A plethora of epidemiological and animal studies show that consumption of cruciferous vegetables may lower the risk for variety of cancers [1
]. It is suggested that the cancer-protective effects of cruciferous vegetables can be associated with the presence of glucosinolates, which are cleaved to biologically active compounds, such as indoles and isothiocyanates [5
Another group of cruciferous-derived phytochemicals, the indole phytoalexins, have attracted scientists’ interest because of their ability to modulate processes involved in oncogenic transformation, such as alterations of cell cycle control, apoptosis evasion and inhibition of different signalling pathways [6
Generally, phytoalexins are low molecular weight secondary metabolites biosynthesized de novo
by plants in response to stress caused by biotic or abiotic factors [9
]. Although phytoalexins are part of general defense mechanisms used to ward off plant invaders, their chemical diversity suggest substantially broader biological activities. In addition to their antimicrobial activity, some phytoalexins also possess antiinflammatory [11
], antioxidant [12
], antiproliferative [13
], as well as anticancer [15
Indole phytoalexins are structurally unique, sulfur-containing natural products isolated from plants of the family Cruciferae (syn. Brassicaceae). Besides their antimicrobial properties, several indole phytoalexins also exhibit antiproliferative/anticancer activity [17
Brassinin ([3-(S-methyldithiocarbamoyl) aminomethyl indole]), first isolated from Chinese cabbage [22
], is an indole phytoalexin with demonstrated antiproliferative/anticancer activity. Mehta and co-workers [23
] documented dose-dependent inhibition of 7,12-dimethylbenz[a]anthracene (DMBA)-induced preneoplastic lesion formation by brassinin and cyclobrassinin in a mouse mammary gland organ culture model. Later, Csomós et al.
] showed antiproliferative effects of brassinin, isobrassinin and isobrassinin derivatives in different cancer cell types. Recently, Izutani et al.
] described the ability of brassinin to inhibit cell growth in human colon cancer cells by arresting the cell cycle at the G1
phase via increased expression of p21 and p27. In the last decade we have also documented the antiproliferative effects of brassinin or its derivatives in different cancer cells [25
Although the precise mechanism(s) of the antiproliferative activity of brassinin and its derivatives still remain unknown, inhibition of indoleamine 2,3-dioxygenase and inhibition of PI3K/Akt/mTOR signalling pathways may interfere with cancer cell survival and proliferation [7
]. However, so far there is no published information about the antiproliferative molecular mechanisms of homobrassinin on cancer cells.
It is well known that oxidative stress may play role in the cytotoxicity of different natural compounds [32
]. Recently, it was documented that the antiproliferative effect of some indole phytoalexins may be associated with ROS production [34
] or glutathione depletion [19
], which may lead to imbalance between antioxidant and prooxidant factors. This prompted us to explore the role of ROS in the antiproliferative effects of brassinin and its derivatives. Our results demonstrate that homobrassinin (K1
) is the most active in inhibiting the growth of Caco2 cells among the compounds studied. Effect of K1
is associated with ROS production leading to mitochondrial dysfunction, caspase 3 activation and apoptosis induction. The role of ROS in K1
-induced cell death was analysed by intracellular ROS generation and ROS scavenger experiments. These findings generate a rationale for in vivo
efficacy studies with this compound in preclinical cancer models.
3. Experimental Section
3.1. Test Compounds
), homobrassinin (K1
), 1-[(1R,2S,5R)-menthoxycarbonyl]brassinin (K49
); 1-[(1R,2S,5R)-8-phenylmenthoxycarbonyl]brassinin (K170
). The synthesis of tested compounds was described in the previous studies: 1
3.2. Cell Culture
The human cancer cell lines HCT116 (human colorectal carcinoma), HepG2 (human hepatocellular carcinoma), HeLa (human cervical adenocarcinoma), Jurkat (human leukemic T cell lymphoma) were cultured in RPMI 1640 medium (PAA Laboratories, Pasching, Austria) and Caco2 (human colorectal adenocarcinoma), A549 (human alveolar adenocarcinoma), MCF-7 (human Caucasian breast adenocarcinoma) and MDA-MB-231 (human mammary gland adenocarcinoma) were maintained in growth medium consisting of high glucose Dulbecco’s Modified Eagle Medium (Invitrogen, Carlsbad, CA, USA). Both media were supplemented with a 10% fetal bovine serum (FBS), penicillin (100 IU/mL) and streptomycin (100 μg/mL) (all from Invitrogen). The cells (obtained from the American Tissue Culture Collection, ATCC, Rockville, MD, USA) were maintained under standard tissue culture conditions of 37 °C, 95% air/5% CO2
. Cell viability, estimated by trypan blue exclusion, was greater than 95% before each experiment. Human umbilical vein endothelial cells (HUVECs) were isolated and cultured as previously described by Ivanova et al.
3.3. Growth Inhibition Assay
The antiproliferative effects of compounds were determined using colorimetric microculture assay with the MTT end-point [56
]. Briefly, 3 × 103
cells were plated per well in 96-well polystyrene microplates (Sarstedt AG & Co, Nümbrecht, Germany) in the culture medium containing tested chemicals at final concentrations of 10−4
mol/L or Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid; Fluka, Buchs, Schwitzerland) at final concentrations 300 μM, or at mutual combinations. After 72 h of incubation (for Trolox and mutual combinations also after 24 and 48 h), 10 μL of MTT (5 mg/mL) (Sigma-Aldrich Corporation, St. Louis, MO, USA) were added in each well. After an additional 4 h, during which insoluble formazan was produced, 100 μL of 10% sodium dodecyl sulfate were added in each well and another 12 h were allowed for the dissolution of formazan. The absorbance was measured at 540 nm using the automated uQuant ™ Universal Microplate Spectrophotometer (Biotek, Winooski, VT, USA). The blank-corrected absorbance of the control wells was taken as 100% and the results were expressed as a percentage of the control. All experiments were performed in triplicate. Due to spontaneous apoptosis, HUVEC cells were incubated only 48 h.
3.4. 5-Bromo-2'-Deoxyuridine (BrdU) Cell Proliferation Assay
Cell proliferation activity was directly monitored by quantification of BrdU incorporated into the genomic DNA during cell growth. DNA synthesis was assessed using colorimetric cell proliferation ELISA assay (Roche Diagnostics GmbH, Mannheim, Germany) following the vendor’s protocol. Briefly, 2 × 103 cells/well in 80 µL medium were plated in a 96-well polystyrene microplates (Sarstedt AG & Co, Nümbrecht, Germany). Twenty-four hours after cell seeding different concentrations (10−4–10−6 mol/L) of the compound were added. After 48 h of treatment, cells were incubated with BrdU labeling solution (10 µM final concentration) for another 24 h at 37 °C followed by fixation and incubation with anti-BrdU peroxidase conjugate for an additional 1.5 h at room temperature. Finally, after substrate reaction, the stop solution was added (25 µL 1 M H2SO4) and colour intensity was measured with multi-well microplate ELISA reader at 450 nm (reference wavelength: 690 nm).
3.5. xCELLigence Cell Analysis System
The xCELLigence system is a unique, impedance-based system for cell-based assays, allowing for label-free and real-time monitoring of cellular processes such as cell growth, proliferation, cytotoxicity, adhesion, morphological dynamics and modulation of barrier function. It measures impedance changes in a meshwork of interdigitated gold microelectrodes located at the well bottom (E-plate) or at the bottom side of a microporous membrane (CIM16-plate). These changes are caused by the gradual increase of electrode surface occupation by (proliferated/ migrated/invaded) cells during the course of time and thus can provide an index of cell viability, migration and invasion. This method of quantification is directly proportional to cellular morphology, spreading, ruffling and adhesion quality as well as cell number [57
The xCELLigence RTCA system was initialized, as per manufacturer’s instructions, prior to commencement of the experiment by filling all 16 wells of the E-plate (ACEA Biosciences, San Diego, CA, USA) with the growth medium (100 μL) and equilibrated at room temperature for 30 min. The plate was placed into the single plate (SP) station cradle (housed in a humidified incubator at 37 °C with a 5% CO2 atmosphere) to establish a background reading. Then, Caco2 cells were seeded in E-plates at a density of 2 × 103 cells per well. After 24 h, K1 was added at final concentrations of 1–50 μM and cells were allowed to grow for additional 72 h under label-free conditions. The electrical impedance was measured by the RTCA-integrated software of the xCELLigence system (ACEA Biosciences) as a dimensionless parameter termed CI.
3.6. Experimental Design for Flow Cytometry Analysis
Caco2 cells (3 × 105) were seeded in Petri dishes and cultivated 24 h in a complete medium with 10% FBS. Cells were treated with K1 (c = 10 µM) for 12, 24, 48 and 72 h prior to analysis. The apoptosis, caspase 3 activation and cell cycle parameters were analysed 24, 48 and 72 h after treatment. Changes in MMP and ROS production were also analysed after 12 h of incubation. To evaluate ROS-dependent/independent mechanisms of K1 treatment we used Trolox, a water-soluble analogue of vitamin E, as antioxidant. Trolox (300 μM) was used 1 h before K1 treatment and after as a co-treatment with K1 for 12, 24, 48 and 72 h before ROS and MMP analysis.
3.7. Analysis of Cell Cycle
For flow cytometric analysis (FCM) of the cell cycle, floating and adherent cells were harvested together 24, 48 and 72 h after treatment, washed in cold PBS, fixed in cold 70% ethanol and kept at −20 °C overnight. Prior to analysis, cells were washed twice in PBS, resuspended in staining solution (final concentration 0.1% Triton X-100, 0.5 mg/mL ribonuclease A and 0.025 mg/mL propidium iodide-PI), incubated in the dark at room temperature for 30 min and analysed using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA).
3.8. Annexin V-FITC Labelling
The plasma membrane changes characteristic of apoptosis were analysed by double staining with Annexin V-FITC and PI according to the manufacturer’s instructions. Adherent and floating cells (1 × 105) were harvested together 24, 48 and 72 h after treatment and stained with Annexin V-FITC (BD Biosciences Pharmingen, San Diego, CA, USA) in binding buffer for 15 min, washed, stained with PI for 5 min and thereafter analysed using a BD FACSCalibur flow cytometer. Three populations of cells were observed: viable cells: Annexin V-FITC negative and PI negative; apoptotic cells: Annexin V-FITC positive and PI negative; late apoptotic/necrotic cells: Annexin V-FITC positive and PI positive or and Annexin V-FITC negative and PI positive.
3.9. Measurement of ROS
The intracellular production of ROS was detected with FCM analysis using dihydrorhodamine-123 (DHR-123, Fluka), which reacts with intracellular hydrogen peroxide. The cells treated with an appropriate agent were harvested, washed twice in PBS, and resuspended in PBS. DHR-123 was added at a final concentration of 0.2 μM. The samples were then incubated for 15 min in dark and after incubation samples were placed on ice. Fluorescence was detected with 530/30 (FL-1) optical filter. Forward and side scatters were used to gate the viable populations of cells.
3.10. Detection of MMP
The changes in MMP were analysed with FCM using tetramethylrhodamine ethyl ester per chlorate (TMRE, Molecular Probes, Eugene, OR, USA). The cells were washed with PBS, resuspended in 0.1 μM of TMRE in PBS, and incubated for 30 min at room temperature in the dark. The cells were then washed twice with PBS, resuspended in 500 μM of the total volume, and analysed (1 × 104 cell per sample). Fluorescence was detected with 585/42 (FL-2) optical filter.
3.11. Detection of Active Caspase 3
The changes in caspase 3 activation were analysed with FCM using BD Pharmingen Active Caspase-3 PE MAb Apoptosis kit (BD Bioscience, San Diego, CA, USA). The cells were prepared according to manufactory condition and stained with PE conjugated antibody and incubated for 30 min at room temperature in the dark. The cells were then washed twice with PBS, resuspended in 500 μM of the total volume, and analysed (1 × 104 cell per sample). Fluorescence was detected with 585/42 (FL-2) optical filter.
3.12. DNA Fragmentation Assay
The culture medium was removed from untreated (1 × 106) and treated Caco2 cells (K1 10 μM for 24, 48 and 72 h and 10 μM of cisplatin as positive control) and centrifuged at 1300 rpm for 5 min to collect them. Cells were washed twice with PBS calcium and magnesium free. Then cells were lysed in a lysis buffer containing 10 mmol/L EDTA, 0.5% Triton X-100. Proteinase K (1 mg/mL) was added and cells were incubated at 37 °C for 1 h followed by 10 min incubation at 70 °C. RNase (200 µg/mL) was added and cells were incubated for another 1 h at 37 °C. Samples were transferred to 2% agarose gel and run with 40 V for 3 h. DNA fragments were visualized by a UV illuminator.
3.13. DAPI Staining
Twenty four hours after treatment (K1 10 μM), Caco2 cells grown on cover slips were fixed with 2% paraformaldehyde for 20 min at 4 °C. After incubation, the cells were washed briefly with PBS and incubated at room temperature with SlowFade® Gold antifade reagent with 4',6-diaminidino-2-phenyl-indole, dihydrochloride (DAPI) (Invitrogen) for nuclear visualization. The slides were analysed using fluorescence microscope Leica DMI6000 B (Leica Microsystems, Inc., Bannockburn, IL, USA) and evaluated as percentages of cells with a fragmented nucleus from a minimum of 300 cells.
3.14. RNA Isolation and cDNA Synthesis
Total RNA was isolated from Caco2 cells using the TRI Reagent (Molecular Research Center, Inc., Cincinnati, OH, USA) according to the manufacturer’s instruction. Total RNA quality was verified on an agarose gel. Total RNA (0.5 µg) was reverse transcribed into cDNA by the RevertAidTM H Minus First Strand cDNA synthesis kit (Fermentas GmbH, St. Leon-Rot, Germany) according to the manufacturer’s instruction, and used for quantitative real time PCR.
3.15. Quantitative Real-Time PCR
Quantitative real time PCR analysis was performed in Light Cycler (Roche, Mannheim, Germany) using iQTM SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA, USA) to verify the alterations of α-tubulin, α1-tubulin, β5-tubulin gene expression. The PCR program was initiated by 5 min at 95 °C before 40 thermal cycles, each of 30 s at 95 °C and 45 s at 55 °C. Data were analysed according to the comparative Ct method and were normalized by β-actin expression in each sample. Melting curves for each PCR reaction were generated to ensure the purity of the amplification product.
3.16. Western Blot Analysis
Caco2 cells were treated with compound K1 (10 μM), Trolox (300 μM) and mutual combinations for 24, 48 and 72 h. Protein extracts were obtained using a lysis buffer containing 100 mM Tris (pH 7.4), 1% SDS and 10% glycerol in the presence of PIC (protease inhibitor cocktail), for 30 min on ice. After the insoluble materials were removed by centrifugation at 12,000 g for 10 min at 4 °C, total protein concentrations were quantified using the Pierce® BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Twenty micrograms of total cellular proteins were separated on 10% SDS polyacrylamide gels and electrotransferred onto nitrocellulose membranes (Pall Corporation, Port Washington, NY, USA). Membranes were blocked in 5% skim milk in Tris-buffered saline (TBS) containing 0.1% Tween-20 for 1 h at room temperature to minimize non-specific binding and incubated with the primary antibodies overnight at 4 °C. Immunoblotting was carried out with α Tubulin (E-19), α1c Tubulin (MH-87), β Tubulin (H-235) and β-Actin (C4) Antibody (all from Santa Cruz Biotechnology, Inc. Dallas, Texas, USA). After incubation with primary antibodies, membranes were washed 1 × 5 min with TBS-Tween followed by an incubation of 1 h at room temperature with the corresponding horseradish peroxidase-conjugated secondary antibodies (anti-rabbit IgG-HRP, anti-mouse IgG-HRP, all from Sigma-Aldrich). After washing 4 × 10 min with TBS-Tween expression was detected by chemiluminescence emission using ECL (Thermo Fisher Scientific) and then the blots were exposed to x-ray films.
3.17. Statistical Analysis
Results are expressed as mean ± SD. Statistical analyses of the data were performed using standard procedures, with one-way ANOVA followed by the Bonferroni multiple comparisons test. Differences were considered significant when p values were smaller than 0.05.