NCI-H460 Non-small Cell Lung Cancer Research Articles

In-silico and in-vitro functional validation of imidazole derivatives as potential sirtuin inhibitor.

Epigenetic enzymes can interact with a wide range of genes that actively participate in the progression or repression of a diseased condition, as they are involved in maintaining cellular homeostasis. Sirtuins are a family of Class III epigenetic modifying enzymes that regulate cellular processes by removing acetyl groups from proteins. They rely on NAD+ as a coenzyme in contrast to classical histone deacetylases (HDACs) (Class I, II, and IV) that depend on Zn+ for their activation, linking their function to cellular energy levels. There are seven mammalian sirtuin isoforms (Sirt1-7), each located in different subcellular compartments. Sirtuins have emerged as a promising target, given that inhibitors of natural and synthetic sources are highly warranted. Imidazole derivatives are often investigated as sirtuin regulators due to their ability to interact with the binding site and modulate their activity. Imidazole bestows many possible substitutions on its ring and neighboring atoms to design and synthesize derivatives with specific target selectivity and improved pharmacokinetic properties, optimizing drug development. Ligand preparation, protein preparation, molecular docking, molecular dynamics, density function theory (DFT) analysis, and absorption, distribution, metabolism, and excretion (ADME) analysis were performed to understand the interacting potential and effective stability of the ligand with the protein. RT-PCR and Western blot analyses were performed to understand the impact of ligands on the gene and protein expression of Class III HDAC enzymes. We evaluated the sirtuin inhibition activity of our in-house compound comprised of imidazole derivatives by docking the molecules with the protein data bank. ADME properties of all the compounds used in the study were evaluated, and it was found that all fall within the favorable range of being a potential drug. The molecule with the highest docking score was analyzed using DFT, and the specific compound was used to treat the non-small cell lung cancer (NSCLC) cell lines A549 and NCI-H460. The gene and protein expression data support the in-silico finding that the compound Ethyl 2-[5-(4-chlorophenyl)-2-methyl-1-H-Imidazole-4-yl) acetate has an inhibitory effect on nuclear sirtuins. In conclusion, targeting sirtuins is an emerging strategy to combat carcinogenesis. In this study, we establish that Ethyl 2-[5-(4-chlorophenyl)-2-methyl-1-H-Imidazole-4-yl) acetate possesses a strong inhibitory effect on nuclear sirtuins in NSCLC cell lines.

Cytotoxic Activity of Vitex leucoxylon Aqueous Leaf Extract Against A549 and NCIH-460 Lung Cancer Cell Lines

The primary goal of this research was to determine the cytotoxic potential of an aqueous extract of Vitex leucoxylon aerial parts on the lung cancer A549 and non-small cell lung cancer NCIH-460 cell lines. The Soxhlet apparatus and distilled water were used for the extraction of the medicinal plant. The MTT in vitro assay was used to test the compounds for anticancer activity against two different lines of lung cancer. The cytotoxic activity of V. leucoxylon in the present study was very substantial as measured by its ability to suppress the growth of both cell lines. The IC50 values for A549 and NCI-H460 were determined to be 315.57 and 560.48 μg/mL, respectively. The current research confirmed that V. leucoxylon’s aqueous leaf extract possessed potent anticancer properties. Both the cancer cell lines were significantly inhibited in their ability to differentiate when subjected to the MTT assay. In future, flavonoid compounds in the aqueous extract need to be purified, characterized, and structurally elucidated for in-vivo studies, which could lead to the creation of new drug candidates.

Abstract 3091: Discovery and development of a potent and highly selective WEE1 inhibitor IMP7068

Abstract WEE1 is activated upon DNA damage and regulates the G2/M cell cycle checkpoints. Inhibition of WEE1, in conjunction with genetic alterations and/or a DNA damaging agent, could result in mitotic catastrophe and apoptosis of cancer cells, offering an attractive approach to treat cancer. Small molecule WEE1 inhibitors, such as AZD1775, showed good clinical POC (proof of concept) data in several tumors including ovarian, colon, and uterine carcinoma. We have identified a novel, potent, and highly selective WEE1 inhibitor IMP7068. Herein we will present its discovery and IND enabling in vitro and in vivo studies. IMP7068 exhibits superior WEE1/PLK1 selectivity (>435 fold) compared to AZD1775. IMP7068 also shows potent cytotoxicity against a wide range of cancer cell lines. In addition, IMP7068 has desirable PK profiles with long half-life and high exposure in preclinical species. Anti-tumor efficacy of IMP7068 has been demonstrated in mice CDX models of colorectal LoVo, non-small cell lung cancer NCI-H1299 and a PDX model of uterine UT5318 with good dose-response tumor inhibition and tolerability. IMP7068 is currently in a phase I study to evaluate its safety, tolerability, pharmacokinetics, and anti-tumor activity in patients with advanced solid tumors (ClinicalTrials.gov Identifier: NCT04768868). Citation Format: Sui Xiong Cai, Ning Ma, Xiaozhu Wang, Yangzhen Jiang, Hongxia Zhang, Mingchuan Guo, Ruiyu Zhou, Ye Edward Tian. Discovery and development of a potent and highly selective WEE1 inhibitor IMP7068 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3091.

In vitro antioxidant, anti-inflammatory, and anticancer activities of mixture Thai medicinal plants

BackgroundThe phytochemical study of medicinal plants is rapidly gaining popularity with many pharmacologic effects. This study aims to determine the antioxidant capacity as well as anticancer and antimigration activities of Clear belongs Plus extract (CBL-P) which consisted of five medicinal plants namely, Alpinia galanga, Piper nigrum, Citrus aurantifolia, Tiliacora triandra, and Cannabis sativa on human colon cancer cells SW620 and HCT116 cell lines, and human non-small cell lung cancer cells A549 and NCI-H460 cell lines.MethodsIn this study the dried-plant powder was extracted using 90% ethanol. Additionally, CBL-P was studied antioxidative activity via DPPH and ABTS assays and anti-inflammatory activities using nitric oxide assay using Griess reaction. Antiproliferation and antimigration of CBL-P were investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and scratch assay.ResultsThe results showed that CBL-P had potent antiproliferative activity with IC50 values in a concentration- and time-dependent manners for all four cell lines. CBL-P also possessed potent antimigration activity against all studied cancer cells. CBL-P demonstrated antimigration activity on four different types of cancer cells (A549, NCI-H460, HCT116, and SW620) after 48 h of incubation, with the greatest effect seen at the highest concentration tested (15 μg/mL) in A549 cells (10.23% of wound closure) and NCI-H460 cells (9.16% of wound closure). CBL-P was also effective in reducing migration in HCT116 and SW620 cells, with a range of closure area from 10—50%. In addition, CBL-P had antioxidant activity with IC50 values of 8.549 ± 0.241 mg/mL and 2.673 ± 0.437 mg/mL for DPPH and ABTS assays, respectively. CBL-P also showed anti-inflammatory activity with the best inhibitory activity on NO production at a concentration of 40 μg/mL.ConclusionIn conclusion, the mixture extract possessed antioxidant and anti-inflammatory activity. Furthermore, the mixture plant extract significantly exhibited antiproliferative and antimigration activities on SW620, HCT116, A549, and NCI-H460 cells (P ≤ 0.05). Taken together, our results suggest that medicinal plants may have synergistic effects that could potentially enhance the effectiveness of cancer treatment when used as adjuvants. These findings provide a solid scientific foundation for future efforts to explore the mechanism of action.

Tumor killing by a dietary curcumin mono-carbonyl analog that works as a selective ROS generator via TrxR inhibition

In comparison with normal cells, cancer cells feature intrinsic oxidative stress, thereby being more vulnerable to further production of reactive oxygen species (ROS) by pro-oxidative anticancer agents (PAAs). However, PAAs also inevitably generate ROS in normal cells, resulting in their narrow therapeutic window and toxic side effects that greatly limit their clinical application. To develop PAAs that generate ROS selectively in cancer cells over in normal cells, we rationally designed three series of 21 dietary curcumin 5-carbon mono-carbonyl analogs differentiated by either placement of the cyclohexanone, piperidone, and methylpiperidone linkers, or introduction of electron-withdrawing trifluoromethyl and electron-donating methoxyl groups on its two aromatic rings in the ortho, meta, or para position to the linkers. From the designed molecules, 2c, characterized of the presence of the meta-CF3-substituted mode and the piperidone linker, was identified as a potent selective ROS-generating agent, allowing its ability to kill selectively human non-small cell lung cancer NCI–H460 (IC50 = 0.44 μM) over human normal lung MRC-5 cells with a selectivity index of 32.0. Additionally, it was more potent and selective than the conventional chemotherapeutic agents (5-fluorouracil and camptothecin) did. Mechanistical investigation reveals that by means of its Michael acceptor unit and structure characteristics as described above, 2c could covalently modify the Sec-498 residue of intracellular thioredoxin reductase (TrxR) to generate ROS selectively, resulting in ROS-dependent apoptosis and ferroptosis of NCI–H460 cells. Noticeably, 2c inhibited significantly the growth of NCI–H460 cell xenograft tumor in nude mice without obvious toxicity to liver and kidney. Together, this work highlights a practical strategy of targeting TrxR overexpressed in cancer cells to develop PAAs capable of generating ROS selectively, as evidenced by the example of 2c.

Exosomal epidermal growth factor receptor is involved in HPV-16 E7-induced epithelial-mesenchymal transition of non-small cell lung cancer cells: A driver of signaling in vivo?

ABSTRACT Our previous studies have demonstrated that human papillomavirus (HPV)-16 E7 oncoprotein promoted epithelial-mesenchymal transition (EMT) in non-small cell lung cancer (NSCLC) cells. Moreover, recent studies have found that exosomes can mediate EMT of NSCLC cells and epidermal growth factor receptor (EGFR) is related to the progression of NSCLC. Here, we further investigated the role of exosomal EGFR in HPV-16 E7-induced EMT of NSCLC cells. Our results showed that the exosomes derived from the stable HPV-16 E7-overexpressing A549 and NCI-H460 NSCLC cells (E7 Exo) significantly increased migration, invasion, and proliferation abilities of NSCLC cells as compared with the exosomes derived from empty vector-infected NSCLC cells (ev Exo). Moreover, both in vitro and in vivo results demonstrated that E7 Exo dramatically enhanced EMT of NSCLC cells and promoted the growth of subcutaneous NSCLC xenografts. Additionally, HPV-16 E7 enhanced the expression of EGFR and p-EGFR in both NSCLC cells and exosomes. Furthermore, the inhibition of EGFR activation or exosome secretion suppressed E7 Exo-induced migration, invasion, and EMT of NSCLC. Moreover, 12 kinds of differentially expressed miRNAs between E7 Exo and ev Exo (fold change≥2, P ≤ .05) were screened out, of which 7 miRNAs were up-regulated while 5 miRNAs were down-regulated in A549 E7 Exo. Taken together, our findings suggest that exosomal EGFR is involved in HPV-16 E7-induced EMT of NSCLC cells, which may play a key role in the progression of HPV-related NSCLC.

Abstract 2596: Impact of novel Ku-DNA binding inhibitors on the DNA DSBs-induced DNA damage response

Abstract The DNA-dependent protein kinase (DNA-PK) plays a critical role in the non-homologous end joining (NHEJ) double-strand break (DSB) repair pathway and the DNA damage response (DDR). Consequently, blocking DNA-PK kinase activity is a novel anti-cancer therapeutic strategy in combination with ionizing radiation (IR). Towards developing a new class of DNA-PK inhibitors, our laboratory has exploited the mechanism of DNA-PK activation which requires binding to DNA termini via the Ku 70/80 heterodimer. We have previously reported the development of Ku-DNA binding inhibitors (Ku-DBi’s) that act via this novel mechanism of action to inhibit DNA-PK catalytic kinase activity. Ku-DBi’s display nanomolar activity in vitro, possess cellular DNA-PK and NHEJ inhibitory activity and sensitize non-small cell lung cancer (NSCLC) cells to DSB generating chemotherapeutics bleomycin and etoposide. In this study, we demonstrate that pre-incubation of our novel Ku-DBi’s can potentiate the cellular effects of bleomycin and IR and also induce p53 phosphorylation through the activation of the ATM pathway, which is concomitant with a decrease in DNA-PKcs autophosphorylation events at the S2056 (pS2056) cluster. Using a combination of Western blot and immunofluorescence assays in the NSCLC NCI-H460 and A549 cell lines, Ku-DBi’s treatment in combination with DNA DSBs-inducing agents such as bleomycin and IR, showed a significant reduction of autophosphorylation events of DNA-PKcs at the S2056 cluster compared to DNA DSBs-inducing agent alone. In addition, analysis of phospho-ATM and phospho-p53 protein levels in these NSCLC cell lines, suggested activation of the ATM pathway as a function of Ku-DBi’s treatment followed by bleomycin, evidenced by an increase of phosphorylation of ATM at Ser1981, and a modest increase of p53 phosphorylation at Ser15. Our findings demonstrate that Ku-DBi’s block DNA-DSB dependent DNA-PKcs autophosphorylation, resulting in potentiating cellular sensitivity to bleomycin and IR, and a likely effect on the ATM-dependent signaling pathway. These data are consistent with Ku-DBi’s possessing a novel mechanism of action that abrogates autophosphorylation of DNA-PKcs to impact DSB repair and potentially DDR signaling, becoming in a promising approach as part of an anticancer therapeutic strategy in combination with DNA DSBs-inducing agents. Citation Format: Pamela L. Mendoza-Munoz, Navnath S. Gavande, Pamela S. VanderVere-Carozza, Katherine S. Pawelczak, Joseph R. Dynlacht, Joy E. Garrett, John J. Turchi. Impact of novel Ku-DNA binding inhibitors on the DNA DSBs-induced DNA damage response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2596.

Pirfenidone Sensitizes NCI-H460 Non-Small Cell Lung Cancer Cells to Paclitaxel and to a Combination of Paclitaxel with Carboplatin.

Pirfenidone, an antifibrotic drug, has antitumor potential against different types of cancers. Our work explored whether pirfenidone sensitizes non-small cell lung cancer (NSCLC) cell lines to chemotherapeutic treatments. The cytotoxic effect of paclitaxel in combination with pirfenidone against three NSCLC cell lines (A549, NCI-H322 and NCI-H460) was evaluated using the sulforhodamine B assay. The effects of this combination on cell viability (trypan blue exclusion assay), proliferation (BrdU incorporation assay), cell cycle (flow cytometry following PI staining) and cell death (Annexin V-FITC detection assay and Western blot) were analyzed on the most sensitive cell line (NCI-H460). The cytotoxic effect of this drug combination was also evaluated against two non-tumorigenic cell lines (MCF-10A and MCF-12A). Finally, the ability of pirfenidone to sensitize NCI-H460 cells to a combination of paclitaxel plus carboplatin was assessed. The results demonstrated that pirfenidone sensitized NCI-H460 cells to paclitaxel treatment, reducing cell growth, viability and proliferation, inducing alterations in the cell cycle profile and causing an increase in the % of cell death. Remarkably, this combination did not increase cytotoxicity in non-tumorigenic cells. Importantly, pirfenidone also sensitized NCI-H460 cells to paclitaxel plus carboplatin. This work highlights the possibility of repurposing pirfenidone in combination with chemotherapy for the treatment of NSCLC.

Abstract P001: The anti-angiogenic antibody BD0801 demonstrates better anti-tumor activity than bevacizumab and synergize with chemotherapies in multiple tumor models

Abstract Vascular endothelial growth factor (VEGF) and its receptor (VEGFR) play a fundamental role in tumor growth and angiogenesis. An increasing number of evidences have suggested enhanced anti-tumor activity when combining anti-angiogenic agents with chemotherapies in the clinic. To support the clinical development of BD0801, a humanized rabbit anti-VEGF monoclonal antibody, we assessed anti-tumor activities of BD0801 using a series of preclinical tumor mouse models including human ovarian cancer OVCAR-8 and SK-OV-3, human non-small cell lung cancer (NSCLC) NCI-H460, and human renal cancer Caki-1 models, with bevacizumab being used as a benchmark. We also investigated the pharmacological kinetics (PK) of BD0801 in cynomolgus monkeys. BD0801 or bevacizumab were intravenously injected into tumor bearing mice at 0.8 mg/kg, 2.5 mg/kg or 7.5 mg/kg twice a week. Combinations of chemotherapy and BD0801 or bevacizumab were also investigated in some of the tumor models. BD0801 presented more potent anti-tumor activities than bevacizumab in ovarian cancer, NSCLC and renal cancer mouse models in a dose-dependent manner. The tumor growth inhibition of BD0801 was 2-20%, 34-66%, or 32-84% when dosed at 0.8 mg/kg, 2.5 mg/kg or 7.5 mg/kg, respectively. Besides, combination of 2.5 mg/kg BD0801 and 10 mg/kg paclitaxel presented a statistically stronger antitumor activity in human ovarian cancer models compared to both single treatments (P<0.05). Combination of 2.5 mg/kg BD0801, 10 mg/kg paclitaxel, and 80 mg/kg carboplatin also presented a stronger antitumor activity than BD0801 or chemotherapy alone in the human NSCLC model. In cynomolgus monkeys received a single intravenous injection of BD0801 at 1.5, 5, and 15 mg/kg, the half-life (t1/2) of BD0801 was 39.4-142 h, the clearance (Cl) was 0.321-0.900 mL/h/kg, and the apparent volume of distribution (Vd) was 49.5-64.4 mL/kg. The peak concentrations (Cmax) of BD0801 were proportional to the doses administered. No difference on the PK parameters was observed between female and male monkeys. Taken together, this study has demonstrated BD0801 as a more potent molecule than bevacizumab in some preclinical tumor models, supporting further clinical development plans for BD0801, especially in combination with chemotherapeutics. Citation Format: Liting Xue, Jianxing Tang, Yuyin Ding, Lei Song, Shansen Xu, Yue Huang, Yan Wu, Wenjie Song, Renhong Tang, Wenqing Yang. The anti-angiogenic antibody BD0801 demonstrates better anti-tumor activity than bevacizumab and synergize with chemotherapies in multiple tumor models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P001.

Structural characterisation and antitumor activity against non-small cell lung cancer of polysaccharides from Sanghuangporus vaninii

The medicinal fungus Sanghuangporus vaninii can be cultivated in large scale and has outstanding antitumour activity. In this study, water-soluble S. vaninii polysaccharides (SVPs) were extracted from fruiting bodies. Four polysaccharide sub-fractions (SVP-W, SVP-1, SVP-2 and SVP-3) were isolated, with molecular weights from 90.50 kDa to 261.70 kDa, and all inhibited the proliferation of non-small cell lung cancer cell lines A549, 95-D and NCI-H460, especially the acidic SVP-1. SVP-1 affected cell morphology and colony formation in NCI-H460 cells. It also promoted cell apoptosis following nuclear fluorescence staining and flow cytometry. Methylation and nuclear magnetic resonance analyses revealed that SVP-1 is a heteroglycan with the main chain →4)-β-D-Glcp-(1 → 6)-β-D-Glcp-(1 → 6)-α-D-Galp-(1 → 6)-β-D-Glcp-(1→, and the branched chain α-D-Manp-(1 → 2)-α-D-Manp-(1 → 3)-β-D-Glcp-(1 → 3,6)-β-D-Glcp-(1→. The findings indicate that this natural acidic polysaccharide has potential for non-small cell lung cancer therapy.

In vivo targeting of lentiviral vectors pseudotyped with the Tupaia paramyxovirus H glycoprotein bearing a cell-specific ligand

Despite their exceptional capacity for transgene delivery ex vivo, lentiviral (LV) vectors have been slow to demonstrate clinical utility in the context of in vivo applications. Unresolved safety concerns related to broad LV vector tropism have limited LV vectors to ex vivo applications. Here, we report on a novel LV vector-pseudotyping strategy involving envelope glycoproteins of Tupaia paramyxovirus (TPMV) engineered to specifically target human cell-surface receptors. LV vectors pseudotyped with the TPMV hemagglutinin (H) protein bearing the interleukin (IL)-13 ligand in concert with the TPMV fusion (F) protein allowed efficient transduction of cells expressing the human IL-13 receptor alpha 2 (IL-13Rα2). Immunodeficient mice bearing orthotopically implanted human IL-13Rα2 expressing NCI-H1299 non-small cell lung cancer cells were injected intravenously with a single dose of LV vector pseudotyped with the TPMV H-IL-13 glycoprotein. Vector biodistribution was monitored using bioluminescence imaging of firefly luciferase transgene expression, revealing specific transduction of tumor tissue. A quantitative droplet digital PCR (ddPCR) analysis of lung tissue samples revealed a >15-fold increase in the tumor transduction in mice treated with LV vectors displaying IL-13 relative to those without IL-13. Our results show that TPMV envelope glycoproteins can be equipped with ligands to develop targeted LV vectors for in vivo applications.

Abstract PO-023: Impact of a novel Ku-DNA binding inhibitor on the IR-induced DNA damage response

Abstract The DNA-dependent protein kinase (DNA-PK) plays a critical role in the non-homologous end joining (NHEJ) double stand break (DSB) repair pathway and the DNA damage response (DDR). Consequently, blocking DNA-PK kinase activity is a novel anti-cancer therapeutic strategy in combination with ionizing radiation (IR). Towards developing a series of a new class of DNA-PK inhibitors, our laboratory has exploited the mechanism of DNA-PK activation which requires binding to DNA termini via the Ku 70/80 heterodimer. We recently reported the development of Ku-DNA binding inhibitors (Ku-DBi’s) that act via this novel mechanism of action to inhibit DNA-PK catalytic kinase activity. Ku-DBi’s display nanomolar activity in vitro, possess cellular DNA-PK and NHEJ inhibitory activity and sensitize non-small cell lung cancer (NSCLC) cells to chemotherapeutics bleomycin and etoposide. In this study, we demonstrate that pre-incubation of our novel Ku-DBi’s can potentiate the cellular effects of IR. We demonstrate that the increase in IR sensitivity is preceded by a decrease in DNA-PKcs autophosphorylation events at the S2056 (pS2056) cluster. Using a combination of Western blot and immunofluorescence assays in the NSCLC NCI-H460 cell line, Ku-DBi 245 treatment in combination with IR showed a significant reduction of phosphorylation of DNA-PKcs at S2056 compared to IR alone. Analysis of gH2A.X also showed an inhibition of the DDR as a function of 245 treatment, evidenced by reduction of S139 phosphorylation. These results demonstrate that Ku-DBi 245 blocks DNA-break dependent DNA-PKcs autophosphorylation, resulting in an increased in radiation sensitivity. These data are consistent with Ku-DBi’s possessing a novel mechanism of action that abrogates autophosphorylation of DNA-PKcs and DDR signaling as part of an anticancer therapeutic strategy in combination with IR. Citation Format: Pamela Mendoza-Munoz, Navnath S. Gavande, Pamela S. VanderVere-Carozza, Katherine S. Pawelczak, Joseph R. Dynlacht, Joy Garrett, John J. Turchi. Impact of a novel Ku-DNA binding inhibitor on the IR-induced DNA damage response [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-023.

Cytochalasin H isolated from mangrove-derived endophytic fungus inhibits epithelial-mesenchymal transition and cancer stemness via YAP/TAZ signaling pathway in non-small cell lung cancer cells.

Our previous studies have isolated cytochalasin H (CyH) from endophytic fungus derived from mangrove and found that CyH induced apoptosis and inhibited migration and angiogenesis in non-small cell lung cancer (NSCLC) cells. In this study, we further investigated the effect of CyH on epithelial-mesenchymal transition (EMT) and cancer stemness of A549 and NCI-H460 NSCLC cells and the underlying mechanisms, especially the role of YAP/ TAZ signaling pathway in the process. Our results showed that CyH significantly inhibited invasive ability and the sphere formation of NSCLC cells. The expression of E-cadherin, an EMT epithelial marker, was obviously up-regulated, while the expression of Vimentin and N-cadherin, the EMT mesenchymal markers, was dramatically down-regulated by CyH treatment in NSCLC cells. Moreover, the expression of EMT-associated transcription factors including Slug, Twist1, and Snail1 and stemness markers including Nanog, Sox-2, and Oct-4 was significantly down-regulated by CyH treatment in NSCLC cells. Additionally, CyH significantly down-regulated YAP and TAZ expression and up-regulated LAST1/2 and MST1/2 expression, and CyH inhibited the interaction between YAP and TEAD. Furthermore, YAP knockdown abolished the effect of CyH on the expression of EMT- and stemness-related markers in NSCLC cells. Taken together, these results suggest that CyH inhibits EMT and cancer stemness of NSCLC cells via the regulation of YAP/TAZ signaling pathway.

Establishment and Characterization of a Topotecan Resistant Non-small Cell Lung Cancer NCI-H460/TPT10 Cell Line

While topotecan (TPT) is a first- and second-line chemotherapeutic drug in treating lung cancer, the development of drug resistance in tumors still reserves as a major obstacle to chemotherapeutic success. Therefore, a better understanding of the mechanisms of topotecan resistance is critical. In this study, the first topotecan-resistant human non-small cell lung cancer (NSCLC) cell line, termed NCI-H460/TPT10, was established from the parental NCI-H460 cell line. NCI-H460/TPT10 cells exhibited a 394.7-fold resistance to TPT, and cross-resistance to SN-38, mitoxantrone, and doxorubicin, compared to parental NCI-H460 cells. Overexpression of ABCG2 localized on the cell membrane, but not ABCB1 or ABCC1, was found in NCI-H460/TPT10 cells, indicating that ABCG2 was likely to be involved in topotecan-resistance. This was confirmed by the abolishment of drug resistance in NCI-H460/TPT10 cells after ABCG2 knockout. Moreover, the involvement of functional ABCG2 as a drug efflux pump conferring multidrug resistance (MDR) was indicated by low intracellular accumulation of TPT in NCI-H460/TPT10 cells, and the reversal effects by ABCG2 inhibitor Ko143. The NCI-H460/TPT10 cell line and its parental cell line can be useful for drug screening and developing targeted strategies to overcome ABCG2-mediated MDR in NSCLC.

Screening and Identification of the Peptides Specifically Binding to Human Non-small Cell Lung Cancer NCI-H1299 Cells

背景与目的非小细胞肺癌（non-small cell lung cancer, NSCLC）是最常见的肺癌组织学类型，也是病死率最高的恶性肿瘤之一。多肽作为光学分子成像探针的主体可以实现肿瘤的早期筛查及诊断，提高患者存活率。本研究旨在利用体内噬菌体展示技术筛选与人NSCLC细胞NCI-H1299高度结合的小分子多肽并通过体外实验鉴定其结合特异性。方法制备NCI-H1299细胞荷瘤裸鼠模型，用噬菌体展示环七肽库进行3轮体内筛选后随机挑取噬菌体克隆，免疫组织化学法及酶联免疫吸附法（enzyme-linked immunosorbent assay, ELISA）鉴定噬菌体克隆对NCI-H1299细胞的亲和力。提取阳性单克隆噬菌体DNA测序获得外源多肽氨基酸序列，将重复率最高的序列化学合成多肽并进行异硫氰酸荧光素（fluorescein, FITC）标记，制备光学分子探针，初步鉴定其对NCI-H1299细胞的特异性。结果经3轮体内筛选后噬菌体富集率是首轮的341.3倍；免疫组织化学染色显示随着筛选次数增加，肿瘤组织中结合的噬菌体不断增加，且结合量明显高于正常组织；ELISA结果显示随机挑取的30个噬菌体克隆中20个为阳性克隆，经测序后将重复率最高的序列合成多肽并命名为NSP1；四甲基偶氮唑盐比色法（methyl thiazolyl tetrazolium assay, MTT）、细胞划痕实验表明NSP1不会影响细胞增殖、迁移；流式细胞术、细胞免疫荧光结果表明NSP1可与NCI-H1299细胞特异性结合。结论利用体内噬菌体展示技术成功得到了与肺癌NCI-H1299细胞特异性结合的多肽NSP1，为NSCLC的早期诊断及靶向治疗奠定了研究基础。

A novel orally active microtubule destabilizing agent S-40 targets the colchicine-binding site and shows potent antitumor activity.

The tubulin colchicine binding site has been recognized as an attractive drug target to combat cancer, but none of the candidate drugs have been approved for medical treatment. We recently identified a structurally distinct small molecule S-40 as an oral potent tubulin destabilizing agent. Crystal structure analysis of S-40 in a complex with tubulin at a resolution of 2.4Å indicated that S-40 occupies all 3 zones in the colchicine pocket with interactions different from known microtubule inhibitors, presenting unique effects on assembly and curvature of tubulin dimers. S-40 overcomes paclitaxel resistance and lacks neurotoxicity, which are the main obstacles limiting clinical applications of paclitaxel. Moreover, S-40 harbors the ability to inhibit growth of cancer cell lines as well as patient-derived organoids, induce mitotic arrest and cell apoptosis. Xenograft mouse models of human prostate cancer DU145, non-small cell lung cancer NCI-H1299 and paclitaxel-resistant A549 were strongly restrained without apparent side effects by S-40 oral administration once daily. These findings provide evidence for the development of S-40 as the next generation of orally effective microtubule inhibitors for cancer therapy.

Effect of Huaier aqueous extract on growth and metastasis of human non-small cell lung cancer NCI-H1299 cells and its underlying mechanisms

This study aims to investigate the effect of Huaier aqueous extract on the growth and metastasis of human non-small cell lung cancer NCI-H1299 cells and its underlying mechanisms. MTT assay was used to detect the effect of Huaier aqueous extract on the proliferation of NCI-H1299 cells. Flow cytometry was used to examine the effect of Huaier aqueous extract on the apoptosis, cell cycle, and ROS level of NCI-H1299 cells. Wound healing assay was used to evaluate the effect of Huaier aqueous extract on the migration ability of NCI-H1299 cells. Western blot was used to detect the levels of proteins involving apoptosis, epithelial-mesenchymal transition(EMT), and MAPK signaling pathway in NCI-H1299 cells exposed to Huaier aqueous extract. The results showed that Huaier aqueous extract inhibited the proliferation of NCI-H1299 cells, and induced cell-cycle arrest at the phase S. Huaier aqueous extract promoted the apoptosis of NCI-H1299 cells by down-regulating the expression of anti-apoptotic protein Bcl-2. Moreover, Huaier aqueous extract increased ROS level and induced ferroptosis in NCI-H1299 cells. EMT played a critical role in cancer metastasis. Huaier aqueous extract reduced the migration ability of NCI-H1299 cells by inhibiting EMT of NCI-H1299 cells. In addition, this study revealed that Huaier aqueous extract inhibited MAPK signaling pathway in human non-small cell lung cancer NCI-H1299 cells, which may be one of Huaier's mechanisms in inhibiting growth and metastasis of NCI-H1299 cells. This study provides a new theoretical basis for the clinical treatment of lung cancer with Huaier, and important reference significance for further studies on the anti-tumor mechanisms of Huaier.

ALCAM contributes to brain metastasis formation in non-small-cell lung cancer through interaction with the vascular endothelium.

BackgroundBrain metastasis (BM) in non-small-cell lung cancer (NSCLC) has a very poor prognosis. Recent studies have demonstrated the importance of cell adhesion molecules in tumor metastasis. The aim of our study was to investigate the role of activated leukocyte cell adhesion molecule (ALCAM) in BM formation in NSCLC.MethodsImmunohistochemical analysis was performed on 143 NSCLC primary tumors and BM. A correlation between clinicopathological parameters and survival was developed. Biological properties of ALCAM were assessed in vitro by gene ablation using CRISPR/Cas9 technology in the NCI-H460 NSCLC cell line and in vivo by intracranial and intracardial cell injection of NCI-H460 cells in NMRI-Foxn1nu/nu mice.ResultsALCAM expression was significantly upregulated in NSCLC brain metastasis (P = 0.023) with a de novo expression of ALCAM in 31.2% of BM. Moderate/strong ALCAM expression in both primary NSCLC and brain metastasis was associated with shortened survival. Functional analysis of an ALCAM knock-out (KO) cell line showed a significantly decreased cell adhesion capacity to human brain endothelial cells by 38% (P = 0.045). In vivo studies showed significantly lower tumor cell dissemination in mice injected with ALCAM-KO cells in both mouse models, and both the number and size of BM were significantly diminished in ALCAM depleted tumors.ConclusionsOur findings suggest that elevated levels of ALCAM expression promote BM formation in NSCLC through increased tumor cell dissemination and interaction with the brain endothelial cells. Therefore, ALCAM could be targeted to reduce the occurrence of BM.Key Points1. ALCAM expression associates with poor prognosis and brain metastasis in NSCLC.2. ALCAM mediates interaction of NSCLC tumor cells with brain vascular endothelium.3. ALCAM might represent a novel preventive target to reduce the occurrence of BM in NSCLC.

The role of monoamine oxidase A in HPV-16 E7-induced epithelial-mesenchymal transition and HIF-1α protein accumulation in non-small cell lung cancer cells.

Our previous studies have found that human papillomavirus (HPV)-16 E7 oncoprotein promotes epithelial-mesenchymal transition (EMT) and hypoxia-inducible factor-1α (HIF-1α) protein accumulation in non-small cell lung cancer (NSCLC) cells and monoamine oxidase A (MAOA) is highly expressed in NSCLC tissues. Here, we further explored the role of MAOA in HPV-16 E7-induced EMT and HIF-1α protein accumulation in A549 and NCI-H460 NSCLC cells. Our results showed that HPV-16 E7 enhanced MAOA expression in NSCLC cells. Additionally, MAOA knockout inhibited HPV-16 E7-induced migration, invasion, and EMT, and significantly reduced HPV-16 E7-induced ROS generation and HIF-1α protein accumulation via promoting its degradation. Furthermore, MAOA knockout suppressed HPV-16 E7-induced ERK1/2 activation. In vivo, MAOA knockout inhibited tumor growth, metastasis, and the expression of EMT-related markers and HIF-1α proteins induced by HPV-16 E7 in NCI-H460 NSCLC subcutaneous xenograft and in situ intrapulmonary models of nude mice. Taken together, our findings provide evidence that MAOA plays a key role in EMT and HIF-1α protein accumulation induced by HPV-16 E7 in NSCLC cells, suggesting that MAOA may be a potential therapeutic target for HPV-related NSCLC.

Ganoderic acid DM induces autophagic apoptosis in non-small cell lung cancer cells by inhibiting the PI3K/Akt/mTOR activity

The incidence and mortality of lung cancer are the highest among cancer-related deaths. However, the long-term use of currently available cytotoxic drugs can increase genetic alterations in cancer cells and cause drug-resistance, which significantly limits their usage. Since current systemic treatment options are limited, effective chemotherapeutic agents are urgently needed for non-small cell lung cancer (NSCLC) treatment. In this study, we demonstrated that ganoderic acid DM (GA-DM) could increase apoptosis in A549 and NCI–H460 NSCLC cells. GA-DM treatment decreased the protein expression levels of Bcl-2 and increased the expression levels of Bax, cleaved caspase-3 and cleaved PRAP. Furthermore, GA-DM could promote autophagic flux, and the cytotoxic effect against cancer cells of GA-DM was significantly inhibited by targeted suppression of autophagy, suggesting that autophagy contributed to GA-DM-induced cell death in NSCLC. Moreover, GA-DM clearly induced autophagy by inactivating the PI3K/Akt/mTOR pathway. When overexpression of Akt reactivated Akt/mTOR pathway in A549 or NCI–H460 cells, the increase of autophagy related marker LC3B-II and apoptosis related protein cleaved PARP and cleaved caspase 3 and the ration of apoptotic cells by GA-DM was reversed, suggesting that GA-DM promoted autophagy and apoptosis by inhibiting Akt/mTOR pathway-mediated autophagy induction. In conclusion, our study indicated that GA-DM can induce autophagic apoptosis in NSCLC by inhibiting Akt/mTOR activity. (209 words).