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OBJECTIVE@#To evaluate the apoptosis and cycle arrest effects of Oldenlandia diffusa flavonoids on human gastric cancer cells, determine the action mechanisms in association with the mitochondrial dependent signal transduction pathway that controls production of reactive oxygen species (ROS), and evaluate the pharmacodynamics of a mouse xenotransplantation model to provide a reference for the use of flavonoids in prevention and treatment of gastric cancer.@*METHODS@#Flavonoids were extracted by an enzymatic-ultrasonic assisted method and purified with D-101 resin. Bioactive components were characterized by high-performance liquid chromatography. Cell lines MKN-45, AGS, and GES-1 were treated with different concentrations of flavonoids (64, 96, 128, 160 µg/mL). The effect of flavonoids on cell viability was evaluated by MTT method, and cell nuclear morphology was observed by Hoechst staining. The apoptosis rate and cell cycle phases were measured by flow cytometry, the production of ROS was detected by laser confocal microscope, the mitochondrial membrane potential (MMP) were observed by fluorescence microscope, and the expression of apoptotic proteins related to activation of mitochondrial pathway were measured by immunoblotting. MKN-45 cells were transplanted into BALB/c nude mice to establish a xenograft tumor model. Hematoxylin and eosin staining was used to reveal the subcutaneous tumor tissue. The tumor volume and tumor weight were measured, the expression levels of proliferation markers proliferating cell nuclear antigen (PCNA) and Ki-67 were detected by immunohistochemistry, and the expression levels of CA72-4 were measured by enzyme linked immunosorbent assay.@*RESULTS@#Oldenlandia diffusa flavonoids inhibited proliferation of MKN-45 and AGS human gastric cancer cells, arrested the cell cycle in G1/S phase, induced accumulation of ROS in the process of apoptosis, and altered MMP. In addition, flavonoids increased Apaf-1, Cleaved-Caspase-3, and Bax, and decreased Cyclin A, Cdk2, Bcl-2, Pro-Caspase-9, and Mitochondrial Cytochrome C (P<0.05). The MKN-45 cell mouse xenotransplantation model further clarified the growth inhibitory effect of flavonoids towards tumors. The expression levels of PCNA and Ki-67 decreased in each flavonoid dose group, the expression level of CA72-4 decreased (P<0.05).@*CONCLUSION@#Flavonoids derived from Oldenlandia diffusa can inhibit proliferation and induce apoptosis of human gastric cancer cells by activating the mitochondrial controlled signal transduction pathway.
Subject(s)
Humans , Animals , Mice , Oldenlandia/metabolism , Proliferating Cell Nuclear Antigen , Stomach Neoplasms , Flavonoids/pharmacology , Reactive Oxygen Species/metabolism , Mice, Nude , Ki-67 Antigen , Cell Line, Tumor , Apoptosis , Plant Extracts/pharmacology , Caspases , Cell ProliferationABSTRACT
【Objective】 To investigate the effects of total flavone of oldenlandia diffusa (FOD) on the proliferation and apoptosis of hepatocellular carcinoma (HCC) stem cells sorted from Huh7. 【Methods】 Human HCC cell lines Huh7 was cultured in vitro; CD133 positive (CD133+) stem cells in Huh7 cell line were sorted by flow cytometry, and stem cell markers such as Nanog, Oct4 and Sox2 were tested by Western blotting. CD133+-Huh7 was stimulated by different concentrations (0 μg/mL, 50 μg/mL, 100 μg/mL and 400 μg/mL) of FOD for different time (24 h, 48 h, 72 h and 96 h). CCK8 and plate cell cloning assay were used to detect the effect of FOD on CD133+-Huh7 proliferation while Annexin V-PE/7-AAD was used to detect the effect of FOD on CD133+-Huh7 apoptosis. Western blotting was used to detect the protein expressions of protein 53 (P53), factor associated suicide-Fas-associating protein with a novel death domain (Fas-FADD), B-cell lymphoma-2 (Bcl-2), Cleaved-Caspase3, and Bcl-2 associated X protein (Bax). 【Results】 More than 95% of stem cells were purified for further experiments. Cell proliferation of CD133+-Huh7 was significantly inhibited by FOD, with the significant suppression at the concentration of 100 μg/mL for 72 h compared with negative control group (P<0.05). The apoptosis rate was significantly upregulated than that in the negative control group (P<0.05). The protein expression of Bcl2 decreased while Bax and Cleaved-Caspae3 increased via FAS/FADDD and P53 axis. 【Conclusion】 FOD can significantly inhibit the proliferation and promote the apoptosis of CD133+-Huh7.
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【Objective】 To investigate the effect of total flavone of oldenlandia diffusa(FOD) on the stemness, proliferation and apoptosis of breast cancer(BC) stem cells sorted from MDA-MB-231. 【Methods】 Human BC cell lines MDA-MB-231 was cultured in vitro; MDA-MB-231 was stimulated by different concentrations(0 μg/mL, 100 μg/mL, 200 μg/mL and 400 μg/mL) of FOD for different time (24 h, 48 h and 72 h). CCK8 and plate cell cloning assay were used to detect the effect of FOD on MDA-MB-231 proliferation; CD44+/CD24-MDA-MB-231 cell line were tested by flow cytometry and stem cell markers such as Nanog, Oct4 and Sox2 were tested by Western blotting; Annexin V-PE/7-AAD was used to detect the effect of FOD on MDA-MB-231 apoptosis and Bcl2, cleaved-caspase3 and Bax were tested by Western blotting. 【Results】 Cell proliferation of MDA-MB-231 was significantly inhibited by FOD, with the significant suppression at concentrations of 400 μg/mL for 72 h compared with negative control group(P<0.05). The apoptosis rate was significantly upregulated than the negative control group (P<0.05). The protein expression of Bcl2 decreased while Bax and cleaved-caspae3 increased, and stemness markers such as Nanog, Sox2 and Oct4 decreased in FOD-treated cells. Moverover, Akt-GSK3β-β-catenin axis was inhibited in FOD-treated cells. 【Conclusion】 FOD could significantly inhibit the stemness and proliferation and promote the apoptosis of MDA-MB-231.
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ABSTRACT:Objective To investigate the effects of total flavones of oldenlandia diffusa (FOD)on epithelial-mesenchymal transition in hepatocellular cancer cell line MHCC97-H.Methods TGF-β1 induced EMT in routinely cultured liver cancer cell line MHCC97-H;then MHCC97-H cell was divided into 5 groups:normal control group, TGF-β1 group,TGF-β1 + FOD group,TGF-β1 + 5-FU group,and TGF-β1 + FOD + 5-FU group.After 48 h of treatment,the invasion ability of MHCC97-H cell was detected by Transwell;the proteins of E-cadherin and vimentin were determined by Western blot.Results Compared with the normal form of MHCC97-H cell line,the cell had obvious long fusiform after TGF-β1 induction,and the invasion ability enhanced (P = 0.02 ).But after treatment,the invasion ability of MHCC97-H cell decreased in FOD group and 5-FU group compared with that in TGF-β1 group (P = 0.03,P = 0.02 ),and decreased more significantly in FOD + 5-FU group (P = 0.01 ).The expression of E-cadherin at the protein level decreased significantly (P = 0.01 )in TGF-β1 group,which was abolished in FOD group (P =0.03 )and 5-FU group (P = 0.02 ).The expression of vimentin at the protein level increased significantly (P =0.01)in TGF-β1 group,which was abolished in FOD group (P =0.04)and 5-FU group (P =0.03)and more obviously in FOD+5-FU group (P =0.01).Conclusion FOD can reverse the invasion of MHCC97-H cells in EMT induced by TGF-β1 through decreasing the expression of E-cadherin protein and inhibiting the epithelial-mesenchymal transition of MHCC97-H cell.
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Objective: To explore the effect of exogenous nitric oxide (NO) on seed germination of Oldenlandia diffusa under drought stress and to determine the optimal concentration of sodium nitroprusside (SNP, exterior nitric oxide donor) for alleviating the drought stress. Methods: Using 15% PEG6000 to imitate drought stress, the germination potential, germination rate, germination index, and vigor index of O. diffusa seeds were measured by the treatment of SNP at different concentration. Results: The germination process of O. diffusa seeds was significantly suppressed by drought stress. SNP could significantly promote the germination of O. diffusa seeds under drought stress. SNP (0.3 mmol/L) increased the germination rate and germination potential by 2.78- and 7.77-folds, respectively. In the case of germination index and vigor index, SNP (0.1 and 0.3 mmol/L ) showed the maximum positive impact and there was no significant difference between the two SNP concentration treatments. Conclusion: SNP could alleviate the negative influence of drought stress on the germination process of O. diffusa seeds and improve the drought-resistant activity. Among all the treatments, the concentration of SNP at 0.3 mmol/L leads to the maximum germination rate.
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Objective To explore the inhibitory effect of the ethanol extract of Oldenlandia diffusa on the proliferation of CT-26 colon cancer cells which come from BALB/c mice. Method We determined the inhibitory effect of different concentrations of ethanol extract of Oldenlandia diffusa on CT-26 cells' proliferation by using methyl thiazolyl tetrazolium (MTT), and calculated the 50% inhibiting concentration (IC50) . Results As to the same concentration, the inhibitory effect of the ethanol extract of Oldenlandia diffusa on CT-26 cells was increased with time, for exsample:after treated with 0.08 mg/mL of ethanol extract of Oldenlandia diffusa for 24 h, 48 h and 72 h, the inhibitory rates of CT-26 cells were (16.67 ±9.35)%, (34.66 ±9.23)%and (40.07 ±9.16)%, respectively. After treating CT-26 cancer cells for 24 h, 48 h and 72 h, the IC50 values of the ethanol extract of Oldenlandia diffusa were 0.315,0.155 and 0.115 mg/mL, respectively. In the same treatment time, the inhibitory effect of the ethanol extract of Oldenlandia diffusa on CT-26 cells was increased with the increase of concentration:after treatment for 72 h with different concentrations (0.06 mg/mL,0.08 mg/mL,0.10 mg/mL,0.12 mg/mL, 0.14 mg/mL,0.16 mg/mL,0.18 mg/mL and 0.20 mg/mL) of the ethanol extract of Oldenlandia diffusa,the inhibitory rates of CT-26 cells were (35.46 ±3.59)%, (40.07 ±9.16)%, (40.77 ±6.92)%, (52.81 ±1.87)%, (54.22±2.35)%, (68.72±3.71)%, (70.04±8.03)%and (71.84±3.12)%, respectively. Conclusion The ethanol extract of olenlandia diffusa can inhibit the proliferation of CT-26 colon cancer cells from BALB/c mice in a time and dose dependent manner.
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Objective To explore the inhibitory effect of Oldenlandia diffusa extract on colorectal cancer angiogenesis in BALB/c mice. Methods Thirty-two BALB/c mice with subcutaneous CT26 colon cancer animal model were randomly equally divided into four groups,including the control group (groupⅠ,saline 0.1 mL/(10. d), O. diffusa ethanol extract of 90 mg/(kg.d) (groupⅡ), O. diffusa ethanol extract of 180 mg/(kg.d) (groupⅢ) and O. diffusa ethanol extract of 360 mg/(kg.d) (group Ⅳ) . Each group of mice were treated with intragastric administration of law administration 12 days after vaccination, then stopped and continue fed to 32 days, and the mice were killed. Micro-vascular dense ( MVD) was observed and countered under the microscopy by immunohistory chemistry. Results The murine colon tumor volumes of GroupⅡ,ⅢandⅣwere significantly less than that of groupⅠ,with significant difference ( <0.05) . The tumor microvessel density values of four groups was (7.83±2.87), (5.32±1.27), (1.77±0.70) and (1.87±0.68),respectively. The number of tumor blood vessels in GroupⅡ,Ⅲ and Ⅳ were significantly less than that of Ⅰ group, with significant difference ( <0.05) . Conclusion Within a certain dose range, the ethanol extract of O. diffusa can significantly inhibit the mouse colon cancer and the mechanism may be realated to inhibiting tumor angiogenesis.
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Objective To rapidly separate and identify the chemical components in traditional Chinese herbal medicine of Oldenlandia diffusa and its injection preparations by high performance liquid chromatography-time of flight mass spectrometry (HPLC-TOFMS). Methods An Agilent Zorbax XDB-Q18 column (250 mm×4. 6 mm, 5 μm) was used for separation and identification of chemical components in Oldenlandia diffusa, with a mobile phase of 0. 3% acetic acid (A) and methanol (B) in gradient elution, 0-30 min, 30%-90%B. The flow rate was set at 1. 0 ml/min and the injection volume was 10 μl. The time- of-flight mass spectrometer was equipped with an EIS ion source. The scanning mass range was between m/z 100-1 000. Results The traditional Chinese medicine of Oldenlandia diffusa and its injection preparation were on-line separated and characterized by HPLC-TOFMS, and 11 chemical compounds were identified in Oldenlandia diffusa, 6 compounds in market injection preparation, and 2 compounds in the injection prepared by ourselves. Conclusion Chromatographic demonstration of chemical compounds in Oldenlandia diffusa in one run provides a foundation for the further studying the metabolism and mechanism of Oldenlandia diffusa and its injection preparations.
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Aim To study anti-inflammatory and antibacterial effects of total flavones of oldenlandia diffusa Willd(FOD).Methods The models of dimethylbenzene-induced ear swollen in mice,turpentine-induced granuloma pouch in rats,paw edema caused by egg white in rats,increased vascular permeability by acetic acid in mice and the antibacterial activity in vitro were used to study FOD. Results FOD(15、30 and 60 mg?kg~-1 )show a marked suppressive effect on dimethylbenzene-induced ear swollen and increased vascular permeability in mice. In addition,FOD(12、24 and 48 mg?kg~-1 ) could reduce the paws edema and the granuloma pouch in rats.On the other hand,FOD had significally bacteriostatic effects against eight kinds of bacterial in vitro, in which effect on coccus is superior to that on bacillus.Conclusion FOD has anti-inflammatory and antibacterial effects.
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Aim To investigate the immunomdulatory effects of total flavones of oldenlandia diffusa willd (FOD).Methods In immunosuppressed mice induced by cyclophosphamide, IgM production,proliferation of T and B lymphocytes, levels of IL-2 and INF-? in serum were used to study the specific immunity of FOD. White blood cell count of mice suppressed by cyclophosphamide and cytarabine was used to observe the non-specific immunity of FOD.Results FOD(60 (mg?kg~(-1))) enhanced IgM production in (immunosup-)pressed mice induced by Cy and white blood cell count of mice suppressed by cytarabine and cyclophosphamide ;FOD(15,30 and 60 mg?kg~(-1)) increased proliferation of T and B lymphocytes and enhanced levels of IL-2 and INF-? in serum in immunosuppressed mice induced by cyclophosphamide. Conclusion FOD enhances the specific and non-specific immunity.