Proliferation Of NCI-H1299 Cells Research Articles

Transcriptome Analysis of the Inhibitory Effects of 20(S)-Protopanaxadiol on NCI-H1299 Non-Small Cell Lung Cancer Cells.

Lung cancer seriously threatens human health. To explore the molecular mechanism of 20(S)-Protopanaxadiol (PPD) on human non-small cell lung cancer cells, we investigated the transcriptional profile of PPD-treated NCI-H1299 cells. Cell proliferation, cell cycle, and apoptosis were detected using cell counting kit-8 and flow cytometry, respectively. Differentially expressed genes (DEGs) between PPD-treated and untreated cells were determined using RNA sequencing and bioinformatic analysis. Protein phosphorylation was detected using Western blotting. Data of mRNA expression profiles of lung cancer were from The Cancer Genome Atlas (TCGA) and analyzed using R software version 4.3.1. PPD showed an inhibitory effect on the proliferation of NCI-H1299 cells and induced apoptosis. There were 938 upregulated genes and 466 downregulated genes in PPD-treated cells, and DEGs were primarily enriched in the MAPK signaling pathway. The detection of phosphorylation revealed that the phosphorylation of ERK and p38 MAPK was significantly reduced in PPD-treated cells. Further comparison of PPD-regulated DEGs with clinical data of lung adenocarcinoma demonstrated that most downregulated genes in tumor tissues were upregulated in PPD-treated cells or vice versa. Two PPD-downregulated genes HSPA2 and EFNA2 were associated with patients' overall survival. Therefore, PPD could inhibit NCI-H1299 cells by affecting gene expression and regulating ERK and p38 MAPK pathways.

Experimental study of shRNA Twist gene on proliferation and invasion of lung adenocarcinoma NCI-H1299 cells

Objective To explore the mechanism of shRNA Twist gene on proliferation and invasion of lung adenocarcinoma NCI-H1299 cells. Methods Twist siRNA interference expression vector was constructed and NCI-H1299 cells were divided into three groups: blank control group, negative control group and experimental group. The blank control group was the untreated cell group, while the negative control group was the lentivirus transfected cell group by the blank vector. The experimental group was the lentivirus transfected cell group constructed by the lentivirus interference vector of shRNA Twist. The siRNA interference expression vector of Twist was constructed by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot to detect the expression of Twist. Transwell kit was used to detect cell invasion. Cell counting kit-8 (CCK-8) kit was used to detect cell proliferation. Results ⑴ The titer of lentivirus was detected. The transfection titer of lentivirus vector: shRNA-Twist vector was 3×108 TU/ml. ⑵ The results of qRT-PCR test showed that compared with the negative control group, the mRNA expression of Twist in the experimental group was decreased (q=3.177, P=0.0234). ⑶ The results of Western blot showed that compared with the negative control group, the protein expression of Twist in the experimental group was decreased (q=4.071, P=0.0304). ⑷ The results of Transwell test showed that there was significant statistical difference among the three groups (F=19.472, P=0.000). Compared with the negative control group, the number of cell imigration in the experimental group was decreased (q=3.567, P=0.0318). ⑸ The results of CCK-8 showed that there was significant statistical difference among the three groups (F=20.983, P=0.000). Compared with the negative control group, the proliferation rate in the experimental group was decreased (q=5.272, P=0.0157). Conclusions ShRNA Twist gene can significantly inhibit the proliferation and invasion of lung adenocarcinoma NCI-H1299 cells. Key words: Cell line, tumor; Adenocarcinoma; Lung neoplasms; RNA, small interfering; Cell proliferation; Cell migration assays

Abstract 2204: Novel FAK/ALK/ROS1 inhibitor APG-2449 synergizes with osimertinib in preclinical xenograft models of EGFR-mutant NSCLC

Abstract Focal adhesion kinase (FAK) plays an important role in cell migration, growth factor signaling, cell cycle progression and cellular survival. It had been shown that FAK and SRC family kinases were able to sustain downstream AKT and MAPK signaling under continuous EGFR inhibition driven by osimertinib (AZD9291), a mutant-selective third-generation EGFR inhibitor. Incomplete inhibition of AKT and MAPK was consistently observed even under increased concentrations of osimertinib and led to acquired resistance to osimertinib, implicating the need of combination therapy with FAK/SRC inhibitors to enhance antitumor activity of osimertinib and overcome its resistance. APG-2449 is a novel oral active small-molecular inhibitor that targets FAK, ALK and ROS1. In this study, we investigated the effect of combination treatment with APG-2449 and osimertinib using NSCLC NCI-H1975 cells carrying EGFRL858R/T790M mutation and an osimertinib-resistant patient-derived xenograft (PDX) model carrying EGFRT790M/19del/C797S mutation. In vitro, the combination treatment with APG-2449 and osimertinib synergistically inhibited the proliferation of NCI-H1975 cells. In vivo, APG-2449 significantly enhanced antitumor activity of osimertinib in NCI-H1975 xenograft models, leading to complete or partial tumor regression. More profound antitumor activity of this combination was also demonstrated in the osimeritinib-resistant PDX model in comparison with single agents. In term of mechanism, the combination arm significantly suppressed the on-target phosphorylation of EGFR, FAK and SRC, as well as AKT and ERK in NCI-H1975 xenografts. Collectively, these results suggest that addition of APG-2449 to osimeritinib may enhance antitumor activity and suppress the development of resistance in NSCLC. Citation Format: Guangfeng Wang, Douglas D. Fang, Ping Min, Ran Tao, Chunyang Tang, Shoulai Gu, Li Rui, Jiajun Li, Jingwen Wang, Miaoyi Wu, Yingfeng Li, Dingxiong Chen, Fei Zhang, Kejie Lian, Feifei Zhang, Lvcheng Wang, Rongcheng Xu, Dajun Yang, Yifan Zhai. Novel FAK/ALK/ROS1 inhibitor APG-2449 synergizes with osimertinib in preclinical xenograft models of EGFR-mutant NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2204.

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.

Epigenetic regulation in combination with endocrine therapy reducing the proliferation of NCI-H1975 cells

Objective To investigate the effect of epigenetic regulation in combination with endocrine therapy on proliferation of female lung ardenocarcinoma NCI-H1975 cells.Methods NCI-H1975 cells were treated with histone deacetylase (HDAC) inhibitor trichostatin A (TSA) and DNA methy transferas 1 ( DNMT1 ) inhibitor 5-aza-2' -deoxycytidine (5-AZA-CdR,AZA).The mRNA expression of estrogen receptor alpha (ERα) and progesterone receptor (PR) in the treated cells were detected by using polymerase chain reaction (PCR).The water-soluble tetrazolium salt (WST) methods were used to analyze the cells proliferation.Results After treatment with TSA and AZA,the expression of PR in NCI-H1975 cells was up-regulated ( P ＜0.01 ).The proliferation of the treated NCI-H1975 cells was inhibited by 4-OH Tamoxifen.The favorite concentration of the treatment was 2.5 μmol/L for AZA and 100 μg/L for TSA respectively.Conclusion Epigenetic regulation in combination with endocrine therapy can reduce the proliferation of NCI-H1975 cells. Key words: Lung carcinoma; Progesterone receptor; Epigenetic regulation

Lipid-Soluble Ginseng Extract Induces Apoptosis and G0/G1 Cell Cycle Arrest in NCI-H460 Human Lung Cancer Cells

This study was performed to elucidate the anticancer mechanism of a lipid-soluble ginseng extract (LSGE) by analyzing induction of apoptosis and arrest of cell cycle progression using the NCI-H460 human lung cancer cell line. Proliferation of NCI-H460 cells was potently inhibited by LSGE in a dose-dependent manner. The cell cycle arrest at the G0/G1 phase in NCI-H460 cells was induced by LSGE. The percentage of G0/G1 phase cells significantly increased, while that of S phase cells decreased after treatment with LSGE. The expression levels of cyclin-dependent kinase2 (CDK2), CDK4, CDK6, cyclin D3 and cyclin E related to G0/G1 cells progression were also altered by LSGE. In addition, LSGE-induced cell death occurred through apoptosis, which was accompanied by increasing the activity of caspases including caspase-8, caspase-9 and caspase-3. Consistent with enhancement of caspase activity, LSGE increased protein levels of cleaved caspase-3, caspase-8, caspase-9, and poly-ADP-ribose polymerase (PARP). These apoptotic effects of LSGE were inhibited by the pan-caspase inhibitor Z-VAD-fmk. These findings indicate that LSGE inhibits NCI-H460 human lung cancer cell growth by cell cycle arrest at the G0/G1 phase and induction of caspase-mediated apoptosis.

Adenovirus-Mediated SiRNA Targeting c-Met Inhibits Proliferation and Invasion of Small-Cell Lung Cancer (SCLC) Cells

The hepatocyte growth factor receptor c-Met and its ligand hepatocyte growth factor (HGF) have been reported to be involved in cellular motility, growth, and invasion by activating mitogenic signaling pathways. The overexpression of c-Met gene has been found in many malignant cancers, but the roles of c-Met overexpression in SCLC tumors still remain unclear. The aim of the present study was to explore its roles and potential as a therapeutic target for SCLC. Quantitative real-time RT-PCR and immunohistochemistry assays were performed to detect the expression of c-Met mRNA and protein in SCLC tissue or corresponding non-tumor lung tissue samples. Adenovirus-mediated small interfering RNA (siRNA) was employed to down-regulate the expression of c-Met gene in SCLC cell line (NCI-H446). MTT and colony formation assays were performed to detect in vitro proliferation of NCI-H446 cells. In vitro wound-healing and transwell invasion assays were performed to detect in vitro invasion and metastasis of NCI-H446 cells. Finally, in vivo tumorigenicity and metastasis assays were done to analyze in vivo proliferation and metastasis of NCI-H446 cells in a xenograft model. We showed that the levels of c-Met mRNA expression were significantly higher in SCLC tissue samples (0.97±0.08) than those in corresponding non-tumor lung tissue samples (0.21±0.02; P<0.05). Additionally, the immunostaining of c-Met protein in SCLC tissues was stronger than that in corresponding non-tumor tissues. Adenovirus-mediated siRNA targeting c-Met could significantly down-regulate c-Met expression, and the specific down-regulation of c-Met expression in SCLC cells could strongly inhibit proliferation of SCLC cells both in vitro and in vivo. Moreover, c-Met down-regulation could also reduce invasion capacity in vitro and metastasis capacity in vivo of SCLC cells. Taken together, our results indicated that the overexpression of c-Met gene played an important role in the progression and development of SCLC, and adenovirus-mediated siRNA targeting c-Met could potentially be an experimental approach for SCLC gene therapy.

Fibronectin enhances viability and alters cytoskeletal functions (with effects on the phosphatidylinositol 3-kinase pathway) in small cell lung cancer.

Small cell lung cancer (SCLC) is a rapidly progressive disease with ultimate poor outcome. SCLC has been shown to interact closely with the stromal and extracellular matrix (ECM) components of the diseased host. ECM consists of type I/IV collagen, laminin, vitronectin, and fibronectin (FN) among others. Herein, we investigated the behavior of a SCLC cell line (NCI-H446) on FN-coated surface. Over a course of 72 h, FN (10 micro g/ml) caused both increased survival and proliferation of NCI-H446 cells. Survival under serum-starved conditions increased 1.44-fold and proliferation in the presence of fetal calf serum increased by 1.30-fold. The phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002 reduced both survival and proliferation of NCI-H446 cells (0.48- and 0.27-fold, respectively), even on FN-coated surface. We next determined the effects of FN on cytoskeletal function such as cell motility/morphology and adhesion. Over a course of 24 h, FN reduced aggregation of NCI-H446 cells and induced flattened cellular morphology with neurite-like projections after 1 h, however, in the presence of LY294002, the cells rounded up. Adhesion of NCI-H446 cells also increased with FN (4.47-fold) which was abrogated with LY294002 treatment. This correlated with phosphorylation of the cytoskeletal protein p125FAK, on Tyr397, Tyr861 and Ser843 residues with FN. Even in the presence of LY294002, these serine/tyrosine residues were still phosphorylated on FN-coated surface. In contrast, the focal adhesion protein paxillin was not phosphorylated at Tyr31 with FN. In summary, FN stimulation of SCLC cells leads to enhancement of viability and changes in cytoskeletal function that are partially mediated through the PI3-K pathway.