Induction of apoptosis in human colon carcinoma COLO 205 cells by the recombinant α subunit of C-phycocyanin

The α-subunit of C-phycocyanin (CpcA) was expressed in Escherichia coli and purified. The recombinant CpcA inhibited the growth of human colon carcinoma COLO 205 cells. Typical apoptotic morphological characteristics, such as chromatin condensation and nuclear fragmentation, were observed in CpcA-treated COLO 205 cells by fluorescence microscopy and transmission electron microscopy. Moreover, the apoptotic process was associated with the Bax/Bcl-2 ratio up-regulation, mitochondrial membrane depolarization, cytochrome c release, and caspase-9 activation. These findings indicate that CpcA induced the death of COLO 205 cells through the intrinsic apoptotic pathway.     

ER stress-mediated apoptosis induced by celastrol in cancer cells and important role of glycogen synthase kinase-3β in the signal network

HeLa cells treated with celastrol, a natural compound with inhibitive effect on proteasome, exhibited increase in apoptotic rate and characteristics of apoptosis. To clarify the signal network activated by celastrol to induce apoptosis, both the direct target proteins and undirect target proteins of celastrol were searched in the present study. Proteasome catalytic subunit β1 was predicted by computational analysis to be a possible direct target of celastrol and confirmed by checking direct effect of celastrol on the activity of recombinant human proteasome subunit β1 in vitro. Undirect target-related proteins of celastrol were searched using proteomic studies including two-dimensional electrophoresis (2-DE) analysis and iTRAQ-based LC-MS analysis. Possible target-related proteins of celastrol such as endoplasmic reticulum protein 29 (ERP29) and mitochondrial import receptor Tom22 (TOM22) were found by 2-DE analysis of total cellular protein expression profiles. Further study showed that celastrol induced ER stress and ER stress inhibitor could ameliorate cell death induced by celastrol. Celastrol induced translocation of Bax into the mitochondria, which might be related to the upregulation of BH-3-only proteins such as BIM and the increase in the expression level of TOM22. To further search possible target-related proteins of celastrol in ER and ER-related fractions, iTRAQ-based LC-MS method was use to analyze protein expression profiles of ER/microsomal vesicles-riched fraction of cells with or without celastrol treatment. Based on possible target-related proteins found in both 2-DE analysis and iTRAQ-based LC-MS analysis, protein–protein interaction (PPI) network was established using bioinformatic analysis. The important role of glycogen synthase kinase-3β (GSK3β) in the signal cascades of celastrol was suggested. Pretreatment of LiCL, an inhibitor of GSK3β, could significantly ameliorate apoptosis induced by celastrol. On the basis of the results of the present study, possible signal network of celastrol activated by celastrol leading to apoptosis was predicted.     

AREL1 resists the apoptosis induced by TGF-β by inhibiting SMAC in vascular endothelial cells

Abnormal apoptosis of vascular endothelial cells is an important feature of arteriosclerosis (AS). Here, we induced apoptosis in human umbilical vein endothelial cells (HUVECs) using transforming growth factor-β (TGF-β), and investigated the role of antiapoptotic E3 ubiquitin ligase (AREL1) in the apoptosis of vascular endothelial cells. We proved that AREL1 is downregulated in TGF-β treated HUVECs. The overexpression of AREL1 inhibits the activation of Caspase-3 and Caspase-9 and attenuates cell apoptosis induced by TGF-β. According to the result of coimmunoprecipitation, AREL1 interacts with the proapoptotic proteins the second mitochondria-derived activator of caspases (SMAC) in TGF-β treated HUVECs. In addition, miR-320b inhibits the expression of AREL1, and the overexpression of AREL1 attenuates the apoptosis induced by miR-320b mimics in HUVECs. In conclusion, AREL1 is downregulated by miR-320b. AREL1 overexpression inhibits TGF-β induced apoptosis through downregulating SMAC in vascular endothelial cells. Our study explores pathogenesis regulation mechanism and new biological therapeutic targets for vascular disease.     

Activation of p53 in gastric cancer cells by tripchlorolide specifically induces apoptosis

There are two possible outcomes when DNA damage occurs in normal mammalian cells: either induction of cell-cycle checkpoint which inhibits the progress of the cell cycles as well as activates DNA repair pathways, or activation of apoptosis to eliminate damaged cells. The p53 tumour-suppressor gene plays a key role in selecting these pathways. In our present works, the human gastric cancer cell line AGS was treated with tripchlorolide, a potent antitumor compound purified from a Chinese herb Tripterygium Wilfordii Hook. Single cell gel electrophoresis (Comet assay) showed that the treatment of tripchlorolide resulted in DNA damage in AGS cells. The damaged AGS cells went through apoptosis, which was time- and dose- dependent.     

Rottlerin induces pro-apoptotic endoplasmic reticulum stress through the protein kinase C-δ-independent pathway in human colon cancer cells

Rottlerin, a compound reported to be a PKC δ-selective inhibitor, has been shown to induce growth arrest or apoptosis of human cancer cell lines. In our study, rottlerin dose-dependently induced apoptotic cell death in colon carcinoma cells. Treatment of HT29 human colon carcinoma cells with rottlerin was found to induce a number of signature ER stress markers; phosphorylation of eukaryotic initiation factor-2α (eIF-2α), ER stress-specific XBP1 splicing, and up-regulation of glucose-regulated protein (GRP)-78 and CCAAT/enhancer-binding protein-homologous protein (CHOP). However, suppression of PKC δ expression by siRNA or overexpression of WT-PKC δ and DN-PKC δ did not abrogate the rottlerin-mediated induction of CHOP. These results suggest that rottlerin induces up-regulation of CHOP via PKC δ-independent pathway. Furthermore, down-regulation of CHOP expression using CHOP siRNA attenuated rottlerin-induced apoptosis. Taken together, the present study thus provides strong evidence to support an important role of ER stress response in mediating the rottlerin-induced apoptosis.     

Protective effect of an antithyroid compound against γ-radiation-induced damage in human colon cancer cells

We have previously reported the radioprotective effect of propylthiouracil (PTU) on thyroid cells. The aim of the present study was to analyze whether tumor cells and normal cells demonstrate the same response to PTU. Human colon carcinoma cells were irradiated with γ-irradiation with or without PTU. We evaluated the clonogenic survival, intracellular reactive oxygen species levels, catalase, superoxide dismutase and glutathione peroxidase activities, and apoptosis by nuclear cell morphology and caspase-3 activity assays. Cyclic AMP (cAMP) levels were measured by radioimmunoassay. PTU treatment increased surviving cell fraction at 2 Gy (SF2) from 56.9 ± 3.6 in controls to 75.0 ± 3.5 (p < 0.05) and diminished radiation-induced apoptosis. In addition, we observed that the level of antioxidant enzymes’ activity was increased in cells treated with PTU. Moreover, pretreatment with PTU increased intracellular levels of cAMP. Forskolin (p < 0.01) and dibutyryl cAMP (p < 0.05) mimicked the effect of PTU on SF2. Co-treatment with H89, an inhibitor of protein kinase A, abolished the radioprotective effect of PTU. PTU reduces the toxicity of ionizing radiation by increasing cAMP levels and also possibly through a reduction in apoptosis levels and in radiation-induced oxidative stress damage. We therefore conclude that PTU protects both normal and cancer cells during exposure to radiation in conditions mimicking the radiotherapy.     

Protein S-glutathionylation induced by hypoxia increases hypoxia-inducible factor-1α in human colon cancer cells

Hypoxia is a common characteristic of many types of solid tumors. Intratumoral hypoxia selects for tumor cells that survive in a low oxygen environment, undergo epithelial–mesenchymal transition, are more motile and invasive, and show gene expression changes driven by hypoxia-inducible factor-1α (HIF-1α) activation. Therefore, targeting HIF-1α is an attractive strategy for disrupting multiple pathways crucial for tumor growth. In the present study, we demonstrated that hypoxia increases the S-glutathionylation of HIF-1α and its protein levels in colon cancer cells. This effect is significantly prevented by decreasing oxidized glutathione as well as glutathione depletion, indicating that S-glutathionylation and the formation of protein-glutathione mixed disulfides is related to HIF-1α protein levels. Moreover, colon cancer cells expressing glutaredoxin 1 are resistant to inducing HIF-1α and expressing hypoxia-responsive genes under hypoxic conditions. Therefore, S-glutathionylation of HIF-1α induced by tumor hypoxia may be a novel therapeutic target for the development of new drugs.     

Role of mitochondria in apoptosis induced by the 2‐5A system and mechanisms involved

The 2‐5A system (2-5OAS/RNaseL) is composed of the 2′,5′oligoadenylate synthetase 1 (2-5OAS1) and 2-5A-dependent RNase (RNaseL), enzymes that play a key role in antiviral defence mechanisms. Activation of the 2-5A system by double stranded RNA (dsRNA) induces degradation of ribosomal RNAs and apoptosis in mammalian cells. To obtain further information into the molecular mechanisms by which RNaseL induces apoptosis, we expressed human RNaseL and 2-5OAS in HeLa cells using recombinant vaccinia viruses as vectors and we analysed in detail different biochemical markers of apoptosis. In this expression virus-cell system the activation of RNaseL, as index of rRNA degradation, is an upstream event of apoptosis induction. RNaseL induces apoptosis in a caspase-dependent manner (caspases 8, 9 and 2). At the beginning of apoptosis RNaseL and 2-5OAS are localized in the mitochondria and cytosol fractions, while at the onset of apoptosis both enzymes are largely in mitochondria. The 2-5A system induces the release of Cytochrome c from mitochondria to cytosol in a caspase dependent manner. The onset of apoptosis elicits the disruption of mitochondrial membrane potential (ΔΦm), as well as the generation of reactive oxygen species (ROS). Moreover, the activation of RNaseL induces morphological alterations in the mitochondria. Apoptosis induced by the 2-5A system involves mitochondrial proteins, such as the human anti-apoptotic protein Bcl-2, which blocks both the apoptosis and the change of ΔΦm induced by the activation of RNaseL. These findings provide new insights into the molecular mechanisms of apoptosis induction by the 2-5A system, demonstrating the importance of mitochondria in 2-5OAS/RNaseL-induced apoptosis.     

α1,3 Fucosyltransferase-VII modifies the susceptibility of apoptosis induced by ultraviolet and retinoic acid in human hepatocarcinoma cells

The role of α1,3fucosyltransferase-VII (α1,3 FucT-VII) in cell apoptosis was studied in human hepatocellular carcinoma H7721 cells. After the cells were transfected with α1,3 FucT-VII cDNA, the expression of apoptotic protease, procaspase-3, was decreased, while the anti-apoptotic proteins, phospho-PKB and phospho-Bad were increased as compared with mock (vector) transfected cells, indicating that α1,3FucT-VII is a potential anti-apoptotic factor in H7721 cells. After “α1,3FucT-VII” cells were irradiated by UV to induce apoptosis, the anti-apoptotic potential of α1,3FucT-VII became more apparent, as evidenced by the less apoptotic cell % and active cleaved caspase-3, more phospho-p38 MAPK and JNK (two anti-apoptotic signaling molecules in H7721 cells responsible to UV stress) when compared with the “Mock” cells. In contrast, “α1,3FucT-VII” cells facilitated the apoptosis induced by all-trans retinoic acid (ATRA), which was verified by the greater sub-G1 (apoptotic cells) peak in flow cytometry analysis, more expressions of active caspase-3 and pro-apoptotic protein Bax, as well as less expressions of anti-apoptotic proteins, Bcl-2 and Bcl-X_L. The up regulation of α1,3FucT-VII mRNA and cell surface SLe^x (α1,3FucT-VII product) by UV and down regulation of them by ATRA was speculated to be one of the mechanisms that α1,3FucT-VII decreased and increased the susceptibility of apoptosis induced by UV and ATRA respectively. Hao Wang and Qiu-Yan Wang contributed to this article equally.     

Cytotoxic aggregates of α-lactalbumin induced by unsaturated fatty acid induce apoptosis in tumor cells

The effects of three fatty acids on cytotoxic aggregate formation of Ca²⁺-depleted bovine α-lactalbumin (apo-BLA) have been studied by UV absorbance spectroscopy and transmission electron microscopy. The experimental results demonstrate that two unsaturated fatty acids, oleic acid and linoleic acid, and one saturated fatty acid, stearic acid, induce the intermediate of apo-BLA at pH 4.0-4.5 to form amorphous aggregates in time- and concentration-dependent manners. These aggregates are dissolved under physiological conditions at 37 °C and further characterized by fluorescence spectroscopy, circular dichroism and time-of-flight mass spectrometry. Our data here indicate that the structural characteristics of these aggregates are similar to those of HAMLET/BAMLET (human/bovine α-lactalbumin made lethal to tumor cells), a complex of the partially unfolded α-lactalbumin with oleic acid. Cell viability experiments indicate the aggregates of apo-BLA induced by oleic acid and linoleic acid show significant dose-dependent cytotoxicity to human lung tumor cells of A549 but those induced by stearic acid have no toxicity to tumor cells. Furthermore, the cytotoxic aggregates of apo-BLA induced by both unsaturated fatty acids induce apoptosis of human lung cancer cell line A549, suggesting that such cytotoxic aggregates of apo-BLA could be potential antitumor drugs. The present study provides insight into the mechanism of fatty acid-dependent oligomerization and cytotoxicity of α-lactalbumin, and will be helpful in the understanding of the molecular mechanism of HAMLET/BAMLET formation.     

β-lapachone significantly increases the effect of ionizing radiation to cause mitochondrial apoptosis via JNK activation in cancer cells

Background

β-lapachone (β-lap), has been known to cause NQO1-dependnet death in cancer cells and sensitize cancer cells to ionizing radiation (IR). We investigated the mechanisms underlying the radiosensitization caused by β-lap.

Methodology/Principal Findings

β-lap enhanced the effect of IR to cause clonogenic cells in NQO1⁺-MDA-MB-231 cells but not in NQO1⁻-MDA-MB-231 cells. β-lap caused apoptosis only in NQO1⁺ cells and not in NQO1⁻ cells and it markedly increased IR-induced apoptosis only in NQO1⁺ cells. Combined treatment of NQO1⁺ cells induced ROS generation, triggered ER stress and stimulated activation of ERK and JNK. Inhibition of ROS generation by NAC effectively attenuated the activation of ERK and JNK, induction of ER stress, and subsequent apoptosis. Importantly, inhibition of ERK abolished ROS generation and ER stress, whereas inhibition of JNK did not, indicating that positive feedback regulation between ERK activation and ROS generation triggers ER stress in response to combined treatment. Furthermore, prevention of ER stress completely blocked combination treatment-induced JNK activation and subsequent apoptotic cell death. In addition, combined treatment efficiently induced the mitochondrial translocation of cleaved Bax, disrupted mitochondrial membrane potential, and the nuclear translocation of AIF, all of which were efficiently blocked by a JNK inhibitor. Caspases 3, 8 and 9 were activated by combined treatment but inhibition of these caspases did not abolish apoptosis indicating caspase activation played a minor role in the induction of apoptosis.

Conclusions/Significance

β-lap causes NQO1-dependent radiosensitization of cancer cells. When NQO1⁺ cells are treated with combination of IR and β-lap, positive feedback regulation between ERK and ROS leads to ER stress causing JNK activation and mitochondrial translocation of cleaved Bax. The resultant decrease in mitochondrial membrane leads to translocation of AIF and apoptosis.     

Activation of autophagy and PPARγ protect colon cancer cells against apoptosis induced by interactive effects of butyrate and DHA in a cell type-dependent manner: The role of cell differentiation

The short-chain and n-3 polyunsaturated fatty acids exhibit anticancer properties, and they may mutually interact within the colon. However, the molecular mechanisms of their action in colon cancer cells are still not fully understood. Our study focused on the mechanisms responsible for the diverse effects of sodium butyrate (NaBt), in particular when interacting with docosahexaenoic acid (DHA), in distinct colon cancer cell types, in which NaBt either induces cell differentiation or activates programmed cell death involving mitochondrial pathway. NaBt activated autophagy both in HT-29 cells, which are sensitive to induction of differentiation, and in nondifferentiating HCT-116 cells. However, autophagy supported cell survival only in HT-29 cells. Combination of NaBt with DHA-promoted cell death, especially in HCT-116 cells and after longer time intervals. The inhibition of autophagy both attenuated differentiation and enhanced apoptosis in HT-29 cells treated with NaBt and DHA, but it had no effect in HCT-116 cells. NaBt, especially in combination with DHA, activated PPARγ in both cell types. PPARγ silencing decreased differentiation and increased apoptosis only in HT-29 cells, therefore we verified the role of caspases in apoptosis, differentiation and also PPARγ activity using a pan-caspase inhibitor. In summary, our data suggest that diverse responses of colon cancer cells to fatty acids may rely on their sensitivity to differentiation, which may in turn depend on distinct engagement of autophagy, caspases and PPARγ. These results contribute to understanding of mechanisms underlying differential effects of NaBt, when interacting with other dietary fatty acids, in colon cancer cells.     

Upregulation of Fas-induced apoptosis by interferon-γ accompanied by increased ICE expression in renal cell cancer cells

The integration of Fas/Apo-1 (CD95) by Fas ligand or anti-Fas antibody induces apoptosis, and this system plays a pivotal role for the lysis of target cells by cytotoxic T lymphocytes. Fas-mediated apoptosis is also increased by a prior incubation of Fas-bearing cells with interferon(IFN)-. Interleukin-1- converting enzyme (ICE) and/or CPP32, or other members of ICE family act as direct cell death executors downstream of this mechanism, and a tetrapeptide inhibitor of these cysteine proteases blocks Fas-mediated apoptosis. In this study, we examined the effect of IFN- on Fas-mediated apoptosis in ACHN cells. IFN- augmented apoptosis in a dose dependent manner and reached a plateau at 400 U/ml when exposed for 48 h before the end of culture. The kinetics revealed a significant increase in apoptosis after 24 h. Exposing ACHN cells to IFN- increased pro-ICE expression accompanied with a decrease of pro-CPP32. These results suggest that direct enhancement of ICE expression and/or upregulation of conversion of pro-CPP32 to active form increases Fas-mediated apoptosis by IFN- in ACHN cells.     

β-Carotene-induced apoptosis is mediated with loss of Ku proteins in gastric cancer AGS cells

High dietary intakes and high blood levels of β-carotene are associated with a decreased incidence of various cancers. The anticancer effect of β-carotene is related to its pro-oxidant activity. DNA repair Ku proteins, as a heterodimer of Ku70 and Ku80, play a crucial role in DNA double-strand break repair. Reductions in Ku70/80 contribute to apoptosis. Previously, we showed that reactive oxygen species (ROS) activate caspase-3 which induces degradation of Ku proteins. In the present study, we investigated the mechanism of β-carotene-induced apoptosis of gastric cancer AGS cells by determining cell viability, DNA fragmentation, apoptotic indices (increases in cytochrome c and Bax, decrease in Bcl-2), ROS levels, mitochondrial membrane potential, caspase-3 activity, Ku70/80 levels, and Ku-DNA-binding activity of the cells treated with or without antioxidant N-acetyl cysteine and caspase-3 inhibitor z-DEVED-fmk. As a result, β-carotene induced apoptosis (decrease in cell viability, increases in DNA fragmentation and apoptotic indices) and caspase-3 activation, but decreased Ku70/80 levels and Ku-DNA-binding activity. β-Carotene-induced alterations (increase in caspase-3 activity, decrease in Ku proteins) and apoptosis were inhibited by N-acetyl cysteine and z-DEVED-fmk. Increment of intracellular and mitochondrial ROS levels and loss of mitochondrial membrane potential were suppressed by N-acetyl cysteine, but not by z-DEVED-fmk in β-carotene-treated cells. Therefore, β-carotene-induced increases in ROS and caspase-3 activity may lead to reduction of Ku70/80 levels, which results in apoptosis in gastric cancer cells. Loss of Ku proteins might be the underlying mechanism for β-carotene-induced apoptosis in gastric cancer cells.     

The effect of nucleosides on the induced synthesis of β-galactosidase in non-growing cells ofEscherichia coli

The induced synthesis of β-galactosidase in non-growing cells ofEscherichia coli starving for exogenous carbon and nitrogen sources was stimulated markedly by the addition of any of four nucleosides tested: adenosine, guanosine, cytidine, and uridine. Adenosine was used as a representative of this group of compounds in most experiments. The decrease of ability of the cells to synthesize β-galactosidase, resulting from a prolonged starvation for exogenous carbon and nitrogen, was removed by adenosine. This compound also considerably reduced the inhibitory effect of metabolic poisons on the induced synthesis of β-galactosidase. The blockade of induced β-galactosidase synthesis evoked in aerobically grown cells by anaerobic starvation for exogenous sources of carbon and nitrogen was also significantly reduced by adenosine. The weak transient catabolic repression of induced synthesis of β-galactosidase evoked by glucose in non-growing cells ofEscherichia coli deprived of exogenous carbon and nitrogen sources was prevented by adenosine. The total repression caused by higher glucose concentrations was not influenced by this compound. The results are discussed from the point of view of the role of the energy state ofEscherichia coli cells in the regulation of β-galactosidase synthesis.     

Significant increase in Young׳s modulus of ATDC5 cells during chondrogenic differentiation induced by PAMPS/PDMAAm double-network gel: Comparison with induction by insulin

A double-network (DN) gel, which was composed of poly(2-acrylamido-2-methylpropanesulfonic acid) and poly(N,N′-dimethyl acrylamide) (PAMPS/PDMAAm), has the potential to induce chondrogenesis both in vitro and in vivo. The present study investigated the biomechanical and biological responses of chondrogenic progenitor ATDC5 cells cultured on the DN gel. ATDC5 cells were cultured on a polystyrene surface without insulin (Culture 1) and with insulin (Culture 2), and on the DN gel without insulin (Culture 3). The cultured cells were evaluated using micropipette aspiration for cell Young?s modulus and qPCR for gene expression of chondrogenic and actin organization markers on days 3, 7 and 14. On day 3, the cells in Culture 3 formed nodules, in which the cells exhibited an actin cortical layer inside them, and gene expression of type-II collagen, aggrecan, and SOX9 was significantly higher in Culture 3 than Cultures 1 and 2 (p<0.05). Young?s modulus in Culture 3 was significantly higher than that in Culture 1 throughout the testing period (p<0.05) and that in Culture 2 on day 14 (p<0.01). There was continuous expression of actin organization markers in Culture 3. This study highlights that the cells on the DN gel increased the modulus and mRNA expression of chondrogenic markers at an earlier time point with a greater magnitude compared to those on the polystyrene surface with insulin. This study also demonstrates a possible strong interrelation among alteration of cell mechanical properties, changes in actin organization and the induction of chondrogenic differentiation.