Mechanism Of Apoptotic Cell Death Research Articles

Regulation of efferocytosis by caspase-dependent apoptotic cell death in atherosclerosis.

During the growing process of the atherosclerotic lesions, lipid-filled macrophage foam cells form, accumulate, and ultimately undergo apoptotic death. If the apoptotic foam cells are not timely removed, they may undergo secondary necrosis, and form a necrotic lipid core which renders the plaque unstable and susceptible to rupture. Therefore, the non-lipid-filled fellow macrophages, as the main phagocytic cells in atherosclerotic lesions, need to effectively remove the apoptotic foam cells. In general, in apoptotic macrophages, caspases are the central regulators of several key processes required for their efficient efferocytosis. The processes include the generation of "Find-Me" signals (such as adenosine triphosphate/uridine triphosphate, fractalkine, lysophosphatidylcholine, and sphingosine-1-phosphate) for the recruitment of viable macrophages, generation of the "Eat-Me" signals (for example, phosphatidylserine) for the engulfment process, and, finally, release of anti-inflammatory mediators (including transforming factor β and interleukin-10) as a tolerance-enhancing and an anti-inflammatory response, and for the motile behavior of the apoptotic cell. The caspase-dependent mechanisms are operative also in apoptotic macrophages driving the atherogenesis. In this review, we explore the role of the molecular pathways related to the caspase-dependent events in efferocytosis in the context of atherosclerosis. Understanding of the molecular mechanisms of apoptotic cell death in atherosclerotic lesions is essential when searching for new leads to treat atherosclerosis.

Apoptotic effect of fluoxetine through the endoplasmic reticulum stress pathway in the human gastric cancer cell line AGS.

Gastric cancer is the fourth most common cancer in the world. Fluoxetine (FLX), a selective serotonin reuptake inhibitor, can inhibit the growth of cancer cells by inducing apoptotic cell death through various signaling pathways. This study was aimed to determine the mechanism of apoptotic cell death induced by FLX in AGS cells. MTT assay for cell viability test and colony forming assay was performed for detection of cell proliferation. Western blot analysis was conducted for protein expression. Increased fluorescence intensity and chromatin condensation were observed using DAPI staining. Production of reactive oxygen species (ROS) was measured by DCFDA assay. AGS cell proliferation was remarkedly inhibited by FLX in a dose-dependent manner starting at a concentration of 20 μM. The expression of death receptors was increased, which resulted in elevated expression of activated caspases and cleaved PARP, leading to FLX-induced apoptosis. Moreover, FLX significantly increased production of ROS, and N-acetyl cysteine, which scavenges ROS, attenuated the cytotoxic effects of FLX. In addition, treatment with FLX increased the expression of the endoplasmic reticulum (ER) stress marker, CHOP. P53 protein expression in AGS cells also decreased significantly with FLX treatment. Inhibition of ER stress significantly decreased the expressions of death receptor 5 (DR5), cleaved caspase 3, and cleaved PARP, but not to control levels. FLX-induced apoptosis in AGS involved upregulation of death receptors, ROS generation, and activation of ER stress.

Pt(II) and Pd(II) complexes with a thiazoline derivative ligand: Synthesis, structural characterization, antiproliferative activity and evaluation of pro-apoptotic ability in tumor cell lines HT-29 and U-937

Eluding apoptosis represents the hallmark of tumoral cell behavior. Cisplatin (CisPt) is a very common chemotherapeutic agent to treat cancer by reestablishing apoptotic mechanisms of cell death. However, certain patients acquire resistance to CisPt as well as suffer nephrotoxicity, neurotoxicity, nausea and vomiting. The synthesis of new Pt(II) compounds represents an alternative to CisPt to avoid resistance and undesirable side effects. Pd(II) could be a Pt(II) surrogate given the similarity of coordination chemistry between them, thus widening the spectra of available anticancer drugs. Herein, we have synthesized and characterized two Pt(II) or Pd(II) complexes with TdTn (2-(3,4-dichlorophenyl)imino-N-(2-thiazolin-2-yl)thiazolidine), a thiazoline derivative ligand, with formula [PtCl2(TdTn)] and [PdCl2(TdTn)]. The potential anticancer ability was evaluated in human colon adenocarcinoma HT-29 and human histiocytic lymphoma U-937 cell lines. To that aim, U-937 and HT-29 cells were treated with TdTn, [PtCl2(TdTn)] and [PdCl2(TdTn)] for 24 h. The microscopy monitoring indicated that TdTn, [PtCl2(TdTn)] and [PdCl2(TdTn)] arrested the cell proliferation of U-937 and HT-29 cells with respect to control, in agreement with MTT (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide) analysis. Moreover, it is noteworthy that the ligand by its own showed antiproliferative effects in both cell lines. [PtCl2(TdTn)] and [PdCl2(TdTn)] caused caspase-3 activation in U-937 cells, simultaneously with caspase-9 activation due to complexes; however, in HT-29 caspase-3 activation occurred simultaneously with caspase-8 activation induced by the ligand TdTn. Only metal complexes were able to induce ROS (Reactive Oxygen Species) generation in U-937 cells, but not TdTn. In HT-29 cells neither the metal complexes, nor the ligand induced ROS generation.

Elevation of Cleaved p18 Bax Levels Associated with the Kinetics of Neuronal Cell Death during Japanese Encephalitis Virus Infection.

Japanese encephalitis virus (JEV) infection induces uncontrolled neuronal apoptosis, leading to irreversible brain damage. However, the mechanism of JEV-induced neuronal apoptosis has not been clearly elucidated. This study aimed to investigate both virus replication and neuronal cell apoptosis during JEV infection in human neuroblastoma SH-SY5Y cells. As a result, the kinetic productions of new viral progeny were time- and dose-dependent. The stimulation of SH-SY5Y cell apoptosis was dependent on the multiplicity of infections (MOIs) and infection periods, particularly during the late period of infection. Interestingly, we observed that of full-length Bax (p21 Bax) level started to decrease, which corresponded to the increased level of its cleaved form (p18 Bax). The formation of p18 Bax resulting in cytochrome c release into the cytosol appeared to correlate with JEV-induced apoptotic cell death together with the activation of caspase-3/7 activity, especially during the late stage of a robust viral infection. Therefore, our results suggest another possible mechanism of JEV-induced apoptotic cell death via the induction of the proteolysis of endogenous p21 Bax to generate p18 Bax. This finding could be a new avenue to facilitate novel drug discovery for the further development of therapeutic treatments that could relieve neuronal damage from JEV infection.

Recombinant Pierisin-5 Induces Apoptosis and Differential Expression of Bcl-2, Bax, and p53 in Human Cancer Cells.

Pierisin-5 protein (pie-5) belongs to a family of proteins possessing DNA-dependent ADP-ribosyltransferase activity, which can induce apoptotic cell death. The baculovirus-mediated expression vector system (BEVS) has been commonly used for in vitro expression of heterologous protein subunits for basic scientific research, in addition to the development and production of diagnostics and vaccines. In this study, a new method for the in vitro expression of the cytotoxic protein was established using the baculovirus expression system. The antiproliferative and apoptotic effect of the novel recombinant pierisin-5 protein (rpie-5) was investigated in different human cancer cell lines, such as HeLa, HepG2, and AGS. Cloning, in vitro overexpression, and purification of the rpie-5 protein were performed by using BEVS in Sf21 (Spodoptera frugiperda) insect cell line. The rpie-5 protein exhibits cytotoxicity in all the cell lines, but HeLa (IC50 0.6 μg/mL) was more sensitive when compared with HepG2 (IC50 1.9 μg/mL) and AGS (IC50 3.7 μg/mL) cell lines. The cytotoxic effects of rpie-5 lead to apoptotic cell death in cancer cells and resulted in nuclear fragmentation, enlargement of the nucleus, loss of mitochondrial membrane potential, and finally release of lactose dehydrogenase (LDH) enzyme from the cell membrane. This study reports the molecular mechanism of apoptotic cell death through the upregulation of Bax (Bcl-2 family activating protein-X), Bad, APAF-1 (apoptotic protease activating factor-1), Cyt-c, and caspase-3/9 and the downregulation of Bcl-2 (B-cell lymphoma 2) in rpie-5-treated cancer cells. The study concludes that rpie-5 has p53-independent apoptosis in HepG2 cells and p53-dependent apoptosis in HeLa and AGS cell lines. In the future, this study helps to understand the molecular mechanism of rpie-5 to induction of apoptosis and cell death.

In vitro apoptotic effect of Zinc(II) complex with N-donor heterocyclic ligand on breast cancer cells

Abstract Background The synthesis of new ligand and transition metal complexes have drawn great attention in cancer treatment due to excellent DNA cleavage activities and high antitumor activity. Objective The purpose of this study was to synthesize a new Zn(II) complex with 2,2′-bipyridine (bpy) ligand, [Zn(bpy)2(H2O)]2(ClO4), and to investigate the potential therapeutic activity against breast cancer. Materials and methods Zn(II) complex was obtained and structurally characterized using crystallography and other spectroscopic methods. The cytotoxic and apoptotic effects of Zn(II) complex on MCF7 breast cancer and HUVEC control cells were evaluated by WST-1, Annexin V and cell cycle analysis. Results The spectroscopic data demonstrated that Zn(II) complex was successfully synthesized. Furthermore, Zn(II) complex exhibited significant cytotoxic effect on MCF7 breast cancer and HUVEC cells (p < 0.01). MCF7 and HUVEC cells proliferation was reduced to 11.0% and 52.6%, respectively at 10 μM for 48 h. Additionally, Zn(II) complex significantly induced apoptotic cell death (84.87%) and caused S phase arrest (36.4%) in MCF7 cells at 10 μM (p < 0.01). Conclusions Zn(II) complex has great therapeutic potential for the treatment of breast cancer. However, further studies are warranted to identify the underlying mechanisms of apoptotic cell death and to reduce the toxicity of Zn(II) complex on control cells.

Genetic Aberrations Associated with Photodynamic Therapy in Colorectal Cancer Cells.

Photodynamic therapy (PDT) is a cancer treatment modality that utilizes three components: light (λ 650–750 nm), a photosensitizer (PS) and molecular oxygen, which upon activation renders the modality effective. Colorectal cancer has one of the highest incident rates as well as a high mortality rate worldwide. In this study, a zinc (Zn) metal-based phthalocyanine (ZnPcSmix) PS was used to determine its efficacy for the treatment of colon adenocarcinoma cells (DLD-1 and Caco-2). Photoactivation of the PS was achieved by laser irradiation at a wavelength of 680 nm. Dose responses were performed to establish optimal PS concentration and irradiation fluence. A working combination of 20 µM ZnPcSmix and 5 J/cm2 was used. Biochemical responses were determined after 1 or 24 h incubation post-treatment. Since ZnPcSmix is localized in lysosomes and mitochondria, mitochondrial destabilization analysis was performed monitoring mitochondrial membrane potential (MMP). Cytosolic acidification was determined measuring hydrogen peroxide (H2O2) levels in the cytoplasm. Having established apoptotic cell death induction, an apoptosis PCR array was performed to establish the apoptotic mechanism. In DLD-1 cells, expression of genes included 3 up-regulated and 20 down-regulated genes while in Caco-2 cells, there were 16 up-regulated and 22 down-regulated genes. In both cell lines, in up-regulated genes, there was a combination of pro- and anti-apoptotic genes that were significantly expressed. Gene expression results showed that more tumorigenic cells (DLD-1) went through apoptosis; however, they exhibit increased risk of resistance and recurrence, while less tumorigenic Caco-2 cells responded better to PDT, thus being suggestive of a better prognosis post-PDT treatment. In addition, the possible apoptotic mechanisms of cell death were deduced based on the genetic expression profiling of regulatory apoptotic inducing factors.

The POU-Domain Transcription Factor Oct-6/POU3F1 as a Regulator of Cellular Response to Genotoxic Stress.

DNA damage and the generation of reactive oxygen species (ROS) are key mechanisms of apoptotic cell death by commonly used genotoxic drugs. However, the complex cellular response to these pharmacologic agents remains yet to be fully characterized. Several studies have described the role of transcription factor octamer-1 (Oct-1)/Pit-1, Oct-1/2, and Unc-86 shared domain class 2 homeobox 1 (POU2F1) in the regulation of the genes important for cellular response to genotoxic stress. Evaluating the possible involvement of other POU family transcription factors in these pathways, we revealed the inducible expression of Oct-6/POU3F1, a regulator of neural morphogenesis and epidermal differentiation, in cancer cells by genotoxic drugs. The induction of Oct-6 occurs at the transcriptional level via reactive oxygen species (ROS) and ataxia telangiectasia mutated- and Rad3-related (ATR)-dependent mechanisms, but in a p53 independent manner. Moreover, we provide evidence that Oct-6 may play a role in the regulation of cellular response to DNA damaging agents. Indeed, by using the shRNA approach, we demonstrate that in doxorubicin-treated H460 non-small-cell lung carcinoma (NSCLC) cells, Oct-6 depletion leads to a reduced G2-cell cycle arrest and senescence, but also to increased levels of intracellular ROS and DNA damage. In addition, we could identify p21 and catalase as Oct-6 target genes possibly mediating these effects. These results demonstrate that Oct-6 is expressed in cancer cells after genotoxic stress, and suggests its possible role in the control of ROS, DNA damage response (DDR), and senescence.

Synergistic Effects of Nobiletin and Sorafenib Combination on Metastatic Prostate Cancer Cells

Objective: Herein we, for the first time, investigated a potential synergistic effect of nobiletin (NOB) and sorafenib (SOR) on PC-3 prostate cancer and HUVEC control cell lines.Methods: In order to determine the cytotoxic and apoptotic effects of the combination of NOB and SOR, WST-1, Annexin V, and cell cycle analysis were performed. The potential molecular mechanism of apoptotic cell death was assessed by Bax, Bcl-2, CCDN1, Rb1, and CDKN1A gene expression and acridine orange (AO) and DAPI staining.Results: Our results indicated that NOB and SOR combination had a significant inhibitory effect on the viability of PC-3 cells with less toxicity on HUVEC cells than SOR alone (P < 0.01). NOB and SOR combination significantly caused much more apoptotic cell death and cell cycle arrest at G0/G1 phase by up-regulation of Bax, Rb1, and CDKN1A levels in PC-3 cells (P < 0.01). Therefore, strong synergistic effects between NOB and SOR were analyzed (CI < 1).Conclusion: NOB and SOR combination was more effective than SOR and NOB alone and reduced the exposure time for SOR and NOB in PC-3 cells. Combination strategy is a therapeutic potential to improve efficacy and reduce side-effect of SOR for the treatment of metastatic prostate cancer cells.

What is the chromosomal constitution of blastomeres excluded from the formation of a morphologically normal blastocyst?

Introduction Blastomeres that have arisen at any time throughout cleavage-stage are sometimes excluded from the formation of the blastocyst and are sequestered either between the zona pellucida and the perivitelline space or remain internally during blastocyst formation and are sequestered to the blastocoel cavity. In both ways, once isolated, those cells do not take further part in preimplantation development. It has been demonstrated that excluded cells have poor gap junction communication with the embryo ( Hardy et al., 1996 ). Material and Methods This observational study was based on 40 good or top-quality blastocysts (at least 4BB) with blastomeres excluded from the formation of the blastocyst and sequestered between the zona pellucida and the perivitelline space. They were belonging to 33 patients (mean age: 33.15) applying for preimplantation genetic screening (PGS) due to advanced maternal age (>37). Patients with structural abnormalities or a history of abnormal fetal karyotype were not included in the analysis. The 40 trophectoderm biopsies were prospectively analyzed and the corresponding excluded blastomeres (2- 5/ blastocyst) samples were in parallel diagnosed either by next generation sequencing (NGS) (PGM platform, ThermoFisher) or by array Comparative Genomic Hybridization (aCGH, Illumina). Assisted hatching was done on day-3 and blastocysts having excluded blastomeres in the axis of the zona opening were included in the analysis to decrease manipulation and most importantly, not to harm the blastocyst. Results The PGS analysis of the trophectoderm biopsies revealed 17 chromosomes involved in monosomies, 37 in trisomies, 7 in mosaics and 13 euploids, showing that 32.5% of the blastocysts with excluded blastomeres were euploid. In 13 blastocysts one chromosome was involved in the aneuploidy whereas for 9 blastocysts two chromosomes were implicated. Only three blastocysts were aneuploid for three chromosomes. None but one of the excluded blastomeres had the same diagnosis with the corresponding embryo (trisomy 4). However, in nearly half of the embryos (15/40) at least one chromosomal aneuploidy was shared by both samples; the excluded blastomeres having a more complex diagnosis with a mean of four chromosomes involved in the aneuploidy [for example, a diagnosis of monosomy 7, 14 for the blastocyst and a diagnosis of trisomy 1q, monosomy (3, 4, 7, 14, 16, 19) for the corresponding excluded blastomeres]. Only two samples of excluded blastomeres were euploid although the corresponding blastocyst was aneuploid. Conclusions Morphologically normal blastocysts with excluded blastomeres were diagnosed with aneuploidy affecting a mean of 1.4 chromosomes, whereas excluded blastomeres were chromosomally chaotic (mean of 4.35 aneuploid chromosomes). It may be hypothesized that either the embryo attempts to exclude blastomeres with chaotic aneuploidies from the blastocyst or that the excluded blastomeres have become chromosomally chaotic because of apoptotic cell death mechanisms that have been already initiated in the aneuploid embryo.

Dual Targeting of Cell-Cycle Checkpoint Kinase and Unfolded Protein Response As a Novel Therapeutic Approach for High-Risk Multiple Myeloma with TP53 Deletion or Mutations

Multiple myeloma (MM) is a hematological malignancy characterized by abnormal clonal proliferation of malignant plasma cells. Despite the introduction of novel agents such as proteasome inhibitors, immunomodulatory drugs, and antibodies that have significantly improved clinical outcomes of the patients of MM, most patients eventually relapse and develop drug resistance. In particular, the prognosis of patients harboring either chromosome TP53 deletion or mutations remains very poor, suggesting the prevalence of TP53 abnormalities increases with disease progression. Therefore, novel therapeutic strategies to overcome this unfavorable feature are urgently needed in clinical settings. WEE1 is a cell-cycle checkpoint kinase and a key regulator of DNA damage surveillance pathways. In response to extrinsically induced DNA damage, WEE1 kinase induces cell cycle arrest, allowing damaged DNA to be repaired before the cell undergoes DNA replication in S phase, and preventing cells harboring unrepaired, damaged DNA from mitotic lethality. Furthermore, WEE1 overexpression has been observed in many types of cancers. In addition, our previous studies revealed that monotherapy with AZD1775, a potent and highly selective inhibitor of WEE1, inhibited the proliferation of various MM cell lines irrespective of TP53 status. (Blood 2016; 128: 3256). On the other hand, one of the defining features of MM cells is the production of large amounts of protein, such as immunoglobulin, that must be processed within the endoplasmic reticulum (ER). Due to the accumulation of abundant immunoglobulin in ER, MM cells are constitutively under conditions of ER stress. The unfolded protein response (UPR)-signaling pathway is a cytoprotective mechanism against ER stress, and is therefore activated in MM cells to survive these conditions. Activation of the UPR has been observed in many types of cancers, and loss of TP53 has shown to enhance the UPR. Protein kinase RNA-like endoplasmic reticulum kinase (PERK) is one of three ER transmembrane protein kinases implicated as primary effectors of the UPR. Recent studies have suggested that PERK inhibition resulted in dose-dependent inhibition of tumor growth both in vitro and in vivo. In addition, more recent studies have proposed that PERK induces resistance to cell death elicited by chemotherapy. The combination of WEE1 and PERK inhibitors might thus offer an attractive therapeutic option against this incurable hematological malignancy. Here, we investigated the therapeutic utility of AZD1775 and GSK2606414, a highly selective inhibitor of PERK kinase, alone and in combination in various MM cells including TP53 wild-type (MM1.S) as well as TP53-deficient (KMS-11) and TP53-mutated (U266, RPMI8226, OPM-2) cell lines. AZD1775 and GSK2606414 alone induced dose-dependent cell growth inhibition in all investigated MM cells irrespective of TP53 status. Interestingly, GSK2606414 in combination with AZD1775 inhibited proliferation of all MM cells more effectively than either single agent. Assays for apoptotic cell death demonstrated that AZD1775 in combination with GSK2606414 induced significant and marked apoptotic cell death in MM cells used in this study compared to monotherapy alone. Next, western blotting analysis was performed to address the mechanisms of apoptotic cell death by the treatment of WEE1 and PERK inhibitors in MM cells. GSK2606414 inhibited PERK activation and decreased its downstream substrates (phospho-eIF2a, ATF4, and CHOP). Combination treatment with WEE1 inhibitor and various doses of PERK inhibitor significantly increased PARP and caspase 3 cleavage, CDK1 phosphorylation, and histone H2AX expression. Taken together, these data suggest that combining AZD1775 and GSK2606414 synergistically induced DNA damage and promoted premature mitotic entry, resulting in apoptotic cell death of TP53-deleted or -mutated MM cells. In conclusion, dual targeting of WEE1 and PERK might be a promising therapeutic approach for MM irrespective of TP53 status. DisclosuresTokuhira:Bristol-Myers Squibb: Speakers Bureau; AYUMI Pharmaceutical Corporation: Speakers Bureau; Mitsubishi Tanabe Pharma Corporation: Speakers Bureau; Chugai: Speakers Bureau. Kizaki:Novartis: Speakers Bureau; Bristol-Myers Squibb: Research Funding, Speakers Bureau; Celgene: Research Funding, Speakers Bureau; Nippon Shinyaku,: Research Funding, Speakers Bureau.

6-MOMIPP, a novel brain-penetrant anti-mitotic indolyl-chalcone, inhibits glioblastoma growth and viability.

3-(6-Methoxy-2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propene-1-one (6-MOMIPP) is a novel indole-based chalcone that disrupts microtubules. The present study aims to define the mechanism through which 6-MOMIPP induces cell death and to evaluate the efficacy of the compound in penetrating the blood-brain barrier and inhibiting growth of glioblastoma xenografts. The effects of 6-MOMIPP were evaluated in cultured U251 glioblastoma cells, using viability, flow cytometry, and tubulin polymerization assays. Scintillation proximity and tubulin crosslinking methods were used to identify the binding site of 6-MOMIPP on tubulin, and western blots were performed to define the signaling pathways that contribute to cell death. LC/MS assays were used to study the pharmacokinetic behavior of 6-MOMIPP in mice. Subcutaneous and intracerebral xenograft models were utilized to assess the effects of 6-MOMIPP on growth of U251 glioblastoma in vivo. The findings indicate that 6-MOMIPP targets the colchicine site on β-tubulin. At concentrations ≥ 250nm, 6-MOMIPP induces mitotic arrest, caspase activation and loss of cell viability. Cells are protected by caspase inhibitors, pointing to an apoptotic mechanism of cell death. Loss of cell viability is preceded by activation of Cdk1(Cdc2) and phosphorylation of Bcl-2 and Bcl-xL. Inhibition of both events with a Cdk1 inhibitor prevents cell death. 6-MOMIPP has broad activity against the viability of multiple glioblastoma, melanoma and lung carcinoma cell lines. Viability of normal cells, including differentiated neurons, is not significantly affected at a drug concentration (1µM) that reduces viability in most cancer lines. Pharmacokinetic studies in mice show that concentrations of 6-MOMIPP in the brain mirror those in the plasma, indicating that 6-MOMIPP readily penetrates the blood-brain barrier. Studies with mice bearing human U251 glioblastoma xenografts demonstrate that 6-MOMIPP is effective in suppressing growth of subcutaneous and intracerebral tumors without causing general toxicity. The results indicate that 6-MOMIPP is anovel microtubule disruptor that targets the colchicine binding site on β-tubulin to induce mitotic arrest and cell death. The ability of 6-MOMIPP to penetrate the blood-brain barrier and inhibit growth of glioblastoma xenografts suggests that it warrants further preclinical evaluation as potential small-molecule therapeutic that may have advantages in treating primary and metastatic brain tumors.

In silico and in vitro screening of small molecule Inhibitors against SYT-SSX1 fusion protein in synovial sarcoma

Synovial sarcoma (SS) is characterized by a tumour specific chromosomal translocation t(X;18) (p11;q11) which results in the formation of SYT-SSX1 fusion protein. This fusion protein represents a clear therapeutic target and molecules specifically targeting SYT-SSX1 fusion protein are currently not available. In this study, SYT-SSX1 fusion protein sequence was retrieved from Uniprot and 3D structure was generated using I-TASSER modeling program. A structure based computational screening approach has been employed using Glide docking software to identify potential SYT-SSX1 small molecule inhibitors that bind to the junction region of the fusion protein. The obtained inhibitors were further filtered based on the docking score and ADME/T properties. Ten best fit compounds were chosen for in vitro studies. The anti-proliferative activities of these 10 compounds were screened in Yamato, ASKA (carries SYT-SSX1 fusion protein) and other sarcoma cell lines such as A673, 143B to understand the specificity of inhibition of the chosen compounds. The in vitro activity was compared against HEK293 cell lines. The compound 5-fluoro-3-(1-phenyl-1H-tetraazol-5-yl)-1H-indole (FPTI) was found to be selectively cytotoxic in synovial sarcoma cell lines (Yamato and ASKA) and this compound also showed insignificant anti proliferative activity on other cell lines. Further, target gene expression study confirmed that FPTI treatment down-regulated SYT-SSX1 and modulated its downstream target genes. Cell cycle analysis revealed the involvement of an apoptotic mechanism of cell death. Further experimental validations may elucidate the therapeutic potentials of FPTI against SYT-SSX1 fusion protein.

Rodzaje śmierci komórki

STRESZCZENIEHomeostaza w organizmie utrzymywana jest przez wiele procesów, w tym również dzięki zachowywaniu równowagi między pojawianiem się nowych komórek a ich umieraniem. W pracy opisano mechanizm i szlaki apoptotycznej śmierci komórek oraz zjawisko nekrozy i autofagii. Ponadto opisano rzadziej prezentowane typy umierania komórek, a mianowicie katastrofę mitotyczną, onkozę i pyroptozę. Przebieg tych procesów jest zróżnicowany, co podkreślono w pracy, a także związany z jednej strony z utrzymaniem stabilności tkanek w przebiegu procesów fizjologicznych, a z drugiej z indukcją i progresją procesów patologicznych. W pracy starano się przedstawić podstawowe różnice między opisywanymi procesami i ich znaczenie dla organizmu.

Daphnegiravone D from Daphne giraldii Nitsche induces p38-dependent apoptosis via oxidative and nitrosative stress in hepatocellular carcinoma cells

Daphnegiravone D (DGD), a prenylated flavonoid from Daphne giraldii Nitsche, significantly inhibited cell growth of several cancer cell lines without cytotoxicity on human normal cells. Our previous study showed that DGD could induce apoptosis in hepatocellular carcinoma Hep3B and HepG2 cells, but the detailed mechanism was still unclear. The present study provides that DGD-induced oxidative and nitrosative stress contribute to apoptotic cell death in Hep3B and HepG2 cells. Furthermore, there is a positive loop between oxidative stress and p38 activation, similar result is observed between nitrosative stress and p38. N-Acetylcysteine (NAC), a reactive oxygen species scavenger, could relieve DGD-induced oxidative stress, but exerts little effect on nitrosative stress. In addition, carboxy-PTIO (PTIO, a well-known scavenger of reactive nitrogen species) down-regulates the induction of nitrosative stress without obvious effect on oxidative stress in DGD-treated cells. In conclusion, the induction of oxidative and nitrosative stress could enhance p38-mediated apoptosis in DGD-treated Hep3B and HepG2 cells. Moreover, we speculated that OS and NS could not ultimately affect each other in DGD-treated HCC cells. This study gives a new insight on the mechanism of DGD-induced apoptotic cell death via oxidative and nitrosative stress in HCC cells.

Acute Noise Exposure Is Associated With Intrinsic Apoptosis in Murine Central Auditory Pathway.

Noise that is capable of inducing the hearing loss (NIHL) has a strong impact on the inner ear structures and causes early and most obvious pathophysiological changes in the auditory periphery. Several studies indicated that intrinsic apoptotic cell death mechanisms are the key factors inducing cellular degeneration immediately after noise exposure and are maintained for days or even weeks. In addition, studies demonstrated several changes in the central auditory system following noise exposure, consistent with early apoptosis-related pathologies. To clarify the underlying mechanisms, the present study focused on the noise-induced gene and protein expression of the pro-apoptotic protease activating factor-1 (APAF1) and the anti-apoptotic B-cell lymphoma 2 related protein a1a (BCL2A1A) in the cochlear nucleus (CN), inferior colliculus (IC) and auditory cortex (AC) of the murine central auditory pathway. The expression of Bcl2a1a mRNA was upregulated immediately after trauma in all tissues investigated, whereas the protein levels were significantly reduced at least in the auditory brainstem. Conversely, acute noise has decreased the expression of Apaf1 gene along the auditory pathway. The changes in APAF1 protein level were not statistically significant. It is tempting to speculate that the acoustic overstimulation leads to mitochondrial dysfunction and induction of apoptosis by regulation of proapoptotic and antiapoptotic proteins. The inverse expression pattern on the mRNA level of both genes might reflect a protective response to decrease cellular damage. Our results indicate the immediate presence of intrinsic apoptosis following noise trauma. This, in turn, may significantly contribute to the development of central structural deficits. Auditory pathway-specific inhibition of intrinsic apoptosis could be a therapeutic approach for the treatment of acute (noise-induced) hearing loss to prevent irreversible neuronal injury in auditory brain structures and to avoid profound deficits in complex auditory processing.

Induction of Apoptotic Cell Death on Human Cervix Cancer HeLa cells by Extract from Loranthus yadoriki

Loranthus yadoriki, one of the Korean mistletoe species, has been already known for anti-viral effects, but the molecular basis that it caused apoptosis in cancer cells was not definitely revealed yet. The aim of this study was to estimate the mechanisms of apoptotic cell death of the extract from Loranthus yadoriki (named as ELY) in human cervix HeLa cells. We identified that ELY prevented the proliferation of HeLa cells between 50 and 300 μg/mL which did not affect non-cancerous HaCaT cells. In addition, ELY induced a morphological change and nucleus disruption as well as an accumulation of sub-G1 phase in HeLa cells. The mechanism study, by using western blot analysis, showed that the phosphorylation of Fas-associated death domain (FADD), Bim and Bak was up-regulated by ELY treatment. Furthermore, the expression of cytochrome c and Apaf-1 was increased by ELY treatment. In immunofluorescence staining, the increased intensity of cleaved caspase-3 and cleaved PARP was also observed under ELY treatment. Sequentially, the caspase cascade was activated by ELY from caspase-8 to caspase-3 and from caspase-9 to caspase-3, in both extrinsic and intrinsic pathways. The results of this study demonstrate that ELY has anti-cancer effects on human cervix cancer HeLa cells via caspase cascade in apoptotic signaling pathways.

Synthesis, antiproliferative activity and apoptosis-promoting effects of arene ruthenium(II) complexes with N, O chelating ligands

New half sandwich arene ruthenium(II) complexes of the type [Ru(arene)Cl(L)] (where arene = benzene and p-cymene, L = thiophene benzhydrazone ligands) have been synthesized from the reactions of the neutral precursor [Ru(arene) (μ-Cl) Cl]2 and the corresponding benzhydrazone ligand. All the complexes were completely characterized by elemental analysis and additionally by IR, UV–Vis, 1H NMR and ESI-MS spectroscopic methods. The solid state structures of the complexes 6 and 7 were determined by single-crystal X-ray diffraction analysis, which exhibit typical pseudo-octahedral geometry around the metal centre. The antiproliferative activity of the complexes was evaluated on cancerous (HeLa, MDA-MB-231, and Hep G2) and noncancerous (NIH3T3) cell lines. In general, complexes containing electron releasing OCH3 substituent have potential anticancer activity than those incorporating H, Cl and Br substituents. Moreover, the p-cymene complexes show more cytotoxicity than benzene derivatives, suggesting that the substituent at arene plays a vital role in the biological activity of the compounds. Further, an apoptotic mechanism of cell death in MDA-MB-231 was confirmed by AO-EB, Hoechst 33258 staining and annexin-V/PI double-staining techniques. In addition, the extent of DNA fragmentation in cancer cells was studied by comet assay.

Photodynamic Therapy Mediated by Aloe-Emodin Inhibited Angiogenesis and Cell Metastasis Through Activating MAPK Signaling Pathway on HUVECs.

Photodynamic therapy is a clinically used, minimally invasive therapeutic procedure that involves the application of photosensitizers which can locate in target cells and so be irradiated at a corresponding wavelength. Laser light irradiation activation of photosensitizers generates free reactive oxygen species, which induces selective cytotoxic activity in target cells. Within recent years, aloe-emodin as a photosensitizer has been successfully applied in photodynamic therapy applications. Angiogenesis plays an important role in tumor growth and metastasis; thus, the development of a novel target treatment for angiogenesis is essential in order to improve treatment therapeutics for cancer treatment. An essential step in angiogenesis involves the formation of tube-like structures during matrix degradation, rearrangement, and apoptosis of endothelial cells. In the present study, we investigated the mechanisms of photocytotoxicity induced by aloe-emodin in human umbilical vein endothelial cells. Analysis of cell proliferation results noted a significant decrease in cultured cells which received various concentrations of aloe-emodin and photodynamic therapy–induced light doses. Additionally, mitochondrial mechanisms of apoptotic cell death were observed in aloe-emodin photodynamic therapy–treated cells, as tube formation assays noted angiogenesis suppression after treatment. The capacity of migration and invasion of human umbilical vein endothelial cells was measured using the transwell assay and demonstrated that aloe-emodin photodynamic therapy significantly inhibited the migration and invasion of human umbilical vein endothelial cells. The expression of p38, extracellular signal-regulated kinase, the c-Jun N-terminal kinases, and vascular endothelial growth factor suggested that the cellular metastasis was related to mitogen-activated protein kinase signal pathway. Furthermore, disorganization of F action cytoskeleton components was observed after aloe-emodin photodynamic therapy. Overall, the findings from this study suggest that aloe-emodin photodynamic therapy inhibited angiogenesis and cellular metastasis in human umbilical vein endothelial cells by activating the mitogen-activated protein kinase apoptotic signaling cell death pathway.

Cytotoxic effects of delphinidin in human osteosarcoma cells

IntroductionThe aim of this study was to evaluate whether delphinidin is cytoprotective or cytotoxic in osteosarcoma cell lines, and to elucidate the underlying mechanisms. Materials and methodsThe present study investigated whether apoptosis or autophagy is induced by delphinidin in human osteosarcoma cell lines. Delphinidin was used as the antioxidant, along with two autophagy inhibitors: 3-methyladenine and bafilomycin A1. Cell viability and known autophagic markers, such as LC3-II expression, were evaluated. Reactive oxygen species (ROS) formation and cell cycle analysis were also investigated. ResultsDelphinidin showed concentration-dependent cytotoxicity to osteosarcoma cell. Delphinidin is closely associated with apoptotic cell death mechanisms and pathways related to ROS accumulation. In addition, we observed delphinidin-induced autophagosome formation and increasing levels of LC3-II conversion. However, in spite of delphinidin induced autophagy, the cytotoxic effects induced in the osteosarcoma cells may not be operating via autophagic cell death mechanisms. ConclusionsDelphinidin compromises the cellular protective mechanisms by inhibiting autophagy, permitting ROS to accumulate and finally enhance apoptotic cell death. Our results indicate that delphinidin may play a critical role as a chemotherapeutic agent by preventing the development and progression of osteosarcoma cells.