Chelidonine selectively inhibits the growth of gefitinib-resistant non-small cell lung cancer cells through the EGFR-AMPK pathway.

Tyrosine kinase inhibitors (TKIs) have been widely used for the clinical treatment of patients with non-small cell lung cancer (NSCLC) harboring mutations in the EGFR. Unfortunately, due to the secondary mutation in EGFR, eventual drug-resistance is inevitable. Therefore, to overcome the resistance, new agent is urgently required. Chelidonine, extracted from the roots of Chelidonium majus, was proved to effectively suppress the growth of NSCLC cells with EGFR double mutation. Proteomics analysis indicated that mitochondrial respiratory chain was significantly inhibited by chelidonine, and inhibitor of AMPK effectively blocked the apoptosis induced by chelidonine. Molecular dynamics simulations indicated that chelidonine could directly bind to EGFR and showed a much higher binding affinity to EGFRL858R/T790M than EGFRWT, which demonstrated that chelidonine could selectively inhibit the phosphorylation of EGFR in cells with EGFR double-mutation. In vivo study revealed that chelidonine has a similar inhibitory effect like second generation TKI Afatinib. In conclusion, targeting EGFR and inhibition of mitochondrial function is a promising anti-cancer therapeutic strategy for inhibiting NSCLC with EGFR mutation and TKI resistance.

Cucurbitacin B Induces the Lysosomal Degradation of EGFR and Suppresses the CIP2A/PP2A/Akt Signaling Axis in Gefitinib-Resistant Non-Small Cell Lung Cancer.

Non-small cell lung cancer (NSCLC) patients carrying an epidermal growth factor receptor (EGFR) mutation are initially sensitive to EGFR-tyrosine kinase inhibitors (TKIs) treatment, but soon develop an acquired resistance. The treatment effect of EGFR-TKIs-resistant NSCLC patients still faces challenges. Cucurbitacin B (CuB), a triterpene hydrocarbon compound isolated from plants of various families and genera, elicits anticancer effects in a variety of cancer types. However, whether CuB is a viable treatment option for gefitinib-resistant (GR) NSCLC remains unclear. Here, we investigated the anticancer effects and underlying mechanisms of CuB. We report that CuB inhibited the growth and invasion of GR NSCLC cells and induced apoptosis. The inhibitory effect of CuB occurred through its promotion of the lysosomal degradation of EGFR and the downregulation of the cancerous inhibitor of protein phosphatase 2A/protein phosphatase 2A/Akt (CIP2A/PP2A/Akt) signaling axis. CuB and cisplatin synergistically inhibited tumor growth. A xenograft tumor model indicated that CuB inhibited tumor growth in vivo. Immunohistochemistry results further demonstrated that CuB decreased EGFR and CIP2A levels in vivo. These findings suggested that CuB could suppress the growth and invasion of GR NSCLC cells by inducing the lysosomal degradation of EGFR and by downregulating the CIP2A/PP2A/Akt signaling axis. Thus, CuB may be a new drug candidate for the treatment of GR NSCLC.

Design, synthesis, and biological evaluation of novel 1-oxo-1,2,3,4-tetrahydropyrazino[1,2-a]indole-3-carboxamide analogs in MCF-7 and MDA-MB-468 breast cancer cell lines.

A series of novel 1-oxo-1,2,3,4-tetrahydropyrazino[1,2-a]indole-3-carboxamide analogs were designed and synthesized for developing pyrazinoindolone scaffolds as anti-breast cancer agents. Compounds 1h and 1i, having a furan-2-yl-methylamide and benzylamide group, respectively, exhibited more potent cytotoxicity in MDA-MB-468 triple-negative breast cancer (TNBC) cells than compounds possessing aliphatic groups. Compounds 2a and 2b, as (R)-enantiomers of 1h and 1i, also had inhibitory activity against MDA-MB-468 cells. Moreover, analogs (3a-b and 3d-e) bearing a benzyl group at the N-2 position showed more potent activity than gefitinib, as a potent EFGR-TK inhibitor. Especially, compound 3a exhibited selective cytotoxic activity against MDA-MB-468 cells; it also had a synergistic effect in combination with gefitinib against MDA-MB-468 cells. In addition, we confirmed that compounds 3a and 3d inhibit phosphorylation of Akt in MDA-MB-468 cells using western blot analysis. Therefore, these 1-oxo-1,2,3,4-tetrahydropyrazino[1,2-a]indole-3-carboxamide analogs may be helpful for investigating new anti-TNBC agents.

Cucurbitacin B inhibits TGF-ß1-induced epithelial-mesenchymal transition (EMT) in NSCLC through regulating ROS and PI3K/Akt/mTOR pathways.

BACKGROUND: Lung cancer is the leading cause of cancer mortality worldwide, and most of the patients after treatment with EGF-TKIs develop drug resistance, which is closely correlated with EMT. Cucurbitacin B (CuB) is a natural product of the Chinese herb Cucurbitaceae plant, which has a favorable role in anti-inflammation and anti-cancer activities. However, the effect of CuB on EMT is still far from fully explored. In this study, the inhibition effect of CuB on EMT was investigated. METHODS: In this study, TGF-ß1 was used to induce EMT in A549 cells. MTS assay was used to detect the cell viability of CuB co-treated with TGF-ß1. Wound healing assay and transwell assay were used to determine the migration and invasion capacity of cells. Flow cytometry and fluorescence microscope were used to detect the ROS level in cells. Western blotting assay and immunofluorescence assay were used to detect the proteins expression. Gefitinib was used to establish EGF-TKI resistant NSCLC cells. B16-F10 intravenous injection mice model was used to evaluate the effect of CuB on lung cancer metastasis in vivo. Caliper IVIS Lumina and HE staining were used to detect the lung cancer metastasis of mice. RESULTS: In this study, the results indicated that CuB inhibited TGF-ß1-induced EMT in A549 cells through reversing the cell morphology changes of EMT, increasing the protein expression of E-cadherin, decreasing the proteins expression of N-cadherin and Vimentin, suppressing the migration and invasion ability. CuB also decreased the ROS production and p-PI3K, p-Akt and p-mTOR expression in TGF-ß1-induced EMT in A549 cells. Furthermore, Gefitinib resistant A549 cells (A549-GR) were well established, which has the EMT characteristics, and CuB could inhibit the EMT in A549-GR cells through ROS and PI3K/Akt/mTOR pathways. In vivo study showed that CuB inhibited the lung cancer metastasis effectively through intratracheal administration. CONCLUSION: CuB inhibits EMT in TGF-ß1-induced A549 cells and Gefitinib resistant A549 cells through decreasing ROS production and PI3K/Akt/mTOR signaling pathway. In vivo study validated that CuB inhibits lung cancer metastasis in mice. The study may be supporting CuB as a promising therapeutic agent for NSCLC and Gefitinib resistant NSCLC.

Deoxypodophyllotoxin Inhibits Cell Growth and Induces Apoptosis by Blocking EGFR and MET in Gefitinib-Resistant Non-Small Cell Lung Cancer.

As one of the major types of lung cancer, non-small cell lung cancer (NSCLC) accounts for the majority of cancer-related deaths worldwide. Treatments for NSCLC includes surgery, chemotherapy, and targeted therapy. Among the targeted therapies, resistance to inhibitors of the epidermal growth factor receptor (EGFR) is common and remains a problem to be solved. MET (hepatocyte growth factor receptor) amplification is one of the major causes of EGFR-tyrosine kinase inhibitor (TKI) resistance. Therefore, there exists a need to find new and more efficacious therapies. Deoxypodophyllotoxin (DPT) extracted from Anthriscus sylvestris roots exhibits various pharmacological activities including anti-inflammation and anti-cancer effects. In this study we sought to determine the anti-cancer effects of DPT on HCC827GR cells, which are resistant to gefitinib (EGFR-TKI) due to regulation of EGFR and MET and their related signaling pathways. To identify the direct binding of DPT to EGFR and MET, we performed pull-down, ATP-binding, and kinase assays. DPT exhibited competitive binding with ATP against the network kinases EGFR and MET and reduced their activities. Also, DPT suppressed the expression of p-EGFR and p-MET as well as their downstreat proteins p-ErbB3, p-AKT, and p-ERK. The treatment of HCC827GR cells with DPT induced high ROS generation that led to endoplasmic-reticulum stress. Accordingly, loss of mitochondrial membrane potential and apoptosis by multi-caspase activation were observed. In conclusion, these results demonstrate the apoptotic effects of DPT on HCC827GR cells and signify the potential of DPT to serve as an adjuvant anti-cancer drug by simultaneously inhibiting EGFR and MET.

Multifunctional Lipid-Based Nanoparticles for Codelivery of Anticancer Drugs and siRNA for Treatment of Non-Small Cell Lung Cancer with Different Level of Resistance and EGFR Mutations.

Resistance to chemotherapy, enhanced proliferation, invasion, angiogenesis, and metastasis (RPIAM) represent major obstacles that limit the efficacy of cancer treatment especially in advanced stages of cancer. Overcoming or suppressing RPIAM can dramatically improve the treatment outcome. Non-small cell lung cancer (NSCLC) is frequently diagnosed in an advanced stage and often possesses intrinsic resistance to chemotherapy accompanied by the fast development of acquired resistance during the treatment. Oncogenic receptor tyrosine kinases (TKs), specifically epidermal growth factor (EGF) TKs, play an important role in the activation of MAPK/PI3K/Akt/STAT pathways, finally leading to the development of RPIAM. However, the suppression of EGF-TK by different drugs is limited by various defensive mechanisms and mutations. In order to effectively prevent the development of RPIAM in NSCLC, we formulated and tested a multicomponent and multifunctional cancer targeted delivery system containing Nanostructured Lipid Carriers (NLCs) as vehicles, luteinizing hormone release hormone (LHRH) as a cancer targeting moiety, EFG-TK inhibitor gefitinib and/or paclitaxel as anticancer drug(s), siRNA targeted to EGF receptor (EGFR) mRNA as a suppressor of EGF receptors, and an imaging agent (rhodamine) for the visualization of cancer cells. Experimental data obtained show that this complex delivery system possesses significantly enhanced anticancer activity that cannot be achieved by individual components applied separately.

Identification of Key Genes and Pathways in Gefitinib-Resistant Lung Adenocarcinoma using Bioinformatics Analysis.

Gefitinib resistance is a serious threat in the treatment of patients with non-small cell lung cancer (NSCLC). Elucidating the underlying mechanisms and developing effective therapies to overcome gefitinib resistance is urgently needed. The differentially expressed genes (DEGs) were screened from the gene expression profile GSE122005 between gefitinib-sensitive and resistant samples. GO and KEGG analyses were performed with DAVID. The protein-protein interaction (PPI) network was established to visualize DEGs and screen hub genes. The functional roles of CCL20 in lung adenocarcinoma (LUAD) were examined using gene set enrichment analysis (GSEA). Functional analysis revealed that the DEGs were mainly concentrated in inflammatory, cell chemotaxis, and PI3K signal regulation. Ten hub genes were identified based on the PPI network. The survival analysis of the hub genes showed that CCL20 had a significant effect on the prognosis of LUAD patients. GSEA analysis showed that CCL20 high expression group was mainly enriched in cytokine-related signaling pathways. In conclusion, our analysis suggests that changes in inflammation and cytokine-related signaling pathways are closely related to gefitinib resistance in patients with lung cancer. The CCL20 gene may promote the formation of gefitinib resistance, which may serve as a new biomarker for predicting gefitinib resistance in patients with lung cancer.

In Vitro and in Vivo Efficacy of NBDHEX on Gefitinib-resistant Human Non-small Cell Lung Cancer.

Gefitinib, a first-generation EGFR tyrosine kinase inhibitor (EGFR-TKI), is recommended for treatment of non-small cell lung cancer (NSCLC) patients who harbor activating EGFR mutations. However, the tumors of most patients initially sensitive to gefitinib will develop resistance within several months of therapy. Drug resistance is a major obstacle to NSCLC treatment. The novel glutathione transferase P1 (GSTPi) inhibitor 6-(7-nitro-2, 1, 3-benzoxadiazol-4-ylthio) hexanol (NBDHEX) has recently been shown to be active against tumors. In this study, we investigated the in vitro and in vivo efficacy of NBDHEX against NSCLC. Treatment with NBDHEX inhibited GSTpi enzymatic activity and promoted apoptosis of gefinitb-resistant NSCLC cells. Moreover, NBDHEX reduced tumor growth in mice. These findings indicated that NBDHEX is a good candidate for treatment of NSCLC patients, and that NBDHEX offers a new approach to cancer therapy.

Inhibition of Alternative Cancer Cell Metabolism of EGFR Mutated Non-Small Cell Lung Cancer Serves as a Potential Therapeutic Strategy.

Targeted therapy is an efficient treatment for patients with epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC). Therapeutic resistance invariably occurs in NSCLC patients. Many studies have focused on drug resistance mechanisms, but only a few have addressed the metabolic flexibility in drug-resistant NSCLC. In the present study, we found that during the developing resistance to tyrosine kinase inhibitor (TKI), TKI-resistant NSCLC cells acquired metabolic flexibility in that they switched from dependence on glycolysis to oxidative phosphorylation by substantially increasing the activity of the mitochondria. Concurrently, we found the predominant expression of monocarboxylate transporter 1 (MCT-1) in the TKI-resistant NSCLC cells was strongly increased in those cells that oxidized lactate. Thus, we hypothesized that inhibiting MCT-1 could represent a novel treatment strategy. We treated cells with the MCT-1 inhibitor AZD3965. We found a significant decrease in cell proliferation and cell motility in TKI-sensitive and TKI-resistant cells. Taken together, these results demonstrated that gefitinib-resistant NSCLC cells harbored higher mitochondrial bioenergetics and MCT-1 expression. These results implied that targeting mitochondrial oxidative phosphorylation proteins or MCT-1 could serve as potential treatments for both TKI-sensitive and -resistant non-small cell lung cancer.

Tobacco extracts promote PD-L1 expression and enhance malignant biological differences via mTOR in gefitinib-resistant cell lines.

BACKGROUND: The aim of this study was to investigate whether tobacco extracts could regulate PD-L1 expression and enhance malignant biological differences in gefitinib-resistant cell lines. METHODS: We constructed gefitinib-resistant cells and observed the biological differences in gefitinib-resistant cells. The cells were stimulated with medium containing 5% volume of tobacco extract, and the change in PD-L1 expression and the mammalian target of rapamycin (mTOR) and p-mTOR expression in gefitinib-resistant cells treated with tobacco extracts was observed. We discussed the relationship between PD-L1 and mTOR. RESULTS: Tobacco extracts could promote PD-L1 expression in the cell line. Western blot analysis showed that mTOR and p-mTOR were significantly enhanced in gefitinib-resistant cell lines cultured in the tobacco extracts. The mTOR signaling pathway was involved in PD-L1 expression and in regulating the expression of cytokines IL-6 and IL-23. In addition, the tobacco extracts could promote macrophage migration via mTOR/IL-6. CONCLUSIONS: PD-L1 can transmit inhibitory signals and reduce the proliferation of CD8 + T cells in lymph nodes. Tobacco extracts upregulate PD-L1 expression via mTOR/IL-6. These results imply that lung cancer patients should not smoke and stay away from a smoke environment.

A new epigallocatechin gallate derivative isolated from Anhua dark tea sensitizes the chemosensitivity of gefitinib via the suppression of PI3K/mTOR and epithelial-mesenchymal transition.

The acquired resistance to gefitinib limits its clinical application. Epigallocatechin-3-gallate (EGCG) has been found to enhance the efficacy of gefitinib against resistant. However, the cellular and molecular mechanisms have not been completely illuminated in NSCLC. In this study, a new epigallocatechin gallate derivative (2R,3R-6-methoxycarbonylgallocatechin-3-O-gallate, the following referred to as EGCGD) (1) and three known epigallocatechin gallate compounds including epicatechin-3-O-gallate (2), gallocatechin-3-O-gallate (3) and epigallocatechin-3-O-gallate (4, EGCG) were isolated and identified from Anhua dark tea. The pharmacological studies showed EGCGD was more effective against gefitinib-resistant HCC827-Gef cells compared to that of other three epigallocatechin gallate compounds including EGCG, suggesting that introduction of 6-methoxycarbonyl to EGCG might enhance its antitumor activities. Further study on molecular mechanism showed EGCGD increased the potency of gefitinib against HCC827-Gef cells via suppression of epithelial-Mesenchymal transition (EMT) and dual inhibition of PI3K/mTOR.

MiR-153 inhibits the resistance of lung cancer to gefitinib via modulating expression of ABCE1.

BACKGROUND: Gefitinib-resistance in lung cancers has become an intractable clinical problem. However, the mechanisms underlying this resistance are not fully understood. OBJECTIVE: Present study aims to investigate the roles and underlying mechanism of miR-153 in modulating gefitinib resistance in lung cancers. METHODS: In the present study, genes expression of miR-153, MDR-1 and ABCE1 were detected by qRT-PCR and western blot. The cell viability was examined by MTT assays. The regulation of miR-153 on ABCE1 was examined by luciferase reporter gene assays. The interaction of miR-153 and ABCE1 was detected by gene over-expression and siRNA interference technology. RESULTS: The mRNA level of miR-153 was significantly down-regulated in gefitinib-resistance (GR) tissues and HCC827 cells, while the protein level of ABCE1 was up-regulated in GR tissues and HCC827 cells. Besides, miR-153 over-expression evidently increased miR-153 level and suppressed cell viability and multi drug resistance gene (MDR-1) expression in HCC827/Gef cells, while silence of miR-153 caused adverse alterations in HCC827 cells. Luciferase reporter assay results showed that miR-153 directly targeted ABCE1. Further studies showed that ABCE1 over-expression improved the expression of ABCE1 and MDR-1 and increased cell viability in HCC827/Gef cells, while ABCE1 silencing resulted in contrary trends in HCC827 cells. What's more, miR-153 over-expression inhibited tumorigenesis and ABCE1 expression, while increased miR-153 level in tumor tissues. CONCLUSIONS: MiR-153 regulates gefitinib resistance by modulating expression of ABCE1 in lung cancers. Our findings may provide a worthwhile therapeutic target to reverse gefitinib resistance in lung cancers in the future.

Benzyl Isothiocyanate Induces Apoptotic Cell Death Through Mitochondria-dependent Pathway in Gefitinib-resistant NCI-H460 Human Lung Cancer Cells In Vitro.

BACKGROUND/AIM: Gefitinib is used to treat patients with lung cancer, but in some patients, the disease becomes gefitinib-resistant. Benzyl isothiocyanate (BITC), found in cruciferous vegetables, has shown anticancer activity in many human cancer cell lines. However, the effects of BITC on gefitinib-resistant NCI-H460 lung cancer cells in vitro have not been investigated. MATERIALS AND METHODS: The effects of BITC on gefitinib-resistant NCI-H460 lung cancer cells were investigated in vitro. Flow cytometric assay was used for determining the total viable cell number, apoptotic cell death, the production of reactive oxygen species (ROS) and Ca2+, mitochondriaI membrane potential (Ψm) and caspase-3, -8 and -9 activities. Furthermore, 4', 6-diamidino-2-phenylindole staining was used to examine chromatin condensation in NCI-H460 and NCI-H460/G cells. RESULTS: BITC reduced total viable cell number via the induction of apoptotic cell death, that was also confirmed by annexin V/propidium iodide double staining assay. BITC increased ROS and Ca2+ production, reduced Ψm and increased caspase-3, -8 and -9 activities in both NCI-H460 and NCI-H460/G cells. Western blotting assay also showed that BITC increased expression of cleaved caspase-3 and -9, cytochrome c, BCL2-associated X protein, endonuclease G, poly (ADP-ribose) polymerase, growth arrest and DNA-damage protein 153, caspase-7 and activating transcription factor 6 alpha, but reduced apoptosis-inducing factor and caspase-9, BH3-interacting domain death agonist, calpain 1, glucose-regulated protein 78 and inositol requiring enzyme 1 alpha in NCI-H460/G cells. CONCLUSION: BITC-induced apoptotic cell death appears to occur via caspase- and mitochondria-dependent pathways in both cell lines.

Suppression of Lipogenesis via Reactive Oxygen Species-AMPK Signaling for Treating Malignant and Proliferative Diseases.

AIMS: Systemic diseases often have common characteristics. The aim of this study was to investigate the feasibility of targeting common pathological metabolism to inhibit the progression of malignant and proliferative diseases. RESULTS: Gefitinib-resistant (G-R) nonsmall-cell lung cancer (NSCLC) and rheumatoid arthritis (RA) were studied as conditions representative of malignant and proliferative diseases, respectively. Strong lipogenic activity and high expression of sterol regulatory element-binding protein 1 (SREBP1) were found in both G-R NSCLC cells and synovial fibroblasts from RA patients (RASFs). Berberine (BBR), an effective suppressor of SREBP1 and lipogenesis regulated through reactive oxygen species (ROS)/AMPK pathway, selectively inhibited the growth of G-R NSCLC cells and RASFs but not that of normal cells. It effectively caused mitochondrial dysfunction, activated ROS/AMPK pathway, and finally suppressed cellular lipogenesis and cell proliferation. Addition of ROS blocker, AMPK inhibitor, and palmitic acid significantly reduced the effect of BBR. In an in vivo study, treatment of BBR led to significant inhibition of mouse tumor xenograft growth and remarkably slowed down the development of adjuvant-induced arthritis in rats. Innovation and Conclusion: Targeting ROS/AMPK/lipogenesis signaling pathway selectively inhibited the growth of G-R NSCLC cells and the progress of RASFs in vitro and in vivo, which provides a new avenue for treating malignancies and proliferative diseases. Antioxid. Redox Signal. 28, 339-357.

Novel direct AMPK activator suppresses non-small cell lung cancer through inhibition of lipid metabolism.

Drug resistance is becoming an obstacle in anti-cancer therapies. For target-based therapy of lung cancer, gefitinib, as the first generation of tyrosine kinase inhibitors (TKIs), demonstrated good initial response to the non-small cell lung cancer (NSCLC) patients whom harbors epidermal growth factor receptor (EGFR) mutation. However, within one year, additional EGFR mutation occurred, leading to eventual gefitinib-resistance. Therefore, it is urgently to discover novel effective small molecule inhibitors for those patients. Abnormal energy metabolism is accepted as new cancer hallmark. Recently, a metabolism rate-limiting enzyme 5'-adenosine menophosphate-activated protein kinase (AMPK) has become a promising anti-cancer target. In this study, we have identified a novel direct AMPK agonist, D561-0775 from a compound library by using molecular docking screening technique. We demonstrated that D561-0775 exhibited significant inhibitory effect on gefitinib-resistant NSCLC cell lines but less cytotoxicity on normal cells. Furthermore, D561-0775 demonstrated a remarkable in vitro AMPK enzyme activation effect. Taken together, D561-0775 showed potential anti-cancer activity via inducing apoptosis, cell cycle arrest, suppressing glycolysis and cholesterol synthesis after activation of AMPK in gefitinib-resistant H1975 cells. D561-0775 has provided a new chemical structure that could be developed as cancer drug for gefitinib-resistant NSCLC patients through inhibition lipid metabolism by directly targeting at AMPK directly.