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OBJECTIVE To prepare Gambogic acid （GA） nanocapsules （GA-LNCs） and Neogambogic acid （NGA） nanocapsules（NGA-LNCs），and to evaluate their antidiabetic activities. METHODS Using water as the aqueous phase ，medium- chain triglyceride as the oil phase and polyethylene glycol monostearate as the surfactant ，GA-LNCs and NGA-LNCs were prepared by phase inversion method. Using entrapment efficiency and drug-loading amount as index ，the formulation technologies of above 2 nanocapsules were optimized by simplex lattice design. Its physical and chemical properties were investigated. The diabetic mice model was established. GA-LNCs and NGA-LNCs （1.92 and 2.42 mg/kg respectively ）were given intragastrically ，once a day ，for consecutive 6 weeks. The fasting blood glucose of mice ，the activities of superoxide dismutase （SOD）and glutathione peroxidase （GSH-Px），the contents of malondialdehyde （MDA），total cholesterol （TC），triglyceride （TG），high-density lipoprotein cholesterol（HDL-C）and low-density lipoprotein cholesterol （LDL-C）were all detected. RESULTS The optimal formulation of 2 kinds of nanocapsules included 60％ water，10％ medium-chain triglyceride ，30％ polyethylene glycol monostearate （total amount of the three was 2 g）and 35 mg GA or NGA . The encapsulation efficiencies of GA-LNCs and NGA-LNC obtained by the optimal formulation were （92.01±0.68）％ and（93.12±2.11）％；the drug-loading amount were （0.99±0.21）％ and（1.21±0.22）％， respectively. GA-LNCs and NGA-LNCs were yellow ，homogeneous and transparent liquid without precipitation. They were spherical in microscopic shape ， and had obvious shell- Δ 基金项目：吉林省科技发展计划项目（No.20200404090YY）；吉 membrane structure. The particle sizes were （28.11 ± 9.76） 林省教育厅科学技术研究项目（No.JJKH20210372KJ） ＊硕士研究生 。研究方向 ：植物药 。E-mail：zhanhe0108@163. and（22.06±6.84）nm；Zeta potential were （－4.09±1.00） com and（－17.40±1.32）mV，and polydispersity were 0.93±0.06 # 通信作者：讲师，博士。研究方向：中药有效成分治疗疾病的作 and 0.74±0.12. The results of animal experiments showed that 用机制。E-mail：chenweijia_jlau@163.com both GA-LNCs and NGA-LNCs could sig nificantly increase 中国药房 2022年第33卷第9期 China Pharmacy 2022Vol. 33 No. 9 ·1075· the activities of SOD and GSH-Px and the seru m content of HDL-C （P＜0.05 or P＜0.01）in model mice ，and significantly decreased the fasting blood glucose and the serum contents of MDA ， TC， TG and LDL-C （P＜0.05 or P＜0.01）. CONCLUSIONS GA-LNCs，NGA-LNCs prepared in this study are good in physical and chemical properties and have good anti-diabetes activity.
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Gambogic acid is the main active component of Garcinia hanburyi Hook. f. ，which can inhibit the growth of a variety of tumor cells. However ，its low solubility ，short half-life and poor stability limit its clinical application to a certain extent. In order to improve the above shortcomings and improve the bioavailability of gambogic acid ，many studies have used covalent binding and physical encapsulation methods to obtain a new gambogic acid delivery system with targeting ，high permeability ，stability， biocompatibility，in vivo long circulation and other properties ，such as polymer prodrug delivery system ，anoxic prodrug delivery system，magnetic field responsive prodrug delivery system ，multi environment sensitive prodrug delivery system ，bionic nano drug delivery system. This paper reviews the new drug delivery system and its characteristics of gambogic acid. The results show that the design of drug carrier has greatly improved the defects of gambogic acid ，and the introduction of more responsive groups into gambogic acid drug carrier may make it achieve better antitumor effect.
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Objective To investigate the effect of gambogic acid (GA) on invasion in human gastric carcinoma SGC-7901 cells and its possible mechanism. Methods Cell counting kit-8(CCK-8) assay was performed to detect the effects of GA, inhibitor of nuclear factor kappa-B kinase( IKK) 16 and 5-fluorouracil (5-FU) on cell activity of GES-1 and SGC-7901 cells. Cell invasion was assessed with Transwell invasion assay. Western blotting was used to analyze the protein levels of vimentin, matrix metalloproteinase 2 ( MMP-2) and MMP-9 and protein phosphorylation of IKKα and p65. Results The cell activity was significantly decreased in SGC-7901 cells treated with GA in a dose-dependent manner with a half inhibiton concentration(IC50) value of 1. 89 μmol/L. But GA had no significant influence on cell viability of GES-1 cells. Meanwhile, 5-FU reduced the cell activity of GES-1 and SGC-7901 cells with IC50values of 7.36 μmol/L and 199.57 μmol/L respectively. Low-dose GA and IKK 16 impaired separately the ability of invasion in SGC-7901 cells, and down-regulated the protein levels of MMP-2, MMP-9 and vimentin, and inhibited phosphorylation of IKKot and p65, while a stronger inhibition was showed when the combination of GA and IKK16 was used. Conclusion Low-dose GA might inhibit invasion of SGC-7901 cells via IKKot/p65 signaling pathway.
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@#[Abstract] Objective: To explore whether gambogic acid can enhance the sensitivity of glioma U251 cells to temozolomide and further explore its mechanism. Methods: U251 cells were cultured in vitro and divided into blank control group, gambogic acid treatment group, temozolomide alone treatment group and combined treatment group. The cells survival rates of cells in each group was detected by CCK-8 assay. Flow cytometry was used to detect cell apoptosis and changes in ROS level. Western blotting was used to detect the changes in protein expressions. Results: CCK-8 results showed that the cells survival rates of the four groups after treatment for 24 h were (98.65±3.68)%, (93.58±2.47)%, (66.81±2.39)% and (38.65±4.13)%, respectively. It can be seen that the combined treatment could significantly increase the inhibitory effect of temozolomide on U251 cells. The proportion of apoptotic U251 cells in the combined treatment group was (61.43±2.58)%, which was significantly higher than that of (26.68±1.82)% in the temozolomide-treated group. Combined treatment of gambogic acid and temozolomide could up-regulate ROS level in U251 cells, reduce the expressions of GLUT-3 and p-AKT, and inhibit the GLUT-3/AKT signaling pathway. Conclusion: Gambogic acid combined with temozolomide can enhance the sensitivity of U251 cells to temozolomide by up-regulating ROS level and inhibiting GLUT-3/AKT signaling pathway in U251 cells, and provides a theoretical basis for the application of gambogic acid in the treatment of glioma.
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Objective: The formulation of co-loaded docetaxel(DTX) and gambogic acid(GA) albumin nanoparticles(DTX-GA-BSA NPs) was optimized by central composite design-response surface methodology to prepare DTX-GA-BSA NPs, and its quality was evaluated. The optimal synergistic ratio of DTX and GA was screened by coefficient of drug interaction(CDI). Method: NabTM method was used to prepare DTX-GA-BSA NPs with bovine serum albumin(BSA) as the carrier material. Design-Expert 8.0.6 software was used to design the experiment and process the data, overall desirability(OD) of particle size and polydispersity index(PDI), encapsulation rate were taken as indexes. The particle size and Zeta potential of the nanoparticles were measured. Individual and synergistic inhibitory effects of DTX and GA on the proliferation of MGC-803 and HGC-27 cells were determined by methyl thiazolyl tetrazolium(MTT) assay, respectively. Result: The optimum prescription of DTX-GA-BSA NPs was as follows:BSA concentration of 5 g·L-1, water-oil phase volume ratio of 1:17, drug-loading ratio(mass ration of drug to carrier) of 1:10.The average particle size of DTX-GA-BSA NPs was 135.8 nm and PDI was 0.09, Zeta potential was -21.4 mV. The deviation between the predicted value and the observed value of the model was small, the model had good predictability. For MGC-803 cell, when the concentrations of DTX and GA were 0.004, 0.12 μmol·L-1, respectively(mass ratio of DTX to GA was 1:23), the CDI value was the smallest and the synergistic proliferation inhibition was the most significant. For HGC-27 cell, when the concentrations of DTX and GA were 0.004, 1 μmol·L-1, respectively(mass ratio of DTX to GA was 1:195), the synergistic proliferation inhibition was the most significant. Conclusion: The optimized formulation of DTX-GA-BSA NPs is stable and reliable. The established mathematical model has good predictive ability and practicability. DTX combined with GA has synergistic effect on MGC-803 and HGC-27 cells without concentration dependence.
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As the chemotherapeutic resistance of extranodal NK/T-cell lymphoma (ENKTL) rises year by year, searching for novel chemoprevention compounds has become imminent. Gambogic acid (GA) has recently been shown to have anti-tumor effects, but its role and underling mechanism in ENKTL are rather elusive. In the present study, we showed that GA inhibited the cell growth and potently induced the apoptosis of ENKTL cells in vitro in a time- and concentration-dependent manner. Furthermore, GA induced cell death through endoplasmic reticulum stress (ERS) mediated suppression of Akt signaling pathways and finally the release of the caspase-3 proteases. Overall, our data provided evidences supporting GA as a potential therapeutic agent for ENKTL, which may facilitate further preclinical development of anti-tumor drugs.
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Humans , Apoptosis , Cell Line, Tumor , Cell Proliferation , Endoplasmic Reticulum Stress , Lymphoma, Extranodal NK-T-Cell , Drug Therapy , Genetics , Metabolism , Proto-Oncogene Proteins c-akt , Genetics , Metabolism , Signal Transduction , Xanthones , PharmacologyABSTRACT
As the chemotherapeutic resistance of extranodal NK/T-cell lymphoma (ENKTL) rises year by year, searching for novel chemoprevention compounds has become imminent. Gambogic acid (GA) has recently been shown to have anti-tumor effects, but its role and underling mechanism in ENKTL are rather elusive. In the present study, we showed that GA inhibited the cell growth and potently induced the apoptosis of ENKTL cells in vitro in a time- and concentration-dependent manner. Furthermore, GA induced cell death through endoplasmic reticulum stress (ERS) mediated suppression of Akt signaling pathways and finally the release of the caspase-3 proteases. Overall, our data provided evidences supporting GA as a potential therapeutic agent for ENKTL, which may facilitate further preclinical development of anti-tumor drugs.
Subject(s)
Humans , Apoptosis , Cell Line, Tumor , Cell Proliferation , Endoplasmic Reticulum Stress , Lymphoma, Extranodal NK-T-Cell , Drug Therapy , Genetics , Metabolism , Proto-Oncogene Proteins c-akt , Genetics , Metabolism , Signal Transduction , Xanthones , PharmacologyABSTRACT
Gambogic acid (GA), the main active ingredient in gamboge, has been reported to have good anti-tumor activity with excellent selectivity. However, its clinical application is limited by the poor water solubility. GA nanosuspensions were designed in this study in order to solve this problem. GA nanosuspensions were prepared by microprecipitation method based on pH adjustment. Suitable stabilizer was screened according to the size and polydispersity index (PDI) of the resultant nanosuspensions. Dynamic light scattering method was used to measure the particle size and transmission electron microscopy was used to observe the morphology. The stability was studied in different medium. The drug release was evaluated using a dialysis method. MTT assay was used to assess their cytotoxicity in vitro against cancer cell line. Anti-tumor effect in vivo was investigated on H22-bearing mice. In result, Poloxamer (P188) was found to be a good stabilizer. The resultant GA nanosuspensions (GA-NSps) were 135.9 ±5.1 nm in diameter, with PDI value being 0.26 ±0.01 and the zeta potential being −35.1 ±1.36) mV. GA-NSps were nearly spherical. They were quite stable in various physiological media. GA-NSps exhibited a sustained drug release pattern, with the cumulative release reaching 90.26% within 312 h. In MTT assay, GA-NSps had a stronger cytotoxicity against HepG2 cells than the free drug (IC50, 0.851 8 μg·mL−1 vs 2.104 μg·mL−1, P vs 66.80%, P < 0.01). In summary, we prepared GA-NSps with high drug loading capacity, small particle size and good stability, and provided a solid basis for the effective dosage form of gambogic acid.
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<p><b>OBJECTIVE</b>To investigate the effect of gambogic acid (GA) on the growth and cell death of castrate resistant prostate cancer (PC) with phosphate and tension homology (PTEN) and p53 genes deleted in vitro and ex vivo, and elucidate the underlying possible molecular mechanisms.</p><p><b>METHODS</b>PTEN/p53PC cells and Los Angeles prostate cancer-4 (LAPC-4) cells were treated with GA for 24 h and 48 h, then cell viability was determined by cell proliferation assay. PTEN/p53PC cells organoids number was calculated under GA treatment for 1 week. In addition, cell titer glo assay was performed to analyze 3 dimensional cell viability of patients derived xenografts (PDX) 170.2 organoids. Flow cytometry was used to detect apoptotic cells treated with GA. And confocal image was performed to detect the apoptotic mitochondrial morphological changes. Apoptotic cell death related protein levels were measured through Western blot (WB) in GA treated cells and organoids. The expression levels of mitogen-activated protein kinases (MAPKs) pathway related ribonucleic acid (RNAs) and proteins were analyzed by reverse transcription polymerase chain reaction (RT-PCR) and WB, respectively.</p><p><b>RESULTS</b>The treatment of GA significantly reduced cell viability of PTEN/p53PC cells and LAPC-4 in a time- and concentration-dependent manner. In organoids, GA showed strong inhibition towards organoids' numbers and diameters and continuously led to a complete organoids inhibition with GA 150 nmol/L. Ex vivo results validated that GA 1 μmol/L inhibited 44.6% PDX170.2 organoids growth. As for mechanism, flow cytometry detected continuously increased apoptotic portion under GA treatment from 1.98% to 11.78% (6 h) and 29.94% (8 h, P<0.05). In addition, mitochondrial fragmentation emerged in GA treated cells indicated the mitochondrial apoptotic pathway might be involved. Furthermore, WB detected caspases-3, -9 activation and light chain (LC)-3 conversion with GA treatment. WB revealed decreased activity of MAPK pathway and down-regulation of downstream c-fos oncogene RNA level was detected by RT-PCR before undergoing apoptosis (P<0.05).</p><p><b>CONCLUSION</b>GA was a potent anti-tumor compound as for PTEN/p53PC, which contributed to cell apoptosis via inhibition of the MAPK pathway and c-fos.</p>
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Objective· To identify the effect and potential mechanism of gambogic acid (GA) on natural killer/T-cell lymphoma (NK/TCL) cell lines.Methods · SNK-1,SNK-6 and SNT-8 were incubated with various concentrations of GA for 24 h,and cell viability was detected with CCK-8 assay.Cell apoptosis was examined by Annexin V-FITC/PI staining assay.Levels of proteins regulating cell apoptosis and phosphorylation levels of proteins in key signaling pathways were detected by Western blotting.Results · GA showed a potent effect on reduction of cell viability of NK/fCL cell lines in CCK-8 assay.GA increased the percentages of Annexin V positive cells and induced activation of caspase-3 and caspase-9,cleavage of PARP as well as the reduction of Bcl-xl.GA also inhibited the phosphorylation levels of STAT3 in SNK-1 and SNT-8,and ERK1/2 in SNK-1 and SNK-6 significantly.Conclusion· GA induces cell apoptosis in NK/TCL cell lines SNK-1,SNK-6 and SNT-8.Anti-apoptosis protein Bcl-xl and signaling pathway JAKs/STATs and MEK/MAPK might be involved in this process.
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OBJECTIVE To evaluate the cardiotoxicity of a widen-spectrum antineoplastic drug, gambogic acid, through quantitative multiple cellular phenotypic characterization. METHODS H9c2 cell line was used as a model with doxorubicin (Dox) and amiodarone (Ami) as positive controls, hypaconi?tine as negative control and 0.1% DMSO as normal control. An optimized protocol was established to identify the morphology and function of cell nuclei. The effect of drugs on cell viability, nuclear area (Hoechst33342), mitochondria mass (MitoTracker Deep Red) and cytoplasmic calcium ion mobilization (Rhod2 AM)was studied. EC50 and Z′values were calculated to evaluate the degree of toxicology and to estimate the precision and false-positive rate, respectively. RESULTS Dose-response analysis indicated that EC50 of Dox on cell viability, nuclear area, mitochondrial mass was 0.72, 0.014 and 1.21μmol · L-1, respectively. On the other hand, EC50 of Ami on the parameters of cell viability, nuclear area and mitochon?drial mass was 14.83, 6.72 and 4.54μmol·L-1, respectively with Z′value above 0.5. Hypaconitine decreased the SER ridge of mitochondria. Gambogic acid caused significant mortality of H9c2 cells and induced nuclear shrinkage as Ami did. The EC50 values of cell viability and nuclear area were 0.24 and 1.16 μmol · L- 1. Meanwhile,gambogic acid disturbed the mitochondrial function as indicated by the increased mitochondrial area (EC50=0.44 μmol · L-1), abnormal SER Ridge(EC50=0.99 μmol · L-1) and decreased mitochondrial mass(EC50=1.21 μmol · L- 1). Cellular calcium mobilization was lower than normal (EC50=0.41 μmol · L-1). CONCLUSION The EC50 values of positive controls calculated from our assessment are similar those reported in literature. A multi-parameter and simultaneous evaluation enables a comprehensive analysis of the morphology of nuclei and mitochondria of cardiomyocytes and a preliminary assessment of the mechanisms of toxicity.
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OBJECTIVE Nanotechnology provides a novel strategy for the delivery of anticancer drugs. In this study, titanium dioxide coated gold nanorod (GNR/TiO2) nanostructures were used as the drug carrier for gambogic acid in order to improve its anticancer effect. METHODS Biocompatibility and cellular uptake of GNR/TiO2 nanostructures were studied in human glioblastoma U-87 MG cells. Cell viability was evaluated by ATP assay and calcein AM staining. LysoSensor Green DND-189 and Hoechst 33342 were used to analyze the intracellular location of GNR/TiO2 nanostructures. The in vitro anti-cancer effect of gambogic acid loaded nanoparticles was compared with free drug. RESULTS The results showed that GNR/TiO2 nanostructures are biocompatible, and they are localized at the intracel?lular acidic compartments of endosomes and lysosomes. The intracellular drug content delivered via GNR/TiO2 nanostructures was 6 fold higher than the free form, thus dramatically enhancing the anticancer effect of gambogic acid. Furthermore, mild photothermal therapy also showed synergistic effect with the drug. CONCLUSION Our study suggested that GNR/TiO2 nanostructures can be considered as a promising anticancer drug carrier.
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Objective To explore the effects and mechanism of gambogic acid ( GA) on lipopolysaccharide ( LPS)-induced acute lung injury ( ALI) .Methods ALI model was established by the injection of lipopolysaccharide into the tail vein of mice(4mg/kg, iv).The mice were randomly divided into control group (control), model group (model), GA group(GA), and pretreatment with GA of ALI group (GA+LPS).After six hours, the wet/dry weight ratio ( W/D) of the lung, myeloperoxidase ( MPO) activity in lung tissue , total proteins and number of white blood cells in bronchoalveolar lavage fluid ( BALF) were determined .Tumor necrosis factor-α( TNF-α) and interleukin-1β( IL-1β) were measured by the enzyme-linked immunosorbent assay methods .Results Compared with control group, the wet/dry weight ratio (W/D) of the lung, MPO activity in lung tissue, levels of total pro-teins and number of white blood cells in BALF of LPS group obviously increased , in addition the level of lung tissue TNF-αand IL-1βin LPS group were significantly higher (all P<0.01), while in GA pretreatment groups alleviated all the changes in ALI mice .Conclusions GA pre-treatment attenuated LPS-induced ALI , possibly by inhibiting inflammatory cytokines production so as to decrease the recruitment of neutrophils , reduce pulmonary edema .
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@#According to “2015 State of Innovation” released by Thomson Reuters, China Pharmaceutical University ranked the first in the field of heterocyclic compounds in 2010-2014 among innovation agencies in Asia. In order to fully understand the situation of the new drug research of this university, this paper analyzes its patent application from 2010 to 2014 in the field of heterocyclic compounds. The key applications were summarized. In summary, this university showed its advantage of high filings and high licensing rate in the field of heterocyclic compounds, yet with the shortcomings of low patent applications in PCT. Furthermore, suggestions have been made for the intellectual property management of this university.
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Objective: To optimize the preparation process of gambogic acid (GA) liposomes and study the in vitro and in vivo release. Methods: The detection method of GA was established, using the Box-Behnken experiment design to optimize liposomes formula, GA liposomes were obtained with the highest encapsulation efficiency; Using scanning electron micrographs (SEM) to observe liposome surface morphology, using the dialysis method to study the liposome release in vitro, we also measured the stability of liposome in 15 d; Male Wistar rats were injected with GA or GA liposomes (1 mg/mL) via tail vein, UPLC-MS/MS method was used to determine the drug concentration, and differences in pharmacokinetic parameters of the two drugs were compared. Results: After Box-Behnken optimization, the encapsulation efficiency of liposomes was 92.3%, and the optimized liposomes formula is cholesterol of 440 mg, egg phosphatidylcholine of 1823 mg,,and istearoyl phosphoethanolamine-PEG 2000 of 705 mg, liposomes had uniform particle size and smooth surface; In vitro release results showed that the liposomes could be gentle and slowly release and had a long- term effect. The liposomes were stable keeping in 4 ℃ within 15 d; In in vivo study, the half-life of GA liposome was 9.97 h, 4.43 times of GA; AUC0-24 h of GA liposome was 22.55 μg∙h/mL, 4.73 times of GA. Conclusion: Compared with GA, GA liposome has the characteristics of long-circulating, high blood drug concentration, and could release smoothly.
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Objective To study the effect of gambogic acid on apoptosis and autophagy in human hepatoma cells HepG2, and to detect its possible mechanism. Methods After exposure of HepG2 cells to gambogic acid at different concentration for 24 h, cell proliferation rates was determined by MTT assay, apoptosis rate was detected by the flow cytometry (FCM), formation of autophagic vacuoles was observed by the monodansyl cadaverine (MDC) fluorescence staining, expression level of apoptosis-related proteins Bax, bcl-2 and autophagy related protein Beclin 1 was detected by Western blot. Results HepG2 cell growth was inhibited by the gambogic acid dose dependence. After exposure to gambogic acid at 0, 2.0, 4.0 and 8.0 μmol/L for 24 h, cell apoptosis rate was significantly increased to 5.31 %, 29.18 %, 31.50 % and 46.09 %(P <0.05), MDC average fluorescence intensity was also significantly increased to 6.3 ±1.1, 82.6 ±4.5, 132.9±15.7 and 157.7±9.0 (P<0.01). Western blot showed that gambogic acid could promote the expression of apoptosis protein Bax (0.17 ±0.02, 0.75 ±0.06, 0.78 ±0.05, 0.89 ±0.10, P <0.05), and decrease the expression of anti-apoptosis protein bcl-2 (1.18 ±0.04, 0.90 ±0.06, 0.64 ±0.08, 0.57 ±0.05, P <0.05), meanwhile, it could also increase the expression of autophagy related protein Beclin (0.67±0.03, 0.92±0.04, 0.95±0.07, 1.04±0.06, P<0.05). Conclusion Gambogic acid can inhibit the growth of human hepatoma HepG2 cells by inducing apoptosis and autophagic cell death.
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Objective To develop a double-wavelength HPLC method for the simultaneous determination of neogambogic acid,gambogic acid,( +)-catechin and L-epicatechin in Litong pill. Methods The quantitative analysis was carried out on C18 column(250 mm×4.6 mm,5 μm).A linear elution of methanol-acetonitrile(4: 1)and 0.2% phosphoric acid solution was adopted at the flow rate of 1.1 mL·min-1 ,with the detection wavelength set at 360 nm for neogambogic acid and gambogic acid,and at 280 nm for(+)-catechin and L-epicatechin. Results The linear ranges of neogambogic acid,gambogic acid,(+)-catechin and L-epicatechin were 5.85-117.00 μg·mL-1(r= 0.999 5),8.95-179.00 μg·mL-1(r= 0.999 8),6.90-138.00 μg·mL-1(r= 0.999 7),and 5.30-106.00 μg·mL-1(r = 0.999 9),respectively.The average recoveries were 97.82%(RSD= 1.21%),98.72%(RSD = 1.30%),96.96%(RSD = 0.84%)and 99.26%( RSD = 1.46%)for neogambogic acid, gambogic acid,(+)-catechin and L-epicatechin,respectively. Conclusion The method is simple,accurate,reproducible and may be used for the simultaneous determination of neogambogic acid,gambogic acid,(+)-catechin and L-epicatechin in Litong pill.
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Gambogic acid (GA) is an anticancer agent in phase ‖b clinical trial in China but its mechanism of action has not been fully clarified. The present study was designed to search the possible target-related proteins of GA in cancer cells using proteomic method and establish possible network using bioinformatic analysis. Cytotoxicity and anti-migration effects of GA in MDA-MB-231 cells were checked using MTT assay, flow cytometry, wound migration assay, and chamber migration assay. Possible target-related proteins of GA at early (3 h) and late stage (24 h) of treatment were searched using a proteomic technology, two-dimensional electrophoresis (2-DE). The possible network of GA was established using bioinformatic analysis. The intracellular expression levels of vimentin, keratin 18, and calumenin were determined using Western blotting. GA inhibited cell proliferation and induced cell cycle arrest at G2/M phase and apoptosis in MDA-MB-231 cells. Additionally, GA exhibited anti-migration effects at non-toxic doses. In 2-DE analysis, totally 23 possible GA targeted proteins were found, including those with functions in cytoskeleton and transport, regulation of redox state, metabolism, ubiquitin-proteasome system, transcription and translation, protein transport and modification, and cytokine. Network analysis of these proteins suggested that cytoskeleton-related proteins might play important roles in the effects of GA. Results of Western blotting confirmed the cleavage of vimentin, increase in keratin 18, and decrease in calumenin levels in GA-treated cells. In summary, GA is a multi-target compound and its anti-cancer effects may be based on several target-related proteins such as cytoskeleton-related proteins.
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Humans , Antineoplastic Agents , Pharmacokinetics , Apoptosis , Breast Neoplasms , Drug Therapy , Metabolism , Calcium-Binding Proteins , Genetics , Cell Line, Tumor , Cell Migration Assays , Cell Migration Inhibition , Cell Proliferation , Computational Biology , Methods , Cytoskeleton , Metabolism , Electrophoresis, Gel, Two-Dimensional , Flow Cytometry , Gene Expression , Keratin-18 , Genetics , Oxidation-Reduction , Protein Biosynthesis , Protein Transport , Proteomics , Methods , Transcription, Genetic , Ubiquitin-Specific Proteases , Pharmacokinetics , Vimentin , Genetics , Xanthones , PharmacokineticsABSTRACT
Objective Gambogic acid ( GA) can suppress the growth of multiple tumor cells , including gastric carcinoma , hepatoma , hematologic neoplasms and breast carcinoma , but there have been few reports about its effect on urologic neoplasms .This study was to investigate the possible mechanisms of GA inducing bladder cancer cell apoptosis and cell cycle arrest . Methods We cultured human bladder cancer BIU8-7 cell lines in vitor and treated the cells in the logarithmic growth phase with isotonic saline solu-tion (negative control)or GA at the concentrations of 1.0, 2.0, and 3.0μmol/L, respectively.We determined the expression of the Caspase-3 protein in the tumor tissue using the immunohistochemical S-P method and detected GA-induced apoptosis of the bladder cancer cells and cell cycle changes by flow cytometry . Results The expressions of the Caspase-3 protein were 4.28 ±1.86, 5.03 ± 0.78, and 6.47 ±1.31 in the 1.0, 2.0, and 3.0μmol/L GA groups, respectively, significantly higher than 2.13 ±1.27 in the nega-tive control (P<0.05).Flow cytometry showed a gradual decrease of the cells in the G 0/G1 phase and a gradual increase in the G2/M phase , but no obvious change in the S phase . Conclusion Gambogic acid can promote the apoptosis , arrest the cell cycle , and in-hibit the proliferation of bladder cancer cells by increasing the expression of the Caspase -3 protein.
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Objective To investigate the effects of gambogic acid on proliferation,invasion and protein expression of MMP-2 in LoVo human colon cancer cell line.Methods The CCK-8 assay was used to determine the anti-proliferation ability.Transwell chamber assay was used to study the invasion of LoVo colon cancer cells.Western Blot was applied to examine the protein expression of MMP-2.Results CCK-8 assay showed that after cells treated with 1,2,4 μmol/L GA for 24 h,48 h the inhibition rates were(0.16±0.11)％,(0.24±0.08)％,(0.58±0.01)％,(0.67±0.03)％,(0.79±0.01)％ and (0.27±0.05)％,(0.69±0.09)％,(0.85±0.01)％,(0.87±0.01)％,(0.89±0.01)％,and there had statistically significant differences between control group (0 μmo/L) with experimental group (P＜0.05).Gambogic acid can effectively inhibit LoVo cells invasion at a low-dose concentration,which the numbers of cells that digested the matrigel in control group (0 μmo/L)was 120.50± 8.69 and 69.83 ±4.75 in experimental group (1 μnol/L),P＜ 0.05).Western Blot revealed that Gambogic acid can down-regulate the expression of MMP-2,which the levels of down-regulating the expression of MMP-2 after treated with 1,2,4 umol/L GA were 48.67％,74.72％and 82.70％ (P＜0.05) Conclusion Gambogic acid can effectively inhibit the growth and invasion of the LoVo cells,which may be related with down-regulating the expression of MMP-2.