IJOInternational Journal of Oncology1019-64391791-2423D.A. Spandidos10.3892/ijo.2013.1816ijo-42-04-1452ArticlesChemical constituents of volatile oil from Pyrolae herba and antiproliferative activity against SW1353 human chondrosarcoma cellsCAILIANGLIANG*YEHONGZHI*LIXIHAILINYANPINGYUFANGRONGCHENJIASHOULIHUITINGLIUXIANXIANGAcademy of Integrative Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou, Fujian 350108,
P.R. ChinaCorrespondence to: Professor Xianxiang Liu, Academy of Integrative Medicine, Fujian University of Traditional Chinese Medicine, 1 Huatuo Road, Minhou Shangjie, Fuzhou, Fujian 350108, P.R. China, E-mail: liuxianxiang@163.com
The objective of the present study was to identify chemical constituents of volatile oil from Pyrolae herba (PHVO) and evaluate the antiproliferative activity of PHVO against SW1353 human chondrosarcoma cells. The volatile oil from Pyrolae herba was prepared by hydrodistillation and characterized by gas chromatography-mass spectroscopy (GC-MS). A total of 12 components in PHVO were identified representing 81.62% of the total integrated chromatographic peaks. The major compounds were found to be n-hexadecanoic acid (29.29%), cedrol (17.08%), 6,10,14-trimethyl-2-pentadecanone (9.59%) and cis-9-octadecadienoic acid (8.23%). The antiproliferative activity of PHVO against SW1353 cells was investigated using MTT assay, flow cytometry and western blot analysis. Our results demonstrated that PHVO inhibited SW1353 cell viability in a dose- and time-dependent manner. Furthermore, PHVO treatment decreased the number of cells entering the S phase and caused a reduction in the expression of cyclin D1, cyclin-dependent kinase (CDK)4 and CDK6, whereas it caused an increase in the expression of p21. PHVO demonstrated potent antitumor activity against SW1353 cells, suggesting its potential use as a therapeutic agent in the treatment of chondrosarcoma.
Chondrosarcoma is the most common primary malignant bone tumor in children and adolescents, comprising almost 60% of all bone sarcomas (1,2). To date, the main treatments for patients with osteosarcoma are surgical resection and chemotherapy (3). However, despite advances in chemotherapy and surgery, the survival rate for osteosarcoma has reached a plateau and 40% of osteosarcoma patients eventually succumb to the disease; thus, there is an urgent need for the development of novel therapeutic agents (4).
Over the past few years, natural products have attracted extensive attention due to their relatively few side-effects and have been used in clinical practice for thousands of years as important alternative remedies for a variety of diseases (5–7). Moreover, natural products are an attractive source of new therapeutic candidate compounds, which make an enormous contribution to drug discovery today (8). An analysis of the number of chemotherapeutic agents and their sources indicates that over 60% of approved drugs are derived from natural compounds (9). Several plant-derived compounds are currently successfully employed in disease treatment. For instance, arteannuin, which was isolated from Artemisia annua, has been used for the treatment of malaria and the vinca alkaloid isolated from the periwinkle Catharanthus roseus has been used for the treatment of cancer (10,11). Volatile oils are a type of secondary plant metabolite, and have been shown to exhibit antioxidant and antibacterial activities (12,13).
Pyrolae herba listed in Chinese Pharmacopoeia is commonly used in traditional Chinese medicine for the treatment of cancer, hypertension, coronary heart disease and various inflammatory diseases, including rheumatoid arthritis and arthralgia. Pyrolae herba functions by expelling wind and removing dampness, and promoting blood circulation for the regulation of menstruation and the relief of pain (14,15). In modern pharmacological studies, the active components of Pyrolae herba such as chimaphilin, ursolic acid and hyperoside had previously been shown to possess anti-inflammatory, antioxidant and antibacterial properties (16–21). Recent studies have suggested that ursolic acid inhibits cell proliferation and downregulates the expression of anti-apoptotic and metastatic gene products, potentiating antitumor activity (22–25). However, the chemical constituents and antitumor activity of volatile oil from Pryrolae herba have not yet been reported.
In this study, volatile oil from Pyrolae herba (PHVO) was obtained by hydrodistillation and identified by gas chromatography-mass spectrometry (GC-MS). To further investigate the antiproliferative activity of PHVO against SW1353 cells, MTT assay was used to evaluate its inhibitory activity. Moreover, cell cycle distribution and protein expression levels of cyclin D1, cyclin-dependent kinase (CDK)4, CDK6 and p21 were determined by flow cytometry and western blot analysis, respectively.
Materials and methodsMaterials and reagents
Pyrolae herba was purchased from Jiangxi Herb Co., Ltd., Nanchang, Jiangxi, China (Chinese FDA approval no. 1202001). Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM) and trypsin-EDTA were purchased from HyClone (Logan, UT, USA). MTT was obtained from Sigma-Aldrich (St. Louis, MO, USA). The cycle test kit was purchased from Becton-Dickinson (San Jose, CA, USA). The total protein extraction kit was purchased from Nanjing KeyGen Biotech (Nanjing, China). Polyvinylidene fluoride (PVDF) membrane was purchased from Millipore (Lincoln Park, NJ, USA). Rabbit anti-rat cyclin D1, CDK4, CDK6 and p21, and β-actin HRP secondary goat anti-rabbit antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA).
Extraction of essential oil
A total of 300 g of Pyrolae herba was placed in a 5-l round-bottom flask with 3 l deionized water. The material was soaked for approximately 2 h before it was subjected to hydrodistillation, then Pyrolae herba were distilled for 6 h to collect volatile oil, according to the method recommended by Chinese Pharmacopoeia (26). The essential oil was dried over anhydrous sodium sulfate and stored at −20°C in the dark.
GC-MS analysis
GC-MS analysis was performed on an Agilent 6890N Network GC System, fitted with a HP-5MS capillary column [30 m × 0.25 mm inner diameter (i.d.) × 0.25 μm film thickness; maximum temperature, 350°C], coupled to an Agilent 5975 inert XL Mass Selective Detector. Helium was used as the carrier gas with a flow rate of 1.0 ml/min and the injector was set at 250°C. The GC oven initial temperature was 50°C for 3 min, then programmed to 160°C at 1.5°C/min and increased to 280 at 3°C/min, finally holding at 280°C for 40 min. The ion source temperature was 230°C. MS scanning was carried out at 70 eV over a mass range of 30–500 amu. Samples were injected (1 μl) with a split mode ratio of 50:1. Compounds were identified by comparing their spectral data and retention time with data from NIST Mass Spectral Library.
Cell culture
The SW1353 human chondrosarcoma cell line was obtained from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). SW1353 cells were grown in DMEM containing 10% (v/v) FBS, and 100 U/ml penicillin and 100 μg/ml streptomycin. Cells were grown in a 37°C incubator supplied with 5% CO2.
Measurement of cell viability
Cell viability was determined by MTT assay. SW1353 cells were seeded in 96-well plates at a density of 1×104 cells/well in 0.1 ml medium. The cells were treated with various concentrations of PHVO for 48 h or 100 μg/ml of PHVO for different periods of time. The medium was then removed and 20 μl of 0.5% MTT solution were added to each well followed by incubation for 4 h at 37°C. The purple-blue MTT formazan precipitate was dissolved in 100 μl DMSO. The absorbance of each well was determined at 490 nm using an ELISA reader (BioTek, Model ELX800, Winnoski, VT, USA).
Flow cytometry detection of cell cycle
To determine the effect of PHVO on the cell cycle distribution, SW1353 cells were treated with various concentrations (0, 50, 100, 150 μg/ml) of PHVO for 48 h. The cells were harvested and washed with PBS. The cell concentration was adjusted to 1×106 cells/ml following centrifugation. Solutions A, B and C were added according to the manufacturer’s instructions. DNA content of the cells was measured by FACSCalibur (Becton-Dickinson) flow cytometry system and analyzed using Modfit LT version 3.0 (Verity Software House, Topsham, ME, USA).
Western blot analysis
SW1353 cells were seeded in culture flasks and treated with or without PHVO for 24 h at 37°C. Cells were scraped from the culture, washed twice with PBS and then suspended in 30 μl western blot lysis buffer. The protein concentration was then determined using the bicinchoninic acid (BCA) protein assay (Beyotime Biotechnology, Haimen, China). Samples were loaded with 20 μg of protein and separated by electrophoresis on 12% SDS-polyacrylamide gels. Following electrophoresis, the protein blots were transferred onto a PVDF membrane. The membrane was blocked with 5% skimmed milk in TBST solution and incubated overnight with primary antibodies at 4°C. The membranes were then washed in TBST and exposed to secondary antibodies. Finally, the bands were quantified by scanning densitometry (170–8070; Molecular Imager ChemiDoc XRS System, Bio-Rad, Hercules, CA, USA).
Statistical analysis
All data are the means of three determinations and the data were analyzed using the SPSS 16.0 package. Student’s t-tests and one-way ANOVA test were used to compare the differences among groups and P-values <0.05 were considered to indicate statistically significant differences.
ResultsGC-MS analysis of the oil samples
The results of GC-MS analyses are listed in Table I. GC-MS analysis of the oil samples revealed the presence of 16 different components, 12 of which were listed (Table I), representing 81.62% of the total oil composition from Pyrolae herba. n-Hexadecanoic acid (29.29%) was the main component of the oil, followed by cedrol (17.08%), 6,10,14-trimethyl-2-pentadecanone (9.59%) and cis-9-octadecadienoic acid (8.23%).
PHVO inhibits the proliferation of SW1353 cells
In order to investigate the effect of PHVO on SW1353 cells, we first observed the cell density by phase-contrast microscopy. As shown in Fig. 1, PHVO treatment significantly reduced the cell density compared with the untreated control cells. The effect of PHVO on the viability of SW1353 cells was determined by MTT assay. As shown in Fig. 2, PHVO treatment decreased the cell viability in a dose- and time-dependent manner compared to the untreated control cells (P<0.05). Taken together, these data demonstrate that PHVO inhibits the proliferation of SW1353 cells.
PHVO blocks the progression of SW1353 cells from the G1 to the S phase
In order to examine the effect of PHVO treatment on the progression of SW1353 cells from the G1 to the S phase, we performed flow cytometric analysis of the SW1353 cells treated with 50, 100 and 150 μg/ml PHVO for 48 h. As shown in Fig. 3, the treatment of SW1353 cells with PHVO led to a decrease in the number of cells in the S phase, associated with a concomitant increase in the number of cells in the G1 phase compared to the untreated cells (P<0.05). These results suggest that PHVO exerts growth inhibibitory effects on SW1353 cells by leading to cell cycle arrest at the G1 phase, and thus not allowing the cells to progress further in the cell cycle and proliferate.
PHVO regulates protein expression of cyclin D1, CDK4, CDK6 and p21
To further investigate the mechanism behind the anti-proliferative activities of PHVO, we performed western blot analysis to detect the protein expression of cyclin D1, CDK4, CDK6 and p21 in the PHVO-treated SW1353 cells. The results showed that PHVO treatment decreased the expression of cyclin D1, CDK4 and CDK6 in dose-dependent manner, but increased that of p21 in the SW1353 cells (Fig. 4).
Discussion
Chondrosarcoma is the most common primary bone tumor and the second highest cause of cancer-related mortality in the pediatric age group. To date, surgery and adjuvant chemotherapy are the main therapeutic approaches for patients with osteosarcoma, leading to extensive side-effects and multidrug resistance. Thus, the prognosis of osteosarcoma after surgery is poor (27–29). Therefore, the ultimate goal remains to discover effective agents, which are affordable and have minimal toxicity. In this study, the volatile components from Pyrolae herba were extracted by hydrodistillation and analyzed by GC-MS. The results showed the presence of a wide range of compounds in the volatile oil of Pyrolae herba, including terpenoids, aromatics, long-chain hydrocarbons, alcohols, ketones, acids and esters. Twelve components were identified representing 81.62% of the total integrated chromatographic peaks. Terpenoids and acids constitute more than 58% of the Pyrolae herba volatile oil, with n-hexadecanoic acid (29.29%) and cedrol (17.08%) as the major components.
In the present study, we investigated the antitumor activity of PHVO. MTT assay was used to evaluate the effects of PHVO on SW1353 cell viability. The results showed that PHVO was effective in inhibiting the proliferation of SW1353 cells.
Cell proliferation is primarily governed by the cell cycle (30). The G1/S transition is one of the two main checkpoints of the cell cycle (31), which is responsible for initiation and completion of DNA replication. In addition, cell cycle progression is also a tightly controlled event regulated positively by main cyclin-dependent kinases (CDK4/CDK6) and their cyclin-regulatory subunits (cyclin D1). Cyclin D1 is a positive regulator which binds to CDK4 or CDK6 to control cell cycle progression from the G1 to the S phase (32). An unchecked or hyperactivated cyclin D1/CDK4 complex often leads to uncontrolled cell division and malignancy (33–35). p21, the first identified inhibitor of cyclin/CDK complexes, was independently isolated as a CDK-binding protein (36). The main role of p21 in the cell cycle regulation is performed by inhibiting the activity of cyclin-CDK complexes (37,38). G1 phase lengthening and S phase shortening could lead to cell cycle arrest and eventually to the inhibition of cell proliferation. In this study, cell cycle distribution was examined by flow cytometry. The results demonstrated that PHVO significantly increased the cell population in the G0/G1 phase and reduced that in the S phase. Furthermore, the expression of cell cycle regulating proteins (cyclin D1, CDK4, CDK6 and p21) was detected by western blot analysis. The results demonstrated that PHVO treatment led to the downregulation of cyclin D1, CDK4 and CDK6 and the upregulation of p21. It was suggested that the molecular mechanism behind the PHVO inhibition of SW1353 cell proliferation was cell cycle arrest at the G1/S phase.
The results of this study demonstrate that the volatile oil of Pyrolae herba significantly inhibits the growth of SW1353 cells. Nevertheless, volatile oil is a complex combination of a variety of chemical components, any of which may play a role in inhibiting cell growth. It is not clear as to which of the components contribute most significantly to antitumor activity. Therefore, components of volatile oil from Pyrolae herba should be further investigated in order to elucidate the individual antitumor activity of each component. The antitumor activity of the mixture was then compared with the expected antitumor activity calculated by the sum of efficiencies of each compound separately, relative to their proportions in the mixture. If it was higher, a synergy was pointed out, whereas a lower value was representative of an antagonism. In so doing, it may be helpful to discover more effective antitumor compounds from the volatile oil of Pyrolae herba.
In conclusion, the direct application of volatile oil in the treatment of chondrosarcoma has thus far not been considered. However, the presence of potential antitumor compounds in the volatile oil from Pyrolae herba, as well as the low toxicity of the oil at rather low doses may provide possibilities for further investigations in the field of antitumor compounds.
This study was sponsored by the National Natural Science Foundation of China (no. 81072826), the Natural Science Foundation of Fujian Province (no. 2010Y0032) and the Chen Keji Integrative Medicine Developmental Foundation (no. CKJ2011003).
ReferencesCormierJNPollockRESoft tissue sarcomasCA Cancer J Clin54941092004HeareTHensleyMADell’OrfanoSBone tumors: osteosarcoma and Ewing’s sarcomaCurr Opin Pediatr213653722009TanMLChoongPFDassCROsteosarcoma: conventional treatment vs. gene therapyCancer Biol Ther81061172009GorlickRKhannaCOsteosarcomaJ Bone Miner Res256836912010GordalizaMNatural products as leads to anticancer drugsClin Transl Oncol97677762007JiHFLiXJZhangHYNatural products and drug discoveryEMBO Rep101942002009ZhaoJJiangPZhangWDMolecular networks for the study of TCM pharmacologyBrief Bioinform114174302009RochaABLopesRMSchwartsmannGNatural products in anticancer therapyCurr Opin Pharmacol13643692001CraggGMNewmanDJSnaderKMNatural products in drug discovery and developmentJ Nat Prod6052601997TuYYThe discovery of artemisinin (qinghaosu) and gifts from Chinese medicineNat Med17121712202001NobiliSLippiDWitortEDonniniMBausiLMiniECapaccioliSNatural compounds for cancer treatment and preventionPharmacol Res593653782009SinghHPMittalSKaurSBatishDRKohliRKChemical composition and antioxidant activity of essential oil from residues of Artemisia scopariaFood Chemistry1146426452009StankoKCOrlićSPoliteoOStrikićFKolakIMilosMSatovicZComposition and antibacterial activities of essential oils of seven Ocimum taxaFood Chemistry1191962012010Chinese Pharmacopoeia CommissionPharmacopoeia of the People’s Republic of China1st editionChemical Industry Press2262772005LeeMHLeeJMJunSHThe anti-inflammatory effects of Pyrolae herba extract through the inhibition of the expression of inducible nitric oxide synthase (iNOS) and NO productionJ Ethnopharmacol11249542007LeeSMAnRBMinBSNaMKLeeCHKangSJMaengHYBaeKHA new naphthoquinone from Pyrola japonicaArch Pharm Res245225232001GalvánIJRashedNMJessulatMAntifungal and antioxidant activities of the phytomedicine pipsissewa, Chimaphila umbellataPhytochemistry697387462008KimSJUmJYLeeJYAnti-inflammatory activity of hyperoside through the suppression of nuclear factor-κB activation in mouse peritoneal macrophagesAm J Chin Med391711742011NikolaevSMNikolaevaIGRazuvaevaYGDorzhiewAMAntioxidant activity and flavonoid composition of the new pancreoprotective preparation PancreophytPharm Chem J452792812011TsaiSJYinMCAntioxidative and anti-inflammatory protection of oleanolic acid and ursolic acid in PC12 cellsJ Food Sci73H174H1782008RamosAAWilsonCPCollinsARProtective effects of ursolic acid and luteolin against oxidative DNA damage include enhancement of DNA repair in Caco-2 cellsMutat Res6926112010WangJSRenTNXiTUrsolic acid induces apoptosis by suppressing the expression of FoxM1 in MCF-7 human breast cancer cellsMed Oncol2910152012LimamiYPinonALegerDYMousseauYMoreauJCBeneytoutJLDelageCLiagreBSimonAHT-29 colorectal cancer cells undergoing apoptosis overexpress COX-2 to delay ursolic acid-induced cell deathBiochimie937497572011HuangCYLinCYTsaiCWYinMCInhibition of cell proliferation, invasion and migration by ursolic acid in human lung cancer cell linesToxicol in Vitro25127412802011ShanJZXuanYYRuanSQSunMProliferation-inhibiting and apoptosis-inducing effects of ursolic acid and oleanolic acid on multi-drug resistance cancer cells in vitroChin J Integr Med176076112011Chinese Pharmacopoeia CommissionPharmacopoeia of the People’s Republic of ChinaChemical Industry Press64652000BacciGFerrariSBertoniFRuggieriPPicciPLonghiACasadeiRFabbriNForniCVersariMCampanacciMLong-term outcome for patients with nonmetastatic osteosarcoma of the extremity treated at the istituto ortopedico rizzoli according to the istituto ortopedico rizzoli/osteosarcoma-2 protocol: an updated reportJ Clin Oncol18401640272000StefanoFEmanuelaPAdjuvant and neoadjuvant combination chemotherapy for osteogenic sarcomaCurr Opin Oncol193413462007LamoureuxFTrichetVChipoyCBlanchardFGouinFRediniFRecent advances in the management of osteosarcoma and forthcoming therapeutic strategiesExpert Rev Anticancer Ther71691812007SherrCJCancer cell cyclesScience274167216771996NursePOrdering S phase and M phase in the cell cycleCell795475501994ZhangMXieRHouWWangBShenRWangXWangQZhuTJonasonJHChenDPTHrP prevents chondrocyte premature hypertrophy by inducing cyclin-D1-dependent Runx2 and Runx3 phosphorylation, ubiquitylation and proteasomal degradationJ Cell Sci122138213892009HarakenSAbu-EI-ArdatKDiab-AssafMNiedzwieckiAEI-SabbanMRathMEpigallocatechin-3-gallate induces apoptosis and cell cycle arrest in HTLV-1-positive and -negative leukemia cellsMed Oncol2530392008KesselDLuoYCells in cryptophycin-induced cell-cycle arrest are susceptile to apoptosisCancer Lett15125292000ZafonteBTHulitJAmanatullahDFAlbaneseCWangCGRosenEReutensASparanoJALisantiMPPestellRGCell-cycle dysregulation in breast cancer: breast cancer therapies targeting the cell cycleFront Biosci5d938d9612000HarperJWAdamiGRWeiNKeyomarsiKElledgeSJThe p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinasesCell758058161993ChenJJJacksonPKKirschnerMWDuttaASeparate domains of p21 involved in the inhibition of Cdk kinase and PCNANature3743863881994GoubinFDucommunBIdentification of binding domains on the p21Cip1 cyclin-dependent kinase inhibitorOncogene10228122871995Figures and Table
Effect of PHVO on the morphology of SW1353 cells. (A) Untreated control SW1353 cells. (B–D) SW1353 cells were treated with 50, 100, 150 μg/ml PHVO for 48 h. Morphological changes of SW1353 cells were observed under a phase-contrast microscope. Images were taken at a magnification of ×100.
Effect of PHVO on SW1353 cell viability (A) SW1353 cells were treated with the indicated concentrations of PHVO for 48 h. (B) SW1353 cells were treated with 100 μg/ml of PHVO for 12, 24, 36 and 48 h. SW1353 cell viability was determined by MTT assay. The data were normalized to the viability of the untreated cells (100%). Data are the means ± SD (error bars). *P<0.05, statistically significant vs. untreated cells.
Effect of PHVO on the cell cycle progression of SW1353 cells. (A) SW1353 cells were treated with the indicated concentrations of PHVO for 48 h, stained with PI and analyzed by FACS. (B) Percentage of SW1353 cells in the G0/G1phase after treatment with PHVO. (C) Percentage of SW1353 cells in S phase after treatment with PHVO. The data shown are the means ± SD (error bars) from three independent experiments. *P<0.05 vs. control cells.
Effect of PHVO treatment on the protein expression levels of cyclin D1, CDK4, CDK6 and p21 in SW1353 cells. (A) SW1353 cells were treated with the indicated concentrations of PHVO for 48 h. The protein expression levels of cyclin D1, CDK4, CDK6 and p21 were determined by western blot analysis. β-actin was used as the internal control. (B–E) Densitometric analysis. The data were normalized to the mean protein expression of the untreated controls (100%). *P<0.05 vs. controls.
Characteristics of the components of volatile oil from Pyrolae herba as shown by GC-MS analysis.