2024 Volume 47 Issue 10 Pages 1648-1656
Timosaponin AIII (TAIII), a steroidal saponin isolated from the root of Anemarrhena asphodeloides Bunge, exhibits various pharmacological activities, including anti-cancer properties. TAIII inhibits the migration and invasion of various cancer cell types. However, the mechanism underlying how TAIII regulates the motility of cancer cells remains incompletely understood. In this study, we demonstrate that TAIII disrupted cell–extracellular matrix (ECM) interactions by inhibiting internalization of cell surface proteins, such as integrins. We found that TAIII inhibited cell adhesion on various ECMs. Structure–activity relationship analysis demonstrated that TAIII exhibited unique activity among the saponins from Anemarrhena asphodeloides Bunge and that the number and position of saccharide moieties were important for TAIII to exert its activity. Time lapse imaging revealed that TAIII also suppressed cell spreading on the ECM, membrane ruffling, and lamellipodia formation. Furthermore, we examined integrin β1 behaviors in response to TAIII treatment and found that TAIII blocked its internalization. These findings contribute to delineating the potential molecular mechanisms by which TAIII exerts anti-metastatic activity.
The leading cause of disease-related death in cancer patients is distant metastasis.1) After dissociation from the primary lesion, cancer cells invade surrounding tissues and subsequently enter blood vessels.2) Cancer cells communicate with the microenvironment to enhance their migratory activities.3) In adhesive migration, the extracellular matrix (ECM) provides a scaffold for cancer cells. Cancer cells interact with the ECM through adhesion complexes including integrins4–6) and generate the traction force necessary for migration based on dynamic turnover of the cell adhesion complexes.7) Additionally, cell adhesion complexes serve as a platform where signaling molecules, including several kinases and adaptors, are recruited and initiate the signal transduction involved in cell migration, proliferation, and survival.8,9)
In East Asian countries, Anemarrhena asphodeloides Bunge has been used as a traditional medicine for various diseases, including arthralgia, hematochezia, and hemoptysis.10) Timosaponin AIII (TAIII), a steroidal saponin, is an active component in Anemarrhena asphodeloides Bunge with anti-pyretic, anti-inflammatory, anti-diabetic, and anti-coagulant effects.11) Numerous studies have demonstrated the anti-cancer properties of TAIII in vitro and in vivo.11,12) In particular, TAIII selectively kills various cancer cell types at certain concentrations.13–15) Mechanistically, TAIII triggers apoptosis by inducing cytochrome c release and caspase activation through overproduction of reactive oxygen species and mitochondrial dysfunction.16) Phosphorylation of c-Jun N-terminal kinase (JNK) and p38 by TAIII also causes caspase activation and poly ADP-ribose polymerase cleavage.17) Furthermore, by targeting the machineries that regulate cell cycle progression, TAIII may arrest cancer cells in G1 or G2/M phases of the cell cycle.13)
Compelling evidence indicates that TAIII inhibits cancer cell motility. TAIII modulates the mRNA expression and proteolytic activity of matrix metalloproteinase-2 and -9, which have major roles in cell migration and invasion.18–21) TAIII also modulates expression of microRNAs that suppress metastasis. In breast cancer cell lines, TAIII induces expression of miR-200c/141, a tumor-suppressive microRNA, and suppresses expression of its negative regulator, Bmi1.22) Furthermore, TAIII regulates miR-129-5p expression by inhibiting phosphatidylinositol 3-kinase (PI3K)–AKT signaling in renal carcinoma cells without affecting cell viability.23) Taken together, the current evidence indicates that TAIII exerts an anti-metastatic activity through transcriptional control of migration regulatory factors. However, it is unclear whether TAIII directly controls cellular machineries that generate migratory force and promote cancer cell invasion and metastasis.
In this study, we found that TAIII suppressed cellular events based on cell–ECM interactions, such as cell adhesion and spreading on an ECM. TAIII also inhibited internalization of cell surface proteins, including integrin β1. These findings explain how TAIII exerts its pharmacological effects, such as the anti-metastatic effect.
TAIII, timosaponin AI (TAI), and timosaponin BII (TBII) were purchased from Kanto Kagaku (Tokyo, Japan). Anemarrhenasaponin I (AMS) was from MedChemExpress (Monmouth Junction, NJ, U.S.A.). Sarsasapogenin (SRS) was from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Laminin-332 (LN332), fibronectin (FN), vitronectin (VN), poly L-lysine (PLL) were from ReproCELL (Yokohama, Japan), BD Biosciences (Franklin Lakes, NJ, U.S.A.), FUJIFILM Wako Pure Chemical Corporation, and Cosmo Bio Co., Ltd. (Tokyo, Japan), respectively.
Cell Culture and TransfectionHeLa cells (Kyoto) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS). For transfection, Lipofectamine LTX (Thermo Fisher Scientific, Waltham, MA, U.S.A.) was used in accordance with the manufacturer’s instructions. Expression plasmids for LifeAct-mCherry (Dr. Naoki Watanabe, Kyoto University) and constitutively active Val12 Rac1 have been described previously.24,25)
Cell Attachment Assay96-Well culture plates were coated with 2.5 µg FN, LN332, or VN in phosphate-buffered saline (PBS) overnight at 4 °C and then blocked with bovine serum albumin (BSA). Two point zero microgram PLL was coated overnight at 37 °C. HeLa cells were trypsinized and washed once with DMEM containing 10% FBS. After incubation for 30 min at 37 °C in the presence of TAIII, 8 × 104 cells were plated in each well and incubated for another 30 min. The culture plates were washed with PBS to remove unattached cells and then attached cells were fixed with 4% formaldehyde and stained with 5 mg/mL crystal violet for 10 min. After unbound dye was washed away, the cells were lysed with 2% sodium dodecyl sulfate (SDS) and absorbance at OD 590 was measured.
Cell Spreading AssayHeLa cells transfected with LifeAct-mCherry-expressing plasmids were trypsinized and washed once with DMEM containing 10% FBS. After incubation for 20 min at 37 °C, the cells were seeded on a glass-bottom dish or coverslip coated with FN (10 µg/mL) for time lapse imaging and immunostaining, respectively. Using the acquired phase contrast images, the cell periphery of more than 100 cells in each condition was drawn and the cell spreading area was measured by ImageJ software.
Immunofluorescence Analyses and Time-Lapse MicroscopyImmunofluorescence analyses were performed as described previously26) with minor modifications. Cells grown on cover slips were fixed with 4% formaldehyde in PBS containing 0.1% Triton X-100 for 10 min, blocked for 30 min with 2% dry skim milk in PBS, and then incubated for 1 h with a mouse monoclonal anti-vinculin antibody (hVIN-1; Sigma-Aldrich, St. Louis, MO, U.S.A.) diluted in blocking buffer. After three times washes with PBS for 5 min each, the cells were incubated for 30 min with Alexa Fluor 488-conjugated anti mouse immunoglobulin G (IgG) (Thermo Fisher Scientific) diluted in blocking buffer together with Texas Red™-X Phalloidin (Thermo Fisher Scientific). The cells were then washed with PBS and mounted, and immunofluorescence was observed under a confocal scanning laser microscope (SP8; Leica, Wetzlar, Germany). Images were processed using Adobe Photoshop software.
For time-lapse analysis, HeLa cells seeded on a 3.5-cm glass-bottom culture dish (Asahi Techno Glass Co., Tokyo, Japan) were observed under the confocal scanning laser microscope equipped with a temperature- and CO2-controlled stage-top live cell incubator (Tokai Hit, Fujinomiya, Japan).
Integrin Internalization AssayHeLa cells were plated on a FN-coated glass bottom dish and cultured overnight. Surface integrin β1 was labeled with an Alexa Fluor® 488 anti-human integrin β1 antibody (clone TS2/16, Biolegend) at 4 °C for 60 min. Then, the cells were incubated at 37 °C to allow internalization. Quantification was performed for one of three representative experiments. Mean pixel intensity from each cell was determined using ImageJ software.
Statistical AnalysisResults are presented as the mean ± standard error of the mean (S.E.M.) or standard deviation (S.D.). All statistical analyses were performed using the two-tailed Student’s t test.
In the previous study,25) we screened a natural compound library and identified activities that disturbs the morphological plasticity of the breast cancer cell line induced by LN332-mediated paracrine interactions with the mammary epithelial cell line. In this screening, we also found that TAIII (Fig. 1) exhibited a potential inhibitory activity in cell–ECM interactions. Therefore, we sought to examine the effect of TAIII in detail.
Structures of saponins and sapogenin isolated from Anemarrhena asphodeloides Bunge tested in this study.
Firstly, we performed a cell attachment assay using HeLa cells, a cervical carcinoma cell line, as a general model of cultured cells. HeLa cells were plated on culture dishes coated with FN in the presence of TAIII (Fig. 2a). After incubation for 30 min, cells associating with the ECM were stained with crystal violet and absorbance at OD 590 was measured. As shown in Fig. 2b, TAIII exhibited the significant inhibitory activity in a dose-dependent manner. Moderate inhibitory activity of TAIII was observed at a concentration of 2 µM (0.47 ± 0.06-fold relative to dimethyl sulfoxide (DMSO)), and the activity reached a maximum at 5 µM (0.22 ± 0.02-fold relative to DMSO). Moreover, TAIII suppressed adhesion of HeLa cells to other ECM molecules, such as LN332 and VN, at similar concentrations, while did not influence their attachment on PLL (Fig. 2b). These results demonstrated that TAIII interfered with cell–ECM interactions.
a. Schematic experimental procedure for cell attachment assay. HeLa cells were plated in a 96-well culture plate together with TAIII and incubated for 30 min. Then, cells were fixed and stained with crystal violet, and the absorbance was measured at 590 nm. b. HeLa cells were subjected to cell attachment assay with the indicated ECMs in the presence of TAIII. Each column shows the mean ± S.E.M. of three independent experiments. p-Value compared with cells treated with DMSO indicated as “-.” c. HeLa cells were subjected to cell attachment assay with the indicated saponins. Each column shows the mean ± S.E.M. of three independent experiments. p-Value compared with cells treated with DMSO indicated as “-.”
We further investigated the impact of TAIII on cell adhesion across various cell lines. TAIII markedly suppressed the adhesion of A549 cells, a lung adenocarcinoma cell line; A375 cells, a melanoma cell line; MDA-MB-231 cells, a breast adenocarcinoma cell line; and SH-SY5Y cells, a neuroblastoma cell line, to FN at levels comparable to those observed in HeLa cells (Supplementary Fig. S1). Moreover, TAIII demonstrated its inhibitory effects on the adhesion of MRC-5 cells, a lung fibroblast cell line (Supplementary Fig. S1), underscoring cell type-independent effects of TAIII on cell adhesion.
To determine whether TAIII exhibited its inhibitory activity as a saponin or sapogenin, we examined SRS (Fig. 1), the aglycone of TAIII, in terms of cell adhesion. Unlike TAIII, SRS did not show any inhibitory effects on cell adhesion (Fig. 2c), demonstrating that TAIII exerted its activity as a saponin on cell adhesion. We also evaluated the cell adhesion inhibitory activity of other saponins isolated from Anemarrhena asphodeloides Bunge (Fig. 1). TAI is a deglycosylated TAIII derivative that lacks a glucose on its saccharide moiety. TBII and AMS harbor identical steroid cores and disaccharide moieties to those of TAIII. However, an extra sugar moiety exists in TBII at the end of its steroidal side chain. Unlike TBII, AMS does not harbor any additional sugar moieties at the end of its steroidal side chain, but it has a hydroxyl group linked to the C-15 position of its steroid core. Among these saponins, we found a slight, but significant, inhibitory effect on cell adhesion when cells were treated with 10 µM TAI (Fig. 2c). Conversely, the other saponins showed no significant effects, demonstrating that the effect of saponins from Anemarrhena asphodeloides Bunge on cell adhesion depended on the number and position of saccharide moieties.
TAIII Interferes with Cell Spreading on the ECM and Membrane DynamicsTo confirm the effect of TAIII on cell–ECM interactions, we also examined spreading of TAIII-treated cells on the ECM. HeLa cells transfected with a plasmid carrying LifeAct-mCherry were plated on FN-coated glass-bottom dishes. After the cells had been attached to the dishes, their behavior upon TAIII treatment was examined by time lapse microscopy (Fig. 3a). After attachment to FN, DMSO-treated cells formed membrane ruffles and protrusions, followed by rapid spreading (Fig. 3b, Supplementary Movie 1). We also observed dynamic remodeling of the actin cytoskeleton in response to the interaction of DMSO-treated cells with FN (Fig. 3b, Supplementary Movie 1). Moreover, vinculin-positive structures were scattered along the cell edge (Fig. 3c). Conversely, TAIII-treated cells did not undergo membrane ruffling, and although they adhered to the culture dish after replating and formed filopodia-like protrusions, they did not spread on the dish (Fig. 3b, Supplementary Movie 2). Vinculin-positive structures in TAIII-treated cells were markedly smaller compared with those in DMSO-treated cells (Fig. 3c). Consistent with this finding, the area of TAIII-treated cells (674.2 ± 15.4 and 580.7 ± 27.4 µm2 in 5 and 10 µM TAIII-treated cells, respectively) was significantly smaller than that of DMSO-treated cells (994.1 ± 27.4 µm2), which was dependent on the TAIII concentration (Fig. 3d). These results supported the inhibitory effect of TAIII on cell–ECM interactions.
a. Schematic experimental procedure for the cell-spreading assays in HeLa cells treated with TAIII. b. HeLa cells transfected with LifeAct-mCherry expression plasmid were plated onto FN-coated dish. After attached, cells were treated with 10 µM TAIII and subjected to time lapse imaging. Fluorescence and phase contrast images were taken until 60 min after TAIII treatment. c. HeLa cells subjected to cell-spreading assay were fixed at 60 min after TAIII treatment and stained for vinculin and phalloidin. Bar; 20 µm. d. Quantification of cell spreading area at 60 min after TAIII treatment at the indicated concentrations. n > 60. Each column shows the mean ± S.D. p-Value compared with cells treated with DMSO indicated as “-.”
Because HeLa cells treated with TAIII did not show membrane ruffling or lamellipodia formation upon attachment to FN, we evaluated the effect of TAIII on membrane dynamics. HeLa cells were transfected with plasmids carrying green fluorescent protein (GFP) and LifeAct-mCherry, a widely used actin-binding peptide fused to mCherry,27) and their membrane behaviors were observed by time lapse imaging (Fig. 4a). Based on the GFP and LifeAct-mCherry fluorescence signals, HeLa cells before TAIII treatment continuously underwent membrane ruffling (Fig. 4b, Supplementary Movies 3, 4). However, these membrane structures disappeared 20 min after treatment with TAIII. Membrane ruffling briefly recovered after 30 min, but was completely suppressed until 60 min, followed by cell contraction. The suppression of membrane dynamics upon TAIII treatment was confirmed by kymographic analysis (Fig. 4b). Cell contraction induced by TAIII showed accumulation of vinculin-positive structures at the cell edge and expanded intercellular gaps. The other saponins did not exert such effects (Supplementary Fig. S2), supporting a unique effect of TAIII among saponins isolated from Anemarrhena asphodeloides Bunge.
a. Schematic experimental procedure for time lapse imaging of HeLa cells treated with TAIII. b. HeLa cells transfected with GFP and LifeAct-mCherry expression plasmids. At 16 h post-transfection, the cells were subjected to time lapse imaging in the presence of TAIII treatment (10 µM). Left: representative images of transfected cells after TAIII treatment (0 and 120 min); scale bar: 20 µm. Right: kymograph analyses at dotted lines in the left panels. c. HeLa cells transfected with GFP-tagged constitutively active Rac1 (Val12) and LifeAct-mCherry expression plasmids were subjected to time lapse imaging as in (b). Left: representative images of transfected cells after TAIII treatment (0 and 120 min); scale bar: 20 µm. Right: kymograph analyses at dotted lines in the left panels.
Membrane dynamics, such as membrane ruffling and lamellipodia formation, are regulated by Rac, a Rho family GTPase, which is activated by cell–ECM interactions.28) Thus, we examined the effect of TAIII on membrane dynamics in GFP-tagged, constitutively active Rac1 (GFP-V12 Rac1)-overexpressing HeLa cells. GFP-V12 Rac1 overexpression induced cell spreading and frequent membrane ruffling, followed by marked remodeling of the actin cytoskeleton at the cell edge (Fig. 4c, Supplementary Movies 5, 6). Remarkably, TAIII suppressed membrane ruffling within 20 min after treatment, even in V12 Rac1-expressing cells. Membrane ruffling was disrupted by TAIII treatment, while microspike formation was intact. Kymographic analysis supported the inhibitory effect of TAIII on membrane dynamics of Val12 Rac1-expressing cells (Fig. 4c).
TAIII Inhibits Integrin InternalizationNext, we investigated how TAIII disrupted cell–ECM interactions. Considering the effects of TAIII on cell–ECM interactions, we focused on integrins, which function as a receptor for ECM molecules and play critical roles in cell adhesion, and examined integrin β1 behaviors in response to TAIII treatment. Cell surface integrin β1 was labeled on ice with an Alexa Fluor 488-conjugated antibody, allowed to internalize at 37 °C, and then subjected to time lapse imaging (Fig. 5a). Intracellular fluorescence signals from traced integrin β1, which was transported via endosomes, were observed in cells treated with DMSO up to 20 min after incubation at 37 °C, and its fluorescence intensity increased over time (Figs. 5b, c, Supplementary Movies 7, 8). Conversely, intracellular signals of internalized integrin β1 in TAIII-treated cells were not detected after incubation at 37 °C, in which fluorescent signals of LifeAct-mCherry indicated cell contraction (Figs. 5b, c, Supplementary Movies 9, 10).
a. Schematic experimental procedure for the integrin internalization assay. b. HeLa cells expressing LifeAct-mCherry plated onto FN-coated dish were subjected to the integrin internalization assay. Cells were analyzed by time lapse imaging until 180 min after incubation at 37 °C (tracing). Representative images of transfected cells after DMSO and TAIII-treatment are shown. Scale bar: 20 µm. c. Quantifications of signal intensity of internalized integrin β1 after tracing. Intensity of Alexa Fluor 488 was normalized against levels at onset of tracing (0 min). Data are presented as the mean ± S.E.M. (n = 20). p < 0.005 after 60 min.
We also examined whether TAIII blocked internalization of other cell surface proteins. To this end, we performed a transferrin uptake assay with fluorescence-labeled transferrin (Supplementary Fig. S3a), because the transferrin receptor has been well established as a prototype cell surface receptor that is continuously internalized.29) In control cells, clear intracellular signals from Alexa Fluor 448-labeled transferrin were observed (Supplementary Fig. S3b, Supplementary Movie 11). However, these signals were not detected in TAIII-treated cells (Supplementary Fig. S3b, Supplementary Movie 12). Collectively, the results suggested that the effect of TAIII on the regulation of internalization of cell surface proteins, such as integrin β1, contributed in part to its inhibitory activity against cell adhesion.
We found that TAIII disrupted cell–ECM interactions as indicated by cell adhesion and spreading. TAIII could suppress adhesion of various types of cancer cells, but also noncancerous cells. Furthermore, TAIII suppressed membrane dynamics and induced cell contraction. Although the underlying mechanisms remain incompletely understood, we found that TAIII inhibited internalization of cell surface proteins, including integrin β1, that play a critical role in cell–ECM interactions. Our data provide a possible explanation for how TAIII exerts pharmacological activities, including an anti-metastatic activity, by regulating internalization of cell surface proteins.
Cell–ECM interactions are predominantly mediated by adhesion complexes containing integrins.30) Adhesion complexes containing integrins have substantial interactions with the ECM. Integrins are internalized and sorted to recycling endosomes to be recycle back to the plasma membrane, or targeted to late endosomes and lysosomes for degradation. Dynamic turnover through the endosomal traffic system is required to maintain integrin functions.4–6) Therefore, TAIII could diminish integrin functions by blocking the initial step of their turnover. The fact that TAIII suppressed membrane ruffling and lamellipodia formation induced by overexpression of constitutively active Rac1 might provide an alternative idea that TAIII targets downstream of Rac, such as WAVE2 and IRSp53.31–33) However, it has been reported that pharmacological inhibition of the endocytosis pathway suppresses lamellipodia formation,34) and that integrin-mediated cell adhesion serves as a platform to induce and stabilize lamellipodia formation.35,36) Integrins associate with various adaptor/scaffold proteins, which mediate linkages to the actin cytoskeleton, thereby generating cellular tension to regulate cell morphology.30) Cellular tension associated with adhesion complexes is also required for cell mobility. Because spatiotemporal regulation of adhesion complexes through trafficking systems plays a pivotal role in the regulation of cell migration,35) the effect of TAIII on internalization may contribute to its inhibitory activity against cell migration. A previous report demonstrates that TAIII regulates integrin expression.37) Collectively, TAIII may alter the generation of migratory force by regulating integrins in multifaceted manners. Furthermore, heparan sulfate proteoglycans (HSPGs), such as syndecans, which are ubiquitously expressed in mammalian cells including HeLa cells,38) act as adhesion molecules involved in the regulation of cell–ECM interactions. Therefore, the modulation of HSPG functions by TAIII is likely to contribute to its anti-metastatic activity.
TAIII interfered with cell–ECM interactions not only in several lines of cancer cells, but also noncancerous cells. A recent study has demonstrated that TAIII inhibits platelet aggregation induced by U46619 in vitro and prevents thrombus formation in vivo.39) Anti-platelet and anti-thrombotic activities of TAIII are reported to be mediated by decreased ADP secretion caused by suppression of thromboxane A2 receptor activity and the Gq signaling pathway. Apart from the suppression of ADP secretion, blockade of integrin activation is an important strategy to interfere with platelet crosslinking and platelet-derived thrombus formation. In fact, inhibitors of glycoprotein IIb/IIIa, an integrin complex on platelets, such as abciximab, eptifibatide, and tirofiban, are used clinically.40) Therefore, we speculate that the effects of TAIII on internalization of cell surface protein might, in part, contribute to its anti-platelet and anti-thrombotic activity. TAIII also exerts anti-angiogenesis effects through inactivation of vascular endothelial growth factor (VEGF) signaling.15) Receptor internalization and subsequent cytoplasmic trafficking are required for activation of signaling cascades mediated by proangiogenic growth factors in endothelial cells in vitro and in vivo.41) Our observations might give an explanation to the pharmacological activity of TAIII in the context of endocytic regulation of signal transduction.
An important issue is how TAIII blocks internalization of cell surface proteins. We did not identify targets of TAIII that may be involved in the inhibition of internalization. To our knowledge, little is known about cell surface targets of TAIII. TAIII has been suggested to direct membrane-associating proteins by computational docking approaches,42) implying the existence of TAIII targets on the cell surface in blockade of the endocytic pathway. It has been reported that diosgenin, a steroidal saponin which is one of major constituents in Dioscorea rhizome, interacts with 1,25D3-MARRS (membrane associated, rapid response steroid-binding), a vitamin D receptor, on the cell surface.43) Additionally, digoxin and oubain, well-established cardiac glycosides, bind to Na+, K+-ATPase and show its enzymatic activity.44) These evidences suggest a possibility that TAIII associates with membrane machineries involved in cell surface protein internalization. Moreover, abrogation of endocytic trafficking perturbs internalization kinetics.45) Thus, TAIII may suppress internalization of cell surface cargoes by disrupting the regulatory machineries of vesicular trafficking. For more than two decades, efforts to fully elucidate the endocytic trafficking network have identified various membrane-associated proteins involved in integrin trafficking.46) Identification of the molecular targets of TAIII may further expand our knowledge of the regulatory mechanisms for cargo transport.
Alternatively, TAIII might affect plasma membrane properties, including fluidity. Some kinds of saponins, such as alpha-hederin,47,48) digitonin,49,50) and alpha-tomatine51,52) exert their activities by associating and complexing with cell membrane cholesterol. The activity of ginsenoside Rh2 is enhanced by an interaction with membrane sphingomyelin.53) Cholesterols and sphingolipids accumulate in caveolae microdomains, which are indispensable for caveolin-dependent endocytosis of cargoes, including integrins.54,55) Thus, TAIII might interact with membrane components, thereby inhibiting endocytosis.
The inhibitory effect of TAIII on cell–ECM interactions was unique among the saponins from Anemarrhena asphodeloides Bunge tested in this study. Comparisons of their structures and activities revealed that the disaccharide moiety was required for TAIII to exert its effect on cell–ECM interactions. The association between the structure and biological activity of TAIII has been linked to its cytotoxicity in cancer cell lines.56) A major difficulty in research on bioactive natural compounds is target identification.57) Identifying the specific targets of bioactive natural compounds is of great benefit to clarify their mechanisms of action and optimize compounds to improve their pharmacological activities. Traditionally, chemical probes such as biotin are used to modify proteins and bioactive natural compounds. Regarding TAIII, the galactose moiety would be an attractive target for modification. This strategy would help to identify TAIII targets in the blockade of internalization. Furthermore, fluorescent labeling would be a powerful method to perform high-resolution imaging to reveal colocalization of TAIII with integrins at the peripheral membrane and in trafficking vesicles. It would be necessary to clarify dynamics of TAIII in the plasma membrane and cytoplasm in the inhibition of cell surface protein internalization.
Herbal medicines have been used as therapies for various diseases. Although numerous compounds have been isolated from herbal medicines, it is poorly understood how they exert their pharmacological effects. Here, we found that TAIII disrupted integrin functions, at least in part, by blocking internalization of cell surface proteins, including integrins. Further studies are needed to uncover the mechanisms by which TAIII regulates the internalization of cell surface proteins, which is important to elucidate its pharmacological properties.
We would like to thank Dr. Naoki Watanabe, Kyoto University, for a kind gift of plasmid for LifeAct-mCherry. We also like to thank Mitchell Arico for editing a draft of this manuscript. This study was performed as a part of the Cooperative Research Project with Institute of Natural Medicine, University of Toyama in 2017.
Takeshi Terabayashi was supported by JSPS KAKENHI Grant Number: 19K08776. Toshimasa Ishizaki was supported by JSPS KAKENHI Grant Number: 17K08339.
The authors declare no conflict of interest.
This article contains supplementary materials.
