Treatment Of Non-small Cell Lung Cancer Cells Research Articles

Jiedu Fuzheng decoction improves the proliferation, migration, invasion and EMT of non-small cell lung cancer via the Wnt/β-catenin pathway

ObjectivesThis study aimed to investigate the effect of Jiedu Fuzheng decoction (JFD) in non-small cell lung cancer (NSCLC) and its potential therapeutic mechanism.ResultsWe prepared JFD-medicated serum from rats and treated NSCLC cells (A549 and NCI-H1650) with 0.5, 1, and 2 mg/mL JFD-medicated serum. CCK-8 and colony formation assays were used to detect cell proliferation. Transwell assays showed that JFD attenuated cell migration and invasion. JFD and SKL2001 (Wnt/β-catenin activator) were simultaneously used to treat NSCLC cells to verify that JFD regulated the biological behavior of NSCLC via Wnt/β-catenin signaling. It was found that 2 mg/mL JFD had the most significant effect on the activity of NSCLC cells. JFD attenuated proliferation and metastasis but increased the proportion of apoptotic cells. At the same time, JFD downregulated N-cadherin, vimentin and β-catenin protein expression in cancer cells. SKL2001 could restore the improvement of JFD on proliferation, metastasis and apoptosis.ConclusionThis study confirmed that JFD suppressed the occurrence and development of NSCLC by regulating Wnt/β-catenin signaling and provided a novel therapeutic scheme for NSCLC.

Upregulation of long non‑coding RNA LINC00460 in EGFR‑mutant lung cancer indicates a poor prognosis in patients treated with osimertinib.

The long non-coding RNA (lncRNA) LINC00460 is involved in tumor growth, metastasis and drug resistance. The present study investigated the clinical significance of LINC00460 expression in patients with epidermal growth factor receptor (EGFR) mutation-positive lung cancer treated with osimertinib. Osimertinib-resistant cells we derived from EGFR-mutant non-small-cell lung cancer (NSCLC) cell lines, after which, small interfering RNA (siRNA)-mediated silencing and in vitro-transcribed (IVT), synthetic LINC00460 RNA transfection were used to investigate the effects of LINC00460 expression on acquired resistance to osimertinib. Reverse transcription-quantitative polymerase chain reaction was performed to evaluate LINC00460 expression in 54 samples (RNA extracted from the tumor tissues of 30 cases and cell-free RNA from 24 cases) obtained from patients with EGFR mutation-positive lung cancer who had received osimertinib as the initial treatment. The acquisition of osimertinib resistance increased the expression of LINC00460 in the EGFR-mutant NSCLC cell lines. By contrast, knockdown of LINC00460 in osimertinib-resistant cell lines increased their sensitivity to osimertinib, whereas treatment of NSCLC cells with IVT LINC00460 RNA decreased their sensitivity to osimertinib. The present study examined LINC00460 expression at the primary tumor site and demonstrated that compared with in the low-expression group (n=24), the high-expression group (n=6) had a significantly lower best overall response rate to osimertinib (16.6% vs. 60.0%; P=0.044), significantly shorter median progression-free survival (PFS; 224 days vs. 669 days; P=0.001) and significantly shorter median overall survival (724 days vs. not reached; P=0.011). Moreover, following osimertinib therapy, PFS was significantly shorter for patients with high LINC00460 expression in plasma cell-free RNA (n=12) than for those with low LINC00460 expression (n=12) (median PFS: 655 days vs. 210 days; P=0.020). In conclusion, the upregulation of LINC00460, the expression of which is implicated in osimertinib resistance, in the primary site and plasma of patients with EGFR mutation-positive lung cancer may be associated with a poor prognosis in those treated with osimertinib.

Abstract 2428: Cancer stem cells maintain stemness via autocrine activation of the IFN/JAK/STAT signaling pathway

Abstract Interferons (IFNs) and Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) signaling are best known for their roles in immunity. However, recent work has suggested that both IFNs and the JAK/STAT pathways are associated with tumor growth and progression, as well as the maintenance of cancer stem cell (CSC) populations. To better understand the regulation of the CSC population maintenance by the IFN/JAK/STAT signaling pathway, we enriched CSCs from a panel of non-small cell lung cancer (NSCLC) cell lines by using spheroid culture and determined the phosphorylation of STAT1. We found that sphere cells exhibit increased level of pSTAT1-Y701 compared to their corresponding bulk cells. We further determined the expression level of a few interferon-stimulated genes (ISGs) and found that sphere cells possess enhanced expression of ISG54 and ISG56 genes, which are normally induced by type I IFN (IFN-I), as well as IFNA and IFNB. These results indicate that the IFN-I/STAT1 signaling is highly activated in CSCs, which produce and secrete increased amount of IFN-I. To directly show the effect of IFN-I on the stemness of NSCLC cells, we treated NSCLC cells with IFNβ, and found that IFNβ can significantly enhance the expression of SOX2, a stem cell-specific transcription factor; Inhibition of the JAK signaling blocked both basal and IFNβ-induced SOX2 expression. In addition, knockdown of STAT1 also reduced SOX2 expression, indicating that STAT1 activation plays an important role in mediating IFN-induced enhancement of stemness. Finally, the ChIP assay demonstrated that STAT1 protein can bind to the promoter region of the SOX2 gene to serve as a transcription factor. In summary, our results indicate that CSCs produce and secrete IFN-I via their enhanced JAK/STAT1 signaling. The secreted IFN-I can bind to the receptor on the CSCs, further activate their JAK/STAT1 signaling, promoting the expression of SOX2 and maintenance of stemness of CSCs via an autocrine manner. Citation Format: Aidan Li, Xuetao Bai, Na Li, Shurui Cai, Ananya Banerjee, Kousalya Lavudi, Linzhou Wang, Qianyun Ge, Yajing Yang, Qi-En Wang. Cancer stem cells maintain stemness via autocrine activation of the IFN/JAK/STAT signaling pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2428.

MARVELD3 inhibits the epithelial-mesenchymal transition and cell migration by suppressing the Wnt/β-catenin signaling pathway in non-small cell lung cancer cells.

The epithelial-mesenchymal transition (EMT), featured by abatement of cell-cell contact, is related to exacerbating non-small cell lung cancer (NSCLC) by inducing metastasis. MAL and relevant proteins for vesicle trafficking and membrane link domain 3 (MARVELD3) is a novel tight junction protein participated in the EMT. There is limited information available about the mechanism of MARVELD3 in NSCLC. In this trial, the inhibition effect of MARVELD3 on human NSCLC cells will be discussed. MARVELD3 expression was measured in NSCLC tissues and para-carcinoma tissues. The expression of MARVELD3 and EMT-related genes were examined in transforming growth factor (TGF)-β1-induced NSCLC cells. NSCLC cells with MARVELD3-knockdown and overexpressed were established to analyze the relationship between MARVELD3 and EMT and cell migration. The Wnt/β-catenin pathway expression was also analyzed in NSCLC cell models and clinic species. Lower protein levels of MARVELD3 were observed in NSCLC samples than para-carcinoma specimens, and the decreased expression of MARVELD3 in NSCLC specimens was associated with tumor metastasis. E-Cadherin and MARVELD3 expression was reduced in TGF-β1 treated NSCLC cells, whereas increased Vimentin expression was detected. MARVELD3 changed the EMT-related genes and induced cell migration. In addition, Wnt/β-catenin pathway and target genes, MYC and CCND1, expressions were inhibited in MARVELD3 overexpressed NSCLC cells. TGF-β1 induced EMT in human NSCLC cells can be suppressed by MARVELD3 through Wnt/β-catenin signaling pathway. These results indicate that MARVELD3 might be a potential therapeutic modality useful in the treatment of NSCLC.

Palbociclib Enhances Migration and Invasion of Cancer Cells via Senescence-Associated Secretory Phenotype-Related CCL5 in Non-Small-Cell Lung Cancer.

Palbociclib is the first CDK4/6 inhibitor approved by FDA and has been studied in many types of cancer. However, some studies showed that it could induce epithelial-mesenchymal transition (EMT) of cancer cells. To test the effect of palbociclib on non-small-cell lung cancer (NSCLC) cells, we treated NSCLC cells with different concentrations of palbociclib and detected its effects via MTT, migration and invasion assays, and apoptosis test. Further RNA sequencing was performed in the cells treated with 2 μM palbociclib or control. And Gene Ontology, Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Set Enrichment Analysis (GSEA), and protein-protein interaction network (PPI) were analyzed to explore the mechanism of palbociclib. The results showed that palbociclib significantly inhibited the growth of NSCLC cells and promoted apoptosis of cells, however, enhanced the migration and invasion abilities of cancer cells. RNA sequencing showed that cell cycle, inflammation-/immunity-related signaling, cytokine-cytokine receptor interaction, and cell senescence pathways were involved in the process, and CCL5 was one of the significantly differential genes affected by palbociclib. Further experiments showed that blocking CCL5-related pathways could reverse the malignant phenotype induced by palbociclib. Our results revealed that palbociclib-induced invasion and migration might be due to senescence-associated secretory phenotype (SASP) rather than EMT and suggested that SASP could act as a potential target to potentiate the antitumor effects of palbociclib in cancer treatment.

Transient receptor potential vanilloid 4 promotes the growth of non-small cell lung cancer by regulating Foxp3.

Transient receptor potential vanilloid 4 (TRPV4) participates in malignant tumor. However, the role of TRPV4 in non-small cell lung cancer (NSCLC) remains unclear. In this study, we demonstrated TRPV4 was upregulated in NSCLC tissues and NSCLC cell lines. TRPV4 level in the NSCLC patients and cell lines were detected, and its function was studied both in vivo and vitro. The level of TRPV4 showed a positive correlation with tumor size of NSCLC patients. Activation TRPV4 by agonist GSK1016790A promoted cell proliferation and decreased apoptosis in A549 cells, and these effects were enhanced when the cells have overexpressed TRPV4. Moreover, GSK1016790A induced inhibitory effects on apoptosis of A549 cells was impaired when GSK1016790A used together with TRPV4 selective antagonist HC-067047, or impaired when the cells have already downregulated TRPV4 expression by TRPV4 siRNA. In vivo study, pharmacological inhibition of TRPV4 prevented A549 cells transplanted tumor growth. It was showed Foxp3 level was significantly increased in the NSCLC tissues, and showed a positive correlation with the level of TRPV4. Deactivation of TRPV4 using TRPV4 siRNA or HC-067047 significantly reduced expression of Foxp3 in GSK1016790A treated NSCLC cells. Moreover, downregulation Foxp3 by transfection of Foxp3 siRNA significantly impaired TRPV4 induced NSCLC cells proliferations in vitro. Antitumor effects caused by TRPV4 inhibition in NSCLC might be attributed to the suppression of Foxp3 which induced subsequent cell apoptosis. Thus, pharmacological inhibition of TRPV4 may be a promising option for NSCLC treatment.

OGT regulated O-GlcNacylation promotes migration and invasion by activating IL-6/STAT3 signaling in NSCLC cells

BackgroundO-linked β-N-acetylglucosamine transferase (O-GlcNAc transferase, OGT) is a key enzyme that regulates O-GlcNAc modification, which is significantly up-regulated and participates in the regulation of tumorigenesis. Although previous research indicated that OGT promotes epithelial-mesenchymal transition (EMT) of lung cancer, the underlying molecular mechanisms, especially within the tumor inflammatory microenvironment, require further elucidation. MethodsThe role of the inflammatory signaling Interleukin 6/Signal Transducer and activator of transcription 3 (IL-6/STAT3) in Non-small cell lung cancer (NSCLC) cells A549 were confirmed by Transwell assay, Scratch wound healing assay, Western blot, Immunofluorescence staining, and Nuclear and cytoplasmic extraction experiment. Western blot detected OGT expression and whole protein O-GlcNacylation after IL-6 stimulation in NSCLCs cells. The biological effects and related mechanism of OGT in NSCLC cells were investigated by Western blot, Transwell assay, Immunofluorescence staining and Immunoprecipitation. The up-stream mechanism of OGT expression was explored by employing the specific chemical inhibitors, and the expression and distribution of OGT and phosphorylated STAT3 in NSCLC samples were confirmed by immunohistochemical analysis. ResultsIL-6/STAT3 promoted the migration and invasion of NSCLC cells. IL-6 stimulation elevated OGT expression and the total protein O-GlcNacylation in A549 cells. Silencing OGT by shRNA significantly inhibited the IL-6 induced EMT marker (N-cadherin and Slug) expression, migration and invasion in A549 cells. OGT interacted with and mediated O-GlcNacylation of STAT3, which promoted STAT3 Y705 phosphorylation in IL-6 treated NSCLC cells. OGT expression was positively regulated by NF-κB p65 signaling pathway after IL-6 stimulation, instead of STAT3 signaling. OGT and phosphorylated STAT3 had an obviously higher expression in human NSCLC tissues, and phosphorylated STAT3 was mainly expressed in the nucleus. ConclusionThe above results showed that OGT regulated O-GlcNacylation promoted migration and invasion by activating IL-6/STAT3 signaling in lung cancer.

4Ei-10 interdiction of oncogenic cap-mediated translation as therapy for non-small cell lung cancer.

In order to suppress 5' cap-mediated translation a highly available inhibitor of the interaction between the 5' mRNA cap and the eIF4E complex has been developed. 4Ei-10 is a member of the class of ProTide compounds and has elevated membrane permeability and is a strong active chemical antagonist for eIF4E. Once taken up by cells it is converted by anchimeric activation of the lipophilic 2-(methylthio) ethyl protecting group and after that Hint1 P-N bond cleavage to N7-(p-chlorophenoxyethyl) guanosine 5'-monophosphate (7-Cl-Ph-Ethyl-GMP). Using this powerful interaction, it has been demonstrated that 4Ei-10 inhibits non-small cell lung cancer (NSCLC) cell growth. In addition, treatment of NSCLC cells with 4Ei-10 results in suppression of translation and diminished expression of a cohort of cellular proteins important to maintaining the malignant phenotype and resisting apoptosis such as Bcl-2, survivin, and ornithine decarboxylase (ODC). Finally, as a result of targeting the translation of anti-apoptotic proteins, NSCLC cells are synergized to be more sensitive to the existing anti-neoplastic treatment gemcitabine currently used in NSCLC therapy.

Clinically Relevant Chemotherapeutics Have the Ability to Induce Immunogenic Cell Death in Non-Small Cell Lung Cancer.

The concept of immunogenic cell death (ICD) has emerged as a cornerstone of therapy-induced anti-tumor immunity. To this end, the following chemotherapies were evaluated for their ability to induce ICD in non-small cell lung cancer (NSCLC) cell lines: docetaxel, carboplatin, cisplatin, oxaliplatin and mafosfamide. The ICD hallmarks ATP, ecto-calreticulin, HMGB1, phagocytosis and maturation status of dendritic cells (DCs) were assessed in vitro. Furthermore, an in vivo vaccination assay on C57BL/6J mice was performed to validate our in vitro results. Docetaxel and the combination of docetaxel with carboplatin or cisplatin demonstrated the highest levels of ATP, ecto-calreticulin and HMGB1 in three out of four NSCLC cell lines. In addition, these regimens resulted in phagocytosis of treated NSCLC cells and maturation of DCs. Along similar lines, all mice vaccinated with NSCLC cells treated with docetaxel and cisplatin remained tumor-free after challenge. However, this was not the case for docetaxel, despite its induction of the ICD-related molecules in vitro, as it failed to reject tumor growth at the challenge site in 60% of the mice. Moreover, our in vitro and in vivo data show the inability of oxaliplatin to induce ICD in NSCLC cells. Overall with this study we demonstrate that clinically relevant chemotherapeutic regimens in NSCLC patients have the ability to induce ICD.

Growth inhibitory role of the p53 activator SCH529074 in non‑small cell lung cancer cells expressing mutantp53.

Mutations of p53 occur in approximately 50% of advanced non‑small cell lung cancer (NSCLC) cases, leading to loss of tumor suppressive function and/or gain of p53oncogenic activity. Reactivation of mutantp53 and consequently induction of apoptosis in cancer cells is the goal of p53‑targeted therapy. Recently, several p53mutant reactivating compounds were discovered including SCH529074. However, the role of SCH529074 in NSCLC has not been fully explored. In the present study, the effects of this compound on cell survival, cell cycle progression, induction of apoptosis and modulation of cell signaling in p53mutant NSCLCcells (H1975, H322 and H157) and p53 wild‑type NSCLCcells (A549), was investigated. Cell‑based functional assays, real‑time RT‑qPCR and western blot assays were used. HCT116 [p53 wild‑type (WT)] and HCT116 p53‑/‑ (p53null) were used as control cells. The results demonstrated that SCH529074 treatment caused significant reduction in cell viability and colony formation activity in p53mutant, p53WT and p53‑deficientcells. The treatment of NSCLC cells with SCH529074 resulted in a dose‑dependent induction of apoptosis and G0/G1 cell cycle arrest, which was associated with the activation of caspases(3and7), p53‑independent upregulation of p21 and PUMA as well as increased LC3II, a biomarker of autophagy. The combination treatment with the autophagy inhibitor chloroquine (CQ) and SCH529074 led to decreased cell viability, colony formation and increased induction of apoptosis. The data indicated that SCH529074 may exert its growth inhibitory function in a p53‑independent manner in NSCLCcells.

Circular RNA circ-ABCB10 promotes non-small cell lung cancer proliferation and inhibits cell apoptosis through repressing KISS1.

Recent researches have proved that circular RNAs (circRNAs) act as an important role in many diseases. Our study aims to uncover the role of circ-ABCB10 in the progression of non-small cell lung cancer (NSCLC). Real Time-quantitative Polymerase Chain Reaction (RT-qPCR) was utilized to detect circ-ABCB10 expression in NSCLC patients. Then, we conducted Cell Counting Kit-8 (CCK-8) assay, colony formation assay, Ethynyl deoxyuridine (EdU) incorporation assay, cell cycle assay, and cell apoptosis assay in treated NSCLC cells. Besides, further experiments including RT-qPCR and Western blot assay were performed to explore the potential mechanism in vitro. Circ-ABCB10 expression level was significantly higher in NSCLC samples comparing to that in adjacent tissues. Moreover, functional assays showed that the cell growth ability of NSCLC cells was inhibited after circ-ABCB10 was knocked down. In addition, the cell apoptosis of NSCLC cells was promoted after circ-ABCB10 was knocked down. Also the expression of KISS1 was upregulated by the knockdown of circ-ABCB10. Furthermore, it was found that KISS1 expression was negatively correlated to the circ-ABCB10 expression in NSCLC tissues. Results above indicated that circ-ABCB10 promoted cell proliferation and inhibited cell apoptosis of NSCLC by suppressing KISS1, which suggested that circ-ABCB10 may be a potential therapeutic target in NSCLC.

Cordyceps militaris Exerts Anticancer Effect on Non-Small Cell Lung Cancer by Inhibiting Hedgehog Signaling via Suppression of TCTN3.

This study aimed to investigate the effect of Cordyceps militaris extract on the proliferation and apoptosis of non–small cell lung cancer (NSCLC) cells and determine the underlying mechanisms. We performed a CCK-8 assay to detect cell proliferation, detection of morphological changes through transmission electron microscopy (TEM), annexin V–FITC/PI double staining to analyze apoptosis, and immunoblotting to measure the protein expression of apoptosis and hedgehog signaling–related proteins, with C militaris treated NSCLC cells. In this study, we first found that C militaris reduced the viability and induced morphological disruption in NSCLC cells. The gene expression profiles indicated a reprogramming pattern of genes and transcription factors associated with the action of TCTN3 on NSCLC cells. We also confirmed that the C militaris–induced inhibition of TCTN3 expression affected the hedgehog signaling pathway. Immunoblotting indicated that C militaris–mediated TCTN3 downregulation induced apoptosis in NSCLC cells, involved in the serial activation of caspases. Moreover, we demonstrated that the C militaris negatively modulated GLI1 transcriptional activity by suppressing SMO/PTCH1 signaling, which affects the intrinsic apoptotic pathway. When hedgehog binds to the PTCH1, SMO dissociates from PTCH1 inhibition at cilia. As a result, the active GLI1 translocates to the nucleus. C militaris clearly suppressed GLI1 nuclear translocation, leading to Bcl-2 and Bcl-xL down-regulation. These results suggested that C militaris induced NSCLC cell apoptosis, possibly through the downregulation of SMO/PTCH1 signaling and GLI1 activation via inhibition of TCTN3. Taken together, our findings provide new insights into the treatment of NSCLC using C militaris.

PLEK2 mediates metastasis and vascular invasion via the ubiquitin-dependent degradation of SHIP2 in non-small cell lung cancer.

Metastasis is the leading cause of death for non-small cell lung cancer (NSCLC) patients. However, how lung cancer cells invade blood vessels during metastasis remains unclear. Here, based on bioinformatics analyses, we found that PLEK2 might regulate NSCLC migration and vascular invasion. As little is known about the function of PLEK2 in NSCLC, we aimed to clarify this. We demonstrated that PLEK2 was significantly upregulated in transforming growth factor beta 1 (TGF-β1)-treated NSCLC cells through ELK1 transcriptional activation, highly expressed in NSCLC tissues, and negatively correlated with NSCLC overall survival. Meanwhile, PLEK2 overexpression significantly promoted NSCLC epithelial-to-mesenchymal transition (EMT) and migration, human lung microvascular endothelial cells endothelial-to-mesenchymal transition (EndoMT), and the destruction of vascular endothelial barriers. Moreover, PLEK2 knockdown inhibited TGF-β1-induced EMT and EndoMT. Furthermore, PLEK2 was found to directly interact with SHIP2 and target it for ubiquitination and degradation in NSCLC cells. Next, we confirmed that SHIP2 overexpression inhibits NSCLC EMT, migration and invasion and showed that PLEK2 overexpression can activate SHIP2-associated TGF-β/PI3K/AKT signaling. Our results suggest that PLEK2 could be a novel prognostic marker and potential therapeutic target for NSCLC metastasis and vascular invasion.

Niche origin of mesenchymal stem cells derived microvesicles determines opposing effects on NSCLC: Primary versus metastatic

Novel therapeutic approaches that address the malignant cells in their stroma microenvironment are urgently needed in lung cancer. The stroma resident mesenchymal stem cells (MSCs) interact with cancer cells in diverse ways including microvesicles (MVs) that transfer proteins and RNA species thereby modulating recipient cells' phenotype. Previously, we have demonstrated that MSCs' secretome from the primary non-small cell lung cancer (NSCLC) niche (lung) and metastatic niche (bone marrow (BM)) demonstrate opposite effects on NSCLC cells in a translation initiation (TI) dependent manner. Here, we examined the effect of MVs secreted from BM-MSCs' or lung-MSCs (healthy, NSCLC) to NSCLC phenotype. Briefly, NSCLC cell lines treated with Lung or BM-MSCs' MVs were assayed for viability (WST-1), cell count/death (trypan), migration (scratch), TI status and MAPKs activation (immunoblotting). Corresponding to previous published trends, Lung-MSCs' MVs promoted NSCLC cells' assayed traits whereas, BM-MSCs' MVs suppressed them. Activation of MAPKs and autophagy was registered in lung-MSCs MVs treated NSCLC cell lines only. Furthermore, lung-MSCs' MVs' treated NSCLC cells demonstrated an early (5min) activation of MAPKs and TI factors (peIF4E/peIF4GI) not evident in BM-MSCs MVs treated cells. These observations depict a role for MSCs'-MVs in NSCLC phenotype design and display distinct differences between the primary and metastatic niches that correspond to disease progression.In conclusion, the systemic nature of MVs marks them as attractive therapeutic markers/targets and we propose that identification of specific cargoes/signals that differentiate between MSCs MVs of primary and metastatic niches may introduce fresh therapeutic approaches.

MiR-34c acts as a tumor suppressor in non-small cell lung cancer by inducing endoplasmic reticulum stress through targeting HMGB1.

ObjectiveTo investigate the role of miR-34c in lung cancer.MethodsThe levels of microRNA-34c (miR-34c) expression in non-small cell lung cancer (NSCLC) tissue and cell lines were examined by the qRT-PCR assay. High mobility group box 1 (HMGB1) expression in NSCLC was assessed by immunohistochemical analysis (IHC), qRT-PCR, and Western blot assays. The effects of miR-34c overexpression or HMGB1 knockdown on cell proliferation and apoptosis were evaluated by CCK-8 and flow cytometry analysis, respectively. Cellular reactive oxygen species (ROS) production in NSCLC cells was detected using a ROS kit. The levels of Bax, p-ERK, eIF2α, GADD153, and IRE1α expression in treated NSCLC cells were measured by Western blot assays. In addition, the interaction between miR-34c and HMGB1 was verified by the dual-luciferase reporter assay.ResultsmiR-34c was only slightly expressed, while HMGB1 was highly expressed in NSCLC tissues and cell lines. Overexpression of miR-34c or knockdown of HMGB1 inhibited cell proliferation, promoted cell apoptosis, and induced ER stress in NSCLC cells. In terms of mechanism, miR-34c negatively regulated HMGB1 expression by directly targeting the 3ʹ-untranslated region (UTR) of HMGB1 mRNA. In addition, we proved that HMGB1 overexpression could block the effects of miR-34c on NSCLC cell proliferation, apoptosis, and ER stress.ConclusionmiR-34c may suppress NSCLC tumors by targeting HMGB1 mRNA, promoting endoplasmic reticulum stress, and increasing ROS levels. Our findings suggest that miR-34c has a role in NSCLC.

Sequence-dependent synergistic cytotoxicity of icotinib and pemetrexed in human lung cancer cell lines in vitro and in vivo

BackgroundRecent Clinical trials of administration of epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) in combination with standard first-line chemotherapy have failed to improve survival in patients with advanced NSCLC, However, the sequential treatment with EGFR-TKIs and chemotherapy is expected to improve survival of NSCLC. The aim of this study is to test the antiproliferative effect of pemetrexed combined with icotinib in different sequences on non-small cell lung cancer (NSCLC) cell lines to determine the optimal combination schedule, and subsequently elaborated the potential mechanisms.MethodsSix human lung cancer cell lines with wild-type or mutant EGFR gene were exposed to pemetrexed and icotinib combined in different sequences. Cell proliferation was examined by cell counting kit-8 (CCK-8) and colony formation assay; cell cycle and apoptosis were evaluated by flow cytometry; cell migration and invasion were measured by wound healing and transwell invasion assays respectively; protein expression was by detected by Western blot.ResultsThe growth inhibition effect of pemetrexed combined with icotinib on NSCLC cells were schedule-dependent in vitro and in vivo. Treatment with pemetrexed followed by icotinib (P-I) had significantly stronger anticancer ability than treatment with icotinib followed by pemetrexed (I-P) and concomitant treatment with pemetrexed and icotinib (P + I). Cell cycle analysis revealed that pemetrexed blocked cells in S phase, whereas icotinib arrested cells in G1 phase. We also found that icotinib markedly enhanced the pro-apoptotic activity of pemetrexed via cytochrome-C/Caspase/Bcl-2 signaling pathway. In addition, our results showed that pemetrexed alone increased the levels of p-EGFR, p-AKT and p-MAPK, which were inhibited by icotinib. Finally, we showed that the washout period of icotinib was no less than 96 h.ConclusionsSequential treatment of NSCLC cells with pemetrexed followed by icotinib had powerful antiproliferative effect, and it could become a novel effective combination therapy for NSCLC patients.

Recombinant cell-permeable HOXA9 protein inhibits NSCLC cell migration and invasion.

Previously, it has been reported that homeobox A9 (HOXA9) protein expression is downregulated in lung cancer cells, and that its expression is inversely correlated with the metastatic potential of lung cancer cells both in vitro and in vivo. As such, HOXA9 shows therapeutic potential. The development of therapeutic strategies based on this protein is, however, limited due to its poor membrane permeability. To overcome this problem, we developed a system to deliver HOXA9 protein into non-small cell lung cancer (NSCLC) cells. First, we constructed a delivery vector expressing polyarginine, a cell-penetrating peptide, as well as HOXA9. The resulting recombinant R10-HOXA9 protein was effectively introduced into A549 and NCI-H1299 NSCLC cells. Next, we examined the roles and molecular mechanisms of recombinant R10-HOXA9 in processes involved in tumor progression. To investigate the therapeutic efficacy of the delivery system, we performed cell motility assays using both in vitro and in vivo experimental models. We found that recombinant R10-HOXA9 protein reduced the invasion and migration rate, but not the proliferation rate, of the NSCLC cells tested, both in vitro and in vivo. Treatment of NSCLC cells with recombinant R10-HOXA9 protein led to a significant increase in E-cadherin expression. Conversely, we found that the expression of snail family zinc finger 2 (SLUG), a transcriptional repressor of E-cadherin, was markedly decreased. In an experimental metastatic mouse model, recombinant R10-HOXA9 protein was found to effectively reduce the rate of lung cancer cell motility. Our data suggest that the developed cell-permeable R10-HOXA9 system may serve as a useful tool to prevent NSCLC cell migration and invasion.

Marsdenia tenacissima extract induces apoptosis and suppresses autophagy through ERK activation in lung cancer cells

BackgroundMarsdenia tenacissima is an herb medicine which has been utilized to treat malignant diseases for decades. The M. tenacissima extract (MTE) shows significant anti-proliferation activity against non-small cell lung cancer (NSCLC) cells, but the underlying mechanisms remain unclear. In this study, we explored the potential anti-proliferation mechanisms of MTE in NSCLC cells in relation to apoptosis as well as autophagy, which are two critical forms to control cancer cell survival and death.MethodsThe proliferation of H1975 and A549 cells was evaluated by MTT assay. Cell apoptosis was assessed by Annexin V and PI staining, Caspase 3 expression and activity. Autophagy flux proteins were detected by Western blot with or without autophagy inducer and inhibitor. Endogenous LC3-II puncta and LysoTracker staining were monitored by confocal microscopy. The formation of autophagic vacuoles was measured by acridine orange staining. ERK is a crucial molecule to interplay with cell autophagy and apoptosis. The role of ERK on cell apoptosis and autophagy influenced by MTE was determined in the presence of MEK/ERK inhibitor U0126.ResultsThe significant growth inhibition and apoptosis induction were observed in MTE treated NSCLC cells. MTE induced cell apoptosis coexisted with elevated Caspase 3 activity. MTE also impaired autophagic flux by upregulated LC3-II and p62 expression. Autophagy inducer EBSS could not abolish the impaired autophagic flux by MTE, while it was augmented in the presence of autophagy inhibitor Baf A1. The autophagosome–lysosome fusion was blocked by MTE via affecting lysosome function as evidenced by decreased expression of LAMP1 and Cathepsin B. The molecule ERK became hyperactivated after MTE treatment, but the MEK/ERK inhibitor U0126 abrogated autophagy inhibition and apoptosis induction caused by MTE, suggested that ERK signaling pathways partially contributed to cell death caused by MTE.ConclusionOur results demonstrate that MTE caused apoptosis induction as well as autophagy inhibition in NSCLC cells. The activated ERK is partially associated with NSCLC apoptotic and autophagic cell death in response to MTE treatment. The present findings reveal new mechanisms for the anti-tumor activity of MTE against NSCLC.

Abstract 3449: AGL loss promotes anchorage independent growth of non-small cell lung cancer by activating FAK

Abstract Loss of glycogen debranching enzyme (AGL) has been shown to promote growth of bladder and non-small cell lung cancer. AGL loss predominantly assists anchorage independent growth of non-small cell lung cancer (NSCLC) cells. However the mechanism driving this anchorage independent growth with AGL loss is not well understood. To identify the driver pathways which contributions to increased anchorage independent growth with AGL loss, we carried out a phospho-kinase screen in A549 NSCLC cells with and without AGL expression. The kinase screen identified an increase in phospho-FAK (Tyr 576/577) levels with loss of AGL. The result of the screen was validated using separate independent Western blot experiments in A549 and other NSCLC cell lines which showed an increase in FAK activation at Tyr 576/577 with loss of AGL. We next treated NSCLC cells with and without AGL with FAK specific inhibitor (FAK inhibitor 14). Treatment with FAK inhibitor 14 had a greater cytotoxic effect (p<0.05) on NSCLC cells that have AGL loss. These data suggest that activation of FAK is a major driver of anchorage independent growth in NSCLC cells that have lost AGL expression and inhibition of FAK activity can serve as a therapeutic avenue for NSCLC patients with low tumor AGL expression. Citation Format: Sunny Guin, Craig Richmond. AGL loss promotes anchorage independent growth of non-small cell lung cancer by activating FAK [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3449.

Knockdown of KLF5 suppresses hypoxia-induced resistance to cisplatin in NSCLC cells by regulating HIF-1\u03b1-dependent glycolysis through inactivation of the PI3K/Akt/mTOR pathway

BackgroundHypoxia-mediated chemoresistance has been regarded as an important obstacle in the development of cancer treatment. Knockdown of krüppel-like factor 5 (KLF5) was reported to inhibit hypoxia-induced cell survival and promote cell apoptosis in non-small cell lung cancer (NSCLC) cells via direct regulation of hypoxia inducible factor-1α (HIF-1α) expression. However, the roles of KLF5 in the development of hypoxia-induced cisplatin (DDP) resistance and its underlying mechanism in NSCLC cells remain to be further elucidated.MethodsWestern blot was performed to determine the protein levels of KLF5, P-glycoprotein (P-gp) and HIF-1α in treated NSCLC cells. Cell survival was examined by MTT assay. The effect of KLF5 knockdown on hypoxia-induced glycolysis was assessed by measuring glucose consumption and lactate production. The effect of KLF5 knockdown on the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway was analyzed by western blot.ResultsHypoxia upregulated the expression of KLF5 in NSCLC cells. KLF5 knockdown suppressed hypoxia-induced DDP resistance in NSCLC cells, as demonstrated by the increased cytotoxic effects of DDP and reduced P-gp expression in NSCLC cells in hypoxia. Moreover, KLF5 knockdown inhibited hypoxia-induced HIF-1α expression and glycolysis, and KLF5 knockdown suppressed hypoxia-induced DDP resistance by inhibiting HIF-1α-dependent glycolysis in NSCLC cells. Furthermore, KLF5 knockdown suppressed hypoxia-induced activation of the PI3K/Akt/mTOR pathway in NSCLC cells and KLF5 overexpression promoted hypoxia-induced DDP resistance in NSCLC cells through activation of the PI3K/Akt/mTOR pathway.ConclusionsKLF5 knockdown could suppress hypoxia-induced DDP resistance, and its mechanism may be due to the inhibition of HIF-1α-dependent glycolysis via inactivation of the PI3K/Akt/mTOR pathway.