br essential for the generation
essential for the generation of lamellipodial Calcitriol networks . 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
Time (sec) Benp
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).