|Year : 2017 | Volume
| Issue : 49 | Page : 118-122
Cytotoxic activities of naturally occurring oleanane-, ursane-, and lupane-type triterpenes on HepG2 and AGS cells
Heejung Yang1, Hyun Woo Kim2, Young Choong Kim2, Sang Hyun Sung2
1 College of Pharmacy, Kangwon National University, Chuncheon, Republic of Korea
2 College of Pharmacy and Research Institute of Pharmaceutical Science, Seoul National University, Seoul, Republic of Korea
|Date of Submission||26-May-2015|
|Date of Acceptance||29-Sep-2015|
|Date of Web Publication||06-Jan-2017|
Prof. Sang Hyun Sung
College of Pharmacy and Research Institute of Pharmaceutical Science, Seoul National University, Seoul
Republic of Korea
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: It is well known that the naturally occurring modified triterpenes in plants have a wide diversity of chemical structures and biological functions. The lupane-oleanane-and ursane-type triterpenes are the three major members of natural triterpenes with a wide range of biological properties. A systematic approach is necessary to review their structures and biological activities according to the backbones and the different substituents. Objective: Thirty lupane-(L1-7)oleanane-(O1-14)and ursane-type (U1-9) triterpenes with structural diversity were examined to evaluate their cytotoxic activities against two cancer cell lineshuman hepatocellular carcinoma (HepG2) and AGS cells. Materials and Methods: They were isolated from Hedera helixJuglans sinensisand Pulsatilla koreana using a series of column chromatography methods and were treated to evaluate their cytotoxic activities against HepG2 and AGS human gastric adenocarcinoma cell. Furthertwo triterpenes showing the most potent activities were subjected to the apoptotic screening assay using flow cytometry. Results: The polar groupssuch as an oxo group at C-1a free hydroxyl at C-2C-3or C-23and a carboxylic moiety at C-28as well as the type of backboneexplicitly increased the cytotoxic activity on two cancer cells. O5 and U5 showed significantly the potent cytotoxic activity in comparison to other glycosidic triterpenes. In annexin-V/propidium iodide (PI) staining assaythe percentage of late apoptosis (annexin-V+/PI+) 12 and 24 h after treatment with O5 and U5 at 25 μM increased from 14.5% to 93.1% and from 46.4% to 49.1%respectivelyin AGS cells. The cytotoxicity induced by O5 showed a significant difference compared to U5 for 12 and 24 h. Conclusion: In the studywe can suggest the potent moieties which influence their cytotoxic activities against two cancer cells. The polar groups at C-1C-2C-3C-23and C-28 and the linkage of sugar moieties influenced the different cytotoxic activities.
Keywords: Flow cytometry, lupane, oleanane, triterpene, ursane
|How to cite this article:|
Yang H, Kim HW, Kim YC, Sung SH. Cytotoxic activities of naturally occurring oleanane-, ursane-, and lupane-type triterpenes on HepG2 and AGS cells. Phcog Mag 2017;13:118-22
|How to cite this URL:|
Yang H, Kim HW, Kim YC, Sung SH. Cytotoxic activities of naturally occurring oleanane-, ursane-, and lupane-type triterpenes on HepG2 and AGS cells. Phcog Mag [serial online] 2017 [cited 2022 Oct 7];13:118-22. Available from: http://www.phcog.com/text.asp?2017/13/49/118/196308
- Thirty naturally occurring oleanane-ursane-and lupane-type triterpenes were isolated from Hedera helixJuglans sinensisand Pulsatilla koreana
- An oxoa free hydroxyla carboxylic moietyand the types of aglycone influenced the cell cytotoxicity
- Corosolic acid and α-hederin showed the most potent cytotoxicity via apoptosis.
| Introduction|| |
Triterpenes are a member of terpenes, which are structurally diverse class of natural compounds derived from the combination of C5 isoprene unitsand have been abundantly found in the plant kingdom. They mainly exist in the cyclized form of squalene (30 carbons) which are acyclic hydrocarbons formed by two units of farnesyl diphosphate. Triterpenes are precursors of steroids in both plants and animals which have a wide range of pharmacological propertiessuch as anti-inflammatoryanti-angiogenesisanti-oxidativepro-apoptoticand re-differentiation effects. Especiallylupane-oleanane-and ursane-type triterpenes showed the cytotoxicity against various cancer cell lines and have been considered as promising anti-cancer agents., Their cytotoxic activity on cancer cells is significantly affected by the type of basic backbone and the present number and position of derivativessuch as alkylhydroxylcarboxyland amino acids., Among thesetriterpene glycosides (i.e.triterpenoidal saponins) occurring by the combination of sugar residues to basic backbone also have significant cytotoxic effects on cancer cells.,
To dateit has been mainly demonstrated the structure-activity relationship between synthesized triterpene derivatives on cancer cells.,, We evaluated the cytotoxic activities of naturally occurring lupane-oleanane-and ursane-type triterpenes isolated from Juglans sinensis Dode (Juglandaceae),, Pulsatilla koreana Nakai (Ranunculaceae), and Hedera helix L. (Araliaceae) against human hepatocellular carcinoma (HepG2) and AGS human gastric adenocarcinoma cell. We tried to suggest structurally some key determinants in the structure of triterpene derivatives on the cytotoxicity against cancer cell linesand also explore their preliminary mechanisms through inhibiting cell cycle and inducing apoptosis.
| Materials and Methods|| |
Compounds L1-7O1-14and U1-9 were isolated from J. sinensis Dode (Juglandaceae) (L1O1-4O10-14and U1-9),, P. koreana Nakai (Ranunculaceae) (L2-7O6and O9), and H. helix L. (O5O7and O8) using repeated column chromatography and high-performance liquid chromatography. The structures of these compounds were unequivocally determined by diverse spectroscopic analysessuch as 1D2D nuclear magnetic resonance (NMR) experimentsmass spectrometry analysesas well as by comparison with the literature for the known compounds. The 1H and 13C NMR1H-1H correlation spectroscopyHeteronuclear single quantum coherence spectroscopy (HSQC)and heteronuclear multiple bond correlation spectra were recorded on a Bruker AMX 400 (Bruker BioSpin GmbHKarlsruheGermany) or 500 (Bruker BioSpin GmbHKarlsruheGermany) spectrometer in pyridine-d5. High- and low-resolution FABMS (Fast Atom Bombardment Mass Spectrometry) were obtained on a JEOL JMS-AX505WA. Detection of DNA cycle and apoptosis on hepatic stellate cells-T6 cells was conducted by flow cytometry (BD BiosciencesFACSCaliburFranklin LakesNJUSA).
HepG2 and AGS (human gastric adenoma carcinoma cells) cell lines were purchased from ATCC (ManassasVAUSA). HepG2 and AGS cells were maintained in Dulbecco's modified eagle medium and Roswell Park Memorial Institute 1640 mediumrespectivelysupplemented with 10% (v/v) fetal bovine serumpenicillin (100 U/mL)and streptomycin (100 μg/mL). They were incubated in a humidified atmosphere of 5% CO2 gas at 37°C.
The cytotoxicity of L1-7O1-14and U1-9 against two cancer cell lines was evaluated by modified 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay. All samples were dissolved in dimethyl sulfoxide (final concentration0.1%) and diluted in distilled water. Cells were seeded in 96-well plates at a density of 3 × 104 cells/well for the cytotoxicity assay. After 24 h incubationserum-free medium was changed and two cell lines were treated with vehicle or L1-7O1-14and U1-9 at the concentration of 100 μM for 24 or 48 h and incubated with 2 mg/mL of MTT for 2 h. Reduction of MTT to formazan was assessed in an ELISA plate reader at 540 nm. Doxorubicin (Sigma-AldrichSt. LouisMOUSA) was used as a positive control. Cell viability rate was calculated as the percentage of MTT absorption as follows: % survival = (mean experimental absorbance/mean control absorbance × 100). Data were expressed as the mean of three independent experiments.
Flow cytometry for analyzing apoptosis and caspase-3 activity
AGS cells were seeded in 6-well plates at a density of 6 × 105 cells/well for measuring the DNA cycleapoptosisand caspase-3 using flow cytometry. After 24 hcells were treated with O5 and U5 at 10 and 25 μMrespectively. After 12 or 24 hcells were trypsinizedwashed twice with PBS bufferand centrifuged at room temperature. For evaluating the DNA cyclecell pellets were suspended in ice-cold 70% ethanol for fixation of the stage of DNA cycle at 4°C. After overnightcells were centrifuged and resuspended in 500 μL of PI/RNASE staining buffer (BD PharmingenFranklin LakesNJUSA)incubated at room temperature for 30 minand analyzed. For measuring apoptosis using a fluorescein isothiocyanate (FITC)-Annexin V apoptosis detection kit (BD PharmingenFranklin LakesNJUSA)cells were stained with annexin V and PIsubsequentlyaccording to the manufacturer's manualand measured by flow cytometry. The alteration of caspase-3 activity by O5 and U5 was carried out according to the manufacturer's manual (FITC Active Caspase-3 kitBD PharmingenFranklin LakesNJUSA). All experiments were performed by flow cytometry (BD BiosciencesFACSCaliburFranklin LakesNJUSA).
The evaluation of statistical significance was determined by one-way ANOVA testwith P < 0.001 and P < 0.01 considered to be statistically significant.
| Results and Discussion|| |
Cytotoxic activities of 30 triterpenes against HepG2 and AGS cell lines
We evaluated the cytotoxicity of 30 triterpene derivatives including lupane-(L1-7)oleanane-(O1-14)and ursane-type (U1-9) against HepG2 and AGS cell lines by MTT cell viability assay in 24 and 48 h. All triterpenes were isolated from J. sinensis Dode (Juglandaceae) (L1O1-4O10-14and U1-9),, P. koreana Nakai (Ranunculaceae) (L2-7O6and O9), and H. helix L. (O5O7and O8). Their names and structures are listed and provided in [Table 1] and [Figure 1] respectively.
Relationship of structures and activities of 30 triterpenes
The cytotoxic activity of L1-7O1-14and U1-9 at the concentration of 100 μM against HepG2 and AGS cells for 24 and 48 h was plotted in [Figure 2]. In lupane-type triterpenes (L1-7)L2 including an O-linkage of two glycosides at C-3a carboxylic group at C-17and a hydroxyl group at C-23 showed the most significant cytotoxic activity than other lupane-type triterpenes against two cancer cell lines. The substituents of sugar moieties at C-28 unambiguously decreased the cytotoxicity against cancer cells (L2 vs. L3-7). In additionL1 with a methyl group instead of a carboxylic group at C-17 did not show the potent inhibitory activity compared to L2. These results supported that a free carboxylic acid instead of a methyl or sugar group at C-17 was essential for cytotoxic activity. Similar cytotoxic results were also observed when oleanane-type triterpenes (O1-14) were treated on two cancer cells. The compounds with sugar moieties at C-28 (O3O4O7-9and O12) showed no potent cytotoxic activities compared to those with a free carboxylic acid (O1O2O5O10O11and O13). Interestinglyintroduction of sugar moiety at C-3 did not influence the loss of cytotoxic activity (O5). The presence of a hydroxyl moiety at C-23 could significantly increase cell growth inhibition (O10 vs. O11). In terms of ursane-type triterpenesthe cytotoxic results of HepG2 and AGS cell lines treated with U1-9 generally showed more potent inhibition than lupane- and oleanane-type triterpenes on cell proliferation. In additionsome modificationssuch as hydroxylation at C-2 (U3 vs. U5 and U4 vs. U6) or C-22 (U7 vs. U8)oxidation from hydroxyl to oxo at C-3 (U4 vs. U9)and glycosylation at C-28 (U4 vs. U7)did not significantly change the cell viability. Howeverthe configuration of hydroxyl group at C-3 had an important influence on the anti-tumor activity. U1 and U2 which have α-hydroxyl group at C-3 showed no cytotoxic activity compared to others which have β-hydroxyl group (U3-9). On the basis of the above resultsit was suggested that a free hydroxyl group at C-2C-3and C-23and a free carboxylic group at C-28 were essential for the cell growth inhibitionand the addition of sugar moiety at C-3 or C-28 reduced the cytotoxic effect as the length of glycosides was increased. In comparison between oleanane- and ursane-type triterpenes having same modificationsursane-type triterpenes had slightly more potent cytotoxicity than oleanane-type triterpenes (U3 vs. O1U6 vs. O2and U7 vs. O3) as reported in the literature.,,,
|Figure 2: The cytotoxic activity of L1-7O1-14and U1-9 against HepG2 and AGS cell lines. Cytotoxic effect of L1-7O1-14and U1-9 were evaluated against AGS (a) and HepG2 (b) cells. The cells were treated with samples at the concentration of 100 μM for 24 or 48 h as indicated in the materials and methods. Each point represents a mean ± standard deviation of 3 experiments. Doxorubicin was used as a positive control and showed IC 50 for AGS and 3.7% (24 h) and 1.5% (48 h) for HepG2respectively. a: P < 0.001b: P < 0.05and c: P < 0.01 compared with control|
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Apoptotic activities of O5 and U5 against HepG2 and AGS cell lines
We investigated the changes in progress of apoptosis induced by O5 (IC50 7.6 μM in AGS cells and 15.2 μM in HepG2 cells) and U5 (IC50 11.3 μM in AGS cells and 24.5 μM in HepG2 cells)which showed the most significant inhibitory activity against two cancer cells using the flow cytometry [Figure 3]. While the structure of U5 includes all the factorssuch as ursane-type aglyconea free hydroxyl group at C-2C-3and C-23and a free carboxylic group at C-28which essentially influence the cell viability against cancer cells in the cytotoxic assayO5 as oleanane-type has not only a hydroxyl moiety at C-23 and a free carboxylic acid at C-28but also two sugars at C-3. O5 and U5 at concentrations of 10 and 25 μM were treated in AGS cells for 12 and 24 h. The annexin-V/PI double staining results described that two compoundsO5 and U5showed different aspects on the stage of apoptosis in AGS cells. When treated with 10 and 25 μM for 12 h incubationU5 had much more potent cytotoxicity than O5 ([Figure 3]; b1 vs. d1 and c1 vs. e1). While O5 at 10 μM for 24 h did not induce the significant apoptosis compared to control groupthe population of the apoptotic cells treated with O5 at 25 μM for 24 h in late apoptosis stage (annexin-V+/PI+) was dramatically increased to about 93.1% compared to U5 at the same concentration (49.1%). Our finding suggested that the delayed cytotoxicity of O5 might arise from the alteration of cell permeability by two glycosides at C-3 in structure. In the case of U5the percentage of late apoptotic cells (annexin-V+/PI+) treated with 10 μM has doubled from 20.9% in 12 h to 43.6% in 24 hwhile the number of late apoptotic cells induced by 25 μM showed no significant change as time passed (46.4% in 12 h and 49.1% in 24 h).
|Figure 3: The flow cytometry analysis of the stage of O5 and U5-induced apoptosis in AGS cells. After 12 (a1-e1) or 24 h (a2-e2) incubated with O5 (b and c) and U5 (d and e) at the concentrations of 10 (b and d) and 25 (c and e) μMrespectively. (a1) and (a2) were the negative controls which were treated with the vehicle. Values are represented as percentage. Data are representative of three independent experiments|
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Changes of caspase-3 activity by O5 and U5 in AGS cell line
To further understand the mechanism of O5 and U5-induced apoptosiswe evaluated the caspase-3 activity that plays a pivotal role in the process of apoptosis in AGS cells. O5 and U5 gradually increased the percentage of the caspase-3 activity in a concentration-dependent mannerwhen the cells were incubated with samples for 24 h [Figure 4]. The percentages of the activated caspase-3 treated with O5 and U5 compared to those of the control cells increased gradually with increasing the concentrations from 10 (O5; 41.1% and U5; 45.0%) to 25 μM (O5; 56.6% and U5; 55.2%). These results suggested that active caspase-3 played an important role in executing apoptosis by O5 and U5 in AGS cells.
|Figure 4: The flow cytometry analysis of O5 and U5-induced caspase-3 activity in AGS cells. The cells were incubated for 24 h with vehicle (controlwhite color)O5 or U5 at the indicated concentration (red color). Values are represented as percentage. Data are representative of three independent experiments|
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| Conclusion|| |
In the present studywe examined the cytotoxic activities of 30 lupane-oleanane-and ursane-type triterpenes against HepG2 and AGS cell lines. The types of backbones and simple structural modificationssuch as hydroxylation and glycosylationsignificantly influenced their biological activities. O5an oleanolic acid derivative with hydroxyl group at C-23 and two sugar groups at C-3and U5an ursolic acid derivative with hydroxyl group at C-2were the most potent apoptotic activities on the two cancer cell lines via caspase-3 regulation. Although further studies are needed to clarify the mechanism related with apoptosis by O5 and U5 on the inhibition of cancer cells proliferationthe structure-activity relationship of 30 triterpenes including O5 and U5 might give an idea for developing the therapeutic agents consisting of a triterpenes-concentrated preparation.
This work was supported by the 2014 Research Grant from Kangwon National University.
Financial support and sponsorship
This work was supported by the 2014 Research Grant from Kangwon National University (No. 120140653) and Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of ScienceICT & Future Planning (No. NRF-2015R1C1A1A010553892).
Conflicts of interest
There are no conflicts of interest.
| References|| |
Hill RA, Connolly JD, Triterpenoids. Nat Prod Rep 2012;29:780-818.
Dewick PD, Medicinal Natural Products-A Biosynthetic Approach 2009;3rd edWileyWest Sussex
Liu J, Oleanolic acid and ursolic acid: Research perspectives. J Ethnopharmacol 2005;100:92-4.
Laszczyk MN, Pentacyclic triterpenes of the lupane, oleanane and ursane group as tools in cancer therapy. Planta Med 2009;75:1549-60.
Meng YQ, Liu D, Cai LL, Chen H, Cao B, Wang YZ, The synthesis of ursolic acid derivatives with cytotoxic activity and the investigation of their preliminary mechanism of action. Bioorg Med Chem 2009;17:848-54.
Ma CM, Cai SQ, Cui JR, Wang RQ, Tu PF, Hattori M, et al.
The cytotoxic activity of ursolic acid derivatives. Eur J Med Chem 2005;40:582-9.
Bachran C, Bachran S, Sutherland M, Bachran D, Fuchs H, Saponins in tumor therapy. Mini Rev Med Chem 2008;8:575-84.
Fuchs H, Bachran D, Panjideh H, Schellmann N, Weng A, Melzig MF, et al.
Saponins as tool for improved targeted tumor therapies. Curr Drug Targets 2009;10:140-51.
Huang Z, Fu J, Liu L, Sun Y, Lai Y, Ji H, et al.
Glycosylated diazeniumdiolate-based oleanolic acid derivatives: Synthesis in vitro
and in vivo
biological evaluation as anti-HepG2 agents. Org Biomol Chem 2012;10:3882-91.
Gao J, Li X, Gu G, Liu S, Cui M, Lou HX, Facile synthesis of triterpenoid saponins bearing ß-Glu/Gal-(1>3)-ß-GluA methyl ester and their cytotoxic activities. Bioorg Med Chem Lett 2012;22:2396-400.
Chen L, Wu JB, Lei F, Qian S, Hai L, Wu Y, Synthesis and biological evaluation of oleanolic acid derivatives as antitumor agents. J Asian Nat Prod Res 2012;14:355-63.
Yang H, Jeong EJ, Kim J, Sung SH, Kim YC, Antiproliferative triterpenes from the leaves and twigs of Juglans sinensis
on HSC-T6 cells. J Nat Prod 2011;74:751-6.
Yang H, Cho HJ, Sim SH, Chung YK, Kim DD, Sung SH, et al.
Cytotoxic terpenoids from Juglans sinensis
leaves and twigs. Bioorg Med Chem Lett 2012;22:2079-83.
Yang H, Cho YW, Kim SH, Kim YC, Sung SH, Triterpenoidal saponins of Pulsatilla koreana
roots. Phytochemistry 2010;71:1892-9.
Mosmann T, Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods 1983;65:55-63.
Shao JW, Dai YC, Xue JP, Wang JC, Lin FP, Guo YH, In vitro
and in vivo
anticancer activity evaluation of ursolic acid derivatives. Eur J Med Chem 2011;46:2652-61.
Li J, Guo WJ, Yang QY, Effects of ursolic acid and oleanolic acid on human colon carcinoma cell line HCT15. World J Gastroenterol 2002;8:493-5.
Yan SL, Huang CY, Wu ST, Yin MC, Oleanolic acid and ursolic acid induce apoptosis in four human liver cancer cell lines. Toxicol In Vitro
Shan JZ, Xuan YY, Ruan SQ, Sun M, Proliferation-inhibiting and apoptosis-inducing effects of ursolic acid and oleanolic acid on multi-drug resistance cancer cells in vitro
. Chin J Integr Med 2011;17:607-11.
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;59:755-62.
Pradelli LA, Bénéteau M, Ricci JE, Mitochondrial control of caspase-dependent and -independent cell death. Cell Mol Life Sci 2010;67:1589-97.
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
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