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1.
The application of high pressure (HP) for food preservation requires insight into mechanisms of HP-mediated cell injury and death. The HP inactivation in model beer of Lactobacillus plantarum TMW1.460, a beer-spoiling organism, was investigated at pressures ranging from 200 to 600 MPa. Surviving cells were characterized by determination of (i) cell viability and sublethal injury, (ii) membrane permeability to the fluorescent dyes propidium iodide (PI) and ethidium bromide (EB), (iii) metabolic activity with tetrazolium salts, and (iv) the activity of HorA, an ATP binding cassette-type multidrug resistance transporter conferring resistance to hop compounds. HP inactivation curves exhibited a shoulder, an exponential inactivation phase, and pronounced tailing caused by a barotolerant fraction of the population, about 1 in 10(6) cells. During exponential inactivation, more than 99.99% of cells were sublethally injured; however, no sublethal injury was detected in the barotolerant fraction of the culture. Sublethally injured cells were metabolically active, and loss of metabolic activity corresponded to the decrease of cell viability. Membrane damage measured by PI uptake occurred later than cell death, indicating that dye exclusion may be used as a fail-safe method for preliminary characterization of HP inactivation. An increase of membrane permeability to EB and a reduction of HorA activity were observed prior to the loss of cell viability, indicating loss of hop resistance of pressurized cells. Even mild HP treatments thus abolished the ability of cells to survive under adverse conditions.  相似文献   

2.
The application of high pressure (HP) for food preservation requires insight into mechanisms of HP-mediated cell injury and death. The HP inactivation in model beer of Lactobacillus plantarum TMW1.460, a beer-spoiling organism, was investigated at pressures ranging from 200 to 600 MPa. Surviving cells were characterized by determination of (i) cell viability and sublethal injury, (ii) membrane permeability to the fluorescent dyes propidium iodide (PI) and ethidium bromide (EB), (iii) metabolic activity with tetrazolium salts, and (iv) the activity of HorA, an ATP binding cassette-type multidrug resistance transporter conferring resistance to hop compounds. HP inactivation curves exhibited a shoulder, an exponential inactivation phase, and pronounced tailing caused by a barotolerant fraction of the population, about 1 in 106 cells. During exponential inactivation, more than 99.99% of cells were sublethally injured; however, no sublethal injury was detected in the barotolerant fraction of the culture. Sublethally injured cells were metabolically active, and loss of metabolic activity corresponded to the decrease of cell viability. Membrane damage measured by PI uptake occurred later than cell death, indicating that dye exclusion may be used as a fail-safe method for preliminary characterization of HP inactivation. An increase of membrane permeability to EB and a reduction of HorA activity were observed prior to the loss of cell viability, indicating loss of hop resistance of pressurized cells. Even mild HP treatments thus abolished the ability of cells to survive under adverse conditions.  相似文献   

3.
The metabolism of purine- and pyrimidine nucleotides in pine pollen (Pinus mugo) grown in suspension cultures have been examined. In the ungerminated dehydrated pollen, the presence of ATP has been demonstrated. Incubation of the pollen in a germination medium leads to an exhaustion of the ATP pool, which is restored with the onset of oxygen uptake. By labelling pollen cultures with 32P-orthophosphate, it has been possible to quantitate the nucleotide components of the pollen, and thereby to measure changes in the nucleotide pattern at various growth stages. The most marked changes occur during the initial phase of tube growth when a large increase in the ribonucleoside triphosphate and the sugar nucleotide pools is observed. The contents of ATP and UDP-glucose are further increased if starch synthesis is initiated by the addition of sucrose to the culture medium. In order to determine whether nucleotides in pine pollen are synthesized from de novo pathways or via reutilization pathways, from breakdown products of nucleic acids, pollen was incubated with 14C-labelled precursors of both the de novo and the reutilization pathways. Incorporation experiments established de novo synthesis of ATP and GTP from glycine, and de novo synthesis of CTP and UTP from orotic acid. The operation of pathways for the utilization of exogenous nucleosides was also demonstrated. While uridine, cytidine and adenosine are incorporated into nucleoside triphosphate to a great extent, only minor incorporation of inosine and guanosine is observed. These reutilization pathways might be of importance for the synthesis of nucleotides during tube growth in situ. Addition of inhibitors of glycolysis and oxidative phosphorylation drastically reduces the level of ribonucleoside triphosphates, indicating a rapid turnover of the nucleotide pool.  相似文献   

4.
The viability and various physiological characteristics of individual sporangiospores of Rhizopus oligosporus in tempe starter cultures that had been stored for 8, 10, 16 and 30 months were examined by flow cytometry in combination with fluorescent dyes. Besides live, dead, and dormant spores we distinguished a category of sublethally damaged spores. Results indicated that the shelf-life of tempe starters was not limited by the death of spores, but by sublethal damage to spores as well as by dormancy which can be overcome by resuscitation, respiratory activation. During storage, the number of dormant and sublethally damaged spores increased: the longer the starter cultures were stored, the less dormant spores could still be activated. In contrast, the transition from sublethally damaged (spores that are not able to transform cFDA and emit green fluorescence except by activation treatment) to activated spores did not decrease with longer storage. However, after very long (30 months) storage, sublethally damaged spores could still be activated but could not germinate anymore. The shelf-life of spores in tempe starter is related to the physiological state of spores being sublethally damaged; a mechanism of physiological state transitions of R. oligosporus sporangiospores is proposed.  相似文献   

5.
Chemotherapy can induce anticancer immune responses. In contrast to a widely extended prejudice, apoptotic cell death is often more efficient in eliciting a protective anticancer immune response than necrotic cell death. Recently, we have found that purinergic receptors of the P2X7 type are required for the anticancer immune response induced by chemotherapy. ATP is the endogenous ligand that has the highest affinity for P2X7. Therefore, we investigated the capacity of a panel of chemotherapeutic agents to induce ATP release from cancer cells. Here, we describe that multiple distinct anticancer drugs reduce the intracellular concentration of ATP before and during the manifestation of apoptotic characteristics such as the dissipation of the mitochondrial transmembrane potential and the exposure of phosphatidylserine residues on the plasma membrane. Indeed, as apoptosis progresses, intracellular ATP concentrations decrease, although even advanced-stage apoptotic cells still contain sizeable ATP levels. Only when cells enter secondary necrosis, the ATP concentration falls to undetectable levels. Concomitantly, a wide range of chemotherapeutic agents causes the release of ATP into the extracellular space as they induce tumor cell death. Hence, ATP release is a general correlate of apoptotic cell death induced by conventional anticancer therapies.  相似文献   

6.
Apoptosis and anticancer drug resistance.   总被引:5,自引:0,他引:5  
Anticancer agents induce cancer cell death through apoptosis or necrosis. As anticancer agents at low and high concentrations cause apoptosis and necrosis, respectively, cancer cells may be merely injured by an anticancer agent in apoptosis, and cell death may result from an activation of the internal constituents to induce apoptosis. Therefore, an alternation of apoptotic pathway must change the efficacy of anticancer agents. As an escape of cancer cells from apoptosis seems to be closely associated with the development of anticancer resistance, this report focuses on mechanisms of apoptosis and its association with anticancer resistance. A Bax induces apoptosis mitochondria-dependently, whereas Fas can induce apoptosis mitochondria-independently. An interaction of Bax and Bcl-2 is very important to decide cell life or death, and Bcl-2 phosphorylation may control this interaction: Paclitaxel treatment induced Bcl-2 phosphorylation and typical apoptosis, whereas hyperthermia induced not Bcl-2 phosphorylation but nuclear translocation and failed to induce apoptosis. Moreover, Fas was localized in the cytoplasm of exponentially growing cells and on the cell membrane of confluent cells. We would like to emphasize that it is very important to check the localization of constituents of apoptosis in order to evaluate the susceptibility of cancer cells to apoptosis.  相似文献   

7.
Pyrimidine metabolism was investigated at various stages ofsomatic embryo development of white spruce (Picea glauca). The contribution of thede novo and the salvage pathways of pyrimidine biosynthesis to nucleotide and nucleic acid formation and the catabolism of pyrimidine was estimated by the exogenously supplied [6-14C]orotic acid, an intermediate of thede novo pathway, and with [2-14C]uridine and [2-14C]uracil, substrates of the salvage pathways. Thede novo pathway was very active throughout embryo development. More than 80 percnt; of [6-14C]orotic acid taken up by the tissue was utilized for nucleotide and nucleic acid synthesis in all stages of this process. The salvage pathways of uridine and uracil were also operative. Relatively high nucleic acid biosynthesis from uridine was observed, whereas the contribution of uracil salvage to the pyrimidine nucleotide and nucleic acid synthesis was extremely limited. A large proportion of uracil was degraded as 14CO2, probably via β-ureidopropionate. Among the enzymes of pyrimidine metabolism, orotate phosphoribosyltransferase was high during the initial phases of embryo development, after which it gradually declined. Uridine kinase, responsible for the salvage of uridine, showed an opposite pattern, since its activity increased as embryos developed. Low activities of uracil phosphoribosyltransferase and non-specific nucleoside phosphotransferase were also detected throughout the developmental period. These results suggest that the flux of thede novo and salvage pathways of pyrimidine nucleotide biosynthesisin vivo is roughly controlled by the amount of these enzymes. However, changing patterns of enzyme activity during embryo development that were measuredin vitro did not exactly correlate with the flux estimated by the radioactive precursors. Therefore, other fine control mechanisms, such as the fluctuation of levels of substrates and/or effectors may also participate to the real control of pyrimidine metabolism during white spruce somatic embryo development.  相似文献   

8.
This paper presents a brief review of applications of kinetic simulation of multi-enzyme networks to the study of antimetabolite drugs used as anticancer agents. Kinetic models consist of systems of nonlinear differential equations that describe changes in concentrations of cellular metabolites with respect to time. Such models have been used to predict the effect of changes in activity of enzymes, or changes in enzyme kinetic parameters, on sensitivity to inhibition. Kinetic simulation has provided insight into several aspects of the biochemical pharmacology of antimetabolites, including drug sensitivity and resistance, and drug-drug interactions. Two specific studies are described in detail. The first concerns the importance of the ratio of competing enzymes in determining the selectivity of inhibitors of one of the competing enzymes, studied by a simple model. The second case study examines the effect of alternative biosynthetic pathways, thede novo and salvage pathways of pyrimidine nucleotide biosynthesis, on the selectivity of antipyrimidine drugs, as studied by a detailed model of 27 reactions of pyrimidine metabolism.  相似文献   

9.
Control of pyrimidine biosynthesis was examined in Pseudomonas mucidolens ATCC 4685 and the five de novo pyrimidine biosynthetic enzyme activities unique to this pathway were influenced by pyrimidine supplementation in cells grown on glucose or succinate as a carbon source. When uracil was supplemented to glucose-grown ATCC 4685 cells, activities of four de novo enzymes were depressed which indicated possible repression of enzyme synthesis. To learn whether the pathway was repressible, pyrimidine limitation experiments were conducted using an orotate phosphoribosyltransferase (pyrE) mutant strain identified in this study. Compared to excess uracil growth conditions for the glucose-grown mutant strain cells, pyrimidine limitation of this strain caused aspartate transcarbamoylase, dihydroorotase and dihydroorotate dehydrogenase activities to increase by more than 3-fold while OMP decarboxylase activity increased by 2.7-fold. The syntheses of the de novo enzymes appeared to be regulated by pyrimidines. At the level of enzyme activity, aspartate transcarbamoylase activity in P. mucidolens ATCC 4685 was subject to inhibition at saturating substrate concentrations. Transcarbamoylase activity was strongly inhibited by UTP, ADP, ATP, GTP and pyrophosphate.  相似文献   

10.
The rapid accumulation of knowledge on apoptosis regulation in the 1990s was followed by the development of several experimental anticancer‐ and anti‐ischaemia (stroke or myocardial infarction) drugs. Activation of apoptotic pathways or the removal of cellular apoptotic inhibitors has been suggested to aid cancer therapy and the inhibition of apoptosis was thought to limit ischaemia‐induced damage. However, initial clinical studies on apoptosis‐modulating drugs led to unexpected results in different clinical conditions and this may have been due to co‐effects on non‐apoptotic interconnected cell death mechanisms and the ‘yin‐yang’ role of autophagy in survival versus cell death. In this review, we extend the analysis of cell death beyond apoptosis. Upon introduction of molecular pathways governing autophagy and necrosis (also called necroptosis or programmed necrosis), we focus on the interconnected character of cell death signals and on the shared cell death processes involving mitochondria (e.g. mitophagy and mitoptosis) and molecular signals playing prominent roles in multiple pathways (e.g. Bcl2‐family members and p53). We also briefly highlight stress‐induced cell senescence that plays a role not only in organismal ageing but also offers the development of novel anticancer strategies. Finally, we briefly illustrate the interconnected character of cell death forms in clinical settings while discussing irradiation‐induced mitotic catastrophe. The signalling pathways are discussed in their relation to cancer biology and treatment approaches.  相似文献   

11.
Since 1956, when exogenous uridine and cytidine were found to be necessary for the maintenance of perfused rat brain function, the co-existence of de novo synthesis, salvage pathways and removal of pyrimidine bases in the CNS has been a controversial subject. Here, we review studies on metabolites and enzymes of pyrimidine metabolism through more than 60 years. In view of known and newly-described inherited pyrimidine and purine disorders - some with complex clinical profiles of neurological impairments - we underline the necessity to investigate how the different pathways work together in the developing brain and then sustain plasticity, regeneration and neuro-transmission in the adult CNS. Experimentally, early incorporation studies in animal brain slices and homogenates with radio-labelled nucleosides or precursors demonstrated salvage activity or de novo synthesis. Later, the nucleoside transporters and organic anionic transporters underlying uptake of metabolites and anti-pyrimidine drugs in the CNS were identified. Recently, the expression of de novo enzymes in glial cells and neurons was verified using (immuno) histochemical and in-situ-hybridization techniques. Adult brain was shown to take up or produce all pyrimidine (deoxy) ribonucleosides or, after uptake and phosphorolysis of nucleosides, to make use of ribose for different purposes, including energy. More recently, non-canonical pyrimidine bases (5mC, 5hmC) have been found most notably in brain, pointing to considerable postreplicative DNA metabolism, with the need for pyrimidine-specific enzymes. Even more perspectives are emerging, with advances in genome analysis and in the manipulation of expression from the gene.  相似文献   

12.
Apoptosis is the cell's natural intrinsic regulatory mechanism of normal cells for programmed cell death, which plays an important role in cancer as a classical mechanism of tumor cell death causing minimal inflammation without causing damage to other cells in the vicinity. Induction of apoptosis by activation of caspases is one of the primary targets for cancer treatment. Over the years, a diverse range of natural, synthetic, and semisynthetic compounds and their derivatives have been investigated for their caspase-mediated apoptosis-induced anticancer activities. The review aims to compile the preclinical evidence and highlight the critical mechanistic pathways related to caspase-induced cell apoptosis in cancer treatment. The focus is placed on the key components of the mechanisms, including their chemical nature, and specific attention is given to phytochemicals derived from natural sources and synthetic and semisynthetic compounds. 180+ compounds from the past two decades with potential as anticancer agents are discussed in this review article. By summarizing the current knowledge and advancements in this field, this review provides a comprehensive overview of potential therapeutic strategies targeting apoptosis in cancer cells. The findings presented herein contribute to the ongoing efforts to combat cancer and stimulate further research into the development of effective and targeted anticancer therapies.  相似文献   

13.
14.
Glutamate excitotoxicity may culminate with neuronal and glial cell death. Glutamate induces apoptosis in vivo and in cell cultures. However, glutamate-induced apoptosis and the signaling pathways related to glutamate-induced cell death in acute hippocampal slices remain elusive. Hippocampal slices exposed to 1 or 10 mM glutamate for 1 h and evaluated after 6 h, showed reduced cell viability, without altering membrane permeability. This action of glutamate was accompanied by cytochrome c release, caspase-3 activation and DNA fragmentation. Glutamate at low concentration (10 μM) induced caspase-3 activation and DNA fragmentation, but it did not cause cytochrome c release and, it did not alter the viability of slices. Glutamate-induced impairment of hippocampal cell viability was completely blocked by MK-801 (non-competitive antagonist of NMDA receptors) and GAMS (antagonist of KA/AMPA glutamate receptors). Regarding intracellular signaling pathways, glutamate-induced cell death was not altered by a MEK1 inhibitor, PD98059. However, the p38MAPK inhibitor, SB203580, prevented glutamate-induced cell damage. In the present study we have shown that glutamate induces apoptosis in hippocampal slices and it causes an impairment of cell viability that was dependent of ionotropic and metabotropic receptors activation and, may involve the activation of p38MAPK pathway.  相似文献   

15.
Apoptosis or programmed cell death is a key regulator of physiological growth control and regulation of tissue homeostasis. Tipping the balance between cell death and proliferation in favor of cell survival may result in tumor formation. Moreover, current cancer therapies, e.g. chemotherapy, gamma-irradiation, immunotherapy or suicide gene therapy, primarily exert their antitumor effect by triggering an evolutionary conserved apoptosis program in cancer cells. For example, death receptor signaling has been implied to contribute to the efficacy of cancer therapy. Thus, failure to undergo apoptosis in response to anticancer therapy because of defects in death receptor pathways may result in resistance. Further insights into the mechanisms regulating apoptosis in response to anticancer therapy and how cancer cells evade cell death may provide novel opportunities for targeted therapeutics. Thus, agents designed to selectively activate death receptor pathways may enhance the efficacy of conventional therapies and may even overcome some forms of cancer resistance.  相似文献   

16.
Production of cellulose, a stress response‐mediated process in enterobacteria, is modulated in Escherichia coli by the activity of the two pyrimidine nucleotide biosynthetic pathways, namely, the de novo biosynthetic pathway and the salvage pathway, which relies on the environmental availability of pyrimidine nitrogenous bases. We had previously reported that prevalence of the salvage over the de novo pathway triggers cellulose production via synthesis of the second messenger c‐di‐GMP by the DgcQ (YedQ) diguanylate cyclase. In this work, we show that DgcQ enzymatic activity is enhanced by UTP, whilst being inhibited by N‐carbamoyl‐aspartate, an intermediate of the de novo pathway. Thus, direct allosteric control by these ligands allows full DgcQ activity exclusively in cells actively synthesizing pyrimidine nucleotides via the salvage pathway. Inhibition of DgcQ activity by N‐carbamoyl‐aspartate appears to be favoured by protein‐protein interaction between DgcQ and PyrB, a subunit of aspartate transcarbamylase, which synthesizes N‐carbamoyl‐aspartate. Our results suggest that availability of pyrimidine bases might be sensed, somehow paradoxically, as an environmental stress by E. coli. We hypothesize that this link might have evolved since stress events, leading to extensive DNA/RNA degradation or lysis of neighbouring cells, can result in increased pyrimidine concentrations and activation of the salvage pathway.  相似文献   

17.
In cervical cancer, HPV infection and disruption of mechanisms involving cell growth, differentiation, and apoptosis are strictly linked with tumor progression and invasion. Tumor microenvironment is ATP and adenosine rich, suggesting a role for purinergic signaling in cancer cell growth and death. Here we investigate the effect of extracellular ATP on human cervical cancer cells. We find that extracellular ATP itself has a small cytotoxic effect, whereas adenosine formed from ATP degradation by ectonucleotidases is the main factor responsible for apoptosis induction. The level of P2×7 receptor seemed to define the main cytotoxic mechanism triggered by ATP, since ATP itself eliminated a small subpopulation of cells that express high P2×7 levels, probably through its activation. Corroborating these data, blockage or knockdown of P2×7 only slightly reduced ATP cytotoxicity. On the other hand, cell viability was almost totally recovered with dipyridamole, an adenosine transporter inhibitor. Moreover, ATP-induced apoptosis and signaling—p53 increase, AMPK activation, and PARP cleavage—as well as autophagy induction were also inhibited by dipyridamole. In addition, inhibition of adenosine conversion into AMP also blocked cell death, indicating that metabolization of intracellular adenosine originating from extracellular ATP is responsible for the main effects of the latter in human cervical cancer cells.  相似文献   

18.
Autophagy is an evolutionarily conserved cell survival pathway that enables cells to recoup ATP and other critical biosynthetic molecules during nutrient deprivation or exposure to hypoxia, which are hallmarks of the tumour microenvironment. Autophagy has been implicated as a potential mechanism of resistance to anticancer agents as it can promote cell survival in the face of stress induced by chemotherapeutic agents by breaking down cellular components to generate alternative sources of energy. Disruption of autophagy with chloroquine (CQ) induces the accumulation of ubiquitin‐conjugated proteins in a manner similar to the proteasome inhibitor bortezomib (BZ). However, CQ‐induced protein accumulation occurs at a slower rate and is localized to lysosomes in contrast to BZ, which stimulates rapid buildup of ubiquitinated proteins and aggresome formation in the cytosol. The histone deacetylase (HDAC) inhibitor vorinostat (VOR) blocked BZ‐induced aggresome formation, but promoted CQ‐mediated ubiquitinated protein accumulation. Disruption of autophagy with CQ strongly enhanced VOR‐mediated apoptosis in colon cancer cells. Accordingly, knockdown of the essential autophagy gene Atg7 also sensitized cells to VOR‐induced apoptosis. Knockdown of HDAC6 greatly enhanced BZ‐induced apoptosis, but only marginally sensitized cells to CQ. Subsequent studies determined that the CQ/VOR combination promoted a large increase in superoxide generation that was required for ubiquitinated protein accumulation and cell death. Finally, treatment with the CQ/VOR combination significantly reduced tumour burden and induced apoptosis in a colon cancer xenograft model. Collectively, our results establish that inhibition of autophagy with CQ induces ubiquitinated protein accumulation and VOR potentiates CQ‐mediated aggregate formation, superoxide generation and apoptosis.  相似文献   

19.
Thermal ablation in combination with transarterial chemoembolization (TACE) has been reported to exert a more powerful antitumor effect than thermal ablation alone in hepatocellular carcinoma patients. However, the underlying mechanisms remain unclear. The purpose of the present study was to evaluate whether sublethal hyperthermia encountered in the periablation zone during thermal ablation enhances the anticancer activity of doxorubicin in chronically hypoxic (encountered in the tumor area after TACE) liver cancer cells and to explore the underlying mechanisms. In the present study, HepG2 cells precultured under chronic hypoxic conditions (1% oxygen) were treated in a 42°C water bath for 15 or 30 min, followed by incubation with doxorubicin. Assays were then performed to determine intracellular uptake of doxorubicin, cell viability, apoptosis, cell cycle, mitochondrial membrane potential (MMP), reactive oxygen species (ROS), and total antioxidant capacity. The results confirmed that sublethal hyperthermia enhanced the intracellular uptake of doxorubicin into hypoxic HepG2 cells. Hyperthermia combined with doxorubicin led to a greater inhibition of cell viability and increased apoptosis in hypoxic HepG2 cells as compared with hyperthermia or doxorubicin alone. In addition, the combination induced apoptosis by increasing ROS and causing disruption of MMP. Pretreatment with the ROS scavenger N-acetyl cysteine significantly inhibited the apoptotic response, suggesting that cell death is ROS-dependent. These findings suggested that sublethal hyperthermia enhances the anticancer activity of doxorubicin in hypoxic HepG2 cells via a ROS-dependent mechanism.  相似文献   

20.
TRAIL, a putative anticancer cytokine, induces extrinsic cell death by activating the caspase cascade directly (Type I cells) via the death-inducing signaling complex (DISC) or indirectly (Type II cells) by caspase-8 cleavage of Bid and activation of the mitochondrial cell death pathway. Cancer cells are characterized by their dependence on aerobic glycolysis, which, although inefficient in terms of ATP production, facilitates tumor metabolism. Our studies show that TRAIL-induced cell death is significantly affected by the metabolic status of the cell. Inhibiting glycolysis with 2-deoxyglucose potentiates TRAIL-induced cell death, whereas glucose deprivation can paradoxically inhibit apoptosis. These conflicting responses to glycolysis inhibition are modulated by the balance between the Akt and AMPK pathways and their subsequent downstream regulation of mTORC1. This results in marked changes in protein translation, in which the equilibrium between anti- and pro-apoptotic Bcl-2 family member proteins is decided by their individual degradation rates. This regulates the mitochondrial cell death pathway and alters its sensitivity not only to TRAIL, but to ABT-737, a Bcl-2 inhibitor. Taken together, our studies show that the sensitivity of cancer cells to apoptosis can be modulated by targeting their unique metabolism in order to enhance sensitivity to apoptotic agents.  相似文献   

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