Mechanism of Stemona tuberosa Alkaloids in Inhibiting Proliferation and Inducing Apoptosis of Non-small Cell Lung Cancer NCI-H460 Cells / 中国实验方剂学杂志

ObjectiveTo study the effect and underlying mechanism of Stemona tuberosa alkaloids on the proliferation and apoptosis of human non-small cell lung cancer NCI-H460 cells. MethodNon-small cell lung cancer NCI-H460 cells were divided into a blank group and S. tuberosa alkaloids groups （50， 100， 150， 200， and 250 mg·L-1）. The effect of S. tuberosa alkaloids on the proliferation of human NCI-H460 cells was observed by thiazolyl blue tetrazolium bromide （MTT） assay and colony formation assay. Cell apoptosis was observed by Hoechst 33258 staining and flow cytometry. Real-time fluorescence-based polymerase chain reaction （Real-time PCR） was used to detect the effect of S. tuberosa alkaloids on the mRNA expression of cysteinyl aspartate-specific protease 3 （Caspase-3）， B-cell lymphoma-2 （Bcl-2）， Bcl-2-associated X protein （Bax）， and epidermal growth factor receptor （EGFR）. The protein expression levels of Caspase-3， Bax， Bcl-2， protein kinase B （Akt）， phosphorylated （p-）Akt， EGFR， c-Jun N-terminal kinase （JNK）， p-JNK， p38 mitogen-activated protein kinase （p38 MAPK）， and p-p38 MAPK were measured by Western blot. ResultCompared with the blank group， the S. tuberosa alkaloids groups showed increased inhibition rate on cell proliferation （P<0.01）， reduced number of cell clones formed and the rate of cell clonal formation （P<0.05， P<0.01）， and increased karyopyknosis， cytoplasmic aggregation， and cell apoptosis rate （P<0.01）. The S. tuberosa alkaloids groups at 100， 150， 200， and 250 mg·L-1 showed increased Caspase-3 mRNA expression （P<0.05）， decreased EGFR mRNA expression （P<0.05， P<0.01）， up-regulated protein expression of Caspase-3 and p-JNK （P<0.01）， and down-regulated protein expression of EGFR and p-Akt （P<0.05， P<0.01）. Additionally， compared with the blank group， the S. tuberosa alkaloids groups showed increased expression of Bax mRNA （P<0.01）， decreased expression of Bcl-2 mRNA （P<0.01）， up-regulated protein expression of Bax and p-p38 MAPK （P<0.01）， and down-regulated protein expression of Bcl-2 （P<0.01）. ConclusionsS. tuberosa alkaloids can inhibit proliferation and induce apoptosis of human non-small cell lung cancer NCI-H460 cells， and the mechanism may be related to the inhibition of EGFR protein expression and phosphorylation of Akt protein， as well as the activation of the JNK/p38 MAPK signaling pathway.

In vitro Antiproliferative activity of Melissa officinalis L. (Lamiaceae) leaves essential oil / Actividad antiproliferativa in vitro de aceite esencial de hojas de Melissa officinalis L (Lamiaceae)

In the present study, we investigated the antiproliferative activity of essential oil from leaves of Melissa officinalis L. grown in Southern Bosnia and Herzegovina. In vitro evaluation of antiproliferative activity of the M. officinalis essential oil was carried out on three human tumor cell lines: MCF-7, NCI-H460 and MOLT-4 by MTT assay. M. officinalis essential oil was characterized by high percentage of monoterpenes (77,5%), followed by the sesquiterpene fraction (14,5%) and aliphatic compounds (2,2%). The main constituents of the essential oil of M. officinalis are citral (47,2%), caryophyllene oxide (10,2%), citronellal (5,4%), geraniol (6,6%), geranyl acetate (4,1%) and ß- caryophyllene (3,8%). The essential oil showed significant antiproliferative activity against three cancer cell lines, MOLT-4, MCF-7, and NCI-H460 cells, with GI50 values of <5, 6±2 and 31±17 µg/mL, respectively. The results revealed that M. officinalis L. essential oil has a potential as anticancer therapeutic agent.

En el presente estudio, investigamos la actividad antiproliferativa del aceite esencial de las hojas de Melissa officinalis L. cultivadas en el sur de Bosnia y Herzegovina. La evaluación in vitro de la actividad antiproliferativa del aceite esencial de M. officinalis se llevó a cabo en tres líneas celulares de tumores humanos: MCF-7, NCI-H460 y MOLT-4 utilizando el ensayo de MTT. El aceite esencial de M. officinalis se caracterizó por un alto porcentaje de monoterpenos (77,5%), seguido de la fracción sesquiterpénica (14,5%) y compuestos alifáticos (2,2%). Los principales constituyentes del aceite esencial de M. officinalis fueron citral (47,2%), óxido de cariofileno (10,2%), citronelal (5,4%), geraniol (6,6%), acetato de geranilo (4, 1%), y ß-cariofileno (3,8%). El aceite esencial mostró una actividad antiproliferativa significativa contra las líneas celulares de cáncer MOLT-4, MCF-7 y NCI-H460, con valores GI50 de <5, 6±2 y 31±17 µg/mL, respectivamente. Los resultados revelaron que el aceite esencial de M. officinalis L. tiene potencial como agente terapéutico contra el cáncer.

Mechanism of hsa-miRNA124-3p regulating the proliferation and migration of human lung cancer cell line NCI-H460 / 上海交通大学学报（医学版）

Objective · To study the regulation of hsa-miRNA124-3p on the proliferation and migration of human lung cancer NCI-H460 cells and its mechanism. Methods · Four pairs of lung cancer and para-carcinoma tissues were harvested in clinical and measured for hsa-miRNA124-3p and Krüppellike factor 4 (KLF4) levels. The theoretical binding site of hsa-miRNA124-3p in 3'-UTR of KLF4 was predicted by bioinformatics, and validated by luciferase report assay. NCI-H460 cells were transfected with pshRNA-Sponge-miRNA124 or pshRNA-KLF4, and 48 hours later, the proliferation of NCI-H460 cells after genetic intervention was assayed by the MTT method, and cell migration ability was observed by streak method. Results · For all four pairs of samples tested, hsa-miRNA124-3p was higher in the cancer tissues than in the adjacent tissue (P0.05). The data of cell migration assay showed that the changes of cell migration ability were the same as proliferation activity of the cells in groups 72 h after transfection. Conclusion · Hsa-miRNA124-3p increases the proliferation and migration in NCI-H460 cells via suppressing the expression of KLF4, and reducing the content of miRNA124-3p in NC-H460 cells can inhibit cell proliferation and migration via upregulating KLF4 expression.

Optimization of aqueous extraction for polysaccharides from Astragali Radix and the in vitro antitumor activity / 中成药

AIM To optimize the aqueous extraction for polysaccharides from Astragali Radix and to evaluate the in vitro antitumor activity.METHODS With extraction temperature,extraction time and solid-liquid ratio as influencing factors,extraction rate of polysaccharides as an evaluation index,the extraction was optimized by uniform design.The effect of polysaccharides on the proliferation of non-small cell lung cancer NCI-H460 cells,the apoptosis rate and cell cycle of NCI-H460 cells,and the expressions of Caspase-3,Bax and Bcl-2 were detected by MTT assay,flow cytometry and Western blot,respectively.RESULTS The optimal conditions were determined to be 100 ℃ for extraction temperature,1 h for extraction time,and 1 ∶ 35 for solid-liquid ratio,the extraction rate of polysaccharides was 3.62％.Compared with the control group,the proliferation of NCI-H460 cells was significandy inhibited in a dose-dependent manner (P ＜ 0.01),the S phase ratio,early apoptosis rate,late apoptosis rate and total apoptosis rate were markedly increased (P ＜ 0.01),and the Caspase-3 expression and Bax/Bcl-2 ratio were also obviously increased (P ＜ 0.01) in the polysaccharides group.CONCLUSION This fast,stable and reliable method can be used for the aqueous extraction for polysaccharides from Astragali Radix,which can significantly inhibit the proliferation of NCI-H460 cells and induce apoptosis of NCI-H460 cells.

Optimization of aqueous extraction for polysaccharides from Astragali Radix and the in vitro antitumor activity / 中成药

AIM To optimize the aqueous extraction for polysaccharides from Astragali Radix and to evaluate the in vitro antitumor activity.METHODS With extraction temperature,extraction time and solid-liquid ratio as influencing factors,extraction rate of polysaccharides as an evaluation index,the extraction was optimized by uniform design.The effect of polysaccharides on the proliferation of non-small cell lung cancer NCI-H460 cells,the apoptosis rate and cell cycle of NCI-H460 cells,and the expressions of Caspase-3,Bax and Bcl-2 were detected by MTT assay,flow cytometry and Western blot,respectively.RESULTS The optimal conditions were determined to be 100 ℃ for extraction temperature,1 h for extraction time,and 1 ∶ 35 for solid-liquid ratio,the extraction rate of polysaccharides was 3.62％.Compared with the control group,the proliferation of NCI-H460 cells was significandy inhibited in a dose-dependent manner (P ＜ 0.01),the S phase ratio,early apoptosis rate,late apoptosis rate and total apoptosis rate were markedly increased (P ＜ 0.01),and the Caspase-3 expression and Bax/Bcl-2 ratio were also obviously increased (P ＜ 0.01) in the polysaccharides group.CONCLUSION This fast,stable and reliable method can be used for the aqueous extraction for polysaccharides from Astragali Radix,which can significantly inhibit the proliferation of NCI-H460 cells and induce apoptosis of NCI-H460 cells.

Inhibitory effect of dianhydrogalactitol on DNA TopoⅡ in NCI-H460 cells / 中国药理学通报

Aim To evaluate the antitumor activity of dianhydrogalactitol ( DAG) in vitro, and further clarify its underlying mechanisms. Methods The inhibitory effect of DAG in NCI-H460 cells was detected by CCK-8 assay and colony formation assay. The morphology of cells treated with DAG was observed under optical mi-croscope. Nuclear morphology was captured by fluores-cence microscopy after Hoechst 33342 staining. Real-time PCR was used to analyze the expression level of topoisomerase Ⅱ ( Topo Ⅱ) mRNA. The protein ex-pression level of Topo Ⅱ was detected by Western blot. Additionally, molecular docking approaches were used to predict the interaction between DAG and TopoⅡ. Results DAG exhibited potent antitumor activity in NCI-H460 cells, and inhibited cell proliferation per-sistently. DAG obviously induced nuclear morphologi-cal changes of NCI-H460 cells. Furthermore, DAG could down-regulate the mRNA and protein expression level of Topo Ⅱ detected by Real-time PCR analysis and Western blot, respectively. Molecular docking predicted that DAG could bind to Topo Ⅱ. Conclu-sion DAG can significantly inhibit the proliferation of NCI-H460 cells, and its underlying mechanisms may involve the down-regulation of Topo Ⅱ mRNA and di-rect binding to Topo Ⅱ, leading to cancer cell death.

Anti-tumor activity studies of vinorelbine tartrate liposome / 中国药学杂志

OBJECTIVE: To explore the anti-tumor efficacy of liposomal vinorelbine tartrate injection(NVB-lipo). METHODS: The anti-neoplastic effect of NVB-lipo was evaluated by using mice bearing H22 tumor and nude mice bearing NCI-H460 human xenograft tumor model. RESULTS: After a single intravenous injection at doses of 20, 10, 5 and 2.5 mg · kg-1, the inhibition ratio of H22 tumor were 82.1%, 75.8%, 63.2% and 35.4% respectively, and that of NVB-free(20 and 10 mg · kg-1) were 45.8% and 37.1%. After three times iv injection interval 3-day, NVB-lipo 8 mg · kg-1 could markedly inhibited the growth of NCI-H460 tumor, relative to control and NVB-free (P < 0.05). The anti-tumor effect of NVB-lipo in H22 and NCI-H460 tumor model was much stronger than NVB-free, and was positively correlated with its dose levels. CONCLUSION: At the same dose level, the antineoplastic effects of NVB-lipo is stronger than NVB-free.

Effect of Flavopiridol on Radiation-induced Apoptosis of Human Laryngeal and Lung Cancer Cells / 대한방사선종양학회지

PURPOSE: To investigate the flavopiridol effect on radiation-induced apoptosis and expression of apoptosis- related genes of human laryngeal and lung cancer cells. MATERIALS AND METHODS: A human laryngeal cancer cell line, AMC-HN3 and a human lung cancer cell line, NCI-H460, were used in the study. The cells were divided into four groups according to the type of treatment: 1) control groups; 2) cells that were only irradiated; 3) cells treated only with flavopiridol; 4) cells treated with flavopiridol and radiation simultaneously. The cells were irradiated with 10 Gy of X-rays using a 4 MV linear accelerator. Flavopiridol was administered to the media at a concentration of 100 nM for 24 hours. We compared the fraction of apoptotic cells of each group 24 hours after the initiation of treatment. The fraction of apoptotic cells was detected by measurement of the sub-G1 fractions from a flow cytometric analysis. The expression of apoptosis-regulating genes, including cleaved caspase-3, cleaved PARP (poly (ADP-ribose) polymerase), p53, p21, cyclin D1, and phosphorylated Akt (protein kinase B) were analyzed by Western blotting. RESULTS: The sub-G1 fraction of cells was significantly increased in the combination treatment group, as compared to cells exposed to radiation alone or flavopiridol alone. Western blotting also showed an increased expression of cleaved caspase-3 and cleaved PARP expression in cells of the combination treatment group, as compared with cells exposed to radiation alone or flavopiridol alone. Treatment with flavopiridol down regulated cyclin D1 expression of both cell lines but its effect on p53 and p21 expression was different according to each individual cell line. Flavopiridol did not affect the expression of phophorylated Akt in both cell lines. CONCLUSION: Treatment with flavopiridol increased radiation-induced apoptosis of both the human laryngeal and lung cancer cell lines. Flavopiridol effects on p53 and p21 expression were different according to the individual cell line and it did not affect Akt activation of both cell lines.

bcl-2 antisense oligodeoxynucleotide increases sensitivity of NCI-H460 cell lines to docetaxel and radiation / 第二军医大学学报

Objective:To determine whether the bcl-2 antisense oligonucleotide (ASODN) can increase the sensitivity of NCI-H460 cell lines to docetaxel or radiation. Methods: Docetaxel in combination with bcl-2 ASODN or non-sense oligonucleotide (NSODN) was used to treat NCI-H460 cells, and then IC_~50 of docetaxel against NCI-H460 cells was determined with MTT method. In radiation group, NCI-H460 cells were divided into pure radiation, radiation plus bcl-2 ASODN, and radiation plus NSODN groups. The inhibition rates of cell growth were assayed by MTT, and the expression levels of Bcl-2 protein were assayed by immunofluorescence. Results: IC_~50 in bcl-2 ASODN plus docetaxel, pure docetaxel and NSODN plus docetaxel groups was (0.101?0.009) ?mol/L,(0.183?0.018)?mol/L, and (0.179?0.016)?mol/L, respectively. The differences between the first group and the latter 2 groups were significant (P