The natural triterpene 3β,6β,16β-trihydroxy-lup-20(29)-ene obtained from the flowers of Combretum leprosum induces apoptosis in MCF-7 breast cancer cells
© Viau et al.; licensee BioMed Central Ltd. 2014
Received: 25 March 2014
Accepted: 30 July 2014
Published: 2 August 2014
The 3β, 6β, 16β-trihydroxylup-20(29)-ene (TTHL) is a pentacyclic triterpene obtained from the medicinal plant Combretum leprosum Mart. In folk medicine, this plant is popularly known as mofumbo, cipoaba or mufumbo, and is used to treat several diseases associated with inflammation and pain.
We investigated the antitumor efficacy of TTHL isolated from C. leprosum. The TTHL cytotoxic effect was investigated in MRC5, MCF-7, HepG2, T24, HCT116, HT29, and CACO-2 cells after 24, 48, 72 and 120 h of treatment. The mechanisms of cell death and DNA damage induction were investigated by flow cytometry and comet assay, respectively.
The results indicated that TTHL induced a time- and concentration-dependent growth inhibition in all human cancer cell lines. The cytotoxicity was more pronounced in MCF-7 breast cancer cells, with an IC50 of 0.30 μg/mL at 120 h. We therefore evaluated the cell death mechanism induced by TTHL (IC20, IC50, and IC80) in MCF-7 cells at 24 h. We found that the treatment with IC50 and IC80 TTHL for 24 h induced apoptosis in 14% (IC50) and 52% (IC80) of MCF-7 cells. The apoptosis induced by TTHL was accompanied by increased levels of both cleaved caspase-9 and intracellular ROS. In order to further understand the biological mechanism of TTHL-induced cytotoxicity, we have also investigated its effect on different Saccharomyces cerevisiae yeast strains. The mutant strains sod1 Δ, sod2 Δ, and sod1 Δsod2 Δ, which are deficient in superoxide dismutase antioxidant defenses, were hypersensitive to TTHL, suggesting that its capacity to disturb cellular redox balance plays a role in drug toxicity. Moreover, TTHL induced mutagenicity in the yeast strain XV185-14c.
Taken together, the results suggest that TTHL forms covalent adducts with cellular macromolecules, potentially disrupting cellular function and triggering apoptosis.
KeywordsCombretum leprosum 3β 6β 16β-trihydroxy-lup-20(29)-ene triterpene Antiproliferative activity Cleaved caspase-9 ROS Mitochondrial apoptotic pathway
Medicinal plants have been used since ancient times in virtually all cultures as a source of medicines, and are of great importance to the health of individuals and communities. Traditional medicine is used in all parts of the World and has a rapidly growing economic importance, mainly through the use of medicinal plants, especially in developing countries. The medicinal use of plants of the Combretaceae family is widely described in the scientific literature[4–6]. This family is distributed in 20 genera, with approximately 600 species. The largest genera are Combretum and Terminalia, with about 370 and 200 species, respectively. Members of Combretaceae occur mainly in tropical and subtropical areas, such as Africa and Brazil.
The pentacyclic triterpene TTHL, obtained from the flowers of C. leprosum, presents a pronounced antinociceptive effect, which is dependent on opioid and serotonergic systems, G(i/o) protein activation and the opening of specific K(+) channels. Behavioral and electrographic recordings revealed that pretreatment with TTHL increased the latencies to the first clonic seizure to the tonic-clonic and reduced the duration of the generalized seizures induced by the GABA(A) receptor antagonist pentylenetetrazol.
Although considerable work has been done on the plant for different activities, scientific information on toxicological properties of TTHL is still not available. Since elimination of cancer in early stages is an integral part of chemoprevention, the assessment of antiproliferative properties against cancer cells provides useful insights on the chemo-protective potential of natural products. In this sense, we evaluated the abilities of TTHL to function as an antitumoral drug. Moreover, to further understand the biological mechanism of the cytotoxic effect of TTHL, our group also investigated its effect on different Saccharomyces cerevisiae strains.
Dulbecco’s modified Eagle’s medium (DMEM), low-melting-point agarose (LMP), high-melting-point agarose (HMP), phosphate-buffered saline (PBS; Na2HPO4, KH2PO4 and KCl, pH 7.4), propidium iodide (PI), mitoxantrone (MXT), hydrogen peroxide (H2O2), amino acids and nitrogenated bases were purchased from Sigma (St. Louis, MO, USA). Fetal bovine serum (FBS) and penicillin/streptomycin were obtained from Gibco-BRL (Grand Island, NY, USA). Primary antibody anti-caspase-9 and secondary antibody anti-rabbit IgG (H + L) F(ab’)2 fragment conjugated to Alexa Fluor® 488 were obtained from Cell Signaling Technology (USA) and Invitrogen (Grand Island, NY, USA), respectively. Cell Proliferation Kit II (XTT) was acquired from Roche (Basel, Switzerland). Annexin V-Phycoerythrin (PE) and 7-Amino-Actinomycin (7-AAD) were purchased from BD Biosciences (San Diego, CA). Yeast extract, bacto-peptone, bacto-agar and yeast nitrogen base were obtained from Difco Laboratories (Detroit, MI). All other reagents were of analytical grade.
Plant material and TTHL isolation
Botanical material was collected by Dr. Edilberto Rocha Silveira (Federal University of Ceará, Fortaleza) in May 2007 in a free area of Viçosa, Ceará State, Brazil, and classified by Dr. Afrânio Fernandes (Federal University of Ceará, Fortaleza) as Combretum leprosum Mart. A voucher specimen of this plant was deposited in Herbarium Prisco Bezerra of the Biology Department, Federal University of Ceará, Brazil, under number 12446. All necessary permits were obtained for the harvesting of the flowers.
The isolation of TTHL triterpene has been described by Facundo et al.. Briefly, the dried flowers (2.7 kg) were powdered and extracted with ethanol (5 L), being stirred and macerated at room temperature (24 ± 3°C) for approximately 24 h. This procedure was repeated three times. The solvent was fully evaporated under reduced pressure and the EE (yield 58.3 g) was lyophilized and stored in a freezer at -20°C until use. Part of the EE (32.0 g) was subjected to column chromatography on silica gel, eluting with n-hexane, chloroform (CHCl3), ethyl acetate (EtOAc) and methanol (MeOH). The fraction eluted with CHCl3 was chromatographed on a silica gel column and was eluted with n-hexane-EtOAc, in increasing polarity. The fractions 27–30, eluted with n-hexane-EtOAc (30:70), were combined on the basis of thin layer chromatography (TLC) analysis, and the presence of a white precipitate was observed, which after recrystallization from ethanol was identified as TTHL (2.37 g). The analysis of 1H NMR and 13C NMR spectra showed that TTHL analytical and spectroscopic data fully agreed with their assigned structures, and the chemical purity of TTHL was > 98%[11, 12]. For cell treatments, a stock solution of the TTHL was prepared freshly prior to use, using dimethylsulfoxide (DMSO) as solvent. The appropriate concentrations were obtained by diluting the stock solution in sterile distilled water, and the final concentration of DMSO in the incubation mixture never exceeded 0.1%. Control samples were always treated with the same amount of DMSO (0.1% v/v) as used in the corresponding experiments.
Assays with mammalian cells
The human cell lines MCF-7 (breast adenocarcinoma), HepG2 (hepatoma), T24 (bladder cancer), CACO-2 (colorectal adenocarcinoma) and MRC5 (lung normal fibroblast) were obtained from the Rio de Janeiro Cell Bank (Rio de Janeiro, RJ, Brazil). The HCT116 cell (colorectal carcinoma), and HT29 cell (colorectal adenocarcinoma) were kindly provided by Dr. Annette K. Larsen (Institut National de la Santé et de la Recherche Médicale - INSERM, Paris, França). All cell lines except MCF-7 were grown in DMEM supplemented with 10% or 20% (for CACO-2) FBS, 100 units.mL-1 penicillin and 100 μg.mL-1 streptomycin at 37°C in a humidified atmosphere of 5% CO2. MCF-7 cells were maintained in RPMI-1640 supplemented with 20% FBS at the same conditions described above.
Cell viability assay
The cytotoxic potential of the TTHL was evaluated by the XTT assay in human tumor cell lines as well as in MRC5 normal fibroblasts. Cells (1 × 104 cells) were seeded on 96-well plates in growth medium and incubated overnight. Afterwards, TTHL (0.5, 1.0, 2.5, 5.0, and 10 μg/mL) was added to each well and incubated for 24, 48, 72, and 120 h. Mitoxantrone, a cytostatic anthracenedione that intercalates in DNA and increases the incidence of double-strand breaks by stabilizing the cleavable complex of topoisomerase II and DNA, was used as positive control. At the end of each treatment, cell viability was assessed according to the manufacturer’s instructions. Briefly, after discarding the medium, 1 mL of XTT labeling mixture was added to the cells and incubated for 2 h at 37°C. Absorbance was measured with a SpectraMax reader (Bio-Rad, USA) at a test wavelength of 492 nm (A492) and a reference wavelength of 690 nm (A690). The final result corresponds to A492-A690. The absorbance of negative control cells was set as 100% viability, and the values for treated cells were calculated as a percentage of the control.
All other experiments (comet, cell cycle, apoptosis, ROS production and protein expression assays) were conducted in the breast cancer cell line MCF-7 using IC20 = 0.50 μg/mL (20% inhibitory concentration), IC50 = 1.36 μg/mL (50% inhibitory concentration) and IC80 = 3.70 μg/mL (80% inhibitory concentration) values of TTHL, as determined by XTT assay. Cells (2 × 105 cells/mL) were seeded on 6-well tissue culture plates, and grown for 1 day up to 70–80% confluence before treatment with the test substance. The TTHL was added to medium with FBS to obtain the different concentrations, and cells were treated at 37°C for 24 h in humidified atmosphere containing 5% CO2.
Cell cycle distribution by flow cytometric analysis
Cell cycle distribution determination analysis was performed as previously described. After treatment, cells were trypsinized, centrifuged and resuspended in ice-cold 70% ethanol, and left for 24 h at 4°C. Ethanol-fixed cells were centrifuged, washed with PBS and resuspended in buffer containing 0.2 mg/mL RNAse, 50 μg/mL PI and 0.1% Triton X-100, and incubated for 30 min at room temperature. Samples were analyzed on a FACS Calibur flow cytometer (Becton-Dikinson, San Fransisco, CA) using the CellQuest software. A total of 10,000 events were measured per sample. The data were analyzed to determine the percentage of cells at each phase of the cell cycle (sub-G1, G1, S and G2/M).
Assessment of apoptosis by flow cytometric analysis
Annexin V-PE was used in conjunction with a vital dye, 7-AAD, to distinguish apoptotic (Annexin V-PE positive, 7-AAD negative) from necrotic (Annexin V-PE positive, 7-AAD positive) cells. After treatment, cells were trypsinized, collected and resuspended in 40 μL of binding buffer with 2 μL Annexin V-PE. Cells were incubated for 15 min in the dark at room temperature. After incubation, 160 μL of binding buffer and 2 μL of 7-AAD were added. Cells were incubated for 5 min and additional 200 μL of binding buffer were added. Before analysis, cells were filtered through a cell strainer cap fitted to a polystyrene round bottom flow cytometric tube. Data were collected and analyzed by a FACS Calibur flow cytometer with CellQuest software, in a total of 10,000 events per sample; fluorescence was measured and the percentage of viable, early apoptotic, late apoptotic and necrotic cells was determined.
Quantification of cleaved caspase-9 by flow cytometric analysis
After treatment, cells (1 × 106) were harvested, resuspended in 25 μL PBS and fixed with 4% formaldehyde. After permeabilization and blocking (0.2% Triton X-100 in PBS and 1% BSA), cells were incubated with anti-caspase-9 antibody (diluted 1:1000) for 1 h at room temperature, followed by incubation with anti-rabbit FITC secondary antibody (Uniscience) (diluted 1:1000) for 1 h at room temperature in the dark. A total of 10,000 events were analyzed per sample by FACS Calibur flow cytometer. Fluorescence intensity in arbitrary units was plotted in histograms; the mean fluorescence intensity was calculated using CellQuest software.
Alkaline comet assay
The alkaline comet assay was performed as previously described. Briefly, 10 μL of cell suspension (1 × 104 cells) treated with TTHL were mixed with 90 μL LMP agarose, spread on a normal agarose precoated microscope slide, and placed at 4°C for 5 min to allow for solidification. Cells were lysed in high concentration of salt and detergent (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris with 1% Triton X-100 and 10% DMSO freshly added) for 2 h. Slides were removed from lysing solution and washed three times with PBS. Subsequently, cells were exposed to alkali conditions (300 mM NaOH/1 mM Na2EDTA, pH >13, 30 min, 4°C) to allow DNA unwinding and expression of alkali-labile sites. Electrophoresis was conducted for 25 min at 25 V and 300 mA (94 V/cm). After electrophoresis, the slides were neutralized and silver stained. One hundred cells were scored visually according to the tail length and the amount of DNA present in the tail. Each comet was given an arbitrary value of 0–4 (0, undamaged; 4, maximally damaged), as described by Collins et al.. Damage score was thus assigned to each sample and can range from 0 (completely undamaged: 100 cells × 0) to 400 (with maximum damage: 100 cells × 4). International guidelines and recommendations for the comet assay consider that visual scoring of comets is a well-validated evaluation method, as it is highly correlated with computer-based image analysis[22, 23].
Reactive oxygen species (ROS) detection by flow cytometric analysis
Levels of intracellular ROS were estimated following treatment with TTHL using 2′,7′-dichlorofluorescein diacetate (H2DCFDA, Sigma) as a fluorescent probe. Detection of oxidative stress was done by incubating the cells with 20 μM of H2DCFDA for 20 min at 37°C. Cells were then detached by trypsinization and washed twice with PBS. After filtration through cell strainer cap, cells were analyzed using a FACS Calibur flow cytometer with CellQuest software in accordance with Bass et al.. A total of 10,000 events were measured per sample. DCF fluorescence intensity was shown in arbitrary units.
Assays with S. cerevisiae
Strains, media and treatment
Saccharomyces cerevisiae strains used in this study
Enzymatic defense lacking
MATα: leu2 Δ0 his 3-Δ1 trp1-289 ura 3-52
EG118 (sod1 Δ)
Like EG103, except sod1::URA3
Cu-Zn SOD (cytosolic)
EG110 (sod2 Δ)
Like EG103, except sod2::TRP1
Mn SOD (Mitochondrial)
EG133 (sod1 Δsod2 Δ)
Like EG103, except sod1::URA3 and sod2::TRP1
MATα: ade2-2 his1-798 lys1-1 trp5-48 hom3-10 arg4-17
von Borstel et al. (1971)b
Stationary phase (STAT) cultures were obtained by inoculation of a single colony into liquid YPD. We chose to work in the stationary phase of growth because this resembles most cells of multicellular organisms in important aspects: (i) most energy comes from mitochondrial respiration; (ii) the cells have left the active cell cycle and have entered the Go phase; and (iii) damage accumulates over time[26, 27].
Survival assays in the EG103 background strains
STAT cells (1 × 108 cells/mL) were exposed to TTHL (10–500 μg/mL) and incubated under growth conditions for 1 h in PBS at 30°C. Cells were then washed and treated with H2O2 (5 mM) in PBS for another hour. Suitable aliquots were plated in triplicate on solid YPD (2–3 days, 30°C) and colony-forming units were counted. Sensitivity was expressed as a percentage of survival in relation to the negative control (solvent).
Point and frameshift mutations in the XV185-14c haploid yeast
Cell cultures were grown as described above, exposed to TTHL in concentrations ranging from 10 to 500 μg/mL, and then incubated in PBS for 1 h at 30°C. Two alleles, his1-798 and lys1-1, were used to detect point mutagenesis. The suppressible ochre nonsense mutant allele lys1-1 can be reverted either by locus-specific sequence alteration (true reversion) or by a forward mutation in a suppressor gene. Distinction between true reversions and forward (suppressor) mutations at the lys1-1 locus was performed according to Schuller & Von Borstel, where the reduced adenine content of the SC-lys medium shows true reversions as red and suppressor mutations as white colonies. Survival was determined on SC medium (3–5 days, 30°C) and mutation induction (HIS, LYS or HOM revertants) on media lacking the appropriate amino acid (7–10 days, 30°C). Induction of reversion of point mutation to his1-798, ochre allele lys1-1, and frameshift mutation values were scored per number of surviving cells.
The IC20, IC50, and IC80 values and their 95% confidence intervals (CI 95%) were obtained by nonlinear regression using GraphPad Prism v5 program (Intuitive Software for Science, San Diego, CA, USA). Selective index (SI) was calculated by IC50 in MRC5 cells/IC50 in tumoral cells. All experiments were independently repeated at least three times, with triplicate samples for each treatment. Results are expressed as means ± standard deviation (SD). Data were analyzed by one-way analysis of variance (ANOVA), and means were compared using Tukey test, with P ≤ 0.05 considered as statistically significant.
Effects of TTHL on tumoral cells
Antiproliferative activity of TTHL on human cell lines
IC50 (μg/mL)a ± SD
Time of Treatment (h)
Selectivity index (SI)b/superscript value
8.40 ± 0.28
1.36 ± 0.05 6X
6.50 ± 0.43 1.3X
5.55 ± 0.64 1.5X
7.05 ± 0.21 1X
8.00 ± 0.14 1X
8.55 ± 0.35 MR
7.60 ± 0.14
0.73 ± 0.05 10X
6.10 ± 0.28 1X
5.20 ± 0.42 1.5X
5.87 ± 0.04 1X
7.00 ± 0.42 1X
7.90 ± 0.28 MR
7.40 ± 0.15
0.63 ± 0.04 12X
5.45 ± 0.35 1X
1.09 ± 0.14 7X
5.20 ± 0.28 1X
6.15 ± 0.21 1X
6.35 ± 0.35 1X
6.84 ± 0.10
0.30 ± 0.04 23X
0.88 ± 0.13 8X
0.39 ± 0.10 17X
4.50 ± 0.29 1.5X
5.30 ± 0.28 1X
5.00 ± 0.19 1X
2.88 ± 1.44
0.87 ± 1.59
3.5 ± 0.39
2.50 ± 0.62
0.61 ± 0.67
0.88 ± 2.33
2.4 ± 1.81
Interestingly, our results suggest that TTHL exerted the highest cytotoxicity against MCF-7 cells, with an IC50 value of 1.36 ± 0.05 after 24 h of treatment and a higher potency - six fold more potent in these cells than in normal cells (MRC5). In addition, TTHL also showed a significant inhibitory activity in human cancer cell line T24, but only after 72 h of treatment (Table 2). Comparatively, mitoxantrone, used in this study as a positive control, demonstrated IC50 values ranging from 0.61-3.5 μg/mL in tumor cell lines. Considering both cytotoxic parameters and selectivity index, TTHL displayed the best profile in MCF-7 cells. Therefore, we chose the MCF-7 cells for further experiments to verify the cytotoxic mechanism of TTHL, using concentrations of 0.50 μg/mL, 1.36 μg/mL and 3.70 μg/mL (IC20, IC50 and IC80, respectively).
Cell cycle distribution by flow cytometric analysis
Apoptosis by flow cytometric analysis
Quantification of cleaved caspase-9 by flow cytometric analysis
Intracellular increase of ROS is responsible for apoptosis induced by TTHL in MCF-7 cells
Effects of TTHL on yeast cells
Cytotoxic and mutagenic effects in S. cerevisiae
Cytotoxicity and antioxidant effect of TTHL isolated product from ethnolic extract of C. leprosum in S. cerevisiae
sod1 Δsod2 Δ
100.0 ± 0.0
100.0 ± 0.0
100.0 ± 0.0
100.0 ± 0.0
TTHL 10 μg/mL
100 ± 2.83
92.60 ± 2.83
83.20 ± 3.68**
79.35 ± 2.19**
TTHL 50 μg/mL
87.65 ± 5.16**
89.30 ± 0.85**
72.90 ± 7.07***
67.85 ± 10.54***
TTHL 100 μg/mL
79.45 ± 1.91***
75.65 ± 4.60***
62.25 ± 3.32***
58.15 ± 3.89***
TTHL 500 μg/mL
67.65 ± 3.04***
69.30 ± 3.68***
56.65 ± 1.06***
41.45 ± 4.03***
PCb: H2O2 5 mM
63.03 ± 4.12
16.27 ± 4.88
19.10 ± 4.03
19.33 ± 2.10
Induction of reversion of point mutation for his1-798 , ochre allele lys1-1 , and frameshift mutation ( hom3-10 ) in haploid strain XV185-14c of S. cerevisiae after treatment of TTHL isolated product from ethanolic extract of C. leprosum
STAT cells treated in PBS
4.00 ± 2.83c
10.50 ± 0.71c
4.0 ± 1.41c
20.85 ± 2.48***
49.50 ± 9.19***
13.50 ± 4.95**
4.78 ± 0.63
10.77 ± 4.40
6.11 ± 1.58
5.68 ± 2.22
13.33 ± 1.10
8.80 ± 0.14
9.72 ± 0.55
20.83 ± 1.80
8.49 ± 2.12
14.40 ± 3.40**
30.10 ± 0.21**
11.15 ± 0.92*
Drug discovery from medicinal plants has played an especially important role in the treatment of cancer and, indeed, over the last half century, most new clinical applications of plant secondary metabolites and their derivatives have been applied towards combating cancer. Of all the available anticancer drugs between 1940 and 2002, 40% were natural products or natural product-derived, with another 8% considered natural product mimics[33–35]. Anticancer agents from plants currently in clinical use can be categorized into four main classes of compounds: vinca (or Catharanthus) alkaloids, epipodophyllotoxins, taxanes, and camptothecins.
Alternatively, the pentacyclic triterpenes are a group of promising secondary plant metabolites for cancer treatment. The triterpenes belonging to the lupane, oleanane or ursane groups have the potential to treat cancer by different mechanisms of action. Since Pisha et al. reported that betulinic acid is a highly promising anticancer drug, inducing apoptosis in melanoma cell lines in vitro and in vivo, experimental work has focused on the apoptosis-inducing mechanisms.
Accumulating data indicate that the cytotoxic effect of many chemotherapeutic drugs occurs through programmed cell death (apoptosis)[37, 38]. Hence, the ability of tumor cells to respond and activate the apoptotic program may, in part, determine the success of the therapeutic strategy. It is well documented that apoptosis can be induced by a variety of drugs with diverse chemical structures and different mechanisms of action, and two major routes including the death-receptor pathway and the mitochondrial-pathway have been identified. Apoptosis is a highly regulated process that involves many proteins and genes[40, 41]. It is characterized by cell shrinkage, plasma membrane bebbling, and chromatin condensation. The death program is executed by caspases, which amplify the apoptotic signal and proteolytically process numerous cellular molecules with different functions[40–42].
Caspases, a family of cysteine proteases, are central components of cellular apoptosis[41, 42]. Based on their different functions and structures, caspases are classified into two groups. Caspase-1, -2, -8, -9, and -10 belong to the first group, called initiator caspases, which can autocleave, and activate the second group of caspases, referred to as executioner caspases, which include caspase-3, -6, and -7. Accumulating evidence reveals that inhibition of apoptosis cascades plays an important role in tumor therapy resistance. For example, up-regulation of caspase-8 inhibitors like Flice-like inhibitory protein or inhibition of caspase-8 by Bcl-2 can induce tumor resistance to chemotherapy drugs by decreasing cellular apoptosis. Suppressing the activation of caspase-9 downstream can cause chemotherapy resistance in diffuse large B-cell lymphoma; overexpression of the inhibitor of caspase-3 can activate deoxyribonuclease in human renal carcinoma cells, therefore enhancing their resistance to cytotoxic chemotherapy. These studies strongly suggest that regulating the activity of caspases might be beneficial in tumor chemotherapy.
In order to evaluate the new compound TTHL, obtained from C. Leprosum, a folklore medicinal plant, as a potential therapy for cancer, different concentrations and exposure times were studied against a panel of six human tumoral cell lines: MCF-7, HepG2, T24, HCT116, HT29, CACO-2 (Table 2). We showed that TTHL induces an increase in sub-G1 population (Figure 2), and the induction of apoptosis by TTHL was confirmed by the increasing population labeled with Annexin V (Figure 3). We also observed that there is an increase in the cleavage of caspase-9, a mediator of the execution phase of apoptosis. This suggests a mytochondria-mediated triggering of the apoptotic program in MCF-7 cells that is consistent with the involvement of oxidative stress (Figure 6 and Table 3).
Here we found that TTHL treatment induced an increase in intracelular ROS formation in MCF-7 cell, as measured by DCF oxidation (Figure 6). Corroborationg with these results, TTHL also induced sensitivity in strains without superoxide dismutase enzyme (Table 3), which reinforced the indirect action caused by TTHL through ROS generation in both yeast and mammalian cells. This suggests that the cellular toxicity of TTHL must be related with its capacity of TTHL to produce ROS, and the electrophilicity of TTHL, which enables to form adducts with cellular macromolecules.
Subsequent experiments focused on direct interaction of TTHL with DNA. Chemical compounds bearing planar topologies and electrophilicity are often capable of intercalating between DNA bases. As TTHL is an electrophilic molecule with a polar group at either 3-OH, 6-OH, or 16-OH positions, and was capable of inducing frameshift mutation (Table 4), we suggest TTHL is a weak intercalator mutagen.
In addition to the mutagenicity assay, the comet assay is a sensitive and valuable technique to observe genotoxic damage. The comet assay detects primary (repairable) single and double-strand DNA breaks and alkali-labile sites in the alkaline test version. The comet assay results indicate that TTHL induced DNA strand breaks in MCF-7 cells (Figure 5). Thus, the observed increase in DNA migration in comet assay could be explained by either base damage or strand breaks induction by ROS generation or by direct action of TTHL on DNA. In this way, the TTHL DNA-damaging effect occurs not only by ROS generation but also via DNA intercalation (Table 4).
It is known that various triterpenoids are able to intervene in such processes as DNA repair, cell proliferation, cell differentiation, angiogenesis and apoptosis. The antitumoral effects of ursolic acid, maslinic acid and betulinic acid have been reported to stimulate similar apoptotic mechanisms. Ursolic acid induces apoptosis via the mitochondrial intrinsic pathway with alterations of the Bax/Bcl-2 balance in M4Beu cells and its anti-carcinogenic properties have also been described. Several attempts were undertaken for the derivatization of ursolic acid, seeking to obtain analogs with improved anti-tumor activity. Ma et al. modified the C-3, C-28, C-11 positions of ursolic acid and, among the 23 derivatives they synthesized, 3β-amino derivative was found to be 20 times more potent than the parent ursolic acid in HL-60, Bel-7402 and HeLa cell lines. Usually, compounds with β-oriented hydrogen-bond forming groups at C-3 exhibit more potent cytotoxicity than their α-counterparts. Analyzing the structure-activity relationship (SAR), it appears that a polar group at either 3-OH, 6-OH, or 16-OH positions is essential for the cytotoxic activity of TTHL (Figure 1 and Table 1). Especially, TTHL showed higher activity in antiproliferation assays in MCF-7 cells.
More recently, some studies have shown that maslinic acid, a natural pentacyclic triterpene has anti-cancer capacity in different cell types, including melanoma, liver cancer, astrocytoma, and colon cancer. Specifically in colon malignancies, maslinic acid possesses potent differentiating and anti-proliferation properties, inducing cell-cycle arrest in the G0/G1 phase and apoptosis in colon cancer cells without affecting non-tumoral cells.
The induction of apoptosis by betulinic acid involves several mitochondrial perturbations, such as the release of cytochrome-c and activation of caspase-8. Furthermore, it has been reported that the protein Smac is released during the induction of apoptosis by betulinic acid, whereas with other tumoral cells it provokes the down-regulation of Bcl-2, thus blocking the release of anti-apoptogenic molecules. In addition, the formation of ROS that modulate Bcl-2 and Bax levels during the action of betulinic acid has also been detected.
Other terpenoids, such as amooranin, induce apoptosis in MDA-468 cells via the activation of caspases -9, -3, and -8, the cleavage of Bid and the release of cytochrome-c from the mitochondria, concomitant with the up-regulation of p53 and Bax and down-regulation of Bcl-2. In addition, an increase in the Bax/Bcl-2 ratio and a decrease in mitochondrial membrane potential have been reported as being involved in the induction of apoptosis for alisol B acetate.
This is one of the first studies demonstrating the in vitro antitumor activity of TTHL in MCF-7 cells. The antitumor effect of TTHL can be further enhanced by the use of combined therapy and novel drug delivery systems, thus making it a promising candidate for management of breast cancer patients.
This research was supported by grants from the Brazilian Agencies Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq/INCT/INPeTAm, Grant no. 573695/2008-3), Programa Nacional de Cooperação Acadêmica/Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS - PRONEX/FAPERGS/CNPq, Grant no. 10/0044-3).
- Hoareau L, Da Silva EJ: Medicinal plants: A re-emerging health aid. Electron J Biotechnol. 1999, 2: 56-70.Google Scholar
- Edeoga HO, Okwu DE, Mbaebie BO: Phytochemical constituents of some Nigerian medicinal plants. Afr J Biotechnol. 2005, 4: 685-688.View ArticleGoogle Scholar
- Agra MF, Freitas PF, Barbosa-Filho JM: Synopsis of the plants known as medicinal and poisonous in Northeast of Brazil. Rev Bras Farmacogn. 2007, 17: 114-140.View ArticleGoogle Scholar
- Atindehou KK, Schmid C, Brun R, Koné MW, Traore D: Antitrypanosomal and antiplasmodial activity of medicinal plants from Côte d’Ivoire. J Ethnopharmacol. 2004, 90: 221-227.View ArticleGoogle Scholar
- Muthu C, Ayyanar M, Raja N, Ignacimuthu S: Medicinal plants used by traditional healers in Kancheepuram district of Tamil Nadu. India. J Ethnobiol Ethnomed. 2006, 2: 43-View ArticlePubMedPubMed CentralGoogle Scholar
- Gansané A, Sanon S, Ouattara LP, Traoré A, Hutter S, Ollivier E, Azas N, Traore AS, Guissou IP, Sirima SB, Nebié I: Antiplasmodial activity and toxicity of crude extracts from alternatives parts of plants widely used for the treatment of malaria in Burkina Faso: Contribution for their preservation. Parasitol Res. 2010, 106: 335-340.View ArticlePubMedGoogle Scholar
- Pietrovski EF, Rosa KA, Facundo VA, Rios K, Marques MC, Santo AR: Antinociceptive properties of the ethanolic extract and of the triterpene 3beta,6beta,16beta-trihidroxilup-20(29)-ene obtained from the flowers of Combretum leprosum in mice. Pharmacol Biochem Behav. 2006, 83: 90-99.View ArticlePubMedGoogle Scholar
- De Morais Lima GR, de Sales IR, Caldas Filho MR, de Jesus NZ, de Sousa FH, Barbosa-Filho JM, Cabral AG, Souto AL, Tavares JF, Batista LM: Bioactivities of the genus Combretum (Combretaceae): a review. Molecules. 2012, 17: 9142-9206.View ArticlePubMedGoogle Scholar
- Eloff JN, Katerere DR, McGaw LJ: The biological activity and chemistry of the southern African Combretaceae. J Ethnopharmacol. 2008, 119: 686-699.View ArticlePubMedGoogle Scholar
- Ribeiro SS, de Jesus AM, dos Anjos CS, da Silva TB, Santos AD, de Jesus JR, Andrade MS, Sampaio TS, Gomes WF, Alves PB, Carvalho AA, Pessoa C, de Moraes MO, Pinheiro ML, Prata AP, Blank AF, Silva-Mann R, Moraes VR, Costa EV, Nogueira PC, Bezerra DP: Evaluation of the cytotoxic activity of some Brazilian medicinal plants. Planta Med. 2012, 78: 1601-1606.View ArticlePubMedGoogle Scholar
- Longhi-Balbinot DT, Martins DF, Lanznaster D, Silva MD, Facundo VA, Santos AR: Further analyses of mechanisms underlying the antinociceptive effect of the triterpene 3β, 6β, 16β-trihydroxylup-20(29)-ene in mice. Eur J Pharmacol. 2011, 653: 32-40.View ArticlePubMedGoogle Scholar
- Longhi-Balbinot DT, Lanznaster D, Baggio CH, Silva MD, Cabrera CH: Anti-inflammatory effect of triterpene 3β, 6β, 16β-trihydroxylup-20(29)-ene obtained from Combretum leprosu m Mart & Eich in mice. J Ethnopharmacol. 2012, 142: 59-64.View ArticlePubMedGoogle Scholar
- Lopes LS, Marques RB, Pereira SS, Ayres MC, Chaves MH: Antinociceptive effect on mice of the hydroalcoholic fraction and (-) epicatechin obtained from Combretum leprosum Mart & Eich. Braz J Med Biol Res. 2010, 43: 1184-1192.View ArticlePubMedGoogle Scholar
- McGaw LJ, Eloff JN: Ethnoveterinary use of southern African plants and scientific evaluation of their medicinal properties. J Ethnopharmacol. 2008, 119: 559-574.View ArticlePubMedGoogle Scholar
- McGaw LJ, Rabe T, Sparg SG, Jäger AK, Eloff JN: An investigation on the biological activity of Combretum species. J Ethnopharmacol. 2001, 75: 45-50.View ArticlePubMedGoogle Scholar
- Facundo VA, Andrade CHS, Silveira ER, Braz-Filho R, Hufford C: Triterpenes and flavonoids from Combretum leprosum. Phytochemistry. 1993, 32: 411-515.View ArticleGoogle Scholar
- Della-Pace ID, Rambo LM, Ribeiro LR, Saraiva AL, de Oliveira SM, Silva CR, Villarinho JG, Rossato MF, Ferreira J, de Carvalho LM, de Oliveira Lima F, Furian AF, Oliveria MS, Santos AR, Facundo VA, Fighera MR, Royes LF: Triterpene 3β, 6β, 16β trihidroxilup-20(29)-ene protects against excitability and oxidative damage induced by pentylenetetrazol: the role of Na(+), K(+)-ATPase activity. Neuropharmacology. 2013, 67: 455-464.View ArticlePubMedGoogle Scholar
- Hagemeister F, Cabanillas F, Coleman M, Gregory SA, Zinzani PL: The role of mitoxantrone in the treatment of indolent lymphomas. Oncologist. 2005, 10: 150-159.View ArticlePubMedGoogle Scholar
- Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C: A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods. 1991, 139: 271-279.View ArticlePubMedGoogle Scholar
- Singh NP, McCoy MT, Tice RR, Schneider EL: A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res. 1988, 175: 184-191.View ArticlePubMedGoogle Scholar
- Nadin SB, Vargas-Roig LM, Ciocca DR: A silver staining method for single-cell gel assay. J Histochem Cytochem. 2001, 49: 1183-1186.View ArticlePubMedGoogle Scholar
- Collins AR, Ma AG, Duthie SJ: The kinetics of repair of oxidative DNA damage (strand breaks and oxidised pyrimidines) in human cells. Mut Res. 1995, 336: 69-77.View ArticleGoogle Scholar
- Burlinson B, Tice RR, Speit G, Agurell E, Brendler-Schwaab SY, Collins AR, Escobar P, Honma M, Kumaravel TS, Nakajima M, Sasaki YF, Thybaud V, Uno Y, Vasquez M, Hartmann A: Fourth International Workgroup on Genotoxicity testing: results of the in vivo Comet assay workgroup. Mutat Res. 2007, 627: 31-35.View ArticlePubMedGoogle Scholar
- Bass DA, Parce JW, Dechatelet LR, Szejda P, Seeds MC, Thomas M: Flow cytometric studies of oxidative product formation by neutrophils: a graded response to membrane stimulation. J Immunol. 1983, 130: 1910-1917.PubMedGoogle Scholar
- Burke D, Dawson T, Stearns T: Methods in Yeast Genetics, a CSH Laboratory Course Manual. 2000, Plainview, NY: CSH Laboratory PressGoogle Scholar
- Longo VD, Gralla EB, Valentine JS: Superoxide dismutase activity is essential for stationary phase survival in Saccharomyces cerevisiae. Mitochondrial production of toxic oxygen species in vivo. J Biol Chem. 1996, 271: 12275-12280.View ArticlePubMedGoogle Scholar
- Cyrne L, Martins L, Fernandes L, Marinho HS: Regulation of antioxidant enzymes gene expression in the yeast Saccharomyces cerevisiae during stationary phase. Free Radic Biol Med. 2003, 34: 385-393.View ArticlePubMedGoogle Scholar
- Da Costa Júnior JS, de Almeida AA, Costa JP, das Graças Lopes Citó AM, Saffi J, de Freitas RM: Superoxide dismutase and catalase activities in rat hippocampus pretreated with garcinielliptone FC from Platonia insignis. Pharm Biol. 2012, 50: 453-457.View ArticlePubMedGoogle Scholar
- Schuller RC, Von Borstel RC: Spontaneous mutability in yeast. I. Stability of lysine reversion rates to variation of adenine concentration. Mutat Res. 1974, 24: 17-23.View ArticlePubMedGoogle Scholar
- Acton EM, Narayanan VL, Risbood PA, Shoemaker RH, Vistica DT, Boyd MR: Anticancer specificity of some ellipticinium salts against human brain tumors in vitro. J Med Chem. 1994, 37: 2185-2189.View ArticlePubMedGoogle Scholar
- Ibáñez E, Plano D, Font M, Calvo A, Prior C, Palop JA, Sanmartín C: Synthesis and antiproliferative activity of novel symmetrical alkylthio- and alkylseleno-imidocarbamates. Eur J Med Chem. 2011, 46: 265-274.View ArticlePubMedGoogle Scholar
- Mazumder K, Tanaka K, Fukase K: Cytotoxic activity of ursolic acid derivatives obtained by isolation and oxidative derivatization. Molecules. 2013, 18: 8929-8944.View ArticlePubMedGoogle Scholar
- Butler MS: The role of natural product chemistry in drug discovery. J Nat Prod. 2004, 67: 2141-2153.View ArticlePubMedGoogle Scholar
- Newman DJ, Cragg GM, Snader KM: The influence of natural products upon drug discovery. Nat Prod Rep. 2000, 17: 215-234.View ArticlePubMedGoogle Scholar
- Newman DJ, Cragg GM, Snader KM: Natural products as sources of new drugs over the period 1981–2002. J Nat Prod. 2003, 66: 1022-1037.View ArticlePubMedGoogle Scholar
- Pisha E, Chai H, Lee IS, Chagwedera TE, Farnsworth NR, Cordell GA, Beecher CW, Fong HH, Kinghorn AD, Brown DM, Wani MC, Wall ME, Hieken TJ, Gupta TKD, Pezzuto JM: Discovery of betulinic acid as a selective inhibitor of human melanoma that functions by induction of apoptosis. Nat Med. 1995, 1: 1046-1051.View ArticlePubMedGoogle Scholar
- Jain MV, Paczulla AM, Klonisch T, Dimgba FN, Rao SB, Roberg K, Schweizer F, Lengerke C, Davoodpour P, Palicharla VR, Maddika S, Łos M: Interconnections between apoptotic, autophagic and necrotic pathways: implications for cancer therapy development. J Cell Mol Med. 2013, 17: 12-29.View ArticlePubMedPubMed CentralGoogle Scholar
- Reyes-Zurita FJ, Pachón-Peña G, Lizárraga D, Rufino-Palomares EE, Cascante M, Lupiáñez JA: The natural triterpene maslinic acid induces apoptosis in HT29 colon cancer cells by a JNK-p53-dependent mechanism. BMC Cancer. 2011, 11: 154-View ArticlePubMedPubMed CentralGoogle Scholar
- Grimm D, Wehland M, Pietsch J, Infanger M, Bauer J: Drugs interfering with apoptosis in breast cancer. Curr Pharm Des. 2011, 17: 272-283.View ArticlePubMedGoogle Scholar
- Fiandalo MV, Kyprianou N: Caspase control: protagonists of cancer cell apoptosis. Exp Oncol. 2012, 34: 165-175.PubMedPubMed CentralGoogle Scholar
- Portt L, Norman G, Clapp C, Greenwood M, Greenwood MT: Anti-apoptosis and cell survival: a review. Biochim Biophys Acta. 1813, 2011: 238-259.Google Scholar
- Park HH: Structural features of caspase-activating complexes. Int J Mol Sci. 2012, 2012 (13): 4807-4818.View ArticleGoogle Scholar
- Kim PK, Mahidhara R, Seol DW: The role of caspase-8 in resistance to cancer chemotherapy. Drug Resist Updat. 2001, 4: 293-296.View ArticlePubMedGoogle Scholar
- Cillessen SA, Hess CJ, Hooijberg E, Castricum KC, Kortman P, Denkers F, Vos W, van de Wiel MA, Schuurhuis GJ, Ossenkoppele GJ, Meijer CJ, Oudejans JJ: Inhibition of the intrinsic apoptosis pathway downstream of caspase-9 activation causes chemotherapy resistance in diffuse large B-cell lymphoma. Clin Cancer Res. 2007, 13: 7012-7021.View ArticlePubMedGoogle Scholar
- Hara S, Miyake H, Arakawa S, Kamidono S, Hara I: Over expression of inhibitor of caspase 3 activated deoxyribonuclease in human renal cell carcinoma cells enhances their resistance to cytotoxic chemotherapy in vivo. J Urol. 2001, 166: 2491-2494.View ArticlePubMedGoogle Scholar
- Snyder RD, Arnone MR: Putative identification of functional interactions between DNA intercalating agents and topoisomerase II using the V79 in vitro micronucleus assay. Mutat Res. 2002, 503: 21-35.View ArticlePubMedGoogle Scholar
- Harmand PO, Duval R, Delage C, Simon A: Ursolic acid induces apoptosis through mitochondrial intrinsic pathway and caspase-3 activation in M4Beu melanoma cells. Int J Cancer. 2005, 114: 1-11.View ArticlePubMedGoogle Scholar
- Shishodia S, Majumdar S, Banerjee S, Aggarwal BB: Ursolic acid inhibits nuclear factor-kappaB activation induced by carcinogenic agents through suppression of IkappaBalpha kinase and p65 phosphorylation: correlation with down-regulation of cyclooxygenase 2, matrix metalloproteinase 9, and cyclin D1. Cancer Res. 2003, 63: 4375-4383.PubMedGoogle Scholar
- Ma CM, Cai SQ, Cui JR, Wang RQ, Tu PF, Hattori M, Daneshtalab M: The cytotoxic activity of ursolic acid derivatives. Eur J Med Chem. 2005, 40: 582-589.View ArticlePubMedGoogle Scholar
- Parra A, Rivas F, Martin-Fonseca S, Garcia-Granados A, Martinez A: Maslinic acid derivatives induce significant apoptosis in b16f10 murine melanoma cells. Eur J Med Chem. 2011, 46: 5991-6001.View ArticlePubMedGoogle Scholar
- Lin CC, Huang CY, Mong MC, Chan CY, Yin MC: Antiangiogenic potential of three triterpenic acids in human liver cancer cells. J Agric Food Chem. 2011, 59: 755-762.View ArticlePubMedGoogle Scholar
- Martín R, Carvalho-Tavares J, Ibeas E, Hernández M, Ruiz-Gutierrez V, Nieto ML: Acidic triterpenes compromise growth and survival of astrocytoma cell lines by regulating reactive oxygen species accumulation. Cancer Res. 2007, 67: 3741-3751.View ArticlePubMedGoogle Scholar
- Reyes FJ, Centelles JJ, Lupiáñez JA, Cascante M: (2Alpha,3beta)-2,3-dihydroxyolean-12-en-28-oic acid, a new natural triterpene from Olea europea, induces caspase dependent apoptosis selectively in colon adenocarcinoma cells. FEBS Lett. 2006, 580: 6302-6310.View ArticlePubMedGoogle Scholar
- Fulda S, Debatin KM: Betulinic acid induces apoptosis through a direct effect on mitochondria in neuroectodermal tumors. Med Pediatr Oncol. 2000, 35: 616-618.View ArticlePubMedGoogle Scholar
- Karpova MB, Sanmun D, Henter JI, Smirnov AF, Fadeel B: Betulinic acid, a natural cytotoxic agent, fails to trigger apoptosis in human Burkitt’s lymphoma-derived B-cell lines. Int J Cancer. 2006, 118: 246-252.View ArticlePubMedGoogle Scholar
- Zuco V, Supino R, Righetti SC, Cleris L, Marchesi E, Gambacorti-Passerini C, Formelli F: Selective cytotoxicity of betulinic acid on tumor cell lines, but not on normal cells. Cancer Lett. 2002, 175: 17-25.View ArticlePubMedGoogle Scholar
- Rabi T, Wang L, Banerjee S: Novel triterpenoid 25-hydroxy-3-oxoolean-12-en-28-oic acid induces growth arrest and apoptosis in breast cancer cells. Breast Cancer Res Treat. 2007, 101: 27-36.View ArticlePubMedGoogle Scholar
- Chen HW, Hsu MJ, Chien CT, Huang HC: Effect of alisol B acetate, a plant triterpene, on apoptosis in vascular smooth muscle cells and lymphocytes. Eur J Pharmacol. 2001, 419: 127-138.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6882/14/280/prepub
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.