ABSTRACT
To explore the inhibitory effects of gomisin A (GA) in combination with carboplatin (CBP) on the proliferation, invasion and metastasis of human ovarian cancer Skov3 cells and their mechanisms, and to illustrate the synergistic antitumor effect of GA. Methods: The ovarian cancer Skov3 cells were treated with different concentrations of GA combined with different concentrations of CBP. MTT assay was used to detect the inhibitory rates of proliferation. According to the results, the appropriate concentrations of GA and CBP were conformed. The Skov3 cells were treated with PBS, GA 0.04 μmol · L-1), CBP 16 mg · L-1) and GA 0.04 jumol · L-1) in combination with CBP 16 mg · L-1), respectively, and used as control group, GA group, CBP group and GA+CBP group. After 48 h of treatment, the cell morphology was observed by optical microscope, the cell reproductive ability was determined by clonogenic cell survival assay, and the cell migration ability was measured by cell wound healing test; Transwell experiment was used to detect the cell invasion ability. The expression levels of MMP-2 and MMP-9 mRNA were determined by RT-PCR and the protein expression levels of MMP-2, MMP-9, AKT1 and pAKT1 were determined by Western blotting method. Results: Compared with GA and CBP groups, the inhibitory rate of proliferation of Skov3 cells in GA + CBP group was significantly increased (P<0.01). The concentrations of GA and CBP for the best dose ratio were 0.04 μmol · L-1 and 16 mg · L-1, respectively. The results of clone formation assay showed that the colony formation rates in CBP and GA+CBP groups were decreased compared with control group (P<0.05 or P<0.01); the colony formation rate of cells in GA + CBP group was significantly decreased compared with GA and CBP groups (P<0.01); the wound scratch assay results suggested that the number of metastic Skov3 cells in GA and CBP groups were decreased compared with control group; and the number of metastic Skov3 cells in GA + CBP group was decreased compared with GA and CBP groups. The results of Transwell expriment showed that the capacities of cells passing the ECM in GA and CBP groups were decreased compared with control group; the capacity of cells passing the ECM was decreased compared with GA and CBP groups. The RT-PCR and Western blotting results showed that the expression levels of MMP-2 and MMP-9 mRNA in GA+CBP group were down-regulated compared with GA and CBP groups (P< 0.01), and the expression levels of MMP-2, MMP-9, AKT1 and pAKTl protein were down-regulated (P<0.01). Conclusion: GA can enhance the ability of CBP to inhibit the proliferation, invasion and metastasis of human ovarian cancer Skov3 cells, and thus play a role in decreasing the toxicity and increasing the efficacy of chemotherapy.
ABSTRACT
Objective: To explore the effect of gomisin A (GA) in combination with carboplatin (CBP) on the apoptosis of ovarian cancer Skov3 cells, and to illustrate the synergistic antitumor effect of GA. Methods: The ovarian cancer Skov3 cells were treated with GA combined with CBP. MTT assay was used to detect the inhibitory rates of proliferation and the appropriate concentrations of GA and CBP were selected according to the results. The Skov3 cells were divided into control group, GA (0. 04 μmol · L-1) group, CBP (16 mg · L-1) group and GA (0.04 μmol · L-1) + CBP (16 mg · L-1) group. After 48 h treatment, the cell morphology was observed by microscope, the apoptotic rate was measured by flow cytometry, the apoptotic index (AI) was observed by TUNEL staining, the mitochondrial membrane potential was detected by JC-1 staining, and the mRNA and protein expression levels of apoptosis-related genes (Bax, caspase-3, Stat3) were determined by RT-PCR and Western blotting method. Results: The inhibitory rate of proliferation of Skov3 cells in GA + CBP group (52. 1%) was significantly higher than those in GA and CBP groups (P<0. 01). The concentrations of GA and CBP for the best dose ratio were 0. 04 μmol · L-1 and 16 mg · L-1, respectively. Compared with control group, the cell refractivities were decreased, the cells retracted, and part of cells were suspended in GA group and CBP groups; there were more suspended cells and cell retraction phenomenon was more prominent in GA + CBP group. The results of TUNEL staining and flow cytometry showed that the AI and apoptotic rate of cells in GA + CBP group were significantly increased compared with control group, GA and CBP group (P<0. 05 or P<0. 01); the JC-1 staining results suggested that the mitochondrial membrane potential of Skov3 cells in GA + CBP group was decreased (P<0. 05 or P<0. 01). The RT-PCR and Western blotting results showed that the expression levels of Bax and caspase-3 mRNA and protein were up-regulated (P<0. 05) and the expression levels of Bcl-2 and Stat3 mRNA and protein in GA + CBP group were down-regulated compared with control, GA and CBP groups (P< 0. 05). Conclusion: GA can enhance the ability of CBP to induce the apoptosis of human ovarian cancer Skov3 cells, and its mechanisms may be related to the up-regulation of the Bax and Caspase-3 expressions and the down-reguation of the Bcl-2 expression; GA can synergize the induction of CBP on apoptosis.
ABSTRACT
Objective:To explore the effect of gomisin A (GA) in combination with carboplatin (CBP) on the apoptosis of ovarian cancer Skov3 cells,and to illustrate the synergistic antitumor effect of GA.Methods:The ovarian cancer Skov3 cells were treated with GA combined with CBP.MTT assay was used to detect the inhibitory rates of proliferation and the appropriate concentrations of GA and CBP were selected according to the results.The Skov3 cells were divided into control group,GA (0.04 μmol · L-1) group,CBP (16 mg · L 1) group and GA (0.04 μmol· L-1)+ CBP (16 mg · L-1) group.After 48 h treatment,the cell morphology was observed by microscope,the apoptotic rate was measured by flow eytometry,the apoptotic index (AI) was observed by TUNEL staining,the mitochondrial membrane potential was detected by JC-1 staining,and the mRNA and protein expression levels of apoptosis-related genes (Bax,caspase-3,Stat3) were determined by RT PCR and Western blotting method.Results:The inhibitory rate of proliferation of Skov3 cells in GA + CBP group (52.1％) was significantly higher than those in GA and CBP groups (P＜0.01).The concentrations of GA and CBP for the best dose ratio were 0.04 μmol· L-1 and 16 mg · L-1,respectively.Compared with control group,the cell refractivities were decreased,the cells retracted,and part of cells were suspended in GA group and CBP groups;there were more suspended cells and cell retraction phenomenon was more prominent in GA + CBP group.The results of TUNEL staining and flow cytometry showed that the AI and apoptotic rate of cells in GA + CBP group were significantly increased compared with control group,GA and CBP group (P＜0.05 or P＜0.01);the JC-1staining results suggested that the mitochondrial membrane potential of Skov3 cells in GA + CBP group was decreased (P＜0.05 or P＜0.01).The RT-PCR and Western blotting results showed that the expression levels of Bax and caspase-3 mRNA and protein were up-regulated (P＜0.05) and the expression levels of Bcl-2 and Stat3mRNA and protein in GA + CBP group were down-regulated compared with control,GA and CBP groups (P＜0.05).Conclusion:GA can enhance the ability of CBP to induce the apoptosis of human ovarian cancer Skov3 cells,and its mechanisms may be related to the up-regulation of the Bax and Caspase-3 expressions and the down-reguation of the Bcl-2 expression;GA can synergize the induction of CBP on apoptosis.
ABSTRACT
Objective:To explore the inhibitory effects of gomisin A(GA)in combination with carboplatin (CBP)on the proliferation,invasion and metastasis of human ovarian cancer Skov3 cells and their mechanisms,and to illustrate the synergistic antitumor effect of GA.Methods:The ovarian cancer Skov3 cells were treated with different concentrations of GA combined with different concentrations of CBP.MTT assay was used to detect the inhibitory rates of proliferation.According to the results,the appropriate concentrations of GA and CBP were conformed.The Skov3 cells were treated with PBS,GA(0.04 μmol·L-1),CBP(16 mg·L-1)and GA (0.04 μmol·L-1)in combination with CBP(16 mg·L-1),respectively,and used as control group,GA group, CBP group and GA+CBP group.After 48 h of treatment,the cell morphology was observed by optical microscope, the cell reproductive ability was determined by clonogenic cell survival assay,and the cell migration ability was measured by cell wound healing test;Transwell experiment was used to detect the cell invasion ability.The expression levels of MMP-2 and MMP-9 mRNA were determined by RT-PCR and the protein expression levels of MMP-2,MMP-9,AKT1 and pAKT1 were determined by Western blotting method.Results:Compared with GA and CBP groups,the inhibitory rate of proliferation of Skov3 cells in GA + CBP group was significantly increased (P<0.01).The concentrations of GA and CBP for the best dose ratio were 0.04 μmol·L-1and 16 mg·L-1, respectively.The results of clone formation assay showed that the colony formation rates in CBP and GA+CBP groups were decreased compared with control group(P<0.05 or P<0.01);the colony formation rate of cells in GA + CBP group was significantly decreased compared with GA and CBP groups(P<0.01);the wound scratch assay results suggested that the number of metastic Skov3 cells in GA and CBP groups were decreased compared with control group;and the number of metastic Skov3 cells in GA+ CBP group was decreased compared with GA and CBP groups.The results of Transwell expriment showed that the capacities of cells passing the ECM in GA and CBP groups were decreased compared with control group;the capacity of cells passing the ECM was decreased compared with GA and CBP groups.The RT-PCR and Western blotting results showed that the expression levels of MMP-2 and MMP-9 mRNA in GA+CBP group were down-regulated compared with GA and CBP groups(P<0.01),and the expression levels of MMP-2,MMP-9,AKT1 and pAKT1 protein were down-regulated(P<0.01).Conclusion:GA can enhance the ability of CBP to inhibit the proliferation,invasion and metastasis of human ovarian cancer Skov3 cells,and thus play a role in decreasing the toxicity and increasing the efficacy of chemotherapy.
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Objective: Multi-type resource chemical constituents in Schizandrae Fructus residues were analyzed in the process of Shengmai Injection production, in order to provide the scientific basis for Schizandrae Fructus in the further process of industrialization. Methods: The lignan components were analysed and evaluated by HPLC-UV method. After using NaOH to extract sample, the BCA method was adopted to measure the mass fraction of total protein and take bovine serum albumin as the reference. Using UV-Vis spectrophotometer to measure the mass fraction and constitutes of neutral polysaccharide and acidic polysaccharide, respectively. The equipment for raw fiber determination was taken to gauge the crude fiber content of Schizandrae Fructus residues. Results: The mass fractions of schisandrin A, schizandrin B, schizandrin C, gomisin A, gomisin B, and schisantherin A were 1.442 4, 3.788 0, 1.350 9, 4.399 3, 3.231 3, and 0.505 3 mg/g, respectively. Compared with the original medicinal materials, the technology utilization rate of gomisin A was 20.84% during the process of Shengmai Injection production. But the gomisin B virtually was unused and remained in the residues. The mass fractions of schisandrin A, schizandrin B, schizandrin C, and schisantherin A were higher than that of the original medicinal material. The available macromolecular substances are proteins, polysaccharide, and crude fiber. The content of total proteins was 14.69%. The mass fractions of neutral polysaccharide and acidic polysaccharide were 3.82% and 1.31%, respectively. The analysis on crude fiber showed that the mass fraction of crude fiber in Schizandrae Fructus residues was 43.80%. Conclusion: The analysis shows that Schizandrae Fructus residues contain many resource chemical components in the process of Shengmai Injection production, such as lignins, protein, saccharides, and crude fiber. The strategy for recycling and possible way is proposed exploringly based on the available resource chemical components of Schizandrae Fructus residues and water-extracting technology. It provides the reference for the utilization of waste resource in further process of industralization, promoting the resource conservation, and development of environmental protection, realizing the harmonious coexistence between the economy and ecology.
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Objective: To establish a UPLC-MS/MS analytical method for the simultaneous analysis of ginsenosides (ginsenosides Rb1, Re, Rg1, Rc, Rd, Rf, Rg3, F2, and notoginsenoside R1) and lignans (gomisin A, schisandrol B, deoxyschizandrin, and schisandrin B) in Yiqi Fumai Injection (freeze-dried) (YFI), and measure the contents of these constituents in YFI. Methods: Quantitative research of 13 components in YFI was done by reversed-phase liquid chromatography on a C18 column using a gradient elution (0.1% formic acid in water and 0.1% formic acid in methanol). A triple quadrupole mass spectrometer operating in positive electrospray ionization mode with multiple reaction monitoring was used. Results: Thirteen components in YFI have good linear relationship, precision, stability, and repeatability according to the requirements of the methodology determination. The recoveries were 98.28%-101.08%. The 13 components in three batches of YFI were determined by UPLC-MS/MS method. Conclusion: The developed UPLC-MS/MS method is simple, sensitive, and accurate, and has good repeatability. The 13 components in YFI could be rapidly and accurately quantified by UPLC-MS/MS, which provides the helpful information for the comprehensive quality evaluation of YFI.
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Objective To optimize the extract technology of active lignins from the fruits of Schisandra chinensis.Methods The content of schizandrin,gomisin A,and deoxyschizandrin were selected as standards to evaluate the efficiency of smashing tissue extraction (STE).Solid-liquid ratio,extracting times,ethanol concentration,and extracting time were investigated through orthogonal test.Results The optimized conditions for STE were ten times amount of 80％ EtOH,extracting for three times,and 2 min for each time.Conclusion STE could obtain relatively higher yield,simplicity of operation,and benefit for environment protection.It could be better choice for the extraction ofS.chinensis.
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Gomisin A possesses a hepatic function-facilitating property in liver-injured rats. Its preventive action on carbon tetrachloride-induced cholestasis is due to maintenance of the function of the bile acids-independent fraction. To investigate alterations in gene expression after gomisin A treatment on injured rat liver, DNA microarray analyses were performed on a Rat 44K 4-Plex Gene Expression platform with duplicated reactions after gomisin A treatment. We identified 255 up-regulated and 230 down-regulated genes due to the effects of gomisin A on recovery of carbon tetrachloride-induced rat liver damage. For functional characterization of these genes, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes biochemical pathways analyses were performed. Many up-regulated or down-regulated genes were related to cell cycle or focal adhesion and cell death genes, respectively. Our microarray experiment indicated that the liver repair mechanism induced by gomisin A was strongly associated with increased gene expressions related to cell cycle and suppression of the gene expression related in cell death.