H1299 Cells Research Articles

AXL-Mediated Drug Resistance in ALK-Rearranged NSCLC Enhanced by GAS6 From Macrophages and MMP11 Positive Fibroblasts.

Anaplastic lymphoma kinase (ALK) rearranged non-small cell lung cancer (NSCLC) shows marked tumor shrinkage by ALK-tyrosine kinase inhibitors (TKIs). However, tumors almost inevitably relapse owing to the development of acquired resistance. Resistance mechanisms include secondary ALK mutations and the activation of bypass pathways, such as cMET, cKIT, or EGFR, though some remain unknown. In this study, we analyzed alectinib-resistant patient samples and identified a significant increase in AXL expression in the tumor, and a high level of GAS6, the ligand for AXL, in the pleural effusion. AXL-overexpressing H3122 ALK-rearranged NSCLC cells exhibited partial resistance to alectinib, which was enhanced by GAS6 supplementation but could be overcome by the ALK/AXL inhibitor gilteritinib. Moreover, GAS6-overexpressing NIH3T3 cells and AXL-expressing H3122 cells were subcutaneously injected into the left and right sides of nude mice simultaneously, followed by alectinib treatment. The supply of GAS6 from NIH3T3 may have accelerated tumor relapse under alectinib treatment. However, even without GAS6-overexpressing NIH3T3, AXL-overexpressing H3122 tumor relapsed within 1 month possibly due to increased host mouse Gas6 expression. Single-cell RNA sequencing revealed that specific cancer-associated fibroblasts (CAFs) and a subset of tumor-associated macrophages (TAMs) are the primary sources of Gas6 in the tumor microenvironment (TME). During alectinib treatment, TAMs increased their infiltration into the TME, whereas CAFs altered their expression patterns, substantially upregulating Mmp11. These findings suggest that AXL expression in resistant cancer cells, combined with increased Gas6 production in the TME, contributes to enhanced ALK-TKI resistance.

The crosstalk between SND1 and PDCD4 is associated with chemoresistance of non-small cell lung carcinoma cells

Lung cancer is the leading cause of cancer-related deaths worldwide. Non-small cell lung cancer (NSCLC) is highly resistant to chemo- or radiation therapy, which poses a huge challenge for treatment of advanced NSCLC. Previously, we demonstrated the oncogenic role of Tudor Staphylococcal nuclease (TSN, also known as Staphylococcal nuclease domain-containing protein 1, SND1), in regulating chemoresistance in NSCLC cells. Here, we showed that silencing of SND1 augmented the sensitivity of NSCLC cells to different chemotherapeutic drugs. Additionally, the expression of PDCD4 (a tumor suppressor highly associated with lung cancer) in NSCLC cells with low endogenous levels was attenuated by SND1 silencing, implying that SND1 might function as a molecular regulator upstream of PDCD4. PDCD4 is differentially expressed in various NSCLC cells. In the NSCLC cells (A549 and H23 cells) with low expression of PDCD4, despite the downregulation of PDCD4, silencing of SND1 still led to sensitization of NSCLC cells to treatment with different chemotherapeutic agents by the inhibition of autophagic activity. Thus, a novel correlation interlinking SND1 and PDCD4 in the regulation of NSCLC cells concerning chemotherapy was revealed, which contributes to understanding the mechanisms of chemoresistance in NSCLC.

Monotropein represses the proliferation and angiogenesis via the AKT/NF-κB pathway in lung cancer.

Monotropein (Mon) is an iridoid glycosides extracted from Morinda officinalis F.C. How. (Rubiaceae) that shows anticancer effects on colorectal cancer. This study aimed to investigate the therapeutic effect of Mon on lung cancer. The viability, apoptosis, invasion, and tube formation of A549 and H1299 were determined by CCK8 assay, flow cytometry, transwell assay, and tube formation assay. NF-κB expression in the nucleus was detected by immunofluorescent staining. In vivo assay, BALB/c nude mice were divided into a sham group and a 2mg/kg Mon group. For the Mon group, mice were orally administered with 2mg/kg Mon. The duration of the animal study was 35days, and the tumor formation and angiogenesis were investigated. Compared with the control (Mon 0μM) group, Mon dramatically repressed the viability, invasion, tube formation, proliferation marker Ki67, N-cadherin, and VEGFA expressions in A549 cells (Mon of 25, 50, and 100µM) and H1299 cells (Mon of 50 and 100µM). Mon dramatically promoted cell apoptosis, cleaved caspase 3, and E-cadherin expressions. Besides, Mon remarkably downregulated p-AKT and p-NF-κB protein expressions. Moreover, AKT agonist SC79 reversed the anticancer effects of 100µM Mon on A549 cells. Furthermore, Mon markedly suppressed tumor growth, decreased N-cadherin, VEGFA, p-AKT, and p-NF-κB expressions, increased apoptosis and E-cadherin expression, and SC79 reversed the inhibition effects of Mon in vivo. Thus, Mon is a potential antitumor natural drug, and more signaling pathways involved in Mon-modulated lung cancer progression are worth testing for further investigation.

The off‑target effect of loratadine triggers autophagy‑mediated apoptosis in lung adenocarcinoma cells by deactivating JNK, p38, and STAT3 signaling through both PP2A‑dependent and independent pathways.

Lung adenocarcinoma (LUAD) is a typical inflammation‑associated cancer, and anti‑inflammatory medications can be valuable in cancer therapy. Loratadine, a histamine receptor H1 (HRH1) antagonist, shows both anti‑inflammatory and anticancer properties. The present study aimed to evaluate impacts of loratadine on LUAD cells as well as in a LUAD xenograft mouse model, and explore underlying mechanisms. Mechanistic investigations were conducted through using western blotting, flow cytometry, immunohistochemistry, acridine orange staining, TUNEL assays, and in silico analyses of loratadine‑modulated genes in LUAD specimens. It was observed that loratadine inhibited LUAD cell proliferation and colony formation by inducing autophagy‑mediated apoptotic cell death independently of HRH1. In a LUAD xenograft model, loratadine decreased tumor proliferation and angiogenesis while enhancing autophagy and apoptosis. Mechanistically, loratadine induced protein phosphatase 2A (PP2A) activation to deactivate c‑Jun N‑terminal kinase (JNK)1/2 and p38 in H23 and PC9 LUAD cells. Additionally, loratadine inhibited signal transducer and activator of transcription 3 (STAT3) activation via a PP2A‑independent pathway. Furthermore, the combination of loratadine with inhibitors for JNK, p38 and STAT3 all enhanced proliferation inhibition of loratadine alone in both cell lines. In the clinic, patients with LUAD expressing high PP2A had favorable prognoses. The present study suggests that loratadine can be used as a PP2A activator for LUAD treatment, and the combination of repurposing loratadine with inhibitors of STAT3, JNK and p38 would be an effectively strategy for inhibiting LUAD growth.

Utilizing TP53 hotspot mutations as effective predictors of gemcitabine treatment outcome in non-small-cell lung cancer

TP53 mutations are recognized to correlate with a worse prognosis in individuals with non-small cell lung cancer (NSCLC). There exists an immediate necessity to pinpoint selective treatment for patients carrying TP53 mutations. Potential drugs were identified by comparing drug sensitivity differences, represented by the half-maximal inhibitory concentration (IC50), between TP53 mutant and wild-type NSCLC cell lines using database analysis. In addition, clinical data from NSCLC patients were collected to evaluate both their TP53 status and their response to gemcitabine, thereby facilitating further validation. Subsequently, NSCLC cell lines with different TP53 status (A549 and H1299) were subjected to gemcitabine treatment to investigate the association between TP53 mutations and gemcitabine response. According to the dataset, NSCLC cell lines carrying TP53 mutations displayed heightened sensitivity to gemcitabine. From a clinical standpoint, patients exhibiting TP53 hotspot mutations demonstrated prolonged overall survival upon gemcitabine treatment. In vitro, overexpressing various hotspot TP53 mutations significantly sensitized H1299 cells to gemcitabine. Moreover, the knockdown of TP53 in A549 cells notably augmented sensitivity to gemcitabine treatment, as evidenced by cell viability and reproductive cell death assays. Conversely, the overexpression of wild-type TP53 in H1299 cells led to an increased resistance against gemcitabine. Gemcitabine is a treatment option for patients with non-small cell lung cancer (NSCLC) who carry TP53 hotspot mutations. This potential effectiveness might arise from its ability to disrupt DNA damage repair processes, leading to G2/M phase cell cycle arrest or an augmentation of mitotic abnormalities, eventually cause cell death. As a result, when planning treatment strategies for NSCLC patients possessing TP53 hotspot mutations, gemcitabine should be considered to incorporate into the indication.

Downregulation of AATK enhances susceptibility to ferroptosis by promoting endosome recycling in gefitinib-resistant lung cancer cells.

Ferroptosis has been characterised by disruption of the cell membrane through iron-related lipid peroxidation. However, regulation of iron homeostasis in lung cancer cells that are resistant to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) remains unclear. Transcriptome analysis identified a significant downregulation of apoptosis-associated tyrosine kinase (AATK) mRNA expression in gefitinib-resistant PC9 (PC9-GR) cells, which were found to be more susceptible to ferroptosis inducers. An in-depth analysis of publicly available datasets revealed that downregulation of AATK mRNA was associated with lymph node metastasis and poor prognosis in patients with lung adenocarcinoma. Knockdown of AATK-sensitised PC9, HCC827, and H441 cells to the ferroptosis inducer RSL3, whereas ectopic expression of AATK reduced RSL3-induced cell death in PC9-GR and HCC827-GR cells. Compared to PC9 cells, PC9-GR cells exhibited higher transferrin uptake, endosome recycling rate, and increased intracellular iron levels. Blocking iron transport reduced RSL3-induced ferroptosis in PC9-GR cells. Mechanistic studies showed that AATK localised to both early and recycling endosomes. Knockdown of AATK facilitated endosome recycling and elevated intracellular ferrous iron (Fe2+) levels in PC9 cells. Conversely, ectopic expression of AATK delayed endosome recycling and reduced intracellular Fe2+ levels in PC9-GR cells. Inhibition of AATK downregulation-induced iron accumulation decreased RSL3-induced ferroptosis. Taken together, our study indicates that the downregulation of AATK contributes to endosome recycling and iron accumulation, leading to an increased susceptibility to ferroptosis in EGFR-TKI-resistant lung cancer cells. © 2025 The Pathological Society of Great Britain and Ireland.

Abstract P017: Circulating tumor DNA kinetics identify prodynorphin signaling as a target to radiosensitize non-small cell lung cancer

Abstract Introduction: Definitive chemoradiation therapy (CRT) is essential for controlling locoregionally advanced non-small cell lung cancer (NSCLC); however, up to 30% of patients experience local recurrences within the radiation field. Analyzing favorable responders to treatment could enable identification of genetic alterations associated with radiosensitivity, but standard imaging cannot distinguish residual disease from inflammation and fibrosis during treatment to assess treatment response. We hypothesized that circulating tumor DNA (ctDNA) kinetics during CRT could identify genetic alterations associated with radiosensitivity that could be validated in preclinical models and harnessed to develop new combination therapies with radiotherapy (RT). Methods: We applied cancer personalized profiling by deep sequencing (CAPP-Seq) ctDNA analysis to pre-CRT and mid-treatment (mid-CRT) plasma samples from 61 patients with Stage II-III NSCLC. We identified ctDNA rapid responders as the 10 patients with the largest decrease in ctDNA concentration mid-CRT without local progression and determined the prevalence of genetic alterations in rapid responders versus slow responders using Fisher’s exact tests corrected for multiple hypothesis testing. To investigate the therapeutic potential of targeting PDYN during RT, we performed clonogenic assays on isogeneic PDYN CRISPR-Cas9 knockout and wildtype H1299 and SW1573 human NSCLC cell lines and H1299 cells treated with prodynorphin-neutralizing antibodies. Results: Mid-CRT log-fold change in ctDNA concentration was significantly associated with progression-free survival (P=0.02) in Stage II-III NSCLC. Mutations in PDYN, which encodes the opioid peptide precursor protein prodynorphin, were observed in 30% of ctDNA rapid responders and 0% of ctDNA slow responders (adjusted P=0.04). In NSCLC patients from TCGA treated with RT, the cumulative incidence of local failure was significantly lower in tumors with PDYN mutations (0% vs. 17% at 3 years, P<0.001). PDYN knockout (PDYNNULL) radiosensitized human NSCLC cancer cells by clonogenic assay, and extracellular administration of prodynorphin or its cleavage product dynophin A partially restored radioresistance in PDYNNULL cells. Prodynorphin cleavage products have been reported to signal through opioid receptors and to antagonize N-methyl-D-aspartate receptor (NMDAR) signaling. Although treatment of wildtype H1299 cells with opioid receptor antagonists did not affect radiosensitivity, the NMDAR antagonist MK-801 partially restored RT resistance in PDYNNULL cells. Finally, prodynorphin-neutralizing antibodies significantly reduced clonogenic survival of human NSCLC cells. Conclusions: ctDNA kinetics enable the identification of patients responding favorably to treatment and putative targets to enhance the efficacy of RT. Targeting prodynorphin may enhance the radiosensitivity of NSCLC by increasing NMDAR signaling. Citation Format: Ziwei Wang, Angela B. Hui, Ash A. Alizadeh, Maximilian Diehn, Everett J. Moding.Circulating tumor DNA kinetics identify prodynorphin signaling as a target to radiosensitize non-small cell lung cancer.[abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Targeted Therapies in Combination with Radiotherapy; 2025 Jan 26-29; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(2_Suppl):Abstract nr P017

Multi-SpinX: An advanced framework for automated tracking of mitotic spindles and kinetochores in multicellular environments.

SpinX, an AI-guided spindle tracking software, allows the 3-dimensional (3D) tracking of metaphase spindle movements in mammalian cells. Using over 900 images of dividing cells, we create the Multi-SpinX framework to significantly expand SpinX's applications: a) to track spindles and cell cortex in multicellular environments, b) to combine two object tracking (spindle with kinetochores marked by centromeric probes) and c) to extend spindle tracking beyond metaphase to prometaphase and anaphase stages where spindle morphology is different. We have used a human-in-the-loop approach to assess our optimisation steps, to manually identify challenges and to build a robust computational pipeline for segmenting kinetochore pairs and spindles. Spindles of both H1299 and RPE1 cells have been assessed and validated for use through Multi-SpinX, and we expect the tool to be versatile in enabling quantitative studies of mitotic subcellular dynamics.

Design, synthesis and antitumour activity of pyrimidine derivatives as novel selective EGFR kinase inhibitors.

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, often linked to overexpression or abnormal activation of the epidermal growth factor receptor (EGFR). The issue of developing resistance to third-generation EGFR kinase inhibitors, such as osimertinib, underscores the urgent need for new therapies to overcome this resistance. Our findings revealed that compound A8 exhibits 88.01% kinase inhibition efficacy against the EGFRL858R/T790M mutation at a concentration of 0.1 μM, with an IC50 value of 5.0 nM. Moreover, its selectivity for this double mutation is 29.5, surpassing that of osimertinib. Most notably, A8 demonstrates an inhibitory activity of 2.9 nM against the EGFRL858R/T790M/C797S triple mutation, outperforming the benchmark drug osimertinib. Furthermore, compound A8 has demonstrated strong antiproliferative effects against H1975 cells, and its activity was better than osimertinib. The mechanism by which compound A8 operates as a selective EGFRL858R/T790M inhibitor was confirmed through a series of cell migration, apoptosis, and cell cycle assays. This lays the foundation for the development of a new structural type of EGFR kinase inhibitors.

MicroRNA-150-3p enhances the antitumour effects of CGP57380 and is associated with a favourable prognosis in non-small cell lung cancer

MicroRNA (miRNA) dysregulation has been identified in several carcinomas, including non-small cell lung cancer (NSCLC), and is known to play a role in the development and progression of this disease. We initially conducted a miRNA microarray analysis, which revealed that the MNK inhibitor CGP57380 increased the expression of miR-150-3p. A similar analysis was performed using data from The Cancer Genome Atlas (TCGA). Cell proliferation, colony formation and migration assays were validated in A549 and H157 cells treated with miR-150-3p mimics. Quantitative polymerase chain reaction (qPCR) was then used to detect potential target genes. We observed significant downregulation of miR-150-3p in NSCLC samples compared with normal samples (P = 0.035). High miR-150-3p expression was associated with longer overall survival (P = 0.005), as determined via a tissue microarray (TMA). These results were validated in the TCGA and revealed that miR-150-3p was expressed at low levels in NSCLC tissues (P < 0.0001) and that patients with high miR-150-3p expression had a better prognosis (P = 0.042). Moreover, the combination of miR-150-3p and CGP57380 exerted a synergistic inhibitory effect on colony formation, growth, and migration and induced apoptosis in NSCLC cell lines. We investigated the potential targets of miR-150-3p and successfully validated six potential target genes through qPCR analysis. High miR-150-3p expression may enhance the response to immunotherapy, cisplatin and gemcitabine. In summary, this study underscores the promising therapeutic implications of combining miR-150-3p and CGP57380 for NSCLC treatment. Additionally, this study provides valuable insights into the molecular mechanisms underlying the effects of this treatment.

IL-17 triggers PD-L1 gene transcription in NSCLC cells via TRIM31-dependent MEF2C K63-linked polyubiquitination

BackgroundNon-small cell lung cancer (NSCLC) is a disease related to inflammation. Proinflammatory cytokines such as interleukin 17 (IL-17) can induce cancer cell proliferation, metastasis and immune escape. Although NSCLC immune escape is partly due to the interaction between PD-1 and PD-L1 and PD-L1 expression can be upregulated in cancer cells upon stimulation with IL-17, the underlying mechanism of IL-17-triggered PD-L1 gene transcription in NSCLC cells remains elusive.MethodsRT‒PCR, real-time PCR, and IB were used to assess the levels of PD-L1, MEF2C, and TRIM31 in NSCLC tissues as well as in IL-17–stimulated H1299 or PC9 cells. Bioinformatics analysis, luciferase assays, and ChIP were utilized to investigate the transcriptional mechanism of the PD-L1 gene. Co-IP/IB was used to examine the interaction between MEF2C and PD-L1, including MEF2C ubiquitination. IHC staining was carried out to analyse the expression of IL-17RA, MEF2C, TRIM31, and PD-L1 in NSCLC tissue arrays. The corresponding plasmids were constructed and identified. An isograft model was used to verify the findings in vitro.ResultsPD-L1, MEF2C and TRIM31 expression levels were increased in NSCLC tissues and NSCLC cells exposed to IL-17. Mechanistically, MEF2C could bind to the − 778 to -475 nt and − 336 to -97 nt regions of the PD-L1 promoter. TRIM31 could mediate MEF2C K63-linked polyubiquitination at Lys 25, increasing MEF2C recruitment to the PD-L1 promoter and PD-L1 gene transcription. MEF2C, TRIM31 or PD-L1 gene silencing effectively suppressed MEF2C K63-linked polyubiquitination, PD-L1 induction and NSCLC growth in mice inoculated with Lewis lung cancer (LLC) cells transfected with the corresponding shRNA and treated with IL-17.ConclusionIL-17 induces PD-L1 gene transcription in NSCLC cells through TRIM31-dependent MEF2C K63-linked polyubiquitination.

OCT4 promotes lung cancer progression through upregulation of VEGF-correlated chemokine-1.

Embryonic development and tumor genesis share numerous similarities, with OCT4 standing out as a pivotal transcription factor in embryonic development. Expression of OCT4 is associated with poor prognosis of lung adenocarcinoma. VEGF-correlated chemokine-1 (VCC-1), also known as C-X-C motif chemokine ligand 17 (CXCL17), has been suggested to play a role in promoting tumor angiogenesis and metastasis. In the present study, we show a positive correlation between OCT4 expression levels and tumor metastatic potential, where an increase in OCT4 expression parallels an upregulation of VCC-1 in lung cancer. This relationship was substantiated through DNA microarray analysis and further confirmed by tissue staining of clinical lung cancer samples, demonstrating a positive correlation between OCT4 and VCC-1 expression. In A549 and H1299 human lung cancer cells, modulations in OCT4 expression directly influenced VCC-1 levels, as evidenced by the reporter assay of the VCC-1 promoter, indicating the regulatory role of OCT4 in transactivating VCC-1 expression. Furthermore, enhanced VCC-1 expression in H1299 cells promoted transforming growth factor-β (TGF-β) secretion, contributing to lung cancer cell aggressiveness. Additionally, VCC-1 secretion by H1299 cells could attract THP-1 macrophages, further implicating its role in tumor progression. NOD/SCID mice inoculated with VCC-1-knockdown A549 lung cancer cells exhibited significantly smaller tumors than those inoculated with control cells. On the basis of these findings, we highlight the importance of the OCT4-VCC-1 axis in lung cancer progression. Our findings also provide therapeutic targets for lung cancer.

Cancer-associated fibroblasts induce almonertinib resistance in non-small cell lung cancer

BackgroundAlmonertinib is the initial third-generation EGFR-TKI in China, but its resistance mechanism is unknown. Cancer-associated fibroblasts (CAFs) are essential matrix components in the tumor microenvironment, but their impact on almonertinib resistance is unknown. This study aimed to explore the correlation between CAFs and almonertinib resistance in non-small cell lung cancer (NSCLC).MethodsThe anti-cancer effects of almonertinib on NSCLC cells, as well as the reversal of these effects mediated by CAFs, were validated through phenotypic experiments. Differential gene expression analysis, along with GO and KEGG enrichment analyses, was performed to predict the potential mechanisms underlying resistance to third-generation EGFR-TKIs. Finally, qPCR and Western blot analyses were used to explore the signaling pathways by which CAFs induce resistance to almonertinib in NSCLC cells.ResultsOur findings revealed that almonertinib significantly suppressed the invasion, migration, and proliferation of EGFR T790M-mutant NSCLC cells. TGF-β1 successfully induced the differentiation of CAFs and upregulated the expression of CAF markers, including α-SMA and fibroblast activation protein (FAP). Exposure of H1975 cells to almonertinib increased TGF-β1 secretion. Additionally, CAFs enhanced the survival of almonertinib-treated NSCLC cells, whereas normal fibroblasts (NFs) exerted the opposite effect. qPCR analysis demonstrated that the expression of the core molecules of the Hippo pathway, YAP and TAZ, was lower in A549 cells than in H1975 cells, and CAF intervention further reduced YAP/TAZ expression in H1975 cells. Western blot analysis confirmed a significant reduction in YAP/TAZ protein levels in cancer cells treated with CAF-conditioned medium (CAF-CM) compared to those treated with normal control-conditioned medium (NC-CM). Finally, we demonstrated that CAFs induced resistance to almonertinib in NSCLC cells, potentially through a mechanism involving YAP/TAZ.ConclusionThis study demonstrated that H1975 cells stimulated by almonertinib promoted the accumulation of CAFs in NSCLC cells, likely through increased secretion of TGF-β1. The accumulation of CAFs enhanced the survival of NSCLC cells undergoing almonertinib treatment and induced drug resistance. Additionally, the mechanism underlying CAF-induced drug resistance in NSCLC cells was potentially linked to the activation of the YAP/TAZ signaling pathway.

Endoglin as a predictive biomarker for pemetrexed sensitivity in non-small-cell lung cancer: a cellular study.

Based on our previous research, which demonstrated that elevated plasma endoglin (ENG) levels in lung cancer patients were associated with a better prognosis, increased sensitivity to pemetrexed, and enhanced tumor suppression, this study aims to validate these findings at the cellular level. The focus is on membrane and extracellular ENG and their influence on drug response and tumor cell behavior in non-small cell lung cancer (NSCLC) cells. The correlation between ENG expression and pemetrexed-induced cytotoxicity in eight human non-squamous subtype NSCLC cell lines was analyzed. ENG in A549 and H1975 cells was knocked down using shRNA. MTT assay, cell cycle assay, western blot analysis, and boyden chamber assay were used to detect the effect of ENG on pemetrexed-induced cytotoxicity, cell cycle distribution, and cell migration. The expression of membrane ENG was positively correlated with pemetrexed-induced cytotoxicity in human NSCLC cells. Compared to pemetrexed-sensitive A549 cells, the A549/a400 (pemetrexed-resistant subline) cells exhibited a reduced accumulation of cells in the S phase, making them less susceptible to cell death. ENG knockdown also alleviated pemetrexed-induced S phase arrest and regulated G1/S phase-related proteins (p53, p21, CDK2, and Cyclin A). Additionally, co-treatment with recombinant ENG enhanced pemetrexed-induced migration inhibition in the sensitive cel1 line and cytotoxicity in the resistance cell line. The present results strengthened our prior clinical findings, showing that higher membrane ENG expression enhances pemetrexed-induced cytotoxicity and S phase arrest, which may involve the ENG-p21 pathway. Additionally, microenvironmental ENG enhanced the anti-migration of pemetrexed. These findings highlight the potential of ENG as a biomarker and therapeutic target, opening new avenues to improve the outcomes of non-squamous cell NSCLC treatment.

Molecular mechanisms of transcription factor KLF4-mediated immune infiltration influencing lung adenocarcinoma invasion.

Lung adenocarcinoma (LUAD) is associated with an increasing incidence and mortality rate while existing treatment strategies continue to exhibit considerable limitation. Studies have demonstrated that upregulation of KLF4 gene inhibits LUAD progression, but its underlying mechanisms remain elusive. The present research explored roles and mechanisms of KLF4 and the NF-κB pathway in LUAD. Lentiviral vectors encoding KLF4 were constructed and transduced into H1299 and A549 cells to generate stable cell lines. These stable cell lines were then injected into BALB/c mice to establish a LUAD model. Subsequently, RNA sequencing, HE staining, immunohistochemistry, ELISA, Western blotting, and flow cytometry were employed to investigate the effects of KLF4 on tumor growth, invasion, immune cell infiltration, and related signaling pathways. Finally, dual-luciferase and in vivo mouse experiments were conducted to validate the molecular mechanisms. KLF4 significantly reduced tumor cell invasion while promoted tumor cell necrosis. Transcriptomic sequencing identified CXCR2 as a target gene and the NF-κB signaling pathway associated with immune infiltration regulation. KLF4 downregulated NF-κB2 and CXCR2 expression, concomitantly decreasing tumor cell invasiveness but increasing levels of CD4+ and CD8+ T cells and macrophages. NF-κB and CXCR2 play an important role in KLF4-mediated immune infiltration, thereby inhibiting tumor invasion and promoting tumor cell apoptosis in mice.

Exploring the Anticancer Potential of MonoHER (7-Mono-O-(β-Hydroxyethyl)-Rutoside): Mitochondrial-Dependent Apoptosis in HepG2 Cells.

Flavonoids are a group of polyphenols, abundantly present in our diet. Although, based on their chemoprotective effects, intake of flavonoids is associated with a high anticancer potential as evidenced in in vitro and in vivo models, the molecular mechanism is still elusive. This study explores the antiproliferative and cytotoxic effects of the semi-synthetic flavonoid MonoHER (7-mono-O-(β-hydroxyethyl)-rutoside) in vitro on cancer cells. HepG2 liver, MCF7 breast, and H1299 lung cancer cells were grown under ambient conditions with or without MonoHER exposure. CCK8 assay was used to assess cell viability. Apoptosis, JC-1, and mitochondrial mass were determined using flow cytometry and confocal analysis. The effects of monoHER on apoptosis proteins were detected by confocal microscopy analysis and Western blot. It was found that MonoHER can reduce HepG2 cells' and MCF7 cells' viability, but not H1299 cells', and induced apoptosis only in HepG2 cells. MonoHER has the potential to enhance the expression of caspase-9 and caspase-3, to damage mitochondria, and to provoke the release of cytochrome C from the mitochondria. MonoHER can inhibit cell growth and induce apoptosis especially in HepG2 human liver cancer cells by triggering the mitochondrial signal transduction pathway, leading to the release of cytochrome C in the cytoplasm and the subsequent activation of caspase-9 and caspase-3. Future research should further explore MonoHER's mechanism of action, efficacy, and potential for clinical translation.

Transcriptome analysis reveals significant discrepancies between two in vitro models of host-trematode interaction.

Cell models emulating an in vitro parasitic infection can greatly improve our understanding of helminthiases. Nonetheless, it remains challenging to select an appropriate in vitro model to study molecular pathogenesis of infections by trematodes having a complex life cycle. Therefore, adequate models are in high demand. The epidemiologically important foodborne trematode Opisthorchis felineus parasitizes bile ducts of fish-eating mammals, including humans. The human infection leads to chronic inflammation and biliary intraepithelial neoplasia, which is considered precancerous. This study was aimed at evaluating two useful in vitro research tools based on human cholangiocytes' (H69 cells') response to the trematode: coculture with live worms or incubation with parasite-derived excretory-secretory products (ESPs). We assessed H69 cells' proliferation, migration rate, cell cycle shift, and cytokine production. We also conducted genome-wide transcriptome analysis to identify affected cascades of regulatory signaling events. We demonstrated significant discrepancies between the two in vitro models of host-parasite interactions. Although differences between the two models in cell proliferation and cell migration rate were weak, there were substantial differences in the production and release of cytokines IL-6, IL-4, and TNF. A total of 144 genes in H69 cells were found to be differentially expressed after coculture with live worms, whereas 537 genes were differentially expressed after exposure to ESPs. Transcriptomic analysis revealed only 11 common upregulated genes and six common downregulated genes. Functional enrichment analysis of the gene sets also revealed some striking differences between the in vitro models. Our data will contribute to a deeper understanding of biliary neoplasia associated with liver fluke infection. This study underscores the importance of choosing an appropriate in vitro model to accurately emulate host-parasite interactions. The data also highlight the need for further investigation into the pathogenesis of the precancerous biliary lesions associated with liver fluke infection.

Revealing 5-(3,5-difluorobenzyl)-1H-indazole as the active pharmacophore of ALK/ROS1 dual inhibitors through theoretical calculations and biological activity evaluations

Anaplastic lymphoma kinase (ALK) and tyrosine protein kinase (ROS1) are recognized as driver genes in lung cancer, with dual inhibition of both targets offering a promising approach to enhance therapeutic outcomes in non-small cell lung cancer (NSCLC). Although numerous ALK/ROS1 inhibitors have received FDA approval, detailed research into the essential active structural motifs within these inhibitors remains limited. Addressing this gap, the current study employed computer-aided drug design (CADD) methodologies, incorporating bioisosteric and conformational similarity principles to design and synthesize 31 dual-target 2-morpholinobenzamide derivatives. These derivatives each include the 5-(3,5-difluorobenzyl)-1H-indazole, 4-benzylmorpholine, and thiophene moieties. Based on docking binding energies, we proposed that 5-(3,5-difluorobenzyl)-1H-indazole may represent a key pharmacophore for ALK/ROS1 activity. Subsequent kinase and cellular assays validated this hypothesis, with compound X4 exhibiting optimal inhibitory activity against both ALK and ROS1 kinases and lung cancer cell lines, achieving IC50 values of 0.512 µM (ALK), 0.766 µM (ROS1), and 0.034 ± 0.002 µM (H2228). In vitro antitumor assays demonstrated dose-dependent induction of apoptosis in H2228 cells by X4. Western blot (WB) analysis further confirmed that X4 effectively suppresses the expression of p-ALK and p-ERK. Importantly, X4 exhibited high specificity in targeting ALK and ROS1 across a range of kinases. In an H2228 xenograft model, X4 achieved a tumor inhibition rate of 54.71 %, underscoring its considerable potential in ALK/ROS1 inhibition.

Characterization and evaluation of the cytotoxic, antioxidant, and anti-human lung cancer properties of copper nanoparticles green-synthesized by fennel extract following the PI3K/AKT/Mtor signaling pathway.

This work established the cytotoxic, antioxidant and anticancer effects of copper nanoparticles (CuNPs) manufactured with fennel extract, especially on non-small cell lung cancer (NSCLC) as well. CuNPs caused cytotoxicity in a dose-dependent manner for two NSCLC cell lines, A549 and H1650. At 100 μg/ml, CuNPs reduced cell viability to 70% in A549 cells and 65% in H1650 cells. which showed a cytotoxic effect (p<0. 05). Lactate dehydrogenase (LDH) was correspondingly present in a high proportion in the cells, demonstrated upon testing. Together with their cytotoxic properties, CuNPs demonstrated high antioxidative activity. When the concentration of the nano particles was high (100 μg/ml), the ratio of reactive oxygen species (ROS) was reduced as much as 50%, which in turn suggested antioxidant activity. There was plenty of evidence that CuNPs had anti-cancer potential; this has been shown by the effect of the molecules on the PI3K/AKT/mTOR pathway, which was one of the pathways crucial for cancer survival. Western blot analysis and qRT-PCR results indicated a widespread degradation of the proteins in this pathway upon CuNP exposure. Interestingly, there was a declined phosphorylation up to 75% of PI3K, AKT, and mTOR at 100 μg/ml (p<0. 001). In summary, these findings illustrated the mechanisms behind the therapeutic effect of CuNPs, thus making them good targets for the NSCLC treatment. CuNPs have cytotoxic and antioxidant capacity, as well as significant alterations in lung cancers pathway, and therefore they can be considered as anti-cancer candidates.

Drimane-type sesquiterpenoids and triterpenoids from the whole plant of Limonium sinense with their antiproliferative and anti-inflammatory activities.

Saline-tolerant medicinal plants possess novel chemical constituents with high bioactivity because of their unique secondary metabolic pathways. Limonium sinense, an aquatic plant found in the coastal wetlands of the Yellow River Delta, was collected and studied in the present work. Ten drimane-type sesquiterpenoids and four triterpenoids, including six new ones (sinenseines A-F), were isolated from a whole plant of L. sinense for the first time. Their structures, including the absolute configurations, were determined by analyzing the comprehensive spectroscopic data. In addition, twelve terpenoids, including nine sesquiterpenoids, were identified using UPLC-MS/MS and GNPS methods. All isolates were evaluated for their antiproliferative and anti-inflammatory activities. Compounds 2-4, 6, 13, and 14 showed moderate anti-tumor effects on A549, H1299, HepG2 and A2780 cells with IC50 values ranging from 35.2 ± 2.0 to 90.5 ± 3.1 μM. Furthermore, compound 1 exhibited significant anti-inflammatory activity with an IC50 value of 8.3 ± 1.2 μM against NO production in LPS-induced RAW 264.7 macrophages.