Mitochondrial Targeting Research Articles

Abstract P32: Cell Type-specific Genetic Therapy of Cancer, Virus Infection and Mitochondrial Disease

Abstract Cell type-specific gene delivery which restricts gene transfer to therapy-relevant cells, hence reducing unwanted side effects caused by the ectopic expression of transgenes, remains one of the biggest challenges. To mitigate potential off-target effects, we developed a programmable cell type-specific gene therapy using an RNA trans-splicing-based approach for gene replacement therapy or suicide gene therapy that can selectively express therapeutic proteins or suicide signals based on the cell’s endogenous pre-mRNA expression profile. Using the liver as a medical target organ, we developed a hepatocellular carcinoma (HCC)-specific trans-splicing based Herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) suicide gene therapy approach. We rationally designed suicide RNAs that can target multiple HCC-related pre-mRNA biomarkers thereby increasing the overall concentration of targetable pre-mRNA. For delivery, non-viral redosable dumbbell-shaped DNA vectors were designed, manufactured, and tested in tissue culture cells, in 3D patient-derived xenograft spheroids and in mouse model. For cell-type specific delivery, the conjugation of dumbbell vectors with GalNAc3 residues enabled targeted delivery into hepatoblastoma-derived human tissue culture cells. To deliver nucleic acids into the mitochondria of human cells, we studied the long non-coding β2.7 RNA of the human cytomegalovirus which was previously reported to colocalise with mitochondrial complex I. Using thermodynamic profiling, we identified four thermodynamically conserved structural subdomains within the β2.7 RNA that are responsible for its mitochondrial targeting activity. A vector comprising eight mitochondrial targeting domains exhibited a three times higher mitochondrial targeting activity as compared with the parental β2.7 RNA. The amalgamation of mitochondrial targeting RNA and trans-splicing allows cell type-specific transcription of mRNA which can be delivered into mitochondria for mitochondrial gene therapy. Trans-splicing based gene therapy represent a technology that is highly translational offering promising perspectives towards suicide gene therapy of any cancer types or incurable infections with integrating viruses and may enable cell type-specific mitochondrial gene therapy. Citation Format: Pei She Loh (Rachel), Sushmita Poddar, Kaustav Chatterjee, Chiu Jing Wen, Derrick Wee Zan Liang, Fritz Lai, Chen Qingfeng, Volker Patzel. Cell Type-specific Genetic Therapy of Cancer, Virus Infection and Mitochondrial Disease [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P32.

Abstract P23: Mitochondrial Delivery of Functional Nucleic Acids for Targeting of Mitochondrial Dysfunction

Abstract Mitochondria is the main power supply for cellular activity and a key player for the regulation of cellular metabolism, and they contain their own DNA. Critical proteins for mitochondrial functions are not only encoded by nuclear DNA (nDNA) but also by mitochondrial DNA (mtDNA). Abnormalities in mitochondrial functions could lead to diseases including neurodegeneration, diabetes, and cancer progression. The ability for a non-coding RNA (β2.7) of human cytomegalovirus (CMV) to localize to the host mitochondria revealed its potential to be used as a tool for mitochondria gene targeting. Using computational methods, our lab identified four thermodynamically conserved structural subdomains (D1 to D4) within the β2.7 RNA. While all four domains exhibited mitochondrial uptake potential, sub-domain D3 exhibited the highest absolute mitochondrial targeting activity and sub-domain D2 the highest activity per nucleotide. Further modifications on different sub-domain alignments found that maximum uptake efficiency was achieved by tandem repeats of 4 copies of D3 domains followed by 4 copies of D2 domains (abbreviated as D3 × 4_D2 × 4). Delivery of a GFP-coding mRNA or antisense RNA targeting mitochondrial genes mt-ATP6 and mt-ATP8 both confirmed its ability to co-deliver functional RNA into mitochondria. To cleave and destroy mutated mitochondrial genomes for future gene therapeutic applications, we explore mitochondrial delivery of the CRISPR-Cas9 system. The mRNAs coding for the wild-type Cas9 as well as more specific Cas9 variants were fused to the mitochondrial delivery vector (D3 × 4_D2 × 4). Latest RT-qPCR on mitochondrial RNA showed delivery of Cas9 mRNA into mitochondria of HepG2 cells. Currently, we investigate co-delivery of single-guide RNA and quantify Cas9 expression in mitochondria. In an alternative approach we deliver sequences coding for functional mitochondrial protein. Examples are the mRNAs coding for mitochondrial proteins mt-ND1, -ND4 or -ND6 which are frequently mutated is patients with Leber’s hereditary optic neuropathy (LHON). Citation Format: Jinglin Ruan, Volker Patzel. Mitochondrial Delivery of Functional Nucleic Acids for Targeting of Mitochondrial Dysfunction [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P23.

Evolutionary Changes in Primate Glutamate Dehydrogenases 1 and 2 Influence the Protein Regulation by Ligands, Targeting and Posttranslational Modifications

There are two paralogs of glutamate dehydrogenase (GDH) in humans encoded by the GLUD1 and GLUD2 genes as a result of a recent retroposition during the evolution of primates. The two human GDHs possess significantly different regulation by allosteric ligands, which is not fully characterized at the structural level. Recent advances in identification of the GDH ligand binding sites provide a deeper perspective on the significance of the accumulated substitutions within the two GDH paralogs. In this review, we describe the evolution of GLUD1 and GLUD2 after the duplication event in primates using the accumulated sequencing and structural data. A new gibbon GLUD2 sequence questions the indispensability of ancestral R496S and G509A mutations for GLUD2 irresponsiveness to GTP, providing an alternative with potentially similar regulatory features. The data of both GLUD1 and GLUD2 evolution not only confirm substitutions enhancing GLUD2 mitochondrial targeting, but also reveal a conserved mutation in ape GLUD1 mitochondrial targeting sequence that likely reduces its transport to mitochondria. Moreover, the information of GDH interactors, posttranslational modification and subcellular localization are provided for better understanding of the GDH mutations. Medically significant point mutations causing deregulation of GDH are considered from the structural and regulatory point of view.

Cytosolic RNA binding of the mitochondrial TCA cycle enzyme malate dehydrogenase (MDH2).

Several enzymes of intermediary metabolism have been identified to bind RNA in 2 cells, with potential consequences for the bound RNAs and/or the enzyme. In this 3 study, we investigate the RNA-binding activity of the mitochondrial enzyme malate 4 dehydrogenase 2 (MDH2), which functions in the tricarboxylic acid (TCA) cycle and 5 the malate-aspartate shuttle. We confirmed in cellulo RNA-binding of MDH2 using 6 orthogonal biochemical assays and performed enhanced crosslinking and 7 immunoprecipitation (eCLIP) to identify the cellular RNAs associated with endogenous 8 MDH2. Surprisingly, MDH2 preferentially binds cytosolic over mitochondrial RNAs, 9 although the latter are abundant in the milieu of the mature protein. Subcellular 10 fractionation followed by RNA-binding assays revealed that MDH2-RNA interactions 11 occur predominantly outside of mitochondria. We also found that a cytosolically-12 retained N-terminal deletion mutant of MDH2 is competent to bind RNA, indicating that 13 mitochondrial targeting is dispensable for MDH2-RNA interactions. MDH2 RNA 14 binding increased when cellular NAD+ levels (MDH2's co-factor) was 15 pharmacologically diminished, suggesting that the metabolic state of cells affects RNA 16 binding. Taken together, our data implicate an as yet unidentified function of MDH2 17 binding RNA in the cytosol.

Monitoring the Mitochondrial Viscosity Changes During Cuproptosis with Iridium(III) Complex Probe via In Situ Phosphorescence Lifetime Imaging.

Cuproptosis is a novel copper-dependent form of programmed cell death, displaying important regulatory functions in many human diseases, including cancer. However, the relationship between the changes in mitochondrial viscosity, a key factor associated with cellular malfunction, and cuproptosis is still unclear. Herein, we prepared a phosphorescent iridium (Ir) complex probe for precisely monitoring the changes of mitochondrial viscosity during cuprotosis via phosphorescence lifetime imaging. The Ir complex probe possessed microsecond lifetimes (up to 1 μs), which could be easily distinguished from cellular autofluorescence to improve the imaging contrast and sensitivity. Benefiting from the long phosphorescence lifetime, excellent viscosity selectivity, and mitochondrial targeting abilities, the Ir complex probe could monitor the increase in the mitochondrial viscosity during cuproptosis (from 46.8 to 68.9 cP) in a quantitative manner. Moreover, through in situ fluorescence imaging, the Ir complex probe successfully monitored the increase in viscosity in zebrafish treated with lipopolysaccharides or elescolomol-Cu2+, which were well-known cuproptosis inducers. We anticipate that this new Ir complex probe will be a useful tool for in-depth understanding of the biological effects of mitochondrial viscosity during cuproptosis.

Exploration of Novel Meso-C═N-BODIPY-Based AIE Fluorescent Rotors with Large Stokes Shifts for Organelle-Viscosity Imaging.

The research on fluorescent rotors for viscosity has attracted extensive interest to better comprehend the close relationships of microviscosity variations with related diseases. Although scientists have made great efforts, fluorescent probes for cellular viscosity with both aggregation-induced emissions (AIEs) and large Stokes shifts to improve sensing properties have rarely been reported. Herein, we first report four new meso-C═N-substituted BODIPY-based rotors with large Stokes shifts, investigate their viscosity/AIE characteristics, and perform cellular imaging of the viscosity in subcellular organelles. Interestingly, the meso-C═N-phenyl group-substituted probe 6 showed an obvious 594 nm fluorescence enhancement in glycerol and a moderate 650 nm red AIE emission in water. Further, on attaching CF3 to the phenyl group, a similar phenomenon was observed for 7 with red-shifted emissions, attributed to the introduction of a phenyl group, which plays a key role in the red AIE emissions and large Stokes shifts. Comparatively, for phenyl-group-free probes, both the meso-C═N-trifluoroethyl group and thiazole-substituted probes (8 and 9) exhibited good viscosity-responsive properties, while no AIE was observed due to the absence of phenyl groups. For cellular experiments, 6 and 9 showed good lysosomal and mitochondrial targeting properties, respectively, and were further successfully used for imaging viscosity through the preincubation of monensin and lipopolysaccharide (LPS), indicating that C═N polar groups potentially work as rotatable moieties and organelle-targeting groups, and the targeting difference might be ascribed to increased charges of thiazole. Therefore, in this study, we investigated the structural relationships of four meso-C═N BODIPY-based rotors with respect to their viscosity/AIE characteristics, subcellular-targeting ability, and cellular imaging for viscosity, potentially serving as AIE fluorescent probes with large Stokes shifts for subcellular viscosity imaging.

Construction of a mitochondrial targeted H2O2 fluorescence probe with a large Stokes shift and its application in bacterial infection

Macrophages are crucial innate immune system cells that have the ability to generate large amounts of H2O2 in the cytoplasm to combat germs. Macrophage mitochondria also produce H2O2 to participate in the antimicrobial response when infected by bacteria. However, visualization of H2O2 production in the mitochondria of bacterially infected macrophages is extremely challenging due to the lack of appropriate research methods. Here, by developing a new mitochondrial-targeting fluorescent H2O2 probe, combined with confocal fluorescence imaging, we report for the first time that H2O2 production in the mitochondria of bacterially infected macrophages (Raw264.7 cells) was visualized with a fluorescent probe. In addition, the developed probe has accurate mitochondrial targeting ability, large Stokes shift, fast response capability, high selectivity and sensitivity (detection limit of 4.346 × 10−7 M), and the probe can be used to detect H2O2 in living cells and zebrafish. Therefore, this work is expected to be used to further reveal the biological function of H2O2 in subcellular mitochondria.

Abstract 4730: Pharmacodynamic determinants of anti tumor activity of SHMT2 inhibitors

Abstract These studies investigate factors that impact the antitumor activity of SHMT2 targeting including drug accumulation, metabolism, expression of one carbon (C1) related enzymes, and tumor hypoxia. Mitochondrial C1 metabolism is upregulated in a variety of cancer types including leukemias and solid tumors. The mitochondrial C1 enzymes serine hydroxymethyltransferase (SHMT) 2 and 5,10-methylene tetrahydrofolate (THF) dehydrogenase 2 (MTHFD2) are among the top 5 overexpressed metabolic enzymes in tumors vs normal tissues in a wide range of tumor types. Thus, SHMT2 and MTHFD2 are potential therapeutic targets for cancer, sparing normal tissues that are less reliant on mitochondrial C1 metabolism. SHMT2 metabolizes serine and THF to produce glycine and 5,10-methylene THF; MTHFD2 metabolizes 5,10-methylene THF to NADH and 10-formyl THF which is converted to formate by MTHFD1L. Thus, mitochondrial C1 metabolism is the principal source of C1 units for cellular biosynthesis and provides the majority of glycine for protein, purine and glutathione synthesis. We previously discovered novel 5-substituted pyrrolo[3,2-d]pyrimidine antifolate compounds which exhibit primary inhibition at mitochondrial SHMT2 along with cytosolic SHMT1 and de novo purine biosynthesis at glycinamide ribonucleotide and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferases. The lead compound AGF347 is taken up into cells by the facilitative transporters reduced folate carrier (SLC19A1) and the proton coupled folate transporter (SLC46A1) and like folate cofactors is metabolized to polyglutamate (PG) forms by folylpolyglutamate synthase (FPGS) which promotes retention in cytosolic and mitochondrial compartments and target enzyme engagement. We investigated the impact of folate transporter expression on metabolism to PGs for SHMT2 targeted antifolates. Folate transporter expression inversely correlated with anti-tumor efficacy of SHMT2 targeted antifolates and overexpression of FPGS resulted in dramatically increased sensitivities. However, for pyrazolopyran inhibitors of SHMT2 (e.g. SHIN1) which enter cells by diffusion and are not metabolized to polyglutamates, increased transporter and FPGS levels dramatically decreased anti-tumor activity. As SHMT2 is a target of HIF1α, we studied the impact of multitargeting SHMT2 and purine biosynthesis by pyrrolo[3,2-d]pyrimidine antifolates under hypoxic conditions. Our results establish that multi-targeting SHMT2 and purine biosynthesis preserves anti-tumor activity under hypoxia. Collectively, our results demonstrate that SHMT2 targeted pyrrolo[3,2-d]pyrimidine antifolates exhibit increased antitumor efficacies in cells expressing low folate transporter expression and is further enhanced by increased FPGS activity. Additionally, we find multitargeting of SHMT2 and de novo purine biosynthesis manifests as antitumor activity even under hypoxic conditions. Citation Format: Mathew Joseph Schneider, Carrie O'Connor, Xun Bao, Md. Junayed Nayeen, Zhanjun Hou, Jing Li, Aleem Gangjee, Larry Matherly. Pharmacodynamic determinants of anti tumor activity of SHMT2 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4730.

Mitochondrial P-JNK target, SAB (SH3BP5), in regulation of cell death.

Cell death occurs in various circumstances, such as homeostasis, stress response, and defense, via specific pathways and mechanisms that are regulated by specific activator-induced signal transductions. Among them, Jun N-terminal kinases (JNKs) participate in various aspects, and the recent discovery of JNKs and mitochondrial protein SAB interaction in signal regulation of cell death completes our understanding of the mechanism of sustained activation of JNK (P-JNK), which leads to triggering of the machinery of cell death. This understanding will lead the investigators to discover the modulators facilitating or preventing cell death for therapeutic application in acute or chronic diseases and cancer. We discuss here the mechanism and modulators of the JNK-SAB-ROS activation loop, which is the core component of mitochondria-dependent cell death, specifically apoptosis and mitochondrial permeability transition (MPT)-driven necrosis, and which may also contribute to cell death mechanisms of ferroptosis and pyroptosis. The discussion here is based on the results and evidence discovered from liver disease models, but the JNK-SAB-ROS activation loop to sustain JNK activation is universally applicable to various disease models where mitochondria and reactive oxygen species contribute to the mechanism of disease.

Mitochondrial extracellular vesicles, autoimmunity and myocarditis.

For many decades viral infections have been suspected as 'triggers' of autoimmune disease, but mechanisms for how this could occur have been difficult to establish. Recent studies have shown that viral infections that are commonly associated with viral myocarditis and other autoimmune diseases such as coxsackievirus B3 (CVB3) and SARS-CoV-2 target mitochondria and are released from cells in mitochondrial vesicles that are able to activate the innate immune response. Studies have shown that Toll-like receptor (TLR)4 and the inflammasome pathway are activated by mitochondrial components. Autoreactivity against cardiac myosin and heart-specific immune responses that occur after infection with viruses where the heart is not the primary site of infection (e.g., CVB3, SARS-CoV-2) may occur because the heart has the highest density of mitochondria in the body. Evidence exists for autoantibodies against mitochondrial antigens in patients with myocarditis and dilated cardiomyopathy. Defects in tolerance mechanisms like autoimmune regulator gene (AIRE) may further increase the likelihood of autoreactivity against mitochondrial antigens leading to autoimmune disease. The focus of this review is to summarize current literature regarding the role of viral infection in the production of extracellular vesicles containing mitochondria and virus and the development of myocarditis.

Dual cytoplasmic-mitochondrial engineering for improved patchoulol production in Komagataella phaffii

Mitochondria are a potential sub-organelle for the synthesis of terpenoids due to its abundant acetyl-CoA, cofactors, and compact compartment for targeting metabolic pathways and enzymes. Hence, conducting systematic investigations and harnessing the subcellular compartment of mitochondria may offer a viable avenue for enhancing terpenoids biosynthesis in yeast and other eukaryotic cells. Previous endeavors to boost terpenoids production in Komagataella phaffii mainly focused on diverse metabolic engineering strategies in the cytoplasm. Numerous studies have demonstrated that reconstructing specific metabolic pathways within mitochondria can lead to higher production yields than engineering the same pathways in the cytoplasm. In our study, we initially identified 6 well-performed mitochondrial targeting sequences (MTSs) through colocalization observation of EGFP and a mitochondrial red fluorescent probe via fluorescence microscopy. These MTSs were selected for the assembly of complete patchoulol biosynthesis pathways in the mitochondria, which resulted in a 1.7-fold increase in patchoulol production compared to the original strain. Furthermore, dual regulation of the cytoplasm and mitochondria was implemented in the P6H4＋PM2 strain to further enhance patchoulol synthesis to 109 mg/L, representing a 1.0-fold and 10.7-fold greater yield than that of the strains engineered solely in the cytoplasm or mitochondria, respectively. This study offers valuable guidance for effectively utilizing mitochondria to achieve higher patchoulol and other sesquiterpenes through dual cytoplasmic-mitochondrial engineering in K. phaffii.

Ursolic acid loaded tri-block copolymer nanoparticles based on triphenylphosphine for mitochondria-targeted cancer therapy

A novel biodegradable amphiphilic triblock copolymer, polyphosphate, polyethylene glycol, and polylactic acid (PAEEP-PEG-PLLA), was synthesized by twice ring-opening polymerization and triphenylphosphine (TPP) was grafted onto the block copolymer to synthesize a carrier material TPP-PAEEP-PEG-PLLA, which was identified by 1H-nuclear magnetic resonance (1H-NMR) spectroscopy. The TPP-PAEEP-PEG-PLLA nanoparticles encapsulated with ursolic acid (UA) were prepared by the emulsion-solvent evaporation method and characterized by dynamic light scattering. The mitochondrial targeting ability of fluorescently labeled nanoparticles was evaluated by laser confocal microscopy. The average particle size and surface charge of the UA -loaded nanoparticle solution were 180.07 ± 1.67 nm and +15.57 ± 1.33 mV, respectively. The biocompatibility of nanoparticles was briefly evaluated by erythrocyte hemolysis assay. In vitro cell proliferation assay and scratch migration assay were performed to compare the difference in anti-tumor effect between UA and UA nanoparticles. The results showed that TPP-modified triblock copolymers had good mitochondrial targeting and improved the low bioavailability of UA, and UA nanoparticles exhibited more pronounced anti-tumor capabilities. In summary, the results suggested that our UA nanoparticles were a promising drug-targeted delivery system for the treatment of tumors.

Fibroblast growth factor 7 protects osteoblasts against oxidative damage through targeting mitochondria.

The pathophysiology of osteoporosis is significantly influenced by the impaired functioning of osteoblasts, which is particularly caused by oxidative stress. Nevertheless, the underlying mechanisms responsible for this phenomenon are still not well understood. The objective of this study was to elucidate the impact of fibroblast growth factor 7 (FGF7) on the behavior of osteoblasts under conditions of oxidative stress. The osteoblast-like MC3T3 cells were pretreated with recombinant FGF7 in the presence of oxidative stress induced by hydrogen peroxide (H2 O2 ). We first provided the evidence that the endogenous FGF7 was significantly increased in osteoblasts in response to the increased H2 O2 levels. Recombined FGF7 demonstrated a remarkable capacity to resist the detrimental effects of H2 O2 -induced oxidative stress, including the increase in cell apoptosis, decrease in osteoblast viability, and impairment in osteogenic differentiation capacity, on osteoblasts. Furthermore, we extensively explored the mechanism underlying these protective effects and discovered a remarkable modulation of reactive oxygen species (ROS) homeostasis in H2 O2 -treated cells following the pronounced expression of FGF7, which significantly differed from the control group. Additionally, we observed that FGF7 exerted partial preservation on both the morphology and function of mitochondria when exposed to oxidative stress conditions. Furthermore, FGF7 exhibited the ability to enhance the activation of the p38/MAPK signaling pathway while concurrently suppressing the JNK/MAPK signaling pathway in response to oxidative stress. These results underscore the promising role and underlying mechanisms of FGF7 in preserving osteoblast homeostasis in the face of oxidative stress.

Mitochondrial-Targeted Metal-Phenolic Nanoparticles to Attenuate Intervertebral Disc Degeneration: Alleviating Oxidative Stress and Mitochondrial Dysfunction.

As intervertebral disc degeneration (IVDD) proceeds, the dysfunctional mitochondria disrupt the viability of nucleus pulposus cells, initiating the degradation of the extracellular matrix. To date, there is a lack of effective therapies targeting the mitochondria of nucleus pulposus cells. Here, we synthesized polygallic acid-manganese (PGA-Mn) nanoparticles via self-assembly polymerization of gallic acid in an aqueous medium and introduced a mitochondrial targeting peptide (TP04) onto the nanoparticles using a Schiff base linkage, resulting in PGA-Mn-TP04 nanoparticles. With a size smaller than 50 nm, PGA-Mn-TP04 possesses pH-buffering capacity, avoiding lysosomal confinement and selectively accumulating within mitochondria through electrostatic interactions. The rapid electron exchange between manganese ions and gallic acid enhances the redox capability of PGA-Mn-TP04, effectively reducing mitochondrial damage caused by mitochondrial reactive oxygen species. Moreover, PGA-Mn-TP04 restores mitochondrial function by facilitating the fusion of mitochondria and minimizing their fission, thereby sustaining the vitality of nucleus pulposus cells. In the rat IVDD model, PGA-Mn-TP04 maintained intervertebral disc height and nucleus pulposus tissue hydration. It offers a nonoperative treatment approach for IVDD and other skeletal muscle diseases resulting from mitochondrial dysfunction, presenting an alternative to traditional surgical interventions.

Regulation of metastatic potential by drug repurposing and mitochondrial targeting in colorectal cancer cells

BackgroundIncreased mitochondrial activities contributing to cancer cell proliferation, invasion, and metastasis have been reported in different cancers; however, studies on the therapeutic targeting of mitochondria in regulating cell proliferation and invasiveness are limited. Because mitochondria are believed to have evolved through bacterial invasion in mammalian cells, antibiotics could provide an alternative approach to target mitochondria, especially in cancers with increased mitochondrial activities. In this study, we investigated the therapeutic potential of bacteriostatic antibiotics in regulating the growth potential of colorectal cancer (CRC) cells, which differ in their metastatic potential and mitochondrial functions.MethodsA combination of viability, cell migration, and spheroid formation assays was used to measure the effect on metastatic potential. The effect on mitochondrial mechanisms was investigated by measuring mitochondrial DNA copy number by qPCR, biogenesis (by qPCR and immunoblotting), and functions by measuring reactive oxygen species, membrane potential, and ATP using standard methods. In addition, the effect on assembly and activities of respiratory chain (RC) complexes was determined using blue native gel electrophoresis and in-gel assays, respectively). Changes in metastatic and cell death signaling were measured by immunoblotting with specific marker proteins and compared between CRC cells.ResultsBoth tigecycline and tetracycline effectively reduced the viability, migration, and spheroid-forming capacity of highly metastatic CRC cells. This increased sensitivity was attributed to reduced mtDNA content, mitochondrial biogenesis, ATP content, membrane potential, and increased oxidative stress. Specifically, complex I assembly and activity were significantly inhibited by these antibiotics in high-metastatic cells. Significant down-regulation in the expression of mitochondrial-mediated survival pathways, such as phospho-AKT, cMYC, phospho-SRC, and phospho-FAK, and upregulation in cell death (apoptosis and autophagy) were observed, which contributed to the enhanced sensitivity of highly metastatic CRC cells toward these antibiotics. In addition, the combined treatment of the CRC chemotherapeutic agent oxaliplatin with tigecycline/tetracycline at physiological concentrations effectively sensitized these cells at early time points.ConclusionAltogether, our study reports that bacterial antibiotics, such as tigecycline and tetracycline, target mitochondrial functions specifically mitochondrial complex I architecture and activity and would be useful in combination with cancer chemotherapeutics for high metastatic conditions.

Autophagy sustains mitochondrial respiration and determines resistance to BRAFV600E inhibition in thyroid carcinoma cells

ABSTRACT BRAFV600E is the most prevalent mutation in thyroid cancer and correlates with poor prognosis and therapy resistance. Although selective inhibitors of BRAFV600E have been developed, more advanced tumors such as anaplastic thyroid carcinomas show a poor response in clinical trials. Therefore, the study of alternative survival mechanisms is needed. Since metabolic changes have been related to malignant progression, in this work we explore metabolic dependencies of thyroid tumor cells to exploit them therapeutically. Our results show that respiration of thyroid carcinoma cells is highly dependent on fatty acid oxidation and, in turn, fatty acid mitochondrial availability is regulated through macroautophagy/autophagy. Furthermore, we show that both lysosomal inhibition and the knockout of the essential autophagy gene, ATG7, lead to enhanced lipolysis; although this effect is not essential for survival of thyroid carcinoma cells. We also demonstrate that following inhibition of either autophagy or fatty acid oxidation, thyroid tumor cells compensate oxidative phosphorylation deficiency with an increase in glycolysis. In contrast to lipolysis induction, upon autophagy inhibition, glycolytic boost in autophagy-deficient cells is essential for survival and, importantly, correlates with a higher sensitivity to the BRAFV600E selective inhibitor, vemurafenib. In agreement, downregulation of the glycolytic pathway results in enhanced mitochondrial respiration and vemurafenib resistance. Our work provides new insights into the role of autophagy in thyroid cancer metabolism and supports mitochondrial targeting in combination with vemurafenib to eliminate BRAFV600E-positive thyroid carcinoma cells. Abbreviations: AMP: adenosine monophosphate; ATC: anaplastic thyroid carcinoma; ATG: autophagy related; ATP: adenosine triphosphate; BRAF: B-Raf proto-oncogene, serine/threonine kinase; Cas9: CRISPR-associated protein; CREB: cAMP responsive element binding protein; CRISPR: clustered regularly interspaced short palindromic repeats; 2DG: 2-deoxyglucose; FA: fatty acid; FAO: fatty acid oxidation; FASN: fatty acid synthase; FCCP: trifluoromethoxy carbonyl cyanide phenylhydrazone; LAMP1: lysosomal associated membrane protein 1; LIPE/HSL: lipase E, hormone sensitive type; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; OCR: oxygen consumption rate; OXPHOS: oxidative phosphorylation; PRKA/PKA: protein kinase cAMP-activated; PTC: papillary thyroid carcinoma; SREBF1/SREBP1: sterol regulatory element binding transcription factor 1.

Mitochondrial targeting improves the selectivity of singlet-oxygen cleavable prodrugs in NMIBC treatment.

Mitochondria play an essential role in cancer treatment by providing apoptotic signals. Hexyl aminolevulinate, an FDA-approved diagnosis for non-muscle invasive bladder cancer, induces the production of protoporphyrin IX (PpIX) preferentially by mitochondria in cancer cells. Photosensitizer PpIX upon illumination can release active chemotherapy drugs from singlet oxygen-activatable prodrugs. Prodrugs placed close enough to PpIX formed in mitochondria can improve the antitumor efficiency of PpIX-PDT. The preferred uptake of prodrugs by cancer cells and tumors can further enhance the selective damage of cancer cells over non-cancer cells and surrounding normal tissues. Mitochondriotropic prodrugs of anticancer drugs, such as paclitaxel and SN-38, were synthesized using rhodamine, a mitochondrial-targeting moiety. Invitro, the mitochondrial targeting helped achieve preferential cellular uptake in cancer cells. In RT112 cells (human bladder cancer cells), intracellular prodrug concentrations were 2-3 times higher than the intracellular prodrug concentrations in BdEC cells (human bladder epithelial cells), after 2 h incubation. In an orthotopic rat bladder tumor model, mitochondria-targeted prodrugs achieved as much as 34 times higher prodrug diffusion in the tumor area compared to the nontumor bladder area. Overall, mitochondria targeting made prodrugs more effective in targeting cancer cells and tumors over non-tumor areas, thereby reducing nonspecific toxicity.

Identification of a novel MT-ND3 variant and restoring mitochondrial function by allotopic expression of MT-ND3 gene

Mitochondrial diseases are caused by nuclear, or mitochondrial DNA (mtDNA) variants and related co-factors. Here, we report a novel m.10197G > C variant in MT-ND3 in a patient, and two other patients with m.10191 T > C. MT-ND3 variants are known to cause Leigh syndrome or mitochondrial complex I deficiency. We performed the functional analyses of the novel m.10197G > C variant that significantly lowered MT-ND3 protein levels, causing complex I assembly and activity deficiency, and reduction of ATP synthesis. We adapted a previously described re-engineering technique of delivering mitochondrial genes into mitochondria through codon optimization for nuclear expression and translation by cytoplasmic ribosomes to rescue defects arising from the MT-ND3 variants. We constructed mitochondrial targeting sequences along with the codon-optimized MT-ND3 and imported them into the mitochondria. To achieve the goal, we imported codon-optimized MT-ND3 into mitochondria in three patients with m.10197G > C and m.10191 T > C missense variants in the MT-ND3. Nuclear expression of the MT-ND3 gene partially restored protein levels, complex I deficiency, and significant improvement of ATP production indicating a functional rescue of the mutant phenotype. The codon-optimized nuclear expression of mitochondrial protein and import inside the mitochondria can supplement the requirements for ATP in energy-deficient mitochondrial disease patients.

OXPHOS-targeted nanoparticles for boosting photodynamic therapy against hypoxia tumor

Hypoxia as an inherent feature in tumors is firmly associated with unsatisfactory clinical outcomes of photodynamic therapy (PDT) since the lack of oxygen leads to ineffective reactive oxygen species (ROS) productivity for tumor eradication. In this study, an oxidative phosphorylation (OXPHOS) targeting nanoplatform was fabricated to alleviate hypoxia and enhance the performance of PDT by encapsulating IR780 and OXPHOS inhibitor atovaquone (ATO) in triphenylphosphine (TPP) modified poly(ethylene glycol) methyl ether-block-poly(L-lactide-co-glycolide) (mPEG-PLGA) nanocarriers (TNPs/IA). ATO by interrupting the electron transfer in OXPHOS could suppress mitochondrial respiration of tumor cells, economising on oxygen for the generation of ROS. Benefiting from the mitochondrial targeting function of TPP, ATO was directly delivered to its site of action to obtain highlighted effect at a lower dosage. Furthermore, positioning the photosensitizer IR780 to mitochondria, a more vulnerable organelle to ROS, was a promising method to attenuate the spatiotemporal limitation of ROS caused by its short half-life and narrow diffusion radius. As a result, TNPs/IA exhibited accurate subcellular localization, lead to the collapse of ATP production by damaging mitochondrion and elicited significant antitumor efficacy via oxygen-augmented PDT in the HeLa subcutaneous xenograft model. Overall, TNPs/IA was a potential strategy in photodynamic eradication of tumors.

Enhanced Mitochondrial Targeting and Inhibition of Pyroptosis with Multifunctional Metallopolyphenol Nanoparticles in Intervertebral Disc Degeneration (Small 13/2024)

Enhanced Mitochondrial Targeting and Inhibition of Pyroptosis with Multifunctional Metallopolyphenol Nanoparticles in Intervertebral Disc Degeneration (Small 13/2024)