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BACKGROUND@#The anti-tumor effect of pigment epithelium-derived factor (PEDF) has been widely confirmed. However, the anti-tumor effect of its peptides is rarely reported. This study aims to investigate the effects of PEDF and its peptides on the apoptosis and migration of non-small cell lung cancer (NSCLC).@*METHODS@#In this study, A549 cells and H1299 cells were selected as the research object, and the cells were divided into normal group, PEDF treatment group, 34 peptide treatment group, 44 peptide treatment group and 34+44 peptide treatment group by administering different drugs at the same concentration to the cells. The proliferation activity of cells in each group was detected by CCK-8 method; the migration ability of cells was detected by scratch test; the expression levels of apoptosis related proteins such as protein kinase 3 (RIP3) and cleaved-caspase-3 were detected by Western blot; the expression levels of epithelial mesenchymal transition (EMT) markers in each group, such as cadherin (E-cadherin) and α-smooth muscle actin (α-SMA) were detected by Western blot; the apoptosis rate of each group was detected by flow cytometry.@*RESULTS@#The results of CCK-8 showed that PEDF and its peptides could inhibit cell proliferation, and the inhibitory effect of 34+44 peptide was the strongest (P<0.05); Observation under the microscope found that PEDF and its peptides can inhibit the proliferation and mesenchymal transformation of A549 cells and H1299 cells, and the inhibitory effect of the 34+44 peptide group is the most obvious; Western blot indicated that compared with other groups, the expressions of cleaved-caspase-3 and RIP3 in 34+44 peptide group were significantly higher (P<0.05), and the expressions of EMT protein E-cadherin were higher, the expression of α-SMA decreased (P<0.05); The results of flow cytometry showed that the apoptosis rate of 34+44 peptide group was significantly higher than those of other groups (P<0.05); The scratch test showed that compared with all the other groups, the healing rate of 34+44 peptide group was the lowest (P<0.05).@*CONCLUSIONS@#34+44 combination peptide can better promote the apoptosis of NSCLC, inhibit the migration of NSCLC, and thereby inhibit the growth of NSCLC.
Subject(s)
Humans , Apoptosis , Cadherins/genetics , Carcinoma, Non-Small-Cell Lung/genetics , Caspase 3 , Cell Line, Tumor , Cell Movement , Cell Proliferation , Epithelial-Mesenchymal Transition , Eye Proteins , Lung Neoplasms/genetics , Nerve Growth Factors , Peptides/pharmacology , Serpins , SincalideABSTRACT
BACKGROUND@#Non-small cell lung cancer (NSCLC) is the most common histological type of lung cancer, and one of the malignant tumor with the highest mortality. As the main part of the optical molecular imaging probe, peptide can realize the early screening and diagnosis of tumor and improve the survival rate of patients. The aim of this study was to screen the small-molecule peptide that highly binds to NSCLC NCI-H1299 cells using in vivo phage display technology and to identify their binding specificity by in vitro experiment.@*METHODS@#To prepare a tumor-bearing nude mouse model of NCI-H1299 cells, after 3 rounds of in vivo screening with Ph.D.-C7CTM Peptide Library, phage clones were randomly picked, using immunohistochemistry and enzyme-linked immunosorbent assay (ELISA) to identify the affinity of phage clones to NCI-H1299 cells. The positive monoclonal phages DNA was extracted and sequenced to obtain the amino acid sequence of the peptides. The peptides with the highest repetition rate was chemically synthesized and labeled with fluorescein (FITC) to prepare optical molecular probe. We preliminary identified the specificity of the probe binding to lung cancer cells by in vitro experiment.@*RESULTS@#After three rounds of in vivo screening, the phages enrichment rate was 341.3 times compared with the first round. Immunohistochemical staining showed that with the increase of screening times, the phages binding to tumor tissues continued to increase, and the binding amount was significantly higher than normal tissues; ELISA results showed that 20 clones among the 30 randomly selected phage clones were positive. After sequencing, the peptide with the highest repetition rate was synthesized and named NSP1; Methyl thiazolyl tetrazolium assay (MTT) and would healing assay showed that NSP1 will not affect cell proliferation and migration. Flow cytometry and immunofluorescence showed specific binding of NSP1 to NCI-H1299 cells.@*CONCLUSIONS@#We successfully obtained the peptide NSP1 that specifically binds to lung cancer NCI-H1299 cells by in vivo phage display, which provide a theoretical basis for NSCLC early diagnosis and targeted therapy.
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Objective To study whether carvacrol can cause apoptosis in non-small cell lung cancer (NSCLC) cell line NCI-H1299, and explore its possible molecular mechanism. Methods NCI-H1299 cells were treated with different concentrations of carvacrol (20, 40, 60 and 80μmol/L) for 24 or 48 h. The viability of cells was evaluated by MTT assay, apoptosis was analyzed by flow cytometry (FCM) and the effect of carvacrol on metastasis of NCI-H 1299 was analyzed by Transwell assay. The expression level of caspase-9, MMP-9 and TIMP-1 were detected by quantitative realtime-PCR and Western blot assay. Results After treatment with carvacrol, the viability of NCI-H1299 cells was suppressed dramatically (P0.05). After being incubated with carvacrol for 24 h, FCM analysis indicated that carvacrol effectively induced apoptosis in NCI-H1299 cells (P0.05). The ability of invasion was decreased (40.67±3.63 vs. 76.00±5.78). Carvacrol inhibited the protein and mRNA expression levels of caspase-9, but increased the expression of MMP-9 and TIMP-1 (P<0.05). Conclusion Carvacrol can induce apoptosis of NCI-H1299 cells and inhibit their invasion, which may be associated with up-regulation of caspase-9 expression and down-regulation of MMP-9 expression.
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AIM:To investigate the effect of signal transducer and activator of transcription 1 ( STAT 1 ) on proliferation and interferon-β(IFN-β) sensitivity of human non-small-cell lung cancer H1299 cells.METHODS:STAT1 or EGFP gene was transfected into H1299 cells by the lentiviral vectors system.The cell number was counted under a mi-croscope and cell proliferation was tested by MTT assay.In addition, the cells transfected with STAT1 and EGFP were trea-ted with IFN-βand cell viability was measured by MTT assay.The protein levels of p-STAT1, ICAM-1 and PCNA were de-tected by Western blot.RESULTS: Over-expression of STAT1 inhibited H1299 cell proliferation (P<0.05).H1299 cells transfected with STAT1 gene had a higher sensitivity to IFN-βthan the control cells transfected with EGFP ( P <0.05).Overexpression of STAT1 increased the protein level of p-STAT1, and reduced IACM-1 expression in H1299 cells. Moreover, STAT1 enhanced STAT1 phosphorylation and downregulated the expression of PCNA in H1299 cells treated with IFN-β.CONCLUSION:STAT1 inhibits the proliferation and enhances the IFN-βsensitivity of non-small-cell lung cancer H1299 cells.
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Objective To explore the effects of knocking down ULF gene on the apoptosis of non-small-cell carcinoma H1299 cell after treatment with etoposide.Methods Three ULF small interfering RNA(siRNA)sequences and one negative control siRNA sequence were designed and synthesized, and then individually transfected into H1299 cell via lentivirus.The interference efficiency of ULF-siRNA were screened by real-time PCR and Western blotting.Then the most target siRNA was used for apoptosis assay after treatment with etoposide,MTT assay for H1299 cell proliferation,flow cytometry for cell cycle distribution. Results The expression of ULF gene and its protein ULF were down-regulated in H1299 cell when transfected with ULF-siRNA,and ULF-siRNA-1 was the most effective one,which had the highest inhibition rate(80%)of ULF expression.Compared with negative control group,ULF-siRNA group showed an obvious apoptosis after treatment with etoposide,and the inhibition rate of was higher than control group,which was positively correlated with etoposide dose,the difference was statistically significant(P<0.05 ).Flow cytometry showed that compared with the control group,G0/G1 cell cycle in ULF-siRNA group was increased,and S phase cells was decreased,the differences were all statistically significant(P<0.05).Conclusion Down-regulation ULF protein expression through treatment with etoposide can induce apoptosis of non-small-cell carcinoma H1299 cells,and inhibit cell proliferation,which lead to cell cycle arrest.ULF gene may become the new target of gene therapy for cancer.
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Objective To investigate the influences of miR-34a on the radiosensitivity of H1299 cells.Methods CCK-8 kit was used to examine the viability of H1299 cells which were exposed to different doses (0,2,4,6 and 8 Gy) of 60Co γ-rays after transient transfection of pre-miR-34a.Apoptosis rate and cell cycle were measured with flow cytometry.The expression levels of miR-34a target genes,bcl-2,bax,CDK4,CDK6 and cyclinD1 were analyzed by real-time PCR.Results Compared to the control group of negative transfection,the cell viability in pre-miR-34a transfection group decreased significantly after irradiation at0,2,4,6,8 Gy (t=-2.39,-3.12,-4.98,-4.03,-3.06,P＜0.05) in a dose-dependent manner.After being irradiated with 6 Gy γ-rays,the apoptotic rate in pre-miR-34a transfection group was significantly increased (t =7.06,P ＜ 0.05) together with an accumulation of G0/G1 phase (t =3.94,P ＜ 0.05) and a reduction of S phase (t =6.23,P ＜ 0.05).The gene expression levels of bcl-2,CDK4 and CDK6 in pre-miR-34a transfection group were respectively decreased (t =3.39,12.88,6.21,P ＜ 0.05) of negative control.cyclinD1 was also decreased but no significance,while bax was increased to 1.94 times of negative control (t =-4.35,P ＜ 0.05) together with a decrease of bcl-2/bax.Conclusions miR-34a could promote cell apoptosis,induce G0/G1 phase accumulation,suppress cell activity,and in turn increase the radiosensitivity of H1299 cells.
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The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor family of cytokines. TRAIL selectively induces apoptotic cell death in various tumors and cancer cells, but it has little or no toxicity in normal cells. Agonism of TRAIL receptors has been considered to be a valuable cancer-therapeutic strategy. However, more than 85% of primary tumors are resistant to TRAIL, emphasizing the importance of investigating how to overcome TRAIL resistance. In this report, we have found that nemadipine-A, a cell-permeable L-type calcium channel inhibitor, sensitizes TRAIL-resistant cancer cells to this ligand. Combination treatments using TRAIL with nemadipine-A synergistically induced both the caspase cascade and apoptotic cell death, which were blocked by a pan caspase inhibitor (zVAD) but not by autophagy or a necrosis inhibitor. We further found that nemadipine-A, either alone or in combination with TRAIL, notably reduced the expression of survivin, an inhibitor of the apoptosis protein (IAP) family of proteins. Depletion of survivin by small RNA interference (siRNA) resulted in increased cell death and caspase activation by TRAIL treatment. These results suggest that nemadipine-A potentiates TRAIL-induced apoptosis by down-regulation of survivin expression in TRAIL resistant cells. Thus, combination of TRAIL with nemadipine-A may serve a new therapeutic scheme for the treatment of TRAIL resistant cancer cells, suggesting that a detailed study of this combination would be useful.