H1299 Non-small Cell Lung Cancer Cells Research Articles

A Novel 3-Benzyloxychromone From Celastrus orbiculatus Thunb. Exhibits Anticancer Effects on Non-Small Cell Lung Cancer Cells via GSK-3β-Dependent c-Jun/ATF2 Pathway.

Celastrus orbiculatus Thunb. is a vine used as a traditional Chinese medicinal herb. In this study, we focused on the anticancer cytotoxicity and underlying mechanism of previously unreported 3-oxygen-substituted isoflavone analogue (3-benzyloxychromone, 3-Boc) from the herb. Initially, we established cell line-derived xenograft mouse model using H1299 non-small cell lung cancer (NSCLC) cells and found that the ethanol crude extracts of the stem part of C. orbiculatus (200 mg/kg) could substantially suppress the growth of xenograft tumors in athymic nu/nu mice. We compared 3-Boc with three other flavonoid analogues isolated from the stem part of C. orbiculatus. Among these, 3-Boc showed the most potent cytotoxicity against H1299 and H1975 NSCLC cells. Colony formation, EdU incorporation and Annexin V-FITC/PI apoptosis assays demonstrated that 3-Boc induced growth inhibition primarily by inhibiting DNA replication and inducing apoptotic death of NSCLC cells. Structure-based target prediction and MD simulation suggested that 3-Boc potentially suppressed the activity of glycogen synthase kinase-3β (GSK-3β) by interacting with the ATP-binding site. Western blot analysis indicated that 3-Boc triggered the phosphorylation of Serine 21 of GSK-3α or Serine 9 of GSK-3β in a time- and dose-dependent manner. To investigate the dependency of GSK-3β, we established GSK-3β knockout in H1299 cells. Depletion of GSK-3β enhanced 3-Boc-induced cytotoxicity compared with wild-type counterparts through activated c-Jun/ATF2 signaling pathway. Altogether, our study highlights the anticancer potential of C. orbiculatus and the discovery of novel 3-oxygen-substituted chromone from the herb, which may have important implications for screening promising modulators of GSK-3β and related signaling pathways in the treatment of cancer.

Bazedoxifene Inhibits Cell Viability, Colony-Forming Activity, and Cell Migration in Human Non-Small Cell Lung Cancer Cells and Improves the Treatment Efficacy of Paclitaxel and Gemcitabine.

Bazedoxifene is a third-generation selective estrogen receptor modulator that inhibits the IL6/IL6R/GP130 signaling pathway by inhibiting IL6-induced homodimerization of GP130. Considering that the IL6/IL6R/GP130 signaling pathway is important in tumorigenesis and metastasis, bazedoxifene is thought to have an antitumor effect, which has been proven preliminarily in breast cancer and pancreatic cancer but has not yet been studied in non-small cell lung cancer (NSCLC). This study is aimed at evaluating the antitumor effect of bazedoxifene in NSCLC. A549 and H1299 NSCLC cell lines were employed and exposed to various concentrations of bazedoxifene, paclitaxel, gemcitabine, and their combinations for cell viability, colony formation, and wound healing assays to demonstrate the antitumor effect of bazedoxifene with or without paclitaxel or gemcitabine. MTT cell viability, colony formation, and wound healing assays showed that bazedoxifene was capable of inhibiting cell viability, colony formation, and cell migration in a dose-dependent manner. In addition, bazedoxifene was capable of working with paclitaxel or gemcitabine synergistically to inhibit cell viability, colony formation, and cell migration. This study demonstrated the potential antitumor effect of bazedoxifene and its ability to improve the treatment efficacy of paclitaxel and gemcitabine.

Efficacy of GluN2B-Containing NMDA receptor antagonist for antitumor and antidepressant therapy in non-small cell lung cancer

Non-small cell lung cancer (NSCLC) is the predominant subtype of lung cancer. Evidence suggests that the ionotropic glutamate receptor N-methyl-D-aspartate (NMDA) receptor, a critical molecule in the central nervous system, is expressed in NSCLC. However, the specific expression patterns, subcellular localization, functional modulation, and pathological implications of NMDA receptor subtypes in NSCLC have not been fully elucidated. In this study, we employed a multi-disciplinary approach, combining biochemical and molecular biology with electrophysiological recordings and behavioral assays, to investigate these aspects. We reveal the expression of GluN2B-containing NMDA receptors in A549 and H460 NSCLC cell lines and the induction of NMDA receptor-mediated currents by glutamate in A549 cells. Furthermore, the GluN2B-specific inhibitors ifenprodil and Ro 25–6981 significantly reduced cell viability and migration, while promoting apoptosis. Importantly, intraperitoneal administration of ifenprodil in nude mice inhibited the growth of subcutaneous tumors derived from A549 and H460 cells and ameliorated depression-like behaviors. These findings underscore the potential antiproliferative effects of ifenprodil and Ro 25–6981 and suggest that GluN2B-containing NMDA receptors may represent novel therapeutic targets for NSCLC, with the added benefit of potential antidepressant action.

Adiponectin Receptor Agonist AdipoRon Inhibits Proliferation and Drives Glycolytic Dependence in Non-Small-Cell Lung Cancer Cells.

Despite the countless therapeutic advances achieved over the years, non-small-cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide. To this primacy contribute both non-oncogene addicted and advanced NSCLCs, in which conventional therapies are only partially effective. The adiponectin receptor agonist AdipoRon has revealed antiproliferative action in different cancers, including osteosarcoma and pancreatic cancer. Herein, we investigated its potential anticancer role in NSCLC for the first time. We proved that AdipoRon strongly inhibits viability, growth and colony formation in H1299 and A549 NSCLC cells, mainly through a slowdown in cell cycle progression. Along with the biological behaviors, a metabolic switching was observed after AdipoRon administration in NSCLC cells, consisting of higher glucose consumption and lactate accumulation. Remarkably, both 2-Deoxy Glucose and Oxamate glycolytic-interfering agents greatly enhanced AdipoRon's antiproliferative features. As a master regulator of cell metabolism, AMP-activated protein kinase (AMPK) was activated by AdipoRon. Notably, the ablation of AdipoRon-induced AMPK phosphorylation by Compound-C significantly counteracted its effectiveness. However, the engagement of other pathways should be investigated afterwards. With a focus on NSCLC, our findings further support the ability of AdipoRon in acting as an anticancer molecule, driving its endorsement as a future candidate in NSCLC therapy.

Amyloid Beta Leads to Decreased Acetylcholine Levels and Non-Small Cell Lung Cancer Cell Survival via a Mechanism That Involves p38 Mitogen-Activated Protein Kinase and Protein Kinase C in a p53-Dependent and -Independent Manner.

Several studies have shown an inverse correlation between the likelihood of developing a neurodegenerative disorder and cancer. We previously reported that the levels of amyloid beta (Aβ), at the center of Alzheimer's disease pathophysiology, are regulated by acetylcholinesterase (AChE) in non-small cell lung cancer (NSCLC). Here, we examined the effect of Aβ or its fragments on the levels of ACh in A549 (p53 wild-type) and H1299 (p53-null) NSCLC cell media. ACh levels were reduced by cell treatment with Aβ 1-42, Aβ 1-40, Aβ 1-28, and Aβ 25-35. AChE and p53 activities increased upon A549 cell treatment with Aβ, while knockdown of p53 in A549 cells increased ACh levels, decreased AChE activity, and diminished the Aβ effects. Aβ increased the ratio of phospho/total p38 MAPK and decreased the activity of PKC. Inhibiting p38 MAPK reduced the activity of p53 in A549 cells and increased ACh levels in the media of both cell lines, while opposite effects were found upon inhibiting PKC. ACh decreased the activity of p53 in A549 cells, decreased p38 MAPK activity, increased PKC activity, and diminished the effect of Aβ on those activities. Moreover, the negative effect of Aβ on cell viability was diminished by cell co-treatment with ACh.

Abstract 4687: Primary study on the anticancer role of BAM15 in lung cancer cells

Abstract Background: Lung cancer remains the leading cause of cancer-related death worldwide. Non-small cell lung cancer (NSCLC) accounts for about 80-85% of all lung cancer cases. BAM15, a mitochondrial uncoupler, is a potential therapeutic agent for many diseases, including obesity, diabetes, nonalcoholic fatty liver disease, Sepsis and cancers. However, there is no report on the anti-tumor role of BAM15 in NSCLC. Methods: To investigate the direct anti-cancer effects of BAM15 in NSCLC, we examined its inhibitory role on cell proliferation of NSCLC cells using the Cell Counting Kit-8(CCK8), colony forming assays and EdU staining assays. The impact of BAM15 on the cell cycle and cell apoptosis of NSCLC cells was analyzed by flow cytometric. A xenotransplanted NSCLC model in mice were used to test the anticancer role of BAM15. Result: Using the CCK8 assay, we found that the inhibitory role of BAM15 on the proliferation and colony formation of NSCLC cells is dose-dependent. The half maximal inhibitory concentration (IC50) values for cell proliferation were 2.71, 4.74, and 9.50 μM for A549, PC9, and H1299 NSCLC cells, respectively. The EdU staining assay demonstrated that BAM15 treatment significantly inhibited DNA synthesis in NSCLC cells. Flow cytometric analysis revealed that BAM15 arrested cell cycle at G2 phase in NSCLC cells and caused a striking increase in the percentage of apoptotic cells in NSCLC cells. The xenotransplanted NSCLC experiment indicated that BAM15 could significantly suppressed the growth of NSCLC tumors compared with the control tumors. Conclusions: Our study demonstrates that BAM15 significantly inhibits cell proliferation and promotes apoptosis in NSCLC cells, indicating that BAM15 is a potential therapeutic candidate for the treatment of NSCLC. Citation Format: Mei-yin Zhang, Yue-Ning Wang, Yu-Feng Zhou, Shuo-Cheng Wang, Shi-Juan Mai, Hui-Yun Wang. Primary study on the anticancer role of BAM15 in lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4687.

Sufentanil Suppresses Cell Carcinogenesis Via Targeting miR-186-5p/HMGB1 Axis and Wnt/β-Catenin Pathway in Non-Small-Cell Lung Cancer.

Sufentanil is a common opioid anesthetic agent, which exerts anti-cancer properties in several cancer types. However, its action mechanisms in non-small cell lung cancer (NSCLC) are unclear. Therefore, the present study investigated the pharmacological effect of sufentanil on miRNAs in NSCLC treatment. In this study, after treatment with sufentanil, the proliferation, migration, invasion and apoptosis of A549 and H1299 NSCLC cell lines were measured by cell counting kit-8 (CCK-8) assay, colony formation assay, transwell assays and flow cytometry. Quantitative real time polymerase chain reaction (qRT-PCR) was utilized to detect the expression of miR-186-5p and high mobility group box-1 (HMGB1), and their interaction was analyzed using luciferase reporter assay. The proteins of HMGB1, and apoptosis- and Wnt/β-catenin pathway-related factors were detected by western blot. It was demonstrated that sufentanil significantly upregulated miR‑186‑5p to restrict NSCLC cell proliferation, migration, invasion, and boost apoptosis in vitro. Mechanically, miR-186-5p interacted with HMGB1 and negatively regulated HMGB1 in NSCLC cells. Furthermore, rescue assay showed that sufentanil exerted antitumor activities by upregulating miR-186-5p, which targeted HMGB1 and restrained Wnt/β-catenin signal pathway in NSCLC cells. In conclusion, these results suggested that sufentanil disrupts the oncogenicity of NSCLC cells by regulating miR-186-5p/HMGB1/β-catenin axis, providing a promising implication for the anti-oncogenic effect of sufentanil.

Knockdown of EIF4G1 in NSCLC induces CXCL8 secretion.

Non-small cell lung cancer (NSCLC) is the most common type of lung tumor; however, we lack effective early detection indicators and therapeutic targets. Eukaryotic translation initiation factor 4 gamma 1 (EIF4G1) is vital to initiate protein synthesis, acting as a scaffolding protein for the eukaryotic protein translation initiation factor complex, EIF4F, which regulates protein synthesis together with EIF4A, EIF4E, and other translation initiation factors. However, EIF4G1's function in NSCLC cancer is unclear. Herein, transcriptome sequencing showed that knockdown of EIF4G1 in H1299 NSCLC cells upregulated the expression of various inflammation-related factors. Inflammatory cytokines were also significantly overexpressed in NSCLC tumor tissues, among which CXCL8 (encoding C-X-C motif chemokine ligand 8) showed the most significant changes in both in the transcriptome sequencing data and tumor tissues. We revealed that EIF4G1 regulates the protein level of TNF receptor superfamily member 10a (TNFRSF10A) resulting in activation of the mitogen activated protein kinase (MAPK) and nuclear factor kappa B (NFκB) pathways, which induces CXCL8 secretion, leading to targeted chemotaxis of immune cells. We verified that H1299 cells with EIF4G1 knockdown showed increased chemotaxis compared with the control group and promoted increased chemotaxis of macrophages. These data suggested that EIF4G1 is an important molecule in the inflammatory response of cancer tissues in NSCLC.

Carnosic Acid against Lung Cancer: Induction of Autophagy and Activation of Sestrin-2/LKB1/AMPK Signalling.

Non-small cell lung cancer (NSCLC) represents 80% of all lung cancer cases and is characterized by low survival rates due to chemotherapy and radiation resistance. Novel treatment strategies for NSCLC are urgently needed. Liver kinase B1 (LKB1), a tumor suppressor prevalently mutated in NSCLC, activates AMP-activated protein kinase (AMPK) which in turn inhibits mammalian target of rapamycin complex 1 (mTORC1) and activates unc-51 like autophagy activating kinase 1 (ULK1) to promote autophagy. Sestrin-2 is a stress-induced protein that enhances LKB1-dependent activation of AMPK, functioning as a tumor suppressor in NSCLC. In previous studies, rosemary (Rosmarinus officinalis) extract (RE) activated the AMPK pathway while inhibiting mTORC1 to suppress proliferation, survival, and migration, leading to the apoptosis of NSCLC cells. In the present study, we investigated the anticancer potential of carnosic acid (CA), a bioactive polyphenolic diterpene compound found in RE. The treatment of H1299 and H460 NSCLC cells with CA resulted in concentration and time-dependent inhibition of cell proliferation assessed with crystal violet staining and 3H-thymidine incorporation, and concentration-dependent inhibition of survival, assessed using a colony formation assay. Additionally, CA induced apoptosis of H1299 cells as indicated by decreased B-cell lymphoma 2 (Bcl-2) levels, increased cleaved caspase-3, -7, poly (ADP-ribose) polymerase (PARP), Bcl-2-associated X protein (BAX) levels, and increased nuclear condensation. These antiproliferative and proapoptotic effects coincided with the upregulation of sestrin-2 and the phosphorylation/activation of LKB1 and AMPK. Downstream of AMPK signaling, CA increased levels of autophagy marker light chain 3 (LC3), an established marker of autophagy; inhibiting autophagy with 3-methyladenine (3MA) blocked the antiproliferative effect of CA. Overall, these data indicate that CA can inhibit NSCLC cell viability and that the underlying mechanism of action of CA involves the induction of autophagy through a Sestrin-2/LKB1/AMPK signaling cascade. Future experiments will use siRNA and small molecule inhibitors to better elucidate the role of these signaling molecules in the mechanism of action of CA as well as tumor xenograft models to assess the anticancer properties of CA in vivo.

Unveiling Novel ERCC1-XPF Complex Inhibitors: Bridging the Gap from In Silico Exploration to Experimental Design.

Modifications in DNA repair pathways are recognized as prognostic markers and potential therapeutic targets in various cancers, including non-small cell lung cancer (NSCLC). Overexpression of ERCC1 correlates with poorer prognosis and response to platinum-based chemotherapy. As a result, there is a pressing need to discover new inhibitors of the ERCC1-XPF complex that can potentiate the efficacy of cisplatin in NSCLC. In this study, we developed a structure-based virtual screening strategy targeting the inhibition of ERCC1 and XPF interaction. Analysis of crystal structures and a library of small molecules known to act against the complex highlighted the pivotal role of Phe293 (ERCC1) in maintaining complex stability. This residue was chosen as the primary binding site for virtual screening. Using an optimized docking protocol, we screened compounds from various databases, ultimately identifying more than one hundred potential inhibitors. Their capability to amplify cisplatin-induced cytotoxicity was assessed in NSCLC H1299 cells, which exhibited the highest ERCC1 expression of all the cell lines tested. Of these, 22 compounds emerged as promising enhancers of cisplatin efficacy. Our results underscore the value of pinpointing crucial molecular characteristics in the pursuit of novel modulators of the ERCC1-XPF interaction, which could be combined with cisplatin to treat NSCLC more effectively.

Shikonin reactivates TSGs GADD45B and PPP3CC to block NSCLC cell proliferation and migration through JNK/P38/MAPK signaling pathways

BackgroundShikonin, a natural naphthoquinone compound extracted from the Chinese traditional herbal medicine “Lithospermum erythrorhizon”, possesses antitumor activity against various cancer types. Tumor-suppressor genes (TSGs) negatively regulate cell growth, proliferation, and differentiation, thereby inhibiting tumor formation. However, the molecular mechanism of action of shikonin on TSGs in non–small-cell lung cancer (NSCLC) remains unclear.MethodsThe inhibitory effect of shikonin on the proliferation and migration abilities of lung cancer cells were measured by Cell Counting Kit 8 (CCK8) and wound healing assays. The alteration of genes by shikonin treatment was detected by mRNA high-throughput sequencing and further confirmed by qPCR and western blotting experiments. The dominant functions of the upregulated genes were analyzed by GO and KEGG profiling.ResultsShikonin inhibited the proliferation and migration of A549 and H1299 NSCLC cells in a dose-dependent manner. mRNA high-throughput sequencing revealed a total of 1794 upregulated genes in shikonin-treated NSCLC cells. Moreover, bioinformatic analysis of GO and KEGG profiling revealed that the up-regulated genes were mostly involved in the JNK/P38/MAPK signaling pathway, among which the expression of GADD45B and PPP3CC was significantly enhanced. Finally, we confirmed that GADD45B and PPP3CC were indeed upregulated in JNK/P38/MAPK pathway.ConclusionsTaken together, these results suggested that shikonin might affect the expression of GADD45B and PPP3CC through the JNK/P38/MAPK pathway, therefore exerting an inhibitory effect on the proliferation and migration of cancer cells. To our knowledge, this is the first study reporting the role of shikonin in upregulating TSGs to activate the JNK/P38/MAPK signaling pathways in NSCLC.

The Ethanolic Extract of Dictyopteris Divaricata Induces Apoptosis in Non-Small Cell Lung Cancer Cells by Inhibiting STAT3 Activity

Dictyopteris divaricata (DD) has been reported to exert diverse pharmacological activities, including anti-inflammatory, antioxidant, and anticancer effects. In this study, we aimed to investigate the anticancer potential of the ethanolic extract of DD (EDD) in non-small cell lung cancer (NSCLC) cells and to explore the underlying mechanism. EDD significantly suppressed cell proliferation in H1299, PC9, and H1975 NSCLC cells. EDD treatment increased the proportion of Annexin V-positive cells and cells in sub-G1 phase, indicating the induction of apoptosis. This observation was further supported by the presence of fragmented nuclei and increased expression of cleaved PARP and cleaved caspase-3 in NSCLC cells following EDD treatment. Mechanistically, EDD decreased the phosphorylation levels of signal transducer and activator of transcription 3 (STAT3) and Src. Transfection of constitutively activated STAT3 into H1975 cells partially attenuated EDD-induced apoptosis, highlighting the contribution of STAT3 inhibition to the anticancer activity of EDD. In addition, we identified fucosterol as a major constituent of EDD that exhibited similar anticancer potential in NSCLC cells. Taken together, our results demonstrate that EDD induces apoptosis in NSCLC cells by inhibiting STAT3 activity. We propose EDD as a potential candidate for the development of therapies targeting NSCLC.

Linker optimization and activity validation of a cell surface vimentin targeted homo-dimeric peptoid antagonist for lung cancer stem cells

Epithelial-to-mesenchymal transition (EMT) endows epithelia-derived cancer cells with properties of stem cells that govern cancer invasion and metastasis. Vimentin is one of the best studied EMT markers and recent reports indicate that vimentin interestingly translocated onto cell surface under various tumor conditions. We recently reported a cell surface vimentin (CSV) specific peptoid antagonist named JM3A. We now investigated the selective antagonist activity of the optimized homo-dimeric version of JM3A, JM3A-L2D on stem-like cancer cells or cancer stem cells (CSCs) over normal cells in non-small cell lung cancer (NSCLC). Homo-dimerization of JM3A provided the avidity effect and improved the biological activity compared to the monomeric version. We first optimized the central linker length of the dimer by designing seven JM3A derivatives with varying linker lengths/types and evaluated the anti-cancer activity using the standard MTS cell viability assay. The most optimized derivative contains a central lysine linker and two glycines, named JM3A-L2D, which displayed 100 nM vimentin binding affinity (Kd) with an anti-cancer activity (IC50) of 6.7 µM on H1299 NSCLC cells. This is a 190-fold improvement in binding over the original JM3A. JM3A-L2D exhibited better potency on high vimentin-expressing NSCLC cells (H1299 and H460) compared to low vimentin-expressing NSCLC cells (H2122). No activity was observed on normal bronchial HBEC3-KT cells. The anti-CSC activity of JM3A-L2D was evaluated using the standard colony formation assay and JM3A-L2D disrupted the colony formation with IC50 ∼ 400 nM. In addition, JM3A-L2D inhibited cell migration activity at IC50 ∼ 2 µM, assessed via wound healing assay. The underlying mechanism of action seems to be the induction of apoptosis by JM3A-L2D on high-vimentin expressing H1229 and H460 NSCLC cells. Our optimized highly CSV selective peptoid has the potential to be developed as an anti-cancer drug candidate, especially considering the high serum stability and economical synthesis of peptoids.

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.

Targeting the aryl hydrocarbon receptor (AhR) with BAY 2416964: a selective small molecule inhibitor for cancer immunotherapy

BackgroundThe metabolism of tryptophan to kynurenines (KYN) by indoleamine-2,3-dioxygenase or tryptophan-2,3-dioxygenase is a key pathway of constitutive and adaptive tumor immune resistance. The immunosuppressive effects of KYN in the tumor...

Kaempferol promotes non-small cell lung cancer cell autophagy via restricting Met pathway

BackgroundKaempferol is extracted from Hedyotis diffusa, exerting an obvious anti-cancer effect. Here in the present study, we explored the anti-cancer effects and mechanism of kaempferol in non-small cell lung cancer cell (NSCLC). PurposeOur objective is to figure out the molecular mechanism by which kaempferol promotes autophagy in NSCLC cells. Study designA549 and H1299 NSCLC cell lines were used for in vitro experiments. And BALB/c nude mice of NSCLC were used to perform in vivo experiments. MethodsFor in vitro experiments, CCK-8 and EdU assay was used to observe the effect of kaempferol on NSCLC cell proliferation. Confocal microscopy of mCherry-EGFR-LC3 assay and electron microscopy assay were used to detect NSCLC cell autophagy. Protein expression was determined using Western blot, and mRNA expression was determined using qRT-PCR. Flow cytometry was performed to detect the cell apoptosis. For in vivo experiments, a subcutaneously implanted tumor model in BALB/C nude mice was performed using human NSCLC cell line A549-Luc. The kaempferol effect on NSCLC mice model was detected by measuring the tumor weight and bioluminescence intensity. Immunohistochemistry was done to measure the key protein expression from mice tumor tissues. ResultsOur results confirmed that kaempferol inhibited NSCLC cell proliferation significantly. And it promoted NSCLC cell autophagy, leading to NSCLC cell death. Interestingly, Met-was greatly inhibited at both protein and mRNA levels. Meanwhile, PI3K/AKT/mTOR signaling pathway was inhibited accordingly. Furthermore, overexpressing Met-reversed the effect of kaempferol on NSCLC cell viability and cell autophagy with significance. Finally, the above effect and pathway were validated using the xenograft model. ConclusionKaempferol may exert its anti-NSCLC effect by promoting NSCLC cell autophagy. Mechanistically, Met-and its downstream PI3K/AKT/mTOR signaling pathway were involved in the process, which provides a novel mechanism how kaempferol functions in inhibiting NSCLC.

Inhibition of non-homologous end joining mitigates paclitaxel resistance resulting from mitotic slippage in non-small cell lung cancer

ABSTRACT Mitotic slippage, which enables cancer cells to bypass cell death by transitioning from mitosis to the G1 phase without undergoing normal cytokinesis, is one likely mechanism of paclitaxel (PTX) resistance. DNA double-strand breaks (DSBs) in the G1 phase are mainly repaired through non-homologous end joining (NHEJ). Therefore, inhibiting NHEJ could augment the PTX-induced cytotoxicity by impeding the repair of PTX-induced DSBs during the G1 phase following mitotic slippage. We aimed to evaluate the effects of NHEJ inhibition on mitotic slippage after PTX treatment in non-small cell lung cancer (NSCLC). H1299, A549, H1975, and H520 NSCLC cell lines were employed. In addition, A-196 and JQ1 were used as NHEJ inhibitors. H1299 cells were PTX-resistant and exhibited an increased frequency of mitotic slippage upon PTX treatment. NHEJ inhibitors significantly augmented the PTX-induced cytotoxicity, DSBs, and apoptosis in H1299 cells. The newly generated PTX-resistant cells were even more prone to mitotic slippage following PTX treatment and susceptible to the combined therapy. Docetaxel further demonstrated synergistic effects with the NHEJ inhibitor in PTX-resistant cells. NHEJ inhibition may overcome intrinsic or acquired PTX resistance resulting from mitotic slippage by synergistically increasing the cytotoxic effects of antimitotic drugs in NSCLC.

Synergism Antiproliferative Effects of Apigenin and Naringenin in NSCLC Cells

Non-small cell lung cancer (NSCLC) is one of the leading cancer killers. Apigenin (Api) and Naringenin (Nar) are natural bioactive substances obtained in various vegetables and fruits, possessing anti-tumor effects across multiple studies. This study investigated the latent synergistic antiproliferative functions of Api and Nar in A549 and H1299 NSCLC cells. Cell viability was determined after incubating with different concentrations of Api, Nar, or the combination of Api and Nar (CoAN) for 24 h. Analysis using the CompuSyn software revealed that the CI value of each combined dose was < 1, depicting that the two drugs had a synergistic inhibitory effect. The CoAN (A:N = 3:2) group with the lowest CI value was selected for subsequent experiments. The IC50 of CoAN (A:N = 3:2) was used to determine the cell cycle, the expression ratio of Bax to Bcl2, Caspase 3 activity, and mitochondrial function to assess oxidative stress and apoptosis. The results established that CoAN treatment caused significant cytotoxicity with cell cycle arrest at G2/M phases. Furthermore, CoAN significantly enhanced mitochondria dysfunction, elevated oxidative stress, and activated the apoptotic pathway versus Api or Nar alone groups. Thus, the CoAN chemotherapy approach is promising and deserves further research.

Nifuroxazide induces the apoptosis of human non‑small cell lung cancer cells through the endoplasmic reticulum stress PERK signaling pathway.

The aim of the present study was to investigate the molecular mechanism of nifuroxazide (NFZ) in the induction of apoptosis of NCI-H1299 human non-small cell lung cancer (NSCLC) cells through the reactive oxygen species (ROS)/Ca2+/protein kinase R-like ER kinase (PERK)-activating transcription factor 4 (ATF4)-DNA damage inducible transcript 3 (CHOP) signaling pathway. Morphological changes of cells were observed by microscopy, and the apoptosis and intracellular ROS levels of cells were observed by inverted fluorescence microscopy. Cell viability after the addition of the PERK inhibitor, GSK2606414, were detected by Cell Counting Kit-8 assay. Annexin V-FITC was used to detect cell apoptosis, Brite 670 was used to detect intracellular ROS and Fura Red AM was used to detect Ca2+ content. Western blotting was used to detect PERK, phosphorylated (P)-PERK, ATF4, CHOP, P-Janus kinase 2 and P-signal transducer and activator of transcription 3 expression levels. Compared with the dimethyl sulfoxide control group, NFZ inhibited the survival activity in the H1299 NSCLC cell line, in a time- and dose-dependent manner. However, GSK2606414 inhibited the NFZ-induced apoptosis of H1299 cells. GSK2606414 also inhibited the increase in ROS and Ca2+ in H1299 cells induced by NFZ. Western blotting results demonstrated that NFZ significantly increased the expression levels of P-PERK, ATF4 and CHOP, whereas GSK2606414 significantly reduced the NFZ-induced increase in these protein expression levels. In conclusion, NFZ may induce the apoptosis of H1299 NSCLC cells through the ROS/Ca2+/PERK-ATF4-CHOP signaling pathway.

Validation of an orthotopic non-small cell lung cancer mouse model, with left or right tumor growths, to use in conformal radiotherapy studies.

Orthotopic non-small cell lung cancer (NSCLC) mice models are important for establishing translatability of in vitro results. However, most orthotopic lung models do not produce localized tumors treatable by conformal radiotherapy (RT). Here we report on the performance of an orthotopic mice model featuring conformal RT treatable tumors following either left or right lung tumor cell implantation. Athymic Nude mice were surgically implanted with H1299 NSCLC cell line in either the left or right lung. Tumor development was tracked bi-weekly using computed tomography (CT) imaging. When lesions reached an appropriate size for treatment, animals were separated into non-treatment (control group) and RT treated groups. Both RT treated left and right lung tumors which were given a single dose of 20 Gy of 225 kV X-rays. Left lung tumors were treated with a two-field parallel opposed plan while right lung tumors were treated with a more conformal four-field plan to assess tumor control. Mice were monitored for 30 days after RT or after tumor reached treatment size for non-treatment animals. Treatment images from the left and right lung tumor were also used to assess the dose distribution for four distinct treatment plans: 1) Two sets of perpendicularly staggered parallel opposed fields, 2) two fields positioned in the anterior-posterior and posterior-anterior configuration, 3) an 180° arc field from 0° to 180° and 4) two parallel opposed fields which cross through the contralateral lung. Tumor volumes and changes throughout the follow-up period were tracked by three different types of quantitative tumor size approximation and tumor volumes derived from contours. Ultimately, our model generated delineable and conformal RT treatable tumor following both left and right lung implantation. Similarly consistent tumor development was noted between left and right models. We were also able to demonstrate that a single 20 Gy dose of 225 kV X-rays applied to either the right or left lung tumor models had similar levels of tumor control resulting in similar adverse outcomes and survival. And finally, three-dimensional tumor approximation featuring volume computed from the measured length across three perpendicular axes gave the best approximation of tumor volume, most closely resembled tumor volumes obtained with contours.