Pro-apoptotic Bax Protein Research Articles

Proteins from Lignosus tigris with selective apoptotic cytotoxicity towards MCF7 cell line and suppresses MCF7-xenograft tumor growth.

BackgroundLignosus tigris, a recently discovered species of the unique Lignosus family, has been traditionally used by the indigenous communities in Peninsular Malaysia to treat various ailments and as an alternative medicine for cancer treatment. The L. tigris cultivar sclerotia (Ligno TG-K) was found to contain numerous bioactive compounds with beneficial biomedicinal properties and the sclerotial extract exhibited potent antioxidant activity. However, the anticancer property of the Ligno TG-K including in vitro and in vivo antitumor effects as well as its anticancer active compounds and the mechanisms has yet to be investigated.MethodsThe cytotoxicity of the Ligno TG-K against human breast (MCF7), prostate (PC3) and lung (A549) adenocarcinoma cell lines was evaluated using MTT cytotoxicity assay. The cytotoxic mechanisms of the active high molecular weight proteins (HMWp) fraction were investigated through detection of caspases activity and apoptotic-related proteins expression by Western blotting. The in vivo antitumor activity of the isolated HMWp was examined using MCF7 mouse xenograft model. Shotgun LC-MS/MS analysis was performed to identify the proteins in the HMWp.Results and DiscussionCold water extract of the sclerotia inhibited proliferation of MCF7, A549 and PC3 cells with IC50 ranged from 28.9 to 95.0 µg/mL. Bioassay guided fractionation of the extract revealed that HMWp exhibited selective cytotoxicity against MCF7 cells via induction of cellular apoptosis by the activation of extrinsic and intrinsic signaling pathways. HMWp activated expression of caspase-8 and -9 enzymes, and pro-apoptotic Bax protein whilst inhibiting expression of tumor survivor protein, Bcl-2. HMWp induced tumor-cell apoptosis and suppressed growth of tumor in MCF-7 xenograft mice. Lectins, serine proteases, RNase Gf29 and a 230NA deoxyribonuclease are the major cytotoxic proteins that accounted for 55.93% of the HMWp.ConclusionThe findings from this study provided scientific evidences to support the traditional use of the L. tigris sclerotia for treatment of breast cancer. Several cytotoxic proteins with high abundance have been identified in the HMWp of the sclerotial extract and these proteins have potential to be developed into new anticancer agents or as adjunct cancer therapy.

Effect of methoxy stilbenes\u2014analogs of resveratrol\u2014on the viability and induction of cell cycle arrest and apoptosis in human myeloid leukemia cells

The present study aimed to evaluate the cytotoxicity and its mechanism of five synthetic methoxy stilbenes, namely 3,4,4ʹ-trimethoxy, 3,4,2ʹ-trimethoxy, 3,4,2ʹ,4ʹ-tetramethoxy, 3,4,2ʹ,6ʹ-tetramethoxy, and 3,4,2ʹ,4ʹ,6ʹ-pentamethoxy-trans-stilbenes (MS), in comparison with resveratrol (RSV). Human promyelocytic (HL-60) and monocytic leukemia (THP-1) cells were treated with the tested compounds for 24 h, and cytotoxicity, cell cycle distribution, and apoptosis were evaluated. Significant differences were found in the susceptibility of these cell lines to all stilbenes, including RSV. The THP-1 cells were more resistant to cytotoxic activity of these compounds than HL-60 cells. Among the tested stilbenes, 3,4,4ʹ-tri-MS and 3,4,2ʹ,4ʹ-tetra-MS exhibited higher cytotoxicity toward both cell lines than RSV and the other methoxy stilbenes. This activity might be related to cell cycle arrest at the G2/M phase and induction of apoptosis. In this regard, 3,4,4ʹ-tri-MS and 3,4,2ʹ,4ʹ-tetra-MS at highest concentrations increased the p53 protein level particularly in HL-60 cells. Moreover, treatment with these derivatives increased the ratio of the proapoptotic Bax protein to the antiapoptotic Bcl-xl protein, suggesting the induction of apoptosis through the intrinsic mitochondrial pathway in both cell lines. Further studies are required to fully elucidate the mechanism of these activities.

Induction of Apoptosis by Coptisine in Hep3B Hepatocellular Carcinoma Cells through Activation of the ROS-Mediated JNK Signaling Pathway.

Hepatocellular carcinoma (HCC) has a high mortality rate worldwide, and treatment is very limited due to its high recurrence and low diagnosis rate, and therefore there is an increasing need to develop more effective drugs to treat HCC. Coptisine is one of the isoquinoline alkaloids, and it has various pharmacological effects. However, the evidence for the molecular mechanism of the anticancer efficacy is still insufficient. Therefore, this study investigated the antiproliferative effect of coptisine on human HCC Hep3B cells and identified the action mechanism. Our results showed that coptisine markedly increased DNA damage and apoptotic cell death, which was associated with induction of death receptor proteins. Coptisine also significantly upregulated expression of proapoptotic Bax protein, downregulated expression of anti-apoptotic Bcl-2 protein, and activated caspase-3, -8, and -9. In addition, coptisine remarkably increased the generation of reactive oxygen species (ROS), loss of mitochondrial membrane potential (MMP), and release of cytochrome c into the cytoplasm. However, N-acetylcysteine (NAC), a ROS scavenger, significantly attenuated the apoptosis-inducing effect of coptisine. It is worth noting that coptisine significantly upregulated phosphorylation of ROS-dependent c-Jun N-terminal kinase (JNK), whereas treatment with JNK inhibitor could suppress an apoptosis-related series event. Taken together, our results suggest that coptisine has an anticancer effect in Hep3B cells through ROS-mediated activation of the JNK signaling pathway.

The Effects of Calcium-β-Hydroxy-β-Methylbutyrate on Aging-Associated Apoptotic Signaling and Muscle Mass and Function in Unloaded but Nonatrophied Extensor Digitorum Longus Muscles of Aged Rats.

Beta-hydroxy-beta-methylbutyrate (HMB), a naturally occurring leucine metabolite, has been shown to attenuate plantar flexor muscle loss and increase myogenic stem cell activation during reloading after a period of significant muscle wasting by disuse in old rodents. However, it was less clear if HMB would alter dorsiflexor muscle response to unloading or reloading when there was no significant atrophy that was induced by unloading. In this study, we tested if calcium HMB (Ca-HMB) would improve muscle function and alter apoptotic signaling in the extensor digitorum longus (EDL) of aged animals that were unloaded but did not undergo atrophy. The EDL muscle was unloaded for 14 days by hindlimb suspension (HS) in aged (34-36 mo.) male Fisher 344 × Brown Norway rats. The rats were removed from HS and allowed normal cage ambulation for 14 days of reloading (R). Throughout the study, the rats were gavaged daily with 170 mg of Ca-HMB or water 7 days prior to HS, then throughout 14 days of HS and 14 days of recovery after removing HS. The animals' body weights were significantly reduced by ~18% after 14 days of HS and continued to decline by ~22% during R as compared to control conditions; however, despite unloading, EDL did not atrophy by HS, nor did it increase in mass after R. No changes were observed in EDL twitch contraction time, force production, fatigue resistance, fiber cross-sectional area, or markers of nuclear apoptosis (myonuclei + satellite cells) after HS or R. While HS and R increased the proapoptotic Bax protein abundance, BCL-2 abundance was also increased as was the frequency of TUNEL-positive myonuclei and satellite cells, yet muscle mass and fiber cross-sectional area did not change and Ca-HMB treatment had no effect reducing apoptotic signaling. These data indicate that (i) increased apoptotic signaling preceded muscle atrophy or occurred without significant EDL atrophy and (ii) that Ca-HMB treatment did not improve EDL signaling, muscle mass, or muscle function in aged rats, when HS and R did not impact mass or function.

Sensing the Generation of Intracellular Free Electrons Using the Inactive Catalytic Subunit of Cytochrome P450s as a Sink.

Cytochrome P450 reductase (CPR) abstracts electrons from Nicotinamide adenine dinucleotide phosphate H (NADPH), transferring them to an active Cytochrome P450 (CYP) site to provide a functional CYP. In the present study, a yeast strain was genetically engineered to delete the endogenous CPR gene. A human CYP expressed in a CPR-null (yRD−) strain was inactive. It was queried if Bax—which induces apoptosis in yeast and human cells by generating reactive oxygen species (ROS)—substituted for the absence of CPR. Since Bax-generated ROS stems from an initial release of electrons, is it possible for these released electrons to be captured by an inactive CYP to make it active once again? In this study, yeast cells that did not contain any CPR activity (i.e., because the yeasts’ CPR gene was completely deleted) were used to show that (a) human CYPs produced within CPR-null (yRD-) yeast cells were inactive and (b) low levels of the pro-apoptotic human Bax protein could activate inactive human CYPs within this yeast cells. Surprisingly, Bax activated three inactive CYP proteins, confirming that it could compensate for CPR’s absence within yeast cells. These findings could be useful in research, development of bioassays, bioreactors, biosensors, and disease diagnosis, among others.

Diosgenin as a Novel Alternative Therapy for Inhibition of Growth, Invasion, and Angiogenesis Abilities of Different Glioblastoma Cell Lines.

Fenugreek (Trigonella foenum-graecum) seeds and roots of wild yam (Dioscorea villosa) possess nutritional and medicinal properties and have been used for centuries in traditional medicine to treat different diseases and inflammatory responses. Diosgenin is a natural steroidal sapogenin extracted from fenugreek and wild yam and it is one of the major bioactive compounds used in the treatment of diabetes, hypercholesterolemia, and inflammation. Recent studies have shown a promising effect of diosgenin as ananti-tumor agent for inhibition of cell proliferation and induction of apoptosis in many cancers such as colon cancer, leukemia, breast cancer, and liver cancer. We examined the effects of different concentrations (5, 10, 15, 20, and 25µM) of diosgenin on proliferation of rat C6 and human T98G glioblastoma cell lines. We noticed that diosgenin had a high inhibitory effect on the growth of both C6 and T98G cell lines. Diosgenin induced the differentiation of glioblastoma cells, as determined by the increasein the expression of the differentiation marker glial fibrillary acidic protein (GFAP); and decreased the dedifferentiation of the cells, as shown by the decreasein the abundance of the dedifferentiation marker proteins Id2, N-Myc, telomerase reverse transcriptase (TERT), and Notch-1. It also induced apoptosis in C6 and T98G cell lines and themolecular mechanisms involved in the induction of apoptosis included increase in pro-apoptotic Bax protein and decrease in anti-apoptotic Bcl-2 protein. Further, the diosgenin-induced suppression of cell migration was correlated with the decrease in expressionof matrix metalloproteinase 2 (MMP2) and MMP9; and the inhibition of angiogenesis, as determined by the tube formation assay, was correlated with a decrease in the protein levels of vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2). In conclusion, diosgenin showed anti-tumoreffects in glioblastoma cells by induction of differentiation and apoptosis and inhibition of migration,invasion, and angiogenesis.

The intrinsic apoptotic pathway lies upstream of oxidative stress in multiple organs

Oxidative stress increases with age in multiple organ systems and is implicated in the development of age-related pathologies in them. Studies from our laboratory show that the intrinsic pathway proapoptotic proteins BAX and caspase-3 (CASP3) lie upstream of mitochondrial production of oxidative stress-inducing reactive species (RS) such as reactive oxygen and reactive nitrogen species (ROS and RNS) in apoptotic and nonapoptotic neurons in cell culture. Our objective in this study was to determine if these findings could be generalized to the development of oxidative stress in nonneuronal tissues in vivo. We first investigated the effect of genetic deletion of Bax on DNA damage in the liver, heart and kidneys of female mice of increasing ages (5, 14, 22 months). The organs of the aged mice showed increased oxidative DNA strand breaks compared to young animals (5 month). Ablation of Bax greatly reduced this damage. We next assessed lipid peroxidation, DNA oxidation, and protein tyrosine nitration to determine whether Casp3 deletion reduces oxidative stress in the hearts, livers, and kidneys of 12-month-old female mice. Lipid peroxides and 8-hydroxy-2’-deoxyguanosine (8-OHdG) levels were much lower in organs from mice with depleted Casp3 than in those of wild type animals. Nitration of protein tyrosine residues, caused by RNS, was also significantly suppressed in the tissues of Casp3 null mice compared to those in wild type mice. Our findings indicate that BAX and CASP3 have a vital role in the generation of oxidative stress in organs of aged mice.

MiR-144-3p increases radiosensibility of gastric cancer cells by targeting inhibition of ZEB1.

This study set out to probe into the effect and mechanism of miR-144-3p on radiosensitivity of gastric cancer (GC) cells. Cancer tissue and paracancerous tissue of GC patients admitted to our hospital were collected, their miR-144-3p expression was tested, GC cells were transfected, and survival and biological behavior of those cells under radiation were detected. After detection, miR-144-3p expression was down-regulated in GC tissue, while ZEB1 was up-regulated. There was no remarkable difference in the survival fraction of cells in each group before receiving radiation, but that of tumor cells decreased obviously (p < 0.05) after radiation exposure. Survival fraction of cells overexpressing miR-144-3p or silencing ZEB1 decreased more obviously, while the inhibition of miR-144-3p or overexpressing ZEB1 was weaker. Biological behavior of cells under 6Gy radiation was detected. It was found that miR-144-3p overexpression or silencing ZEB1 dramatically inhibited the proliferation activity of GC cells under 6Gy radiation, increased the levels of pro-apoptotic Bax and caspase-3 proteins (p < 0.05) and decreased the anti-apoptotic protein Bcl-2 level (p < 0.05), resulting in an increase in the apoptosis rate of cells. miR-144-3p was confirmed to be ZEB1 targeting site by dual luciferase report. Moreover, rescue experiments prove that it can increase the radiosensitivity of GC cells by regulating ZEB1 expression. miR-144-3p expression was down-regulated in GC, and it can increase the radiosensitivity of those cells by inhibiting ZEB1 expression.

Ethanol-extracted Cameroonian propolis: Antiproliferative effects and potential mechanism of action in prostate cancer.

The present study was conducted to evaluate in vitro and in vivo antiproliferative potential of the Cameroonian propolis and to elucidate its underlying mechanism. In vitro, ethanol-extracted propolis (EEP) was tested on cell growth, cell proliferation, cell cycle, cell death mechanism and cell migration. The cell cycle- and apoptosis-regulating proteins were assessed by Western blotting. In vivo the testosterone-induced benign prostatic hyperplasia (BPH) in Wistar rat was used to evaluate the antiproliferative potential of EEP. EEP reduced DU145 and PC3 cell survival with an IC50 of 70 and 22μg/ml respectively. It increased the number of late apoptotic cells, the amount of cells in G0/G1 phase in DU145 and PC3 cells at 50µg/ml. Cell cycle proteins (cdk1, pcdk1 and their related cyclins A and B) were down-regulated in both DU145 and PC3 cells, while cdk2 and pcdk2 were down-regulated only in PC3 cells. The pro-apoptotic Bax protein was up-regulated, while the anti-apoptotic Akt and pAKT, and Bcl-2 proteins were down-regulated. It increased prostate cell adhesion and chemotaxis. EEP reduced prostate weight, volume and epithelial thickness in rats. We demonstrated for the first time that Cameroonian propolis is endowed with in vitro and in vivo antiproliferative properties in the prostate.

Notoginsenoside R1 attenuates sevoflurane-induced neurotoxicity.

BackgroundSevoflurane, a volatile anesthetic, is known to induce widespread neuronal degeneration and apoptosis. Recently, the stress-inducible protein sestrin 2 and adenosine monophosphate-activated protein kinase (AMPK) have been found to regulate the levels of intracellular reactive oxygen species (ROS) and suppress oxidative stress. Notoginsenoside R1 (NGR1), a saponin isolated from Panax notoginseng, has been shown to exert neuroprotective effects. The effects of NGR1 against neurotoxicity induced by sevoflurane were assessed.MethodsSprague-Dawley rat pups on postnatal day 7 (PD7) were exposed to sevoflurane (3%) anesthesia for 6 h. NGR1 at doses of 12.5, 25, or 50 mg/kg body weight was orally administered to pups from PD2 to PD7.ResultsPretreatment with NGR1 attenuated sevoflurane-induced generation of ROS and reduced apoptotic cell counts. Western blotting revealed decreased cleaved caspase 3 and Bad and Bax pro-apoptotic protein expression. NGR1 substantially upregulated nuclear factor erythroid 2-related factor 2 (Nrf2) expression along with increased heme oxygenase-1 (HO-1) and NAD(P)H quinone oxidoreductase-1 levels, suggesting Nrf2 signaling activation. Enhanced sestrin-2 and phosphorylated AMPK expression were noticed following NGR1 pretreatment.ConclusionThis study revealed the neuroprotective effects of NGR1 through effective suppression of apoptosis and ROS via regulation of apoptotic proteins and activation of Nrf2/HO-1 and sestrin 2/AMPK signaling cascades.

The Effect of Phototherapy on Apaptosisin Hyperbilirubinemic Preterm Neonates

Objective: This study aimed to assess the impact of hyperbilirubinemia and Intensive phototherapy on apaptosis in peripheral blood of preterm infants. Patients and Method: The study was conducted on 18 preterm infants with non-hemolytic hyperbilirubinemia and 9 control healthy preterm infants. Apaptosismarkers (anti-apoptotic protein Bcl2, and pro-apoptotic protein BAX) was assessed using ELISA technique. Blood samples wereobtained at enrollment in all infants and after24 hours of exposure to intensive phototherapy in jaundiced infants. Result: Before phototherapy, no significant difference in the serum levels of bcl2 and Bax protein between jaundiced and non-jaundiced preterm infants (P = 0.570, p= 0.329 respectively), After phototherapy The serum levels of Bcl2 decrease while The serum levels of BAX increased significantly (P=0.003, p&lt;0.001 respectively). Conclusion: Hyperbilirubinemia does not influence apaptosis whereas intensive phototherapy do in preterm infants with hyperbilirubinemia.

Neferine, an alkaloid from lotus seed embryo targets HeLa and SiHa cervical cancer cells via pro-oxidant anticancer mechanism.

Apoptosis and autophagy are important processes that control cellular homeostasis and have been highlighted as promising targets for novel anticancer drugs. This study aims to investigate the inhibitory effects and mechanisms of Neferine (Nef), an alkaloid from the lotus seed embryos of Nelumbo nucifera (N. nucifera), as a dual inducer of apoptosis and autophagy through the reactive oxygen species (ROS) activation in cervical cancer cells. Nef and N. nucifera extract suppressed the cell viability of HeLa and SiHa cells in a dose-dependent manner. Importantly, Nef showed minimal toxicity to normal cells. Furthermore, Nef inhibited anchorage-independent growth, colony formation and migration ability of cervical cancer cells. Nef induces mitochondrial apoptosis by increasing pro-apoptotic protein bax, cytochrome-c, cleaved caspase-3 and caspase-9, poly-ADP ribose polymerase (PARP) cleavage, DNA damage (pH2 AX) while downregulating Bcl-2, procaspase-3 and procaspase-9, and TCTP. Of note, apoptotic effect by Nef was significantly attenuated in the presence of N-acetylcysteine (NAC), suggesting pro-oxidant activity of this compound. Nef also promoted autophagy induction through increasing beclin-1, atg-4, atg-5 and atg-12, LC-3 activation, and P 62/SQSTM1 as determined by western blot analysis. Collectively, these results demonstrate that Nef is a potent anticancer compound against cervical cancer cells through inducing apoptosis and autophagic pathway involving ROS.

Nos2 deficiency enhances carbon tetrachloride-induced liver injury in aged mice.

Objective(s):As a multifunctional molecule, NO has different effects on liver injury. The present work aimed to investigate the effects of Nos2 knockout (KO) on acute liver injury in aged mice treated with carbon tetrachloride (CCl4). Materials and Methods:The acute liver injury model was produced by CCl4 at 10 ml/kg body weight in 24-month-old Nos2 KO mice and wild type (WT) mice groups. The histological changes, transaminase and glutathione (GSH) contents, and the expressions of liver function genes superoxide dismutase (SOD2) and butyrylcholinesterase (BCHE), as well as apoptosis- and inflammation-associated genes were detected at 0, 6, 16, 20, 28, and 48 hr, respectively. Results:Compared with WT aged mice, there are more fat droplets in liver tissues of Nos2 KO aged mice, and the serum levels of ALT and AST were elevated in the KO group; in addition, there was a decrease in the expression of SOD2 and BCHE and GSH content at multiple time-points. Furthermore, the expression of apoptosis protein CASPASE-3 was elevated from 20 to 48 hr, the same as CASPASE-9 at 28 and 48 hr and pro-apoptotic protein BAX at 6 and 28 hr, while the expression of apoptosis inhibitory protein BCL2 declined at 6 and 28 hr; at the same time the mRNA expressions of genes related to inflammation were increased at different extents in liver extracts of Nos2 KO aged mice. Conclusion: Nos2 KO exacerbated liver injury probably by elevated oxidative stress, apoptosis and inflammation response in CCl4-induced aged mice liver intoxication model.

HVEM/HIF-1\u03b1 promoted proliferation and inhibited apoptosis of ovarian cancer cells under hypoxic microenvironment conditions

BackgroundOur previous studies showed the expression of herpes virus entry mediator (HVEM) is high in ovarian cancer samples and correlated to the patient clinic pathological features. As we all know, the hypoxic environment is the main feature of tumor. In this work, we explored the role of HVEM in hypoxic ovarian cancer cells and its effects on HIF-1α, a transcription factor responding to hypoxia.MethodsThe expression of HVEM, HIF-1α and apoptosis-related genes was detected by qRT-PCR and western blot. The proliferation and apoptosis of the ovarian cancer cells were determined with the Cell Counting Kit-8 assay and AnnexinV-FITC/PI-stained flow cytometry assay, respectively.ResultsThe expression of HVEM was positively correlated to that of HIF-1α. The expression of HVEM and HIF-1α under hypoxic conditions was higher than that under normoxic conditions, which suggested that the level of HVEM and HIF-1α correlates with prolonged periods of hypoxia in ovarian cancer. The overexpression of HVEM promoted cell proliferation and inhibited cell apoptosis under hypoxic condition. HVEM overexpression elevated the expression of HIF-1α and Bcl-2 (anti-apoptotic protein), and reduced the expression of Bax (pro-apoptotic protein). In addition, overexpression of HVEM activated the AKT/mTOR signaling. Moreover, knockdown of HVEM had the completely opposite effects.ConclusionThese data indicated that HVEM signaling might promote HIF-1α activity via AKT/mTOR signaling pathway and thus to regulate tumor growth in ovarian cancer under the hypoxic conditions. Furthermore, these findings indicate that this molecular mechanism could represent a therapeutic target for ovarian cancer.

Interplay of microRNA‐503 and N‐acetylcysteine in regulating hypoxia‐reoxygenation injury in cardiomyocyte H9C2 cells

microRNAs(miRs) are involved in many cardiovascular diseases including myocardial ischemia / reperfusion injury (IRI). Myocardial IRI can lead to oxidative stress, many miRs are regulated in response to oxidative stress. N‐acetylcysteine (NAC) –a known antioxidant, is reported to have protective effects against myocardial IRI. Previously, we and others observed that cardiac miR‐503 was down‐regulated during myocardial IRI in vivo in rodents or in cardiomyocyte H9C2 cells subjected to hypoxia and reoxygenation (H/R). This study is aimed to explore the interplay of miR503 and NAC in the regulation of hypoxia‐reoxygenation injury and possible molecular mechanisms in cardiomyocyte H9C2 cells. Here, H9C2 cells were subjected to hypoxia for 10 hours followed by reoxygenation 6 hours as simulated myocardial IRI. Results showed that cell viability was decreased and lactate dehydrogenase (LDH) activity was increased upon H/R; Pretreatment with NAC (1mM) to H9C2 cells for 30 hours can improve cell viability and reduce LDH activity in both normoxia and H/R states, while pretreatment with NAC (2mM) decreased cell viability and increased LDH activity. We also found that NAC (1mM) can down‐regulate miR‐503 expression level in H9C2 cells subjected to normoxia, it also down‐regulated miR‐503 expression level upon H/R. In order to inhibit endogenous miR‐503, H9C2 cells was transfected with Antagomir‐503, inhibition of miR‐503 can improve cell viability and reduce LDH activity upon H/R; Western blotting results showed that there was no significant difference among control, HR, HR+negative control of Antagomir‐503(NC) or HR+Antagomir‐503 groups for proapoptotic Bax protein, while anti‐apoptotic Bcl2 protein was increased after transfection of Antagomir‐503 compared with HR+NC. Target‐Scan software showed that Phosphoinositide‐3‐kinase, regulatory subunit 1(PI3K p85) is one of miR‐503 target genes, and P85 protein was decreased after H/R, but it was increased significantly after administration of Antagomir‐503, its down‐stream protein phospho‐AKT (Thr450) was also significantly increased. However, Antagomir‐503 and NAC (1mM) used in combination to H9C2 cells upon H/R, did not provide an additional advantage in protecting cell injury. It is concluded that administration of NAC (1mM) or Antagomir‐503 alone can alleviate cell injury, while the combination of NAC (1mM) and Antagomir‐503 has no synergistic cytoprotective effects, the possible molecular mechanisms need to be further explored.Support or Funding InformationSupported by Hong Kong RGC(17158616 M) and HMRF (05161826)grants.

Effects of triptolide and BET protein inhibitor JQ1 on the proliferation and apoptosis of MLL-rearranged acute myeloid leukemia cells and their mechanisms

Objective To investigate the effects of triptolide (TPL) and BET protein inhibitor JQ1 on proliferation inhibition and apoptosis induction of MLL-rearranged acute myeloid leukemia (AML) cell line MV4-11, and to explore their synergistic mechanisms. Methods MV4-11 cells in logarithmic growth phase were treated with different concentrations (100, 200, 300, and 400 nmol/L) of JQ1, 4 nmol/L TPL or different concentrations of JQ1 combined with 4 nmol/L TPL for 48 h. Cell proliferation was detected by CCK-8 method, apoptosis was detected by flow cytometry (FCM), mitochondrial membrane potential was detected by JC-1 method, and expressions of mitochondrial apoptosis pathway-related proteins were detected by Western blot. Results The 50% inhibitory concentration (IC50) value of MV4-11 cells treated with JQ1 for 48 h was (283.9±10.7) nmol/L. However, 4 nmol/L TPL significantly enhanced the inhibitory effect of JQ1 on proliferation of MV4-11 cells, the IC50 value of MV4-11 cells treated with JQ1 combined with TPL was (148.1±2.6) nmol/L, and the difference was statistically significant (t = 25.31, P = 0.029). The result of FCM assay showed that compared with the JQ1 alone group [(9.6±2.3)%, (12.6±1.4)%, (19.5±3.3)%, and (22.7±2.1)%], 4 nmol/L TPL combined with different concentrations (100, 200, 300, and 400 nmol/L) of JQ1 acted on MV4-11 cells for 48 h, the proportions of apoptotic cells were (16.4±1.9)%, (27.5±2.1)%, (32.9±3.6)%, and (35.5±3.0)%, respectively, the difference was statistically significant (F = 9.25, P < 0.01). After treated with 4 nmol/L TPL and JQ1 for 12 h, the level of cell membrane potential in MV4-11 cells was significantly lower than that of JQ1 single agent group, and the difference was statistically significant (P < 0.05). After treated by 4 nmol/L TPL combined with JQ1 for 24 h, the levels of anti-apoptotic proteins bcl-2 and Mcl-1 decreased, and the level of pro-apoptotic protein bax increased. Conclusion TPL can significantly enhance the proliferation inhibition and apoptosis induction effects of BET protein inhibitor JQ1 on MLL-rearranged AML cells, and the mechanism may be related to enhancing the mitochondrial apoptosis pathway. Key words: Gene rearrangement; Leukemia, myeloid, acute; Triptolide; BET protein inhibitor JQ1

PHYCI_587572: An RxLR Effector Gene and New Biomarker in A Recombinase Polymerase Amplification Assay for Rapid Detection of Phytophthora cinnamomi

Phytophthora cinnamomi is a devastating pathogen causing root and crown rot and dieback diseases of nearly 5000 plant species. Accurate and rapid detection of P. cinnamomi plays a fundamental role within the current disease prevention and management programs. In this study, a novel effector gene PHYCI_587572 was found as unique to P. cinnamomi based on a comparative genomic analysis of 12 Phytophthora species. Its avirulence homolog protein 87 (Avh87) is characterized by the Arg-Xaa-Leu-Arg (RxLR) motif. Avh87 suppressed the pro-apoptotic protein BAX- and elicitin protein INF1-mediated cell death of Nicotiana benthamiana. Furthermore, a recombinase polymerase amplification-lateral flow dipstick detection assay targeting this P. cinnamomi-specific biomarker was developed. While successfully detected 19 P. cinnamomi isolates of a global distribution, this assay lacked detection of 37 other oomycete and fungal species, including P. parvispora, a sister taxon of P. cinnamomi. In addition, it detected P. cinnamomi from artificially inoculated leaves of Cedrus deodara. Moreover, the RPA-LFD assay was found to be more sensitive than a conventional PCR assay, by detecting as low as 2 pg of genomic DNA in a 50-µL reaction. It detected P. cinnamomi in 13 infested soil samples, while the detection rate was 46.2% using PCR. Results in this study indicated that PHYCI_587572 is a unique biomarker for detecting P. cinnamomi. Although PHYCI_587572 was identified as an effector gene based on the RxLR motif of Avh87 and the avirulence activity on Nicotiana, its exact genetic background and biological function on the natural hosts of P. cinnamomi warrant further investigations.

Hypoxia-induced elevated NDRG1 mediates apoptosis through reprograming mitochondrial fission in HCC.

Hypoxia, as a form of stress, plays a critical role in oncogenesis, including metabolic reprogramming. Mitochondrial, the centers of energy production, re-balance mitochondria dynamic to maintain cell survival during high levels of environmental stresses. NDRG1 is a hypoxia-inducible protein that is involved in various human cancers, including HCC. However, little is known about whether NDRG1 participants in the quality control of mitochondrial in times of stress. Here, we firstly showed that how NDRG1 exerted its role through mediating mitochondrial dynamic in HCC cells under hypoxia. Initially, we identified that NDRG1 expression varies with oxygen content. NDRG1 silencing notably induced cell apoptosis under hypoxia, while no obviously change of wildtype cells in hypoxia compared with that in normoxia. Further analysis revealed that NDRG1 silencing in HCC cells led to increase of pro apoptotic protein BAX and decrease in anti-apoptotic proteins Bcl-2 and Bclx, which meant mitochondrial damage were induced. In the analysis of mitochondria, we found that more released cytochrome c located in cytosolic with NDRG1 knockdown in hypoxia, which may be due to mitochondria division. And the following experiment proved that more fragmented mitochondria were presented in NDRG1 silencing cells, as well as destroyed mitochondrial membrane potential with evidence by JC-1 was verified. Moreover, these trends could be reversed by Mdivi1. Further research showed that NDRG1 silencing disrupt hypoxia-enhanced aerobic glycolysis through effectively decreased glucose uptake, lactate output and ECAR value. In sum, we provide the first direct evidence that NDRG1-driven change in mitochondrial dynamics and aerobic glycolysis maintain cells survival in HCC during hypoxia.

Evaluation of the Effect of Prazosin Treatment on α-2c Adrenoceptor and Apoptosis Protein Levels in the Predator Scent-Induced Rat Model of Post-Traumatic Stress Disorder

The predator scent-induced (PSI) stress model is a rat model used to mimic post-traumatic stress disorder (PTSD) symptoms in humans. There is growing evidence that prazosin, which blocks α-1 and is approved by the FDA as an anti-hypertensive drug, can potentially be of use in the treatment of PTSD-related sleep disorders. The aim of this study was to investigate the role of prazosin treatment on behavioral parameters (freezing time, total transitions, and rearing frequency measured from the open field; anxiety index, total entries and time spent in open arms calculated from the elevated plus maze), apoptotic proteins and α-2c-AR in fear memory reconsolidation in the PSI stress rat model. We used western blot analysis to determine the effect of prazosin (0.5mg/kg/ip) on α-2c-AR and apoptotic protein expression changes in the frontal cortex, hippocampus, and amygdala. It was determined that in the stress group, there was increased freezing time and anxiety index, and decreased rearing frequency, total transitions, total entries, and time spent in open arms compared to the control groups. Following PSI-stress, pro-apoptotic (bax) protein expression levels increased and α-2c AR and anti-apoptotic protein (bcl-2) levels decreased in investigated all brain regions. The majority of stress-induced changes were recovered with prazosin treatment. The results of our study may potentially be useful in understanding the effect of prazosin treatment, given the fact that the amygdala, frontal cortex, and hippocampus regions are affected for stress conditions.

Protective Effects of the Soluble Epoxide Hydrolase Inhibitor 1-Trifluoromethoxyphenyl-3-(1-Propionylpiperidin-4-yl) Urea in a Rat Model of Permanent Middle Cerebral Artery Occlusion.

Acute ischemic stroke is a serious disease that endangers human health. In our efforts to develop an effective therapy, we previously showed that the potent, highly selective inhibitor of soluble epoxide hydrolase called 1-trifuoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU) protects the brain against focal ischemia in rats. Here we explored the mechanism of TPPU action by assessing whether it could preserve blood-brain barrier integrity and reduce apoptosis in the brain during permanent middle cerebral artery occlusion in male Sprague-Dawley rats. TPPU administration at the onset of stroke and once daily thereafter led to smaller infarct volume and brain edema as well as milder neurological deficits. TPPU significantly inhibited the activity of soluble epoxide hydrolase and matrix metalloproteases 2 and 9, reducing 14,15-DHET levels, while increasing expression of tight junction proteins. TPPU decreased numbers of apoptotic cells by down-regulating the pro-apoptotic proteins BAX and Caspase-3, while up-regulating the anti-apoptotic protein BCL-2. Our results suggest that TPPU can protect the blood-brain barrier and reduce the apoptosis of brain tissue caused by ischemia.