To assess cell death pathways in response to magnetic hyperthermia.
Whether responses of cells to extracellular environments affect the induction of apoptotic cell death is poorly understood. The current study aimed to unravel the different effects of culture media employed in vitro as extracellular environments on the susceptibility of cells to apoptosis. We found that apoptosis is stimulated to the higher levels by culturing human HeLa cells in Opti-MEM with unknown components, a medium that is specifically used for transfections, than by culturing cells in Dulbecco’s modified Eagle’s medium, a medium that is generally used for maintenance of cells. We showed that apoptosis is suppressed partially by culturing cells in heat-treated Opti-MEM, implicating a heat-sensitive component(s) in stimulating the apoptotic response of cells. Thus, different extracellular environments may contribute to different responses of cells to apoptosis, and this should be considered to evaluate the incidences of apoptotic cell death and could be applied to develop an efficient treatment for curing diseases such as cancer.
Apoptosis has been implicated in compensatory proliferation signaling (CPS), whereby dying cells induce proliferation in neighboring cells as a means to restore homeostasis. The nature of signaling between apoptotic cells and their neighboring cells remains largely unknown. Here we show that a fraction of apoptotic cells produce and release CrkI-containing microvesicles (distinct from exosomes and apoptotic bodies), which induce proliferation in neighboring cells upon contact. We provide visual evidence of CPS by videomicroscopy. We show that purified vesicles in vitro and in vivo are sufficient to stimulate proliferation in other cells. Our data demonstrate that CrkI inactivation by ExoT bacterial toxin or by mutagenesis blocks vesicle formation in apoptotic cells and inhibits CPS, thus uncoupling apoptosis from CPS. We further show that c-Jun amino-terminal kinase (JNK) plays a pivotal role in mediating vesicle-induced CPS in recipient cells. CPS could have important ramifications in diseases that involve apoptotic cell death.
Synaptic activity increases the resistance of neurons to diverse apoptotic insults; however, the underlying mechanisms remain less well understood. Zinc promotes cell survival under varied conditions, but the role of synaptically released zinc in the activity-dependent anti-apoptotic effect is unknown. Using cultured hippocampal slices and primary neurons we show that a typical apoptosis inducer-staurosporine (STP) was able to cause concentration-dependent apoptotic cell death in brain slices; Enhanced synaptic activity by bicuculline (Bic)/4-Aminopyridine (AP) treatment effectively prevented neurons from STP-induced cell apoptosis, as indicated by increased cell survival and suppressed caspase-3 activity. Application of Ca-EDTA, a cell membrane-impermeable zinc chelator which can efficiently capture the synaptically released zinc, completely blocked the neuronal activity-dependent anti-apoptotic effect. Same results were also observed in cultured primary hippocampal neurons. Therefore, our results indicate that synaptic activity improves neuronal resistance to apoptosis via synaptically released zinc.
We demonstrate that cortical interneurons derived from ventral eminences, including the caudal ganglionic eminence, undergo programmed cell death. Moreover, with the exception of VIP interneurons, this occurs in a manner that is activity-dependent. In addition, we demonstrate that, within interneurons, Calcineurin, a calcium-dependent protein phosphatase, plays a critical role in sequentially linking activity to maturation (E15-P5) and survival (P5-P20). Specifically, embryonic inactivation of Calcineurin results in a failure of interneurons to morphologically mature and prevents them from undergoing apoptosis. By contrast, early postnatal inactivation of Calcineurin increases apoptosis. We conclude that Calcineurin serves a dual role of promoting first the differentiation of interneurons and, subsequently, their survival.
Cerebral ischemia causes severe brain injury and results in selective neuronal death through programmed cell death mechanisms, including apoptosis and autophagy. Minimizing neuronal injury has been considered a hot topic among clinicians. The present study elucidated the effect of thymosin β4 (Tβ4) on neuronal death induced by cerebral ischemia/reperfusion in PC12 cells that were subjected to oxygen‑glucose deprivation and reoxygenation (OGD/R). The survival, apoptotic and autophagy rates of PC12 cells were investigated. Tβ4 pre‑conditioning prior to OGD/R was performed to evaluate PC12‑cell viability and the protective mechanisms of Tβ4. Tβ4 significantly increased cell survival after OGD/R. Tβ4 inhibited the release of lactate dehydrogenase, downregulated malondialdehyde and upregulated the activities of glutathione peroxidase and superoxide dismutase. In addition, Tβ4 attenuated OGD/R‑associated decreases in the expression of P62 and the anti‑apoptotic protein B‑cell lymphoma‑2, as well as the upregulation of autophagy mediators, including autophagy‑related protein‑5 and the ratio of microtubule‑associated protein 1 light chain 3 (LC3) II vs. LC3 I. These results suggested that Tβ4 effectively inhibits cell apoptosis and autophagy induced by OGD/R. To the best of our knowledge, the present study was the first to report on the antioxidant, anti‑apoptotic and anti‑autophagic effects of Tβ4 in neuronal‑like PC12 cells. These results suggested that Tβ4 may be explored as a potential treatment for cerebral ischemia.
Neutrophils play a key role in host defenses and have recently been implicated in the pathogenesis of autoimmune diseases by various mechanisms, including formation of neutrophil extracellular traps through a recently described distinct form of programmed cell death called NETosis. Techniques to assess and quantitate NETosis in an unbiased, reproducible, and efficient way are lacking, considerably limiting the advancement of research in this field. We optimized and validated, a new method to automatically quantify the percentage of neutrophils undergoing NETosis in real time using the IncuCyte ZOOM imaging platform and the membrane-permeability properties of two DNA dyes. Neutrophils undergoing NETosis induced by various physiological stimuli showed distinct changes, with a loss of multilobulated nuclei, as well as nuclear decondensation followed by membrane compromise, and were accurately counted by applying filters based on fluorescence intensity and nuclear size. Findings were confirmed and validated with the established method of immunofluorescence microscopy. The platform was also validated to rapidly assess and quantify the dose-dependent effect of inhibitors of NETosis. In addition, this method was able to distinguish among neutrophils undergoing NETosis, apoptosis, or necrosis based on distinct changes in nuclear morphology and membrane integrity. The IncuCyte ZOOM platform is a novel real-time assay that quantifies NETosis in a rapid, automated, and reproducible way, significantly optimizing the study of neutrophils. This platform is a powerful tool to assess neutrophil physiology and NETosis, as well as to swiftly develop and test novel neutrophil targets.
Based on the model considering the influences of mitochondria, a further theoretical study on the dynamic behaviors of calcium signals is made. First of all, the reason for the generation and disappearance of calcium oscillations is verified in theory. Second, an analysis on the model considering the influences of mitochondria and the model neglecting the influences of mitochondria is carried out. Third, β (representing calcium leak) is introduced and it can be found that with the increase of β, the Hopf bifurcation points of system move towards the decreasing direction of μ (representing stimulus intensity) and calcium oscillations region gradually decreases. Forth, the study on τh (representing relaxation time) indicates that with the increase of τh, the second Hopf bifurcation point of system moves towards the increasing direction of μ and calcium oscillations region gradually increases. Under certain stimulus intensity, when relaxation time increases, calcium oscillation peak rises rapidly and the period increases obviously. Fifth, two-parameter bifurcation diagram of Vm1 (representing mitochondria activity) and μ contains three regions: stable region, oscillation region and unstable region. When the parameters fall in the unstable region Ca2+ gather towards mitochondria and further lead to cell apoptosis. With the increase of Vm1, calcium oscillations region shrinks gradually. Vm1 and μ both play a key role in regulating cell apoptosis. Only when Vm1 and μ are high enough can cells enter into programmed cell death and the higher Vm1 is, the lower the stimulus intensity required by cell apoptosis is.
The aims of this study were to identify a robust apoptosis marker suitable for both quantification and back-to-back analyses of programmed cell death and to define specific upstream targets for apoptosis in corneal cells.
The expression of microRNA‑206 (miR‑206) is aberrantly induced in steroid‑induced avascular necrosis of femoral head (SANFH). Therefore, investigating the function of miR‑206 in SANFH and uncovering the functional mechanism associated with the condition will promote the understanding and treatment of the disease. The purpose of the present study was to investigate the pro‑osteoclasteogenic effect of miR‑206 that occurs through regulation of programmed cell death 4 (PDCD4). The expression of miR‑206 and PDCD4 was analyzed in the clinical SANFH specimens. The level of miR‑206 and PDCD4 was regulated in human osteoblast lineage hFOB1.19 and the effect of different treatments on cell viability, proliferation, apoptosis and differentiation potential of osteoblasts were analyzed with a Cell Counting kit‑8, 5‑ethynyl‑2'‑deoxyuridine staining, flow cytometry and Hoechst staining. The expression of miR‑206 was upregulated while PDCD4 was downregulated in the SANFH specimens. Induced expression of miR‑206 decreased cell viability and proliferation, while apoptosis was induced. At the molecular level, overexpression of miR‑206 inhibited the expression of PDCD4, alkaline phosphatase (ALP) and B‑cell lymphoma 2 (Bcl‑2), and increased the expression of apoptosis regulator Bcl2‑X‑associated protein (Bax). Inhibiting the expression of miR‑206 increased cell viability and proliferation but had no effect on cell apoptosis, as detected by flow cytometry and Hoechst staining. However, at the molecular level, inhibiting the expression of miR‑206 induced expression of PDCD4, ALP and Bcl‑2, while it decreased the expression of Bax. Additionally, knockdown of PDCD4 blocked the effect of miR‑206 inhibition on hFOB1.19 cells, representing a PDCD4‑dependent manner of miR‑206 in inducing apoptosis of osteoblasts. Therefore, miR‑206 promoted the onset of SANFH by inducing apoptosis and suppressed the proliferation of osteoblasts, which was dependent on the inhibition of PDCD4.