Asiatic Acid, Corosolic Acid, and Maslinic Acid Interfere with Intracellular Trafficking and N-Linked Glycosylation of Intercellular Adhesion Molecule-1

Abstract

The pentacyclic triterpenoid ursolic acid was previously shown to inhibit the intracellular trafficking of intercellular adhesion molecule-1 (ICAM-1) from the endoplasmic reticulum (ER) to the Golgi apparatus. In the present study, we further investigated the biological activities of three pentacyclic triterpenoids closely related to ursolic acid on the interleukin 1α-induced expression and intracellular trafficking of ICAM-1. In human lung adenocarcinoma A549 cells, asiatic acid, corosolic acid, and maslinic acid interfered with the intracellular transport of ICAM-1 to the cell surface. Endoglycosidase H-sensitive glycans were linked to ICAM-1 in asiatic acid-, corosolic acid-, and maslinic acid-treated cells. Unlike corosolic acid, asiatic acid and maslinic acid increased the amount of the ICAM-1 protein. Moreover, asiatic acid increased the co-localization of ICAM-1 with calnexin (an ER marker), but not GM130 (a cis-Golgi marker). Asiatic acid, corosolic acid, and maslinic acid inhibited yeast α-glucosidase activity, but not Jack bean α-mannosidase activity. These results indicate that asiatic acid, corosolic acid, and maslinic acid interfere with the intracellular transport of ICAM-1 to the cell surface and cause the accumulation of ICAM-1 linked to endoglycosidase H-sensitive glycans.

Cell-surface adhesion molecules play an essential role in the regulation of immune and inflammatory responses.¹⁾ As one of most important adhesion molecules, intercellular adhesion molecule-1 (ICAM-1) is mainly up-regulated by the transcription factor nuclear factor κB (NF-κB) in response to inflammatory cytokines.²⁾ ICAM-1 interacts with multiple molecules, including lymphocyte function-associated antigen-1 and Macrophage-1 antigen.³⁾ As a post-translation modification, ICAM-1 is highly glycosylated with N-linked glycans.³⁾ ICAM-1 is up-regulated on vascular endothelial cells during inflammatory responses and is required for the recruitment of leukocytes and their transmigration to inflammation sites.^4,5) In addition, ICAM-1 has been reported to play a role in cancer metastasis.⁶⁾

Pentacyclic triterpenoids are often found as secondary metabolites of plants.^7,8) Ursolic acid, oleanolic acid, and betulinic acid are major pentacyclic triterpenoids, and have been reported to exhibit many biological activities, such as anti-inflammatory and anti-cancer activities.^7–12) We previously reported that ursolic acid prevents the intracellular trafficking and causes the accumulation of ICAM-1 linked with high-mannose-type glycans in the endoplasmic reticulum (ER).¹³⁾ In contrast, betulinic acid and oleanolic acid did not affect the intracellular transport of ICAM-1 to the cell surface, but interfered with the N-linked glycan modification of ICAM-1.¹⁴⁾ These findings suggest that pentacyclic triterpenoids possess multiple targets in the intracellular trafficking of glycoproteins and their post-translational modifications.

Asiatic acid, corosolic acid, and maslinic acid (Fig. 1A) are pentacyclic triterpenoids closely related to ursolic acid, and have been shown to exhibit diverse biological activities, such as anti-inflammatory and anti-tumor activities.^15–18) In the present study, we further investigated the biological activities of these pentacyclic triterpenoids on the inflammatory cytokine-induced expression and intracellular trafficking of as well as post-translational modifications to ICAM-1. We found that asiatic acid, corosolic acid, and maslinic acid interfered with the intracellular trafficking and caused the accumulation of ICAM-1 linked to endoglycosidase H (Endo H)-sensitive glycans. In addition, we showed that asiatic acid and maslinic acid, but not corosolic acid, increased the expression of the ICAM-1 protein.

Fig. 1. Corosolic Acid and Maslinic Acid Decreased IL-1α-Induced Expression of Cell-Surface ICAM-1

(A) Structures of asiatic acid, corosolic acid, and maslinic acid. (B–D) A549 cells were preincubated with asiatic acid (B), corosolic acid (C), or maslinic acid (D) for 1 h, and then incubated with (+; filled circles) or without (–; open circles) IL-1α (0.25 ng/mL) for 6 h in the presence or absence of asiatic acid, corosolic acid, or maslinic acid at the indicated concentrations as the final concentrations. The expression of cell-surface ICAM-1 was measured by cell-ELISA. Cell-surface ICAM-1 (%) is shown as the mean±S.E. of five (B) and three (C and D) independent experiments. * p<0.05, ** p<0.01, and *** p<0.001, significantly different from the control.

MATERIALS AND METHODS

Cell Culture

Human lung adenocarcinoma A549 cells (JCRB 0076) were provided by the National Institutes of Biomedical Innovation, Health and Nutrition JCRB Cell Bank (Osaka, Japan). A549 cells were cultured in RPMI 1640 medium (Thermo Fisher Scientific, Grand Island, NY, U.S.A.) supplemented with heat-inactivated fetal calf serum (Nichirei Bioscience, Tokyo, Japan) and penicillin–streptomycin mixed solution (Nacalai Tesque, Kyoto, Japan).

Reagents

Asiatic acid (Tokyo Chemical Industry, Tokyo, Japan), corosolic acid (Sigma-Aldrich, St. Louis. MO, U.S.A.; Abcam, Cambridge, U.K.), maslinic acid (Sigma-Aldrich), ursolic acid (Sigma-Aldrich), and 1-deoxymannojirimycin hydrochloride (Santa Cruz Biotechnology, Dallas, TX, U.S.A.) were commercially obtained. Human interleukin-1α (IL-1α) was kindly provided by Dainippon Pharmaceutical (Osaka, Japan). Human IL-1β was purchased from Genzyme Diagnostics (Cambridge, MA, U.S.A.).

Cell-Enzyme-Linked Immunosorbent Assay (ELISA)

Cell-ELISA was performed basically as described previously.¹³⁾ A549 cells were washed twice with phosphate-buffered saline (PBS) and then fixed with 1% paraformaldehyde-PBS. Fixed cells were incubated serially with a mouse anti-ICAM-1 antibody (clone 15.2; Leinco Technologies, St. Louis, MO, U.S.A.) and horseradish peroxidase (HRP)-conjugated anti-mouse immunoglobulin G (IgG) (H+L) antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, U.S.A.), followed by the HRP colorimetric reaction. Cell-surface ICAM-1 expression was evaluated by measuring absorbance at 415 or 450 nm.

Flow Cytometry

A549 cells were harvested and washed with PBS, followed by fixation with 1% paraformaldehyde-PBS. Fixed cells were stained with a mouse anti-ICAM-1 antibody (clone 15.2) or mouse IgG1 isotype control (MOPC-21; BioLegend, San Diego, CA, U.S.A.) for 1 h and then with a fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG antibody (Jackson ImmunoResearch) for 30 min. Stained cells were analyzed using the Guava EasyCyte Plus™ System (Merck Millipore, Darmstadt, Germany).

Cell Viability Assays

Crystal violet staining and the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay were performed basically as described previously.^13,14,19) Cells were incubated with MTT for the last 2 h of the incubation. Cells were washed with PBS and stained with crystal violet. Absorbance at 570 or 595 nm was measured.

Western Blotting

The preparation of cell lysates and Western blotting were performed as described previously.^13,19) Cells were washed with PBS and lyzed by 1% Triton X-100 lysis buffer. Cytoplasmic fractions were collected by centrifugation (15300×g, 5 min) as supernatants. Precipitates were washed twice with lysis buffer and prepared as nuclear fractions. Primary antibodies for β-actin (AC-15, Sigma-Aldrich; 2F3, Wako Pure Chemical Industries, Ltd., Osaka, Japan), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (6C5; Santa Cruz Biotechnology), ICAM-1 (clone 28; BD Biosciences, San Jose, CA, U.S.A.), lamin A/C (E-1; Santa Cruz Biotechnology), and RelA (C-20; Santa Cruz Biotechnology) were used. Protein bands were analyzed by ImageQuant LAS 4000 mini (GE Healthcare Japan, Tokyo, Japan).

Quantitative PCR

The preparation of total RNA, cDNA synthesis, and real-time PCR were performed as described previously.¹⁹⁾ The amounts of ICAM-1 mRNA and β-actin mRNA were calculated by primer-specific standard curves.

Glycosidase Digestion

Glycosidase digestion was performed basically as described previously.^13,14) Endo H and the peptide, N-glycosidase F (PNGase F) were obtained from New England Biolabs (Ipswich, MA, U.S.A.). Cell lysates (20 µg) were heated at 100°C for 10 min and then incubated with or without Endo H or PNGase F for 1 h, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting.

Confocal Microscopy

Confocal microscopy was performed basically as described previously.¹³⁾ Cells were grown on coverslips coated with Cellmatrix^® type I-C (Nitta Gelatin, Osaka, Japan). Cells were fixed with 4% paraformaldehyde-PBS and washed with PBS. Fixed cells were stained with a mouse anti-ICAM-1 antibody (clone 15.2) together with a rabbit anti-calnexin antibody (EPR3632, Abcam) or rabbit anti-GM130 antibody (EP892Y, Abcam), and then an Alexa Fluor 488-conjugated anti-mouse IgG antibody (A-11029; Thermo Fisher Scientific) or Alexa Fluor 594-conjugated anti-rabbit IgG antibody (A-11037; Thermo Fisher Scientific). Cells were observed with the confocal laser scanning microscope system FV 10i (Olympus, Tokyo, Japan). Regarding the quantification of the co-localization of ICAM-1 with calnexin or GM130, at least 50 cells of five different images per one experiment were evaluated.

Glycosidase Assays

Glycosidase assays were performed as described previously.^13,14) Yeast α-glucosidase and Jack bean (Canavalia ensiformis) α-mannosidase were obtained from Oriental Yeast (Osaka, Japan) and Sigma-Aldrich, respectively. p-Nitrophenyl-α-D-glucopyranoside and 4-nitrophenyl-α-D-mannopyranoside were used for substrates of α-glucosidase and α-mannosidase, respectively. Absorbance at 405 nm was measured.

Statistical Analysis

The significance of differences was evaluated by a one-way ANOVA followed by Tukey’s test for multiple comparisons unless otherwise specified.

RESULTS

Corosolic Acid and Maslinic Acid Inhibited the Expression of Cell-Surface ICAM-1 Induced by IL-1α

In response to pro-inflammatory cytokines, vascular endothelial cells up-regulate cell-surface adhesion molecules, such as ICAM-1. We have used human lung adenocarcinoma A549 cells as a model cell line for vascular endothelial cells because they are responsive to multiple pro-inflammatory cytokines (e.g. tumor necrosis factor-α (TNF-α), IL-1α, and IL-1β) and express a detectable amount of cell-surface ICAM-1 by cell-ELISA when stimulated.^19,20) We previously showed that ursolic acid inhibited the IL-1α-induced expression of cell-surface ICAM-1 in A549 cells and human umbilical vein endothelial cells.¹³⁾ In order to investigate the inhibitory activities of pentacyclic triterpenoids structurally related to ursolic acid, A549 cells were preincubated with them for 1 h and then incubated with IL-1α for 6 h, followed by cell-ELISA to measure the expression of cell-surface ICAM-1. Asiatic acid did not markedly affect the expression of cell-surface ICAM-1 at concentrations up to 100 µM (Fig. 1B). Corosolic acid down-regulated the IL-1α-induced expression of cell-surface ICAM-1 in a dose-dependent manner and completely at 60 µM (Fig. 1C). Maslinic acid reduced the expression of cell-surface ICAM-1 at higher concentrations than corosolic acid (Fig. 1D). A flow cytometric analysis confirmed that corosolic acid and maslinic acid decreased the amount of cell-surface ICAM-1 at a concentration of 100 µM, while asiatic acid at 100 µM did not markedly affect the expression of cell-surface ICAM-1 (Figs. 2A, B).

Fig. 2. A Flow Cytometric Analysis Confirmed That Corosolic Acid and Maslinic Acid Decreased IL-1α-Induced Expression of Cell-Surface ICAM-1

(A and B) A549 cells were preincubated with asiatic acid, corosolic acid, or maslinic acid or without compounds (Control) for 1 h, and then incubated with (+) or without (−) IL-1α (0.25 ng/mL) for 6 h in the presence or absence of asiatic acid, corosolic acid, or maslinic acid at 100 µM as the final concentration. A549 cells were stained with an anti-human ICAM-1 antibody (black lines) or isotype control antibody (gray lines) and FITC-conjugated secondary antibody, and then subjected to a flow cytometric analysis. Results are representative of three independent experiments (A). ICAM-1⁺ cells (%) in the M1 region is shown as the mean±S.E. of three independent experiments (B). *** p<0.001, significantly different from the control.

MTT-reducing activity was used to evaluate the viability of A549 cells. Asiatic acid, corosolic acid, and maslinic acid did not decrease MTT-reducing activity at concentrations up to 100 µM (Figs. 3A–C). Crystal violet staining was then used to measure adherent cells. Asiatic acid only slightly decreased the number of adherent cells at 100 µM (Fig. 3D). In contrast, corosolic acid reduced the number of adherent cells in a dose-dependent manner (Fig. 3E). Maslinic acid did not affect the number of adherent cells up to 80 µM and decreased it partially at 100 µM (Fig. 3F). These results indicate that asiatic acid only weakly affected the viability of A549 cells at concentrations up to 100 µM, while corosolic acid and maslinic acid preferentially inhibited the expression of cell-surface ICAM-1 more than reducing the number of adherent cells.

Fig. 3. Asiatic Acid, Corosolic Acid, and Maslinic Acid Did Not Affect MTT-Reducing Activity

(A–F) A549 cells were preincubated with asiatic acid (A and D), corosolic acid (B and E), or maslinic acid (C and F) for 1 h, and then incubated with (+; filled circles) or without (–; open circles) IL-1α (0.25 ng/mL) for 6 h in the presence or absence of asiatic acid, corosolic acid, or maslinic acid at the indicated concentrations as the final concentrations. MTT-reducing activity (%) is shown as the mean±S.E. of three independent experiments (A–C). Crystal violet (%) is shown as the mean±S.E. of three independent experiments (D–F). * p<0.05, ** p<0.01, and *** p<0.001, significantly different from the control.

Asiatic Acid and Maslinic Acid Up-Regulated the Expression of the ICAM-1 Protein Induced by IL-1α and IL-1β

A549 cells were preincubated with pentacyclic triterpenoids for 1 h and then incubated with IL-1α for 6 h. ICAM-1 was detected as multiple bands in IL-1α-stimulated 549 cells, possibly due to heterogeneous glycosylation. As a common feature, the molecular weight of ICAM-1 was decreased by asiatic acid, corosolic acid, and maslinic acid (Figs. 4A–C). Moreover, we found that asiatic acid and maslinic acid increased the amount of the ICAM-1 protein, particularly at 100 and 60 µM, respectively (Figs. 4A, C). In contrast, corosolic acid decreased the amount of the ICAM-1 protein at concentrations more than 80 µM (Fig. 4B). A quantitative comparison of the cell-surface ICAM-1 and ICAM-1 proteins indicated that asiatic acid, corosolic acid, and maslinic acid interfered with the intracellular transport of ICAM-1 to the cell surface.

Fig. 4. Asiatic Acid and Maslinic Acid Increased the Amount of the ICAM-1 Protein Induced by IL-1α, Whereas Corosolic Acid Decreased It

(A–C) A549 cells were preincubated with asiatic acid (A), corosolic acid (B), or maslinic acid (C) for 1 h, and then incubated with (+) or without (−) IL-1α (0.25 ng/mL) for 6 h in the presence or absence of asiatic acid, corosolic acid, or maslinic acid at the indicated concentrations as the final concentrations. Cell lysates were analyzed by Western blotting. Blots were representative of four (A) and three (B and C) independent experiments. The amounts of the ICAM-1 protein relative to β-actin are shown as the mean±S.E. of four (A) and three (B and C) independent experiments. * p<0.05, ** p<0.01, and *** p<0.001, significantly different from the control.

IL-1α and IL-1β transmit intracellular signals via the IL-1 receptor.²¹⁾ We previously showed that IL-1β up-regulated the expression of ICAM-1 in A549 cells.²⁰⁾ A549 cells were preincubated with pentacyclic triterpenoids for 1 h and then incubated with IL-1β for 6 h. In IL-1β-stimulated A549 cells, asiatic acid and maslinic acid increased the amount of the ICAM-1 protein, whereas it was decreased by corosolic acid (Fig. 5).

Fig. 5. Asiatic Acid and Maslinic Acid Increased the Amount of the ICAM-1 Protein Induced by IL-1β, Whereas Corosolic Acid Decreased It

A549 cells were preincubated with asiatic acid, corosolic acid, or maslinic acid for 1 h, and then incubated with (+) or without (−) IL-1β (1 ng/mL) for 6 h in the presence or absence of asiatic acid, corosolic acid, or maslinic acid at the indicated concentrations as the final concentrations. Cell lysates were analyzed by Western blotting. Blots were representative of three independent experiments. The amounts of the ICAM-1 protein relative to β-actin are shown as the mean±S.E. of three independent experiments. ** p<0.01, and *** p<0.001, significantly different from the control.

Asiatic Acid, Corosolic Acid and Maslinic Acid Exerted Different Effects on ICAM-1 Transcription Induced by IL-1α

To evaluate the expression of ICAM-1 mRNA, A549 cells were preincubated with asiatic acid, corosolic acid, and maslinic acid for 1 h, and stimulated with IL-1α for 2 h, followed by the preparation of total RNA, cDNA synthesis, and real-time PCR. Asiatic acid only slightly increased IL-1α-induced ICAM-1 mRNA expression (Fig. 6A). Corosolic acid partially reduced IL-1α-induced ICAM-1 mRNA expression (Fig. 6B). In contrast, maslinic acid increased IL-1α-induced ICAM-1 mRNA expression at 60–100 µM (Fig. 6C). These results indicated that asiatic acid, corosolic acid and maslinic acid exerted different effects on IL-1α-induced ICAM-1 transcription.

Fig. 6. Asiatic Acid, Corosolic Acid and Maslinic Acid Exert Different Effects on the IL-1α-Induced Expression of ICAM-1 mRNA

(A–C) A549 cells were preincubated with asiatic acid (A), corosolic acid (B), or maslinic acid (C) for 1 h, and then incubated with (+) or without (−) IL-1α (0.25 ng/mL) for 2 h in the presence or absence of asiatic acid, corosolic acid, or maslinic acid at the indicated concentrations as the final concentrations. The expression of ICAM-1 mRNA normalized to β-actin mRNA was measured by real-time PCR. ICAM-1 mRNA (%) is shown as the mean±S.E. of three independent experiments. * p<0.05, ** p<0.01, and *** p<0.001, significantly different from the control.

ICAM-1 is mainly regulated by the NF-κB transcription factor.²⁾ To examine the effects of asiatic acid on the NF-κB signaling pathway, A549 cells were treated with asiatic acid for 1 h and then stimulated with IL-1α for 30 min, followed by Western blotting using an antibody specific to the NF-κB subunit RelA (also known as p65). Upon the IL-1α stimulation, a part of RelA was translocated from the cytoplasm to the nucleus (Fig. 7). Asiatic acid did not markedly affect the nuclear translocation of RelA and the amount of RelA in the cytoplasm at concentrations up to 100 µM (Fig. 7). These results are consistent with asiatic acid not greatly affecting IL-1α-induced ICAM-1 mRNA expression (Fig. 6A).

Fig. 7. Asiatic Acid Did Not Affect the IL-1α-Induced Nuclear Translocation of the NF-κB Subunit RelA

A549 cells were preincubated with asiatic acid for 1 h, and then incubated with (+) or without (−) IL-1α (0.25 ng/mL) for 30 min in the presence or absence of asiatic acid at the indicated concentrations as the final concentrations. Nuclear and cytoplasmic lysates were analyzed by Western blotting. Blots were representative of five independent experiments. The protein levels of nuclear RelA and cytoplasmic RelA relative to lamin A/C and GAPDH, respectively, are shown as the mean±S.E. of five independent experiments. *** p<0.001, significantly different from the control.

Endo H-Sensitive Glycans Were Mainly Linked to ICAM-1 in Asiatic Acid-, Corosolic Acid-, and Maslinic Acid-Treated Cells

In order to investigate whether asiatic acid, corosolic acid, and maslinic acid affect N-linked glycan modifications, cell lysates were digested by PNGase F and Endo H. Major ICAM-1 bands migrated mostly at the same molecular weight on SDS-PAGE when A549 cells were treated with asiatic acid, corosolic acid, maslinic acid, and ursolic acid (Fig. 8). ICAM-1 was cleaved into the same sizes by PNGase F in control cells as well as asiatic acid-, corosolic acid-, maslinic acid-, and ursolic acid-treated cells (Fig. 8). Moreover, ICAM-1 was digested to the same major band by Endo H in asiatic acid-, corosolic acid-, maslinic acid-, and ursolic acid-treated cells, while minor Endo H-resistant bands remained in asiatic acid- and maslinic acid-treated cells (Fig. 8). Since high-mannose-type and hybrid-type glycans are sensitive to Endo H digestion, these results indicate that asiatic acid, corosolic acid, and maslinic acid cause the accumulation of ICAM-1 linked to high-mannose-type and/or hybrid-type glycans.

Fig. 8. Asiatic Acid, Corosolic Acid, and Maslinic Acid Decreased the Molecular Weight of ICAM-1, Which Was Linked to Endo H-Sensitive Glycans

A549 cells were preincubated with asiatic acid, corosolic acid, maslinic acid, or ursolic acid or without compounds (Control) for 1 h, and then incubated with IL-1α (0.25 ng/mL) for 6 h in the presence or absence of these compounds. The final concentrations used were: asiatic acid (100 µM), corosolic acid (50 µM), maslinic acid (50 µM), and ursolic acid (50 µM). Cell lysates were treated with (+) or without (−) PNGase F or Endo H, and then analyzed by Western blotting. Blots were representative of at least three independent experiments.

Asiatic Acid Increased Co-localization of ICAM-1 with Calnexin, but Not GM130

We previously showed that ursolic acid inhibited intracellular trafficking from the ER to the Golgi apparatus and caused the accumulation of Endo H-sensitive ICAM-1 in the ER.¹³⁾ Asiatic acid at 100 µM did not markedly affect the expression of cell-surface ICAM-1, but up-regulated the expression of the ICAM-1 protein by more than two-fold (Figs. 1B, 4A), implying that asiatic acid increased the amount of intracellular ICAM-1. In order to examine the subcellular localization of ICAM-1, A549 cells were preincubated with asiatic acid for 1 h and then incubated with IL-1α for 6 h. A549 cells were stained for ICAM-1, together with the cis-Golgi marker GM130 or ER marker calnexin. In control A549 cells, ICAM-1 was detected more abundantly at areas outside of calnexin and GM130, although some ICAM-1 co-localized with either GM130 or calnexin, indicating that ICAM-1 was mostly transported to the cell surface (Figs. 9A–D). In asiatic acid-treated A549 cells, the co-localization of ICAM-1 with calnexin was markedly increased, while that of ICAM-1 with GM130 was unaffected (Figs. 9A–D). It is important to note that asiatic acid did not affect the morphology of the Golgi apparatus, but altered that of the ER to smaller or dot-like shapes (Figs. 9A, B). These results indicate that asiatic acid increases the amount of ICAM-1 in the ER.

Fig. 9. Asiatic Acid Augmented the Localization of ICAM-1 in the ER

(A–D) A549 cells were preincubated with or without asiatic acid for 1 h, and then incubated with or without IL-1α (0.25 ng/mL) for 6 h in the presence or absence of asiatic acid (100 µM) as the final concentrations. Cells were stained for ICAM-1, together with the cis-Golgi marker GM130 (A and C) or ER marker calnexin (B and D). Data are representative of three independent experiments (A and B). The co-localization of ICAM-1 with GM130 or calnexin is shown as the mean±S.E. of three independent experiments (C and D). The Student’s t-test was used to evaluate the significance of differences. *** p<0.001, ns: not significant.

Asiatic Acid Delayed the Expression of Cell-Surface ICAM-1

We performed time–course experiments on the expression of cell-surface ICAM-1 upon an IL-1α stimulation. In control A549 cells, cell-surface ICAM-1 steadily increased 1–3 h after the IL-1α stimulation and then gradually reached a plateau after 3–6 h (Fig. 10). Asiatic acid at 100 µM decreased the amount of cell-surface ICAM-1 during 2–4 h, but not 5–6 h (Fig. 10). These results indicate that asiatic acid delays the expression of cell-surface ICAM-1.

Fig. 10. Asiatic Acid Delayed the Expression of Cell-Surface ICAM-1

A549 cells were preincubated with (squares) or without (circles) asiatic acid for 1 h, and then incubated with (filled symbols) or without (open symbols) IL-1α (0.25 ng/mL) for the indicated times in the presence or absence of asiatic acid (100 µM). Cell-surface ICAM-1 (%) is shown as the mean±S.E. of three independent experiments. ** p<0.01 and *** p<0.001.

Asiatic Acid, Corosolic Acid, and Maslinic Acid Inhibit α-Glucosidase, but Not α-Mannosidase

We previously showed that ursolic acid inhibited yeast α-glucosidase, but not Jack bean α-mannosidase.^13,14) Asiatic acid, corosolic acid, and maslinic acid inhibited yeast α-glucosidase activity in a dose-dependent manner (Figs. 11A–C). The IC₅₀ values of asiatic acid, corosolic acid, and maslinic acid were calculated to be 24.4±3.0, 8.5±2.5, and 19.0±3.1 µM (mean±standard error (S.E.) of three independent experiments), respectively. Unlike 1-deoxymannojirimycin, Jack bean α-mannosidase was not inhibited by asiatic acid, corosolic acid, maslinic acid, or ursolic acid at 100 µM (Fig. 11D).

Fig. 11. Asiatic Acid, Corosolic Acid, and Maslinic Acid Inhibited α-Glucosidase Activity, but Not α-Mannosidase Activity

(A–C) Yeast α-glucosidase was preincubated with asiatic acid (A), corosolic acid (B), or maslinic acid (C) at room temperature for 30 min and then incubated with p-nitrophenyl-α-D-glucopyranoside (0.5 mM) at 37°C for 15 min in the presence of asiatic acid, corosolic acid, or maslinic acid at the final concentrations. α-Glucosidase activity (%) is shown as the mean±S.E. of three independent experiments. *** p<0.001, significantly different from the control. (D) Jack bean α-mannosidase was preincubated with asiatic acid, corosolic acid, maslinic acid, ursolic acid, or 1-deoxymannojirimycin or without compounds (Control) at room temperature for 30 min and then incubated with 4-nitrophenyl-α-D-mannopyranoside (0.5 mM) at 37°C for 15 min in the presence of these compounds (100 µM) as the final concentrations. α-Mannosidase activity (%) is shown as the mean±S.E. of three independent experiments. *** p<0.001, significantly different from the control.

DISCUSSION

Pentacyclic triterpenoids have been reported to exhibit various biological activities.^{7–12,15–18)} As unique biological activities, we previously showed that ursolic acid inhibited the transport of ICAM-1 from the ER to the Golgi apparatus, and that betulinic acid and oleanolic acid did not inhibit the intracellular transport of ICAM-1, but affected its post-translational glycosylation most likely at the step of ER α-glucosidases.^13,14) In the present study, we revealed that asiatic acid, corosolic acid, and maslinic acid interfered with the intracellular transport of ICAM-1 and caused the accumulation of ICAM-1 linked to Endo H-sensitive glycans, which are assumed to be high-mannose-type and/or hybrid-type glycans. Moreover, we showed that asiatic acid and maslinic acid up-regulated the expression of the ICAM-1 protein, whereas corosolic acid down-regulated it at higher concentrations. Thus, these pentacyclic triterpenoids exert common and distinct effects on the protein expression and post-translational glycosylation of ICAM-1 and its intracellular transport to the cell surface.

A previous study reported that asiatic acid, corosolic acid, and maslinic acid inhibited α-glucosidase from Saccharomyces cerevisiae at 30.03±0.41, 3.53±0.27, and 5.52±0.19 µg/mL, respectively,²²⁾ which were calculated to be 61, 7.5, and 12 µM, respectively. In another study, the IC₅₀ values of asiatic acid and corosolic acid on yeast α-glucosidase were found to be 100.2±0.2 and 17.2±0.9 µM, respectively.²³⁾ Consistent with these findings, we showed that asiatic acid, corosolic acid, and maslinic acid inhibited yeast α-glucosidase, indicating that corosolic acid inhibits yeast α-glucosidase more strongly than asiatic acid and maslinic acid. We previously demonstrated that betulinic acid and oleanolic acid inhibited α-glucosidase.¹⁴⁾ Betulinic acid and oleanolic acid affected N-linked glycan modifications to ICAM-1 most likely at the step of ER α-glucosidases.¹⁴⁾ However, in contrast to asiatic acid-, corosolic acid-, maslinic acid-, and ursolic acid-treated cells, ICAM-1 proteins were insensitive to Endo H digestion in betulinic acid- and oleanolic acid-treated cells.¹⁴⁾ Thus, in addition to ER α-glucosidases, asiatic acid, corosolic acid, maslinic acid, and ursolic acid appear to inhibit additional processes that cause the accumulation of the Endo H-sensitive glycans of ICAM-1.

Corosolic acid is known to exhibit various biological activities, including anti-inflammatory and anti-cancer activities.¹⁶⁾ We showed that corosolic acid inhibited the expression of cell-surface ICAM-1 and decreased the molecular weight of ICAM-1. Moreover, it appeared that corosolic acid partially inhibited the mRNA expression of ICAM-1. In response to inflammatory cytokines, ICAM-1 is mainly up-regulated by NF-κB-dependent transcription.²⁾ Previous studies reported that corosolic acid inhibited the NF-κB signaling pathway in several types of cells.^24–27) Thus, the down-regulation of ICAM-1 protein expression by corosolic acid appears to be attributed partly to the inhibition of the ICAM-1 mRNA expression or the NF-κB signaling pathway. Corosolic acid is a close structural derivative of ursolic acid, which possesses an extra hydroxyl group at the C-2 position. We previously reported that ursolic acid inhibited the expression of cell-surface ICAM-1 without reducing its protein level at concentrations of 30–50 µM.^13,14) ICAM-1 proteins in corosolic acid- and ursolic acid-treated cells were observed to be nearly the same size and equally sensitive to Endo H digestion. Therefore, corosolic acid and ursolic acid appear to share similar biological activities.

We found that corosolic acid reduced the number of adherent cells, but did not decrease MTT-reducing activity during the 7-h incubation in A549 cells. A previous study reported that corosolic acid induced apoptotic cell death in A549 cells in a 12-h and longer incubation.²⁸⁾ We speculate that the difference between the present results and previous finding may be attributed to the incubation time; however, culture conditions may also influence the sensitivity of A549 cells to corosolic acid.

Maslinic acid up-regulated the expression of the ICAM-1 protein, particularly at concentrations of 60 µM, whereas it augmented ICAM-1 mRNA expression at 60–100 µM. Maslinic acid has been shown to inhibit the NF-κB signaling pathway.^29–34) Furthermore, it decreased TNF-α-induced ICAM-1 expression.²⁹⁾ In contrast to these findings, maslinic acid appeared to up-regulate the mRNA and protein expression of ICAM-1, at least in A549 cells. Further experiments are needed to elucidate the mechanisms by which maslinic acid augments the IL-1α-induced mRNA and protein expression of ICAM-1.

Asiatic acid up-regulated the expression of the ICAM-1 protein, particularly at concentrations of 100 µM. In contrast to maslinic acid, asiatic acid only slightly increased ICAM-1 mRNA expression or did not markedly affect the RelA nuclear translocation. Previous studies reported that asiatic acid inhibited the NF-κB signaling pathway in various types of cells.^35–40) Asiatic acid was found to decrease TNF-α-induced ICAM-1 expression.⁴¹⁾ Thus, asiatic acid appears to exert a slight effect on the IL-1α-induced ICAM-1 transcription, at least in A549 cells, and it may rather have affected translation or post-translational processes in order to up-regulate ICAM-1 protein levels. ICAM-1 possesses multiple N-linked glycosylation sites.³⁾ In the ER, high-mannose-type sugar chains are transferred to newly-synthesized proteins and ER α-glucosidases I and II then sequentially remove glucose residues, which regulate the association with malectin, calnexin, and calreticulin.^42–44) These processes regulate the protein folding, quality control, and ER-associated degradation of glycoproteins.^42–44) Castanospermine, as an ER α-glucosidase inhibitor, did not increase the amount of the ICAM-1 protein,¹⁴⁾ suggesting that the inhibition of ER α-glucosidases does not cause an increase in the amount of the ICAM-1 protein. Further investigations are needed in order to elucidate the mechanisms by which asiatic acid and maslinic acid up-regulate the expression of the ICAM-1 protein.

A comparison between total ICAM-1 protein and cell-surface ICAM-1 indicated that asiatic acid, corosolic acid, and maslinic acid interfere with the intracellular trafficking of ICAM-1. In particular, asiatic acid at 100 µM increased the localization of the ICAM-1 protein in the ER and delayed the expression of cell-surface ICAM-1. We previously reported that ursolic acid at 50 µM induced the fragmentation of the Golgi apparatus,¹³⁾ which appears to account for the inhibition of glycoprotein transport from the ER to the Golgi apparatus. In asiatic acid-treated cells, the ER was changed to smaller or dot-like structures, while the Golgi apparatus remained unaltered. This may explain why ICAM-1 remains longer in the ER, but is ultimately transported to the cell surface in asiatic acid-treated cells. We previously reported that tunicamycin caused an unglycosylated form of ICAM-1 and inhibited its expression to the cell surface.¹³⁾ Castanosperimine (an inhibitor of ER α-glucosidases I and II), 1-deoxymannojirimycin (an inhibitor of Golgi α-mannosidase I), and swainosonine (an inhibitor of Golgi α-mannosidase II) did not inhibit the expression of cell surface ICAM-1, but decreased its molecular weight.^13,14) Thus, the inhibition of α-glucosidases or α-mannosidases may be dispensable for the cell surface expression of the ICAM-1 protein.

In conclusion, we herein revealed that asiatic acid, corosolic acid, and maslinic acid interfered with the intracellular trafficking of ICAM-1 to the cell surface. Moreover, asiatic acid and maslinic acid increased the amount of the ICAM-1 protein, while corosolic acid inhibited the expression of the ICAM-1 protein. Cell-surface adhesion molecules, such as ICAM-1, play an essential role in inflammatory responses and cancer metastasis.^4–6) Glycosylation regulates the function of endothelial adhesion molecules, such ICAM-1.⁴⁵⁾ Hence, the inhibition of cell-surface adhesion molecules is considered to serve as anti-inflammatory and anti-cancer agents. Pentacyclic triterpenoids are known to be major components in many plants and are composed of a large number of structural derivatives.^7,8) Pentacyclic triterpenoids are assumed to target multiple cellular proteins and exhibit diverse biological activities. Further structure–activity relationships and the identification of target proteins may be important for the development of more effective anti-inflammatory and anti-cancer agents.

Acknowledgments

This work was partly supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant number 16H04910 (to T.K.).

Conflict of Interest

The authors declare no conflict of interest.

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