A Missense Variant in AIFM1 Caused Mitochondrial Dysfunction and Intolerance to Riboflavin Deficiency.

AIFM1 is a mitochondrial flavoprotein involved in caspase-independent cell death and regulation of respiratory chain complex biogenesis. Mutations in the AIFM1 gene have been associated with multiple clinical phenotypes, but the effectiveness of riboflavin treatment remains controversial. Furthermore, few studies explored the reasons underlying this controversy. We reported a 7-year-old boy with ataxia, sensorimotor neuropathy and muscle weakness. Genetic and histopathological analyses were conducted, along with assessments of mitochondrial function and apoptosis level induced by staurosporine. Riboflavin deficiency and supplementation experiments were performed using fibroblasts. A missense c.1019T > C (p. Met340Thr) variant of AIFM1 was detected in the proband, which caused reduced expression of AIFM1 protein and mitochondrial dysfunction as evidenced by downregulation of mitochondrial complex subunits, respiratory deficiency and collapse of ΔΨm. The proportion of apoptotic cells in mutant fibroblasts was lower than controls after induction of apoptosis. Riboflavin deficiency resulted in decreased AIFM1 protein levels, while supplementation with high concentrations of riboflavin partially increased AIFM1 protein levels in variant fibroblasts. In addition, mitochondrial respiratory function of mutant fibroblasts was partly improved after riboflavin supplementation. Our study elucidated the pathogenicity of the AIFM1 c.1019T > C variant and revealed mutant fibroblasts was intolerant to riboflavin deficiency. Riboflavin supplementation is helpful in maintaining the level of AIFM1 protein and mitochondrial respiratory function. Early riboflavin treatment may serve as a valuable attempt for patients with AIFM1 variant.

Medicinal Herbs and Their Derived Ingredients Protect against Cognitive Decline in In Vivo Models of Alzheimer's Disease.

Alzheimer's disease (AD) has pathological hallmarks including amyloid beta (Aß) plaque formation. Currently approved single-target drugs cannot effectively ameliorate AD. Medicinal herbs and their derived ingredients (MHDIs) have multitarget and multichannel properties, engendering exceptional AD treatment outcomes. This review delineates how in in vivo models MHDIs suppress Aß deposition by downregulating ß- and γ-secretase activities; inhibit oxidative stress by enhancing the antioxidant activities and reducing lipid peroxidation; prevent tau hyperphosphorylation by upregulating protein phosphatase 2A expression and downregulating glycogen synthase kinase-3ß expression; reduce inflammatory mediators partly by upregulating brain-derived neurotrophic factor/extracellular signal-regulated protein kinase 1/2-mediated signaling and downregulating p38 mitogen-activated protein kinase (p38 MAPK)/c-Jun N-terminal kinase (JNK)-mediated signaling; attenuate synaptic dysfunction by increasing presynaptic protein, postsynaptic protein, and acetylcholine levels and preventing acetylcholinesterase activity; and protect against neuronal apoptosis mainly by upregulating Akt/cyclic AMP response element-binding protein/B-cell lymphoma 2 (Bcl-2)-mediated anti-apoptotic signaling and downregulating p38 MAPK/JNK/Bcl-2-associated x protein (Bax)/caspase-3-, Bax/apoptosis-inducing factor-, C/EBP homologous protein/glucose-regulated protein 78-, and autophagy-mediated apoptotic signaling. Therefore, MHDIs listed in this review protect against Aß-induced cognitive decline by inhibiting Aß accumulation, oxidative stress, tau hyperphosphorylation, inflammation, synaptic damage, and neuronal apoptosis in the cortex and hippocampus during the early and late AD phases.

Realgar (As4S4), a traditional Chinese medicine, induces acute promyelocytic leukemia cell death via the Bcl-2/Bax/Cyt-C/AIF signaling pathway in vitro.

Acute promyelocytic leukemia (APL) is a specific subtype of acute myelogenous leukemia (AML) characterized by the proliferation of abnormal promyelocytes. Realgar, a Chinese medicine containing arsenic, can be taken orally. Traditional Chinese medicine physicians have employed realgar to treat APL for over a thousand years. Therefore, realgar may be a promising candidate for the treatment of APL. Nevertheless, the underlying mechanism behind realgar therapy is largely unclear. The present study aimed to investigate the effect of realgar on cell death in the APL cell line (NB4) in vitro and to elucidate the underlying mechanism. In this study, after APL cells were treated with different concentrations of realgar, the cell survival rate, apoptotic assay, morphological changes, ATP levels and cell cycle arrest were assessed. The expression of Bcl-2, Bax, Cytochrome C (Cyt-C) and apoptosis-inducing factor (AIF) at the mRNA and protein levels were also measured by immunofluorescence, quantitative PCR (qPCR) and Western blotting. We found that realgar could significantly inhibit APL cell proliferation and cell death in a time- and dose-dependent manner. Realgar effectively decreased the ATP levels in APL cells. Realgar also induced APL cell cycle arrest at the S and G2/M phases. Following realgar treatment, the mRNA and protein levels of Bcl-2 were significantly downregulated, whereas the levels of Bax, Cyt-C, and AIF were significantly upregulated. In summary, realgar can induce APL cell death via the Bcl-2/Bax/Cyt-C/AIF signaling pathway, suggesting that realgar may be an effective therapeutic for APL.

Effects of moxibustion at bilateral Feishu (BL13) and Xinshu (BL15) combined with benazepril on myocardial cells apoptosis index and apoptosis-related proteins cytochrome c and apoptosis-inducing factor in rats with chronic heart failure.

OBJECTIVE: To observe the effects of moxibustion at bilateral Feishu (BL13) and Xinshu (BL15) combined with benazepril on myocardial cells apoptosis index, the expression levels of apoptosis-related proteins cytochrome c (Cyt-C) and apoptosis-inducing factor (AIF) in chronic heart failure (CHF) rats. METHODS: Sixty-five rats were randomly divided into normal group () and model-I group (). After modeling, CHF rats in model-I group were divided into model group, moxibustion group, benazepril group, moxibustion plus benazepril group (abbreviated as aibei group, the same below), 10 rats in each group. Echocardiogram index was examined by echocardiography. Hemodynamic indices were measured by rat cardiac function meter. Serum B-type brain natriuretic peptide (BNP) was detected by enzyme-linked immunosorbent assay. Myocardial cells apoptosis index was detected by terminal-deoxynucleoitidyl transferase mediated nick end labeling staining. Pathological changes of myocardial tissues were observed by hematoxylin and eosin staining. The expression levels of Cyt-C and AIF in myocardial tissues were detected by Western blot. RESULTS: Compared with normal group, ejection fraction and left ventricular diameter shortening rate in model-Ⅰ group were significantly reduced, myocardial cells of rats in model group exhibited unclear transverse striations, cells swellings and vacuoles, cardiac functions were deteriorated, serum BNP level, myocardial cells apoptosis index, and the expression levels of Cyt-C and AIF were significantly increased. Compared with model group, myocardial cells of rats in moxibustion group, benazepril group, and aibei group were dyed more evenly, muscle fibers were arranged relatively neatly, cardiac functions were improved, serum BNP level, myocardial cells apoptosis index, and the expression levels of Cyt-C and AIF were significantly decreased. Compared with aibei group, cardiac functions were worsened, myocardial cells apoptosis index, and the expression levels of Cyt-C and AIF were increased. CONCLUSION: Moxibustion at bilateral Feishu (BL13) and Xinshu (BL15) combined with benazepril could improve CHF better than moxibustion at bilateral Feishu (BL13) and Xinshu (BL15) or benazepril alone. The mechanisms might be that they can inhibit the expressions of Cyt-C and AIF, and inhibit the apoptosis of cardiomyocytes.

Auriculasin enhances ROS generation to regulate colorectal cancer cell apoptosis, ferroptosis, oxeiptosis, invasion and colony formation.

Colorectal cancer (CRC) is one of the most common malignant tumors in the digestive system, and Chinese herbal medicine plays an important role in tumor treatment. The in-depth study of auriculasin isolated from Flemingia philippinensis showed that auriculasin promoted reactive oxygen species (ROS) generation in a concentration-dependent manner; when ROS scavenger NAC was added, the effects of auriculasin in promoting ROS generation and inhibiting cell viability were blocked. Auriculasin induced CRC cell apoptosis, led to mitochondrial shrinkage, and increased the intracellular accumulation of Fe2+ and MDA. When auriculasin and NAC were added simultaneously, the levels of apoptosis, Fe2+ and MDA returned to the control group levels, indicating that auriculasin activated apoptosis and ferroptosis by inducing ROS generation. In addition, auriculasin promoted the expression of Keap1 and AIFM1, but significantly reduced the phosphorylation level of AIFM1, while NAC significantly blocked the regulation of Keap1 and AIFM1 by auriculasin, which indicates that auriculasin can also induce oxeiptosis through ROS. When Z-VAD-FMK, Ferrostatin-1, Keap1 siRNA, PGAM5 siRNA and AIFM1 siRNA were added respectively, the inhibitory effect of auriculasin on cell viability was significantly weakened, indicating that auriculasin inhibits cell viability by inducing apoptosis, ferroptosis and oxeiptosis. Auriculasin also inhibited the invasion and clone forming ability of CRC cells, while NAC blocked the above effects of auriculasin. Therefore, auriculasin can promote CRC cell apoptosis, ferroptosis and oxeiptosis by inducing ROS generation, thereby inhibiting cell viability, invasion and clone formation, indicating that auriculasin has a significant antitumor effect.

[Lycium barbarum polysaccharide reduces testicular spermatogenic injury in Immp2l－/－mice through GPX4 and AIF pathways].

OBJECTIVE: To investigate the protective effect of Lycium barbarum polysaccharide (LBP) against testicular spermatogenic injury in mice with oxidative stress (OS) and its mechanism. METHODS: A unique OS model was made in 1.5-month-old mice with mitochondrial inner membrane-like peptide-2 mutation (Immp2l－/－), which were fed with water (the negative control group) or LBP in water at the concentration of 20 mg/kg (the LBP intervention group), and wild-type Immp2l＋/＋ mice used as normal controls and fed with water only. Then all the mice were sacrificed at 13 months old and the testis tissue harvested for observation of pathological changes by HE staining, measurement of routine semen parameters, and detection of the apoptosis of spermatogenic cells by TUNEL and the expression levels of glutathione peroxidase 4 (GPX4) and apoptosis-inducing factor (AIF) by immunohistochemistry and Western blot. RESULTS: Thinned testicular cortex was observed in the negative controls, with evident vacuolar degeneration and reduced numbers of germ cells and elongated spermatids in the lumen of the seminiferous tubules, but all these pathological changes were improved and the germ cells at different levels orderly arranged in the LBP intervention group. Compared with the normal controls, the mice in the negative control group showed dramatically reduced sperm count (ï¼»72.89 ± 8.28ï¼½ vs ï¼»20.78 ± 1.45ï¼½ ×106, P<0.01) and the percentages of progressively motile sperm (PMS) (ï¼»58.62 ± 6.15ï¼½% vs ï¼»18.37 ± 2.67ï¼½%, P<0.01) and morphologically normal sperm (MNS) (ï¼»65.81 ± 7.69ï¼½% vs ï¼»20.33 ± 3.17ï¼½%, P<0.01) and increased apoptosis of spermatogenic cells (ï¼»1.45 ± 0.43ï¼½% vs ï¼»7.14 ± 0.78ï¼½%, P<0.01). LBP intervention, however, significantly increased the sperm count (ï¼»45.25 ± 3.39ï¼½ ×106, P<0.05), PMS (ï¼»36.34 ± 4.56ï¼½%, P<0.05) and MNS (ï¼»38.72 ± 3.63ï¼½%, P<0.05) and decreased the apoptosis of spermatogenic cells (ï¼»2.28 ± 0.07ï¼½%, P<0.01). The mice in the LBP intervention group, in comparison with the negative controls, exhibited remarkably up-regulated expression of GPX4 (2.75 ± 0.48 vs 1.43 ± 0.17, P<0.05) and down-regulated expression of AIF (2.43 ± 0.15 vs 1.35 ± 0.51, P<0.05). CONCLUSIONS: Lycium barbarum polysaccharide at 20 mg/kg can reduce testicular spermatogenic injury in Immp2l－/－ mice with oxidative stress through GPX4 and AIF pathways.

[Effect of hot water extract of Korean ginseng on neuroblastoma cell parthanatos].

OBJECTIVE: To explore the effect of pretreatment of neuroblastoma cells with hot water extract of Korean ginseng on MNNG-induced parthanatos and its mechanism. METHODS: Neuroblastoma SH-SY5Y cells were pretreated with 1 mg/L hot water extract of Korean ginseng before induction with 250 µmol/L MNNG for 1 h or 4 h. CCK-8 and cell flow cytometry were used to detect cell survival rate. Western blotting was used to detect the changes in poly(ADP-ribose) (PAR) expression in the treated cells. Immunofluorescence assay was used to detect nuclear distribution of apoptosis-inducing factor (AIF), and flow cytometry was used to detect the level of reactive oxygen species (ROS) in the cells. RESULTS: Compared with the blank control cells, MNNG-treated SH-SY5Y cells showed significantly decreased survival rate as the concentration of MNNG and the stimulation time increased (P < 0.05). Stimulation with MNNG also resulted in significantly increased expression of PAR protein in the cells (P < 0.05). Pretreatment of the cells with hot water extract of Korean ginseng obviously inhibited MNNG-induced cell death and significantly reduced AIF expression and nucleation in the cells (P < 0.05). MNNG stimulation significantly increased ROS level in the cells, which was decreased significantly by pretreatment of the cells with the extract (P < 0.05). CONCLUSIONS: Pretreatment with hot water extract of Korean ginseng reduces MNNG-induced parthanatos and ROS production in SH-SY5Y cells.

Cytotoxic mechanisms of primin, a natural quinone isolated from Eugenia hiemalis, on hematological cancer cell lines.

Considering the high morbidity and mortality rates associated with hematological malignancies and the frequent development of drug resistance by these diseases, the search for new cytotoxic agents is an urgent necessity. The new compounds should present higher efficiency and specificity in inducing tumor cell death, be easily administered and have little or negligible adverse effects. Quinones have been reported in the literature by their several pharmacological properties, including antitumor activity, thus, the aim of this study was to investigate the cytotoxic effect of primin, a natural quinone, on hematological malignancies cell lines. Primin was highly cytotoxic against the three cell lines included in this study (K562, Jurkat and MM.1S) in a concentration- and time-dependent manner, as demonstrated by the MTT method. The compound triggered an apoptotic-like cell death, as observed by ethidium bromide/acridine orange staining, DNA fragmentation and phosphatidylserine exposure after labeling with Annexin V. Both intrinsic and extrinsic apoptosis are involved in cell death induced by primin, as well as the modulation of cell proliferation marker KI-67. The activation of intrinsic apoptosis appears to be related to a decreased Bcl-2 expression and increased Bax expression. While the increase in FasR expression signals activate extrinsic apoptosis. The results suggest that primin is a promising natural molecule that could be used in hematological malignancies therapy or as prototypes for the development of new chemotherapics.

The poly-ADP ribose polymerase-1/apoptosis-inducing factor pathway may help mediate the protective effect of electroacupuncture on early brain injury after subarachnoid hemorrhage.

Subarachnoid hemorrhage (SAH) is a clinically common, acute, critical cerebrovascular disease associated with high mortality. Here, we investigated the effects of electroacupuncture on early brain injury after SAH. We successfully established a Sprague-Dawley rat model of the SAH model, and randomly divided the rats into four groups: sham-operated group, SAH group, positive control group, and electroacupuncture group. Electroacupuncture effectively decreased the number of transferase UTP nick end labeling-positive cells and extent of DNA fragmentation compared with the control, indicating a decrease in apoptosis. Moreover, electroacupuncture decreased the expression of proteins involved in the poly-ADP ribose polymerase-1/apoptosis-inducing factor (PARP-1/AIF) pathway in vivo, and the difference was statistically significant (P < 0.05). Treatment with electroacupuncture resulted in a significant improvement in neurological function. It inhibited the increase in blood-brain barrier permeability by regulating the protein expression of matrix metalloproteinase-9, occludin, and claudin-5. Additionally, electroacupuncture limited the development of cerebral edema and microglial activation in early brain injury after SAH. In conclusion, electroacupuncture can ameliorate early brain injury after SAH, and this may occur via inhibition of the PARP-1/AIF pathway.

Involvement of PARP-1/AIF Signaling Pathway in Protective Effects of Gualou Guizhi Decoction Against Ischemia-Reperfusion Injury-Induced Apoptosis.

Cerebral ischemia-reperfusion injury is a complex pathophysiological process. Poly(ADP-ribose) (PAR) polymerase-1 (PARP-1)/apoptosis-inducing factor (AIF) signaling pathway-mediated apoptosis is one of the non-caspase-dependent cell death programs that are widely present in neurological diseases such as stroke. In our study, we aimed to conduct further research on the effects of Gualou Guizhi decoction (GLGZD) on the PARP-1/AIF signaling pathway in cell apoptosis after ischemia-reperfusion injury caused by middle cerebral artery occlusion (MCAO). The results showed that GLGZD administration for 7 days significantly ameliorated MCAO-induced neurological damage, limb paralysis and the pathological state of the ischemic cortex. GLGZD exerted its effects by significantly reducing the volume of ischemic cerebral infarction, increasing the number of Nissl-positive cells, and reducing neuronal apoptosis. Furthermore, Western blot analysis showed that GLGZD significantly inhibited the total protein expression of PARP-1, PAR, AIF and endonuclease G (Endo G) in the ischemic cortex and significantly increased the total protein expression of heat-shock protein 70 (Hsp70). On the one hand, the expression of PARP-1, AIF and Endo G protein in the nucleus significantly decreased while the expression of PAR nucleoprotein significantly upregulated. On the other hand, compared with the MCAO model group, the GLGZD-treated group showed a significantly reduced protein expression of PAR in mitochondria and significantly increased protein expression of mitochondrial AIF and Endo G. It was concluded that GLGZD had good therapeutic effects in MCAO model rats. These effects were closely related to GLGZD-mediated inhibition of ischemia-induced neuronal apoptosis by regulation of protein expression and translocation in the PARP-1/AIF signaling pathway.

Modified Xiaoyao San ameliorates depressive-like behaviors by triggering autophagosome formation to alleviate neuronal apoptosis.

Major depressive disorder (MDD) affects ˜16% of the world population. Chronic stressors contribute to reduced hippocampal volumes and increase the risk of developing MDD. Our previous work showed that XYS ameliorates social isolation and chronic unpredictable mild stress (CUMS) induced depressive-like behaviors in rats by regulating hypothalamic-pituitary-adrenal hyperactivation, locus coeruleus -norepinephrine activity and kynurenine/5-hydroxytryptamin balance. Here, we report that CUMS & isolation-treated mice exhibit depressive-like behaviors and show a phenotype of mixed apoptosis/autophagy characteristic in mice hippocampus in vivo. Modified Xiaoyao San (MXS) significantly ameliorates CUMS & social isolation-induced anhedonia, loss of interests, psychomotor retardation and behavioral despair. It suppresses the apoptosis by downregulaing condensation of heterochromatin and reducing hippocampal TdT-mediated dUTP Nick-End Labeling (TUNEL)-positive cells. MSX significantly inhibits mitochondrial outer membrane permeabilization (MOMP) reduces the release of cytochrome C and the shift of apoptosis inducing factor (AIF) from mitochondria to nucleus. Further, it stimulates the formation of autophagosomes and activates the expression of Atg5 and LC3II. Combined silencing of Atg5 and Atg7 dampens MOMP and impaired the anti-apoptotic effects of MXS. In conclusion, MXS ameliorates depressive-like behaviors by triggering autophagy to alleviate neuronal apoptosis. MXS is an effective supplement for MDD treatment, and can be harnessed to enhance autophagy and synergize with antidepressant action.

Berberine Induces Cell Apoptosis through Cytochrome C/Apoptotic Protease-Activating Factor 1/Caspase-3 and Apoptosis Inducing Factor Pathway in Mouse Insulinoma Cells.

OBJECTIVE: To investigate apoptotic effects of berberine, a significant alkaloids component existing in Rhizoma coptidis, and its possible acting mechanism in insulinoma cells. METHODS: Different concentrations of berberine were used to treat mouse insulinoma (MIN6) cells for various period of time. The viability and apoptosis of the cells were analyzed using methylthiazolyldiphenvl-tetrazolium bromide assay, flow cytometry and enzyme-linked immuno sorbent assay. Changes in the relating pro- and anti-apoptosis proteins were detected by western-blotting. RESULTS: The half-maximal inhibitory concentration (IC50) of berberine was 5.7 µmol/L on MIN6 cells viability for 16 h. Berberine caused a 20% reduction (P<0.05) in cell number after only 4-h incubation; which reached 50% after 24 h (P<0.01). Berberine treatment for 16 h significantly increased the level of DNA fragmentation. The flow cytometry showed the apoptotic rate increased 2.9- and 4.6-fold after treating with berberine (5 µmol/L) for 8 and 16 h, while 3- and 8.7-fold after 10 µmol/L treatment for 8 and 16 h (P<0.01). Berberine treatment dramatically elevated the expression ratio of Bax to Bcl-2. Meanwhile, berberine notably increased the apoptosis-inducing factors and cytochrome C transforming from the mitochondria to the cytoplasm. Apoptotic protease-activating factor 1 (Apaf-1) was subsequently activated after cytochrome C release. Furthermore, caspase-3 and poly adenosine diphosphate-ribose polymerase were also activated to trigger apoptosis cascade. CONCLUSION: High concentration (5 and 10 µmol/L) of berberine could induce the apoptosis of MIN6 cells through cytochrome C/Apaf-1/caspase-3 and apoptosis inducing factor (AIF) pathway.

Alpha lipoic acid attenuates cadmium-induced nephrotoxicity via the mitochondrial apoptotic pathways in rat.

Alpha lipoic acid (α-LA), a potent antioxidant, is protective against acute nephrotoxicity. In the present study, the attenuation of cadmium (Cd)-induced kidney injury by α-LA on was investigated in a rat model. Exposure to 50 mg/L Cd for 12 weeks increased kidney index and Cd content, malondialdehyde (MDA) levels, and histological damage to the renal cortex, and decreased the activities of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH). Treatment with 50 mg/L Cd also damaged renal cell mitochondria and nuclei, and activated the mitochondrial apoptosis pathway, indicated by increased gene and protein expression/activation of caspase-9, caspase-3, poly ADP-ribose polymerase (PARP) and Bcl-2 adenovirus E1a nineteen kilodalton interacting protein 3 (BNIP3), and translocation of cytochrome c (cyt c), apoptosis-inducing factor (AIF), and endonuclease G (Endo G). However, simultaneous supplementation with α-LA (50 mg/kg·bw) protected kidney cells from Cd-induced cytotoxicity by reducing MDA levels and Cd content, restoring endogenous enzyme activities, renewing mitochondrial function, and preventing activation of the mitochondria apoptosis pathway.

Auriculasin-induced ROS causes prostate cancer cell death via induction of apoptosis.

Recent studies have demonstrated that natural agents targeting the accumulation of reactive oxygen species (ROS) that selectively kill, leaving normal cells undamaged, can suppress prostate cancer. Here, we show that auriculasin, derived from Flemingia philippinensis, induces significant cell death and apoptosis via ROS generation in prostate cancer cells. Auriculasin treatment resulted in selective apoptotic cell death in LNCaP prostate cancer cells, characterized by DNA fragmentation, accumulation of sub-G1 cell population, cleavage of poly (ADP-ribose) polymerase (PARP), regulation of Bax/Bcl-2 ratio, increase of cytosolic apoptosis-inducing factor (AIF) and endonuclease G (EndoG), in addition to inhibiting tumor growth in a xenograft mouse model. Interestingly, auriculasin-induced apoptosis did not result in caspase-3, -8, and -9 activations. We found that auriculasin treatment decreased phosphorylation of AKT/mTOR/p70s6k in a dose- and time-dependent manner. Further, cellular ROS levels increased in LNCaP cells treated with auriculasin and blocking ROS accumulation with ROS scavengers resulted in inhibition of auriculasin-induced PARP cleavage, AIF increase, upregulation of Bax/Bcl-2 ratio, and decrease in AKT/mTOR phosphorylation. Taken together, these data suggest that auriculasin targets ROS-mediated caspase-independent pathways and suppresses PI3K/AKT/mTOR signaling, which leads to apoptosis and decreased tumor growth.

Supplementary choline attenuates olive oil lipid emulsion-induced enterocyte apoptosis through suppression of CELF1/AIF pathway.

Enterocyte apoptosis induced by lipid emulsions is a key cause of intestinal atrophy under total parenteral nutrition (TPN) support, and our previous work demonstrated that olive oil lipid emulsion (OOLE) could induce enterocyte apoptosis via CUGBP, Elav-like family member 1 (CELF1)/ apoptosis-inducing factor (AIF) pathway. As TPN-associated complications are partially related to choline deficiency, we aimed to address whether choline supplementation could attenuate OOLE-induced enterocyte apoptosis. Herein we present evidence that supplementary choline exhibits protective effect against OOLE-induced enterocyte apoptosis both in vivo and in vitro. In a rat model of TPN, substantial reduction in apoptotic rate along with decreased expression of CELF1 was observed when supplementary choline was added to OOLE. In cultured Caco-2 cells, supplementary choline attenuated OOLE-induced apoptosis and mitochondria dysfunction by suppressing CELF1/AIF pathway. Compared to OOLE alone, the expression of CELF1 and AIF was significantly decreased by supplementary choline, whereas the expression of Bcl-2 was evidently increased. No obvious alterations were observed in Bax expression and caspase-3 activation. Mechanistically, supplementary choline repressed the expression of CELF1 by increasing the recruitment of CELF1 mRNA to processing bodies, thus resulting in suppression of its protein translation. Taken together, our data suggest that supplementary choline exhibits effective protection against OOLE-induced enterocyte apoptosis, and thus, it has the potential to be used for the prevention and treatment of TPN-induced intestinal atrophy.

Olive Oil-Supplemented Lipid Emulsion Induces CELF1 Expression and Promotes Apoptosis in Caco-2 Cells.

BACKGROUND AND AIMS: Parenterally-administered lipid emulsion (LE) is a key cause of enterocyte apoptosis under total parenteral nutrition, yet the pathogenesis has not been fully understood. CUGBP, Elav-like family member 1 (CELF1) has been recently identified as a crucial modulator of apoptosis, and thus this study sought to investigate its role in the LE-induced apoptosis in vitro. METHODS: Caco-2 cells were used as an in vitro model. The cells were treated with varying LEs derived from soybean oil, olive oil or fish oil, and changes in the apoptosis and CELF1 expression were assessed. Rescue study was performed using transient knockdown of CELF1 with specific siRNA prior to LE treatment. Regulation of CELF1 by LE treatment was studied using quantitative real-time PCR and Western blotting. RESULTS: All the LEs up-regulated CELF1expression and induced apoptosis, but only olive oil-supplemented lipid emulsion (OOLE)-induced apoptosis was attenuated by depletion of CELF1. Up-regulation of apoptosis-inducing factor (AIF) was involved in OOLE-induced CELF1 dependent apoptosis. The protein expression of CELF1 was up-regulated by OOLE in a dose- and time-dependent manner, but the mRNA expression of CELF1 was unchanged. Analysis by polysomal profiling and nascent protein synthesis revealed that the regulation of CELF1 by OOLE treatment was mediated by directly accelerating its protein translation. CONCLUSION: OOLE-induces apoptosis in Caco-2 cells partially through up-regulation of CELF1.

Ramalin-Mediated Apoptosis Is Enhanced by Autophagy Inhibition in Human Breast Cancer Cells.

Breast cancer, the most commonly diagnosed cancer in women worldwide, is treated in various ways. Ramalin is a chemical compound derived from the Antarctic lichen Ramalina terebrata and is known to exhibit antioxidant and antiinflammatory activities. However, its effect on breast cancer cells remains unknown. We examined the ability of ramalin to induce apoptosis and its mechanisms in MCF-7 and MDA-MB-231 human breast cancer cell lines. Ramalin inhibited cell growth and induced apoptosis in both cell lines in a concentration-dependent manner. By upregulating Bax and downregulating Bcl-2, ramalin caused cytochrome c and apoptosis-inducing factor to be released from the mitochondria into the cytosol, thus activating the mitochondrial apoptotic pathway. In addition, activated caspase-8 and caspase-9 were detected in both types of cells exposed to ramalin, whereas ramalin activated caspase-3 only in the MDA-MB-231 cells. Ramalin treatment also increased the levels of LC3-II and p62. Moreover, the inhibition of autophagy by 3-methyladenine or Atg5 siRNA significantly enhanced ramalin-induced apoptosis, which was accompanied by a decrease in Bcl-2 levels and an increase in Bax levels. Therefore, autophagy appears to be activated as a protective mechanism against apoptosis in cancer cells exposed to ramalin. These findings suggest that ramalin is a potential anticancer agent for the treatment of patients with non-invasive or invasive breast cancer.

TPGS/Phospholipids Mixed Micelles for Delivery of Icariside II to Multidrug-Resistant Breast Cancer.

The biggest challenge for the treatment of multidrug resistant cancer is to deliver a high concentration of anticancer drugs to cancer cells. Icariside II is a flavonoid from Epimedium koreanum Nakai with remarkable anticancer properties, but poor solubility and significant efflux from cancer cells limited its clinical use. In our previous study, a self-assembled mixture of micelles (TPGS-Icariside II-phospholipid complex) was successfully constructed, which could substantially increase the solubility of Icariside II and inhibit the efflux on Caco-2 cells. In this study, we evaluate the anticancer effect of the mixed micelles encapsulating Icariside II (Icar-MC) on MCF-7/ADR, a multidrug-resistant breast cancer cell line. The cellular uptake of the micelles was confirmed by fluorescent coumarin-6-loaded micelles. The IC50 of Icar-MC in MCF-7/ADR was 2-fold less than the free drug. The in vitro study showed Icar-MC induced more apoptosis and lactate dehydrogenase release. Intravenous injection of Icar-MC into nude mice bearing MCF-7/ADR xenograft resulted in a better antitumor efficacy compared with the administration of free drug, without causing significant body weight changes in mice. The antitumor effect was further verified by magnetic resonance imaging and immunohistochemical assays for Ki-67, a proliferative indicator. Moreover, Icar-MC treatment also elevated Bax/Bcl-2 ratio and the expressions of cleaved caspase-3, -8, -9 and AIFM1 in tumors. This study suggests that phospholipid/TPGS mixed micelles might be a suitable drug delivery system for Icariside II to treat multidrug resistant breast cancer.

Deoxypodophyllotoxin triggers parthanatos in glioma cells via induction of excessive ROS.

Parthanatos is a new form of programmed cell death that is regulated by hyper-activated PARP-1, and is emerging as a new strategy to kill cancer cells. Deoxypodophyllotoxin (DPT) is a natural chemical that is found to induce cancer cell death, in which the role of parthanatos is unknown. Thus, we investigated this issue in this study by using glioma cell lines and mice model of xenograft glioma. We found that DPT induced glioma cell death in vitro and inhibited the growth of xenograft glioma in vivo, which was accompanied with parthanatos-related biochemical events including expressional upregulation of PARP-1, cytoplasmic accumulation of PAR polymer, and nuclear translocation of AIF. In vitro study revealed that genetic knockdown of PARP-1 with small interfering RNA attenuated DPT-induced elevation in the cytoplasmic PAR-polymer and the nuclear AIF, as well as protected glioma cells against the toxicity of DPT. Further, antioxidant NAC, as well as PARP-1 inhibitor 3AB, not only alleviated the overproduction of ROS caused by DPT, but also reversed the above-mentioned biochemical events, maintained mitochondrial membrane potential and rescued glioma cells death. Therefore, we demonstrated that deoxypodophyllotoxin triggered parthanatos in glioma cells via induction of excessive ROS.

Apoptosis-inducing Factor (AIF) and Its Family Member Protein, AMID, Are Rotenone-sensitive NADH:Ubiquinone Oxidoreductases (NDH-2).

Apoptosis-inducing factor (AIF) and AMID (AIF-homologous mitochondrion-associated inducer of death) are flavoproteins. Although AIF was originally discovered as a caspase-independent cell death effector, bioenergetic roles of AIF, particularly relating to complex I functions, have since emerged. However, the role of AIF in mitochondrial respiration and redox metabolism has remained unknown. Here, we investigated the redox properties of human AIF and AMID by comparing them with yeast Ndi1, a type 2 NADH:ubiquinone oxidoreductase (NDH-2) regarded as alternative complex I. Isolated AIF and AMID containing naturally incorporated FAD displayed no NADH oxidase activities. However, after reconstituting isolated AIF or AMID into bacterial or mitochondrial membranes, N-terminally tagged AIF and AMID displayed substantial NADH:O2 activities and supported NADH-linked proton pumping activities in the host membranes almost as efficiently as Ndi1. NADH:ubiquinone-1 activities in the reconstituted membranes were highly sensitive to 2-n-heptyl-4-hydroxyquinoline-N-oxide (IC50 = ∼1 µm), a quinone-binding inhibitor. Overexpressing N-terminally tagged AIF and AMID enhanced the growth of a double knock-out Escherichia coli strain lacking complex I and NDH-2. In contrast, C-terminally tagged AIF and NADH-binding site mutants of N-terminally tagged AIF and AMID failed to show both NADH:O2 activity and the growth-enhancing effect. The disease mutant AIFΔR201 showed decreased NADH:O2 activity and growth-enhancing effect. Furthermore, we surprisingly found that the redox activities of N-terminally tagged AIF and AMID were sensitive to rotenone, a well known complex I inhibitor. We propose that AIF and AMID are previously unidentified mammalian NDH-2 enzymes, whose bioenergetic function could be supplemental NADH oxidation in cells.