H1299 Cells Research Articles

TRAIL predisposes non-small cell lung cancer to ferroptosis by regulating ASK-1/JNK1 pathway

ObjectiveOur current study aimed to assess the relationship between TNF-related apoptosis-inducing ligand (TRAIL) and ferroptosis in non-small cell lung cancer (NSCLC) development.MethodsThe expression of TRAIL was detected by western blot, RT-qRCR and immunohistochemistry. The viability of NSCLC cells was analyzed by CCK-8 kit. The migration and invasion of NSCLC cells were detected by wound healing assay and transwell assay, respectively. Labile iron pool (LIP) was detected based on the calcein-acetoxymethyl ester method. Ferrous iron (Fe2+) and iron levels were assessed by detection kits. The levels of superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) were measured using corresponding detection kits. Mice tumor xenograft models were used for the in vivo research.ResultsThe expression of TRAIL was reduced in H1299, NCL-H1395, and A549 cells compared with BEAS-2B cells. The up-regulation of TRAIL expression significantly reduced cell viability, invasion, and migration of H1299 and A549 cells. TRAIL reduced the expression of ferroptosis-related genes (FTH1, GPX4, and SLC7A11), increased the levels of LIP, iron, and Fe2+, and promoted lipid peroxidation, thereby predisposing NSCLC cells to ferroptosis. TRAIL up-regulated the expression of phosphate modification of ASK-1 and JNK. ASKI-1 inhibitor GS-4977 attenuated the effects of TRAIL on the viability, migration, invasion, and ferroptosis of H1299 cells. Furthermore, TRAIL further suppressed tumor growth and ferroptosis in mice tumor xenograft models.ConclusionWe indicated that overexpression of TRAIL induced ferroptosis in NSCLC cells and exerted anti-tumor effects. Mechanistically, TRAIL promoted ferroptosis by the activation of the ASK-1/JNK1 pathway. Our results may provide new therapeutic strategies for NSCLC.

Rational design and synthesis of 2,4-dichloro-6-methyl pyrimidine derivatives as potential selective EGFRT790M/L858R inhibitors for the treatment of non-small cell lung cancer.

Many patients with non-small cell lung cancer (NSCLC) initially benefit from epidermal growth factor receptor (EGFR) targeted therapy. Unfortunately, varying degrees of resistance or side effects eventually develop. Overcoming and preventing the resistance and side effects of EGFR inhibitors has become a hot topic of research today. Based on the previous studies on AZD-9291, we designed and synthesized two series of 2,4-dichloro-6-methylpyrimidine derivatives, 19 compounds in total, as potential inhibitors of the EGFR kinase. The most promising compound, L-18, showed better inhibitory activity (81.9%) and selectivity against EGFRT790M/L858R kinase. In addition, L-18 showed strong antiproliferative activity against H1975 cells with an IC50 value of 0.65 ± 0.06 μM and no toxicity to normal cells (LO-2). L-18 was able to dose-dependently induce the apoptosis of H1975 cells and produced a cell-cycle-blocking effect, and it can also dose-dependently inhibit the migration and invasion of H1975 cells. L-18 also showed in vivo anticancer efficacy in H1975 cells xenograft mice. We also performed a series of in vivo and in vitro toxicological evaluations of compound L-18, which did not cause obvious injury in mice during administration. These results suggest that L-18 may be a promising drug candidate that warrants further investigation.

Demethylzeylasteral inhibits proliferation, migration and invasion and promotes apoptosis of non-small cell lung cancer cells by inhibiting the AKT/CREB signaling pathway

To investigate the mechanism underlying the inhibitory effects of Demethylzeylasteral (T-96) on non-small cell lung cancer (NSCLC) cells. We first examined the effects of different concentrations (1, 3, 10, and 30 μmol/L) of demethylzeylasteral on morphology and cell number of A549 and H1299 cells. The changes in proliferation, cell viability, migration, invasion, and apoptosis of A549 and H1299 cells following demethylzeylasteral treatment were detected using clone formation, CCK-8, cell scratch, Transwell, and flow cytometric assays, and the effect of SC79 treatment against demethylzeylasteral-induced cell apoptosis was assessed. Western blotting was performed to detect the changes in expressions of E-cadherin, N-cadherin, vimentin, Bax, Bcl-2 and cleaved caspase-3 and phosphorylation of AKT/CREB in demethylzeylasteral-treated A549 and H1299 cells and the cellular expressions of apoptotic proteins following treatment with both demethylzeylasteral and SC79. T-96 treatment caused elongation of the cell body and widening of the intercellular space and significantly inhibited cell viability, proliferation, migration and invasion of A549 and H1299 cells (P < 0.05). Flow cytometry showed that demethylzeylasteral induced apoptosis in both A549 and H1299 cells, whereas SC79 treatment obviously attenuated its pro-apoptotic effect (P < 0.05). Western blotting revealed up-regulated expressions of Bax and cleaved caspase-3 proteins and lowered Bcl-2 expression level in demethylzeylasteral-treated A549 and H1299 cells, but cotreatment with SC79 obviously attenuated the expressions of the apoptotic proteins. T-96 significantly up-regulated the expression level of E-cadherin, down-regulated the expressions of N-cadherin and vimentin, and inhibited the phosphorylation of AKT and CREB in the two cell lines (P < 0.05). T-96 inhibits the proliferation, migration and invasion and induces apoptosis of NSCLC cells possibly by inhibiting the AKT/CREB signaling pathway.

Effect of photodynamic therapy mediated by hematoporphyrin derivatives on small cell lung cancer H446 cells and bronchial epithelial BEAS-2B cells

To investigate the effects of photodynamic therapy (PDT) mediated by hematoporphyrin derivatives (HPD) on the proliferation of small cell lung cancer H446 cells and bronchial epithelial BEAS-2B cells. H446 cells and BEAS-2B cells were cultured in vitro with different concentrations of HPD(0, 5, 10, 12, 15, 20 μg/mL) for 4 h, and then irradiated with 630 nm laser with different energy densities (0, 25, 50, 75, 100 mW/cm2). Cell viability of H446 cells and BEAS-2B cells were detected by CCK8 assay. The cell apoptosis was observed with Annexin V-FTTC/PI double staining and Hoechst 33258. The RT-PCR examination was applied to detect the transcriptional changes of the mRNA of Bax、Bcl-2, and Caspase-9. The results of CCK8 showed that when the HPD was 15 μg/mL and the laser power density reached 50 mW/cm2, the cell viability was significantly decreased compared with the black control group. Hoechst 33258 staining showed that with the increase of HPD concentration, the cell density was reduced, and apoptotic cells increased. Flow cytometry assay revealed that the apoptotic rates of the HPD-PDT group of H446 cells and BEAS-2B cells were significantly different from those of the blank control group. The RT-PCR examination showed that the expression levels of Bax and Caspase-9 mRNA in the HPD-PDT group were up-regulated, while the expression levels of Bcl-2 mRNA were down-regulated significantly. HPD-PDT can inhibit H446 cells and BEAS-2B cells growth. The mechanism may be related to up-regulating the expression levels of Bax and Caspase-9 mRNA and down-regulating the expression levels of Bcl-2 mRNA.

Synthesis and preclinical evaluation of [11C]EAI045 as a PET tracer for imaging tumors expressing mutated epidermal growth factor receptor

BackgroundMutations in the epidermal growth factor receptor (EGFR) kinase domain are common in non-small cell lung cancer. Conventional tyrosine kinase inhibitors target the mutation site in the ATP binding pocket, thereby inhibiting the receptor's function. However, subsequent treatment resistance mutations in the ATP binding site are common. The EGFR allosteric inhibitor, EAI045, is proposed to have an alternative mechanism of action, disrupting receptor signaling independent of the ATP-binding site. The antibody cetuximab is hypothesized to increase the number of accessible allosteric pockets for EAI045, thus increasing the potency of the inhibitor. This work aimed to gain further knowledge on pharmacokinetics, the EGFR mutation-targeting potential, and the influence of cetuximab on the uptake by radiolabeling EAI045 with carbon-11 and tritium.Results2-(5-fluoro-2-hydroxyphenyl)-2-((2-iodobenzyl)amino)-N-(thiazol-2-yl)acetamide and 2-(5-fluoro-2-hydroxyphenyl)-N-(5-iodothiazol-2-yl)-2-(1-oxoisoindolin-2-yl)acetamide were synthesized as precursors for the carbon-11 and tritium labeling of EAI045, respectively. [11C]EAI045 was synthesized using [11C]CO in a palladium-catalyzed ring closure in a 10 ± 1% radiochemical yield (decay corrected to end of [11C]CO2 production), > 97% radiochemical purity and 26 ± 1 GBq/µmol molar activity (determined at end of synthesis) in 51 min. [3H]EAI045 was synthesized by a tritium-halogen exchange in a 0.2% radiochemical yield, 98% radiochemical purity, and 763 kBq/nmol molar activity. The ability of [11C]EAI045 to differentiate between L858R/T790M mutated EGFR expressing H1975 xenografts and wild-type EGFR expressing A549 xenografts was evaluated in female nu/nu mice. The uptake was statistically significantly higher in H1975 xenografts compared to A549 xenografts (0.45 ± 0.07%ID/g vs. 0.31 ± 0.10%ID/g, P = 0.0166). The synergy in inhibition between EAI045 and cetuximab was evaluated in vivo and in vitro. While there was some indication that cetuximab influenced the uptake of [3H]EAI045 in vitro, this could not be confirmed in vivo when tumor-bearing mice were administered cetuximab (0.5 mg), 24 h prior to injection of [11C]EAI045.ConclusionsEAI045 was successfully labeled with tritium and carbon-11, and the in vivo results indicated [11C]EAI045 may be able to distinguish between mutated and non-mutated EGFR in non-small cell lung cancer mouse models. Cetuximab was hypothesized to increase EAI045 uptake; however, no significant effect was observed on the uptake of [11C]EAI045 in vivo or [3H]EAI045 in vitro in H1975 xenografts and cells.

Synthesis, characterization, and anticancer properties of Ag nanoparticles derived from walnut leaves tested on cells of L929, MCF-7 and H1299

In this study, Ag nanoparticles (Ag NPs) were synthesized using walnut leaves according to a straightforward method. The synthesized Ag NPs were characterized by advanced analytical techniques such as XRD, TEM, UV–Vis, FESEM, EDS and FTIR. The synthesized Ag NPs tested on some cells to detect its antiproliferative effects. The antiproliferative effects of Ag NPs derived from walnut leaves were investigated on L929, MCF-7 and H1299 cells. The characterization studies showed that Ag NPs were well formed and the cell studies revealed that the synthesized Ag NPs have considerable anticancer effects. The mean particle size of Ag NPs was found to be 18.76 nm by TEM analyses. FESEM analyses revealed that Ag NPs contained Ag, C, and O elements and FTIR analyses revealed these elements bound to each other with C–O bounds. The amount composition of Ag NPs was detected to be 85 % by EDS analyses. The antiproliferative effect of AgNPs obtained from walnut leaf extract on human breast cancer cell line (MCF-7), human lung cancer cell line (H1299) and mouse fibroblast cell line (L929) was determined by MTT cell viability assay method. While the silver nanoparticles synthesized within the scope of the study did not show any toxic effect on the mouse fibroblast cell line (L929) that we used for control purposes, it was observed that it was highly effective against MCF-7 and H1299 cells. Apoptosis and necrosis index studies were calculated by cell counting from randomly selected areas and these studies revealed that the synthesized silver nanoparticles suppressed cell viability by inducing apoptosis in cells. Significantly suppressing the viability of cancer cells proves that silver nanoparticles obtained from walnut leaves can be a new treatment approach for cancer treat.

Structures and Tumor Cell Lines Proliferation Activities of Triterpenes Isolated from Astilbe grandis.

A total of seven compounds, including four triterpene acids and three triterpene lactones, were isolated from the ethanolic extract of the roots of Astilbe grandis Stapf ex Wils. Two of the triterpene lactones (1-2) were never reported before and compounds 3-5 were isolated for the first time from the plant. The structures of these compounds were all identified by spectroscopic analysis. Compounds 1-2 were analyzed by 2D NMR and their absolute configurations were determined using experimental CD in comparison with calculated ECD values. The structure of compound 1 was also further confirmed by single crystal X-ray diffraction analysis. The cytotoxicity of compounds 1-7 on A549, Caco-2, H460 and Skov-3 tumor cells were all evaluated using CCK-8. They all exhibited positive inhibitory effects on Caco-2 tumor cells with IC50 less than10 μM, while the inhibitory effects on H460 tumor cells were more moderate. Unfortunately, they displayed little apparent cytotoxicity to the other two types of cells.

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.

Discovery of Pyroptosis-inducing Drugs and Antineoplastic Activity based on the ROS/ER Stress/Pyroptosis Axis.

Pyroptosis, a cell death process triggered by chemotherapy drugs, has emerged as a highly promising mechanism for combating tumors in recent years. As the lead of new drugs, natural products play an important role in the discovery of anticancer drugs. Compared to other natural products, the medicine food homologous natural products (MFHNP) exhibit a superior safety profile. Among a series of MFHNP molecular skeletons, this study found that only benzylideneacetophenone (1) could induce cancer cell pyroptosis. However, the anti-cancer activity of 1 remains to be improved. This study aimed to find a pyroptosis inducer with highly effective antitumor activity by modifying the chalcone structure. To examine the effect of the Michael receptor in compound 1 on the induction of pyroptosis, several analogs were synthesized by modifying the Michael acceptor. Subsequently, the anticancer activity was tested by MTT assay, and morphological indications of pyroptosis were observed in human lung carcinoma NCI-H460 and human ovarian cancer CP-70 cell lines. Furthermore, to improve the activity of the chalcone skeleton, the anticancer group 3,4,5- trimethoxyphenyl was incorporated into the phenyl ring. Subsequently, compounds 2-22 were designed, synthesized, and screened in human lung cancer cells (NCI-H460, H1975, and A549). Additionally, a quantitative structure-activity relationship (QSAR) model was established using the eXtreme Gradient Boosting (XGBoost) machine learning library to identify the pharmacophore. Furthermore, both in vitro and in vivo experiments were conducted to investigate the molecular mechanisms of pyroptosis induced by the active compound. α, β-unsaturated ketone was the functional group of the chalcone skeleton and played a pivotal role in inducing cancer cell pyroptosis. QSAR models showed that the regression coefficients (R2) were 0.992 (A549 cells), 0.990 (NCI-H460 cells), and 0.998 (H1975 cells). Among these compounds, compound 7 was selected to be the active compound. Moreover, compound 7 was found to induce pyroptosis in lung cancer cells by upregulating the expression of CHOP by increasing the ROS level. Furthermore, it effectively suppressed the growth of lung cancer xenograft tumors. Compound 7 exhibits antineoplastic activity by regulating the ROS/ER stress/pyroptosis axis and is a kind of promising pyroptosis inducer.

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.

Design, Synthesis, and Biological Evaluation of Dual Inhibitors of EGFRL858R/T790M/ACK1 to Overcome Osimertinib Resistance in Nonsmall Cell Lung Cancers.

Activation of the alternative pathways and abnormal signaling transduction are frequently observed in third-generation EGFR-TKIs (epidermal growth factor receptor tyrosine kinase inhibitors)-resistant patients. Wherein, hyperphosphorylation of ACK1 contributes to EGFR-TKIs acquired resistance. Dual inhibition of EGFRL858R/T790M and ACK1 might improve therapeutic efficacy and overcome resistance in lung cancers treatment. Here, we identified a EGFRL858R/T790M/ACK1 dual-targeting compound 21a with aminoquinazoline scaffold, which showed excellent inhibitory activities against EGFRL858R/T790M (IC50 = 23 nM) and ACK1 (IC50 = 263 nM). The cocrystal and docking analysis showed that 21a occupied the ATP binding pockets of EGFRL858R/T790M and ACK1. Moreover, 21a showed potent antiproliferative activities against the H1975 cells, MCF-7 cells and osimertinib-resistant cells AZDR. Further, 21a showed significant antitumor effects and good safety in ADZR xenograft-bearing mice. Taken together, 21a was a potent dual inhibitor of EGFRL858R/T790M/ACK1, which is deserved as a potential lead for overcoming acquired resistance to osimertinib during the EGFR-targeted therapy.

Interaction between NSCLC Cells, CD8+ T-Cells and Immune Checkpoint Inhibitors Potentiates Coagulation and Promotes Metabolic Remodeling-New Cues on CAT-VTE.

Cancer-associated thrombosis (CAT) and venous thromboembolism (VTE) are frequent cancer-related complications associated with high mortality; thus, this urges the identification of predictive markers. Immune checkpoint inhibitors (ICIs) used in cancer immunotherapy allow T-cell activation against cancer cells. Retrospective studies showed increased VTE following ICI administration in some patients. Non-small cell lung cancer (NSCLC) patients are at high risk of thrombosis and thus, the adoption of immunotherapy, as a first-line treatment, seems to be associated with coagulation-fibrinolysis derangement. We pharmacologically modulated NSCLC cell lines in co-culture with CD8+ T-cells (TCD8+) and myeloid-derived suppressor cells (MDSCs), isolated from healthy blood donors. The effects of ICIs Nivolumab and Ipilimumab on NSCLC cell death were assessed by annexin V and propidium iodide (PI) flow cytometry analysis. The potential procoagulant properties were analyzed by in vitro clotting assays and enzyme-linked immunosorbent assays (ELISAs). The metabolic remodeling induced by the ICIs was explored by 1H nuclear magnetic resonance (NMR) spectroscopy. Flow cytometry analysis showed that TCD8+ and ICIs increase cell death in H292 and PC-9 cells but not in A549 cells. Conditioned media from NSCLC cells exposed to TCD8+ and ICI induced in vitro platelet aggregation. In A549, Podoplanin (PDPN) levels increased with Nivolumab. In H292, ICIs increased PDPN levels in the absence of TCD8+. In PC-9, Ipilimumab decreased PDPN levels, this effect being rescued by TCD8+. MDSCs did not interfere with the effect of TCD8+ in the production of TF or PDPN in any NSCLC cell lines. The exometabolome showed a metabolic remodeling in NSCLC cells upon exposure to TCD8+ and ICIs. This study provides some insights into the interplay of immune cells, ICIs and cancer cells influencing the coagulation status. ICIs are important promoters of coagulation, benefiting from TCD8+ mediation. The exometabolome analysis highlighted the relevance of acetate, pyruvate, glycine, glutamine, valine, leucine and isoleucine as biomarkers. Further investigation is needed to validate this finding in a cohort of NSCLC patients.

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.

Small molecule inhibitor azd1480 reverses radiotherapy resistance in NSCLC by targeting JAK2/STAT3 pathway

Purpose: To investigate the effect of small molecule inhibitor AZD1480 on radiotherapy resistance in non-small cell lung cancer (NSCLC), and the involvement of Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway in the process.&#x0D; Methods: Radiation-resistant cell lines A549-20F, A549-30F, A549-40F and H460, H46040F, H460- 40F, and H460-40F were established, and expressions of proteins related to JAK/STAT pathway, mitogen-activated protein (MAPK) pathway and transforming growth factor-β (TGF- β) were assayed. The JAK2V617FH460 overexpression cell line and JAK2H460 cell line were established, and expressions of ATGL and CPT1A were compared. The H460 cells and H460-40F cells were treated with JAK2 small molecule inhibitor AZD1480, and the expressions of ATGL, CPT1A and JAK2/STAT3 pathway-related proteins were compared. The survival and proliferation of cell lines were also compared. &#x0D; Results: The JAK/STAT pathway was significantly enriched, and MAPK and TGF-β were up-regulated. In H460 cells, JAK2/STAT3 route was obvious, suggesting that radiotherapy activated JAK2/STAT3 pathway in NSCLC cells. Significant down-regulations of p- JAK2Y1007, JAK2, p-STAT3s727, STAT3, ATGL and CPT1A proteins expressions were seen in H460 + AZD1480 group, relative to H460 group, but protein levels of p-JAK2Y1007, JAK2, p-STAT3s727, STAT3, ATGL and CPT1A were significantly lower in H460-40F + AZD1480 group than in H460-40F group (p &lt; 0.05). The survival and proliferation rates were significantly lower in A549 + AZD1480 group than in A549 and A549-40F groups (p &lt; 0.05).&#x0D; Conclusion: Radiotherapy up-regulates the expressions of ATGL and CPT1A in NSCLC cells by activating the JAK2/STAT3 pathway, while AZD1480, a small molecule inhibitor, reverses the radiation resistance of NSCLC by targeting JAK2/STAT3 pathway and key enzymes of lipid metabolism. Therefore, azd1480 may enhance clinical treatment efficacy in NSCLC patients by reducing radiation resistance.

A Short Post-Reattachment Ultrasensitive Window of Time in Human Cancer Cells as Therapeutic Target of Prolonged Low-Dose Administration of Specific Compounds.

Prolonged low-dose administration (PLDA) of several FDA-approved drugs for noncancer conditions or dietary compounds is associated with a lower incidence of specific types of cancers and with the lower formation of metastasis. However, the underlying mechanism is unknown; there is a discrepancy between the concentration of drugs needed to kill cancer cells in vitro and the actual serum levels (10 and >1000 times lower) found in patients. In this study, we evaluated the hypothesis that clonogenicity may be the target of PLDA. We compared the effect of nigericin (NIG) and menadione (MEN) on the human A549 and H460 lung and MCF-7 and MDA-MB-231 breast cancer cell lines using routine MTT and colony forming assays (CFA). The ability of both NIG and MEN to eliminate 100% of cancer cells was at least 2-10 times more potent in CFA compared to MTT assays. Our results revealed the existence of a short post-reattachment window of time when cancer cells growing at low density are more sensitive to PLDA of specific drugs likely by targeting clonogenic rather than proliferation pathways. This short ultrasensitive window of time (SUSWoT) was cell- and drug-type specific: the SUSWoT for NIG was present in H460, A549, and MDA-MB-231 cells but not evident in MCF-7 cells. Conversely, a similar SUSWoT for MEN was present in MCF-7, MDA-MD-231, and A549 cells but not evident in H460 cells. Our findings partially explain the decreased incidence of specific types of cancer by PLDA of FDA-approved drugs (or dietary compounds) for noncancer conditions.

Identification of New EGFR Inhibitors by Structure-Based Virtual Screening and Biological Evaluation.

Epidermal growth factor receptor (EGFR) inhibitors have been used in clinical for the treatment of non-small-cell lung cancer for years. However, the emergence of drug resistance continues to be a major problem. To identify potential inhibitors, molecular docking-based virtual screening was conducted on ChemDiv and Enamine commercial databases using the Glide program. After multi-step VS and visual inspection, a total of 23 compounds with novel and varied structures were selected, and the predicted ADMET properties were within the satisfactory range. Further molecular dynamics simulations revealed that the reprehensive compound ZINC49691377 formed a stable complex with the allosteric pocket of EGFR and exhibited conserved hydrogen bond interactions with Lys 745 and Asp855 of EGFR over the course of simulation. All compounds were further tested in experiments. Among them, the most promising hit ZINC49691377 demonstrated excellent anti-proliferation activity against H1975 and PC-9 cells, while showing no significant anti-proliferation activity against A549 cells. Meanwhile, apoptosis analysis indicated that the compound ZINC49691377 can effectively induce apoptosis of H1975 and PC-9 cells in a dose-dependent manner, while having no significant effect on the apoptosis of A549 cells. The results indicate that ZINC49691377 exhibits good selectivity. Based on virtual screening and bioassays, ZINC4961377 can be considered as an excellent starting point for the development of new EGFR inhibitors.

NPM3 as an Unfavorable Prognostic Biomarker Involved in Oncogenic Pathways of Lung Adenocarcinoma via MYC Translational Activation.

The nucleoplasmin/nucleophosmin (NPM) family was previously regarded as a critical regulator during disease development, and its mediation in carcinogenesis has achieved intensive attention recently. However, the clinical importance and functional mechanism of NPM3 in lung adenocarcinoma (LUAD) have not been reported yet. This study aimed to investigate the role and clinical significance of NPM3 in the development and progression of LUAD, including the underlying mechanisms. The expression of NPM3 in pan-cancer was analyzed via GEPIA. The effect of NPM3 on prognosis was analyzed by the Kaplan-Meier plotter and the PrognoScan database. In vitro, cell transfection, RT-qPCR, CCK-8 assay, and wound healing assay were employed to examine the role of NPM3 in A549 and H1299 cells. Gene set enrichment analysis (GSEA) was performed using the R software package to analyze the tumor hallmark pathway and KEGG pathway of NPM3. The transcription factors of NPM3 were predicted based on the ChIP-Atlas database. Dual-luciferase reporter assay was applied to verify the transcriptional regulatory factor of the NPM3 promoter region. The NPM3 expression was found to be markedly higher in the LUAD tumor group than the normal group and to be positively correlated with poor prognosis, tumor stages, and radiation therapy. In vitro, the knockdown of NPM3 greatly inhibited the proliferation and migration of A549 and H1299 cells. Mechanistically, GSEA predicted that NPM3 activated the oncogenic pathways. Further, the NPM3 expression was found to be positively correlated with cell cycle, DNA replication, G2M checkpoint, HYPOXIA, MTORC1 signaling, glycolysis, and MYC targets. Besides, MYC targeted the promoter region of NPM3 and contributed to the enhanced expression of NPM3 in LUAD. The overexpression of NPM3 is an unfavorable prognostic biomarker participating in oncogenic pathways of LUAD via MYC translational activation and it contributes to tumor progression. Thus, NPM3 could be a novel target for LUAD therapy.

Tumor-Associated Macrophage-Derived TGF-β1 Activates GLI2 via the Smad2/3 Signaling Pathway to Affect Cisplatin Resistance in Lung Adenocarcinoma.

Transforming growth factor-β1 (TGF-β1) is an immunosuppressive cytokine that is highly expressed in the tumor microenvironment (TME) of lung adenocarcinoma (LUAD). TGF-β1 plays important roles in regulating tumor metastasis and chemotherapy resistance. However, the specific molecular mechanisms by which TGF-β1 regulates cisplatin resistance in the TAM of LUAD remain unclear. THP-1 induced macrophages were co-cultured with A549 and H1975 cells, and subsequently transfected with silencing TGF-β1 (siTGF-β1), GLI2 (siGLI2), a GLI2 overexpression plasmid, and their negative controls. Cellular activity was measured by CCK-8 and colony formation assays. Cell apoptosis was evaluated by flow cytometry and TUNEL staining. Transwell assays were performed to assess cell migration and invasion capabilities. The levels of Smad2/3, GLI2, cyclin D, and cyclin E expression were evaluated by qPCR, western blotting, and immunofluorescence methods. TGF-β1 levels were determined by ELISA. Macrophages suppressed the apoptosis and promoted the migration and invasion of LUAD cells. TAM siTGF-β1 downregulated the Smad2/3 signaling pathways and GLI2 expression, deceased cell proliferation, and promoted apoptosis. SiGLI2 increased apoptosis and decreased the proliferation of LUAD cell lines. GLI2 decreased cisplatin resistance in LUAD cells. High expression of TGF-β1 in the TAM positively activates GLI2 expression via the Smad2/3 pathway, which subsequently regulates cyclin D and cyclin E expression, and promotes the cisplatin resistance of LUAD.

Cancer sensitizing effect of deazaflavin analogs is associated with increased intracellular drug accumulation

As part of our efforts geared towards developing mechanism-based cancer sensitizing agents, we have previously synthesized and characterized novel deazaflavin analogs as potent tyrosyl DNA phosphodiesterase 2 (TDP2) inhibitors for combination treatments with topoisomerase II (TOP2) poisons. Interestingly, the sensitizing effect of a few analogs toward TOP2 poison etoposide (ETP) was associated with a significant increase in intracellular drug accumulation, which could be an alternative mechanism to boost the clinical efficacy of ETP in cancer chemotherapies. Hence, we evaluated more deazaflavin TDP2 inhibitors for their impact on drug retention in cancer cells. We found that all but one tested TDP2 inhibitors substantially increased the ETP retention in DT40 cells. Particularly, we identified an exceptionally potent analog, ZW-1226, which at 3 nM increased the intracellular ETP by 13-fold. Significantly, ZW-1226 also stimulated cellular accumulation of two other anticancer drugs, TOP2 poison teniposide and antifolate pemetrexed, and produced an effect more pronounced than those of ABC transporter inhibitors verapamil and elacridar in human leukemic CCRF-CEM cells toward ETP. Lastly, ZW-1226 potentiated the action of ETP in the sensitive human CCRF-CEM cells and a few resistant non-small-cell lung cancer cells, including H460 and H838 cells. Collectively, the results of this study strongly suggest that deazaflavin analog ZW-1226 could be an effective cancer sensitizing agent which warrants further investigation.

Tanshinone IIA inhibits proliferation and migration by downregulation of the PI3K/Akt pathway in small cell lung cancer cells

BackgroundSmall cell lung cancer (SCLC) is the most malignant lung cancer type. Due to the high rates of metastasis and drug resistance, effective therapeutic strategies remain lacking. Tanshinone IIA (Tan IIA) has been reported to exhibit anti-tumor activity. Therefore, this study investigated the ability and underlying mechanism of Tan IIA to inhibit the metastasis and proliferation of SCLC.MethodsH1688 and H446 cells were treated in vitro with Tan IIA (0, 1, 2 and 4 µM) or LY294002 (10 µM) for 24, 48, 72 h. H1688 and H446 cell migration was evaluated in wound healing and transwell migration assays. RNA-sequencing helped assess gene expression. BALB/c nude mice were injected with H1688 cells and treated with the Tan IIA group (10 mg/kg/day) or a control. Expression of E-cadherin, vimentin and PI3K/Akt signaling pathway proteins in tumors and H1688 was investigated by immunohistochemical analysis and western blot.ResultsTan IIA inhibited H1688 and H446 cell proliferation without inducing apoptosis and suppressed H1688 and H446 cell migration. E-cadherin expression was increased, while vimentin expression was reduced after administration of Tan IIA. RNA-sequencing revealed that some genes related with the PI3K/Akt signaling pathway were altered using Tan IIA treatment. Furthermore, western blot helped detect PI3K and p-Akt expression was also reduced by Tan IIA treatment. Tan IIA inhibited tumor growth in vivo. Moreover, Tan IIA increased tumoral expression of E-cadherin accompanied by PI3K and p-Akt downregulation.ConclusionTan IIA suppresses SCLC proliferation and metastasis by inhibiting the PI3K/Akt signaling pathway, thereby highlighting the potential of Tan IIA as a new and relatively safe drug candidate to treat SCLC.