Expression Of Several Apoptosis-related Genes Research Articles

Effects of Ibuprofen and Diclofenac Pre-Treatment on Viability and Apoptosis Processes in Human Dental Pulp Stem Cells.

Background and Objectives: Stem cell-based regeneration strategies have shown therapeutic efficacy in various fields of regenerative medicine. These include bone healing after bone augmentation, often complicated by pain, which is managed by using nonsteroidal anti-inflammatory drugs (NSAIDs). However, information is limited about how NSAIDs affect the therapeutic potential of stem cells. Materials and Methods: We investigated the effects of ibuprofen and diclofenac on the characteristics, morphology, and immunophenotype of human mesenchymal stromal cells isolated from the dental pulp (DPSCs) and cultured in vitro, as well as their effects on the expression of angiogenic growth factors (VEGFA and HGF) and selected genes in apoptosis signalling pathways (BAX, BAK, CASP3, CASP9, and BCL2). Results: Ibuprofen and diclofenac significantly reduced the viability of DPSCs, while the expression of mesenchymal stem cell surface markers was unaffected. Both ibuprofen and diclofenac treatment significantly upregulated the expression of HGF, while the expression of VEGFA remained unchanged. Ibuprofen significantly altered the expression of several apoptosis-related genes, including the upregulation of CASP9 and BCL2, with decreased CASP3 expression. BAK, CASP3, CASP9, and BCL2 expressions were significantly increased in the diclofenac-treated DPSCs, while no difference was demonstrated in BAX expression. Conclusions: Our results suggest that concomitant use of the NSAIDs ibuprofen or diclofenac with stem cell therapy may negatively impact cell viability and alter the expression of apoptosis-related genes, affecting the efficacy of stem cell therapy.

Dual roles of α1,4-galactosyltransferase1 in spermatogenesis of Drosophila melanogaster.

Spermatogenesis is critical for insect reproduction and the process is regulated by multiple genes. Glycosyltransferases have been shown to participate in the development of Drosophila melanogaster; however, their role in spermatogenesis is still unclear. In this study, we found that α1,4-galactosyltransferase1 (α4GT1) was expressed at a significantly higher level in the testis than in the ovary of Drosophila. Importantly, the hatching rate was significantly decreased when α4GT1 RNA interference (RNAi) males were crossed with w1118 females, with only a few mature sperm being present in the seminal vesicle of α4GT1 RNAi flies. Immunofluorescence staining further revealed that the individualization complex (IC) in the testes from α4GT1 RNAi flies was scattered and did not move synchronically, compared with the clustered IC observed in the control flies. Terminal deoxyribonucleotide transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay showed that apoptosis signals in the sperm bundles of α4GT1 RNAi flies were significantly increased. Moreover, the expression of several individualization-related genes, such as Shrub, Obp44a and Hanabi, was significantly decreased, whereas the expression of several apoptosis-related genes, including Dronc and Drice, was significantly increased in the testes of α4GT1 RNAi flies. Together, these results suggest that α4GT1 may play dual roles in Drosophila spermatogenesis by regulating the sperm individualization process and maintaining the survival of sperm bundles.

Apoptotic Effects of Drug Targeting Conjugates Containing Different GnRH Analogs on Colon Carcinoma Cells

The wide range of cellular target reactions (e.g., antitumor) of gonadotropin-releasing hormone (GnRH) variants provides the possibility to develop multifunctional GnRH conjugates. The aim of our work was to compare the cytotoxic/apoptotic activity of different GnRH-based, daunorubicin (Dau)-linked conjugates with or without butyrated Lys in position 4 (4Lys(Bu)) at a molecular level in a human colorectal carcinoma cell line. Cell viability was measured by impedimetry, cellular uptake and apoptosis were studied by flow cytometry, and the expression of apoptosis-related genes was analyzed by qRT-PCR. The modification with 4Lys(Bu) resulted in an increased cytotoxic and apoptotic effects and cellular uptake of the GnRH-I and GnRH-III conjugates. Depending on the GnRH isoform and the presence of 4Lys(Bu), the conjugates could regulate the expression of several apoptosis-related genes, especially tumor necrosis factor (TNF), tumor protein p53 (TP53) and the members of growth-factor signaling. The stronger cytotoxicity of GnRH-I and GnRH-III conjugates containing 4Lys(Bu) was associated with a stronger inhibitory effect on the expression of growth-factor signaling elements in comparison with their 4Ser counterparts, in which the upregulation of TP53 and caspases (e.g., CASP9) seemed to play a more important role. We were able to provide further evidence that targeting the GnRH receptor could serve as a successful therapeutic approach in colon cancer, and GnRH-III-[4Lys(Bu),8Lys(Dau=Aoa)] proved to be the best candidate for this purpose.

Engineering the periodontal ligament in hyaluronan-gelatin-type I collagen constructs: upregulation of apoptosis and alterations in gene expression by cyclic compressive strain.

To engineer constructs of the periodontal ligament (PDL), human PDL cells were incorporated into a matrix of hyaluronan, gelatin, and type I collagen (COLI) in sample holders (13×1 mm) of six-well Biopress culture plates. The loading dynamics of the PDL were mimicked by applying a cyclic compressive strain of 33.4 kPa (340.6 gm/cm(2)) to the constructs for 1.0 s every 60 s, for 6, 12, and 24 h in a Flexercell FX-4000C Strain Unit. Compression significantly increased the number of nonviable cells and increased the expression of several apoptosis-related genes, including initiator and executioner caspases. Of the 15 extracellular matrix genes screened, most were upregulated at some point after 6-12 h deformation, but all were downregulated at 24 h, except for MMPs1-3 and CTGF. In culture supernatants, matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of metalloproteinases-1 (TIMP-1) protein levels were upregulated at 24 h; receptor activator of nuclear kappa factor B (RANKL), osteoprotegerin (OPG) and fibroblast growth factor-2 (FGF-2) were unchanged; and connective tissue growth factor (CTGF) not detected. The low modulus of elasticity of the constructs was a disadvantage-future mechanobiology studies and tissue engineering applications will require constructs with much higher stiffness. Since the major structural protein of the PDL is COLI, a more rational approach would be to permeabilize preformed COLI scaffolds with PDL-populated matrices.

Abstract 2591: Global gene expression regulated by phenethyl isothiocyanate in human colon cancer SW480 cells.

Abstract Phenethyl isothiocyanate (PEITC), a promising chemopreventive compound originated from cruciferous vegetables, is known to inhibit proliferation of colon cancer cells through inducing apoptosis and cell cycle arrest. Although exact mechanisms are unclear, it seems that PEITC may have multiple molecular targets and play multiple roles in a variety of events. Some of our recent studies indicate that a key mechanism of apoptosis induction by PEITC may be through covalent modification of its target proteins, such as tubulin. Numerous studies also reported that PEITC may induce apoptotic signals through gene transcriptional regulation. However, systematic studies on PEITC-induced gene expression profile, particularly in colon cancer cells, are rare. In this study, we examined overall gene expression profile of SW480 cells at different time points after PEITC treatment using cDNA microarray. Results showed that PEITC induced significant changes in expression of several apoptosis-related genes, such as BIM, DEDD2, GADD45B, HSPB8, SYK, PIK3C2A, CTNNB1, LGALS8, and NOXA. Furthermore, these changes were confirmed by real-time polymerase chain reaction at transcript level and immunoblot at protein level. Considering significant roles of these genes in signaling transduction leading to downstream events, such as apoptosis, the findings of this study may shed lights on the molecular mechanisms by which PEITC exert its anti-carcinogenic effects. (Supported by NCI grant CA100853) Citation Format: Lixin Mi, Xiantao Wang, Xin Li, Fung-Lung Chung. Global gene expression regulated by phenethyl isothiocyanate in human colon cancer SW480 cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2591. doi:10.1158/1538-7445.AM2013-2591

EVI-1 modulates leukemogenic potential and apoptosis sensitivity in human acute lymphoblastic leukemia

The transcriptional regulator ecotropic viral integration site-1 (EVI-1) has mainly been studied for its role in myeloid malignancies, in which high EVI-1 levels are associated with particularly aggressive disease. The role of EVI-1 in lymphoid cells, however, is largely unknown. Here we show that EVI-1 is indeed expressed in lymphoid malignancies such as acute lymphoblastic leukemia (ALL) and a subset of chronic lymphocytic leukemia. Expression data from pediatric ALL further suggest that high EVI-1 levels are associated with poor prognosis. Suppression of EVI-1 expression by RNA interference reduces cell growth and enhances apoptosis sensitivity in response to various stimuli in lymphoblastic leukemia cells. At the molecular level, EVI-1 modulates expression of several apoptosis-related genes (such as BCL2, BCL-x, XIAP, NOXA, PUMA, TRAIL-R1). Furthermore, EVI-1 knockdown strongly impairs in vivo engraftment of lymphoblastic leukemia cells upon transplantation in immune-permissive NOD/SCID/IL2Rγ(null) mice, conferring a survival benefit when compared with mice transplanted with control cells. Thus, our data show that EVI-1 is expressed not only in myeloid but also in lymphoid leukemias, and contributes to the leukemogenic potential and apoptosis resistance of ALL cells.

Reduction in programmed cell death and improvement in functional outcome of transient focal cerebral ischemia after administration of granulocyte-macrophage colony-stimulating factor in rats

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a potent hematopoietic growth factor that both enhances the survival and drives the differentiation and proliferation of myeloid lineage cells. Recent studies have suggested that GM-CSF has a neuroprotective effect against CNS injury. In this paper, the authors investigated the neuroprotective effect of GM-CSF on neuron survival and locomotor behavior in a rat model of focal cerebral ischemic injury. To understand its neuroprotective effect in vitro, GM-CSF was administered to a glutamate-induced excitotoxicity neuronal injury cell culture model that mimics the pathophysiology of focal hypoxic cerebral injury. In the animal study, the authors prepared a rat focal cerebral ischemia model by occluding the unilateral middle cerebral artery. They then examined the effects of GM-CSF administration on changes in infarct volume, apoptosis-related gene expression, and improvement in locomotor behavior. Treatment with GM-CSF significantly increased cell viability in a cell culture model of glutamate-induced neuronal injury. Furthermore, in vivo administration of GM-CSF at 60 microg/kg body weight daily for 5 consecutive days beginning immediately after injury decreased infarction volume, altered the expression of several apoptosis-related genes (Bcl-2, Bax, caspase 3, and p53), and improved locomotor behavior in the focal cerebral ischemia model. The GM-CSF had neuroprotective effects in in vitro and in vivo experiments and resulted in decreased infarction volume and improved locomotor behavior. Although the specific mechanism involved in stroke recovery was not fully elucidated as it was not the primary focus of this study, administration of GM-CSF appeared to decrease the extent of neuronal apoptosis by modulating the expression of several apoptosis-related genes such as Bcl-2, Bax, caspase 3, and p53. Further investigations are necessary to better understand the role of GM-CSF on neural regeneration during the recovery phase of a stroke, as well as the intracellular signal transduction pathways that mediate neuroprotection.

Apoptosis related genes expressed in cultured Fallopian tube epithelial cells infected in vitro with Neisseria gonorrhoeae.

Infection of the Fallopian tubes (FT) by Neisseria gonorrhoeae (Ngo) can lead to acute salpingitis, an inflammatory condition resulting in damage primarily to the ciliated cells, with loss of ciliary activity and sloughing of the cells from the epithelium. Recently, we have shown that Ngo infection induced apoptosis in FT epithelium cells by a TNF-alpha dependent mechanism that could contribute to the cell and tissue damage observed in gonococcal salpingitis. To investigate the apoptosis-related genes expressed during apoptosis induction in cultured FT epithelial cells infected in vitro by Ngo. In the current study, we used cDNA macroarrays and real time PCR to identify and determine the expression levels of apoptosis related genes during the in vitro gonococci infection of FT epithelial cells. Significant apoptosis was induced following infection with Ngo. Macroarray analysis identified the expression of multiple genes of the TNF receptor family (TNFRSF1B, -4, -6, -10A, -10B and -10D) and the Bcl-2 family (BAK1, BAX, BLK, HRK and MCL-1) without differences between controls and infected cells. This lack of difference was confirmed by RT-PCR of BAX, Bcl-2, TNFRS1A (TNFR-I) and TNFRSF1B (TNFR-II). Several genes related to apoptosis are expressed in primary cultures of epithelial cells of the human Fallopian tube. Infection with Ngo induces apoptosis without changes in the pattern of gene expression of several apoptosis-related genes. RESULTS strongly suggest that Ngo regulates apoptosis in the FT by post-transcriptional mechanisms that need to be further addressed.

Apoptosis related genes expressed in cultured Fallopian tube epithelial cells infected in vitro with Neisseria gonorrhoeae

Infection of the Fallopian tubes (FT) by Neisseria gonorrhoeae (Ngo) can lead to acute salpingitis, an inflammatory condition resulting in damage primarily to the ciliated cells, with loss of ciliary activity and sloughing of the cells from the epithelium. Recently, we have shown that Ngo infection induced apoptosis in FT epithelium cells by a TNF-alpha dependent mechanism that could contribute to the cell and tissue damage observed in gonococcal salpingitis.To investigate the apoptosis-related genes expressed during apoptosis induction in cultured FT epithelial cells infected in vitro by Ngo.In the current study, we used cDNA macroarrays and real time PCR to identify and determine the expression levels of apoptosis related genes during the in vitro gonococci infection of FT epithelial cells.Significant apoptosis was induced following infection with Ngo. Macroarray analysis identified the expression of multiple genes of the TNF receptor family (TNFRSF1B, -4, -6, -10A, -10B and -10D) and the Bcl-2 family (BAK1, BAX, BLK, HRK and MCL-1) without differences between controls and infected cells. This lack of difference was confirmed by RT-PCR of BAX, Bcl-2, TNFRS1A (TNFR-I) and TNFRSF1B (TNFR-II).Several genes related to apoptosis are expressed in primary cultures of epithelial cells of the human Fallopian tube. Infection with Ngo induces apoptosis without changes in the pattern of gene expression of several apoptosis-related genes. RESULTS strongly suggest that Ngo regulates apoptosis in the FT by post-transcriptional mechanisms that need to be further addressed.

Effect of Combined Therapy With Low-Dose 5-Aza-2′-Deoxycytidine and Irinotecan on Colon Cancer Cell Line HCT-15

Aberrant promoter hypermethylation is an epigenetic change that silences the expression of crucial genes, resulting in inactivation of the apoptotic pathway in various cancers. This hypermethylation can be restored by the demethylating agent 5-aza-2'-deoxycytidine (DAC). DAC might increase the tumor sensitivity to chemotherapy through demethylation and restoration of gene expression. We investigated the effect of combined therapy with DAC and irinotecan (CPT-11) on the human colon cancer cell line HCT-15. Human colon cancer cell line HCT-15 was treated with DAC and/or CPT-11 both in vitro and in vivo. The changes in mRNA expression of several apoptosis-related genes were investigated by reverse transcriptase-polymerase chain reaction (PCR). Promoter methylation was detected by methylation-specific PCR and combined bisulfite restriction analysis. Suppression of tumor growth was observed during the treatment with DAC and/or CPT-11 and apoptosis in the tumors was investigated by TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) assay. Promoter methylation of p14ARF, p16 INK4a, BNIP3, and XAF1 was confirmed, and DAC restored mRNA expression of these genes. Demethylation and restoration of gene expression was observed with low-dose DAC, and demethylation status was sustained for several weeks. Combined therapy with DAC and CPT-11 produced marked suppression in tumor growth compared with DAC or CPT-11 alone, both in vitro and in vivo. Pretreatment with low-dose DAC may have the potential to be used as a "biosensitizer" of DNA-damaging agents such as CPT-11 when the apoptotic pathway is inactivated as a result of aberrant promoter methylation in the cancer.

Sodium selenite induces apoptosis in cultured cortical neurons with special concomitant changes in expression of the apoptosis-related genes

Sodium selenite was used to examine whether selenium compound is able to trigger apoptotic degeneration in cultured cortical neurons in vitro and to explore the detailed changes in expression of the related genes during the apoptotic processes using molecular biological and flow cytometric examinations. The results indicated that: (1) cortical neurons treated with sodium selenite with different dosages (0.0008, 0.004, 0.0200, 0.1000, and 0.5000 μM) and different exposure times (2, 4, 24, and 48 h) exhibited dose- and time-dependent apoptotic processes as revealed by typical DNA ladder formation detected by agarose gel electrophoresis; (2) the internucleosomal DNA fragmentation detected by flow cytometric examination showed a prominent peak of hypodiploid DNA contents as early as 4 h after exposure of 0.1 μM sodium selenite; (3) the DNA fragmentation induced by sodium selenite as revealed by the above two examinations could be blocked by aurintricarboxylic acid; (4) the transcriptions of mRNAs related to bcl-2, bax, c-fos, p53, and acetylcholinesterase (AChE) genes, as detected by RT-PCR assays, showed down-regulation for bcl-2 and up-regulation for bax, c-fos, p53, and AChE genes after exposure of sodium selenite. This study suggests that the sodium selenite is effective for inducing apoptosis in cultured cortical neurons and that relevant changes in expression of several apoptosis-related genes might further our understanding of the mechanism(s) that initiates and maintains the apoptotic processes.

Role of Transcription Factors in Angiogenesis

Angiogenesis is a complex phenomenon that requires at least migration, proliferation, and tubular morphogenesis of endothelial cells (ECs). Some genes are expressed in ECs during these processes, and therefore the regulation of gene expression in ECs is critical. Increasing evidence suggests that the Ets family of transcription factors plays an important role in angiogenesis. We observed that Ets-1, a prototype of the Ets family of transcription factors, promoted angiogenesis by inducing the expression of matrix metalloproteinases and integrin beta3 in ECs, and the elimination of the transactivation activity of Ets-1 by a dominant negative molecule inhibited angiogenesis. Apoptosis, a term used to describe the terminal morphological and biochemical events seen in programmed cell death, is critical for the development or reconstitution of multicellular organs. Apoptosis of ECs is observed at the initiation of angiogenesis, at the branching or communication with newly formed vessels, and at the regression of neo-vessels. The Ets family of transcription factors is generally thought to be anti-apoptotic. However, there are conflicting reports on the role of Ets-1 in apoptosis. We examined the role of Ets-1 in apoptosis of ECs and found that Ets-1 was pro-apoptotic to ECs by modulating the expression of several apoptosis-related genes.

Estrogen and NGF synergistically protect terminally differentiated, ERalpha-transfected PC12 cells from apoptosis.

The potential cytoprotective effects of the gonadal steroid estrogen, acting via its receptor (ER) on target neurons, are of considerable interest in aging, disease, and trauma. In this study, we examined the effects of estrogen on a prototypical neuronal-like cell, namely, nerve growth factor (NGF) differentiated PC12 cells when these cells are placed into an apoptosis-inducing environment. A clonal line of PC12 cells stably transfected to express full-length rat ERalpha mRNA and protein (PCER cells), as well as control cells (vector DNA transfected: PCCON cells), were differentiated into neurite bearing cells by 14 days of NGF treatment. Reverse transcription polymerase chain reaction (RT-PCR) and immunocytochemistry for ERalpha revealed that terminally differentiated PCER cells continue to show robust ER mRNA and protein expression after differentiation. PCCON cells do not express ERalpha either before or after NGF-induced differentiation. After differentiation, serum and NGF withdrawal from wild type PC12 cells is well known to induce cell death via apoptotic mechanisms. We challenged differentiated PCER and PCCON cells with serum-free media and assessed the effects of various treatments on cell survival and apoptosis using trypan blue staining and quantitative TUNEL staining. When differentiated PCER cells were treated with 17beta-estradiol (E2) plus NGF during a 2-day serum-free media challenge, cell survival was significantly greater, and the apoptotic index was significantly lower, than when PCER cells were treated with NGF only during the challenge. No additional improvement in cell survival, and no further reduction in apoptosis, was observed in non-ER expressing cells (PCCON) by E2 plus NGF treatment over that obtained by NGF alone. The effects of E2 on the expression of several apoptosis-related genes in this system were also examined. We found that E2 treatment of differentiated PCER cells in serum-free media increased the levels of Bcl-XL mRNA and reduced the levels of BAD mRNA relative to those in vehicle-treated PCER cells, and also relative to those in PCCON cells. Thus, estrogen's protective effects on PCER cells appear to, at least partly, involve a modulation of apoptosis-regulating genes.

Apoptotic cell death and related gene expression in metastatic tumors of AKR lymphomas of varying malignancy.

Resistance to apoptosis may be related to tumor progression, due to the implications it might have on both tumor mass and genetic instability. We compared the tendency to spontaneous apoptosis and the proliferative capacity of metastatic growths of several AKR lymphoma variants (TAU-45, TAU-47, TAU-44, TAU-33, TAU-42 and TAU-46, in the order of increasing metastatic potential). We further compared the expression of several apoptosis-related genes. Cell proliferative capacity did not appear to determine malignant behavior since, on the whole, a decrease in S + G2M fraction was observed with increasing malignancy. Sensitivity to apoptotic cell death decreased with increasing malignancy when comparing the TAU-45, TAU-47, TAU-44 and TAU-33 variants, suggesting a role of reduced apoptosis in this T-cell lymphoma. An increase in Bcl-2 content with increasing aggressiveness among these variants, implicates this protein in this tumor progression-related resistance to apoptosis. However, the two variants of highest malignancy, TAU-42 and TAU-46, did not follow the same trend, since they displayed a relatively high content in apoptotic cells and a low Bcl-2 content. Fas receptor expression did not correlate with tendency to apoptosis, indicating that malignant behavior in the AKR lymphoma does not depend on CD95/Fas/APO1 downregulation. Overexpression of p53 was observed only in one of the variants of lowest malignancy.