Synergistic effect of cAMP and palmitate in promoting altered mitochondrial function and cell death in HepG2 cells

Saturated free fatty acids (FFAs), e.g. palmitate, have long been shown to induce toxicity and cell death in various types of cells. In this study, we demonstrate that cAMP synergistically amplifies the effect of palmitate on the induction of cell death in human hepatocellular carcinoma cell line, HepG2 cells. Elevation of cAMP level in palmitate-treated cells led to enhanced mitochondrial fragmentation, mitochondrial reactive oxygen species (ROS) generation and mitochondrial biogenesis. Mitochondrial fragmentation precedes mitochondrial ROS generation and mitochondrial biogenesis, and may contribute to mitochondrial ROS overproduction and subsequent mitochondrial biogenesis. Fragmentation of mitochondria also facilitated the release of cytotoxic mitochondrial proteins, such as Smac, from the mitochondria and subsequent activation of caspases. However, cell death induced by palmitate and cAMP was caspase-independent and mainly necrotic.     

Neuroprotective strategies targeting apoptotic and necrotic cell death for stroke

It has been a major challenge to develop effective therapeutics for stroke, a leading cause of death and serious debilitation. Intensive research in the past 15 years have implicated many regulators and the related mechanisms by which neuronal cell death is regulated. It is now clear that even a brief ischemic stroke may trigger complex cellular events that lead to both apoptotic and necrotic neuronal cell death in a progressive manner. Although efforts at developing specific chemical inhibitors for validated targets have been successful for in vitro enzymatic assays, the development of some of such inhibitors into human therapy has been often hindered by their in vivo bioavailability profile. Considerations for the ability to chemically target a cellular mechanism in manner compatible with disease targets in vivo might be emphasized early in the development process by putting a priority on identifying key targets that can be effectively targeted chemically. Thorough interrogation of cellular pathways by saturation chemical genetics may provide a novel strategy to identify multiple key molecular entities that can be targeted chemically in order to select a target suitable for the treatment of intended human diseases such as stroke.     

Proteolytic mechanisms in necrotic cell death and neurodegeneration

Programmed neuronal cell death is required during development to achieve the accurate wiring of the nervous system. However, genetic or accidental factors can lead to the premature, non-programmed death of neurons during adult life. Inappropriate death of cells in the nervous system is the cause of multiple neurodegenerative disorders. Pathological neuronal death can occur by apoptosis, by necrosis or by a combination of both. Necrotic cell death underlies the pathology of devastating neurological diseases such as neurodegenerative disorders, stroke or trauma. However, little is known about the molecular mechanisms that bring about necrotic cell death. Proteases play crucial roles in neuron degeneration by exerting both regulatory and catabolic functions. Elevated intracellular calcium is the most ubiquitous feature of neuronal death with the concomitant activation of cysteine calcium-dependent proteases, calpains. Calpains and lysosomal, catabolic aspartyl proteases, play key roles in the necrotic death of neurons. In this review, we survey the recent literature on the role of cysteine and aspartyl proteases in necrosis and neurodegeneration, aiming to delineate common proteolytic mechanisms mediating cellular destruction.     

Apoptosis-suppressing and autophagy-promoting effects of calpain on oridonin-induced L929 cell death

Calpain, calcium-dependent cysteine protease, is reported here to impose the crucial influence on oridonin-induced L929 cell apoptosis and autophagy. We found that inhibition of calpain increased oridonin-induced Bax activation, cytochrome c release and PARP cleavage, indicating that calpain plays an anti-apoptotic role in oridonin-induced L929 cell apoptosis. To explore this potential anti-apoptotic mechanism, we inhibited calpain and proteasome activity in oridonin-induced L929 cell apoptosis, and discovered that the inducible IκBα proteolysis was partially blocked by the inhibition of either calpain or proteasome, but completely blocked by the inhibition of both. It demonstrated that calpain and proteasome were two distinct pathways participating in IκBα degradation. To further study the role of calpain in oridonin-induced L929 cell autophagy, we discovered that calpain inhibitor decreased oridonin-induced autophagy, as well as Beclin 1 activation and the conversion from LC3-I to LC3-II. Moreover, Inhibition of autophagy by 3-MA increased oridonin-induced apoptosis. In conclusion, besides suppressing apoptosis, calpain promotes autophagy in oridonin-induced L929 cell death, and inhibition of autophagy might contribute to up-regulation of apoptosis.     

Alterations of extracellular calcium elicit selective modes of cell death and protease activation in SH-SY5Y human neuroblastoma cells

The role of intracellular Ca2+ homeostasis in mechanisms of neuronal cell death and cysteine protease activation was investigated in SH-SY5Y human neuroblastoma cells. Cells were incubated in 2 mM EGTA to lower intracellular Ca2+ or 5 mM CaCl2 to raise it. Cell death and activation of calpain and caspase-3 were measured. Both EGTA and excess CaCl2 elicited cell death. EGTA induced DNA laddering and an increase in caspase-3-like, but not calpain, activity. Pan-caspase inhibitors protected against EGTA-, but not CaCl2-, induced cell death. Conversely, excess Ca2+ elicited necrosis and activated calpain but not caspase-3. Calpain inhibitors did not preserve cell viability. Ca2+ was the death-mediating factor, because restoration of extracellular Ca2+ protected against cell death induced by EGTA and blockade of Ca2+ channels by Ni2+ protected against that induced by high Ca2+. We conclude that the EGTA treatment lowered intracellular Ca2+ and elicited caspase-3-like protease activity, which led to apoptosis. Conversely, excess extracellular Ca2+ entered Ca2+ channels and increased intracellular Ca2+ leading to calpain activation and necrosis. The mode of cell death and protease activation in response to changing Ca2+ were selective and mutually exclusive, demonstrating that these are useful models to individually investigate apoptosis and necrosis.     

A natural phenylpropionate derivative from Mirabilis himalaica inhibits cell proliferation and induces apoptosis in HepG2 cells

Bioactivity-guided study led to the isolation of a natural phenylpropionate derivative, (E)-3-(4-hydroxy-2-methoxyphenyl)-propenoic acid 4-hydroxy-3-methoxyphenyl ester from the roots of Mirabilis himalaica. Cellular analysis showed that compound 1 specifically inhibited the cancer cell growth through the S phase arrest. Mechanistically, compound 1 was able to induce the apoptosis in HepG2 cells through mitochondrial apoptosis pathway in which Bcl-2 and p53 were required. Interestingly, the cellular phenotype of compound 1 were shown specifically in cancer cells originated from hepatocellular carcinoma (HepG2) while compromised influence by compound 1 were detected within the normal human liver cells (L-02). Consistently, the in vivo inhibitory effects of compound 1 on tumor growth were validated by the in xenograft administrated with HepG2 cells. Our results provided a novel compound which might serve as a promising candidate and shed light on the therapy of the hepatocellular carcinoma.     

Modeling suppression of cell death by Bcl-2 over-expression in myeloma NS0 6A1 cells

A novel population-balance model was employed to evaluate the suppression of cell death in myeloma NS0 6A1 cells metabolically engineered to over-express the apoptotic suppressor Bcl-2. The model is robust in its ability to simulate cell population dynamics in batch suspension culture and in response to thymidine-induced growth inhibition: 89% of simulated cell concentrations are within two standard deviations of experimental data. Kinetic rate constants in model equations suggest that Bcl-2 over-expression extends culture longevity from 6 days to at least 15 days by suppressing the specific rate of early apoptotic cell formation by more than 6-fold and necrotic cell formation by at least 3-fold, despite nearly a 3-fold decrease in initial cell growth rate and no significant change in the specific rate of late apoptotic cell formation. This computational analysis supports a mechanism in which Bcl-2 is a common mediator of early apoptotic and necrotic events occurring at rates that are dependent on cellular factors accumulating over time. The model has current application to the rational design of cell cultures through metabolic engineering for the industrial production of biopharmaceuticals.     

Sal B对肝癌细胞株HepG2的促凋亡作用

目的揭示丹酚酸B(Salvianolic Acid B,Sal B)对肝癌细胞株HepG2的杀伤作用。方法用不同浓度的丹酚酸B处理HepG2细胞,37℃培养24h。用RT-PCR检测促凋亡基因Bax的转录水平,并用流式细胞术检测细胞凋亡的水平。结果①100μmol/L、50μmol/L、25μmol/L等浓度的Sal B处理都能使HepG2细胞促凋亡基因bax的转录水平升高,其中100μmol/L处理组最为明显。②不同浓度的Sal B处理都能使HepG2细胞发生凋亡,其中100μmol/L处理组最为明显。结论 Sal B有促进HepG2细胞凋亡的作用。     

Transglutaminase 2 promotes both caspase-dependent and caspase-independent apoptotic cell death via the calpain/Bax protein signaling pathway

Transglutaminase 2 (TG2) is a versatile protein that is implicated in significant biological processes, including cell death and degenerative diseases. A possible role of TG2 in the apoptotic death of cancer cells induced by photodynamic therapy (PDT) was suggested recently; however, the mechanism by which TG2 regulates apoptotic responses to PDT remains to be elucidated. In this study, we investigated the key signaling pathways stimulated during apoptotic cell death following PDT and whether inhibition of TG2 activation using pharmacological approaches and siRNAs affects the signaling pathways. PDT caused the release of both cytochrome c and apoptosis-inducing factor (AIF) by damaging mitochondria, which resulted in caspase-dependent and caspase-independent apoptotic cell death, respectively. Released AIF translocated to the nucleus and, synergistically with the caspase-dependent pathway, led to apoptotic cell death. Both the caspase cascade and the activation of AIF following PDT were mediated by TG2 activation. In addition, PDT-activated calpain was responsible for the sequential events of Bax translocation, the collapse of ΔΨ(m), caspase-3 activation, and AIF translocation, all of which were provoked by TG2 activation. Together, these results demonstrate that PDT with a chlorin-based photosensitizer targets TG2 by activating calpain-induced Bax translocation, which induces apoptotic cell death through both caspase-dependent and AIF-mediated pathways. Moreover, these results indicate that TG2 may be a possible therapeutic target for PDT treatment of cancer.     

Molecular mechanisms of echinocystic acid-induced apoptosis in HepG2 cells

Echinocystic acid (EA), a natural triterpone enriched in various herbs, has been showed to have cytotoxic activity in some cancer cells, and is used for medicinal purpose in many Asian countries. In the present study, we found that EA could induce apoptosis in human HepG2 cells, as characterized by DNA fragmentation, activation of caspase-3, -8, and -9, and PARP cleavage. The efficacious induction of apoptosis was observed at 45 microM for 24 h. Molecular data showed that EA induced the truncation of Bid protein and reduction of Bcl-2 protein. EA also caused the loss of mitochondrial membrane potential (DeltaPsi(m)) and cytochrome c release from mitochondria to cytosol. Moreover, EA could activate c-Jun NH(2)-terminal kinase (JNK) and p38 kinase, and JNK-specific inhibitor SP600125 and p38 kinase-specific inhibitor SB200235 could block serial molecular events of EA-induced apoptosis such as Bid truncation, Bcl-2 reduction, cytochrome c release, caspase activation, and DNA fragmentation in HepG2 cells. These findings indicate that JNK- and p38 kinase-mediated mitochondrial pathways might be involved in EA-induced apoptosis and enhance our understanding of the anticancer function of EA in herbal medicine.     

Serglycin proteoglycan promotes apoptotic versus necrotic cell death in mast cells

The mechanisms that govern whether a cell dies by apoptosis or necrosis are not fully understood. Here we show that serglycin, a secretory granule proteoglycan of hematopoietic cells, can have a major impact on this decision. Wild type and serglycin(-/-) mast cells were equally sensitive to a range of cell death-inducing regimens. However, whereas wild type mast cells underwent apoptotic cell death, serglycin(-/-) cells died predominantly by necrosis. Investigations of the underlying mechanism revealed that cell death was accompanied by leakage of secretory granule compounds into the cytosol and that the necrotic phenotype of serglycin(-/-) mast cells was linked to defective degradation of poly(ADP-ribose) polymerase-1. Cells lacking mouse mast cell protease 6, a major serglycin-associated protease, exhibited similar defects in apoptosis as observed in serglycin(-/-) cells, indicating that the pro-apoptotic function of serglycin is due to downstream effects of proteases that are complex-bound to serglycin. Together, these findings implicate serglycin in promoting apoptotic versus necrotic cell death.     

Involvement of catalase in the apoptotic mechanism induced by apigenin in HepG2 human hepatoma cells

Apigenin has been reported to inhibit proliferation of cancer cells; however, the mechanism underlying its action is not completely understood. Here, we evaluated the effects of apigenin on the levels of expression and activity of antioxidant enzymes, and the involvement of ROS in the mechanism of cell death induced by apigenin in HepG2 human hepatoma cells. Upon treatment with apigenin, HepG2 cells displayed a reduction in cell viability in a dose- and time-dependent manner, and some morphological changes. In addition, apigenin treatment induced ROS generation and significantly decreased the mRNA levels and activity of catalase and levels of intracellular GSH. On the other hand, apigenin treatment did not alter the expression or activity levels of other antioxidant enzymes. Addition of exogenous catalase significantly reduced the effects of apigenin on HepG2 cell death. We also demonstrated that HepG2 cells are more sensitive to apigenin-mediated cell death than are primary cultures of mouse hepatocytes, suggesting a differential toxic effect of this agent in tumor cells. Our results suggest that apigenin-induced apoptosis in HepG2 cells may be mediated by a H₂O₂-dependent pathway via reduction of the antioxidant defenses.     

紫草素对人肝癌HepG2细胞增殖的抑制及诱导凋亡作用

目的:观察紫草素抑制人肝癌HepG2细胞增殖及凋亡诱导的作用。方法:用不同浓度的紫草素处理HepG2细胞,MTT检测紫草素对HepG2细胞生长增殖的抑制作用;比色法测定Caspase-3酶活性;Western blot法检测磷酸化Akt蛋白(pAkt)的表达。结果:紫草素能够抑制人肝癌HepG2细胞的增殖,并呈浓度、时间依赖性,紫草素与HepG2细胞作用24小时后Caspase-3酶活性显著增强,显示紫草素诱导的调亡作用随时间的延长而增加;同时,紫草素处理HepG2细胞后,随着药物浓度的增加,磷酸化Akt蛋白表达下降。结论:紫草素可抑制人肝癌细胞HepG2的增殖,诱导HepG2细胞凋亡,凋亡机制可能与紫草素抑制PI3K/Akt信号途径有关。     

Role of undecan-2-one on ethanol-induced apoptosis in HepG2 cells

Based on the reduced expression of ethanol-oxidizing enzymes in human hepatocellular carcinoma (HepG2) cells, we analyzed the role of nonoxidative metabolites in ethanol-induced apoptosis in HepG2 cells. For this purpose, an analysis of volatile metabolites of ethanol using ion-mobility spectrometry and gas chromatography–mass spectrometry was performed. HepG2 cells exposed to 1 mmol/L ethanol exhibited significant synthesis of undecan-2-one compared to untreated cells. Undecan-2-one is a fatty acid ethyl ester metabolite synthesized through a nonoxidative pathway. Undecan-2-one had a dose-dependent cytotoxic effect on HepG2 cells as shown by release of lactate dehydrogenase (LDH). The most notable finding of this study was the potentiation of ethanol-induced apoptosis demonstrated by an increased apoptotic rate induced by undecan-2-one in ethanol-treated HepG2 cells. The data presented in this study contribute to the better understanding of the molecular mechanisms of ethanol exposure at low concentration in HepG2 cells, a human hepatocellular carcinoma-derived cell line.     

The algal metabolite yessotoxin affects heterogeneous nuclear ribonucleoproteins in HepG2 cells

The dinoflagellate metabolite yessotoxin (YTX) is produced by several species of algae and accumulates in marine food chains, leading to concerns about possible affects on aquaculture industries and human health. In mice used for toxicity testing, YTX is lethal by the intraperitoneal route, but is considerably less toxic when orally administered. The mode of action of YTX and its potential effect on humans is unclear and we therefore conducted the first proteomic analysis of the effects of this compound. We used 2‐DE to examine protein changes in HepG2 cell cultures exposed to 1.4 μM YTX for 3, 12.5, 18 and 24 h. After selecting proteins that changed more than three‐fold after YTX exposure, 55 spots were deemed significantly affected by the toxin (p<0.05). Major groups of affected proteins include members from the heterogeneous nuclear ribonucleoprotein (hnRNP), lamin, cathepsin and heat shock protein families that often are associated with apoptosis. We therefore confirmed apoptosis using Annexin‐V‐FLUOS staining of phosphatidylserine exposed at the surface of apoptotic cells. Ingenuity pathways analysis also indicated effects on pathways involved in protein processing, cell cycling and cell death.     

The apoptotic effect of sarsasapogenin from Anemarrhena asphodeloides on HepG2 human hepatoma cells

Sarsasapogenin, a kind of mainly effective components of Anemarrhena asphodeloides Bunge (Liliaceae) has the effects of being anti-diabetes and improving memory. However, there are few reports focusing on its anti-tumor effects. In this study, the sarsasapogenin was extracted from rhizomes of A. asphodeloides Bunge and applied to inhibit HepG2 human hepatoma cells. MTT assay showed that sarsasapogenin induced a distinct dose- and time-dependent diminution of cell viability with IC(50) of 42.4+/-1.0microg/ml for 48h. Furthermore, sarsasapogenin-induced apoptosis of HepG2 cells was verified by Hoechst 33258 staining, electron microscopy, DNA fragmentation and PI staining. Flow cytometry analysis showed that sarsasapogenin-induced cell apoptosis was through arrest of cell cycle in G(2)/M phase. Hence we proposed that sarsasapogenin could be used as an anti-liver cancer drug for future studies.     

Distinction of apoptotic and necrotic cell death by in situ labelling of fragmented DNA

The occurrence and spatial distribution of intracellular DNA fragmentation was investigated by in situ 3 end labelling of DNA breaks in K562 cells treated in such a way to cause either apoptotic or necrotic cell death. The localisation of DNA breaks was examined by confocal laser microscopy and compared with the electron-microscopic appearance of the cells. In addition, the number of cells with fragmented DNA was counted and compared with the number of dead cells, as determined by the nigrosin dye exclusion test. Apoptosis was induced by cultivation of the cells in the presence of actinomycin D. Cells undergoing apoptosis were characterised by massive intracellular DNA fragmentation that was highly ordered into successive steps. Cells in early stages of the apoptotic process had DNA breaks diffusely distributed in the entire nucleus, except the nucleolus, with crescent-like accumulations beyond the nuclear membrane. In the more advanced stages, the nucleus was transformed into many round bodies with intense labelling. Intracellular accumulations of fragmented DNA corresponded exactly to electron-dense chromatin seen in the electron microscope, whereas diffuse DNA breaks had no morphological correlate at the ultrastructural level. In necrosis induced by ionomycin, NaN₃, or rapid freezing combined with thawing, no DNA fragmentation occurred at the onset of cell death, but appeared 24 h later. This fragmentation was not characterised by a unique morphology, but represented the breakdown of the chromatin in the configuration remaining after cell death. Therefore, apoptosis is characterised by DNA fragmentation that proceeds in a regular orderly sequence at the beginning of cell death, and can be detected by in situ 3end labelling of DNA breaks.     

Major cell death pathways at a glance

Cell death is a crucial process during development, homeostasis and immune regulation of multicellular organisms, and its dysregulation is associated with numerous pathologies. Cell death is often induced upon pathogen infection as part of the defense mechanism, and pathogens have evolved strategies to modulate host cell death. In this review, we will discuss the molecular mechanisms and physiological relevance of four major types of programmed cell death, namely apoptosis, necrosis, autophagic cell death and pyroptosis.