br After washing with PBS twice the attached cells were
After washing with PBS twice, the attached Thapsigargin were fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. The fixed cells were permeabilized with 0.2% Triton X-100 for 10 min and blocked with 1% BSA in PBS for 1 h. The cells were incubated with anti-Cortactin (Abcam, ab33333) antibody followed by an Alexa Fluor 647-conjugated donkey anti-mouse IgG antibody (Thermo Fisher Scientific Inc., A-31571). For F-actin staining, the cells were incubated with Alexa Fluor 488 phalloidin (Cell Signaling Technology, Danvers, MA, USA,
8878). The nuclei were counter-stained with 2 μg/mL DAPI (4′, 6′-diamino-2-phenylindole) (Santa Cruz Biotechnology, sc-3598) in PBS for 2 min. All images were acquired on a laser scanning confocal mi-croscope (LSM 510 META, Carl Zeiss Vision, St Cloud, MN, USA) and analyzed with LSM Version 3.2 software (Carl Zeiss Vision).
2.14. Statistical analysis
All experiments were performed at least twice and multiple samples represent biological (not technological) replicates. All animal experi-ments were performed using randomly assigned mice without in-vestigator blinding. Statistical analyses were performed using Excel. Statistical significance was tested using two-tailed t-tests. Values of P < 0.05, P < 0.01 and P < 0.001 are denoted by *, ** and ***, respectively.
3.1. Benproperine is an effective anti-migratory and anti-metastatic drug
Metastasis is a complex process involving the physical translocation of tumor cells from a primary tumor to distant tissues [4,26]. We fo-cused on the physical dissemination step to block metastasis and sought anti-migratory compounds from a library of 719 FDA-approved drugs or clinically tested compounds using cell migration assay. Benp was identified as an active compound, which is currently used as an anti-tussive drug (Fig. 1A) [27,28]. In a variety of cell types, Benp inhibited cell viability in a dose-dependent manner (Fig. 1B). Interestingly, more than 80% of cell viability was maintained when treated with Benp up to a concentration of 20 μM (Fig. 1B). Benp significantly inhibited the migration of various types of cancer cells (Fig. 1C, D). Benp inhibited the migration and invasion of DLD-1 and AsPC-1 cells with IC50 values of 1–2 μM (Fig. 1E and F).
As shown in Fig. 2A, Benp strongly suppressed pancreatic tumor cell metastasis into the major organs, such as the spleen, liver, kidney, and colon, compared to the vehicle control (45.8% inhibition). In addition, Benp also inhibited primary pancreatic tumor growth compared to the vehicle control (47.7% inhibition) without body weights change (Fig. 2B and C). To investigate whether Benp inhibits pancreatic cancer cell metastasis to the lung, we injected luciferase-expressing AsPC-1 cells into the lateral tail vein of the mice. The Benp-treated group showed a marked decrease in the lung metastasis of AsPC-1 cells (56.1% inhibition) (Fig. 2D). Luciferase-expressing HCT-116 or DLD-1 cells were implanted into murine spleens to establish colon cancer cell metastasis to the liver. Benp significantly suppressed the liver metas-tasis of HCT-116 cells by 78.9% and DLD-1 cells by 78.2% (Fig. 2E and F). Our data suggest that Benp can be an effective anti-metastatic drug to block the primary and metastatic growth of pancreatic and colorectal cancers.
3.2. Identification of Arp2/3 complex proteins as targets of Benproperine
To identify the target proteins of Benp, we eluted the bound proteins to Benp-tagged Sepharose beads and resolved them using SDS-PAGE (Fig. 3A). We identified several proteins from gel bands (Elution 2) by mass spectrometry (Fig. 3B) and selected 6 proteins, such as ARPC2, ACTR3, CTSD, NEBL, ARFIP1, and PLIN3 that were involved in cell migration. The expression of each of the 6 candidate genes was knocked down with a specific siRNA and the effect on cell migration was ana-lyzed. Arp3 or ARPC2 knockdown significantly decreased cell migration to a greater extent than that of the other binding proteins (Fig. 3C).
During cell migration, Arp2/3 complex-mediated branching is es-sential for the generation of lamellipodial actin networks [29,30]. To elucidate whether Benp has the same activities as Arp2/3 inhibitors, we treated cells with Benp or Arp2/3 inhibitors (CK666 or CK869). In comparison with Arp2/3 inhibitors, Benp much strongly inhibited cancer cell migration without cytotoxicity (Fig. 3D). These results suggest that ARPC2 inhibitor has a stronger inhibitory effects on cancer cell migration than that of Arp2/3 inhibitors. Biochemical Pharmacology 163 (2019) 46–59
3.3. Abolished anti-migratory activity of Benproperine by mutating Phe225 in ARPC2