The cytotoxicity effect of 7-hydroxy-3,4-dihydrocadalene from Heterotheca inuloides and semisynthetic cadalenes derivates towards breast cancer cells: involvement of oxidative stress-mediated apoptosis

Reagents and chemicals

RPMI 1640 medium, gentamicin, trypsin, phosphate-buffered saline (PBS) and fetal bovine serum (FBS) were acquired from Gibco (Invitrogen, Carlsbad, CA, USA). Isolated compounds 7-hydroxy-3,4-dihydrocadalene (>98.0%), 7-(phenylcarbamate)-3,4-dihydrocadalene (77% yield) and 7-(phenylcarbamate)-cadalene (79% yield) were generously provided by Dr. Guillermo Delgado Lamas (Instituto de Química, UNAM). Their isolation or preparation and structural characterization are described by Egas et al. (2017).

Dimethyl Sulphoxide (DMSO), H₂O₂, 2′,7′-Dichlorofluorescein Diacetate (DCFH-DA), 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), Bovine Serum Albumin (BSA), 2-thiobarbituric acid, antimycin A, oligomycin, rotenone, RIPA buffer, phosphatase inhibitor cocktails, bicinchoninic acid reagent, Tris Buffered Saline-Tween 20, GAPDH antibody (G-8795), caspase 3 (sc-7148) and Bcl-2 (sc-492) antibodies were purchased from Sigma-Aldrich (St. Louis, MO, USA). Protease inhibitor cocktail from Roche (Lewes, UK). Polyvinylidene difluoride (PVDF) membranes from GE Healthcare (Buckinghamshire, UK).

Cell culture

MCF7 (#HTB-22) cell line, derived from human breast adenocarcinoma, was obtained from the American Type Culture Collection (ATCC®, Manassas, VA, USA). It was maintained under standard conditions: RPMI-1640, 2.0 g/L NaHCO₃, supplemented with 10% FBS and gentamicin 50 mg/mL.

HEK 293 (CRL-1573) cell line, human embryonic kidney, was obtained from ATCC® (Manassas, VA, USA). It was grown in DMEM medium (Caisson Laboratories, Inc., Smithfield, UT, USA) with 10% v/v phenol red of inactivated FBS, 44 mM NaHCO₃, 1 mM sodium pyruvate and penicillin 100 I.U.−100 μg/mL streptomycin−25 ng/mL amphotericin B, pH 7.4. Both cell lines were incubated at 37 °C and 5% CO₂.

MTT assay: cell viability (mitochondrial activity) determination

Cells were treated with isolated compounds (0.1, 1, 5, 10, 25, 50 and 100 µM) for 24, 48 and 72 h. After treatments, culture media was replaced by MTT (100 μL) dissolved in RPMI media (0.33 mg/mL final concentration) per well and incubated at 37 °C with 5% CO₂ for 1 h in dark. After incubating, supernatant was removed and replaced with DMSO to dissolve formazan crystals. Absorbance value was measured in a microplate reader (SPECTROstar Nano; BMG Labtech, Ortenberg, Germany) at 570 nm. IC₂₅ and IC₅₀ values were determined using GraphPad Prism 7.00 (GraphPad Software, La Jolla, CA, USA). Percentage of cell viability was calculated in comparison with control cells (Mosmann, 1983).

In silico assay

DataWarrior software (v4.7.2; https://openmolecules.org/datawarrior/) was used to predict the physicochemical properties molecular weight (M_W), partition coefficient molecular weight (logP), aqueous solubility distribution coefficient at pH = 7.4 (logD), number of hydrogen donors (HBD), number of hydrogen bond acceptors (HBA), topological polar surface area (TPSA) and number of links that can be rotated (n_Rot) of isolated compounds.

Trypan blue dye exclusion assay: cell proliferation quantification

Proliferation was evaluated using trypan blue dye exclusion assay at different incubation times (0, 2, 4, 8, 24, 48 and 72 h) employing IC₅₀ (55.24 μM) and IC₅₀/2 (27.62 μM) value of 7-hydroxy-3,4-dihydrocadalene. Cells were trypsinized, centrifuged and stained with trypan blue dye (0.4% w/v in PBS). The number of viable cells and dead cells were counted on a Neubauer hemocytometer under an inverted microscope. The percentage of cell proliferation was calculated by comparing with control cells.

Wound-healing assay: cell migration measure

Cells were seeded in triplicate in six-well plates under standard conditions until 90–100% confluence was reached, followed by wounding the monolayer by scratching with a micropipette tip, then cells were treated with H₂O₂ 1.0 mM, IC₅₀ (55.24 μM) and IC₅₀/2 (27.62 μM) of 7-hydroxy-3,4-dihydrocalene compound in RPMI medium supplemented with 1% FBS. Wound areas were monitored at four different times (0, 24, 48, and 72 h) using the Cytation 5 cell imager (BioTek Instruments, Inc., Winooski, VT, USA). To determine the migration rate of the cells, the wound areas were quantified using the ImageJ software, (Schneider, Rasband & Eliceiri, 2012). The variation between the percentage of cell migration (as a function of time) was calculated using the following formula:

$$\mathrm{\%}\mathrm{M}\mathrm{i}\mathrm{g}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}=100-[(\mathrm{A}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{o}\mathrm{f}\mathrm{i}\mathrm{n}\mathrm{j}\mathrm{u}\mathrm{r}\mathrm{y}\mathrm{t}\mathrm{i}\mathrm{m}\mathrm{e}\mathrm{t}/\mathrm{A}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{o}\mathrm{f}\mathrm{i}\mathrm{n}\mathrm{j}\mathrm{u}\mathrm{r}\mathrm{y}\mathrm{t}\mathrm{i}\mathrm{m}\mathrm{e}\mathrm{z}\mathrm{e}\mathrm{r}\mathrm{o})(100)]$$

2′,7′-dichlorofluorescein diacetate (DCFH-DA) assay: intracellular ROS detection

Intracellular ROS were detected by fluorescent compound DCFH-DA. Cells were treated with 1.0 mM H₂O₂, IC₅₀ (55.2 μM) and IC₅₀/2 (27.6 μM) of 7-hydroxy-3,4-dihydrocadalene for 48 h. Then, culture media was replaced with DCFH-DA (20 μM dissolved in glucose 1.8 mg/mL PBS) per well and incubated in the dark for 30 min. DCF fluorescence intensity was measured using an FLx800 microplate fluorescence reader (Bio-Tek Instruments, Inc., Winooski, VT, USA) at 480 and 530 nm excitation and emission wavelengths, respectively (LeBel, Ischiropoulos & Bondy, 1992).

Pellet harvest and total extract

Mechanical scraping was employed to collect adherent MCF7 cells after treatments. Then, cells were centrifugated (1,500 rpm for 5 min at 37 °C) and supernatants were removed. Pellets were lyzed adding lysis buffer (25 mM TrisHCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% Triton X-100, 1 mg/mL leupeptin, 0.5 mM PMSF and 1 mg/mL pepstatin) and put on ice for 20 min. Finally, cell lysates were centrifugated (2,500 rpm for 10 min at 4 °C) and supernatants were collected to following assays.

Bicinchoninic acid assay: protein measure

Total protein concentrations were measured as described by Smith et al. (1985). BCA/CuSO₄ reactive solution (1:20) was added to each cell lysates and incubated in the dark for 30 min at 37 °C. Absorbance was measured with a SPECTROstar Nano microplate reader (BMG Labtech, Ortenberg, Germany) at a wavelength of 550 nm.

TBARS assay: lipid peroxidation determination

Lipid peroxidation was measured colorimetrically as described by Uchiyama & Mihara (1978). Cell pellets, stored at −80 °C, were thawed at room temperature and then, they were homogenized in thiobarbituric acid reactive substances (TBARS) solution (28.2 mM 2-TBA in 18.5 mL of TCA 16%-HCl 0.27 N). Then, the mixtures were heated for 10 min at 100 °C, and subsequently cooled in ice to stop the reaction. Supernatants of each sample were separated by centrifugation (3,000 rpm for 10 min at 4 °C) and immediately loaded into a SPECTROstar Nano microplate reader (BMG Labtech, Ortenberg, Germany) for absorbance measurement at 530 nm. TBARS values were expressed as nmol/mg protein.

Caspase-3/9 activity assay

Fluorometric assays of caspase-3 and caspase-9 enzymes were performed in accordance with the instructions provided by ALEXIS Biochemicals kits (Enzo Life Sciences Inc., San Diego, CA, USA). This enzymatic assay was based on hydrolysis of the peptide substrates of caspases-3/9 (Ac-DEVD 20 μM and Ac-LEHD 20 μM, respectively) conjugated to a fluorochrome (7-amido-4-methylcoumarin or AMC) at the terminal carbon of Asp, resulting in the release of the fluorescent motif. Cell lysates (20 μg) were used for each treatment. After incubation for 1 h at 37 °C, fluorescence was determined using a FLx800 microplate reader at excitation and emission wavelengths of 380 and 460 nm, respectively.

Immunoblotting

Western blot assays were performed using standard techniques. Primary antibodies were anti-caspase-3, Bcl-2 and GAPDH from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). The secondary antibody was anti-rabbit IgG, HRP-linked (Antibody #7074; Cell Signaling Technology, Danvers, MA, USA).

Cell respirometry

The oxygen consumption experiments were performed using a high resolution respirometry equipment O2k meter (Oroboros Instruments, Innsbruck, Austria) described by Aparicio-Trejo et al. (2019). The respiratory parameters were defined as: (A) Routine respiration, corresponding to oxygen consumption in presence only of cells in culture medium. (B) Leak of the respiration, corresponding to cellular oxygen consumption in presence of 15 μM oligomycin. (C) RCI corresponding to the ratio basal/leak. (D) Respiration attributable to OXPHOS (P) corresponding to: Routine-Leak. All parameters were corrected by subtracting non-mitochondrial respiration resulting from the addition of 15 μM rotenone and 15 μM antimycin A and normalized by cells number.

Statistical analysis

All the data were analyzed and plotted using GraphPad Prism software version 7.00 for Windows (GraphPad Software, San Diego, CA, USA) and Microsoft Office Excel version 16.17 for macOS, while the statistical analysis was performed through the one- or two-way ANOVA test of the Tukey, Dunnett or Sidak multiple comparisons tests. The values p < 0.05 were considered statistically significant.

Effect on cell viability

Initially, we investigated the effect of 7-hydroxy-3,4-dihydrocadalene from Heterotheca inuloides and the semi-synthetic cadinanes derivatives 7-(phenylcarbamate)-3,4-dihydrocadalene and 7-(phenylcarbamate)-cadalene on MCF7 breast cancer cell line. As shown in Fig. 2A, the natural product 7-hydroxy-3,4-dihydrocadalene reduced MCF7 cell viability in a concentration and time-dependent manner. The highest cytotoxicity was found at 48 and 72 h of exposure. Significant differences were observed between treatments at 24 and 48 h and, at 24 and 72 h, but not significant differences between treatments at 48 and 72 h. The inhibitory concentration (IC₅₀) values for 7-hydroxy-3,4-dihydrocadalene were 55.24 µM at 48 h and 52.83 µM at 72 h (Fig. 2A). On the other hand, the cytotoxic potency of the semisynthetic derivatives 7-(phenylcarbamate)-3,4-dihydrocadalene and 7-(phenylcarbamate)-cadalene were remarkably much lower than the natural product. As shown in Fig. 2B, the compound 7-(phenylcarbamate)-3,4-dihydrocadalene did not show a clear trend of MCF7 cell viability reduction dependently on the concentration or the time. However, the compound 7-(phenylcarbamate)-cadalene slightly inhibited MCF7 cell growth with different concentrations, 10 and 50 µM at 72 h of exposure (Fig. 2C). It was not possible for the semisynthetic compounds to determine the IC₅₀ values under the experimental conditions established in this work.

As a positive control we used (Z)-4-hydroxytamoxifen an estrogen receptor inhibitor (highly expressed in MCF7), obtaining an IC₅₀ of 16.50 µM at 24 h (Fig. 2D). Finally, non-tumoral cells from kidney (HEK 293) were treated with 7-hydroxy-3,4-dihydrocadalene for 72 h without effect (Fig. 2E).

Physicochemical properties with cytotoxic potential

As shown in Table 1, the molecular weight of the three studied compounds was less than 350 Da, being especially lower for the natural product (216.3 g/mol). Moreover, none of the studied compounds have ionizable groups as evidenced the identical values for logP and logD. Furthermore, the values of logS were −4.23 for 7-hydroxy-3,4-dihydrocadalene, −6.65 for 7-(phenylcarbamate)-3,4-dihydrocadalene and −7.47 for 7-(phenylcarbamate)-cadalene. Regarding the number of hydrogen bond donors, this is identical for the three compounds (1), whereas the number of hydrogen acceptors is three for semisynthetic compounds and one for the natural product. There are also differences in TPSA and nRot between the natural product and the semisynthetic compounds.

Effects on morphological changes and cell migration

Continuing with the study of the cytotoxic activity of the natural product 7-hydroxy-3,4-dihydrocadalene, we evaluated its effect on MCF7 proliferation and cell morphology with IC₅₀ and IC₅₀/2 concentrations for 48 h. As shown in Fig. 3A, IC₅₀ value significantly reduced cell proliferation in a concentration and time dependent manner. Moreover, morphological changes (cells are rounded off, adhesion capacity decreases, and membrane is blebbing) were observed after treatments with IC₅₀ value of 7-hydroxy-3,4-dihydrocadalene and H₂O₂ (1 mM as positive control) (Fig. 3B).

The effect of the natural product on cell migration (wound-healing assay) was analyzed using a multimodal plate reader to monitor the motility of the treated cells with 1 mM H₂O₂, IC₅₀ (48 h) and IC₅₀/2 of the natural compound. Fig. 3C illustrates a representative time course of the promotion or inhibition of MCF7 monolayer healing exposed to each treatment, while Fig. 3D shows the quantification of cell migration as a function of time and treatment where IC₅₀ of 7-hydroxy-3,4-dihydrocadalene significantly inhibits MCF7 migration at 24, 48 and 72 h of treatment exposition. Apparently after 72 h of treatment with 1 mM H₂O₂ cells were completely detached and it was not able to be quantified.

Effect on reactive oxygen species (ROS) and lipid peroxidation levels

Next, we evaluated whether the natural product 7-hydroxy-3,4-dihydrocadalene could generate intracellular ROS using the DCFH-DA assay. H₂O₂ was used as positive control (1 mM for 24 h). As shown in Fig. 4A, 7-hydroxy-3,4-dihydrocadalene significantly increased the intracellular ROS levels at IC₅₀ concentration (182% compared to control cells) like that of H₂O₂ (194.9% compared to control cells). Significant differences were also found between IC₅₀ and IC₅₀/2 treatments.

Moreover, we explore the effect of 7-hydroxy-3,4-dihydrocadalene on lipid peroxidation using the thiobarbituric acid reactive substance (TBARS) method. As shown in Fig. 4B, lipid peroxidation levels significantly increased at IC₅₀ concentration of 7-hydroxy-3,4-dihydrocadalene (0.52 nmol/mg protein) compared to control cells (0.11 nmol/mg protein), IC₅₀/2 (0.23 nmol/mg protein) and even the positive control hydrogen peroxide (0.24 nmol/mg protein) (Fig. 4B).

Effect on cell death through apoptosis

To corroborate if the cytotoxic effect of 7-hydroxy-3,4-dihydrocadalene was apoptosis pathway mediated, caspase 3 and caspase 9 were evaluated as the effector enzymes that initiate the point of no return in the apoptotic death cascade. As shown in Fig. 5A, 7-hydroxy-3,4-dihydrocadalene increased significantly both caspase 9 and caspase 3 activities (values of 118.42% and 24.17%, respectively compared to control cells) like the H₂O₂ (values of 115.08% for caspase 9 and 136.25% for caspase 3). However, no effect was observed on caspases 3 and 9 at the IC₅₀/2 concentration. Moreover, we measured caspase 3 and Bcl-2 levels by immunoblotting after 7-hydroxy-3,4-dihydrocadalene treatments (IC₅₀ and IC₅₀/2) and H₂O₂. The WB analysis in Fig. 5B showed that the expression of caspase 3 with H₂O₂ increased and it was more evident when treating the cells with 7-hydroxy-3,4-dihydrocadalene at IC₅₀ concentration. Slightly differences of Bcl-2 levels were observed but not significant.

Effect on mitochondrial bioenergetic metabolism

To determine if ROS production increases is related to mitochondrial bioenergetic metabolism alterations, the respiratory parameters including routine and leak respirations, respiration attributable to OXPHOS (P) and RCI were evaluated. As shown in Fig. 6, MCF7 cells were treated with 7-hydroxy-3,4-dihydrocadalene at IC₅₀ for 48 h and a drastically decreased in P and slightly reduction in RCI were observed. This reduction was more marked than that produced by H₂O₂ treatment.