The Impact of Inadequate Exposure to Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitors on the Development of Resistance in Non-Small-Cell Lung Cancer Cells.

Epidermal growth factor receptor (EGFR)-mutant non-small-cell lung cancer (NSCLC) patients treated with EGFR-tyrosine kinase inhibitors (TKIs) inevitably develop resistance through several biological mechanisms. However, little is known on the molecular mechanisms underlying acquired resistance to suboptimal EGFR-TKI doses, due to pharmacodynamics leading to inadequate drug exposure. To evaluate the effects of suboptimal EGFR-TKI exposure on resistance in NSCLC, we obtained HCC827 and PC9 cell lines resistant to suboptimal fixed and intermittent doses of gefitinib and compared them to cells exposed to higher doses of the drug. We analyzed the differences in terms of EGFR signaling activation and the expression of epithelial-mesenchymal transition (EMT) markers, whole transcriptomes byRNA sequencing, and cell motility. We observed that the exposure to low doses of gefitinib more frequently induced a partial EMT associated with an induced migratory ability, and an enhanced transcription of cancer stem cell markers, particularly in the HCC827 gefitinib-resistant cells. Finally, the HCC827 gefitinib-resistant cells showed increased secretion of the EMT inducer transforming growth factor (TGF)-ß1, whose inhibition was able to partially restore gefitinib sensitivity. These data provide evidence that different levels of exposure to EGFR-TKIs in tumor masses might promote different mechanisms of acquired resistance.

Gefitinib metabolism-related lncRNAs for the prediction of prognosis, tumor microenvironment and drug sensitivity in lung adenocarcinoma.

The complete compound of gefitinib is effective in the treatment of lung adenocarcinoma. However, the effect on lung adenocarcinoma (LUAD) during its catabolism has not yet been elucidated. We carried out this study to examine the predictive value of gefitinib metabolism-related long noncoding RNAs (GMLncs) in LUAD patients. To filter GMLncs and create a prognostic model, we employed Pearson correlation, Lasso, univariate Cox, and multivariate Cox analysis. We combined risk scores and clinical features to create nomograms for better application in clinical settings. According to the constructed prognostic model, we performed GO/KEGG and GSEA enrichment analysis, tumor immune microenvironment analysis, immune evasion and immunotherapy analysis, somatic cell mutation analysis, drug sensitivity analysis, IMvigor210 immunotherapy validation, stem cell index analysis and real-time quantitative PCR (RT-qPCR) analysis. We built a predictive model with 9 GMLncs, which showed good predictive performance in validation and training sets. The calibration curve demonstrated excellent agreement between the expected and observed survival rates, for which the predictive performance was better than that of the nomogram without a risk score. The metabolism of gefitinib is related to the cytochrome P450 pathway and lipid metabolism pathway, and may be one of the causes of gefitinib resistance, according to analyses from the Gene Set Enrichment Analysis (GSEA), Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Immunological evasion and immunotherapy analysis revealed that the likelihood of immune evasion increased with risk score. Tumor microenvironment analysis found most immune cells at higher concentrations in the low-risk group. Drug sensitivity analysis found 23 sensitive drugs. Twenty-one of these drugs exhibited heightened sensitivity in the high-risk group. RT-qPCR analysis validated the characteristics of 9 GMlncs. The predictive model and nomogram that we constructed have good application value in evaluating the prognosis of patients and guiding clinical treatment.

Discovery of gefitinib-1,2,3-triazole derivatives against lung cancer via inducing apoptosis and inhibiting the colony formation.

A series of 20 novel gefitinib derivatives incorporating the 1,2,3-triazole moiety were designed and synthesized. The synthesized compounds were evaluated for their potential anticancer activity against EGFR wild-type human non-small cell lung cancer cells (NCI-H1299, A549) and human lung adenocarcinoma cells (NCI-H1437) as non-small cell lung cancer. In comparison to gefitinib, Initial biological assessments revealed that several compounds exhibited potent anti-proliferative activity against these cancer cell lines. Notably, compounds 7a and 7j demonstrated the most pronounced effects, with an IC50 value of 3.94 ± 0.17 µmol L-1 (NCI-H1299), 3.16 ± 0.11 µmol L-1 (A549), and 1.83 ± 0.13 µmol L-1 (NCI-H1437) for 7a, and an IC50 value of 3.84 ± 0.22 µmol L-1 (NCI-H1299), 3.86 ± 0.38 µmol L-1 (A549), and 1.69 ± 0.25 µmol L-1 (NCI-H1437) for 7j. These two compounds could inhibit the colony formation and migration ability of H1299 cells, and induce apoptosis in H1299 cells. Acute toxicity experiments on mice demonstrated that compound 7a exhibited low toxicity in mice. Based on these results, it is proposed that 7a and 7j could potentially be developed as novel drugs for the treatment of lung cancer.

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.

Parkin Enhances Gefitinib-induced Anoikis in HeLa Cervical Cancer Cells.

BACKGROUND/AIM: Gefitinib exhibits anticancer activity against cervical cancer cells via anoikis, a type of apoptosis induced by cell detachment from the extracellular matrix. Previous studies have reported that Parkin expression affects the efficacy of anticancer drugs. However, the impact of Parkin expression on the therapeutic effects of gefitinib in human cervical cancer remains unclear. Thus, this study aimed to evaluate whether Parkin over-expression improves the therapeutic effects of gefitinib against HeLa cervical cancer cells. MATERIALS AND METHODS: Cell viability and apoptotic death of HeLa cells were measured by trypan blue dye exclusion assay and flow cytometry. Cell detachment, adhesion, spreading, and cell-cell interaction were observed by inverted microscopy. Alteration of adhesion-related molecules was evaluated by confocal microscopy and western blot assay. RESULTS: Parkin expression potentiated gefitinib-induced cell detachment by affecting the organization of the actin cytoskeleton. In addition, Parkin expression induced a further reduction in the reattachment of and interaction between detached cells. The therapeutic efficacy of low-dose gefitinib combined with Parkin expression was equivalent to that of high-dose gefitinib alone. CONCLUSION: Parkin expression promotes gefitinib-induced anoikis, consequently increasing the efficacy of gefitinib against HeLa human cervical cancer cells. Based on our results, we propose that Parkin can be used to increase the anti-cancer effect of gefitinib on cervical cancer cells.

Genome-wide CRISPR screens identify the YAP/TEAD axis as a driver of persister cells in EGFR mutant lung cancer.

Most lung cancer patients with metastatic cancer eventually relapse with drug-resistant disease following treatment and EGFR mutant lung cancer is no exception. Genome-wide CRISPR screens, to either knock out or overexpress all protein-coding genes in cancer cell lines, revealed the landscape of pathways that cause resistance to the EGFR inhibitors osimertinib or gefitinib in EGFR mutant lung cancer. Among the most recurrent resistance genes were those that regulate the Hippo pathway. Following osimertinib treatment a subpopulation of cancer cells are able to survive and over time develop stable resistance. These 'persister' cells can exploit non-genetic (transcriptional) programs that enable cancer cells to survive drug treatment. Using genetic and pharmacologic tools we identified Hippo signalling as an important non-genetic mechanism of cell survival following osimertinib treatment. Further, we show that combinatorial targeting of the Hippo pathway and EGFR is highly effective in EGFR mutant lung cancer cells and patient-derived organoids, suggesting a new therapeutic strategy for EGFR mutant lung cancer patients.

Establishment of a novel microfluidic co-culture system for simultaneous analysis of multiple indicators of gefitinib sensitivity in colorectal cancer cells.

The therapeutic effect of gefitinib on colorectal cancer (CRC) is unclear, but it has been reported that stromal cells in the tumor microenvironment may have an impact on drug sensitivity. Herein, we established a microfluidic co-culture system and explored the sensitivity of CRC cells co-cultured with cancer-associated fibroblasts (CAFs) to gefitinib. The system consisted of a multichannel chip and a Petri dish. The chambers in the chip and dish were designed to continuously supply nutrients for long-term cell survival and create chemokine gradients for driving cell invasion without any external equipment. Using this system, the proliferation and invasiveness of cells were simultaneously evaluated by quantifying the area of cells and the migration distance of cells. In addition, the system combined with live cell workstation could evaluate the dynamic drug response of co-cultured cells and track individual cell trajectories in real-time. When CRC cells were co-cultured with CAFs, CAFs promoted CRC cell proliferation and invasion and reduced the sensitivity of cells to gefitinib through the exosomes secreted by CAFs. Furthermore, the cells that migrated out of the chip were collected, and EMT-related markers were determined by immunofluorescent and western blot assays. The results demonstrated that CAFs affected the response of CRC cells to gefitinib by inducing EMT, providing new ideas for further research on the resistance mechanism of gefitinib. This suggests that targeting CAFs or exosomes might be a new approach to enhance CRC sensitivity to gefitinib, and our system could be a novel platform for investigating the crosstalk between tumor cells and CAFs and understanding multiple biological changes of the tumor cells in the tumor microenvironment.

Gefitinib Induces Apoptosis in NSCLC Cells by Promoting Glutaminolysis and Inhibiting the MEK/ERK Signaling Pathway.

BACKGROUND: Over 80% of lung cancer cases constitute non-small cell lung cancer (NSCLC), making it the most prevalent type of lung cancer globally and the leading cause of cancer-related deaths. The treatment of NSCLC patients with gefitinib has demonstrated promising initial efficacy. However, the underlying mechanism remains unclear. This study aims to investigate how gefitinib affects the mitogen-activated protein kinase kinase (MEK)/extracellular regulated protein kinases (ERK) signaling pathway-mediated growth and death of NSCLC cells. METHODS: In this study, the NSCLC cell line A549 was cultured in vitro and divided into a control group and a gefitinib group. The viability of the A549 cells was assessed using the methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. Flow cytometry was employed to detect apoptosis in A549 cells, and the expression of glutamate dehydrogenase (GDH1) mRNA in these cells was determined using real-time quantitative PCR (RT-PCR). Western blotting was utilized to evaluate the protein expression levels of key components in the MEK/ERK signaling pathway, including phospho-MEK1/2, MEK1/2, phospho-ERK1/2, and ERK1/2. Additionally, intracellular glutamine content in A549 cells was measured using a colorimetric method. RESULTS: In contrast to the control group, the proliferation of A549 cells, the transcription level of glutamate dehydrogenase (GDH1), the intracellular glutamine content, and the protein expression levels of phospho-MEK1/2 and phospho-ERK1/2 were significantly lower in the gefitinib group. Moreover, apoptosis markedly increased. CONCLUSIONS: Gefitinib expedites apoptosis and diminishes proliferation in the NSCLC cell line A549 by downregulating the epidermal growth factor receptor (EGFR)/MEK/ERK signaling pathway. This effect is accomplished by fostering the expression of GDH1 to augment glutaminolysis in A549 cells.

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.

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.

L-Methionine accentuates anti-tumor action of Gefitinib in Gefitinib-resistant lung adenocarcinoma: Role of EGFR/ERK/AKT signaling and histone H3K36me2 alteration.

Adenocarcinoma, the predominant subtype of non-small cell lung cancer (NSCLC), poses a significant clinical challenge due to its prevalence and aggressive nature. Gefitinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor is often susceptible to development of resistance despite being the preferred treatment option for NSCLC. In this study, we investigated the potential of L-Methionine in enhancing the cytotoxicity of Gefitinib and preventing resistance development. In vitro experiment employing the H1975 cell line demonstrated a notable enhancement in cytotoxic efficacy when L-Methionine (10 mM) was combined with Gefitinib, as indicated by a substantial reduction in IC50 values (155.854 ± 1.87 µM vs 45.83 ± 4.83 µM). Complementary in vivo investigations in a lung cancer model corroborated these findings. Co-administration of L-Methionine (100 mg/kg and 400 mg/kg) with Gefitinib (15 mg/kg) for 21 days exhibited marked improvements in therapeutic efficacy, which was observed by macroscopic and histopathological assessments. Mechanistic insights revealed that the enhanced cytotoxicity of the combination stemmed from the inhibition of the EGFR, modulating the downstream cascade of ERK/AKT and AMPK pathways. Concurrently inhibition of p-AMPK-α by the combination also disrupted metabolic homeostasis, leading to the increased production of reactive oxygen species (ROS). Notably, L-Methionine, functioning as a methyl group donor, elevated the expression of H3K36me2 (an activation mark), while reducing the p-ERK activity. Our study provides the first evidence supporting L-Methionine supplementation as a novel strategy to enhance Gefitinib chemosensitivity against pulmonary adenocarcinoma.

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.

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.

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.

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.

[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.

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.

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].

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.