Specific inhibitor of Notch­3 enhances the sensitivity of NSCLC cells to gemcitabine.

Notch­3 is a receptor of the Notch signaling pathway and plays an important role in regulating self­renewal, differentiation and apoptosis in cancer cells. Overexpression of Notch­3 has been proved to be associated with resistance to gemcitabine (GEM) and poor patient prognosis for various malignant tumors. In the present study, two non­small cell lung cancer (NSCLC) cell lines, H1299 and A549, were induced with GEM for two months and then were treated with various concentrations of a Notch signaling blocker, N­[N­(3,5­difluorophenacetyl)­L­alanyl]­S­phenylglycine t­butyl ester (DAPT), with the goal of reducing expression of Notch intracellular domain 3 (NICD3). Both cell lines were subsequently treated with either DAPT or DAPT combined with GEM and then viability, apoptosis, colony formation and cell count assays were performed. DAPT treatment effectively downregulated the expression of NICD3 in both cell lines. DAPT combined with GEM also significantly reduced the percentage of viable cells in both cell lines, while increasing the percentage of apoptotic cells, compared with GEM alone. In the clonogenicity assays, the combination of DAPT and GEM led to a decrease in clone numbers and significantly greater inhibition of the H1299 and A549 cells compared to treatment with DAPT or GEM alone. Meanwhile, levels of the apoptosis­related proteins, Bcl­2 and Bax, were found to be affected by the various treatments. Thus Notch­3 appears to be a promising target for gene therapy and DAPT is able to mediate a strong antitumor effect in NSCLC cells that overexpress Notch­3. Further studies of a combined treatment regimen with DAPT and GEM are warranted and may provide greater efficacy and safety in the treatment of NSCLC patients.

Increased expression of Toll-like receptors 4 and 9 in human lung cancer.

BACKGROUND: It has been reported lately that Toll-like receptors (TLRs) play an essential role in the activation of innate immunity, and TLRs are expressed in a large number of immune cells like B-lymphocytes, monocytes, plasmacytoid dendritic cells and at low levels in human respiratory cells as well as epithelial cells. In the present study, we investigated whether there is a relationship between the expression of TLR4 or TLR9 and the clinical or pathological changes in human lung cancer. METHOD: Protein expression of TLR4 and TLR9 was assessed by using immunohistochemistry and western blotting. mRNA expressions of TLR4 and TLR9 were detected by reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: High TLR4 and TLR9 mRNA signal intensity was found in the majority of lung cancer specimens. In contrast, tumor-free lung tissue showed lower signal intensity. Consistently, the low amount of TLR4 and TLR9 protein expression was found in tumor-free lung tissue, while they were strongly expressed in lung cancer tissue. In addition, we found for the first time that the differentiation degree of tumor cells was positively correlated with the expression level of TLR4. There was no relationship between the expressions of TLR4 or TLR9 and patients' age, gender, smoking, the histological type of tumor, lymph node metastasis, and tumor node metastases (TNM) stage. CONCLUSIONS: We found that both mRNA and protein levels of TLR4 and TLR9 were strongly expressed in lung cancer tissue. In addition, we reported for the first time a positive correlation between the expression level of TLR4 and malignancy of lung cancer. These results suggested that TLR4 and TLR9 may be involved in the development of lung cancer which may have the potentials for the treatment of this malignant tumor.

[Mechanism of regulation of leptin expression in human lung adenocarcinoma cells by hypoxia inducible factor-1alpha: a preliminary study].

OBJECTIVE: To investigate the regulation of leptin expression in human lung adenocarcinoma cells by hypoxia inducible factor-1 (HIF-1alpha) and the mechanism thereof. METHODS: Lung adenocarcinoma tissues were collected from 42 patients during operation and samples of corresponding adjacent lung tissue were obtained from 16 of the 42 specimens. The expression levels of HIF-1alpha and leptin were detected by immunohistochemical staining. Human lung adenocarcinoma cells of the line A549 cells were cultured for 0, 12, 24, and 48 h respectively, exposed to hypoxia induced by CoCl2. Other A549 cells were treated with GL331, a kind of HIF-1alpha inhibitor, of the concentrations of 0, 5, 10, or 20 micromol/L under normoxic condition for 24 h, and then were exposed to hypoxia induced by CoCl2 for 24 h. RT-PCR and Western-blotting were used to examine the mRNA and protein expression levels of HIF-1alpha and leptin in different groups. RESULTS: Immunohistochemistry showed that the positive rates of HIF-1alpha and leptin in the lung adenocarcinoma tissue were 57.1% and 69.0% respectively, both significantly higher than those in the adjacent normal lung tissues (18.8% and 25.0% respectively, both P<0.01). The protein expression levels of HIF-1alpha in the hypoxia 0, 12, 24, and 48 h groups were 0.314+/-0.030, 0.552+/-0.027, 0.743+/-0.015, and 0.799+/-0.010 respectively, and the protein expression levels of leptin were 0.398+/-0.016, 0.633+/-0.036, 0.796+/-0.008, and 0.942+/-0.088 respectively, increasing in a time dependent manner too. The mRNA expression levels of leptin in the 4 hypoxia groups were 0.144+/-0.009, 0.336+/-0.017, 0.524+/-0.013, and 0.671+/-0.021 respectively, increasing in a time dependent manner, however, there was no significant differences in the mRNA expression levels of HIF-1alpha among the groups exposed to hypoxia for different courses of time (all P>0.05). The protein expression levels of HIF-1alpha and leptin, and the mRNA expression levels of leptin in the groups exposed to hypoxia for different courses of time were all significantly higher than those of the control (0 h hypoxia) group (all P<0.01). The mRNA and protein expression levels of leptin in the A549 cells exposed to GL331 and hypoxia were decreased dose-dependently. CONCLUSION: The expression of leptin is up-regulated by hypoxia and regulated by HIF-1alpha.