Mitochondria In A549 Cells Research Articles

Novel bibenzyl compound 8Ae induces apoptosis and inhibits glycolysis by detaching hexokinase 2 from mitochondria in A549 cells

In this paper, we investigated the anticancer effect and the mechanism of our newly synthesized bibenzyl 8Ae against human lung cancer A549 cells. Compound 8Ae could induce apoptosis by inhibiting the glycolysis in A549 cells. Hexokinase 2 (HK2), the first key enzyme in glycolysis process, was significantly down-regulated by 8Ae. Besides, compound 8Ae induced HK2 dissociated from mitochondria to cytosol, which could be induced by inhibiting the phosphorylation of Akt. In addition, 8Ae could induce mitochondrial-mediated apoptosis, and mitochondrial membrane potential (MMP) was decreased. After 8Ae treatment, the Bax/Bcl-2 ratio was increased and cytochrome c (Cyt c) was release from mitochondria to cytosol. Molecular docking indicated that 8Ae have an interaction with HK2 by extending into acitve pockets of the protein to form stable hydrogen bonds. Additionally, 8Ae had significantly improved pharmacokinetic properties through the prediction, comparison, and analysis of the ADMET properties of 8Ae and moscatilin (MST). Taken together, 8Ae might inhibit glycolysis by stimulating the shedding of HK2 from mitochondria and promoting mitochondria-regulated apoptosis to inhibit the proliferation of A549 cells. This article provides a research basis for bibenzyl compounds as new small molecule drugs for lung cancer.

Fluorinated High-Valent Sn(IV) Porphyrins Show Remarkable Photodynamic Activity in Cancer Cells.

In recent years, Sn(IV) porphyrins have proven to be excellent choice as photosensitizers for photodynamic therapy. This work reports the synthesis, characterization and photodynamic activity of four high-valent fluorinated Sn(IV) porphyrins having different numbers of F-atoms in the peripheral of meso-phenyl groups viz. (Dichloro)meso-tetrakis(4-fluorophenylporphyrinato)stannic(IV), [Sn(IV)FTPP(Cl)2] or Sn1; (Dichloro)meso-tetrakis(2,4-difluorophenylporphyrinato)stannic(IV), [Sn(IV)2,4-FTPP(Cl)2] or Sn2; (Dichloro)meso-tetrakis(2,6-difluorophenylporphyrinato)stannic(IV), [Sn(IV)2,6-FTPP(Cl)2] or Sn3 and (Dichloro)meso-tetrakis(4-trifluoromethylphenylporphyrinato)stannic(IV), [Sn(IV)CF3TPP(Cl)2] or Sn4. The solid-state structure of Sn1 has been determined by single crystal X-ray diffraction analysis. The increasing number of F-atoms attached to the meso-phenyl positions of the porphyrin framework results in increase of their lipophilicity, singlet oxygen quantum yield (ΦΔ) and photocytotoxicity in A549 (human lung adenocarcinoma cells), MCF-7 and MDA-MB-231 (human breast adenocarcinoma) cells. Sn4 predominantly localize in the mitochondria of A549 cells. The light-induced cell death by the Sn(IV) porphyrins in A549 cells occur primarily via apoptosis.

Cu(II) flavonoids as potential photochemotherapeutic agents.

Flavonoids, naturally derived polyphenolic compounds, have received significant attention due to their remarkable biochemical properties that offer substantial health benefits to humans. In this work, a series of six Cu(II) flavonoid complexes of the formulation [Cu(L1)(L2)](ClO4) where L1 is 3-hydroxy flavone (HF1, 1 and 4), 4-fluoro-3-hydroxy flavone (HF2, 2 and 5), and 2,6-difluoro-3-hydroxy flavone (HF3, 3 and 6); L2 is 1,10-phenanthroline (phen, 1-3) and 2-(anthracen-1-yl)-1H-imidazo[4,5-f][1,10]phenanthroline (aip, 4-6) were successfully synthesized, fully characterized and also evaluated for their in vitro photo-triggered cytotoxicity in cancer cells. The single-crystal X-ray diffraction structure of complex 2 shows square pyramidal geometry around the Cu(II) center. The complexes 1-6 showed quasi-reversible cyclic voltammetric responses for the Cu(II)/Cu(I) couple at ∼-0.230 V with a very large ΔEp value of ∼350-480 mV against the Ag/AgCl reference electrode in DMF-0.1 M tetrabutylammonium perchlorate (TBAP) at a scan rate of 50 mV s-1. The complexes were found to have considerable binding propensity for human serum albumin (HSA) and calf thymus DNA (ct-DNA). The complexes displayed remarkable dose-dependent photocytotoxicity in visible light (400-700 nm) in both A549 (human lung cancer) and MCF-7 (human breast cancer) cell lines while remaining significantly less toxic in darkness. They were found to be much less toxic to HPL1D (immortalized human peripheral lung epithelial) normal cells compared to A549 and MCF-7 cancer cells. Upon exposure to visible light, they generate reactive oxygen species, which are thought to be the main contributors to the death of cancer cells. In the presence of visible light, the complexes predominantly elicit an apoptotic mode of cell death. Complex 6 preferentially localizes in the mitochondria of A549 cells.

Melittin kills A549 cells by targeting mitochondria and blocking mitophagy flux

ABSTRACT 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.

Co(II) complexes of curcumin and a ferrocene-based curcuminoid: a study on photo-induced antitumor activity.

Co(II) complexes having a ferrocene-based curcuminoid (Fc-curH) ligand viz. [Co(L)2(Fc-cur)]ClO4 (1, 2), where L is phenanthroline base, namely, 1,10-phenanthroline (phen in 1) and dipyrido[3,2-a:2',3'-c]phenazine (dppz in 2) have been synthesized, characterized and evaluated as photochemotherapeutic agents in vitro. The corresponding Co(II) complexes of the naturally occurring polyphenol curcumin (curH), namely, [Co(L)2(cur)]ClO4 (3, 4), where L is phen (in 3) and dppz (in 4) were synthesized and their photo-induced anticancer activities compared with their ferrocene containing counterparts 1 and 2. The Co(II) acetylacetonato complex viz. [Co(phen)2(acac)]ClO4 (5) was structurally characterized through X-ray crystallography and used as control for cellular experiments. The Co(II) complexes having ferrocene-based curcuminoid are remarkably stable at physiological condition with higher lipophilicity compared to their curcumin analogues. The complexes display significant binding propensity to calf thymus (ct) DNA and human serum albumin (HSA). The complexes 1-4 display remarkable visible light induced cytotoxicity with the ferrocenyl analogues showing more phototoxic index (PI). The Co(II) curcumin complexes localize in the nucleus and mitochondria of A549 cells. The primary cell death mechanism is believed to be apoptotic in nature induced by light assisted generation of reactive oxygen species (ROS).Graphic abstract.

Cr (VI) induced mitophagy via the interaction of HMGA2 and PARK2

Chromium (Cr) (VI) is a proven toxin, mutagen and carcinogen. Here, the role of high mobility group A2 (HMGA2) mediating Cr (VI)-induced mitophagy was investigated. Cr (VI)-treatment caused the formation of double membrane autophagic vesicles (AVs) engulfing mitochondria and increased the expression of PINK1, PARK2, LC3 as well as HMGA2 particularly in mitochondria in A549 cells. Silencing of HMGA2 by siRNA decreased expression of PINK1, PARK2 and LC3 II especially in mitochondria, while over-expression of HMGA2 increased the expression of them in A549 cells. It indicated that HMGA2 played a critical role in Cr (VI)-induced mitophagy. Most importantly, the results of co-immunoprecipitation showed for the first time that HMGA2 could bind to PARK2 in mitochondria to activate the mitophagy pathway. In BALB/c mice, Cr (VI) increased the expression of PINK1 and PARK2 in lung tissues. Furthermore, over-expression of HMGA2 in BALB/c mice by transfection of plasmid HMGA2 significantly increased the levels of PINK1, PARK2 and LC3 II in lung tissues. Collectively, our data demonstrated that HMGA2 plays an important role in Cr (VI)-induced mitophagy through direct interaction with PARK2 in A549 cells and lung tissue.

Berberine derivatives with a long alkyl chain branched by hydroxyl group and methoxycarbonyl group at 9-position show improved anti-proliferation activity and membrane permeability in A549 cells.

Berberine (BBR) exhibits diverse bioactivities, including anticancer activity; but its poor druggability limits its applications. In this study, we designed and synthesized a series of 9-O position modified BBR derivatives aiming to improve its cell permeability and anticancer activity, utilizing a long alkyl chain branched by hydroxyl group and methoxycarbonyl group. Among these compounds, B10 showed 3.6-fold higher intracellular concentration than BBR, as well as 60-fold increased anti-proliferation activity against human lung cancer A549 cells compared with BBR. Treatment with B10 (1, 2 μM) induced apoptosis of A549 cells. Further investigations showed that B10 treatment dose-dependently affected mitochondrial functions, including oxygen consumption rate (OCR), mitochondrial membrane potential (MMP) and the morphology of mitochondria in A549 cells. Therefore, this work offers a new way for BBR structural modification through improving cell membrane permeability to affect mitochondrial functions and potential anti-tumor therapy in the future.

Enrichment of novel quinazoline derivatives with high antitumor activity in mitochondria tracked by its self-fluorescence

In novel synthetic 28 4-arylamino-6-fluoro quinazoline derivatives, compound 3a displayed the most remarkable inhibitory activities against tumor cells (IC50 values ranging between 0.71 and 2.30 μM) in vitro. Importantly, 3a obviously inhibited the proliferation and metastasis of A549 cells in a zebrafish xenograft model, while also mediated cell apoptosis and G0/G1-phase cell cycle arrest in A549 cells. Interestingly, 3a had excellent fluorescence at 439 nm (λex = 375 nm) in DMSO and at 428 nm (λex = 372 nm) in 0.5% DMSO-phosphate buffer, and the self-fluorescent characteristic revealed 3a itself accumulates in the mitochondria of A549 cells, which suggested the antitumor process of 3a may involve the mitochondrial apoptotic pathway. The hypothesis was verified by the increase of the intracellular reactive oxygen species, the decrease of mitochondrial membrane potential, the release of cytochrome C from the mitochondria into the cytoplasm, and the cascade activation of caspase-9 and caspase-3 when A549 cells were treated with 3a. This work has great implications for further development of anticancer agents that can be enriched in mitochondria and can be tracked in real-time in biological systems due to the ideal fluorescence.

A mitochondria-targeted fluorescent probe based on coumarin-pyridine derivatives for hypochlorite imaging in living cells and zebrafish.

Hypochlorite plays a critical role in various physiological processes and is involved in many diseases. Thus, real-time, rapid, and accurate monitoring of hypochlorite has important medical and physiological significance. Herein, a novel coumarin-pyridine derivative (CPD) probe was designed and synthesized, which exhibited fantastic advantages, such as a rapid response (within 10 s), naked eye recognition, large Stokes shift (185 nm), dual-channel detection, and high selectivity and sensitivity toward OCl- (detection limit 0.012 μM, S/N = 3). Furthermore, the current CPD probe was successfully used to image OCl- in the mitochondria of both A549 cells and zebrafish, which further demonstrated its suitability for practical applications in biological systems.

Abstract A67: Jarid1b protects from metformin-induced chemosensitization to cisplatin through p53 downregulation in NSCLC

Abstract Background: Instead of nontransformed cells, tumor cells use the glycolytic pathway as their main energetic source, even when oxygen is fully available. Recently, a new tumor subpopulation that is characterized by the overexpression of the histone demethylase Jarid1b has been found in melanomas and other tumors. Jarid1b-high cells use the mitochondria as the main energy source and are more resistant to chemotherapy. Also, Jarid1b-high cells have downregulation of p53. Changes in the metabolic profile of chemoresistant tumor cells could restore chemosensitization to drugs that induce high levels of oxidative stress, such as cisplatin. A possible strategy to modify the metabolism of tumor cells that uses the mitochondria as the main energy source is to use metformin. Many reports shows that patients who use metformin as treatment for type II diabetes have less incidence of cancer in general. However, many other reports show that metformin is unable to improve cancer treatment when used as an adjuvant therapy. Results from our group show that the use of metformin as a chemosensitizing agent depends on p53 status. When p53 is present, metformin can sensitize cells to cisplatin. Cells that lose p53 expression, like Jarid1b-high cells, or p53 KO cells are resistant to the combination of metformin and cisplatin. Aim: To evaluate when metformin can be used as a chemosensitizing agent to cisplatin. Methods: The human NSCLC A549 (p53 WT) and H1299 (p53 KO) and the colon carcinoma cells HCT 116 (p53 WT) and HCT 116 (CRISPR to p53) were used in this study. A549 cells were pretreated with low dose of cisplatin (PT-Pop) to induce Jarid1b overexpression and p53 downregulation. Results: Metformin sensitized A549 cells to cisplatin, leading to high levels of cell death. On the PT-Pop, metformin was not able to induce chemosensitization to cisplatin due to Jarid1b overexpression and p53 downregulation. Also, on the p53 KO cells H1299, metformin was not able to induce chemosensitization to cisplatin. Jarid1b inhibition during pretreatment with low dose of cisplatin on A549 cells restores p53 expression, allowing metformin to chemosensitize the A549 PT-Pop to cisplatin. Overexpression of p53 on the Jarid1b-high cells in the PT-Pop also restores the ability of metformin to sensitize these cells to cisplatin. Metformin sensitized the p53 WT HCT 116 cells to cisplatin, but not the HCT 116 p53 KO cells. Metformin translocates p53 to the mitochondria in A549 cells and the inhibition of the p53 compartmentalization to the mitochondria protects from metformin-induced chemosensitization to cisplatin. Metformin does not lead to ROS increase on A549 cells that were not pretreated with low dose of cisplatin. Only on PT-Pop, metformin was able to increase ROS levels. In the PT-Pop, where Jarid1b was inhibited by PBIT, metformin was able to increase even more ROS levels on A549 cells. Also, in NSCLC, metformin was able to increase lactate production only on A549 cells that was not pretreated with low dose of cisplatin. In the PT-Pop of A549 cells and in H1299 cells, metformin could not increase lactate production. Conclusion: Metformin needs the presence of p53 in NSCLC and colon cancer cells to lead to chemosensitization to cisplatin. Citation Format: Tharcisio Citrangulo Tortelli, Jr., Rodrigo Esaki Tamura, Michele Tatiana Pereira Tomitao, Silvina Odete Bustos, Renata de Freitas Saito, Sarah Milani de Moraes Leandrini, Mayara D'Aurea Jacomassi, Ulysses Ribeiro, Jr., Bryan Erik Strauss, Roger Chammas. Jarid1b protects from metformin-induced chemosensitization to cisplatin through p53 downregulation in NSCLC [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A67.

Emergence of Highly Pathogenic Avian Influenza A(H5N1) Virus PB1-F2 Variants and Their Virulence in BALB/c Mice.

Influenza A viruses (IAVs) express the PB1-F2 protein from an alternate reading frame within the PB1 gene segment. The roles of PB1-F2 are not well understood but appear to involve modulation of host cell responses. As shown in previous studies, we find that PB1-F2 proteins of mammalian IAVs frequently have premature stop codons that are expected to cause truncations of the protein, whereas avian IAVs usually express a full-length 90-amino-acid PB1-F2. However, in contrast to other avian IAVs, recent isolates of highly pathogenic H5N1 influenza viruses had a high proportion of PB1-F2 truncations (15% since 2010; 61% of isolates in 2013) due to several independent mutations that have persisted and expanded in circulating viruses. One natural H5N1 IAV containing a mutated PB1-F2 start codon (i.e., lacking ATG) was 1,000-fold more virulent for BALB/c mice than a closely related H5N1 containing intact PB1-F2. In vitro, we detected expression of an in-frame protein (C-terminal PB1-F2) from downstream ATGs in PB1-F2 plasmids lacking the well-conserved ATG start codon. Transient expression of full-length PB1-F2, truncated (24-amino-acid) PB1-F2, and PB1-F2 lacking the initiating ATG in mammalian and avian cells had no effect on cell apoptosis or interferon expression in human lung epithelial cells. Full-length and C-terminal PB1-F2 mutants colocalized with mitochondria in A549 cells. Close monitoring of alterations of PB1-F2 and their frequency in contemporary avian H5N1 viruses should continue, as such changes may be markers for mammalian virulence. Although most avian influenza viruses are harmless for humans, some (such as highly pathogenic H5N1 avian influenza viruses) are capable of infecting humans and causing severe disease with a high mortality rate. A number of risk factors potentially associated with adaptation to mammalian infection have been noted. Here we demonstrate that the protein PB1-F2 is frequently truncated in recent isolates of highly pathogenic H5N1 viruses. Truncation of PB1-F2 has been proposed to act as an adaptation to mammalian infection. We show that some forms of truncation of PB1-F2 may be associated with increased virulence in mammals. Our data support the assessment of PB1-F2 truncations for genomic surveillance of influenza viruses.

Selective metabolic effects of gold nanorods on normal and cancer cells and their application in anticancer drug screening

Cetyltrimethylammonim bromide coated gold nanorods (Au NRs) has a potential to become anti-cancer nano-drugs. Previously, the comparative responses of human alveolar adenocarcinoma epithelial cells (A549) and normal bronchial epithelial cells (16HBE) exposed to Au NRs have been characterized. It has been shown that Au NRs are translocated from the lysosome to the mitochondria in A549 cells but not in normal 16HBE cell lines. However, the molecular information during this cellular translocation remains largely undetermined. Here, we have used a metabonomic technique to comparatively analyze the time-dependent metabolic changes in Au NRs-induced A549 and 16HBE. We found that Au NRs exposure caused a disruption in the intracellular environment of both A549 and 16HBE cells, which metabolically manifested in the reduction of lactate levels in both cell lines. In addition, Au NRs induced oxidative stress in both cells lines. However, the 16HBE cells are more able to offset the oxidative stress than the A549 cells; this is because de novo GSH synthesis is triggered in Au NRs treated 16HBE cells but not in A549 cells, and the conversion of GSH to GSSG is more profound in 16HBE cells compared to A549 cells. The severe oxidative stress induces damage to mitochondria in A549 cells, leading to cell death, which is evident in the marked reduction in the levels of nucleosides and nucleotides. Furthermore, significantly elevated levels of amino acids are likely due to stress hormones being produced in Au NRs treated cells. These findings provide comprehensive molecular information on the distinctive intracellular localization, cellular uptake and translocation of Au NRs in normal and tumor cells, highlighting the value of metabonomics in assessing biological effects of nano-drugs.

WWOX-mediated apoptosis in A549 cells mainly involves the mitochondrial pathway

The human WWOX gene, known as WW domain-containing oxidoreductase, is located on 16q23.3-24.1, a chromosome region that spans the common fragile site, FRA16D. Abnormal transcripts or even loss of expression are frequently found in a number of cancer cell types, including breast, ovarian, prostate and lung cancer cells. It has therefore been proposed that the WWOX gene encodes a candidate tumor suppressor, possibly a pro-apoptotic protein. However, the mechanism behind this is not entirely clear. In the present study, we examined the pro-apoptotic action of WWOX using transient expression in A549 cells. We observed that the ectopic expression of WWOX caused apoptosis in A549 cells. We further observed procaspase-3 and procaspase-9 activation and the release of cytochrome C from the mitochondria in A549 cells transfected with pcDNA3.0-WWOX. These data indicate that WWOX induces apoptosis in A549 cells via the mitochondrial pathway.

Proapoptotic Action of p53-Tom5 in p53-Resistant A549 Human Non-small Cell Lung Cancer Cells through Direct Mitochondrial Dysfunction

Transcription-dependent apoptosis triggered by p53 hardly occurs in alternative reading frame (ARF)-null cancer cells. Loss of ARF leads to hyperactivation of murine double minute 2 (MDM2), resulting in the degradation of p53. In the present study, A549 (ARF-null) human non-small lung cancer cells were transfected with a plasmid DNA encoding human wild-type p53 and the mitochondrial transmembrane domain of Tom5 (p53-Tom5) for delivering p53 to mitochondria. As a result, p53-Tom5 exclusively localized at mitochondria in A549 cells and suppressed the proliferation of them, whereas wild-type p53 did not. In addition, mitochondrial dysfunction and release of cytochrome c were induced by p53-Tom5 in A549 cells. These data suggest that p53-Tom5 suppressed the proliferation of A549 cells through direct mitochondrial dysfunction.

Abstract 73: Mitochondrial 10-formyltetrahydrofolate dehydrogenase: A novel enzyme in folate metabolism

Abstract Cytosolic 10-formyltetrahydrofolate dehydrogenase (FDH, Aldh1L1) is an important regulator of intracellular folate pools, which displays antiproliferative effects in cancer cells. We have identified a gene at the chromosome locus 12q24.11 in the human genome, the product of which has 87% sequence similarity with cytosolic FDH. This protein has an extra amino-terminal sequence of 22 amino acid residues enriched in arginines, which is predicted to be a mitochondrial translocation signal. The mitochondrial targeting function of the leader has been confirmed in Cos7 cells: green fluorescent protein (GFP)-tagged at the amino-terminus with the leader, localizes to mitochondria. Transfection of Cos7 or A549 cell lines with a construct, in which GFP has been introduced between the leader sequence and the rest of the putative mitochondrial FDH (mtFDH), has also shown mitochondrial localization, suggesting that the identified gene encodes a mitochondrial enzyme. To evaluate the abundance of mtFDH, we have measured its mRNA levels in a wide array of human tissues by real-time PCR, and compared them to the levels of mRNA that encode cytosolic FDH. While cytosolic FDH mRNA is highest in liver, kidney and pancreas, mtFDH mRNA is most highly expressed in pancreas, heart and brain, but not in liver or kidney. In contrast to the cytosolic enzyme, which is non detectable in human cancer cell lines, the presence of mtFDH mRNA was demonstrated in A549 and PC3 cells by conventional and real-time PCR. The presence of the endogenous enzyme in mitochondria of A549 cells has been further confirmed using specific polyclonal antibody generated against purified recombinant mtFDH. We have also shown that recombinant mtFDH, similar to the cytosolic enzyme, catalyzes NADP+-dependent oxidation of the 10-formyltetrahydrofolate to tetrahydrofolate and CO2. Thus, the enzyme is a likely source of CO2 production in mitochondria and we propose that it plays an essential role in distribution of one-carbon groups between cytosolic and mitochondrial compartments of the cell. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 73.