Non-small Cell Lung Cancer Cells Research Articles

CircSEC24A induces KLF8 expression to promote the malignant progression of non-small cell lung cancer by regulating miR-1253.

This study aimed to analyze the role of circSEC24A in non-small cell lung cancer (NSCLC) and its underlying mechanism. RNA levels of circSEC24A, microRNA-1253 (miR-1253), and KLF transcription factor 8 (KLF8) were detected by quantitative real-time polymerase chain reaction. Protein expression was analyzed by western blot or immunohistochemistry assay. Cell proliferation and apoptosis were investigated by colony formation assay, 5-ethynyl-2'-deoxyuridine assay, and flow cytometry analysis. Glycolysis was evaluated by commercial kits. Dual-luciferase reporter assay and RNA immunoprecipitation assay were conducted to identify the associations among circSEC24A, miR-1253, and KLF8. Xenograft mouse model assay was used to evaluate the effect of circSEC24A on tumor tumorigenesis. CircSEC24A and KLF8 were upregulated, while miR-1253 was downregulated in NSCLC. CircSEC24A knockdown inhibited proliferation and glycolysis but induced the apoptosis of NSCLC cells. CircSEC24A acted as a miR-1253 sponge and regulated NSCLC cell malignancy by targeting miR-1253. KLF8 was identified as a target of miR-1253, and its overexpression attenuated miR-1253-induced effects in NSCLC cells. Besides, circSEC24A upregulated KLF8 by sponging miR-1253. Further, circSEC24A knockdown suppressed NSCLC cell tumorigenesis in vivo. CircSEC24A silencing inhibited NSCLC cell malignancy through the miR-1253/KLF8 pathway, providing a potential therapeutic target for NSCLC.

Xanthohumol overcomes osimertinib resistance via governing ubiquitination-modulated Ets-1 turnover

Non-small cell lung cancer (NSCLC) is a prevalent and fatal malignancy with a significant global impact. Recent advancements have introduced targeted therapies like tyrosine kinase inhibitors (TKIs) such as osimertinib, which have improved patient outcomes, particularly in those with EGFR mutations. Despite these advancements, acquired resistance to TKIs remains a significant challenge. Hence, one of the current research priorities is understanding the resistance mechanisms and identifying new therapeutic targets to improve therapeutic efficacy. Herein, we identified high expression of c-Met in osimertinib-resistant NSCLC cells, and depletion of c-Met significantly inhibited the proliferation of osimertinib-resistant cells and prolonged survival in mice, suggesting c-Met as an attractive therapeutic target. To identify effective anti-tumor agents targeting c-Met, we screened a compound library containing 641 natural products and found that only xanthohumol exhibited potent inhibitory effects against osimertinib-resistant NSCLC cells. Moreover, combination treatment with xanthohumol and osimertinib sensitized osimertinib-resistant NSCLC cells to osimertinib both in vitro and in vivo. Mechanistically, xanthohumol disrupted the interaction between USP9X and Ets-1, and inhibited the phosphorylation of Ets-1 at Thr38, promoting its degradation, thereby targeting the Ets-1/c-Met signaling axis and inducing intrinsic apoptosis in osimertinib-resistant NSCLC cells. Overall, the research highlights the critical role of targeting c-Met to address osimertinib resistance in NSCLC. By demonstrating the efficacy of xanthohumol in overcoming resistance and enhancing therapeutic outcomes, this study provides valuable insights and potential new strategies for improving the clinical management of NSCLC.

STRAP Knockdown Inhibits Migration and Growth of Non-Small Cell Lung Cancer.

Serine-threonine kinase receptor-associated protein (STRAP) regulates cell proliferation and apoptosis by binding to many target proteins and plays an important regulatory role in tumor development. We studied the effects of STRAP on non-small cell lung cancer (NSCLC) in vivo and in vitro in order to elucidate possible mechanisms underlying the regulatory effects of this protein. The levels of STRAP in NSCLC tissues and cells were determined by quantitative reverse transcription PCR, immunohistochemical staining, and Western blotting. In in vitro experiments, A549 and HCC827 cells were transfected with small interfering RNA (siRNA) to knockdown STRAP (si-STRAP) or with negative control sequence; cell migration and invasion were detected by scratch and Transwell assays, respectively. The expression levels of X-linked inhibitor of apoptosis protein (XIAP), caspase-3, and caspase-9 were determined by Western blotting. In addition, we analyzed changes of tumor volume in a nude mouse NSCLC model. STRAP was highly expressed in NSCLC tissues and cells, but its expression was significantly suppressed in A549 and HCC827 cells transfected with si-STRAP. STRAP knockdown resulted in a significant inhibition of migration and invasion of A549 and HCC827 cells. It also significantly reduced the expression of XIAP and elevated expression of caspase-3 and caspase-9. In nude mice with tumor originated from transplanted A549 cells, inhibition of STRAP expression retarded the tumor growth. Overall, these findings indicate that STRAP is overexpressed in NSCLC, while knockdown of STRAP gene inhibits the growth of NSCLC.

Cytochalasin H enhances sensitivity to gefitinib in non-small-cell lung cancer cells through inhibiting EGFR activation and PD-L1 expression

In our previous study, we have isolated cytochalasin H (CyH) from endophytic fungus derived from mangrove plant and found that CyH inhibited the proliferation of non-small cell lung cancer (NSCLC) cells. Recently, epidermal growth factor receptor (EGFR) activation and programmed cell death 1 ligand (PD-L1) expression have been demonstrated to mediate NSCLC resistance to gefitinib, first-generation EGFR tyrosine kinase inhibitor (EGFR-TKI). Here, we further investigated the effect of CyH on EGFR activation, PD-L1 expression, and gefitinib sensitivity in NSCLC cell lines, A549 (wild-type EGFR), HCC827 (EGFR mutation), and NCI-H1975 (dual EGFR mutations and acquired gefitinib resistance) and animal model. Our results showed that CyH significantly inhibited EGFR activation and PD-L1 expression in NSCLC cells. Additionally, CyH dramatically promoted the inhibitory effect of gefitinib on the proliferation of A549 and HCC827 cells, and enhanced the sensitivity to gefitinib in NCI-H1975 cells. Moreover, CyH increased the inhibitory effect of gefitinib on EGFR activation and PD-L1 expression in HCC827 and NCI-H1975 cells. Animal experiments further demonstrated that CyH significantly promoted the inhibitory effect of gefitinib on the growth of NSCLC and the expression of Ki-67, p-EGFR, and PD-L1 in NCI-H1975 NSCLC xenograft tumors of nude mice. Furthermore, CyH inhibited the activation of JAK3/STAT signaling pathway. Taken together, our findings suggest that CyH promotes the sensitivity to gefitinib in NSCLC cells through the inhibition of EGFR activation and PD-L1 expression.

Development of a novel N14-substituted antitumor evodiamine derivative with inhibiting heat shock protein 70 in non-small cell lung cancer

Notwithstanding the latest advancements in anticancer therapy, non-small cell lung cancer (NSCLC) remains a prominent contributor to cancer-associated mortality worldwide. Therefore, effective anti-cancer agents are required for the treatment of NSCLC. We previously demonstrated that the natural alkaloid evodiamine efficiently suppressed lung cancer cells and lung cancer stem-like cell populations by suppressing heat shock protein 70 (Hsp70). This finding inspired us to formulate evodiamine-based anti-cancer compounds against NSCLC. In this study, we synthesized a series of evodiamine derivatives with substitutions at the N14 position. EV206 was chosen for further study because it was the most effective among the 22 evodiamine derivatives at stopping H1299 cell growth. EV206 treatment efficiently suppressed cell viability and colony formation in both attached cells and in soft agar, even in those carrying drug resistance, by inducing apoptosis. The effectiveness of EV206 is approximately ten times greater than that of evodiamine. Normal cell viability was marginally affected by EV206 treatment. Additionally, EV206 efficiently decreased the cancer stem cell (CSC) population in the NSCLC cells. EV206 reduced the growth of H460 xenograft tumors without exhibiting toxic effects. These data implied that EV206 has the potential to be an effective Hsp70-targeting anticancer drug with low toxicity.

The oncogenic axis YAP/MYC/EZH2 impairs PTEN tumor suppression activity enhancing lung tumorigenicity

The tumor suppressor PTEN (phosphatase and tensin homolog deleted in chromosome 10) is genetically deleted or downregulated in many cancer types. Loss of PTEN protein expression is frequently found in lung cancer while genetic alterations are less abundant. PTEN expression is regulated at multiple genetic and epigenetic levels and even partial reduction of its expression increases cancer occurrence. We show that YAP and TAZ cooperate with EZH2, and MYC to transcriptionally repress onco-suppressor genes, including PTEN, in non-small cell lung cancer (NSCLC) cells. YAP/TAZ-EZH2-MYC transcriptional regulators form a nuclear complex that represses PTEN transcription, while their combinatorial targeting restores PTEN expression, attenuates NSCLC cell growth, and prevents compensatory responses induced by single treatments. Datasets analysis of NSCLC patients revealed that PTEN expression is negatively correlated to YAP/TAZ, EZH2 and MYC and that low expression of PTEN is predictive of poor prognosis, especially at earlier stages of the disease. These findings highlight the repressive role of the YAP/TAZ-EZH2-MYC axis on tumor-suppressor genes and offer a potential therapeutic strategy for lung cancer patients with low PTEN levels.

Cisplatin-resistance and aggressiveness are enhanced by a highly stable endothelin-converting enzyme-1c in lung cancer cells

BackgroundLung cancer constitutes the leading cause of cancer mortality. High levels of endothelin-1 (ET-1), its cognate receptor ETAR and its activating enzyme, the endothelin-converting enzyme-1 (ECE-1), have been reported in several cancer types, including lung cancer. ECE-1 comprises four isoforms, which only differ in their cytoplasmic N-terminus. Protein kinase CK2 phosphorylates the N-terminus of isoform ECE-1c, increasing its stability and leading to enhanced invasiveness in glioblastoma and colorectal cancer cells, which is believed to be mediated by the amino acid residue Lys-6, a conserved putative ubiquitination site neighboring the CK2-phosphorylated residues Ser-18 and Ser-20. Whether Lys-6 is linked to the acquisition of a cancer stem cell (CSC)-like phenotype and aggressiveness in human non–small cell lung cancer (NSCLC) cells has not been studied.MethodsIn order to establish the role of Lys-6 in the stability of ECE-1c and its involvement in lung cancer aggressiveness, we mutated this residue to a non-ubiquitinable arginine and constitutively expressed the wild-type (ECE-1cWT) and mutant (ECE-1cK6R) proteins in A549 and H1299 human NSCLC cells by lentiviral transduction. We determined the protein stability of these clones alone or in the presence of the CK2 inhibitor silmitasertib, compared to ECE-1cWT and mock-transduced cells. In addition, the concentration of secreted ET-1 in the growth media was determined by ELISA. Expression of stemness genes were determined by Western blot and RT-qPCR. Chemoresistance to cisplatin was studied by MTS viability assay. Migration and invasion were measured through transwell and Matrigel assays, respectively, and the side-population was determined using flow cytometry.ResultsECE-1cK6R displayed higher stability in NSCLC cells compared to ECE-1cWT-expressing cells, but ET-1 secreted levels showed no difference up to 48 h. Most importantly, ECE-1cK6R promoted expression of the stemness genes c-Myc, Sox-2, Oct-4, CD44 and CD133, which enhance cellular self-renewal capability. Also, the ECE-1cK6R-expressing cells showed higher cisplatin chemoresistance, correlating with an augmented side-population abundance due to the increased expression of the ABCG2 efflux pump. Finally, the ECE-1cK6R-expressing cells showed enhanced invasiveness, which correlated with the regulated expression of known EMT markers.ConclusionsOur findings suggest an important role of ECE-1c in lung cancer. ECE-1c is key in a non-canonical ET-1-independent mechanism which triggers a CSC-like phenotype, leading to enhanced lung cancer aggressiveness. Underlying this mechanism, ECE-1c is stabilized upon phosphorylation by CK2, which is upregulated in many cancers. Thus, phospho-ECE-1c may be considered as a novel prognostic biomarker of recurrence, as well as the CK2 inhibitor silmitasertib as a potential therapy for lung cancer patients.

Inhibition of Chk1 with Prexasertib Enhances the Anticancer Activity of Ciclopirox in Non-Small Cell Lung Cancer Cells

Lung cancer is a leading cause of cancer-related deaths worldwide. Non-small cell lung cancer (NSCLC) is the most prevalent lung cancer subtype. Ciclopirox olamine (CPX), an off-patent fungicide, has been identified as a new anticancer agent. Prexasertib (PRE), a Chk1 inhibitor, is in phase 1/2 clinical trials in various tumors. The anticancer effect of the combination of CPX with PRE on NSCLC cells is unknown. Here, we show that CPX is synergistic with PRE in inhibiting cell proliferation and inducing apoptosis of NSCLC (A549 and A427) cells. Combined treatment with CPX and PRE significantly increased the cell population in the G1/G0 and sub-G1 phases, compared to the single treatment with CPX or PRE. Concurrently, the combined treatment downregulated the protein levels of cyclins (A, B1), cyclin-dependent kinases 4, 6, 2 (CDK4, CDK6, CDK2), cell division cycle 25 B, C (Cdc25B, Cdc25C), and upregulated the protein levels of the CDK inhibitors p21 and p27, leading to decreased phosphorylation of Rb. In addition, the combined treatment increased DNA damage, evidenced by increased expression of γH2AX. In line with this, the combined treatment induced more apoptosis than either single treatment. This was associated with increased expression of DR4, DR5, Fas, and FADD and decreased expression of survivin, resulting in activation of caspase 8 and caspase 3 as well as cleavage of poly (ADP ribose) polymerase (PARP). Taken together, the results suggest that inhibition of Chk1 with PRE can enhance the anticancer activity of CPX at least partly by decreasing cell proliferation and increasing apoptosis in NSCLC cells.

3D cultivation of non-small-cell lung cancer cell lines using four different methods

PurposeThe aim of this study was to set up reliable and reproducible culture conditions for 3D tumoroids derived from non-small cell lung cancer (NSCLC) cell lines to enable greater opportunity for successful cultivation of patient-derived samples.MethodsFour NSCLC cell lines, two adenocarcinomas (A549, NCI-H1975) and two squamous cell carcinomas (HCC-95, HCC-1588), were first cultured in traditional 2D settings. Their expected expression profiles concerning TTF-1, CK7, CK5, and p40 status were confirmed by immunohistochemistry (IHC) before the generation of 3D cultures. Tumoroids were established in the hydrogel GrowDex®-T, Nunclon™ Sphera™ flasks, BIOFLOAT™ plates, and Corning® Elplasia® plates. Western blot was used to verify antigen protein expression. Hematoxylin-eosin staining was used to evaluate the cell morphology in the 2D and 3D cultures. Mutational analysis of KRAS and EGFR by PCR on extracted DNA from 3D tumoroids generated from cells with known mutations (A549; KRAS G12S mutation, NCI-H1975; EGFR L858R/T790M mutations).ResultsWe successfully established 3D cultures from A549, NCI-H1975, HCC-95, and HCC-1588 with all four used cultivation methods. The adenocarcinomas (A549, NCI-H1975) maintained their original IHC features in the tumoroids, while the squamous cell carcinomas (HCC-95, HCC-1588) lost their unique markers in the cultures. PCR analysis confirmed persistent genetic changes where expected.ConclusionThe establishment of tumoroids from lung cancer cell lines is feasible with various methodologies, which is promising for future tumoroid growth from clinical lung cancer samples. However, analysis of relevant markers is a prerequisite and may need to be validated for each model and cell type.

MAT1A activation of glycolysis to promote NSCLC progression depends on stabilizing CCND1

Non-small cell lung cancer (NSCLC) remains a cause for concern as the leading cause of cancer-related death worldwide. Amidst ongoing debates on the role and mechanisms of methionine adenosyltransferase 1A (MAT1A) in cancer, our study sheds light on its significance in NSCLC. Leveraging TCGA database and immunohistochemical staining, we systematically analyzed MAT1A expression in NSCLC, uncovering its marked upregulation. To unravel the functional and mechanistic underpinnings, we implemented stable knockdown of MAT1A in NSCLC cell lines. Our findings converged to demonstrate that suppression of MAT1A expression effectively impeded the proliferation and migratory capabilities of NSCLC cells, while concurrently enhancing apoptosis. Mechanistically, we discovered that MAT1A depletion accelerated the degradation of CCND1, a key cell cycle regulator, through S-phase kinase-associated protein 2 (SKP2)-mediated ubiquitination. Notably, CCND1 emerged as a crucial MAT1A partner, jointly orchestrating glycolytic metabolism in NSCLC cells. This intricate interplay suggests that MAT1A promotes NSCLC progression by safeguarding CCND1 protein stability and activating glycolytic pathways, thereby sustaining tumorigenesis. In summary, our study not only identifies MAT1A as a prognostic marker for poor survival in NSCLC patients but also elucidates its mechanistic contributions to cancer progression. These findings pave the way for the development of targeted therapies aimed at disrupting the deleterious MAT1A-CCND1-glycolysis axis in NSCLC.

MYL9 binding with MYO19 suppresses epithelial-mesenchymal transition in non-small cell lung cancer.

The elusive function of myosin light chain 9 (MYL9) in cancer is an area ripe for further investigation. Bioinformatics was utilized to compare the expression levels of MYL9 in non-small cell lung cancer (NSCLC) and normal tissues. Gene set enrichment analysis (GSEA) was employed to investigate the pathways associated with MYL9. BioGRID database was utilized to screen for potential targets of MYL9. The expression of MYL9 and myosin 19 (MYO19) mRNA was quantified using quantitative reverse transcriptase PCR. Cell migration was assessed using a scratch wound healing assay. Protein levels of MYL9, MYO19, and epithelial-mesenchymal transition (EMT) biomarkers were examined using western blot (WB). EpCAM expression in different cell groups was profiled using flow cytometry analysis. Co-immunoprecipitation assays were performed to determine the binding affinity between MYL9 and MYO19. Additionally, direct protein interaction between MYL9 and MYO19 was explored using a GST-pull-down assay. In NSCLC patients, MYL9 was significantly downregulated in vivo and in cell cultures, showing high enrichment in the EMT pathway. Scratch assays indicated its inhibitory effect on cell migration. Western blotting revealed that MYL9 suppresses EMT marker protein expression in NSCLC cells. Flow cytometry showed that MYL9 reduced EpCAM levels on the cell surface. MYO19 was identified as a potential target of MYL9 through CoIP and GST-pull-down assays. Rescue experiments demonstrated that MYO19 enhances cell migration, EMT marker expression, and EpCAM levels, but these effects were countered by MYL9 overexpression. MYL9 impedes the migration and EMT in NSCLC cells by binding to MYO19.

Ultrasound-Driven Nanomachine for Enhanced Sonodynamic Therapy of Non-Small-Cell Lung Cancer.

Non-small-cell lung cancer (NSCLC) is the most prevalent type of lung cancer, and there is an urgent need for developing novel therapies. Sonodynamic therapy exhibits exceptional tissue penetration and minimal harm to healthy tissue, making it extremely promising for cancer treatment. The efficacy of SDT is limited by the intricate immunological microenvironment and the resistance to tumor treatment. This study developed targeted nanoparticles that use ultrasound to concentrate on treating NSCLC. The hybrid targeted nanoparticles utilize gold nanoparticles as their fundamental component, with the outside modified with engineered macrophage exosomes and the aptamer S11e to specifically target NSCLC. Ultrasound could effectively eliminate tumors in NSCLC cells by destroying lysosomes via targeted nanoparticles. Simultaneously, fragmented tumor antigens could effectively activate dendritic cell cells to recruit T cells. This method has significant efficacy in suppressing the development of NSCLC and exhibits potential for therapeutic application.

Nerolidol inhibits proliferation and triggers ROS-facilitated apoptosis in lung carcinoma cells via the suppression of MAPK/STAT3/NF-κB and P13K/AKT pathways.

Lung cancer (LC) is the leading cause of malignancy-related mortalities globally, and the existing treatment interventions are associated with harmful side effects. In the current study, we evaluated the anti-tumor efficiency of nerolidol (NRD) on human non-small cell lung cancer (NSCLC) cells. Nerolidol is a sesquiterpene alcohol extracted from the essential oils of aromatic flora with known anti-cancer activities. The latent action of NRD on antiproliferative and apoptotic effects in A549 cells is uncertain. Thus, our work is designed to explore the antiproliferative and apoptotic actions of NRD (20 and 25 μM/mL) against A549 cells. The activity of NRD on A549 cell cytotoxicity, intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP), apoptosis, anti-apoptotic proteins, and MAPK/TAT3/NF-κB and P13K/AKT signaling pathways were assessed using MTT tests, dichlorodihydrofluorescein diacetate (DCFH-DA), dual acridine orange/ethidium bromide (AO/EB), DAPI, Rh-123, reverse transciption polymerase chain reaction (RT-PCR), and western blot analyses. We found that NRD could inhibit NSCLC cell viability through elevated intracellular ROS and MMP loss and elicited apoptosis in a quantity-dependent manner. Similarly, NRD can reduce inflammatory cytokines and anti-apoptotic elements, as well as trigger apoptotic signaling pathways. Our data established that NRD decreases A549 cell proliferation through ROS-mediated apoptosis, triggering the MAPK/STAT3/NF-κB and P13K/AKT pathways, suggesting that NRD is a possible protective remedy for NSCLC.

Bee venom prompts the inhibition of gefitinib on proliferation, migration, and invasion of non-small cell lung cancer cells via EGFR-mediated autophagy

It has been confirmed that bee venom (BV) can inhibit tumor metastasis of lung cancer cells induced by epidermal growth factor, suggesting the inhibitory role of BV on the regulation of epidermal growth factor receptor (EGFR), and may synergistically promote the anti-lung cancer effect of EGFR tyrosine kinase inhibitor gefitinib. This paper aims to ascertain the therapeutic potentials of BV combined with gefitinib against non-small cell lung cancer (NSCLC) in vitro. As results, the content of the main component melittin in air-dried BV was determined by HPLC. Subsequently, it was found that BV significantly inhibited the proliferation of NSCLC PC-9 and NCI-H1299 cells, but not generated apparent toxicity to human normal lung epithelial BEAS-2B cells. Meanwhile, the combination of BV and gefitinib also significantly inhibited the proliferation of these two cells, and suppressed the migration and invasion of PC-9 cells. By bioinformatics analysis and molecular docking, it was predicted that the main component melittin in BV could act on the cell membrane and transmembrane protein EGFR. Ultimately, Western blot assays showed BV alone or combined with gefitinib significantly decreased the protein expression of phosphorylated EGFR (p-EGFR) and the protein expression ratio of p-EGFR to EGFR, and increased the protein expression ratio of LC3-II to LC3-I in PC-9 cells or epidermal growth factor-activated PC-9 cells. The results demonstrated that BV could prompt the inhibition of gefitinib on proliferation, migration, and invasion of NSCLC cells via EGFR-mediated autophagy, showing the synergistic anti-NSCLC potential when combined with gefitinib.

To Reveal the Potential Mechanism of Quercetin against NSCLC Based on Network Pharmacology and Experimental Validation.

This study aimed to initially clarify the potential mechanism of quercetin in the treatment of non-small cell lung cancer (NSCLC) based on network pharmacology, molecular docking and in vitro experiments. TCMSP, SwissTargetPrediction, TCMIP, STITCH, and ETCM databases were applied to obtain the targets of quercetin. NSCLC-related targets were retrieved from GeneCards, OMIM, PharmGKB, TTD, and NCBI databases. Their intersection targets were imported into the STRING database to construct a protein-protein interaction (PPI) network and core targets were identified through the Cytoscape 3.10.0 soft and the CytoHubba tool. Furthermore, Gene Ontology (GO) functional analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were performed on the intersection targets. A compound-targetspathways network was subsequently constructed to screen for key targets and pathways. Molecular docking was performed with Discovery Studio software to verify the interactions between quercetin and core targets. In vitro validations were conducted employing CCK-8 assays, flow cytometry, and Western blotting (WB). 193 potential targets of quercetin for treating NSCLC were obtained. The top ten core targets identified within the PPI network included TP53, HSP90AA1, AKT1, JUN, SRC, EGFR, ACTB, TNF, MAPK1, and VEGFA. GO analysis yielded 2319 items, and KEGG analysis resulted in 211 enriched pathways. Molecular docking results demonstrated a high affinity of quercetin towards the core targets. Based on the compound-targets-pathways network and molecular docking, the PI3K/AKT/P53 pathway and its key-related proteins (PIK3R1, AKT1, and TP53) were selected for further validation. Quercetin(20 and 40 μg/mL) significantly decreased the viability of A549 NSCLC cells but not BEAS-2B normal cells via CCK-8 assays. Flow cytometry and WB analyses further corroborated that quercetin could promote apoptosis of A549 cells by downregulating and upregulating the expression of Bcl-2 and Bax (P<0.05), respectively. Notably, quercetin did not significantly alter the total protein levels of PI3K, AKT, and P53 but downregulated the phosphorylation levels of PI3K and AKT (P<0.05) and upregulated the phosphorylation level of P53 (P<0.05). Quercetin exhibits therapeutic potential in NSCLC by regulating the PI3K/AKT/P53 pathway to promote cell apoptosis.

GOLPH3 inhibition overcomes cisplatin resistance by restoring the glutathione/reactive oxygen species balance in the A549 non‑small cell lung cancer cell line.

Cisplatin resistance is common in non‑small cell lung cancer (NSCLC); however, the molecular mechanisms remain unclear. The present study aimed to identify a new function of Golgi phosphoprotein 3 (GOLPH3) in NSCLC‑associated cisplatin resistance. Using A549 human NSCLC cells and the cisplatin‑resistant variant, stable cell lines with GOLPH3 knockdown or overexpression were established using lentiviral vectors. Through Cell Counting Kit‑8 and EdU assays, it was revealed that knockdown of GOLPH3 significantly enhanced cisplatin sensitivity in NSCLC cells. Specifically, flow cytometric analysis showed that GOLPH3 knockdown promoted apoptosis and G2‑phase cell cycle arrest in A549 cells. Mechanistically, intracellular reactive oxygen species (ROS) and glutathione (GSH) levels were measured using assay kits, and it was demonstrated that GOLPH3 knockdown decreased intracellular GSH levels, and further attenuated intracellular cisplatin efflux and GSH/ROS imbalance. In addition, tumor‑sphere formation assays verified that GOLPH3 knockdown mitigated the stem cell‑like phenotype of NSCLC cells. In conclusion, the present findings indicated the relevance of GOLPH3 in NSCLC‑associated cisplatin resistance, and thus targeting GOLPH3 may be developed into a combination therapy to overcome cisplatin resistance.

Hypoxia drives estrogen receptor β-mediated cell growth via transcription activation in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is a highly malignant tumor with a poor prognosis. Hypoxia conditions affect multiple cellular processes promoting the adaptation and progression of cancer cells via the activation of hypoxia-inducible factors (HIF) and subsequent transcription activation of their target genes. Preliminary studies have suggested that estrogen receptor β (ERβ) might play a promoting role in the progression of NSCLC. However, the precise mechanisms, particularly its connection to HIF-1α-mediated modulation under hypoxia, remain unclear. Our findings demonstrated that the overexpression of ERβ, not ERα, increased cell proliferation and inhibition of apoptosis in NSCLC cells and xenografts. Tissue microarray staining revealed a strong correlation between the protein expression of HIF-1α and ERβ. HIF-1α induced ERβ gene transcription and protein expression in CoCl2-induced hypoxia, 1% O2 incubation, or HIF-1α overexpressing cells. ChIP identified HIF-1α binding to a hypoxia response element in the ESR2 promoter. The suppression of HIF-1α and ERβ both in vitro and in vivo effectively reduced the tumor growth, thus emphasizing the promising prospects of targeting HIF-1α and ERβ as a therapeutic approach for the treatment of NSCLC. KEY MESSAGES: ERβ, not ERα, increases cell proliferation and inhibition of apoptosis in NSCLC cells and xenografts. A strong correlation exists between the protein expression of HIF-1α and ERβ. HIF-1α induced ERβ gene transcription and protein expression in hypoxic cells via binding to HRE in the ESR2 promoter. The suppression of HIF-1α and ERβ both in vitro and in vivo effectively reduced the NSCLC tumor growth.

Modeling compound lipid homeostasis using stable isotope tracing.

Lipids represent the most diverse pool of metabolites found in cells, facilitating compartmentation, signaling, and other functions. Dysregulation of lipid metabolism is linked to disease states such as cancer and neurodegeneration. However, limited tools are available for quantifying metabolic fluxes across the lipidome. To directly measure reaction fluxes encompassing compound lipid homeostasis, we applied stable isotope tracing, high-resolution mass spectrometry, and network-based isotopologue modeling to non-small cell lung cancer (NSCLC) models. Compound lipid metabolic flux analysis (CL-MFA) enables the concurrent quantitation of fatty acid synthesis, elongation, headgroup assembly, and salvage reactions within virtually any biological system. Here, we resolve liver kinase B1 (LKB1)-mediated regulation of sphingolipid recycling in NSCLC cells and precision-cut lung slice cultures. We also demonstrate that widely used tissue culture conditions drive cells to upregulate fatty acid synthase flux to supraphysiological levels. Finally, we identify previously uncharacterized isozyme specificity of ceramide synthase inhibitors, highlighting the molecular detail revealed by CL-MFA.

A Novel Lipid Nanoparticle NBF-006 Encapsulating Glutathione S-Transferase P siRNA for the Treatment of KRAS-driven Non-small Cell Lung Cancer.

Non-small cell lung cancer (NSCLC) accounts for approximately 85% of lung cancers, and KRAS mutations occur in 25-30% of NSCLC. Our approach to developing a therapeutic with the potential to target KRAS mutant NSCLC was to identify a new target involved in modulating signaling proteins in the RAS pathway. Glutathione S-Transferase P (GSTP) known as a Phase II detoxification enzyme has more recently been identified as a modulator of MAP kinase-related cell-signaling pathways. Therefore, developing a GSTP siRNA may be an effective therapeutic approach to treat KRAS mutant NSCLC. The lead drug product candidate (NBF-006) is a proprietary siRNA-based lipid nanoparticle (LNP) comprising GSTP siRNA (NDT-05-1040). Here, studies using a panel of KRAS mutant NSCLC cell lines demonstrated that NDT-05-1040 is a very potent and selective GSTP siRNA inhibitor. Our Western blot analysis showed that NDT-05-1040 effectively decreased the phosphorylation of MAPK and PI3K pathway components while upregulating apoptotic signaling cascade. Our in vivo studies revealed statistically significant higher distribution of NBF-006 to the lungs and tumor as compared to liver. In the subcutaneous and orthotopic tumor models, NBF-006 led to a statistically significant and dose dependent anti-tumor growth inhibition. Further, quantitative image analysis of PCNA and PARP staining showed that NBF-006 decreased proliferation and induced apoptosis, respectively, in tumors. Additionally, in a surgically implanted orthotopic lung tumor model, the survival rate of the NBF-006 treatment group was significantly prolonged (P <0.005) as compared to the vehicle control group. Together, these preclinical studies supported advancement of NBF-006 into clinical studies.

Immuno-PET Imaging of EGFR with 64Cu-NOTA Panitumumab in Subcutaneous and Metastatic Nonsmall Cell Lung Cancer Xenografts.

Objective: About 65-90% of nonsmall cell lung cancer (NSCLC) express the epithelial growth factor receptor (EGFR) as a transmembrane protein that is activated by binding of specific ligands, including epidermal growth factor and transforming growth factor α (TGFα). Identifying EGFR as an oncogene has led to the development of anticancer therapeutics directed against EGFR, including the full-length human IgG2 monoclonal antibody panitumumab. The main goal of the present study was to investigate 64Cu-labeled panitumumab with immuno-PET in subcutaneous and metastatic EGFR-positive NSCLC xenografts. Methods: Bifunctional chelating agent 2-S-(4-isothiocyanatobenzyl)-1,4,7-triazacyclo-nonane-1,4,7-triacetic acid (NOTA-NCS) was attached to panitumumab. The number of chelators per panitumumab was determined using matrix-assisted laser desorption/ionization (MALDI) mass spectroscopy. The incorporation efficiency of 64Cu into NOTA-panitumumab was measured by using radio-TLC. EGFR-expressing epithelial-like H1299-luc+ NSCLC cells were used for in vitro and in vivo experiments. Cell uptake of [64Cu]Cu-NOTA-panitumumab was measured in the presence and absence of panitumumab. Subcutaneous and metastatic H1299-luc tumor models were grown in male NSG mice. The presence of tumors at lung and metastatic sites was analyzed by [18F]FLT PET. Immuno-PET with [64Cu]Cu-NOTA-panitumumab was performed as static PET imaging at 2, 24, and 48 h postinjection in both tumor models. Proof-of-target was confirmed by blocking experiments with panitumumab. Detailed ex vivo biodistribution experiments were performed in both animal tumor models to confirm biodistribution profiles obtained by immuno-PET imaging. Results: MALDI analysis confirmed the attachment of ∼1.5 NOTA per antibody. Radiolabeling efficiency with [64Cu]CuCl2 was 93.8 ± 5.7% and a molar activity of 0.65 MBq/μg. Cellular uptake studies with [64Cu]Cu-NOTA-panitumumab in H1299 cells demonstrated increasing uptake over time, reaching 29.1 ± 2.9% radioactivity(Bq)/mg protein (n = 3) and plateauing at 45 min. Addition of 25 μg of panitumumab reduced radioligand uptake to 1.22 ± 0.06% radioactivity/mg protein (n = 3). PET imaging revealed high uptake of [64Cu]Cu-NOTA-panitumumab in subcutaneous tumors: Standardized uptake values (SUV)mean reached 4.70 ± 0.42 and 5.37 ± 0.40 (n = 5) after 24 and 48 h postinjection, respectively. Administration of 1 mg panitumumab reduced tumor uptake significantly to 1.94 ± 0.22 and 1.66 ± 0.08 (n = 4; p < 0.001). In the metastatic model, the following SUVmean were analyzed from liver and lung lesions: 5.55 ± 0.34 and 6.28 ± 0.46 (both n = 23 lesions from 6 mice) after 24 and 48 h postinjection, which was also significantly reduced to 2.53 ± 0.39 and 2.31 ± 0.15 (both n = 16 lesions from 4 mice; p < 0.001) after injection of 1 mg panitumumab. Detailed ex vivo biodistribution confirmed immuno-PET analysis in both models. Panitumumab reduced radioactivity uptake into subcutaneous tumors from 11.01 ± 0.72 (n = 4) to 3.67 ± 0.33% ID/g (n = 5; p < 0.001), and in metastatic liver lesions from 29.44 ± 8.14 (n = 4) to 8.35 ± 1.30% ID/g (n = 5; p < 0.001), respectively. Conclusions: [64Cu]Cu-NOTA-panitumumab was successfully used for immuno-PET imaging of EGFR-expressing subcutaneous and metastatic NSCLC tumors. This result represents the basis for developing radiotheranostics for targeting EGFR in cancers and for selecting the right patients for the right treatment at the right time.