H460 Cell Lines Research Articles

Abstract 5446: Discovery of a novel IGF2BP1 inhibitor, based on lead optimization through structure-activity relationship studies, as metastasis-specific therapeutic agent

Abstract IGF2BP1 is a multifunctional RNA-binding protein that regulates the stability, localization and translation of its mRNA targets. High levels of IGF2BP1 expression have been shown to be associated with poor prognosis in patients with variety of cancer types. Given the correlation between elevated IGF2BP1 expression and poor clinical outcomes, the specific activation of IGF2BP paralogs in a wide variety of cancers, and the effectiveness of preventing metastasis in animal models by reducing IGF2BP1 activity, therapies directed at inhibiting IGF2BP1 function constitute a potentially powerful approach for fighting cancer. In addition, IGF2BP1’s pleiotropic effect on multiple pro-tumorigenic and pro-metastatic pathways makes it a promising target, as this may avoid the development of resistance, associated with targeted agents in general. Furthermore, specific inhibitors of IGF2BP1 would be expected to have minimal side effects since; i) IGF2BP1 is expressed at very low levels in normal adult tissues, and ii) adult mice with an inducible whole-mouse knockout of IGF2BP1 are healthy. To identify an efficient IGF2BP1 inhibitor, a fluorescent polarization (FP)-based high throughput screen of over 100,000 small molecules was performed, and the most promising candidates were further validated in an array of in vitro and cell-based assays leading to the identification a lead compound, “7773,” that selectively inhibited IGF2BP1 RNA binding and a variety of its cellular functions. To further optimize ‘7773’ and create more selective, effective and safe small-molecule inhibitors of IGF2BP1 that could be developed clinically as cancer therapeutics, we conducted a SAR study based on the lead ‘7773’ structure. Novel 27 compounds were designed and synthesized and evaluated for IGF2BP1 inhibition using our novel cell-based split-luciferase assay, which led to the identification of 6 compounds that performed similar or better than ‘7773’. Cell-based wound healing assay revealed that one of these selected compounds, AVJ16, was especially (>14 times than “7773”) effective in inhibiting cell migration in H1299 cell line that express high levels of endogenous IGF2BP1, but not in LKR-M cells that express very low levels of IGF2BP1. The specificity of AVJ16 was further confirmed in LKR-M cells that ectopically express IGF2BP1 - these cells become sensitive to AVJ16 upon overexpression of IGF2BP1 (but not GFP). Together our data provide strong evidence for AVJ16 to be effective and selective in inhibiting IGF2BP1 function by interfering with its ability to bind target RNAs. Citation Format: Amandeep Singh, Arun Sharma, Vladimir Spiegelman, Vikash Singh, Nadav Wallis, Omer Elimelech, Joel Yisraeli. Discovery of a novel IGF2BP1 inhibitor, based on lead optimization through structure-activity relationship studies, as metastasis-specific therapeutic agent [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5446.

Abstract 1088: Therapeutic targeting of sphingosine kinase 2 in tyrosine kinase inhibitor resistance and non-small cell lung cancer tumorigenicity

Abstract It is anticipated that there will be 235,760 cases and 131,880 deaths due to lung cancer in the US in 2021. Tyrosine Kinase Inhibitors (TKIs) such as Erlotinib (ER) and Osimertinib (OR) are currently being used to treat lung cancer patients with EGFR mutations; however, patients develop resistance to these drugs within 10-18 months. Sphingosine kinase 2 (SK2), which is upregulated in Non-Small Cell Lung Cancer (NSCLC), phosphorylates sphingosine to sphingosine-1-phosphate (S1P), promoting cancer cell proliferation. The sphingolipid rheostat controls the cell fate by maintaining a balance between pro-survival and pro-apoptotic sphingolipids, which is regulated by SK2. However, the role of SK2 in TKI resistance is not established. To determine if SK2 causes TKI resistance, we have studied its role in NSCLC and TKI resistance. We hypothesize that SK2 may be involved in inducing TKI- resistance in NSCLC cells and is positively correlated with tumor progression. We studied SK2 in both drug resistant (OR and ER) and drug sensitive (parental) H358, H2170, H3255 and PC9 NSCLC cell lines. Our study investigated SK2 expression in drug resistant cells versus parental cells using qPCR and Western Blotting. The effects of SK2 inhibitor, Opaganib, on NSCLC cell proliferation was studied through an MTT assay. We have also studied its expression in early- and late-stage tumors using Immunohistochemistry (IHC). In drug resistant cells, qPCR studies showed that SK2 was upregulated in H2170 by 1.30-fold (ER) and 1.5-fold (OR); in H358 by 1.36-fold (ER) and 1.17-fold (OR); in PC9 by 3.3-fold (ER) and 1.8-fold (OR) and in H3255 by 4.3-fold (OR) compared to parental cells. A western blotting experiment demonstrated that SK2 was upregulated significantly by 1.7-fold and 1.5-fold in H358OR and H2170OR cells, respectively, compared to parental cells. These results suggest that SK2 may play a role in TKI resistance, as displayed by SK2 upregulation in TKI-resistant cells. MTT assay data showed 71% inhibition of cell growth by Osimertinib and SK2 inhibitor in H2170OR. The inhibition due to the combination of the drug and inhibitor was more than additive by 9% compared to the drug and inhibitor individually. This indicates SK2s potential to overcome TKI- Resistance. To analyze SK2 expression in cancer progression, IHC experiments were performed on 63 early and 48 late stage lung cancer patient sections. We observed upregulation of SK2 in late- stage lung cancer (p<0.05), indicating its role in cancer progression. In conclusion, SK2 may be a key biomarker in combating TKI-resistance and play a role in cancer progression in NSCLC. Thus, SK2 may be a promising candidate for molecular targeted therapy against NSCLC. Citation Format: Namrata Umeshchandra Dube, Katie Lam, Neelu Puri. Therapeutic targeting of sphingosine kinase 2 in tyrosine kinase inhibitor resistance and non-small cell lung cancer tumorigenicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1088.

Abstract 802: Development of replication protein A (RPA) targeted PROTACs for the treatment of lung and ovarian cancers

Abstract Despite remarkable advances within the field of molecular cancer therapeutics over the preceding decades, DNA-damaging agents retain a central role in the pharmacotherapy of neoplastic diseases. The clinical response to DNA-damaging therapies is varied within and across cancer subtypes where a robust DNA damage response (DDR) predicts recalcitrance. Replication protein A (RPA) is the predominant human single-stranded DNA (ssDNA)-binding protein, playing essential roles in DNA replication, repair, recombination, and the DNA-damage response (DDR). Inhibition of the RPA−DNA interaction is an approach to cancer drug discovery that holds potential to provide single-agent activity and/or synergy with existing therapeutics. Proteolysis targeting chimera (PROTAC) based drug design presages a paradigm shift within drug discovery as PROTACs begin to carve out a space in the clinical landscape. The PROTAC platform is particularly suited for drugging overexpressed proteins such as RPA because iterative target degradation improves drug:target stoichiometry and precludes RPA’s role in protein scaffolding. Herein, we describe the synthesis and biological evaluation of an RPA targeted PROTAC library conceived by conjugation of the diaryl pyrazole RPA inhibitor TDRL-551 and an E3-ligand via a linker of variable length and composition. Residing within the beyond rule of five (bRo5) chemical space, careful consideration was given to minimize the liability to cell permeability imposed by the high molecular weight of PROTACs. Excitingly, cellular uptake across the compound library was broadly improved relative to the unconjugated warhead TDRL-551. Similarly, RPA engagement by the PROTAC series was widely unhindered and, in many cases, improved relative to the warhead TDRL-551. A subset of the compound library unified by flexible linkers and alkyne attachment of the E3-ligand (typified by GL-3311) display low single-digit micromolar IC50 values in viability assays across H460, A549, and A2780 cell lines. Citation Format: Jeremy M. Kelm, Jitender Dev Gaddameedi, Pamela S. VanderVere-Carozza, Amirreza Samarbakhsh, Nivisa Vakeesan, Hussein W. Kansou, Sara Serafimovski, Katherine S. Pawelczak, John J. Turchi, Navnath S. Gavande. Development of replication protein A (RPA) targeted PROTACs for the treatment of lung and ovarian cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 802.

Abstract 839: YES1 is a novel druggable oncogene in small cell lung cancer

Abstract Small cell lung cancer (SCLC) is a high-grade neuroendocrine subtype of lung cancer. This tumor is characterized by its aggressiveness, refractoriness to therapies, poor prognosis, and early dissemination. The first line of treatment consists of platinum-based chemotherapy, and although patients initially respond very well to this therapy, they promptly develop resistance. Currently, there are no targeted therapies approved for the treatment of SCLC patients. Here, we identified YES1, a non-receptor tyrosine kinase, as a novel targetable candidate with a relevant role in SCLC. In silico analysis revealed that YES1 is overexpressed in SCLC patients compared to non-malignant lung tissues. In a multi-institutional cohort of 80 SCLC patients, 31% of cases showed high YES1 protein expression and 26% of all specimens had YES1 gain/amplification (CNV≥2.5), analyzed by immunohistochemistry and FISH, respectively. In addition, high expression of YES1 predicted both lower overall survival (OS) and progression free survival (PFS) (n=80). In accordance, YES1 mRNA expression significantly correlated with YES1 CNV in SCLC cell lines. We knocked-down YES1 in two human SCLC cell lines (DMS53 and H209) with high expression and amplification of YES1. Depletion of YES1 almost totally abrogated cell proliferation and the formation of cell line-derived organoids in 3D. Accordingly, inhibition of YES1 arrested the cell cycle and promoted cell apoptosis in vitro. Consistent with these results, knockdown of YES1 promoted a dramatic reduction of subcutaneous tumor growth in both models with high levels of YES1, leading to a rate of partial regression of 54% and 23% complete regression. A metastasis assay, performed by intracardiac injection of H209 cells, showed that inhibition of YES1 led to complete disappearance of metastatic lung, leg and liver tumors in 4 out of 5 mice. Gene set enrichment analysis (GSEA) of RNA sequencing data performed with YES1-depleted DMS53 cells revealed that cells lacking YES1 had a downregulation in DNA replication and DNA repair signatures. We next explored the effect of pharmacological inhibition of YES1 using the multityrosine kinase inhibitor dasatinib and the selective YES1 inhibitor CH6953755. Dasatinib and CH6953755 effectively reduced cell proliferation in vitro at doses <1000 nM in the YES-high lines in contrast to the H69 cell line (low expression of YES1, >10µM). More interestingly, dasatinib and CH6953755 decreased DMS53, H209 and a high-YES1 PDX (YU-16)-derived organoid growth. Pharmacological inhibition of YES1 significantly reduced the tumor growth of high-YES1 models: DMS53 and H209 xenografts, and TP135 and YU-16 PDXs, in contrast to low-YES1 models (H69 xenograft and YU-34 PDX). Our results provide evidence that YES1 is a new druggable oncogenic target in SCLC and that its inhibition could be a promising therapeutic strategy for a subset of patients with high YES1 expression or copy number gain/amplification. Citation Format: Esther Redin, Eva M. Garrido-Martin, Karmele Valencia, Miriam Redrado, Jose Luis Solorzano, Rafael Carias, Franscisco Exposito, Diego Serrano, Nerea Otegui, Luis Paz Ares, Katerina Politi, Luis M. Montuenga, Alfonso Calvo. YES1 is a novel druggable oncogene in small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 839.

Abstract 1537: Understanding role of uniquely enriched RNAs carried in non-small cell lung cancer derived extracellular vesicles and dynamics of their selective export

Abstract The dynamic interaction mediated by extracellular vesicles (EVs) between cancer cells and microenvironment has been shown to regulate cancer progression. Specifically, tumor derived EVs alter the phenotypes of recipient cells by delivering functional biomolecules including RNAs (EV-RNA). Studies from our lab reveal the impact of NSCLC derived EVs (Calu6 and H358) in promoting invasion of non-tumorigenic cells (BEAS-2B) and disruption of an epithelial barrier. We further determined that of all the major macromolecules contained in EVs, EV-RNA is a significant contributor to the observed phenotypes. Additionally, our data supports dysregulation of RNA subsets enclosed in NSCLC cell-derived EVs in comparison with non-tumorigenic EVs. Interestingly, RNA sequencing analysis revealed preferential enrichment of several small RNAs (<200nt), especially miRNAs, in EVs isolated from NSCLC cell lines which suggest the presence of precise mechanisms involved in loading and export of RNAs into EVs. We shortlisted a cohort of uniquely enriched miRNAs in EVs derived from Calu6 and H358 cell lines to understand their combinatorial effects on non-tumorigenic recipient cells. Amongst the shortlisted candidates miR-100, miR-10b, miR-21, miR-155 and miR-486 stand out as potential contributors to NSCLC-EV mediated function. To understand the dynamics of export of miRNAs into EVs, we transfected Calu6 cells with one of the uniquely enriched fluorescently labelled candidate miRNA (miR-451) and tracked its export into EVs. We first verified that inclusion of a fluorophore to the RNA does not impair RNA loading into EVs. To set the experiment up, one EV-enriched RNA and one Cell-enriched RNA (as a control) conjugated with different fluorophores were transfected into cells, and cellular and EV fluorescence were monitored. Interestingly, cellular fluorescence corresponding to the EV-enriched RNA diminished, while fluorescence corresponding to the Cell-enriched RNA was retained. Fluorescence signal for EV-enriched RNA showed retention in EVs isolated 48 hours after transfection. Validation of release of candidate miRNA into EVs was performed by inhibiting biosynthesis of EVs after treatment with nSMase inhibitor Gw4869. Inhibition of EV release caused EV-enriched RNA to be retained by the cells which was confirmed following flow cytometry analysis of treated cells. Despite clear evidence that dysregulated EV-RNA subsets enclosed in cancer cell-derived EVs can modulate the cellular microenvironment, research showing dynamics and mechanisms of their export is limiting. Hence, there is a critical need to reveal mechanistic details of RNA export into EVs. Resulting data from this study is expected to not only reveal functional EV-RNA subsets and dynamics of their loading but will ultimately point to novel targets for future therapeutic intervention. Citation Format: Humna Hasan, Nadia A. Lanman, Sagar Utturkar, Andrea L. Kasinski. Understanding role of uniquely enriched RNAs carried in non-small cell lung cancer derived extracellular vesicles and dynamics of their selective export [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1537.

Design, synthesis and biological evaluation of novel osimertinib derivatives as reversible EGFR kinase inhibitors

A series of osimertinib derivatives without acrylamide groups were synthesized and their inhibitory rates against L858R/T790M/C797S mutated EGFR kinase and antiproliferation activities against non-small cell lung cancer cell lines (A549, H1975) were evaluated. The preferred compounds were selected and their in vitro inhibitory activities against various EGFR kinases (wild-type, L858R/T790M, L858R/T790M/C797S) and c-Met kinase were tested. Compound 9h showed remarkable inhibitory activity against the wild type (IC50 = 29 nM), L858R/T790M mutant type (IC50 = 10 nM) and L858R/T790M/C797S mutant type (IC50 = 242 nM) as reversible EGFR kinase inhibitor, which was selected to further perform the AO/EB staining assays, cell cycle distribution assays and wound-healing assays on A549 and/or H1975 cell lines. The results showed dose-dependent activities of the induction of cell apoptosis, G1/G0-phase arrestation and inhibition of migration. Compound 22a showed remarkable inhibitory activity against the L858R/T790M/C797S mutant EGFR kinase (IC50 = 137 nM), which was nearly three times compared to osimertinib (IC50 = 410 nM). It's worth noting that 22a exhibited excellent kinase selectivity against the L858R/T790M/C797S mutant EGFR kinase rather than the wild-type, which reached 5.4 times and far more than the 0.012 times of osimertinib. Additionally, molecular docking analyses were performed to explain the action modes between the compounds and the corresponding EGFR kinases. In conclusion, compounds 9h and 22a have been demonstrated as promising candidates and worth further study.

Green supported Cu nanoparticles on modified Fe3O4 nanoparticles using thymbra spicata flower extract: Investigation of its antioxidant and the anti-human lung cancer properties

In recent days, the green synthesized nanomagnetic biocomposites have been evolved with tremendous potential as the future biological agents. This has encouraged us to design and synthesis of a novel Cu NPs supported Thyme flower extract modified magnetic nanomaterial (Fe 3 O 4 / Thyme -Cu). It was meticulously characterized using advanced analytical techniques like FT-IR, FESEM, TEM, EDX, VSM, XRD and ICP-OES. After the characterization, the synthesized Fe 3 O 4 / Thyme -Cu nanocomposite was engaged in biological assays like study of anti-oxidant properties by DPPH mediated free radical scavenging test using BHT as a reference molecule. Thereafter, on having a significant IC50 value in radical scavenging assay, we extended the bio-application of the desired nanocomposite in anticancer study of A549, Calu6 and H358 human lung cell lines in-vitro through MTT assay. They had very low cell viability and high anti-human lung cancer activities dose-dependently against A549, Calu6 and H358 cell lines without any cytotoxicity on the normal cell line (MRC-5). The IC50 of Fe 3 O 4 / Thyme -Cu nanocomposite was 124, 265, and 181 µg/mL against A549, Calu6 and H358 cell lines, respectively. Maybe significant anti-human lung cancer potentials of Fe 3 O 4 / Thyme -Cu nanocomposite against common lung cancer cell lines are related to their antioxidant activities. So, these results suggest that synthesized Fe 3 O 4 / Thyme -Cu nanocomposite as a chemotherapeutic nanomaterial have a suitable anticancer activity against lung cell lines.

Oak gum mediated green synthesis of silver nanoparticles under ultrasonic conditions: Characterization and evaluation of its antioxidant and anti-lung cancer effects

Herein, we represent the bio-synthesis of silver nanoparticles (Ag NPs) employing Oak gum as the green template, an efficient natural and non-toxic reductant and stabilizer based on its phytochemicals by using ultrasonic irradiation. The characterization of as-synthesized Ag NPs was performed through Fourier transformed infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy dispersive X-ray spectroscopy (EDS), elemental mapping, UV–Vis and X-ray diffraction (XRD). After the characterization, the synthesized Ag NPs/O. Gum was engaged in biological assays like study of anti-oxidant properties by DPPH mediated free radical scavenging test using MeOH and BHT as reference molecules. Thereafter, on having a significant IC 50 value in radical scavenging assay, we extended the bio-application of the desired nanocomposite in anticancer study of A549, Calu6 and H358 human lung cell lines in-vitro through MTT assay. They had very low cell viability and high anti-human lung cancer activities dose-dependently against the cell lines without any cytotoxicity on the normal cell line (MRC-5). The IC 50 of Ag NPs/O. Gum was found 161.25, 289.26 and 235.29 µg/mL against A549, Calu6 and H358 cell lines, respectively. Maybe significant anti-human lung cancer potentials of Ag NPs/O. Gum against common lung cancer cell lines are related to their antioxidant activities. So, these results suggest that synthesized Ag NPs/O. Gum as a chemotherapeutic nanomaterial have a suitable anticancer activity against lung cell lines.

Cyclophilin a represses reactive oxygen species generation and death of hypoxic non-small-cell lung cancer cells by degrading thioredoxin-interacting protein

ABSTRACT Cyclophilin A (cypA) is overexpressed in many types of carcinomas, including non-small-cell lung cancer (NSCLC). However, the effect of anoxia, a critical feature of the carcinoma cell microenvironment, on cypA expression in NSCLC is unknown. Here, formaldehyde-fixed and paraffin-embedded samples were collected from 60 subjects with NSCLC. The protein expression levels of cypA and hypoxia-inducible factor-1α (HIF-1α) were evaluated using immunohistochemistry. Kaplan–Meier analysis showed that subjects with high cypA expression had remarkably shorter progression-free survival than those with low cypA expression. Furthermore, cypA expression levels were significantly related to HIF-1α expression levels (Spearman’s correlation = 0.34, P < 0.0001). To further assess the effect of cypA, an anoxic carcinoma cell model was established. CypA expression was remarkably upregulated in H1299 and A549 cell lines under hypoxic conditions. Overexpression of cypA restored hypoxia-impaired cell growth and prevented reactive oxygen species (ROS) production and cell death in hypoxic A549 and H1299 cells. However, these phenotypes were not altered by the inactive R55A mutant of cypA. Mechanistic studies demonstrated that cypA can bind to and degrade the tumor suppressor protein TXNIP in H1299 and A549 cells. Restored TXNIP expression in cypA-overexpressed and hypoxic NSCLC cells led to increased ROS levels and apoptotic cell numbers and decreased cell growth compared with cypA-overexpressed and hypoxic NSCLC cells. These findings indicate that anoxia results in an increase in cypA expression in NSCLC. Additionally, cypA served as an oncogene during hypoxia by interacting with TXNIP.

Bone morphogenetic protein signaling regulation of AMPK and PI3K in lung cancer cells and C. elegans

BackgroundBone morphogenetic protein (BMP) is a phylogenetically conserved signaling pathway required for development that is aberrantly expressed in several age-related diseases including cancer, Alzheimer’s disease, obesity, and cardiovascular disease. Aberrant BMP signaling in mice leads to obesity, suggesting it may alter normal metabolism. The role of BMP signaling regulating cancer metabolism is not known.MethodsTo examine BMP regulation of metabolism, C. elegans harboring BMP gain-of-function (gof) and loss-of-function (lof) mutations were examined for changes in activity of catabolic and anabolic metabolism utilizing Western blot analysis and fluorescent reporters. AMP activated kinase (AMPK) gof and lof mutants were used to examine AMPK regulation of BMP signaling. H1299 (LKB1 wild-type), A549 (LKB1 lof), and A549-LKB1 (LKB1 restored) lung cancer cell lines were used to study BMP regulation of catabolic and anabolic metabolism. Studies were done using recombinant BMP ligands to activate BMP signaling, and BMP receptor specific inhibitors and siRNA to inhibit signaling.ResultsBMP signaling in both C. elegans and cancer cells is responsive to nutrient conditions. In both C. elegans and lung cancer cell lines BMP suppressed AMPK, the master regulator of catabolism, while activating PI3K, a regulator of anabolism. In lung cancer cells, inhibition of BMP signaling by siRNA or small molecules increased AMPK activity, and this increase was mediated by activation of LKB1. BMP2 ligand suppressed AMPK activation during starvation. BMP2 ligand decreased expression of TCA cycle intermediates and non-essential amino acids in H1299 cells. Furthermore, we show that BMP activation of PI3K is mediated through BMP type II receptor. We also observed feedback signaling, as AMPK suppressed BMP signaling, whereas PI3K increased BMP signaling.ConclusionThese studies show that BMP signaling suppresses catabolic metabolism and stimulates anabolic metabolism. We identified feedback mechanisms where catabolic induced signaling mediated by AMPK negatively regulates BMP signaling, whereas anabolic signaling produces a positive feedback regulation of BMP signing through Akt. These mechanisms were conserved in both lung cancer cells and C. elegans. These studies suggest that aberrant BMP signaling causes dysregulation of metabolism that is a potential mechanism by which BMP promotes survival of cancer cells.

Glycoprotein Receptor CEACAM5-Targeted Intraoperative Molecular Imaging Tracer in Non-Small Cell Lung Cancer

BackgroundIntraoperative molecular imaging has emerged as a potential tool in addressing challenges faced during lung cancer surgery by localizing small lesions, ensuring negative margins, and identifying synchronous cancers. Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) glycoprotein has emerged as a potential target in fluorescent labeling of non-small cell lung cancer given the high antigen density in tumor cells and absence of expression in normal parenchyma. The goal of our study was to determine whether anti-CEACAM5 targeted near-infrared fluorochrome could be a suitable target in non-small cell lung cancer. MethodsThe CEACAM5 expression was evaluated in AB-12 (known negative control), HT29 (known positive control), and H460 (non-small cell lung cancer) cell lines by polymerase chain reaction. SGM-101, a CEACAM5 antibody, coupled with a BM-104 near-infrared fluorescent tracer was evaluated with dose escalation, in vitro cellular localization, and immunofluorescence microscopy. Subsequently, in vivo validation was performed in 52 athymic nude xenografts. ResultsPolymerase chain reaction analysis demonstrated 3000x relative expression of CEACAM5 in HT-29 cells compared with AB-12. The H460 cells showed 1000x relative expression compared with AB12 (P < .05). Both HT29 and H460 cells showed tracer internalization with signal to background ratio of 4.5 (SD 0.34) whereas there was minimal uptake by AB12 cells with signal to background ratio 1.1 (SD 0.1; P < .05). There was linear fluorescence increase with increasing tracer dosing in receptor expressing cell lines. In preclinical models, HT-29 and H460 cells lines produced near-infrared fluorescence with average tumor to background ratio of 3.89 (SD 0.25) irrespective of tumor size compared with no fluorescence by AB12 tumors (P < .05). The CEACAM5 expressing tumors had excellent dye uptake compared with AB12 tumors. ConclusionsCEACAM5 serves as a possible receptor for targeted intraoperative molecular imaging resections in lung cancer. This study sets a path for evaluation of CEACAM5 targets in future clinical trials.

Design, semi-synthesis and bioactivity evaluation of novel podophyllotoxin derivatives as potent anti-tumor agents

In this study, a series of potential candidate molecules with excellent antitumor activity targeting tubulin and PTEN/PI3K/Akt signaling pathway was synthesized by modifying the molecule structure of podophyllotoxin (PPT) at the C-4 position via a structure-guided drug design approach. MTT assay results indicated that compound 12c had stronger anti-proliferative activities against HGC-27, MCF-7 and H460 cell lines than etoposide (VP-16), especially for HGC-27 (12c: IC50 = 0.89 ± 0.023 μM; PPT: IC50 = 6.54 ± 0.69 μM, VP-16: IC50 = 2.66 ± 0.28 μM) with lower affect in healthy human cells (293 T and GES-1). Further pharmacological analysis exhibited that 12c could bind the tubulin at the colchicine site and disrupt the dynamic equilibrium of microtubules. Moreover, 12c also suppressed the expressions/activities of matrix metalloprotease (MMP)−2, vimentin and up-regulation E-cadherin suggesting that 12c could block the epithelial-mesenchymal transition (EMT). The increased cell survival and invasion/migration were associated with the inactivation of PTEN/PI3K/Akt, 12c could regulate this pathway and cascade influence on the mitochondrial pathway, eventually, leading to the cell apoptosis. Thus, 12c may have the potential to become a candidate molecule in gastric cancer clinical treatment.

FHL1 Overexpression as A Inhibitor of Lung Cancer Cell Invasion via Increasing RhoGDIß mRNA Expression.

Objective Four and a half Lin-11, Isl-1, Mac-3 (LIM) protein 1 (FHL1) is one of the FHL protein family, which is regarded as a tumor suppressor in the multiple malignant tumors. In this study, we aimed to explore the regulatory effects and mechanisms of FHL1 on lung cancer cell invasion. Materials and Methods In this experimental study, bioinformatics analysis of FHL1 transcripts in human lung adenocarcinomas of TCGA database was performed. Quantitative real-time polymerasechain reaction (PCR) was performed to detect FHL1 mRNA expression in 15 paired human lung cancer tissues and their adjacent normal lung tissues, or lung cancer cell lines (A549 and H1299) in comparison with human bronchial epithelial cell line (Beas- 2B). Moreover, western blot was used to analyze FHL1 and rho GDP-dissociation inhibitor beta (RhoGDIβ) protein expression in the indicated cell lines. Also, transwell assays were employed to measure the migrated, and invaded of indicated cell lines. Results FHL1 transcripts were downregulated in the human lung adenocarcinoma. The impaired FHL1 transcripts were positively correlated with advanced tumor node metastasis (TNM) stage. Moreover, as compared to the adjacent normal lung tissues, FHL1 mRNA was low expressed in 15 paired human lung cancer tissues than their adjacent normal lung tissues. Besides, FHL1 mRNA and protein expression were also reduced in H1299 and A549 cell lines in comparison with Beas-2B cell line. Overexpressed FHL1 protein inhibited the invasive ability of H1299 and A549 cell lines. Mechanically, FHL1 protein overexpression increased the RhoGDIβ protein and mRNA abundance, while knockdown of RhoGDIβ protein, completely restored the invasion ability of A549 (Flag-FHL1) cell line. ConclusionOur findings indicated that as a key FHL1 downstream regulator, RhoGDIβ is in charge of FHL1 inhibiting lung cancer cell invasion abilities, providing a critical insight into understanding the role of FHL1 for lung cancer development.

Preclinical studies for improving radiosensitivity of non-small cell lung cancer cell lines by combining glutaminase inhibition and senolysis

Glutamine metabolism, known as glutaminolysis, is abnormally activated in many cancer cells with KRAS or BRAF mutations or active c-MYC. Glutaminolysis plays an important role in the proliferation of cancer cells with oncogenic mutations. In this study, we characterized radiation-induced cell death, which was enhanced by glutaminolysis inhibition in non-small cell lung cancer A549 and H460 cell lines with KRAS mutation. A clonogenic survival assay revealed that treatment with a glutaminase inhibitor, CB839, enhanced radiosensitivity. X-irradiation increased glutamate production, mitochondrial oxygen consumption, and ATP production, whereas CB839 treatment suppressed these effects. The data suggest that the enhancement of glutaminolysis-dependent energy metabolism for ATP production is important for survival after X-irradiation. Evaluation of the cell death phenotype revealed that glutaminolysis inhibitory treatment with CB839 or a low-glutamine medium significantly promoted the proliferation of β-galactosidase-positive and IL-6/IL-8 secretory cells among X-irradiated tumor cells, corresponding to an increase in the senescent cell population. Furthermore, treatment with ABT263, a Bcl-2 family inhibitor, transformed senescent cells into apoptotic cells. The findings suggest that combination treatment with a glutaminolysis inhibitor and a senolytic drug is useful for efficient radiotherapy.

Effects of Long Noncoding RNA TUG1 (Taurine Up-Regulated Gene 1) on Growth and Metastasis of the Non-Small Cell Lung Cancer and Its Mechanism

Objective: Our research was to discuss effects and mechanism of lncRNA TUG1 in NSCLC by vitro study. Methods: A549 and H1299 cells were divided into NC, pcDNA 3.1 and lncRNA TUG1 groups. Measuring cell proliferation using CCK-8 assay, cell apoptosis by flow cytometry, invasion cell number by transwell and wound healing rate by wound healing assay. Relative gene and protein expressions by RT-qPCR and WB assay. Results: Compared with NC group, the cell proliferation rate, invasion cell number and wound healing rate were significantly depressed in A549 and H1299 cell lines (P &lt; 0.001, respectively). By RT-qPCR and WB assay, lncRNA TUG1 gene expression were significantly increased (P &lt; 0.001, respectively); E-cadherin gene and protein expression were significantly up-regulation, and N-cadherin and Vimentin gene and protein expressions were significantly depressed compared with those of NC group in A549 and H1299 cell lines (P &lt; 0.001, respectively). Conclusion: lncRNA TUG1 had effects to suppress NSCLC cell biological activities by regulation EMT relative gene and proteins expression in vivo study.

Design, Synthesis, and Biological Evaluation of 2-Anilino-4-Triazolpyrimidine Derivatives as CDK4/HDACs Inhibitors.

PurposeTo enhance the cytotoxicities of our obtained CDK4 inhibitors and get CDK4/HDACs inhibitors with potent enzymatic inhibitory and anti-proliferative activities.MethodsA series of novel CDK4/HDACs inhibitors were designed and synthesized by incorporating the HDAC pharmacophores (hydroxylamine or o-diaminoaniline) into the basic structure of our newly obtained 2-anilino-4-triazolpyrimidine based CDK4 inhibitors. The enzymatic inhibitory (HDAC1, CDK2, CDK4, and CDK6) activities and cytotoxicities of these compounds were evaluated. Moreover, HDAC isoforms inhibitory activity, cell cycle arrest assay, cell apoptosis analysis, cell migration, and cell colony formation assay were performed for the representative compound 11k.ResultsMost of these compounds showed excellent HDAC1 inhibitory activities (IC50s: 0.68~244.5 nM) and anti-proliferative activities against cancer cell lines. Some compounds displayed potent CDK4 inhibitory activities and a certain selectivity towards CDK2 and CDK6. Compound 11k exhibited potent enzymatic (CDK4: IC50=23.59 nM; HDAC1: IC50=61.11 nM; HDAC2: IC50=80.62 nM; HDAC6: IC50=45.33 nM) and anti-proliferative activities against H460, MDA-MB-468, HCT116, and HepG2 cell lines with IC50 values 1.20, 1.34, 2.07, and 2.66 μM, respectively. Further mechanistic studies revealed that compound 11k could arrest the cell cycle in G0/G1 phase and induce apoptosis in HCT116 and MDA-MB-468 cells. In addition, compound 11k significantly inhibited the migration and cell colony formation of H460 and HCT116 cells.ConclusionThis study suggested that the incorporation of the HDAC pharmacophore into CDK4 inhibitor scaffold to design CDK/HDAC inhibitors might be a tractable strategy to enhance the antitumor potency of compounds.

Sufentanil inhibits the proliferation and epithelial mesenchymal transition of lung cancer cells through Wnt/beta-catenin signaling pathway

ABSTRACT Lung cancer is the most common malignancy and leading cause of cancer-related death. Sufentanil is a commonly used opioid anesthetic in clinics. This study aimed to explore the effects of sufentanil on the malignant behavior of lung cancer cells. H460 and H1299 lung cancer cell lines were selected for in vitro experiments. The MTT assay was conducted to detect cell viability. Proliferation ability was determined by colony formation and EdU assays. Transwell assays were performed to measure migration and invasion abilities. Western blotting was used to detect the expression of related proteins. LiCl was used to activate the Wnt/β-catenin signaling pathway. Sufentanil decreased the proliferation, migration, and invasion of H460 and H1299 cells. The protein expression levels of vimentin, N-cadherin, β-catenin, c-Myc, and MMP2 were downregulated, while those of E-cadherin and ZO-1 were upregulated after sufentanil treatment. LiCl treatment reversed the effects of sufentanil on H460 and H1299 cells. Sufentanil inhibited the proliferation, migration, invasion, and epithelial–mesenchymal transition of lung cancer cells by regulating the Wnt/β-catenin signaling pathway.

Metformin Enhances TKI-Afatinib Cytotoxic Effect, Causing Downregulation of Glycolysis, Epithelial-Mesenchymal Transition, and EGFR-Signaling Pathway Activation in Lung Cancer Cells.

The combination of metformin and TKIs for non-small cell lung cancer has been proposed as a strategy to overcome resistance of neoplastic cells induced by several molecular mechanisms. This study sought to investigate the effects of a second generation TKI afatinib, metformin, or their combination on three adenocarcinoma lung cancer cell lines with different EGFRmutation status. A549, H1975, and HCC827 cell lines were treated with afatinib, metformin, and their combination for 72 h. Afterwards, several parameters were assessed including cytotoxicity, interactions, apoptosis, and EGFR protein levels at the cell membrane and several glycolytic, oxidative phosphorylation (OXPHOS), and EMT expression markers. All cell lines showed additive to synergic interactions for the induction of cytotoxicity caused by the tested combination, as well as an improved pro-apoptotic effect. This effect was accompanied by downregulation of glycolytic, EMT markers, a significant decrease in glucose uptake, extracellular lactate, and a tendency towards increased OXPHOS subunits expression. Interestingly, we observed a better response to the combined therapy in lung cancer cell lines A549 and H1975, which normally have low affinity for TKI treatment. Findings from this study suggest a sensitization to afatinib therapy by metformin in TKI-resistant lung cancer cells, as well as a reduction in cellular glycolytic phenotype.

Potential benefit of osimertinib plus bevacizumab in leptomeningeal metastasis with EGFR mutant non-small-cell lung cancer

BackgroundEGFR-mutant non-small cell lung cancer (NSCLC) is prone to leptomeningeal metastasis (LM) after Tyrosine kinase inhibitors (TKIs) treatment. Our previous study suggested that osimertinib plus bevacizumab was safe and effective in LM from EGFR-mutant NSCLC. This study aimed to compare the efficacy of osimertinib plus bevacizumab with osimertinib in EGFR-mutant NSCLC patients with LM.MethodsWe retrospectively reviewed the data from 27 LM patients with EGFR-mutant NSCLC who received osimertinib with or without bevacizumab at the Second Affiliated Hospital of Nanchang University. Next, we investigated the antitumor efficacy of osimertinib plus bevacizumab in an LM xenograft model using the H1975 (EGFR exon20 T790M and exon21 L858R) cell line. We examined the ability of osimertinib plus bevacizumab compared with osimertinib to penetrate the blood–brain barrier (BBB) and explored the potential mechanism.ResultsOur retrospective study observed the improved survival of LM patients in osimertinib plus bevacizumab group. The median overall survival (OS) of the patients who received osimertinib and bevacizumab (n = 16) compared with osimertinib group (n = 11) was 18.0 months versus 13.7 months (log-rank test, p = 0.046, HR = 2.867, 95% CI 1.007–8.162). The median intracranial Progression-free Survival (iPFS) was 10.6 months versus 5.5 months (log-rank test, p = 0.037, HR = 3.401, 95% CI 1.079–10.720). In the LM xenograft model with H1975 cells, the combined treatment significantly increased the effective intracranial concentration of osimertinib, modulated the level of E-cadherin and downregulated the levels of EGFR and downstream signaling pathways including p-AKT and reduced tumor microvessel density (TMD), indicated that combined osimertinib with bevacizumab may exhibit a synergistic effect in EGFR-mutant LM model possibly by modulating the level of E-cadherin.ConclusionsOur findings indicate the potential benefit of osimertinib plus bevacizumab in LM with EGFR-mutant NSCLC, and more larger sample size research are still needed.

Formulation of Chitosan-Coated Brigatinib Nanospanlastics: Optimization, Characterization, Stability Assessment and In-Vitro Cytotoxicity Activity against H-1975 Cell Lines.

The purpose of the current study was to develop Brigatinib (BGT)-loaded nanospanlastics (BGT-loaded NSPs) (S1-S13) containing Span 60 with different edge activators (Tween 80 and Pluronic F127) and optimized based on the vesicle size, zeta potential (ZP), and percent entrapment efficiency (%EE) using Design-Expert® software. The optimum formula was recommended with desirability of 0.819 and composed of Span-60:Tween 80 at a ratio of 4:1 and 10 min as a sonication time (S13). It showed predicted EE% (81.58%), vesicle size (386.55 nm), and ZP (−29.51 mv). The optimized nanospanlastics (S13) was further coated with chitosan and further evaluated for Differential Scanning Calorimetry (DSC), X-ray Diffraction (XRD), in vitro release, Transmission Electron Microscopy (TEM), stability and in-vitro cytotoxicity studies against H-1975 lung cancer cell lines. The DSC and XRD revealed complete encapsulation of the drug. TEM imagery revealed spherical nanovesicles with a smooth surface. Also, the coated formula showed high stability for three months in two different conditions. Moreover, it resulted in improved and sustained drug release than free BGT suspension and exhibited Higuchi kinetic release mechanism. The cytotoxic activity of BGT-loaded SPs (S13) was enhanced three times in comparison to free the BGT drug against the H-1975 cell lines. Overall, these results confirmed that BGT-loaded SPs could be a promising nanocarrier to improve the anticancer efficacy of BGT.