title: Inhibition of VDAC1 prevents Ca²⁺-mediated oxidative stress and apoptosis induced by 5-aminolevulinic acid mediated sonodynamic therapy in THP-1 macrophages.
abstract: Ultrasound combined with endogenous protoporphyrin IX derived from 5-aminolevulinic acid (ALA-SDT) is known to induce apoptosis in multiple cancer cells and macrophages. Persistent retention of macrophages in the plaque has been implicated in the pathophysiology and progression of atherosclerosis. Here we investigated the effects of inhibition of voltage-dependent anion channel 1 (VDAC1) on ALA-SDT-induced THP-1 macrophages apoptosis. Cells were pre-treated with VDAC1 inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) disodium salt for 1 h or downregulated VDAC1 expression by small interfering RNA and exposed to ultrasound. Cell viability was assessed by MTT assay, and cell apoptosis along with necrosis was evaluated by Hoechst 33342/propidium iodide staining and flow cytometry. Levels of cytochrome c release was assessed by confocal microscope and Western blot. The levels of full length caspases, caspase activation, and VDAC isoforms were analyzed by Western blot. Intracellular reactive oxygen species generation, mitochondrial membrane permeability, and intracellular Ca(2+) [Ca(2+)]i levels were measured with fluorescent probes. We confirmed that the pharmacological inhibition of VDAC1 by DIDS notably prevented ALA-SDT-induced cell apoptosis in THP-1 macrophages. Additionally, DIDS significantly inhibited intracellular ROS generation and apoptotic biochemical changes such as inner mitochondrial membrane permeabilization, loss of mitochondrial membrane potential, cytochrome c release and activation of caspase-3 and caspase-9. Moreover, ALA-SDT elevated the [Ca(2+)]i levels and it was also notably reduced by DIDS. Furthermore, both of intracellular ROS generation and cell apoptosis were predominately inhibited by Ca(2+) chelating reagent BAPTA-AM. Intriguingly, ALA-treatment markedly augmented VDAC1 protein levels exclusively, and the downregulation of VDAC1 expression by specific siRNA also significantly abolished cell apoptosis. Altogether, these results suggest that VDAC1 plays a crucial role in ALA-SDT-induced THP-1 macrophages apoptosis, and targeting VDAC1 is a potential way regulating macrophages apoptosis, a finding that may be relevant to therapeutic strategies against atherosclerosis.
authors.affiliation: Department of Cardiology, The First Affiliated Hospital, Cardiovascular Institute, Harbin Medical University, 23 Youzheng Street, Harbin, 150001, Heilongjiang, China.
authors.forename: Haibo
authors.initials: H
authors.lastname: Chen
authors.forename: Weiwei
authors.initials: W
authors.lastname: Gao
authors.forename: Yang
authors.initials: Y
authors.lastname: Yang
authors.forename: Shuyuan
authors.initials: S
authors.lastname: Guo
authors.forename: Huan
authors.initials: H
authors.lastname: Wang
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.forename: Shuisheng
authors.initials: S
authors.lastname: Zhang
authors.forename: Qi
authors.initials: Q
authors.lastname: Zhou
authors.forename: Haobo
authors.initials: H
authors.lastname: Xu
authors.forename: Jianting
authors.initials: J
authors.lastname: Yao
authors.forename: Zhen
authors.initials: Z
authors.lastname: Tian
authors.forename: Bicheng
authors.initials: B
authors.lastname: Li
authors.forename: Wenwu
authors.initials: W
authors.lastname: Cao
authors.forename: Zhiguo
authors.initials: Z
authors.lastname: Zhang
authors.forename: Ye
authors.initials: Y
authors.lastname: Tian
mesh_terms: D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000622:Aminolevulinic Acid / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D065096:Calcium Chelating Agents / Q000494:pharmacology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D008264:Macrophages / Q000187:drug effects* / Q000378:metabolism / Q000528:radiation effects*; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D008928:Mitochondria / Q000378:metabolism; D051336:Mitochondrial Membranes / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects*; D011524:Protoporphyrins / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism; D013016:Sound; D050995:Voltage-Dependent Anion Channel 1 / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D002121:Calcium Channel Blockers; D065096:Calcium Chelating Agents; D011524:Protoporphyrins; D017382:Reactive Oxygen Species; C494762:VDAC1 protein, human; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D000622:Aminolevulinic Acid; D050995:Voltage-Dependent Anion Channel 1; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D002118:Calcium