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  • br A The structure of MPT G B The

    2020-07-31


    (A) The structure of MPT0G211. (B) The human triple-negative breast cancer cell line MDA-MB-231 and estrogen receptor-positive breast cancer cell line MCF-7 (1 × 106) were incubated with or without MPT0G211 or tubastatin A (10 μM) for indicated periods. (C) MDA-MB-231 and MCF-7 cells were treated with MPT0G211, tubastatin A or SAHA (0.1–10 μM) for 24 h, after which whole-cell extracts were subjected to western blotting for the indicated proteins. Acetyl-α-tubulin and acetyl-histone H3 levels in western blots were determined using ImageQuant (Molecular Dynamics, USA). The results are shown as means ± standard errors of the means from three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the control group.
    diarrhea), thrombocytopenia, and anorexia [10]. The further finding that pan-HDAC inhibitors can augment chemotherapy-induced DNA damage in cardiomyocytes [11] highlights the need to identify more selective inhibitors.
    The HDAC family comprises four classes (I, II, III, and IV) in which isoforms grouped according to their homology to yeast histone deace-tylases and localization within cells [7,12]. The isoform HDAC6 loca-lizes predominantly to the cytoplasm, where it modulates the acetyla-tion of non-histone proteins, including transcription factors (such as p53, FOXP3), heat shock protein 90 (Hsp90), tubulin, and cortactin [13,14]. In a recent study by Rey et al., HDAC6 was identified as an important regulator of cytoskeletal remodeling and cell migration and a required element for two-dimensional matrix proteolysis through ex-periments involving the small interfering RNA-mediated silencing of protein expression in the highly invasive TNBC cell line MDA-MB-231 [15]. In another mechanistic study, HDAC6 was shown to regulate AZD-5991 and/or microtubule dynamics by deacetylating cortactin, which in turn affects lamellipodia formation or focal adhesion [16]. Furthermore, mice lacking HDAC6 develop normally but exhibit greatly elevated levels of tubulin acetylation in multiple organs [17].
    Given its unique structure and function, HDAC6 has been con-sidered as a cancer therapeutic target, and a few inhibitors selective for this subtype have been reported. One such inhibitor is tubastatin-A, which showed antitumor effects when combined with COX-2 inhibitor [18]; another HDAC6 inhibitor ACY-1215 is currently being subjected to clinical trials as part of a combination treatment for multiple mye-loma [19]. However, few studies have evaluated the anti-metastatic effects and related mechanisms of selective HDAC6 inhibitors. Our previous studies have shown MPT0G211 is a selective HDAC6 inhibitor which suppresses growth of multiple myeloma cells [20] and amelio-rates tau phosphorylation and cognitive deficits in an Alzheimer's dis-ease model [21]. In this study, we examined the anti-metastatic activity of MPT0G211 against MDA-MB-231 cells and found treatment with this agent significantly inhibited TNBC cell migration both in vitro and in vivo via regulation of the Hsp90–aurora-A–cofilin–F-actin and cortactin pathways. In addition, the combination of AZD-5991 MPT0G211 with paclitaxel more significantly inhibited breast cancer cell migration relative to either agent alone. From a translational perspective, these findings have implications for improving anticancer treatment efficacy.
    2. Abbreviations
    TNBC triple negative breast cancer
    HAT histone acetyltransferase
    HDAC histone deacetylase
    HSP90 heat shock protein 90
    3. Materials and methods
    MPT0G211 and vorinostat (SAHA) (purities > 98%) were synthe-sized by Professor Jing-Ping Liou. Tubastatin A was purchased from Merck (Whitehouse Station, NJ, USA). Non-conjugated primary anti-bodies against HDAC6 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA); acetyl-histone 3, histone 3, acetyl-α-tubulin, α-tubulin, Hsp90, phospho-cofilin, cofilin, SSH1 and acetyl-lysine were obtained from Cell Signaling Technology (Danvers, MA, USA). Aurora- 
    A and β-actin were obtained from Epitomics Inc. (Burlingame, CA, USA). Cortactin and Acetyl-cortactin were purchased from Merck Millipore (Danvers, MA, USA). Acti-Stain™ 488 Fluorescent Phalloidin was purchased from Cytoskeleton Inc. (Denver, CO, USA). Horseradish peroxidase (HRP)-conjugated secondary anti-mouse or anti-rabbit IgG antibodies were obtained from Jackson ImmunoResearch Inc. (West Grove, PA, USA). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise stated.
    MDA-MB-231 and MCF-7 human breast cancer cells were obtained from Prof. Shiow-Lin Pan (Graduate Institute of Cancer Biology and Drug Discovery, Taipei Medical University, Taiwan) and cultured in RPMI 1640 medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum; 100 U/mL of penicillin and 100 μg/mL of strepto-mycin were also added. All cells were maintained at 37 °C in a humi-dified atmosphere of 5% CO2 in air. All cells at passages three to ten were used in this study.