Anti-invasive and Anti-tumor Effects of Dryopteris crassirhizoma Extract by Disturbing Actin Polymerization

Abstract

Aim: To evaluate the anti-invasive effect of ethanol extracts of rhizome of Dryopteris crassirhizoma (EEDC) in matrix invasion and formation of functional invadopodia and to determine the anti-tumor effect of EEDC in a mouse model of mandibular invasion by gingival squamous cell carcinoma (SCC).

Methods: The rhizome of D crassirhizoma was extracted in ethanol. The anti-invasive effect of EEDC was analyzed with a Matrigel-coated transwell invasion and 3D culture system. Crucial factors related to the control of cancer cell invasion by EEDC were determined using a human protease array. Molecular evidence supporting the anti-invasive effect of EEDC in oral SCC (OSCC) cells used an invadopodia-mediated extracellular matrix (ECM) degradation; an in vivo athymic mouse model was also provided.

Results: EEDC treatment (10 µg/mL) suppressed transwell migration and invasion of HSC-3 OSCC cells without cytotoxicity. Decreased levels of matrix metalloprotease (MMP)-7, kalikrein 10, cathepsin V, MMP-2, and cathepsin D were also found in EEDC-treated HSC-3 cells based on human protease array. The anti-invasive effects of EEDC involved the suppression of invadopodia-mediated ECM degradation via inhibition of globular-actin elongation. The anti-invasive effect resulting from disturbance of functional invadopodia formation by EEDC was observed even at a low concentration of 5 µg/mL. The phosphorylation of cortactin involved in functional invadopodia formation was decreased at EEDC concentrations that inhibited invadopodia formation. The anti-tumor effect of EEDC was also observed in a mouse xenograft model. Administration of EEDC resulted in inhibition of tumor growth and progression.

Conclusions: EEDC represents a potential anti-invasive and anti-tumor agent in cancer control.

Keywords: actin polymerization; extracellular matrix; invadopodia; invasion; xenograft.

Figure 1.

EEDC inhibits invasion of HSC-3 cells: (A) cytotoxicity of EEDC to NGF, IGF, or HSC-3 was determined by the MTT assay. DMSO treatment was used as control. (B) Matrigel-coated transwell invasion was performed with or without EEDC. Changes in invasion activity of HSC-3 cells were detected by membrane staining with hematoxylin. Results represent the mean ± SE of 3 independent experiments. *P <.01 versus without EEDC control. (C) Cell motility assay was performed in confluent monolayer cell after wounding with a sterile pipette tip. EEDC was added to complete media, and images were captured after 12 hours of incubation. (D) Invasion into dermal equivalent was performed in matrix of type I-A collagen. EEDC was added to complete media. Media was replaced with fresh media containing EEDC every third day. Invasion pattern was observed with paraffin-embedded matrix section by hematoxylin and eosin staining. (E) Protease modulated by EEDC in HSC-3 cells: culture medium was harvested and analyzed by Proteome Profiler Human Protease Array. Altered factors are indicated with rectangles and circled numbers. (F) Inhibitory effects of EEDC on mRNA expression levels of MMP-7, kalikrein 10, cathepsin V, MMP-2, and cathepsin D by RT-PCR. Abbreviations: DMSO, dimethyl sulfoxide; EEDC, ethanol extracts of rhizome of the Dryopteris crassirhizoma; IGF, immortalized gingival fibroblast; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; MMP, matrix metalloproteinase; NGF, normal gingival fibroblast; RT-PCR, reverse transcription polymerase chain reaction.

Figure 2.

EEDC impairs ECM degradation through inhibiting invadopodia formation of HSC-3 cells: (A) Cells on gelatin-coated slips were fixed and double stained with actin (phalloidin, red) and cortactin (green) specific antibodies to identify invadopodia. Merged images show colocalization between actin and cortactin (arrow, original magnification 400×). (B) Cells were cultured on FITC (fluorescein isothiocyanate)-conjugated gelatin-coated slips (green) for 12 hours with or without EEDC, and F-actin was visualized with phalloidin (red). Degraded matrix regions are dark (original magnification 400×). A total of 350 cells were counted to quantify the number of invadopodia foci. The dark area was measured for gelatin degradation compared with the control. Results represent the mean ± standard error of 3 independent experiments. *P <.01 versus control. (C) Cells were treated with EEDC for 1 hour and analyzed by western blotting with specific antibody for total and phosphor(Y466) cortactin. Abbreviations: ECM, extracellular matrix; EEDC, ethanol extracts of rhizome of the Dryopteris crassirhizoma.

Figure 3.

EEDC disturbs actin polymerization: In vitro actin polymerization assay was performed with or without EEDC in HSC-3 cells. G-buffer containing pyrene-actin and ATP was analyzed as positive control. DMSO was added as sample control instead of EEDC. This experiment was repeated 3 times independently, yielding similar results. Abbreviations: ATP, adenosine triphosphate; DMSO, dimethyl sulfoxide; EEDC, ethanol extracts of rhizome of the Dryopteris crassirhizoma; G-actin, globular-actin.

Figure 4.

EEDC inhibits tumor growth and progression: (A) HSC-3 cells were injected into the masseter muscle of athymic nude mice. Tumor growth was observed for 5 weeks. EEDC was orally administered 3 times a week at a dose of 5 mg/PBS/kg body weight. The head and neck region of mice were scanned using micro–computed tomography. The graph represents tumor volumes in each group. *P <.01 versus PBS group. (B) Immunohistochemical staining for total- and phosphor-cortactin were also performed (original magnification 100×). Abbreviations: EEDC, ethanol extracts of rhizome of the Dryopteris crassirhizoma; PBS, phosphate buffered saline.