Hypoxic A549 Cells Research Articles

NF-κB in the Radiation Response of A549 Non-Small Cell Lung Cancer Cells to X-rays and Carbon Ions under Hypoxia.

Cellular hypoxia, detectable in up to 80% of non-small cell lung carcinoma (NSCLC) tumors, is a known cause of radioresistance. High linear energy transfer (LET) particle radiation might be effective in the treatment of hypoxic solid tumors, including NSCLC. Cellular hypoxia can activate nuclear factor κB (NF-κB), which can modulate radioresistance by influencing cancer cell survival. The effect of high-LET radiation on NF-κB activation in hypoxic NSCLC cells is unclear. Therefore, we compared the effect of low (X-rays)- and high (12C)-LET radiation on NF-κB responsive genes' upregulation, as well as its target cytokines' synthesis in normoxic and hypoxic A549 NSCLC cells. The cells were incubated under normoxia (20% O2) or hypoxia (1% O2) for 48 h, followed by irradiation with 8 Gy X-rays or 12C ions, maintaining the oxygen conditions until fixation or lysis. Regulation of NF-κB responsive genes was evaluated by mRNA sequencing. Secretion of NF-κB target cytokines, IL-6 and IL-8, was quantified by ELISA. A greater fold change increase in expression of NF-κB target genes in A549 cells following exposure to 12C ions compared to X-rays was observed, regardless of oxygenation status. These genes regulate cell migration, cell cycle, and cell survival. A greater number of NF-κB target genes was activated under hypoxia, regardless of irradiation status. These genes regulate cell migration, survival, proliferation, and inflammation. X-ray exposure under hypoxia additionally upregulated NF-κB target genes modulating immunosurveillance and epithelial-mesenchymal transition (EMT). Increased IL-6 and IL-8 secretion under hypoxia confirmed NF-κB-mediated expression of pro-inflammatory genes. Therefore, radiotherapy, particularly with X-rays, may increase tumor invasiveness in surviving hypoxic A549 cells.

β-Caryophyllene Inhibits Endothelial Tube Formation by Modulating the Secretome of Hypoxic Lung Cancer Cells-Possible Role of VEGF Downregulation.

β-Caryophyllene (BCP), a bicyclic sesquiterpene that is a component of the essential oils of various spice and food plants, has been described as a selective CB2 cannabinoid receptor agonist. In the present study, the effect of BCP on angiogenesis was investigated. It was found that conditioned media (CM) from BCP-treated hypoxic A549 lung cancer cells exhibited a concentration-dependent inhibitory effect on human umbilical vein endothelial cell (HUVEC) tube formation induced by CM from vehicle-treated hypoxic A549 cells. There was an associated concentration-dependent decrease in the proangiogenic factor vascular endothelial growth factor (VEGF) in the CM, with both BCP inhibitory effects (tube formation, VEGF secretion) being CB2 receptor-dependent. A reduction of the transcription factor hypoxia-inducible factor 1α (HIF-1α) was furthermore detected. The antiangiogenic and VEGF-lowering properties of BCP were confirmed when CM from another lung cancer cell line, H358, were tested. When directly exposed to HUVECs, BCP showed no significant effect on tube formation, but at 10 µM, impaired VEGF receptor 2 (VEGFR2) phosphorylation triggered by recombinant VEGF in a CB2 receptor-independent manner. In summary, BCP has a dual antiangiogenic effect on HUVECs, manifested in the inhibition of tube formation through modulation of the tumor cell secretome and additionally in the inhibition of VEGF-induced VEGFR2 activation. Because the CB2 agonist has no psychoactive properties, BCP should continue to be evaluated preclinically for further antitumor effects.

Roles of hypoxic environment and M2 macrophage-derived extracellular vesicles on the progression of non-small cell lung cancer

BackgroundHypoxia contributes to the development of invasive and metastatic cancer cells, and is detrimental to cancer treatment. This study aimed to explore the molecular mechanisms by which hypoxic microenvironments affect hypoxic non-small cell lung cancer (NSCLC) development and the effects of M2 macrophage-derived extracellular vesicles (EVs) on NSCLC cells.MethodsA549 cells were cultured in an anoxic incubator for 48 h to construct hypoxic A549 cells, and then normal and hypoxic A549 cells were harvested for RNA sequencing. Next, THP-1 cells were used to induce M2 macrophages, and EVs were isolated from THP-1 cells and M2 macrophages. Cell counting kit-8 and transwell assays were used to determine the viability and migration of hypoxic A549 cells, respectively.ResultsAfter sequencing, 2426 DElncRNAs and 501 DEmiRNAs were identified in normal A549 cells and hypoxic A549 cells. These DElncRNAs and DEmiRNAs were significantly enriched in “Wnt signaling pathway,” “Hippo signaling pathway,” “Rap1 signaling pathway,” “calcium signaling pathway,” “mTOR signaling pathway,” and “TNF signaling pathway.” Subsequently, ceRNA networks consisting of 4 lncRNA NDRG1 transcripts, 16 miRNAs and 221 target mRNAs were built, and the genes in the ceRNA networks were significantly associated with “Hippo signaling pathway” and “HIF-1 signaling pathway.” EVs were successfully extracted from THP-1 cells and M2 macrophages, and M2 macrophage-derived EVs significantly enhanced the viability and migration of hypoxic A549 cells. Finally, M2 macrophage-derived EVs further upregulated the expression of NDRG1-009, NDRG1-006, VEGFA, and EGLN3, while downregulating miR-34c-5p, miR-346, and miR-205-5p in hypoxic A549 cells.ConclusionsM2 macrophage-derived EVs may worsen the progression of NSCLC in a hypoxic microenvironment by regulating the NDRG1-009-miR-34c-5p-VEGFA, NDRG1-006-miR-346-EGLN3, NDRG1-009-miR-205-5p-VEGFA, and Hippo/HIF-1 signaling pathways.

Cyclophilin a represses reactive oxygen species generation and death of hypoxic non-small-cell lung cancer cells by degrading thioredoxin-interacting protein

ABSTRACT Cyclophilin A (cypA) is overexpressed in many types of carcinomas, including non-small-cell lung cancer (NSCLC). However, the effect of anoxia, a critical feature of the carcinoma cell microenvironment, on cypA expression in NSCLC is unknown. Here, formaldehyde-fixed and paraffin-embedded samples were collected from 60 subjects with NSCLC. The protein expression levels of cypA and hypoxia-inducible factor-1α (HIF-1α) were evaluated using immunohistochemistry. Kaplan–Meier analysis showed that subjects with high cypA expression had remarkably shorter progression-free survival than those with low cypA expression. Furthermore, cypA expression levels were significantly related to HIF-1α expression levels (Spearman’s correlation = 0.34, P < 0.0001). To further assess the effect of cypA, an anoxic carcinoma cell model was established. CypA expression was remarkably upregulated in H1299 and A549 cell lines under hypoxic conditions. Overexpression of cypA restored hypoxia-impaired cell growth and prevented reactive oxygen species (ROS) production and cell death in hypoxic A549 and H1299 cells. However, these phenotypes were not altered by the inactive R55A mutant of cypA. Mechanistic studies demonstrated that cypA can bind to and degrade the tumor suppressor protein TXNIP in H1299 and A549 cells. Restored TXNIP expression in cypA-overexpressed and hypoxic NSCLC cells led to increased ROS levels and apoptotic cell numbers and decreased cell growth compared with cypA-overexpressed and hypoxic NSCLC cells. These findings indicate that anoxia results in an increase in cypA expression in NSCLC. Additionally, cypA served as an oncogene during hypoxia by interacting with TXNIP.

FDA approved L-type channel blocker Nifedipine reduces cell death in hypoxic A549 cells through modulation of mitochondrial calcium and superoxide generation

As hypoxia is a major driver for the pathophysiology of COVID-19, it is crucial to characterize the hypoxic response at the cellular and molecular levels. In order to augment drug repurposing with the identification of appropriate molecular targets, investigations on therapeutics preventing hypoxic cell damage is required. In this work, we propose a hypoxia model based on alveolar lung epithelial cells line using chemical inducer, CoCl2 that can be used for testing calcium channel blockers (CCBs). Since recent studies suggested that CCBs may reduce the infectivity of SARS-Cov-2, we specifically select FDA approved calcium channel blocker, nifedipine for the study. First, we examined hypoxia-induced cell morphology and found a significant increase in cytosolic calcium levels, mitochondrial calcium overload as well as ROS production in hypoxic A549 cells. Secondly, we demonstrate the protective behaviour of nifedipine for cells that are already subjected to hypoxia through measurement of cell viability as well as 4D imaging of cellular morphology and nuclear condensation. Thirdly, we show that the protective effect of nifedipine is achieved through the reduction of cytosolic calcium, mitochondrial calcium, and ROS generation. Overall, we outline a framework for quantitative analysis of mitochondrial calcium and ROS using 3D imaging in laser scanning confocal microscopy and the open-source image analysis platform ImageJ. The proposed pipeline was used to visualize mitochondrial calcium and ROS level in individual cells that provide an understanding of molecular targets. Our findings suggest that the therapeutic value of nifedipine may potentially be evaluated in the context of COVID-19 therapeutic trials.

MicroRNA-130a targeting hypoxia-inducible factor 1 alpha suppresses cell metastasis and Warburg effect of NSCLC cells under hypoxia

MicroRNAs have been demonstrated to play critical role in the development of non-small cell lung cancer (NSCLC) and hypoxia is a common hallmark of NSCLC. MiRNA-130a-3p (miR-130a) is a well-known tumor suppressor, and we intended to explore the role and mechanism of miR-130a in NSCLC cells under hypoxia. We used real-time quantitative polymerase chain reaction method to measure miR-130a expression, and found that miR-130a was downregulated in human NSCLC tumors and cell lines (A549 and H1299), accompanied with upregulation of hypoxia-inducible factor 1 alpha (HIF1A), a marker of hypoxia. Besides, miR-130a low expression was associated with tumor burden and poor overall survival. Moreover, miR-130a expression was even downregulated in hypoxia-treated A549 and H1299 cells. Ectopic expression of miR-130a suppressed Warburg effect, migration and invasion in hypoxic A549 and H1299 cells, as evidenced by decreased glucose consumption, lactate production, hexokinase 2 expression, and numbers of migration cells and invasion cells analyzed by commercial glucose and lactate assay kits, western blotting and transwell assays. Furthermore, overexpression of miR-130a restrained xenograft tumor growth of A549 cells in mice. However, recovery of HIF1A could reverse the suppressive effect of miR-130a overexpression on cell migration, invasion and Warburg effect in hypoxic A549 and H1299 cells. Mechanically, dual-luciferase reporter assay, RNA immunoprecipitation and RNA pull-down assay confirmed a target relationship between miR-130a and HIF1A. Collectively, we demonstrated an anti-tumor role of miR-130a in NSCLC cells under hypoxia through targeting HIF1A, suggesting a potential target for the interfering of NSCLC.

Targeted Myoglobin Delivery as a Strategy for Enhancing the Sensitivity of Hypoxic Cancer Cells to Radiation.

SummaryThe effectiveness of cancer radiotherapy is frequently hindered by the hypoxia of the tumor microenvironment. Direct delivery of oxygen to hypoxic tumor tissues is an attractive strategy to overcome this hypoxia-associated radioresistance. Herein, we report the generation of submicron-sized particles comprising myoglobin fused to the crystal-forming domain of Cry3Aa protein for the targeted delivery of oxygen to cancer cells. We demonstrate that myoglobin-containing particles were successfully produced in Bacillus thuringiensis with the assistance of the Cry3Aa domain I. Furthermore, these particles could be genetically modified to incorporate the cell penetrating peptide TAT and cell targeting peptide A549.1, resulting in particles that exhibited improved cellular uptake and targeting toward A549 cells. Notably, these myoglobin-containing particles increased the intracellular oxygen levels of A549 cells and thereby sensitized them to radiation. These findings suggest that the targeted delivery of O2-bound myoglobin could be an effective approach to enhance the efficacy of radiotherapy.

Radiosensitizing effects of Cyclocaryapaliurus polysaccharide on hypoxic A549 and H520 human non-small cell lung carcinoma cells.

Cyclocarya paliurus (CP) polysaccharide (CPP) is a chemical component contained in CP, which has been reported to possess significant hypoglycemic activity. The present study aimed to investigate the radiosensitizing effect and underlying mechanisms of CPP on hypoxic A549 and H520 human non-small cell lung carcinoma cells. Cell viability, apoptosis and proliferation were determined using Cell Counting kit-8 assay, flow cytometry and colony formation assay, respectively. mRNA and protein expression levels were determined by reverse transcription-quantitative PCR and western blot analysis, respectively. The results suggested that CPP markedly inhibited the viability of hypoxic A549 and H520 cells. In response to combined treatment with CPP and radiation, hypoxic A549 and H520 cells exhibited enhanced apoptosis; in addition, cell proliferation was suppressed and the expression levels of hypoxia-inducible factor-1α, survivin and cleaved caspase-3 were modified. Furthermore, CPP in combination with radiation affected the mammalian target of rapamycin (mTOR)/Akt/phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) pathway. These findings indicated that CPP may enhance the radiosensitivity of hypoxic A549 and H520 cells; this effect may be associated with inhibition of the mTOR/Akt/PI3K pathway. The potential radiosensitizing effects of CPP on hypoxic A549 and H520 cells suggested that CPP may be an effective target for treatment of non-small cell lung carcinoma.

Aspirin inhibits hypoxia-mediated lung cancer cell stemness and exosome function

BackgroundEpidemiological studies have illustrated that regular aspirin consumption may decrease the risk of non-small cell lung cancer (NSCLC). The present study aims to investigate the mechanism of aspirin-induced inhibition of NSCLC development during hypoxia. MethodsA549 cells were pre-treated with the vehicle control or aspirin and then subjected to hypoxic culture. Cell viability was monitored by CCK-8 assay, and flow cytometry was performed to detect cell cycle distributions, apoptosis, and proportion of cancer stem cells (CSCs). Flow cytometric cell sorting was used to separate CSCs. Quantitative reverse transcription–polymerase chain reaction and Western blot were used to detect the mRNA and protein levels of stem cell markers and the related signaling molecules. The abundance of prostaglandin E2 was detected by enzyme-linked immunosorbent assay. Exosomes in the cell culture medium were isolated using ExoQuick, and the number of exosomes was quantified by the EXOCET exosome quantification assay kit. Cell migration and angiogenesis were monitored by transwell migration assay and in vitro angiogenesis experiments. ResultsAspirin inhibited cell proliferation and induced G2/M cell cycle arrest in hypoxic A549 cells; it also inhibited hypoxia-enhanced stemness in both A549 and ALDH+ cells. The drug reduced hypoxia-enhanced numbers of exosomes in A549 cells and exerted negative effects on the hypoxia-mediated up-regulation of exosomal HIF-1α/COX-2 and expression of exosomal miR-135b and miR-210. While hypoxic-induced exosomes can promote the proliferation, migration, and angiogenesis of other A549 cells, aspirin can weaken this promotion by reducing the amount of exosome secreted and changing exosome contents. ConclusionsAspirin inhibits the hypoxia-induced stemness, hypoxic-mediated exosome release, and malignant paracrine effects of A549 cells.

YC-1 Prevents Tumor-Associated Tissue Factor Expression and Procoagulant Activity in Hypoxic Conditions by Inhibiting p38/NF-κB Signaling Pathway.

Tissue factor (TF) expressed in cancer cells has been linked to tumor-associated thrombosis, a major cause of mortality in malignancy. Hypoxia is a common feature of solid tumors and can upregulate TF. In this study, the effect of YC-1, a putative inhibitor of hypoxia-inducible factor-1α (HIF-1α), on hypoxia-induced TF expression was investigated in human lung cancer A549 cells. YC-1 selectively prevented hypoxia-induced TF expression and procoagulant activity without affecting the basal TF levels. Surprisingly, knockdown or pharmacological inhibition of HIF-1α failed to mimic YC-1′s effect on TF expression, suggesting other mechanisms are involved. NF-κB, a transcription factor for TF, and its upstream regulator p38, were activated by hypoxia exposure. Treatment of hypoxic A549 cells with YC-1 prevented the activation of both NF-κB and p38. Inhibition of p38 suppressed hypoxia-activated NF-κB, and inhibited TF expression and activity to similar levels as treatment with an NF-κB inhibitor. Furthermore, stimulation of p38 by anisomycin reversed the effects of YC-1. Taken together, our results suggest that YC-1 prevents hypoxia-induced TF in cancer cells by inhibiting the p38/NF-κB pathway, this is distinct from the conventional anticoagulants that systemically inhibit blood coagulation and may shed new light on approaches to treat tumor-associated thrombosis.

Chloramphenicol Induces Autophagy and Inhibits the Hypoxia Inducible Factor-1 Alpha Pathway in Non-Small Cell Lung Cancer Cells.

Chloramphenicol is an inexpensive and excellent bactericidal antibiotic. It is used to combat anaerobic infections in the Third World countries, whereas its systemic application has been abandoned in developed countries. However, in recent years, clinicians have reintroduced chloramphenicol in clinical practice. In this study, chloramphenicol was found to repress the oxygen-labile transcription factor, hypoxia inducible factor-1 alpha (HIF-1α), in hypoxic A549 and H1299 cells. Furthermore, it suppressed the mRNA levels of vascular endothelial growth factor (VEGF) and glucose transporter 1, eventually decreasing VEGF release. Chloramphenicol initiated the autophagy pathway in treated cells, as observed by the increase in formation of Atg12-Atg5 conjugates, and in beclin-1 and LC3-II levels. The chloramphenicol-mediated HIF-1α degradation was completely reverted by autophagic flux blockage. In HIF-1α-overexpressing cells, the formation of HIF-1α/SENP-1 (Sentrin/SUMO-specific protease 1) protein complex seemed to facilitate the escape of HIF-1α from degradation. Chloramphenicol inhibited HIF-1α/SENP-1 protein interaction, thereby destabilizing HIF-1α protein. The enhancement in HIF-1α degradation due to chloramphenicol was evident during the incubation of the antibiotic before hypoxia and after HIF-1α accumulation. Since HIF-1α plays multiple roles in infections, inflammation, and cancer cell stemness, our findings suggest a potential clinical value of chloramphenicol in the treatment of these conditions.

TRAIL overexpression co-regulated by Egr1 and HRE enhances radiosensitivity of hypoxic A549 cells depending on its apoptosis inducing role.

Ionizing radiation can upregulate the expression levels of TRAIL and enhance tumor cell apoptosis. While Early growth response1 (Egr1) gene promoter has radiation inducible characteristics, the expression for exogenous gene controlled by Egr1 promoter could be enhanced by ionizing radiation, but its efficiency is limited by tissue hypoxia. Hypoxia response elements (HREs) are important hypoxic response regulatory sequences and sensitivity enhancers. Therefore, we chose TRAIL as the gene radiotherapy to observe whether it is regulated by Egr1 and HER and its effects on A549 cells and its mechanism. The pcDNA3.1-Egr1-TRAIL (pc-E-hsT) and pcDNA3.1-HRE/Egr1-TRAIL (pc-H/E-hsT) plasmids containing Egr1-hsTRAIL and HRE/Egr1-hsTRAIL were transfected into A549 cells, the cells were treated by hypoxia and radiation. The TRAIL mRNA in the cells and protein concentration in the culture supernatants were measured by RT-PCR and ELISA, respectively. Mean lethal dose D0value was evaluated with colony forming assay. The cell apoptotic rates were analyzed by FCM and TUNEL assay. Expression of DR4, DR5 and cleaved caspase-3 proteins were analyzed by western blotting. It showed that TRAIL mRNA expression and TRAIL concentration all significantly increased under hypoxia and/or radiation. D0value of pc-H/E‑hsT transfected cells under hypoxia was lowest, indicating more high radiosensitivity. Hypoxia could not cause the pc-E-hsT transfected cell apoptotic rate increase, but there were promoting effects in pc-H/E-hsT transfected cells. DR4 had not obvious change in pc-E-hsT and pc-H/E-hsT transfected cells under normoxic and hypoxic condition, otherwise, DR5 and cleaved caspase-3 increased mostly in pc-H/E-hsT transfected cells under hypoxic condition. TRAIL overexpression was co-regulated by Egr1 and HRE. TRAIL might promote hypoxic A549 cell radiosensitivity and induce apoptosis depending on DR5 to caspase-3 pathways.

Lysyl oxidase mediates hypoxia-induced radioresistance in non-small cell lung cancer A549 cells.

Hypoxia-induced radioresistance has been well known as the main obstacle in cancer radiotherapy. Lysyl oxidase (LOX) was previously demonstrated to play an important role in hypoxia-induced biological behaviors, such as metastasis and angiogenesis, through hypoxia-inducible factor-1α (HIF-1α), which is an important contributing factor to radioresistance in tumor cells. However, how LOX plays a role in hypoxia-induced radioresistance has yet to be determined. Here, we found that LOX expression was in accordance with HIF-1α expression, and LOX expression at the mRNA and protein level, and enzymatic activity were remarkably upregulated in the hypoxic A549 cells, compared with normoxic A549 cells. Inhibition of LOX resulted in the reduction of the ability to repair double-stranded breaks (DSBs), promotion of apoptosis, relief of G2/M cycle arrest, and eventually reduction of hypoxia-induced radioresistance in the hypoxic A549 cells. This suggests that LOX may play an important role in hypoxia-induced radioresistance. Together, our results might suggest a novel potential therapeutic target in the management of non-small cell lung cancer (NSCLC).

LW6, a hypoxia-inducible factor 1 inhibitor, selectively induces apoptosis in hypoxic cells through depolarization of mitochondria in A549 human lung cancer cells.

Hypoxia-inducible factor 1 (HIF-1) activates the transcription of genes that act upon the adaptation of cancer cells to hypoxia. LW6, an HIF-1 inhibitor, was hypothesized to improve resistance to cancer therapy in hypoxic tumors by inhibiting the accumulation of HIF-1α. A clear anti-tumor effect under low oxygen conditions would indicate that LW6 may be an improved treatment strategy for cancer in hypoxia. In the present study, the HIF-1 inhibition potential of LW6 on the growth and apoptosis of A549 lung cancer cells in association with oxygen availability was evaluated. LW6 was observed to inhibit the expression of HIF-1α induced by hypoxia in A549 cells at 20 mM, independently of the von Hippel-Lindau protein. In addition, at this concentration, LW6 induced hypoxia-selective apoptosis together with a reduction in the mitochondrial membrane potential. The intracellular reactive oxygen species levels increased in LW6-treated hypoxic A549 cells and LW6 induced a hypoxia-selective increase of mitochondrial O2•−. In conclusion, LW6 inhibited the growth of hypoxic A549 cells by affecting the mitochondria. The inhibition of the mitochondrial respiratory chain is suggested as a potentially effective strategy to target apoptosis in cancer cells.

Ultrasensitive near-infrared fluorescence-enhanced probe for in vivo nitroreductase imaging.

Nitroreductase (NTR) can be overexpressed in hypoxic tumors, thus the selective and efficient detection of NTR is of great importance. To date, although a few optical methods have been reported for the detection of NTR in solution, an effective optical probe for NTR monitoring in vivo is still lacking. Therefore, it is necessary to develop a near-infrared (NIR) fluorescent detection probe for NTR. In this study, five NIR cyanine dyes with fluorescence reporting structure decorated with different nitro aromatic groups, Cy7-1-5, have been designed and explored for possible rapid detection of NTR. Our experimental results presented that only a para-nitro benzoate group modified cyanine probe (Cy7-1) could serve as a rapid NIR fluorescence-enhanced probe for monitoring and bioimaging of NTR. The structure-function relationship has been revealed by theoretical study. The linker connecting the detecting and fluorescence reporting groups and the nitro group position is a key factor for the formation of hydrogen bonds and spatial structure match, inducing the NTR catalytic ability enhancement. The in vitro response and mechanism of the enzyme-catalyzed reduction of Cy7-1 have been investigated through kinetic optical studies and other methods. The results have indicated that an electro-withdrawing group induced electron-transfer process becomes blocked when Cy7-1 is catalytically reduced to Cy7-NH2 by NTR, which is manifested in enhanced fluorescence intensity during the detection process. Confocal fluorescence imaging of hypoxic A549 cells has confirmed the NTR detection ability of Cy7-1 at the cellular level. Importantly, Cy7-1 can detect tumor hypoxia in a murine hypoxic tumor model, showing a rapid and significant enhancement of its NIR fluorescence characteristics suitable for fluorescence bioimaging. This method may potentially be used for tumor hypoxia diagnosis.

Effects of hypoxia/radiation dual-sensitive promoter-Smac plasmid combined with X-rays irradiation on proliferation of hypoxic A549 cells

Objective To construct recombinant expression vector of Smac gene mediated by hypoxia/radiation dual-sensitive promoter (HRE/Egrl),and observe the expression characteristics in A549 cells under normoxic and hypoxic conditions after transfection,and the inhibitory effects on proliferation under hypoxic condition.Methods The plasmid pcDNA3.1-Egrl-Smac and pcDNA3.1-HRE/Egrl-Smac were successfully constructed,and transfected into A549 cells.The expression levels of mRNA and protein were detected by using real-time quantitative polymerase chain reaction (Real-time PCR) and Western blotting,respectively.Cell proliferation changes were measured by methyl thiazol tetrazolium (MTT) as-say.Results Under normoxic condition,as compared with control group,Smac mRNA was significantly increased in A549 cells irradiated with 2 Gy or transfected and irradiated with 2 Gy (P ＜ 0.05).Hypoxia could significantly enhance the effects (P ＜ 0.05).At the same time,Smac protein had similar expression characteristics.Under normoxic and hypoxic conditions,at the same time point,as compared with control group,proliferation was significantly decreased in A549 cells irradiated with 2 Gy or transfected and irradiated with 2 Gy (P ＜ 0.05),and proliferation in each group under hypoxic condition was more lower than that in each group under normoxic condition (P ＜ 0.05).Conclusion Both hypoxia and radiation could enhance the Smac expression in A549 cells,and combination of hypoxia and 2 Gy irradiation could significantly inhibit proliferation of A549 cells. Key words: X-rays ; Hypoxia; Lung adenocarcinoma; Proliferation

Effect of YC-1 on improving hypoxia and radiosensitizing human lung adenocarcinoma cellsin vitro

Objective To investigate the radiosensitizing effect of 3-(5'-hydroxy-2'-furyl)-1-benzyl indazole ( YC-1 ) on hypoxic human adenocarcinoma cell line A549.Methods MTT assay was used to test the inhibitory effect of YC-1 on proliferation of A549 cells.Clonogenic assay was performed to determine the radiosensitizing effect of YC-1 on hopxic A549 cells.Single-hit multi-target model was used to plot survival curve and calculate sensitization enhancement ratio (SER).The cell cycle and apoptosis were measured by flow cytometry.Results The proliferation of A549 cells was inhibited by YC-1 in a time-dose-dependent manner.In normoxic and hypoxic cells,the IC20 was 16.7 μmol/L and 39.2 μmol/L at 24 h,respectively.In the group of hypoxia plus YC-1,SERD0 and SERDq were 1.11 and 1.26,respectively.In hypoxia,YC-1combined with 2 Gy irradiation could induce cell apoptosis and prolong G2 + M phase arrest ( ( 30.17 ±1.21 )％ ∶ ( 15.44 ±0.96) ％,P =0.000; (21.56 ±0.47 )％ ∶ (6.16 ±0.16)％,P =0.000).Concinsions YC-1 could enhance the radiosensitivity of hypoxic A549 cells. Key words: 3-(5'-hydroxy-2'-furyl)-1-benzyl indazole; Radiosensitivity agents; A549 cell line

Arsenic trioxide inhibits the growth of human lung cancer cell lines via cell cycle arrest and induction of apoptosis at both normoxia and hypoxia

Arsenic trioxide (As(2)O(3)) has been established to be an effective agent for treating acute promyleocytic leukemia. Laboratory data suggest that As(2)O(3) induces apoptosis of several solid tumor cells including lung cancer cells. Regions of tissue hypoxia often arise in aggressive solid tumors, and hypoxic tumors exhibit augmented invasiveness and metastatic ability in several malignancies. Furthermore, hypoxia may impair the treatment efficiency; therefore, we studied the cytotoxic effect of As(2)O(3) on human lung adenocarcinoma cell lines A549 and A549/R (resistant to vincristine, adriamycin and mitomycin etc.) grown under normoxic and hypoxic (1% oxygen) conditions. At both normoxia and hypoxia, 5, 10 and 15 microM As(2)O(3) induced evident growth inhibition and apoptosis in A549 cells as well as A549/R cells after 48 hours of exposure. In contrast, the conventional chemotherapeutic drug vincristine showed lowered efficiency in hypoxic A549 cells. As(2)O(3) induced G(2)/M cell cycle arrest in both normoxic and hypoxic A549 cells. As(2)O(3) significantly decreased the messenger RNA (mRNA) levels of Cyclin B(1) and survivin and the protein levels of Cyclin B(1), phospho-CDC(2) (Thr 161) and survivin in both normoxic and hypoxic A549 cells. Together, our findings indicated that As(2)O(3) significantly inhibited the proliferation of lung cancer cells via G(2)/M cell cycle arrest and induction of apoptosis at both normoxia and hypoxia, and the induction of apoptosis was associated with down regulation of survivin.

Radiolytic activation of a cytarabine prodrug possessing a 2-oxoalkyl group: one-electron reduction and cytotoxicity characteristics

An anti-tumour agent of cytarabine (ara-C) was conjugated with a 2-oxopropyl group at the N(4) position to obtain a radiation-activated prodrug (oxo-ara-C) that targeted hypoxic tumour tissues with selective cytotoxicity. The parent anti-tumour agent, ara-C, was confirmed to be released from oxo-ara-Cvia one-electron reduction upon hypoxic X-ray treatment. The prodrug oxo-ara-C had dramatically reduced cytotoxicity against human lung adenocarcinoma A549 cells relative to ara-C because of the effect of 2-oxopropyl substituent. In contrast, X-ray treatment of hypoxic A549 cells containing oxo-ara-C enhanced the cytotoxic effect, indicating that toxic ara-C was preferentially released in hypoxic cells via radiolytic one-electron reduction by hydrated electrons (e(aq)(-)).

Hypoxia-inducible-factor 2 alpha mediates cisplatin resistance in the lung adenocarcinoma cell line A549

10573 Background: Hypoxia in solid tumors is associated with cancer progression, metastasis and resistance to chemotherapy. The hypoxia inducible factors (HIF-1a and HIF-2a) are key players in the response to hypoxia affecting angiogenesis, proliferation and apoptosis. Here we investigated the role of HIF-1a and HIF-2a in cellular resistance to cisplatin in non small lung cancer cells (A549). Results: Hypoxia reduced the efficiency of cisplatin treatment on A549 cells via enhancement of drug efflux activity. In addition, hypoxia induced the hypoxia-responsive-element (HRE) reporter gene assay, demonstrating the transcriptional activity of HIF-1a and HIF-2a. Administration of cisplatin further increased the reporter activity under both normoxic and hypoxic conditions. This correlated with the induction of HIF-2a protein expression upon cisplatin treatment in normoxia and hypoxia, whereas HIF-1a was suppressed. Additionally, cisplatin induced nuclear accumulation of HIF-2a. Suppression of HIF-2a by RNA interference potentiated the sensitivity to cisplatin treatment in hypoxic A549 cells. In vivo growth delay assay gave analogues results for subcutaneous A549 tumors treated with cisplatin and suppression of HIF-2a. In comparison, other cytostatic drugs such as doxorubicin and gemcitabine differed with respect to HIF signalling. Doxorubicin induced both HIF-1a and HIF- 2a. Cellular sensitivity to doxorubicin was increased via suppression of HIF-1a and HIF-2a. Gemcitabine appeared to act independent of HIF. The underlying mechanism of hypoxia-induced resistance was due to increased mRNA expression of drug resistance proteins such as MDR1, MRP1 and LRP. These were found to be HIF-2a but not HIF-1a dependent. Conclusion: This data identified HIF-2a as a key player in the hypoxia induced cisplatin resistance and show on the molecular level that HIF-2a is a credible target for therapeutic strategies. No significant financial relationships to disclose.