Deficiency in apoptosis-inducing factor recapitulates chronic kidney disease via aberrant mitochondrial homeostasis

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein with dual roles in redox signaling and programmed cell death. Deficiency in AIF is known to result in defective oxidative phosphorylation (OXPHOS), via loss of complex I activity and assembly in other tissues. Because the kidney relies on OXPHOS for metabolic homeostasis, we hypothesized that a decrease in AIF would result in chronic kidney disease (CKD). Here, we report that partial knockdown of Aif in mice recapitulates many features of CKD, in association with a compensatory increase in the mitochondrial ATP pool via a shift toward mitochondrial fusion, excess mitochondrial reactive oxygen species production, and Nox4 upregulation. However, despite a 50% lower AIF protein content in the kidney cortex, there was no loss of complex I activity or assembly. When diabetes was superimposed onto Aif knockdown, there were extensive changes in mitochondrial function and networking, which augmented the renal lesion. Studies in patients with diabetic nephropathy showed a decrease in AIF within the renal tubular compartment and lower AIFM1 renal cortical gene expression, which correlated with declining glomerular filtration rate. Lentiviral overexpression of Aif1m rescued glucose-induced disruption of mitochondrial respiration in human primary proximal tubule cells. These studies demonstrate that AIF deficiency is a risk factor for the development of diabetic kidney disease.

Bioprospecção de polissacarídeos sulfatados de macroalgas marinhas do litoral do Rio Grande do Norte: caracterização de uma heterofucana extraída da alga marron sargassum filipendula que induz apoptose em células hela

The coast of Rio Grande do Norte has more than 100 species of seaweed, mostly unexplored regarding their pharmacological potential. The sulfated polysaccharides (PS) are by far the more seaweed compounds studied, these present a range of biological properties, such as anticoagulant activity, anti-inflammatory, antitumor and antioxidant properties. In this study, we extract sulfated polysaccharide rich-extracts of eleven algae from the coast of Rio Grande do Norte (Dictyota cervicornis; Dictiopterys delicatula; Dictyota menstruallis; Dictyota mertensis; Sargassum filipendula; Spatoglossum schröederi; Gracilaria caudata; Caulerpa cupresoides; Caulerpa prolifera; Caulerpa sertularioides e Codim isthmocladum), and these were evaluated for the potential anticoagulant, antioxidant and antiproliferative. All polysaccharide extracts showed activity for anticoagulant, antioxidant and/or antiproliferative activity, especially D. delicatula and S. filipendula, which showed the most prominent pharmacological potential, thereby being chosen to have their sulfated polysaccharides extracted. By fractionating method were obtained six fractions rich in sulfated polysaccharides to the algae D. delicatula (DD-0,5V, DD-0, 7V, DD-1,0v, DD-1,3v, DD-1,5v and DD-2,0) and five fractions to the alga S. filipendula (SF-0,5V, SF-0,7V, SF-1,0v, SF-1,5v and SF-2,0v). For the anticoagulant assay only the fractions of D. delicatula showed activity, with emphasis on DD-1, 5v that presented the most prominent activity, with APTT ratio similar to clexane® at 0.1 mg/mL. When evaluated the antioxidant potential, all fractions showed potential in all tests (total antioxidant capacity, hydroxyl and superoxide radicals scavenging, ferrous chelation and reducing power), however, the ability to chelate iron ions appears as the main mechanism antioxidant of sulfated polysaccharides from seaweed. In antiproliferative assay, all heterofucanas showed dose-dependent activity for the inhibition of cell proliferation of HeLa, however, with the exception of SF-0,7V, SF- 1,0v and SF-1,5v, all fractions showed antiproliferative activity against MC3T3, a normal cell line. The heterofucana SF-1,5V had its antiproliferative mechanism of action evaluated. This heterofucan induces apoptosis in HeLa cells by a pathway caspase independent, promoting the release of apoptosis Inducing Factor (AIF) in the cytosol, which in turn induces chromatin condensation and DNA fragmentation into 50Kb fragments. These results are significant in that they provide a mechanistic framework for further exploring the use of SF-1.5v as a novel chemotherapeutics against human cervical cancer.

Bioprospecção de polissacarídeos sulfatados de macroalgas marinhas do litoral do Rio Grande do Norte: caracterização de uma heterofucana extraída da alga marron Sargassum filipendula que induz apoptose em células HeLa

The coast of Rio Grande do Norte has more than 100 species of seaweed, mostly unexplored regarding their pharmacological potential. The sulfated polysaccharides (PS) are by far the more seaweed compounds studied, these present a range of biological properties, such as anticoagulant activity, anti-inflammatory, antitumor and antioxidant properties. In this study, we extract sulfated polysaccharide rich-extracts of eleven algae from the coast of Rio Grande do Norte (Dictyota cervicornis; Dictiopterys delicatula; Dictyota menstruallis; Dictyota mertensis; Sargassum filipendula; Spatoglossum schröederi; Gracilaria caudata; Caulerpa cupresoides; Caulerpa prolifera; Caulerpa sertularioides e Codim isthmocladum), and these were evaluated for the potential anticoagulant, antioxidant and antiproliferative. All polysaccharide extracts showed activity for anticoagulant, antioxidant and/or antiproliferative activity, especially D. delicatula and S. filipendula, which showed the most prominent pharmacological potential, thereby being chosen to have their sulfated polysaccharides extracted. By fractionating method were obtained six fractions rich in sulfated polysaccharides to the algae D. delicatula (DD-0,5V, DD-0, 7V, DD-1,0v, DD-1,3v, DD-1,5v and DD-2,0) and five fractions to the alga S. filipendula (SF-0,5V, SF-0,7V, SF-1,0v, SF-1,5v and SF-2,0v). For the anticoagulant assay only the fractions of D. delicatula showed activity, with emphasis on DD-1, 5v that presented the most prominent activity, with APTT ratio similar to clexane® at 0.1 mg/mL. When evaluated the antioxidant potential, all fractions showed potential in all tests (total antioxidant capacity, hydroxyl and superoxide radicals scavenging, ferrous chelation and reducing power), however, the ability to chelate iron ions appears as the main mechanism antioxidant of sulfated polysaccharides from seaweed. In antiproliferative assay, all heterofucanas showed dose-dependent activity for the inhibition of cell proliferation of HeLa, however, with the exception of SF-0,7V, SF- 1,0v and SF-1,5v, all fractions showed antiproliferative activity against MC3T3, a normal cell line. The heterofucana SF-1,5V had its antiproliferative mechanism of action evaluated. This heterofucan induces apoptosis in HeLa cells by a pathway caspase independent, promoting the release of apoptosis Inducing Factor (AIF) in the cytosol, which in turn induces chromatin condensation and DNA fragmentation into 50Kb fragments. These results are significant in that they provide a mechanistic framework for further exploring the use of SF-1.5v as a novel chemotherapeutics against human cervical cancer.

Proteasome inhibitors trigger NOXA-mediated apoptosis in melanoma and myeloma cells

Patients with metastatic melanoma or multiple myeloma have a dismal prognosis because these aggressive malignancies resist conventional treatment. A promising new oncologic approach uses molecularly targeted therapeutics that overcomes apoptotic resistance and, at the same time, achieves tumor selectivity. The unexpected selectivity of proteasome inhibition for inducing apoptosis in cancer cells, but not in normal cells, prompted us to define the mechanism of action for this class of drugs, including Food and Drug Administration-approved bortezomib. In this report, five melanoma cell lines and a myeloma cell line are treated with three different proteasome inhibitors (MG-132, lactacystin, and bortezomib), and the mechanism underlying the apoptotic pathway is defined. Following exposure to proteasome inhibitors, effective killing of human melanoma and myeloma cells, but not of normal proliferating melanocytes, was shown to involve p53-independent induction of the BH3-only protein NOXA. Induction of NOXA at the protein level was preceded by enhanced transcription of NOXA mRNA. Engagement of mitochondrial-based apoptotic pathway involved release of cytochrome c, second mitochondria-derived activator of caspases, and apoptosis-inducing factor, accompanied by a proteolytic cascade with processing of caspases 9, 3, and 8 and poly(ADP)-ribose polymerase. Blocking NOXA induction using an antisense (but not control) oligonucleotide reduced the apoptotic response by 30% to 50%, indicating a NOXA-dependent component in the overall killing of melanoma cells. These results provide a novel mechanism for overcoming the apoptotic resistance of tumor cells, and validate agents triggering NOXA induction as potential selective cancer therapeutics for life-threatening malignancies such as melanoma and multiple myeloma.

Involvement of mitochondrial and B-RAF/ERK signaling pathways in berberine-induced apoptosis in human melanoma cells

The natural isoquinoline alkaloid berberine exhibits a wide spectrum of biological activities including antitumor activity, but its mechanism of action remains to be fully elucidated. Here, we report that berberine induced apoptosis in human melanoma cells, through a process that involved mitochondria and caspase activation. Berberine-induced activation of a number of caspases, including caspases 3, 4, 7, 8, and 9. Pan-caspase inhibitor, z-VAD-fmk, and caspase-8 and caspase-9 inhibitors prevented apoptosis. Berberine also led to the generation of the p20 cleavage fragment of BAP31, involved in directing proapoptotic signals between the endoplasmic reticulum and the mitochondria. Treatment of SK-MEL-2 melanoma cells with berberine induced disruption of the mitochondrial transmembrane potential, release of cytochrome c and apoptosis-inducing factor from the mitochondria to the cytosol, generation of reactive oxygen species (ROS), and a decreased ATP/ADP ratio. Overexpression of bcl-xL by gene transfer prevented berberine-induced cell death, mitochondrial transmembrane potential loss, and cytochrome c and apoptosis-inducing factor release, but not ROS generation. N-acetyl-L-cysteine inhibited the production of ROS, but did not abrogate the berberine-induced apoptosis. Inhibition of extracellular signal-regulated kinase (ERK) phosphorylation, by using the mitogen-activated protein kinase/ERK kinase inhibitor PD98059, and reduction of B-RAF levels by silencing RNA induced cell death of SK-MEL-2 cells, and diminished the berberine concentration required to promote apoptosis. These data show that berberine-induced apoptosis in melanoma cells involves mitochondria and caspase activation, but ROS generation was not essential. Our results indicate that inhibition of B-RAF/ERK survival signaling facilitates the cell death response triggered by berberine. © 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins.

Novel titanocene anti-cancer drugs and their effect on apoptosis and the apoptotic pathway in prostate cancer cells

Advanced prostate cancer is not curable by current treatment strategies indicating a significant need for new chemotherapeutic options. Highly substituted ansatitanocene compounds have shown promising cytotoxic activity in a range of cancers. The objectives of this study are to examine the effects of these titanocene compounds on prostate cancer cells. Prostate cell lines were treated with three novel titanocene compounds and compared to titanocene dichloride and cisplatin. Percent apoptosis, viability and cell cycle were assessed using propidium iodide DNA incorporation with flow cytometry. Cytochrome C was assessed by western blotting of mitochondrial and cytoplasmic fractions. Apoptosis Inducing Factor was assessed by confocal microscopy. These novel compounds induced more apoptosis compared to cisplatin in a dose dependent manner. Compound Y had the most significant effect on cell cycle and apoptosis. Despite the release of cytochrome C from the mitochondrial fraction there was no inhibition of apoptosis with the pan caspase inhibitor, ZVAD-FMK. AIF was shown to translocate from the cytosol to the nucleus mediating a caspase independent cell death. Bcl-2 over expressing PC-3 cells, which were resistant to cisplatin induced apoptosis, underwent apoptosis following treatment with all the titanocene compounds. This study demonstrates possible mechanisms by which these novel titanocene compounds can mediate their apoptotic effect in vitro. The fact that they can induce more apoptosis than cisplatin in advanced cancer cell lines would confer an advantage over cisplatin. They represent exciting new agents with future potential for the treatment of advanced prostate cancer.

Brain-derived neurotrophic factor protects against multiple forms of brain injury in bacterial meningitis

BACKGROUND Brain-derived neurotrophic factor (BDNF) blocks activation of caspase-3, reduces translocation of apoptosis-inducing factor (AIF), attenuates excitotoxicity of glutamate, and increases antioxidant enzyme activities. The mechanisms of neuroprotection suggest that BDNF may be beneficial in bacterial meningitis. METHODS To assess a potentially beneficial effect of adjuvant treatment with BDNF in bacterial meningitis, 11-day-old infant rats with experimental meningitis due to Streptococcus pneumoniae or group B streptococci (GBS) were randomly assigned to receive intracisternal injections with either BDNF (3 mg/kg) or equal volumes (10 mu L) of saline. Twenty-two hours after infection, brains were analyzed, by histomorphometrical examination, for the extent of cortical and hippocampal neuronal injury. RESULTS Compared with treatment with saline, treatment with BDNF significantly reduced the extent of 3 distinct forms of brain cell injury in this disease model: cortical necrosis in meningitis due to GBS (median, 0.0% [range, 0.0%-33.7%] vs. 21.3% [range, 0.0%-55.3%]; P<.03), caspase-3-dependent cell death in meningitis due to S. pneumoniae (median score, 0.33 [range, 0.0-1.0] vs. 1.10 [0.10-1.56]; P<.05), and caspase-3-independent hippocampal cell death in meningitis due to GBS (median score, 0 [range, 0-2] vs. 0.88 [range, 0-3.25]; P<.02). The last form of injury was associated with nuclear translocation of AIF. CONCLUSION BDNF efficiently reduces multiple forms of neuronal injury in bacterial meningitis and may hold promise as adjunctive therapy for this disease.

Expression of B-nerve growth factor in rabbit male tract and seminal plasma

Nerve growth factor (NGF) has been recently identified as an ovulation inductor factor (OIF) in the seminal plasma (SP) (Ratto et al. PNAS 2012; 109:15042-7). The presence of OIF in rabbit has been suggested but this protein has not yet been identified. Our aim was to study the mRNA expression in the rabbit male reproductive tract and to identify the protein β-NGF in the SP.

Par4 is a coactivator for a splice isoform-specific transcriptional activation domain in WT1

The Wilms’ tumor suppressor protein WT1 is a transcriptional regulator involved in differentiation and the regulation of cell growth. WT1 is subject to alternative splicing, one isoform including a 17–amino acid region that is specific to mammals. The function of this 17–amino acid insertion is not clear, however. Here, we describe a transcriptional activation domain in WT1 that is specific to the WT1 splice isoform that contains the 17–amino acid insertion. We show that the function of this domain in transcriptional activation is dependent on a specific interaction with the prostate apoptosis response factor par4. A mutation in WT1 found in Wilms’ tumor disturbs the interaction with par4 and disrupts the function of the activation domain. Analysis of WT1 derivatives in cells treated to induce par4 expression showed a strong correlation between the transcription function of the WT1 17–amino acid insertion and the ability of WT1 to regulate cell survival and proliferation. Our results provide a molecular mechanism by which alternative splicing of WT1 can regulate cell growth in development and disease.

A Novel Anticancer Agent, 8-Methoxypyrimido4 `,5 `: 4,5]thieno(2,3-b) Quinoline-4(3H)-One Induces Neuro 2a Neuroblastoma Cell Death through p53-Dependent, Caspase-Dependent and Independent Apoptotic Pathways

Neuroblastoma is the most common cancer in infants and fourth most common cancer in children. Despite recent advances in cancer treatments, the prognosis of stage-IV neuroblastoma patients continues to be dismal which warrant new pharmacotherapy. A novel tetracyclic condensed quinoline compound, 8-methoxypyrimido 4 `,5 `: 4,5] thieno(2,3-b) quinoline-4(3H)-one (MPTQ) is a structural analogue of an anticancer drug ellipticine and has been reported to posses anticancer property. Study on MPTQ on neuroblastoma cells is very limited and mechanisms related to its cytotoxicity on neuroblastoma cells are completely unknown. Here, we evaluated the anticancer property of MPTQ on mouse neuro 2a and human SH-SY5Y neuroblastoma cells and investigated the mechanisms underlying MPTQ-mediated neuro 2a cell death. MPTQ-mediated neuro 2a and SH-SY5Y cell deaths were found to be dose and time dependent. Moreover, MPTQ induced cell death reached approximately 99.8% and 90% in neuro 2a and SH-SY5Y cells respectively. Nuclear oligonucleosomal DNA fragmentation and Terminal dUTP Nick End Labelling assays indicated MPTQ-mediated neuro 2a cell death involved apoptosis. MPTQ-mediated apoptosis is associated with increased phosphorylation of p53 at Ser15 and Ser20 which correlates with the hyperphosphorylation of Ataxia-Telangiectasia mutated protein (ATM). Immunocytochemical analysis demonstrated the increased level of Bax protein in MPTQ treated neuro 2a cells. MPTQ-mediated apoptosis is also associated with increased activation of caspase-9, -3 and -7 but not caspase-2 and -8. Furthermore, increased level of caspase-3 and cleaved Poly ( ADP Ribose) polymerase were observed in the nucleus of MPTQ treated neuro 2a cells, suggesting the involvement of caspase-dependent intrinsic but not extrinsic apoptotic pathway. Increased nuclear translocation of apoptosis inducing factor suggests additional involvement of caspase-independent apoptosis pathway in MPTQ treated neuro 2a cells. Collectively, MPTQ-induced neuro 2a cell death is mediated by ATM and p53 activation, and Bax-mediated activation of caspase-dependent and caspase-independent mitochondrial apoptosis pathways.

Interaction of Leukocyte Elastase Inhibitor/L-DNase II with BCL-2 and BAX

Leukocyte Elastase Inhibitor (LEI, also called serpin B1) is a protein involved in apoptosis among other physiological processes. We have previously shown that upon cleavage by its cognate protease, LEI is transformed into L-DNase II, a protein with a pro-apoptotic activity. The caspase independent apoptotic pathway, in which L-DNase II is the final effector, interacts with other pro-apoptotic molecules like Poly-ADP-Ribose polymerase (PARP) or Apoptosis Inducing Factor (AIF). The screening of LEI/L-DNase II interactions showed a possible interaction with several members of the BCL-2 family of proteins which are known to have a central role in the regulation of caspase dependent cell death. In this study, we investigated the regulation of LEI/L-DNase II pathway by two members of this family of proteins: BAX and BCL-2, which have opposite effects on cell survival. We show that, in both BHK and HeLa cells, LEI/L-DNase II can interact with BCL-2 and BAX in apoptotic and non-apoptotic conditions. These proteins which are usually thought to be anti-apoptotic and pro-apoptotic respectively, both inhibit the L-DNase II pro-apoptotic activity. These results give further insight in the regulation of caspase-independent pathways and highlight the involvement of the intracellular environment of a given protein in the determinism of its function. They also add a link between caspase-dependent and independent pathways of apoptosis.

Redox-sensitive prosurvival and proapoptotic protein expression in the myocardial remodeling post-infarction in rats

In this study, we investigated the oxidative stress influence in some prosurvival and proapoptotic proteins after myocardial infarction (MI). Male Wistar rats were divided in two groups: Sham-operated (control) and MI. MI was induced by left coronary artery occlusion. 28-days after surgery, echocardiographic, morphometric, and hemodynamic parameters were evaluated. Redox status (reduced to oxidized glutathione ratio, GSH/GSSG) and hydrogen peroxide levels (H(2)O(2)) were measured in heart tissue. The p-ERK/ERK, p-Akt/Akt, p-mTOR/mTOR and p-GSK-3 beta/GSK-3 beta ratios, as well as apoptosis-inducing factor (AIF) myocardial protein expression were quantified by Western blot. MI group showed an increase in cardiac hypertrophy (23%) associated with a decrease in ejection fraction (38%) and increase in left ventricular end-diastolic pressure (82%) when compared to control, characterizing ventricular dysfunction. Redox status imbalance was seen in MI animals, as evidenced by the decrease in the GSH/GSSG ratio (30%) and increased levels of H(2)O(2) (45%). This group also showed an increase in the ERK phosphorylation and a reduction of Akt and mTOR phosphorylation when compared to control. Moreover, we showed a reduction in the GSK-3 beta phosphorylation and an increase in AIF protein expression in MI group. Taken together, our results show increased H(2)O(2) levels and cellular redox imbalance associated to a higher p-ERK and AIF immunocontent, which would contribute to a maladaptive hypertrophy phenotype.

Células-tronco mesenquimais de equinos: isolamento, cultivo e caracterização

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

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

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Inhibition of tissue transglutaminase sensitizes TRAIL-resistant lung cancer cells through upregulation of death receptor 5

Tissue transglutaminase (TG2) is implicated in cellular processes such as apoptosis and cell migration. Its acyl transferase activity cross-links certain proteins, among them transcription factors were described. We show here that the TG2 inhibitor KCC009 reversed resistance to tumor necrosis factor-related apoptosis-inducing factor (TRAIL) in lung cancer cells. Sensitization required upregulation of death receptor 5 (DR5) but not of death receptor 4. Upregulation of DR5 involved the first intron of the DR5 gene albeit it was independent from p53 and nuclear factor kappa B. In conclusion, inhibition of tissue transglutaminase provides an interesting strategy for sensitization to TRAIL-induced apoptosis in p53-deficient lung cancer cells.