Molecular signal transduction in vascular cell apoptosis

INTRODUCTIONApoptosis represents a model of genetically pro--grammed ce1l death and a major mechanism bywhiCh tissue removes unwanted, aged or damagedce1ls. Although cells of mammalian tissues consist ofa broad dtwsity of phenotypes and g6notypes, during the developmeat of apoptosis, all cell types un-dergo similar morphological alteratiOns include chro-matin compaction and margination, nuclear conden-sation and fragmentation, and cell body sbIinkageand b1ebbingf1l. Characteristic apopto…     

Mechanisms of autophagy and apoptosis: Recent developments in breast cancer cells

Autophagy,the pathway whereby cell components are degraded by lysosomes,is involved in the cell response to environmental stresses,such as nutrient deprivation,hypoxia or exposition to chemotherapeutic agents.Under these conditions,which are reminiscent of certain phases of tumor development,autophagy either promotes cell survival or induces cell death. This strengthens the possibility that autophagy could be an important target in cancer therapy,as has been proposed.Here,we describe the regulation of survival and death by autophagy and apoptosis,especially in cultured breast cancer cells.In particular,we discuss whether autophagy represents an apoptosis-independent process and/or if they share common pathways. We believe that understanding in detail the molecular mechanisms that underlie the relationships between autophagy and apoptosis in breast cancer cells could improve the available treatments for this disease.     

Insulin-like growth factors induce apoptosis as well as proliferation in LIM 1215 colon cancer cells

The insulin-like growth factor (IGF) system plays an important role in cell proliferation and survival. However, more recently, a small number of studies have shown that IGFs induce apoptosis in some cells. Our initial studies showed this occurred in LIM 1215 colon cancer cells but not RD rhabdomyosarcoma cells. IGFs induced both proliferation and apoptosis in LIM 1215 cells, and the induction of apoptosis was dose-dependent. [R54, R55]IGF-II, which binds to the IGF-I receptor with normal affinity but does not bind to the IGF-II receptor, induced apoptosis to the same extent as IGF-II, whereas [L27]IGF-II, which binds to the IGF-I receptor with 1000-fold reduced affinity, had no effect on apoptosis. These results suggest that the IGF-I receptor is involved in induction of apoptosis. Western blot analyses demonstrated that Akt and Erk1/2 were constitutively activated in RD cells. In contrast, phosphorylation of Akt and Erk1/2 were transient and basal expression of Akt protein was lower in LIM 1215 cells. Analysis of apoptosis-related proteins showed that IGFs decreased pro-caspase-3 levels and increased expression of pro-apoptotic Bad in LIM 1215 cells. IGFs co-activate proliferative and apoptotic pathways in LIM 1215 cells, which may contribute to increased cell turnover. Since high turnover correlates with poor prognosis in colorectal cancer, this study provides further evidence for the role of the IGF system in its progression.     

Bcl-2, survivin and variant CD44 v7-v10 are downregulated and p53 is upregulated in breast cancer cells by progesterone: Inhibition of cell growth and induction of apoptosis

Progesterone inhibits the proliferation of normal breast epithelial cells in vivo, as well as breast cancer cells in vitro. But the biologic mechanism of this inhibition remains to be determined. We explored the possibility that an antiproliferative activity of progesterone in breast cancer cell lines is due to its ability to induce apoptosis. Since p53, bcl-2 and survivin genetically control the apoptotic process, we investigated whether or not these genes could be involved in the progesterone-induced apoptosis.We found a maximal 90% inhibition of cell proliferation with T47-D breast cancer cells after exposure to 10 M progesterone for 72 h. Control progesterone receptor negative MDA-231 cancer cells were unresponsive to 10 M progesterone. The earliest sign of apoptosis is translocation of phosphatidylserine from the inner to the outer leaflet of the plasma membrane and can be monitored by the calcium-dependent binding of annexin V in conjunction with flow cytometry. After 24 h of exposure to 10 M progesterone, cytofluorometric analysis of T47-D breast cancer cells indicated 43% were annexin V-positive and had undergone apoptosis and no cells showed signs of cellular necrosis (propidium iodide negative). After 72 h of exposure to 10 M progesterone, 48% of the cells had undergone apoptosis and 40% were annexin V positive/propidium iodide positive indicating signs of necrosis. Control untreated cancer cells did not undergo apoptosis. Evidence proving apoptosis was also demonstrated by fragmentation of nuclear DNA into multiples of oligonucleosomal fragments.After 24 h of exposure of T47-D cells to either 1 or 10 M progesterone, we observed a marked down-regulation of protooncogene bcl-2 protein and mRNA levels. mRNA levels of survivin and the metastatic variant CD44 v7-v10 were also downregulated. Progesterone increased p53 mRNA levels.These results demonstrate that progesterone at relative high physiological concentrations, but comparable to those seen in plasma during the third trimester of human pregnancy, exhibited a strong antiproliferative effect on breast cancer cells and induced apoptosis.     

Anticancer drugs induce necrosis of human endothelial cells involving both oncosis and apoptosis

The endothelium is the first physiological barrier between blood and tissues and can be injured by physical or chemical stress, particularly by the drugs used in cancer therapy. We found that four anticancer agents: etoposide, doxorubicin, bleomycin and paclitaxel induced apoptosis in human umbilical vein endothelial cells (HUVECs) (as judged by DNA fragmentation) with a time- and concentration-dependent decrease in bcl-2 protein but without the involvement of p53. As revealed by immunoblotting, bax protein was expressed in HUVECs treated with 1 mg/ml etoposide whereas bcl-2 protein disappeared. Oncosis occurred parallel to apoptosis with the release of lactate dehydrogenase into the supernatant, and, for doxorubicin and etoposide with the inversion of the distribution of angiotensin I-converting enzyme between supernatant and cells. Among the four tested anticancer drugs, only doxorubicin induced an oxidative stress, with significative malondialdehyde production. Thus, human endothelial cells in confluent cultures seem to be in an equilibrium of resistance to apoptosis related to bcl-2 expression; this equilibrium can be disrupted by a chemical stress, such as the antiproliferative drugs known as pro-apoptotic for tumour cells. For doxorubicin and bleomycin, this cellular toxicity can be related to their unwanted effects in human cancer therapy. Low doses of doxorubicin, paclitaxel or etoposide, however, could induce apoptosis of endothelial cells of new vessels surrounding the tumour, thus leading to specific vessel regression with minimal toxic effects for the endothelium of the other vessels. These findings provide evidence of relationships between endothelial toxicity of anticancer drugs and the key role of bcl-2 for resistance of endothelium cells toward apoptosis; moreover lack of p53 and bax in quiescent cells contributes to resistance of endothelial cells to DNA-damaging agents.     

Elimination of extrachromosomal c-myc genes by hydroxyurea induces apoptosis

Hydroxyurea (HU) increases extrachromosomal DNA elimination in tumor cell lines. The c-myc oncogene is one of the many relevant amplified genes contained within the extrachromosomal DNA compartment. Spontaneous loss of amplified copies of c-myc induces terminal differentiation and apoptosis in the human HL-60 leukemia cell lines. In the present study, we evaluate HU's ability to induce apoptosis by eliminating extrachromosomally located c-myc oncogene in human tumor cell lines. The consequences of eliminating extrachromosomal DNA by HU were explored in two different cell lines using the TdT assay and acridine orange/ethidium bromide labeling. COLO 320 clone 3 and COLO 320 clone 21 cell lines contain the same number of amplified copies of c-myc oncogene, but located respectively on extrachromosomal DNA, and intrachromosomally in homogeneously staining regions. HU induced apoptosis in the COLO 320 clone 3 cell line by a time and concentration dependent mechanism but could not induce apoptosis in the COLO 320 clone 21 cell line. These results suggested that HU-induced apoptosis in COLO 320 cell lines depends on elimination of extrachromosomal amplified copies of the c-myc oncogene. The ability of HU to eliminate extrachromosomally amplified copies of the c-myc oncogene and to induce apoptosis should be considered when targeting malignancies with amplification of the c-myc oncogene in an extrachromosomal site.     

Inactivation of Myc-induced p53-dependent apoptosis in human tumors

The Myc family of oncoproteins promote cell growth and are frequently overexpressed in human tumors. However, Myc can also trigger cell death by apoptosis. This is at least in part mediated via the ARF-p53 pathway. Myc activation leads to a selection for inactivation of ARF or p53, allowing cell survival and tumor progression. Restoration of p53-dependent apoptosis by various means is an attractive approach for new cancer therapy.     

The role of radiation-induced apoptosis as a determinant of tumor responses to radiation therapy

Ionizing radiation is an effective means of killing tumor cells. Approximately 50% of all American cancer patients are treated with radiotherapy at some time during the course of their disease, making radiation one of the most widely used cytotoxic therapies. Currently, much effort is focused on understanding the molecular pathways that regulate tumor cell survival following radiotherapy, with the long term goal of developing novel therapeutic strategies for specifically sensitizing tumors to radiation. At present, there is particular interest in the role of tumor cell apoptotic potential as a regulator of both intrinsic and extrinsic determinants of the response of tumors to radiation therapy. Here we review what is currently known about the role of apoptosis as a mechanism of tumor cell killing by ionizing radiation and the relative contribution of apoptosis to cellular radiosensitivity and the ability to control human cancers using radiotherapy. The following topics will be discussed: (1) radiation-induced apoptosis in normal and malignant cells, (2) clinical findings with respect to apoptosis in human cancers treated with radiotherapy, (3) the contribution of apoptosis to intrinsic radiosensitivity in vitro, (4) the relevance of apoptosis to treatment outcome in experimental tumor models in vivo and (5) the potential of exploiting apoptosis as a means to improve the therapeutic efficacy of radiotherapy.     

Identifying pathways affected by cancer mutations

Mutations in 15 cancers, sourced from the COSMIC Whole Genomes database, and 297 human pathways, arranged into pathway groups based on the processes they orchestrate, and sourced from the KEGG pathway database, have together been used to identify pathways affected by cancer mutations. Genes studied in ≥ 15, and mutated in ≥ 10 samples of a cancer have been considered recurrently mutated, and pathways with recurrently mutated genes have been considered affected in the cancer. Novel doughnut plots have been presented which enable visualization of the extent to which pathways and genes, in each pathway group, are targeted, in each cancer. The ‘organismal systems’ pathway group (including organism-level pathways; e.g., nervous system) is the most targeted, more than even the well-recognized signal transduction, cell-cycle and apoptosis, and DNA repair pathway groups. The important, yet poorly-recognized, role played by the group merits attention. Pathways affected in ≥ 7 cancers yielded insights into processes affected.     

Modulation of death receptor-mediated apoptosis by CK2

Protein kinase CK2 has long been known to be involved in cell growth and proliferation. Recent work has also implicated its role in the suppression of apoptosis. We originally documented that removal of survival or growth stimuli resulted in rapid loss of CK2 from the nuclear matrix and chromatin which preceded induction of apoptosis. Further, we demonstrated that overexpression of CK2 in cells promotes suppression of drug-mediated apoptosis. In the present work, we have extended these observations to demonstrate that CK2 can influence apoptosis mediated via the death receptors. Overexpression of CK2 resulted in suppression of apoptosis mediated by TNF-α, TRAIL, and Fas-L in cells responsive to these ligands, whereas downregulation of CK2 resulted in augmentation of apoptosis mediated by these ligands. To our knowledge, this is the first report to show that receptor-mediated apoptosis can be modulated by changes in CK2 in prostate cancer cells. Based on our previous observations together with the evidence presented here, we propose that CK2 has an impact on the process of apoptosis mediated by diverse type of mechanisms thus playing a global role in regulation of apoptotic activity in cells.     

Oxidative stress signalling in the apoptosis of Jurkat T-lymphocytes

Within the first 24 h after castration of an adult male rat, the vascular system of the ventral prostate gland undergoes a degenerative process that drastically reduces blood flow to the tissue. Since the vascular degeneration precedes the loss of the prostatic epithelium (by apoptosis), we have proposed that the onset of epithelial cell apoptosis in this tissue is caused by an ischemic/hypoxic environment resulting from the loss of blood flow. In order to further evaluate the extent to which ischemia/hypoxia might be a factor in apoptosis of the prostate epithelium after castration, we analyzed for biomarkers of cellular hypoxia in rat ventral prostates during the first 3 days following castration. Ventral prostate tissues removed from hypoxyprobe-1-treated adult male rats (uncastrated controls; surgically castrated for 24, 48 or 72 h, or sham-castrated for equivalent times) were directly analyzed for evidence of hypoxia by in situ immunohistochemical evaluation of hypoxyprobe-1 adduct formation in the prostate cells. Protein extracts from these tissues were also tested for expression of the 120 kDa hypoxia-inducible factor-1-alpha (HIF-1-alpha) protein as well as for expression of mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) proteins using a Western blot assay. The tyrosine phosphorylation status of the latter signaling molecules was also evaluated by Western blotting using anti-tyrosine phosphate antibodies. Our results showed that epithelial cells of the rat ventral prostate stained positively for hypoxyprobe-1 adducts at all times after castration, whereas cells in control tissues were unstained by this procedure. In addition, the prostatic expression of HIF-1-alpha protein was increased approximately 20-fold at 48 h after castration compared to control tissues. Finally, although prostatic MAPK and JNK protein expression was unaltered during the early period after castration, phosphorylation of the JUN kinase protein was significantly elevated, indicating that this stress-activated cellular signaling pathway becomes more active subsequent to castration. These results support our proposal that early castration-induced degeneration and constriction of the vascular system of the rat ventral prostate gland leads to reduced oxygenation of prostatic epithelial cells and the activation of hypoxic cellular signaling in these cells through upregulation of HIF-1-alpha expression and stimulation of the JUN kinase signaling pathway.     

CD38 expression enhances sensitivity of lymphoma T and B cell lines to biochemical and receptor-mediated apoptosis

CD38 has been widely characterised both as an ectoenzyme and as a receptor. In the present paper, we investigated the role of CD38 as possible modulator of apoptosis. CD38-positive (CD38(+)) and negative (CD38(-)) fractions, obtained by sorting CD38(+) cells from lymphoma T (Jurkat) and lymphoma B (Raji) and by transfecting lymphoma LG14 and myeloid leukemia K562 cell lines, were used. Cellular subpopulations were exposed to different triggers (H(2)O(2), UV-B, alpha-TOS and hrTRAIL) and the extent of apoptosis was determined by Annexin V-FITC/PI assay. Our data showed that, in lymphoma cells, propensity to apoptosis was significantly linked to CD38 expression and that, remarkably, such response was independent of the nature of the trigger used. Inhibition of CD38 expression by antisense oligonucleotides treatment resulted in CD38-silenced fractions which were as prone to apoptosis as CD38(-) ones. Notably, susceptibility of K562 to apoptosis-inducing challenges was not affected by CD38 expression.     

Adenovirus-Mediated Gene Transfer of Inhibitors of Apoptosis Proteins Delays Apoptosis in Cerebellar Granule Neurons

Abstract : The inhibitor of apoptosis (IAP) family of anti-apoptotic genes, originally discovered in baculovirus, exists in animals ranging from insects to humans. Here, we investigated the ability of IAPs to suppress cell death in both a neuronal model of apoptosis and excitotoxicity. Cerebellar granule neurons undergo apoptosis when switched from 25 to 5 m M potassium, and excitotoxic cell death in response to glutamate. We examined the endogenous expression of four members of the IAP family, X chromosome-linked IAP (XIAP), rat IAP1 (RIAP1), RIAP2, and neuronal apoptosis inhibitory protein (NAIP), by semiquantitative reverse PCR and immunoblot analysis in cultured cerebellar granule neurons. Cerebellar granule neurons express significant levels of RIAP2 mRNA and protein, but expression of RIAP1, NAIP, and XIAP was not detected. RIAP2 mRNA content and protein levels did not change when cells were switched from 25 to 5 m M potassium. To determine whether ectopic expression of IAP influenced neuronal survival after potassium withdrawal or glutamate exposure, we used recombinant adenoviral vectors to target XIAP, human IAP1 (HIAP1), HIAP2, and NAIP into cerebellar granule neurons. We demonstrate that forced expression of IAPs efficiently blocked potassium withdrawal-induced N -acetly-Asp-Glu-Val-Asp-specific caspase activity and reduced DNA fragmentation. However, neurons were only protected from apoptosis up to 24 h after potassium withdrawal, not at later time points suggesting that IAPS delay but do not block apoptosis in cerebellar granule neurons. In contrast, treatment with 100 μ M or 1 m M glutamate did not induce caspase activity and adenoviral-mediated expression of IAPs had no influence on subsequent excitotoxic cell death.     

Celastrol inhibits gastric cancer growth by induction of apoptosis and autophagy

Recently, the interest in natural products for the treatment of cancer is increasing because they are the pre-screened candidates. In the present study, we demonstrate the therapeutic effect of celastrol, a triterpene extracted from the root bark of Chinese medicine on gastric cancer. The proliferation of AGS and YCC-2 cells were most sensitively decreased in six kinds of gastric cancer cell lines after the treatment with celastrol. Celastrol inhibited the cell migration and increased G1 arrest in cell-cycle populations in both cell lines. The treatment with celastrol significantly induced autophagy and apoptosis and increased the expression of autophagy and apoptosis-related proteins. We also found an increase in phosphorylated AMPK following a decrease in all phosphorylated forms of AKT, mTOR and S6K after the treatment with celastrol. Moreover, gastric tumor burdens were reduced in a dose-dependent manner by celastrol administration in a xenografted mice model. Taken together, celastrol distinctly inhibits the gastric cancer cell proliferation and induces autophagy and apoptosis. [BMB Reports 2014; 47(12): 697-702]     

A novel histone deacetylase inhibitor Chidamide induces apoptosis of human colon cancer cells

Many studies have demonstrated that histone deacetylase (HDAC) inhibitors induce various tumor cells to undergo apoptosis, and such inhibitors have been used in different clinical trials against different human cancers. In this study, we designed and synthesized a novel HDAC inhibitor, Chidamide. We showed that Chidamide was able to increase the acetylation levels of histone H3 and to inhibit the PI3K/Akt and MAPK/Ras signaling pathways, which resulted in arresting colon cancer cells at the G1 phase of the cell cycle and promoting apoptosis. As a result, the proliferation of colon cancer cells was suppressed in vitro. Our data support the potential application of Chidamide as an anticancer agent in treating colon cancer. Future studies are needed to demonstrate its in vivo efficacy.     

光动力疗法对体外培养人胰腺癌细胞株PANC-1作用

目的:探讨血卟啉注射液(Hematoporphyrin Derivative,HPD)光动力疗法(Photodynamic Therapy,PDT)对体外培养的人胰腺癌细胞株PANC-1的生物作用。方法:实验分为4组,空白对照组、单纯HPD组、单纯光照组及HPD+PDT组。采用MTT法检测光动力作用后细胞的存活率,并用Annexin V-FITC/P I双染法检测其凋亡率。结果:在光敏剂浓度为5mg/L,光照剂量为10J/cm2时,光动力对PANC-1细胞达到最佳的实验效果,与对照组相比差异有显著性。在此实验参数条件下,流式细胞术(FCM)检测各组人胰腺癌细胞PANC-1凋亡率:HPD+PDT实验组达(36.40±4.21)%,明显高于单纯HPD(6.76±0.44)%,单纯PDT组(8.30±0.32)%及空白组(5.00±0.53)%三个对照组(P<0.05),三个对照组间差异无统计学意义(P>0.05)。结论:PDT光动力作用对体外培养人胰腺癌细胞PANC-1有明确抑制效应,并与HPD浓度及光照强度相关。     

Human Gyrovirus Apoptin shows a similar subcellular distribution pattern and apoptosis induction as the chicken anaemia virus derived VP3/Apoptin

The chicken anaemia virus-derived protein Apoptin/VP3 (CAV-Apoptin) has the important ability to induce tumour-selective apoptosis in a variety of human cancer cells. Recently the first human Gyrovirus (HGyV) was isolated from a human skin swab. It shows significant structural and organisational resemblance to CAV and encodes a homologue of CAV-Apoptin/VP3. Using overlapping primers we constructed a synthetic human Gyrovirus Apoptin (HGyV-Apoptin) fused to green fluorescent protein in order to compare its apoptotic function in various human cancer cell lines to CAV-Apoptin. HGyV-Apoptin displayed a similar subcellular expression pattern as observed for CAV-Apoptin, marked by translocation to the nucleus of cancer cells, although it is predominantly located in the cytosol of normal human cells. Furthermore, expression of either HGyV-Apoptin or CAV-Apoptin in several cancer cell lines triggered apoptosis at comparable levels. These findings indicate a potential anti-cancer role for HGyV-Apoptin.     

Human amniotic epithelial cells induce apoptosis of cancer cells: a new anti-tumor therapeutic strategy

Background aimsAmniotic membrane (AM), the innermost layer of human placenta, is composed of a single layer of epithelial cells, a basement membrane and an avascular stroma. The AM has many functions and properties, among which angiogenic modulatory and immunoregulatory effects are applicable in cancer therapy. Because these functions belong to amniotic epithelial cells, in this study we compared the anti-cancer effect of amniotic epithelial cells and the whole AM.MethodsThe effect of the AM and the amniotic epithelial cells on cancer cell apoptosis was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay, terminal deoxynucleotidyl transferase dUTP nick end labeling assay and immunocytochemistry. The effect of the AM on angiogenesis in conditions both with and without epithelial cells was also evaluated using rat aortic ring assay.ResultsThere was a decrease in cancer cell viability after adding either AM or amniotic epithelial cell supernatant to cancer cells. A significant increase in caspase-3 and caspase-8 expression in cancer cells treated with amniotic epithelial cell supernatant was observed. The recorded media also demonstrated the possible induction of apoptosis in cancer cells treated with the amniotic epithelial cell supernatant. In the aorta ring assay, the AM showed an anti-angiogenic effect in the presence of its epithelial cells; however, this effect was altered to initiate angiogenesis when amniotic epithelial cells were removed from the AM.ConclusionsThese results suggest that amniotic epithelial cells, with their anti-angiogenic effect and induction of apoptosis, are candidates for cancer therapeutic agents in the near future.     

Fenofibrate reduces cisplatin-induced apoptosis by inhibiting the p53/Puma/Caspase-9 pathway and the MAPK/Caspase-8 pathway rather than by promoting autophagy in murine renal proximal tubular cells

The main lesion of cisplatin nephrotoxicity is damage to proximal tubular cells due to increased apoptosis via the mitochondrial and death receptor pathways, which may be alleviated by appropriate promotion of autophagy. Fenofibrate, a peroxisome proliferator-activated receptor-alpha (PPAR-α) activator, is recently reported to promote autophagy as well as protect against cisplatin nephrotoxicity, although the mechanisms were only partially analyzed. Here, the detailed mechanisms of these putative protective effects were investigated in a murine renal proximal tubular (mProx) cell line. Fenofibrate attenuated cisplatin-induced apoptosis of mProx cells based on flow cytometry. As for the mitochondrial apoptotic pathway, the reagent reduced cisplatin-stimulated caspase-3 activation by decreasing the phosphorylation of p53, JNK, and 14-3-3, cytosolic and mitochondrial Puma accumulation, cytochrome C release to the cytosol, and resulting cytosolic caspase-9 activation. Fenofibrate also decreased cisplatin-stimulated activation of caspases-8 by suppressing MAPK and NFkB pathways and reducing the gene expression of TNF-α, TL1A, and Fas, main mediators of the death receptor apoptotic pathway. Autophagy defined by p62 reduction and an increase in LC3 II/I was promoted by fenofibrate in mProx cells under starvation. Autophagy inhibition using 3-MA further increased basal and cisplatin-induced caspase-3 and -8 activation, but had no influence on the inhibitory effects of fenofibrate on caspase activation. In conclusion, our study suggests fenofibrate to be a candidate agent to mitigate cisplatin nephrotoxicity by inhibiting the mitochondrial and death apoptotic pathways rather than by promoting autophagy.