cells with capsaicin increased the activity of calpain 1 and 2 by threefold relative to untreated SCLC cells. Such calpain activation, in response to capsaicin, was downstream of the TRPV6 receptor. Taken together, our data provide insights into the mechanism underlying the apoptotic activity of capsaicin in human SCLCs.

Topics: Animals; Anticarcinogenic Agents; Antineoplastic Agents; Apoptosis; Calcium; Calcium Channels; Calpain; Capsaicin; Cell Line, Tumor; Cell Proliferation; Heterografts; Humans; Lung Neoplasms; Male; Mice, Nude; Neoplasm Transplantation; Signal Transduction; Small Cell Lung Carcinoma; TRPV Cation Channels

We investigated the role of the ubiquitously expressed calpain 2 isoform in breast tumor cell growth, migration, signaling, and tumorigenesis. RNAi-mediated knockdown of the capn2 transcript was used to manipulate expression of the catalytic subunit of calpain 2 in the AC2M2 mouse mammary carcinoma cell line. Stable knockdown of capn2 correlated with reduced in vitro proliferation rates, soft agar colony formation efficiency, and migration rates, indicating roles for calpain 2 in mitogenesis, survival, and motogenesis. Biochemical analysis showed increased levels of protein phosphatase 2A and reduced levels of activated Akt in calpain 2-deficient cells, and this correlated with increased levels of the FoxO3a target gene product p27(Kip1), a key regulator of cell proliferation. Calpain 2 deficiency in the AC2M2 cells correlated with enhanced nuclear localization of FoxO3a, consistent with it being in a derepressed state capable of regulating transcriptional targets. Orthotopically engrafted calpain 2 knockdown AC2M2 cells generated tumors with reduced growth rates and enhanced in vivo expression of p27(Kip1). In summary, calpain 2 deficiency correlated with reduced Akt activity, increased protein phosphatase 2A levels, derepression of FoxO3a, and enhanced expression of the p27(Kip1) tumor suppressor. These observations argue that calpain 2 promotes tumor cell growth both in vitro and in vivo through the PI3K-Akt-FoxO-p27(Kip1) signaling cascade. Inhibition of calpain 2 might therefore provide therapeutic benefits in the treatment of cancer.

Topics: Animals; Blotting, Western; Calpain; Cell Line, Tumor; Cell Movement; Cell Nucleus; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p27; Cytoplasm; Female; Forkhead Box Protein O3; Forkhead Transcription Factors; Lung Neoplasms; Mammary Neoplasms, Animal; Mice; Mice, Nude; Microscopy, Fluorescence; Proto-Oncogene Proteins c-akt; RNA Interference; Signal Transduction; Tumor Burden

Lung cancer is one of the most common fatal diseases in the developed world. The disease is rarely cured by currently available therapies, with an overall survival rate of ∼10%. Characterizing novel proteins that offer crucial insights into the processes of lung tumour invasion and metastasis may therefore provide much-needed prognostic markers, and influence therapeutic strategies. Aberrant function of the integrin family of heterodimeric cell surface receptors is a common theme in cancer--investigation into novel integrin activity regulators may offer crucial insights into the processes of tumour invasion and metastasis and may reveal insights into potential therapeutic targets. We previously described that depletion of the novel multi-transmembrane domain protein Fam38A, located at the endoplasmic reticulum (ER), inactivates endogenous beta1 integrin affinity, reducing cell adhesion. We now show that depletion of Fam38A, also now known as Piezo1, causes anchorage independence and a switch to a reduced integrin-dependent mode of cell migration/invasion, a novel phenotype for this integrin-regulating protein. Normal lung epithelial cells show increased rates of migration by 2D time-lapse microscopy and increased capacity to invade into matrigel, despite having decreased integrin affinity. We confirm greatly depleted Fam38A expression in small cell lung cancer (SCLC) lines where a form of reduced integrin-dependent migration, i.e. amoeboid migration, is a known phenotype. We propose that loss of Fam38A expression may cause increased cell migration and metastasis in lung tumours.

Topics: Actin Cytoskeleton; Calpain; Cell Adhesion; Cell Line, Tumor; Cell Movement; Gene Expression; Gene Knockdown Techniques; Humans; Integrins; Ion Channels; Loss of Heterozygosity; Lung Neoplasms; Microfilament Proteins; RNA, Small Interfering; Small Cell Lung Carcinoma; Tensins

Caspase-8 is a proapoptotic protease that suppresses neuroblastoma metastasis by inducing programmed cell death. Paradoxically, caspase-8 can also promote cell migration among nonapoptotic cells; here, we show that caspase-8 can promote metastasis when apoptosis is compromised. Migration is enhanced by caspase-8 recruitment to the cellular migration machinery following integrin ligation. Caspase-8 catalytic activity is not required for caspase-8-enhanced cell migration; rather, caspase-8 interacts with a multiprotein complex that can include focal adhesion kinase and calpain 2 (CPN2), enhancing cleavage of focal adhesion substrates and cell migration. Caspase-8 association with CPN2/calpastatin disrupts calpastatin-mediated inhibition of CPN2. In vivo, knockdown of either caspase-8 or CPN2 disrupts metastasis among apoptosis-resistant tumors. This unexpected molecular collaboration provides an explanation for the continued or elevated expression of caspase-8 observed in many tumors.

Topics: Alstrom Syndrome; Animals; Calcium-Binding Proteins; Calpain; Caspase 8; Cell Line, Tumor; Cell Movement; Focal Adhesion Protein-Tyrosine Kinases; Focal Adhesions; Humans; Lung Neoplasms; Mice; Mice, Transgenic; Neoplasm Metastasis; Neuroblastoma; Talin

Focal adhesion kinase (FAK) is overexpressed in a variety of cancers, such as breast, colon, prostate, ovary, and lung cancers. However, the mechanism by which extracellular matrix fibronectin stimulates lung cancer cell migration and invasion through FAK remains to be investigated.. The signalling pathways in fibronectin-mediated lung cancer cell migration and invasion were examined using western blotting. The metastasis function was detected by wound healing, migration and invasion assays. Further, RNA interference and kinase inhibitors were also used to study the downstream signals.. In this study, we examined the FAK signalling pathways in relation to calpain-2 and RhoA in fibronectin-mediated lung cancer cell migration and invasion. We found that A549 lung epithelial cells stimulated by fibronectin showed increased phosphorylation of FAK and its downstream targets, Src, ERK1/2, phosphatidylinositol 3'-kinase (PI3K), and Akt. Consistent with this observation, depletion of FAK by siRNA resulted in the inhibition of Src, ERK1/2, PI3K, and Akt activity. In addition, the Src inhibitor, PP2, blocked the phosphorylation of FAK, ERK1/2, PI3K, and Akt. Conversely, inhibition of MEK1/2 using PD98059 reduced the expression of matrix metalloproteinase-9 (MMP9) and calpain-2. The PI3K inhibitor, LY294002, further blocked the expression of MMP9 and RhoA. Inhibition of both MEK1/2 and PI3K caused reduced cell migration and invasion.. Our data suggest that fibronectin-mediated activation of FAK that leads to lung cancer metastasis could occur through ERK or PI3K/Akt regulation of MMP9/calpain-2 or MMP9/RhoA activity, respectively.

Topics: Adenocarcinoma; Calpain; Cell Movement; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Fibronectins; Focal Adhesion Kinase 1; Humans; Lung Neoplasms; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; rhoA GTP-Binding Protein; Signal Transduction

Cisplatin, an effective anticancer agent, can induce tumor cell apoptosis via caspase-dependent and-independent pathways. However, the precise mechanism that regulates the pathways remains unclear. In this study, we showed that micro-calpain mediated both caspase-dependent and-independent pathways during cisplatin-induced apoptosis in human lung adenocarcinoma cells. After cisplatin treatment, calpain activation, as measured by a fluorescent substrate, was an early event, taking place well before apoptosis inducing factor (AIF) release and caspase-9/-3 activation. Confocal imaging of cells transfected with AIF-GFP demonstrated that AIF release occurred about 9 hr after cisplatin treatment. The increase of micro-calpain activity proved to be a crucial event in the apoptotic machinery, as demonstrated by the significant protection of cell death in samples suppressed the endogenous micro-calpain expression level, as well as cotreated with the calpain inhibitors, calpeptin and PD150606. Inhibition of mu-calpain not only significantly reduced caspase-9/-3 activities but also completely blocked AIF redistribution. Our study also showed that endogenous mitochondrial micro-calpain could directly induce the truncation and release of AIF, while caspases and cathepsins were not necessary for this process. In conclusion, the study demonstrated that activation of micro-calpain played an essential role in regulating both caspase-dependent and AIF-mediated caspase-independent apoptotic pathways in cisplatin-induced apoptosis.

Topics: Adenocarcinoma; Antineoplastic Agents; Apoptosis; Apoptosis Inducing Factor; Blotting, Western; Calpain; Caspases; Cisplatin; Flow Cytometry; Humans; Lung Neoplasms; Membrane Potential, Mitochondrial; Mitochondria; RNA, Small Interfering; Subcellular Fractions; Tumor Cells, Cultured

Cisplatin is an efficient anticancer agent. Cisplatin-based chemotherapy is believed to involve different signal transduction pathways, among which calpain activation has been proposed as an important factor in the induced apoptosis. In our study, based on real-time single cell analysis, we investigated the molecular involvement of calpain in cisplatin-induced apoptosis in living human lung adenocarcinoma cells. After cisplatin treatment, calpain was activated, resulting in Bid cleavage at 4-5 hr, followed by Bid translocation and cytochrome c release, leading to cell death. Calpeptin and PD150606, specific inhibitors of calpain, blocked Bid activation completely; however, cytochrome c release was delayed by more than 2 hr, which was associated with the delay of caspase-3 activation and cell death. Remarkably, calpain-mediated release of cytochrome c and cell death was significantly compromised in the Bid knockdown cells. Z-IETD-fmk and Z-VDVAD-fmk were used to block the activation of caspase-8 and caspase-2, respectively; however, the progression of apoptosis were not affected, suggesting that caspase-8 and caspase-2 were not involved in this experimental model. Taken together, the data demonstrate that calpain mediated cisplatin-induced apoptosis in human lung adenocarcinoma cells through activating Bid, which then regulated the mitochondrial apoptotic pathway. The delays of cytochrome c release, caspase-3 activation and subsequent cell death by inactivating calpain or silencing Bid exclude other earlier or parallel pathways, strongly suggesting that the calpain-mediated pathway is the kinetically earliest one, which dominates the cisplatin-induced apoptosis.

Topics: Adenocarcinoma; Antineoplastic Agents; Apoptosis; BH3 Interacting Domain Death Agonist Protein; Blotting, Western; Calpain; Caspases; Cisplatin; Cytochromes c; Humans; Lung Neoplasms; Mitochondria; RNA, Small Interfering; Signal Transduction; Tumor Cells, Cultured

Nicotine is a major component in cigarette smoke that activates the growth-promoting pathways to facilitate the development of lung cancer. However, it is not clear whether nicotine affects cell motility to facilitate tumor metastasis. Here we discovered that nicotine potently induces phosphorylation of both mu- and m-calpains via activation of protein kinase Ciota (PKCiota), which is associated with accelerated migration and invasion of human lung cancer cells. Purified PKCiota directly phosphorylates mu- and m-calpains in vitro. Overexpression of PKCiota results in increased phosphorylation of both mu- and m-calpains in vivo. Nicotine also induces activation of c-Src, which is a known PKCiota upstream kinase. Treatment of cells with the alpha(7) nicotinic acetylcholine receptor inhibitor alpha-bungarotoxin can block nicotine-induced calpain phosphorylation with suppression of calpain activity, wound healing, cell migration, and invasion, indicating that nicotine-induced calpain phosphorylation occurs, at least in part, through a signaling pathway involving the upstream alpha(7) nicotinic acetylcholine receptor. Intriguingly, depletion of PKCiota by RNA interference suppresses nicotine-induced calpain phosphorylation, calpain activity, cell migration, and invasion, indicating that PKCiota is a necessary component in nicotine-mediated cell motility signaling. Importantly, nicotine potently induces secretion of mu- and m-calpains from lung cancer cells into culture medium, which may have potential to cleave substrates in the extracellular matrix. These findings reveal a novel role for PKCiota as a nicotine-activated, physiological calpain kinase that directly phosphorylates and activates calpains, leading to enhanced migration and invasion of human lung cancer cells.

Topics: Bungarotoxins; Calpain; Cell Movement; Cytoplasm; Humans; Isoenzymes; Lung Neoplasms; Neoplasm Invasiveness; Nicotine; Phosphorylation; Protein Kinase C; Wound Healing

Cancer cells can invade three-dimensional matrices by distinct mechanisms, recently defined by their dependence on extracellular proteases, including matrix metalloproteinases. Upon treatment with protease inhibitors, some tumour cells undergo a 'mesenchymal to amoeboid' transition that allows invasion in the absence of pericellular proteolysis and matrix degradation. We show here that in HT1080 cells, this transition is associated with weakened integrin-dependent adhesion, consistently reduced cell surface expression of the alpha2beta1 integrin collagen receptor and impaired signalling downstream, as judged by reduced autophosphorylation of focal adhesion kinase (FAK). On examining cancer cells that use defined invasion strategies, we show that distinct from mesenchymal invasion, amoeboid invasion is independent of intracellular calpain 2 proteolytic activity that is usually needed for turnover of integrin-linked adhesions during two-dimensional planar migration. Moreover, an inhibitor of Rho/ROCK signalling, which specifically impairs amoeboid-like invasion, restores cell surface expression of alpha2beta1 integrin, downstream FAK autophosphorylation and calpain 2 sensitivity--features of mesenchymal invasion. These findings link weakened integrin function to a lack of requirement for calpain 2-mediated integrin adhesion turnover during amoeboid invasion. In keeping with the need for integrin adhesion turnover, mesenchymal invasion is uniquely sensitive to Src inhibitors. Thus, the need for a major pathway that controls integrin adhesion turnover defines and distinguishes cancer cell invasion strategies.

Topics: Base Sequence; Calpain; Carcinoma, Non-Small-Cell Lung; Cell Adhesion; Cell Line, Tumor; Fibrosarcoma; Flow Cytometry; Humans; Integrins; Lung Neoplasms; Mesoderm; Mutation, Missense; Neoplasm Invasiveness; RNA, Messenger; RNA, Small Interfering; src-Family Kinases

The mu- and m-calpains are major members of the calpain family that play an essential role in regulating cell motility. We have recently discovered that nicotine-activated protein kinase C iota enhances calpain phosphorylation in association with enhanced calpain activity and accelerated migration and invasion of human lung cancer cells. Here we found that specific disruption of protein phosphatase 2A (PP2A) activity by expression of SV40 small tumor antigen up-regulates phosphorylation of mu- and m-calpains whereas C2-ceramide, a potent PP2A activator, reduces nicotine-induced calpain phosphorylation, suggesting that PP2A may function as a physiological calpain phosphatase. PP2A co-localizes and interacts with mu- and m-calpains. Purified, active PP2A directly dephosphorylates mu- and m-calpains in vitro. Overexpression of the PP2A catalytic subunit (PP2A/C) suppresses nicotine-stimulated phosphorylation of mu- and m-calpains, which is associated with inhibition of calpain activity, wound healing, cell migration, and invasion. By contrast, depletion of PP2A/C by RNA interference enhances calpain phosphorylation, calpain activity, cell migration, and invasion. Importantly, C2-ceramide-induced suppression of calpain phosphorylation results in decreased secretion of mu- and m-calpains from lung cancer cells into culture medium, which may have potential clinic relevance in controlling metastasis of lung cancer. These findings reveal a novel role for PP2A as a physiological calpain phosphatase that not only directly dephosphorylates but also inactivates mu- and m-calpains, leading to suppression of migration and invasion of human lung cancer cells.

Topics: Calpain; Cell Line, Tumor; Cell Movement; Ceramides; Gene Expression Regulation; Humans; Lung Neoplasms; Neoplasm Invasiveness; Nicotine; Phosphoprotein Phosphatases; Phosphorylation; Protein Phosphatase 2; Protein Transport; Up-Regulation

Activation of Src kinase plays important roles in the development of many neoplasias. Most of the previous Src studies focused on the deregulation of Src kinase activity. The deregulated Src protein synthesis and stability in mediating malignant phenotypes of cancer cells, however, have been neglected. While investigating the signal transduction pathways contributing to ErbB2-mediated metastasis, we found that ErbB2-activated breast cancer cells that had higher metastatic potentials also had increased Src activity compared with ErbB2 low-expressing cells. The increased Src activity in ErbB2-activated cells paralleled higher Src protein levels, whereas Src RNA levels were not significantly altered. Our studies revealed two novel mechanisms that are involved in Src protein up-regulation and activation by ErbB2: (a) ErbB2 increased Src translation through activation of the Akt/mammalian target of rapamycin/4E-BP1 pathway and (b) ErbB2 increased Src stability most likely through the inhibition of the calpain protease. Furthermore, inhibition of Src activity by a Src-specific inhibitor, PP2, or a Src dominant-negative mutant dramatically reduced ErbB2-mediated cancer cell invasion in vitro and metastasis in an experimental metastasis animal model. Together, activation of ErbB2 and downstream signaling pathways can lead to increased Src protein synthesis and decreased Src protein degradation resulting in Src up-regulation and activation, which play critical roles in ErbB2-mediated breast cancer invasion and metastasis.

Topics: Adaptor Proteins, Signal Transducing; Animals; Breast Neoplasms; Calpain; Carrier Proteins; Cell Cycle Proteins; Enzyme Activation; Enzyme Stability; Eukaryotic Initiation Factors; Female; Genes, Dominant; Humans; Lung Neoplasms; Mice; Mice, Inbred ICR; Mice, SCID; Neoplasm Invasiveness; Phosphoprotein Phosphatases; Phosphoproteins; Phosphorylation; Protein Biosynthesis; Protein Kinases; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins pp60(c-src); Receptor, ErbB-2; Signal Transduction; TOR Serine-Threonine Kinases; Tumor Cells, Cultured

Mounting evidence indicates that cigarette smoking not only promotes tumorigenesis but also may increase the spread of cancer cells in the body. However, the intracellular mechanism(s) by which cigarette smoking promotes metastasis of human lung cancer remains enigmatic. Nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is an important component in cigarette smoke and is formed by nitrosation of nicotine. mu- and m-calpain (calpain I and calpain II) are major members of the calpain family, which are ubiquitously expressed in both small cell lung cancer and non-small cell lung cancer cells. Our findings indicated that NNK potently induces phosphorylation of both mu- and m-calpain in association with their activation and increased migration as well as invasion of lung cancer cells. Treatment of cells with PD98059 blocked phosphorylation of m- and mu-calpain and resulted in suppression of NNK-induced cell migration and invasion. p44 MAPK/extracellular signal-regulated kinase 1 (ERK1) and p42 MAPK/ERK2 were activated by NNK, co-localized with mu- and m-calpain in cytoplasm, and directly phosphorylated mu- and m-calpain in vitro. These findings suggest a role for the ERK1/2 kinases as NNK-activated physiological calpain kinases. Specific knock-down of mu- and/or m-calpain expression by RNA interference blocked NNK-stimulated migration and invasion, suggesting that mu- and m-calpain may act as required targets in a NNK-induced metastatic signaling pathway. Furthermore, NNK promotes secretion of active mu- and m-calpain from lung cancer cells through vesicles, which may have the potential to cleave substrates in the extracellular matrix. Thus, NNK-induced cell migration and invasion may occur, at least in part, through a novel mechanism involving phosphorylation of calpains that leads to their activation and secretion, which may contribute to metastasis and/or progression of lung cancer.

Topics: Blotting, Western; Calpain; Carcinogens; Cell Line, Tumor; Cell Movement; Disease Progression; Dose-Response Relationship, Drug; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Flavonoids; Gene Silencing; Genetic Vectors; Humans; Immunoprecipitation; Lung Neoplasms; Microscopy, Fluorescence; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Models, Biological; Neoplasm Invasiveness; Neoplasm Metastasis; Nicotiana; Nitrosamines; Phosphorylation; RNA Interference; RNA, Small Interfering; Signal Transduction

Bax is cleaved by calpain at aspartate 33 (Asp33) to yield p18 Bax during stress-induced apoptosis. To assess the role of p18 Bax in apoptosis, an ecdysone-inducible expression system was generated. Similar levels of wild-type (WT) and noncleavable Asp33Ala (Asp-->Ala) Bax are induced in 293 cells while expression of N-terminal-deleted p18 (Delta1-33) Bax remains low (20% of full-length p21 Bax) due to a reduced half-life (2 hours versus 12 hours for p21 Bax) resulting from increased sensitivity to cathepsin-like proteolytic degradation. Expression of p18 Bax is enhanced to levels comparable to p21 Bax when induction is carried out in the presence of cathepsin inhibitors, Z-Phe-Gly-NHO-Bz or N-Acetyl-Leu-Leu-Met-CHO. Compared with WT Bax, expression of similar levels of p18 Bax and, surprisingly, Asp33Ala Bax more potently induces apoptosis as indicated by increased cytochrome c release, caspase-9/-3 activation, and DNA fragmentation, potentially due to their increased homo-oligomerization in mitochondrial membranes. Studies in A-549, U-937, K-562, and HL-60 cells confirm that inhibition of Bax cleavage results in 25% to 35% reduction of drug-induced apoptosis, while inhibition of p18 Bax degradation enhances apoptosis by 25% to 40%. Results indicate that although cleavage to p18 Bax is not required for Bax to initiate apoptosis, p18 Bax potently accelerates the apoptotic process.

Topics: Animals; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Calpain; Caspase 3; Caspase 9; Caspases; Cathepsins; Cell Membrane; Cytochrome c Group; Ecdysterone; Endopeptidases; Etoposide; Gene Expression; HL-60 Cells; Humans; Interleukin-3; K562 Cells; Kidney; Lung Neoplasms; Mice; Mitochondria; Mutagenesis; Peptide Fragments; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; U937 Cells

Ubiquitous calpains (mu- and m-calpain) have been repeatedly implicated in apoptosis, but the underlying mechanism(s) remain(s) to be elucidated. We examined ionomycin-induced cell death in LCLC 103H cells, derived from a human large cell lung carcinoma. We detected hallmarks of apoptosis such as membrane blebbing, nuclear condensation, DNA ladder formation, caspase activation, and poly-(ADP-ribose)polymerase cleavage. Apoptosis was prevented by preincubation of the cells with the calpain inhibitor acetyl-calpastatin 27-peptide and the caspase inhibitor Z-DEVD-fmk, implicating both the calpains and caspases in the apoptotic process. The apoptotic events correlated in a calpastatin-inhibitable manner with Bid and Bcl-2 decrease and with activation of caspases-9, -3, and -7. In vitro both ubiquitous calpains cleaved recombinant Bcl-2, Bid, and Bcl-x(L) at single sites truncating their N-terminal regions. Binding studies revealed diminished interactions of calpain-truncated Bcl-2 and Bid with immobilized intact Bcl-2 family proteins. Moreover, calpain-cleaved Bcl-2 and Bid induced cytochrome c release from isolated mitochondria. We conclude that ionomycin-induced calpain activation promotes decrease of Bcl-2 proteins thereby triggering the intrinsic apoptotic pathway.

Topics: Apoptosis; Calcium; Calpain; Carcinoma, Large Cell; Cell Nucleus; Cell Separation; Cells, Cultured; Cytochrome c Group; Cytoplasm; DNA; Enzyme Activation; Flow Cytometry; Humans; Ionomycin; Ionophores; Lung Neoplasms; Mitochondria; Models, Molecular; Oligopeptides; Poly(ADP-ribose) Polymerases; Protein Binding; Proto-Oncogene Proteins c-bcl-2; Time Factors; Tumor Cells, Cultured

Expression of several molecular determinants of apoptosis was analyzed in 10 untreated small cell (SCLC) and 6 untreated non-small cell (NSCLC) lung carcinoma cell lines. Although SCLC lines were more prone to spontaneous apoptosis compared with NSCLC lines, the former showed higher Bcl-2 expression and a higher Bcl-2/Bax ratio. In order to understand this apparent contradiction, the expression of pro-caspases as well as calpain was analyzed in these cell lines at the protein and mRNA levels. No differences in protein level of pro-caspases-2, -3, -7, and -9 and of calpain were detected between the SCLC and the NSCLC lines, but a striking difference in pro-caspase-8 expression was noted. All 6 NSCLC, but only 2 of the 10 SCLC lines, expressed pro-caspase-8 protein. Further experiments using the RNase protection assay indicated that the lack of pro-caspase-8 expression at the mRNA level was characteristic for SCLC. Using the same experimental approach, we found that SCLC cell lines in addition to pro-caspase-8 were deficient in mRNA expression of pro-caspases-1, -4, and -10, suggesting a different caspase-activating cascade in SCLC compared with NSCLC. This first systematic characterization of pro-caspase expression in lung cancer surprisingly showed that SCLC, which are more prone to undergo spontaneous apoptosis, are deficient in several pro-caspases and have a high Bcl-2/Bax ratio. Thus, the propensity of SCLC cells to undergo apoptosis cannot be explained only by the expression of factors involved in regulation or execution of apoptosis.

Topics: Apoptosis; Calpain; Carcinoma, Non-Small-Cell Lung; Carcinoma, Small Cell; Caspases; Enzyme Activation; Enzyme Precursors; Gene Expression Regulation, Neoplastic; Humans; Isoenzymes; Lung Neoplasms; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Tumor Cells, Cultured

BALB/cBy (BALB) and CXB H mice responded similarly to a single intraperitoneal injection of butylated hydroxytoluene (BHT). This transient pneumotoxicity was characterized by an elevated lung weight and alveolar damage. These changes were accompanied by alterations in the calcium second messenger pathway, namely, two- to five-fold decreases in the activities and pulmonary content of protein kinase C alpha (PKC alpha) and calcium-dependent protease isozyme II (calpain II). BALB and CXB H mice varied in their responsiveness to a chronic (4-6 weekly injections) BHT regimen. CXB H mice became tolerant to BHT and all of the above parameters had returned to control values when examined after the last injection. Chronic BHT administration also failed to enhance lung tumor multiplicity in CXB H mice when the first BHT injection was preceded by the carcinogen, urethane. In contrast, the additional BHT doses potentiated the pathological and biochemical alterations in BALB mice above that found with acute treatment. This included a chronic inflammatory response characterized by the presence of activated macrophages, and a lung tumor multiplicity that was 3-fold greater than in BALB mice receiving urethane alone. One BHT injection increased calpain II mRNA in both strains (1.5- to 3-fold); mRNA declined following multiple BHT injections in BALB mice, but remained elevated in CXB H mice. Studies with the cytochrome P450 inhibitor, piperonyl butoxide, indicated that metabolism of BHT was required for both its acute and chronic effects. We conclude the following: (i) Differences between inbred mice in their susceptibility to chronic pneumotoxicant exposure may exist even when they respond similarly to an acute exposure of the same agent; (ii) A chronic inflammatory state involving a high concentration of activated macrophages may play an important role in tumor enhancement by a non-carcinogen; (iii) The protein contents of PKC alpha and calpain II decrease during BHT-induced lung damage.

Topics: Animals; Blotting, Northern; Blotting, Western; Butylated Hydroxytoluene; Calpain; Drug Interactions; Female; Lung; Lung Neoplasms; Macrophages, Alveolar; Male; Mice; Mice, Inbred BALB C; Mice, Inbred Strains; Organ Size; Piperonyl Butoxide; Protein Kinase C; RNA, Messenger; Species Specificity