2015, Vol.26 
b Beijing Key Laboratory of Microanalytical Methods and Instrumentation, Department of Chemistry, Tsinghua University, Beijing 100084, China;
c Department of Bioengineering, School of Engineering, The University of Tokyo, Tokyo 113-8656, Japan
Glioblastoma multiforme (GBM) is the most common malignant primary tumor of the brain and associated with a median survival of about one year[1].This continuing poor prognosis is due to therapeutic resistance and tumor relapse[2].Glioma stem cells (GSCs) are considered responsible for drug resistance and glioma relapse in glioblastoma multiforme (GBM)[3, 4, 5, 6].SU3 glioma cell is a highly invasive glioma stem cell line and is also a new progenitor cell line from the patients with glioblastoma multiforme which gets much attention recently[7].It is of great significance to study the chemotherapy of SU3 glioma cell which represents the aggressive biological behavior of glioblastoma multiforme.Temozolomide has demonstrated activity against recurrent glioma[8, 9, 10].However,the prognosis of glioblastoma multiforme is still poor[11, 12, 13].Laboratory studies and clinical trials are investigating whether it might be possible to further increase the anticancer potency of temozolomide by combining it with other pharmacologic agents.
Paclitaxel is a new class of microtubule stabilizing agents with perfect antiglioma activity[14].However there are still few studies about the pharmacologic effects of paclitaxel on the SU3 glioma cell.In general,paclitaxel usually binds to microtubules and promotes microtubules hyper-stabilization,and induced glioma cell death in the chemotherapy[14, 15, 16].So far in clinical trials,the chemotherapy of breast,ovarian and lung cancer by paclitaxel has been successfully achieved[17, 18, 19].Moreover,paclitaxel has been investigated about the efficiency in the therapy of oesophageal,head and neck cancers[20, 21, 22].Clinical studies have shown that paclitaxel in combination with an alkylating agent synergistically inhibits many types of tumors[23].Moreover,paclitaxel with the alkylating-like chemotherapy drug cisplatin has clear synergistic anti-tumor effects against malignant glioma cells in vitro[24].Temozolomide is an orally active alkylating agent and the first-line chemotherapy agent used for treating glioma[25, 26].However,it is unclear whether the paclitaxel can enhance the cytotoxic response of SU3 glioma cells to temozolomide.6-O-methylguanine (6-O-MeG) plays a key role in this process[26, 27].Quantification of 6-O-MeG may help to determine the cytotoxic response of SU3 cell following exposure to the temozolomide in combination of paclitaxel.Thus,it is greatly demanded to develop modern analytical technique to study the drug treatment and molecular mechanism in the biomedical researches.
During the past decades,liquid chromatography-mass spectrometry (LC-MS) has been widely extended to environment analysis,food safety,and biomedical research,because it may provide many information about the nature of the generated molecular.For example,Chen et al.[28]developed a microfluidic chip coupled with mass spectrometry platform to study the drug metabolism of breast cancers and its biological mechanism.Zhang et al.[29]presented an approach of mass spectrometry sensor to monitor the drug absorption of liver cells,which was recognized as a powerful tool to investigate the cell biology at the molecular level.However,it is still greatly significant to extend its application in the biomedical events that is relevant to human healthcare.
In this paper,we develop a LC-MS method to analyze the pharmacological effects of paclitaxel on SU3 glioma cells,and to study the effects of the concentrations and incubation periods of paclitaxel on cell viability.The induced cell apoptosis and the enhanced cytotoxic response in SU3 glioma cells is an important problem in the chemotherapy but not studied before.Cell-specific fluorescence dyes were used to evaluate the effects of anticancer drugs on the viability,apoptosis and ROS level of SU3 glioma cells.We further developed the LC-MS method to directly detect 6-O-MeG and 7-methylguanine (7-MeG).
2.Experimental 2.1.Apparatus and chemicalsAll chemicals were of analytical grade and were used as received.Paclitaxel was purchased from Puyihua company (China,Beijing),and the temozolomide (TMZ) was generously provided by Tianlishi Corp (Tianjin,China).The standard solutions of 1 mg/mL paclitaxel and of 15.5 mg/mL temozolomide were prepared by DMSO.The work solution was gradually diluted by DMSO.A Live/Dead assay kit (Calcein-AM/EthD-1) was obtained from Invitrogen (CA,USA).Trichlorosilane,dimethylsulfoxide (DMSO),acetonitrile,formic acid and paraformaldehyde were obtained from Sigma (St.Louis,MO).TIANamp Genomic DNA kit (DP 304-02) was bought from Tiangen Corp (Beijing,China).Spectrophotometer (NanoDrop 2000) was obtained from Thermo Scientific (USA).PC hilic S5(2.0 mm × 150 mm) column was from Shiseido Co.,Ltd.6-O-MeG and 7-MeG were purchased from Sigma-Aldrich (St.Louis,MO,USA).Hoechst 33342 was from Invitrogen (CA,USA).Generation of reactive oxygen species (ROS) was analyzed by dihydroethidium (DHE,Beijing,China).Glutathione (GSH) was detected by naphthalene-2,3-dicarboxaldehyde (NDA).SU3 cell line,which was provided by The Second Affiliated Hospital of Soochow University,was from the patients with GBM.
2.2.Cell culture and paclitaxel treatmentSU3 cells were cultured in DMEM medium with 10% fetal bovine serum,100 mg/mL penicillin,and 100 mg/mL streptomycin.The cells were incubated in humidified air plus 5% CO2 at 37 ℃.For the control,1.0 × 105 cells were plated in each cultured dish.The medium was removed 24 h after the plating and fresh medium containing paclitaxel and 0.1% DMSO was applied.In order to obtain a concentration-dependent survival curve,cells were incubated for different times with the final concentrations of paclitaxel ranging from 1 ng/mL to 1 μg/mL.This proved to be constant for all three experiments.
2.3.Cell viability assay and determination of apoptosisThe cytotoxicity of paclitaxel on SU3 cells was evaluated by seeding 5000 cells per pore.Following three days of continuous exposure to various concentration of paclitaxel cell viability was examined with a Cell Counting Kit-8(CCK-8) assay.The CCK-8 assay was done with a Microplate reader.The absorbance was measured with a test wavelength of 450 nm and a reference wavelength of 630 nm.The percentage of viable cells at each concentration of paclitaxel was calculated.The cell viability was calculated by blank DMEM containing 0.1% DMSO as control.Cell apoptosis was detected by 5 μg/mL hoechst 33342(HOE) labeling for 20 min at 37 ℃[30, 31].The cell apoptosis was determined by FACS flow cytometer and was confirmed by morphological evaluation of the samples via fluorescence microscopy.Finally,each experiment was repeated at least three times.
2.4.ROS and GSH assayTo detect ROS,SU3 cells were labeled by 10 μmol/L DHE for 20 min at 37 ℃ after incubation with various concentrations of paclitaxel under 24 h,48 h,and 72 h.GSH analysis for SU3 cells that were treated by paclitaxel were performed with 10 μL 10 mmol/L NDA at 37 ℃ 15 min.2.0 × 105 cells were counted to obtain a statistically relevant number.
2.5.Paclitaxel in combination of temozolomide treatment and DNA extractionFor drug treatment,cells were divided into two groups.One was treated with 0 μmol/L,200 μmol/L,400 μmol/L and 600 μmol/L temozolomide,the other was treated with 0.5 ng/mL paclitaxel in combination with 0 μmol/L,200 μmol/L,400 μmol/L and 600 μmol/L temozolomide.After the cells were cultured for 72 h,cell viability was examined with a CCK-8 assay,and then the total DNA was isolated and purified from SU3 glioma cells by TIANamp Genomic DNA kit (DP 304-02).DNA yield was qualified by spectrophotometer.DNA solution 20 μL was mixed 20 μL HPLC grade water and 4 μL formic acid (90% in water) was added to the mixture.After mixing,the mixture was heating at 85 ℃ for 60 min by digital dry baths and then cool down to room temperature.The hydrolytes were used for quantitation of 6-O-MeG by LC/MS immediately.
2.6.LC/ESI-MS/MS conditionsAnalysis was performed on a PC hilic S5 2.0 mm × 150 mm column from Shiseido Co Ltd.Various combination of organic solvents including acetonitrile and methanol,and HPLC-grade water or HPLC-grade water containing acid or basic compounds were investigated to optimize the mobile phase for sensitivity,speed,and peak shape.The best conditions of the mobile phase A was achieved with 10 mmol ammonium hydroxide containing 0.1% formic acid,and the mobile phase B was acetonitrile containing 0.1 formic acid (B).When the gradient data was composed by 30% A and 70% B,the peak was best.The mobile phase system was used for optimizing the settings of the electrospray source parameter.Quantitation was performed with multiple reaction monitoring (MRM) mode and the collision energy was 30 eV.The mass transitions (precursor to product) monitored were 166.2 > 149.2 for 6-O-MeG and 7-MeG.A dwell time of 100 ms was used for each transition.The quadrupoles Q1 and Q3 were set on unit resolution.20 μl of sample was injected per each analysis.Finally,each experiment was repeated at least three times.For the metabolic analysis,the data was also calculated with an average of six times.
2.7.Statistical analysisEpidata entry is used for simple or programmed data entry and data documentation.SPSS 19.0 was used for statistical analysis.Student's t-test is applied to estimate the normally distributed population and Mann-Whitney U test is used to evaluate the nonnormal distributions.A value of p < 0.05 is considered significant.
3.Results and discussion 3.1.The effects of paclitaxel on cell density,viability and apoptosisAlthough paclitaxel has been widely used in various cancer treatment,the cytotoxicity of paclitaxel for SU3 cell line is still unidentified,which hinders further clinical application.In this work,we investigated the effects of paclitaxel on the growth of SU3 glioma cells.Fig.1 shows the viabilities of SU3 cells analyzed by a Live/Dead assay kit (Calcein AM/EthD-1).The cell densities were gradually decreased under the stimulation of paclitaxel compared with control experiments,and the dead cells increased,suggesting that paclitaxel was effective to inhibit SU3 glioma cells.After treatment of paclitaxel,we found that the SU3 cells become larger.The reason is that the cytoskeleton of SU3 has been severely destroyed and the cell edge became fuzzy.Large hollows of cell organelles and lots of vesicles were found.Furthermore,the live SU3 cells gradually decreased with increasing time,and the dead SU3 cells increased with the increasing of drug concentrations.Quantitative statistics of pharmacological effects were also conducted.Fig.2 shows the survival curves of SU3 cells treated by paclitaxel with different concentrations and different times.It showed that the cell viabilities of SU3 cells were decreased under paclitaxel treatment in time-dependent and concentrationdependent manners.These results illustrated that SU3 cells were sensitive to paclitaxel,and different half maximal inhibitory concentration (IC50) could be deduced from the time-dependent curve.It also indicated that paclitaxel could be an effective strategy for the study of SU3 cells in vitro.
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| Fig. 1.Fluorescent images of SU3 cells for the investigation of cell density.The red dash indicated cell damage and cell shrink.The concentration of paclitaxel was 5.00 ng/mL. | |
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| Fig. 2.Curves of cell viability analyzed by CCK8 kit.The cell viabilities were detected with different concentrations (1-100 ng/mL) at 1-5 days. | |
Cell apoptosis was evaluated by HOE staining and observed by fluorescence microscope.SU3 cells were exposed in 5.0 ng/mL paclitaxel solution with 0.1% DMSO for three days.Then,cells were stained by a fluorescent dye HOE,because HOE could bind to DNA in the nucleus and exhibits fluorescence signal depending on the ratio of dye and base pairs.As shown in Fig.3,a normal cell apoptosis was found in control experiment during 5 days.In contrast,severe cell apoptosis (even death) occurred under the treatment of paclitaxel.The cells also had shrinking nucleuses and extensive microtubule networks,which was caused by the formation of microtubule aggregated and the cell debris increased with prolonging medication time.These results indicated that thecell membrane permeability was increased,and the structure of the chromosomal DNA was changed,demonstrating that cells underwent gradually apoptosis.Hence paclitaxel was effective for the treatment of SU3 glioma cells in vitro though the sensitivity of SU3 glioma cells to paclitaxel in vivo might be lower,possibly owing to the blood brain barrier.
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| Fig. 3.The images of cell apoptosis by treatment with time-dependent paclitaxel (5.00 ng/mL) for 5 days. | |
Reactive oxygen species (ROS) are naturally generated by aerobic metabolism of cells,which are related to many diseases including cancers.Because high level of ROS can stimulate cell growth and proliferation,ROS is significant for maintaining cancer phenotypes.Meanwhile,high level of ROS promotes cellular damage by DNA damage,enzyme inactivation,protein oxidation,and so on[32, 33, 34].GSH is the most abundant free thiol in eukaryotic cells.It can maintain a stable intracellular redox environment to get proper functions of cellular proteins[35, 36, 37].High levels of ROS alters GSH redox state,which leads to trigger the unfolding protein response and cell apoptosis.Therefore,the levels of ROS and GSH are two typical markers for investigation of drug toxicity in vitro.In our experiments,two specific fluorescence probes,DHE (λex=535 nm,λem=610 nm) and NDA (λex=460 nm,λem=530 nm),were specially used to measure intracellular ROS and GSH,respectively.As shown in Fig.4,with the increase of paclitaxel incubation time,the quantity of ROS increased while the amount of GSH decreased in SU3 cells.In control experiments,the ROS level was at a low level because it could be neutralized byantioxidant system such as GSH and antioxidant enzymes.But this balance was broken in the presence of paclitaxel.Moreover,the intensity of ROS and GSH were little altered during 24 h,demonstrating that SU3 cells were vulnerable to oxidative stress induced by exogenous ROS-generating agents.In addition,the levels of ROS and GSH were simultaneously reduced after 4 days because lots of cells were dead.Combined with the results of cell apoptosis,we concluded that paclitaxel induced ROS production in gliomas led to DNA damage and cell apoptosis.ROS generation of SU3 cells was associated with the treatment of paclitaxel.ROS could activate NF-κB,stabilize HIF-1,and promote VEGF expression,thus it was relative to the drug resistance[38, 39, 40].Furthermore,ROS could also alter cell viability and cell density.Our results showed that the level of ROS was changed by very low concentration of paclitaxel,which could be related to low paclitaxel resistance for SU3 cells.
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| Fig. 4.Intracellular ROS generation (A) and GSH reduction (B) along with time of 5.00 ng/mL paclitaxel. | |
Previous studies reported that 6-O-MeG and 7-MeG are the two important adducts of the cells after being treated by TMZ[26, 41].After the SU3 cells exposed to temozolomide,the DNA was extracted and acid-hydrolyzed.In this work,LC-MS was used for quantitation of 6-O-MeG and 7-MeG (Fig.5A).Firstly,various combination of organic solvents including acetonitrile and methanol,and HPLC-grade water or HPLC-grade water containing acid or basic compounds were investigated with a view to optimizing the mobile phase for sensitivity,speed,and peak shape.The final combined optimized conditions were used for quantitation of 6-O-MeG and 7-MeG in DNA hydrolytes (Fig.5A).Under the optimized conditions,the analytes of 6-O-MeG and 7-MeG have a retention time 2.48 min and 2.99 min (Fig.5A).The typical MRM ion chromatogram was show in Fig.5B and C.Then,a series of calibrated standards for 6-O-MeG and 7-MeG were prepared at various concentrations.The calibration curves obtained were linear over the concentration ranges of 0.05-2.0 ng/mL for 6-OMeG (Y=9.49 × 105X+2.42 × 104;R2=0.9998).For 7-MeG,the calibration curves over the concentration ranges of 0.125-5.0 ng/mL (Y=4.72 × 104X+2.21 × 103;R2=0.9996)(Fig.5D and E).
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| Fig. 5.LC-MS method for quantitation of 6-O-MeG and 7-MeG.(A) DNA adducts were got from SU3 cell and were used for quantitation of 6-O-MeG and 7-MeG.(B,C) Positive ESI product ion spectra of[M+H]+ions of 6-O-MeG and 7-MeG.(D,E) The linear analysis of 6-O-MeG and 7-MeG. | |
To determine the effectiveness of paclitaxel in combination of temozolomide treatment on the SU3 glioma cells,we evaluated the effects of various concentrations of temozolomide in combination with 0.5 ng paclitaxel on the proliferation of SU3 cells using CCK-8 which is a sensitive nonradioactive colorimetric assay kit.As shown in Fig.6A,we found that paclitaxel can enhance the sensitivity of SU3 glioma cell to temozolomide.To further investigate the mechanism of this phenomenon,6-O-MeG and 7-MeG,which play a key role in the response of tumor cells to temozolomide,are qualitatively and quantitatively analyzed by LC-MS.The amounts of 6-O-MeG and 7-MeG from SU3 glioma cells were successfully analyzed by LC/MS (Fig.6B),and we found that the paclitaxel can increase the amount of 6-O-MeG and 7-MeG in SU3 glioma cell (Fig.6C and D).Hence,we proposed that the paclitaxel can enhance the sensitivity of SU3 glioma cell to temozolomide.
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| Fig. 6.Analysis of 6-O-MeG and 7-MeG at different concentration of temozolomide in combination with paclitaxel using LC-MS.(A) The results of CCK-8 on the SU3 glioma cells.(B) Analysis of sample from SU3 glioma cell by LC-MS in MRM mode.(C,D) Quantitation of 6-O-MeG and 7-MeG in DNA adducts from SU3 glioma cells.*means p < 0.05. | |
In this work,the combination of LC-MS analysis and cellspecific fluorescence dyes enables us to study the effects of paclitaxel on cell viability,ROS and GSH,the amount changes of biomarkers in glioblastoma multiforme derived cell line SU3.We concluded that paclitaxel reduced cell density and viability,promote cell apoptosis,and increase the level of ROS.Our results show that the concentrations of paclitaxel in vitro are different from that in vivo.In vivo concentrations that are relevant for a clinical application is much higher than in vitro.The paclitaxel concentration obtained in the plasma of patients treated with this cytostatic drug ranges from 1.0 μmol/L to 10 μmol/L.These in vivo concentrations are higher than those used in vitro due to absorption,digestion,and degradation of drug in vivo.Thus,this study demonstrates that the LC-MS could be used as an effective tool to study the mechanism of paclitaxel effection on SU3 cells.It expects a useful guidance to employ the analytical tools for solving the biomedical issues.
AcknowledgmentsWe thank Dr.Yan-Li Guo,Qian Yang (The Frontier Science Department of Shiseido China Co.,Ltd.),Ying Zhang and Qiushui Chen (Department of Chemistry,Tsinghua University) for assistance with MS analysis.We also thank Jun Dong,Xinliang DAI of the neurosurgery department of The Second Affiliated Hospital of Soochow University for assistance with cell culture.The work was supported by National Nature Science Foundation of China (Nos.214350002,81373373) and CERS-China Equipment and Education Resources System (No.CERS-1-75).
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