Proliferation and Invasion of Melanoma Are Suppressed by a Plant Protease Inhibitor, Leading to Downregulation of Survival/Death-Related Proteins

Cell adhesion and migration are crucial for cancer progression and malignancy. Drugs available for the treatment of metastatic melanoma are expensive and unfit for certain patients. Therefore, there is still a need to identify new drugs that block tumor cell development. We investigated the effects of Enterolobium contortisiliquum trypsin inhibitor (EcTI), a protease inhibitor, on cell viability, cell migration, invasion, cell adhesion, and cell death (hallmarks of cancer) in vitro using human melanoma cells (SK-MEL-28 and CHL-1). Although EcTI did not affect non-tumor cells, it significantly inhibited the proliferation, migration, invasion, and adhesion of melanoma cells. Investigation of the underlying mechanisms revealed that EcTI triggered apoptosis and nuclear shrinkage, increased PI uptake, activated effector caspases-3/7, and produced reactive oxygen species (ROS). Furthermore, EcTI disrupted the mitochondrial membrane potential, altered calcium homeostasis, and modified proteins associated with survival and apoptosis/autophagy regulation. Acridine orange staining indicated acidic vesicular organelle formation upon EcTI treatment, demonstrating a cell death display. Electronic microscopy corroborated the apoptotic pattern by allowing the visualization of apoptotic bodies, mitochondrial cristae disorganization, and autophagic vesicles. Taken together, these results provide new insights into the anti-cancer properties of the natural EcTI protein, establishing it as a promising new therapeutic drug for use in melanoma treatment

Eleusine coracana Gaertn.

原著和名: シコクビエ科名: イネ科 = Gramineae採集地: 高知県 香美郡 土佐山田町 北組 (土佐 土佐山田町 北組)採集日: 1984/9/8採集者: 萩庭丈壽整理番号: JH039976国立科学博物館整理番号: TNS-VS-98997

Effect of steroid hormones (E₂ and P₄) on the viability of leiomyoma and myometrial adjacent cells.

<p>(A) Leiomyoma cells (5 × 10³) and myometrial adjacent cells were treated with E₂ (100 nmol/L) and P4 (100 nmol/L) for 24 h in 96-well microtiter plates. Cell viability was assessed by the MTT reduction test. (B) Phase contrast–confluent culture of leiomyoma and myometrial adjacent cells after treat with E₂ (100 nmol/L) and P₄ (100 nmol/L). The statistical significance was evaluated using one-way ANOVA followed by the Tukey's test. A p-value of ≤0.05 was considered to indicate significance (*). These experiments were performed with cultured primary cells from specimens collected from patients.</p

Detection of signaling phosphoproteins by Immunoblot analysis in leiomyoma and myometrial adjacent cells.

<p>Leiomyoma and myometrial adjacent cells (5 x 10⁵) were treated with E₂ (100 nmol/L) and P₄ (100 nmol/L) for 16 h in 6-well microtiter plates; lysate proteins were separated by 10% SDS-PAGE and electro-transferred to nitrocellulose membranes. Membranes were blocked and incubated with rabbit primary antibodies, (A and C) anti-phospho-Src (Tyr-416), anti-Src, anti-phospho-FAK (Tyr-397), anti-FAK, anti- phospho-Erk1/2 MAPK, anti-Erk1/2 MAPK, (E) anti-p130Cas Y165, (F) anti-p130Cas Y410, (H and J) anti-phospho-Akt, anti-Akt, and anti-β-actin. (B, D, G, I, and L) Graph bars represent the densitometric analyses of the immunoblotting results. The results are represented as band intensities in arbitrary units relative to the respective total phopho-proteins load and total control (β-actin) load. Antibody binding was visualized by chemiluminescence, and the relative levels of these proteins were determined by the densitometric analyses. These experiments were performed with cultured primary cells from specimens collected from patients (*p<0.01).</p

Immunophenotype of leiomyoma and myometrial cells from women with myoma uterine.

<p>Adhered and spread cells after tissue disaggregation in collagenase following with primary explants. Cells are shown under phase contrast microscopy and indirect immunofluorescence for phalloidin, fibronectin-integrin β1/CD29, vimentin, and DAPI (blue, for nuclei). (A-B) Phase contrast in the confluent culture of leiomyoma cells after three days. (A) Low density (B) high density (magnification, ×400). Mycoplasma contamination was not observed in any of the processed tissues. (C) Log-phase growth rate by cell counting–growth characteristics of leiomyoma cells, myometrial adjacent cells, and co-cultured myometrial adjacent (as feeders) with leiomyoma cells (on plastic surface). (D-F) Analysis of myometrial markers by confocal microscopy; vimentin and fibronectin integrin β1/CD29. (D) Cytoskeletal organization (Phalloidin, Alexa-594-red); (E) integrin β1/CD29 (FITC-488, green); and (F) Co-localization integrin β1(FITC-488, green) and vimentin (Alexa-594-red). Bar, 10 μm.</p

Cell viability by the MTT assay.

<p>(A and B) Leiomyoma and myometrial adjacent cells (1 × 10⁴) were maintained in serum deprivation with different concentrations of FBS (0%-10%) for 24 and 48 h in 96-well microtiter plates. (C) Cell viability of uterine leiomyoma cells and myometrial adjacent cells cultured in two concentrations of FBS (2% and 10%); cells (5 × 10³) were seed on plastic and on a collagen type I coated plates. (D and E) Phase contrast of confluent culture of leiomyoma cells and myometrial adjacent cells on collagen type I coated plates. The morphology of leiomyoma cells is not altered; these cells were less spread than the myometrial adjacent cells when seeded onto collagen type I-coated plates. Mycoplasma contamination was not observed in any of the processed tissues.</p

Characteristics of Samples Used in the Study.

<p>Characteristics of Samples Used in the Study.</p