Non-small Cell Lung Cancer Cell Lines Research Articles

Epigallocatechin gallate modulates ferroptosis through downregulation of tsRNA-13502 in non-small cell lung cancer

Ferroptosis, an iron-dependent regulated cell death mechanism, holds significant promise as a therapeutic strategy in oncology. In the current study, we explored the regulatory effects of epigallocatechin gallate (EGCG), a prominent polyphenol in green tea, on ferroptosis and its potential therapeutic implications for non-small cell lung cancer (NSCLC). Treatment of NSCLC cell lines with varying concentrations of EGCG resulted in a notable suppression of cell proliferation, as evidenced by a reduction in Ki67 immunofluorescence staining. Western blot analyses demonstrated that EGCG treatment led to a decrease in the expression of glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 (SLC7A11) while increasing the levels of acyl-CoA synthetase long-chain family member 4 (ACSL4). These molecular changes were accompanied by an increase in intracellular iron, malondialdehyde (MDA), and reactive oxygen species (ROS), alongside ultrastructural alterations characteristic of ferroptosis. Through small RNA sequencing and RT-qPCR, transfer RNA-derived small RNA 13502 (tsRNA-13502) was identified as a significant target of EGCG action, with its expression being upregulated in NSCLC tissues compared to adjacent non-tumorous tissues. EGCG was found to modulate the ferroptosis pathway by downregulating tsRNA-13502 and altering the expression of key ferroptosis regulators (GPX4/SLC7A11 and ACSL4), thereby promoting the accumulation of iron, MDA, and ROS, and ultimately inducing ferroptosis in NSCLC cells. This study elucidates EGCG’s multifaceted mechanisms of action, underscoring the modulation of ferroptosis as a viable therapeutic approach for enhancing NSCLC treatment outcomes.

Frequency-Dependent Ecological Interactions Increase the Prevalence, and Shape the Distribution, of Preexisting Drug Resistance

The evolution of resistance remains one of the primary challenges for modern medicine, from infectious diseases to cancers. Many of these resistance-conferring mutations often carry a substantial fitness cost in the absence of treatment. As a result, we would expect these mutants to undergo purifying selection and be rapidly driven to extinction. Nevertheless, preexisting resistance is frequently observed from drug-resistant malaria to targeted cancer therapies in non-small-cell lung cancer (NSCLC) and melanoma. Solutions to this apparent paradox have taken several forms, from spatial rescue to simple mutation supply arguments. Recently, in an evolved resistant NSCLC cell line, we found that frequency-dependent ecological interactions between ancestor and resistant mutant ameliorate the cost of resistance in the absence of treatment. Here, we hypothesize that frequency-dependent ecological interactions in general play a major role in the prevalence of preexisting resistance. We combine numerical simulations with robust analytical approximations to provide a rigorous mathematical framework for studying the effects of frequency-dependent ecological interactions on the evolutionary dynamics of preexisting resistance. First, we find that ecological interactions significantly expand the parameter regime under which we expect to observe preexisting resistance. Next, even when positive ecological interactions between mutants and ancestors are rare, these resistant clones provide the primary mode of evolved resistance because even weak positive interaction leads to significantly longer extinction times. We then find that even in the case where mutation supply alone is sufficient to predict preexisting resistance, frequency-dependent ecological forces still contribute a strong evolutionary pressure that selects for increasingly positive ecological effects (negative frequency-dependent selection). Finally, we genetically engineer several of the most common clinically observed resistance mechanisms to targeted therapies in NSCLC, a treatment notorious for preexisting resistance. We find that each engineered mutant displays a positive ecological interaction with their ancestor. As a whole, these results suggest that frequency-dependent ecological effects can play a crucial role in shaping the evolutionary dynamics of preexisting resistance. Published by the American Physical Society 2024

Remodelin delays non-small cell lung cancer progression by inhibiting NAT10 via the EMT pathway.

Lung cancer remains the foremost reason of cancer-related mortality, with invasion and metastasis profoundly influencing patient prognosis. N-acetyltransferase 10 (NAT10) catalyzes the exclusive N (4)-acetylcytidine (ac4C) modification in eukaryotic RNA. NAT10 dysregulation is linked to various diseases, yet its role in non-small cell lung cancer (NSCLC) invasion and metastasis remains unclear. Our study delves into the clinical significance and functional aspects of NAT10 in NSCLC. We investigated NAT10's clinical relevance using The Cancer Genome Atlas (TCGA) and a group of 98 NSCLC patients. Employing WB, qRT-PCR, and IHC analyses, we assessed NAT10 expression in NSCLC tissues, bronchial epithelial cells (BECs), NSCLC cell lines, and mouse xenografts. Further, knockdown and overexpression techniques (siRNA, shRNA, and plasmid) were employed to evaluate NAT10's effects. A series of assays were carried out, including CCK-8, colony formation, wound healing, and transwell assays, to elucidate NAT10's role in proliferation, invasion, and metastasis. Additionally, we utilized lung cancer patient-derived 3D organoids, mouse xenograft models, and Remodelin (NAT10 inhibitor) to corroborate these findings. Our investigations revealed high NAT10 expression in NSCLC tissues, cell lines and mouse xenograft models. High NAT10 level correlated with advanced T stage, lymph node metastasis and poor overall survive. NAT10 knockdown curtailed proliferation, invasion, and migration, whereas NAT10 overexpression yielded contrary effects. Furthermore, diminished NAT10 levels correlated with increased E-cadherin level whereas decreased N-cadherin and vimentin expressions, while heightened NAT10 expression displayed contrasting results. Notably, Remodelin efficiently attenuated NSCLC proliferation, invasion, and migration by inhibiting NAT10 through the epithelial-mesenchymal transition (EMT) pathway. Our data underscore NAT10 as a potential therapeutic target for NSCLC, presenting avenues for targeted intervention against lung cancer through NAT10 inhibition.

Updated study results of novel FAK/ALK/ROS1 inhibitor APG-2449 in patients (pts) with non-small-cell lung cancer (NSCLC) resistant to second-generation ALK inhibitors.

3124 Background: ALK inhibitors increase FAK pathway gene expression in ALK+ NSCLC cell lines, with the highest induced expression in drug-tolerant persister cells. This suggests that FAK pathway activation is involved in ALK inhibitor-induced early adaptive tolerance in ALK+ NSCLC. Investigational APG-2449 is a novel, orally active FAK inhibitor and a third-generation (3G) ALK/ROS1 tyrosine kinase inhibitor (TKI) with potent activity in preclinical models. APG-2449 is well tolerated at the recommended phase 2 dose (RP2D) and showed preliminary efficacy in pts resistant to 2G ALK TKIs. Here, we provide further safety and efficacy results and demonstrate associations between this therapy and FAK signaling pathways. Methods: After the RP2D was determined as 1,200 mg daily (QD), pts with NSCLC were enrolled into 2 dose expansion cohorts. Cohort 1 included pts who were resistant to 2G ALK TKIs. Cohort 2 included those who were ALK or ROS1 TKI naïve. Results: As of December 9, 2023, 144 pts (median [range] age, 53 [21-78] years; 53.5% female) with NSCLC, mesothelioma, or ovarian cancer were treated with APG-2449 at doses of 150 to 1,500 mg. A total of 129 (89.6%) pts had treatment-related adverse events (TRAEs), the most frequent of which were elevated serum creatinine (49.3%), ALT (42.4%), and AST (36.1%); nausea (28.5%); vomiting (23.6%); diarrhea (22.9%); and decreased leukocyte count (22.2%). In all, 20 (13.9%) TRAEs were grade ≥ 3. Of pts with TKI-naïve NSCLC (n = 36) treated at the RP2D, the overall response (ORR) and disease control rates (DCR = CR + PR + SD) were 68.2% (15/22) and 90.1% (20/22) in ROS1 TKI-naïve pts; and 78.6% (11/14) and 100% (14/14) in ALK TKI-naïve pts. Median progression-free survival (mPFS) in ALK TKI-naïve pts was not reached. Of 22 pts with NSCLC resistant to 2G ALK inhibitors and without targetable bypass gene mutations (e.g., KRAS G12C, BRAF V600E), 9 had PRs (9/22; 40.9%) and the mPFS during APG-2449 treatment at RP2D was 11.86 months. Nine of 12 pts in the RP2D group achieved intracranial PR, resulting in an intracranial ORR of 75.0%. Before APG-2449 administration, 17 tumor tissue samples were collected from pts with NSCLC resistant to 2G ALK inhibitors (in the RP2D group), and IHC was used to assess protein levels of phosphorylated FAK (pFAK). The PFS of pts treated with APG-2449 correlated with pFAK levels in baseline tumor tissue, and pts with higher pFAK levels were more likely to benefit from APG-2449 treatment. Conclusions: APG-2449 demonstrated preliminary efficacy in pts with NSCLC whose disease was TKI naïve and resistant to 2G ALK inhibitors, especially in brain metastases. High pFAK expression levels in baseline tumor tissue correlated with improved APG-2449 treatment responses in pts with NSCLC resistant to 2G ALK inhibitors. Internal study identifier: APG2449XC101. Clinical trial information: NCT03917043 .

Identification of Circular RNAs as Biomarker Candidates in Lung Cancer Treatment.

Lung cancer is the most common malignancy and among the leading cause of cancer death worldwide. Therefore, there is an important need for biomarkers that can be used in the early diagnosis of the disease and in the follow-up of treatment. Circular RNAs (circRNAs) have a covalently closed circular structure that lacks 3' and 5' polar ends and is resistant to RNAase enzymes. Due to these properties, they can be stably found in body fluids. Therefore, they can serve as potential biomarkers in the diagnosis, monitoring of therapeutic response and prognosis of cancer. In our study, we aimed to investigate the expression levels of circRNA molecules in the treatment of lung cancer and to determine those that have the potential to be biomarkers. In this in vitro study, expression levels of 163 circRNAs were investigated in A549 cells, a non-small cell lung cancer cell line, before and after treatment with carboplatin and pemetrexed. Total RNA isolation and cDNA synthesis were performed after treatments. Expression levels of circRNA genes were determined by RT-qPCR method with the designed divergent primer sequences. The study revealed the characterisation of differentially expressed circRNAs by treatment in lung cancer cells. Of them, hsa_circ_0001320 is not expressed in cancer cells, is expressed only after treatment, and increased the level of its expression in response to combination therapy. As a result, while carboplatin, pemetrexed, and combined drug applications changed the expression levels of some circRNAs in lung cancer cells, some circRNAs were expressed only after treatment. In treatment follow-up and management, hsa_circ_0001320 has been identified as potential biomarker candidate.

The thioredoxin system determines CHK1 inhibitor sensitivity via redox-mediated regulation of ribonucleotide reductase activity

Checkpoint kinase 1 (CHK1) is critical for cell survival under replication stress (RS). CHK1 inhibitors (CHK1i’s) in combination with chemotherapy have shown promising results in preclinical studies but have displayed minimal efficacy with substantial toxicity in clinical trials. To explore combinatorial strategies that can overcome these limitations, we perform an unbiased high-throughput screen in a non-small cell lung cancer (NSCLC) cell line and identify thioredoxin1 (Trx1), a major component of the mammalian antioxidant-system, as a determinant of CHK1i sensitivity. We establish a role for redox recycling of RRM1, the larger subunit of ribonucleotide reductase (RNR), and a depletion of the deoxynucleotide pool in this Trx1-mediated CHK1i sensitivity. Further, the TrxR inhibitor auranofin, an approved anti-rheumatoid arthritis drug, shows a synergistic interaction with CHK1i via interruption of the deoxynucleotide pool. Together, we show a pharmacological combination to treat NSCLC that relies on a redox regulatory link between the Trx system and mammalian RNR activity.

Synergistic anticancer effects of SMYD2 inhibitor BAY-598 and doxorubicin in non-small cell lung cancer

BackgroundNon-small cell lung Cancer (NSCLC) persists as a lethal neoplastic manifestation, exhibiting a diminished 5-year survival rate, partially attributable to chemotherapeutic resistance. This investigative endeavor aimed to elucidate the synergistic antineoplastic effects and underlying mechanisms of the SMYD2 inhibitor BAY-598 and the chemotherapeutic agent doxorubicin (DOX) in NSCLC. MethodsThe human non-small cell lung cancer cell lines A549 and H460 were subjected to treatment regimens involving BAY-598 and/or DOX. Cellular viability, apoptotic events, invasive capacity, and migratory potential were evaluated through the implementation of CCK-8 assays, flow cytometric analyses, and Transwell assays, respectively. Protein expression levels were quantified via Western blot analyses. An in vivo xenograft murine model was established to assess therapeutic efficacy. ResultsBAY-598 and DOX synergistically suppressed the viability, invasiveness, and migratory capabilities of NSCLC cells. Co-treatment Promoting cell apoptosis and cell cycle arrest. Additionally, Furthermore, co-administration significantly inhibited cell migration and invasion. Mechanistic studies revealed coordinately inhibited JAK-STAT signaling upon combination treatment. In vivo study further validated the synergistic antitumor efficacy of BAY-598 and DOX against NSCLC xenografts. ConclusionsOur findings demonstrate that BAY-598 potentiates the anti-cancer effects of DOX in non-small cell lung cancer cells by modulating the JAK/STAT signaling pathway as a synergistic strategy. The combination holds promise as an emerging therapeutic strategy for NSCLC. Further optimization and validation are warranted to promote its translational potential.

Downregulation of hsa_circTLK1 represses non-small cell lung cancer progression by regulating miR-876-3p/SRSF7 axis

BackgroundThis study clarified the expression of cicrTLK1 in non-small cell lung cancer (NSCLC) and explored its role in cancer growth, metastasis and immune escape, providing a potential molecular target and theoretical basis for NSCLC treatment. MethodsThe expression levels of circTLK1, miR-876-3p and SRSF7 were determined by RT-qPCR assay. The localization of circTLK1 in NSCLC cells was determined by FISH assay. EdU and cell plate clone formation assay were applied to explore cell proliferation. Wound healing test and Transwell assay were applied to measure the migration and invasion ability. Cell apoptosis rate was detected by FCM assay. Western blotting assay was adopted to measure the protein expression of SRSF7. Dual-luciferase reporter gene assay was applied to assess the interaction between miR-876-3p and circTLK1, and between miR-876-3p and SRSF7. The ability of cirTLK1 to regulate tumor formation in vivo was examined by tumor transplantation experiments in nude mice. ResultsThe relative expression of circTLK1 was increased in NSCLC cell lines. Knockdown of circTLK1 prohibited the proliferation, migration, and invasion, and promoted apoptosis rate, but miR-876-3p inhibitor reversed the effects of circTLK1 knockdown. In addition, silencing of circTLK1 overtly restrained the growth of transplanted tumors in vivo, and inhibited immune escape. In addition, circTLK1 interacted with miR-876-3p, and SRSF7 was concluded to be the target gene of miR-876-3p. ConclusionIn this study, we researched the inhibitory effect of circTLK1knockdown on NSCLC progression and immune escape, and further elucidated the potential regulatory mechanism of circTLK1/miR876-3p/SRSF7 axis.

Chemosensitizing effect of pentoxifylline in sensitive and multidrug-resistant non-small cell lung cancer cells.

Aim: Multidrug resistance (MDR) is frequent in non-small cell lung cancer (NSCLC) patients, which can be due to its fibrotic stroma. This work explores the combination of pentoxifylline, an anti-fibrotic and chitinase 3-like-1 (CHI3L1) inhibitor drug, with conventional chemotherapy to improve NSCLC treatment. Methods: The effect of pentoxifylline in the expression levels of P-glycoprotein (P-gp), CHI3L1 and its main downstream proteins, as well as on cell death, cell cycle profile, and P-gp activity was studied in two pairs of sensitive and MDR counterpart NSCLC cell lines (NCI-H460/NCI-H460/R and A549/A549-CDR2). Association studies between CHI3L1 gene expression and NSCLC patients' survival were performed using The Cancer Genome Atlas (TCGA) analysis. The sensitizing effect of pentoxifylline to different drug regimens was evaluated in both sensitive and MDR NSCLC cell lines. The cytotoxicity of the drug combinations was assessed in MCF10A non-tumorigenic cells. Results: Pentoxifylline slightly decreased the expression levels of CHI3L1, β-catenin and signal transducer and activator of transcription 3 (STAT3), and caused a significant increase in the G1 phase of the cell cycle in both pairs of NSCLC cell lines. A significant increase in the % of cell death was observed in the sensitive NCI-H460 cell line. TCGA analysis revealed that high levels of CHI3L1 are associated with low overall survival (OS) in NSCLC patients treated with vinorelbine. Moreover, pentoxifylline sensitized both pairs of sensitive and MDR NSCLC cell lines to the different drug regimens, without causing significant toxicity to non-tumorigenic cells. Conclusion: This study suggests the possibility of combining pentoxifylline with chemotherapy to increase NSCLC therapeutic response, even in cases of MDR.

Overexpression of lncRNA FEZF1-AS1 promotes progression of non-small cell lung cancer via the miR-130a-5p/CCND1 axis

To explore the molecular mechanism by which FEZF1-AS1 overexpression promotes progression of nonsmall cell lung cancer (NSCLC) via the miR-130a-5p/CCND1 axis. TCGA database was used to analyze FEZF1-AS1 expression levels in NSCLC. FEZF1-AS1 expression was detected by qRT-PCR in clinical specimens of NSCLC tissues and NSCLC cell lines, and its correlation with clinical features of the patients were analyzed. The binding sites of FEZF1-AS1 with hsa-miR-130a-5p and those of hsa-miR-130a-5p with CCND1 were predicted. CCK8 assay, clone formation assay, scratch assay, and Transwell assay were employed to examine the effects of FEZF1-AS1 knockdown and hsa-miR-130a-5p inhibitor on proliferation, invasion, and migration abilities of lung cancer cell lines. Dual luciferase assay was used to verify the binding of FEZF1-AS1 with hsa-miR-130a-5p and the binding of hsa-miR-130a-5p with CCND1. Western blotting was performed to detect the changes in CCND1 protein expression level in H1299 and H358 cells following FEZF1-AS1 knockdown and treatment with hsa-miR-130a-5p inhibitor. FEZF1-AS1 was highly expressed in NSCLC tissues in close correlation with lymph node metastasis and also in H1299 and H358 cell lines (all P < 0.05). FEZF1-AS1 knockdown obviously reduced proliferation, migration, and invasion abilities of NSCLC cells (P < 0.05). Dual luciferase assay confirmed the binding of hsa-miR-130a-5p with FEZF1-AS1 and CCND1 (P < 0.05), and hsa-miR-130a-5p inhibitor significantly inhibited proliferation, migration, and invasion of NSCLC cells (P < 0.05). FEZF1-AS1 knockdown significantly reduced CCND1 protein expression in NSCLC cells, and this effect was strongly inhibited by treatment with hsa-miR-130a-5p inhibitor (P < 0.05). FEZF1-AS1 is highly expressed in NSCLC tissue in close correlation with lymph node metastasis to promote cancer progression through the miR-130a-5p/CCND1 axis.

Evaluation of drug resistance for EGFR-TKIs in lung cancer via multicellular lung-on-a-chip

Drug resistance to irreversible epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) is a primary factor affecting their therapeutic efficacy in human non-small cell lung cancer (NSCLC). NSCLC cells can undergo epithelial-mesenchymal transition (EMT) induced by many factors in the tumour microenvironment (TME), which plays a crucial role in tumour drug resistance. In this study, a multicellular lung-on-a-chip that can realise the cell co-culture of the human non-small cell lung cancer cell line HCC827, human foetal lung fibroblasts (HFL-1), and human umbilical vein endothelial cells (HUVECs) is prepared. The TME was simulated on the chip combined with perfusion and other factors, and the drug evaluation of osimertinib was performed to explore the drug resistance mechanism of EGFR-TKIs. In the early stages, a two-dimensional static cell co-culture was achieved by microchip, and the results showed that HFL-1 cells could be transformed into cancer-associated fibroblasts (CAFs), and HCC827 cells could undergo EMT, both of which were mediated by Interleukin-6 (IL-6). Vimentin (VIM) and Alpha Skeletal Muscle Actin (a-SMA) expression of HFL-1 was upregulated, whereas E-cadherin (E-cad) expression of HCC827 was down-regulated. Further, N-cadherin (N-cad) expression of HCC827 was upregulated. In both the static cell co-culture and multicellular lung-on-a-chip, HCC827 cells with CAFs co-culture or IL-6 treatment developed resistance to osimertinib. Further use of the IL-6 antibody inhibitor tocilizumab could reverse EGFR-TKI resistance to a certain extent. Combination therapy with tocilizumab and EGFR-TKIs may provide a novel therapeutic strategy for overcoming EGFR-TKI resistance caused by EMT in NSCLC. Furthermore, the lung-on-a-chip can simulate complex TME and can be used for evaluating tumour resistance and exploring mechanisms, with the potential to become an important tool for personalised diagnosis, treatment, and biomedical research.

Bcl-2 dependent modulation of Hippo pathway in cancer cells

IntroductionBcl-2 and Bcl-xL are the most studied anti-apoptotic members of Bcl-2 family proteins. We previously characterized both of them, not only for their role in regulating apoptosis and resistance to therapy in cancer cells, but also for their non-canonical functions, mainly including promotion of cancer progression, metastatization, angiogenesis, and involvement in the crosstalk among cancer cells and components of the tumor microenvironment. Our goal was to identify transcriptional signature and novel cellular pathways specifically modulated by Bcl-2.MethodsWe performed RNAseq analysis of siRNA-mediated transient knockdown of Bcl-2 or Bcl-xL in human melanoma cells and gene ontology analysis to identify a specific Bcl-2 transcriptional signature. Expression of genes modulated by Bcl-2 and associated to Hippo pathway were validated in human melanoma, breast adenocarcinoma and non-small cell lung cancer cell lines by qRT-PCR. Western blotting analysis were performed to analyse protein expression of upstream regulators of YAP and in relation to different level of Bcl-2 protein. The effects of YAP silencing in Bcl-2 overexpressing cancer cells were evaluated in migration and cell viability assays in relation to different stiffness conditions. In vitro wound healing assays and co-cultures were used to evaluate cancer-specific Bcl-2 ability to activate fibroblasts.ResultsWe demonstrated the Bcl-2-dependent modulation of Hippo Pathway in cancer cell lines from different tumor types by acting on upstream YAP regulators. YAP inhibition abolished the ability of Bcl-2 to increase tumor cell migration and proliferation on high stiffness condition of culture, to stimulate in vitro fibroblasts migration and to induce fibroblasts activation.ConclusionsWe discovered that Bcl-2 regulates the Hippo pathway in different tumor types, promoting cell migration, adaptation to higher stiffness culture condition and fibroblast activation. Our data indicate that Bcl-2 inhibitors should be further investigated to counteract cancer-promoting mechanisms.

Disruption of NADPH homeostasis by total flavonoids from Adinandra nitida Merr. ex Li leaves triggers ROS-dependent p53 activation leading to apoptosis in non-small cell lung cancer cells

Ethnopharmacological relevanceAdinandra nitida Merr. ex Li leaves serve as a herbal tea and hold a significant role in traditional Chinese medicine, being applied to assist in tumor treatment. Flavonoids present the primary bioactive constituents in Adinandra nitida Merr. ex Li leaves. Aim of the studyTo explore the potential of total flavonoids from Adinandra nitida Merr. ex Li Leaves (TFAN) in inhibiting non-small cell lung cancer (NSCLC) and further elucidate the underlying mechanisms. Materials and methodsHuman NSCLC cell lines and normal lung cell line were employed to assess the impact of TFAN (0–160 μg/mL for 24, 28 and 72 h) on cell proliferation in vitro. Immunofluorescence (IF) staining gauged p53 expression changes in NSCLC cells under TFAN present condition (150 μg/mL for 24 h). In vivo study utilized NSCLC cell derived xenograft tumors in nude mice, administering TFAN orally (200 and 400 mg/kg) for 14 days. Immunohistochemistry assessed Cleaved Caspase 3 expression change in A549 xenograft tumors treated with TFAN (400 mg/kg for 14 days). RNA-seq and KEGG analysis identified gene expression changes and enriched processes in A549 xenograft tumors treated with TFAN. CM-H2DCFDA and metabolomics assessed ROS level and GSH/GSSG pool changes in A549 cells under TFAN present condition. Cell viability assay and IF staining assessed A549 cell proliferation and p53 expression changes under H2O2-induced oxidative stress (0–40 μM for 24 h) and TFAN present conditions. GSEA and N-Acetyl-L-cysteine (NAC) rescue (0–1 μM for 24 h) analyzed the impact of TFAN on GSH de novo synthesis. NADPH/NADP+ pool measurement and NADPH rescue (0–10 μM for 24 h) analyzed the impact of TFAN on GSH salvage synthesis. GC-FID and HPLC-MS were utilized to detect ethanol and ethyl acetate residues, and to characterize the chemical constituents in TFAN, respectively. The total flavonoid content of TFAN was determined using a 330 nm wavelength. ResultsTFAN significantly inhibited A549 cells (wild-type p53) but not NCI–H1299 cells (p53-deficient), NCI–H596 cells (p53-mutant) or BEAS-2B in vitro. IF staining validated p53 genotype for the cell lines and revealed an increase in p53 expression in A549 cells after TFAN treatment. In vivo, TFAN selectively inhibited A549 xenograft tumor growth without discernible toxicity, inducing apoptosis evidenced by Cleaved Caspase 3 upregulation. RNA-seq and KEGG analysis suggested ROS biosynthesis was involved in TFAN-induced p53 activation in A549 cells. Elevated ROS level in TFAN-treated A549 cells were observed. Moreover, TFAN sensitized A549 cells to H2O2-induced oxidative stress, with higher p53 expression. Additionally, A549 cells compensated with GSH de novo synthesis under TFAN present condition, confirmed by GSEA and NAC rescue experiment. TFAN disrupted NADPH homeostasis to impair GSH salvage biosynthesis, supported by NADPH/NADP+ change and NADPH rescue experiment. The chemical constituents of TFAN, with acceptable limits for ethanol and ethyl acetate residues and a total flavonoid content of 68.87%, included Catechin, Epicatechin, Quercitroside, Camellianin A, and Apigenin. ConclusionThe disruption of NADPH homeostasis by TFAN triggers ROS-dependent p53 activation that leads to apoptotic cell death, ultimately suppressing NSCLC growth. These findings offer potential therapeutic implications of Adinandra nitida Merr. ex Li leaves in combating NSCLC.

PARP inhibitor synthetic lethality in ATM biallelic mutant cancer cell lines is associated with BRCA1/2 and RAD51 downregulation.

Ataxia telangiectasia-mutated (ATM) kinase is a central regulator of the DNA damage response (DDR) signaling pathway, and its function is critical for the maintenance of genomic stability in cells that coordinate a network of cellular processes, including DNA replication, DNA repair, and cell cycle progression. ATM is frequently mutated in human cancers, and approximately 3% of lung cancers have biallelic mutations in ATM, i.e., including 3.5% of lung adenocarcinomas (LUAD) and 1.4% of lung squamous cell carcinomas (LUSC). We investigated the potential of targeting the DDR pathway in lung cancer as a potential therapeutic approach. In this context, we examined whether ATM loss is synthetically lethal with niraparib monotherapy. This exploration involved the use of hATM knockout (KO) isogenic cell lines containing hATM homozygous (-/-) and heterozygous (+/-) generated via CRISPR/Cas9 gene knockout technology in DLD-1, a human colorectal adenocarcinoma cell line. Subsequently, we extended our investigation to non-small cell lung cancer (NSCLC) patient derived xenograft (PDX) models for further validation of poly ADP-ribose polymerase inhibitor (PARPi) synthetic lethality in ATM mutant NSCLC models. Here, we demonstared that biallelic hATM deletion (-/-) in DLD-1 impairs homologous recombination (HR) repair function and sensitizes cells to the PARPi, niraparib. Niraparib also caused significant tumor regression in one-third of the NSCLC PDX models harboring deleterious biallelic ATM mutations. Loss of hATM (-/-) was concomitantly associated with low BRCA1 and BRCA2 protein expression in both the hATM (-/-) DLD-1 cell line and PARPi-sensitive ATM mutant NSCLC PDX models, suggesting a downstream effect on the impairment of HR-mediated DNA checkpoint signaling. Further analysis revealed that loss of ATM led to inhibition of phosphorylation of MRN (Mre11-Rad50-NBS1) complex proteins, which are required for ATM-mediated downstream phosphorylation of p53, BRCA1, and CHK2. Taken together, our findings highlight that the synthetic lethality of niraparib in ATM-deficient tumors can be regulated through a subsequent effect on the modulation of BRCA1/2 expression and its effect on HR function.

Mechanistic insights into Yifei Sanjie pill's regulation of EMT to enhance gefitinib treatment effect in NSCLC by in silico analysis and experimental validation

Ethnopharmacological relevanceThe Yi-Fei San-Jie pill (YFSJ) is a well-known Chinese medicine that has been used to treat non-small cell lung cancer in China for decades. Aim of the studyPrevious studies have shown that YFSJ combined with gefitinib can effectively inhibit the proliferation of gefitinib-resistant non-small cell lung cancer (NSCLC) cell lines by promoting apoptosis and autophagy, but the molecular biological mechanisms involved and whether YFSJ combined with gefitinib can have synergistic effects still need to be further explored. Thus, the present study aimed to establish an in silico and experimental framework to decipher the underlying mechanism by which YFSJ augments the efficacy of gefitinib in treating NSCLC. Materials and methodsIntegrated approaches, including microarray analysis, network pharmacology, RNA sequencing, bioinformatics algorithm analysis and in vivo and in vitro experiments, were applied to elucidate the underlying mechanism. ResultsAnalysis of microarray datasets indicated that gefitinib may play a role in the regulation of the epithelial–mesenchymal transition (EMT) of PC9 cells. EMT-related Gene Ontology (GO) terms and the MAPK pathway were found to be enriched in the differentially expressed genes (DEGs), and a decreasing trend was observed in the EMT score. Network pharmacology analysis revealed that the potential NSCLC-related targets of YFSJ also showed enrichment in EMT-related GO terms and the MAPK pathway. Experimental findings demonstrated that combined YFSJ-treated serum and gefitinib treatment significantly inhibited PC9 cell migration and invasion. In addition, the combined treatment dramatically reduced the tumour volume in an animal model. The effectiveness of the combination treatment surpassed that of gefitinib alone in both cell and animal experiments. RNA sequencing analysis revealed significant enrichment of DEGs in EMT-related GO terms for the gefitinib treatment group, YFSJ treatment group, and combination treatment group compared to the control group. Notably, the negative regulation of EMT showed significant enrichment in the DEGs of the combination treatment group. The MAPK pathway was significantly enriched among the different groups. Moreover, combined treatment with YFSJ and gefitinib may exert synergistic anti-NSCLC effects by inhibiting the p-p38 MAPK/GSK3β signalling axis, subsequently suppressing downstream EMT processes. ConclusionCombined treatment with YFSJ and gefitinib could enhance the sensitivity of NSCLC cells to gefitinib by suppressing EMT through the EGFR/p-p38 MAPK/GSK3β signalling axis. YFSJ may serve as an important adjunctive medication for NSCLC patients receiving gefitinib treatment in clinical practice.

Circ_0006949 as a potential non-invasive diagnosis biomarker promotes the proliferation of NSCLC cells via miR-4673/GLUL axis

The 5-year survival for non-small cell lung cancer (NSCLC) remains <20 %, primarily due to the early symptoms of lung cancer are inconspicuous. Prompt identification and medical intervention could serve as effective strategies for mitigating the death rate. We therefore set out to identify biomarkers to help diagnose NSCLC. CircRNA microarray and qRT-PCR reveal that sputum circ_0006949 is a potential biomarker for the early diagnosis and therapy of NSCLC, which can enhance the proliferation and clone formation, regulate the cell cycle, and accelerate the migration and invasion of NSCLC cells. Circ_0006949 and miR-4673 are predominantly co-localized in the cytoplasm of NSCLC cell lines and tissues; it upregulates GLUL by adsorption of miR-4673 through competing endogenous RNAs mechanism. The circ_0006949/miR-4673/GLUL axis exerts pro-cancer effects in vitro and in vivo. Circ_0006949 can boost GLUL catalytic activity, and they are highly expressed in NSCLC tissues and correlate with poor prognosis. In summary, circ_0006949 is a potential biomarker for the early diagnosis and therapy of NSCLC. This novel sputum circRNA is statistically more predictive than conventional serum markers for NSCLC diagnosis. Non-invasive detection of patients with early-stage NSCLC using sputum has shown good potential for routine diagnosis and possible screening.

Profiling of BCLxL Protein Complexes in Non-Small Cell Lung Cancer Cells via Multiplexed Single-Molecule Pull-Down and Co-Immunoprecipitation.

We introduce multiplexed single-molecule pull-down and co-immunoprecipitation, named m-SMPC, an analysis tool for profiling multiple protein complexes within a single reaction chamber using single-molecule fluorescence imaging. We employed site-selective conjugation of biotin and fluorescent dye directly onto the monoclonal antibodies, which completed an independent sandwich immunoassay without the issue of host cross-reactivity. We applied this technique to profile endogenous B-cell lymphoma extra-large (BCLxL) complexes in non-small cell lung cancer (NSCLC) cells. Up to three distinct BCLxL complexes were successfully detected simultaneously within a single reaction chamber without fluorescence signal crosstalk. Notably, the NSCLC cell line EBC-1 exhibited high BCLxL-BAX and BCLxL-BAK levels, which closely paralleled a strong response to the BCLxL inhibitor A-1331852. This streamlined method offers the potential for quantitative biomarkers derived from protein complex profiling, paving the way for their application in protein complex-targeted therapies.

HPLC, NMR Based Characterization, Antioxidant and Anticancer Activities of Chemical Constituents from Therapeutically Active Fungal Endophytes.

Fungi generate different metabolites some of which are intrinsically bioactive and could therefore serve as templates for drug development. In the current study, six endophytic fungi namely Aspergillus flavus, Aspergillus tubigenesis, Aspergillus oryzae, Penicillium oxalicum, Aspergillus niger, and Aspergillus brasiliensis were isolated and identified from the medicinal plant, Silybum marianum. These endophytic fungi were identified through intra transcribed sequence (ITS) gene sequencing. The bioactive potentials of fungal extracts were investigated using several bioassays such as antibacterial activity by well-diffusion, MIC, MBC, anti-biofilm, antioxidant, and haemolysis. The Pseudomonas aeruginosa PAO1 was used to determine the antibiofilm activity. The ethyl acetate extract of Aspergillus flavus showed strong to moderate efficacy against Staphylococcus aureus, Escherichia coli, P. aeruginosa, and Bacillus spizizenii. Aspergillus flavus and Aspergillus brasiliensis exhibited significant antibiofilm activity with IC50 at 4.02 and 3.63 mg/ml, while A. flavus exhibited maximum antioxidant activity of 50.8%. Based on HPLC, LC-MS, and NMR experiments kojic acid (1) and carbamic acid (methylene-4, 1-phenylene) bis-dimethyl ester (2) were identified from A. flavus. Kojic acid exhibited DPPH free radical scavenging activity with an IC50 value of 99.3 μg/ml and moderate activity against ovarian teratocarcinoma (CH1), colon carcinoma (SW480), and non-small cell lung cancer (A549) cell lines. These findings suggest that endophytic fungi are able to produce promising bioactive compounds which deserve further investigation.

T790M mutation sensitizes non-small cell lung cancer cells to radiation via suppressing SPOCK1

BackgroundApproximately 50% of patients harbor the T790M mutation after developing first-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) resistance. Evidence has showed the major treatment failure is local relapses and limited metastases. Several studies have demonstrated the value of radiotherapy in metastatic non-small cell lung cancer (NSCLC) with the EGFR T790M mutation after the development of TKI resistance. The aim of this study was to explore the role of radiation in T790M-mutant NSCLC and the value of early radiotherapy for NSCLC with T790M-mediated EGFR-TKI resistance. MethodsGefitinib-resistant NSCLC cell lines were established via stepwise exposure to increasing concentrations of gefitinib (PC-9-GR). Droplet digital PCR was used to determine the relative T790M subclone abundance. In vitro and in vivo models were established using different mixtures of PC-9-GR and PC-9 cells. Differentially expressed genes were identified using RNA sequencing. Two research models were constructed (salvage and prophylactic radiotherapy) to determine the effects of early radiotherapy on gefitinib-resistant cells. ResultsPC-9-GR cells exhibited higher radiosensitivity than PC-9 cells (sensitivity enhancement ratio = 1.5). Salvage radiation reduced the number of T790M-mutant subclones, and the relative T790M abundance was significantly lower than that without radiation at 90 days (10.94% vs. 21.54%). Prophylactic radiation prevented the development of T790M subclones. These results were also confirmed in vivo. qRT-PCR revealed threefold elevation of miR-1243 in PC-9-GR cells, and the increased radiosensitivity of PC-9-GR cells was inhibited when miR-1243 was knocked down. RNA sequencing revealed that SPOCK1 was downregulated in PC-9-GR cells. Interestingly, bioinformatic analysis showed that SPOCK1 was a target gene of miR-1243. SPOCK1 knockdown markedly increased the radiosensitivity of PC-9 cells. ConclusionGefitinib-resistant NSCLC with the T790M mutation had higher radiosensitivity than that without the mutation, possibly mediated by SPOCK1. Early radiotherapy can eliminate T790M subclones, providing evidence for the benefit of early local treatment in patients with TKI-resistant NSCLC.

Cuproptosis-related gene LIPT1 as a prognostic indicator in non-small cell lung cancer: Functional involvement and regulation of ATOX1 expression.

Non-small cell lung cancer (NSCLC) is a leading cause of cancer-related deaths, necessitating a deeper understanding of novel cell death pathways like cuproptosis. This study explored the relevance of cuproptosis-related genes in NSCLC and their potential prognostic significance. We analyzed the expression of 16 cuproptosis-related genes in 1017 NSCLC tumors and 578 Genotype-Tissue Expression (GTEx) normal samples from The Cancer Genome Atlas (TCGA) to identify significant genes. A risk model and prognostic nomogram were employed to identify the pivotal prognostic gene. Further in vitro experiments were conducted to investigate the functions of the identified genes in NSCLC cell lines. LIPT1, a gene for lipoate-protein ligase 1 enzyme, emerged as the central prognostic gene with decreased expression in NSCLC. Importantly, elevated LIPT1 levels were associated with a favorable prognosis for NSCLC patients. Overexpression of LIPT1 inhibited cell growth and enhanced apoptosis in NSCLC. We confirmed that LIPT1 downregulates the copper chaperone gene antioxidant 1 (ATOX1), thereby impeding NSCLC progression. Our study identified LIPT1 as a valuable prognostic biomarker in NSCLC as it elucidates its tumor-inhibitory role through the modulation of ATOX1. These findings offered insights into the potential therapeutic targeting of LIPT1 in NSCLC, contributing to a deeper understanding of this deadly disease.