Non-small Cell Lung Carcinoma Cells Research Articles

βIII-tubulin suppression enhances the activity of Amuvatinib to inhibit cell proliferation in c-Met positive non-small cell lung cancer cells.

Non-Small Cell Lung Carcinoma (NSCLC) remains a leading cause of cancer death. Resistance to therapy is a significant problem, highlighting the need to find new ways of sensitising tumour cells to therapeutic agents. βIII-tubulin is associated with aggressive tumours and chemotherapy resistance in a range of cancers including NSCLC. βIII-tubulin expression has been shown to impact kinase signalling in NSCLC cells. Here, we sought to exploit this interaction by identifying co-activity between βIII-tubulin suppression and small-molecule kinase inhibitors. To achieve this, a forced-genetics approach combined with a high-throughput drug screen was used. We show that activity of the multi-kinase inhibitor Amuvatinib (MP-470) is enhanced by βIII-tubulin suppression in independent NSCLC cell lines. We also show that this compound significantly inhibits cell proliferation among βIII-tubulin knockdown cells expressing the receptor tyrosine kinase c-Met. Together, our results highlight that βIII-tubulin suppression combined with targeting specific receptor tyrosine kinases may represent a novel therapeutic approach for otherwise difficult-to-treat lung carcinomas.

Long Non-Coding RNA LINC01929 Facilitates Cell Proliferation and Metastasis as a Competing Endogenous RNA Against MicroRNA miR-1179 in Non-Small Cell Lung Carcinoma.

Introduction: Non-small cell lung carcinoma (NSCLC) constitutes most lung cancers and has a poor prognosis. LncRNAs are a potential repository for the discovery of cancer prognostic markers. This study explored the role of LINC01929 in NSCLC, both the clinical prognostic significance and the mechanism of its influence on cells. Materials and Methods: LINC01929 levels in 143 pairs of NSCLC tissues and non-cancerous tissues were detected by RT-qPCR. Kaplan-Meier curves and multivariate Cox regression assays were generated for evaluating the prognostic values of LINC01929. To evaluate the cellular function, an XTT assay and transwell invasion assays were performed. Results: LINC01929 was up-regulated in NSCLC tissues compared with healthy tissues. A positive correlation was observed between LINC01929 expression level and tumor T (p = 0.002) or N stage (p = 0.010). Patients with higher LINC01929 levels had shorter overall survival (p = 0.009). Compared with other factors, high LINC01929 expression was significantly associated with poor survival in univariate Cox analysis (HR: 2.485, 95%CI: 1.220–5.060, p = 0.012). After multivariate Cox regression assays, LINC01929 was a independent prognostic factor (HR: 3.021, 95%CI: 1.377–6.628, p = 0.006). miR-1179 was a target miRNA of LINC01929. Inhibited expression of LINC01929 significantly reduced the proliferation, migration, and invasion of NSCLC cells by targeting miR-1179. Discussion: This study revealed the upregulation of LINC01929 in NSCLC. This study supports previous studies showing LINC01929 as a potential prognostic factor for NSCLC.

Targeting the SREBP-1/Hsa-Mir-497/SCAP/FASN Oncometabolic Axis Inhibits the Cancer Stem-like and Chemoresistant Phenotype of Non-Small Cell Lung Carcinoma Cells.

Background: Lung cancer remains a leading cause of cancer-related death, with an annual global mortality rate of 18.4%. Despite advances in diagnostic and therapeutic technologies, non–small cell lung carcinoma (NSCLC) continues to be characterized by a poor prognosis. This may be associated with the enrichment of cancer stem cells (CSCs) and the development of chemoresistance—a double-edged challenge that continues to impede the improvement of long-term outcomes. Metabolic reprogramming is a new hallmark of cancer. Sterol regulatory element-binding proteins (SREBPs) play crucial regulatory roles in the synthesis and uptake of cholesterol, fatty acids, and phospholipids. Recent evidence has demonstrated that SREBP-1 is upregulated in several cancer types. However, its role in lung cancer remains unclear. Objective: This study investigated the role of SREBP-1 in NSCLC biology, progression, and therapeutic response and explored the therapeutic exploitability of SREBP-1 and SREBP-1-dependent oncometabolic signaling and miRNA epigenetic regulation. Methods: We analyzed SREBP-1 levels and biological functions in clinical samples and the human NSCLC cell lines H441 and A549 through shRNA-based knock down of SREBP function, cisplatin-resistant clone generation, immunohistochemical staining of clinical samples, and cell viability, sphere-formation, Western blot, and quantitative PCR assays. We conducted in-silico analysis of miRNA expression in NSCLC samples by using the Gene Expression Omnibus (GSE102286) database. Results: We demonstrated that SREBP-1 and SCAP are highly expressed in NSCLC and are positively correlated with the aggressive phenotypes of NSCLC cells. In addition, downregulation of the expression of tumor-suppressing hsa-miR-497-5p, which predictively targets SREBP-1, was observed. We also demonstrated that SREBP-1/SCAP/FASN lipogenic signaling plays a key role in CSCs-like and chemoresistant NSCLC phenotypes, especially because the fatostatin or shRNA targeting of SREBP-1 significantly suppressed the viability, cisplatin resistance, and cancer stemness of NSCLC cells and because treatment induced the expression of hsa-miR-497. Conclusion: Targeting the SREBP-1/hsa-miR-497 signaling axis is a potentially effective anticancer therapeutic strategy for NSCLC.

LY2874455 and Abemaciclib Reverse FGF3/4/19/CCND1 Amplification Mediated Gefitinib Resistance in NSCLC.

Non-small cell lung carcinoma (NSCLC) patients who initially received tyrosine kinase inhibitor (TKI) therapy often acquired resistance via multiple complex mechanisms. The amplification of FGF3/4/19/CCND1 on chromosome 11q13 was found in many cancers with TKI resistance. However, the role of these amplifications in TKI-resistant NSCLC remains uncovered. Here, we generated the FGF3/4/19/CCND1 amplification model in the NSCLC cell lines PC-9 and HCC827. Upregulation of FGF3/4/19/CCND1 strongly promoted cell proliferation and gefitinib resistance in NSCLC cells. To find out the potential therapeutic strategies, we screened the combination of inhibitors against the FGF/FGFR signaling pathway and the CCND1/CDK4 complex and revealed that gefitinib combined with LY2874455 and abemaciclib exhibited the most effective inhibition of resistance in vitro and in vivo. Mechanistically, FGFs/CCND1 activated the MAPK pathway, which was abolished by the combination drugs. Our study provides a rationale for clinical testing of dual targeting FGFR and CCND1 with LY2874455 and abemaciclib in NSCLC patients who harbored FGF3/4/19/CCND1 amplification.

Euphorbiasteroid Abrogates EGFR and Wnt/β-Catenin Signaling in Non-Small-Cell Lung Cancer Cells to Impart Anticancer Activity.

EGFR and Wnt/β-catenin signaling pathways play a prominent role in tumor progression in various human cancers including non-small-cell lung carcinoma (NSCLC). Transactivation and crosstalk between the EGFR and Wnt/β-catenin pathways may contribute to the aggressiveness of cancers. Targeting these oncogenic pathways with small molecules is an attractive approach to counteract various types of cancers. In this study, we demonstrate the effect of euphorbiasteroid (EPBS) on the EGFR and Wnt/β-catenin pathways in NSCLC cells. EPBS induced preferential cytotoxicity toward A549 (wildtype EGFR-expressing) cells over PC-9 (mutant EGFR-expressing) cells. EPBS suppressed the expression of EGFR, Wnt3a, β-catenin, and FZD-1, and the reduction in β-catenin levels was found to be mediated through the activation of GSK-3β. EPBS reduced the phosphorylation of GSK-3βS9 with a parallel increase in β-TrCP and phosphorylation of GSK-3βY216. Lithium chloride treatment increased the phosphorylation of GSK-3βS9 and nuclear localization of β-catenin, whereas EPBS reverted these effects. Forced expression or depletion of EGFR in NSCLC cells increased or decreased the levels of Wnt3a, β-catenin, and FZD-1, respectively. Overall, EPBS abrogates EGFR and Wnt/β-catenin pathways to impart its anticancer activity in NSCLC cells.

Recapitulated Crosstalk between Cerebral Metastatic Lung Cancer Cells and Brain Perivascular Tumor Microenvironment in a Microfluidic Co-Culture Chip.

Non‐small cell lung carcinoma (NSCLC), which affects the brain, is fatal and resistant to anti‐cancer therapies. Despite innate, distinct characteristics of the brain from other organs, the underlying delicate crosstalk between brain metastatic NSCLC (BM‐NSCLC) cells and brain tumor microenvironment (bTME) associated with tumor evolution remains elusive. Here, a novel 3D microfluidic tri‐culture platform is proposed for recapitulating positive feedback from BM‐NSCLC and astrocytes and brain‐specific endothelial cells, two major players in bTME. Advanced imaging and quantitative functional assessment of the 3D tri‐culture model enable real‐time live imaging of cell viability and separate analyses of genomic/molecular/secretome from each subset. Susceptibility of multiple patient‐derived BM‐NSCLCs to representative targeted agents is altered and secretion of serpin E1, interleukin‐8, and secreted phosphoprotein 1, which are associated with tumor aggressiveness and poor clinical outcome, is increased in tri‐culture. Notably, multiple signaling pathways involved in inflammatory responses, nuclear factor kappa‐light‐chain‐enhancer of activated B cells, and cancer metastasis are activated in BM‐NSCLC through interaction with two bTME cell types. This novel platform offers a tool to elucidate potential molecular targets and for effective anti‐cancer therapy targeting the crosstalk between metastatic cancer cells and adjacent components of bTME.

Silencing of lncRNA CRNDE attenuates nonsmall-cell lung cancer progression by mediating the miR-455-3p/HDAC2 axis.

Nonsmall-cell lung carcinoma (NSCLC) is one of the deadliest malignancies in the world. LncRNAs are confirmed to be involved in the progression of NSCLC. Meanwhile, lncRNA CRNDE is known to be upregulated in NSCLC; however, the mechanism by which CRNDE regulates the tumourigenesis of NSCLC remains unclear. To test the function of CRNDE in NSCLC, cell proliferation, invasion, and migration were investigated by colony formation and Transwell assays, respectively. qPCR and Western blotting were applied to test gene and protein levels. In addition, the relationship among CRNDE, miR-455-3p, and HDAC2 was explored by dual-luciferase and RIP assays. The data revealed that the expression of CRNDE was upregulated in NSCLC tissues, while miR-455-3p was downregulated. CRNDE knockdown inhibited the viability, migration and invasion of NSCLC cells or epidermal growth factor receptor gene (EGFR)-mutant NSCLC cells. Moreover, inhibition of miR-455-3p exhibited the opposite effect. CRNDE bound with miR-455-3p, and HDAC2 was found to be targeted by miR-455-3p. Meanwhile, miR-455-3p downregulation reversed the effect of CRNDE knockdown on NSCLC cell function. Furthermore, miR-455-3p notably inhibited the growth and invasion of NSCLC cells via downregulation of HDAC2. Knockdown of CRNDE attenuated NSCLC progression via modulation of the miR-455-3p/HDAC2 axis. Thus, those findings might provide a novel strategy against NSCLC.

MO136: Histological Findings in Checkpoint Inhibitor-Induced Nephropathy

Abstract BACKGROUND AND AIMS Immune checkpoint inhibitors (CPIs)—a landmark in cancer therapy—has been occasionally associated with immune-related nephropathy [1]. Acute interstitial nephritis is considered to be the most common underlying histological entity, according to recent literature [2]. METHOD We report on six patients, 58–73 years old, three women and three men who presented with acute kidney disease, while on immunotherapy with CPIs. Five of them carried the diagnosis of non-small cell lung carcinoma (NSCLC) and one the diagnosis of renal cell carcinoma. All received proton pump inhibitors and experienced one or more of immune-related adverse events (dermatitis, arthritis, colitis, hepatitis and pneumonitis), prior to nephropathy. All underwent kidney biopsy. RESULTS Two female patients, both with NSCLC, developed heavy nephrotic syndrome (serum albumin: 1.7–1.8 mg/dL and 24-h urine protein: 12–16 g), after 23–28 months on pembrolizumab monotherapy, which was withdrawn. In the first patient, kidney biopsy demonstrated histology consistent with a lupus like membranous nephropathy (Figure 1). She received corticosteroids with partial recovery. Rechallenge with pembrolizumab was attempted and well tolerated. Renal biopsy of the second patient demonstrated a secondary AA (+) amyloidosis (Figure 2). She showed increased levels of SAA (114 mg/L) and IL-1b (22.9 pg/mL). She received colchicine and moderate dose of corticosteroids with partial remission. Two other patients, with NSCLC and clear cell renal carcinoma respectively, demonstrated renal biopsy findings consistent with acute interstitial nephritis (serum creatinine 1.5–1.6 mg/dL). The first one was on pembrolizumab along with cisplatin and pemetrexed, while the second one was on pembrolizumab along with axitinib. Both received prednisolone (10–20 mg for 3 months approximately). Pembrolizumab was restarted thereafter. Their renal function remained stable. Finally, two patients with NSCLC, both men, disclosed acute renal failure (serum creatinine: 3.6- 5 mg/dL) without considerable albuminuria. Acute tubular injury (ATI) was the predominant histological finding (along with focal and mild interstitial inflammation). Both were on combined chemo- and immune- therapy. One on ipilimumab and nivolumab along with platinum and pemetrexed and the other on pembrolizumab and pemetrexed. ATI was attributed most probably to pemetrexed which was withdrawn. Corticosteroids were promptly stopped in the first patient. The second patient showed gradual improvement of his renal function without corticosteroid therapy. CONCLUSION CPIs can induce nephropathy other than acute interstitial nephritis. Renal biopsy showed diverse and unusual histological findings. Renal biopsy along with the clinical setting was helpful in choosing appropriate therapy and deciding on CPI's continuation or withdrawal.

Cytotoxic Alkylynols of the Sponge Cribrochalina vasculum: Structure, Synthetic Analogs and SAR Studies.

A series of twenty-three linear and branched chain mono acetylene lipids were isolated from the Caribbean Sea sponge Cribrochalina vasculum. Seventeen of the compounds, 1–17, are new, while six, 18–23, were previously characterized from the same sponge. Some of the new acetylene-3-hydroxy alkanes 1, 6, 7, 8, 10 were tested for selective cytotoxicity in non-small cell lung carcinoma (NSCLC) cells over WI-38 normal diploid lung fibroblasts. Compound 7, presented clear tumor selective activity while, 1 and 8, showed selectivity at lower doses and 6 and 10, were not active towards NSCLC cells at all. The earlier reported selective cytotoxicity of some acetylene-3-hydroxy alkanes (scal-18 and 23), in NSCLC cells and/or other tumor cell types were also confirmed for 19, 20 and 22. To further study the structure activity relationships (SAR) of this group of compounds, we synthesized several derivatives of acetylene-3-hydroxy alkanes, rac-18, scal-S-18, R-18, rac-27, rac-32, R-32, S-32, rac-33, rac-41, rac-42, rac-43, rac-45, rac-48 and rac-49, along with other 3-substituted derivatives, rac-35, rac-36, rac-37, rac-38, rac-39 and rac-40, and assessed their cytotoxic activity against NSCLC cells and diploid fibroblasts. SAR studies revealed that the alcohol moiety at position 3 and its absolute R configuration both were essential for the tumor cell line selective activity while for its cytotoxic magnitude the alkyl chain length and branching were of less significance.

Study on the Mechanism of Action of Paclitaxel-Loaded Polylactic-co-glycolic Acid Nanoparticles in Non-Small-Cell Lung Carcinoma Cells.

Objective To study effective carriers that can enhance the antitumor effect of paclitaxel (PTX). Methods PTX-loaded polylactic-co-glycolic acid (PLGA) nanoparticles (NPs) (PTX-PLGA NPs), constructed using the emulsification solvent evaporation method, were characterized by scanning electron microscopy and dynamic light scattering. Non-small-cell lung carcinoma (NSCLC) cells were divided into the dimethyl sulfoxide (DMSO) group, PLGA NPs group, PTX group, and PTX-PLGA NPs group. Cell viability was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), cell apoptosis was determined by flow cytometry, and cell migration and invasion were assessed using Transwell assay. Results PTX-PLGA NPs were smooth in the surface and spherical in shape, with a particle size of 268 ± 1.3 nm. Both PTX and PTX-PLGA NPs could effectively inhibit the activity of A549 and H1650 cells. At 12 and 24 h, PTX-PLGA NPs presented weaker inhibition on the activity of NSCLC cells than PTX, but at 48 and 72 h, PTX-PLGA NPs presented stronger inhibition. Compared with PTX, PTX-PLGA NPs were more effective in enhancing apoptosis and inhibiting migration and invasion of NSCLC cells. Conclusion With good sustained release and the ability to promote cellular uptake, PTX-PLGA NPs can strongly inhibit the malignant activities of NSCLC cells, which can be used as a promising drug carrier.

Evodiamine suppresses the progression of non-small cell lung carcinoma via endoplasmic reticulum stress-mediated apoptosis pathway in vivo and in vitro.

BackgroundEvodiamine (EVO) is one of the major components isolated from Evodia rutaecarpa (Juss.). Recent studies have shown that EVO has an anti-cancer effect. However, the pharmacological mechanism by which EVO impacts cancer is still poorly understood.ObjectivesThis study focused on asking the anti-cancer effect of EVO in human non-small cell lung carcinoma (NSCLC), and in particular to investigate whether EVO acts via modulating the endoplasmic reticulum stress (ERS)-mediated apoptosis pathway.Materials and MethodsA Lewis lung carcinoma (LLC) tumor-bearing mouse model was treated with low-dose EVO (5 mg/kg) and high-dose EVO (10 mg/kg) intraperitoneally for 14 d. The effects of EVO on tumor growth, apoptosis, and ERS were assessed. In addition, NSCLC A549 and LLC cells were treated with EVO in vitro. The effects of EVO on cell proliferation, apoptosis, and ERS were investigated. Finally, 4-phenylbutyric acid (4-PBA), an ERS inhibitor, was used to validate whether EVO induced apoptosis of NSCLC cells by modulating ERS.ResultsEVO treatment significantly inhibited tumor growth in LLC tumor-bearing mice. H&E staining indicated that EVO treatment reduced the number of tumor cells and the nucleo-plasmic ratio. Immunostaining showed that EVO treatment significantly decreased the expression of Ki-67. TUNEL staining revealed that EVO induced apoptosis in the tumor. Likewise, EVO treatment up-regulated the expression of apoptosis-related genes and proteins and increased activation of the ERS pathway in the tumor. Additionally, EVO inhibited cell proliferation and increased cell apoptotic rates in A549 and LLC cells. EVO also increased the expression levels of genes and proteins associated with ERS-mediated apoptosis pathway in vitro. The effects of EVO on apoptosis were abolished by 4-PBA treatment.ConclusionsOur study demonstrated that EVO suppresses the progression of NSCLC by modulating the ERS-mediated apoptosis pathway.

Recombinant human erythropoietin accelerated the proliferation of non-small cell lung cancer cell lines and reduced the expression of VEGF, HIF-1α, and PD-L1 under a simulated hypoxic environment in vitro.

BackgroundAs erythropoietin (EPO) has been used to treat anemia in cancer patients, negative controversy has continued. Unfortunately, its effects on non‐small‐cell lung carcinoma (NSCLC) cell lines are uncertain and the phenomenon of inducing immune escape of tumor cells remains to be explored. This study aimed to provide an important basis for the application of exogenous EPO in the treatment of tumor‐associated anemia.MethodsCells were cultured in 1% O2, 5% CO2, and 94% N2 to simulate a hypoxic environment of the tumor. A549 cell line (lower expression EPOR) and NCI‐H838 cell line (higher expression EPOR) were treated with 2 and 8 U/ml recombinant human EPO (rhEPO). CCK‐8 method was used to determine the logarithmic growth phase of the cells and to detect cell proliferation. The expression levels of VEGF, HIF‐1α, and PD‐L1 were determined by western blot. One‐way ANOVA was used for statistical analysis between groups, with p < 0.05 indicating a significant difference.ResultsHypoxia itself could decrease the survival rate of NSCLC cells. Under the hypoxic condition, rhEPO induced tumor cells proliferation, especially in the NCI‐H838 cell line, where 2 U/ml rhEPO increased the total number of surviving cells (Hypoxia + rhEPO 2 U/ml vs. Hypoxia, p < 0.05). Western blot analysis showed that hypoxia upregulated the expression of VEGF, HIF‐1α, and PD‐L1 in NSCLC cell lines (Normoxia vs. Hypoxia, p < 0.05), but may not be dependent on the expression levels of EPOR. RhEPO decreased the expression levels of VEGF and HIF‐1α. In the A549 cell line, it depended on the concentration of rhEPO and was particularly obvious in HIF‐1α (Hypoxia vs. Hypoxia + rhEPO 2 U/ml vs. Hypoxia + rhEPO 8 U/ml, p < 0.05). A low concentration of rhEPO may not reduce VEGF expression. In the NCI‐H838 cell line, the effect of rhEPO on VEGF was more obvious, but it may be independent of rhEPO concentrations. The downregulation of PD‐L1 expression by rhEPO was only presented in the A549 cell line and required higher rhEPO concentrations (Hypoxia + rhEPO 8 U/ml vs. Hypoxia&Hypoxia + rhEPO 2 U/ml, p < 0.05).ConclusionThe effect of prolonged high concentrations of rhEPO under hypoxic conditions resulted in accelerated cells proliferation of non‐small‐cell lung cancer and was independent of EPOR expression levels on the cell lines surface. Hypoxia resulted in increased expression of VEGF, HIF‐1α, and PD‐L1 on the NSCLC cell lines. Under normoxic conditions, rhEPO did not affect the expression of VEGF, HIF‐1α, and PD‐L1; but under hypoxic conditions, the application of rhEPO reduced the expression of VEGF, HIF‐1α, and PD‐L1, producing an impact on the biological behavior of tumor cells.

Integrated Network Pharmacology and Experimental Verification to Explore the Molecular Mechanism of Hedysarum Multijugum Maxim–Curcumae Rhizoma Herb Pair for Treating Non-Small Cell Lung Cancer

BackgroundHedysarum Multijugum Maxim–Curcumae Rhizoma (HMMCR), a well-known herb pair in traditional Chinese medicine (TCM), has been widely used for the treatment of various cancers. However, the active components of HMMCR and the underlying mechanism of HMMCR for non-small-cell lung carcinoma (NSCLC) remain unclear.MethodsActive ingredients of HMMCR were detected by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). On this basis, potential targets of HMMCR were obtained from SwissTargetPrediction database. NSCLC-related targets were collected from four public databases (GeneCards, OMIM, TTD, and PharmGkb). The drug ingredients–disease targets network was visualized. The hub targets between HMMCR and NSCLC were further analyzed by protein–protein interaction (PPI), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Subsequently, the results predicted by network pharmacology were further validated via in vitro experiments.ResultsA total of 181 compounds were identified from the aqueous extract of HMMCR. Through network analysis, a compound–target network including 153 active ingredients of HMMCR and 756 HMMCR-NSCLC co-targets was conducted; 6 crucial compounds and 62 hub targets were further identified. The results of KEGG enrichment analysis showed that PI3K/Akt signaling pathway may be the critical pathway of HMMCR in the treatment of NSCLC. The in vitro experiments indicated that HMMCR inhibits the proliferation and migration of NSCLC cells via inactivation of the PI3K/Akt signaling pathway, consistent with the results predicted by network pharmacology.ConclusionIntegrating LC-ESI-MS/MS, network pharmacology approach, and in vitro experiments, this study shows that HMMCR has vital therapeutic effect on NSCLC through multi-compound, multi-target, and multi-pathway, which provides a rationale for using HMMCR for the treatment of NSCLC.

Bisdemethoxycurcumin sensitizes the response of cisplatin resistant non-small cell lung carcinoma cell lines by activating apoptosis and autophagy

Lung cancer belongs to the most frequent and deadliest cancer types worldwide, non-small cell lung carcinoma (NSCLC) being the most frequent type. Development of chemoresistance in NSCLC patients is common and responsible for bad outcome. Curcuminoids are naturally occurring substances with prominent cytotoxic effects in different cancer cells. Here we analyzed influence of bisdemethoxycurcumin (BDMC) on phenotype and molecular mechanisms in cisplatin-sensitive NSCLC cell lines (A549 and H460) and their cisplatin-resistant counterparts. NSCLC cell lines were exposed to BDMC and analyzed by cell viability, proliferation, and motility assays, as well as fluorescence-activated cell sorting. Immunoblotting was assessed to detect apoptosis and autophagy. Colony-formation assay and multicellular tumor spheroid model were used to investigate the effects of BDMC. Expression levels of different Hedgehog-pathway genes were determined by RT-qPCR analysis. We identified substantial cytotoxic effects of BDMC on NSCLC cells in general and on cisplatin-resistant NSCLC cells in special. BDMC markedly decreased the cell viability by inducing apoptosis and autophagy in a cell-type specific manner. BDMC emphasized cisplatin-induced cell death and inhibited cell cycle progression of cisplatin-resistant NSCLC cells. Scratch-closure, colony formation, and multicellular spheroid growth in cisplatin-resistant NSCLC cell lines were inhibited by BDMC. Expression profile analyses of different Hedgehog-pathway regulatory genes showed that Gli1, the mean transcriptional regulator of this pathway, was markedly decreased upon the BDMC treatment, this decrement being most prominent in cisplatin-resistant cells. Our data identified BDMC as a potent substance that may be suitable for combined cisplatin-based therapy in cisplatin-resistant subpopulation of NSCLC patients.

Sanguinarine Represses the Growth and Metastasis of Non-small Cell Lung Cancer by Facilitating Ferroptosis.

Ethnopharmacological relevance: Sanguinarine (SAG), a natural benzophenanthridine alkaloid derived from the root of Sanguinaria canadensis Linn. (Bloodroot), possesses a potential anticancer activity. Lung carcinoma is the chief cause of malignancy-related mortality in China. Non-small cell lung carcinoma (NSCLC) is the main subtype of lung carcinoma and accounts for about eighty-five percent of this disease. Current treatment in controlling and curing NSCLC remains deficient. The role and underlying mechanism of SAG in repressing the growth and metastasis of NSCLC were explored. The role of SAG in regulating the proliferation and invasion of NSCLC cells was evaluated in vitro and in a xenograft model. After treatment with SAG, Fe2+ concentration, reactive oxygen species (ROS) levels, malondialdehyde (MDA), and glutathione (GSH) content in NSCLC cells were assessed to evaluate the effect of SAG on facilitating ferroptosis. SAG exhibited a dose- and time-dependent cytotoxicity in A549 and H3122 cells. SAG treatment effectively repressed the growth and metastasis of NSCLC in a xenograft model. We, for the first time, verified that SAG triggered ferroptosis of NSCLC cells, as evidenced by increased Fe2+ concentration, ROS level, and MDA content, and decreased GSH content. Mechanistically, SAG decreased the protein stability of glutathione peroxide 4 (GPX4) through E3 ligase STUB1-mediated ubiquitination and degradation of endogenous GPX4. GPX4 overexpression restored the proliferation and invasion of NSCLC cells treated with SAG through inhibiting ferroptosis. SAG inhibits the growth and metastasis of NSCLC by regulating STUB1/GPX4-dependent ferroptosis.

MiR-148a promotes cell sensitivity through downregulating SOS2 in radiation-resistant non-small cell lung cancer cells.

Non-small cell lung carcinoma (NSCLC) is the most common type of lung cancer; however, radioresistance is a significant barrier in NSCLC radiotherapy. MicroRNA (miR)-148a has been reported to be a tumor suppressor in various types of cancer, including NSCLC. In the present study, the potential role of miR-148a in regulating radiosensitivity of NSCLC cells was investigated. Serum miR-148a expression was evaluated by reverse transcription-quantitative PCR in patients with NSCLC and healthy controls. The effects of miR-148a on cell viability, migration and invasion were assessed by Cell Counting Kit-8 and Transwell assays in radiation-resistant NSCLC cells. Serum miR-148a was downregulated in patients with NSCLC compared with healthy controls and its expression was significantly increased after radiotherapy. By contrast, miR-148a expression was decreased in the radioresistant patients compared with the radiosensitivity patients. Additionally, miR-148a overexpression inhibited the cell proliferation, migration and invasion of radiation-resistant NSCLC cells. In addition, miR-148a had putative binding site with Son of Sevenless 2 (SOS2) and negatively regulated SOS2 expression. Silencing SOS2 expression significantly suppressed miR-148a inhibitor-induced increase in radiosensitivity in NSCLC. In conclusion, the results of the present study suggested that miR-148a could enhance the radiosensitivity of NSCLC cells through targeting SOS2, thus providing potential therapeutic targets to improve radiotherapy in NSCLC.

Anthrax toxin receptor 2 is a potential therapeutic target for non-small cell lung carcinoma with MET exon 14 skipping mutations

Although MET tyrosine kinase inhibitors (TKIs) are generally effective against non-small cell lung carcinoma (NSCLC) with MET exon 14 skipping mutations (METΔex14), resistance to MET TKIs can occur, indicating the need to develop other therapeutic options. We found that Hs-746 T cells, which harbor METΔex14 plus amplification, were able to survive and grow in the absence of MET signaling, exhibiting primary resistance to MET TKIs. We also found a moderately positive correlation between MET and anthrax toxin receptor 2 (ANTXR2) mRNA expression in NSCLC cell lines using data from the Cancer Dependency Map database. As expected, Hs-746 T cells were positive for ANTXR2 expression. We used an antibody–drug conjugate (ADC) analog in the form of an anti-ANTXR2 monoclonal antibody, H8R23, conjugated to DT3C recombinant protein which consists of diphtheria toxin (DT) lacking the receptor-binding domain but containing the C1, C2, and C3 domains of streptococcal protein G (3C). H8R23-DT3C conjugates, which function in vitro like an ADC, induced Hs-746 T cells to undergo apoptosis, resulting in decreased viability. These findings collectively suggest that an ADC targeting ANTXR2 could be effective for the treatment of METΔex14-positive NSCLC.

ING2-WTAP is a potential therapeutic target in non-small cell lung cancer

Non-small-cell lung carcinoma (NSCLC) is the most common kind of lung cancer, which is also the largest cause of cancer-linked deaths globally. A lack of effective prognostic biomarkers contributes to the poor prognosis of NSCLC. For better patient outcomes, NSCLC diagnosing, prognostic, and predictive biomarkers are critically needed. A component of the chromatin-modulatory complex that dampens gene expression, ING2 (inhibitor of growth family member 2), has been linked to cellular mechanisms that enhance tumor repression. Nevertheless, its role in human NSCLC is unclear. Herein, we observed that ING2 downregulation in NSCLC tissues correlates with poor NSCLC prognosis. Functional analysis illustrated that ING2 dampened growth, infiltration along with metastasis and blocked apoptosis of NSCLC cells in vitro, as well as in vivo. The mechanism might be implicated in the EMT (epithelial-mesenchymal transition) process. Mechanistically, we found that ING2 silencing suppresses the expressions of Wilms tumor 1-associated protein (WTAP) and that WTAP expression negatively correlates with ING2 expression. Moreover, rescue experiments illustrated that WTAP overexpression partially counteracts the effect of ING2 silencing on proliferation. Finally, we found that elevated WTAP levels correlate with poor NSCLC prognosis. These results highlight the prognostic and therapeutic potential of ING2-WTAP in NSCLC.

Heteronemin and tetrac derivatives suppress non-small cell lung cancer growth via ERK1/2 inhibition

The most common cancer, lung cancer, causes deaths worldwide. Most lung cancer patients have non-small cell lung carcinomas (NSCLCs) with a poor prognosis. The chemotherapies frequently cause resistance therefore search for new effective drugs for NSCLC patients is an urgent and essential issue. Deaminated thyroxine, tetraiodothyroacetic acid (tetrac), and its nano-analogue (NDAT) exhibit antiproliferative properties in several types of cancers. On the other hand, the most abundant secondary metabolite in the sponge Hippospongia sp., heteronemin, shows effective cytotoxic activity against different types of cancer cells. In the current study, we investigated the anticancer effects of heteronemin against two NSCLC cell lines, A549 and H1299 cells in vitro. Combined treatment with heteronemin and tetrac derivatives synergistically inhibited cancer cell growth and significantly modulated the ERK1/2 and STAT3 pathways in A549 cells but only ERK1/2 in H1299 cells. The combination treatments induce apoptosis via the caspases pathway in A549 cells but promote cell cycle arrest via CCND1 and PCNA inhibition in H1299 cells. In summary, these results suggest that combined treatment with heteronemin and tetrac derivatives could suppress signal transduction pathways essential for NSCLC cell growth. The synergetic effects can be used potentially as a therapeutic procedure for NSCLC patients.

Hippocalcin-like 1 is a key regulator of LDHA activation that promotes the growth of non-small cell lung carcinoma.

Hippocalcin-like 1 (HPCAL1), a neuronal calcium sensor protein family member, has been reported toregulate cancer growth. As yet, however, the biological functions of HPCAL1 and its molecular mechanisms have not been investigated in non-small cell lung carcinoma (NSCLC). HPCAL1 expression in NSCLC samples was detected using immunohistochemistry, Western blotting and RT-PCR. The anticancer effects of HPCAL1 knockdown were determined by MTT, soft agar, cell cycle, oxygen consumption and reactive oxygen species assays. The effect of HPCAL1 knockdown on in vivo tumor growth was assessed using NSCLC cancer patient-derived xenograft models. Potentially interacting protein partners of HPCAL1 were identified using IP-MS/MS, immunoprecipitation and Western blottingassays. Metabolic alterations resulting from HPCAL1 knockdown were investigated using non-targeted metabolomics and RNA sequencing analyses. We found thatHPCAL1 is highly expressed in NSCLC tissues and is positively correlated with low survival rates and AJCC clinical staging in lung cancer patients. Knockdown of HPCAL1 strongly increased oxygen consumption rates andthe production of reactive oxygen species. HPCAL1 knockdown also inhibited NSCLC cell growth and patient-derived NSCLC tumor growth in vivo. Mechanistically, we found that HPCAL1 can directly bindto LDHA and enhance SRC-mediated phosphorylation of LDHA at tyrosine 10. The metabolomics and RNA sequencing analyses indicated thatHPCAL1 knockdown reduces amino acid levels and induces fatty acid synthesis through regulating the expression of metabolism-related genes. Additionally, rescued cells expressing wild-type or mutant LDHA in HPCAL1 knockdown cells suggest that LDHAmay serve as the main substrate of HPCAL1. Our data indicate that the effect of HPCAL1 knockdown on reducing SRC-mediated LDHA activity attenuates NSCLC growth. Our findings reveal novel biological functions and a mechanismunderlying the role of HPCAL1 in NSCLC growth in vitro and in vivo.