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  • br essential for the generation

    2020-08-18


    essential for the generation of lamellipodial Calcitriol networks [35]. As shown in Fig. 6B, the DLD-1 cells after Benp treatment changed to smaller and shrunken cells with a narrow band of F-actin at the cell periphery. In the cells stained for cortactin, lamellipodia were reduced 
    in DLD-1 cells by both Benp and CK869, showing disruption of the structure of the lamellipodial actin networks by Arp2/3 inhibition (Fig. 6B). We also observed that the morphology of various cancer cells including melanoma cancer cell A375P after Benp treatment was
    Merge
    Cortactin (Cortactin/F-actin/Nucleus)
    DMSO
    Actin
    Actin+Benp
    Actin+Arp2/3,VCA+Benp
    Time (sec) Benp
    DMSO Benp
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    Cortactin
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    Fig. 6. Inhibition of the initiation of actin polymerization and the formation of lamellipodia by Benproperine. (A) Arp2/3-dependent actin polymerization stimulated with VCA in the presence of Benp (10 μM) or CK-869 (50 μM) (n = 2). The reactions contain 0.4 mg/mL actin, either alone (actin) or with 40 nM Arp2/3 and 80 nM VCA. (B) F-actin and cortactin staining in DLD-1 cells that were treated with DMSO, Benp (10 μM), or CK869 (20 μM) for 6 h. Arrows indicate lamellipodia at cell edges. Scale bars, 20 μm. (C) F-actin and cortactin staining in various cells that were treated with DMSO or Benp (10 μM) for 6 h. Arrows indicate lamellipodia at cell edges. Scale bars, 20 μm. The data represent means ± s.d.
    Fig. 7. Inhibition of cancer cell migration in ARPC2-knockdown cells. (A) Immunoblotting for ARPC2 and β-actin from AsPC-1 cells that stably expressed scrambled shRNA or ARPC2 shRNA. (B) Representative images of migrated AsPC-1 (Vector), and shARPC2 cells and quantification of the migrated cells. Scale bars, 100 μm. (C) F-actin and cortactin staining in AsPC-1 Vector and shARPC2 cells that were treated with DMSO or Benp (10 μM) for 6 h. Arrows indicate lamellipodia at cell edges. Scale bars, 20 μm. (D) Representative bioluminescence images from AsPC-1 cells that stably expressed scrambled shRNA or ARPC2 shRNA in the whole body. (E) Bioluminescence images of the lungs from luciferase-expressing AsPC-1 cells that stably expressed scrambled shRNA or ARPC2 shRNA (n = 6 per group). The color scale indicates radiance (×101 photons/s/sr). (F) Quantification of photon flux in the lungs at the indicated points. The data represent means ± s.d.; comparisons were performed with t-tests (two groups); **P < 0.01.
    changed to smaller and shrunken cells with a narrow band of F-actin at the cell periphery (Fig. 6C). Our data show that Benp attenuates the rate of action polymerization nucleation by impairing Arp2/3 function.
    3.6. Validation of ARPC2 as an anti-metastatic target
    To provide a proof-of-concept that ARPC2 is an effective anti-me-tastatic target, we performed an in vivo metastasis assay using AsPC-1 cancer cells with stably down-regulated ARPC2 expression in response to a specific shRNA. The ARPC2 shRNA decreased the ARPC2 levels by 62.0% compared to the control shRNA-expressing cells (Fig. 7A). ARPC2 down-regulation inhibited AsPC-1 cell migration by 48.0% compared to the control shRNA-expressing cells (Fig. 7B). We next examined the overall structure of the F-actin cytoskeleton in ARPC2-knockdown AsPC-1 cells. Interestingly, shARPC2 cells displayed re-duced cortactin-rich lammelipodium at cell edges (Fig. 7C). Benp treatment caused a pronounced lamellipodial disruption in control cells, whereas no detectable change in lamellipodia was observed in ARPC2-kockdown cells (Fig. 7C).
    To investigate whether ARPC2 is involved in pancreatic cancer cell metastasis, AsPC-1-luciferase cells with shRNA for ARPC2 were injected into the lateral tail veins of BALB/c mice. Metastasis was monitored by bioluminescence imaging of luciferase activity for 4 weeks. shRNA-mediated depletion of ARPC2 in the AsPC-1 cells significantly sup-pressed lung metastasis in mice compared to cells that were treated with vector control (92.9% inhibition) (Fig. 7D–F). These results further confirm that ARPC2 inhibition by Benp or knockdown by shRNA can effectively block cancer metastasis; as a result, ARPC2 is a promising anti-cancer therapeutic target.
    3.7. Inhibition of cancer cell migration without anti-migratory effects on normal cells
    DLD-1 and AsPC-1 cancer cells, as well as immortalized MCF-10A and HFF cells, were treated with Benp for the indicated times, and cell viability was analyzed using WST-1. Treating cancer cells with Benp significantly decreased cell viability after 3 days, whereas Benp did not affect that of immortalized cells (Fig. 8A). In addition, Benp did not inhibit the migration and invasion of non-transformed human mam-mary epithelial MCF-10A cells (Fig. 8B and C). However, Arp2/3 in-hibitors suppressed the migration of both cancer and normal cells without cytotoxicity (Fig. 8B). Furthermore, Benp did not affect cor-tactin-rich lamellipodium, however, CK869 significantly disrupted la-mellipodia formation in MCF-10A cells (Fig. 8D).