Human Non-small Lung Cancer A549 Research Articles

Abstract 888: Elucidating the mechanisms of eATP-induced changes in cell growth, energy metabolism, and cancer stem cells in human non-small lung cancer A549 cells

Abstract The tumor microenvironment (TME) has concentrations of extracellular ATP (eATP) 103-104 times higher than normal healthy tissues, implying that eATP may have important roles in tumor development and progression. Our previous study, cited by a Nature Reviews Cancer paper, showed that the extracellular ATP (eATP) is internalized by tumors via macropinocytosis, a type of endocytosis, to increase intracellular ATP (iATP) levels and support tumor cells growth, drug resistance, and cancer cell movement by initiating epithelial to mesenchymal transition (EMT). Recently, our lab found that eATP functions in promoting cancer cell growth, metastasis are similar to those of TGF-β, a well-known EMT and cancer stem cells (CSCs) inducer. Increasing reports state that the EMT and CSCs share some molecular links, but the detailed mechanisms and inductor of them are still far from clear. In this report, eATP’s function in CSCs induction was investigated first. Result shows that eATP treatment significantly induces the protein expression level of CSC related makers, increases cell colony numbers, and cell populations with CSC markers expressed in human lung cancer A549 cells. In addition, RNA-sequencing analysis reveals the gene Stanniocalcin 1 (STC1) is significantly upregulated in both eATP and TGF-β treatments. STC1 gene has been reported to be a competitive biomarker in the tracking of cancers’ progressing. Besides, STC1 exhibits concentration-dependent stimulation effects on electron transportation chain (ETC) and respiration. However, the detail function of STC1 gene in eATP induced cell growth is unknown. To find out the mechanism, CRISPR-Cas9 technique was used to generate STC1 gene targeted knockout (STC1 KO) A549 cells. STC1 KO cells show slower cell growth rate, lower intracellular ATP level and lower oxygen consumption rate (OCR) when compared with normal A549 cells in vitro. These results strongly indicate STC1 gene involved in eATP induced cell growth by alteration iATP level and metabolic profile. Overall, this study not only unravels new role of eATP in CSC induction, but also provides new mechanism of eATP induced cell growth by altering the protein expression level of STC1. Citation Format: Jingwen Song, Xiaozhuo Chen. Elucidating the mechanisms of eATP-induced changes in cell growth, energy metabolism, and cancer stem cells in human non-small lung cancer A549 cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 888.

Screening of Apoptosis Pathway-Mediated Anti-Proliferative Activity of the Phytochemical Compound Furanodienone against Human Non-Small Lung Cancer A-549 Cells.

Furanodienone (FDN), a major bioactive component of sesquiterpenes produced from Rhizoma Curcumae, has been repeatedly acknowledged for its intrinsic anticancer efficacy against different types of cancer. In this study, we aimed to investigate the cytotoxic potential of furanodienone against human lung cancer (NSCLC A549) cells in vitro, as well as its underlying molecular mechanisms in the induction of apoptosis. Herein, we found that FDN significantly inhibited the proliferation of A549 cells in a dose-dependent manner. In addition, treatment with FDN potentially triggered apoptosis in A549 cells via not only disrupting the nuclear morphology, but by activating capsase-9 and caspase-3 with concomitant modulation of the pro- and antiapoptotic gene expression as well. Furthermore, FDN revealed its competence in inducing cell cycle arrest at G0/G1 phase in A549 cells, which was associated with decreased expression of cyclin D1 and cyclin-dependent kinase 4 (CDK4), along with increased expression of CDK inhibitor p21Cip1. Intriguingly, FDN treatment efficiently downregulated the Wnt signaling pathway, which was correlated with increased apoptosis, as well as cell cycle arrest, in A549 cells. Collectively, FDN might represent a promising adjuvant therapy for the management of lung cancer.

A Mesoporous Gd-MOF with Lewis Basic Sites for 5-Fu Delivery and Inhibition of Human Lung Cancer Cells In Vivo and In Vitro

A hydrolytically stable mesoporous Gd(III)-MOF {[Gd2(TATAB)2]·6 DMF}n (1) has been successfully prepared by reaction of Gd(NO3)3·6H2O with a flexible ligand 4,4′,4′′-s-triazine-1,3,5-triyltri-p-aminobenzoate (H3TATAB) under solvothermal conditions. Further, the nanostructure 1 could be obtained via an ultrasonic treatment method. Nitrogen adsorption measurements revealed the presence of mesoporosity as well as a moderate high BET surface areas in the activated nanostructure 1 (1a), which was further used in the 5-Fu loading with the uptake capacity of 47 wt%. Meanwhile, the controlled release of 5-Fu in a simulated human body with liquid phosphate-buffered saline solution was realized. In the biological study, the anti-proliferation activity of 5-Fu@1a on A549 human non-small lung cancer (NSCLC) cells after 5-Fu@1a exposure was evaluated by the CCK-8 preformation. The effect of 5-Fu@1a on A549 NSCLC cell migration and invasion ability was detected by transwell assay. The mechanism was revealed by the detection of the NLRP3 inflammasome activation level in A549 through western blot and ELISA assay. For further explore the inhibitory effect of 5-Fu@1a, the anti-tumor ability was evaluated in the in vivo xenograft model.

Enhanced treatment effect of nanoparticles containing cisplatin and a GSH-reactive probe compound

Cisplatin is a highly effective antitumor drug, which can kill cancer cells by crossing-linking DNA and inhibiting transcription, but this process is limited by the combination of cisplatin and many endogenous nucleophiles, such as glutathione (GSH). Thus, when cisplatin enter cells, it is potentially vulnerable to cytoplasmic inactivation by GSH. To settle this bottleneck, we designed and synthesized a probe compound (Probe 1) and fabricated pH-responsed cisplatin, Probe 1-loaded lipid-polymer hybrid NanoParticles (CPNPs) using a single-step sonication method. Probe 1 can specifically bind to GSH, thus avoiding the combination of GSH and cisplatin, and enhancing the pharmacological activity of cisplatin. In vitro studies have suggested CPNPs, compared with cisplatin, loaded lipid-polymer hybrid NanoParticles CNPs (Not contain Probe 1), could efficiently kill MCF-7 human breast cancer cells and A549 human nonsmall lung cancer cell. Hence, the CPNPs provided a new idea for treating cancer.

The Apoptotic and Anti-apoptotic Effects of Pendimethalin and Trifluralin on A549 Cells In Vitro.

Pendimethalin and trifluralin are commonly used in many countries to control broadleaf weeds and grassy weed species because of their inhibitor effects on growth and cell division. In this study, we examined the apoptotic and anti-apoptotic potentials of pendimethalin and trifluralin on A549 human non-small lung cancer cells with several concentrations in vitro. The expression levels of apoptosis-related genes BCL-2, BAX, CAS3, CAS9, P53, BIRC, and PPIA were examined using quantitative RT-PCR after 24 h treatment of 1, 10, 50, 100 and 500 μM pendimethalin and trifluralin. The effects of pendimethalin were found more repressive than trifluralin on all studied concentrations. Twenty-four hours' exposure with 100 μM pendimethalin and trifluralin altered the gene expressions, suppressing apoptosis and allowing cancer cells to grow and proliferate. Care should be taken not to exceed the permissible values and residue limits in food during pendimethalin and trifluralin use in order to reduce the possible carcinogenic effects on humans.

Projection-Based 3D Printing of Cell Patterning Scaffolds with Multiscale Channels.

To fully actualize artificial, cell-laden biological models in tissue engineering, such as 3D organoids and organs-on-a-chip systems, cells need to be patterned such that they can precisely mimic natural microenvironments in vitro. Despite increasing interest in this area, patterning cells at multiscale (∼10 μm to 10 mm) remains a significant challenge in bioengineering. Here, we report a projection-based 3D printing system that achieves rapid and high-resolution fabrication of hydrogel scaffolds featuring intricate channels for multiscale cell patterning. Using this system, we were able to use biocompatible poly(ethylene glycol)diacrylate in fabricating a variety of scaffold architectures, ranging from regular geometries such as serpentine, spiral, and fractal-like to more irregular/intricate geometries, such as biomimetic arborescent and capillary networks. A red food dye solution was able to freely fill all channels in the scaffolds, from the trunk (>1100 μm in width) to the small branch (∼17 μm in width) without an external pump. The dimensions of the printed scaffolds remained stable over 3 days while being immersed in Dulbecco's phosphate-buffered saline at 37 °C, and a penetration analysis revealed that these scaffolds are suitable for metabolic and nutrient transport. Cell patterning experiments showed that red fluorescent protein-transfected A549 human nonsmall lung cancer cells adhered well in the scaffolds' channels, and showed further attachment and penetration during cell culture proliferation.

Nrf2 mediates the resistance of human A549 and HepG2 cancer cells to boningmycin, a new antitumor antibiotic, in vitro through regulation of glutathione levels.

NF-E2-related factor 2 (Nrf2) is a transcription factor and a pivotal factor in the induction of the cell defense system. Recent reports show that Nrf2 plays critical roles in tumor heterogeneity and drug resistance. In the present study we investigated whether and how Nrf2 mediated the resistance of human cancer cells to boningmycin (BON), a new antitumor antibiotic of the bleomycin family. We showed that in the expression levels of Nrf2 in human non-small lung cancer A549 cells were much higher than those in human hepatoblastoma HepG2 cells, and their resistance to BON was opposite to Nrf2 expression (the IC50values of BON in A549 cells and HepG2 cells were 5.97 and 0.61 μmol/L, respectively). Similar results were observed with the anticancer agent cisdiamminedichloroplatinum (DDP), which was used as a positive control. In A549 cells, Nrf2 mRNA knockdown significantly increased their susceptibilities to BON and DDP. An enhanced resistance to BON and DDP was observed in HepG2 cells after overexpression of the wild-type Nrf2 protein. Treatment with a specific Nrf2 inhibitor, luteolin, significantly sensitized A549 cells to BON and DDP and increased BON- or DDP-induced apoptosis. The total levels of glutathione (GSH), the final product of the Nrf2 signaling pathway, were much higher in A549 cells than those in HepG2 cells. Supplementation of GSH in HepG2 cells significantly decreased their susceptibility to BON and DDP, wheras depleting GSH with the specific inhibitor L-buthionine sulfoximine in A549 cells significantly increased their susceptibility to BON and DDP. Our results demonstrate that Nrf2 mediates the resistance to BON through regulating glutathione levels in A549 cells and HepG2 cells.

Cyclooxygenase inhibitors combined with deuterium-enriched water augment cytotoxicity in A549 lung cancer cell line via activation of apoptosis and MAPK pathways.

Objective(s):Combination chemotherapy is a rational strategy to increase patient response and tolerability and to decrease adverse effects and drug resistance. Recently, the use of non-steroidal anti-inflammatory drugs (NSAIDs) has been reported to be associated with reduction in occurrence of a variety of cancers including lung cancer. On the other hand, growing evidences suggest that deuterium-enriched water (DEW, D2O) and deuterium-depleted water (DDW) play a role both in treatment and prevention of cancers. In the present study, we examined the effects of DEW and DDW in combination with two NSAIDs, celecoxib and indomethacin, on A549 human non-small lung cancer cell to identify novel treatment options.Materials and Methods:The cytotoxicity of celecoxib or indomethacin, alone and in combination with DDW and DEW was determined. The COX-2, MAPK pathway proteins, the anti-apoptotic Bcl2 and pro-apoptotic Bax proteins and caspase-3 activity were studied for cytotoxic combinations.Results:Co-administration of selective and non-selective COX-2 inhibitors with DEW led to a remarkable increase in cytotoxicity and apoptosis of A549 cells. These events were associated with activation of p38 and JNK MAPKs and decreasing pro-survival proteins Bcl-2, COX-2 and ERK1/2. Furthermore, the combination therapy activated caspase-3, and the apoptosis mediator, and disabled poly ADP-ribose polymerase (PARP), the key DNA repair enzyme, by cleaving it.Conclusion:The combination of DEW with NSAIDs might be effective against lung cancer cells by influence on principal cell signalling pathways, and this has a potential to become a candidate for chemotherapy.

Structural characterization of a polysaccharide from the flower buds of Tussilago farfara, and its effect on proliferation and apoptosis of A549 human non-small lung cancer cell line

In present study, we purified a polysaccharide, TFPB1, from the flower buds of Tussilago farfara using DEAE-cellulose 52 anion-exchange and Sephacryl S-300 HR gel filtration chromatography. TFPB1 was a homogeneous polysaccharide with a molecular weight of 37.8kDa and composed of rhamnose, galacturonic acid, glucose, galactose, and arabinose, in a ratio of 13:13:1:7:12. Methylation and NMR results demonstrated that TFPB1 contained a rhamnogalacturonan I backbone consisting of a repeat disaccharide unit →4)-α-D-GalAp-(1→2)-α-L-Rhap-(1→, substituted by various type II arabinogalactan branches including terminal galactose, (1→3)-β-D-galactan and (1→5)-α-L-arabinan, attached to the O-4 of (1→2)-α-L-Rhap. TFPB1 was found to inhibit cell proliferation of A549 cells and induce cell apoptosis in vitro. Furthermore, TFPB1 downregulated the phosphorylation of Akt, and upregulated caspase-3, Fas, FasL, and Bax expression, but downregulated Bcl-2 expression. Therefore, TFPB1 exhibited anti-proliferative and anti-apoptotic effect partly depending on the suppression of Akt signaling pathway. These findings provided us a potential chemotherapeutic strategy for the treatment of human non-small cell lung cancer.

Screening of antiproliferative activity mediated through apoptosis pathway in human non-small lung cancer A-549 cells by active compounds present in medicinal plants

Objective: To explore the antiproliferative activity and apoptosis in cells caused by active compounds present in plants using different techniques. Methods: We investigated the antiproliferative effects of methanolic extracts from different parts of seven plants on A-549 (lung cancer) cells and primary cell culture (chick embryo fibroblast cells, as normal cells) using MTT assay and the potent plant was fractioned further. All these fractions were screened again for anti-proliferative activity. DNA fragmentation and DAPI staining were used to study apoptosis. Quantitative real-time was used to investigate the expression of apoptotic-related genes. LC-MS and 1H-NMR techniques were used to identify the active compounds present. EnzCheck caspase-3 assay kit was used to measure caspase-3 activity. Results: Methanolic extract of Vitex negundo (V. negundo) was selected as a potent fraction. Among all fractions screened, ethylacetate fraction of V. negundo was selected as the most potent antiproliferative fraction and phytochemical analysis of the extract revealed the presence of secondary metabolites. Ethaylacetate fraction of V. negundo was found to cause characteristic apoptotic morphological changes and generation of ROS in A-549 cells. Ethaylacetate fraction of V. negundo also induced apoptosis in A-549 which was supported by DNA fragmentation and DAPI staining. To investigate the molecular mechanism behind the cytotoxic effect of ethaylacetate fraction of V. negundo, quantitative real-time PCR was used to measure expression levels of p53, bax, bcl2, casp-3 and casp-9. Using LC-MS and 1H-NMR techniques, cytotoxic compounds (luteolin and p-hydroxy benzoic acid) were identified which increased casp-3 activity in a dose and time-dependent manner in A-549 treated cells. Conclusions: It is concluded from the present study that V. negundo is capable of triggering growth-inhibitive and apoptosis effects in A-549 cells, signifying that V. negundo may possesses anti-lung cancer activity.

Levobuipivacaine-Induced Dissemination of A549 Lung Cancer Cells

While anaesthetics are frequently used on cancer patients during surgical procedures, their consequence on cancer progression remains to be elucidated. In this study, we sought to investigate the influence of local anesthetics on lung cancer cell dissemination in vitro and in vivo. A549 human non-small lung cancer cells were treated with various local anaesthetics including ropivacaine, lidocaine, levobupivacaine and bupivacaine. Cell barrier property was assessed using an electric cell-substrate impedance sensing (ECIS) system. The epithelial-to-mesenchymal transition (EMT) of treated cells was studied by immunofluorescence staining. In vitro and in vivo cancer cell dissemination were investigated.Gene expression microarray and quantitative real-time PCR (qrt-PCR) assays were used to identify the genes responsible for levobupivacaine-mediated cancer cell dissemination.The results illustrated that only levobupivacaine induced EMT in the treated cells and also caused the dissemination of cancer cells in vitro. In addition, after intravenous injection, levobupivacaine encouraged cancer cell dissemination in vivo. Gene expression microarray, qrt-PCR and immunoblotting revealed that after levobupivacaine treatment, the hypoxia-inducible factor (HIF)- 2α gene was upregulated in cancer cells. Our findings suggest that levobupivacaine may induce A549 lung cancer cell dissemination both in vitro and in vivo. More specifically, HIF-2α signaling possibly contributes to levobupivacaine-mediated A549 lung cancer cell dissemination.

Genistein induces apoptosis by stabilizing intracellular p53 protein through an APE1-mediated pathway

Genistein (GEN) has been previously shown to have a proapoptotic effect on cancer cells through a p53-dependent pathway, the mechanism of which remains unclear. One of its intracellular targets, APE1, protects against apoptosis under genotoxic stress and interacts with p53. In this current study, we explored the mechanism of the proapoptotic effect of GEN by examining the APE1–p53 protein–protein interaction. We initially showed that the p53 protein level was elevated in GEN-treated human non-small lung cancer A549 cells and cervical cancer HeLa cells. By examining both protein synthesis and degradation, we found that GEN enhances p53 intracellular stability by interfering with the interaction of APE1 and p53, which provided a plausible explanation for how GEN initiates apoptosis. Furthermore, we found that the interaction between APE1 and p53 is important for the degradation of p53 and is dependent on the redox domain of APE1 by utilizing the redox domain mutant APE1 C65A. Our data suggest that the degradation of wild-type p53 is blocked when the redox domain of APE1 is masked or interrupted. Based on this evidence, we hereby report a novel mechanism of p53 degradation through an APE1-mediated, redox-dependent pathway.

Matrine Reduces Proliferation of Human Lung Cancer Cells by Inducing Apoptosis and Changing miRNA Expression Profiles

Matrine, a main active component extracted from dry roots of Sophora flavecens , has been reported to exert antitumor effects on A549 human non-small lung cancer cells, but its mechanisms of action remain unclear. To determine effects of matrine on proliferation of A549 cells and assess possible mechanisms, MTT assays were employed to detect cytotoxicity, along with o flow cytometric analysis of DNA content of nuclei of cells following staining with propidium iodide to analyze cell cycle distribution. Western blotting was performed to determined expression levels of Bax, Bcl-2, VEGF and HDAC1, while a microarray was used to assessed changes of miRNA profiles. In the MTT assay, matrine suppressed growth of human lung cancer cell A549 in a dose- and time- dependent manner at doses of 0.25-2.5 mg/ml for 24h, 48h or 72h. Matrine induced cell cycle arrest in G0/G1 phase and decreased the G2/M phase, while down-regulating the expression of Bcl2 protein, leading to a reduction in the Bcl-2/Bax ratio. In addition, matrine down regulated the expression level of VEGF and HDAC1 of A549 cells. Microarray analysis demonstrated that matrine altered the expression level of miRNAs compared with untreated control A549 cells. In conclusion, matrine could inhibit proliferation of A549 cells, providing useful information for understanding anticancer mechanisms.

β-ionone의 인체 비소폐암세포 A-549에 대한 anti-proliferative 효과

<TEX>${\beta}$</TEX>-Ionone의 인체 비소폐암세포 A-549 cells (human non-small lung cancer A-549 cell)에 대한 anti-proliferative effect에 관한 연구를 수행하였다. A-549 cell에 다양한 농도의 <TEX>${\beta}$</TEX>-ionone (1, 5, 10, and 15 <TEX>${\mu}M$</TEX>)을 2, 4, 6일 간 처리하고, 배양 2일에 A-549 cell의 생육억제와 관련되는 biochemical marker를 측정하였다. <TEX>${\beta}$</TEX>-Ionone은 A-549 cell 생육을 dose와 time 의존적으로 저해하였다. <TEX>${\beta}$</TEX>-Ionone 배양 2일의 <TEX>$IC_{50}$</TEX>은 5.0 <TEX>${\mu}g/ml$</TEX>이었다. <TEX>${\beta}$</TEX>-Ionone은 농도 의존적으로 apoptosis를 유도하였다. <TEX>${\beta}$</TEX>-Ionone은 p53, p21 및 Bax protein 수준을 증가시켰으나, Bcl-2 protein 발현은 억제시켰다. <TEX>${\beta}$</TEX>-ionone은 cytosol cytochrome c 함량을 증가시켰고, caspase-9과 caspase-3 효소 활성증가를 유도하였다. 또한, <TEX>${\beta}$</TEX>-ionone은 <TEX>$cPLA_2$</TEX>와 COX-2 protein level을 감소시켰다. 이와 같은 결과는 <TEX>${\beta}$</TEX>-ionone의 A-549 cell에 대한 생육억제효과는 Bax와 Bcl-2 gene 발현을 reciprocal regulation하여 유도한 apoptosis와 <TEX>$cPLA_2$</TEX> 및 COX-2 protein 발현 억제에 기인함을 의미한다. The anti-proliferative activity of <TEX>${\beta}$</TEX>-ionone was investigated on human non-small lung cancer A-549 cells (designated A-549 cells). A-549 cells were treated with various concentrations of <TEX>${\beta}$</TEX>-ionone (1, 5, 10, and 15 <TEX>${\mu}M$</TEX>) for two, four, and six days. Biochemical markers related to the growth inhibition of A-549 cells by <TEX>${\beta}$</TEX>-ionone were measured at the second day of incubation. <TEX>${\beta}$</TEX>-Ionone inhibited the growth of A-549 cells by dose-and time-dependent manners, resulting in an <TEX>$IC_{50}$</TEX> of 5.0 <TEX>${\mu}g/ml$</TEX> at the second day of incubation. <TEX>${\beta}$</TEX>-Ionone induced apoptosis by a dose-dependent manner. <TEX>${\beta}$</TEX>-Ionone increased levels of p53, p21, and Bax proteins, but suppressed expression of the Bcl-2 protein. Similarly, <TEX>${\beta}$</TEX>-ionone enhanced cytochrome c release from the mitochondria to the cytosol, and induced activation of caspase-9 and -3. Additionally, <TEX>${\beta}$</TEX>-ion-one reduced <TEX>$cPLA_2$</TEX> and COX-2 protein levels. These results suggest that the <TEX>${\beta}$</TEX>-ionone inhibits the proliferation of A-549 cells through reciprocal regulation of Bax and Bcl-2 gene expression and suppression of <TEX>$cPLA_2$</TEX> and COX-2 protein expressions.