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Pike 2011 Biochim Biophys Acta

From Bioblast
Publications in the MiPMap
Pike LS, Smift AL, Croteau NJ, Ferrick DA, Wu M (2011) Inhibition of fatty acid oxidation by etomoxir impairs NADPH production and increases reactive oxygen species resulting in ATP depletion and cell death in human glioblastoma cells. https://doi.org/10.1016/j.bbabio.2010.10.022

» Biochim Biophys Acta 1807:726-34. PMID: 21692241 Open Access

Pike LS, Smift AL, Croteau NJ, Ferrick DA, Wu M (2011) Biochim Biophys Acta

Abstract: Normal differentiated cells rely primarily on mitochondrial oxidative phosphorylation to produce adenosine triphosphate (ATP) to maintain their viability and functions by using three major bioenergetic fuels: glucose, glutamine and fatty acids. Many cancer cells, however, rely on aerobic glycolysis for their growth and survival, and recent studies indicate that some cancer cells depend on glutamine as well. This altered metabolism in cancers occurs through oncogene activation or loss of tumor suppressor genes in multiple signaling pathways, including the phosphoinositide 3-kinase and Myc pathways. Relatively little is known, however, about the role of fatty acids as a bioenergetic fuel in growth and survival of cancer cells. Here, we report that human glioblastoma SF188 cells oxidize fatty acids and that inhibition of fatty acid β-oxidation by etomoxir, a carnitine palmitoyltransferase 1 inhibitor, markedly reduces cellular ATP levels and viability. We also found that inhibition of fatty acid oxidation controls the NADPH level. In the presence of reactive oxygen species scavenger tiron, however, ATP depletion is prevented without restoring fatty acid oxidation. This suggests that oxidative stress may lead to bioenergetic failure and cell death. Our work provides evidence that mitochondrial fatty acid oxidation may provide NADPH for defense against oxidative stress and prevent ATP loss and cell death.

Bioblast editor: Gnaiger E

Communicated by Gnaiger E (2023-01-17)
Red arrow: TCA cycle corrected in original Figure 1a

Critical

Pike et al (2011) is cited with critical consideration of off-target side effects of etoxomir:
  • Silva FSG, Komlodi T, Garcia-Souza LF, Bento G, Doerrier C, Oliveira PJ, Gnaiger E (2019) Can fatty acid oxidation be specifically blocked by the CPT1 inhibitor etomoxir? - »Bioblast link«
  • Goretzki A, Lin YJ, Zimmermann J, Rainer H, Junker AC, Wolfheimer S, Vieths S, Scheurer S, Schülke S. Role of Glycolysis and Fatty Acid Synthesis in the Activation and T Cell-Modulating Potential of Dendritic Cells Stimulated with a TLR5-Ligand Allergen Fusion Protein. Int J Mol Sci. 2022 Oct 21;23(20):12695. https://doi.org/10.3390/ijms232012695. PMID: 36293550
Ref 12: "Inhibition of fatty acid metabolism was achieved through pretreatment with either cerulenin to block fatty acid synthase [11] or etomoxir to block fatty acid oxidation by inhibiting carnitine palmitoyltransferase 1 [12]. .. Cerulenin slightly reduced the expression levels of CD40 and CD69 by approx. 60% and 50%, respectively, while etomoxir had no effect on the expression levels of the investigated surface markers. .. This effect was abolished when the cells were either pretreated with the mTOR-inhibitor rapamycin or the inhibitors of fatty acid metabolism cerulenin or etomoxir. .. We further analyzed the contribution of fatty acid metabolism to the observed activation of mDCs by rFlaA:Betv1. Metabolically, only inhibition of fatty acid synthesis by cerulenin slightly reduced glycolysis (while having no effect on OCR). Moreover, cerulenin pretreatment dose-dependently suppressed the rFlaA:Betv1-induced IL-10- and IL-12p70 secretion, while IL-1β secretion remained unchanged. In contrast, etomoxir pretreatment slightly reduced mitochondrial oxygen consumption and interfered with antimicrobial activity otherwise observed in supernatants of rFlaA:Betv1-stimulated mDCs, but had no impact on the investigated ECAR, cytokine secretion, or expression of co-stimulatory molecules. Etomoxir concentrations greater than 5 µM were recently shown to induce mitochondrial stress associated with ROS production in human T cells independently of its effect on carnitine palmitoyltransferase 1 (CPT1a) [29]. On the basis of these results, similar off-target effects of etomoxir in mDCs cannot be excluded. Therefore, our results obtained in mDCs pre-treated with etomoxir have to be interpreted cautiously. .. 10, 50, or 100 µM etomoxir"
  • Sainero-Alcolado L, Liaño-Pons J, Ruiz-Pérez MV, Arsenian-Henriksson M. Targeting mitochondrial metabolism for precision medicine in cancer. Cell Death Differ. 2022 Jul;29(7):1304-1317. doi: 10.1038/s41418-022-01022-y. Epub 2022 Jul 13. PMID: 35831624
Ref 81: "Etomoxir further induces cell death of glioblastoma cells in vitro [81] and delays tumor apparition and progression in a glioblastoma mouse model [82]. However, toxicity, off-target effects on ETC Complex I, and the high doses needed, limits its potential for clinical applications."
  • Yao CH, Liu GY, Wang R, Moon SH, Gross RW, Patti GJ (2018) Identifying off-target effects of etomoxir reveals that carnitine palmitoyltransferase I is essential for cancer cell proliferation independent of β-oxidation. PLoS Biol 16:2003782.
Ref 15: It is common in cancer studies to use etomoxir at hundreds of micromolar concentrations [5, 15, 18, 25, 26]. .. Given that studies evaluating the role of CPT1 in cancer have commonly used concentrations of etomoxir at the hundreds of micromolar or even 1 mM level [15],
Here we show that when FAO was reduced approximately 90% by pharmacological inhibition of carnitine palmitoyltransferase I (CPT1) with low concentrations of etomoxir, the proliferation rate of various cancer cells was unaffected. Efforts to pharmacologically inhibit FAO more than 90% revealed that high concentrations of etomoxir (200 μM) have an off-target effect of inhibiting complex I of the electron transport chain.

Belief in a paradigm

Pike et al (2011) is cited many times without sufficiently taking into account off-target side effects of etoxomir:
  • Gaca-Tabaszewska M, Bogusiewicz J, Bojko B. Metabolomic and Lipidomic Profiling of Gliomas-A New Direction in Personalized Therapies. Cancers (Basel). 2022 Oct 14;14(20):5041. doi: 10.3390/cancers14205041. PMID: 36291824
"Many studies, including those focused on gliomas, have proposed FAO as a very good potential target for new drugs. As such, researchers have considered etomoxir as a CPT-1a inhibitor, as it slows the proliferation of glioma cells and prolongs the patient’s time of survival [73]. .. Promising results have been obtained from clinical trials examining the combined use of etomoxir and reradiation to target hypoxia and improve treatment efficiency for cancers such as lung adenocarcinoma or prostate cancer [76]. In addition, Taib et al. [77] observed the significant inhibition of cell proliferation when etomoxir was applied alongside oleic acid in GBM and astrocytic cells [77]. To date, most studies in this area have employed in vitro experiments using gliomas or other cancer cells; however, the literature also contains clinical trials and animal studies demonstrating the inhibitory effects of etomoxir treatment in neurodegenerative diseases, such as Parkinson’s or Alzheimer’s disease [78]. Thus, we can assume that BBB crossing is an issue that can be overcome in the case of etomoxir and that more trials examining the use of this drug to treat gliomas are forthcoming."
  • Shim JK, Choi S, Yoon SJ, Choi RJ, Park J, Lee EH, Cho HJ, Lee S, Teo WY, Moon JH, Kim HS, Kim EH, Cheong JH, Chang JH, Yook JI, Kang SG (2022) Etomoxir, a carnitine palmitoyltransferase 1 inhibitor, combined with temozolomide reduces stemness and invasiveness in patient-derived glioblastoma tumorspheres. Cancer Cell Int 22:309. doi: 10.1186/s12935-022-02731-7. PMID: 36221088
"The importance of fatty acid oxidation (FAO) in the bioenergetics of glioblastoma (GBM) is being realized. Etomoxir (ETO), a carnitine palmitoyltransferase 1 (CPT1) inhibitor exerts cytotoxic effects in GBM, which involve interrupting the FAO pathway. We hypothesized that FAO inhibition could affect the outcomes of current standard temozolomide (TMZ) chemotherapy against GBM."
  • Wang CY, Wang CH, Mai RT, Chen TW, Li CW, Chao CH (2022) Mutant p53-microRNA-200c-ZEB2-Axis-Induced CPT1C Elevation Contributes to Metabolic Reprogramming and Tumor Progression in Basal-Like Breast Cancers. Front Oncol 12:940402. doi: 10.3389/fonc.2022.940402. PMID: 35936710
"To quantify the FAO activity which contributes to total cellular oxygen consumption, a “basal ratio” (the ratio between FAO-mediated OCR and total OCR) was assigned by the following equation: [(basal respiration of the vehicle group–basal respiration of the ETO group)/basal respiration of the vehicle group] x 100 %. As shown in Figure 1G , the basal ratio of control cells is around 13%, which means FAO contributes to 13 % of cellular oxygen consumption, whereas it was at least doubled in Mutp53-bearing MCF12A cells. .. to understand whether FAO is involved in Mutp53-promoted tumor properties, we suppressed cellular FAO activity with etomoxir (ETO), the pan-CPT1 inhibitor. The p53-R273H and p53-R280K MCF-12A cells were pretreated with ETO (50 and 100 μmol/L) for seven days, .."
  • Jiang N, Xie B, Xiao W, Fan M, Xu S, Duan Y, Hamsafar Y, Evans AC, Huang J, Zhou W, Lin X, Ye N, Wanggou S, Chen W, Jing D, Fragoso RC, Dugger BN, Wilson PF, Coleman MA, Xia S, Li X, Sun LQ, Monjazeb AM, Wang A, Murphy WJ, Kung HJ, Lam KS, Chen HW, Li JJ (2022) Fatty acid oxidation fuels glioblastoma radioresistance with CD47-mediated immune evasion. Nat Commun 13:1511. doi: 10.1038/s41467-022-29137-3. PMID: 35314680
"Inhibition of FAO by CPT1 inhibitor etomoxir or CRISPR-generated CPT1A−/−, CPT2−/−, ACAD9−/− cells demonstrate that FAO-derived acetyl-CoA upregulates CD47 transcription via NF-κB/RelA acetylation. Blocking FAO impairs tumor growth and reduces CD47 anti-phagocytosis. Etomoxir combined with anti-CD47 antibody synergizes radiation control of regrown tumors with boosted macrophage phagocytosis. These results demonstrate that enhanced fat acid metabolism promotes aggressive growth of GBM with CD47-mediated immune evasion. .. cells treated with CPT1A inhibitor etomoxir (ET; 200 µM, 24 h). .. To inhibit FAO, CPT1 inhibitor etomoxir was added into the testing wells 0.5 h before detection."
  • Wu Y, Li Y, Lv G, Bu W (2022) Redox dyshomeostasis strategy for tumor therapy based on nanomaterials chemistry. Chem Sci 13:2202-17. doi: 10.1039/d1sc06315d. PMID: 35310479
Ref 62: "Besides azobenzene, etomoxir, a carnitine palmitoyltransferase 1 inhibitor, can also decrease NADPH levels, thereby reducing the GSH content by inhibiting fatty acid oxidation.62"


Labels: MiParea: Respiration  Pathology: Cancer 


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