Apoptosis-promoting Effects Research Articles

Enhancement of Activation of Caspases by Presenilin 1 Gene Mutations and its Inhibition by Secretase Inhibitors

Presenilin 1 (PS1) gene mutations are the major causes of early-onset familial Alzheimer's disease. Acceleration of apoptosis is one of the major pathogenic mechanisms of PS1 mutants, and PS1 mutants have also been reported to induce overproduction of amyloid-beta protein 42. Here, we investigated aberrancy in activation of initiator caspases related to two PS1 gene mutations, I143T and G384A. Acceleration of apoptosis, elevation of caspase-3/7 activity, and significant increases in caspase-4, -8 and -9 activities during apoptosis induced by several agents were found in these mutant PS1-transfected cells. Interestingly, thapsigargin treatment enhanced caspase-4 and -9 activities in I143T-mutant PS1-transfected cells, while hydrogen peroxide treatment enhanced caspase-4, -8 and -9 activities in G384A-mutant PS1-transfected cells, indicating diverse apoptosis-promoting effects of PS1 gene mutations. In addition, treatment with a beta-secretase inhibitor or gamma-secretase inhibitor significantly attenuated the effects of the PS1 mutants on caspase-3/7 activation and recovered cell viability. Our present data suggest that these PS1 mutants accelerate the activation of initiator caspases and promote apoptosis, which may be associated, at least in part, with amyloid-beta production.

Apoptosis-promoting effects of forkhead box O3a in human gastric cancer SGC7901 cells

Apoptosis-promoting effects of forkhead box O3a in human gastric cancer SGC7901 cells

Induction of apoptosis by Drosophila Myc

Myc proteins are essential regulators of cellular growth and proliferation during normal development. Activating mutations in myc genes result in excessive growth and are frequently associated with human cancers. At the same time, forced expression of Myc sensitizes vertebrate cells towards different pro-apoptotic stimuli. Recently, the ability of overexpressed Myc to induce cell-autonomous apoptosis has been shown to be evolutionarily conserved in Drosophila Myc (dMyc). Here, we show that dMyc induced apoptosis is accompanied by the induction of Drosophila p53 mRNA, but that dp53 activity is not essential for dMyc's ability to induce apoptosis. Conversely, larvae carrying a hypomorphic dmyc mutation are more resistant to the apoptosis-promoting effects of X-irradiation. These data suggest that the control of apoptosis is a physiological function of Myc and that dMyc might play a role in the response to DNA damage.

MAP kinase‐dependent, NF‐κB‐independent regulation of inhibitor of apoptosis protein genes by TNF‐α

The inhibitor of apoptosis protein (IAP) family of molecules regulates apoptotic processes triggered by various stimuli. However, the mechanisms involved in the regulation of the IAP genes are not fully understood. In this report, we examined roles of nuclear factor-kappaB (NF-kappaB) and mitogen-activated protein (MAP) kinases in tumor necrosis factor-alpha (TNF-alpha)-induced expression of IAP genes. In human endothelial cells, TNF-alpha induced c-IAP1 and c-IAP2, but not XIAP and TIAP/Survivin, at the transcriptional level. Inactivation of NF-kappaB by overexpression of a super-repressor mutant of IkappaBalpha did not affect the induction of IAPs by TNF-alpha. In contrast, extracellular signal-regulated kinase, p38 MAP kinase, and c-Jun N-terminal kinase were activated after stimulation with TNF-alpha, and inhibition of each kinase by PD098059, SB203580, curcumin, or SP600125 substantially attenuated the TNF-alpha-induced c-IAP1 and c-IAP2 expression. To examine whether the MAP kinases-mediated induction of IAPs contributes to survival of TNF-alpha-exposed cells, cells were pretreated with MAP kinase inhibitors and stimulated with TNF-alpha. Treatment with kinase inhibitors alone did not induce apoptosis but enhanced markedly TNF-alpha-triggered apoptosis. Furthermore, overexpression of either c-IAP1 or c-IAP2 diminished the apoptosis-promoting effects of MAP kinase inhibitors. These data indicated that TNF-alpha induced expression of c-IAP1 and c-IAP2 via MAP kinases, but not via NF-kappaB, and that MAP kinases participated in the inhibition of apoptosis by induction of c-IAPs in TNF-alpha-stimulated endothelial cells.

The N-terminal 26-residue fragment of human programmed cell death 5 protein can form a stable α-helix having unique electrostatic potential character

PDCD5-(1-26) is a N-terminal 26-residue fragment of human PDCD5 (programmed cell death 5) protein. PDCD5 is an important novel protein that regulates both apoptotic and non-apoptotic programmed cell death. The conformation of PDCD5 protein is a stable helical core consisting of a triple-helix bundle and two dissociated terminal regions. The N-terminal region is ordered and contains abundant secondary structure. Overexpression and purification of the N-terminal 26-residure fragment, PDCD5-(1-26), was performed in this study to better understand its tertiary structure. The spectroscopic studies using CD and hetero- and homo-nuclear NMR methods determine a stable alpha-helix formed by Asp3-Ala19 of PDCD5-(1-26). The N-terminal residues Asp3-Ala19 of PDCD5 were then affirmed to have the capacity to form a stable alpha-helix independently of the core of the protein. Analysis of the helical peptide of PDCD5-(1-26) indicates that the surface of this well-formed alpha-helix has a unique electrostatic potential character. This may provide an environment for the N-terminal alpha-helix of PDCD5 to serve as an independent functional entity of the protein. The apoptosis activity assay shows that the deletion of the N-terminal alpha-helix of PDCD5 significantly attenuates the apoptosis-promoting effects on HL-60 cells induced by serum withdrawal.

Apoptosis-promoting effect of Panax notoginseng extracts on MNNG-transformed GES-1 cells

To study the apoptosis-promoting effect of the serum from Panax notoginseng extracts-fed dog on precancerous gastric cells by means of flow cytometry. In the experiment, we adopted eternalized human gastric mucosa epithelium GES-1 cells transformed by N-methyl-N'-nitro-N-nitroso-guanidine (MNNG) (MC cells) as the model of precancerous lesions for study in vitro. We took the serum of a dog before and at two different points of time (2 and 6 hours) after feeding the dog with Panax notoginseng extracts for experiment. The MC cells were cultured in mediums with different concentrations of the medicated serum at 2- or 6-hour point of time for 72 hours. By means of flow cytometry, we examined the apoptosis-promoting effects of the serums on the MC cells. The medicated serums at these 2 points of time had significant effects in promoting MC cell apoptosis. The proportions of apoptotic cells in culture mediums with medicated serums had a significant increase as compared with those in culture mediums with non-medicated serums (serum obtained before administration of extracts to the dog) under the same conditions (P<0.05). The number of MC cells in G(0)/G(1)phase was decreased (P<0.05) and that in G(2)/M phase increased (P<0.05), while no consistent changes were observed in S phase. The medicated serums obtained at the two different points of time have significant apoptosis-promoting effects on MC cells. They decrease the number of MC cells in G(0)/G(1) phase and increase the number of MC cells in G(2)/M phase. This is probably responsible for the effects of Panax notoginseng extracts in inhibiting the proliferation of MC cells and promoting its apoptosis.

In this issue

The matrix metalloproteases, or MMPs, are a family of zinc-dependent proteolytic enzymes that degrade the extracellular matrix. As such, they are important during cell migration, angiogenesis and wound healing. However, they are also necessary for the invasion and migration of tumor cells. Therefore, MMP expression is often upregulated in invasive metastatic cancers. MMP-19 lacks several structural features present in other MMP subclasses, notably the Asp, Tyr and Gly residues close to the zinc-binding site, the fibronectin-like domain, the transmembrane domain and the furin-activation sequence. On pages 709–716 of this issue, Impola et al. demonstrate yet another unique feature of MMP-19: unlike some of its family members, it is present in normal, proliferating, but not migrating keratinocytes; it disappears from these cells over the course of neoplastic dedifferentiation. Because MMP-19 is induced by TNF-α, because it degrades many basal membrane components, including type IV collage, laminin-1 and nidogen, and because it is present in normal cells, they hypothesize that MMP-19 plays a role in normal tissue remodeling such as that which occurs during wound repair. Because the intrauterine environment may play a role in the development of adult diseases, Leong et al. endeavor to characterize the relationship between birth weight (as a measure of said intrauterine environment) and adult obesity (as a known risk factor for cancer). They collected information on birth weight, adult height and adult weight from randomly selected women aged 50–79 living in the states of Massachusetts, New Hampshire, and Wisconsin via telephone interviews. In their paper on pages 789–791 of this issue, they report their results that both low and high birth weights are associated with higher adult body mass index, and they conclude that fetal experience may influence adult obesity and therefore contribute to cancer risk. Glutathione-S-transferases (GSTs) catalyze the glutathione-dependent detoxification of several chemotherapeutic agents or their metabolites. Therefore, Sweeney et al. hypothesized that polymorphisms resulting in a reduction or elimination of enzymatic activity may modify the effectiveness of chemotherapy and thus predict differences in treatment outcomes. GSTA1 is highly active in the glutathione conjugation of metabolites of cyclophosphamide (CP), a nitrogen mustard chemotherapeutic drug often used in combination chemotherapy for breast cancer. A newly recognized polymorphism, the GSTA1*B allele, reduces hepatic expression of this enzyme. On pages 810–814 of this issue, they report that GSTA1*B/*B homozygotes receiving combination chemotherapy for breast cancer had a longer overall survival during the first 5 years following their diagnosis, probably because their reduced ability to detoxify CP means that they received a higher effective dose. However, after 5 years there was no evidence of a continued survival difference based on genotype. Similar results were reported for patients with the GST1105Val polymorphism who received combination chemotherapy for advanced colorectal cancer. Overall survival among breast cancer patients treated by chemotherapy, by genotype at the GSTA1 proximal promoter polymorphism. Micrometastases are one of the worst problems facing cancer patients who undergo successful resection of their primary tumors, for even if no metastases are detected at the time of surgery, micrometastases may have sprouted and can become manifest years later. The lungs are one of the most common sites for these micrometastases. MMPs play a pivotal role in metastasis by promoting tumor invasion and angiogenesis; thus, a number of inhibitors (MMPIs) have been developed to counteract them. ONO-4817 is a selective spectrum MMPI that inhibits the gelatinases MMP-2 and MMP-9, but not MMP-1, by binding reversibly to the zinc-binding region of these MMPs. On pages 822–828, Yamamoto et al. test its efficacy in combination with a cytotoxic anticancer drug, docetaxel (DOC), against established lung micrometastases. They injected nude mice with either PC14PE6 cells, human non-small-cell lung adenocarcinoma cells, or H226 cells, human squamous cell carcinoma cells, both of which express MMP-1, MMP-2 and/or MMP-9. The mice were then treated with DOC and ONO-4817 after micrometastases had been established. They found that the combination therapy significantly suppressed the tumor burden and prolonged the survival of the mice. Effect of treatment with ONO-4817 and DOC on the survival of nude mice inoculated with PC14PE6 cells. Expression of the RARβ subtype of the retinoic acid receptor (RARβ) is selectively lost in most cultured cancer cell lines and premalignant and malignant lesions, implying that its loss is associated with human cancer development. Its loss also renders the cells resistant to the growth inhibitory effects of retinoic acid treatment. In their report on pages 833–839 of this issue, Teraishi et al. report that recombinant adenovirus-mediated gene transfer of the G1 phase cell cycle inhibitor p21 into H1299 human non-small-cell lung cancer cells and DLD-1 human colorectal cancer cells transcriptionally upregulates RARβ expression, thereby rendering these cells more sensitive to the apoptosis-promoting effects of treatment with all-trans-retinoic acid. Transcriptional activation of the RARβ promoter by Ad5CMV-p21 infection.

Nifedipine induces apoptosis in cultured vascular smooth muscle cells from spontaneously hypertensive rats

Apoptosis (programmed cell death) of smooth muscle cells (SMC) in blood vessels is an essential process involved in the control of vessel wall structure. Several antihypertensive drugs currently used in therapy may exert their pharmacological effects by promoting SMC apoptosis. The biochemical events which regulate SMC apoptosis in the vessel wall are complex, and not well understood. We therefore investigated whether treatment of cultured SMC from normotensive Wistar-Kyoto rats (WKY) and from spontaneously hypertensive rats (SHR) with selected antihypertensive drugs would induce SMC apoptosis. We treated aortic SMC from WKY and SHR in vitro with the L-type Ca 2+ channel antagonist, nifedipine; with the nitric oxide donor, sodium nitroprusside (SNAP); with forskolin (an activator of adenylyl cyclase); or with thapsigargin (a selective inhibitor of the sarcoplasmic reticulum (SR), Ca 2+-ATPase); and compared their apoptosis-promoting effects in SMC derived from the two strains of rats. SMC were derived from the thoracic aorta of 3-4-week-old WKY and SHR, and were used in passages 7–10. Apoptotic cells were detected by in-situ end labeling using the terminal deoxynucleotide transferase-mediated dUTP-nick end-labeling (TUNEL) method, and by morphological examination. We found that: 1) Treatment of cultured aortic SMC with the L-type Ca 2+ channel antagonist, nifedipine (5 × 10 −5 M) for 24 hours induced a significantly higher level of apoptosis in SHR cells than in SMC from WKY. Cells from WKY, following exposure to nifedipine for 72 hours, exhibited a similar response to the cells from SHR treated for 24 hours. This was detectable by both morphological criteria as well as DNA labeling by the TUNEL technique. 2) Similar treatment of these cells with thapsigargin (1 × 10 −7 M) led to morphological alterations characteristic of apoptotic cells in SMC from both WKY and SHR, and cells from SHR but not WKY were labeled by the TUNEL technique at 24 hours. The TUNEL method did however identify cells from both WKY and SHR as apoptotic after 48 and 72 hours of treatment. 3) The addition of SNAP, or forskolin to the cultured SMC induced significant, but low levels of apoptosis in WKY SMC only. This selective apoptosis-promoting effect of nifedipine in SHR SMC may result from differences in the control of intracellular Ca 2+ between the two strains of cells, or it may indicate that the signaling pathways which regulate apoptosis are different in SMC from the normotensive and the hypertensive rats. Our findings imply that SMC apoptosis may be a selective target for pharmacological intervention in hypertension.

Pathways through which a regimen of melatonin and retinoic acid induces apoptosis in MCF-7 human breast cancer cells.

It has been established that melatonin (Mlt) and retinoic acid, individually, inhibit the proliferation of the estrogen receptor-alpha (ER alpha)-positive MCF-7 breast cancer cell line. Our laboratory has previously demonstrated that Mlt and all-trans-retinoic acid (atRA) not only inhibit the proliferation, but also induce apoptosis of MCF-7 cells when used in a sequential regimen of Mlt followed 24 h later by atRA. Using this same MCF-7 breast cancer cell line, we investigated the potential pathways through which apoptosis is being induced. We found that treatment of MCF-7 cells with Mlt for 24 h before the addition of atRA decreased the protein levels of the death suppressor, Bcl-2, and increased, although with different time courses, the levels of the death promoters, Bax and Bak; however, there was no change in the levels of the tumor suppressor gene, p53. MCF-7 cells treated sequentially with Mlt and atRA also demonstrated an enhanced sensitivity to the apoptotic effects of atRA, which did not appear to be due to increased expression of the retinoic acid receptors, RAR alpha or RXR alpha, but rather to enhanced transcriptional activity of the RAR alpha. These data suggest that the sequential treatment regimen of Mlt and atRA may induce apoptosis by modulation of members of the Bcl-2 family of proteins. Thus, this combinatorial regimen, which reduces the concentration of atRA needed for clinical efficacy while enhancing its anti-tumorigenic activity, could be of great therapeutic benefit, and may, in fact, specifically induce the regression of established breast tumors due to its apoptosis-promoting effects.