Amphiregulin promotes BAX inhibition and resistance to gefitinib in non-small-cell lung cancers.

Molecular resistance mechanisms affecting the efficiency of receptor tyrosine kinase inhibitors such as gefitinib in non-small-cell lung cancer (NSCLC) cells are not fully understood. Amphiregulin (Areg) overexpression has been proposed to predict NSCLC resistance to gefitinib and we have established that Areg-overexpressing H358 NSCLC cells resist apoptosis. Here, we demonstrate that Areg prevents gefitinib-induced apoptosis in NSCLC cells. We show that H358 cells are resistant to gefitinib in contrast to H322 cells, which do not overexpress Areg. Inhibition of Areg expression by small-interfering RNAs (siRNAs) restores gefitinib sensitivity in H358 cells, whereas addition of recombinant Areg confers resistance in H322 cells. Areg knockdown overcomes resistance to gefitinib and induced apoptosis in NSCLC H358 cells in vitro and in vivo. Under gefitinib treatment, Areg decreases the expression of the proapoptotic protein BAX, inhibits its conformational change and its mitochondrial translocation. Thus, in the presence of Areg, gefitinib-mediated apoptosis is reduced because BAX is sequestered in the cytoplasm. This suggests that treatments using epidermal growth factor receptor (EGFR) inhibitors may be poorly efficient in patients with elevated levels of Areg. These findings indicate the need for inhibition of Areg to enhance the efficiency of the EGFR inhibitors in patients suffering NSCLC.

Amphiregulin promotes resistance to gefitinib in nonsmall cell lung cancer cells by regulating Ku70 acetylation.

Multiple molecular resistance mechanisms reduce the efficiency of receptor tyrosine kinase inhibitors such as gefitinib in non-small cell lung cancer (NSCLC). We previously demonstrated that amphiregulin (Areg) inhibits gefitinib-induced apoptosis in NSCLC cells by inactivating the proapoptotic protein BAX. In this part of the investigation, we studied the molecular mechanisms leading to BAX inactivation. We show that Areg prevents gefitinib-mediated acetylation of Ku70. This augments the BAX-Ku70 interaction and therefore prevents BAX-mediated apoptosis. Accordingly, Areg or Ku70 knock down restore BAX activation and apoptosis in gefitinib-treated H358 cells in vitro. In addition, overexpression of the histone acetyltransferase (HAT) CREB-binding protein (CBP) or treatments with histone deacetylase (HDAC) inhibitors sensitize H358 cells to gefitinib. Moreover, a treatment with vorinostat, a HDAC inhibitor strongly sensitized tumors to gefitinib in vivo. These findings suggest new prospects in combining both HDAC and epidermal growth factor receptor inhibitors for the treatment of NSCLC.

Inhibition of apoptosis by amphiregulin via an insulin-like growth factor-1 receptor-dependent pathway in non-small cell lung cancer cell lines.

The reciprocal activation of amphiregulin (AR) and insulin-like growth factor-1 (IGF1) pathways has been shown to induce inhibition of serum deprivation apoptosis in non-small cell lung cancer (NSCLC) cell lines H358 and H322. We demonstrated that AR activated the IGF1 receptor (IGF1-R), which in turn induced the secretion of AR and IGF1. Transactivation of the IGF1-R by AR is independent of its binding to EGFR. Thus, AR can inhibit apoptosis in NSCLC cells through an IGF1-R-dependent pathway.

Side-effects of a systemic injection of linear polyethylenimine-DNA complexes.

BACKGROUND: Systemic administration of linear polyethylenimine-DNA complexes (L-PEI/DNA) results in transient expression of the transgene in the lung. This study analyzes the side-effects associated with L-PEI-mediated transfection. METHODS: Mice weighing from 16 to 25 g received increasing amounts of L-PEI/DNA intravenously. Gene expression was evaluated using luciferase as a reporter gene. Toxicity was evaluated by monitoring the appearance of shock after injection, the survival of the animals, and the microscopic damage in the tissues. Adherence of blood cells and endothelium activation were observed after CD11-b and von Willebrand immunostaining. Anti-aggregant treatments were used in order to prevent the formation of thrombi. RESULTS: Increasing the quantity of L-PEI/DNA resulted in a marked augmentation of the luciferase activity in the lung, but was associated with liver necrosis and death. Lethality was reached at lower doses in older mice, suggesting an age influence. Transfection was associated with activation of the lung endothelium and increased adhesion of small aggregates containing platelets and CD11-b-positive cells, without the appearance of large thrombi and of lung injury. Anti-aggregant treatments (aspirin, EDTA, heparin or clopidogrel) decreased the L-PEI-mediated transfection, supporting the hypothesis that platelets participate in the blocking of DNA complexes in the lung capillaries. CONCLUSION: This study demonstrates that L-PEI/DNA activates the lung endothelium and forms small aggregates, a side-effect linked to the transfection efficiency.