Mitochondrial division inhibitor (mdivi-1) induces extracellular matrix (ECM)-detachment of viable breast cancer cells by a DRP1-independent mechanism.

Increasing evidence supports the hypothesis that cancer progression is under mitochondrial control. Mitochondrial fission plays a pivotal role in the maintenance of cancer cell homeostasis. The inhibition of DRP1, the main regulator of mitochondrial fission, with the mitochondrial division inhibitor (mdivi-1) had been associated with cancer cell sensitivity to chemotherapeutics and decrease proliferation. Here, using breast cancer cells we find that mdivi-1 induces the detachment of the cells, leading to a bulk of floating cells that conserved their viability. Despite a decrease in their proliferative and clonogenic capabilities, these floating cells maintain the capacity to re-adhere upon re-seeding and retain their migratory and invasive potential. Interestingly, the cell detachment induced by mdivi-1 is independent of DRP1 but relies on inhibition of mitochondrial complex I. Furthermore, mdivi-1 induces cell detachment rely on glucose and the pentose phosphate pathway. Our data evidence a novel DRP1-independent effect of mdivi-1 in the attachment of cancer cells. The generation of floating viable cells restricts the use of mdivi-1 as a therapeutic agent and demonstrates that mdivi-1 effect on cancer cells are more complex than anticipated.

The ER-mitochondria Ca2+ signaling in cancer progression: Fueling the monster.

Cancer is a leading cause of death worldwide. All major tumor suppressors and oncogenes are now recognized to have fundamental connections with metabolic pathways. A hallmark feature of cancer cells is a reprogramming of their metabolism even when nutrients are available. Increasing evidence indicates that most cancer cells rely on mitochondrial metabolism to sustain their energetic and biosynthetic demands. Mitochondria are functionally and physically coupled to the endoplasmic reticulum (ER), the major calcium (Ca2+) storage organelle in mammalian cells, through special domains known as mitochondria-ER contact sites (MERCS). In this domain, the release of Ca2+ from the ER is mainly regulated by inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs), a family of Ca2+ release channels activated by the ligand IP3. IP3R mediated Ca2+ release is transferred to mitochondria through the mitochondrial Ca2+ uniporter (MCU). Once in the mitochondrial matrix, Ca2+ activates several proteins that stimulate mitochondrial performance. The role of IP3R and MCU in cancer, as well as the other proteins that enable the Ca2+ communication between these two organelles is just beginning to be understood. Here, we describe the function of the main players of the ER mitochondrial Ca2+ communication and discuss how this particular signal may contribute to the rise and development of cancer traits.

Keeping zombies alive: The ER-mitochondria Ca2+ transfer in cellular senescence.

Cellular senescence generates a permanent cell cycle arrest, characterized by apoptosis resistance and a pro-inflammatory senescence-associated secretory phenotype (SASP). Physiologically, senescent cells promote tissue remodeling during development and after injury. However, when accumulated over a certain threshold as happens during aging or after cellular stress, senescent cells contribute to the functional decline of tissues, participating in the generation of several diseases. Cellular senescence is accompanied by increased mitochondrial metabolism. How mitochondrial function is regulated and what role it plays in senescent cell homeostasis is poorly understood. Mitochondria are functionally and physically coupled to the endoplasmic reticulum (ER), the major calcium (Ca2+) storage organelle in mammalian cells, through special domains known as mitochondria-ER contacts (MERCs). In this domain, the release of Ca2+ from the ER is mainly regulated by inositol 1,4,5-trisphosphate receptors (IP3Rs), a family of three Ca2+ release channels activated by a ligand (IP3). IP3R-mediated Ca2+ release is transferred to mitochondria through the mitochondrial Ca2+ uniporter (MCU), where it modulates the activity of several enzymes and transporters impacting its bioenergetic and biosynthetic function. Here, we review the possible connection between ER to mitochondria Ca2+ transfer and senescence. Understanding the pathways that contribute to senescence is essential to reveal new therapeutic targets that allow either delaying senescent cell accumulation or reduce senescent cell burden to alleviate multiple diseases.

Cdkn1a transcript variant 2 is a marker of aging and cellular senescence.

Cellular senescence is a cell fate response characterized by a permanent cell cycle arrest driven primarily the by cell cycle inhibitor and tumor suppressor proteins p16Ink4a and p21Cip1/Waf1. In mice, the p21Cip1/Waf1 encoding locus, Cdkn1a, is known to generate two transcripts that produce identical proteins, but one of these transcript variants is poorly characterized. We show that the Cdkn1a transcript variant 2, but not the better-studied variant 1, is selectively elevated during natural aging across multiple mouse tissues. Importantly, mouse cells induced to senescence in culture by genotoxic stress (ionizing radiation or doxorubicin) upregulated both transcripts, but with different temporal dynamics: variant 1 responded nearly immediately to genotoxic stress, whereas variant 2 increased much more slowly as cells acquired senescent characteristics. Upon treating mice systemically with doxorubicin, which induces widespread cellular senescence in vivo, variant 2 increased to a larger extent than variant 1. Variant 2 levels were also more sensitive to the senolytic drug ABT-263 in naturally aged mice. Thus, variant 2 is a novel and more sensitive marker than variant 1 or total p21Cip1/Waf1 protein for assessing the senescent cell burden and clearance in mice.

In the Right Place at the Right Time: Regulation of Cell Metabolism by IP3R-Mediated Inter-Organelle Ca2+ Fluxes.

In the last few years, metabolism has been shown to be controlled by cross-organelle communication. The relationship between the endoplasmic reticulum and mitochondria/lysosomes is the most studied; here, inositol 1,4,5-triphosphate (IP3) receptor (IP3R)-mediated calcium (Ca2+) release plays a central role. Recent evidence suggests that IP3R isoforms participate in synthesis and degradation pathways. This minireview will summarize the current findings in this area, emphasizing the critical role of Ca2+ communication on organelle function as well as catabolism and anabolism, particularly in cancer.

Ca2+ transfer to mitochondria: a spark of life in unexpected conditions.

The inositol 1,4,5-triphosphate receptor (InsP3R)-mediated calcium (Ca2+) transfer to mitochondria is important to maintain mitochondrial respiration and bioenergetics in normal and cancer cells, even though cancer cells have defective oxidative phosphorylation (OXPHOS). Here, we discuss how tumor mitochondria could become a feasible therapeutic target to treat tumors that depend on reductive carboxylation.

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

Inhibition of InsP3R with Xestospongin B Reduces Mitochondrial Respiration and Induces Selective Cell Death in T Cell Acute Lymphoblastic Leukemia Cells.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy whose chemoresistance and relapse persist as a problem despite significant advances in its chemotherapeutic treatments. Mitochondrial metabolism has emerged as an interesting therapeutic target given its essential role in maintaining bioenergetic and metabolic homeostasis. T-ALL cells are characterized by high levels of mitochondrial respiration, making them suitable for this type of intervention. Mitochondrial function is sustained by a constitutive transfer of calcium from the endoplasmic reticulum to mitochondria through the inositol 1,4,5-trisphosphate receptor (InsP3R), making T-ALL cells vulnerable to its inhibition. Here, we determine the bioenergetic profile of the T-ALL cell lines CCRF-CEM and Jurkat and evaluate their sensitivity to InsP3R inhibition with the specific inhibitor, Xestospongin B (XeB). Our results show that T-ALL cell lines exhibit higher mitochondrial respiration than non-malignant cells, which is blunted by the inhibition of the InsP3R. Prolonged treatment with XeB causes T-ALL cell death without affecting the normal counterpart. Moreover, the combination of XeB and glucocorticoids significantly enhanced cell death in the CCRF-CEM cells. The inhibition of InsP3R with XeB rises as a potential therapeutic alternative for the treatment of T-ALL.

Cancer cells with defective oxidative phosphorylation require endoplasmic reticulum-to-mitochondria Ca2+ transfer for survival.

Spontaneous Ca2+ signaling from the InsP3R intracellular Ca2+ release channel to mitochondria is essential for optimal oxidative phosphorylation (OXPHOS) and ATP production. In cells with defective OXPHOS, reductive carboxylation replaces oxidative metabolism to maintain amounts of reducing equivalents and metabolic precursors. To investigate the role of mitochondrial Ca2+ uptake in regulating bioenergetics in these cells, we used OXPHOS-competent and OXPHOS-defective cells. Inhibition of InsP3R activity or mitochondrial Ca2+ uptake increased α-ketoglutarate (αKG) abundance and the NAD+/NADH ratio, indicating that constitutive endoplasmic reticulum (ER)-to-mitochondria Ca2+ transfer promoted optimal αKG dehydrogenase (αKGDH) activity. Reducing mitochondrial Ca2+ inhibited αKGDH activity and increased NAD+, which induced SIRT1-dependent autophagy in both OXPHOS-competent and OXPHOS-defective cells. Whereas autophagic flux in OXPHOS-competent cells promoted cell survival, it was impaired in OXPHOS-defective cells because of inhibition of autophagosome-lysosome fusion. Inhibition of αKGDH and impaired autophagic flux in OXPHOS-defective cells resulted in pronounced cell death in response to interruption of constitutive flux of Ca2+ from ER to mitochondria. These results demonstrate that mitochondria play a fundamental role in maintaining bioenergetic homeostasis of both OXPHOS-competent and OXPHOS-defective cells, with Ca2+ regulation of αKGDH activity playing a pivotal role. Inhibition of ER-to-mitochondria Ca2+ transfer may represent a general therapeutic strategy against cancer cells regardless of their OXPHOS status.

Concerted Action of AMPK and Sirtuin-1 Induces Mitochondrial Fragmentation Upon Inhibition of Ca2+ Transfer to Mitochondria.

Mitochondria are highly dynamic organelles constantly undergoing fusion and fission. Ca2+ regulates many aspects of mitochondrial physiology by modulating the activity of several mitochondrial proteins. We previously showed that inhibition of constitutive IP3R-mediated Ca2+ transfer to the mitochondria leads to a metabolic cellular stress and eventually cell death. Here, we show that the decline of mitochondrial function generated by a lack of Ca2+ transfer induces a DRP-1 independent mitochondrial fragmentation that at an early time is mediated by an increase in the NAD+/NADH ratio and activation of SIRT1. Subsequently, AMPK predominates and drives the fragmentation. SIRT1 activation leads to the deacetylation of cortactin, favoring actin polymerization, and mitochondrial fragmentation. Knockdown of cortactin or inhibition of actin polymerization prevents fragmentation. These data reveal SIRT1 as a new player in the regulation of mitochondrial fragmentation induced by metabolic/bioenergetic stress through regulating the actin cytoskeleton.

MTOR-independent autophagy induced by interrupted endoplasmic reticulum-mitochondrial Ca2+ communication: a dead end in cancer cells.

The interruption of endoplasmic reticulum (ER)-mitochondrial Ca2+ communication induces a bioenergetic crisis characterized by an increase of MTOR-independent AMPK-dependent macroautophagic/autophagic flux, which is not sufficient to reestablish the metabolic and energetic homeostasis in cancer cells. Here, we propose that upon ER-mitochondrial Ca2+ transfer inhibition, AMPK present at the mitochondria-associated membranes (MAMs) activate localized autophagy via BECN1 (beclin 1). This local response could prevent the proper interorganelle communication that would allow the autophagy-derived metabolites to reach the necessary anabolic pathways to maintain mitochondrial function and cellular homeostasis. Abbreviations: 3MA: 3-methyladenine; ADP: adenosine diphosphate; AMP: adenosine monophosphate; ATG13: autophagy related 13; ATG14: autophagy related 14; ATP: adenosine triphosphate; BECN1: beclin 1; Ca2+: calcium; DNA: deoxyribonucleic acid; ER: endoplasmic reticulum; GEF: guanine nucleotide exchange factor; ITPR: inositol 1,4,5-trisphosphate receptor; MAMs: mitochondria-associated membranes; MCU: mitochondrial calcium uniporter; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; OCR: oxygen consumption rate; PtdIns3K: class III phosphatidylinositol 3-kinase; RB1CC1/FIP200: RB1 inducible coiled-coil 1; RPTOR: regulatory associated protein of MTOR complex 1; RYRs: ryanodine receptors; STK11/LKB1: serine/threonine kinase 11; TCA: tricarboxylic acid; TSC2: TSC complex subunit 2; ULK1: unc-51 like autophagy activating kinase 1; V-ATPase: vacuolar-type H+-ATPase; VDAC: voltage dependent anion channel; XeB: xestospongin B.

FR58P1a; a new uncoupler of OXPHOS that inhibits migration in triple-negative breast cancer cells via Sirt1/AMPK/ß1-integrin pathway.

Highly malignant triple-negative breast cancer (TNBC) cells rely mostly on glycolysis to maintain cellular homeostasis; however, mitochondria are still required for migration and metastasis. Taking advantage of the metabolic flexibility of TNBC MDA-MB-231 cells to generate subpopulations with glycolytic or oxidative phenotypes, we screened phenolic compounds containing an ortho-carbonyl group with mitochondrial activity and identified a bromoalkyl-ester of hydroquinone named FR58P1a, as a mitochondrial metabolism-affecting compound that uncouples OXPHOS through a protonophoric mechanism. In contrast to well-known protonophore uncoupler FCCP, FR58P1a does not depolarize the plasma membrane and its effect on the mitochondrial membrane potential and bioenergetics is moderate suggesting a mild uncoupling of OXPHOS. FR58P1a activates AMPK in a Sirt1-dependent fashion. Although the activation of Sirt1/AMPK axis by FR58P1a has a cyto-protective role, selectively inhibits fibronectin-dependent adhesion and migration in TNBC cells but not in non-tumoral MCF10A cells by decreasing ß1-integrin at the cell surface. Prolonged exposure to FR58P1a triggers a metabolic reprograming in TNBC cells characterized by down-regulation of OXPHOS-related genes that promote cell survival but comprise their ability to migrate. Taken together, our results show that TNBC cell migration is susceptible to mitochondrial alterations induced by small molecules as FR58P1a, which may have therapeutic implications.

Mitochondria and Calcium Regulation as Basis of Neurodegeneration Associated With Aging.

Age is the main risk factor for the onset of neurodegenerative diseases. A decline of mitochondrial function has been observed in several age-dependent neurodegenerative diseases and may be a major contributing factor in their progression. Recent findings have shown that mitochondrial fitness is tightly regulated by Ca2+ signals, which are altered long before the onset of measurable histopathology hallmarks or cognitive deficits in several neurodegenerative diseases including Alzheimer's disease (AD), the most frequent cause of dementia. The transfer of Ca2+ from the endoplasmic reticulum (ER) to the mitochondria, facilitated by the presence of mitochondria-associated membranes (MAMs), is essential for several physiological mitochondrial functions such as respiration. Ca2+ transfer to mitochondria must be finely regulated because excess Ca2+ will disturb oxidative phosphorylation (OXPHOS), thereby increasing the generation of reactive oxygen species (ROS) that leads to cellular damage observed in both aging and neurodegenerative diseases. In addition, excess Ca2+ and ROS trigger the opening of the mitochondrial transition pore mPTP, leading to loss of mitochondrial function and cell death. mPTP opening probably increases with age and its activity has been associated with several neurodegenerative diseases. As Ca2+ seems to be the initiator of the mitochondrial failure that contributes to the synaptic deficit observed during aging and neurodegeneration, in this review, we aim to look at current evidence for mitochondrial dysfunction caused by Ca2+ miscommunication in neuronal models of neurodegenerative disorders related to aging, with special emphasis on AD.

Autophagy protects against neural cell death induced by piperidine alkaloids present in Prosopis juliflora (Mesquite).

Prosopis juliflora is a shrub that has been used to feed animals and humans. However, a synergistic action of piperidine alkaloids has been suggested to be responsible for neurotoxic damage observed in animals. We investigated the involvement of programmed cell death (PCD) and autophagy on the mechanism of cell death induced by a total extract (TAE) of alkaloids and fraction (F32) from P. juliflora leaves composed majoritary of juliprosopine in a model of neuron/glial cell co-culture. We saw that TAE (30 µg/mL) and F32 (7.5 µg/mL) induced reduction in ATP levels and changes in mitochondrial membrane potential at 12 h exposure. Moreover, TAE and F32 induced caspase-9 activation, nuclear condensation and neuronal death at 16 h exposure. After 4 h, they induced autophagy characterized by decreases of P62 protein level, increase of LC3II expression and increase in number of GFP-LC3 cells. Interestingly, we demonstrated that inhibition of autophagy by bafilomycin and vinblastine increased the cell death induced by TAE and autophagy induced by serum deprivation and rapamycin reduced cell death induced by F32 at 24 h. These results indicate that the mechanism neural cell death induced by these alkaloids involves PCD via caspase-9 activation and autophagy, which seems to be an important protective mechanism.

Autophagy protects against neural cell death induced by piperidine alkaloids present in Prosopis juliflora (Mesquite)

ABSTRACT Prosopis juliflora is a shrub that has been used to feed animals and humans. However, a synergistic action of piperidine alkaloids has been suggested to be responsible for neurotoxic damage observed in animals. We investigated the involvement of programmed cell death (PCD) and autophagy on the mechanism of cell death induced by a total extract (TAE) of alkaloids and fraction (F32) from P. juliflora leaves composed majoritary of juliprosopine in a model of neuron/glial cell co-culture. We saw that TAE (30 µg/mL) and F32 (7.5 µg/mL) induced reduction in ATP levels and changes in mitochondrial membrane potential at 12 h exposure. Moreover, TAE and F32 induced caspase-9 activation, nuclear condensation and neuronal death at 16 h exposure. After 4 h, they induced autophagy characterized by decreases of P62 protein level, increase of LC3II expression and increase in number of GFP-LC3 cells. Interestingly, we demonstrated that inhibition of autophagy by bafilomycin and vinblastine increased the cell death induced by TAE and autophagy induced by serum deprivation and rapamycin reduced cell death induced by F32 at 24 h. These results indicate that the mechanism neural cell death induced by these alkaloids involves PCD via caspase-9 activation and autophagy, which seems to be an important protective mechanism.

One-electron reduction of 6-hydroxydopamine quinone is essential in 6-hydroxydopamine neurotoxicity.

6-Hydroxydamine has widely been used as neurotoxin in preclinical studies related on the neurodegenerative process of dopaminergic neurons in Parkinson's disease based on its ability to be neurotoxic as a consequence of free radical formation during its auto-oxidation to topaminequinone. We report that 50-µM 6-hydroxydopamine is not neurotoxic in RCSN-3 cells derived from substantia nigra incubated during 24 h contrasting with a significant sixfold increase in cell death (16 ± 2 %; P < 0.001) was observed in RCSN-3NQ7 cells expressing a siRNA against DT-diaphorase that silence the enzyme expression. To observe a significant cell death in RCSN-3 cells induced by 6-hydroxydopamine (24 ± 1 %; P < 0.01), we have to increase the concentration to 250 µm while a 45 ± 2 % cell death (P < 0.001) was observed at this concentration in RCSN-3NQ7 cells. The cell death induced by 6-hydroxydopamine in RCSN-3NQ7 cells was accompanied with a (i) significant increase in oxygen consumption (P < 0.01), (ii) depletion of reduced glutathione and (iii) a significant decrease in ATP level (P < 0.05) in comparison with RCSN-3 cells. In conclusion, our results suggest that one-electron reduction of 6-hydroxydopamine quinone seems to be the main reaction responsible for 6-hydroxydopamine neurotoxic effects in dopaminergic neurons and DT-diaphorase seems to play an important neuroprotective role by preventing one-electron reduction of topaminequinone.