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Antiproliferative activity and apoptosis-inducing mechanism of constituents from Toona sinensis on human cancer cells

Shengjie Yang12, Qi Zhao12, Hongmei Xiang12, Minjie Liu12, Qiuyun Zhang12, Wei Xue12, Baoan Song12* and Song Yang12*

Author Affiliations

1 State-Local Joint Laboratory for Comprehensive Utilization of Biomass, State Key Laboratory Breeding Base of Green Pesticide and Agricultural Bioengineering, Key Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of Education, Guizhou University, Guiyang, 550025, P.R. China

2 Ctr for R&D of Fine Chemicals, Guizhou University, Huaxi St, Guiyang, 550025, China

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Cancer Cell International 2013, 13:12  doi:10.1186/1475-2867-13-12


The electronic version of this article is the complete one and can be found online at: http://www.cancerci.com/content/13/1/12


Received:16 December 2012
Accepted:4 February 2013
Published:9 February 2013

© 2013 Yang et al.; licensee BioMed Central Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

Natural products, including plants, microorganisms and marines, have been considered as valuable sources for anticancer drug discovery. Many Chinese herbs have been discovered to be potential sources of antitumor drugs.

Methods

In the present study, we investigated the antitumor efficacy of the compounds isolated from Toona sinensis, an important herbal medicine. The inhibitory activities of these compounds were investigated on MGC-803, PC3, A549, MCF-7, and NIH3T3 cells in vitro by MTT assay. The mechanism of the antitumor action of active compounds was investigated through AO/EB staining, Hoechst 33258 staining, TUNEL assay, flow cytometry analysis, and western blotting analysis.

Results

Fifteen compounds were isolated from the roots of Toona sinensis. Betulonic acid (BTA) and 3-oxours-12-en-28-oic acid (OEA) isolated from the plant inhibited the proliferation of MGC-803 and PC3 cells, with IC50 values of 17.7 μM and 13.6 μM, 26.5 μM and 21.9 μM, respectively. Both could lead to cell apoptosis, and apoptosis ratios reached 27.3% and 24.5% in MGC-803 cells at 72 h after treatment at 20 μM, respectively. Moreover, the study of cancer cell apoptotic signaling pathway indicated that both of them could induce cancer cell apoptosis through the mitochondrial pathway, involving the expressions of p53, Bax, caspase 9 and caspase 3.

Conclusions

The study shows that most of the compounds obtained from Toona sinensis could inhibit the growth of human cancer cells. Furthermore, BTA and OEA exhibited potent antitumor activities via induction of cancer cell apoptosis.

Keywords:
Toona sinensis; Antiproliferation; Apoptosis; Pathway

Background

Among the conventional antitumor cytotoxic chemotherapies, many compounds are derived from natural products [1-3]. Over 60% of the current anticancer drugs have their origin in one way or another from natural sources [4,5]. Natural compounds had attracted considerable attention as cancer chemopreventive agents and also as cancer therapeutics [6,7]. As cancer cells have evolved multiple mechanisms to resist the induction of programmed cell death (apoptosis), the modulation of apoptosis signaling pathways by natural compounds have been demonstrated to constitute a key event in these antitumor activities [8,9]. Toona sinensis, an important herb medicine, belongs to the Meliaceae family which comprises approximately 50 genera and 1400 species throughout the world [10], and is widely distributed in China except Xinjiang and Inner Mongolia Autonomous Regions. The objective of present study was to evaluate the potency of the components from the plant for growth inhibiting of human cancer cell lines and to study their antitumor mechanism. Fifteen compounds were isolated from the plant, and these compounds were bioassayed on human gastric cancer cell line MGC-803, prostatic cancer cell line PC3, lung cancer cell line A549, breast cancer cell line MCF-7, and mouse embryonic fibroblast cell line NIH3T3 in vitro by MTT assay. Interestingly, it was found that betulonic acid (BTA) and 3-oxours-12-en-28-oic acid (OEA) had the potent inhibitory activities against MGC-803 and PC3 cell lines, and were less toxic on normal cells than on the investigated cancer cell lines. Also, BTA and OEA are betulinic acid (BA) and ursolic acid (UA) derivatives, respectively. BA and UA are naturally occurring pentacyclic triterpenoids which are widely distributed in the plant kingdom [11,12]. It was found that BA could inhibit growth of cancer cells [13,14], without affecting normal cells [15,16], and it was a highly selective growth inhibitor of human melanoma, neuroectodermal and malignant tumor cells [17]. UA has also been reported to show significant cytotoxicity against some tumor cell lines [13,18-21]. There are a few reports on the anticancer effects of BTA and OEA on various tumor cells recently. Some studies have shown that BTA could inhibit the growth of various types of human tumor cell lines, including SGC-7901, HepG-2 [22], LNCaP, and DU-145 [23] cells. In 1999, Min et al. found that OEA possessed antitumor activity on A549, SK-OV-3, SK-MEL-2, XF498, and HCT15 cells, with low IC50 values (< 5 μg/mL) [18]. However, no report was found on the antitumor mechanism of the two compounds. Thus, the mechanism of action needs to be further clarified. Further investigation of BTA and OEA was carried out on MGC-803 and PC3 cells, and experimental results of fluorescent staining and flow cytometry analysis indicated that the two compounds could induce cell apoptosis. In addition, the mechanism underlying apoptosis of BTA and OEA was also investigated in this study. To the best of our knowledge, this is the first report on apoptosis inducing of BTA and OEA in MGC-803 and PC3 cells.

Methods

Plant material

Fresh samples of Toona sinensis were collected from Bijie, Guizhou Province in China, in August 2011. Prof. Qingde Long, Department of Medicine, Guiyang Medical University, identified the plant material. A voucher specimen was deposited at Guiyang Medical University, Guiyang, China.

Cell culture

MGC-803, PC3, A549. MCF-7, and NIH3T3 cell lines were obtained from the Institute of Biochemistry and Cell Biology, China Academy of Science. MGC-803 is human gastric cancer cell line, PC3 is prostatic cancer cell line, A549 is lung cancer cell line, MCF-7 is breast cancer cell line, and NIH3T3 is mouse embryonic fibroblast cell line. The entire cancer cell lines were maintained in the RPMI 1640 medium and NIH3T3 was maintained in the DMEM medium. They were supplemented with 10% heat-inactivated fetal bovine serum (FBS) in a humidified atmosphere of 5% CO2 at 37°C. All cell lines were maintained at 37°C in a humidified 5% carbon dioxide and 95% air incubator.

MTT assays

The antitumor activities of the compounds were determined by MTT assay. All tested compounds were dissolved in DMSO and subsequently diluted in the culture medium before treatment of the cultured cells. When the cells were 80-90% confluent, they were harvested by treatment with a solution containing 0.25% trypsin, thoroughly washed and resuspended in supplemented growth medium. Cells (1×104/well) were plated in 100 μL of medium/well in 96-well plate. After incubations overnight, the cells were treated with different concentrations of extracts or compounds for 72 h. Thereafter, 100 μL of MTT (Beyotime Co., Jiangsu, China) solution was added to each well and then incubated for 4 h. The colored MTT-formazan crystals which were produced from MTT were dissolved in SDS for 12 h. And then the OD values were measured at 595 nm with a microplate reader (BIO-RAD, model 680), which is directly proportional to the number of living cells in culture [24-26].

AO/EB staining

The active compounds were investigated for apoptotic activity by AO/EB staining. When the cells were 80-90% confluent, they were harvested by treatment with a solution containing 0.25% trypsin, thoroughly washed and resuspended in supplemented growth medium. The cells were seeded in 6-well tissue culture plates (5×104 cell/mL, 0.6 mL/well). After incubations overnight, the medium was removed and replaced with fresh medium plus 10% FBS and then supplemented with compounds (20 μmol/L). After the treatment period, 20 μL of the AO/EB dye mix (Beyotime Co., Shanghai, China) were added to each well, and the apoptotic cells were viewed and counted under the fluorescent microscope (OLYMPUS Co., Tokyo Met, Japan) [27,28].

Hoechst 33258 staining

Morphological assessment of apoptotic cells was performed using Hoechst 33258 staining method. The cells were seeded in 6-well tissue culture plates (5×104 cell/mL, 0.6 mL/well). After incubations overnight, the medium was removed and replaced with fresh medium plus 10% FBS and then supplemented with compounds (20 μmol/L) for a certain range of treatment time. The culture medium containing compounds was removed, and the cells were fixed in 4% paraformaldehyde for 10 min. The cells were washed twice with PBS, and were consequently stained with 0.5 mL of Hoechst 33258 staining (Beyotime Co., Jiangsu, China) for 5 min. The stained nuclei were washed twice with PBS, and were consequently observed under an IX71SIF-3 fluorescence microscope at 350 nm excitation and 460 nm emissions [29].

TUNEL assay

The cells (5×104 cell/mL, 0.6 mL/well) were seeded in 6-well tissue culture plates. Following incubation, the medium was removed and replaced with fresh medium plus 10% FBS and then supplemented with compounds (20 μmol/L). TUNEL assays were performed using a colorimetric TUNEL apoptosis assay kit according to the manufacturer’s instructions. (1) After the treatment period, cells were washed with 1×PBS and fixed in 4% paraformaldehyde for 40 min. The cells were washed once with PBS, and were consequently permeabilized with immunol staining wash buffer for 2 min on ice. (2) The cells were rewashed once with PBS, and were consequently incubated in 0.3% H2O2 in methanol at room temperature for 20 min to inactivate the endogenous peroxidases, after which the cells were washed thrice with PBS. (3) The cells were incubated with 2 μL of TdT-enzyme and 48 μL of Biotin-dUTP per specimen for 60 min at 37°C. The cells were terminated for 10 min, and were consequently incubated with streptavidin-HRP (50 μL per specimen) conjugate diluted at 1:50 in sample diluent for 30 min. (4) The cells were washed three times with PBS, and were consequently incubated with diaminobenzidine solution (200 μL per specimen) for 10 min. At last, the cells were rewashed twice with PBS, and were consequently imaged under an XDS-1B inverted biological microscope [30].

Flow cytometry analysis

Prepared MGC-803 cells (1×106/mL) were washed twice with cold PBS and then re-suspended gently in 500 μL binding buffer. Thereafter, cells were stained in 5 μL Annexin V-FITC and shaked well. Finally, 5 μL PI was added to these cells and incubated for 20 min in a dark place, analyzed by FACS Calibur, Becton Dickinson [31,32].

Caspase 3 enzyme assay

Cells were collected after treatment with BTA and OEA at 2.5, 5, and 10 μM for 12 h, respectively. Prepared MGC-803 cells (1×106/mL, 5 ml) were washed twice with cold PBS. Then, 100 μL of lysis buffer was added to the cells for 25 min on ice and centrifuged at 16000 g for 15 min. 80 μL of reaction buffer and 10 μL of Ac-DEVED-pNA were added to 10 μL of supernatant liquid. After incubating at 37°C for 2–3 h in darkness, the absorbance was measured at 405 nm, with the lysis buffer and reaction buffer as control

Caspase 9 enzyme assay

Cells were collected after treatment with BTA and OEA at 2.5, 5, and 10 μM for 12 h, respectively. Prepared MGC-803 cells (1×106/mL, 5 ml) were washed twice with cold PBS. Then, 100 μL of lysis buffer was added to the cells for 25 min on ice and centrifuged at 16000 g for 15 min. 80 μL of reaction buffer and 10 μL of Ac-LEHD-pNA were added to 10 μL of supernatant liquid. After incubating at 37°C for 2–3 h in darkness, the absorbance was measured at 405 nm, with the lysis buffer and reaction buffer as control.

Western botting analysis

Cells were collected after treatment with BTA and OEA at 2.5, 5, and 10 μM for 12 h, respectively. Western blotting analysis was performed as previously described [33], using the following antibodies at dilutions of 1:500 to 1:1000: anti-p53, anti-Bax, and anti-β actin (Cell signaling technology, Beverly, MA).

Statistical analysis

All statistical analyses were performed using SPSS 10.0, and the data were analyzed using one-way ANOVA. The mean separations were performed using the least significant difference method. Each experiment was performed in triplicate, and all experiments were run thrice and yielded similar results. Measurements from all the replicates were combined, and the treatment effects were analyzed.

Results and discussion

The roots of Toona sinensis collected from Guizhou province were studied, and fifteen compounds were isolated from the plants. The extraction and purification process of the compounds from the plant and their NMR data are presented in Additional file 1.

Additional file 1. The extraction and purification process of the compounds from the plant and their NMR data.

Format: DOC Size: 86KB Download file

This file can be viewed with: Microsoft Word ViewerOpen Data

The potential effect of the compounds from Toona sinensis was investigated on the viability of MGC-803, PC3, A549, MCF-7, and NIH3T3 cells by MTT assay, with ADM (Adriamycin) being used as the positive control and culture medium containing 0.1% DMSO used as the negative control. The inhibitory percentage of cancer cells was treated with 20 μmol/L of each compound for 72 h. The results are summarized in Table  1. It could be seen from Table  1 that both of BTA and OEA showed potent antitumor activities against MGC-803 and PC3 cell lines. The inhibitory ratios of BTA and OEA at 72 h after treatment were 56.1% and 45.2% against MGC-803 cells, 63.4% and 42.5% against PC3 cells, 22.1% and 23.6% against NIH3T3 normal cell line, respectively. In addition, BTA also had good activities against MCF-7 cells, with inhibitory ratio of 51.2%. Thus, the two compounds were less toxic on normal cells than on the investigated cancer cell lines.

Table 1. Antitumor activities of the isolated compounds on the proliferation of different cell lines

To best of our knowledge, the two compounds, BTA and OEA, were obtained from Toona sinensis for the first time. It was also found to have the greatest potency against the growth of human cancer cell lines and little toxic effect on NIH3T3 cells among the isolated constituents. Further experiments found that proliferation of these four cancer cells were significantly inhibited by BTA and OEA in a concentration-dependent manner, as shown in Figure 1A and 1B. The IC50 values of BTA and OEA on MGC-803 and PC3 cells were determined to be 17.7 μM and 13.6 μM, 26.5 μM and 21.9 μM, respectively, all of which were lower than that on NIH3T3 cells (IC50 > 50 μM) by MTT assay. On this occasion, the two compounds were both less toxic on normal cells than on the investigated cancer cell lines and much selective to cancer cells.

thumbnailFigure 1. Effect of BTA and OEA on proliferation of tumor cells. Data are presented as means ± SD, n = 4

Apoptosis is a physiological pattern of cell death characterized by morphological features and extensive DNA fragmentation [34]. Thus, to determine whether the grown inhibitory activities of BTA and OEA were related to the induction of apoptosis, the morphological changes of MGC-803 and PC3 cells were investigated using acridine orange/ethidium bromide (AO/EB) staining and Hoechst 33258 staining, and Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay to confirm cell apoptosis. Moreover, the apoptosis ratios induced by BTA and OEA caused apoptosis in MGC-803 cells were quantitatively assessed by flow cytometry (FCM). Interestingly, whether the cancer cell apoptosis by the two compounds was though the mitochondrial pathway was also studied.

AO is taken up by both viable and non-viable cells and emits green fluorescence if intercalated into double stranded nucleic acid (DNA), and EB is taken up only by non-viable cells and emits red fluorescence by intercalation into DNA. Thus, live cells have a normal green nucleus, whereas the early apoptotic cells are bright green nucleus with condensed or fragmented chromatin and the late apoptotic cells display condensed and fragmented orange chromatin [35]. With HCPT as positive control, the BTA and OEA at 20 μM for 24, 48 h were detected via AO/EB staining. As can be seen in Figure 2, early apoptotic cells with yellow dots and late apoptotic cells with orange dots in MGC-803 and PC3 cell nuclei in positive control, and the cells treated with BTA and OEA had changed. Yellow and orange dots in MGC-803 and PC3 cells showed early and late apoptotic cells, and the appearance of little red cells indicated that BTA and OEA were low cytotoxicity. Therefore, it can be concluded that BTA and OEA could induce apoptosis without any significant cytotoxicity.

thumbnailFigure 2. Results from the AO/EB staining. For MGC-803 cells group, A: negative control; B: positive control, treated with HCPT (20 μM) for 48 h; C, D: treated with BTA (20 μM) for 24, 48 h; E, F: treated with OEA (20 μM) for 24, 48 h. For PC3 cells group, A’: negative control; B’: positive control, treated with HCPT (20 μM) for 48 h; C’, D’: treated with BTA (20 μM) for 24, 48 h; E’, F’: treated with OEA (20 μM) for 24, 48 h

Hoechst 33258 staining is used to visualize nuclear changes and apoptotic body formation that are characteristic of apoptosis. And it showed apoptosis in all four types of cells, which were characterized by cytoplasmic and nuclear shrinkage, chromatin condensation and apoptosis body [36]. With HCPT as positive control, the BTA and OEA at 20 μM for 24, 48 h were detected via Hoechst 33258 staining. As shown in Figure 3, cells treated with the negative control were normally blue. The cells of the negative group were normal blue. However, the HCPT group appeared compact condensed, and crescent-shaped. The cells exhibited strong blue fluorescence, revealing the typical apoptosis characteristics. The cells treated with BTA and OEA had changed, and cells nuclei appeared to be highly condensed and crescent-shaped. These findings demonstrate that BTA and OEA could induce apoptosis against MGC-803 and PC3 cell lines, consistent with the results for the previous AO/EB double staining.

thumbnailFigure 3. Results from the Hoechst 33258 staining. For MGC-803 cells group, A: negative control; B: positive control, treated with HCPT (20 μM) for 48 h; C, D: treated with BTA (20 μM) for 24, 48 h; E, F: treated with OEA (20 μM) for 24, 48 h. For PC3 cells group, A’: negative control; B’: positive control, treated with HCPT (20 μM) for 48 h; C’, D’: treated with BTA (20 μM) for 24, 48 h; E’, F’: treated with OEA (20 μM) for 24, 48 h

In addition, TUNEL, one of the popular methods to investigate the apoptosis induction, identified apoptotic cells in situ via the detection of DNA fragmentation, due to the degradation of DNA after the activation of Ca/Mg-dependent endonucleases. This DNA cleavage leads to strand breaks within the DNA, and could be identified by terminal deoxynucleotidyl transferase that catalyzed the addition of biotin-dUTP. The biotin-labeled cleavage sites were then detected by reaction with streptavidin-HRP and visualized by diaminobenzidine, as indicated by a brown color [37]. With HCPT as positive control, the BTA and OEA at 20 μM for 24, 48 h were detected via TUNEL assay. As shown in Figure 4, the cells treated with BTA, OEA and HCPT appear as brown precipitates. Therefore, it can be further concluded that BTA and OEA could induced apoptosis in MGC-803 and PC3 cells. The results were identical with the previous experiment.

thumbnailFigure 4. Results from the TUNEL assay. For MGC-803 cells group, A: negative control; B: positive control, treated with HCPT (20 μM) for 48 h; C, D: treated with BTA (20 μM) for 24, 48 h; E, F: treated with OEA (20 μM) for 24, 48 h. For PC3 cells group, A’: negative control; B’: positive control, treated with HCPT (20 μM) for 48 h; C’, D’: treated with BTA (20 μM) for 24, 48 h; E’, F’: treated with OEA (20 μM) for 24, 48 h

The apoptosis ratios induced by BTA and OEA in MGC-803 cells were quantitatively assessed by FCM. In early apoptotic cells, phosphatidylserine (PS) which distributed inside the lipid bilayer in the normal cells was transferred from the inside of the cell membrane to the outside. Annexin V, a Ca2+ dependent phospholipid-binding protein with a high affinity for PS, was used to detect early apoptotic cells. PI (Propidine Iodide) was a red fluorescent dye and stained cells that had lost membrane integrity. Cells stained with Annexin V-FITC and PI were classified as necrotic cells (the upper left quadrant; Annexin/PI+), late apoptotic cells (the upper right quadrant; Annexin+/PI+), intact cells (the lower left quadrant; Annexin/PI) or early apoptotic cells (the lower right quadrant; Annexin+/PI) [38]. As shown in Figure 5A, BTA and OEA could induce apoptosis in MGC-803 cells. Apoptosis ratios (including the early and late apoptosis ratios) for BTA and OEA were obtained after 72 h of treatment at a concentration of 20 μM, with the highest apoptosis ratios being 27.3% and 24.5%, respectively. Furthermore, as shown in Figure 5B, the apoptosis of MGC-803 cells which treated with BTA and OEA increased gradually in a time-dependent manner.

thumbnailFigure 5. Flow cytometry analysis. A: the apoptosis ratios of MGC-803 cells treated with BTA and OEA (20 μM) assessed by flow cytometry. B: flow cytometry analysis for apoptosis inducing activities of BTA and OEA on MGC-803 cells, a: control; b, c, and d: treated with BTA (20 μM); e, f, and g: treated with OEA (20 μM)

p53 could induce apoptosis after DNA damage in cancer cells [39], while the pro-apoptotic bcl-2 family member, Bax was a candidate mediator of p53-induced apoptosis [40]. The bcl-2 family divided into pro-survival members such as Bcl-2, Bcl-XL, Bcl-w, and CED 9 and pro-apoptotic members such as Bax, Bad, and Bid [41]. On this occasion, these opposing family members could heterodimerize and the relative ratio of the pro-survival vs. pro-apoptotic members may determine whether the cell lives or dies [42]. The anti-apoptotic members appear to function by inhibiting the release of cytochrome c from the mitochondria or by inhibiting Apaf-1 directly [43]. Cytochrome c acts as a co-factor with ATP for the activation of Apaf-1 which then activates caspase 9, an “initiator caspase”, and caspase 9 can then in turn activate caspase 3 [44]. As shown in Figure 6A, 6B, and 6C, when MGC-803 cells were treated with BTA and OEA at different concentrations after 12 h, the caspase 3/9, p53, and Bax were activated significantly. Thus, the results revealed that the BTA and OEA could induce mitochondria pathway mediated cell apoptosis in MGC-803 cell line (Figure 6D).

thumbnailFigure 6. Levels of caspases, p53, and Bax. A: activation of caspase 3 in MGC-803 cells; B: activation of caspase 9 in MGC-803 cells; C: western blot analysis of p53 and Bax in MGC-803 cells; D: cell apoptosis was mediated by the mitochondria pathway

Conclusions

In conclusion, studies on the chemical constituents from Toona sinensis, and their biological activities have assumed significance for the rational development and utilization of this plant. In this study, fifteen compounds were isolated and identified. Meanwhile, the tumor cell growth inhibition effects of these constituents on MGC-803, PC3, A549 and MCF-7 cells were carried out by MTT assay. Among these compounds, BTA and OEA, which were isolated from Toona sinensis, showed potent activities on MGC-803 and PC3 cell lines in a dose-dependent manner. The IC50 values of BTA and OEA on MGC-803 and PC3 cells were determined to be 17.7 μM and 13.6 μM, 26.5 μM and 21.9 μM, respectively, all of which were lower than that on NIH3T3 cells (IC50 > 50 μM). The apoptosis inducing activities of BTA and OEA on MGC-803 and PC3 cell lines were investigated through AO/EB staining, Hoechst 33258 staining, and TUNEL assay. In addition, the apoptosis ratios induced by BTA and OEA caused apoptosis of MGC-803 cells were quantitatively assessed by flow cytometry, with apoptosis ratios of 27.3% and 24.5% after 72 h of treatment at 20 μM, respectively. Interestingly, the BTA and OEA induced cell apoptosis through the mitochondrial pathway in MGC-803 cells. Our findings have implied that BTA and OEA has potential therapeutic value for treatment of cancer.

Competing interest

The authors declare there are not any competing interests.

Authors’ contribution

SY designed the experiments and carried out most of the bioassay experiments. QZ and HX took part in the compound structural elucidation and bioassay experiments. ML took part of the bioassay experiments. QZ and WX carried out some structure elucidation experiments. Prof. BS and Prof. SY are the co-corresponding authors for this work. All authors read and approved the final manuscript.

Acknowledgements

The authors wish to thank the National Key Program for Basic Research (Nos.2010CB126105, 2010CB134504), the National Natural Science Foundation of China (Nos. 21132003, 21172048), Guizhou Province S&T Program (No. 20103052) for the financial support.

References

  1. Demain AL, Vaishnav P: Natural products for cancer chemotherapy.

    Microb Biotechnol 2011, 4:687-699. PubMed Abstract | Publisher Full Text OpenURL

  2. Massaoka MH, Matsuo AL, Figueiredo CR, Farias CF, Girola N, Arruda DC, Scutti JAB, Romoff P, Favero OA, Ferreira MJP, Lago JHG, Travassos LR: Jacaranone induces apoptosis in melanoma cells via ROS-mediated downregulation of Akt and p38 MAPK activation and displays antitumor activity in vivo.

    PLoS One 2012, 7:1-11. OpenURL

  3. Patel B, Prakash R, Yasir M, Sattwik das: Natural bioactive compound with anticancer potential.

    Int J Adv Pharm Sci 2010, 1:32-41. Publisher Full Text OpenURL

  4. Cragg GM, Newman J: Nature: a vital source of leads for anticancer drug development.

    Phytochem Rev 2009, 8:313-331. Publisher Full Text OpenURL

  5. Cragg GM, Newman J: Plants as a source of anti-cancer and anti-HIV agents.

    Ann Appl Biol 2003, 143:127-133. Publisher Full Text OpenURL

  6. Nobili S, Lippi D, Witort E, Donnini M, Bausi L, Mini E, Capaccioli S: Natural compounds for cancer treatment and prevention.

    Pharmacol Res 2009, 59:365-378. PubMed Abstract | Publisher Full Text OpenURL

  7. Fulda S, Debatin KM: Sensitization for tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by the chemopreventive agent resveratrol.

    Cancer Res 2004, 64:337-346. PubMed Abstract | Publisher Full Text OpenURL

  8. Fulda S: Modulation of apoptosis by natural products for cancer therapy.

    Planta Med 2010, 76:1075-1079. PubMed Abstract | Publisher Full Text OpenURL

  9. Solary E, Droin N, Bettaieb A, Corcos L, Dimanche-Boitrel MT, Garrido C: Positive and negative regulation of apoptotic pathways by cytotoxic agents in hematological malignancies.

    Leukemia 2000, 14:1833-1849. PubMed Abstract | Publisher Full Text OpenURL

  10. Castellanos L, Correa RS, Martinez E, Calderon JS: Oleanane triterpenoids from Cedrela montana (Meliaceae).

    Z Naturforsch C 2002, 57:575-578. PubMed Abstract OpenURL

  11. Kommera H, Dittrich S, Kalbitz J, Dräger B, Mueller T, Paschke R, Kalud-erovic’GN: Carbamate derivatives of betulinic acid and betulin with selective cytotoxic activity.

    Bioorg Med Chem Lett 2010, 20:3409-3412. PubMed Abstract | Publisher Full Text OpenURL

  12. Rao VS, de Melo CL, Queiroz MGR, Lemos TLG, Menezes DB, Melo TS, Santos FA: Ursolic acid, a pentacyclic triterpene from Sambucus australis, prevents abdominal adiposity in mice fed a high-fat diet.

    J Med Food 2011, 14:1375-1382. PubMed Abstract | Publisher Full Text OpenURL

  13. Ryu SY, Choi SU, Lee SH, Lee CO, No Z, Ahn JW: Antitumor triterpenes from medicinal plants.

    Arch Pharm Res 1994, 17:375-377. Publisher Full Text OpenURL

  14. Cichewicz RH, Kouzi SA: Chemistry, biological activity, and chemotherapeutic potential of betulinic acid for the prevention and treatment of cancer and HIV infection.

    Med Res Rev 2004, 24:90-114. PubMed Abstract | Publisher Full Text OpenURL

  15. Chintharlapalli S, Papineni S, Lei P, Pathi S, Safe S: Betulinic acid inhibits colon cancer cell and tumor growth and induces proteasome-dependent and -independent downregulation of specificity proteins (Sp) transcription factors.

    BMC Cancer 2011, 11:371-383. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text OpenURL

  16. Fulda S: Betulinic acid for cancer treatment and prevention.

    Int J Mol Sci 2008, 9:1096-1107. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  17. Pisha E, Chai H, Lee IS, Chagwedera TE, Farnsworth NR, Cordell GA, Beecher CW, Fong HH, Kinghorn AD, Brown DM: Discovery of betulinic acid as a selective inhibitor of human melanoma that functions by induction of apoptosis.

    Nat Med 1995, 1:1046-1051. PubMed Abstract | Publisher Full Text OpenURL

  18. Min BS, Kim YH, Lee SM, Jung HJ, Lee JS, Na MK, Lee CO, Lee JP, Bae K: Cytotoxic Triterpenes from Crataegus pinnatifida.

    Arch Pharm Res 2000, 23:155-158. PubMed Abstract | Publisher Full Text OpenURL

  19. 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. PubMed Abstract | Publisher Full Text OpenURL

  20. Kim DK, Baek JH, Kang CM, Yoo MA, Sung JW, Chung HY, Kim ND, Choi YH, Lee SH, Kim KW: Apoptotic activity of ursolic acid may correlate with the inhibition of initiation of DNA replication.

    Int J Cancer 2000, 87:629-836. PubMed Abstract | Publisher Full Text OpenURL

  21. Andersson D, Liu JJ, Nilsson A, Duan RD: Ursolic acid inhibits proliferation and stimulates apoptosis in HT29 cells following activation of alkaline sphingomyelinase.

    Anticancer Res 2003, 23:3317-3322. PubMed Abstract OpenURL

  22. Zhang X, Li H, Jin Y, Fang G: Effects of betulonic acid on SGC-7901, HepG-2 and mice of bearing S180 tumor cells.

    Nat Prod Res Dev 2009, 21:766-770. OpenURL

  23. Saxena BB, Zhu L, Hao M, Kisilis E, Katdare M, Oktem O, Bomshteyna A, Rathnam P: Boc-lysinated-betulonic acid: a potent, anti-prostate cancer agent.

    Bioorg Med Chem 2006, 14:6349-6358. PubMed Abstract | Publisher Full Text OpenURL

  24. Guo L, Wu JZ, Han T, Cao T: Chemical composition, antifugal and antitumor properties of ether extracts of Scapania verrucosa Heeg. and its endophytic fungus Chaetomium fusiforme.

    Molecules 2008, 13:2114-2125. PubMed Abstract | Publisher Full Text OpenURL

  25. Dellai A, Deghrigue M, Laroche-Clary A, Masour HB, Chouchane N, Robert J, Bouraoui A: Evaluation of antiproliferative and anti-inflammatory activities of methanol extract and its fractions from the Mediterranean sponge.

    Cancer Cell Int 2012, 12:18. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text OpenURL

  26. Kjellström J, Oredsson SM, Wennerberg J: Increased toxicity of a trinuclear Pt-compound in a human squamous carcinoma cell line by polyamine depletion.

    Cancer Cell Int 2012, 12:20. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text OpenURL

  27. Wei HB, Hu BG, Han XY, Zheng ZH, Wei B, Huang JL: Effect of all-trans retinoic acid on drug sensitivity and expression of survivin in LoVo cells.

    Chin Med J 2008, 4:331-335. OpenURL

  28. Jiang Z, Wu W, Qian M: Cellular damage and apoptosis along with changes in NF-kappa B expression were induced with contrast agent enhanced ultrasound in gastric cancer cells and hepatoma cells.

    Cancer Cell Int 2012, 12:8. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text OpenURL

  29. Holmquist G: Hoechst 33258 fluorescent staining of Drosophila chromosomes.

    Chromosoma 1975, 49:333-356. PubMed Abstract OpenURL

  30. Liu MC, Yang SJ, Jin LH, Hu DY, Wu ZB, Yang S: Chemical constituents of the ethyl acetate extract of Belamcanda chinensis (L.) DC roots and their antitumor activities.

    Molecules 2012, 5:6156-6169. OpenURL

  31. Orozco AF, Lewis DE: Flow cytometric analysis of circulating microparticles in plasma.

    Cytom A 2010, 77:502-514. OpenURL

  32. Ishikawa J, Takahashi Y, Hazawa M, Fukushi Y, Yoshizawa A, Kashiwakura I: Suppressive effects of liquid crystal compounds on the growth of U937 human leukemic monocyte lymphoma cells.

    Cancer Cell Int 2012, 12:3. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text OpenURL

  33. Liu J, Uematsu H, Tsuchida N, Ikeda MA: Essential role of caspase-8 in p53/p73-dependent apoptosis induced by etoposide in head and neck carcinoma cells.

    Mol Cancer 2011, 10:1-13. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text OpenURL

  34. Collins JA, Schandl CA, Young KK, Vesely J, Willingham MC: Major DNA fragmentation is a late event in apoptosis.

    J Histochem Cytochem 1997, 45:923-934. PubMed Abstract | Publisher Full Text OpenURL

  35. Liu MC, Yang SJ, Jin LH, Hu DY, Xue W, Song BA, Yang S: Synthesis and cytotoxicity of novel ursolic acid derivatives containing an acyl piperazine moiety.

    Eur J Med Chem 2012, 58:128-135. PubMed Abstract | Publisher Full Text OpenURL

  36. Wu J, Yi WS, Jin LH, Hu DY, Song BA: Antiproliferative and cell apoptosis-inducing activities of compounds from Buddleja davidii in MGC-803 cells.

    Cell Div 2012, 7:1-20. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text OpenURL

  37. Xu XQ, Gao XH, Jin LH, Yuan K, Hu DY, Song BA, Yang S: Antiproliferation and cell apoptosis inducing bioactivities of constituents from Dysosma versipellis in PC3 and Bcap-37 cell lines.

    Cell Div 2011, 6:1-14. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text OpenURL

  38. Yuan K, Song BA, Jin LH, Xu S, Hu DY, Xu XQ, Yang S: Synthesis and biological evaluation of novel 1-aryl, 5-(phenoxy-substituted) aryl-1,4-pentadien-3-one derivatives.

    Med Chem Commn 2011, 2:585-589. Publisher Full Text OpenURL

  39. Cui H, Schroering A, Ding HF: p53 mediates DNA damaging drug-induced apoptosis through a caspase-9-dependent pathway in SH-SY5Y neuroblastoma cells.

    Mol Cancer Ther 2002, 1:679-686. PubMed Abstract | Publisher Full Text OpenURL

  40. Juin P, Hunt A, Littlewood T, Griffiths B, Swigart BL, Korsmeyer S, Evan G: c-Myc functionally cooperates with Bax to induce apoptosis.

    Mol Cell Biol 2002, 22:6158-6169. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  41. Brunelle JK, Letai A: Control of mitochondrial apoptosis by the Bcl-2 family.

    J Cell Sci 2009, 122:437-441. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  42. Basu A, Haldar S: The relationship between Bcl2, Bax and p53: consequences for cell cycle progression and cell death.

    Mol Hum Reprod 1998, 1998:1099-1109. OpenURL

  43. Gross A, McDonnell JM, Korsmeyer SJ: BCL-2 family members and the mitochondria in apoptosis.

    Gene Dev 1999, 13:1899-1911. PubMed Abstract | Publisher Full Text OpenURL

  44. Rodriguez J, Lazebnik Y: Caspase-9 and APAF-1 form an active holoenzyme.

    Gene Dev 1999, 13:3179-3184. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL