Induction of mitochondrial toxicity by non-steroidal anti-inflammatory drugs (NSAIDs): The ultimate trade-off governing the therapeutic merits and demerits of these wonder drugs.

Non-steroidal anti-inflammatory drugs (NSAIDs) are most extensively used over-the-counter FDA-approved analgesic medicines for treating inflammation, musculoskeletal pain, arthritis, pyrexia and menstrual cramps. Moreover, aspirin is widely used against cardiovascular complications. Owing to their non-addictive nature, NSAIDs are also commissioned as safer opioid-sparing alternatives in acute trauma and post-surgical treatments. In fact, therapeutic spectrum of NSAIDs is expanding. These "wonder-drugs" are now repurposed against lung diseases, diabetes, neurodegenerative disorders, fungal infections and most notably cancer, due to their efficacy against chemoresistance, radio-resistance and cancer stem cells. However, prolonged NSAID treatment accompany several adverse effects. Mechanistically, apart from cyclooxygenase inhibition, NSAIDs directly target mitochondria to induce cell death. Interestingly, there are also incidences of dose-dependent effects where NSAIDs are found to improve mitochondrial health thereby suggesting plausible mitohormesis. While mitochondria-targeted effects of NSAIDs are discretely studied, a comprehensive account emphasizing the multiple dimensions in which NSAIDs affect mitochondrial structure-function integrity, leading to cell death, is lacking. This review discusses the current understanding of NSAID-mitochondria interactions in the pathophysiological background. This is essential for assessing the risk-benefit trade-offs of NSAIDs for judiciously strategizing NSAID-based approaches to manage pain and inflammation as well as formulating effective anti-cancer strategies. We also discuss recent developments constituting selective mitochondria-targeted NSAIDs including theranostics, mitocans, chimeric small molecules, prodrugs and nanomedicines that rationally optimize safer application of NSAIDs. Thus, we present a comprehensive understanding of therapeutic merits and demerits of NSAIDs with mitochondria at its cross roads. This would help in NSAID-based disease management research and drug development.

Plasmodium falciparum Alba6 exhibits DNase activity and participates in stress response.

Alba domain proteins, owing to their functional plasticity, play a significant role in organisms. Here, we report an intrinsic DNase activity of PfAlba6 from Plasmodium falciparum, an etiological agent responsible for human malignant malaria. We identified that tyrosine28 plays a critical role in the Mg2+ driven 5'-3' DNase activity of PfAlba6. PfAlba6 cleaves both dsDNA as well as ssDNA. We also characterized PfAlba6-DNA interaction and observed concentration-dependent oligomerization in the presence of DNA, which is evident from size exclusion chromatography and single molecule AFM-imaging. PfAlba6 mRNA expression level is up-regulated several folds following heat stress and treatment with artemisinin, indicating a possible role in stress response. PfAlba6 has no human orthologs and is expressed in all intra-erythrocytic stages; thus, this protein can potentially be a new anti-malarial drug target.

NSAID targets SIRT3 to trigger mitochondrial dysfunction and gastric cancer cell death.

Gastric cancer (GC) is a deadly malignancy that demands effective therapeutic intervention capitalizing unique drug target/s. Here, we report that indomethacin, a cyclooxygenase non-selective non-steroidal anti-inflammatory drug, arrests GC cell growth by targeting mitochondrial deacetylase Sirtuin 3 (SIRT3). Interaction study revealed that indomethacin competitively inhibited SIRT3 by binding to nicotinamide adenine dinucleotide (NAD)-binding site. The Cancer Genome Atlas data meta-analysis indicated poor prognosis associated with high SIRT3 expression in GC. Further, transcriptome sequencing data of human gastric adenocarcinoma cells revealed that indomethacin treatment severely downregulated SIRT3. Indomethacin-induced SIRT3 downregulation augmented SOD2 and OGG1 acetylation, leading to mitochondrial redox dyshomeostasis, mtDNA damage, respiratory chain failure, bioenergetic crisis, mitochondrial fragmentation, and apoptosis via blocking the AMPK/PGC1α/SIRT3 axis. Indomethacin also downregulated SIRT3 regulators ERRα and PGC1α. Further, SIRT3 knockdown aggravated indomethacin-induced mitochondrial dysfunction as well as blocked cell-cycle progression to increase cell death. Thus, we reveal how indomethacin induces GC cell death by disrupting SIRT3 signaling.

Nuclease activity of Plasmodium falciparum Alba family protein PfAlba3.

Plasmodium falciparum Alba domain-containing protein Alba3 (PfAlba3) is ubiquitously expressed in intra-erythrocytic stages of Plasmodium falciparum, but the function of this protein is not yet established. Here, we report an apurinic/apyrimidinic site-driven intrinsic nuclease activity of PfAlba3 assisted by divalent metal ions. Surface plasmon resonance and atomic force microscopy confirm sequence non-specific DNA binding by PfAlba3. Upon binding, PfAlba3 cleaves double-stranded DNA (dsDNA) hydrolytically. Mutational studies coupled with mass spectrometric analysis indicate that K23 is the essential residue in modulating the binding to DNA through acetylation-deacetylation. We further demonstrate that PfSir2a interacts and deacetylates K23-acetylated PfAlba3 in favoring DNA binding. Hence, K23 serves as a putative molecular switch regulating the nuclease activity of PfAlba3. Thus, the nuclease activity of PfAlba3, along with its apurinic/apyrimidinic (AP) endonuclease feature identified in this study, indicates a role of PfAlba3 in DNA-damage response that may have a far-reaching consequence in Plasmodium pathogenicity.

Honokiol, an inducer of sirtuin-3, protects against non-steroidal anti-inflammatory drug-induced gastric mucosal mitochondrial pathology, apoptosis and inflammatory tissue injury.

BACKGROUND AND PURPOSE: Mitochondrial oxidative stress, inflammation and apoptosis primarily underlie gastric mucosal injury caused by the widely used non-steroidal anti-inflammatory drugs (NSAIDs). Alternative gastroprotective strategies are therefore needed. Sirtuin-3 pivotally maintains mitochondrial structural integrity and metabolism while preventing oxidative stress; however, its relevance to gastric injury was never explored. Here, we have investigated whether and how sirtuin-3 stimulation by the phytochemical, honokiol, could rescue NSAID-induced gastric injury. EXPERIMENTAL APPROACH: Gastric injury in rats induced by indomethacin was used to assess the effects of honokiol. Next-generation sequencing-based transcriptomics followed by functional validation identified the gastroprotective function of sirtuin-3. Flow cytometry, immunoblotting, qRT-PCR and immunohistochemistry were used measure effects on oxidative stress, mitochondrial dynamics, electron transport chain function, and markers of inflammation and apoptosis. Sirtuin-3 deacetylase activity was also estimated and gastric luminal pH was measured. KEY RESULTS: Indomethacin down-regulated sirtuin-3 to induce oxidative stress, mitochondrial hyperacetylation, 8-oxoguanine DNA glycosylase 1 depletion, mitochondrial DNA damage, respiratory chain defect and mitochondrial fragmentation leading to severe mucosal injury. Indomethacin dose-dependently inhibited sirtuin-3 deacetylase activity. Honokiol prevented mitochondrial oxidative damage and inflammatory tissue injury by attenuating indomethacin-induced depletion of both sirtuin-3 and its transcriptional regulators PGC1α and ERRα. Honokiol also accelerated gastric wound healing but did not alter gastric acid secretion, unlike lansoprazole. CONCLUSIONS AND IMPLICATIONS: Sirtuin-3 stimulation by honokiol prevented and reversed NSAID-induced gastric injury through maintaining mitochondrial integrity. Honokiol did not affect gastric acid secretion. Sirtuin-3 stimulation by honokiol may be utilized as a mitochondria-based, acid-independent novel gastroprotective strategy against NSAIDs.

Emerging role of mitochondrial DAMPs, aberrant mitochondrial dynamics and anomalous mitophagy in gut mucosal pathogenesis.

Gastroduodenal inflammation and ulcerative injuries are increasing due to expanding socio-economic stress, unhealthy food habits-lifestyle, smoking, alcoholism and usage of medicines like non-steroidal anti-inflammatory drugs. In fact, gastrointestinal (GI) complications, associated with the prevailing COVID-19 pandemic, further, poses a challenge to global healthcare towards safeguarding the GI tract. Emerging evidences have discretely identified mitochondrial dysfunctions as common etiological denominators in diseases. However, it is worth realizing that mitochondrial dysfunctions are not just consequences of diseases. Rather, damaged mitochondria severely aggravate the pathogenesis thereby qualifying as perpetrable factors worth of prophylactic and therapeutic targeting. Oxidative and nitrosative stress due to endogenous and exogenous stimuli triggers mitochondrial injury causing production of mitochondrial damage associated molecular patterns (mtDAMPs), which, in a feed-forward loop, inflicts inflammatory tissue damage. Mitochondrial structural dynamics and mitophagy are crucial quality control parameters determining the extent of mitopathology and disease outcomes. Interestingly, apart from endogenous factors, mitochondria also crosstalk and in turn get detrimentally affected by gut pathobionts colonized during luminal dysbiosis. Although mitopathology is documented in various pre-clinical/clinical studies, a comprehensive account appreciating the mitochondrial basis of GI mucosal pathogenesis is largely lacking. Here we critically discuss the molecular events impinging on mitochondria along with the interplay of mitochondria-derived factors in fueling mucosal damage. We specifically emphasize on the potential role of aberrant mitochondrial dynamics, anomalous mitophagy, mitochondrial lipoxidation and ferroptosis as emerging regulators of GI mucosal pathogenesis. We finally discuss about the prospect of mitochondrial targeting for next-generation drug discovery against GI disorders.

Rab7 of Plasmodium falciparum is involved in its retromer complex assembly near the digestive vacuole.

BACKGROUND: Intracellular protein trafficking is crucial for survival of cell and proper functioning of the organelles; however, these pathways are not well studied in the malaria parasite. Its unique cellular architecture and organellar composition raise an interesting question to investigate. METHODS: The interaction of Plasmodium falciparum Rab7 (PfRab7) with vacuolar protein sorting-associated protein 26 (PfVPS26) of retromer complex was shown by coimmunoprecipitation (co-IP). Confocal microscopy was used to show the localization of the complex in the parasite with respect to different organelles. Further chemical tools were employed to explore the role of digestive vacuole (DV) in retromer trafficking in parasite and GTPase activity of PfRab7 was examined. RESULTS: PfRab7 was found to be interacting with retromer complex that assembled mostly near DV and the Golgi in trophozoites. Chemical disruption of DV by chloroquine (CQ) led to its disassembly that was further validated by using compound 5f, a heme polymerization inhibitor in the DV. PfRab7 exhibited Mg2+ dependent weak GTPase activity that was inhibited by a specific Rab7 GTPase inhibitor, CID 1067700, which prevented the assembly of retromer complex in P. falciparum and inhibited its growth suggesting the role of GTPase activity of PfRab7 in retromer assembly. CONCLUSION: Retromer complex was found to be interacting with PfRab7 and the functional integrity of the DV was found to be important for retromer assembly in P. falciparum. GENERAL SIGNIFICANCE: This study explores the retromer trafficking in P. falciparum and describes amechanism to validate DV targeting antiplasmodial molecules.

Indomethacin impairs mitochondrial dynamics by activating the PKCζ-p38-DRP1 pathway and inducing apoptosis in gastric cancer and normal mucosal cells.

The subcellular mechanism by which nonsteroidal anti-inflammatory drugs (NSAIDs) induce apoptosis in gastric cancer and normal mucosal cells is elusive because of the diverse cyclooxygenase-independent effects of these drugs. Using human gastric carcinoma cells (AGSs) and a rat gastric injury model, here we report that the NSAID indomethacin activates the protein kinase Cζ (PKCζ)-p38 MAPK (p38)-dynamin-related protein 1 (DRP1) pathway and thereby disrupts the physiological balance of mitochondrial dynamics by promoting mitochondrial hyper-fission and dysfunction leading to apoptosis. Notably, DRP1 knockdown or SB203580-induced p38 inhibition reduced indomethacin-induced damage to AGSs. Indomethacin impaired mitochondrial dynamics by promoting fissogenic activation and mitochondrial recruitment of DRP1 and down-regulating fusogenic optic atrophy 1 (OPA1) and mitofusins in rat gastric mucosa. Consistent with OPA1 maintaining cristae architecture, its down-regulation resulted in EM-detectable cristae deformity. Deregulated mitochondrial dynamics resulting in defective mitochondria were evident from enhanced Parkin expression and mitochondrial proteome ubiquitination. Indomethacin ultimately induced mitochondrial metabolic and bioenergetic crises in the rat stomach, indicated by compromised fatty acid oxidation, reduced complex I- associated electron transport chain activity, and ATP depletion. Interestingly, Mdivi-1, a fission-preventing mito-protective drug, reversed indomethacin-induced DRP1 phosphorylation on Ser-616, mitochondrial proteome ubiquitination, and mitochondrial metabolic crisis. Mdivi-1 also prevented indomethacin-induced mitochondrial macromolecular damage, caspase activation, mucosal inflammation, and gastric mucosal injury. Our results identify mitochondrial hyper-fission as a critical and common subcellular event triggered by indomethacin that promotes apoptosis in both gastric cancer and normal mucosal cells, thereby contributing to mucosal injury.

Hydrazonophenol, a Food Vacuole-Targeted and Ferriprotoporphyrin IX-Interacting Chemotype Prevents Drug-Resistant Malaria.

The rapid emergence of resistance against frontline antimalarial drugs essentially warrants the identification of new-generation antimalarials. Here, we describe the synthesis of ( E)-2-isopropyl-5-methyl-4-((2-(pyridin-4-yl)hydrazono)methyl)phenol (18), which binds ferriprotoporphyrin-IX (FeIII-PPIX) ( Kd = 33 nM) and offers antimalarial activity against chloroquine-resistant and sensitive strains of Plasmodium falciparum in vitro. Structure-function analysis reveals that compound 18 binds FeIII-PPIX through the -C═N-NH- moiety and 2-pyridyl substitution at the hydrazine counterpart plays a critical role in antimalarial efficacy. Live cell confocal imaging using a fluorophore-tagged compound confirms its accumulation inside the acidic food vacuole (FV) of P. falciparum. Furthermore, this compound concentration-dependently elevates the pH in FV, implicating a plausible interference with FeIII-PPIX crystallization (hemozoin formation) by a dual function: increasing the pH and binding free FeIII-PPIX. Different off-target bioassays reduce the possibility of the promiscuous nature of compound 18. Compound 18 also exhibits potent in vivo antimalarial activity against chloroquine-resistant P. yoelii and P. berghei ANKA (causing cerebral malaria) in mice with negligible toxicity.

Macrophage migration inhibitory factor regulates mitochondrial dynamics and cell growth of human cancer cell lines through CD74-NF-κB signaling.

The indispensable role of macrophage migration inhibitory factor (MIF) in cancer cell proliferation is unambiguous, although which specific roles the cytokine plays to block apoptosis by preserving cell growth is still obscure. Using different cancer cell lines (AGS, HepG2, HCT116, and HeLa), here we report that the silencing of MIF severely deregulated mitochondrial structural dynamics by shifting the balance toward excess fission, besides inducing apoptosis with increasing sub-G0 cells. Furthermore, enhanced mitochondrial Bax translocation along with cytochrome c release, down-regulation of Bcl-xL, and Bcl-2 as well as up-regulation of Bad, Bax, and p53 indicated the activation of a mitochondrial pathway of apoptosis upon MIF silencing. The data also indicate a concerted down-regulation of Opa1 and Mfn1 along with a significant elevation of Drp1, cumulatively causing mitochondrial fragmentation upon MIF silencing. Up-regulation of Drp1 was found to be further coupled with fissogenic serine 616 phosphorylation and serine 637 dephosphorylation, thus ensuring enhanced mitochondrial translocation. Interestingly, MIF silencing was found to be associated with decreased NF-κB activation. In fact, NF-κB knockdown in turn increased mitochondrial fission and cell death. In addition, the silencing of CD74, the cognate receptor of MIF, remarkably increased mitochondrial fragmentation in addition to preventing cell proliferation, inducing mitochondrial depolarization, and increasing apoptotic cell death. This indicates the active operation of a MIF-regulated CD74-NF-κB signaling axis for maintaining mitochondrial stability and cell growth. Thus, we propose that MIF, through CD74, constitutively activates NF-κB to control mitochondrial dynamics and stability for promoting carcinogenesis via averting apoptosis.

Acute mental stress induces mitochondrial bioenergetic crisis and hyper-fission along with aberrant mitophagy in the gut mucosa in rodent model of stress-related mucosal disease.

Psychological stress, depression and anxiety lead to multiple organ dysfunctions wherein stress-related mucosal disease (SRMD) is common to people experiencing stress and also occur as a side effect in patients admitted to intensive care units; however the underlying molecular aetiology is still obscure. We report that in rat-SRMD model, cold restraint-stress severely damaged gut mitochondrial functions to generate superoxide anion (O2•-), depleted ATP and shifted mitochondrial fission-fusion dynamics towards enhanced fission to induce mucosal injury. Activation of mitophagy to clear damaged and fragmented mitochondria was evident from mitochondrial translocation of Parkin and PINK1 along with enhanced mitochondrial proteome ubiquitination, depletion of mitochondrial DNA copy number and TOM 20. However, excess and sustained accumulation of O2•--generating defective mitochondria overpowered the mitophagic machinery, ultimately triggering Bax-dependent apoptosis and NF-κB-intervened pro-inflammatory mucosal injury. We further observed that stress-induced enhanced serum corticosterone stimulated mitochondrial recruitment of glucocorticoid receptor (GR), which contributed to gut mitochondrial dysfunctions as documented from reduced ETC complex 1 activity, mitochondrial O2•- accumulation, depolarization and hyper-fission. GR-antagonism by RU486 or specific scavenging of mitochondrial O2•- by a mitochondrially targeted antioxidant mitoTEMPO ameliorated stress-induced mucosal damage. Gut mitopathology and mucosal injury were also averted when the perception of mental stress was blocked by pre-treatment with a sedative or antipsychotic. Altogether, we suggest the role of mitochondrial GR-O2•--fission cohort in brain-mitochondria cross-talk during acute mental stress and advocate the utilization of this pathway as a potential target to prevent mitochondrial unrest and gastropathy bypassing central nervous system.

Selective scavenging of intra-mitochondrial superoxide corrects diclofenac-induced mitochondrial dysfunction and gastric injury: A novel gastroprotective mechanism independent of gastric acid suppression.

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used to treat multiple inflammatory diseases and pain but severe gastric mucosal damage is the worst outcome of NSAID-therapy. Here we report that mitoTEMPO, a mitochondrially targeted superoxide (O2-) scavenger protected as well as healed gastric injury induced by diclofenac (DCF), the most commonly used NSAID. Common existing therapy against gastric injury involves suppression of gastric acid secretion by proton pump inhibitors and histamine H2 receptor antagonists; however, dyspepsia, vitamin B12 deficiency and gastric microfloral dysbalance are the major drawbacks of acid suppression. Interestingly, mitoTEMPO did not inhibit gastric acid secretion but offered gastroprotection by preventing DCF-induced generation of O2- due to mitochondrial respiratory chain failure and by preventing mitochondrial oxidative stress (MOS)-mediated mitopathology. MitoTEMPO even restored DCF-stimulated reduced fatty acid oxidation, mitochondrial depolarization and bioenergetic crisis in gastric mucosa. MitoTEMPO also prevented the activation of mitochondrial pathway of apoptosis and MOS-mediated proinflammatory signaling through NF-κB by DCF. Furthermore, mitoTEMPO when administered in rats with preformed gastric lesions expedited the healing of gastric injury and the healed stomach exhibited its normal physiology as evident from gastric acid and pepsin secretions under basal or stimulated conditions. Thus, in contrast to the existing antiulcer drugs, mitochondrially targeted O2- scavengers like mitoTEMPO may represent a novel class of gastroprotective molecules that does not affect gastric acid secretion and may be used in combination with DCF, keeping its anti-inflammatory action intact, while reducing its gastrodamaging effects.