Apoptosis

Letter to the editor regarding article, "Role of glycogen synthase kinase following myocardial infarction and ischemia–reperfusion"

Role of glycogen synthase kinase following myocardial infarction and ischemia–reperfusion

17-Aminogeldanamycin selectively diminishes IRE1α-XBP1s pathway activity and cooperatively induces apoptosis with MEK1/2 and BRAF V600E inhibitors in melanoma cells of different genetic subtypes Abstract Outcomes of melanoma patient treatment remain unsatisfactory despite accessibility of oncoprotein-targeting drugs and immunotherapy. Here, we reported that 17-aminogeldanamycin more potently activated caspase-3/7 in BRAFV600E melanoma cells than geldanamycin, another inhibitor of heat shock protein 90 (HSP90). 17-aminogeldanamycin alleviated self-triggered compensatory increase in HSP70 mRNA level and induced endoplasmic reticulum (ER) stress, which was followed by selective diminution of cytoprotective IRE1α-XBP1s pathway activity of unfolded protein response (UPR), inhibition of ERK1/2 activity and induction of apoptosis. Concomitantly, ATF6/p50 level and expression of PERK-dependent genes, CHOP and BIM, remained unaltered. This might result from an inframe deletion in EIF2AK3 leading to a PERKL21del variant revealed by whole-exome sequencing in melanoma cell lines. 17-aminogeldanamycin exhibited similar activity in NRASQ61R melanoma cells that harbored a heterozygous inactivating variant of NAD(P)H:quinone oxidoreductase 1 (NQO1P187S). In addition, 17-aminogeldanamycin acted cooperatively with trametinib (an inhibitor of MEK1/2) and vemurafenib (an inhibitor of BRAFV600E) in induction of apoptosis in melanoma cell lines as evidenced by in-cell caspase-3/7 activation and PARP cleavage that occurred earlier compared with either drug used alone. As trametinib and vemurafenib did not significantly affect HSP70 and GRP78 transcript levels, cooperation of MEK/BRAFV600E inhibitors and 17-aminogeldanamycin might result from a concurrent inhibition of the RAS/RAF/MEK/ERK cascade and IRE1α-dependent signaling, and cell-intrinsic ER homeostasis can determine the extent of the drug cooperation. Our study indicates that 17-aminogeldanamycin takes several advantages compared with other HSP90-targeting compounds, and can complement activity of BRAF/MEK inhibitors in melanoma cells of different genetic subtypes.

Copper-imidazo[1,2- a ]pyridines induce intrinsic apoptosis and modulate the expression of mutated p53, haem-oxygenase-1 and apoptotic inhibitory proteins in HT-29 colorectal cancer cells Abstract Metastatic colorectal cancer responds poorly to treatment and is a leading cause of cancer related deaths. Worldwide, chemotherapy of metastatic colorectal cancer remains plagued by poor efficacy, development of resistance and serious adverse effects. Copper-imidazo[1,2-a]pyridines were previously shown by our group to be selectively active against several cancer cell lines, with three complexes, JD46(27), JD47(29), and JD88(21), showing IC50 values between 0.8 and 1.8 μM against HT-29 cells. Here, we report that treatment with the copper complexes resulted in fragmented nuclei suggestive of apoptotic cell death, which was confirmed by increased annexin V binding and caspase-3/7 activity. The copper complexes caused a loss of mitochondrial membrane potential and increased caspase-9 activity. The absence of caspase-8 activity indicated activation of the intrinsic pathway. Proteomic analysis revealed that copper-imidazo[1,2-a]pyridines decreased the expression of phosphorylated forms of p53 [phospho-p53(S15), phospho-p53(S46) and phospho-p53(S392)]. The expression of inhibitor of apoptosis proteins, XIAP, cIAP1, livin, and the antiapoptotic proteins, Bcl-2 and Bcl-x, was decreased. HO/HMOX/HSP32, expression was notably increased, which suggested the accumulation of reactive oxygen species. Increased expression of TRAIL-R2/DR5 death receptor indicated the possible dual activation of both the extrinsic and intrinsic apoptotic pathways; however, caspase-8 activation could not be demonstrated. In conclusion, the copper-imidazo[1,2-a]pyridines were effective inducers of apoptotic cell death at low micromolar concentrations and changed the expression levels of proteins important for cell survival and cell death. These copper complexes may be useful tools to better understand the complexity of signalling networks in cancer cell death in response to cell stress.

Matrix metalloproteinase 9 induces keratinocyte apoptosis through FasL/Fas pathway in diabetic wound Abstract Apoptosis is a mechanism to remove unwanted cells in the tissue. In diabetic wound, which is characterized by delayed healing process, excessive apoptosis is documented and plays a crucial role. Matrix metalloproteinase 9 (MMP9), which is elevated in non-healed diabetic wound, is necessary for healing process but its abnormality resulted in a delayed healing. The classical function of MMP9 is the degradation of extracellular matrix (ECM). However, there is some literature evidence that MMP9 triggers cell apoptosis. Whether the excessive MMP9 contributes to epidermis cell apoptosis in delayed healing diabetic wound and the underlying mechanisms is not clear. In this study, we aimed to explore whether MMP9 induced keratinocyte apoptosis and investigate the plausible mechanisms. Our in vitro study showed that advanced glycation end products (AGEs) induced keratinocyte apoptosis and enhanced MMP9 level. Besides, MMP9, both intra-cellular expressions and extra-cellular supplement, promoted cell apoptosis. Further, MMP9 resulted in an increased expression of FasL, other than Fas and p53. These findings identified a novel effect that MMP9 exerted in delayed diabetic wound healing, owing to a pro-apoptotic effect on keratinocyte, which was mediated by an increase of FasL expression. This study increases understanding of elevated MMP9 which is involved in diabetic wound repair and offers some insights into novel future therapies.

Berberine mitigates IL-21/IL-21R mediated autophagic influx in fibroblast-like synoviocytes and regulates Th17/Treg imbalance in rheumatoid arthritis Abstract In our previous study, we explored the therapeutic effect of berberine (BBR) against IL-21/IL-21R mediated inflammatory proliferation of adjuvant-induced arthritic fibroblast-like synoviocytes (AA-FLS) through the PI3K/Akt pathway. The current study was designed to explore the therapeutic potential of BBR (15–45 µM) against IL-21/IL-21R mediated autophagy in AA-FLS mediated through PI3K/Akt signaling and Th17/Treg imbalance. Upon IL-21 stimulation, AA-FLS expressed elevated levels of autophagy-related 5 (Atg5), Beclin-1 and LC3-phosphatidylethanolamine conjugate 3-II (LC3-II) through the utilization of p62 and inhibition of C/EBP homologous protein (CHOP). BBR (15–45 µM) inhibited autophagy in AA-FLS cells mediated through PI3K/Akt signaling via suppressing autophagic elements, p62 sequestration and induction of CHOP in a dose-dependent manner. Moreover, IL-21 promoted the uncontrolled proliferation of AA-FLS through induction of B cell lymphoma-2 (Bcl-2) and diminished expression of Bcl-2 associated X protein (BAX) via PI3K/Akt signaling. BBR inhibited the proliferation of AA-FLS via promoting apoptosis through increased expression of BAX and diminished Bcl-2 transcription factor levels. Furthermore, T cells stimulated with IL-21 induced CD4+ CD196+ Th17 cells proliferation through RORγt activation mediated in a PI3K/Akt dependent manner. BBR inhibited the proliferation of Th17 cells through downregulation of RORγt in a concentration-dependent manner. BBR also promoted the differentiation of CD4+ CD25+ Treg cells through induction of forkhead box P3 (Foxp3) activation via aryl hydrocarbon receptor (AhR) and upregulation of cytochrome P450 family 1, subfamily A, polypeptide 1 (CYP1A1). Collectively, we conclude that BBR might attenuate AA-FLS proliferation through inhibition of IL-21/IL-21R dependent autophagy and regulates the Th17/Treg imbalance in RA.

CNOT3 contributes to cisplatin resistance in lung cancer through inhibiting RIPK3 expression Abstract Chemotherapeutic resistance always results in poor clinical outcomes of cancer patients and its intricate mechanisms are large obstacles in overcoming drug resistance. CCR4-NOT transcription complex subunit 3 (CNOT3), a post-translational regulator, is suggested to be involved in cancer development and progression. However, its role in chemotherapeutic resistance is not well understood. In this study, after screening the CNOT3mRNA in a cancer microarray database called Oncomine and examining the expression levels of CNOT3 mRNA in normal tissues and lung cancer tissues, we found that CNOT3 was up-regulated in lung cancer tissues. Besides, its high-expression was associated with poor prognosis of lung cancer patients. We also found higher expression level of CNOT3 and lower expression level of receptor-interacting protein kinase 3 (RIPK3) in cisplatin-resistant A549 (A549/DDP) cells, and knocking down CNOT3 expression could sensitize A549/DDP cells to cisplatin-induced apoptosis. We demonstrated that CNOT3 depletion up-regulated the expression level of RIPK3 and the enhanced apoptosis was mediated by the elevated RIPK3 to further trigger Caspase 8 activation. Taken together, our results reveal a role of CNOT3 in cisplatin resistance of lung cancer and provide a potential target for lung cancer therapy.

Recombinant human lactoferrin induces apoptosis, disruption of F-actin structure and cell cycle arrest with selective cytotoxicity on human triple negative breast cancer cells Abstract Breast cancer is the most frequently diagnosed cancer among women worldwide. Here, recombinant human lactoferrin (rhLf) expressed in Pichia pastoris was tested for its potential cytotoxic activity on a panel of six human breast cancer cell lines. The rhLf cytotoxic effect was determined via a live-cell HTS imaging assay. Also, confocal microscopy and flow cytometry protocols were employed to investigate the rhLf mode of action. The rhLf revealed an effective CC50 of 91.4 and 109.46 µg/ml on non-metastatic and metastatic MDA-MB-231 cells, with favorable selective cytotoxicity index values, 11.68 and 13.99, respectively. Moreover, rhLf displayed satisfactory SCI values on four additional cell lines, MDA-MB-468, HCC70, MCF-7 and T-47D (1.55–3.34). Also, rhLf provoked plasma membrane blebbing, chromatin condensation and cell shrinkage in MDA-MB-231 cells, being all three apoptosis-related morphological changes. Also, rhLf was able to shrink the microfilaments, forming a punctuated cytoplasmic pattern in both the MDA-MB-231 and Hs-27 cells, as visualized in confocal photomicrographs. Moreover, performing flow cytometric analysis, rhLf provoked significant phosphatidylserine externalization, cell cycle arrest in the S phase and apoptosis-induced DNA fragmentation in MDA-MB-231 cells. Hence, rhLf possesses selective cytotoxicity on breast cancer cells. Also, rhLf caused apoptosis-associated morphologic changes, disruption of F-actin cytoskeleton organization, phosphatidylserine externalization, DNA fragmentation, and arrest of the cell cycle progression on triple-negative breast cancer MDA-MB-231 cells. Overall results suggest that rhLf is using the apoptosis pathway as its mechanism to inflict cell death. Findings warranty further evaluation of rhLf as a potential anti-breast cancer drug option.

Endothelin-1-mediated cerebral ischemia in mice: early cellular events and the role of caspase-3 Abstract Over the past 30 years a number of animal models of cerebral ischemic injury have been developed. Middle cerebral artery occlusion (MCAO) in particular reproduces both ischemic and reperfusion elements and is widely utilized as a model of ischemic stroke in rodents. However substantial variability exists in this model even in clonal inbred mice due to stochastic elements of the cerebral vasculature. Models such as MCAO thus exhibit significant irreducible variabilities with respect to their zone of injury as well as inducing a sizable volume of injury to the cerebrum with damage to sub-cortical structures, conditions not typically seen for the majority of human clinical strokes. An alternative model utilizes endothelin-1 application focally to cerebral vasculature, resulting in an ischemic reperfusion injury which more closely mimics that seen in human clinical stroke. In order to further define this model we demonstrate that intra-cortical administration of ET-1 results in a highly reproducible pattern of tissue injury which is limited to the cerebral cortex, characterizing the early cellular and molecular events which occur during the first 24 h post-injury. In addition we demonstrate that caspase-3 is both necessary and sufficient to regulate a majority of cortical cell death observed during this period. The enhanced survival effects seen upon genetic deletion of caspase-3 appear to arise as a result of direct modification of cell autonomous PCD signaling as opposed to secondary effectors such as granulocyte infiltration or microglia activation. Taken together these findings detail the early mechanistic features regulating endothelin-1-mediated ischemic injury.

Cellular responses of BRCA1 -defective HCC1937 breast cancer cells induced by the antimetastasis ruthenium(II) arene compound RAPTA-T Abstract An organometallic ruthenium(II) arene compound, Ru(η6-toluene)(PTA)Cl2 (PTA = 1,3,5-triaza-7-phosphaadamantane), termed RAPTA-T, exerts promising antimetastatic properties. In this study, the effects of RAPTA-T on BRCA1-defective HCC1937 breast cancer cells have been investigated, and compared to its effects on BRCA1-competent MCF-7 breast cancer cells. RAPTA-T showed a very low cytotoxicity against both tested cells. Ruthenium is found mostly in the cytoplasmic compartment of both cells. Flow cytometric analysis reveals that the compound arrests the growth of both cells by triggering the G2/M phase that led to the induction of apoptosis. At equimolar concentrations, RAPTA-T causes much more cellular BRCA1 damage in HCC1937 than in MCF-7 cells, suppressing the expression of BRCA1 mRNA in both cell lines with the subsequent down-regulation of the BRCA1 protein. Interestingly, RAPTA-T exhibits an approximately fivefold greater ability to suppress the expression of the BRCA1 protein in HCC1937 than in MCF-7 cells. These data provide insights into the molecular mechanisms by which RAPTA-T exerts its effects on BRCA1-associated breast cancer cells.

Alexandros Sfakianakis
Anapafseos 5 . Agios Nikolaos
Crete.Greece.72100
2841026182

6948891480

alsfakia@gmail.com