ABSTRACT
This study explored the role of radiation-induced autophagy in low-dose hyperradiosensitivity (HRS) in the human lung cancer cell line A549. A549 cells, either treated with an autophagic inhibitor 3-methyladenine (3-MA), or with a vehicle control, were irradiated at different low doses (≤0.5 Gy). The generation of autophagy was examined by laser scanning confocal microscopy. Western blotting was used to detect the expression of microtubule-associated protein l light chain 3B II (LC3B-II). Flow cytometry (FCM) and clonogenic assays were used to measure the fraction of surviving cells at the low irradiation doses. Our results showed that there was a greater inhibition of autophagic activity, but a higher degree of low-dose HRS in A549 cells treated with 3-MA than in control group. Our data demonstrated that radiation-induced autophagy is correlated with HRS in A549 cells, and is probably one of the mechanisms underlying HRS.
Subject(s)
Humans , Adenine , Pharmacology , Autophagy , Radiation Effects , Blotting, Western , Cell Line, Tumor , Cell Survival , Radiation Effects , Dose-Response Relationship, Radiation , Flow Cytometry , Green Fluorescent Proteins , Genetics , Metabolism , Lung Neoplasms , Genetics , Metabolism , Pathology , Microscopy, Confocal , Microscopy, Electron, Transmission , Microtubule-Associated Proteins , Genetics , Metabolism , Phagosomes , Radiation Effects , Radiation Tolerance , Radiation EffectsABSTRACT
The effects of class I PI3K inhibitor NVP-BKM120 on cell proliferation, cell cycle distribution, cellular apoptosis, phosphorylation of several proteins of the PI3K/AKT signaling pathway and the mRNA expression levels of HIF1-α, VEGF and MMP9 in the acquired gefitinib resistant cell line H1975 were investigated, and whether NVP-BKM120 can overcome the acquired resistance caused by the EGFR T790M mutation and the underlying mechanism were explored. MTT assay was performed to detect the effect of gefitinib, NVP-BKM120, NVP-BKM120 plus 1 μmol/L gefitinib on growth of H1975 cells. The distribution of cell cycle and apoptosis rate of H1975 cells were examined by using flow cytometry. The mRNA expression levels of tumor-related genes such as HIF1-α, VEGF and MMP9 were detected by using real-time quantitative PCR. Western blotting was used to detect the expression level of phosphorylated proteins in the PI3K/AKT signaling pathway, such as Ser473-p-AKT, Ser235/236-p-S6 and Thr70-p-4E-BP1, as well as total AKT, S6 and 4E-BP1. The results showed that the NVP-BKM120 could inhibit the growth of H1975 cells in a concentration-dependent manner, and H1975 cells were more sensitive to NVP-BKM120 than gefitinib (IC50:1.385 vs. 15.09 μmol/L respectively), whereas combination of NVP-BKM120 and gefitinib (1 μmol/L) did not show more obvious effect than NVP-BKM120 used alone on inhibition of cell growth (P>0.05). NVP-BKM120 (1 μmol/L) increased the proportion of H1975 cells in G0-G1 phase and the effect was concentration-dependent, and 2 μmol/L NVP-BKM120 promoted apoptosis of H1975 cells. There was no significant difference in the proportion of H1975 cells in G0-G1 phase and apoptosis rate between NVP-BKM120-treated alone group and NVP-BKM120 plus genfitinib (1 μmol/L)-treated group or between DMSO-treated control group and gefitinib (1 μmol/L)-treated alone group (P>0.05 for all). It was also found that the mRNA expression levels of these genes were down-regulated by NVP-BKM120 (1 μmol/L), and NVP-BKM120 (1 μmol/L) or NVP-BKM120 (1 μmol/L) plus gefitinib (1 μmol/L) obviously inhibited the activation of Akt, S6 and 4E-BP1 as compared with control group, but single use of gefitinib (1 μmol/L) exerted no significant effect. These data suggested that NVP-BKM120 can overcome gefitinib resistance in H1975 cells, and the combination of NVP-BKM120 and gefitinib did not have additive or synergistic effects. It was also concluded that NVP-BKM120 could overcome the acquired resistance to gefitinib by down-regulating the phosphorylated protein in PI3K/AKT signal pathways in H1975 cells, but it could not enhance the sensitivity of H1975 cells to gefitinib.
Subject(s)
Humans , Adenocarcinoma , Metabolism , Aminopyridines , Pharmacology , Antineoplastic Agents , Pharmacology , Apoptosis , Cell Line, Tumor , Cell Proliferation , Drug Resistance, Neoplasm , Hypoxia-Inducible Factor 1, alpha Subunit , Genetics , Metabolism , Lung Neoplasms , Metabolism , Matrix Metalloproteinase 9 , Genetics , Metabolism , Morpholines , Pharmacology , Phosphatidylinositol 3-Kinases , Quinazolines , Pharmacology , RNA, Messenger , Genetics , Metabolism , Vascular Endothelial Growth Factor A , Genetics , MetabolismABSTRACT
The effects of class I PI3K inhibitor NVP-BKM120 on cell proliferation, cell cycle distribution, cellular apoptosis, phosphorylation of several proteins of the PI3K/AKT signaling pathway and the mRNA expression levels of HIF1-α, VEGF and MMP9 in the acquired gefitinib resistant cell line H1975 were investigated, and whether NVP-BKM120 can overcome the acquired resistance caused by the EGFR T790M mutation and the underlying mechanism were explored. MTT assay was performed to detect the effect of gefitinib, NVP-BKM120, NVP-BKM120 plus 1 μmol/L gefitinib on growth of H1975 cells. The distribution of cell cycle and apoptosis rate of H1975 cells were examined by using flow cytometry. The mRNA expression levels of tumor-related genes such as HIF1-α, VEGF and MMP9 were detected by using real-time quantitative PCR. Western blotting was used to detect the expression level of phosphorylated proteins in the PI3K/AKT signaling pathway, such as Ser473-p-AKT, Ser235/236-p-S6 and Thr70-p-4E-BP1, as well as total AKT, S6 and 4E-BP1. The results showed that the NVP-BKM120 could inhibit the growth of H1975 cells in a concentration-dependent manner, and H1975 cells were more sensitive to NVP-BKM120 than gefitinib (IC50:1.385 vs. 15.09 μmol/L respectively), whereas combination of NVP-BKM120 and gefitinib (1 μmol/L) did not show more obvious effect than NVP-BKM120 used alone on inhibition of cell growth (P>0.05). NVP-BKM120 (1 μmol/L) increased the proportion of H1975 cells in G0-G1 phase and the effect was concentration-dependent, and 2 μmol/L NVP-BKM120 promoted apoptosis of H1975 cells. There was no significant difference in the proportion of H1975 cells in G0-G1 phase and apoptosis rate between NVP-BKM120-treated alone group and NVP-BKM120 plus genfitinib (1 μmol/L)-treated group or between DMSO-treated control group and gefitinib (1 μmol/L)-treated alone group (P>0.05 for all). It was also found that the mRNA expression levels of these genes were down-regulated by NVP-BKM120 (1 μmol/L), and NVP-BKM120 (1 μmol/L) or NVP-BKM120 (1 μmol/L) plus gefitinib (1 μmol/L) obviously inhibited the activation of Akt, S6 and 4E-BP1 as compared with control group, but single use of gefitinib (1 μmol/L) exerted no significant effect. These data suggested that NVP-BKM120 can overcome gefitinib resistance in H1975 cells, and the combination of NVP-BKM120 and gefitinib did not have additive or synergistic effects. It was also concluded that NVP-BKM120 could overcome the acquired resistance to gefitinib by down-regulating the phosphorylated protein in PI3K/AKT signal pathways in H1975 cells, but it could not enhance the sensitivity of H1975 cells to gefitinib.
ABSTRACT
This study explored the role of radiation-induced autophagy in low-dose hyperradiosensitivity (HRS) in the human lung cancer cell line A549. A549 cells, either treated with an autophagic inhibitor 3-methyladenine (3-MA), or with a vehicle control, were irradiated at different low doses (≤0.5 Gy). The generation of autophagy was examined by laser scanning confocal microscopy. Western blotting was used to detect the expression of microtubule-associated protein l light chain 3B II (LC3B-II). Flow cytometry (FCM) and clonogenic assays were used to measure the fraction of surviving cells at the low irradiation doses. Our results showed that there was a greater inhibition of autophagic activity, but a higher degree of low-dose HRS in A549 cells treated with 3-MA than in control group. Our data demonstrated that radiation-induced autophagy is correlated with HRS in A549 cells, and is probably one of the mechanisms underlying HRS.
ABSTRACT
<p><b>OBJECTIVE</b>To evaluate the efficacy of zoledronic acid combined with local radiotherapy for limited metastatic bone cancer.</p><p><b>METHODS</b>Forty-five patients with limited bone metastatic cancers were randomly divided into two groups: 23 in the combination group who received intravenously administration of zoledronic acid and local radiotherapy, the other 22 in the radiotherapy alone group who underwent local radiotherapy only.</p><p><b>RESULTS</b>The response rate of pain relief was 91.3% in the combination group versus 86.4% in the radiotherapy alone group, without statistically significant difference between two groups (P > 0.05). However, the recalcification rate was significantly higher in the combination therapy group (52.2%) than that in radiotherapy alone group (22.7% P < 0.01), and the proportion of patients with new bone metastasis formation was significantly lower in the combination group (13.0%) than that in the radiotherapy alone group (40.9%, P < 0.05). The common side-effects were transient pyrexia and nausea.</p><p><b>CONCLUSION</b>Zoledronic acid combined with local radiotherapy is effective in relieving pain, improving bone recalcification and reducing the formation of new bone metastasis.</p>