mRNA Expression Levels Of Caspase-3 Research Articles

The Association between Splenocyte Apoptosis and Alterations of Bax, Bcl-2 and Caspase-3 mRNA Expression, and Oxidative Stress Induced by Dietary Nickel Chloride in Broilers

Two hundred and forty avian broilers were equally divided into four groups, and raised with a corn-soybean basal diet or the same diet supplemented with 300, 600, 900 mg/kg NiCl2 for 42 days. Numbers or percentages of apoptotic splenocytes by flow cytometry (FCM) and TUNEL were higher (p < 0.05 or p < 0.01) in the 300, 600 and 900 mg/kg groups than those in the control group. Results measured by qRT-PCR and ELISA showed that mRNA expression and contents were significantly higher (p < 0.05 or p < 0.01) in Bax and Caspase-3, and were significantly lower (p < 0.05 or p < 0.01) in Bcl-2 of the 300, 600 and 900 mg/kg groups. Also, the SOD, CAT and GSH-Px activities, and the ability to inhibit hydroxyl radical, and GSH contents were significantly decreased (p < 0.05 or p < 0.01), and MDA contents were increased (p < 0.05 or p < 0.01) in all groups. In conclusion, dietary NiCl2 in excess of 300 mg/kg caused apoptosis, altered Bax, Bcl-2 and Caspase-3 mRNA expression levels and contents, and induced oxidative stress in the spleen. Also, splenocyte apoptosis was closely related to the alternations of Bax, Bcl-2 and Caspase-3 mRNA expression, and oxidative damage. The splenic immunity and blood filtration functions were impaired in broilers.

Reversal effect of monoisoamyl dimercaptosuccinic acid (MiADMSA) for arsenic and lead induced perturbations in apoptosis and antioxidant enzymes in developing rat brain

Oxidative stress (OS) has been implicated in the pathophysiology of many neurodegenerative disorders. Several studies have shown that exposure to arsenic (As) and lead (Pb) produces oxidative stress, one of the most noted molecular mechanisms for the neurotoxicity of these metals. In the present study, we examined the effect of combined exposure to these metals (As and Pb) on the activity levels of antioxidant enzymes and apoptotic marker enzymes in brain regions (cerebral cortex, hippocampus and cerebellum) of rats at postnatal day (PND) 21, 28 and 3 months age and compared the toxicity levels with individual metals (As or Pb). Further, we also evaluated the therapeutic efficacy of a chelating agent, monoisoamyl dimercaptosuccinic acid (MiADMSA) against arsenic and lead induced developmental neurotoxicity. Pregnant rats were exposed to sodium meta-arsenite (50ppm) and lead acetate (0.2%) individually, and in combination (As=25ppm+Pb=0.1%) via drinking water throughout perinatal period (GD 6 to PND 21). MiADMSA (50mg/kg, orally through gavage) was given for three consecutive days to the PND 18 pups (i.e., PND 18 to PND 20). Exposure to metal mixture resulted in a significant decrease in the activity levels of antioxidant enzymes such as manganese-superoxide dismutase (Mn-SOD), Cu/Zn superoxide dismutase (Cu/Zn-SOD), catalase (CAT) and glutathione peroxidase (GPx) while the malondialdehyde (MDA) levels and mRNA expression levels of caspase-3 and caspase-9 were significantly increased in all the three brain regions. The observed alterations were greater with exposure to metal mixture than individual metals (As or Pb) and the changes were more prominent at PND 28 and greater in cerebral cortex than hippocampus and cerebellum. Interestingly, chelation therapy with MiADMSA showed significant recovery in antioxidant enzymes, lipid peroxidation and gene expression levels of caspase-3 and caspase-9. From these findings, it can be concluded that combined exposure to As and Pb showed an additive effect on antioxidant enzymes than individual metal exposure and chelation therapy with MiADMSA significantly reversed the As and Pb induced apoptosis and oxidative stress, a major contributing factor to neurotoxicity.

Apoptosis of U937 Cells Induced by Hematoporphyrin Monomethyl Ether-Mediated Sonodynamic Action

The present study aims to investigate apoptosis of U937 cells induced by hematoporphyrin monomethyl ether (HMME)-mediated sonodynamic therapy (SDT). HMME concentration was kept constant at 10 μg/mL. Tumor cells suspended in serum-free RPM1640 were exposed to ultrasound at 1.1 MHz for up to 60 seconds with an intensity of 1 W/cm(2) in the presence and absence of HMME. The viability of cells was determined by the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltertrazolium bromide tetrazolium (MTT) test. Apoptosis was analyzed using a flow cytometer with Annexin V-PE/7-ADD staining as well as fluorescence microscopy with 4'-6-diamidino-2-phenylindole (DAPI) staining. The DNA damage of U937 cells, intracellular reactive oxygen species (ROS), and mitochondria membrane potential (MMP) were also analyzed by a flow cytometer after exposures. Western blotting and reverse transcriptase-polymerase chain reaction were used to analyze the protein and mRNA expression level of caspase-3 and poly(ADP-ribose) polymerase (PARP). Fluorescent imaging revealed that HMME mainly localized in the mitochondria. MTT assay showed 55.6% of cell survival at 4 hours post-SDT. Flow cytometric analysis displayed a significant increase in the early- and late-apoptotic cell populations (35.6%) of U937 cells by HMME-mediated SDT. Compared with the control, ultrasound-alone, and HMME-alone groups, the intracellular ROS and the MMP loss were greatly increased in the combined SDT group. Obvious nuclear condensation was also found with DAPI staining, and the DNA fragment increased to 33.9% at 2 hours post-SDT treatment. Immunofluorescent staining indicated obvious Bax translocation after SDT. Western blot showed visible enhancement of caspase-3 and PARP cleavage. In addition, caspase-3 and PARP mRNA expression of U937 cells increased remarkably after SDT treatment. The findings demonstrated that HMME-mediated sonodynamic action (HMME-SDT) significantly induced apoptosis of U937 cells, suggesting that HMME may be a good sonosensitizer, and HMME-SDT might be a potential therapeutic strategy for cancer treatment.

Ursolic acid induces apoptosis via Akt/NF-κB signaling suppression in T24 human bladder cancer cells

The Akt/NF-κB pathway is involved in numerous anti‑apoptotic and drug resistance events which occur in various types of bladder cancer. The present study investigated the role of ursolic acid in the regulation of anti-apoptotic Akt and NF-κBp65 signaling. T24 human bladder cancer cells were treated with ursolic acid at final concentrations of 12.5, 25 or 50µmol/l for 48h. Quantitative PCR (qPCR) and western blotting were performed to detect mRNA and protein expression, respectively. The results showed that anti-apoptotic phospho-Akt1 (pAkt1), phospho-IκBα (pIκBα), NF-κBp65 and Bcl-2 were inhibited and pro-apoptotic caspase-3 was upregulated in a dose‑dependent manner. A 50µmol/l dose of ursonic acid decreased the mRNA expression levels of anti-apoptotic NF-κBp65 and Bcl-2 0.17 (8.9/52.6)-fold and 0.22 (9.5/42.3)‑fold, respectively. The pro-apoptotic caspase-3mRNA expression levels were upregulated 4.78(38.7/8.1)-fold. The anti-apoptotic pAkt1, pIκBα, NF-κBp65 and Bcl-2 protein levels were downregulated to 5.1 (blot grayscales vs. control at 32.3), 3.2 (vs.24.2), 8.5 (vs.45.1) and 9.2 (vs.40.3). The protein levels of pro-apoptotic caspase-3 were upregulated to 20.7 (vs.4.7). The proliferative activity of T24cells treated with 12.5, 25.0 and 50.0µmol/l ursolic acid was significantly reduced compared with that of control cells (83.8, 56.2 and 31.5 vs. 97.6%, respectively, P<0.05 for each). In conclusion, ursolic acid is important in inducing apoptosis via the suppression of Akt/NF-κB signaling in T24 human bladder cancer cells and this occurs in a dose-dependent manner. Ursolic acid may therefore serve as a naturally occurring candidate drug for the prevention and treatment of bladder cancer.

Effects of trichloroethylene on hepatotoxicity in cytochrome 2E1-silenced hepatocytes

To prepare cytochrome (CYP)2E1-silenced hepatocytes by lentivirus-mediated RNA interference technology and to investigate the hepatotoxicity of trichloroethylene (TCE) in CYP2E1-silenced hepatocytes. Short hairpin RNA fragments were designed and synthesized and were then ligated into the lentiviral vector; single colonies were screened; the plasmid was extracted after PCR and sequence identification and then transferred into L02 hepatocytes; the CYP2E1-silenced hepatocytes were selected; real-time quantitative PCR and Western blot were used to evaluate the interference effects. The obtained CYP2E1-silenced hepatocytes, as well as normal L02 hepatocytes, were treated with TCE (0, 0.25, 0.50, 1.00, 2.00, and 4.00 mmol/L). The cell viability and half maximal inhibitory concentration (IC50) of TCE were measured; the apoptotic rate of cells was measured by flow cytometry; the mRNA expression levels of apoptosis genes and oncogenes were measured by real-time quantitative PCR. The IC50s of TCE for L02 hepatocytes and CYP2E1-silenced hepatocytes were 15.1 mmol/L and 23.6 mmol/L, respectively. The apoptotic rate increased as the dose of TCE rose in the two types of cells; the CYP2E1-silenced hepatocytes hada significantly lower apoptotic rate than L02 hepatocytes when they were exposed to 2.0 and 4.0 mmol/L TCE (P < 0.05 or P < 0.01). The mRNA expression level of bcl-2 (anti-apoptosis gene) in CYP2E1-silenced hepatocytes was 15% ∼ 60% higher than that in L02 hepatocytes (P < 0.01), while the mRNA expression levels of caspase-3 and caspase-9 (apoptosis genes) in CYP2E1-silenced hepatocytes were 30% ∼ 60% lower than those in L02 hepatocytes (P < 0.01). The mRNA expression level of p53 (cancer suppressor gene) in CYP2E1-silenced hepatocytes was 81 - 278% higher than that in L02 hepatocytes (P < 0.01), while the mRNA expression levels of c-fos and k-ras (oncogenes) in CYP2E1-silenced hepatocytes were 20-68% lower than those in L02 hepatocytes (P < 0.01). CYP2E1-silenced cells can be successfully prepared by lentivirus-mediated RNA interference technology. Silencing CYP2E1 gene can reduce the hepatotoxicity of TCE and inhibit the expression of some apoptosis genes and oncogenes, suggesting that CYP2E1 gene plays an important role in TCE metabolism and is related to the hepatotoxicity of TCE.

Human Neonatal Neutrophils Are Resistant to Apoptosis with Lower Caspase-3 Activity

The incidence of neonatal inflammatory diseases remains high despite improved strategies for dealing with infection. Neutrophils are believed to play a significant role in neonatal inflammatory diseases due to their secretion of harmful mediators. Neutrophils rapidly undergo apoptosis following activation; dysregulation of the neutrophil apoptotic pathway may be an underlying mechanism of neonatal inflammatory disorders. In this study, we determined whether neonatal neutrophils are intrinsically resistant to apoptosis relative to their adult counterparts. Twelve healthy full-term newborn infants and 12 healthy adult volunteers, aged 20 to 45 years, were enrolled in this study. Neutrophils were isolated from umbilical cord blood or fresh venous peripheral blood, and neutrophils of each subject were cultured for up to 48 hours. Flow cytometric analysis revealed that spontaneous apoptosis of adult neutrophils increased with culture time (23% ± 2% at 12 h, 49% ± 4% at 24 h and 76% ± 3% at 48 h), whereas the frequency of apoptosis was significantly lower in neutrophils from neonates (9% ± 2% at 12 h, 30% ± 4% at 24 h and 49% ± 3% at 48 h) (p < 0.01 for each time point). Importantly, the expression levels of caspase-3 mRNA and a precursor form of caspase-3 protein were lower in neonatal neutrophils than adult neutrophils, as judged by RT-PCR and Western blot analyses. Moreover, caspase-3 activity was lower in neonatal neutrophils, compared to adult neutrophils. These findings suggest that neonatal neutrophils are intrinsically apoptosis-resistant, which may be due to the low expression of caspase-3.

The effect of mild hypothermia on mice cerebral ischemia-reperfusion nerve cell apoptosis

Objective To observe the treatment effect of mild hypothermia on cerebral ischemiareperfusion nerve cell apoptosis, and study its mechanism from the molecular level. Methods SD rats were employed to produce cerebral ischemia-reperfusion injury model, then were divided into mild-hypothermia treatment group and control group. The cell apoptosis was detected by TUNEL mothod, during the mild hypothermia 24 h, 48 h, 72 h, and 24 h, 48 h, 72 h, 96 h and 120 h after rewarming, and RT-PCR method was used to detect the mRNA expression apoptosis gene Caspase-3, Fas and Bcl-2 at all the above time points. Results The apoptosis cell number during mild hypothermia therapy in mild hypothermia group was less than the control group (P＜0. 05), then increased gradually after rewarming, till to 72 h there was no significant difference between two groups (P ＞ 0. 05). And the mRNA expression level of Caspase-3, Fas and Bcl-2 during mild hypothermia therapy in mild hypothermia group were lower than the control group (P ＜ 0. 05), and increased with rewarming, till to 72 h after rewarming there was no significant difference between two groups (P＞0.05). Conclusion Mild hypothermia can reduce the number of nerve cell apoptosis in the process of cerebral ischemia and reperfusion, but the apoptosis rate rise rapidly with rewarming, it suggests that nerve cells will be rescued as soon as possible during mild hypothermia. Key words: Hypothermia therapy; Apoptosis; Gene expression; Rats

Regulation of apoptosis in nucleus pulposus cells by optimized exogenous Bcl‐2 overexpression

Although the etiology of intervertebral disc degeneration is poorly understood, one possible approach to regulate the process of intervertebral disc degeneration may include the inhibition of apoptosis. We investigated the anti-apoptotic effects of bcl-2 in nucleus pulposus cells to enhance disc cell survival. Rat nucleus pulposus cells were transfected in vitro with a codon optimized rat bcl-2 gene. Forty-eight hours after transfection, cells were cultured in serum-deprived medium. After serum withdrawal, the cells were evaluated for bcl-2 protein levels and cell apoptosis. To investigate the effects of bcl-2 overexpression on the final apoptotic pathways and on basic genes important for nucleus pulposus homeostasis, mRNA levels of caspase-3, type II collagen, and aggrecan were also quantified. Nucleus pulposus cells were successfully transfected with codon optimized bcl-2 gene, which effectively reduced serum starvation-induced cell apoptosis. Overexpression of bcl-2 also reduced the mRNA expression level of caspase-3. mRNA levels of type II collagen and aggrecan were significantly higher in bcl-2 transfected groups compared to control plasmid vector groups after serum withdrawal. We firstly showed that bcl-2 overexpression in intervertebral disc cells was effective in preventing in vitro apoptotic cell death, indicating the potential advantages of this therapeutic approach in regulating disc degeneration.

Distinct Regulation of Intrinsic Apoptosis in Benign and Malignant Thyroid Tumours

Aberrations in the control of apoptosis represent a central feature of thyroid carcinogenesis. However, little is known about the regulation of components of the intrinsic apoptosis pathway in the thyroid. Using a real-time PCR approach we investigated the mRNA expression levels of Caspase3, Caspase3 s, xIAP, Bad, and beta-actin in a panel of 79 thyroid tumours. Additionally, we assessed the activation status of Caspase3 by immunohistochemistry. In the present study, we provide first evidence for a deregulation of the intrinsic apoptosis pathway on the transcriptional and post-transcriptional level. Thus, malignant thyroid tumours revealed a significant downregulation of the proapoptotic Bad. In contrast Caspase3 s, an alternative splice variant of Caspase3 with anti-apoptotic characteristics, was upregulated in follicular and anaplastic cancers. Moreover, papillary thyroid tumours revealed a significant upregulation of Caspase3 mRNA. On the post-translational level, thyroid malignancies featured an impairment in the activation of Caspase3, since activated Caspase3 accumulated exclusively in the cytoplasm of thyroid cancer cells, whereas follicular adenoma and normal thyroid tissues showed no cytoplasmatic but nuclear Caspase3 distribution. Further knowledge on apoptosis-deregulation during thyroid carcinogenesis might confer diagnostic and therapeutic benefits in the management of thyroid cancer.

Hepatocyte Growth Factor‐Modulated Rat Leydig Cell Functions

Hepatocyte growth factor (HGF) regulates many cellular functions acting through c-Met, its specific tyrosine kinase receptor. We previously reported that in prepuberal rats HGF is secreted by the peritubular myoid cells during the entire postnatal testicular development and by the Sertoli cells only at puberty. We have also demonstrated that germ cells at different stages of development express c-Met and that HGF modulates germ cell proliferation and apoptosis. In the present article, we extend our study to the interstitial compartment of the testis and demonstrate that the c-Met protein is present on Leydig cells. The receptor is functionally active as demonstrated by the detected effects of HGF. We report in this article that HGF significantly increases the amount of testosterone secreted by the Leydig cells and decreases the number of Leydig cells undergoing apoptosis. The antiapoptotic effect of HGF is mediated by caspase-3 activity because the amount of the active fragment of the enzyme is decreased in Leydig cells cultured in the presence of HGF. However, treatment with the growth factor does not modify the expression levels of caspase-3 mRNA. These data indicate that HGF regulates the functional activities of Leydig cells. Interestingly, the steroidogenetic activity of the cells is increased by HGF in cultured explants of testicular tissues as well as the antiapoptotic effect of HGF. Therefore, our data indicate that HGF has a crucial role in the regulation of male fertility.

Effects of Panax notoginseng saponins on mRNA expressions of interleukin-1β, its correlative factors and cysteinyl-aspartate specific protease after cerebral ischemia-reperfusion in rats

To investigate the effects of Panax notoginseng saponins (PNS) on mRNA expressions of interleukin-1 beta (IL-1 beta), interleukin-1 receptor type I (IL-1RI), interleukin-1 receptor antagonist (IL-1ra), intercellular adhesion molecule-1 (ICAM-1), cysteinyl-aspartate specific protease-1 (caspase-1), caspase-3 and caspase-8 after cerebral ischemia-reperfusion in rats. Focal cerebral ischemia reperfusion in rats was induced by the method of nylon monofilament via the internal carotid artery. PNS was administered intraperitoneally respectively five minutes before cerebral ischemia and twelve hours after cerebral ischemia. After cerebral ischemia for two hours followed by reperfusion for twenty two hours, the mRNA expressions of IL-1 beta, IL-1RI, IL-1ra, ICAM-1, caspase-1, caspase-3 and caspase-8 in brain tissue were determined by reverse transcription polymerase chain reaction assay. After cerebral ischemia for two hours followed by reperfusion for twenty two hours, the mRNA expression levels of IL-1 beta, IL-1RI, IL-1ra, ICAM-1, caspase-1, caspase-3 and caspase-8 in brain tissue in the untreated group were obviously elevated as compared to those in the sham-operation group (P<0.05 or P<0.01). The mRNA expression levels of IL-1 beta, IL-1RI, IL-1ra in brain tissue in the PNS group were lower than those in the untreated group, but higher than those in the sham-operation group, and without statistical differences as compared to those in the sham-operation group and in the untreated group (P>0.05). The mRNA expression level of caspase-3 in brain tissue in the PNS group was significantly lower than that in the untreated group (P<0.05), but PNS had no effect on the mRNA expression levels of ICAM-1, caspase-1 and caspase-8 in brain tissue. PNS can inhibit the mRNA expression of caspase-3, slightly inhibit the mRNA expressions of IL-1 beta and its correlative inflammatory factors in brain tissue. The protective effects of PNS on cerebral injury induced by ischemia-reperfusion may be related to inhibiting the mRNA expressions of caspase-3, IL-1 beta and its correlative inflammatory factors in brain tissue.

Elevated caspase-3 and Fas mRNA expression in jejunum of adult rats during subclinical zinc deficiency

The programmed cell death—so-called apoptosis—is a physiological process occurring in all multicellular organisms to control cell-number homeostasis. Nevertheless, increase of apoptotic cell death in different organs can lead to pathological alterations. As zinc is a potent inhibitor of apoptosis, we investigated the influence of zinc deficiency on mRNA expression levels of caspase-3 and Fas in adult rats. For this purpose, 24 adult rats fed a Zn-deficient diet for up to 29 days were compared to seven animals in the control group. After 1, 2, 4, 7, 11, 16, 22 and 29 days of treatment three animals were sacrificed ( n=24). Total RNA extraction from thymus, liver, jejunum and colon was carried out. Samples were reverse transcribed and subjected to real-time PCR. Relative quantification of caspase-3 and Fas mRNA expression was achieved on the basis of normalisation by glycerolaldehyd-3-phosphate-dehydrogenase mRNA expression levels in all samples. In jejunum, up to day 11 the relative mRNA expression of the respective genes decreased. A significant increase in caspase-3 and Fas expression was found from day 11 of zinc deficiency onward. In contrast, mRNA expression in liver and colon remained unaffected, whereas thymus showed a slight but not significant increase in the expression of these genes. This study provides the first evidence that even moderate zinc deficiency in an adult, non-growing rat model is able to elevate mRNA expression levels of factors involved in early stages of apoptosis.