Effects of Tepary bean (Phaseolus acutifolius) protease inhibitor and semipure lectin fractions on cancer cells

Abstract

Some natural and synthetic protease inhibitors (PI), such as the Bowman-Birk PI from soybean, have anticancer effects. We previously purified and characterized a Bowman-Birk-type PI from Tepary bean (Phaseolus acutifolius) seeds (TBPI). A semipure protein fraction containing this inhibitor, when tested its in vitro effect on transformed cells, showed a differential cytotoxic effect, as well as an increase in cell attachment to culture dishes. In this article we report that lectins were responsible for the cytotoxic effect previously observed, exhibiting a differential, antiproliferative effect on nontransformed cells and on different lineages of cancer cells. Although the purified TBPI lacked cytotoxicity, it was found to be responsible for the increase in cell adhesion, decreasing culture dishes' extracellular matrix degradation, leading to a decrease of the in vitro cell invasion capacity. This effect coincided with the suppression of Matrix Metalloproteinase-9 activity. These results indicate that Tepary bean seeds contain at least 2 different groups of bioactive proteins with distinct effects on cancer cells.

FIG. 1.

Protein fractionation. A: Tepary lectin-rich fraction (TLRF) and Tepary protease inhibitors fraction (TPIF) obtained after molecular weight exclusion chromatography. B: SDS-PAGE profile of the protein fractions after gel exclusion chromatography. Molecular markers (M), TLRF (1), TLRF stained for glycoprotein detection (2), TPIF (3). C: TLRF ionic exchange chromatography. D: SDS-PAGE profile of the protein fractions after ionic exchange chromatography. Molecular markers (M), Tepary lectin fraction (TLF) (1), and TLF stained for glycoprotein (2). E: Tepary bean protease inhibitor (TBPI) RP-HPLC chromatogram. Two peaks, with retention times of 43 min (P1) and 47.4 min (P2), showing protease inhibitor activity. F: SDS-PAGE profile of TBPI isoforms (P1 and P2) using 13.5% polyacrylamide gels loaded with 20 μg of protein per lane (Color figure available online).

FIG. 2.

Effects of Tepary lectin-rich fraction (TLRF), Tepary lectin fraction (TLF), and Tepary bean protease inhibitor (TBPI) on cell proliferation. Non-transformed or transformed fibroblasts were seeded (1 × 10⁴ cells/well) in 24-well plates with Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% calf serum. After 48 h, the medium was removed and different concentrations of (A) TLRF, (B) TLF, or (C) TBPI were added in 0.5% bovine serum albumin (BSA) serum-free medium. Treatment with DMEM containing 0.5% BSA was included as a control. After 72 h, cells were counted using a hemocytometer. Asterisks show statistically significant differences (Dunnett, P ≤ 0.05) with respect to controls, (∗) for transformed cells and (∗∗) for non-transformed cells.

FIG. 3.

Effect of Tepary lectin fraction (TLF) on cancer cells proliferation. A: Dose-response curve for 72 h TLF treatment on human cancer cells. Cells were seeded (3 × 10⁴ cells/well) in 24-well plates with Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% calf serum. After 48 h, the medium was removed and different concentrations of TLF were added in 0.5% bovine serum albumin (BSA) serum-free medium. Treatment with DMEM containing 0.5% BSA was included as a control. After 72 h, cells were counted using a hemocytometer. Small letters show statistically significant differences (Tukey, P × 0.05) with respect to controls in each case. B: IC₅₀ for human cancer cells treated with TLF. IC₅₀ was calculated by linear regression of the logarithm for TLF (AU/mL) vs proliferation percentage (Color figure available online).

FIG. 4.

Effect of Tepary bean protease inhibitor (TBPI) on cell adhesion. NIH 3T3/v-mos fibroblasts were seeded (1 × 10⁴ cells/well) with Dulbecco's modified Eagle's medium with 5% calf serum. After 24 h, the medium was changed and supplemented with 1% bovine serum albumin (BSA)/1.5% calf serum as a control, while adding 0.027 μg protein/mL (equivalent to 250 IU/mL) of TBPI to the experimental group. Every third day, 2 wells of each treatment were fixed with absolute ethanol until cells reached confluence. Fixed wells were stained with carmine/ethanol-HCl, and absorbance of the extracted colorant was determined at 531 nm. The asterisk shows a statistically significant difference with respect to control cells for each day (t student, P ≤ 0.05).

FIG. 5.

Effect of Tepary bean protease inhibitor (TBPI) on cell invasion. NIH 3T3/v-mos fibroblasts were seeded (2 × 10⁴ cells/well) on Matrigel-coated inserts with Dulbecco's modified Eagle's medium supplemented with 1% bovine serum albumin/1.5% calf serum as a control while adding 0.027 μg protein/mL (equivalent to 250 IU/mL) of TBPI for the experimental group. After 5 days incubation, the inserts were removed and the cells were incubated 5 more days and counted with a hemocytometer. The asterisk shows a statistically significant difference with respect to wild type transformed fibroblasts (t student, P ≤ 0.05).

FIG. 6.

SDS-PAGE profile of extracellular matrix degradation assay. A: NIH 3T3/v-mos fibroblasts were seeded (1 × 10⁴ cells/well) with Dulbecco's modified Eagle's medium with 5% calf serum. After 24 h, the medium was changed and supplemented with 1% bovine serum albumin/1.5% calf serum as a control while adding 0.027 μg Protein/mL (equivalent to 250 IU/mL) of TBPI to the experimental group. When confluence was reached, extracellular matrix was extracted and analyzed by SDS-PAGE at 5% polyacrylamide gel loaded with 20 μg of protein per lane, (1) non-cultured dishes, (2) untreated cells, and (3) treated cells. B: The histogram represents the band intensities evaluated in terms of relative proportion respect to pixel number by a densitometry analyzer.

FIG. 7.

Gelatin zymography of conditioned media of 3T3/v-mos fibroblasts treated and no treated with Tepary bean protease inhibitor (TBPI). M = molecular mass markers; S = control cells conditioned media (CM); SI = control cells CM pre-incubated with 10 μg of TBPI before running the gel; T = TBPI treated cells CM; TI = TBPI treated cells CM preincubated with 10 μg of TBPI before running the gel; SE = control cells CM incubated with 5 mM EDTA before running the gel; TE = TBPI treated cells CM incubated with 5 mM EDTA before running the gel; A: Activity in the absence of Triton. B: Activity in the presence of Triton.