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PubMed2006

Activation of caspase-1 dependent interleukins in developmental brain trauma.

Focal mechanical cortical trauma triggers diffuse apoptotic neurodegeneration in the developing rat brain which is associated with invasion of brain tissue with inflammatory mediators. We hypothesized that caspase-1 and the two caspase-1-processed cytokines, interleukin (IL)-1beta and IL-18, are involved in trauma-induced neuronal cell death in the developing brain. 7-day-old Wistar rats or C57/BL6 mice were subjected to head trauma using a weight drop device. Animals were sacrificed at defined time points following trauma and brains were processed for histology and molecular analyses. Neuronal cell death in the immature brain peaked at 12-24 h and was accompanied by a marked increase of mRNA and protein levels for caspase-1, IL-1beta and IL-18 within 2 to 12 h following the injury. Caspase-1 levels were elevated for 72 h, whereas IL-1beta decreased earlier at 48 h. IL-18 remained high over a period of 3 days and decreased to normal levels by day 7 after the injury. Intraperitoneal injection of recombinant human IL-18-binding protein (IL-18BP), a specific inhibitor of IL-18, attenuated traumatic brain injury. Mice deficient in IL-18 (IL-18-/-) were protected against trauma-induced brain damage. These findings indicate that IL-18 is involved in trauma-induced neuronal cell death in the immature rodent brain and might serve as a potential therapeutic target.

Animals↗
PubMed2000

Induction of mitochondrial manganese superoxide dismutase confers resistance to apoptosis in acute myeloblastic leukaemia cells exposed to etoposide.

We investigated the possible roles of mitochondrial manganese superoxide dismutase (MnSOD) and bcl-2 in etoposide-induced cell death in acute myeloblastic leukaemia (AML) using two subclones of the OCI/AML-2 cell line, the etoposide-sensitive (ES) and the etoposide-resistant (ER), as models. Cell death after 24 h exposure to 10 micromol/l etoposide was about 60% and 70% in the ES subclone and about 20% and 25% in the ER subclone, when analysed by trypan blue and annexin V respectively. Cytochrome c efflux from mitochondria to cytosol was observed after 4 h of exposure in both subclones, whereas the activation of caspase-3 was not detectable until after 12 h of exposure in the ES subclone and 24 h of exposure in the ER subclone, using Western blotting. The decrease in mitochondrial membrane potential, when analysed by the JC-1 probe fluorocytometrically, also appeared to take place later in the ER than in the ES subclone. Both subclones showed evident basal expression of MnSOD and bcl-2 by Western blotting. Etoposide caused a potent induction of MnSOD, more than 400% at 12 h, in the ER but not in the ES subclone. No significant change in bcl-2 expression could be observed in either of the subclones during exposure to etoposide when analysed by Western blotting or flow cytometry. In conclusion, we suggest that MnSOD might have a special role in the protection of AML cells against etoposide-induced cell death. Although unable to influence the cytochrome c efflux to cytosol, MnSOD might prevent the disruption of mitochondrial membrane potential, which evidently leads to cell death by releasing various activators of apoptosis.

Antineoplastic Agents, Phytogenic↗
PubMed2001

Involvement of caspases in apoptotic cell death of murine macrophages infected with Actinobacillus actinomycetemcomitans.

Infection of murine macrophages in vitro with periodontopathic bacterium Actinobacillus actinomycetemcomitans induces apoptotic cell death. In this study, we investigated the involvement of caspases in apoptotic cell death of A. actinomycetemcomitans-infected macrophages. Two peptide inhibitors of caspases, benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethyl ketone (Z-VAD-FMK) and benzyloxycarbonyl-Asp-Glu-Val-Asp (OMe)-fluoromethyl ketone (Z-DEVD-FMK), inhibited apoptotic cell death of murine macrophage cell line J774.1 infected with A. actinomycetemcomitans. During the process of apoptosis, interleukin-1beta (IL-1beta) was detected in the culture supernatants of J774.1 cells. IL-1beta secretion was blocked by the caspase-1 inhibitor, Z-VAD-FMK, indicating that caspase-1 is involved in not only the induction of apoptosis but also the IL-1beta secretion from A. actinomycetemcomitans-infected J774.1 cells. Immunoblot analysis revealed that the infection of A. actinomycetemcomitans to J774.1 cells induced the cleavage of retinoblastoma protein (Rb), suggesting that caspase-3 was activated by A. actinomycetemcomitans infection. The cytosol from A. actinomycetemcomitans-infected J774.1 cells induced Rb proteolysis in vitro, which was inhibited by the caspase-3 inhibitor, Z-DEVD-FMK. Furthermore, caspase-3-like activity was markedly increased in J774.1 cells infected with A.actinomycetemcomitans between 12 h and 24 h, which was subsequently inhibited by the addition of caspase-3 inhibitor, Z-DEVD-FMK. These findings indicate that caspase-3 induces apoptosis in J774.1 cells infected with A. actinomycetemcomitans. Taken together, these results suggest that caspase-1 and caspase-3 are involved in the induction of apoptosis in A. actinomycetemcomitans-infected macrophages.

Aggregatibacter actinomycetemcomitans↗
PubMedDate not supplied

Ellagic acid induced p53/p21 expression, G1 arrest and apoptosis in human bladder cancer T24 cells.

It is well known that dietary phenolic compounds can elicit vital cellular responses such as cytotoxicity, cell cycle arrest and apoptosis by activating a cascade of molecular events. Ellagic acid is one of these phenolic compounds, but the exact mechanism of its action is still unclear. The objective of this study was to investigate ellagic acid-induced cell cycle arrest and apoptosis in T24 human bladder cancer cells in vitro. Assays were performed to determine cell viability, cell cycle arrest, apoptosis, caspases-3 activity and gene expression, measured by flow cytometric assay, polymerase chain reaction (PCR) and determination of caspase-3 activity. Ellagic acid significantly reduced the viable cells, induced G0/G1-phase arrest of the cell cycle and apoptosis. Ellagic acid also increased p53 and p21 and decreased CDK2 gene expression, that may lead to the G0/G1 arrest of T24 cells. Ellagic acid also promoted caspase-3 activity after exposure for 1, 3, 6, 12 and 24 h, which led to induction of apoptosis. Furthermore, the ellagic acid-induced apoptosis on T24 cells was blocked by the broad-spectrum caspase inhibitor (z-VAD-fmk).

Aged↗
PubMed2005

Memantine attenuates staurosporine-induced activation of caspase-3 and LDH release in mouse primary neuronal cultures.

Developmental aspects of pro- and antiapoptotic action of some NMDA receptor antagonists in the central nervous system have been postulated. In order to further elucidate this problem, we investigated effect of memantine, an uncompetitive NMDA receptor antagonist and staurosporine alone and in combination on caspase-3 activity and lactate dehydrogenase (LDH) release in primary hippocampal, neocortical and striatal cell cultures on 7 and 12 days in vitro. The data showed that the vulnerability of neuronal cells to induction of caspase-3 activity by staurosporine was higher on 7 DIV than on 12 DIV, whereas staurosporine-mediated LDH release increased with days in vitro in striatal culture only. A specific inhibitor of caspase-3, AcDEVDCHO (60 microM), completely abolished the effect of staurosporine on this enzyme's activity, but only partially attenuated staurosporine-induced LDH release in hippocampal cells. Memantine alone (0.05-2.0 microM) did not induce any cytotoxic effect but attenuated the staurosporine-induced caspase-3 activity and LDH release in hippocampal cultured neurons on each investigated day in vitro. In striatal culture, memantine had a moderate inhibitory effect on staurosporine-evoked LDH release only on 7 DIV with no significant influence on caspase-3 activity. As for neocortical cultures, memantine partially inhibited staurosporine-induced neuronal injury only on 7 DIV. These data showed that the induction of caspase-3 activity by staurosporine was more profound in immature cells, however, the staurosporine neurotoxicity, as reflected by LDH release, only partially depended on caspase-3 activation and stage of cell development. Furthermore, memantine attenuated staurosporine-induced apoptosis more efficiently in hippocampal cultures than in neocortical and striatal ones, which points to tissue specificity of effects of this neuroprotectant.

Analysis of Variance↗
PubMed2003

Immunohistochemical detection of apoptosis, proliferation and inducible nitric oxide synthase in rat urothelium damaged by cyclophosphamide treatment.

The present study was conducted to investigate cell death, proliferation and inducible nitric oxide synthase (iNOS) immunoreactivity in rat urothelium within 24 h after a single intraperitoneal dose of cyclophosphamide (CP). Necrotic cells were identified predominantly in the superficial cell layer from 1 h until 6 h after CP injection, most of them desquamating from the urothelium into the lumen of the urinary bladder. Active caspase-3 immunohistochemistry revealed apoptotic cells from 12 h until 24 h after CP injection. The apoptotic index reached a peak at 18 h and then rapidly dropped. Simultaneously with the decrease of apoptosis, the proliferation index increased from 18 h until 24 h after CP treatment. Immunoreaction to iNOS was first detected at 6 h in basal and intermediate cells. Later, iNOS immunoreactivity became stronger and was present in all cell layers. Our results suggest that the destruction of rat urothelium during 24 h after CP administration is due not only to necrosis, but also to apoptosis. The first 6 h are characterised by necrotic changes and no iNOS immunoreactivity. Thereafter, apoptosis and iNOS immunoreactivity are observed within the damaged urothelium. At 24 h after CP injection, iNOS immunoreactivity is still present, but proliferation prevails over cell death, enabling the urothelium to start regeneration.

Animals↗
PubMed2005

Activation of nuclear factor-kappaB signaling pathway by interleukin-1 after hypoxia/ischemia in neonatal rat hippocampus and cortex.

Perinatal hypoxia/ischemia (HI) is a common cause of neurological deficits in children. Interleukin-1 (IL-1) activity has been implicated in HI-induced brain damage. However, the mechanisms underlying its action in HI have not been characterized. We used a 7-day-old rat model to elucidate the role of nuclear factor-kappaB (NF-kappaB) activation in HI stimulation of IL-1 signaling. HI was induced by permanent ligation of the left carotid artery followed by 90 min of hypoxia (7.8% O(2)). Using ELISA assays, we observed increased cell death and caspase 3 activity in hippocampus and cortex 3, 6, 12, 24 and 48 h post-HI. IL-1beta protein expression increased, beginning at 3 h after HI and lasting until 24 h post-HI in hippocampus and 12 h post-HI in cortex. Intracerebroventricular injection of 2 microg IL-1 receptor antagonist (IL-1Ra) 2 h after HI significantly reduced cell death and caspase 3 activity. Electrophoretic mobility shift assay analyses of hippocampus and cortex after HI for NF-kappaB activity showed increased p65/p50 DNA-binding activity at 24 h post-HI. Western blot analyses showed significant nuclear translocation of p65. Protein expression levels of two known inflammatory agents, inducible nitric oxide synthase and cycloxygenase 2, known to be transcriptionally regulated by NF-kappaB, also increased at 24 h after HI. All these HI-induced changes were reversed by IL-1Ra blockade of IL-1 signaling, consistent with IL-1 triggering of inflammatory apoptotic outcomes via NF-kappaB transcriptional activation. The observed increase in cytoplasmic phosphorylated inhibitor kappaBalpha (IkappaBalpha) and nuclear translocation of Bcl-3 24 h after HI was also significantly attenuated by IL-1Ra blockade, suggesting that HI-induced IL-1 activation of NF-kappaB is via both the degradation of IkappaBalpha and the nuclear translocation of Bcl-3.

Animals↗
PubMed2004

Patterns of uterine cellular proliferation and apoptosis in the implantation site of the rat during pregnancy.

During gestation, the balance between cell proliferation and death is crucial for successful embryo implantation and maintenance of pregnancy. The uterine endometrium responds to blastocyst implantation with extensive proliferation and differentiation of stromal cells into decidual cells, forming the antimesometrial and mesometrial decidua, which regress by apoptosis. In the latter region it is also observed the growth of metrial gland. To elucidate the events underlying this tissue remodelling we investigated the spatial and temporal pattern of expression of the proliferating cell nuclear antigen (PCNA) and localized the apoptotic cells, by the TUNEL assay and by the expression of active caspase-3. We found that PCNA is expressed at high levels during decidualization until day 12 of gestation declining thereafter abruptly. On the contrary, the appearance of apoptotic cells was detected, by the TUNEL and active caspase-3 expression, in the mesometrial decidua on day 12, increasing from days 14 to 16 in the decidua and metrial gland. In the antimesometrial decidua apoptosis was observed from early to day 12 of pregnancy. However, on day 13 only cell debris and neutrophils were observed, indicating also the presence of necrosis. These results suggest that decidual cells undergo, in distinct regions and at different stages of pregnancy, cell death by apoptosis and secondary necrosis.

Animals↗
PubMed2000

Pro-caspase-8 is predominantly localized in mitochondria and released into cytoplasm upon apoptotic stimulation.

The recruitment and cleavage of pro-caspase-8 to produce the active form of caspase-8 is a critical biochemical event in death receptor-mediated apoptosis. However, the source of pro-caspase-8 available for activation by apoptotic triggers is unknown. In human fibroblasts and mouse clonal striatal cells, confocal microscopy revealed that pro-caspase-8 immunofluorescence was colocalized with cytochrome c in mitochondria and was also distributed diffusely in some nuclei. Biochemical analysis of subcellular fractions indicated that pro-caspase-8 was enriched in mitochondria and in nuclei. Pro-caspase-8 was found in the intermembrane space, inner membrane, and matrix of mitochondria after limited digestion of mitochondrial fractions, and this distribution was confirmed by immunogold electron microscopy. Pro-caspase-8 and cytochrome c were released from isolated mitochondria that were treated with an inhibitor of the ADP/ATP carrier atractyloside, which opens the mitochondria permeability transition pore. Release was blocked by the mitochondria permeability transition pore inhibitor cyclosporin A (CsA). After clonal striatal cells were exposed for 6 h to an apoptotic inducer tumor necrosis factor-alpha (TNF-alpha), mitochondria immunoreactive for cytochrome c and pro-caspase-8 became clustered at perinuclear sites. Pro-caspase-8 and cytochrome c levels decreased in mitochondrial fractions and increased, along with pro-caspase-8 cleavage products, in the cytoplasm of the TNF-alpha-treated striatal cells. CsA blocked the TNF-alpha-induced release of pro-caspase 8 but not cytochrome c. Internucleosomal DNA fragmentation started at 6 h and peaked 12 h after TNF-alpha treatment. These results suggest that pro-caspase-8 is predominantly localized in mitochondria and is released upon apoptotic stimulation through a CsA-sensitive mechanism.

Apoptosis↗
PubMed2002

Interleukin-18 involvement in hypoxic-ischemic brain injury.

Inflammation is a critical factor for development of hypoxic-ischemic (HI) brain injury. Interleukin-18 (IL-18) is a proinflammatory cytokine expressed in microglia and processed by caspase-1. Our aim was to characterize the expression of IL-18 and its receptor in relation to caspase-1 and IL-1beta after HI and to evaluate to what extent IL-18 contributes to HI brain injury. Seven-day-old rats were subjected to HI, and brain tissue was sampled at different time points (3 hr to 14 d) after insult. The mRNA for IL-18 and caspase-1 were analyzed with reverse transcriptase PCR, protein was analyzed by Western blot (IL-18, caspase-1) or ELISA (IL-1beta), and the regional distribution was assessed by immunohistochemistry. HI was also induced in C57BL/6 mice, and brain injury in IL-18-deficient animals was compared with that in wild-type animals. The expression of mRNA/protein for caspase-1 and IL-18 in brain homogenates increased progressively at 12 hr to 14 d after HI, whereas IL-1beta peaked at 8 hr. A widespread expression of caspase-1 and IL-18 protein in microglia was found in the HI hemisphere. The IL-18 receptor was expressed on neurons of the cerebral cortex and thalamus. IL-1beta was primarily found in microglia in the habenular nucleus of the thalamus. The infarct volume was reduced by 21% (p = 0.01), and the neuropathology score was significantly decreased in the cerebral cortex (-35%), hippocampus (-22%), striatum (-18%), and thalamus (-17%) in mice with IL-18 deficiency compared with wild-type mice. In conclusion, we found that IL-18 expression in microglia was markedly increased after HI and that IL-18 appears to be important for the development of HI brain injury.

Animals↗
PubMed2005

Apoptosis of hepatocytes caused by Punta Toro virus (Bunyaviridae: Phlebovirus) and its implication for Phlebovirus pathogenesis.

Experimental infection of hamsters with Punta Toro virus (PTV) produces a disease with clinical and pathological similarities to the severe human hemorrhagic fever caused by Rift Valley fever virus (RVFV), thus providing an animal model for RVFV pathogenesis. In this model, hepatocytic apoptosis is the main pathological component of liver injuries that are responsible for severe hemorrhagic manifestations. To further elucidate whether viral replication in hepatocytes directly causes apoptosis, we studied the morphological and biochemical changes of apoptosis in HepG2 cells at different time points after PTV infection. Cellular viability began to decrease 12 hours after infection compared with controls. Caspases 3/7 were activated significantly at 48 and 72 hours after infection, and phosphatidylserine translocation and DNA fragmentation were also detected at 48 and 72 hours. Cell cycle analysis by flow cytometry showed that infected HepG2 cells were arrested at G(0)/G(1) phase. Furthermore, virus titer increased with apoptosis progression, suggesting that viral replication is necessary for the apoptotic process. These results indicate that PTV infection alone, without a secondary inflammatory cellular reaction, induces hepatocytic apoptosis and suggest that future therapeutics for RVFV hemorrhagic disease might target inhibition of cellular apoptotic pathways during the acute infection.

Animals↗
PubMed2003

A comparison of gene expression changes in response to diethylstilbestrol treatment in wild-type and p53+/- hemizygous knockout mice using focussed arrays.

We have investigated the hepatic response of female C57BL/6J wild-type and p53(+/-) hemizygous mice to genotoxic levels of diethylstilbestrol (DES) using cell cycle and apoptosis-focussed cDNA expression arrays. DES induced the expression of 12 genes (bad, bax, bcl-x, caspase-1, p53, cyclin D3, GADD45, p21, p15, p27, p57 and Skp1) and down-regulated the expression of eight genes (bcl-2, caspase-2, caspase-7, caspase-8, E124, iNOS, mdm2 and NFkappab1) at twofold or greater levels. Taken together, these changes were strongly reflective of the induction of apoptosis in the livers of DES-treated mice. Of those genes showing the greatest changes in response to DES, p53, p21 and p57 were expressed at 2.1, 1.7 and 1.6 times greater (respectively) in wild-type mice as compared with p53(+/-) hemizygous mice. Differences in p53, p21 and bax expression were confirmed by RT-PCR and we conclude that the compromised response of p53(+/-) mice is likely to play a central role in the earlier appearance of tumours in this model, following exposure to genotoxic carcinogens.

Animals↗
PubMed2004

Bone morphogenetic proteins and neurotrophins provide complementary protection of septal cholinergic function during phosphatase inhibitor-induced stress.

Cultures of embryonic rat septum were exposed for 24-48 h to 2-5 nm okadaic acid (OA), an inhibitor of pp1A and pp2A phosphatases. This stress killed approximately 75% of neurons. A neurotrophin (NT) combination (nerve growth factor and brain-derived neurotrophic factor, each 100 ng/mL) plus a bone morphogenetic protein (BMP6 or BMP7, 5 nm) reduced the death of both cholinergic and non-cholinergic neurons, and preserved choline acetyltransferase (ChAT) activity assayed 2-6 days post-stress. This NT + BMP combination preserved ChAT activity better than either NTs or BMPs alone, and was effective even if trophic factor addition was delayed until 12 h after stress onset. A general caspase inhibitor (qVD-OPH, 10 micro g/mL) also increased survival of stressed cholinergic neurons, but its protection of ChAT activity was shorter lived than that produced by the NT + BMP combination. Neither the NT + BMP combination nor the caspase inhibitor reduced the OA-induced increase in tau phosphorylation. These findings indicate that NTs and BMPs have synergistic protective effects against an OA stress, and suggest that at least some of these protective effects occur upstream of caspase activation.

Acetylcholinesterase↗
PubMed2005

Organ-specific increase in mutation accumulation and apoptosis rate in CuZn-superoxide dismutase-deficient mice.

Reactive oxygen species have been implicated as a cause of cancer and aging in mammals. Mice deficient for the antioxidant enzyme CuZn-superoxide dismutase (Sod1) have a decreased life span and an elevated incidence of liver cancer. To test the hypothesis that the cancer-prone phenotype in such mice is due to accelerated spontaneous mutation accumulation, we crossed these mutants with mice harboring a neutral lacZ mutation reporter gene. At 2 months of age, the lacZ mutation frequency in the liver of the hybrid animals was already twice as high as in littermate controls of the same age. This difference in mutation frequency increased to >3-fold at 6 months of age, after which it did not increase any further. Characterization of the mutation spectra in liver of the Sod1-null mice indicated mainly GC-to-TA transversions and GC-to-AT transitions, signature mutations of oxidative stress. The accelerated mutation accumulation in liver was accompanied by an increased frequency of apoptotic cells, as indicated by an increase in both terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling- and caspase 3-stained cells at 6 and 12 months of age. In kidney, an elevated mutation frequency above controls of approximately 2.5-fold was found not earlier than at 6 months. No increased mutation accumulation was observed in brain or spleen. These results support the hypothesis, that oxidative stress is an important causal factor of cancer in mammals.

Animals↗
PubMed2003

Induction of apoptosis in vascular cells by plasminogen activator inhibitor-1 and high molecular weight kininogen correlates with their anti-adhesive properties.

Plasminogen activator inhibitor-1 (PAI-1) and two-chain high molecular weight kininogen (HKa) exert anti-adhesive properties in vitronectin-dependent cell adhesion. Here, the hypothesis was tested that these anti-adhesive components promote apoptosis in vascular cells. PAI-1 or HKa induced a 2- to 3-fold increase in apoptosis of human umbilical-vein endothelial cells (HUVEC) and vascular smooth muscle cells (VSMC) adherent to vitronectin, as determined by annexin V-FACS assay, similar to alphav-integrin inhibitor cyclo-(Arg-Gly-Asp-D-Phe-Val)-peptide (cRGDfV). Apoptosis occurred after 12 h incubation and was attributable to caspase 3 activation that in turn induced DNA fragmentation. Induction of apoptosis strongly correlated with the anti-adhesive effect of PAI-1 and HKa on these cells. In contrast, PAI-1 and HKa did not affect fibronectin-dependent adhesion or cell survival. uPA did not influence apoptosis in vitronectin- or fibronectin-adherent cells. In atherosclerotic vessel sections, congruent distribution of vitronectin, PAI-1, HK, and of components of the urokinase plasminogen activator/receptor system with apoptotic cells lining foam cell lesions was demonstrated by immunostaining. These results indicate that inhibition of vitronectin-dependent cell adhesion through PAI-1 and HKa correlates with apoptosis induction in vascular cells mediated through the caspase 3 pathway. Co-distribution of apoptosis with plasminogen activation system components in atherosclerosis exemplifies the significance of anti-adhesive mechanisms and apoptosis for tissue remodeling, such as in neointima development.

Apoptosis↗
PubMed2005

Induction of apoptosis in chicken oviduct cells by C2-ceramide.

The chicken oviduct is a dynamic organ that produces secretory proteins such as ovalbumin and its cells undergo cell proliferation and differentiation. There has been no study of the cellular mechanism involved in cell death in the chicken oviduct. Therefore, this study has focused on the study of apoptosis in primary oviduct cells. Because ceramide is known to activate apoptosis in tumor cells and is produced in the oviduct, we used an exogenous ceramide analog to induce cell death. The viability of ceramide-treated chicken oviduct cells decreased in a dose-dependent manner and apoptotic cells were detected by staining with annexin V. The expression of apoptosis-related genes was assessed by RT-PCR and bcl-2 mRNA was found to decrease after exposure to ceramide while Bcl-x mRNA increased 12 h post-treatment. In addition, caspase-3 was expressed strongly in the early stages of apoptosis, while caspase-1 and -9 transcripts increased at later times. We conclude that ceramide induces apoptosis in oviduct-derived primary cells via a caspase- and bcl-2- dependent pathway.

Animals↗
PubMed2005

[Study of triptolide-induced apoptosis in MUTZ-1 cells and its allied mechanism].

To investigate the apoptotic effect of triptolide on MDS cell line MUTZ-1 cells and its mechanism, MUTZ-1 cells were incubated with indicated concentrations of triptolide. The growth of MUTZ-1 cells was observed by MTT assay and apoptosis was detected by DNA fragmentation analysis and flow cytometry using Annexin V-FITC/PI staining. The gene and protein expressions were determined by reverse transcription polymerase chain reaction (RT-PCR) and Western blot, respectively. The results showed that MUTZ-1 cell viability in presence of triptolide decreased markedly in a dose- and time-dependent manner. The growth-inhibitory IC50 value for triptolide treatment was 55.06 ng/ml. A DNA ladder pattern of internucleosomal fragmentation was observed. The translocation of phosphatidylserine at the outer surface of the cell plasma membrane could be induced by triptolide and its level increased following the augmentation of the drug concentration. Treatment of MUTZ-1 cells with triptolide for 12 hours resulted in the activation of caspase-3, cleavage of PARP and decrease of c-IAP2 mRNA. The expressions of pro-caspase 3 and c-IAP2 were inversely correlated with the incidence of apoptosis. (r = -0.907, P = 0.000; r = -0.919, P = 0.000 respectively). In conclusion, Triptolide inhibits MUTZ-1 cell growth by inducing apoptosis. The apoptotic effect of triptolide in MUTZ-1 cells is mediated by the caspase-3 activation and PARP cleavage. Moreover, the activation of caspase-3 may be associated with the down-regulation of c-IAP2.

Antineoplastic Agents, Alkylating↗
PubMed2004

Proteomic analysis of antiproliferative effects by treatment of 5-fluorouracil in cervical cancer cells.

The global effects of 5-fluorouracil (FU) on cervical carcinoma cells were analyzed using an efficient proteomic method. More than 50 proteins showed a significant change in 5-FU-treated cervical carcinoma cells compared to control cells. Among them, 34 proteins have been identified by employing two-dimensional gel electrophoresis and MALDI-TOF-MS using peptide mass fingerprinting. In results, 22 proteins were upregulated (CIDE-B [cell death-inducing DFFA-like effector B], caspase-3, caspase-8, Apo-1/CD95 (Fas), etc.) and 12 proteins were downregulated (mitotic checkpoint protein BUB3, myc proto-oncogene protein [c-myc], src substrate cortactin, transforming protein p21A, etc.) by 5-FU treatment in HeLa cervical carcinoma cells as determined by spot volume (P <0.05). Our experiments showed that 5-FU engaged the mitochondrial apoptotic pathway involving cytosolic cytochrome c release and subsequent activation of caspase-9 and caspase-3 as well as the membrane death receptor (DR)-mediated apoptotic pathway involving activation of caspase-8 with an Apo-1/CD95 (Fas)-dependent fashion. In addition, we could observe reduction of HPV-18 E6/E7 gene expression and activation of p53, pRb, and p21waf1 proteins by 5-FU treatment in HeLa cervical carcinoma cells. In conclusion, we suggest that 5-FU suppresses the growth of cervical cancer cells not only by antiproliferative effect but also antiviral regulation. Our findings may offer new insights into the mechanism of anticancer effect affected by 5-FU treatment in cervical cancer cells and its mode of action.

Antimetabolites, Antineoplastic↗