Upregulation of heat shock proteins and the promotion of damage-associated molecular pattern signals in a colorectal cancer model by modulated electrohyperthermia

In modulated electrohyperthermia (mEHT) the enrichment of electric field and the concomitant heat can selectively induce cell death in malignant tumors as a result of elevated glycolysis, lactate production (Warburg effect), and reduced electric impedance in cancer compared to normal tissues. Earlier, we showed in HT29 colorectal cancer xenografts that the mEHT-provoked programmed cell death was dominantly caspase independent and driven by apoptosis inducing factor activation. Using this model here, we studied the mEHT-related cell stress 0-, 1-, 4-, 8-, 14-, 24-, 48-, 72-, 120-, 168- and 216-h post-treatment by focusing on damage-associated molecular pattern (DAMP) signals. Significant cell death response upon mEHT treatment was accompanied by the early upregulation (4-h post-treatment) of heat shock protein (Hsp70 and Hsp90) mRNA levels. In situ, the treatment resulted in spatiotemporal occurrence of a DAMP protein signal sequence featured by the significant cytoplasmic to cell membrane translocation of calreticulin at 4 h, Hsp70 between 14 and 24 h and Hsp90 between 24- and 216-h post-treatment. The release of high-mobility group box1 protein (HMGB1) from tumor cell nuclei from 24-h post-treatment and its clearance from tumor cells by 48 h was also detected. Our results suggest that mEHT treatment can induce a DAMP-related signal sequence in colorectal cancer xenografts that may be relevant for promoting immunological cell death response, which need to be further tested in immune-competent animals.     

Extracellular ATP induces cell death in human intestinal epithelial cells

Background

Extracellular ATP is an endogenous signaling molecule released by various cell types and under different stimuli. High concentrations of ATP released into the extracellular medium activate the P2X7 receptor in most inflammatory conditions. Here, we seek to characterize the effects of ATP in human intestinal epithelial cells and to evaluate morphological changes in these cells in the presence of ATP.

Methods

We treated human intestinal epithelial cells with ATP and evaluated the effects of this nucleotide by scanning and transmission electron microscopy analysis and calcium measurements. We used flow cytometry to evaluate apoptosis. We collected human intestinal explants for immunohistochemistry, apoptosis by the TUNEL approach and caspase-3 activity using flow cytometry analyses. We also evaluated the ROS production by flow cytometry and NO secretion by the Griess technique.

Results

ATP treatment induced changes characteristic of cell death by apoptosis and autophagy but not necrosis in the HCT8 cell line. ATP induced apoptosis in human intestinal explants that showed TUNEL-positive cells in the epithelium and in the lamina propria. The explants exhibited a significant increase of caspase-3 activity when the colonic epithelial cells were incubated with IFN-gamma followed by ATP as compared to control cells. In addition, it was found that antioxidants were able to inhibit both the ROS production and the apoptosis induced by ATP in epithelial cells.

General significance

The activation of P2X7 receptors by ATP induces apoptosis and autophagy in human epithelial cells, possibly via ROS production, and this effect might have implications for gut inflammatory conditions.     

ROS-induced autophagy in cancer cells assists in evasion from determinants of immunogenic cell death

Calreticulin surface exposure (ecto-CALR), ATP secretion, maturation of dendritic cells (DCs) and stimulation of T cells are prerequisites for anticancer therapy-induced immunogenic cell death (ICD). Recent evidence suggests that chemotherapy-induced autophagy may positively regulate ICD by favoring ATP secretion. We have recently shown that reactive oxygen species (ROS)-based endoplasmic reticulum (ER) stress triggered by hypericin-mediated photodynamic therapy (Hyp-PDT) induces bona fide ICD. However, whether Hyp-PDT-induced autophagy regulates ICD was not explored. Here we showed that, in contrast to expectations, reducing autophagy (by ATG5 knockdown) in cancer cells did not alter ATP secretion after Hyp-PDT. Autophagy-attenuated cancer cells displayed enhanced ecto-CALR induction following Hyp-PDT, which strongly correlated with their inability to clear oxidatively damaged proteins. Furthermore, autophagy-attenuation in Hyp-PDT-treated cancer cells increased their ability to induce DC maturation, IL6 production and proliferation of CD4⁺ or CD8⁺ T cells, which was accompanied by IFNG production. Thus, our study unravels a role for ROS-induced autophagy in weakening functional interaction between dying cancer cells and the immune system thereby helping in evasion from ICD prerequisites or determinants.     

Heat stress: an inducer of programmed cell death in Chlorella saccharophila

Programmed cell death (PCD) has been recognized as a fundamental cellular process conserved in metazoans, plants and yeast. However, the cellular mechanisms leading to PCD have not been fully elucidated in unicellular organisms. Evidence is presented that heat stress induces PCD in Chlorella saccharophila cells. Our results demonstrate that heat shock triggers a PCD pathway occurring with characteristics features such as chromatin condensation, DNA fragmentation, cell shrinkage and detachment of the plasma membrane from the cell wall, and suggest the presence of caspase 3-like activity. The caspase 3 inhibitor Ac-DEVD-CHO gave significant protection against heat shock-induced cell death. Moreover, a reduction in photosynthetic pigment contents associated with alteration of chloroplast morphology and a fairly rapid disappearance of the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit and the light-harvesting complex of PSII have been observed. The timing of events in the signaling cascade associated with the C. saccharophila heat shock PCD response is discussed. Insights into this field may have general implications for understanding the pathway of cell death in unicellular green algae.     

Clinical and pathological significance of programmed cell death 1 (PD-1)/programmed cell death ligand 1 (PD-L1) expression in high grade serous ovarian cancer

We investigated programmed cell death 1 (PD-1) / programmed cell death ligand 1 (PD-L1) expression in high grade serous ovarian cancer (HGSOC) and its relationship to tumor infiltrating lymphocytes (TIL) and prognosis. Formalin fixed paraffin embedded (FFPE) samples of 94 HGSOC cases were included in the study. Immunohistochemical analysis (CD3, CD4, CD8, PD-1 and PD-L1) was performed. Samples were analyzed for expression of immune proteins in the peritumoral stromal and intratumoral areas, scored, and expression was correlated with overall survival, stage, and age. PD-L1 staining ratio with a score greater than 0 was found to have lower survival. There were two positive staining patterns, patchy/diffuse and patchy/focal patterns, in 24 (25.5%) cases. Considering the threshold value ≥5%, we demonstrated that the PD-L1 positive cancer cell membrane immunoreactivity rate and patchy/diffuse PD-L1 expression were 9.6% (n = 9). There was statistically significant relationship between high PD-1 scores and PD-L1 cases of ≥ 5%. A statistically significant difference was found between PD-L1 staining and survival in patients with a threshold ≥ 5%. However an appropriate rate for treatment was determined in 9.6% cases. There was a statistically significant correlation between PD-1 positive TIL score and intratumoral CD3, peritumoral stromal CD3, intratumoral CD4 and intratumoral CD8 positive cells. Survival was lower in cases with higher PD-L1 positive stromal TIL score.     

Non-apoptotic programmed cell death induced by a copper(II) complex in human fibrosarcoma cells

A₀, a Cu(II) thioxotriazole complex, produces severe cytotoxic effects on HT1080 human fibrosarcoma cells with a potency comparable to that exhibited by cisplatin. A₀ induced a characteristic series of changes, hallmarked by the formation of eosin- and Sudan Black-B-negative vacuoles. No evidence of nuclear fragmentation or caspase-3 activation was detected in cells treated with A₀ which, rather, inhibited cisplatin-stimulated caspase-3 activity. Membrane functional integrity, assessed with calcein and propidium iodide, was spared until the late stages of the death process induced by the copper complex. Vacuoles were negative to the autophagy marker monodansylcadaverine and their formation was not blocked by 3-methyladenine, an inhibitor of autophagic processes. Negativity to the extracellular marker pyranine excluded vacuole derivation from the extracellular fluid. Ultrastructural analysis indicated that A₀ caused the appearance of many electronlight cytoplasmic vesicles, possibly related to the endoplasmic reticulum, which progressively enlarge and coalesce to form large vacuolar structures that eventually fill the cytoplasm. It is concluded that A₀ triggers a non-apoptotic, type 3B programmed cell death (Clarke in Anat Embryol (Berl) 181:195–213, 1990), characterized by an extensive cytoplasmic vacuolization. This peculiar cytotoxicity pattern may render the employment of A₀ to be of particular interest in apoptosis-resistant cell models.     

Protein kinase C Inhibitors selectively modulate dynamics of cell adhesion molecules and cell death in human colon cancer cells

During development of colon cancer, Protein Kinase Cs (PKCs) are involved in regulation of many genes controlling several cellular mechanisms. Here, we examined the changes in cell adhesion molecules and PKCs for colorectal cancer progression. We identified that PKCs affected expression of EpCAM, claudins, tetraspanins. Treatment with low concentrations of PKC inhibitors resulted in decreased cell viability. In addition, immunoblotting and qRT-PCR analysis showed that apoptosis was inhibited while autophagy was induced by PKC inhibition in colon cancer cells. Furthermore, we observed decreased levels of intracellular Reactive Oxygen Species (ROS), lipid peroxidation and protein carbonyl, confirming the ROS-induced apoptosis. Taken together, our results reveal that PKC signalling modulates not only cell adhesion dynamics but also cell death-related mechanisms.

Abbreviations: PKC: Protein Kinase C; EpCAM: Epithelial cell adhesion molecule; FBS: fetal bovine serum; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide); CAM: cell adhesion molecule; ROS: reactive oxygen species     

Calcium-dependent and independent mechanisms of capsaicin receptor (TRPV1)-mediated cytokine production and cell death in human bronchial epithelial cells

Activation of the capsaicin receptor (VR1 or TRPV1) in bronchial epithelial cells by capsaicinoids and other vanilloids promotes pro-inflammatory cytokine production and cell death. The purpose of this study was to investigate the role of TRPV1-mediated calcium flux from extracellular sources as an initiator of these responses and to define additional cellular pathways that control cell death. TRPV1 antagonists and reduction of calcium concentrations in treatment solutions attenuated calcium flux, induction of interleukin-6 and 8 gene expression, and IL-6 secretion by cells treated with capsaicin or resiniferatoxin. Most TRPV1 antagonists also attenuated cell death, but the relative potency and extent of protection did not directly correlate with inhibition of total calcium flux. Treatment solutions with reduced calcium content or chelators had no effect on cytotoxicity. Inhibitors of arachidonic acid metabolism and cyclo-oxygenases also prevented cell death indicating that TRPV1 agonists disrupted basal arachidonic acid metabolism and altered cyclo-oxygenase function via a TRPV1-dependent mechanism in order to produce toxicity. These data confirm previous results demonstrating calcium flux through TRPV1 acts as a trigger for cytokine production by vanilloids, and provides new mechanistic insights on mechanisms of cell death produced by TRPV1 agonists in respiratory epithelial cells.     

Effects of Huangqi （Hex） on Inducing Cell Differentiation and Cell Death in K562 and HEL Cells

InterestintraditionalChineseherbalremedieshasboomedin the western countries. It is very important to study theirmolecular mechanisms and purify effective compoundswith new knowledge and new techniques to meet a greatneed for human health. Ephedrine, the f…     

Nuclear localized protein-1 (Nulp1) increases cell death of human osteosarcoma cells and binds the X-linked inhibitor of apoptosis protein

Nuclear localized protein-1 (Nulp1) is a recently identified gene expressed in mouse and human tissues particularly during embryonic development. Nulp1 belongs to the family of basic helix-loop-helix (bHLH) proteins that are important in development. The precise function of Nulp1 in cells is however not known. We observed that overexpression of Nulp1 induces a large increase in cell death of human osteosarcoma Saos2 cells with DNA fragmentation. In mouse N2A neuroblastoma cells Nulp1 affected cell proliferation and sensitized cells towards death induced by staurosporine. Staining using a novel antibody localized Nulp1 mainly to the cell nucleus and to some extent to the cytoplasm. Nulp1 binds the X-linked inhibitor of apoptosis protein (XIAP) and this interaction was increased during cell death. These results indicate that Nulp1 plays a role in cell death control and may influence tumor growth.     

Ultrastructural changes accompanying secretion and cell death in the molting glands of an insect (Oncopeltus)

During the fifth (last) larval instar of Oncopeltus fasciatus, morphological changes in the molting glands associated with ecdysone secretion include an increase in cytoplasmic volume relative to that of the nucleus, increased amounts of rough endoplasmic reticulum and mitochondria, and the formation of deep infoldings of the plasma membrane. On the sixth day of the fifth instar large electron-lucent areas become apparent beneath the basement membrane; however, the glands remain intact until the seventh (last) day of the instar when a dramatic fragmentation of the cytoplasm, and condensation and fragmentation of the nucleus are observed. It is likely that such changes occur rapidly, just prior to the time of ecdysis to an adult. Cell death in the molting glands of Oncopeltus is markedly different from that described for the molting glands of other insect species in that autophagic vacuoles are not observed prior to a complete loss of cellular integrity.     

Targeted inhibition of Polo‐like kinase 1 by a novel small‐molecule inhibitor induces mitotic catastrophe and apoptosis in human bladder cancer cells

Bladder cancer is a common cancer with particularly high recurrence after transurethral resection. Despite improvements in neoadjuvant chemotherapy, the outcome of patients with advanced bladder cancer has changed very little. In this study, the anti‐tumour activities of a novel Polo‐like kinase 1 (PLK1) inhibitor (RO3280) was evaluated in vitro and in vivo in the bladder carcinoma cell lines 5637 and T24. MTT assays, colony‐formation assays, flow cytometry, cell morphological analysis and trypan blue exclusion assays were used to examine the proliferation, cell cycle distribution and apoptosis of bladder carcinoma cells with or without RO3280 treatment. Moreover, real‐time RT‐PCR and Western blotting were used to detect the expressions of genes that are related to these cellular processes. Our results showed that RO3280 inhibited cell growth and cell cycle progression, increased Wee1 expression and cell division cycle protein 2 phosphorylation. In addition, RO3280 induced mitotic catastrophe and apoptosis, increased cleaved PARP (poly ADP‐ribose polymerase) and caspase‐3, and decreased BubR1 expression. The in vivo assay revealed that RO3280 retarded bladder cancer xenograft growth in a nude mouse model. Although further laboratory and pre‐clinical investigations are needed to corroborate these data, our demonstration of bladder cancer growth inhibition and dissemination using a pharmacological inhibitor of PLK1 provides new opportunities for future therapeutic intervention.     

Apoptotic and non-apoptotic modes of programmed cell death in MCF-7 human breast carcinoma cells

Apoptosis is a specific mode of programmed cell death (PCD), recognized by characteristic morphological and molecular changes. Here we present evidence for a non-apoptotic type of PCD in human MCF-7 breast carcinoma cells. We used TNF-alpha and tyrphostin AG213 to induce apoptotic and non-apoptotic cell death respectively in vitro. Microscopic and immunohistochemical studies, together with DNA analysis and flow cytometric analysis of p53 and bcl-2 oncogene expression, revealed some novel characteristics of non-apoptotic cell death. We show here for the first time some of the biochemical features of an experimentally induced non-apoptotic PCD and emphasize the distinct biochemical events leading to apoptotic and non-apoptotic PCD.     

AT 101 induces early mitochondrial dysfunction and HMOX1 (heme oxygenase 1) to trigger mitophagic cell death in glioma cells

In most cases, macroautophagy/autophagy serves to alleviate cellular stress and acts in a pro-survival manner. However, the effects of autophagy are highly contextual, and autophagic cell death (ACD) is emerging as an alternative paradigm of (stress- and drug-induced) cell demise. AT 101 ([-]-gossypol), a natural compound from cotton seeds, induces ACD in glioma cells as confirmed here by CRISPR/Cas9 knockout of ATG5 that partially, but significantly rescued cell survival following AT 101 treatment. Global proteomic analysis of AT 101-treated U87MG and U343 glioma cells revealed a robust decrease in mitochondrial protein clusters, whereas HMOX1 (heme oxygenase 1) was strongly upregulated. AT 101 rapidly triggered mitochondrial membrane depolarization, engulfment of mitochondria within autophagosomes and a significant reduction of mitochondrial mass and proteins that did not depend on the presence of BAX and BAK1. Conversely, AT 101-induced reduction of mitochondrial mass could be reversed by inhibiting autophagy with wortmannin, bafilomycin A₁ and chloroquine. Silencing of HMOX1 and the mitophagy receptors BNIP3 (BCL2 interacting protein 3) and BNIP3L (BCL2 interacting protein 3 like) significantly attenuated AT 101-dependent mitophagy and cell death. Collectively, these data suggest that early mitochondrial dysfunction and HMOX1 overactivation synergize to trigger lethal mitophagy, which contributes to the cell killing effects of AT 101 in glioma cells.

Abbreviations: ACD, autophagic cell death; ACN, acetonitrile; AT 101, (-)-gossypol; BAF, bafilomycin A₁; BAK1, BCL2-antagonist/killer 1; BAX, BCL2-associated X protein; BH3, BCL2 homology region 3; BNIP3, BCL2 interacting protein 3; BNIP3L, BCL2 interacting protein 3 like; BP, Biological Process; CCCP, carbonyl cyanide m-chlorophenyl hydrazone; CC, Cellular Component; Con, control; CQ, chloroquine; CRISPR, clustered regularly interspaced short palindromic repeats; DMEM, Dulbecco’s Modified Eagle Medium; DTT, 1,4-dithiothreitol; EM, electron microscopy; ER, endoplasmatic reticulum; FACS, fluorescence-activated cell sorting; FBS, fetal bovine serum; FCCP, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; GO, Gene Ontology; HAcO, acetic acid; HMOX1, heme oxygenase 1; DKO, double knockout; LC-MS/MS, liquid chromatography coupled to tandem mass spectrometry; LPL, lipoprotein lipase, MEFs, mouse embryonic fibroblasts; mPTP, mitochondrial permeability transition pore; MTG, MitoTracker Green FM; mt-mKeima, mito-mKeima; MT-ND1, mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 1; PBS, phosphate-buffered saline; PE, phosphatidylethanolamine; PI, propidium iodide; PRKN, parkin RBR E3 ubiquitin protein ligase; SDS, sodium dodecyl sulfate; SQSTM1/p62, sequestome 1; STS, staurosporine; sgRNA, single guide RNA; SILAC, stable isotope labeling with amino acids in cell culture; TFA, trifluoroacetic acid, TMRM, tetramethylrhodamine methyl ester perchlorate; WM, wortmannin; WT, wild-type     

Exopolymer production as a function of cell permeability and death in a diatom (Thalassiosira weissflogii) and a cyanobacterium (Synechococcus elongatus)

Exopolymer particles are found throughout the ocean and play a significant biogeochemical role in carbon cycling. Transparent exopolymer particles (TEP) are composed of acid polysaccharides, and Coomassie staining particles (CSP) are proteins. TEPs have been extensively studied in the ocean, while CSP have been largely overlooked. The objective of this research was to determine the role of stress and cell permeability in the formation of TEP and CSP. The diatom Thalassiosira weissflogii and cyanobacterium Synechococcus elongatus were grown in batch cultures and exposed to hydrogen peroxide (0, 10, and 100 μM) as an environmental stressor. There was no correlation between TEP and CSP concentrations, indicating that they are different populations of particles rather than different chemical components of the same particles. CSP concentrations were not affected by hydrogen peroxide concentration and did not correlate with indicators of stress and cell death. In contrast, TEP concentrations in both taxa were correlated with a decrease in the effective quantum yield of photosystem II, increased activity of caspase‐like enzymes, and an increase in the proportion of the population with permeable cell membranes, indicating that TEP production was associated with the process of cell death. These data show that different environmental factors and physiological processes affected the production of TEP and CSP by phytoplankton. TEP and CSP are separate populations of exopolymer particles with potentially different biogeochemical roles in the ocean.     

Overexpression of Bax inhibitor suppresses the fungal elicitor-induced cell death in rice (Oryza sativa L) cells

Treatment of suspension-cultured cells of rice (Oryza sativa L.) with cell wall extract of rice blast fungus (Magnaporthe grisea) elicits a rapid generation of H2O2, alkalinization of culture medium, and eventual cell death. To elucidate genes involved in these processes, we exploited SAGE (Serial Analysis of Gene Expression) technique for the molecular analysis of cell death in suspension-cultured cells treated with the elicitor. Among the downregulated genes in the elicitor-treated cells, a BI-1 gene coding for Bax inhibitor was identified. Transgenic rice cells overexpressing Arabidopsis BI-1 gene showed sustainable cell survival when challenged with M. grisea elicitor. Thus, the plant Bax inhibitor plays a functional role in regulating cell death in the rice cell culture system.     

Activation of recombinant human TRPV1 receptors expressed in SH-SY5Y human neuroblastoma cells increases [Ca(2+)](i), initiates neurotransmitter release and promotes delayed cell death

The transient receptor potential (TRP) vanilloid receptor subtype 1 (TRPV1) is a ligand-gated, Ca(2+)-permeable ion channel in the TRP superfamily of channels. We report the establishment of the first neuronal model expressing recombinant human TRPV1 (SH-SY5Y(hTRPV1)). SH-SY5Y human neuroblastoma cells were stably transfected with hTRPV1 using the Amaxa Biosystem (hTRPV1 in pIREShyg2 with hygromycin selection). Capsaicin, olvanil, resiniferatoxin and the endocannabinoid anandamide increased [Ca(2+)](i) with potency (EC(50)) values of 2.9 nmol/L, 34.7 nmol/L, 0.9 nmol/L and 4.6 micromol/L, respectively. The putative endovanilloid N-arachidonoyl-dopamine increased [Ca(2+)](i) but this response did not reach a maximum. Capsaicin, anandamide, resiniferatoxin and olvanil mediated increases in [Ca(2+)](i) were inhibited by the TRPV1 antagonists capsazepine and iodo-resiniferatoxin with potencies (K(B)) of approximately 70 nmol/L and 2 nmol/L, respectively. Capsaicin stimulated the release of pre-labelled [(3)H]noradrenaline from monolayers of SH-SY5Y(hTRPV1) cells with an EC(50) of 0.6 nmol/L indicating amplification between [Ca(2+)](i) and release. In a perfusion system, we simultaneously measured [(3)H]noradrenaline release and [Ca(2+)](i) and observed that increased [Ca(2+)](i) preceded transmitter release. Capsaicin treatment also produced a cytotoxic response (EC(50) 155 nmol/L) that was antagonist-sensitive and mirrored the [Ca(2+)](I) response. This model displays pharmacology consistent with TRPV1 heterologously expressed in standard non-neuronal cells and native neuronal cultures. The advantage of SH-SY5Y(hTRPV1) is the ability of hTRPV1 to couple to neuronal biochemical machinery and produce large quantities of cells.