Objective To explore the mechanism of puerarin inhibiting the proliferation,invasion,and migration of non-small cell lung cancer cells. Methods A549 cells were cultured and treated with different concentrations of puerarin.The inhibition rate (IR) on cell proliferation was detected by CCK-8,and qRT-PCR was performed to detect the mRNA levels of miR-490 and denticleless E3 ubiquitin protein ligase(DTL).Double luciferase reporter assay was employed to identify the targets of miR-490 and DTL based on the establishment of NC mimic group,miR-490 mimic group,NC inhibitor group,and miR-490 inhibitor group.The cells treated by 20 μmol/L puerarin were classified into six groups:DMSO,puerarin,puerarin+NC inhibitor,puerarin+miR-490 inhibitor,puerarin+miR-490 inhibitor+Si-NC,and puerarin+miR-490 inhibitor+Si-DTL.Transwell was used to detect cell migration and invasion.Western blotting was performed to detect the protein levels of epithelial-mesenchymal transition-related markers E-cadherin,N-cadherin,and Vimentin. Results With the increase in puerarin concentration,the IR gradually elevated (F=105.375,P<0.001),miR-490 expression gradually increased (F=32.919,P<0.001),and DTL expression gradually decreased (F=116.120,P<0.001).Compared with NC mimic group,miR-490 mimic group had decreased luciferase activity (t=7.762,P=0.016),raised miR-490 mRNA level (t=13.319,P<0.001),and declined DTL mRNA level (t=7.415,P=0.002).Compared with those in NC inhibitor group,miR-490 demonstrated decreased mRNA level (t=9.523,P=0.001) and DTL presented increased mRNA level (t=11.305,P<0.001) in miR-490 inhibitor group.Western blotting showed that the protein level of DTL was higher in NC mimic group (t=7.953,P=0.001) than in miR-490 mimic group and higher in miR-490 inhibitor group than in NC inhibitor group (t=10.552,P<0.001).Compared with DMSO group,puerarin group showed up-regulated mRNA level of miR-490 (t=10.255,P=0.001) while down-regulated mRNA level of DTL (t=6.682,P=0.003).Compared with those in puerarin+NC inhibitor group,the mRNA level of miR-490 declined (t=10.995,P<0.001) while that of DTL raised (t=12.478,P<0.001) in puerarin+miR-490 inhibitor group.The mRNA level of miR-490 had no significant difference between puerarin+miR-490 inhibitor+Si-NC group and puerarin+miR-490 inhibitor+Si-DTL group (t=1.081,P=0.341),and that of DTL was lower in the latter group (t=14.321,P<0.001).The protein level of DTL was higher in puerarin+miR-490 inhibitor group than in puerarin+NC inhibitor group (t=11.423,P<0.001),and lower in puerarin+miR-490 inhibitor+Si-DTL group than in puerarin+miR-490 inhibitor+Si-NC group (t=12.080,P<0.001).Compared with DMSO group,puerarin group showed inhibited cell proliferation (F=129.27,P<0.001).The activity of cell proliferation was higher in puerarin+miR-490 inhibitor group than in puerarin+NC inhibitor group (F=75.12,P<0.001),and higher in puerarin+miR-490 inhibitor+Si-NC group than in puerarin+miR-490 inhibitor+Si-DTL group (F=52.59,P<0.001).Compared with DMSO group,puerarin group had suppressed cell migration (t=8.963,P=0.001).The cell migration ability was higher in puerarin+miR-490 inhibitor group than in puerarin+NC inhibitor group (t=12.117,P<0.001) and higher in puerarin+miR-490 inhibitor+Si-NC group than in puerarin+miR-490 inhibitor+Si-DTL group (t=12.934,P<0.001).Puerarin group showed weakened cell invasion ability compared with DMSO group (t=4.710,P=0.009).The cell invasion ability was higher in puerarin+miR-490 inhibitor group than in puerarin+NC inhibitor group (t=13.264,P<0.001) and lower in puerarin+miR-490 inhibitor+Si-DTL group than in puerarin+miR-490 inhibitor+Si-NC group (t=13.476,P<0.001).Compared with DMSO group,puerarin group showed up-regulated protein level of E-cadherin (t=7.137,P=0.002) while down-regulated protein levels of N-cadherin (t=8.828,P=0.001) and vimentin (t=6.594,P=0.003).Compared with those in puerarin+NC inhibitor group,the protein level of E-cadherin (t=12.376,P<0.001) decreased while those of N-cadherin (t=13.436,P<0.001) and vimentin (t=11.467,P<0.001) increased in puerarin+miR-490 inhibitor group.Compared with puerarin+miR-490 inhibitor+Si-NC group,puerarin+miR-490 inhibitor+Si-DTL group up-regulated the protein level of E-cadherin (t=13.081,P<0.001) while down-regulated the protein levels of N-cadherin (t=10.835,P<0.001) and vimentin (t=11.862,P<0.001). Conclusion Puerarin could inhibit the proliferation,invasion,and migration of non-small cell lung cancer cells by up-regulating miR-490 and down-regulating DTL.
Carcinoma, Non-Small-Cell Lung/pathology* ; Cell Line, Tumor ; Cell Movement/drug effects* ; Cell Proliferation/drug effects* ; Humans ; Isoflavones/pharmacology* ; Lung Neoplasms ; MicroRNAs/metabolism* ; Ubiquitin-Protein Ligases/metabolism*

Apoptosis/drug effects* ; Cell Line, Tumor ; Cell Proliferation ; Humans ; Pentacyclic Triterpenes/pharmacology* ; Stomach Neoplasms/pathology*

Proteasome inhibitors have shown remarkable success in the treatment of hematologic neoplasm. There has been a lot of attention to applying these drugs for solid tumor treatment. Recent preclinical study has signified the effectiveness on cell proliferation inhibition in lung adenocarcinoma treated by carfilzomib (CFZ), a second generation proteasome inhibitor. However, no insight has been gained regarding the mechanism. In this study, we have systematically investigated the CFZ functions in cell proliferation and growth, cell cycle arrest, and apoptosis in lung adenocarcinoma cells. Flow cytometry experiments showed that CFZ significantly induced G2/M cell cycle arrest and apoptosis in lung adenocarcinoma. MTS and colony formation assays revealed that CFZ substantially inhibited survival of lung adenocarcinoma cells. All results were consistently correlated to the upregulation expression of Gadd45a, which is an important gene in modulating cell cycle arrest and apoptosis in response to physiologic and environmental stresses. Here, upregulation of Gadd45a expression was observed after CFZ treatment. Knocking down Gadd45a expression suppressed G2/M arrest and apoptosis in CFZ-treated cells, and reduced cytotoxicity of this drug. The protein expression analysis has further identified that the AKT/FOXO3a pathway is involved in Gadd45a upregulation after CFZ treatment. These findings unveil a novel mechanism of proteasome inhibitor in anti-solid tumor activity, and shed light on novel preferable therapeutic strategy for lung adenocarcinoma. We believe that Gadd45a expression can be a highly promising candidate predictor in evaluating the efficacy of proteasome inhibitors in solid tumor therapy.
Adenocarcinoma of Lung/pathology* ; Apoptosis/drug effects* ; Cell Cycle Checkpoints/drug effects* ; Cell Cycle Proteins/genetics* ; Cell Line, Tumor ; Forkhead Box Protein O3/physiology* ; Gene Expression Regulation, Neoplastic/drug effects* ; Humans ; Lung Neoplasms/pathology* ; Oligopeptides/pharmacology* ; Proto-Oncogene Proteins c-akt/physiology* ; Up-Regulation

To explore the mechanism of β-carboline alkaloids inhibiting the migration and invasion of SGC-7901 cells and its correlation with FAK gene expression,CCK-8 method was used to determine the inhibitory rate of β-carboline alkaloids on the proliferation of gastric cancer SGC-7901 cells under different concentrations.The effect of β-carboline alkaloids on the migration and invasion of SGC-7901 cells was used by Transwell compartment.Detection of mRNA and protein expression of FAK genes were used by qRT-PCR and Western blot.Then si-FAK-1051 recombinant plasmid was transfected into SGC-7901 cells.FAK gene silencing effect was identified by qRT-PCR and Western blot technique again.Finally,the effects of FAK gene silencing on proliferation and migration of gastric cancer SGC-7901 cells were detected by CCK-8 kit and Transwell chamber assay respectively.With the increase of the concentration ofβ-carboline alkaloids,the inhibitory rate of SGC-7901 cells in human gastric cancer cells increased gradually,with IC5013.364 mg·L-1.The number of SGC-7901 cells of Transwell compartment in the positive experimental group(5-FU,5 mg·L-1) and the β-carboline alkaloids group decreased significantly(P<0.01) and the number of SGC-7901 cells in the β-carboline alkaloids group was significantly lower than that in the positive experimental group(P<0.01).Compared with the blank control group,the mRNA and protein expression level of FAK genes in the positive experimental group was significantly lower than that in the experimental group of β-carboline alkaloids(P<0.05).After transfection of si-FAK-1051 into gastric cancer SGC-7901 cells,the expression of mRNA and protein of FAK gene was significantly down regulated(P<0.05).SGC-7901 cell proliferation and cell migration ability also decreased significantly(P<0.05).β-carboline alkaloids are more effective than 5-FU in inhibiting migration and invasion of gastric cancer SGC-7901 cells,and the mechanism may be related to the inhibition of mRNA and protein expression of FAK gene by β-carboline alkaloids.
Alkaloids ; pharmacology ; Carbolines ; pharmacology ; Cell Line, Tumor ; Cell Movement ; drug effects ; Cell Proliferation ; Focal Adhesion Kinase 1 ; genetics ; Gene Expression Regulation, Neoplastic ; Gene Silencing ; Humans ; Neoplasm Invasiveness ; Stomach Neoplasms ; drug therapy ; pathology

OBJECTIVE: To investigate the effects of methanol-ethyl acetate partitioned fractions from (MEDS) on the proliferation and apoptosis of human non-small cell lung cancer H1975 cells. METHODS: The systemic solvent extraction method was used to preliminary separation of the effective fractions in the methanol extract of . The cytotoxicity of each extract (5, 10, 20, 40, and 80 μg/mL) was tested using MTT assay. Colony cloning method was used to assess the effect of different concentrations of methanol-ethyl acetate partitioned fractions from MEDS (5, 10, 20, 40, and 80 μg/ mL) on the proliferation of H1975 cells. Flow cytometric analysis with Annexin V-FITC/PI staining was performed to detect the apoptosis of the cells after treatment with different concentrations of MEDS fractions (10, 20, and 40 μg/mL). Western blotting was used to evaluate the effects of MEDS fractions on the expressions of apoptosis-related proteins Akt, Bax, and Bcl-2. The anti-tumor activity of 100 mg/kg MEDS fractions was tested in a nude mouse model bearing H1975 cell xenografts. RESULTS: MTT assay and colony forming experiment showed that MEDS fractions significantly inhibited the proliferation of H1975 cells in a dose-and time-dependent manner ( < 0.05). The results of flow cytometry showed that MEDS fractions induced obvious apoptosis of H1975 cells in a concentration-dependent manner ( < 0.05). MEDS fractions also significantly decreased the expressions of Bcl-2 and Akt protein and increased the protein expression of Bax ( < 0.05). In the tumor-bearing nude mouse model, MEDS fractions showed potent anti-tumor effects with a low toxicity to affect the body weight and organs of the mice. CONCLUSIONS The methanol-ethyl acetate partitioned fractions from MEDS show potent anti-tumor activity both and , suggesting their value as promising therapeutic agents against lung cancer.
Acetates ; Animals ; Antineoplastic Agents, Phytogenic ; isolation & purification ; pharmacology ; Apoptosis ; drug effects ; Carcinoma, Non-Small-Cell Lung ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Heterografts ; Humans ; Lung Neoplasms ; pathology ; Methanol ; Mice ; Mice, Nude ; Plant Extracts ; isolation & purification ; pharmacology

OBJECTIVE: To study the effects of the overexpression of autophagy-related gene 3 (ATG3) on autophagy and salinomycin-induced apoptosis in breast cancer cells and explore the underlying mechanisms. METHODS: We used the lentivirus approach to establish a breast cancer cell line with stable overexpression of ATG3. Western blotting, immunofluorescence staining and transmission electron microscopy were used to analyze the effect of ATG3 overexpression on autophagy in breast cancer MCF-7 cells. Using the AKT/mTOR agonists SC79 and MHY1485, we analyzed the effect of AKT/mTOR signal pathway activation on ATG3 overexpression-induced autophagy. Western blotting and flow cytometry were used to analyze the effect of autophagy on apoptosis of the ATG3-overexpressing cells treated with salinomycin and 3-MA (an autophagy inhibitor). RESULTS: In ATG3-overexpressing MCF-7 cells, ATG3 overexpression obviously promoted autophagy, inhibited the AKT/mTOR signaling pathway, significantly weakened salinomycin-induced apoptosis ( < 0.01), caused significant reduction of the levels of the pro-apoptotic proteins cleaved-caspase 3 ( < 0.01) and Bax ( < 0.05), and enhanced the expression of the anti-apoptotic protein Bcl-2 ( < 0.05). The inhibition of autophagy obviously weakened the inhibitory effect of ATG3 overexpression on salinomycin-induced apoptosis. CONCLUSIONS ATG3 overexpression promotes autophagy possibly by inhibiting the AKT/mTOR signaling pathway to decrease salinomycin-induced apoptosis in MCF-7 cells, suggesting that autophagy induction might be one of the mechanisms of drug resistance in breast cancer cells.
Acetates ; pharmacology ; Apoptosis ; drug effects ; genetics ; Autophagy ; drug effects ; Autophagy-Related Proteins ; metabolism ; Benzopyrans ; pharmacology ; Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Drug Resistance, Neoplasm ; Female ; Gene Expression Regulation ; Humans ; MCF-7 Cells ; Morpholines ; pharmacology ; Proto-Oncogene Proteins c-akt ; antagonists & inhibitors ; metabolism ; Pyrans ; pharmacology ; TOR Serine-Threonine Kinases ; antagonists & inhibitors ; metabolism ; Triazines ; pharmacology ; Ubiquitin-Conjugating Enzymes ; metabolism

OBJECTIVE: To investigate the interaction between interleukin-17 (IL-17) and interferon-γ (IFN-γ) and how their interaction affects the growth of mouse hepatoma Hepa1-6 cells. METHODS: Hepa1-6 cells treated with IL-17 and IFN-γ either alone or in combination were examined for changes in cell proliferation using MTT assay and in cell cycle distribution using flow cytometry. Western blotting was used to detect the protein expression levels of proliferating cell nuclear antigen (PCNA), cyclin D1, P21 and P16 and the phosphorylation of p38MAPK, ERK1/2 and Stat1 in the cells. RESULTS: Compared with control group, IFN-γ treatment obviously inhibited the growth and proliferation of Hepa1-6 cells, induced cell cycle arrest at G0/G1 phase, reduced the protein expression of PCNA and cyclin D1, and increased the protein expression of P21. IL-17 alone had no effect on the growth of Hepa1-6 cells. In the combined treatment, IL-17 significantly antagonized the effects of IFN-γ. Compared with those treated with IFN-γ alone, the cells with the combined treatment showed significantly decreased G0/G1 cell population, increased the protein expressions of PCNA and cyclin D1, and decreased the protein expression of P21. IL-17 significantly inhibited IFN-γ-induced phosphorylation of p38MAPK and ERK1/2 without affecting the phosphorylation of Stat1. CONCLUSIONS IL-17 obviously reverses the antitumor effects of IFN-γ to promote the proliferation of mouse hepatoma cells and accelerate the development of hepatocellular carcinoma.
Animals ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cyclin D1 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; Interferon-gamma ; antagonists & inhibitors ; Interleukin-17 ; pharmacology ; Liver Neoplasms ; metabolism ; pathology ; Mice ; Neoplasm Proteins ; metabolism ; Proliferating Cell Nuclear Antigen ; metabolism

Diffuse intrinsic pontine glioma (DIPG) is the main cause of brain tumor-related death among children. Until now, there is still a lack of effective therapy with prolonged overall survival for this disease. A typical strategy for preclinical cancer research is to find out the molecular differences between tumor tissue and para-tumor normal tissue, in order to identify potential therapeutic targets. Unfortunately, it is impossible to obtain normal tissue for DIPG because of the vital functions of the pons. Here we report the human fetal hindbrain-derived neural progenitor cells (pontine progenitor cells, PPCs) as normal control cells for DIPG. The PPCs not only harbored similar cell biological and molecular signatures as DIPG glioma stem cells, but also had the potential to be immortalized by the DIPG-specific mutation H3K27M in vitro. These findings provide researchers with a candidate normal control and a potential medicine carrier for preclinical research on DIPG.
Animals ; Brain Stem Neoplasms ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Cellular Senescence ; Female ; Glioma ; genetics ; metabolism ; pathology ; Histones ; genetics ; Humans ; Mice, Inbred NOD ; Mice, SCID ; Neoplasm Transplantation ; Neoplastic Stem Cells ; drug effects ; metabolism ; pathology ; Neural Stem Cells ; drug effects ; metabolism ; pathology ; Pons ; embryology ; metabolism ; pathology ; Primary Cell Culture

BACKGROUND: Lung cancer is one of the most deadly cancers in the world for human. In recent years, the effect of targeted therapy has become increasingly significant. Apatinib is a multi-target anti-tumor drug that is currently under study. The purpose of this study is to investigate the effects of Apatinib on the biological characteristics of lung cancer cells and its possible mechanism. METHODS: Lung cancer cell lines H1299 and H3255 were cultured in vitro. The effects of Apatinib on proliferation, migration and invasion of H1299 and H3255 cells were detected by cell proliferation assays wound healing assays and Transwell assays. The protein expression related to cancer angiogenesis and invasion was detected by Western blot. RESULTS: Apatinib significantly inhibited the proliferation, migration and invasion of H1299 and H3255 in a concentration-dependent manner. Western blot showed that with the increasing of drug concentration, VEGF, VEGFR2, N-cadherin, MMP9, MMP2 and Vimentin were down-regulated, and E-cadherin were up-regulated. CONCLUSIONS Apatinib can inhibit the invasion and migration of lung adenocarcinoma cells H1299 and H3255. By regulation of epithelial-mesenchymal transition and the expression of matrix metalloproteinase-related proteins.
Antineoplastic Agents ; pharmacology ; Cell Line, Tumor ; Cell Movement ; drug effects ; Humans ; Lung Neoplasms ; pathology ; Neoplasm Invasiveness ; Pyridines ; pharmacology

BACKGROUND: Lung cancer is one of the common malignant tumors that impair human health. With the development of epigenetics, the researchers found that enhancer of Zeste homolog 2 (EZH2) is highly expressed in lung cancer tissue and its expression is closely related to the prognosis. EZH2 inhibitor can also enhance the sensitivity of tumor cells to a variety of anti-tumor drugs. The purpose of this study is to investigate the effect of combination of EZH2 inhibitor and gefitinib on the proliferation, apoptosis and migration of Gefitinib-resistant lung cancer cells. METHODS: PC9 and PC9/AB2 cells were used for this study. CCK-8 and EdU experiment were used to detect combined treatment on cell viability and proliferation activity; Wound healing assay and Transwell chamber experiment were used to determine the effects of combination therapy on cell migration ability; Flow cytometry was used to detect the effect of combination therapy on EZH2 and apoptosis; Western blot was used to observe the effect of combination therapy on epidermal growth factor receptor (EGFR) signaling pathway-related proteins expression. RESULTS: In gefitinib-resistant cell line PC9/AB2, gefitinib combined with EZH2 inhibitor GSK343 can significantly inhibit cell viability, reduce cell migration and increase cell apoptosis. At the same time, combination therapy can significantly inhibit the expression of EZH2 and phosphorylation EGFR proteins. CONCLUSIONS The combination of EZH2 inhibitor GSK343 and gefitinib sensitize PC9/AB2 cell to gefitinib response. This study also suggests that synergistic therapy plays a role in the reversal of EGFR-tyrosine kinase inhibitor (EGFR-TKIs) resistance in lung cancer.
Antineoplastic Agents ; pharmacology ; Cell Line, Tumor ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Drug Resistance, Neoplasm ; drug effects ; Drug Synergism ; Enhancer of Zeste Homolog 2 Protein ; antagonists & inhibitors ; ErbB Receptors ; antagonists & inhibitors ; Gefitinib ; pharmacology ; Humans ; Lung Neoplasms ; pathology ; Protein Kinase Inhibitors ; pharmacology