Decrease In Mitochondrial Potential Research Articles

Synthesis, modification and cytotoxic properties of new 7-amino-5-arylazolo[1,5-a]pyrimidine-6-carbonitriles

Azolo[1,5-a]pyrimidine is one of the most promising scaffold in the development of potent anticancer agents. However, the structural motifs in such development are often limited to 5,7-substituted azolo[1,5-a]pyrimidines due to the well-known synthesis route and existing drugs (e.g., Dinaciclib) on their basis. To expand the understanding of rare substituent combinations in this research field, a new series of 7-amino-5-aryl-6-cyanoazolo[1,5-a]pyrimidines with various azole moieties were synthesized and studied on A172, Rd, Hos and HEK293 cell lines. The obtained derivatives were further modified by acylation to introduce an additional pharmacophore moiety. During the study of anticancer activity, compounds with a pronounced cytotoxic effect were found. The analysis of activity against the embryonal rhabdomyosarcoma (Rd) cell line suggested a mechanism of cytotoxic action causing a significant decrease in mitochondrial potential in neoplastic cells.

Nano-fenretinide demonstrates remarkable activity in acute promyeloid leukemia cells

Acute promyelocytic leukemia (APL) is characterized by rearrangements of the retinoic acid receptor, RARα, which makes all-trans retinoic acid (ATRA) highly effective in the treatment of this disease, inducing promyelocytes differentiation. Current therapy, based on ATRA in combination with arsenic trioxide, with or without chemotherapy, provides high rates of event-free survival and overall survival. However, a decline in the drug activity, due to increased ATRA metabolism and RARα mutations, is often observed over long-term treatments. Furthermore, dedifferentiation can occur providing relapse of the disease. In this study we evaluated fenretinide, a semisynthetic ATRA derivative, encapsulated in nanomicelles (nano-fenretinide) as an alternative treatment to ATRA in APL. Nano-fenretinide was prepared by fenretinide encapsulation in a self-assembling phospholipid mixture. Physico-chemical characterization was carried out by dinamic light scattering and spectrophotometry. The biological activity was evaluated by MTT assay, flow cytometry and confocal laser-scanning fluorescence microscopy. Nano-fenretinide induced apoptosis in acute promyelocytic leukemia cells (HL60) by an early increase of reactive oxygen species and a mitochondrial potential decrease. The fenretinide concentration that induced 90–100% decrease in cell viability was about 2.0 µM at 24 h, a concentration easily achievable in vivo when nano-fenretinide is administered by oral or intravenous route, as demonstrated in previous studies. Nano-fenretinide was effective, albeit at slightly higher concentrations, also in doxorubicin-resistant HL60 cells, while a comparison with TK6 lymphoblasts indicated a lack of toxicity on normal cells. The results indicate that nano-fenretinide can be considered an alternative therapy to ATRA in acute promyelocytic leukemia when decreased efficacy, resistance or recurrence of disease emerge after protracted treatments with ATRA.

Effect of brassinosteroid esters with salicylic, succinic, and indolylacetic acids on the tumor cell growth

The aim of this work is to study the effect of new derivatives of brassinosteroids – their esters with salicylic, succinic, and indolylacetic acids on the growth of tumor cells, on their distribution by cell cycle phases and determination of the type of cell death. It was shown that the activity of the new synthesized compounds differed significantly depending on the substitution nature in the steroid nucleus, amounting to <10 µM in the case of epibrassinolide and epicastasterone salicylates, simultaneously causing the cycle arrest in the G0/G1 phase, a mitochondrial potential decrease, and apoptosis of tumor cells. The results obtained indicate the high activity of salicylates, which effectively suppress the growth of the all studied types of tumors, and the prospects of finding new antitumor agents in this series of compounds.

Particulate Matter Induces Oxidative Stress and Ferroptosis in Human Lung Epithelial Cells.

Numerous toxicological studies have highlighted the association between urban particulate matter (PM) and increased respiratory infections and lung diseases. The adverse impact on the lungs is directly linked to the complex composition of particulate matter, initiating reactive oxygen species (ROS) production and consequent lipid peroxidation. Excessive ROS, particularly within mitochondria, can destroy subcellular organelles through various pathways. In this study, we confirmed the induction of ferroptosis, an iron-dependent cell death, upon exposure to an urban PM using RT-qPCR and signaling pathway analysis. We used KRISS CRM 109-02-004, the certified reference material for the analysis of particulate matter, produced by the Korea Research Institute of Standards and Science (KRISS). To validate that ferroptosis causes lung endothelial toxicity, we assessed intracellular mitochondrial potential, ROS overproduction, lipid peroxidation, and specific ferroptosis biomarkers. Following exposure to the urban PM, a significant increase in ROS generation and a decrease in mitochondrial potential were observed. Furthermore, it induced hallmarks of ferroptosis, including the accumulation of lipid peroxidation, the loss of antioxidant defenses, and cellular iron accumulation. In addition, the occurrence of oxidative stress as a key feature of ferroptosis was confirmed by increased expression levels of specific oxidative stress markers such as NQO1, CYP1B1, FTH1, SOD2, and NRF. Finally, a significant increase in key ferroptosis markers was observed, including xCT/SLC7A11, NQO1, TRIM16, HMOX-1, FTL, FTH1, CYP1B1, CHAC1, and GPX4. This provides evidence that elevated ROS levels induce oxidative stress, which ultimately triggers ferroptosis. In conclusion, our results show that the urban PM, KRISS CRM, induces cellular and mitochondrial ROS production, leading to oxidative stress and subsequent ferroptosis. These results suggest that it may induce ferroptosis through ROS generation and may offer potential strategies for the treatment of lung diseases.

Mitochondria as a key target of molecular hydrogen

The aim of the work was to systematize the data on the biologically significant effects of molecular hydrogen to uncover the mechanisms of its effect on the human body. The paper analyzes the literature on the effect of molecular hydrogen administered in the form of inhalation and hydrogenenriched water on the human body, on laboratory mammals (rats, mice), and on model cell systems in vitro. As a result, a mechanism has been proposed according to which, in addition to the already known effect of hydrogen in neutralizing highly reactive oxygen species, there is at least one other group of molecules that are the target of molecular hydrogen in the body. These are the porphyrins, which are part of the hemoproteins, in particularly the cytochromes of the mitochondrial respiratory chain. In the presence of high concentrations of carbon dioxide, which is formed in the tricarboxylic acid cycle in the mitochondrial matrix, hydrogen damages some of the hemes as a result of covalent binding of the CO group to them. At low doses of hydrogen, this causes a moderate decrease in mitochondrial potential and stimulates the adaptive response of the body, including activation of the transcription factor Nrf2, expression of the heme oxygenase and antioxidant defense enzymes, mitophagy, and renewal of the mitochondrial population in the cell.Conclusion. Molecular hydrogen is an adaptogen that causes mitochondrial hormesis – the renewal and strengthening of the body’s bioenergetic and antioxidant systems.

Effect of 5-aminolevulinic Acid on Mitochondrial Activity.

Mitochondrial dysfunction is considered an important mechanism in the pathogenesis of various diseases. Therefore, mitochondria are currently being considered as subjects for targeted therapies, particularly, phototherapy using 5-aminolevulinic acid. This study aimed to investigate the activity of mitochondria in cells with different mutation loads. The study was conducted using 11 cybrid lines obtained from the THP-1 cell line (a human monocytic leukemia cell line) and platelets of patients with different mitochondrial mutations. Our results illustrate that 5-aminolevulinic acid was metabolized equally in all cell lines, however, there was a significant decrease in mitochondrial potential, which differed among lines. The results of this study can be used to develop a personalized therapeutic approach based on different mitochondrial activities.

Isoliquiritigenin Protects Neuronal Cells against Glutamate Excitotoxicity.

It is considered that glutamate excitotoxicity may be a major factor in the pathological death of neurons and mediate the development of neurodegenerative diseases in humans. Here, we show that isoliquiritigenin (ILG) at a concentration of 0.5-5 µM protects primary neuroglial cell culture from glutamate-induced death (glutamate 100 µM). ILG (1 µM) prevented a sharp increase in [Ca2+]i and a decrease in mitochondrial potential (ΔΨm). With the background action of ILG (1-5 µM), there was an increase in oxygen consumption rate (OCR) in response to glutamate, as well as in reserve respiration. The neuroprotective effect of ILG (5 µM) was accompanied by an increase in non-mitochondrial respiration. The results show that ILG can protect cortical neurons from death by preventing the development of calcium deregulation and limiting mitochondrial dysfunction caused by a high dose of glutamate. We hypothesize that ILG will be useful in drug development for the prevention or treatment of neurodegenerative diseases accompanied by glutamate excitotoxicity.

Impact of post-Chernobyl radiation on flow cytometry parameters of human sperm.

The aim of this study was to analyse the long-term radiation effects on human sperm. In total, 104 samples of male donors from 2 regions of Ukraine were tested. Group 1 consisted of 32 donors from the Ivano-Frankivsk region, group 2 included 72 volunteers from the Zhytomyr region. The average age of donors in both groups was 35 ± 6 years (range 24-49). To assess the level of apoptosis, membrane mitochondrial potential, concentration of reactive oxygen species, and ploidy of sperm, flow cytometry was performed. The individual equivalent dose of group 1 was < 0.4 mSv and of group 2 ≥ 0.4 mSv. Live spermatozoa with signs of apoptosis were significantly higher (p < 0.05) in group 2 in comparison to group 1 (15.6% and 11.2%, respectively). Spermatozoa without violating integrity were 73.2% in group 1 and approximately 16% higher than the indices of group 2. The percentage of dead necrotic spermatozoa was twice as high in men with a predicted equivalent dose of ≥ 0.4 mSv than in comparison group. A higher percentage of spermatozoa with low mitochondrial membrane potential, di- and tetraploid was found in group 2. An equivalent individual dose of ≥ 0.4 mSv can cause a decrease in mitochondrial potential, an increase in the production of spermatozoa with pathological ploidy, as well as to provoke increasing apoptosis in cells.

Pyrroloquinoline quinone (PQQ) protects mitochondrial function of HEI-OC1 cells under premature senescence

The aim of this study was to investigate the effects of pyrroloquinoline quinone (PQQ), an oxidoreductase cofactor, on the H2O2-induced premature senescence model in HEI-OC1 auditory cells and to elucidate its mechanism of action in vitro. Cells were treated with PQQ for 1 day before H2O2 (100 μM) exposure. Mitochondrial respiratory capacity was damaged in this premature senescence model but was restored in cells pretreated with PQQ (0.1 nM or 1.0 nM). A decrease in mitochondrial potential, the promotion of mitochondrial fusion and the accelerated movement of mitochondria were all observed in PQQ-pretreated cells. The protein expression of sirtuin 1 (SIRT1) and peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) were significantly decreased under H2O2 exposure while they were increased with PQQ pretreatment, and PGC-1α acetylation was significantly decreased. In conclusion, PQQ has a protective effect on the premature senescence model of HEI-OC1 auditory cells and is associated with the SIRT1/PGC-1α signaling pathway, mitochondrial structure, and mitochondrial respiratory capacity.

C-Myc Protein Level Affected by Unsymmetrical Bisacridines Influences Apoptosis and Senescence Induced in HCT116 Colorectal and H460 Lung Cancer Cells.

Unsymmetrical bisacridines (UAs) are highly active antitumor compounds. They contain in their structure the drugs previously synthesized in our Department: C-1311 and C-1748. UAs exhibit different properties than their monomer components. They do not intercalate to dsDNA but stabilize the G-quadruplex structures, particularly those of the MYC and KRAS genes. Since MYC and KRAS are often mutated and constitutively expressed in cancer cells, they can be used as therapeutic targets. Herein, we investigate whether UAs can affect the expression and protein level of c-Myc and K-Ras in HCT116 and H460 cancer cells, and if so, what are the consequences for the UAs-induced cellular response. UAs did not affect K-Ras, but they strongly influenced the expression and translation of the c-Myc protein, and in H460 cells, they caused its full inhibition. UAs treatment resulted in apoptosis, as confirmed by the morphological changes, the presence of sub-G1 population and active caspase-3, cleaved PARP, annexin-V/PI staining and a decrease in mitochondrial potential. Importantly, apoptosis was induced earlier and to a greater extent in H460 compared to HCT116 cells. Moreover, accelerated senescence occurred only in H460 cells. In conclusion, the strong inhibition of c-Myc by UAs in H460 cells may participate in the final cellular response (apoptosis, senescence).

Microbial BMAA elicits mitochondrial dysfunction, innate immunity activation, and Alzheimer\u2019s disease features in cortical neurons

BackgroundAfter decades of research recognizing it as a complex multifactorial disorder, sporadic Alzheimer’s disease (sAD) still has no known etiology. Adding to the myriad of different pathways involved, bacterial neurotoxins are assuming greater importance in the etiology and/or progression of sAD. β-N-Methylamino-l-alanine (BMAA), a neurotoxin produced by some microorganisms namely cyanobacteria, was previously detected in the brains of AD patients. Indeed, the consumption of BMAA-enriched foods has been proposed to induce amyotrophic lateral sclerosis-parkinsonism-dementia complex (ALS-PDC), which implicated this microbial metabolite in neurodegeneration mechanisms.MethodsFreshly isolated mitochondria from C57BL/6 mice were treated with BMAA and O2 consumption rates were determined. O2 consumption and glycolysis rates were also measured in mouse primary cortical neuronal cultures. Further, mitochondrial membrane potential and ROS production were evaluated by fluorimetry and the integrity of mitochondrial network was examined by immunofluorescence. Finally, the ability of BMAA to activate neuronal innate immunity was quantified by addressing TLRs (Toll-like receptors) expression, p65 NF-κB translocation into the nucleus, increased expression of NLRP3 (Nod-like receptor 3), and pro-IL-1β. Caspase-1 activity was evaluated using a colorimetric substrate and mature IL-1β levels were also determined by ELISA.ResultsTreatment with BMAA reduced O2 consumption rates in both isolated mitochondria and in primary cortical cultures, with additional reduced glycolytic rates, decrease mitochondrial potential and increased ROS production. The mitochondrial network was found to be fragmented, which resulted in cardiolipin exposure that stimulated inflammasome NLRP3, reinforced by decreased mitochondrial turnover, as indicated by increased p62 levels. BMAA treatment also activated neuronal extracellular TLR4 and intracellular TLR3, inducing p65 NF-κB translocation into the nucleus and activating the transcription of NLRP3 and pro-IL-1β. Increased caspase-1 activity resulted in elevated levels of mature IL-1β. These alterations in mitochondrial metabolism and inflammation increased Tau phosphorylation and Aβ peptides production, two hallmarks of AD.ConclusionsHere we propose a unifying mechanism for AD neurodegeneration in which a microbial toxin can induce mitochondrial dysfunction and activate neuronal innate immunity, which ultimately results in Tau and Aβ pathology. Our data show that neurons, alone, can mount inflammatory responses, a role previously attributed exclusively to glial cells.

Toxicity Mechanism of Low Doses of NaGdF₄:Yb3+,Er3+ Upconverting Nanoparticles in Activated Macrophage Cell Lines.

Gadolinium-doped nanoparticles (NPs) are regarded as promising luminescent probes. In this report, we studied details of toxicity mechanism of low doses of NaGdF4-based fluorescent nanoparticles in activated RAW264.7, J774A.1 macrophages. These cell lines were specifically sensitive to the treatment with nanoparticles. Using nanoparticles of three different sizes, but with a uniform zeta potential (about −11 mV), we observed rapid uptake of NPs by the cells, resulting in the increased lysosomal compartment and subsequent superoxide induction along with a decrease in mitochondrial potential, indicating the impairment of mitochondrial homeostasis. At the molecular level, this led to upregulation of proapoptotic Bax and downregulation of anti-apoptotic Bcl-2, which triggered the apoptosis with phosphatidylserine externalization, caspase-3 activation and DNA fragmentation. We provide a time frame of the toxicity process by presenting data from different time points. These effects were present regardless of the size of nanoparticles. Moreover, despite the stability of NaGdF4 nanoparticles at low pH, we identified cell acidification as an essential prerequisite of cytotoxic reaction using acidification inhibitors (NH4Cl or Bafilomycin A1). Therefore, approaching the evaluation of the biocompatibility of such materials, one should keep in mind that toxicity could be revealed only in specific cells. On the other hand, designing gadolinium-doped NPs with increased resistance to harsh conditions of activated macrophage phagolysosomes should prevent NP decomposition, concurrent gadolinium release, and thus the elimination of its toxicity.

Canthin-6-one induces cell death, cell cycle arrest and differentiation in human myeloid leukemia cells

BackgroundCanthin-6-one is a natural product isolated from various plant genera and from fungi with potential antitumor activity. In the present study, we evaluate the antitumor effects of canthin-6-one in human myeloid leukemia lineages. MethodsKasumi-1 lineage was used as a model for acute myeloid leukemia. Cells were treated with canthin-6-one and cell death, cell cycle and differentiation were evaluated in both total cells (Lin+) and leukemia stem cell population (CD34+CD38−Lin−/low). ResultsAmong the human lineages tested, Kasumi-1 was the most sensitive to canthin-6-one. Canthin-6-one induced cell death with apoptotic (caspase activation, decrease of mitochondrial potential) and necrotic (lysosomal permeabilization, double labeling of annexin V/propidium iodide) characteristics. Moreover, canthin-6-one induced cell cycle arrest at G0/G1 (7μM) and G2 (45μM) evidenced by DNA content, BrdU incorporation and cyclin B1/histone 3 quantification. Canthin-6-one also promoted differentiation of Kasumi-1, evidenced by an increase in the expression of myeloid markers (CD11b and CD15) and the transcription factor PU.1. Furthermore, a reduction of the leukemic stem cell population and clonogenic capability of stem cells were observed. ConclusionsThese results show that canthin-6-one can affect Kasumi-1 cells by promoting cell death, cell cycle arrest and cell differentiation depending on concentration used. General significanceCanthin-6-one presents an interesting cytotoxic activity against leukemic cells and represents a promising scaffold for the development of molecules for anti-leukemic applications, especially by its anti-leukemic stem cell activity.

Acquired resistance to venetoclax (ABT-199) in t(14;18) positive lymphoma cells.

The chromosomal translocation t(14;18) in follicular lymphoma (FL) is a primary oncogenic event resulting in BCL-2 over-expression. This study investigates activity of the BH3 mimetic venetoclax (ABT-199), which targets BCL-2, and mechanisms of acquired resistance in FL.The sensitivity of FL cells to venetoclax treatment correlated with BCL-2/BIM ratio. Cells with similar expression of anti-apoptotic proteins, but with higher levels of BIM were more sensitive to the treatment. Venetoclax induced dissociation of BCL-2/BIM complex and a decrease in mitochondrial potential. Interestingly the population of cells that survived venetoclax treatment showed increased p-ERK1/2 and p-BIM (S69), as well as a decrease in total BIM levels. Venetoclax resistant cells initially showed elevated levels of p-AKT and p-Foxo1/3a, a dissociation of BIM/BCL-2/BECLIN1 complex, and a decrease in SQSTM1/p62 level (indicating increased autophagy) together with a slight decline in BIM expression. After stable resistant cell lines were established, a significant reduction of BCL-2 levels and almost total absence of BIM was observed.The acquisition of these resistance phenotypes could be prevented via selective ERK/AKT inhibition or anti-CD20 antibody treatment, thus highlighting possible combination therapies for FL patients.

Mitochondrial bioenergetics and posthepatectomy liver dysfunction.

Liver regeneration requires an enormous energy supply. Experimental evidence suggests that mitochondrial function is of paramount importance for liver regeneration. However, this has not been investigated in the clinical setting. We aimed to: (i) evaluate changes in mitochondrial function during hepatectomy, especially after hepatic pedicle clamping; and (ii) correlate these changes with postoperative hepatocellular function and clinical outcome. Prospective study of thirty patients undergoing hepatectomy. Measurement of mitochondrial membrane potential, respiration and adenosine triphosphate content in intra-operative liver biopsies performed in nonresected parenchyma. Correlation of findings with duration of hepatic pedicle clamping, postoperative markers of hepatocellular necrosis and function (aminotransferases, arterial lactate, international normalized ratio, bilirubin), and morbidity. Longer hepatic pedicle clamping was associated with worse mitochondrial depolarization (r = -0·519; P = 0·011) and longer lag phase (r = 0·568; P = 0·006). Higher postoperative peak aminotransferases, international normalized ratio and bilirubin correlated with worse mitochondrial function (P < 0·05). After major hepatectomy, mitochondrial respiration correlated with postoperative arterial lactate clearance (r = 0·756; P = 0·049). Mitochondrial bioenergetic parameters were significantly decreased in patients with liver-specific morbidity and postoperative liver failure (P < 0·05). On multivariate analysis, decrease in mitochondrial potential was an independent risk factor for liver-specific morbidity (OR = 13·7; P = 0·043). Worse lag phase was highly predictive of posthepatectomy liver failure (area under the curve: 0·933; P = 0·008). There is a relationship between mitochondrial function, duration of hepatic pedicle clamping and clinical outcome after hepatectomy. Mitochondrial bioenergetics can potentially translate into clinical practice, assisting in earlier diagnosis of postoperative liver dysfunction, and as a target for future pharmacological therapies.

Mitochondrial dependent oxidative stress in cell culture induced by laser radiation at 1265 nm.

Photodynamic therapy is the main technique applied for surface carcinoma treatment. This technique employs singlet oxygen generated via a laser excited photosensitizer as a main damaging agent. However, prolonged sensitivity to intensive light, relatively low tissue penetration by activating light the cost of photosensitizer (PS) administration can limit photodynamic therapy applications. Early was reported singlet oxygen generation without photosensitizer induced by a laser irradiation at the wavelength of 1250-1270 nm. Here, we study the dynamics of oxidative stress, DNA damage, changes of mitochondrial potential, and mitochondrial mass induced by a laser at 1265 nm have been studied in HCT-116 and CHO-K cells. Laser irradiation of HCT-116 and CHO-K cells has induced a dose-dependent cell death via increasing intracellular reactive oxygen species (ROS) concentration, increase of DNA damage, decrease of mitochondrial potential, and reduced glutathione. It has been shown that, along with singlet oxygen generation, the increase of the intracellular ROS concentration induced by mitochondrial damage contributes to the damaging effect of the laser irradiation at 1265 nm.

Mitochondrial morphology and dynamics in Triticum aestivum roots in response to rotenone and antimycin A.

Mitochondria are dynamic organelles, capable of fusion and fission as a part of cellular responses to various signals, such as the shifts in the redox status of a cell. The mitochondrial electron transport chain (ETC.) is involved in the generation of reactive oxygen species (ROS), with complexes I and III contributing the most to this process. Disruptions of ETC. can lead to increased ROS generation. Here, we demonstrate the appearance of giant mitochondria in wheat roots in response to simultaneous application of the respiratory inhibitors rotenone (complex I of mitochondrial ETC.) and antimycin A (complex III of mitochondrial ETC.). The existence of such megamitochondria was temporary, and following longer treatment with inhibitors mitochondria resumed their conventional size and oval shape. Changes in mitochondrial morphology were accompanied with a decrease in mitochondrial potential and an unexpected increase in oxygen consumption. Changes in mitochondrial morphology and activity may result from the fusion and fission of mitochondria induced by the disruption of mitochondrial ETC. Results from experiments with the inhibitor of mitochondrial fission Mdivi-1 suggest that the retarded fission may facilitate plant mitochondria to appear in a fused shape. The processes of mitochondrial fusion and fission are involved in the regulation of the efficacy of the functions of the respiratory chain complexes and ROS metabolism during stresses. The changes in morphology of mitochondria, along with the changes in their functional activity, can be a part of the strategy of the plant adaptation to stresses.

Targeting Lon Protease Activity Promotes Growth Inhibition and Apoptosis in Acute Myeloid Leukemia Cell Lines

INTRODUCTIONAcute myeloid leukemia (AML) is a progressive malignant disorder of the myeloid lineage of hematopoietic cells, where overall three-year survival is below 20% for patients above 65 years (1). Therapy resistance or relapsed are the main causes of mortality among these patients. The functional changes that affect mitochondria may contribute to the development of cancer (2). The mitochondrial Lon, ubiquitously expressed in human cells and tissues, seems to be involved in protein quality control and stress response pathways in mitochondria. Several stress conditions induce an upregulation of Lon that may promote tumorigenesis by inhibiting apoptosis and promoting cell proliferation, transformation and angiogenesis (3). The role of Lon in AML has not been described yet. Our study aims to investigate if the activity of the protease may be linked to AML leukemogenesis and drug resistance. In particular we tested Lon protease activity inhibitors to clarify a new pathogenetic mechanism in controlling drug responsiveness in this haematological disorder.METHODSAML cell lines Kasumi-1 (acute myeloblastic leukemia), MOLM-13 (acute monocytic leukemia) and HL-60 (acute promyelocytic leukemia) were used to perform the experiments. Lon expression was measured by RT-PCR and immunoblotting. Cell-growth was quantified by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Reactive oxygen species (ROS) were evaluated by Nitro Blue Tetrazolium (NBT) assay. For cell cycle analysis, cells were fixed with ethanol, resuspended in phosphate-buffered saline (PBS) containing 0.1mg/mL RNase A, NP40 0,15% and 100mg/mL of propidium iodide (PI) and analysed by using flow cytometry and Modfit software. The early and late apoptosis was assessed with Annexin V-FITC Apoptosis Detection kit. To measure the mitochondrial potential, JC-1 Mitochondrial Membrane Potential assay kit was used.RESULTSLon protease was expressed at high levels in AML cell lines compared with resting or activated peripheral blood mononuclear cells and monocytes, but less than other cancer cell lines such as K562 (human erythromyeloblastoid leukemia), HDLM-2 (Hodgkin lymphoma, nuclear sclerosing), SHSY5Y (neuroblastoma), MCF7 (breast carcinoma). Based on previous works (4), which show that 2-Cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) and its derivates inhibit the Lon protease activity, we evaluated the effect of two inhibitors on cell growth and the induction of apoptosis. The leukemic cell lines were treated with CDDO and CDDO-Me (CDDO methyl-ester) at different concentrations for 24, 48 and 72h. Both compounds inhibited the cell proliferation in a dose- and time-dependent manner and induced an increase of ROS. We observed a time-dependent increase in the percentage of Annexin V+ cells after treatment with CDDO (1,25µM) and CDDO-Me (0,25µM). The induction of apoptosis after CDDO exposure was important only at 72h, while we observed a percentage of apoptosis over 60% starting from 24h of CDDO-Me treatment. Interestingly, CDDO induced a time-dependent arrest in G2/M phase in MOLM-13 cells and in G0/G1 phase in Kasumi-1 and HL-60 cells. Instead the CDDO-Me induced an alteration of all cell cycle phases, probably for its cytotoxicity. Moreover CDDO-Me caused a higher decrease in mitochondrial potential (Dψm) than CDDO in all cell lines.CONCLUSIONIn conclusion, these findings show that Lon protease inhibitors promote growth inhibition and apoptosis and support the concept that mitochondrial Lon may represent a novel anticancer drug target.REFERENCES1. Burnett A, Wetzler M, Lowenberg B. Therapeutic advances in acute myeloid leukemia. J Clin Oncol. 2011, 29:487-494.2. Gogvadze V, Orrenius S, Zhivotovsky B. Mitochondria in cancer cells: what is so special about them? Trends Cell Biol. 2008, 18(4):165-73.3. Ngo JK, Pomatto LC, Davies KJ. Upregulation of the mitochondrial Lon Protease allows adaptation to acute oxidative stress but dysregulation is associated with chronic stress, disease, and aging. Redox Biol. 2013, 1(1):258-64.4. Bernstein SH, Venkatesh S, Li M, Lee J, Lu B, Hilchey SP, Morse KM, Metcalfe HM, Skalska J, Andreeff M, Brookes PS, Suzuki CK. The mitochondrial ATP-dependent Lon protease: a novel target in lymphoma death mediated by the synthetic triterpenoid CDDO and its derivatives. Blood. 2012, 119(14):3321-9. DisclosuresMartinelli:Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy; ARIAD: Consultancy.

Effect of highly mineralized natural water on redox processes in HaCaT keratinocytes.

We studied ROS production by HaCaT keratinocytes, the state of transmembrane mitochondrial potential, and activation of transcription factor Nrf2 in response to brine exposure. It was demonstrated that this exposure induces rapid but moderate decrease in mitochondrial potential, stimulates ROS production, and leads to activation of transcription factor Nrf2.

Abstract B161: SLC1A5 inactivation induces apoptosis-mediated cells death in non-small cell lung cancer.

Abstract Introduction: We recently reported that solute carrier family A1 member 5 (SLC1A5) controls glutamine (GLN) uptake and modulates cell growth, oxidative stress and mTOR signalling pathway. SLC1A5 is a transmembrane, high affinity glutamine transporter that is overexpressed and located at the plasma membrane in non-small cell lung cancer (NSCLC). GLN deprivation has been shown to induce cell death in several types of cancers, however the exact mechanism by which SLC1A5 targeting affects NSCLC survival remains unknown. We hypothesize that inhibition of SLC1A5 deprives cells from GLN which induces autophagy which eventually leads to cell starvation and ultimately apoptotic cell death. Methods: To test our hypothesis we targeted SLC1A5 by siRNA or by its specific inhibitor, GPNA, in a panel of 6 NSCLC lung cancer cell lines (express high level of SLC1A5). For comparison 2 human bronchial epithelial cell lines (HBEs) (express low level of SLC1A5) were used. The effects of targeting SLC1A5 on growth, GLN uptake, ATP level, autophagy, and cell death were examined. Markers of autophagy and cell death were analysed using western blot, nuclear staining and cell cycle analysis. Results: SLC1A5 inactivation by GPNA or by siRNA resulted in a significant decrease in cellular GLN uptake in NSCLC cell lines, while HBE cell lines were unaffected. Decrease in GLN uptake was accompanied by an increase in autophagy as evident by decrease in cell size, increase of LC3-II and decrease if LC3-I markers of autophagy. In addition, we observed a significant drop in ATP levels by 2 folds with increasing doses of GPNA. Markers of internal apoptotic pathway that include decrease of mitochondrial potential and cleaved caspases-3 and 9 were detected in response to increasing doses of GPNA. No change was observed in external apoptotic markers, caspases 8 or 1. In addition a 2-10 fold increase (based on the cell line) of sub-G1 was observed in NSCLC cell lines but not in HBE cell lines. Conclusion: Our results show that targeting SLC1A5 in NSCLC cells induces apoptotic cell death. Cells starve due to GLN deprivation and undergo autophagy which leads to a decrease in ATP level resulting in apoptotic cell death. These findings suggest that targeting SLC1A5 may have therapeutic implications in NSCLC. Grant funding: Lung Cancer Research Foundation Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B161. Citation Format: Mohamed Hassanein, Jun Qian, Megan Hoeksema, Marie Jacobovitz, Fredrick T. Harris, Bradford Harris, Pierre P. Massion. SLC1A5 inactivation induces apoptosis-mediated cells death in non-small cell lung cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B161.