SMAC exhibits anti-tumor effects in ECA109 cells by regulating expression of inhibitor of apoptosis protein family.

BACKGROUND: The poor prognosis and rising incidence of esophageal cancer highlight the need for improved therapeutics that are essential prior to treatment. LCL161 is an SMAC (second mitochondrial activator of caspases) mimic and inhibitor of apoptosis protein (IAP) antagonist which exhibits anti-tumor effects and improves the chemical sensitivity of many cancers. AIM: To ascertain the effects and mechanisms of the SMAC analog LCL161 on esophageal cancer cells. METHODS: MTT assay and TUNEL assay were used to detect cell proliferation and apoptosis, respectively. Western blot analysis was used to study the molecular mechanisms of LCL161-induced death of ECA109 cells. RESULTS: LCL161 decreased ECA109 cell proliferation in dose- and time-dependent manner and induced apoptosis of ECA109 cells in a dose-dependent manner. Also, LCL161 induced a significant decrease in the expression of the XIAP and significant increase in the expression of Caspase-3. In addition, Bax increased significantly with increasing concentrations of LCL161, and the relative expression of Bax was significantly different between groups. CONCLUSION: These findings support the hypothesis that LCL161 can inhibit proliferation and induce apoptosis in esophageal cancer cells by regulating the expression of IAP family members, suggesting that it has potential to be an effective treatment for esophageal squamous cell carcinoma.

Alterations of fecal bacterial communities in patients with lung cancer.

Emerging evidence suggests the microbiome may affect a number of diseases, including lung cancer. However, the direct relationship between gut bacteria and lung cancer remains uncharacterized. In this study, we directly sequenced the hypervariable V1-V2 regions of the 16S rRNA gene in fecal samples from patients with lung cancer and healthy volunteers. Unweighted principal coordinate analysis (PCoA) revealed a clear difference in the bacterial community membership between the lung cancer group and the healthy control group. The lung cancer group had remarkably higher levels of Bacteroidetes, Fusobacteria, Cyanobacteria, Spirochaetes, and Lentisphaerae but dramatically lower levels of Firmicutes and Verrucomicrobia than the healthy control group (P < 0.05). Despite significant interindividual variation, eight predominant genera were significantly different between the two groups. The lung cancer group had higher levels of Bacteroides, Veillonella, and Fusobacterium but lower levels of Escherichia-Shigella, Kluyvera, Fecalibacterium, Enterobacter, and Dialister than the healthy control group (P < 0.05). Most notably, correlations between certain specific bacteria and serum inflammatory biomarkers were identified. Our findings demonstrated an altered bacterial community in patients with lung cancer, providing a significant step in understanding the relationship between gut bacteria and lung cancer. To our knowledge, this is the first study to evaluate the correlations between certain specific bacteria and inflammatory indicators. To better understand this relationship, further studies should investigate the underlying mechanisms of gut bacteria in lung cancer animal models.

Effect of spleen tyrosine kinase on nonsmall cell lung cancer.

AIM OF STUDY: To investigate the anti.tumor effect of spleen tyrosine kinase. (Syk) on the human nonsmall cell lung cancer cells in vitro and its mechanism. MATERIALS AND METHODS: In this study, we constructed a eukaryotic expression vector pcDNA3.1D/V5-His-TOPO/Syk and transfected it into human nonsmall cell lung cancer cells A549. Then, we not only analyzed the expression of Syk in transfected cells and its invasion but also the expression of matrix metalloproteinase-9 (MMP-9). RESULTS: The results showed that overexpressed Syk significantly inhibited A549 cell invasive ability by decreasing the expression of MMP-9. CONCLUSION: The overexpressed Syk plays an important role in tumor invasion and metastasis, and a negative role in human nonsmall cell lung cancer cells.

Reversal of Chemoresistance in Human Lung Cancer Cell Line A549/Taxol by Synthetic Second Mitochondria-Derived Activator of Caspase Peptide.

BACKGROUND: Chemoresistance is a leading cause of treatment failure in advanced lung cancer, including that with the extensively prescribed taxol. Recently, a series of structurally unique second mitochondria-derived activators of caspase (Smac) that act as antagonists of inhibitor of apoptosis proteins (IAPs) have been discovered, exhibiting the ability of inducing enhanced apoptosis of various cancer cell types when combined with chemotherapy. In the present study, we synthesized the second mitochondria-derived activator of caspase peptide (Smac-N7 for short) and explored its capacity in combination with taxol in vitro. METHODS: The sensitivity assay and reversal ability of Smac-N7 were tested by MTT. Flow cytometry was used to analyze apoptosis of cells with Annexin V/PI double staining technique. Cell cloning ability was performed to reflect its biological behavior in each group. RESULTS: Concentrations with inhibitory rates < 10% were selected as the reversal value of Smac-N7 peptide using MTT. The reversal folds were 2.52, 3.26, 3.67, and 5.4 in taxol + Smac-N7 (0.0390625, 0.078125, 0.15625, 0.3125 µg/mL, respectively), and concentrations of Smac-N7 and reversal folds appeared in an obvious positive correlation (r(s) = 1, p = 0.000). Apoptosis analyzed at 48 hours by flow cytometry showed the apoptotic rates in taxol and 0.0390625, 0.078125, 0.15625, and 0.3125 µg/mL Smac-N7 + taxol groups were 15.4 ± 1.09%, 20.8% ± 2.18%, 28.4% ± 4.17%, 37.64% ± 6.41%, and 46.6% ± 7.76%, respectively. Concentrations of Smac-N7 appeared to have negative correlations with PE and SF (r(s) = -1, p < 0.05), which showed that the cells' cloning ability in 0.3125 µg/mL Smac-N7 + taxol group was worse than that of other groups. CONCLUSIONS: When combined with taxol, 0.3125 µg/mL Smac-N7 peptide may significantly increase taxol-induced apoptosis in chemoresistant A549/taxol lung cells at 48 hours, and is potentially useful as a reversal agent in lung cancer therapy.

Effect of Second mitochondria-derived activator of caspase in combination with Docetaxel on lung cancer cell A549.

This study was to investigate inhibiting effect of structurally unique Second mitochondria-derived activator of caspase (Smac) in combination with Docetaxel on lung cancer cell line A549. Results showed that the expression of Smac in transfected A549 cells was higher than the control cells both at mRNA level and protein level (P<0.05). Smac over-expression induced a little apoptosis, however, when treated with Docetaxel together, the cells showed a higher apoptosis rate. The apoptosis rate was significantly increased in Smac + Docetaxel group when compared with that in Smac group and Docetaxel group (P<0.05). Cells cloning ability in Smac + Docetaxel group was worse than that of other groups (P<0.05), cell mass formed in relatively small quantities and sparse location. Thus, over-expression of Smac increases the sensitivity of lung cancer A549 cells to Docetaxel treatment, and transfection of Smac to tumor cells might provide a potential therapy modality.

Inhibitory effects of syk transfection on lung cancer cell invasion.

OBJECTIVE: Spleen tyrosine kinase (Syk) is closely related to tumor invasion and metastasis, and has been shown to have potential inhibitory effects in tumors. In this study, we constructed a eukaryotic expression vector for Syk and analyzed its effects on invasive ability of the A549 non-small cell lung cancer cell line in vitro. METHODS: A fragment of Syk was obtained by RT-PCR from human lung cancer cells and cloned into the expression vector pLNCXSyk. After restriction endonuclease digestion, PCR and DNA sequencing confirmation, the recombinant Syk expression plasmid was transfected into A549 human lung cancer cells using lipofectamine protocols. After selection, the cells stably expressed Syk. Detection of Syk expression of the cells by RT-PCR, and invasive ability were examined. RESULTS: The eukaryotic expression plamid pLNCXSyk was constructed and expressed stably in the A549 human lung cancer cells. The RT-PCR results showed that Syk mRNA expression was upregulated significantly (P<0.05). Lower invasion through a basal membrane were apparent after transfection (P<0.05). CONCLUSIONS: A eukaryotic expression plasmid to cause Syk expression in lung cancer cells can obviously inhibit their invasive ability in vitro.

[Study of the expression and function of Syk and VEGF-C in the development of non-small cell lung cancer].

OBJECTIVE: To explore the role of Syk and VEGF-C in the development of NSCLC. METHODS: Transfered these genes(eukaryotic expression vector pcDNA3.1-VEGF-C and pLNCX-syk)into A549 cells with the liposomes. Tested the expression of VEGF-C mRNA and Syk mRNA with RT-PCR.Investigated the cell invasion assay with transwell chamber in vitro. Analysis the expression of VEGF-C proteins in A549 cells and detect Syk and VEGF-C proteins of 81 NSCLC tissue samples with immunohistochemical staining. RESULTS: Higher expression of VEGF-C was revealed in VEGF-C-construct-transfected A549 cell than that in controls through RT-PCR (P < 0.05) and immunohistochemistry(P < 0.01).RT-PCR also revealed that Syk expression was higher in Syk-construct-transfected cells than in controls (P < 0.05). The cell invading experiments showed that there was more invaded cells in both transfected terms than in controls (P < 0.05). The expression of Syk protein in NSCLC tissue were significantly lower than that in the normal lung tissue (P < 0.05), while the expression of VEGF-C protein in NSCLC tissue were significantly higher than that in the normal lung tissue(P < 0.05). The expression of Syk and VEGF-C has a negative correlationship (r = -1.000, P = 0.019). CONCLUSION: The expression of Syk and VEGF-C has a negative correlationship in NSCLC tissue, VEGF-C-construct-transfected A549 cells are more aggressive than Syk-construct-transfected cells. And they may cooperated with each other in the development of NSCLC.

[Relation of vascular endothelial growth factor-D expression to microvessel density, microlymphatic vessel density, and lymph-node metastasis of lung adenocarcinoma].

OBJECTIVE: To investigate the relationship of expression of vascular endothelial growth factor (VEGF)-D to microvessel density (MVD), microlymphatic vessel density (MLVD), and lymph node metastasis in lung adenocarcinoma. METHODS: Fresh specimens of lung adenocarcinoma were collected form 48 patients with lung adenocarcinoma, 28 males and 20 females, aged 35 - 78, during operation. Semi-quantitative RT-PCR was used to detect the mRNA expression of VEGF-D, and immunohistochemistry was used to detect the protein expression of VEGF-D, CD34 (marker of MVD), and VEGF receptor (VEGFR)-3 (marker of MLV). RESULTS: The expression of VEGF-D mRNA in the lung cancer was significantly higher than that in the normal lung tissue, and the expression of VEGF-D protein in the cancerous invasive edge was significantly higher than that in the center of cancerous tissues. Twenty-one of the 29 lung adenocarcinoma patients at stages III - IV were VEGF-D positive, a proportion significantly higher than that of the patients at stages I - II (7/19, P = 0.015). The MLVD of the VEGF-D positive group was 10.25 +/- 2.50, significantly higher than that of the VEGF-D negative group (6.8 +/- 2.2, P < 0.01). Twenty-three of the 28 VEGF-D positive patients showed lymph node metastasis, a proportion significantly higher than that of the VEGF-D negative group (9/20, P = 0.012). CONCLUSION: VEGF-D is related with the lymphangiogenesis and lymph node metastasis in lung adenocarcinoma, but not with angiogenesis.