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Int J Oncol
2014 Oct 01;454:1430-6. doi: 10.3892/ijo.2014.2574.
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Hypoxia induces resistance to ALK inhibitors in the H3122 non-small cell lung cancer cell line with an ALK rearrangement via epithelial-mesenchymal transition.
Kogita A
,
Togashi Y
,
Hayashi H
,
Sogabe S
,
Terashima M
,
De Velasco MA
,
Sakai K
,
Fujita Y
,
Tomida S
,
Takeyama Y
,
Okuno K
,
Nakagawa K
,
Nishio K
.
Abstract
Patients with non-small cell lung cancer (NSCLC) with echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) rearrangements generally respond to ALK inhibitors such as crizotinib. However, some patients with EML4-ALK rearrangements respond poorly to crizotinib. Hypoxia is involved in the resistance to chemotherapeutic treatments in several cancers, and we investigated the association between the responses to ALK inhibitors and hypoxia. Sensitivity of the H3122 NSCLC cell line (EML4-ALK rearrangement) to ALK inhibitors (crizotinib or alectinib) was investigated during a normoxic or hypoxic state using an MTT assay. We found that the cell line was resistant to the inhibitors during hypoxia. Hypoxia mediated morphologic changes, including cell scattering and the elongation of the cell shape, that are characteristic of the epithelial-mesenchymal transition (EMT). A migration assay demonstrated that the number of migrating cells increased significantly during hypoxia, compared with during normoxia. Regarding EMT-related molecules, the expressions of slug, vimentin, and fibronectin were increased while that of E-cadherin was decreased by hypoxia. In addition, hypoxia inducible factor 1A-knockdown cancelled the hypoxia-induced EMT and resistance. Our findings indicate that hypoxia induces resistance to ALK inhibitors in NSCLC with an EML4-ALK rearrangement via the EMT.
Figure 1. Sensitivity of the H3122 cell line to ALK inhibitors (crizotinib and alectinib). To examine the sensitivity of ALK inhibitors, we used an MTT assay. The experiment was performed in triplicate. (A) Growth inhibitory effect of crizotinib. The H3122 cell line was sensitive to crizotinib under a normoxic state, compared with a hypoxic state. The graphs, mean of independent triplicate experiments; error bars, SD. (B) IC50 of ALK inhibitors. The IC50 values of both inhibitors in the H3122 cell lines were significantly higher under hypoxia than under normoxia (crizotinib, *P=0.028 and alectinib, *P=0.0035). *P<0.05.
Figure 2. Western blot analyses of a downstream signal and apoptosis-related molecules in the H3122 cell line. (A) Downstream signal. The cells were incubated under normoxia or hypoxia for 48 h, then crizotinib was added three hours before the sample collection. The concentrations of crizotinib were 0, 0.01, 0.1 and 1 μM. The phosphorylation of AKT and ERK was dose-dependently reduced by crizotinib under normoxia. Under hypoxia, however, the phosphorylation was less reduced by crizotinib. β-actin was used as an internal control. (B) Apoptosis-related molecules. The samples were collected 48 h after DMSO (control) or crizotinib stimulation under normoxia or hypoxia. The concentration of crizotinib was 0.1 μM. The expression of both cleaved PARP and cleaved caspase-3 was elevated by crizotinib under normoxia, whereas neither expression level was elevated under hypoxia. β-actin was used as an internal control.
Figure 3. Morphologic change and migration of the H3122 cell line. (A) Morphologic change. Hypoxia time-dependently induced morphologic changes that are characteristic of the EMT, including cell scattering and an elongation of the cell shape. Scale bar, 20 μm. (B) Migration. The migration assays were performed using the Boyden chamber method. After incubation for 48 h under normoxia or hypoxia, the cells that had migrated to the outer side of the membranes were fixed and stained with crystal violet staining solution, then counted using a light microscope. The experiment was performed in triplicate. Under hypoxia, the number of migrating cells was significantly higher than that under normoxia (8.67±3.5 vs. 1.33±1.53/Field, *P=0.026). Columns, mean of independent triplicate experiments; error bars, SD; *P<0.05.
Figure 4. Real-time RT-PCR and western blot analyses for EMT-related molecules. (A) Real-time RT-PCR for EMT-related genes. To evaluate whether hypoxia mediates the EMT in H3122 cells, changes in the mRNA expression levels of EMT-related genes were evaluated using real-time RT-PCR. The experiment was performed in triplicate. Hypoxia upregulated SLUG mRNA expression (48 h, *P=0.030 and 96 h, *P=0.0053). E-cadherin mRNA expression was clearly downregulated under hypoxia (48 h, *P=0.035 and 96 h, *P=0.042). The expression of mesenchymal marker VIM and FN1 mRNA was also upregulated (48 h, *P=0.025 and *P=0.079, respectively and 96 h, *P=0.031 and *P=0.044, respectively). Columns, mean of independent triplicate experiments; error bars, SD; *P<0.05. (B) Western blot analyses for EMT-related molecules. Consistent with the mRNA changes, hypoxia time-dependently upregulated the protein expression of slug, fibronectin, and vimentin and downregulated the expression of E-cadherin in the cell line, along with HIF1α upregulation. β-actin was used as an internal control.
Figure 5. HIF1A-knockdown effect on the H3122 cell line under hypoxia. To investigate whether HIF1α was a key factor in hypoxia-induced EMT and resistance to ALK inhibitors, HIF1A was knocked down using specific siRNA (H3122/si-HIF1A) or a control (H3122/si-Scr). (A) Morphologic change. Both H3122/si-HIF1A and H3122/si-Scr cell lines were incubated for 48 h under a hypoxic state. HIF1A-knockdown abolished the hypoxia-induced morphologic changes (si-HIF1A), whereas the control-siRNA did not (si-Scr). Scale bar, 20 μm. (B) Western blot analyses for EMT-related molecules. Before the sample collection, both the H3122/si-HIF1A and the H3122/si-Scr cell lines were incubated for 48 h under hypoxia. HIF1A-knockdown cancelled the hypoxia-induced downregulation of E-cadherin and the upregulation of slug, vimentin, and fibronectin. β-actin was used as an internal control. (C) Growth inhibitory effect of crizotinib. To examine the sensitivity, we used an MTT assay during a hypoxic state. The experiment was performed in triplicate. Under hypoxia, the H3122/si-HIF1A cell line was more sensitive to crizotinib, compared with the H3122/si-Scr cell line. The graphs, mean of independent triplicate experiments; error bars, SD.
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