Radiosensitivity Of Non-small Cell Lung Cancer Cells Research Articles

Epidermal growth factor receptor gene mutations in non-small-cell lung cancer cells are associated with increased radiosensitivity in vitro.

IntroductionThere is still lack of specific biomarkers in predicting the radiosensitivity of non-small cell lung cancer (NSCLC) patients in clinic. Previous studies have shown that the EGFR gene status may correlate with radiosensitivity of NSCLC. However, the underlying mechanisms remain unknown. The aim of this study was to further investigate the correlation between EGFR mutation status and the NSCLC cell radiosensitivity and to explore the possible cellular mechanism.MethodsEight NSCLC cell lines with different EGFR gene status were irradiated by linear accelerator, and the radiosensitivity between the cell lines was compared by cell colony formation assay and cell proliferation assay. Cell cycle and apoptosis were analysed by flow cytometry. Radiosensitivity-related protein expression was detected by Western blotting.ResultsIn the present study, we found that NSCLC cell lines with the epidermal growth factor receptor (EGFR) gene mutations were more sensitive to X-ray irradiation than those with wild-type EGFR (P<0.05). No difference in radiosensitivity was observed between NSCLC cells with EGFR exon19 deletion (Del 19) mutation and exon 21 point mutation at position 858 (L858R) with or without T790M mutation (P<0.05), as well as between NSCLC cells with EGFR mutation and those with acquired EGFR-tyrosine kinase inhibitors (TKIs) resistance. Mechanistically, EGFR mutations promoted NSCLC cell apoptosis in response to X-ray irradiation through the upregulation of proapoptotic protein Bax and downregulation of anti-apoptotic proteins such as Bcl-2 and DNA-dependent protein kinase catalytic subunit. In addition, phosphorylated histone (γ-H2AX) foci assay showed that EGFR mutations sustained irradiation-induced DNA damage.ConclusionTaken together, our study demonstrates that EGFR mutations are closely associated with the increased sensitivity of NSCLC cell lines to X-ray irradiation and that EGFR mutation status is a potentially useful indicator to evaluate the effectiveness of radiotherapy in the treatment of NSCLC.

GSK-3\u03b2 inhibits autophagy and enhances radiosensitivity in non-small cell lung cancer

BackgroundRadiotherapy is one of the most common and effective treatment methods for cancer, and improving the radiosensitivity of tumor tissues during the treatment process is vital. We report the mechanisms of glycogen synthase kinase 3 (GSK-3) β-regulated autophagy and the effects of autophagy on radiosensitivity in non-small cell lung cancer (NSCLC).MethodImmunohistochemical staining was performed to determine GSK-3β tissue expression in 89 NSCLC patients with follow-up data and the expression status of GSK-3β and autophagy in NSCLC tissues after X-ray radiotherapy. Western blots were used to quantitate changes in autophagy-related protein expression after A549 cells were treated with GSK-3β inhibitors and after H460 cells were transfected with GSK-3β mutants with different activities and X-ray irradiated. Clonogenic assays were used to measure the effect of autophagy on cellular proliferation.ResultsGSK-3β expression positively correlated with NSCLC differentiation (P < 0.05), and GSK-3β negativity was associated with a better prognosis in 89 NSCLC patients. After X-ray irradiation, the expression levels of GSK-3β and p62 were decreased in NSCLC tissues, and the expression levels of the autophagy-related protein LC3 were increased. A549 and H460 cells were selected as representative GSK-3β-high and GSK-3β-low expression cell lines. After transfecting H460 cells with different GSK-3β mutants [wild type GSK-3β (GSK-3β-WT), constitutively active GSK-3β (GSK-3β-S9A), and catalytically inactive GSK-3β (GSK-3β-K85R)] and subjecting these cells to X-ray irradiation, AMPK and LC3 expression levels decreased, and p62 expression levels increased. These effects were particularly significant for the GSK-3β-S9A mutant. In A549 cells, after GSK-3β inhibition and X-ray irradiation, AMPK and LC3 protein expression levels increased. Moreover, when autophagy was inhibited, cell proliferation decreased.ConclusionOur studies revealed that GSK-3β expression is associated with NSCLC differentiation, and patients with GSK-3β-negative tumors had a better prognosis. X-ray irradiation inhibited GSK-3β expression and promoted autophagy. Therefore, GSK-3β inhibits autophagy and enhances the radiosensitivity of NSCLC cells.

MicroRNA-148b inhibits proliferation and the epithelial-mesenchymal transition and increases radiosensitivity in non-small cell lung carcinomas by regulating ROCK1.

Non-small cell lung cancer (NSCLC) accounts for ~80% of all types of lung cancer, which has the highest morbidity and mortality of all types of cancer worldwide. It is important to identify novel biomarkers and the molecular mechanism of NSCLC to improve current treatments of NSCLC. The present study aimed to investigate the effect of miR-148b expression on the proliferation, epithelial-mesenchymal transition (EMT) and radiosensitivity of NSCLC cells. It was demonstrated that miR-148b expression was significantly decreased in NSCLC tissues and cell lines. A549 cells were then transfected with a miR-148b mimic and a miR-148b inhibitor. Transfection with the miR-148b mimic decreased proliferation whereas transfection with the miR-148b inhibitor increased the proliferation of A549 cells. Additionally, the miR-148b mimic increased E-cadherin expression and decreased N-cadherin and vimentin expression. By contrast, transfection with the miR-148b inhibitor decreased E-cadherin expression and increased N-cadherin and vimentin expression. Irradiation-induced cell death was significantly promoted by the miR-148b mimic but inhibited by the miR-148b inhibitor. The miR-148b mimic significantly decreased the expression of Rho-associated protein kinase 1 (ROCK1) and it was demonstrated that overexpression of ROCK1 significantly inhibited the effects of miR-148b on cell proliferation, the EMT and irradiation-induced cell death. Therefore, the current study revealed that miR-148b inhibited NSCLC cell proliferation and the EMT, and increased the radiosensitivity of NSCLC cells by inhibiting ROCK1 expression. Therefore, miR-148b/ROCK1 signaling may be a novel therapeutic target to inhibit the growth of NSCLC cells and enhance the effects of radiotherapy to treat patients with NSCLC.

Genistein promotes ionizing radiation-induced cell death by reducing cytoplasmic Bcl-xL levels in non-small cell lung cancer

Genistein (GEN) has been previously reported to enhance the radiosensitivity of cancer cells; however, the detailed mechanisms remain unclear. Here, we report that GEN treatment inhibits the cytoplasmic distribution of Bcl-xL and increases nuclear Bcl-xL in non-small cell lung cancer (NSCLC). Interestingly, our in vitro data show that ionizing radiation IR treatment significantly increases IR-induced DNA damage and apoptosis in a low cytoplasmic Bcl-xL NSCLC cell line compared to that of high cytoplasmic Bcl-xL cell lines. In addition, clinical data also show that the level of cytoplasmic Bcl-xL was negatively associated with radiosensitivity in NSCLC. Furthermore, we demonstrated that GEN treatment enhanced the radiosensitivity of NSCLC cells partially due to increases in Beclin-1-mediated autophagy by promoting the dissociation of Bcl-xL and Beclin-1. Taken together, these findings suggest that GEN can significantly enhance radiosensitivity by increasing apoptosis and autophagy due to inhibition of cytoplasmic Bcl-xL distribution and the interaction of Bcl-xL and Beclin-1 in NSCLC cells, respectively.

MiR-144-5p Enhances the Radiosensitivity of Non-Small-Cell Lung Cancer Cells via Targeting ATF2.

MicroRNAs (miRNAs or miRs) regulate gene expression at the posttranscriptional level and are involved in many biological processes such as cell proliferation and migration, stem cell differentiation, inflammation, and apoptosis. In particular, miR-144-3p is downregulated in various cancers, and its overexpression inhibits the proliferation and metastasis of cancer cells. However, the role of miR-144-5p in non-small-cell lung cancer (NSCLC), especially radiosensitivity, is unknown. In this study, we found that miR-144-5p was downregulated in NSCLC clinical specimens as well as NSCLC cell lines exposed to radiation. Enhanced expression of miR-144-5p promoted the radiosensitivity of NSCLC cells in vitro and A549 cell mouse xenografts in vivo. Furthermore, we identified activating transcription factor 2 (ATF2) as the direct and functional target of miR-144-5p using integrated bioinformatics analysis and a luciferase reporter assay. In addition, restoration of ATF2 expression inhibited miR-144-5p-induced NSCLC cell sensitivity to radiation in vitro and in vivo. Our findings suggest that deregulation of the miR-144-5p/ATF2 axis plays an important role in NSCLC cell radiosensitivity, thus representing a new potential therapeutic target for NSCLC.

Inhibiting polo-like kinase 1 enhances radiosensitization via modulating DNA repair proteins in non-small-cell lung cancer.

To assure faithful chromosome segregation, cells make use of the spindle assembly checkpoint, which can be activated in aneuploid cancer cells. In this study, the efficacies of inhibiting polo-like kinase 1 (PLK1) on the radiosensitization of non-small-cell lung cancer (NSCLC) cells were studied. Clonogenic survival assay was performed to identify the effects of the PLK1 inhibitor on radiosensitivity within NSCLC cells. Mitotic catastrophe assessment was used to measure the cell death and histone H2AX protein (γH2AX) foci were utilized to assess the DNA double-strand breaks (DSB). The transcriptome was analyzed via unbiased profiling of microarray expression. The results showed that the postradiation mitotic catastrophe induction and the DSB repair were induced by PLK1 inhibitor BI-6727, leading to an increase in the radiosensitivity of NSCLC cells. BI-6727 in combination with radiation significantly induced the delayed tumor growth. PLK1-silenced NSCLC cells showed an altered mRNA and protein expression related to DNA damaging, replication, and repairing, including the DNA-dependent protein kinase (DNAPK) and topoisomerase II alpha (TOPO2A). Furthermore, inhibition of PLK1 blocked 2 important DNA repair pathways. To summarize, our study showed PLK1 kinase as an option in the therapy of NSCLC.

Long Noncoding RNA GAS5 Inhibits Tumorigenesis and Enhances Radiosensitivity by Suppressing miR-135b Expression in Non-Small Cell Lung Cancer.

Growth arrest-specific transcript 5 (GAS5) has been demonstrated to correlate with clinicopathological characteristics and serve as a tumor suppressor in non-small cell lung cancer (NSCLC). However, the underlying mechanism of the competing endogenous RNA (ceRNA) regulatory network involving GAS5 in NSCLC remains to be elucidated. In this study, qRT-PCR results showed that GAS5 was downregulated and miR-135b was upregulated in NSCLC tissues and cells. The expressions of GAS5 and miR-135b changed inversely in response to irradiation. Gain-of-function experiments revealed that GAS5 overexpression and miR-135b downregulation significantly suppressed tumorigenesis by repressing cell proliferation and invasion, and enhanced the radiosensitivity of NSCLC cells by reducing colony formation rates. Luciferase reporter assay confirmed that GAS5 could directly target miR-135b and negatively regulate its expression. Moreover, rescue experiments demonstrated that miR-135b upregulation markedly abolished GAS5 overexpression-induced tumorigenesis inhibition and radiosensitivity improvement. Furthermore, xenograft model analysis validated that GAS5 overexpression suppressed tumor growth and improved radiosensitivity of NSCLC cells in vivo. Taken together, GAS5 inhibits tumorigenesis and enhances radiosensitivity by suppressing miR-135b expression in NSCLC cells, deepening our understanding of the mechanism of miRNA–lncRNA interaction and providing a novel therapeutic strategy for NSCLC.

Effect of STAT-3-mediated non-dependent VEGFR-2 pathway on radiosensitivity of non-small cell lung cancer cells

Objective To investigate the effect of signal transducer and activator of transcription-3(STAT-3)-mediated non-dependent vascular endothelial growth factor receptor-2(VEGFR-2) pathway on the radiosensitivity of non-small cell lung cancer (NSCLC) cells and its possible mechanism. Methods NSCLC cells were divided into control group, apatinib (VEGFR-2 inhibitor) group, apatinib+ S3I-201(STAT-3 inhibitor) group, radiation (RT) group, RT+ apatinib group, and RT+ apatinib+ S3I-201 group. Q-PCR and Western blot were used to measure the mRNA and protein expression of VEGFR-2, STAT-3, other proteins involved in the signaling pathway, hypoxia-inducible factor 1α(HIF-1α), and cyclin D1. The cellular radiosensitivity was determined by colony-forming assay and cell apoptosis and cell cycle distribution were evaluated by flow cytometry. Results For Calu-1 cells, there was no significant difference in the expression of VEGFR-2 mRNA and STAT-3 mRNA between the apatinib group and the control group (P>0.05); the apatinib group had significantly lower expression of HIF-1α mRNA and cyclin D1 mRNA than the control group (P<0.05); the apatinib+ S3I-201 group had significantly lower mRNA and phosphorylated protein expression of VEGFR-2, STAT-3, and related downstream target proteins than the control group (F=304.54, P<0.01; F=118.99, P<0.01, F=144.34, P<0.01; F=529.66, P<0.01); compared with the control group, the apatinib+ S3I-201 group and the apatinib group showed increases in apoptosis rate and proportion of cells in G2+ M phase, and the apatinib+ S3I-201 group had significantly greater increases than the apatinib group (F=72.37, P<0.01). Compared with Calu-1 cells, the radiosensitizing effect of apatinib on A549 cells was limited (SER[sensitizer enhancement ratio]=1.39). The radiosensitizing effect of apatinib+ S3I-201 on A549 cells was significantly higher than that of apatinib (SER: 1.72 vs. 1.39, P=0.000). Conclusions The inhibition of STAT-3 can enhance the radiosensitivity of NSCLC cells. When VEGFR-2 is inhibited by apatinib, activated STAT-3 regulates the expression of cyclin D1 directly or indirectly to affect the radiosensitivity of NSCLC cells. The inhibition of VEGFR-2 and STAT-3 has a good radiosensitizing effect on NSCLC cells. Key words: Protein express; Lung meoplasms cell line; Radiosensitivity

Selective ATP hydrolysis inhibition in F1Fo ATP synthase enhances radiosensitivity in non-small-cell lung cancer cells (A549).

BackgroundF1Fo-ATP synthase (F1Fo-ATPase) is a reversibly rotary molecular machine whose dual functions of synthesizing or hydrolyzing ATP switch upon the condition of cell physiology. The robust ATP-hydrolyzing activity occurs in ischemia for maintaining the transmembrane proton motive force of mitochondria inner membrane, but the effect of F1Fo-ATPase on X-ray response of non-small-cell lung cancer (NSCLC) cells is unknown.Methods and FindingsWe studied whether ATP hydrolysis affected X-ray radiation induced cell death. NSCLC cells (A549) were pretreated with BTB06584 (BTB), an elective ATP hydrolysis inhibitor, followed by X-ray radiation. Cell viability and clonogenic survival were markedly decreased, clear indications of enhanced radiosensitivity through BTB incubation. Additionally, ATP5α1 was upregulated in parallel with elevated ATP hydrolytic activity after X-ray radiation, showing an increased mitochondrial membrane potential (ΔΨm). ATP hydrolysis inhibition led to collapse of ΔΨm suggesting ATP hydrolytic activity could enhance ΔΨm after X-ray radiation. Furthermore, we also demonstrated that apoptosis was pronounced with the prolonged collapse of ΔΨm due to hydrolysis inhibition by BTB incubation.ConclusionOverall, these findings supported that ATP hydrolysis inhibition could enhance the radiosensitivity in NSCLC cells (A549) after X-ray radiation, which was due to the collapse of ΔΨm.

Association of ribosomal protein S6 kinase 1 with cellular radiosensitivity of non-small lung cancer

Purpose: Ribosomal S6 kinase 1 (S6K1) plays an important role in cell proliferation, protein translation and cell survival. This study investigated the possibility of using S6K1 as a new target in the radiotherapy of non-small cell lung cancer (NSCLC) and its potential mechanism.Materials and methods: shRNA interference technology was applied to silence the expression of S6K1 in A549 and H460, and clonogenic assay was performed to measure the radiosensitizing effects. DNA repair was monitored by γH2AX foci formation. Cell cycle and apoptosis were measured by flow cytometry assays. Protein expression was analyzed by Western blotting. S6K1 and lentivirus containing programmed cell death 4 (PDCD4), a substrate of S6K1, were co-transfected into cells and radiosensitivity was also detected.Results: S6K1 knockdown significantly enhanced the radiosensitivity of NSCLC cells. The notable findings in response to this combined treatment were prolonged delay in radiation-induced DNA DSB repair, robust G2/M checkpoint arrest, increased apoptosis and upregulation of PDCD4. Moreover, PDCD4 knockdown reversed the radiosensitizing effects of S6K1 inhibition in NSCLC.Conclusions: S6K1 is a promising tumor-specific target for the enhancement of NSCLC radiosensitivity and its effects may be mediated by increased expression of PDCD4.

MiR-30a radiosensitizes non-small cell lung cancer by targeting ATF1 that is involved in the phosphorylation of ATM.

Increasing number of studies report that microRNAs play important roles in radiosensitization. miR-30a has been proved to perform many functions in the development and treatment of cancer, and it is downregulated in non-small cell lung cancer (NSCLC) tissues and cells. This study was conducted to understand if miR-30a plays a role in the radiosensitivity of NSCLC cells. Radiosensitivity was examed by colony survival assay and tumor volume changing in vitro and in vivo, respectively. Bioinformatic analysis and luciferase reporter assays were used to distinguish the candidate target of miR-30a. qRT-PCR and western blotting were carried out to detect the relative expression of mRNAs and proteins. Cell cycle and cell apoptosis were determined by flow cytometry. Our results illustrated miR-30a could increase the radiosensitivity of NSCLC, especially in A549 cell line. In vivo experiment also showed the potential radiosensitizing possibility of miR-30a. Further exploration validated that miR-30a was directly targeting activating transcription factor 1 (ATF1). In studying the ataxia-telangiectasia mutated (ATM) associated effects on cell radiosensitivity, we found that miR-30a could reduce radiation induced G2/M cell cycle arrest and may also affect radiation induced apoptosis. Together, our results demonstrated that miR-30a may modulate the radiosensitivity of NSCLC through reducing the function of ATF1 in phosphorylation of ATM and have potential therapeutic value.

Ursolic acid sensitizes radioresistant NSCLC cells expressing HIF-1α through reducing endogenous GSH and inhibiting HIF-1α.

In previous studies, the present authors demonstrated that effective sensitization of ionizing radiation-induced death of tumor cells, including non-small cell lung cancer (NSCLC) cells, could be produced by oleanolic acid (OA), a pentacyclic triterpenoid present in plants. In the present study, it was investigated whether ursolic acid (UA), an isomer of OA, had also the capacity of sensitizing radioresistant NSCLC cells. The radioresistant cell line H1299/M-hypoxia inducible factor-1α (HIF-1α) was established by transfection with a recombinant plasmid expressing mutant HIF-1α (M-HIF-1α). Compared with parental H1299 cells and H1299 cells transfected with empty plasmid, H1299/M-HIF-1α cells had lower radiosensitivity. Following the use of UA to treat NSCLC cells, elevation of the radiosensitivity of cells was observed by MTT assay. The irradiated H1299/M-HIF-1α cells were more sensitive to UA pretreatment than the irradiated cells with empty plasmid and control. The alteration of DNA damage in the irradiated cells was further measured using micronucleus (MN) assay. The combination of UA treatment with radiation could induce the increase of cellular MN frequencies, in agreement with the change in the tendency observed in the cell viability assay. It was further shown that the endogenous glutathione (GSH) contents were markedly attenuated in the differently irradiated NSCLC cells with UA (80 µmol/l) pretreatment through glutathione reductase/5,5′-dithiobis-(2-nitrob-enzoic acid) (DTNB) recycling assay. The results revealed that UA treatment alone could effectively decrease the GSH content in H1299/M-HIF-1α cells. In addition, the inhibition of HIF-1α expression in radioresistant cells was confirmed by western blotting. It was then concluded that UA could upregulate the radiosensitivity of NSCLC cells, and in particular reduce the refractory response of cells expressing HIF-1α to ionizing radiation. The primary mechanism is associated with reduction of endogenous GSH and inhibition of high expression of intracellular HIF-1α. UA should therefore be deeply studied as a potential radiosensitizing reagent for NSCLC radiotherapy.

Inhibition of microRNA-155 sensitizes lung cancer cells to irradiation via suppression of HK2-modulated glucose metabolism.

MicroRNAs (miRNAs) are small non-coding regulatory RNAs, which are involved in the post-transcriptional regulation of gene expression. miRNA (miR)-155, which has previously been reported to be overexpressed in lung cancer, is correlated with poor patient prognosis. The present study aimed to investigate the effects of miR‑155 on the radiosensitivity of human non‑small cell lung cancer (NSCLC) cells. To explore the roles of miRNAs in the regulation of irradiation sensitivity of human lung cancer cells, the expressions of miR‑155 in response to irradiation, have been studied by RT‑qPCR, and the putative direct target of miR‑155 was identified by western blot and luciferase assays. The results of the present study revealed that the expression of miR‑155 was induced by irradiation, thus suggesting a positive correlation between miR‑155 and radiosensitivity. Furthermore, overexpression of miR‑155 rendered lung cancer cells resistant to irradiation. In addition, hexokinase2 (HK2) was identified as an indirect target of miR‑155; exogenous overexpression of miR‑155 upregulated the expression of HK2, whereas inhibition of miR‑155 by antisense miRNA suppressed HK2 expression. In addition, HK2‑modulated glucose metabolism was significantly upregulated by overexpression of miR‑155. Notably, inhibition of miR‑155 sensitized lung cancer cells to irradiation via suppression of glucose metabolism. In conclusion, the present study reported a novel function for miR‑155 in the regulation of NSCLC cell radiosensitivity, thus suggesting that miR‑155 may be considered a therapeutic target for the development of anticancer drugs.

Effects of HERV-R env Knockdown in Combination with Ionizing Radiation on Apoptosis-Related Gene Expression in A549 Lung Cancer Cells

Radiotherapy has played a key role in the management of non–small-cell lung cancer (NSCLC). However, the use of radiotherapy in treating NSCLC is limited because of the intrinsic radiation resistance of tumor cells and injury to adjacent normal tissues. Many oncogenes are reported to be involved in radioresistance. Thus, novel moleculartargeting approaches to enhance the radiosensitivity of NSCLC cells are required to improve the therapeutic efficiency of radiotherapy. In this study, we report that expression of the human endogenous retrovirus-R (HERV-R) env gene is greatly elevated in γ-irradiation resistant A549 cells compared with radiation sensitive H460 cells. In addition, the HERV-R env gene was significantly increased in A549 cells after treatment with γ-irradiation. HERV-R env knockdown by siRNA in irradiated A549 cells led to overexpression of TP53 mRNA, followed by significant elevation in the levels of CDKN1A mRNA. Moreover, the expression of the apoptosis-related FAS-1 gene was increased, whereas the expression levels of the anti-apoptotic gene BCL2 were significantly decreased in the A549 cells in which the HERV-R env was suppressed by γ-irradiation. These results suggest that knockdown of HERV-R env with γ-irradiation causes cell cycle disturbances, which in turn induces apoptosis. In conclusion, the combination of HERV-R env knockdown and γ-irradiation has the potential to improve the therapeutic efficiency of radiotherapy for NSCLC.

Knocking Down Nucleolin Expression Enhances the Radiosensitivity of Non-Small Cell Lung Cancer by Influencing DNA-PKcs Activity.

Nucleolin (C23) is an important anti-apoptotic protein that is ubiquitously expressed in exponentially growing eukaryotic cells. In order to understand the impact of C23 in radiation therapy, we attempted to investigate the relationship of C23 expression with the radiosensitivity of human non-small cell lung cancer (NSCLC) cells. We investigated the role of C23 in activating the catalytic subunit of DNA-dependent protein kinase (DNA- PKcs), which is a critical protein for DNA double-strand breaks (DSBs) repair. As a result, we found that the expression of C23 was negatively correlated with the radiosensitivity of NSCLC cell lines. In vitro clonogenic survival assays revealed that C23 knockdown increased the radiosensitivity of a human lung adenocarcinoma cell line, potentially through the promotion of radiation-induced apoptosis and adjusting the cell cycle to a more radiosensitive stage. Immunofluorescence data revealed an increasing quantity of γ-H2AX foci and decreasing radiation-induced DNA damage repair following knockdown of C23. To further clarify the mechanism of C23 in DNA DSBs repair, we detected the expression of DNA-PKcs and C23 proteins in NSCLC cell lines. C23 might participate in DNA DSBs repair for the reason that the expression of DNA-PKcs decreased at 30, 60, 120 and 360 minutes after irradiation in C23 knockdown cells. Especially, the activity of DNA-PKcs phosphorylation sites at the S2056 and T2609 was significantly suppressed. Therefore we concluded that C23 knockdown can inhibit DNA-PKcs phosphorylation activity at the S2056 and T2609 sites, thus reducing the radiation damage repair and increasing the radiosensitivity of NSCLC cells. Taken together, the inhibition of C23 expression was shown to increase the radiosensitivity of NSCLC cells, as implied by the relevance to the notably decreased DNA-PKcs phosphorylation activity at the S2056 and T2609 clusters. Further research on targeted C23 treatment may promote effectiveness of radiotherapy and provide new targets for NSCLC patients.

Elevated Serum HGF Is Associated With Radioresistance and Local Recurrence in Non-Small Cell Lung Cancer

Hepatocyte growth factor (HGF), a c-Met ligand involved in cancer tumorigenesis and metastasis, mediates resistance to radiation and drives resistance to ErbB1 inhibitors such as erlotinib. Here we tested whether plasma HGF levels correlate with outcomes after definitive radiation for non-small cell lung cancer (NSCLC) and evaluated the role of HGF in radiosensitivity of NSCLC cells with high or low c-Met expression.

Rhamnetin and Cirsiliol Induce Radiosensitization and Inhibition of Epithelial-Mesenchymal Transition (EMT) by miR-34a-mediated Suppression of Notch-1 Expression in Non-small Cell Lung Cancer Cell Lines

Radioresistance is a major cause of decreasing the efficiency of radiotherapy for non-small cell lung cancer (NSCLC). To understand the radioresistance mechanisms in NSCLC, we focused on the radiation-induced Notch-1 signaling pathway involved in critical cell fate decisions by modulating cell proliferation. In this study, we investigated the use of Notch-1-regulating flavonoid compounds as novel therapeutic drugs to regulate radiosensitivity in NSCLC cells, NCI-H1299 and NCI-H460, with different levels of radioresistance. Rhamnetin and cirsiliol were selected as candidate Notch-1-regulating radiosensitizers based on the results of assay screening for activity and pharmacological properties. Treatment with rhamnetin or cirsiliol reduced the proliferation of NSCLC cells through the suppression of radiation-induced Notch-1 expression. Indeed, rhamnetin and cirsiliol increased the expression of tumor-suppressive microRNA, miR-34a, in a p53-dependent manner, leading to inhibition of Notch-1 expression. Consequently, reduced Notch-1 expression promoted apoptosis through significant down-regulation of the nuclear factor-κB pathway, resulting in a radiosensitizing effect on NSCLC cells. Irradiation-induced epithelial-mesenchymal transition was also notably attenuated in the presence of rhamnetin and cirsiliol. Moreover, an in vivo xenograft mouse model confirmed the radiosensitizing and epithelial-mesenchymal transition inhibition effects of rhamnetin and cirsiliol we observed in vitro. In these mice, tumor volume was significantly reduced by combinational treatment with irradiation and rhamnetin or cirsiliol compared with irradiation alone. Taken together, our findings provided evidence that rhamnetin and cirsiliol can act as promising radiosensitizers that enhance the radiotherapeutic efficacy by inhibiting radiation-induced Notch-1 signaling associated with radioresistance possibly via miR-34a-mediated pathways.

SHP1-mediated cell cycle redistribution inhibits radiosensitivity of non-small cell lung cancer

BackgroundRadioresistance is the common cause for radiotherapy failure in non-small cell lung cancer (NSCLC), and the degree of radiosensitivity of tumor cells is different during different cell cycle phases. The objective of the present study was to investigate the effects of cell cycle redistribution in the establishment of radioresistance in NSCLC, as well as the signaling pathway of SH2 containing Tyrosine Phosphatase (SHP1).MethodsA NSCLC subtype cell line, radioresistant A549 (A549S1), was induced by high-dose hypofractionated ionizing radiations. Radiosensitivity-related parameters, cell cycle distribution and expression of cell cycle-related proteins and SHP1 were investigated. siRNA was designed to down-regulate SHP1expression.ResultsCompared with native A549 cells, the proportion of cells in the S phase was increased, and cells in the G0/G1 phase were consequently decreased, however, the proportion of cells in the G2/M phase did not change in A549S1 cells. Moreover, the expression of SHP1, CDK4 and CylinD1 were significantly increased, while p16 was significantly down-regulated in A549S1 cells compared with native A549 cells. Furthermore, inhibition of SHP1 by siRNA increased the radiosensitivity of A549S1 cells, induced a G0/G1 phase arrest, down-regulated CDK4 and CylinD1expressions, and up-regulated p16 expression.ConclusionsSHP1 decreases the radiosensitivity of NSCLC cells through affecting cell cycle distribution. This finding could unravel the molecular mechanism involved in NSCLC radioresistance.

Phosphorylation of Ribosomal Protein S3 and Antiapoptotic TRAF2 Protein Mediates Radioresistance in Non-small Cell Lung Cancer Cells

Radioresistance is considered as a main factor restricting efficacy of radiotherapy. However, the exact molecular mechanism of radioresistance has not been explained yet. In this study, to elucidate radioresistance mechanism in lung cancer, we compared radiation responses in two types of non-small cell lung cancer (NSCLC) cells with different radiosensitivity and identified key molecules conferring radioresistance. In radioresistant NSCLC cells, ionizing radiation (IR) led to casein kinase 2α (CK2α)- and PKC-mediated phosphorylation of rpS3 and TRAF2, respectively, which induced dissociation of rpS3-TRAF2 complex and NF-κB activation, resulting in significant up-regulation of prosurvival genes (cIAP1, cIAP2, and survivin). Also, dissociated phospho-rpS3 translocated into nucleus and bound with NF-κB complex (p65 and p50), contributing to p65 DNA binding property and specificity. However, in radiosensitive NSCLC cells, IR-mediated rpS3 phosphorylation was not detected due to the absence of CK2α overexpression. Consequently, IR-induced rpS3-TRAF2 complex dissociation, NF-κB activation, and prosurvival gene expression were not presented. Taken together, our findings revealed a novel radioresistance mechanism through functional orchestration of rpS3, TRAF2, and NF-κB in NSCLC cells. Moreover, we provided the first evidence for the function of rpS3 as a new TRAF2-binding protein and demonstrated that phosphorylation of both rpS3 and TRAF2 is a key control point of radioresistance in NSCLC cells. These results suggest that regulation of rpS3 and TRAF2 in combination with radiotherapy could have high pharmacological therapeutic potency for radioresistance of NSCLC.

Overexpression of STAT3 Potentiates Growth, Survival, and Radioresistance of Non-Small-Cell Lung Cancer (NSCLC) cells

Activation of signal transducer and activator of transcription 3 (STAT3) play important roles in tumorigenesis and tumor progression. The overexpression of STAT3 has been found in various malignancies including non-small-cell lung carcinoma (NSCLC). The purpose of this study was to explore the correlation between overexpression of STAT3 gene and growth, survival, and radiosensitivity of NSCLC cells. Subclones using vector-based short hairpin RNA (shRNA) were established. RT-PCR and Western blot assays were performed to detect the expression of STAT3 mRNA and protein in untransfected or stably transfected NSCLC cells. Then, MTT and soft agar colony assays were performed to determine the effect of STAT3 inhibition on in vitro growth of NSCLC cells. Hoechst staining assay was performed to analyze the effect of STAT3 inhibition on apoptosis of NSCLC cells. Additionally, clonogenic survival assays were performed to detect the effect of STAT3 inhibition on in vitro radiosensitivity of NSCLC cells. Finally, to examine the effect of pSUPER-shSTAT3 on proliferation and radiosensitivity in vivo, a subcutaneous (s.c.) tumor formation assay in nude mice was performed. We successfully established two stable transfected cell lines (A549/shSTAT3 and SK-MES-1/shSTAT3) in which the expression of STAT3 mRNA and protein was down-regulated. Those two stable subclones showed a significantly dramatic reduction in colony-forming ability and proliferation not only in vitro but also in vivo. The apoptotic rates of A549/shSTAT3 and SK-MES-1/shSTAT3 cells increased to 19.2% and 16.4%, respectively. Moreover, shRNA-mediated STAT3 inhibition could also significantly enhance radiosensitivity of NSCLC cells both in vitro and in vivo. Together, the overexpression of STAT3 is correlated with growth, survival, and radioresistance of NSCLC cells, and STAT3 might be a molecular therapeutic target for gene therapy or radiosensitization of NSCLC.