Inhaled H2 or CO2 Do Not Augment the Neuroprotective Effect of Therapeutic Hypothermia in a Severe Neonatal Hypoxic-Ischemic Encephalopathy Piglet Model.

Hypoxic-ischemic encephalopathy (HIE) is still a major cause of neonatal death and disability as therapeutic hypothermia (TH) alone cannot afford sufficient neuroprotection. The present study investigated whether ventilation with molecular hydrogen (2.1% H2) or graded restoration of normocapnia with CO2 for 4 h after asphyxia would augment the neuroprotective effect of TH in a subacute (48 h) HIE piglet model. Piglets were randomized to untreated naïve, control-normothermia, asphyxia-normothermia (20-min 4%O2-20%CO2 ventilation; Tcore = 38.5 °C), asphyxia-hypothermia (A-HT, Tcore = 33.5 °C, 2-36 h post-asphyxia), A-HT + H2, or A-HT + CO2 treatment groups. Asphyxia elicited severe hypoxia (pO2 = 19 ± 5 mmHg) and mixed acidosis (pH = 6.79 ± 0.10). HIE development was confirmed by altered cerebral electrical activity and neuropathology. TH was significantly neuroprotective in the caudate nucleus but demonstrated virtually no such effect in the hippocampus. The mRNA levels of apoptosis-inducing factor and caspase-3 showed a ~10-fold increase in the A-HT group compared to naïve animals in the hippocampus but not in the caudate nucleus coinciding with the region-specific neuroprotective effect of TH. H2 or CO2 did not augment TH-induced neuroprotection in any brain areas; rather, CO2 even abolished the neuroprotective effect of TH in the caudate nucleus. In conclusion, the present findings do not support the use of these medical gases to supplement TH in HIE management.

The Role of Caspase-3, Apoptosis-Inducing Factor, and B-cell Lymphoma-2 Expressions in Term Premature Rupture of Membrane.

OBJECTIVE: To determine the role of caspase-3, apoptosis-inducing factor (AIF), and B-cell lymphoma-2 (Bcl-2) expressions in term premature rupture of membrane (PROM). METHODS: An analytic observational study with case-control design was conducted, involving 52 subjects (37-42 weeks of gestation) who were divided into 2 groups: 26 cases of term delivery with PROM, and 26 controls of term delivery without PROM. The expressions of caspase-3, AIF, and Bcl-2 in the amniotic membrane were determined by immunohistochemistry. Data were analyzed using the chi-squared test. The risk of PROM was expressed by odds ratio (OR). RESULTS: There were no significant differences in age, parity and body mass index between the two groups (p > 0.05). High caspase-3 and AIF expressions increased the risk of PROM 17.64 times (OR = 17.64; 95% CI = 4.44-70.07; p = 0.001) and 9.45 times (OR = 9.45; 95% CI= 2.62-34.07; p = 0.001), respectively, while low Bcl-2 expression increased 10.39 times (OR = 10.39; 95% CI = 2.73-39.56; p = 0.001)the risk of PROM . CONCLUSION: High caspase-3 and AIF expressions and low Bcl-2 expression were risk factors for term PROM. Caspase-dependent and independent pathways of apoptosis were involved in the mechanism of PROM in term pregnancy.

The role of caspase-3, apoptosis-inducing factor, and B-cell lymphoma-2 expressions in term premature rupture of membrane

Abstract Objective To determine the role of caspase-3, apoptosis-inducing factor (AIF), and Bcell lymphoma-2 (Bcl-2) expressions in term premature rupture of membrane (PROM). Methods An analytic observational study with case-control design was conducted, involving 52 subjects (37-42 weeks of gestation) who were divided into 2 groups: 26 cases of term delivery with PROM, and 26 controls of term delivery without PROM. The expressions of caspase-3, AIF, and Bcl-2 in the amniotic membrane were determined by immunohistochemistry. Data were analyzed using the chi-squared test. The risk of PROM was expressed by odds ratio (OR). Results There were no significant differences in age, parity and body mass index between the two groups (p > 0.05). High caspase-3 and AIF expressions increased the risk of PROM 17.64 times (OR = 17.64; 95% CI = 4.44-70.07; p = 0.001) and 9.45 times (OR = 9.45; 95% CI= 2.62-34.07; p = 0.001), respectively, while low Bcl-2 expression increased 10.39 times (OR = 10.39; 95% CI = 2.73-39.56; p = 0.001)the risk of PROM . Conclusion High caspase-3 and AIF expressions and low Bcl-2 expression were risk factors for term PROM. Caspase-dependent and independent pathways of apoptosis were involved in the mechanism of PROM in term pregnancy.

A brain-specific isoform of apoptosis-inducing factor 2 attenuates ischemia-induced oxidative stress in HT22 cells.

Apoptosis-inducing factor (AIF) is a family of conserved mitochondrial flavoproteins that have both vital and lethal functions in cells. The function and regulation of AIF-1, the original described and most abundant isoform, has been extensively studied, whereas three other AIF isoforms have not been further characterized. Here, we investigated the role of AIF-2, a brain-specific isoform of AIF, in an in vitro ischemia model in neuronal HT22 cells. We showed that AIF-2 was constitutively expressed in HT22 cells, and the oxygen and glucose deprivation (OGD) did not alter AIF-2 expression. Downregulation of AIF-2 with specific siRNA aggravated OGD-induced lactate dehydrogenase (LDH) release, apoptosis and loss of cell viability, whereas overexpression of AIF-2 through lentivirus transfection exerted the opposite effects. In OGD-treated cells, AIF-2 overexpression promoted the endogenous antioxidant enzyme activities, preserved mitochondrial membrane potential (MMP), inhibited cytochrome c release, and thereby prevented reactive oxygen species (ROS) generation and lipid peroxidation. In addition, AIF-2 significantly prevented the OGD-induced AIF-1 translocation from cytoplasm to the nuclei. In view of these considerations, AIF-2 might represent an ideal strategy to avoid AIF-1 associated neurotoxicity, and could be tested against brain ischemia in animal models.

Neuronal Effects of Sugammadex in combination with Rocuronium or Vecuronium.

Rocuronium (ROC) and Vecuronium (VEC) are the most currently used steroidal non-depolarizing neuromuscular blocking (MNB) agents. Sugammadex (SUG) rapidly reverses steroidal NMB agents after anaesthesia. The present study was conducted in order to evaluate neuronal effects of SUG alone and in combination with both ROC and VEC. Using MTT, CASP-3 activity and Western-blot we determined the toxicity of SUG, ROC or VEC in neurons in primary culture. SUG induces apoptosis/necrosis in neurons in primary culture and increases cytochrome C (CytC), apoptosis-inducing factor (AIF), Smac/Diablo and Caspase 3 (CASP-3) protein expression. Our results also demonstrated that both ROC and VEC prevent these SUG effects. The protective role of both ROC and VEC could be explained by the fact that SUG encapsulates NMB drugs. In BBB impaired conditions it would be desirable to control SUG doses to prevent the excess of free SUG in plasma that may induce neuronal damage. A balance between SUG, ROC or VEC would be necessary to prevent the risk of cell damage.

Roles of hypoxia inducible factor-1α in the temporomandibular joint.

OBJECTIVE: Temporomandibular joint osteoarthritis (TMJ-OA) is a degenerative disease characterized by permanent cartilage loss. Articular cartilage is maintained in a low-oxygen environment. The chondrocyte response to hypoxic conditions involves expression of hypoxia inducible factor 1α (HIF-1α), which induces chondrocytes to increase expression of vascular endothelial growth factor (VEGF). Here, we investigated the role of HIF-1α in mechanical load effects on condylar cartilage and subchondral bone in heterozygous HIF-1α-deficient mice (HIF-1α+/-). DESIGN: Mechanical stress was applied to the TMJ of C57BL/6NCr wild-type (WT) and HIF-1α+/- mice with a sliding plate for 10 days. Histological analysis was performed by HE staining, Safranin-O/Fast green staining, and immunostaining specific for articular cartilage homeostasis. RESULTS: HIF-1α+/- mice had thinner cartilage and smaller areas of proteoglycan than WT controls, without and with mechanical stress. Mechanical stress resulted in prominent degenerative changes with increased expression of HIF-1α, VEGF, and the apoptosis factor cleaved Caspase-3 in condylar cartilage. CONCLUSION: Our results indicate that HIF-1α may be important for articular cartilage homeostasis and protective against articular cartilage degradation in the TMJ under mechanical stress condition, therefore HIF-1α could be an important new therapeutic target in TMJ-OA.

Inhibition of calpain on oxygen glucose deprivation-induced RGC-5 necroptosis.

The purpose of this study was to investigate the effect of inhibition of calpain on retinal ganglion cell-5 (RGC-5) necroptosis following oxygen glucose deprivation (OGD). RGC-5 cells were cultured in Dulbecco's-modified essential medium and necroptosis was induced by 8-h OGD. PI staining and flow cytometry were performed to detect RGC-5 necrosis. The calpain expression was detected by Western blotting and immunofluorescence staining. The calpain activity was tested by activity detection kit. Flow cytometry was used to detect the effect of calpain on RGC-5 necroptosis following OGD with or without N-acetyl-leucyl-leucyl-norleucinal (ALLN) pre-treatment. Western blot was used to detect the protein level of truncated apoptosis inducing factor (tAIF) in RGC-5 cells following OGD. The results showed that there was an up-regulation of the calpain expression and activity following OGD. Upon adding ALLN, the calpain activity was inhibited and tAIF was reduced following OGD along with the decreased number of RGC-5 necroptosis. In conclusion, calpain was involved in OGD-induced RGC-5 necroptosis with the increased expression of its downstream molecule tAIF.

An Ingenol Derived from Euphorbia kansui Induces Hepatocyte Cytotoxicity by Triggering G0/G1 Cell Cycle Arrest and Regulating the Mitochondrial Apoptosis Pathway in Vitro.

Natural product lingenol, a purified diterpenoid compound derived from the root of Euphorbia kansui, exerts serious hepatotoxicity; however, the molecular mechanisms remain to be defined. In the present study, cell counting Kit-8 (CCK-8), inverted phase contrast microscope and flow cytometry were used to demonstrate that lingenol significantly inhibited L-O2 cells proliferation, and induced cell cycle arrest and apoptosis. Moreover, the results investigated that lingenol markedly disrupted mitochondrial functions by high content screening (HCS). In addition, the up-regulation of cytochrome c, AIF and Apaf-1 and activation of caspases were found in L-O2 cells detected by Western blotting and ELISA assay, which was required for lingenol activation of cytochrome c-mediated caspase cascades and AIF-mediated DNA damage. Mechanistic investigations revealed that lingenol significantly down-regulated the Bcl-2/Bax ratio and enhanced the reactive oxygen species (ROS) in L-O2 cells. These data collectively indicated that lingenol modulation of ROS and Bcl-2/Bax ratio led to cell cycle arrest and mitochondrial-mediated apoptosis in L-O2 cells in vitro. All of these results will be helpful to reveal the hepatotoxicity mechanism of Euphorbia kansui and to effectively guide safer and better clinical application of this herb.

Basal metabolic state governs AIF-dependent growth support in pancreatic cancer cells.

BACKGROUND: Apoptosis-inducing factor (AIF), named for its involvement in cell death pathways, is a mitochondrial protein that regulates metabolic homeostasis. In addition to supporting the survival of healthy cells, AIF also plays a contributory role to the development of cancer through its enzymatic activity, and we have previously shown that AIF preferentially supports advanced-stage prostate cancer cells. Here we further evaluated the role of AIF in tumorigenesis by exploring its function in pancreatic cancer, a disease setting that most often presents at an advanced stage by the time of diagnosis. METHODS: A bioinformatics approach was first employed to investigate AIF mRNA transcript levels in pancreatic tumor specimens vs. normal tissues. AIF-deficient pancreatic cancer cell lines were then established via lentiviral infection. Immunoblot analysis was used to determine relative protein quantities within cells. Cell viability was measured by flow cytometry; in vitro and Matrigel™ growth/survival using Coulter™ counting and phase contrast microscopy; and glucose consumption in the absence and presence of Matrigel™ using spectrophotometric methods. RESULTS: Archival gene expression data revealed a modest elevation of AIF transcript levels in subsets of pancreatic tumor specimens, suggesting a possible role in disease progression. AIF expression was then suppressed in a panel of five pancreatic cancer cell lines that display diverse metabolic phenotypes. AIF ablation selectively crippled the growth of cells in vitro in a manner that directly correlated with the loss of mitochondrial respiratory chain subunits and altered glucose metabolism, and these effects were exacerbated in the presence of Matrigel™ substrate. This suggests a critical metabolic role for AIF to pancreatic tumorigenesis, while the spectrum of sensitivities to AIF ablation depends on basal cellular metabolic phenotypes. CONCLUSIONS: Altogether these data indicate that AIF supports the growth and survival of metabolically defined pancreatic cancer cells and that this metabolic function may derive from a novel mechanism so far undocumented in other cancer types.

Pre-administration of BAX-inhibiting peptides decrease the loss of the nigral dopaminergic neurons in rats.

AIMS: In this study, we investigated the effects of Bax-inhibiting peptide (Bip)-V5, an anti-apoptosis membrane-permeable peptide, on the 6-hydroxydopamine (6-OHDA) induced Parkinson's disease (PD) model rats. MATERIALS AND METHODS: Rats were randomly divided into five groups: Control, 6-OHDA only, Vehicle+6-OHDA, zVAD+6-OHDA, and V5+6-OHDA, that rats were preadministrated with different reagents before 6-OHDA administration. KEY FINDINGS: The result showed that intrastriatal preadministration of Bip-V5 significantly decreased the amphetamine-induced rotation of the 6-OHDA model rats and the loss of the nigral dopaminergic (DA) neurons. Moreover, Bip-V5 intrastriatal preadministration not only significantly decreased the expression of activated caspase 9 and activated caspase 3 but also decreased the enhanced expression of AIF and its nuclear translocation in the SNpc. The results in our study provide the first experimental evidence that both caspase-dependent and AIF-dependent apoptosis pathways are involved in the loss of the nigral DA neurons caused by intrastriatal administration of 6-OHDA, and intrastriatal preadministration of Bip-V5 can inhibit the above two apoptosis pathways to protect the nigral DA neurons. SIGNIFICANCE: Our results provide a new idea that Bax-inhibiting peptide may be a promising preventive or therapeutic method for PD.

An Involvement of PI3-K/Akt Activation and Inhibition of AIF Translocation in Neuroprotective Effects of Undecylenic Acid (UDA) Against Pro-Apoptotic Factors-Induced Cell Death in Human Neuroblastoma SH-SY5Y Cells.

Undecylenic acid (UDA), a naturally occurring 11-carbon unsaturated fatty acid, has been used for several years as an economical antifungal agent and a nutritional supplement. Recently, the potential usefulness of UDA as a neuroprotective drug has been suggested based on the ability of this agent to inhibit µ-calpain activity. In order to verify neuroprotective potential of UDA, we tested protective efficacy of this compound against cell damage evoked by pro-apoptotic factors (staurosporine and doxorubicin) and oxidative stress (hydrogen peroxide) in human neuroblastoma SH-SY5Y cells. We showed that UDA partially protected SH-SY5Y cells against the staurosporine- and doxorubicin-evoked cell death; however, this effect was not connected with its influence on caspase-3 activity. UDA decreased the St-induced changes in mitochondrial and cytosolic AIF level, whereas in Dox-model it affected only the cytosolic AIF content. Moreover, UDA (1-40 µM) decreased the hydrogen peroxide-induced cell damage which was connected with attenuation of hydrogen peroxide-mediated necrotic (PI staining, ADP/ATP ratio) and apoptotic (mitochondrial membrane potential, caspase-3 activation, AIF translocation) changes. Finally, we demonstrated that an inhibitor of PI3-K/Akt (LY294002) but not MAPK/ERK1/2 (U0126) pathway blocked the protection mediated by UDA in all tested models of SH-SY5Y cell injury. These in vitro data point to UDA as potentially effective neuroprotectant the utility of which should be further validated in animal studies.

Antitumour activity of 3-nitropropionic acid from Phomopsis sp. and optimization of fermentation conditions.

UNLABELLED: In this study, 3-nitropropionic acid (3-NPA) was separated and purified from endophytic fungi belonging to Phomopsis sp. and its cytotoxicity was determined by MTT assay. Treatment with 3-NPA for 24 h resulted in a dose-dependent apoptosis in MCF-7 cells. Through quantitative detection of the genes that are closely related to the Bcl-2 signalling pathway, there was an increased expression of p53 and Bax and a decreased expression of Bcl-2, which indicated apoptosis in these cells. Meanwhile, the overexpression of PARA (poly ADP-ribose polymerase) and apoptosis inducing factor (AIF) also suggested that 3-NPA induced cellular apoptosis through a caspase-3-independent pathway in caspase-3-deficient MCF-7 cells. The fermentation condition was also improved to produce more 3-NPA: glucose as a carbon source and yeast extract as a nitrogen source, fermentation for 8 days at 32°C and a solution environment of pH 5·0. Under these conditions, the yield of 3-NPA was increased to 529 mg l(-1) compared with 410 mg l(-1) under traditional fermentation conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: 3-Nitropropionic acid is a mitochondrial inhibitor and has some useful bioactivities such as antibacterial activity. In this paper we found that 3-NPA also has obvious cytotoxicity, so we studied its antitumour activity and tried to determine the antitumour molecular mechanism, opening a new perspective for potential antitumour prodrug development. As 3-NPA is often obtained from natural products with a low yield, in order to overcome the disadvantage of an endophytic fungi source of 3-NPA, we optimized the fermentation conditions for 3-NPA in Phomopsis sp. to obtain the maximum production of 3-NPA.

Annonaceous acetogenin mimic AA005 induces cancer cell death via apoptosis inducing factor through a caspase-3-independent mechanism.

BACKGROUND: Annonaceous acetogenins are a family of natural products with antitumor activities. Annonaceous acetogenin mimic AA005 reportedly inhibits mammalian mitochondrial NADH-ubiquinone reductase (Complex I) and induces gastric cancer cell death. However, the mechanisms underlying its cell-death-inducing activity are unclear. METHODS: We used SW620 colorectal adenocarcinoma cells to study AA005 cytotoxic activity. Cell deaths were determined by Trypan blue assay and flow cytometry, and related proteins were characterized by western blot. Immunofluorescence and subcellular fractionation were used to evaluate AIF nuclear translocation. Reactive oxygen species were assessed by using redox-sensitive dye DCFDA. RESULTS: AA005 induces a unique type of cell death in colorectal adenocarcinoma cells, characterized by lack of caspase-3 activation or apoptotic body formation, sensitivity to poly (ADP-ribose) polymerase inhibitor Olaparib (AZD2281) but not pan-caspase inhibitor Z-VAD.fmk, and dependence on apoptosis-inducing factor (AIF). AA005 treatment also reduced expression of mitochondrial Complex I components, and leads to accumulation of intracellular reactive oxygen species (ROS) at the early stage. Blocking ROS formation significantly suppresses AA005-induced cell death in SW620 cells. Moreover, blocking activation of RIP-1 by necroptosis inhibitor necrotatin-1 inhibits AIF translocation and partially suppresses AA005-induced cell death in SW620 cells demonstrating that RIP-1 protein may be essential for cell death. CONCLUSIONS: AA005 may trigger the cell death via mediated by AIF through caspase-3 independent pathway. Our work provided new mechanisms for AA005-induced cancer cell death and novel clues for cancer treatment via AIF dependent cell death.

Nitro-oleic acid attenuates OGD/R-triggered apoptosis in renal tubular cells via inhibition of Bax mitochondrial translocation in a PPAR-γ-dependent manner.

BACKGROUND: Nitroalkene derivatives of oleic acid (OA-NO2) serve as high-affinity ligand for PPAR-γ, which regulates apoptosis, oxidation and inflammation and plays a central role in ischemia-reperfusion injury. In the present study, we elucidated the protective mechanisms of OA-NO2 against renal ischemia-reperfusion injury. METHODS: HK-2 cells were subjected to oxygen and glucose deprivation followed by re-oxygenation (OGD/R) to mimic renal ischemia-reperfusion injury. Cell apoptosis was analyzed by flow cytometry. Bax mitochondrial translocation, cytochrome c and apoptosis-inducing factor (AIF) cytosolic leakage and Akt/Gsk 3ß phosphorylation were evaluated by Western blotting. Bax activation was visualized by immunocytochemistry. GW9662 and siRNA transfection were employed to examine the involvement of PPAR-γ. RESULTS: OGD/R injury promoted mitochondrial translocation and activation of Bax, leakage of cytochrome c and AIF, subsequent caspase-3 activation, and eventually cell apoptosis. Pre-incubation with OA-NO2 (1.25 µM, 45min) inhibited Bax activation and blocked apoptotic cascade, while the protective effects were negated by GW9662 or PPAR-γ siRNA. Moreover, OA-NO2 restored Akt and Gsk 3ß phosphorylation in a PPAR-γ-dependent way. CONCLUSION: These findings suggest that OA-NO2 attenuates OGD/R-induced apoptosis by inhibiting Bax translocation and activation and the subsequent mitochondria-dependent apoptotic cascade in a PPAR-γ dependent manner.

Antitumor activity of irradiated riboflavin on human renal carcinoma cell line 786-O.

Riboflavin (vitamin B2) is a precursor for coenzymes involved in energy production, biosynthesis, detoxification, and electron scavenging. Previously, we demonstrated that irradiated riboflavin (IR) has potential antitumoral effects against human leukemia cells (HL60), human prostate cancer cells (PC3), and mouse melanoma cells (B16F10) through a common mechanism that leads to apoptosis. Hence, we here investigated the effect of IR on 786-O cells, a known model cell line for clear cell renal cell carcinoma (CCRCC), which is characterized by high-risk metastasis and chemotherapy resistance. IR also induced cell death in 786-O cells by apoptosis, which was not prevented by antioxidant agents. IR treatment was characterized by downregulation of Fas ligand (TNF superfamily, member 6)/Fas (TNF receptor superfamily member 6) (FasL/Fas) and tumor necrosis factor receptor superfamily, member 1a (TNFR1)/TNFRSF1A-associated via death domain (TRADD)/TNF receptor-associated factor 2 (TRAF) signaling pathways (the extrinsic apoptosis pathway), while the intrinsic apoptotic pathway was upregulated, as observed by an elevated Bcl-2 associated x protein/B-cell CLL/lymphoma 2 (Bax/Bcl-2) ratio, reduced cellular inhibitor of apoptosis 1 (c-IAP1) expression, and increased expression of apoptosis-inducing factor (AIF). The observed cell death was caspase-dependent as proven by caspase 3 activation and poly(ADP-ribose) polymerase-1 (PARP) cleavage. IR-induced cell death was also associated with downregulation of v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homologue (avian)/protein serine/threonine kinase B/extracellular signal-regulated protein kinase 1/2 (Src/AKT/ERK1/2) pathway and activation of p38 MAP kinase (p38) and Jun-amino-terminal kinase (JNK). Interestingly, IR treatment leads to inhibition of matrix metalloproteinase-2 (MMP-2) activity and reduced expression of renal cancer aggressiveness markers caveolin-1, low molecular weight phosphotyrosine protein phosphatase (LMWPTP), and kinase insert domain receptor (a type III receptor tyrosine kinase) (VEGFR-2). Together, these results show the potential of IR for treating cancer.

AIF downregulation and its interaction with STK3 in renal cell carcinoma.

Apoptosis-inducing factor (AIF) plays a crucial role in caspase-independent programmed cell death by triggering chromatin condensation and DNA fragmentation. Therefore, it might be involved in cell homeostasis and tumor development. In this study, we report significant AIF downregulation in the majority of renal cell carcinomas (RCC). In a group of RCC specimens, 84% (43 out of 51) had AIF downregulation by immunohistochemistry stain. Additional 10 kidney tumors, including an oxyphilic adenoma, also had significant AIF downregulation by Northern blot analysis. The mechanisms of the AIF downregulation included both AIF deletion and its promoter methylation. Forced expression of AIF in RCC cell lines induced massive apoptosis. Further analysis revealed that AIF interacted with STK3, a known regulator of apoptosis, and enhanced its phosphorylation at Thr180. These results suggest that AIF downregulation is a common event in kidney tumor development. AIF loss may lead to decreased STK3 activity, defective apoptosis and malignant transformation.

The effects of high thoracic epidural anesthesia on sympathetic activity and apoptosis in experimentally induced congestive heart failure.

OBJECTIVE: To evaluate the effect of high thoracic epidural analgesia (HTEA) in congestive heart failure (CHF). DESIGN: Rat model of CHF. SETTING: Harbin Medical University, Harbin, Heilongjiang, China. PARTICIPANTS: One hundred thirty-five rats. INTERVENTIONS: HTEA involved 5 times daily injections of 0.1% lidocaine at the T3-T4 level. MEASUREMENTS AND MAIN RESULTS: The authors examined myocardial norepinephrine (NE), angiotensin II (Ang II), endothelin-1 (ET1), and tumor necrosis factor-α (TNF-α) concentrations 2, 4, and 6 weeks after the start of HTEA. They also examined histologic changes in heart tissue and myocardial expression of apoptosis-inducing factor (AIF) and poly (ADP-ribose) polymerase (PARP). Sham rats were used as a control. In the time course, myocardial NE, Ang II, ET1, and TNF-α concentrations were significantly higher in the CHF group compared with the HTEA and sham groups (p< 0.05). Similarly, PARP and AIF protein expression levels were significantly higher in the CHF group compared with the HTEA and sham groups (p< 0.05). Microscopy revealed pronounced damage to myocardial cell structures in the CHF group; this damage clearly was reduced in the HTEA group. In addition, cardiac function evaluation indicated treatment with HTEA resulted in similar heart function as animals that did not have surgically induced CHF. CONCLUSIONS: The findings suggest that HTEA induces changes in sympathetic nervous system, renin-angiotensin system, endothelial, and inflammatory process activity involved in CHF.

Hepatocyte growth factor and HER2/neu downregulate expression of apoptosis-inducing factor in non-small cell lung cancer.

Our previous study showed that patients with advanced stages of non-small cell lung cancer (NSCLC) were frequently detected with upregulation of hepatocyte growth factor (HGF). In vitro, HGF reduced expression of apoptosis-inducing factor (AIF) and cisplatin sensitivity in NSCLC cells. The effect of HGF was via HGF receptor (c-MET) and the downstream effector, focal adhesion kinase (FAK). In this study, we determined the prognostic value of AIF in NSCLC patients. AIF expression was determined by immunohistochemistry and immunoblotting. Our data show that AIF expression was associated with better prognosis. Expression of AIF inversely correlated with that of positive NSCLC markers, e.g., dihydrodiol dehydrogenase (DDH), c-MET, short oncostatin M receptor (OSMRs), matrix metalloproteinase (MMP)-1, and HER2/neu, which were closely associated with drug resistance, tumor recurrence, metastasis and poor prognosis. Noteworthy, silence of HER2/neu gene expression increases AIF level and drug sensitivity. Addition of HGF inhibits AIF expression in HER2/neu-silenced cells. These results suggested that both HGF and HER2/neu affect drug resistance by regulating AIF expression in NSCLC.

[Construction and expression of the eukaryotic expression vector containing AIF gene].

OBJECTIVE: To construct the eukaryotic express vector containing apoptosis-inducing factor (AIF) gene and to study its expression in A549 cells. METHODS: According to the GenBank AIF mRNA sequence, specific primers to amplify AIF gene from lung carcinoma cell line A549 by RT-PCR was designed. The amplified AIF gene fragment was cloned into plasmid pUC-T by TA cloning, then double enzyme digestion and DNA sequencing were used to identifying the positive recombinant AIF-pUC-T. The target fragment was retrieved and cloned into the eukaryotic express vector pcDNA3.1(+). The positive recombinant AIF-pcDNA3.1(+) was transfected into A549 cells, and expression of AIF gene was verified by RT-PCR and Western blot. RESULTS: AIF target gene was successfully amplified and cloned into the pUC-T. The target fragment was retrieved and cloned into the eukaryotic express vector pcDNA3.1(+), and it was completely coincided with the AIF sequence in GenBank suggested by cells transfected with AIF-pcDNA3. 1(+) was much higher than that of control cells which was not transfected with AIF-pcDNA3.1(+). CONCLUSION: The AIF eukaryotic expression vector AIF-pcDNA3.1(+) is successfully constructed in A549 cells and it could be experimental foundations for further study of AIF gene.

Crude saponins from Platycodon grandiflorum induce apoptotic cell death in RC-58T/h/SA#4 prostate cancer cells through the activation of caspase cascades and apoptosis-inducing factor.

Saponins are a major active component of Platycodon grandiflorum (P. grandiflorum) and are known to induce apoptosis in metastatic prostate cancer cell lines. However, thus far, no research has been conducted on the anticancer activity of saponins in RC-58T/h/SA#4 primary prostate cancer cells. In this study, we show that the treatment of prostate cancer cells with saponins extracted from P. grandiflorum (SPG) inhibits cell proliferation in a dose-dependent manner. SPG significantly induced apoptotic cell death, resulting in an increase in the sub-G1 apoptotic cell population, apoptotic DNA fragmentation and morphological changes. Pre-treatment with a caspase inhibitor modestly attenuated the SPG-induced increase in the sub-G1 cell population, suggesting that caspases play a role in SPG-induced apoptosis. Moreover, SPG-induced apoptosis was associated with changes in caspase activity, the upregulation of the apoptotic protein, Bax and the downregulation of the anti-apoptotic protein, Bcl-2. Furthermore, the caspase-independent mitochondrial apoptosis factor, apoptosis-inducing factor (AIF) was upregulated following SPG treatment. These findings indicate that SPG exerts its anticancer effects on RC-58T/h/SA#4 primary prostate cancer cells through mitochondrial caspase-dependent and -independent apoptotic pathways.