bims-mibica 2023-12-03 papers

bims-mibica

Biomed News

on Mitochondrial bioenergetics in cancer

Issue of 2023‒12‒03
29 papers selected by
Kelsey Fisher-Wellman, East Carolina University

Osteopontin induces mitochondrial biogenesis in deadherent cancer cells.
Fatty acid elongation regulates mitochondrial β-oxidation and cell viability in prostate cancer by controlling malonyl-CoA levels.
SIRT5-mediated ME2 desuccinylation promotes cancer growth by enhancing mitochondrial respiration.
Inhibition of mitochondria induces apoptosis and reduces telomere length and activity in acute myeloid leukemia stem cells.
Adipose triglyceride lipase is a therapeutic target in advanced prostate cancer that promotes metabolic plasticity.
Efficient elimination of MELAS-associated m.3243G mutant mitochondrial DNA by an engineered mitoARCUS nuclease.
Cryptotanshinone suppresses ovarian cancer via simultaneous inhibition of glycolysis and oxidative phosphorylation.
Hypermetabolic state is associated with circadian rhythm disruption in mouse and human cancer cells.
Glutathione promotes the synergistic effects of venetoclax and azacytidine against myelodysplastic syndrome‑refractory anemia by regulating the cell cycle.
Reductive carboxylation of glutamine as a potential target in acute myeloid leukemia.
Histone deacetylase inhibitor pracinostat suppresses colorectal cancer by inducing CDK5-Drp1 signaling-mediated peripheral mitofission.
Mitochondrial DNA editing with mitoARCUS.
In vitro anticancer effect of azithromycin targeting hypoxic lung cancer cells via the inhibition of mitophagy.
Targeting ABCA12-controlled ceramide homeostasis inhibits breast cancer stem cell function and chemoresistance.
Mitochondria dysfunction induced by decyl-TPP mitochondriotropic antioxidant based on caffeic acid AntiOxCIN6 sensitizes cisplatin lung anticancer therapy due to a remodeling of energy metabolism.
Metabolomic analyses uncover an inhibitory effect of niclosamide on mitochondrial membrane potential in cholangiocarcinoma cells.
TP-0184 inhibits FLT3/ACVR1 to overcome FLT3 inhibitor resistance and hinder AML growth synergistically with venetoclax.
Standardized assays to monitor drug sensitivity in hematologic cancers.
Mitochondrial E3 ligase MARCH5 is a safeguard against DNA-PKcs-mediated immune signaling in mitochondria-damaged cells.
SOHO State of the Art Updates and Next Questions: Understanding and Overcoming Venetoclax Resistance in Hematologic Malignancies.
Copper(II) complexes with plumbagin and bipyridines target mitochondria for enhanced chemodynamic cancer therapy.
Enhanced TP53 reactivation disrupts MYC transcriptional program and overcomes venetoclax resistance in acute myeloid leukemias.
Different impact of vitamin D on mitochondrial activity and morphology in normal and malignant keratinocytes, the role of genomic pathway.
Metabolic control of mitophagy.
Organic Selenium induces ferroptosis in pancreatic cancer cells.
Melittin kills A549 cells by targeting mitochondria and blocking mitophagy flux.
COQ4 is required for the oxidative decarboxylation of the C1 carbon of Coenzyme Q in eukaryotic cells.
Thioflavin T indicates mitochondrial membrane potential in mammalian cells.
MITF regulates IDH1 and NNT and drives a transcriptional program protecting cutaneous melanoma from reactive oxygen species.

Oncotarget. 2023 Dec 01. 14 957-969

Osteopontin induces mitochondrial biogenesis in deadherent cancer cells.

Gulimirerouzi Fnu, Georg F Weber.

  Metastasizing cells display a unique metabolism, which is very different from the Warburg effect that arises in primary tumors. Over short time frames, oxidative phosphorylation and ATP generation are prominent. Over longer time frames, mitochondrial biogenesis becomes a pronounced feature and aids metastatic success. It has not been known whether or how these two phenomena are connected. We hypothesized that Osteopontin splice variants, which synergize to increase ATP levels in deadherent cells, also increase the mitochondrial mass via the same signaling mechanisms. Here, we report that autocrine Osteopontin does indeed stimulate an increase in mitochondrial size, with the splice variant -c being more effective than the full-length form -a. Osteopontin-c achieves this via its receptor CD44v, jointly with the upregulation and co-ligation of the chloride-dependent cystine-glutamate transporter SLC7A11. The signaling proceeds through activation of the known mitochondrial biogenesis inducer PGC-1 (which acts as a transcription coactivator). Peroxide is an important intermediate in this cascade, but surprisingly acts upstream of PGC-1 and is likely produced as a consequence of SLC7A11 recruitment and activation. In vivo, suppression of the biogenesis-inducing mechanisms leads to a reduction in disseminated tumor mass. This study confirms a functional connection between the short-term oxidative metabolism and the longer-term mitochondrial biogenesis in cancer metastasis - both are induced by Osteopontin-c. The results imply possible mechanisms and targets for treating cancer metastasis.

Keywords:  anchorage independence; metabolism; metastasis; mitochondrial mass; peroxide

DOI:  https://doi.org/10.18632/oncotarget.28540
Biochem Biophys Res Commun. 2023 Nov 18. pii: S0006-291X(23)01367-0. [Epub ahead of print]691 149273

Fatty acid elongation regulates mitochondrial β-oxidation and cell viability in prostate cancer by controlling malonyl-CoA levels.

Julia S Scott, Lake-Ee Quek, Andrew J Hoy, Johannes V Swinnen, Zeyad D Nassar, Lisa M Butler.

  Recently, the fatty acid elongation enzyme ELOVL5 was identified as a critical pro-metastatic factor in prostate cancer, required for cell growth and mitochondrial homeostasis. The fatty acid elongation reaction catalyzed by ELOVL5 utilizes malonyl-CoA as the carbon donor. Here, we demonstrate that ELOVL5 knockdown causes malonyl-CoA accumulation. Malonyl-CoA is a cellular substrate that can inhibit fatty acid β-oxidation in the mitochondria through allosteric inhibition of carnitine palmitoyltransferase 1A (CPT1A), the enzyme that controls the rate-limiting step of the long chain fatty acid β-oxidation cycle. We hypothesized that changes in malonyl-CoA abundance following ELOVL5 knockdown could influence mitochondrial β-oxidation rates in prostate cancer cells, and regulate cell viability. Accordingly, we find that ELOVL5 knockdown is associated with decreased mitochondrial β-oxidation in prostate cancer cells. Combining ELOVL5 knockdown with FASN inhibition to increase malonyl-CoA abundance endogenously enhances the effect of ELOVL5 knockdown on prostate cancer cell viability, while preventing malonyl-CoA production rescues the cells from the effect of ELOVL5 knockdown. Our findings indicate an additional role for fatty acid elongation, in the control of malonyl-CoA homeostasis, alongside its established role in the production of long-chain fatty acid species, to explain the importance of fatty acid elongation for cell viability.

Keywords:  Fatty acid elongation; Fatty acid oxidation; Malonyl-CoA; Prostate cancer

DOI:  https://doi.org/10.1016/j.bbrc.2023.149273
Cell Death Differ. 2023 Nov 25.

SIRT5-mediated ME2 desuccinylation promotes cancer growth by enhancing mitochondrial respiration.

Peng Teng, Kaisa Cui, Surui Yao, Bojian Fei, Feng Ling, Chaoqun Li, Zhaohui Huang.

Mitochondrial malic enzyme 2 (ME2), which catalyzes the conversion of malate to pyruvate, is frequently upregulated during tumorigenesis and is a potential target for cancer therapy. However, the regulatory mechanism underlying ME2 activity is largely unknown. In this study, we demonstrate that ME2 is highly expressed in human colorectal cancer (CRC) tissues, and that ME2 knockdown inhibits the proliferation of CRC cells. Furthermore, we reveal that ME2 is succinylated and identify Sirtuins 5 (SIRT5) as an ME2 desuccinylase. Glutamine deprivation directly enhances the interaction of SIRT5 with ME2 and thus promotes SIRT5-mediated desuccinylation of ME2 at lysine 346, activating ME2 enzymatic activity. Activated ME2 significantly enhances mitochondrial respiration, thereby counteracting the effects of glutamine deprivation and supporting cell proliferation and tumorigenesis. Additionally, the levels of succinylated ME2 at K346 and SIRT5 in CRC tissues, which are negatively correlated, are associated with patient prognosis. These observations suggest that SIRT5-catalyzed ME2 desuccinylation is a key signaling event through which cancer cells maintain mitochondrial respiration and promote CRC progression under glutamine deficiency conditions, offering the possibility of targeting SIRT5-mediated ME2 desuccinylation for CRC treatment.

DOI: https://doi.org/10.1038/s41418-023-01240-y
Cell Biochem Funct. 2023 Nov 28.

Inhibition of mitochondria induces apoptosis and reduces telomere length and activity in acute myeloid leukemia stem cells.

Behnaz Valipour, Sahar Davari, Raheleh Farahzadi, Shahram Pourrasol, Niloofar Mehran, Khadijeh Dizaji Asl, Seyed Mansour Altaha, Zahra Hojjati, Hojjatollah Nozad Charoudeh.

  Acute myeloid leukemia (AML) is a highly lethal hematological malignancy in adults and children. Abnormal proliferation of leukemia stem cells (LSC) with CD34+ and CD38- phenotypes are the main clinical features of AML. Patients with AML face drug resistance and treatment failure due to a default in stem and progenitor cells. Therefore, defining LSC properties is necessary for targeting leukemia-initiating cells. Mitochondrial mass and activity increase in AML initiating cells compared with normal stem cells. This idea has offered the inhibition of the mitochondrial translation machinery to reduce the number of leukemia-initiating cells in patients with AML Tigecycline is an FDA-approved microbial antibiotic that inhibits oxidative phosphorylation in mitochondria, resulting in the suppression of leukemia cell proliferation with little toxicity to normal cells. Thus, the present study was conducted to evaluate whether LSC is influenced by mitochondrial inhibition. We measured the IC50 of tigecycline in KG-1a AML cell lines. KG-1a AML cell lines were separated into CD34+ and CD34- cells by MACS. In the following, these cells were treated with 20 µM (IC50) tigecycline. The expression of Annexin/PI, Caspase 3, apoptotic genes (BCL2, BCLX, BAX, BAD, and P53) and proteins (P53, BAX, BCL2 and Caspase 9) was evaluated in CD34+ , CD34- and KG-1a AML cells. In addition, the telomere length and expression of hTERT were evaluated in this study. The results indicated that BCl2 (gene and protein) and BCLX gene dramatically decreased. In addition, BAD, BAX, and P53 gene and protein expression significantly increased in CD34+ AML cells compared to CD34- AML cells. The results also suggested that tigecycline induced intrinsic (Cleaved-caspase 9/Pro-Caspase 9 ratio) and p53-mediated apoptosis. Furthermore, hTERT gene expression and telomere length decreased in the tigecycline-treated groups. Taken together, our findings indicate that inhibition of mitochondrial activity with tigecycline can induce apoptosis in cancer stem cells and can be used as a novel method for cancer therapy.

Keywords:  acute myeloid leukemia; apoptosis; leukemia stem cells; mitochondria; tigecycline

DOI:  https://doi.org/10.1002/cbf.3888
Cancer Res. 2023 Dec 01.

Adipose triglyceride lipase is a therapeutic target in advanced prostate cancer that promotes metabolic plasticity.

Dominik Awad, Pham Hong Anh Cao, Thomas L Pulliam, Meredith Spradlin, Elavarasan Subramani, Tristen V Tellman, Caroline F Ribeiro, Riccardo Muzzioli, Brittany E Jewell, Hubert Pakula, Jeffrey J Ackroyd, Mollianne M Murray, Jenny J Han, Mei Leng, Antrix Jain, Badrajee Piyarathna, JIngjing Liu, Xingzhi Song, Jianhua Zhang, Albert R Klekers, Justin M Drake, Michael M Ittmann, Cristian Coarfa, David Piwnica-Worms, Mary C Farach-Carson, Massimo Loda, Livia S Eberlin, Daniel E Frigo.

Lipid metabolism plays a central role in prostate cancer. To date, the major focus has centered on de novo lipogenesis and lipid uptake in prostate cancer, but inhibitors of these processes have not benefited patients. Better understanding of how cancer cells access lipids once they are created or taken up and stored could uncover more effective strategies to perturb lipid metabolism and treat patients. Here, we identified that expression of adipose triglyceride lipase (ATGL), an enzyme that controls lipid droplet homeostasis and a previously suspected tumor suppressor, correlates with worse overall survival in men with advanced, castration-resistant prostate cancer (CRPC). Molecular, genetic, or pharmacological inhibition of ATGL impaired human and murine prostate cancer growth in vivo and in cell culture or organoids under conditions mimicking the tumor microenvironment. Mass spectrometry imaging demonstrated ATGL profoundly regulates lipid metabolism in vivo, remodeling membrane composition. ATGL inhibition induced metabolic plasticity, causing a glycolytic shift that could be exploited therapeutically by co-targeting both metabolic pathways. Patient-derived phosphoproteomics identified ATGL serine 404 as a target of CAMKK2-AMPK signaling in CRPC cells. Mutation of serine 404 did not alter the lipolytic activity of ATGL but did decrease CRPC growth, migration, and invasion, indicating that non-canonical ATGL activity also contributes to disease progression. Unbiased immunoprecipitation/mass spectrometry suggested that mutation of serine 404 not only disrupts existing ATGL protein interactions but also leads to new protein-protein interactions. Together, these data nominate ATGL as a therapeutic target for CRPC and provide insights for future drug development and combination therapies.

DOI: https://doi.org/10.1158/0008-5472.CAN-23-0555
Nat Metab. 2023 Nov 30.

Efficient elimination of MELAS-associated m.3243G mutant mitochondrial DNA by an engineered mitoARCUS nuclease.

Wendy K Shoop, Janel Lape, Megan Trum, Alea Powell, Emma Sevigny, Adam Mischler, Sandra R Bacman, Flavia Fontanesi, Jeff Smith, Derek Jantz, Cassandra L Gorsuch, Carlos T Moraes.

Nuclease-mediated editing of heteroplasmic mitochondrial DNA (mtDNA) seeks to preferentially cleave and eliminate mutant mtDNA, leaving wild-type genomes to repopulate the cell and shift mtDNA heteroplasmy. Various technologies are available, but many suffer from limitations based on size and/or specificity. The use of ARCUS nucleases, derived from naturally occurring I-CreI, avoids these pitfalls due to their small size, single-component protein structure and high specificity resulting from a robust protein-engineering process. Here we describe the development of a mitochondrial-targeted ARCUS (mitoARCUS) nuclease designed to target one of the most common pathogenic mtDNA mutations, m.3243A>G. mitoARCUS robustly eliminated mutant mtDNA without cutting wild-type mtDNA, allowing for shifts in heteroplasmy and concomitant improvements in mitochondrial protein steady-state levels and respiration. In vivo efficacy was demonstrated using a m.3243A>G xenograft mouse model with mitoARCUS delivered systemically by adeno-associated virus. Together, these data support the development of mitoARCUS as an in vivo gene-editing therapeutic for m.3243A>G-associated diseases.

DOI: https://doi.org/10.1038/s42255-023-00932-6
Biomed Pharmacother. 2023 Nov 30. pii: S0753-3322(23)01754-7. [Epub ahead of print]170 115956

Cryptotanshinone suppresses ovarian cancer via simultaneous inhibition of glycolysis and oxidative phosphorylation.

Tong Wang, Mengmeng Zhang, Muhammad Khan, Jingjing Li, Xiao Wu, Tonghui Ma, Yongming Li.

  Ovarian cancer is one of the most lethal cancers in female reproductive system due to heterogeneity and lack of effective treatment. Targeting aerobic glycolysis, a predominant energy metabolism of cancer cells has been recognized a novel strategy to overcome cancer cell growth. However, the capability of cancer cells to undergo metabolic reprogramming guarantees their survival even when glycolysis is inhibited. Here in this study, we have shown that Cryptotanshinone (CT), a lipid-soluble bioactive anticancer molecule of Salvia miltiorrhiza, inhibits both glycolysis and oxidative phosphorylation (OXPHOS) in ovarian cancer cells leading to growth suppression and apoptosis induction. Our mechanistic study revealed that CT decreased glucose uptake and lactate production, and inhibited the kinase activity of LDHA and HK2. The molecular docking study showed that CT could directly bind with GLUT1, LDHA, HK2, PKM2 and complex-1. The immunoblotting data showed that CT decreased the expression of aberrantly activated glycolytic proteins includingGLUT1, LDHA, HK2, and PKM2. Besides, we found that CT inhibited mitochondrial ComplexⅠ activity, decreased the ratio of NAD+/NADH, and suppressed the generation of ATP and induced activation of AMPK, which controls energy-reducing processes. These in vitro findings were further validated using xenograft model. The findings of in vivo studies were in line with in vitro studies. Taken together, CT effectively suppressed glycolysis and OXPHOS, inhibited growth and induced apoptosis in ovarian cancer cells both in vitro and in vivo study models.

Keywords:  Apoptosis; Cryptotanshinone; Glycolysis; Ovarian cancer; Oxidative phosphorylation

DOI:  https://doi.org/10.1016/j.biopha.2023.115956
bioRxiv. 2023 Nov 13. pii: 2023.11.08.566310. [Epub ahead of print]

Hypermetabolic state is associated with circadian rhythm disruption in mouse and human cancer cells.

Daniel Maxim Iascone, Xue Zhang, Patricia Bafford, Clementina Mesaros, Yogev Sela, Samuel Hofbauer, Shirley L Zhang, Kieona Cook, Pavel Pivarshev, Ben Z Stanger, Stewart Anderson, Chi V Dang, Amita Sehgal.

Crosstalk between cellular metabolism and circadian rhythms is a fundamental building block of multicellular life, and disruption of this reciprocal communication could be relevant to degenerative disease, including cancer. Here, we investigated whether maintenance of circadian rhythms depends upon specific metabolic pathways, particularly in the context of cancer. We found that in adult mouse fibroblasts, ATP levels were a major contributor to overall levels of a clock gene luciferase reporter, although not necessarily to the strength of circadian cycling. In contrast, we identified significant metabolic control of circadian function in an in vitro mouse model of pancreatic adenocarcinoma. Metabolic profiling of a library of congenic tumor cell clones revealed significant differences in levels of lactate, pyruvate, ATP, and other crucial metabolites that we used to identify candidate clones with which to generate circadian reporter lines. Despite the shared genetic background of the clones, we observed diverse circadian profiles among these lines that varied with their metabolic phenotype: the most hypometabolic line had the strongest circadian rhythms while the most hypermetabolic line had the weakest rhythms. Treatment of these tumor cell lines with bezafibrate, a peroxisome proliferator-activated receptor (PPAR) agonist shown to increase OxPhos, decreased the amplitude of circadian oscillation in a subset of tumor cell lines. Strikingly, treatment with the Complex I antagonist rotenone enhanced circadian rhythms only in the tumor cell line in which glycolysis was also low, thereby establishing a hypometabolic state. We further analyzed metabolic and circadian phenotypes across a panel of human patient-derived melanoma cell lines and observed a significant negative association between metabolic activity and circadian cycling strength. Together, these findings suggest that metabolic heterogeneity in cancer directly contributes to circadian function, and that high levels of glycolysis or OxPhos independently disrupt circadian rhythms in these cells.

DOI: https://doi.org/10.1101/2023.11.08.566310
Exp Ther Med. 2023 Dec;26(6): 574

Glutathione promotes the synergistic effects of venetoclax and azacytidine against myelodysplastic syndrome‑refractory anemia by regulating the cell cycle.

Xiaobo Wang, Lihua Yuan, Bo Lu, Dongjun Lin, Xiaojun Xu.

  Azacitidine is a DNA methyltransferase inhibitor that has been used as a singular agent for the treatment of myelodysplastic syndrome-refractory anemia with excess blast-1 and -2 (MDS-RAEB I/II). However, recurrence and overall response rates following this treatment remain unsatisfactory. The combination of azacitidine and venetoclax has been used for the clinical treatment of a variety of hematological diseases due to the synergistic killing effect of the two drugs. Venetoclax is a BCL-2 inhibitor that can inhibit mitochondrial metabolism. In addition, azacitidine has been shown to reduce the levels of myeloid cell leukemia 1 (MCL-1) in acute myeloid leukemia cells. MCL-1 is an anti-apoptotic protein and a potential source of resistance to venetoclax. However, the mechanism underlying the effects of combined venetoclax and azacitidine treatment remains to be fully elucidated. In the present study, the molecular mechanism underlying the impact of venetoclax on the efficacy of azacitidine was investigated by examining its effects on cell cycle progression. SKM-1 cell lines were treated in vitro with 0-2 µM venetoclax and 0-4 µM azacytidine. After 24, 48 and 72 h of treatment, the impact of the drugs on the cell cycle was assessed by flow cytometry. Following drug treatment, changes in cellular glutamine metabolism pathways was analyzed using western blotting (ATF4, CHOP, ASCT2, IDH2 and RB), quantitative PCR (ASCT2 and IDH2), liquid chromatography-mass spectrometry (α-KG, succinate and glutathione) and ELISA (glutamine and glutaminase). Venetoclax was found to inhibit mitochondrial activity though the alanine-serine-cysteine transporter 2 (ASCT2) pathway, which decreased glutamine uptake. Furthermore, venetoclax partially antagonized the action of azacitidine through this ASCT2 pathway, which was reversed by glutathione (GSH) treatment. These results suggest that GSH treatment can potentiate the synergistic therapeutic effects of venetoclax and azacitidine combined treatment on a myelodysplastic syndrome-refractory anemia cell line at lower concentrations.

Keywords:  azacitidine; glutathione; mitochondrial glutamine metabolism; myelodysplastic syndrome-refractory anemia; venetoclax

DOI:  https://doi.org/10.3892/etm.2023.12274
Oncotarget. 2023 Dec 01. 14 947-948

Reductive carboxylation of glutamine as a potential target in acute myeloid leukemia.

Alessia Roma, Lawrence D Goodridge, Paul A Spagnuolo.



Keywords:  acute myeloid leukemia; complex II; metabolism; mitochondria; reductive carboxylation

DOI:  https://doi.org/10.18632/oncotarget.28474
J Pharm Anal. 2023 Oct;13(10): 1168-1182

Histone deacetylase inhibitor pracinostat suppresses colorectal cancer by inducing CDK5-Drp1 signaling-mediated peripheral mitofission.

Xiao-Ling Liang, Lan Ouyang, Nan-Nan Yu, Zheng-Hua Sun, Zi-Kang Gui, Yu-Long Niu, Qing-Yu He, Jing Zhang, Yang Wang.

  Divisions at the periphery and midzone of mitochondria are two fission signatures that determine the fate of mitochondria and cells. Pharmacological induction of excessively asymmetric mitofission-associated cell death (MFAD) by switching the scission position from the mitochondrial midzone to the periphery represents a promising strategy for anticancer therapy. By screening a series of pan-inhibitors, we identified pracinostat, a pan-histone deacetylase (HDAC) inhibitor, as a novel MFAD inducer, that exhibited a significant anticancer effect on colorectal cancer (CRC) in vivo and in vitro. Pracinostat increased the expression of cyclin-dependent kinase 5 (CDK5) and induced its acetylation at residue lysine 33, accelerating the formation of complex CDK5/CDK5 regulatory subunit 1 and dynamin-related protein 1 (Drp1)-mediated mitochondrial peripheral fission. CRC cells with high level of CDK5 (CDK5-high) displayed midzone mitochondrial division that was associated with oncogenic phenotype, but treatment with pracinostat led to a lethal increase in the already-elevated level of CDK5 in the CRC cells. Mechanistically, pracinostat switched the scission position from the mitochondrial midzone to the periphery by improving the binding of Drp1 from mitochondrial fission factor (MFF) to mitochondrial fission 1 protein (FIS1). Thus, our results revealed the anticancer mechanism of HDACi pracinostat in CRC via activating CDK5-Drp1 signaling to cause selective MFAD of those CDK5-high tumor cells, which implicates a new paradigm to develop potential therapeutic strategies for CRC treatment.

Keywords:  Acetylation; CDK5; Drp1; HDAC inhibitor; Mitochondrial fission; Pracinostat

DOI:  https://doi.org/10.1016/j.jpha.2023.06.005
Nat Metab. 2023 Nov 30.

Mitochondrial DNA editing with mitoARCUS.

Denisa Hathazi, Rita Horvath.

DOI: https://doi.org/10.1038/s42255-023-00933-5
Oncol Lett. 2024 Jan;27(1): 12

In vitro anticancer effect of azithromycin targeting hypoxic lung cancer cells via the inhibition of mitophagy.

Kazutoshi Toriyama, Takashi Okuma, Shinji Abe, Hiroyuki Nakamura, Kazutetsu Aoshiba.

  Solid tumors are predisposed to hypoxia, which induces tumor progression, and causes resistance to treatment. Hypoxic tumor cells exploit auto- and mitophagy to facilitate metabolism and mitochondrial renewal. Azithromycin (AZM), a widely used macrolide, inhibits autophagy in cancer cells. The aim of the present study was to determine whether AZM targeted hypoxic cancer cells by inhibiting mitophagy. Lung cancer cell lines (A549, H1299 and NCI-H441) were cultured for up to 72 h under normoxic (20% O2) or hypoxic (0.3% O2) conditions in the presence or absence of AZM (≤25 µM), and the cell survival, autophagy flux and mitophagy flux were evaluated. AZM treatment reduced cell survival under hypoxic conditions, caused mitolysosome dysfunction with raised lysosomal pH and impaired the efficient removal of hypoxia-damaged mitochondria, eventually inducing apoptosis in the cancer cells. The cytotoxic effect of AZM under hypoxic conditions was abolished in mitochondria-deficient A549 cells (ρ° cells). The present study demonstrated that AZM reduced lung cancer cell survival under hypoxic conditions by interfering with the efficient removal of damaged mitochondria through mitophagy inhibition. Thus, AZM may be considered as a promising anticancer drug that targets the mitochondrial vulnerability of hypoxic lung cancer cells.

Keywords:  AZM; TME; autophagy; hypoxia; mitophagy

DOI:  https://doi.org/10.3892/ol.2023.14146
Sci Adv. 2023 Dec;9(48): eadh1891

Targeting ABCA12-controlled ceramide homeostasis inhibits breast cancer stem cell function and chemoresistance.

Jihong Cui, John R Christin, Julie A Reisz, Francesca Isabelle Cendali, Rahul Sanawar, Marcelo Coutinho De Miranda, Angelo D'Alessandro, Wenjun Guo.

Cancer stem cells (CSCs) drive tumor growth, metastasis, and chemoresistance. While emerging evidence suggests that CSCs have a unique dependency on lipid metabolism, the functions and regulation of distinct lipid species in CSCs remain poorly understood. Here, we developed a stem cell factor SOX9-based reporter for isolating CSCs in primary tumors and metastases of spontaneous mammary tumor models. Transcriptomic analyses uncover that SOX9high CSCs up-regulate the ABCA12 lipid transporter. ABCA12 down-regulation impairs cancer stemness and chemoresistance. Lipidomic analyses reveal that ABCA12 maintains cancer stemness and chemoresistance by reducing intracellular ceramide abundance, identifying a CSC-associated function of ABCA subfamily transporter. Ceramide suppresses cancer stemness by inhibiting the YAP-SOX9 signaling pathway in CSCs. Increasing ceramide levels in tumors enhances their sensitivity to chemotherapy and prevents the enrichment of SOX9high CSCs. In addition, SOX9high and ABCA12high cancer cells contribute to chemoresistance in human patient-derived xenografts. These findings identify a CSC-suppressing lipid metabolism pathway that can be exploited to inhibit CSCs and overcome chemoresistance.

DOI: https://doi.org/10.1126/sciadv.adh1891
Biochem Pharmacol. 2023 Nov 28. pii: S0006-2952(23)00546-4. [Epub ahead of print] 115953

Mitochondria dysfunction induced by decyl-TPP mitochondriotropic antioxidant based on caffeic acid AntiOxCIN6 sensitizes cisplatin lung anticancer therapy due to a remodeling of energy metabolism.

Ricardo Amorim, Carina C Magalhães, Sofia Benfeito, Fernando Cagide, Ludgero C Tavares, Katia Santos, Vilma A Sardão, Sandipan Datta, Gino A Cortopassi, Inês Baldeiras, John G Jones, Fernanda Borges, Paulo J Oliveira, José Teixeira.

  The pharmacological interest in mitochondria is very relevant since these crucial organelles are involved in the pathogenesis of multiple diseases, such as cancer. In order to modulate cellular redox/oxidative balance and enhance mitochondrial function, numerous polyphenolic derivatives targeting mitochondria have been developed. Still, due to the drug resistance emergence in several cancer therapies, significant efforts are being made to develop drugs that combine the induction of mitochondrial metabolic reprogramming with the ability to generate reactive oxygen species, taking into consideration the varying metabolic profiles of different cell types. We previously developed a mitochondria-targeted antioxidant (AntiOxCIN6) by linking caffeic acid to lipophilic triphenylphosphonium cation through a 10-carbon aliphatic chain. The antioxidant activity of AntiOxCIN6 has been documented but how the mitochondriotropic compound impact energy metabolism of both normal and cancer cells remains unknown. We demonstrated that AntiOxCIN6 increased antioxidant defense system in HepG2 cells, although ROS clearance was ineffective. Consequently, AntiOxCIN6 significantly decreased mitochondrial function and morphology, culminating in a decreased capacity in complex I-driven ATP production without affecting cell viability. These alterations were accompanied by an increase in glycolytic fluxes. Additionally, we demonstrate that AntiOxCIN6 sensitized A549 adenocarcinoma cells for CIS-induced apoptotic cell death, while AntiOxCIN6 appears to cause metabolic changes or a redox pre-conditioning on lung MRC-5 fibroblasts, conferring protection against cisplatin. We propose that length and hydrophobicity of the C10-TPP + alkyl linker play a significant role in inducing mitochondrial and cellular toxicity, while the presence of the antioxidant caffeic acid appears to be responsible for activating cytoprotective pathways.

Keywords:  Antioxidant defenses; Glycolysis; Lung cancer therapy; Metabolic remodeling; Mitochondrial (dys)homeostasis; Mitochondriotropic phenolic antioxidants; Reactive oxygen species

DOI:  https://doi.org/10.1016/j.bcp.2023.115953
PeerJ. 2023 ;11 e16512

Metabolomic analyses uncover an inhibitory effect of niclosamide on mitochondrial membrane potential in cholangiocarcinoma cells.

Thanaporn Kulthawatsiri, Yingpinyapat Kittirat, Jutarop Phetcharaburanin, Jittima Tomacha, Bundit Promraksa, Arporn Wangwiwatsin, Poramate Klanrit, Attapol Titapun, Watcharin Loilome, Nisana Namwat.

  Background: Niclosamide is an oral anthelminthic drug that has been used for treating tapeworm infections. Its mechanism involves the disturbance of mitochondrial membrane potential that in turn inhibits oxidative phosphorylation leading to ATP depletion. To date, niclosamide has been validated as the potent anti-cancer agent against several cancers. However, the molecular mechanisms underlying the effects of niclosamide on the liver fluke Opisthorchis viverrini (Ov)-associated cholangiocarcinoma (CCA) cell functions remain to be elucidated. The aims of this study were to investigate the effects of niclosamide on CCA cell proliferation and on metabolic phenoconversion through the alteration of metabolites associated with mitochondrial function in CCA cell lines.Materials and Methods: The inhibitory effect of niclosamide on CCA cells was determined using SRB assay. A mitochondrial membrane potential using tetramethylrhodamine, ethyl ester-mitochondrial membrane potential (TMRE-MMP) assay was conducted. Liquid chromatography-mass spectrometry-based metabolomics was employed to investigate the global metabolic changes upon niclosamide treatment. ATP levels were measured using CellTiter-Glo® luminescent cell viability assay. NAD metabolism was examined by the NAD+/NADH ratio.
Results: Niclosamide strongly inhibited CCA cell growth and reduced the MMP of CCA cells. An orthogonal partial-least square regression analysis revealed that the effects of niclosamide on suppressing cell viability and MMP of CCA cells were significantly associated with an increase in niacinamide, a precursor in NAD synthesis that may disrupt the electron transport system leading to suppression of NAD+/NADH ratio and ATP depletion.
Conclusion: Our findings unravel the mode of action of niclosamide in the energy depletion that could potentially serve as the promising therapeutic strategy for CCA treatment.

Keywords:  Cholangiocarcinoma; LC-MS; Metabolomics; Niclosamide

DOI:  https://doi.org/10.7717/peerj.16512
Leukemia. 2023 Nov 25.

TP-0184 inhibits FLT3/ACVR1 to overcome FLT3 inhibitor resistance and hinder AML growth synergistically with venetoclax.

Anudishi Tyagi, Appalaraju Jaggupilli, Stanley Ly, Bin Yuan, Fouad El-Dana, Venkatesh L Hegde, Vivek Anand, Bijender Kumar, Mamta Puppala, Zheng Yin, Stephen T C Wong, Alexis Mollard, Hariprasad Vankayalapati, Jason M Foulks, Steven L Warner, Naval Daver, Gautam Borthakur, V Lokesh Battula.

We identified activin A receptor type I (ACVR1), a member of the TGF-β superfamily, as a factor favoring acute myeloid leukemia (AML) growth and a new potential therapeutic target. ACVR1 is overexpressed in FLT3-mutated AML and inhibition of ACVR1 expression sensitized AML cells to FLT3 inhibitors. We developed a novel ACVR1 inhibitor, TP-0184, which selectively caused growth arrest in FLT3-mutated AML cell lines. Molecular docking and in vitro kinase assays revealed that TP-0184 binds to both ACVR1 and FLT3 with high affinity and inhibits FLT3/ACVR1 downstream signaling. Treatment with TP-0184 or in combination with BCL2 inhibitor, venetoclax dramatically inhibited leukemia growth in FLT3-mutated AML cell lines and patient-derived xenograft models in a dose-dependent manner. These findings suggest that ACVR1 is a novel biomarker and plays a role in AML resistance to FLT3 inhibitors and that FLT3/ACVR1 dual inhibitor TP-0184 is a novel potential therapeutic tool for AML with FLT3 mutations.

DOI: https://doi.org/10.1038/s41375-023-02086-6
Cell Death Discov. 2023 Dec 01. 9(1): 435

Standardized assays to monitor drug sensitivity in hematologic cancers.

Pilar Ayuda-Durán, Johanne U Hermansen, Mariaserena Giliberto, Yanping Yin, Robert Hanes, Sandra Gordon, Heikki Kuusanmäki, Andrea M Brodersen, Aram N Andersen, Kjetil Taskén, Krister Wennerberg, Jorrit M Enserink, Sigrid S Skånland.

The principle of drug sensitivity testing is to expose cancer cells to a library of different drugs and measure its effects on cell viability. Recent technological advances, continuous approval of targeted therapies, and improved cell culture protocols have enhanced the precision and clinical relevance of such screens. Indeed, drug sensitivity testing has proven diagnostically valuable for patients with advanced hematologic cancers. However, different cell types behave differently in culture and therefore require optimized drug screening protocols to ensure that their ex vivo drug sensitivity accurately reflects in vivo drug responses. For example, primary chronic lymphocytic leukemia (CLL) and multiple myeloma (MM) cells require unique microenvironmental stimuli to survive in culture, while this is less the case for acute myeloid leukemia (AML) cells. Here, we present our optimized and validated protocols for culturing and drug screening of primary cells from AML, CLL, and MM patients, and a generic protocol for cell line models. We also discuss drug library designs, reproducibility, and quality controls. We envision that these protocols may serve as community guidelines for the use and interpretation of assays to monitor drug sensitivity in hematologic cancers and thus contribute to standardization. The read-outs may provide insight into tumor biology, identify or confirm treatment resistance and sensitivity in real time, and ultimately guide clinical decision-making.

DOI: https://doi.org/10.1038/s41420-023-01722-5
Cell Death Dis. 2023 Dec 01. 14(12): 788

Mitochondrial E3 ligase MARCH5 is a safeguard against DNA-PKcs-mediated immune signaling in mitochondria-damaged cells.

June Heo, Yeon-Ji Park, Yonghyeon Kim, Ho-Soo Lee, Jeongah Kim, Soon-Hwan Kwon, Myeong-Gyun Kang, Hyun-Woo Rhee, Woong Sun, Jae-Ho Lee, Hyeseong Cho.

Mitochondrial dysfunction is important in various chronic degenerative disorders, and aberrant immune responses elicited by cytoplasmic mitochondrial DNA (mtDNA) may be related. Here, we developed mtDNA-targeted MTERF1-FokI and TFAM-FokI endonuclease systems to induce mitochondrial DNA double-strand breaks (mtDSBs). In these cells, the mtDNA copy number was significantly reduced upon mtDSB induction. Interestingly, in cGAS knockout cells, synthesis of interferon β1 and interferon-stimulated gene was increased upon mtDSB induction. We found that mtDSBs activated DNA-PKcs and HSPA8 in a VDAC1-dependent manner. Importantly, the mitochondrial E3 ligase MARCH5 bound active DNA-PKcs in cells with mtDSBs and reduced the type І interferon response through the degradation of DNA-PKcs. Likewise, mitochondrial damage caused by LPS treatment in RAW264.7 macrophage cells increased phospho-HSPA8 levels and the synthesis of mIFNB1 mRNA in a DNA-PKcs-dependent manner. Accordingly, in March5 knockout macrophages, phospho-HSPA8 levels and the synthesis of mIFNB1 mRNA were prolonged after LPS stimulation. Together, cytoplasmic mtDNA elicits a cellular immune response through DNA-PKcs, and mitochondrial MARCH5 may be a safeguard to prevent persistent inflammatory reactions.

DOI: https://doi.org/10.1038/s41419-023-06315-9
Clin Lymphoma Myeloma Leuk. 2023 Oct 21. pii: S2152-2650(23)02150-X. [Epub ahead of print]

SOHO State of the Art Updates and Next Questions: Understanding and Overcoming Venetoclax Resistance in Hematologic Malignancies.

Mark Forsberg, Marina Konopleva.

  The discovery of Venetoclax (VEN) has transformed the therapeutic landscape of acute myeloid leukemia (AML) and chronic lymphocytic leukemia (CLL). However, the response is heterogeneous with 10% to 50% of newly diagnosed AML patients not responding to hypomethylating agent (HMA) and VEN. Furthermore, up to 40% of responding patients relapse shortly. This review discusses the mechanism of action of Venetoclax and the major mechanisms of inherent and acquired resistance to VEN. VEN is highly specific to BCL-2 binding, as such other antiapoptotic proteins in BCL-2 family induce resistance. These antiapoptotic proteins can also be upregulated via a number of compensatory cell signaling pathways including PI3K/AKT/mTOR, the MAPK/ERK pathway, and mutant FLT3-ITD. Mutations can occur in BCL-2 and BAX proteins, or they can be silenced by TP53 mutations and other epigenetic changes. Changes to mitochondrial structure and metabolism can induce resistance. Key metabolic regulators include OXPHOS and alternative amino acid metabolism. Finally microenvironmental factors can influence VEN responses. This paper evaluates subsets of AML by differentiation, histology, cytogenetics and molecular markers and their different responses to VEN; with spliceosome mutations, ASXL1, NPM1 and IDH1/2 being favorable while others such as FLT3, TP53 and BCL-2 mutations being less responsive. Currently intensive multiagent chemotherapy and Venetoclax combinations such as 7+3+VEN are favored in fit younger AML patients. However, with resistant patients' subsets targeted combination therapies are becoming an increasingly attractive option. We explore the incorporation of non-BCL-2 inhibitors, next-generation BCL-2 and multi-protein agents, other inhibitors most prominently FLT-3 inhibitors in addition to Venetoclax, and other novel approaches for resolving Venetoclax resistance.

Keywords:  AML; BCL-2; CLL; Drug-resistance; Venetoclax

DOI:  https://doi.org/10.1016/j.clml.2023.10.006
J Inorg Biochem. 2023 Nov 21. pii: S0162-0134(23)00314-8. [Epub ahead of print]251 112432

Copper(II) complexes with plumbagin and bipyridines target mitochondria for enhanced chemodynamic cancer therapy.

Hai-Qun Zhang, Xing Lu, Hong Liang, Zhen-Feng Chen.

  The combination of mitochondrial targeting and chemodynamic therapy is a promising anti-cancer strategy. Three mitochondria targeting copper(II) complexes (Cu1-Cu3) with plumbagin and bipyridine ligands for enhanced chemodynamic therapy were synthesized and characterized. Their anti-proliferative activity to HeLa cells was higher than that of cisplatin, and their toxicity to normal cells was low. Cellular uptake and distribution studies indicated that Cu1 and Cu3 were mainly accumulated in mitochondria. The mechanism studies showed that Cu1 and Cu3 converted intracellular H2O2 into toxic hydroxyl radicals by consuming glutathione, leading to mitochondrial dysfunction. Treatment with the copper complex caused ER stress and cell arrest in the S phase which resulted in apoptosis. In vivo, Cu1 and Cu3 effectively inhibited the growth of HeLa xenograft tumors without obvious toxic and side effects.

Keywords:  Antitumor activity; Chemodynamic therapy; Copper complex; Mitochondrial targeting; Plumbagin

DOI:  https://doi.org/10.1016/j.jinorgbio.2023.112432
Sci Adv. 2023 Dec;9(48): eadh1436

Enhanced TP53 reactivation disrupts MYC transcriptional program and overcomes venetoclax resistance in acute myeloid leukemias.

Yuki Nishida, Jo Ishizawa, Edward Ayoub, Rafael Heinz Montoya, Lauren B Ostermann, Muharrem Muftuoglu, Vivian R Ruvolo, Tallie Patsilevas, Darah A Scruggs, Shayaun Khazaei, Po Yee Mak, Wenjing Tao, Bing Z Carter, Steffen Boettcher, Benjamin L Ebert, Naval G Daver, Marina Konopleva, Takahiko Seki, Kensuke Kojima, Michael Andreeff.

The tumor suppressor TP53 is frequently inactivated in a mutation-independent manner in cancers and is reactivated by inhibiting its negative regulators. We here cotarget MDM2 and the nuclear exporter XPO1 to maximize transcriptional activity of p53. MDM2/XPO1 inhibition accumulated nuclear p53 and elicited a 25- to 60-fold increase of its transcriptional targets. TP53 regulates MYC, and MDM2/XPO1 inhibition disrupted the c-MYC-regulated transcriptome, resulting in the synergistic induction of apoptosis in acute myeloid leukemia (AML). Unexpectedly, venetoclax-resistant AMLs express high levels of c-MYC and are vulnerable to MDM2/XPO1 inhibition in vivo. However, AML cells persisting after MDM2/XPO1 inhibition exhibit a quiescence- and stress response-associated phenotype. Venetoclax overcomes that resistance, as shown by single-cell mass cytometry. The triple inhibition of MDM2, XPO1, and BCL2 was highly effective against venetoclax-resistant AML in vivo. Our results propose a novel, highly translatable therapeutic approach leveraging p53 reactivation to overcome nongenetic, stress-adapted venetoclax resistance.

DOI: https://doi.org/10.1126/sciadv.adh1436
Free Radic Biol Med. 2023 Nov 29. pii: S0891-5849(23)01134-6. [Epub ahead of print]

Different impact of vitamin D on mitochondrial activity and morphology in normal and malignant keratinocytes, the role of genomic pathway.

Anna M Olszewska, Joanna I Nowak, Oliwia Król, Damian Flis, Michał A Zmijewski.

  Deregulation of mitochondria activity is one of the hallmarks of cancerogenesis and an important target for cancer therapy. Therefore, we compared the impact of an active form of vitamin D3 (1,25(OH)2D3) on mitochondrial morphology and bioenergetics in human squamous cell carcinoma (A431) and immortalized HaCaT keratinocytes. It was shown that mitochondria of cancerous A431 cells differ from that observed in HaCaT keratinocytes in terms of network, morphology, bioenergetics, glycolysis, and mitochondrial DNA copy number, while treatment of A431 with 1,25(OH)2D3 partially eliminates these differences. Furthermore, mitochondrial membrane potential, basal respiration, and mitochondrial reactive oxygen species production were decreased in A431 cells treated with 1,25(OH)2D3. Additionally, the expression and protein level of mitophagy marker PINK1 was significantly increased in A431 1,25(OH)2D3 treated cells, but not observed in treated HaCaT cells. Knockout of VDR (vitamin D receptor) or RXRA (binding partner retinoid X receptor) partially altered mitochondrial morphology and function as well as mitochondrial response to 1,25(OH)2D3. Transcriptomic analysis on A431 cells treated with 1,25(OH)2D3 revealed modulation of expression of several mitochondrial-related genes involved in mitochondrial depolarization, mitochondrial protein translation (i.e. LYRM9, MARS2), and fusion-fission (OPA1, FIS1, MFN1 and 2), however, none of the genes coded by mitochondrial DNA was affected. Interestingly, in silico analyses of nuclear-encoded mitochondrial genes revealed that they are rather activated by the secondary genomic response to 1,25(OH)2D3. Taken together, 1,25(OH)2D3 remodels mitochondrial architecture and bioenergetics through VDR-dependent and only partially RXRA-dependent activation of the genomic pathway, thus outlining a new perspective for anticancer properties of vitamin D3 in relation to mitochondria in squamous cell carcinoma.

Keywords:  Fusion/fission; Keratinocytes; Mitochondria; Squamous cell carcinoma; vitamin D

DOI:  https://doi.org/10.1016/j.freeradbiomed.2023.11.033
Eur J Clin Invest. 2023 Dec 01. e14138

Metabolic control of mitophagy.

Andreas Zimmermann, Frank Madeo, Abhinav Diwan, Junichi Sadoshima, Simon Sedej, Guido Kroemer, Mahmoud Abdellatif.

  Mitochondrial dysfunction is a major hallmark of ageing and related chronic disorders. Controlled removal of damaged mitochondria by the autophagic machinery, a process known as mitophagy, is vital for mitochondrial homeostasis and cell survival. The central role of mitochondria in cellular metabolism places mitochondrial removal at the interface of key metabolic pathways affecting the biosynthesis or catabolism of acetyl-coenzyme A, nicotinamide adenine dinucleotide, polyamines, as well as fatty acids and amino acids. Molecular switches that integrate the metabolic status of the cell, like AMP-dependent protein kinase, protein kinase A, mechanistic target of rapamycin and sirtuins, have also emerged as important regulators of mitophagy. In this review, we discuss how metabolic regulation intersects with mitophagy. We place special emphasis on the metabolic regulatory circuits that may be therapeutically targeted to delay ageing and mitochondria-associated chronic diseases. Moreover, we identify outstanding knowledge gaps, such as the ill-defined distinction between basal and damage-induced mitophagy, which must be resolved to boost progress in this area.

Keywords:  AMPK; NAD; acetyl-CoA; ageing; ageing-related disease; metabolism; mitophagy; spermidine

DOI:  https://doi.org/10.1111/eci.14138
Redox Biol. 2023 Nov 20. pii: S2213-2317(23)00363-4. [Epub ahead of print]68 102962

Organic Selenium induces ferroptosis in pancreatic cancer cells.

Roberta Noè, Noemi Inglese, Patrizia Romani, Thauan Serafini, Carlotta Paoli, Beatrice Calciolari, Marco Fantuz, Agata Zamborlin, Nicoletta C Surdo, Vittoria Spada, Martina Spacci, Sara Volta, Maria Laura Ermini, Giulietta Di Benedetto, Valentina Frusca, Claudio Santi, Konstantinos Lefkimmiatis, Sirio Dupont, Valerio Voliani, Luca Sancineto, Alessandro Carrer.

  Pancreatic ductal adenocarcinoma (PDA) cells reprogram both mitochondrial and lysosomal functions to support growth. At the same time, this causes significant dishomeostasis of free radicals. While this is compensated by the upregulation of detoxification mechanisms, it also represents a potential vulnerability. Here we demonstrate that PDA cells are sensitive to the inhibition of the mevalonate pathway (MVP), which supports the biosynthesis of critical antioxidant intermediates and protect from ferroptosis. We attacked the susceptibility of PDA cells to ferroptotic death with selenorganic compounds, including dibenzyl diselenide (DBDS) that exhibits potent pro-oxidant properties and inhibits tumor growth in vitro and in vivo. DBDS treatment induces the mobilization of iron from mitochondria enabling uncontrolled lipid peroxidation. Finally, we showed that DBDS and statins act synergistically to promote ferroptosis and provide evidence that combined treatment is a viable strategy to combat PDA.

Keywords:  Dibenzyl diselenide (DBDS); Ferroptosis; Mevalonate pathway (MVP); Pancreatic cancer; Selenorganic compounds

DOI:  https://doi.org/10.1016/j.redox.2023.102962
Redox Rep. 2023 Dec;28(1): 2284517

Melittin kills A549 cells by targeting mitochondria and blocking mitophagy flux.

Xuan Li, Zheng Li, Yu-Qi Meng, Hui Qiao, Ke-Rong Zhai, Zhen-Qing Li, Shi-Lin Wei, Bin Li.

  Melittin, a naturally occurring polypeptide found in bee venom, has been recognized for its potential anti-tumor effects, particularly in the context of lung cancer. Our previous study focused on its impact on human lung adenocarcinoma cells A549, revealing that melittin induces intracellular reactive oxygen species (ROS) burst and oxidative damage, resulting in cell death. Considering the significant role of mitochondria in maintaining intracellular redox levels and ROS, we further examined the involvement of mitochondrial damage in melittin-induced apoptosis in lung cancer cells. Our findings demonstrated that melittin caused changes in mitochondrial membrane potential (MMP), triggered mitochondrial ROS burst (Figure 1), and activated the mitochondria-related apoptosis pathway Bax/Bcl-2 by directly targeting mitochondria in A549 cells (Figure 2). Further, we infected A549 cells using a lentivirus that can express melittin-Myc and confirmed that melittin can directly target binding to mitochondria, causing the biological effects described above (Figure 2). Notably, melittin induced mitochondrial damage while inhibiting autophagy, resulting in abnormal degradation of damaged mitochondria (Figure 5). To summarize, our study unveils that melittin targets mitochondria, causing mitochondrial damage, and inhibits the autophagy-lysosomal degradation pathway. This process triggers mitoROS burst and ultimately activates the mitochondria-associated Bax/Bcl-2 apoptotic signaling pathways in A549 cells.

Keywords:  A549 cells; Melittin; ROS; apoptosis; autophagy; mitochondria damage; mitophagy; mitophagy flux

DOI:  https://doi.org/10.1080/13510002.2023.2284517
bioRxiv. 2023 Nov 13. pii: 2023.11.13.566839. [Epub ahead of print]

COQ4 is required for the oxidative decarboxylation of the C1 carbon of Coenzyme Q in eukaryotic cells.

Ludovic Pelosi, Laura Morbiato, Arthur Burgardt, Fiorella Tonello, Abigail K Bartlett, Rachel M Guerra, Katayoun Kazemzadeh Ferizhendi, Maria Andrea Desbats, Bérengère Rascalou, Marco Marchi, Luis Vázquez-Fonseca, Caterina Agosto, Giuseppe Zanotti, Morgane Roger-Margueritat, María Alcázar-Fabra, Laura García-Corzo, Ana Sánchez-Cuesta, Plácido Navas, Gloria Brea-Calvo, Eva Trevisson, Volker F Wendisch, David J Pagliarini, Leonardo Salviati, Fabien Pierrel.

Coenzyme Q (CoQ) is a redox lipid that fulfills critical functions in cellular bioenergetics and homeostasis. CoQ is synthesized by a multi-step pathway that involves several COQ proteins. Two steps of the eukaryotic pathway, the decarboxylation and hydroxylation of position C1, have remained uncharacterized. Here, we provide evidence that these two reactions occur in a single oxidative decarboxylation step catalyzed by COQ4. We demonstrate that COQ4 complements an Escherichia coli strain deficient for C1 decarboxylation and hydroxylation and that COQ4 displays oxidative decarboxylation activity in the non-CoQ producer Corynebacterium glutamicum . Overall, our results substantiate that COQ4 contributes to CoQ biosynthesis, not only via its previously proposed structural role, but also via oxidative decarboxylation of CoQ precursors. These findings fill a major gap in the knowledge of eukaryotic CoQ biosynthesis, and shed new light on the pathophysiology of human primary CoQ deficiency due to COQ4 mutations.

DOI: https://doi.org/10.1101/2023.11.13.566839
Biophys Rep (N Y). 2023 Dec 13. 3(4): 100134

Thioflavin T indicates mitochondrial membrane potential in mammalian cells.

Emily Skates, Hadrien Delattre, Zoe Schofield, Munehiro Asally, Orkun S Soyer.

The fluorescent benzothiazole dye thioflavin T (ThT) is widely used as a marker for protein aggregates, most commonly in the context of neurodegenerative disease research and diagnosis. Recently, this same dye was shown to indicate membrane potential in bacteria due to its cationic nature. This finding prompted a question whether ThT fluorescence is linked to the membrane potential in mammalian cells, which would be important for appropriate utilization of ThT in research and diagnosis. Here, we show that ThT localizes into the mitochondria of HeLa cells in a membrane-potential-dependent manner. Specifically, ThT colocalized in cells with the mitochondrial membrane potential indicator tetramethylrhodamine methyl ester (TMRM) and gave similar temporal responses as TMRM to treatment with a protonophore, carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP). Additionally, we found that presence of ThT together with exposure to blue light (λ = 405 nm), but neither factor alone, caused depolarization of mitochondrial membrane potential. This additive effect of the concentration and blue light was recapitulated by a mathematical model implementing the potential-dependent distribution of ThT and its effect on mitochondrial membrane potential through photosensitization. These results show that ThT can act as a mitochondrial membrane potential indicator in mammalian cells, when used at low concentrations and with low blue light exposure. However, it causes dissipation of the mitochondrial membrane potential depending additively on its concentrations and blue light exposure. This conclusion motivates a re-evaluation of ThT's use at micromolar range in live-cell analyses and indicates that this dye can enable future studies on the potential connections between mitochondrial membrane potential dynamics and protein aggregation.

DOI: https://doi.org/10.1016/j.bpr.2023.100134
bioRxiv. 2023 Nov 14. pii: 2023.11.10.564582. [Epub ahead of print]

MITF regulates IDH1 and NNT and drives a transcriptional program protecting cutaneous melanoma from reactive oxygen species.

Elisabeth Roider, Alexandra I T Lakatos, Alicia M McConnell, Poguang Wang, Alina Mueller, Akinori Kawakami, Jennifer Tsoi, Botond L Szabolcs, Anna A Ascsillán, Yusuke Suita, Vivien Igras, Jennifer A Lo, Jennifer J Hsiao, Rebecca Lapides, Dorottya M P Pál, Anna S Lengyel, Alexander Navarini, Arimichi Okazaki, Othon Iliopoulos, István Németh, Thomas G Graeber, Leonard Zon, Roger W Giese, Lajos V Kemeny, David E Fisher.

Microphthalmia-associated transcription factor (MITF) plays pivotal roles in melanocyte development, function, and melanoma pathogenesis. MITF amplification occurs in melanoma and has been associated with resistance to targeted therapies. Here, we show that MITF regulates a global antioxidant program that increases survival of melanoma cell lines by protecting the cells from reactive oxygen species (ROS)-induced damage. In addition, this redox program is correlated with MITF expression in human melanoma cell lines and patient-derived melanoma samples. Using a zebrafish melanoma model, we show that MITF decreases ROS-mediated DNA damage in vivo . Some of the MITF target genes involved, such as IDH1 and NNT , are regulated through direct MITF binding to canonical enhancer box (E-BOX) sequences proximal to their promoters. Utilizing functional experiments, we demonstrate the role of MITF and its target genes in reducing cytosolic and mitochondrial ROS. Collectively, our data identify MITF as a significant driver of the cellular antioxidant state.One Sentence Summary: MITF promote melanoma survival via increasing ROS tolerance.

DOI: https://doi.org/10.1101/2023.11.10.564582