Mitochondrial Bcl-2 Research Articles

TiF4 and NaF varnishes induce low levels of apoptosis in murine and human fibroblasts through mitochondrial Bcl-2 family and death receptor signalling

ObjectivesThis study evaluated the level and mechanism of apoptosis in human gingival fibroblasts (HGF) and murine fibroblasts (NIH/3T3) treated with a titanium tetrafluoride (TiF4) varnish compared those treated with a sodium fluoride (NaF) varnish. MethodsCells were treated with a TiF4, NaF (both 2.45%F) or placebo varnish for 6 h and were then examined using the TUNEL method. The activities of caspase-3, -8 and -9 were assessed. cDNA for Bax, Bad, Bcl-2 and Fas-L was amplified by quantitative PCR. Bax, Bcl-2 and Fas-L were further detected by western blot analysis. ResultsBoth fluorides similarly increased the percentage of apoptosis, while they failed to activate caspases. The Bax/Bcl-2 gene expression ratio was not altered by either fluoride treatment regardless of the type of cell. NaF varnish increased the amplification of the Fas-L gene in NIH/3T3 and HGF cells, while treatment with the TiF4 varnish resulted in a lower Bad/Bcl-2 expression ratio compared to that of the control for NIH/3T3 cells, but not for HGF cells. No effect of the fluorides was detected in the protein analysis. ConclusionsNaF and TiF4, at the studied conditions, similarly induce a low level of apoptosis, with consequent modest activation of the Bcl-2 and Fas-l-dependent signalling pathways. Generally, HGF cells are more susceptible to the fluoride effect than NIH/3T3 cells.

Sodium tanshinone IIA sulfonate protects ARPE-19 cells against oxidative stress by inhibiting autophagy and apoptosis

Oxidative stress in retinal pigment epithelium (RPE) is considered to be a major contributor to the development and progression of age-related macular degeneration (AMD). Previous investigations have shown that sodium tanshinone IIA sulfonate (STS) can alleviate oxidative stress in haemorrhagic shock-induced organ damage and cigarette smoke-induced chronic obstructive pulmonary disease in mice. However, whether STS has a protective effect in ARPE-19 cells under oxidative stress and its exact mechanisms have not yet been fully elucidated. In the present study, we utilized H2O2 to establish an oxidative stress environment. Our findings show that STS activated the PI3K/AKT/mTOR pathway to inhibit autophagy and diminished the expression of the autophagic proteins Beclin 1, ATG3, ATG7 and ATG9 in ARPE-19 cells under oxidative stress. Detection of the intrinsic apoptosis-related factors BAX, mitochondrial membrane potential (MMP), caspase-9, caspase-3 and BCL-2, as well as the extrinsic apoptosis-related factors c-FLIP, v-FLIP and caspase-8, confirmed that STS inhibited the intrinsic and extrinsic apoptotic pathways, and attenuated apoptosis in ARPE-19 cells under oxidative stress conditions. These findings shed new light on the protective effects of STS in ARPE-19 cells and its mechanisms under oxidative stress to provide novel and promising therapeutic strategies for AMD.

Rab14 overexpression regulates gemcitabine sensitivity through regulation of Bcl-2 and mitochondrial function in pancreatic cancer.

Rab family protein Rab14 has been implicated in the development of human cancers. To date, its expression pattern, biological function, and potential mechanism in pancreatic cancer have not been explored. In this study, we analyzed Rab14 expression in 103 cases of pancreatic cancer tissues using immunohistochemistry (IHC) and found that Rab14 was overexpressed in 41/103 cases (39.8%). Rab14 overexpression correlated with the advanced stage. Moreover, elevated Rab14 levels indicated poor prognosis of patients with pancreatic cancers. We used BxPC-3 and Capan-2 respectively for plasmid and siRNA transfection. MTT and colony formation assays showed that Rab14 transfection increased cell proliferation and colony formation in BxPC-3 cells. Rab14 siRNA knockdown inhibits proliferation and colony formation ability in Capan-2 cell line. Cell cycle analysis showed that Rab14 facilitated cell cycle progression. Matrigel invasion assay showed that Rab14 promoted BxPC-3 cell invasion while its depletion inhibited Capan-2 cell invasion. In addition, MTT and AnnexinV/PI analysis demonstrated that overexpression of Rab14 reduced gemcitabine sensitivity which conversely was increased by Rab14 knockdown. We also demonstrated that Rab14 upregulated mitochondrial membrane potential (MMP) while its depletion downregulated MMP during gemcitabine treatment. In addition, western blotting revealed that Rab14 overexpression upregulated cyclin D1, cyclin A, cyclin E, p-Rb, and Bcl-2 and downregulated p21. Rab14 also downregulated caspase3, PARP cleavage, and cytochrome c release. In conclusion, our data indicated that Rab14 was overexpressed in pancreatic cancer and promotes growth and gemcitabine resistance, possibly through regulation of mitochondrial function and Bcl-2.

Adapentpronitrile, a New Dipeptidyl Peptidase-IV Inhibitor, Ameliorates Diabetic Neuronal Injury Through Inhibiting Mitochondria-Related Oxidative Stress and Apoptosis.

Our previous studies indicated that adapentpronitrile, a new adamantane-based dipeptidyl peptidase-IV (DPP-IV) inhibitor, has a hypoglycemic effect and ameliorates rat pancreatic β cell dysfunction in type 2 diabetes mellitus through inhibiting DPP-IV activity. However, the effect of adapentpronitrile on the neurodegenerative diseases has not been studied. In the present study, we first found that adapentpronitrile significantly ameliorated neuronal injury and decreased amyloid precursor protein (APP) and amyloid beta (Aβ) expression in the hippocampus and cortex in the high fat diet/STZ rat model of diabetes. Furthermore, adapentpronitrile significantly attenuated oxidative stress, downregulated expression of the pro-apoptotic proteins BAX, cytochrome c, caspase-9, and caspase-3, and upregulated expression of the anti-apoptotic protein Bcl-2, although there was no effect on GLP-1R expression. At 30 min post-injection of adapentpronitrile (50 mg/kg) via the tail vein, its concentration in normal rat brain was 0.2034 ± 0.0094 μg/g. Subsequently, we further confirmed the neuroprotective effects and mechanism of adapentpronitrile in HT22 cells treated with high glucose (HG) and aluminum maltolate [Al(mal)3] overload, respectively. Our results showed significant decreases in mitochondrial membrane potential (MTP) and Bcl-2 expression, accompanied by a significant increase in apoptosis, reactive oxygen species (ROS) generation, and the expression of pro-apoptotic proteins in HT22 cells exposed to these stimuli. Adapentpronitrile treatment protected against neuronal injury, suppressed ROS generation, and reduced MTP and mitochondrial apoptosis in HT22 cells; however, DPP-IV activity was not detected. Our results suggest that adapentpronitrile protects against diabetic neuronal injury, at least partially, by inhibiting mitochondrial oxidative stress and the apoptotic pathway in a DPP-IV-independent manner.

SGLT2 Inhibitor Ipragliflozin Induces Breast Cancer Apoptosis via Membrane Hyperpolarization and Mitochondria Dysfunction

Currently, cancer is one of major cause of death in patients with type 2 diabetes. We have previously reported the anti-prostate and anti-breast cancer effect of GLP-1R agonist Exendin-4 (Diabetes 2014, Endocrinology 2017). In the present study, we examined the anti-cancer effect of SGLT2 inhibitor ipragliflozin (Ipra) using a breast cancer model. In human breast cancer cell line, MCF-7 cells, SGLT2 expression was detected using both RT-PCR and immunohistochemistry. 1-50nM Ipra significantly and dose-dependently suppressed the growth curve of MCF-7 cells. BrdU assay revealed that Ipra attenuates the proliferation rate of MCF-7 in a dose dependent manner. Further, apoptosis was also induced by Ipra in Tunel assay. Because the anti-breast cancer effect of Ipra was completely canceled by knocking down of SGLT2, this effect could be induced by SGLT2 inhibition by Ipra. We next measured membrane potential and whole cell current using the patch clamp technique. When we treated MCF-7 cell with Ipra or glucose free medium, membrane hyperpolarization was observed. In addition, the replacement of sodium with NMDG and knock-down of SGLT2 by siRNA suppressed glucose induced whole cell current of MCF-7 cell, suggesting that Ipra inhibits sodium and glucose incorporation through SGLT2. Further, mitochondrial membrane protein Bcl-2 was decreased and Bax was increased in western blotting, and JC-1 fluorescence was significantly increased, suggesting the change of mitochondrial membrane potential. These data suggest that SGLT2 inhibitor Ipra induces apoptosis in the breast cancer cell via membrane hyperpolarization and mitochondria dysfunction. Disclosure S. Komatsu: None. T. Nomiyama: Research Support; Self; MSD K.K., Sanofi-Aventis, Boehringer Ingelheim Pharmaceuticals, Inc., Takeda Pharmaceuticals, Japan.T. Numata: None.T. Kawanami: None.Y. Hamaguchi: None.T. Tanaka: None.R. Inoue: None. T. Yanase: Research Support; Self; MSD K.K., Boehringer Ingelheim Pharmaceuticals, Inc., Takeda Pharmaceuticals, Japan.

Expression of Bcl-2 and Bad in hippocampus of status epileptic rats and molecular mechanism of intervened recombinant human erythropoietin

Injury of hippocampal neurons in status epilepticus (SE) SD rats kindled by pentylenetetrazol (PTZ) were studied, and the changes of apoptosis neurons, protein expression of Bad and Bcl-2 alone and combined application of phosphatidyl inositol 3-kinase (PI3K) inhibitor LY294002 and recombinant human erythropoietin (rHuEpo) were evaluated for the possible mechanisms of rHuEpo. The SE rats kindled by the PTZ were randomly divided into normal control group [normal saline (NS)], model group (PTZ + NS), rHuEpo treated group (PTZ + rHuEpo), LY294002 treated group (PTZ + LY294002 + rHuEpo) and LY294002 control group (rHuEpo + PTZ + DMSO). Apoptosis of hippocampal neurons was detected by TUNEL method; expression of phosphorylation protein kinase B (p-PKB/p-Akt), Bcl-2 and Bad were detected by immunohistochemistry; the expression of Bcl-2 mRNA, Bad mRNA in hippocampal neurons of rats were detected through reverse transcription polymerase chain reaction (RT-PCR); the expression of Akt, p-Akt and Bcl-2, Bad protein in hippocampal neurons of rats were detected by western blotting. The amount of apoptotic neurons was less in the rHuEpo treated group and the LY294002 control group than in the LY294002 treated group (P<0.05). The expression of p-Akt protein and Bcl-2 protein increased while the Bad protein decreased significantly in the rHuEpo treated group and the LY294002 control group compared with the LY294002 treated group (P<0.05). The expression of Bad protein and Bad mRNA in hippocampus increased while the p-Akt, Bcl-2, Bcl-2 mRNA decreased significantly in the LY294002 treated group compared with the rHuEpo treated group (P<0.05). The PI3K/Akt signaling pathway is one of the pathways of rHuEpo neuroprotective effects and was confirmed from both the of positive and negative aspects. rHuEpo regulates the expression of mitochondrial apoptotic pathway related factors Bad and Bcl-2 to inhibit apoptosis and promotes neuronal survival.

Procyanidins B2 reverses the T-2 toxin-induced mitochondrial apoptosis in TM3 Leydig cells

Procyanidins B2 (PB2) was found to block T-2 toxin-induced reproductive toxicity and may be used as a dietary preventive agent. We investigated the mechanism of PB2 in blocking the T-2 toxin-induced apoptosis in Leydig cells in conjunction with analysis of T-2 toxin induced proapoptotic responses in Leydig cells including induction of oxidative stresses. The stress inducible proteins ASK, p38 and JNK were increased in response to T-2 toxin and the induction was attenuated by PB2. Furthermore, we showed that T-2 toxin suppressed mitochondrial membrane potential, reduced the levels of the mitochondrial respiratory chain complex I-V, adversely affected the level of mitochondrial proteins CypD, Bax, Bak, Bcl-2, Bcl-xl, Bax/Bcl-2 and cyt c and these adverse effects were counteracted by PB2 in dose-dependent manner. In summary, our results showed great potential of PB2 in preventing T-2 toxin-induced toxicity in Leydig cells through its potent antioxidant property.

Hederacolchiside A1 suppresses proliferation of tumor cells by inducing apoptosis through modulating PI3K/Akt/mTOR signaling pathway

ObjectiveHederacolchiside A1, exhibits cytostatic and cytotoxic activity against various cancer cells in vitro, however, the mechanism is not well understood. MethodsIn this study, Hederacolchiside A1 from Pulsatilla chinensis was isolated and tested its anticancer activity and mechanism. Hederacolchiside A1 could inhibit proliferation of A549, SMMC-7721, BEL-7402, and MCF-7 cells by MTT assay. Investigations of apoptosis of treated cancer cells were identified in hederacolchiside A1 by flow cytometric analysis of annexin V expression. ResultsBased on the results of western blotting and JC-1 staining, hederacolchiside A1 reduced the mitochondrial membrane potential and Bcl-2 protein levels, whereas cleaved caspase-3 was higher. Furthermore, hederacolchiside A1 effectively inhibited the phosphorylations of phosphatidylinositol 3 kinase (PI3K), protein kinase B (Akt), and mammalian target of rapamycin (mTOR). In vivo study showed that hederacolchiside A1 (3.0, 4.5, and 6.0 mg/kg, ip) could significantly inhibit the weight of tumor in an H22 xenograft model. Similar inhibitory activities were observed when the compound (3.25, 7.5, and 15.0 mg/kg, ig) was tested in nude mice xenograft tumor models using human breast carcinoma MCF-7 cells. ConclusionThese data indicated that hederacolchiside A1 suppressed the proliferation of human tumor cells by inducing apoptosis through modulating the PI3K/Akt/mTOR signaling pathway.

Chemopreventive action of Imatinib, a tyrosine kinase inhibitor in the regulation of angiogenesis and apoptosis in rat model of lung cancer

The present study explored the events of angiogenesis and apoptosis in 7,12-dimethyl benz(a)anthracene (DMBA)-induced lung cancer in rat and its chemoprevention with Imatinib, a receptor tyrosine kinase inhibitor. Further, it includes lipopolysaccharide (LPS) mediating inflammation along with DMBA for the promotion of lung carcinogenesis. The animals received a single intratracheal instillation of DMBA (20mg/kg body weight) in olive oil and LPS (0.6mg/kg body weight) to induce tumors in 16weeks. Besides morphology and histology of the lung tissues, RT-PCR, western blots, and immunofluorescence were performed for the expression of apoptotic and angiogenic proteins. Apoptosis was studied by mitochondrial Bcl-2/Bax ratio and staining with thedyes Acridine orange/ethidium bromide of the isolated Broncho epithelial cells. Also, mitochondrial membrane potential (ΔΨM) was studied by JC-1. The study revealed that the expression of VEGF, MMP-2, MMP-9, and the chemokine MCP-1 to be very high in DMBA and DMBA + LPS groups, while Bcl-2 also shows an elevated expression. These results were restored with Imatinib treatment. The pro-apoptotic proteins, Bax, Bad, Apaf-1, and Caspase-3 were highly diminished in DMBA and DMBA + LPS groups which were recovered with Imatinib treatment.

Lup-20(29)-en-3\u03b2,28-di-yl-nitrooxy acetate affects MCF-7 proliferation through the crosstalk between apoptosis and autophagy in mitochondria

Betulin (BT), a pentacyclic lupine-type triterpenoid natural product, possesses antitumor activity in various types of cancers. However, its clinical development was discouraged due to its low biological activities and poor solubility. We prepared lup-20(29)-en-3β,28-di-yl-nitrooxy acetate (NBT), a derivative of BT, that was chemically modified at position 3 of ring A and C-28 by introducing a NO-releasing moiety. This study mainly explored the mechanism of NBT in treating breast cancer through the crosstalk between apoptosis and autophagy in mitochondria. NBT possessed a potent antiproliferative activity in MCF-7 cells both in vitro and in vivo. Mechanically, NBT affected cell death through the mitochondrial apoptosis pathway and autophagy. NBT induced cell cycle arrest in the G0/G1 phase by decreasing the expression of cyclin D1. It also induced mitochondrial apoptosis by increasing the expression of Bax, caspase-9, and poly(ADP-ribose) polymerase and mitochondrial membrane potential loss and leaks of cytochrome c (Cyt C) from mitochondria in MCF-7 cells and decreasing the expression of mitochondrial Bcl-2. We further demonstrated whether chloroquine (CQ), which inhibits the degradation of autophagosome induced by NBT, affects the proliferation of MCF-7 cells compared with NBT. The experiments inferred that the combination of NBT and CQ significantly promoted MCF-7 cell mitochondria to divide and Cyt C to be released from mitochondria to the cytoplasm, resulting in an increased apoptosis rate. The in vivo experiments showed that NBT inhibited the growth of MCF-7 tumor via the apoptosis pathway, and its effect was similar to 5-fluorouracil.

Icariin modulates mitochondrial function and apoptosis in high glucose-induced glomerular podocytes through G protein-coupled estrogen receptors.

Podocyte apoptosis in glomerular lesions has been found to have a dominant role in the progression of diabetic nephropathy. The present research aimed to explore the beneficial effect of icariin on diabetic podocytes by interfering in the process of apoptosis. Podocyte apoptosis was significantly exacerbated after high glucose treatment, with the level of reactive oxygen species (ROS) increasing simultaneously. Here, we demonstrated that icariin, which is a G protein-coupled estrogen receptor 1 (GPER) agonist, inhibited podocyte apoptosis by reducing ROS, maintaining the integrity of mitochondrial membranes. Moreover, the stabilization of mitochondria by icariin was reversed when GPER was knocked down in podocytes. Meanwhile, icariin inhibited the caspase cascade in podocyte apoptosis by promoting Bcl-2 expression and mitochondrial translocation. The above findings at least partly elucidated the mechanism by which icariin stabilized podocytes by inducing the mitochondrial Bcl-2 translocation and therefore preventing downstream apoptosis.

MnTMPyP inhibits paraquat-induced pulmonary epithelial-like cell injury by inhibiting oxidative stress.

To investigate the protective effect and underlying mechanism of the superoxide dismutase mimic, manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin pentachloride (MnTMPyP), on paraquat (PQ)-induced lung alveolar epithelial-like cell injury. Lung alveolar epithelial-like cells (A549) were pretreated with 10 μM MnTMPyP for 1.5 hr and then cultured with or without PQ (750 uM) for 24 hr. Cell survival was determined using the MTT assay. Apoptosis, mitochondrial transmembrane potential, reactive oxygen species (ROS) production, and Ca2+ levels were measured using flow cytometry. Glutathione reductase activity (GR activity) and caspase-3 activation were determined using spectrophotometry. Expression of the apoptosis proteins, Bcl-2 and Bax, and the endoplasmic reticulum (ER) stress proteins, glucose regulatory protein 78 (Grp78) and C/EBP homologous protein (CHOP), was measured using Western blot analysis. Cell viability, mitochondrial membrane potential, GR activity, and Bcl-2 expression were decreased, but apoptosis, ROS production, caspase-3 activity, cytoplasmic Ca2+ levels, and Bax, Grp78 and CHOP expression were all increased in the PQ group compared to the control group. There were no statistically significant changes in the MnTMPyP group. Cell viability, GR activity, mitochondrial membrane potential, and Bcl-2 protein expression were all increased, while apoptosis, ROS production, cytoplasmic Ca2+ levels, caspase-3 activity, and Bax, Grp78 and CHOP expression were all significantly reduced in the MnTMPyP group compared to PQ group. MnTMPyP effectively reduced PQ-induced lung epithelial-like cell injury, and the underlying mechanism is related to antagonism of PQ-induced oxidative stress.

Immunosuppressant drug tacrolimus induced mitochondrial nephrotoxicity, modified PCNA and Bcl-2 expression attenuated by Ocimum basilicum L. in CD1 mice.

Tacrolimus (TAC) is used sporadically as an immunosuppressive agent for organ transplantation, but its clinical used is limited due to its marked nephrotoxicity. Ocimum basilicum L. (Lamiaceae) (OB) had been shown to possess antioxidant, anti-inflammatory and nephroprotective activity, and effective at improving renal inflammation and glomerular. In our study, we aim to evaluate the efficacy of the OB against TAC-induced mitochondrial nephrotoxicity in CD1 mice. Mice were randomly divided into four groups. Group 1 (control group); administered orally with normal saline (1 mL/kg) for two weeks; Group 2 (OB extract treated-group) (500 mg/kg b.wt) gavaged once/day for two weeks; Group 3 (TAC-treated group) (3 mg/kg b.wt, administered ip once a day for two weeks); and Group 4; (TAC plus OB extract treated-group). Tacrolimus-induced nephrotoxicity was assessed biochemically and histopathologically. The OB extract was high in phenolic content (50.3 mg/g of gallic acid equivalent), total flavonoids (14.5 mg/g CE equivalent). The potential antioxidant efficacy of the extract (IC50) was 24.5 μg/mL. OB pretreatment significantly improved the TAC-induced changes in biochemical markers of nephrotoxicity for instance blood urea nitrogen (BUN), creatinine, total protein, and albumin (P < 0.01, when compared with TAC treated group). Also, it significantly restored the increase activities of TBARS, protein carbonyl (PC) (P < 0.001, when compared to healthy control group) and decreased activities of nonprotein thiol (NP-SH) levels, Mn-superoxide dismutase (Mn-SOD) and glutathione peroxidase (GPx) antioxidants of mitochondria. The nephroprotective efficacy of the OB leaves extract was further evident by histopathological analysis together with the PCNA-ir and Bcl2. The upshot of the present study revealed that the OB possessed significant antioxidant and nephroprotective activity and had a preventive effect on the biochemical alterations and histological changes in TAC-treated mice.

Di-2-pyridylhydrazone Dithiocarbamate Butyric Acid Ester Exerted Its Proliferative Inhibition against Gastric Cell via ROS-Mediated Apoptosis and Autophagy.

Diversified biological activities of dithiocarbamates have attracted widespread attention; improving their feature or exploring their potent action of mechanism is a hot topic in medicinal research. Herein, we presented a study on synthesis and investigation of a novel dithiocarbamate, DpdtbA (di-2-pyridylhydrazone dithiocarbamate butyric acid ester), on antitumor activity. The growth inhibition assay revealed that DpdtbA had important antitumor activity for gastric cancer (GC) cell lines (IC50 = 4.2 ± 0.52 μM for SGC-7901, 3.80 ± 0.40 μM for MGC-803). The next study indicated that growth inhibition is involved in ROS generation in mechanism; accordingly, the changes in mitochondrial membrane permeability, apoptotic genes, cytochrome c, bax, and bcl-2 were observed, implying that the growth inhibition of DpdtbA is involved in ROS-mediated apoptosis. On the other hand, the upregulated p53 upon DpdtbA treatment implied that p53 could also mediate the apoptosis. Yet the excess generation of ROS induced by DpdtbA led to cathepsin D translocation and increase of autophagic vacuoles and LC3-II, demonstrating that autophagy was also a contributor to growth inhibition. Further investigation showed that DpdtbA could induce cell cycle arrest at the G1 phase. This clearly indicated the growth inhibition of DpdtbA was via triggering ROS formation and evoking p53 response, consequently leading to alteration in gene expressions that are related to cell survival.

Protective effects of PACAP against lactacystin-induced PC12 cell apoptosis due to prevention of mitochondrial damage and endoplasmic reticulum stress

Background: Pituitary Adenylate Cyclase Activating Peptide (PACAP) has a significant protective effect on nerve cell damage induced by several neurotoxins. The ubiquitin proteasome inhibitor lactacystin, one new neurotoxin, could cause cell damage by inducing dopaminergic cells apoptosis. Whether PACAP has a protective effect on lactacystin-induced cytotoxic model has not been reported. Aims: The present study was designed to explore whether PACAP can effectively inhibit the lactacystininduced dopaminergic apoptosis. Methods: Pheochromocytes (PC12 cells) differentiated by nerve growth factor as neurons were treated with lactacystin (20 μmol/l) for 24 h, to establish the Parkinson's disease cells model. Groups are set up as control group, lactacystin group, PACAP1-27 intervention group, PACAP1-27 and PACAP6-27 cointervention group. Results: Treatment with lactacystin can make the cell vitality obviously decline. Compared with control group, cell morphology presented obviously damaged change in lactacystin group. After treatment with lactacystin, the expression of mitochondrial bcl-2 dramaticly decreased, expression of mitochondrial bax had no change, the rate of bcl-2/bax dramaticly decreased, and thus expression of caspase-3 increased. Meanwhile, caspase-12, the characteristic marker of endoplasmic reticulum stress, increased significantly in lactacystin group. However, these changes could be partly reversed by treatment with PACAP1-27. This protective effect of PACAP1-27 was negated by PACAP6-27, a receptor antagonist of PACAP1-27. Conclusion: Lactacystin led to cell damage by inducing mitochondrial damage and endoplasmic reticulum stress; PACAP1-27 played a protective role by regulating the mitochondrial and endoplasmic reticulum related dual signal pathway. As one PACAP1-27 receptor antagonist, PACAP6-27 attenuated this effect of PACAP1-27.

Pterostilbene Reduces Acetaminophen-Induced Liver Injury by Activating the Nrf2 Antioxidative Defense System via the AMPK/Akt/GSK3β Pathway

Background/Aims: Pterostilbene (Pts), a natural dimethylated analog of resveratrol from blueberries, exerts antioxidative and anti-apoptotic effects in various diseases. This study aims to investigate the protective effects and mechanism of Pts against acetaminophen (APAP)-induced hepatotoxicity in vivo. Methods: C57BL/6 mice were treated with APAP or APAP+Pts. HepG2 cells were used to further explore the underlying mechanisms in vitro. The effects of Pts on hepatotoxicity were measured by commercial kits, Hematoxylin and Eosin (H&E) straining, TUNEL assay, Western blot analysis, and Flow cytometry assay. Results: In vivo, Pts treatment effectively protected against APAP-induced severe liver injury by decreasing the lethality rate, the serum alanine transaminase (ALT) and aspartate aminotransferase (AST) levels, liver histological injury, liver malondialdehyde (MDA) formation and myeloperoxidase (MPO) levels and by increasing liver glutathione (GSH) and superoxide dismutase (SOD) levels. Moreover, in Pts-treated mice, the nuclear factor-erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway was activated; however, APAP-induced c-Jun NH2-terminal kinase (JNK) activation, mitochondrial Bcl-2 Associated X Protein (Bax) translocation, apoptosis-inducing factor (AIF) levels and cytochrome c release were attenuated. In vitro, Pts was found to reverse hydrogen peroxide (H₂O₂) -induced cytotoxicity, reactive oxygen species (ROS) production and apoptosis that depended on Nrf2 activation. Moreover, Pts induced a dose-dependent increase in the phosphorylation of AMP-activated protein kinase (AMPK), serine/threonine kinase (Akt), and glycogen synthase kinase 3β (GSK3β) in HepG2 cells. Moreover, Pts protect against APAP or H₂O₂-induced toxicity were effectively attenuated or abolished in HepG2 Nrf2-/- cells and Nrf2-/- mice. Conclusion: Our data suggest that Pts protects against APAP-induced toxicity by activating Nrf2 via the AMPK/Akt/GSK3β pathway.

Calpain and JNK pathways participate in isoflurane – induced nucleus translocation of apoptosis-inducing factor in the brain of neonatal rats

Recent studies have demonstrated that volatile anesthetic causes caspase-dependent neuroapoptosis and persistent cognitive deficits in young animals. Apoptosis-inducing factor (AIF) can trigger apoptosis by caspase-independent pathway. Whether isoflurane induces neuroapoptosis by activation of AIF and its possible mechanism are underdetermined. Rats at postnatal day 7 were exposed to 1.1% isoflurane for 4 h and the expression of AIF, cytochrome c, caspase-3, μ-calpain, m-calpain, Bcl-2 and Bax in the mitochondrial, cytosolic, and nuclear fraction, as well as the number of both AIF and TUNEL positive neurons in the cortices of rats were measured. Moreover, the effects of calpain inhibitor MDL-28170 or JNK inhibitor SP600125 on isoflurane-induced AIF release, caspase activation and cognitive deficits were assessed. We found isoflurane activated CytC-caspase-3 dependent apoptosis pathway mainly in the early phase (0–6 h after exposure). Moreover, isoflurane activated mitochondrial μ-calpain, induced AIF truncation during early phase and activated m-calpain, induced AIF release from the mitochondria to cytosol and translocation into the nucleus in the late phase (6–24 h after exposure). MDL-28170 attenuated the isoflurane–induced mitochondrial AIF truncation, release and nuclear translocation, but did not change the expression of cleaved-caspase-3 and mitochondrial Bax and Bcl-2 proteins. SP600125 attenuated isoflurane-induced neuroapoptosis by inhibiting both AIF and caspase-3 pathways and reduced cognitive impairment in neonatal rats. This is the first study to provide the evidence that isoflurane induced AIF-dependent neuroapoptosis by activation of mitochondrial μ-calpain and m-calpain in neonatal rats. JNK inhibition reversed isoflurane-induced neuroapoptosis and subsequent long-term neurocognitive impairment, acting via inhibiting activation of both AIF and caspase-3 pathways.

The Influential Role of BCL2 Family Members in Synovial Sarcomagenesis.

Synovial sarcomas are deadly soft tissue malignancies associated with t(X;18) balanced chromosomal translocations. Expression of the apoptotic regulator BCL2 is prominent in synovial sarcomas and has prompted the hypothesis that synovial sarcomagenesis may depend on it. Herein, it is demonstrated that Bcl2 overexpression enhances synovial sarcomagenesis in an animal model. Furthermore, we determined increased familial clustering of human synovial sarcoma patients with victims of other BCL2-associated malignancies in the Utah Population Database. Conditional genetic disruption of Bcl2 in mice also led to reduced sarcomagenesis. Pharmacologic inhibition specific to BCL2 had no demonstrable efficacy against human synovial sarcoma cell lines or mouse tumors. However, targeting BCLxL in human and mouse synovial sarcoma with the small molecule BH3 domain inhibitor, BXI-72, achieved significant cytoreduction and increased apoptotic signaling. Thus, the contributory role of BCL2 in synovial sarcomagenesis does not appear to render it as a therapeutic target, but mitochondrial antiapoptotic BCL2 family members may be.Implications: The association of BCL2 expression with synovial sarcoma is found to fit with a subtle, but significant, impact of its enhanced presence or absence during early tumorigenesis. However, specific pharmacologic inhibition of BCL2 does not demonstrate a persistent dependence in fully developed tumors. Conversely, inhibition of the BCL2 family member BCLxL resulted in nanomolar potency against human synovial sarcoma cell lines and 50% tumor reduction in a genetically engineered mouse model. Mol Cancer Res; 15(12); 1733-40. ©2017 AACR.

Human biliverdin reductase regulates the molecular mechanism underlying cancer development.

Human biliverdin reductase (hBVR), is an enzyme, that converts biliverdin to bilirubin, and has been implicated in epithelial-mesenchymal transition (EMT) in breast cancer. However, few studies have investigated the role of hBVR in cell apoptosis in other cancers. Therefore, the aim of this study was to investigate the effects of hBVR in several lines of cancer cells. The results of this study showed that hBVR expression was up-regulated in breast, lung, and liver cancers, but not ovarian cancer. Moreover, hBVR inhibition by small interfering RNA (si-hBVR) attenuated cell survival and increased caspase-3 protein expression, which was mediated by the ERK1/2 signaling pathway in relative cancer cells. In addition, the mitochondrial membrane potential and mitochondrial membrane proteins Bcl-2 and BAX were found to be necessary for activation of the mitochondria dependent apoptosis pathway. Collectively, these findings indicated, for the first time, that the anti-apoptotic effect of hBVR in cancers which portend new strategies for developing novel biomarkers and effective treatment ways for cancer patients.

Chemical characterization of cytotoxic indole acetic acid derivative from mulberry fruit (Morus alba L.) against human cervical cancer

The fruit of the white mulberry tree (Morus alba L.) is a multiple fruit with a sweet flavor commonly consumed around the world. Chemical investigation of the fruits led to the isolation of two indole acetic acid derivatives (1 −2) including a new compound, which turned out to be an isolation artifact, 3S-(β-D-glucopyranosyloxy)-2,3-dihydro-2-oxo-1H-indole-3-acetic acid butyl ester (1), along with five known compounds (3 −7). Compounds 2 and 7 were newly identified from mulberry fruit. The new isolation artifact (1) exhibited cytotoxic effect on human cervical cancer Hela cells in a dose-dependent manner. Compound 1 activated caspase-8, caspase-9, and caspase-3, followed by cleavage of PARP, a substrate of caspase-3, in a dose-dependent manner. Simultaneous alterations in protein expression of mitochondrial factors Bax, BID and Bcl-2 were also observed. A comparison between compounds 1 and 2 led to a structure-activity relationship analysis of the cytotoxic effect. These results suggest that compound 1 could be beneficial in human cervical cancer treatment, and provide a theoretical basis for further application of compound 1.