Anticancer mechanism of 7-α-hydroxyfrullanolide on microtubules and computational prediction of its target binding in triple-negative breast cancer cells

Plant material preparation and 7HF isolation

Dried G. maderaspatana (L.) Poir plants were purchased from the Chaokromper Drug Store (Bangkok, Thailand). The plant material was prepared, and 7HF was isolated as described previously (Chimplee et al., 2022). The chemical structure of 7HF is shown in Fig. 1A. 7HF was stored at −20 °C.

Cancer cell culture

The MDA-MB-468 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) with condition as described previously (Chimplee et al., 2019; Chimplee et al., 2022).

7HF uptake assay

7HF (125.85–4,027.06 µM) dilutions in dimethyl sulfoxide (DMSO) were scanned to identify the maximum absorbance (λ_max) between 190 and 1,100 nm, using a UV-visible spectrophotometer (Shimadzu, Kyoto, Japan). The λ_max was obtained at 268 nm and at a concentration of 1,006.77 µM (Fig. 1B and Fig. S1). To test whether 7HF was able to pass through the TNBC cells, the cells were treated with 1,006.77 µM 7HF. MDA-MB-468 cells (3 × 10⁴ cells/well) were seeded on 96-well plate and incubated with the DMEM medium overnight. Then, 100 µL of 1,006.77 µM 7HF was used to treat the TNBC cells for 0.25–24 h at 37 °C in a 5% CO₂ incubator. In case of 7HF cells-free medium, 1,006.77 µM of 7HF was prepared with only medium. After incubation, the remaining of 7HF supernatant in cells culture medium and cells-free medium was immediately collected without washing with buffer. The absorbance of 7HF was then measured at 268 nm and the absorbance was plotted versus time (Fig. 1D and Table S2). The mean absorbance values of 7HF were subtracted from the absorbance value of only medium and DMSO-supplemented medium (in cells-free medium), and cells medium and cells-DMSO medium (in cells-culture). The experiment was performed in triplicate. In addition, a concentration curve of 7HF was plotted on a linear scale at 268 nm and concentrations ranging from 125.85 to 4,027.06 µM (Fig. 1C and Table S1). The linear regression coefficients were calculated using the general equation of the calibration curve (y = ax + b; a = 0.1646, b =  − 0.039, and R² = 0.9861) (Fig. 1C).

Immunofluorescence (IF) assay

The effect of 7HF on MT dynamics in the TNBC cells was visualized using indirect IF, with certain modifications (Duangmano et al., 2012). In brief, MDA-MB-468 cells (1.5 × 10⁵ cells/well) were seeded on cover slips and incubated at 37 °C in a 5% CO₂ incubator overnight. After washing with phosphate-buffered saline (PBS), the TNBC cells were treated with 6, 12, and 24 µM 7HF for 1, 12, and 24 h. The 7HF concentrations were obtained from 0.5X, 1X, and 2X its IC₅₀ by MTT assay at 72 h, as reported previously (Chimplee et al., 2022). Paclitaxel (PTX), vincristine (VCR), and doxorubicin (DOX), 200 nM each, were also used to treat the TNBC cells for 24 h, with minor adaptation (Blajeski et al., 2002; Mukhtar, Adhami & Mukhtar, 2014). The control was untreated cells with indicated concentrations of 7HF and chemotherapies. Then, the cells were fixed with 4% formaldehyde, permeabilized with 0.5% Triton X-100, and blocked in 1% bovine serum albumin (BSA) in PBS. After washing the cover slips, the untreated and treated cells were stained overnight with anti- α-tubulin antibody (Cell Signaling Technology, Inc., Danvers, MA, USA; 1:50) and DAPI (Sigma-Aldrich, St. Louis, MO, USA; 1:1,000), followed by incubation with Alexa Fluor 488-conjugated goat anti-IgG rabbit secondary polyclonal antibody (GE Healthcare Ltd., Little Chalfont, Bu, UK; 1:200) for 1 h. Finally, the stained coverslips were mounted with 50% glycerol and sealed on a glass slide. The slides were visualized under an inverted fluorescence microscope (EVOS, Thermo Fisher Scientific Inc.).

Tubulin polymerization assay in cell-free system

The effects of 7HF on tubulin polymerization were analyzed using the tubulin polymerization assay kit (Cytoskeleton Inc., Denver, CO, USA). The assay was performed following the manufacturer’s instructions. Briefly, 2 mg/mL tubulin was diluted using tubulin polymerization buffer. After pre-warming of empty dark 96-well plate at 37 °C in a Spark microplate reader (Tecan, Männedorf, Switzerland) for 5 min, 5 µL 7HF (6, 12, and 24 µM), PTX (3 µM), VCR (3 µM), DOX (3 µM), and tubulin control were added, and the plate was incubated for 1 min, after which 50 µL of tubulin reaction reagent was manually injected into the microplate reader immediately. The effect of reagents on tubulin polymerization was then measured at 360 and 450 nm at 37 °C, every minute up to 1 h in kinetic mode.

Cell proliferation assay using fluorescence-activated cell sorting (FACS)

To study the effect of MT dynamics on DNA damage in 7HF-treated cells, we first treated TNBC cells with anti-bromodeoxyuridine (BrdU) and then incubated them with propidium iodide (PI) to stain the newly synthesized DNA. The protocol for this experiment was adapted from Cecchini, Amiri & Dick (2012) and Zhu (2012), with some modifications. The MDA-MB-468 cells (1 × 10⁶ cells/plate) were cultured and pulsed with 25 µM BrdU (Cayman Chemical, Ann Arbor, MI, USA) for 30 min. Untreated and 7HF-treated cells (6, 12, and 24 µM) were harvested after 12 and 24 h. Then, 1 × 10⁶ cells were counted. After centrifugation (7,000 rpm, 5 mins), the cell pellets were resuspended in 2 mM EDTA, fixed with 70% EtOH, and stored overnight at −20 °C. Then, the cells were harvested and resuspended in washing buffer (0.5% BSA in PBS). After centrifugation (10,000, 2 mins), the cell pellets were resuspended in 2 M HCl and sodium borate (pH 8.5) solution and incubated for 30 min. The cell pellets were washed with washing buffer containing 0.2% Tween-20. After centrifugation (10,000 rpm, 2 mins), anti-BrdU primary antibody (20 µL) and RNase (200 µg/mL) were added, and the cells were incubated overnight at 4 °C. Then, Alexa Fluor 488-conjugated goat-anti-IgG mouse secondary antibody (Thermo Fisher Scientific, Inc.; 50 µL of 50 µg/mL) and PI (Sigma-Aldrich; 20 µg/mL) were added, and the cells were incubated for 1 h prior to injection in the FACS machine (Becton Dickinson, San Jose, CA, USA). Density plots were generated using the CellQuestPro software (Becton Dickinson).

Western blot analysis

To determine the effect of 7HF on the expression of proteins involved in MT dynamics and DNA damage response (DDR) pathways, MDA-MB-468 cells (3 × 10⁵ cells/well or 1 × 10⁶ cells/plate) were grown at 37 °C in a 5% CO₂ incubator overnight. The cells were treated with 6 µM 7HF for 0, 12, 24, and 48 h. For optimizing 7HF concentration, the cells were incubated for 24 h in the presence of 0, 6, (or 12), and 24 µM 7HF. Chemiluminescence-based western blotting was performed as described previously (Chimplee et al., 2022). Briefly, 50 µg protein was separated electrophoretically 12% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and transferred onto nitrocellulose membrane (Chimplee et al., 2022). The blots were blocked and incubated overnight with 1;1,000 dilutions of various primary antibodies (anti-cyclin B1, anti-p-Cdk1 (Tyr15), anti-Bub3, and anti-β-actin; Cell Signaling Technology, Inc.) at 4 °C. After washing, the blots were incubated with horseradish peroxidase-conjugated anti-rabbit IgG (GE Healthcare Ltd; 1:5,000) for 1 h.

For fluorescence-based western blotting, the protein samples were extracted using radioimmunoprecipitation assay buffer (Tris pH 7.5, NaCl, EDTA, EGTA, NP-40, sodium deoxycholate, sodium pyrophosphate, β-glycerophosphate, and protease inhibitor cocktail) and quantified using the Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA). Then, 50 µg protein was separated on SDS-PAGE gels (8%, 12%, and 15% variation with molecular weight of protein) and blotted onto nitrocellulose membranes at 100 V for 2 h. Apart from phosphoproteins for which BSA was used for blocking, the blots were blocked with blocking buffer (5% low-fat dry milk in Tris buffered saline) for 1 h. After washing, the blots were incubated with various primary antibodies for Rb, p-Rb (Ser780), Chk2, p-Chk2 (Ser516), and β-actin (Cell Signaling Technology, Inc.), Chk1, p-Chk1 (Ser345) (kindly provided by Prof. Dr. Carl Smythe), and p-H2AX (Ser139) (EMD Millipore Corp., Billerica, MA, USA) overnight 4 °C. The following antibody concentrations were used: 1:500 for anti-p-Chk2, 1:1,000 for anti-p-Rb, anti-Chk1, anti-p-Chk1, and anti-p-H2AX, 1:2,500 for anti-Rb and anti-Chk2, and 1:5,000 for anti-β actin. The blots were incubated for 1 h in 1:1,000 dilutions of secondary IgGs with various hosts and fluorophore labels, including anti-sheep (for 680 nm), anti-mouse (for 800 nm), and anti-rabbit (for 680 nm) antibodies (LI-COR Biosciences, Lincoln, NE, USA). Finally, the protein bands were observed using a chemiluminescent detection kit (Thermo Fisher Scientific, Inc.) and the Fusion FX western blot imaging system (Vilber Lourmat Sté, Collégien, France). The relative protein expression was quantified using the Fusion Capt Advanced Quantitation Analysis program (Vilber Lourmat Sté). Fluorescence-based protein bands were observed using the Odyssey imaging system (LI-COR Biosciences) and relative expression was calculated using the Image Studio Lite 5.2 program (LI-COR Biosciences). Two-three independent experiments were performed.

Prediction of 7HF drug-likeness

To predict the 7HF drug design, 7HF structure (PubChem CID 11983230) was submitted to the Swiss-absorption, distribution, metabolism and excretion-(ADME) web tool (http://www.swissadme.ch, accessed on 07 December 2021) (Daina, Michielin & Zoete, 2017).

Molecular docking analysis

Molecular docking was done to predict whether 7HF bound to the target protein, as per a previous study with certain modifications (Saechan et al., 2021). The PDB ascension number 5SYF represents the three-dimensional (3D) protein structure of α-, β-tubulin (Kellogg et al., 2017). The AutoDock Tool (ADT) 4.1 was used to eliminate all water molecules. The other co-crystallized ligands were retained. The PDB file format contains all polar hydrogen atoms that emulate hydrogen bond interactions. The 3D structures of 7HF (CID 11983230), pironetin (CID 6438891), PTX (CID 36314), and VCR (ID 5758) were obtained from the PubChem and ChemSpider databases. The SMILES Translator and Structure File Generator (https://cactus.nci.nih.gov/translate/) created PDB files for 7HF, pironetin, PTX, and VCR. All polar hydrogen atoms were added using ADT. Finally, the protein and ligand structures were saved as PDB and Partial Charge (Q) and Atom Type (T) or PDBQT files.

All ligands were docked with the tubulin proteins using AutoDock4 version 4.2 (Morris et al., 2009). Using the SG atom of 315^th-cysteine residue (Cys315) as the interaction point with pironetin, the grid box was centered at an x-y-z position of “329.387 449.699 378.122” with a grid size of 90 × 90 × 90 cubic angstrom (ų) (Prota et al., 2016; Steinmetz & Prota, 2018; Yang et al., 2016). The grid box center of PTX and VCR was as per previous reports (Field Jessica, Díaz José & Miller John, 2013; Gigant et al., 2005). In total, 50 docking runs with a population of 200 were required. The default parameters of the AutoDock4 program were used. The quintuple independent samplings reported the lowest binding energy (ΔG_bind). The drug-tubulin complex structure was visualized using the Visual Molecular Dynamics (VMD) package (Humphrey, Dalke & Schulten, 1996). The ligand-protein interactions were then studied and visualized using the Protein-Ligand Interaction Profiler (PLIP) webtool (Adasme et al., 2021).

Statistical analysis

Each experiment was performed thrice. Data were analyzed using one-way analysis of variance (ANOVA) and two-way ANOVA using GraphPad Prism 5 (GraphPad Software, San Diego, CA, USA). The differences were considered significant at p < 0.05.

7HF uptake in MDA-MB-468 cells

In cells-free medium, 7HF was stable from 15 min to 17 h, and the stability gradually reduced up to 24 h (Fig. 1D and Table S2). In contrast, 7HF could not be detected between 5 h and 17 h in the cells culture medium (Fig. 1D and Table S2). 7HF may have penetrated MDA-MB-468 cells within 5 h and was internalized by 12 h.

Effect of 7HF on microtubule dynamics, tubulin polymerization, and protein expression

To determine the effect of 7HF on spindle MT arrangement, α-tubulin staining (shown in green) was visualized using IF (Fig. 2A). We observed that the spindle MT arrangement differed from that of the control when treated with 6–24 µM 7HF, excepting at 24 h. The radial array of the spindle gradually reduced after 1 h. The fibrous density along the spindle length was differed between control and 12 h 7HF. Radial lengthening of the spindle array was insignificantly observed from that of control at 24 h. We also observed time-dependent changes (1–24 h), i.e., the spindle MT length ranged from short radial arrays to long fiber when treated with 6–24 µM 7HF. Notably, maximum reduction in the number of α-tubulin-presenting cells was observed at 12 h for all 7HF concentrations. Spindle length increased in PTX- and DOX-treated cells in both the fibrous dense bipolar poles and radial arrays (Fig. 2B). The VCR-treated cells showed decrease in spindle length in radial array arrangements. A reduction in α-tubulin-presenting cell aggregation was observed after treatment with all chemotherapeutics (PTX, VCR, and DOX). Additionally, structural changes in nuclear DNA (blue) were observed, with more expansion in 12 µM 7HF treated-cells and minimal shrinkage in PTX- and DOX treated-cells after 24 h (Figs. 2A–2B)

To determine the effect of 7HF on spindle MT formation, α-, β-tubulin polymerization was examined in a cell-free system for 1 h (Fig. 2C and Spreadsheet S1). All concentrations of 7HF (6, 12, and 24 µM) affected tubulin formation. For all 7HF concentrations, tubulin nucleation occurred for 10 min, followed by tubulin growth till 40 min and tubulin equilibrium. After 10 min of nucleation, all 7HF concentrations triggered faster tubulin polymerization than the control. However, tubulin polymerization and relative fluorescence intensity was reduced in a 7HF concentration-dependent manner. Tubulin polymerization was relatively increased by DOX, and PTX and reduced by VCR treatments. PTX stimulated tubulin nucleation within 5 min (fastest among the three drugs), followed by tubulin growth till 40 min, while maximum suppression of tubulin formation was observed in case of VCR.

The expression of proteins involved in MT dynamics at the G2/M phase arrest was examined in 7HF-treated cells both with respect to 7HF concentration and time of incubation (Figs. 2D–2E). For assessing concentration dependence, the cells were incubated with 0, 6, 12, and 24 µM 7HF for 24 h (Fig. 2E and Fig. S2B). Bub3 was significantly upregulated in 6 µM 7HF-treated cells compared to that in the control. Similarly, upregulation of cyclin B1 and p-Cdk1 (Tyr15) was observed in concentration-independent and -dependent manners, respectively. For assessing time dependence, the cells were treated with 6 µM 7HF for 0, 12, 24, and 48 h (Fig. 2D and Fig. S2A). Bub3 was significantly upregulated between 12–24 h. Cyclin B1 was significantly upregulated at 12 h. In addition, p-Cdk1 (Tyr15) was significantly upregulated between 12 and 24 h and gradually downregulated at 48 h. All comparisons were made with the control group.

Effect of 7HF on cell proliferation and expression of DDR proteins

The effect of MT dynamics on cell proliferation and DNA synthesis in 7HF-treated cells was determined using BrdU and PI staining, and FACS analysis. The cells were treated with various concentrations of 7HF (6, 12, and 24 µM) for 12 and 24 h (Figs. 3A–3C and Fig. S3); 7HF-concentration dependence analysis at 12 h (Figs. 3A–3B) revealed that the number of G1 phase cells was significantly higher at 6 µM (45.61%) than that of the control (30.23%). The number of S phase cells increased gradually in a concentration-dependent manner from 6 to 24 µM (29.32%–34.89%), but was lower than that of the control (44.76%). The number of cells in the G2 phase was significantly higher than that in the control (20.07%) and increased in a concentration-dependent manner at 6 µM (26.68%), 12 µM (31.24%), and 24 µM (32.14%). Concentration dependence analysis at 24 h (Figs. 3A and 3C) revealed that the number of cells in the G1 phase was significantly higher than that in the control (33.86%) and increased in a concentration-dependent manner from 6 to 24 µM (51.56–54.87%). The number of S phase cells was clearly lower than that in the control (49.19%) when treated with 6–24 µM 7HF (34.64–24.55%), although the number of cells treated with 24 µM 7HF (22.64%) was higher than that in the control (15.11%). The proportion of cells in the G2 phase appeared independent of time of treatment (12–24 h). In contrast, the proportion of cells in the G1 phase occurred in a time-dependent manner (Figs. 3B–3C). Hence, we concluded that 7HF triggers G2/M and G1 phase arrests in concentration- and time-dependent manner.

7HF treatment significantly increased the number of G1 phase cells at 24 h (Fig. 3C). DDR proteins such as Rb, p-Rb (Ser780), Chk1, p-Chk1 (Ser345), Chk2, p-Chk2 (Ser516), and p-H2AX (Ser139) were subsequently measured in the cells treated with 6 and 24 µM 7HF for 24 h, using western blotting (Fig. 3D and Fig. S4). However, the relative levels of Rb and p-Rb were not detected. Compared to that in the control, Chk1 and p-Chk1 were significantly upregulated, while Chk2 and p-Chk2 were significantly downregulated in a 7HF-concentration-dependent manner in the treated cells. p-H2AX expression was insignificantly differed in 7HF-treated group of 6 and 24 µM.

Drug-likeness properties of 7HF

The Swiss ADME analysis of 7HF revealed its physicochemical properties, pharmacokinetics, drug-likeness, and medicinal properties. 7HF showed predicted drug-likeness, as it followed the Lipinski’s rule of Five (Lipinski et al., 2001; Yang & Hinner, 2015): MW 248.32, 3 H-bond acceptors, 1 H-bond donor, Clog P 2.46, and high gastrointestinal (GI) absorption.

Predicted interaction of 7HF with α- and β-tubulins

Molecular docking was used to predict whether 7HF targeted α- or β-tubulins of MTs. 7HF and pironetin bind to α-tubulin with relative binding energies (ΔG_bind) of −6.77 and −5.45 kcal/mol, respectively (Table 1). However, 7HF, PTX, and VCR bind to β-tubulin with ΔG_bind values of −7.12, −8.05, and −7.09 kcal/mol, respectively (Table 1). 7HF bound to both α- and β-tubulin sites, which was distinct from that observed with pironetin (a known α-tubulin inhibitor) and PTX and VCR (also known β-tubulin inhibitors), as shown in Fig. 4A. Table 1 and Figs. 4B–4C show 7HF-inhibitor interactions in terms of amino acid residues of various tubulin proteins. The initial binding site was at α-tubulin (Fig. 4B). A hydroxyl group (-OH) at C-7 in 7HF interacted with α-tubulin via hydrogen bonds (H-bond) with Ile341 and Phe343. An additional hydrophobic interaction with Lys336 and Phe343 further stabilized the 7HF binding. Compared to 7HF, pironetin formed H-bonds with Cys315, and backbone atoms of Thr257 and Val260. The neighboring amino acids such as Trp346, Ala314, and Lsy352 also supported the binding via hydrophobic interactions.

7HF also interacted with β-tubulin (Table 1 and Fig. 4C). A lactone ring established a salt bridge with Arg215. The C-12 ketone (C =O) formed H-bonds with Asp211. A hydroxyl (-OH) group at C-7 formed H-bonds with Ala298 and Met301. Some amino acids, such as Ala208, Ala298, Asp211, Asp306, and Ile212, exhibited hydrophobic interactions with 7HF. His229 and Arg278 carboxylic acids formed salt bridges to PTX. The PTX-protein complex displayed π–π stacking with His229. Furthermore, hydrophobic interactions with Leu217, Leu219, Leu230, Leu275, Leu 371, Asp226, Ala233, Phe272, and Arg278 contributed to PTX-β-tubulin binding. VCR-tubulin formed a salt bridge at Gln71 tetramine, and its H-bonds were formed by VCR. Amino acids such Asn101, Val182, Phe404, Trp407, and Tyr408 assisted in VCR binding via hydrophobic interactions.