Circ_0001786 facilitates gefitinib resistance and malignant progression in non-small cell lung cancer via miR-34b-5p/SRSF1.

BACKGROUND: Non-small cell lung cancer (NSCLC) is a widespread cancer and gefitinib is a primary therapy for NSCLC patients. Nevertheless, the underlying mechanisms for the progression of acquired drug resistance have not been clarified. The aim of this study was to investigate the role of circular RNA (circ_0001786) in gefitinib-resistant NSCLC. METHODS: Firstly, the expression of circ_0001786, miR-34b-5p and SRSF1 were assayed using qRT-PCR. Subsequently, CCK-8 test was utilized to measure the semi-inhibitory concentration (IC50) of cellular gefitinib. Apoptosis was identified by flow cytometry. At last, dual luciferase assay was applied to prove the binding association between miR-34b-5p, circ_0001786 or SRSF1. RESULTS: Our research disclosed that circ_0001786 was heightened in gefitinib-resistant NSCLC cells and tissues. Knockdown of circ_0001786 restrained IC50 values of gefitinib, attenuated the clonogenic ability and facilitated apoptosis in HCC827-GR and PC9-GR. In addition, circ_0001786 was a molecular sponge for miR-34b-5p. Silencing miR-34b-5p rescued the inhibitory impact of circ_0001786 knockdown on IC50 and cell cloning ability. Moreover, miR-34b-5p directly targeted SRSF1. Importantly, circ_0001786 enhanced gefitinib tolerance and malignant development in NSCLC through miR-34b-5p/SRSF1 pathway. CONCLUSION: This research revealed a novel mechanism by which circ_0001786 enhanced NSCLC resistance to gefitinib by sponging miR-34b-5p and upregulating SRSF1. circ_0001786 was a potential target for improving the treatment of gefitinib-resistant NSCLC patients.

Activation of epidermal growth factor receptors in triple-negative breast cancer cells by morphine; analysis through Raman spectroscopy and machine learning.

Triple negative breast cancer (TNBC) is a very aggressive form of breast cancer, and the analgesic drug morphine has been shown to promote the proliferation of TNBC cells. This article investigates whether morphine causes activation of epidermal growth factor receptors (EGFR), the roles of µ-opioid and EGFR receptors on TNBC cell proliferation and migration. While examining the changes with molecular techniques, we also aimed to investigate the analysis ability of Raman spectroscopy and machine learning-based approach. Effects of morphine on the proliferation and migration of MDA.MB.231 cells were evaluated by MTT and scratch wound-healing tests, respectively. Morphine-induced phosphorylation of the EGFR was analyzed by western blotting in the presence and absence of µ-receptor antagonist naltrexone and the EGFR-tyrosine kinase inhibitor gefitinib. Morphine-induced EGFR phosphorylation and cell migration were significantly inhibited by pretreatments with both naltrexone and gefitinib; however, morphine-increased cell proliferation was inhibited only by naltrexone. While morphine-induced changes were observed in the Raman scatterings of the cells, the inhibitory effect of naltrexone was analyzed with similarity to the control group. Principal component analysis (PCA) of the Raman confirmed the epidermal growth factor (EGF)-like effect of morphine and was inhibited by naltrexone and partly by gefitinib pretreatments. Our in vitro results suggest that combining morphine with an EGFR inhibitor or a peripherally acting opioidergic receptor antagonist may be a good strategy for pain relief without triggering cancer proliferation and migration in TNBC patients. In addition, our results demonstrated the feasibility of the Raman spectroscopy and machine learning-based approach as an effective method to investigate the effects of agents in cancer cells without the need for complex and time-consuming sample preparation. The support vector machine (SVM) with linear kernel automatically classified the effects of drugs on cancer cells with ∼95% accuracy.

Synergistic anti-tumor effects of lenalidomide and gefitinib by upregulating ADRB2 and inactivating the mTOR/PI3K/AKT signaling pathway in lung adenocarcinoma.

Gefitinib is commonly used to be the first-line therapy for advanced non-small cell lung cancer (NSCLC). Therapeutic effect of gefitinib is reduced due to acquired resistance, and combined treatment is recommended. In this research, we planned to explore the impacts of combined treatment of lenalidomide and gefitinib on gefitinib-sensitive or -resistant NSCLC cells. The co-treatment results demonstrated that enhanced antitumor impact on NSCLC cell growth, migration, invasion, cell cycle process and apoptosis. The tumor-bearing mouse models were established using PC9/GR cells. In vivo assays also showed that lenalidomide and gefitinib synergistically inhibited mouse tumor growth along increased the survival of mice. ADRB2 was identified as a lowly expressed gene in PC9/GR cells and LUAD tumor tissues. LUAD patients with high ADRB2 expression were indicated with favorable survival outcomes. Moreover, ADRB2 was upregulated in lenalidomide and/or gefitinib-treated PC9/GR cells. ADRB2 deficiency partially offsets the suppressive impacts of lenalidomide and gefitinib co-treatment on the viability and proliferation of PC9/GR cells. Additionally, lenalidomide and gefitinib cotreatment significantly inactivated the mTOR/PI3K/AKT signaling pathway compared with each treatment alone. Rescue assays were performed to explore whether lenalidomide and gefitinib synergistically inhibited the growth of PC9/GR cells via the PI3K/AKT pathway. PI3K activator SC79 significantly restored reduced cell proliferation, migration and invasion along with elevated cell cycle arrest and apoptosis caused by lenalidomide and gefitinib cotreatment. In conclusion, lenalidomide and gefitinib synergistically suppressed LUAD progression and attenuated gefitinib resistance by upregulating ADRB2 and inactivating the mTOR/PI3K/AKT signaling pathway in lung adenocarcinoma.

YES1 as a potential target to overcome drug resistance in EGFR-deregulated non-small cell lung cancer.

Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) such as gefitinib and osimertinib have primarily been used as first-line treatments for patients with EGFR-activating mutations in non-small cell lung cancer (NSCLC). Novel biomarkers are required to distinguish patients with lung cancer who are resistant to EGFR-TKIs. The aim of the study is to investigate the expression and functional role of YES1, one of the Src-family kinases, in EGFR-TKI-resistant NSCLC. YES1 expression was elevated in gefitinib-resistant HCC827 (HCC827/GR) cells, harboring EGFR mutations. Moreover, HCC827/GR cells exhibited increased reactive oxygen species (ROS) levels compared to those of the parent cells, resulting in the phosphorylation/activation of YES1 due to oxidation of the cysteine residue. HCC827/GR cells showed elevated expression levels of YES1-associated protein 1 (YAP1), NF-E2-related factor 2 (Nrf2), cancer stemness-related markers, and antioxidant proteins compared to those of the parent cells. Knockdown of YES1 in HCC827/GR cells suppressed YAP1 phosphorylation, leading to the inhibition of Bcl-2, Bcl-xL, and Cyclin D1 expression. Silencing YES1 markedly attenuated the proliferation, migration, and tumorigenicity of HCC827/GR cells. Dasatinib inhibited the proliferation of HCC827/GR cells by targeting YES1-mediated signaling pathways. Furthermore, the combination of gefitinib and dasatinib demonstrated a synergistic effect in suppressing the proliferation of HCC827/GR cells. Notably, YES1- and Nrf2-regulated genes showed a positive regulatory relationship in patients with lung cancer and in TKI-resistant NSCLC cell lines. Taken together, these findings suggest that modulation of YES1 expression and activity may be an attractive therapeutic strategy for the treatment of drug-resistant NSCLC.

Chidamide improves gefitinib treatment outcomes in NSCLC by attenuating recruitment and immunosuppressive function of myeloid-derived suppressor cells.

Clinical resistance to EGFR tyrosine kinase inhibitors in non-small-cell lung cancer (NSCLC) remains a significant challenge. Recent studies have indicated that the number of myeloid-derived suppressor cells (MDSCs) increases following gefitinib treatment, correlating with a poor patient response in NSCLC. Our study revealed that gefitinib treatment stimulates the production of CCL2, which subsequently enhances monocyte (M)-MDSC migration to tumor sites. Chidamide, a selective inhibitor of the histone deacetylase subtype, counteracted the gefitinib-induced increase in CCL2 levels in tumor cells. Additionally, chidamide down-regulated the expression of CCR2 in M-MDSCs, inhibiting their migration. Furthermore, chidamide attenuated the immunosuppressive function of M-MDSCs both alone and in combination with gefitinib. Chidamide also alleviated tumor immunosuppression by reducing the number of M-MDSCs in LLC-bearing mice, thereby enhancing the antitumor efficacy of gefitinib. In conclusion, our findings suggest that chidamide can improve gefitinib treatment outcomes, indicating that MDSCs are promising targets in NSCLC.

Gefitinib induces anoikis in cervical cancer cells.

Gefitinib exerts anticancer effects on various types of cancer, such as lung, ovarian, breast, and colon cancers. However, the therapeutic effects of gefitinib on cervical cancer and the underlying mechanisms remain unclear. Thus, this study aimed to explore whether gefitinib can be used to treat cervical cancer and elucidate the underlying mechanisms. Results showed that gefitinib induced a caspase-dependent apoptosis of HeLa cells, which consequently became round and detached from the surface of the culture plate. Gefitinib induced the reorganization of actin cytoskeleton and downregulated the expression of p-FAK, integrin ß1 and E-cadherin, which are important in cell-extracellular matrix adhesion and cell-cell interaction, respectively. Moreover, gefitinib hindered cell reattachment and spreading and suppressed interactions between detached cells in suspension, leading to poly (ADP-ribose) polymerase cleavage, a hallmark of apoptosis. It also induced detachment-induced apoptosis (anoikis) in C33A cells, another cervical cancer cell line. Taken together, these results suggest that gefitinib triggers anoikis in cervical cancer cells. Our findings may serve as a basis for broadening the range of anticancer drugs used to treat cervical cancer. [BMB Reports 2024; 57(2): 104-109].

[Mechanism of n-butanol fraction of Wenxia Formula combining with gefitinib in treating non-small cell lung cancer based on network pharmacology and in vitro experiment].

This study combined network pharmacology, molecular docking, and in vitro experiments to explore the potential mechanism of the active components of the n-butanol fraction of Wenxia Formula(NWXF) combined with gefitinib(GEF) in treating non-small cell lung cancer(NSCLC). Ultra-performance liquid chromatography-quadrupole Orbitrap mass spectrometry(UPLC-Q-Orbitrap MS) was employed to detect the main chemical components of NWXF. The active components of NWXF were retrieved from SwissADME, and the candidate targets of these active components were retrieved from SwissTargetPrediction. Online Mendelian Inheritance in Man(OMIM) and GeneCards were searched for the targets of NSCLC. Cytoscape 3.9.0 and STRING were employed to build the protein-protein interaction(PPI) network with the common targets shared by NWXF and NSCLC. Gene Ontology(GO) annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment were performed in DAVID to predict the potential mechanisms. Finally, molecular docking between the main active ingredients and key targets was conducted in SYBYL-X 2.0. The methyl thiazolyl tetrazolium(MTT) assay was employed to evaluate the inhibitory effects of NWXF and/or GEF on the proliferation of human non-small cell lung cancer cells(A549 and PC-9). Additionally, the impact of NWXF on human embryonic lung fibroblast cells(MRC-5) was assessed. The effectiveness of the drug combination was evaluated based on the Q value. The terminal-deoxynucleoitidyl transferase mediated nick-end labeling(TUNEL) assay was employed to examine the apoptosis of A549 and PC-9 cells treated with NWXF and/or GEF. Quantitative real-time PCR(qRT-PCR) was employed to measure the mRNA levels of epidermal growth factor receptor(EGFR), c-Jun N-terminal kinase(JNK), and Bcl2-associated X protein(Bax) in the A549 and PC-9 cells treated with NWXF and/or GEF. Western blot was employed to determine the protein levels of EGFR, p-EGFR, JNK, p-JNK, and Bax in the A549 and PC-9 cells treated with NWXF and/or GEF. A total of 77 active components, 488 potential targets, and 49 key targets involved in the treatment of NSCLC with NWXF were predicted. The results of GO annotation showed that NWXF may treat NSCLC by regulating the biological processes such as cell proliferation, apoptosis, and protein phosphorylation. KEGG enrichment revealed that the key targets of NWXF in treating NSCLC were enriched in the mitogen-activated protein kinase(MAPK), phosphatidylinositol 3-kinase(PI3K)-protein kinase B(AKT), hypoxia-inducible factor-1(HIF-1), and microRNA-related signaling pathways. Molecular docking results showed that 91.9% of the docking scores were greater than 5, indicating the strong binding capability between main active components and key targets. The cell experiments demonstrated that NWXF combined with GEF synergistically inhibited the proliferation, promoted the apoptosis, decreased p-EGFR/EGFR and p-JNK/JNK values, down-regulated the mRNA levels of EGFR and JNK, and up-regulated the mRNA and protein levels of Bax in A549 and PC-9 cells. In conclusion, NWXF combined with GEF can regulate the EGFR/JNK pathway to promote the apoptosis of NSCLC cells, thus treating NSCLC.

[Combination of shikonin and gefitinib reverses drug resistance in human non-small cell lung cancer and its mechanism].

The occurrence and development of tumors are associated with the cell energy metabolism. Inhibiting energy metabolism of lung cancer cells is an important strategy to overcome drug resistance. Based on the cellular energy metabolism pathway, this study observed the effect of combination of shikonin(SKN) and gefitinib(GFB) on the drug resistance in non-small cell lung cancer and explored the underlying mechanism. The human non-small cell lung cancer line HCC827/GR resistant to gefitinib was used as the cell model in vitro. The CCK-8 assay and flow cytometry were employed to investigate the cell viability and apoptosis, respectively. The high performance liquid chromatography was employed to measure the intracellular accumulation of GFB. A Seahorse XFe96 Analyzer was used to detect the changes of cellular energy metabolism. Western blot was employed to determine the expression of the proteins involved in the drug resistance. The tumor-bearing nude mouse model was used to verify the efficacy of SKN+GFB in overcoming drug resistance in vivo. The results showed that SKN+GFB significantly reduced the IC_(50) of GFB on HCC827/GR cells, with the combination index of 0.628, indicating that the combination of the two drugs had a synergistic effect and promoted cell apoptosis. SKN increased the intracellular accumulation of GFB. SKN+GFB lowered the oxygen consumption rate(OCR) and glycolytic proton efflux rate(GlycoPER) in cell energy metabolism, and down-regulated the overexpression of PKM2, p-EGFR, P-gp, and HIF-1α in drug resistance. The results of reversing drug resistance test in vivo showed that GFB or SKN alone had no significant antitumor effect, while the combination at different doses induced the apoptosis of the tumor tissue and inhibited the expression of PKM2 and P-gp, demonstrating a significant antitumor effect. Moreover, the tumor inhibition rate in the high-dose combination group reached 64.01%. In summary, SKN+GFB may interfere with the energy metabolism to limit the function of HCC827/GR cells, thus reversing the GFB resistance in non-small cell lung cancer.

Nanoplasmonic Detection of EGFR Mutations Based on Extracellular Vesicle-Derived EGFR-Drug Interaction.

Analysis of membrane proteins from extracellular vesicles (EVs) has emerged as an important strategy for molecular cancer diagnosis. The epidermal growth factor receptor (EGFR) is one of the most well-known oncogenic membrane proteins, particularly in non-small cell lung cancer (NSCLC), where targeted therapies using tyrosine kinase inhibitors (TKIs) are often addressed based on EGFR mutation status. Consequently, several studies aimed at analyzing oncogenic membrane proteins have been proposed for cancer diagnosis. However, conventional protein analysis still faces limitations due to the requirement for large sample quantities and extensive post-labeling processes. Here, we develop a nanoplasmonic detection method for EGFR mutations in the diagnosis of NSCLC based on interactions between EGFR loaded in EVs and TKI. Gefitinib is selected as a model TKI due to its strong signals in the surface-enhanced Raman spectroscopy (SERS) and mutation-dependent binding affinity to EGFR. We demonstrate an SERS signal attributed to gefitinib at a higher value in the EGFR exon 19 deletion, both in cells and EVs, compared to wild-type and exon 19 deletion/T790M variants. Furthermore, we observe a significantly higher gefitinib SERS signal in EGFR obtained from exon 19 deletion NSCLC patient plasma-derived EVs compared with those from wild-type and exon 19 deletion/T790M EVs. Since our approach utilizes an analysis of the SERS signal generated by the interaction between oncogenic membrane proteins within EVs and targeted drugs, its diagnostic applicability could potentially extend to other liquid biopsy methods based on EVs.

Leucine Zipper Downregulated in Cancer-1 Interacts with Clathrin Adaptors to Control Epidermal Growth Factor Receptor (EGFR) Internalization and Gefitinib Response in EGFR-Mutated Non-Small Cell Lung Cancer.

The epidermal growth factor receptor (EGFR) is a common driver of non-small cell lung cancer (NSCLC). Clathrin-mediated internalization (CMI) sustains EGFR signaling. AXL is associated with resistance to EGFR-tyrosine kinase inhibitors (TKIs) in EGFR-mutated (EGFRM) NSCLC. We investigated the effects of Leucine zipper downregulated in cancer-1 (LDOC1) on EGFR CMI and NSCLC treatment. Coimmunoprecipitation, double immunofluorescence staining, confocal microscopy analysis, cell surface labelling assays, and immunohistochemistry studies were conducted. We revealed that LDOC1 interacts with clathrin adaptors through binding motifs. LDOC1 depletion promotes internalization and plasma membrane recycling of EGFR in EGFRM NSCLC PC9 and HCC827 cells. Membranous and cytoplasmic EGFR decreased and increased, respectively, in LDOC1 (-) NSCLC tumors. LDOC1 depletion enhanced and sustained activation of EGFR, AXL, and HER2 and enhanced activation of HER3 in PC9 and HCC827 cells. Sensitivity to first-generation EGFR-TKIs (gefitinib and erlotinib) was significantly reduced in LDOC1-depleted PC9 and HCC827 cells. Moreover, LDOC1 downregulation was significantly associated (p < 0.001) with poor overall survival in patients with EGFRM NSCLC receiving gefitinib (n = 100). In conclusion, LDOC1 may regulate the efficacy of first-generation EGFR-TKIs by participating in the CMI of EGFR. Accordingly, LDOC1 may function as a prognostic biomarker for EGFRM NSCLC.

Synthesis and In Vitro Antitumor Activity Evaluation of Gefitinib-1,2,3-Triazole Derivatives.

In this study, 14 structurally novel gefitinib-1,2,3-triazole derivatives were synthesized using a click chemistry approach and characterized by 1H NMR, 13C NMR and high-resolution mass spectrometry (HRMS). Preliminary cell counting kit-8 results showed that most of the compounds exhibit excellent antitumor activity against epidermal growth factor receptor wild-type lung cancer cells NCI-H1299, A549 and NCI-H1437. Among them, 4b and 4c showed the most prominent inhibitory effects. The half maximal inhibitory concentration (IC50) values of 4b were 4.42 ± 0.24 µM (NCI-H1299), 3.94 ± 0.01 µM (A549) and 1.56 ± 0.06 µM (NCI-1437). The IC50 values of 4c were 4.60 ± 0.18 µM (NCI-H1299), 4.00 ± 0.08 µM (A549) and 3.51 ± 0.05 µM (NCI-H1437). Furthermore, our results showed that 4b and 4c could effectively inhibit proliferation, colony formation and cell migration in a concentration-dependent manner, as well as induce apoptosis in H1299 cells. In addition, 4b and 4c exerted its anti-tumor effects by inducing cell apoptosis, upregulating the expression of cleaved-caspase 3 and cleaved-PARP and downregulating the protein levels of Bcl-2. Based on these results, it is suggested that 4b and 4c be developed as potential new drugs for lung cancer treatment.

ELK1/MTOR/S6K1 Pathway Contributes to Acquired Resistance to Gefitinib in Non-Small Cell Lung Cancer.

The development of acquired resistance to small molecule tyrosine kinase inhibitors (TKIs) targeting epidermal growth factor receptor (EGFR) signaling has hindered their efficacy in treating non-small cell lung cancer (NSCLC) patients. Our previous study showed that constitutive activation of the 70 kDa ribosomal protein S6 kinase 1 (S6K1) contributes to the acquired resistance to EGFR-TKIs in NSCLC cell lines and xenograft tumors in nude mice. However, the regulatory mechanisms underlying S6K1 constitutive activation in TKI-resistant cancer cells have not yet been explored. In this study, we recapitulated this finding by taking advantage of a gefitinib-resistant patient-derived xenograft (PDX) model established through a number of passages in mice treated with increasing doses of gefitinib. The dissociated primary cells from the resistant PDX tumors (PDX-R) displayed higher levels of phosphor-S6K1 expression and were resistant to gefitinib compared to cells from passage-matched parental PDX tumors (PDX-P). Both genetic and pharmacological inhibition of S6K1 increased sensitivity to gefitinib in PDX-R cells. In addition, both total and phosphorylated mechanistic target of rapamycin kinase (MTOR) levels were upregulated in PDX-R and gefitinib-resistant PC9G cells. Knockdown of MTOR by siRNA decreased the expression levels of total and phosphor-S6K1 and increased sensitivity to gefitinib in PDX-R and PC9G cells. Moreover, a transcription factor ELK1, which has multiple predicted binding sites on the MTOR promoter, was also upregulated in PDX-R and PC9G cells, while the knockdown of ELK1 led to decreased expression of MTOR and S6K1. The chromatin immunoprecipitation (ChIP)-PCR assay showed the direct binding between ELK1 and the MTOR promoter, and the luciferase reporter assay further indicated that ELK1 could upregulate MTOR expression through tuning up its transcription. Silencing ELK1 via siRNA transfection improved the efficacy of gefitinib in PDX-R and PC9G cells. These results support the notion that activation of ELK1/MTOR/S6K1 signaling contributes to acquired resistance to gefitinib in NSCLC. The findings in this study shed new light on the mechanism for acquired EGFR-TKI resistance and provide potential novel strategies by targeting the ELK1/MTOR/S6K1 pathway.

Phosphorylation of AGO2 by TBK1 Promotes the Formation of Oncogenic miRISC in NSCLC.

Non-small-cell lung cancer (NSCLC) is a highly lethal tumor that often develops resistance to targeted therapy. It is shown that Tank-binding kinase 1 (TBK1) phosphorylates AGO2 at S417 (pS417-AGO2), which promotes NSCLC progression by increasing the formation of microRNA-induced silencing complex (miRISC). High levels of pS417-AGO2 in clinical NSCLC specimens are positively associated with poor prognosis. Interestingly, the treatment with EGFR inhibitor Gefitinib can significantly induce pS417-AGO2, thereby increasing the formation and activity of oncogenic miRISC, which may contribute to NSCLC resistance to Gefitinib. Based on these, two therapeutic strategies is developed. One is jointly to antagonize multiple oncogenic miRNAs highly expressed in NSCLC and use TBK1 inhibitor Amlexanox reducing the formation of oncogenic miRISC. Another approach is to combine Gefitinib with Amlexanox to inhibit the progression of Gefitinib-resistant NSCLC. This findings reveal a novel mechanism of oncogenic miRISC regulation by TBK1-mediated pS417-AGO2 and suggest potential therapeutic approaches for NSCLC.

Tanshinone IIA reverses gefitinib resistance in EGFR-mutant lung cancer via inhibition of SREBP1-mediated lipogenesis.

BACKGROUND AND AIM: Gefitinib resistance is an urgent problem to be solved in the treatment of non-small cell lung cancer (NSCLC). Tanshinone IIA (Tan IIA) is one of the main active components of Salvia miltiorrhiza, which exhibits significant antitumor effects. The aim of this study is to explore the reversal effect of Tan IIA on gefitinib resistance in the epidermal growth factor receptor (EGFR)-mutant NSCLC and the underlying mechanism. EXPERIMENTAL PROCEDURE: CCK-8, colony formation assay, and flow cytometry were applied to detect the cytotoxicity, proliferation, and apoptosis, respectively. The changes in lipid profiles were measured by electrospray ionization-mass spectrometry (MS)/MS. Western blot, real-time q-PCR, and immunohistochemical were used to detect the protein and the corresponding mRNA levels. The in vivo antitumor effect was validated by the xenograft mouse model. KEY RESULTS: Co-treatment of Tan IIA enhanced the sensitivity of resistant NSCLC cells to gefitinib. Mechanistically, Tan IIA could downregulate the expression of sterol regulatory element binding protein 1 (SREBP1) and its downstream target genes, causing changes in lipid profiles, thereby reversing the gefitinib-resistance in EGFR-mutant NSCLC cells in vitro and in vivo. CONCLUSIONS AND IMPLICATIONS: Tan IIA improved gefitinib sensitivity via SREBP1-mediated lipogenesis. Tan IIA could be a potential candidate to enhance sensitivity for gefitinib-resistant NSCLC patients.

Xie-Bai-San increases NSCLC cells sensitivity to gefitinib by inhibiting Beclin-1 mediated autophagosome formation.

BACKGROUND: Autophagy, a cellular process involving lysosomal self-digestion, plays a crucial role in recycling biomolecules and degrading dysfunctional proteins and damaged organelles. However, in non-small cell lung cancer (NSCLC), cancer cells can exploit autophagy to survive metabolic stress and develop resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), which reduce treatment efficacies. Currently, most studies have found that late-stage autophagy inhibitors can hinder EGFR-TKIs resistance, while research on early-stage autophagy inhibitors is still limited. PURPOSE: This study investigates the mechanism via which the Xie-Bai-San (XBS) formula enhances NSCLC cell sensitivity to gefitinib, revealing the relationship between XBS-induced cell death and the inhibition of autophagosome formation. METHODS: Cell viability was assessed using CCK-8 and EdU assays, lentivirus transfection was utilized to generate PC9 cells harboring the PIK3CA E545K mutation (referred to as PC9-M), autophagic flux was monitored using mCherry-GFP-LC3 adenovirus. Protein expression and colocalization were observed through immunofluorescence staining. The interaction between Bcl-2 and Beclin-1 in PC9-GR and PC9-M cells was determined via co-immunoprecipitation (Co-IP) assay, cell apoptosis was assessed by flow cytometry and PI staining, and overall survival analysis of lung adenocarcinoma patients was conducted using the TCGA database. In vivo experiments included a patient-derived xenograft (PDX) model with EGFR and PIK3CA mutations and subcutaneous mice xenografts of NSCLC cell lines (PC9 and PC9-GR). In addition, autophagic vesicles in mouse tumor tissues were observed via transmission electron microscopy analysis. RESULTS: XBS effectively inhibits the proliferation of gefitinib-resistant NSCLC cells and induces apoptosis both in vitro and in vivo. Mechanistically, XBS suppresses gefitinib-induced autophagic flux by inhibiting autophagy through the upregulation of p-mTOR and Bcl-2 and downregulation of Beclin-1. Additionally, XBS enhances the interaction between Bcl-2 and Beclin-1, and the overexpression of Beclin-1 promotes NSCLC cell proliferation and counteracts XBS-induced cell death, while XBS demonstrates minimal impact on autophagosome-lysosome fusion or lysosome function. CONCLUSION: This study reveals a novel role for the XBS formula in impeding autophagy initiation and demonstrates its potential as a candidate drug to counteract autophagy-induced treatment resistance in NSCLC.

Design of balanced dual-target inhibitors of EGFR and microtubule.

Motivated by the clinical success of combining tyrosine kinase inhibitors with microtubule-targeted drugs in antitumor treatment, this paper presents a novel combi-targeting design for dual-target inhibitors, featuring arylformylurea-coupled quinazoline backbones. A series of target compounds (10a-10r) were designed, synthesized, and characterized. Biological assessments demonstrated that 10c notably potentiated ten tumor cell lines in vitro, with IC50 values ranging from 1.04 µM to 7.66 µM. Importantly, 10c (IC50 = 10.66 nM) exhibited superior inhibitory activity against EGFR kinases compared to the reference drug Gefitinib (25.42 nM) and reduced phosphorylated levels of EGFR, AKT, and ERK. Moreover, 10c significantly impeded tubulin polymerization, disrupted the intracellular microtubule network in A549 cells, induced apoptosis, led to S-phase cell cycle arrest, and hindered cell migration. In anticancer evaluation tests using A549 cancer-bearing nude mice models, 10c showed a therapeutic effect similar to Gefitinib, but required only half the dosage (15 mg/kg). These findings indicate that compound 10c is a promising dual-target candidate for anticancer therapy.

Tumor-associated Macrophages Mediate Gefitinib Resistance in Lung Cancer through HGF/c-met Signaling Pathway.

BACKGROUND: The biological behavior of cells changes after they develop drug resistance, and the degree of resistance will be affected by the tumor microenvironment. In this study, we aimed to study the effects of M2 macrophages on gefitinib resistance. METHODS: We polarized THP-1 cells into M0 and M2 macrophages, and conducted various experiments to investigate the effects of M2 macrophages on gefitinib resistance in lung cancer. RESULTS: We found that M2 macrophages promote gefitinib resistance in HCC827 and PC9 cells. In addition, we used ELISA to measure the secretion level of HGF. HGF secretion levels were significantly increased in M2 macrophages. Exogenous HGF remarkably increased the proliferation and invasion in HCC827 and PC9 cells. However, the addition of anti-HGF antibodies abolished the proliferation and invasion of both HCC827 and PC9 cells promoted by M2 macrophages. Furthermore, M2 macrophages or exogenous HGF significantly increased the expression of p-met and p-ERK in HCC827 and PC9 cells, while anti-HGF antibodies diminished the expression of p-met and p-ERK by neutralizing HGF in M2 macrophages. CONCLUSION: Our results revealed that M2 macrophages promote gefitinib resistance by activating ERK and HGF/c-met signaling pathways in HCC827 and PC9 cells. Our findings provide a new therapeutic strategy for gefitinib resistance in lung cancer.

Inhibition of miR-578 through SOCS2-dependent manner reverses gefitinib resistance in NSCLC cells.

BACKGROUND: Nonsmall-cell lung cancer (NSCLC) has emerged as one of the dreadful lung cancers globally due to its increased mortality rates. Concerning chemotherapy, gefitinib has been employed as an effective first-line treatment drug for NSCLC. Nonetheless, the acquired resistance to gefitinib has remained one of the treatment obstacles of NSCLC, requiring improvement in the therapeutic effect of gefitinib. METHODS: Initially, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and Western blotting (WB) analyses were conducted to measure micro-ribose nucleic acid (miRNA, specifically miR-578) and suppressor of cytokine signaling 2 (SOCS2) levels in the clinical samples. Further, NSCLC cell lines resistance to gefitinib, established in vitro, were transfected by miR-578 inhibitor, miR-578 mimic, and si-SOCS2. Similarly, the xenograft mouse model in vivo was constructed to validate the reversing effect of miR-578. RESULTS: Our findings indicated the increased miR-578 expression levels in the gefitinib resistance group. Further, inhibiting the miR-578 expression substantially reversed the gefitinib resistance. In addition, the miR-578 effect was modulated via the SOCS2 expression level. The decreased gefitinib resistance effect of miR-578 was weakened by inhibiting the SOCS2 expression. CONCLUSION: These findings demonstrated that miR-578 effectively abolished gefitinib resistance by regulating the SOCS2 expression within NSCLC cells in vitro and in vivo. Together, these results will undoubtedly support a reference to provide potential molecular therapeutic targets and clinical treatments for treating NSCLC patients.

ZYZ384 suppresses the growth of EGFR-mutant non-small cell lung cancer by activating JNK/MAPK signaling pathway.

The emergency of tyrosine kinase inhibitors has remarkably enhanced the clinical outcomes of cancer therapy, especially the use of EGFR inhibitors for non-small cell lung cancer (NSCLC). However, acquired resistance is inevitable after 8-12 months treatment. New agents or treatments are urgently required to resolve this problem. In this study, we identified that compound ZYZ384 can selectively inhibit the growth of gefitinib-resistant (G-R) lung cancer cells, without affecting that of normal lung epithelial cells. ZYZ384 induced G2 arrest in G-R NSCLC cells, decreasing the expression of Cyclin B1 and increasing the expression of P21. Meanwhile, ZYZ384 also induced apoptosis in NSCLC cells and correspondingly increased the expression of cleaved Caspase 3, 8, and 9 proteins. The expression of p-JNK, p-P38, and p-ERK were also increased in H1975 NSCLC cells treated with ZYZ384. Finally, we observed that the JNK inhibitor effectively reversed the pro-apoptotic effect of ZYZ384. In conclusion, ZYZ384 is a potential therapeutic agent to inhibit the growth of NSCLCs with EGFR mutations through activating JNK, which will help the development of related anticancer drugs.

Tetrahydrobenzothiophene derivatives ameliorate Mia PaCa-2 cell progression and induces apoptosis via inhibiting EGFR2 tyrosine kinase signal.

A series of new thiophene analogues with acarbonitrile-basedmoiety were designed and synthesized via structural optimization. The conjugates were assessed for their in-vitro cytotoxic activity against a human pancreatic cancer cell line (Mia PaCa-2) and among them compound 5b showed IC50 value of 13.37 ± 2.37 µM. The compounds 5b (20 µM & 25 µM) and 7c (30 & 35 µM) also showed reduced clonogenicity, enhanced ROS and decreased mitochondrial membrane potential in Mia PaCa-2 cells. Treatment with these compounds also increased apoptotic population as evident with the double staining assay. Among the evaluated series, compounds 5b, 5g, 7c, and 9a attained a greater inhibitory potency than first generation's reversible EGFR inhibitor, Gefitinib. EGFR2 enzyme inhibitory studies revealed that 5b efficiently and arbitrarily suppressed the development of EGFR2 dependent cells and inhibited the enzymatic activity with an IC50 value of 0.68 µM; interestingly, the most effective molecule 5b with N-methyl piperazine substitution, has 1.29-fold greater potency than well-known EGFR inhibitor Gefitinib and increased Gefitinib's anti-growth impact with 2.04 folds greater against Mia PaCa-2. The in-vitro studies were validated with in-silico docking studies wherein compounds 5b and 7c exhibited binding energies of -8.2 and -7.4 Kcal/mol respectively. The present study reveals that tetrahydrobenzothiophene based analogues could be a promising lead for the evolution of potent chemo preventives over pancreatic cancer.