Apoptosis In Non-small Cell Lung Cancer Cells Research Articles

Carnosic Acid against Lung Cancer: Induction of Autophagy and Activation of Sestrin-2/LKB1/AMPK Signalling.

Non-small cell lung cancer (NSCLC) represents 80% of all lung cancer cases and is characterized by low survival rates due to chemotherapy and radiation resistance. Novel treatment strategies for NSCLC are urgently needed. Liver kinase B1 (LKB1), a tumor suppressor prevalently mutated in NSCLC, activates AMP-activated protein kinase (AMPK) which in turn inhibits mammalian target of rapamycin complex 1 (mTORC1) and activates unc-51 like autophagy activating kinase 1 (ULK1) to promote autophagy. Sestrin-2 is a stress-induced protein that enhances LKB1-dependent activation of AMPK, functioning as a tumor suppressor in NSCLC. In previous studies, rosemary (Rosmarinus officinalis) extract (RE) activated the AMPK pathway while inhibiting mTORC1 to suppress proliferation, survival, and migration, leading to the apoptosis of NSCLC cells. In the present study, we investigated the anticancer potential of carnosic acid (CA), a bioactive polyphenolic diterpene compound found in RE. The treatment of H1299 and H460 NSCLC cells with CA resulted in concentration and time-dependent inhibition of cell proliferation assessed with crystal violet staining and 3H-thymidine incorporation, and concentration-dependent inhibition of survival, assessed using a colony formation assay. Additionally, CA induced apoptosis of H1299 cells as indicated by decreased B-cell lymphoma 2 (Bcl-2) levels, increased cleaved caspase-3, -7, poly (ADP-ribose) polymerase (PARP), Bcl-2-associated X protein (BAX) levels, and increased nuclear condensation. These antiproliferative and proapoptotic effects coincided with the upregulation of sestrin-2 and the phosphorylation/activation of LKB1 and AMPK. Downstream of AMPK signaling, CA increased levels of autophagy marker light chain 3 (LC3), an established marker of autophagy; inhibiting autophagy with 3-methyladenine (3MA) blocked the antiproliferative effect of CA. Overall, these data indicate that CA can inhibit NSCLC cell viability and that the underlying mechanism of action of CA involves the induction of autophagy through a Sestrin-2/LKB1/AMPK signaling cascade. Future experiments will use siRNA and small molecule inhibitors to better elucidate the role of these signaling molecules in the mechanism of action of CA as well as tumor xenograft models to assess the anticancer properties of CA in vivo.

Kinetochore scaffold 1 downregulation suppressed the development of non-small cell lung cancer by inactivating the phosphatidylinositol 3 kinase/protein kinase B (AKT)/nuclear factor-kappa B pathway.

Kinetochore scaffold 1 (KNL1) is indispensable for generating motile micro-tubule attachments and isolating chromosomes. KNL1 is highly expressed in multiple middle-route tissues and promotes tumor development. However, how it functions in non-small cell lung cancer (NSCLC) is unclear. Real-time quantitative PCR (RT-qPCR) and Western blotting (WB) were used to determine KNL1 expression in NSCLC tissues and cells. The sh-KNL1 or oe-KNL1 was transfected into NSCLC cells. The colony formation assay, cell counting kit-8 (CCK-8) assay, and flow cytometry were used to evaluate cell proliferation and apoptosis. A transwell assay was used to monitor invasion and migration. The CCK-8 assay was used to measure NSCLC cell sensitivity to chemotherapy drugs. WB confirmed the protein levels of apoptosis-related proteins, cell cycle-associated proteins, and the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT)/nuclear factor kappaB (NF-κB) pathway. A PI3K/AKT/NF-κB pathway inhibitor was used to intervene in NSCLC cell transfection along with oe-KNL1, thus revealing the function of the pathway in carcinogenicity mediated by KNL1. In result KNL1 expression was substantially increased in NSCLC tissues and cells. High-level KNL1 expression is related to the poor prognosis of NSCLC patients. KNL1 silencing bolstered promoted NSCLC cell apoptosis and inhibited proliferation, cell cycle progression, invasion, and EMT, whereas KNL1 silencing had the opposite effect. KNL1 knockdown increased NSCLC cell sensitivity to chemical drugs. KNL1 promoted PI3K/AKT/NF-κB pathway activation, while PI3K/AKT/NF-κB pathway inhibition weakened the procancer effect mediated by KNL1 overexpression but had little influence on KNL1 levels. We conclude that KNL1 activates the PI3K/AKT/NF-κB pathway to increase NSCLC progression and attenuate NSCLC sensitivity to chemotherapy drugs.

Acacetin inhibited non-small-cell lung cancer (NSCLC) cell growth via upregulating miR-34a in vitro and in vivo

Acacetin, one of the flavonoid compounds, is a natural product found in various plants, including Silver birch, and Damiana. Previous studies showed that acacetin has anti-cancer effects on many kinds of cancer cells, however, the role of and the mechanisms of actions of acacetin on non-small cell lung cancer (NSCLC) cells is still not fully understood. Herein, we found that, in vitro, acacetin inhibited the proliferation, invasion, and migration of NSCLC cells, A549 and H460, in a dose-dependent manner. Meanwhile, flow cytometry assay results showed that acacetin induced G2/M phase cell cycle arrest, and apoptosis of NSCLC cells. In vivo, acacetin suppressed tumor formation of A549-xenografted nude mice model with no obvious toxicities. Western blotting results showed that the protein levels of cell cycle-related proteins cyclin B1, cyclin D, and anti-apoptotic protein Bcl-2 had decreased, while the apoptosis-related protein Bak had increased both in NSCLC cells and in A549-xenografted tumor tissues. For investigating the molecular mechanism behind the biological effects of acacetin on NSCLC, we found that acacetin induced the expression levels of tumor suppressor p53 both in vitro and in vivo. MicroRNA, miR-34a, the direct target of p53, has been shown anti-NSCLC proliferation effects by suppressing the expression of its target gene programmed death ligand 1 (PD-L1). We found that acacetin upregulated the expression levels of miR-34a, and downregulated the expression levels of PD-L1 of NSCLC cells in vitro and of tumors in vivo. In vitro, knockdown p53 expression by siRNAs reversed the induction effects of acacetin on miR34a expression and abolished the inhibitory activity of acacetin on NSCLC cell proliferation. Furthermore, using agomir and antagomir to overexpress and suppress the expression miR-34a in NSCLC cells was also examined. We found that miR-34a agomir showed similar effects as acacetin on A549 cells, while miR-34a antagomir could partially or completely reverse acacetin’s effects on A549 cells. In vivo, intratumor injection of miR-34a antagomir could drastically suppress the anti-tumor formation effects of acacetin in A549-xenografted nude mice. Overall, our results showed that acacetin inhibits cell proliferation and induces cell apoptosis of NSCLC cells by regulating miR-34a.

CircRNA-CPA4 Regulates Cell Proliferation and Apoptosis of Non-small Cell Lung Cancer via the miR-1183/PDPK1 Axis.

Non-small-cell lung cancer (NSCLC) stands as a prevalent subtype of lung cancer, with circular RNAs emerging as key players in cancer development. This study elucidates the role of circRNA-CPA4 in NSCLC. Elevated circRNA-CPA4 expression in NSCLC lines was confirmed through qRT-PCR. Silencing circRNA-CPA4 with shRNA revealed, through CCK-8, colony formation, and flow cytometry assays, a notable constraint on proliferation and promotion of apoptosis in NSCLC cells. Subcellular localization analysis, RNA immunoprecipitation, and expression level assessments were employed to decipher the intricate interplay among miR-1183, circRNA-CPA4, and PDPK1. Results demonstrated heightened circRNA-CPA4 levels in NSCLC, and its knockdown curtailed NSCLC growth in vivo. Acting as a molecular sponge for miR-1183, circRNA-CPA4 regulated PDPK1 expression. Conversely, inhibiting miR-1183 counteracted the impact of circRNA-CPA4 silencing, reinstating NSCLC cell proliferation, and impeding apoptosis. Overall, this study unveils a novel mechanism: circRNA-CPA4 promotes PDPK1 expression by sequestering miR-1183, fostering NSCLC cell proliferation, and hindering apoptosis.

Targeting KCa3.1 channels to overcome erlotinib resistance in non-small cell lung cancer cells

Almost all non-small cell lung cancer (NSCLC) patients initially responding to EGFR tyrosine kinase inhibitors (TKIs) develop acquired resistance. Since KCa3.1 channels, expressed in mitochondria and plasma membrane, regulate similar behavioral traits of NSCLC cells as EGFR, we hypothesized that their blockade contributes to overcoming EGFR-TKI resistance. Meta-analysis of microarray data revealed that KCa3.1 channel expression in erlotinib-resistant NSCLC cells correlates with that of genes of integrin and apoptosis pathways. Using erlotinib-sensitive and –resistant NSCLC cells we monitored the role of mitochondrial KCa3.1 channels in integrin signaling by studying cell-matrix adhesion with single-cell force spectroscopy. Apoptosis was quantified with fluorescence-based assays. The function of mitochondrial KCa3.1 channels in these processes was assessed by measuring the mitochondrial membrane potential and by quantifying ROS production. Functional assays were supplemented by biochemical analyses. We show that KCa3.1 channel inhibition with senicapoc in erlotinib-resistant NSCLC cells increases cell adhesion by increasing β1-integrin expression, that in turn depends on mitochondrial ROS release. Increased adhesion impairs migration of NSCLC cells in a 3D matrix. At the same time, the senicapoc-dependent ROS production induces cytochrome C release and triggers apoptosis of erlotinib-resistant NSCLC cells. Thus, KCa3.1 channel blockade overcomes EGFR-TKI resistance by inhibiting NSCLC motility and inducing apoptosis.

The Ethanolic Extract of Dictyopteris Divaricata Induces Apoptosis in Non-Small Cell Lung Cancer Cells by Inhibiting STAT3 Activity

Dictyopteris divaricata (DD) has been reported to exert diverse pharmacological activities, including anti-inflammatory, antioxidant, and anticancer effects. In this study, we aimed to investigate the anticancer potential of the ethanolic extract of DD (EDD) in non-small cell lung cancer (NSCLC) cells and to explore the underlying mechanism. EDD significantly suppressed cell proliferation in H1299, PC9, and H1975 NSCLC cells. EDD treatment increased the proportion of Annexin V-positive cells and cells in sub-G1 phase, indicating the induction of apoptosis. This observation was further supported by the presence of fragmented nuclei and increased expression of cleaved PARP and cleaved caspase-3 in NSCLC cells following EDD treatment. Mechanistically, EDD decreased the phosphorylation levels of signal transducer and activator of transcription 3 (STAT3) and Src. Transfection of constitutively activated STAT3 into H1975 cells partially attenuated EDD-induced apoptosis, highlighting the contribution of STAT3 inhibition to the anticancer activity of EDD. In addition, we identified fucosterol as a major constituent of EDD that exhibited similar anticancer potential in NSCLC cells. Taken together, our results demonstrate that EDD induces apoptosis in NSCLC cells by inhibiting STAT3 activity. We propose EDD as a potential candidate for the development of therapies targeting NSCLC.

Ginsenoside Rg5 induces NSCLC cell apoptosis and autophagy through PI3K/Akt/mTOR signaling pathway.

Ginsenoside Rg5 (Rg5) is a minor ginsenoside of ginseng and has a strong anti-tumor potential. This study focused on deciphering the function of Rg5 in non-small cell lung cancer (NSCLC) and investigating its related mechanism. After treating human NSCLC cell lines (H1650 and A549) and bronchial epithelial cells (BEAS-2B) with increasing concentration of Rg5, cell viability was examined using methyl thiazolyl tetrazolium (MTT) assay. NSCLC cell proliferation and apoptosis were evaluated by colony formation assay and flow cytometry, respectively. The levels of proteins associated with cell cycle progression, cell apoptosis, and autophagy as well as the key markers in the PI3K/Akt/mTOR pathway were measured using western blot. A xenograft nude mouse model was established to explore the function of Rg5 in vivo. NSCLC cell viability was dose- and time-dependently suppressed after Rg5 treatment. Rg5 restrained NSCLC cell proliferation by inducing G2/M phase arrest via regulation of cell cycle-related genes including p21, cyclin B1, and Cdc2. Additionally, Rg5 promoted caspase-dependent apoptosis in NSCLC cells by regulating the intrinsic mitochondrial signaling pathway. Rg5 induced autophagy via the regulation of autophagy-related proteins. The in vivo experiments revealed the inhibitory impact of Rg5 on xenograft growth. Rg5 also inactivated the PI3K/Akt/mTOR signaling pathway in NSCLC cells and mouse tumors. Rg5 induced autophagy and caspase-dependent apoptosis in NSCLC cells by inhibiting the PI3K/Akt/mTOR signaling pathway, suggesting that Rg5 might become a promising and novel anti-tumor agent for the clinical treatment of NSCLC patients.

Erianin promotes apoptosis and inhibits Akt-mediated aerobic glycolysis of cancer cells.

Highly activated aerobic glycolysis provides the metabolic requirements for tumor cell growth and proliferation. Erianin, a natural product isolated from Dendrobium chrysotoxum Lindl, has been reported to exert antitumor activity in multiple cancers. However, whether Erianin exerts inhibitory effects on aerobic glycolysis and the inherent mechanism remain poorly defined in non-small cell lung cancer (NSCLC). Here, we showed that Erianin inhibited the cell viability and proliferation, and induced apoptosis in NSCLC cells. Moreover, Erianin overtly suppressed aerobic glycolysis via decreasing HK2 expression. Mechanistically, Erianin dose-dependently curbed the Akt-GSK3β signaling pathway phosphorylation activation, which afterwards downregulated HK2 expression. Meanwhile, Erianin inhibited HCC827 tumor growth in vivo. Taken together, our results suggest that the natural product Erianin can suppress aerobic glycolysis and exert potent anticancer effects via the Akt-GSK3β signaling pathway in NSCLC cells.

Juglone triggers apoptosis of non-small cell lung cancer through the reactive oxygen species -mediated PI3K/Akt pathway.

Non-small cell lung cancer (NSCLC) is one of the most common malignancies worldwide, and oxidative stress plays a crucial role in its development. Juglone, a naturally occurring naphthoquinone in J. mandshurica, exhibits significant cytotoxic activity against various cancer cell lines. However, whether the anticancer activity of juglone is associated with oxidative stress remains unexplored. In this study, mouse Lewis lung cancer (LLC) and human non-small cell lung cancer A549 cells were used to explore the anticancer mechanisms of juglone. Juglone inhibited LLC and A549 cells viability, with IC50 values of 10.78 μM and 9.47 μM, respectively, for 24 h, and substantially suppressed the migration and invasion of these two lung cancer cells. Additionally, juglone arrested the cell cycle, induced apoptosis, increased the cleavage of caspase 3 and the protein expression of Bax and Cyt c, and decreased the protein expression of Bcl-2 and caspase-3. Furthermore, juglone treatment considerably increased intracellular reactive oxygen species (ROS) and malondialdehyde (MDA) levels, but suppressed glutathione peroxidase 4 (GPX4) and superoxide dismutase (SOD) activities. It also inhibited the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway, which was attenuated by 1,3-diCQA (an activator of PI3K/Akt). Moreover, N-acetylcysteine (a ROS scavenger) partially reversed the positive effects of juglone in terms of migration, invasion, ROS production, apoptosis, and PI3K/Akt pathway-associated protein expression. Finally, in tumor-bearing nude mouse models, juglone inhibited tumor growth without any apparent toxicity and significantly induced apoptosis in NSCLC cells. Collectively, our findings suggest that juglone triggers apoptosis via the ROS-mediated PI3K/Akt pathway. Therefore, juglone may serve as a potential therapeutic agent for the treatment of NSCLC.

SLL-1A-16 suppresses proliferation and induces autophagy in non-small-cell lung cancer cells via the AKT/mTOR signaling pathway.

Non-small-cell lung cancer (NSCLC), which accounts for approximately eighty-five percent of lung cancer diagnoses worldwide, is a malignancy with high incidence and mortality rates. Among the various antitumor compounds, organic selenium-containing compounds have emerged as a promising class of therapeutic agents for cancer treatment. In the present study, SLL-1A-16, a new organoselenium small molecule, was discovered to exhibit antiproliferative activity against NSCLC both in vitro and in vivo. Treatment with SLL-1A-16 significantly inhibited NSCLC cell proliferation and induced apoptosis and autophagy. Mechanistically, SLL-1A-16 inhibited cell proliferation through G1-S phase arrest by reducing cyclin D1 and CDK4 expression. Additionally, SLL-1A-16 significantly induced apoptosis by upregulating cleaved caspase 3 and Bax expression, while downregulating Bcl-2 levels. Our study also demonstrated that SLL-1A-16 induced autophagy in NSCLC cells by inhibiting the Akt/mTOR pathway. Overall, our findings suggest that SLL-1A-16 could induce cell cycle arrest, apoptosis and autophagy in NSCLC cells by inhibiting the Akt/mTOR signaling pathways, providing a theoretical basis for the potential clinical application of SLL-1A-16 as a chemotherapeutic agent in NSCLC treatment.

MS4A3 Promotes the Chemosensitivity of Lung Cancer via THAP1/EGFR Pathways.

MS4A3 functions as a tumor suppressor in multiple cancer types. However, the roles of MS4A3 in lung cancer are still unknown. Therefore, this study aims to investigate the potentials of MS4A3 in lung cancer. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was carried out to determine mRNA expression. CCK-8 and colony formation assay are conducted to determine cell proliferation. Tube formation assay is performed to determine angiogenesis. Flow cytometry is used to determine cell apoptosis. JASPAR is used to analyze the binding motif of THAP1. Luciferase and ChIP assay are conducted to verify whether MS4A3 can interact with THAP1 to transcriptionally inactivate EGFR. The results showed that MS4A3 is downregulated in non-small-cell lung cancer (NSCLC) patients, which predicts poor clinical outcomes of NSCLC patients. Overexpressed MS4A3 enhances the chemosensitivity of NSCLC cells to osimertinib, whereas MS4A3 knockdown exerts the opposite effects. MS4A3 suppresses the proliferation and angiogenesis and promotes the apoptosis of NSCLC cells. Moreover, MS4A3 upregulates apoptosis-related THAP1 to inactivate EGFR. However, THAP1 knockdown attenuates the effects of MS4A3 and promotes the malignant behavior of NSCLC cells. In conclusion, MS4A3 functions as an anti-tumor gene in NSCLC. MS4A3/THAP1/EGFR signaling enhances the chemosensitivity of lung cancer to EGFR tyrosine kinase inhibitor (TKI).

Mechanism of n-butanol fraction of Wenxia Formula combining with gefitinib in treating non-small cell lung cancer based on network pharmacology and in vitro experiment

This study combined network pharmacology, molecular docking, and in vitro experiments to explore the potential mechanism of the active components of the n-butanol fraction of Wenxia Formula(NWXF) combined with gefitinib(GEF) in treating non-small cell lung cancer(NSCLC). Ultra-performance liquid chromatography-quadrupole Orbitrap mass spectrometry(UPLC-Q-Orbitrap MS) was employed to detect the main chemical components of NWXF. The active components of NWXF were retrieved from SwissADME, and the candidate targets of these active components were retrieved from SwissTargetPrediction. Online Mendelian Inheritance in Man(OMIM) and GeneCards were searched for the targets of NSCLC. Cytoscape 3.9.0 and STRING were employed to build the protein-protein interaction(PPI) network with the common targets shared by NWXF and NSCLC. Gene Ontology(GO) annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment were performed in DAVID to predict the potential mechanisms. Finally, molecular docking between the main active ingredients and key targets was conducted in SYBYL-X 2.0. The methyl thiazolyl tetrazolium(MTT) assay was employed to evaluate the inhibitory effects of NWXF and/or GEF on the proliferation of human non-small cell lung cancer cells(A549 and PC-9). Additionally, the impact of NWXF on human embryonic lung fibroblast cells(MRC-5) was assessed. The effectiveness of the drug combination was evaluated based on the Q value. The terminal-deoxynucleoitidyl transferase mediated nick-end labeling(TUNEL) assay was employed to examine the apoptosis of A549 and PC-9 cells treated with NWXF and/or GEF. Quantitative real-time PCR(qRT-PCR) was employed to measure the mRNA levels of epidermal growth factor receptor(EGFR), c-Jun N-terminal kinase(JNK), and Bcl2-associated X protein(Bax) in the A549 and PC-9 cells treated with NWXF and/or GEF. Western blot was employed to determine the protein levels of EGFR, p-EGFR, JNK, p-JNK, and Bax in the A549 and PC-9 cells treated with NWXF and/or GEF. A total of 77 active components, 488 potential targets, and 49 key targets involved in the treatment of NSCLC with NWXF were predicted. The results of GO annotation showed that NWXF may treat NSCLC by regulating the biological processes such as cell proliferation, apoptosis, and protein phosphorylation. KEGG enrichment revealed that the key targets of NWXF in treating NSCLC were enriched in the mitogen-activated protein kinase(MAPK), phosphatidylinositol 3-kinase(PI3K)-protein kinase B(AKT), hypoxia-inducible factor-1(HIF-1), and microRNA-related signaling pathways. Molecular docking results showed that 91.9% of the docking scores were greater than 5, indicating the strong binding capability between main active components and key targets. The cell experiments demonstrated that NWXF combined with GEF synergistically inhibited the proliferation, promoted the apoptosis, decreased p-EGFR/EGFR and p-JNK/JNK values, down-regulated the mRNA levels of EGFR and JNK, and up-regulated the mRNA and protein levels of Bax in A549 and PC-9 cells. In conclusion, NWXF combined with GEF can regulate the EGFR/JNK pathway to promote the apoptosis of NSCLC cells, thus treating NSCLC.

SP-8356: A Novel Verbenone Derivative Exerts In Vitro Anti-Non-Small Cell Lung Cancer Effects, Promotes Apoptosis via The P53/MDM2 Axis and Inhibits Tumor Formation in Mice.

Non-small cell lung cancer (NSCLC) stands as a prominent contributor to cancer-related fatalities on a global scale, necessitating the search for novel therapeutic agents. SP-8356, a derivative of (1S)-(-)-verbenone, has shown promise as an anticancer agent in preclinical studies. However, specific mechanisms underlying its effects in NSCLC remain to be elucidated. The aim of this research was to explore the in vitro anti-NSCLC effects of SP-8356, elucidate its mechanisms of action, and assess its efficacy in inhibiting tumor formation in a murine model. In this experimental study, NSCLC cell lines were treated with various concentrations of SP- 8356. Cell viability and proliferation were assessed using MTT and colony formation assays, respectively. Cell cycle distribution was analyzed by flow cytometry, and apoptosis was evaluated by determining apoptotic protein expression. Western blot analysis was conducted to assess protein expression levels of the both p53 and MDM2. Additionally, we evaluated efficacy of the SP-8356 in inhibiting tumor formation of the nude mouse model. SP-8356 demonstrated a concentration-dependent inhibition of cell proliferation in the NSCLC cell lines. Flow cytometric analysis showed that SP-8356 led to cell cycle arrest at the G2/M phase, indicating its potential influence on regulating the cell cycle. SP-8356 treatment was associated with the downregulation of CDK1 and Cyclin B1. Additionally, SP-8356 significantly enhanced apoptosis in NSCLC cells. SP-8356 treatment was associated with the downregulation of Bcl-2, while Bax expression was upregulated. Mechanistically, SP-8356 led to accumulation of the p53 protein levels within the NSCLC cells. This accumulation was mediated through inhibition of its negative regulator, MDM2. Using a nude mouse model demonstrated that SP-8356 effectively inhibited tumor formation in vivo. Our findings shed light on the molecular mechanisms underlying anticancer activity of SP-8356 and highlight its potential as a promising therapeutic candidate for NSCLC treatment.

RAMP2-AS1 stabilized RAPM2 mRNA through TIA1 to inhibit the progression of non-small cell lung cancer.

As the most common subtype of lung cancer, non-small cell lung cancer (NSCLC)is responsible for a large proportion of global cancer-caused deaths. The implication of long non-coding RNAs (lncRNAs) as tumor-suppressor or carcinogenic genes in NSCLC has been widely documented. Our study sought to investigate the performance of lncRNA RAMP2 antisense RNA1 (RAMP2-AS1) in NSCLC. GEPIA bioinformatics tool and RT-qPCR were applied for assessing the expression of RAMP2-AS1 and its neighboring gene receptor activity-modifying protein 2 (RAMP2) in NSCLC. Functional assays including CCK-8 assay, colony formation assay as well as caspase-3 activity analysis and Transwell invasion assays were applied for detecting the biological phenotypes of NSCLC cells. Interaction among RAMP2-AS1, RAMP2 and T-cell intracellular antigen 1cytotoxic granule associated RNA binding protein (TIA1) was evaluated by RNA immunoprecipitation and pulldown assays. We found that RAMP2-AS1 and RAMP2 were downregulated in NSCLC. Overexpression of RAMP2-AS1 hampered proliferation and invasion, whereas induced apoptosis of NSCLC cells. Mechanistically, RAMP2-AS1 interacted with TIA1 to stabilize the mRNA of RAMP2. In conclusion, we first uncovered that RAMP2-AS1 stabilized RAPM2 mRNA through TIA1 to inhibit the progression of NSCLC, providing new insight to improve the treatment efficacy of NSCLC.

Proteomic analysis reveals the molecular mechanism of Astragaloside in the treatment of non-small cell lung cancer by inducing apoptosis

BackgroundAstragaloside III (AS III), a saponin-like metabolite derived from the traditional Chinese medicine Astragali Radix, has been shown to be effective in the treatment of cancer and heart failure, and a variety of digestive disorders. However, its molecular mechanism in the treatment of non-small cell lung cancer (NSCLC) is unknown.MethodsHuman lung cancer A549 cells and NCI-H460 cells and a normal human lung epithelial cell BEAS-2B were treated with different concentrations of AS III. CCK-8 and EdU staining were used to determine the anti-proliferative effects of AS III in vitro. Quantitative proteomic analysis was performed on A549 cells treated with the indicated concentrations of AS III, and the expression levels of apoptosis-related proteins were examined by Western blotting.ResultsAS III treatment significantly inhibited proliferation and increased apoptosis in A549 and H460 cells and modulated functional signaling pathways associated with apoptosis and metabolism. At the molecular level, AS III promoted a reduction in the expression of ANXA1 (p < 0.01), with increased levels of cleaved Caspase 3 and PARP 1. In addition, AS III treatment significantly decreased the LC3-I/LC3-II ratio. The results of experiment in vitro showed that AS III promoted NSCLC apoptosis by down-regulating the phosphorylation levels of P38, JNK, and AKT (p < 0.01), inhibiting the expression of Bcl-2 (p < 0.01), and up-regulating the expression of Bax (p < 0.01).ConclusionThese findings provide a mechanism whereby AS III treatment induces apoptosis in NSCLC cells, which may be achieved in part via modulation of the P38, ERK and mTOR signaling pathways.

Shenqi Fuzheng injection restores the sensitivity to gefitinib in non-small cell lung cancer by inhibiting the IL-22/STAT3/AKT pathway

Context Non-small cell lung cancer (NSCLC) is the most common type of lung cancer. Gefitinib is a first-line treatment for NSCLC. However, its effectiveness is hindered by the development of drug resistance. At present, Shenqi Fuzheng injection (SFI) is widely accepted as an adjuvant therapy in NSCLC. Objective This study investigates the molecular mechanism of SFI when combined with gefitinib in regulating cell progression among EGFR-TKI-resistant NSCLC. Materials and methods We established gefitinib-resistant PC9-GR cells by exposing gefitinib escalation from 10 nM with the indicated concentrations of SFI in PC9 cells (1, 4, and 8 mg/mL). Quantitative real-time polymerase chain reaction was performed to assess gene expression. PC9/GR and H1975 cells were treated with 50 ng/mL of interleukin (IL)-22 alone or in combination with 10 mg/mL of SFI. STAT3, p-STAT3, AKT, and p-AKT expression were evaluated using Western blot. The effects on cell proliferation, clonogenicity, and apoptosis in NSCLC cells were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), colony formation and flow cytometry assays. Results SFI treatment alleviated the development of gefitinib resistance in NSCLC. PC9/GR and H1975 cells treated with SFI significantly exhibited a reduction in IL-22 protein and mRNA overexpression levels. SFI effectively counteracted the activation of the STAT3/AKT signaling pathway induced by adding exogenous IL-22 to PC9/GR and H1975 cells. Moreover, IL-22 combined with gefitinib markedly increased cell viability while reducing apoptosis. In contrast, combining SFI with gefitinib and the concurrent treatment of SFI with gefitinib and IL-22 demonstrated the opposite effect. Discussion and Conclusion SFI can be a valuable therapeutic option to address gefitinib resistance in NSCLC by suppressing the IL-22/STAT3/AKT pathway.

Circ-SATB2 (hsa_circ_0008928) and miR-150-5p are regulators of TRIM66 in the regulation of NSCLC cell growth and metastasis of NSCLC cells via the ceRNA pathway.

Circular RNA (circRNA) was an important modulator and potential molecular target of nonsmall cell lung cancer (NSCLC). CircSATB2 was reported to be upregulated in NSCLC. However, the role and mechanism of circSATB2 in NSCLC progression remain to be illustrated. The RNA and protein expression was detected by quantitative real-time polymerase chain reaction, western blot, and immunohistochemistry assay. Cell counting kit-8, cell colony formation, and 5-ethynyl-2'-deoxyuridine assays were applied to assess cell growth. The migrated and invaded cells were examined by transwell assay. Flow cytometry was performed to measure apoptotic cells. The interaction among circSATB2, microRNA-150-5p (miR-150-5p), and tripartite motif-containing protein 66 (TRIM66) was identified by dual-luciferase reporter assay and RNA immunoprecipitation assay. An in vivo experiment was conducted to investigate the effect of circSATB2 on tumor growth. CircSATB2 expression was highly expressed in NSCLC tissues and cell lines. CircSATB2 and TRIM66 silencing both suppressed NSCLC cell growth, migration, and invasion whereas promoted NSCLC cell apoptosis. CircSATB2 acted as a molecular sponge for miR-150-5p, and miR-150-5p interacted with the 3' untranslated region (3'UTR) of TRIM66. Moreover, circSATB2 knockdown-induced effects were partly reversed by TRIM66 overexpression in NSCLC cells. Besides, cirSATB2 expression was negatively correlated with miR-150-5p level and positively correlated with TRIM66 level in NSCLC tumor tissues. CircSATB2 knockdown blocked xenograft tumor growth in vivo. In summary, this study verified that circSATB2 stimulated NSCLC cell malignant behaviors by miR-150-5p/TRIM66 pathway, providing a possible circRNA-targeted therapy for NSCLC.

Hsa-miR-1301-3p Promotes the Proliferation and Migration of Nonsmall Cell Lung Cancer Cells and Reduces Radiosensitivity via Targeting Homeodomain-Only Protein Homeobox.

Background: There is increasing evidence that abnormal expression of microRNAs is involved in the occurrence and progression of tumors. In previous experiments, we found that the content of hsa-miR-1301-3p in tumor tissues of patients with nonsmall cell lung cancer (NSCLC) showed an obvious upward trend compared with that in normal tissues. We performed a detailed study on the impact and underlying mechanism of hsa-miR-1301-3p in NSCLC cells. Methods: The impact of hsa-miR-1301-3p on NSCLC cell proliferation, apoptosis, migration, and invasion was examined using colony formation, flow cytometry, modified Boyden chamber, and wound healing assays. Different doses of radiation were applied to NSCLC cells to investigate their sensitivity to radiotherapy. The potential target gene of hsa-miR-1301-3p was determined by dual-luciferase reporter assay and immunoblotting. Result: hsa-miR-1301-3p was upregulated in NSCLC tissues and cells. hsa-miR-1301-3p effectively promoted the rapid proliferation, migration, and invasion of NSCLC cells, while inhibiting apoptosis. It also induced radioresistance in NSCLC cells. hsa-miR-1301-3p targeted the homeodomain-only protein homeobox (HOPX) mRNA 3' untranslated region and inhibited its transcription in NSCLC cells. Exogenous HOPX overexpression antagonized the mechanism by which hsa-miR-1301-3p regulates NSCLC cell proliferation, metastasis, and apoptosis. Conclusions: hsa-miR-1301-3p plays an oncogenic role in the occurrence and development of NSCLC. By targeting HOPX, hsa-miR-1301-3p can not only promote the proliferation and metastasis of NSCLC cells, but also alleviate apoptosis and reduce radiosensitivity.

ZYZ384 suppresses the growth of EGFR-mutant non-small cell lung cancer by activating JNK/MAPK signaling pathway.

The emergency of tyrosine kinase inhibitors has remarkably enhanced the clinical outcomes of cancer therapy, especially the use of EGFR inhibitors for non-small cell lung cancer (NSCLC). However, acquired resistance is inevitable after 8-12 months treatment. New agents or treatments are urgently required to resolve this problem. In this study, we identified that compound ZYZ384 can selectively inhibit the growth of gefitinib-resistant (G-R) lung cancer cells, without affecting that of normal lung epithelial cells. ZYZ384 induced G2 arrest in G-R NSCLC cells, decreasing the expression of Cyclin B1 and increasing the expression of P21. Meanwhile, ZYZ384 also induced apoptosis in NSCLC cells and correspondingly increased the expression of cleaved Caspase 3, 8, and 9 proteins. The expression of p-JNK, p-P38, and p-ERK were also increased in H1975 NSCLC cells treated with ZYZ384. Finally, we observed that the JNK inhibitor effectively reversed the pro-apoptotic effect of ZYZ384. In conclusion, ZYZ384 is a potential therapeutic agent to inhibit the growth of NSCLCs with EGFR mutations through activating JNK, which will help the development of related anticancer drugs.

MicroRNA-140-3p inhibits proliferation and promotes apoptosis in non-small cell lung cancer by targeting MDIG.

MicroRNAs (miRNAs) are associated with cancer progression. MiR-140-3p is a tumor suppressor. Nevertheless, its function in non-small cell lung cancer (NSCLC) is unclear. MiR-140-3p expression in NSCLC clinical specimens was examined using the TCGA database and real-time PCR. NSCLC cell proliferation and apoptosis were investigated after the miRNA overexpression. Then, mineral dust-induced gene (MDIG) levels in NSCLC clinical specimens were monitored by real-time PCR and western blotting. Bioinformatics predicated the binding of miR-140-3p to MDIG, and their relationship was validated by luciferase reporter assay. The miR-140-3p/MDIG axis was further validated through rescue experiments. The involvement of STAT3 signaling in the actions of miR-140-3p/MDIG axis was investigated. MiR-140-3p was decreased in NSCLC tissues and negatively correlated with MDIG expression. Additionally, it was also lower in high-grade specimens than in low-grade ones. MiR-140-3p restrained cell proliferation, facilitated apoptosis, and inhibited STAT3 signaling in NSCLC. Interestingly, MDIG was a target of this miRNA. Furthermore, MDIG upregulation abolished miR-140-3p's effect on cell proliferation, apoptosis, and STAT3 pathway in NSCLC cells. MiR-140-3p restrained NSCLC development through the regulation of the STAT3 pathway by targeting MDIG. This axis may be a promising target for NSCLC treatment.