Treatment Of A549 Research Articles

Abstract PR015: Discovery of LY4050784 (FHD-909), a selective BRM (SMARCA2) ATPase inhibitor for the treatment of BRG1(SMARCA4) mutant cancers

Abstract BRM (SMARCA2) and BRG1 (SMARCA4) are highly homologous ATPases that are members of the BAF (also known as the mSWI/SNF) chromatin remodeling complex. BRM and BRG1 are mutually exclusive enzymatic subunits of BAF complexes, and functional genomic screens have shown a synthetic lethal relationship between the two genes. BRG1 is frequently mutated in cancer, including in approximately 10% of non-small cell lung carcinomas. Selective inhibition of BRM is a mechanism by which BRG1-mutated cancer cell growth would be affected by losing BRM function, while normal tissues should be spared as they still express functional BRG1. Given that the ATPase domains of BRM and BRG1 are 92% identical, the identification of selective enzymatic inhibitors of BRM has been challenging. Here, we report the discovery of a novel, highly potent compound that selectively inhibits BRM over BRG1 and that also possesses excellent oral pharmacokinetics. LY4050784 (aka FHD-909) inhibits BRM in cell-based transcriptional and proliferation assays and is greater than 30-fold selective over BRG1. It is also selective against other helicases and does not show any significant activity in off-target screening panels. Dosing of mice carrying BRG1-mutant xenografts causes target gene modulation that is correlated with compound exposure. Treatment of A549 and RERF-LC-AI xenografts with LY4050784 led to tumor growth inhibition of 87% and 96%, respectively, at well-tolerated doses, and significant tumor growth inhibition, including regression, was also achieved in BRG1-mutant models NCI-2126 and NCI-1793. The results suggest that our compound has first-in-class potential as a potent and selective BRM ATPase inhibitor for the treatment of BRG1-mutated cancers, and we are planning to file an IND in Q2 of 2024. Citation Format: Janice Y. Lee, Nathan Brooks, Brandon Antonakos, Bryan Perria, Candace Langan, Bonita D. Jones, Robert Stephen Flack, Zhifang Li, David Terry, Ross Wallace, Robert Bondi, Gereint Sis, Ronee Baracani, Maralee McVean, Gabrielle Kolakowski, Dayo Osimboni, Kevin Wilson. Discovery of LY4050784 (FHD-909), a selective BRM (SMARCA2) ATPase inhibitor for the treatment of BRG1(SMARCA4) mutant cancers [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr PR015.

Abstract 3230: Discovery of selective BRM (SMARCA2) ATPase inhibitors for the treatment of BRG1 (SMARCA4) mutant cancers

Abstract BRM (SMARCA2) and BRG1 (SMARCA4) are highly homologous ATPases that are members of the BAF (also known as the mSWI/SNF) chromatin remodeling complex. BRM and BRG1 are mutually exclusive enzymatic subunits of BAF complexes, and functional genomic screens have shown a synthetic lethal relationship between the two genes. BRG1 is frequently mutated in cancer, including in approximately 10% of non-small cell lung carcinomas. Selective inhibition of BRM is a mechanism by which BRG1-mutated cancer cell growth would be affected by losing BRM function, while normal tissues should be spared as they still express functional BRG1. Given that the ATPase domains of BRM and BRG1 are 92% identical, the identification of selective enzymatic inhibitors of BRM has been challenging. Here, we report the discovery of novel, highly potent compounds that selectively inhibit BRM over BRG1 as seen in cell-based transcription and proliferation assays, that also possess excellent oral pharmacokinetics. Our lead compounds inhibit BRM in a cell-based reporter assay with an unbound IC50 of 1 nM and are greater than 20-fold selective over BRG1. They are also selective against other helicases and do not show any significant activity in off-target screening panels. Dosing of mice carrying BRG1-mutant xenografts causes target gene modulation that is correlated with compound exposure. Treatment of A549 and RERF-LC-AI xenografts with lead compounds led to tumor growth inhibition of 87% and 96%, respectively, at well-tolerated doses, and significant tumor growth inhibition was also achieved in multiple other BRG1-mutant models. The results suggest that our compounds have first-in-class potential as potent and selective BRM ATPase inhibitors for the treatment of BRG1-mutated cancers, and we are planning to file an IND in 2024. Citation Format: Janice Y. Lee, Nathan Brooks, Brandon Antonakos, Bryan Perria, Candace Langan, Bonita D. Jones, Robert Stephen Flack, Zhifang Li, David Terry, Ross Wallace, Robert Bondi, Gereint Sis, Ronee Baracani, Maralee McVean, Gabrielle Kolakowski, Dayo Osimboni, Kevin Wilson. Discovery of selective BRM (SMARCA2) ATPase inhibitors for the treatment of BRG1 (SMARCA4) mutant cancers [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 3230.

Trilobolide-6-O-isobutyrate from Sphagneticola trilobata acts by inducing oxidative stress, metabolic changes and apoptosis-like processes by caspase 3/7 activation of human lung cancer cell lines

BackgroundLung cancer, a chronic and heterogeneous disease, is the leading cause of cancer-related death on a global scale. Presently, despite a variety of available treatments, their effectiveness is limited, often resulting in considerable toxicity and adverse effects. Additionally, the development of chemoresistance in cancer cells poses a challenge. Trilobolide-6-O-isobutyrate (TBB), a natural sesquiterpene lactone extracted from Sphagneticola trilobata, has exhibited antitumor effects. Its pharmacological properties in NSCLC lung cancer, however, have not been explored. PurposeThis study evaluated the impact of TBB on the A549 and NCI-H460 tumor cell lines in vitro, examining its antiproliferative properties and initial mechanisms of cell death. MethodsTBB, obtained at 98 % purity from S. trilobata leaves, was characterized using chromatographic techniques. Subsequently, its impact on inhibiting tumor cell proliferation in vitro, TBB-induced cytotoxicity in LLC-MK2, THP-1, AMJ2-C11 cells, as well as its effects on sheep erythrocytes, and the underlying mechanisms of cell death, were assessed. ResultsIn silico predictions have shown promising drug-likeness potential for TBB, indicating high oral bioavailability and intestinal absorption. Treatment of A549 and NCI-H460 human tumor cells with TBB demonstrated a direct impact, inducing significant morphological and structural alterations. TBB also reduced migratory capacity without causing toxicity at lower concentrations to LLC-MK2, THP-1 and AMJ2-C11 cell lines. This antiproliferative effect correlated with elevated oxidative stress, characterized by increased levels of ROS, superoxide anion radicals and NO, accompanied by a decrease in antioxidant markers: SOD and GSH. TBB-stress-induced led to changes in cell metabolism, fostering the accumulation of lipid droplets and autophagic vacuoles. Stress also resulted in compromised mitochondrial integrity, a crucial aspect of cellular function. Additionally, TBB prompted apoptosis-like cell death through activation of caspase 3/7 stressors. ConclusionThese findings underscore the potential of TBB as a promising candidate for future studies and suggest its viability as an additional component in the development of novel anticancer drugs prototypes.

The role of leptin in regulation of the soluble amyloid precursor protein α (sAPPα) levels in lung cancer cell media

Previously, we found that the levels of soluble amyloid precursor protein α (sAPPα) are regulated, in part, by acetylcholinesterase (AChE) in human A549 (p53 wild-type) and H1299 (p53-null) NSCLC cell lines. In this study, we found regulation of sAPPα levels in the media by leptin, a widely recognized obesity-associated adipokine that has recently been shown to play a possible role in cancer signaling. Increased levels of sAPPα, that were accompanied by lower Aβ40/42 levels in the media of A549 and H1299 cells, were detected upon cell incubation with leptin. Conversely, knockdown of leptin or its receptor led to reduced levels of sAPPα and increased levels of Aβ40/42 in the media of A549 and H1299 cells, suggesting that leptin likely shifts APP processing toward the non-amyloidogenic pathway. A549 cell treatment with leptin increased acetylcholine levels and blocked the activities of AChE and p53. Treatment with leptin resulted in increased activation of PKC, ERK1/2, PI3K, and the levels of sAPPα, effects that were reversed by treatment with kinase inhibitors and/or upon addition of AChE to A549 and H1299 cell media. Cell viability increased by treatment of A549 and H1299 cells with leptin and decreased upon co-treatment with AChE and/or inhibitors targeting PKC, ERK1/2, and PI3K. This study is significant as it provides evidence for a likely carcinogenic role of leptin in NSCLC cells via upregulation of sAPPα levels in the media, and highlights the importance of targeting leptin as a potential therapeutic strategy for NSCLC treatment.

Combination of the LARS1 Inhibitor, BC-LI-0186 with a MEK1/2 Inhibitor Enhances the Anti-Tumor Effect in Non-Small Cell Lung Cancer.

The mammalian target of rapamycin complex 1 (mTORC1) regulates cell growth and proliferation by growth factor coordination and amino acid availability. Leucyl-tRNA synthetase 1 (LARS1) senses the intracellular leucine concentration and mediates amino acid-induced activation of mTORC1. Thus, LARS1 inhibition could be useful in cancer treatment. However, the fact that mTORC1 can be stimulated by various growth factors and amino acids suggests that LARS1 inhibition alone has limitations in inhibiting cell growth and proliferation. We investigated the combined effects of BC-LI-0186, a LARS1 inhibitor, and trametinib, an MEK inhibitor, on non-small cell lung cancer (NSCLC). Protein expression and phosphorylation were observed by immunoblotting, and genes differentially expressed between BC-LI-0186-sensitive and -resistant cells were identified by RNA sequencing. The combined effect of the two drugs was inferred from the combination index values and a xenograft model. LARS1 expression was positively correlated with mTORC1 in NSCLC cell lines. BC-LI-0186 treatment of A549 and H460 cells maintained in media supplemented with fetal bovine serum revealed paradoxical phosphorylation of S6 and activation of mitogen- activated protein kinase (MAPK) signaling. Compared with BC-LI-0186-sensitive cells, -resistant cells showed enrichment of the MAPK gene set. The combination of trametinib and BC-LI-0186 inhibited the phosphorylation of S6, MEK, and extracellular signal-regulated kinase and their synergistic effects were confirmed in a mouse xenograft model. The combination of BC-LI-0186 and trametinib inhibited the non-canonical mTORC1-activating function of LARS1. Our study demonstrated a new therapeutic approach for NSCLC without targetable driver mutations.

Synergistic Tumor Inhibition via Energy Elimination by Repurposing Penfluridol and 2-Deoxy-D-Glucose in Lung Cancer.

Simple SummaryDrug repurposing has been effective for discovering novel treatments for cancer. The antipsychotic agent penfluridol was reported to suppress lung cancer growth via ATP energy deprivation. The aim of our study was to investigate how penfluridol influences energy metabolism in lung cancer cells. We observed that penfluridol inhibited mitochondrial oxidative phosphorylation (OXPHOS), but induced glycolysis to compensate for the loss of ATP caused by suppression of mitochondrial OXPHOS. We also confirmed that inhibition of glycolysis by 2-deoxy-D-glucose (2DG) significantly augmented the antitumor effects caused by penfluridol in vitro and in vivo. Our studies provide novel insights into repurposing penfluridol combined with 2-DG for lung cancer treatment.Energy metabolism is the basis for cell growth, and cancer cells in particular, are more energy-dependent cells because of rapid cell proliferation. Previously, we found that penfluridol, an antipsychotic drug, has the ability to trigger cell growth inhibition of lung cancer cells via inducing ATP energy deprivation. The toxic effect of penfluridol is related to energy metabolism, but the underlying mechanisms remain unclear. Herein, we discovered that treatment of A549 and HCC827 lung cancer cells with penfluridol caused a decrease in the total amount of ATP, especially in A549 cells. An Agilent Seahorse ATP real-time rate assay revealed that ATP production rates from mitochondrial respiration and glycolysis were, respectively, decreased and increased after penfluridol treatment. Moreover, the amount and membrane integrity of mitochondria decreased, but glycolysis-related proteins increased after penfluridol treatment. Furthermore, we observed that suppression of glycolysis by reducing glucose supplementation or using 2-deoxy-D-glucose (2DG) synergistically enhanced the inhibitory effect of penfluridol on cancer cell growth and the total amount of mitochondria. A mechanistic study showed that the penfluridol-mediated energy reduction was due to inhibition of critical regulators of mitochondrial biogenesis, the sirtuin 1 (SIRT1)/peroxisome-proliferator-activated receptor co-activator-1α (PGC-1α) axis. Upregulation of the SIRT1/PGC-1α axis reversed the inhibitory effect of penfluridol on mitochondrial biogenesis and cell viability. Clinical lung cancer samples revealed a positive correlation between PGC-1α (PPARGC1A) and SIRT1 expression. In an orthotopic lung cancer mouse model, the anticancer activities of penfluridol, including growth and metastasis inhibition, were also enhanced by combined treatment with 2DG. Our study results strongly support that a combination of repurposing penfluridol and a glycolysis inhibitor would be a good strategy for enhancing the anticancer activities of penfluridol in lung cancer.

Biological Evaluation of New Thienopyridinium and Thienopyrimidinium Derivatives as Human Choline Kinase Inhibitors.

Due to its role in lipid biosynthesis, choline kinase α1 (CKα1) is an interesting target for the development of new antitumor agents. In this work, we present a series of 41 compounds designed based on the well-known and successful strategy of introducing thienopyridine and pyrimidine as bioisosteres of other heterocycles in active antitumor compounds. Notwithstanding the fact that some of these compounds do not show significant enzymatic inhibition, others, in contrast, feature substantially improved enzymatic and antiproliferative inhibition values. This is also confirmed by docking analysis, whereby compounds with longer linkers and thienopyrimidine cationic head have been identified as the most compelling. Among the best compounds is Ff-35, which inhibits the growth of different tumor cells at submicromolar concentrations. Moreover, Ff-35 is more potent in inhibiting CKα1 than other previous biscationic derivatives. Treatment of A549, Hela, and MDA-MB-231 cells with Ff-35 results in their arrest at the G1 phase of the cell cycle. Furthermore, the compound induces cellular apoptosis in a concentration-dependent manner. Altogether, these findings indicate that Ff-35 is a promising new chemotherapeutic agent with encouraging preclinical potential.

TR35 Exerts Anti-tumor Effects by Modulating Mitogen-Activated Protein Kinase and STAT3 Signaling in Lung Cancer Cells.

Cancer is a complex disease extremely dependent on its microenvironment and is highly regulated by a variety of stimuli inside and outside the cell. Evidence suggests that active camel whey fraction (TR35) confer anti-tumor effects in non-small cell lung cancer (NSCLC). However, its exact mechanisms remain elusive. Here, we investigated the mechanisms underlying suppression of NSCLC cell growth and proliferation by TR35. Treatment of A549 and H1299 cells with TR35 suppressed their growth and enhanced apoptosis, as revealed by CCK-8, colony formation and flow cytometric analyses. We find that TR35 suppresses tumor growth in a xenograft nude mouse model without losses in body weight. RNA-seq and KEGG pathway analyses showed that the DEGs were enriched in mitogen-activated protein kinase (MAPK) and Jak-STAT signaling pathways. After test the key factors’ activity associated with these pathways by Immunohistochemical (IHC) staining and western blotting, the activation of JNK phosphorylation and inhibition of p38 and STAT3 phosphorylation was observed both in TR35 treated lung cancer cell and tumor tissue. Taken together, these results showed that TR35 play a significant role in the NSCLC progression in the tumor microenvironment via MAPK and Jak-STAT signaling, highlighting TR35 as a potential therapeutic agent against lung cancer.

Novel N-(aryl/heteroaryl)-2-chlorobenzenesulfonamide derivatives: Synthesis and anticancer activity evaluation

A new series of N-(aryl/heteroaryl)-2-chlorobenzenesulfonamide derivatives 4–21 have been synthesized, and evaluated at the National Cancer Institute (USA) for their in vitro activities against a panel of 60 different human cancer cell lines. Among them, compounds 16, 20 and 21 exhibited remarkable cytotoxic activity against numerous human cancer cell lines. We found that sulfonamide derivative 21 appeared to be more selective than compounds 16 and 20. In comparison to compounds 16 and 20 it showed higher cytotoxic activity against A549 non-small cell lung adenocarcinoma and HCT-116 colon carcinoma cells and was less toxic to HEK-293 human embryonic kidney cells and HaCaT immortalized human keratinocytes. Treatment of A549 and HCT-116 cells with compound 21 resulted in the G0/G1-cell cycle arrest with a concomitant increase in p53 and p21 protein levels. Moreover, compound 21 led to ATP depletion and disruption of the mitochondrial membrane potential in both studied cell lines. Our results suggest that 2,4-dichloro-N-(quinolin-8-yl and/or 1H-indazol-7-yl)benzenesulfonamides serve as novel promising anticancer agents.

Data on cytotoxicity of plant essential oils in A549 and Detroit 551 cells.

To secure the safety for industrial applications of plant essential oils, it is necessary to determine the inhibitory concentration and inhibitory mechanism of cell proliferation in skin cells and lung cells. Considering inhalation through the respiratory system and skin contact of humans with essential oils, we used human lung cancer cells A549 and human skin fibroblasts Detroit 551 cells for all experiments. In this study, we examined IC50 values and protein levels of cell cycle markers (cyclin A, cyclin B, cyclin D, and cyclin E) and apoptosis marker (caspase-3) after exposure to 10 plant essential oils, including Dendranthema indicum (L.) Des Moul, Peucedanum japonicum Thunb, Dendranthema zawadskii var. latilobum (Maxim.) Kitam, Agastache rugosa (Fisch.&Mey.) Kuntze, Vitex rotundifolia L.f, Pinus rigida Mill; Orixa japonica Thunb, Pinus strobus L, Chamaecyparis pisifera (Siebold et Zucc.) Endl. var. filifera Beissn. et Hochst, and Citrus sunki Hort. ex Tanaka. After the treatment of A549 and Detroit 551 cells to varying concentrations of the 10 plant essential oils, IC50 values were determined by CCK analysis, whereas protein expressions of the four cyclins and caspase-3 were identified by Western blotting analysis. We believe that by examining the degree and mechanism of cell proliferation inhibition exerted by essential oils on skin and lung cells of humans, data obtained in this study can provide guidelines for the industrial application of plant essential oils.

Honokiol induces endoplasmic reticulum stress-mediated apoptosis in human lung cancer cells

AimsHonokiol is a hydroxylated biphenyl natural product and displays potent antitumor activity against several cancers including prostate cancer, melanoma, leukemia, and colorectal cancer. The present study was to investigate the in vitro activity of honokiol against A549 and 95-D human lung cancer cells. Main methodsA549 and 95-D cells were used with honokiol treatment. Cell viability was determined by CCK-8 assay. The cell migration and apoptosis were evaluated by wound healing assay and TUNEL staining method respectively. The expressions of ER-related proteins were analyzed by western blot and the CHOP siRNA was used to downregulate the CHOP expression. Key findingsThe results demonstrated that treatment of A549 and 95-D cells with honokiol significantly reduced cell viability in a dose- and time-dependent manner. Furthermore, honokiol treatment decreased cell migration and enhanced cell apoptosis, which is accompanied by the upregulation of the expressions of ER stress-induced apoptotic signaling molecules such as GRP78, phosphorylated PERK, phosphorylated eIF2α, CHOP, Bcl-2, Bax, and cleaved Caspase 9. Honokiol treatment-induced increase of ER stress-related signaling molecules and apoptotic proteins in A549 and 95-D cells were reversed by CHOP siRNA. SignificanceCollectively, we conclude that ER stress may participate in the action of the anticancer activity of honokiol in A549 and 95-D cells and induction of ER stress-related apoptosis may represent a novel therapeutic intervention for human lung cancer.

Bulnesia sarmientoi Supercritical Fluid Extract Exhibits Necroptotic Effects and Anti-Metastatic Activity on Lung Cancer Cells.

Bulnesia sarmientoi (BS) has long been used as an analgesic, wound-healing and anti-inflammatory medicinal plant. The aqueous extract of its bark has been demonstrated to have anti-cancer activity. This study investigated the anti-proliferative and anti-metastatic effects of BS supercritical fluid extract (BSE) on the A549 and H661 lung cancer cell lines. The cytotoxicity on cancer cells was assessed by an MTT assay. After 72 h treatment of A549 and H661 cells, the IC50 values were 18.1 and 24.7 μg/mL, respectively. The cytotoxicity on MRC-5 normal cells was relatively lower (IC50 = 61.1 μg/mL). BSE arrested lung cancer cells at the S and G2/M growth phase. Necrosis of A549 and H661 cells was detected by flow cytometry with Annexin V-FITC/PI double staining. Moreover, the cytotoxic effect of BSE on cancer cells was significantly reverted by Nec-1 pretreatment, and BSE induced TNF-α and RIP-1 expression in the absence of caspase-8 activity. These evidences further support that BSE exhibited necroptotic effects on lung cancer cells. By wound healing and Boyden chamber assays, the inhibitory effects of BSE on the migration and invasion of lung cancer cells were elucidated. Furthermore, the chemical composition of BSE was examined by gas chromatography-mass analysis where ten constituents of BSE were identified. α-Guaiene, (−)-guaiol and β-caryophyllene are responsible for most of the cytotoxic activity of BSE against these two cancer cell lines. Since BSE possesses significant cytotoxicity and anti-metastatic activity on A549 and H661 cells, it may serve as a potential target for the treatment of lung cancer.

Potent anti-cancer activity of Alnus nitida against lung cancer cells; in vitro and in vivo studies

Alnus nitida is used for multiple disorders in norther areas of Pakistan. In this study we have evaluated methanol extract of leaf (ANL) and stem bark (ANB) of A. nitida against two lung cancer cell lines; A-549 and H460 (Human non-small lung cancer cell lines) during in vitro assays for growth inhibition. Treatment with ANL and ANB markedly inhibited the growth of both cancer cell lines. Exposure of A-549 and H460 cancer cell lines to ANL and ANB inhibited cell survival, colony growth and migration of cells. Further, treatment of A-549 and H460 with ANL and ANB indicated alteration in actin fibers after staining with rhodamine-phalloidin. Both extracts cause shrinkage and cell cycle arrest during G1 phase. Treatment of A-549 and H460 cancer cells with ANL and ANB repressed the expression of anti-apoptotic proteins Bcl-2 and Bcl-xL along with downregulation of NFκB, cyclin D1 and PI3-K protein. In addition, intraperitoneal injection of ANL and ANB (10 mg/kg bw and 20 mg/kg bw) to C57BL/6 J mice implanted with B16F10 (Mouse melanoma cancer cell line) cells significantly (p < 0.01) decreased the number of nodules per lung and the level of various proteins reciprocating the in vitro studies. These results suggest that ANL and ANB be explored further for therapeutic use in lung cancer.

Dehydrocostus Lactone Enhances Chemotherapeutic Potential of Doxorubicin in Lung Cancer by Inducing Cell Death and Limiting Metastasis.

BackgroundNatural compounds have been utilized in inhibiting metastasis alone or in combination with other anti-tumor agents. Dehydrocostus lactone (DHC), a natural sesquiterpene lactone, was used to investigate its effect on proliferation of lung cancer cells and on the anti-angiogenic efficacy of doxorubicin.Material/MethodsCell proliferation was assessed by MTT assay and clonogenic assay. Apoptosis and migration were assessed by flow cytometry and wound-healing assay, respectively. Western blotting and qPCR were performed for gene and protein expression analysis. Matrigel plug assay was performed for angiogenesis assessment.ResultsResults of the study show that DHC inhibited the survival and proliferation of lung cancer cells (A549 and H460) and enhanced the growth-inhibitory properties of DOX. Cotreatment of DHC enhanced the apoptosis-inducing effects of DOX by activating caspase-9 and caspase-3 followed by cleavage of PARP. Treatment of A549 and H460 cells with DHC caused suppression of HIF-1α, Akt and pAkt, GSK-3β and pGSK-3β, as well as ERK, pERK, mTOR, and p-mTOR. DHC enhanced the effect of DOX by inhibiting migration of A549 cells as observed by wound-healing assay. DHC caused synergistic inhibition of MMP-2 and MMP-9 genes when treated in combination with DOX. DHC further enhanced the anti-angiogenic properties of DOX in mice implanted with Matrigel plugs. DHC suppressed the proliferation of lung cancer cells and enhanced the anti-angiogenic properties of DOX.ConclusionsThe putative mechanism behind the metastasis-limiting effects of DHC may involve the suppression of Akt/GSK-3β and inhibition of MMP-2 and MMP-9 in lung cancer cells.

Combinatorial treatment with polyI:C and anti-IL6 enhances apoptosis and suppresses metastasis of lung cancer cells.

Activation of TLR3 stimulates cancer cell apoptosis and triggers secretion of inflammatory cytokines. PolyI:C, a TLR3 agonist, activates immune cells and regresses metastatic lung cancer in vivo. Although polyI:C reportedly kills lung carcinomas, the mechanism remains elusive. Here, we demonstrated that polyI:C suppressed the proliferation and survival of metastatic (NCI-H358 and NCI-H292) and non-metastatic (A549) lung cancer cells. Notably, A549, NCI-H292 and NCI-H358 which are inducible by polyI:C, expressed low-to-medium level of TLR3 protein, and were susceptible to polyI:C treatment. By contrast, NCI-H1299, which endogenously expresses high level of TLR3 protein, was insensitive to polyI:C. We showed that polyI:C stimulated pro-inflammatory cytokines associated with survival and metastasis in a cell type-specific manner. While A549 and NCI-H292 released high levels of IL6, IL8 and GRO, the NCI-H358 cells endogenously secretes abundant levels of these cytokines, and was not further induced by polyI:C. Thus, NCI-H358 was resistant to the inhibition of cytokine-dependent metastasis. NCI-H1299, which was unresponsive to polyI:C, did not produce any of the pro-inflammatory cytokines. Treatment of A549 with a combination of polyI:C and anti-IL6 antibody significantly decreased IL6 production, and enhanced polyI:C-mediated killing and suppression of oncogenicity and metastasis. While polyI:C stimulated the phosphorylation of STAT3 and JAK2, blockade of these proteins enhanced polyI:C-mediated suppression of survival and metastasis. Taken together, polyI:C alone provoked apoptosis of lung cancer cells that express low-to-medium levels of functional TLR3 protein. The combinatorial treatment with polyI:C and anti-IL6 enhanced polyI:C-mediated anticancer activities through IL6/JAK2/STAT3 signalling, and apoptosis via TLR3-mediated caspase 3/8 pathway.

Expression of the lncRNA Maternally Expressed Gene 3 (MEG3) Contributes to the Control of Lung Cancer Cell Proliferation by the Rb Pathway

Maternally expressed gene 3 (MEG3, mouse homolog Gtl2) encodes a long noncoding RNA (lncRNA) that is expressed in many normal tissues, but is suppressed in various cancer cell lines and tumors, suggesting it plays a functional role as a tumor suppressor. Hypermethylation has been shown to contribute to this loss of expression. We now demonstrate that MEG3 expression is regulated by the retinoblastoma protein (Rb) pathway and correlates with a change in cell proliferation. Microarray analysis of mouse embryonic fibroblasts (MEFs) isolated from mice with genetic deletion of all three Rb family members (TKO) revealed a significant silencing of Gtl2/MEG3 expression compared to WT MEFs, and re-expression of Gtl2/MEG3 caused decrease in cell proliferation and increased apoptosis. MEG3 levels also were suppressed in A549 lung cancer cells compared with normal human bronchial epithelial (NHBE) cells, and, similar to the TKO cells, re-constitution of MEG3 led to a decrease in cell proliferation and elevated apoptosis. Activation of pRb by treatment of A549 and SK-MES-1 cells with palbociclib, a CDK4/6 inhibitor, increased the expression of MEG3 in a dose-dependent manner, while knockdown of pRb/p107 attenuated this effect. In addition, expression of phosphorylation-deficient mutant of pRb increased MEG3 levels in both lung cancer cell types. Treatment of these cells with palbociclib also decreased the expression of pRb-regulated DNA methyltransferase 1 (DNMT1), while conversely, knockdown of DNMT1 resulted in increased expression of MEG3. As gene methylation has been suggested for MEG3 regulation, we found that palbociclib resulted in decreased methylation of the MEG3 locus similar to that observed with 5-aza-deoxycytidine. Anti-sense oligonucleotide silencing of drug-induced MEG3 expression in A549 and SK-MES-1 cells partially rescued the palbociclib-mediated decrease in cell proliferation, while analysis of the TCGA database revealed decreased MEG3 expression in human lung tumors harboring a disrupted RB pathway. Together, these data suggest that disruption of the pRb-DNMT1 pathway leads to a decrease in MEG3 expression, thereby contributing to the pro-proliferative state of certain cancer cells.

Suppression of reactive oxygen species‐mediated ERK and JNK activation sensitizes dihydromyricetin‐induced mitochondrial apoptosis in human non‐small cell lung cancer

Nonsmall cell lung cancer (NSCLC) is the most common type of lung cancer with a high mortality rate and still remains a therapeutic challenge. A strategy for targeting NSCLC is to identify agents that are effective against NSCLC cells while sparing normal cells. Dihydromyricetin (DHM) is the major flavonoid component derived from Ampelopsis grossedentata, which has a long history of use in medicine. Herein, the molecular mechanisms by which DHM exerts its anticancer effects against NSCLC cells were investigated. Results from MTS, colony formation, Western blot, flow cytometric, and JC-1 mitochondrial membrane potential assays revealed that DHM showed a selective cytotoxic effect against NSCLC cells (A549 and H1975), but not against normal lung (WI-38) fibroblasts, by inducing apoptosis. DHM-induced cell apoptosis occurred through Bcl-w suppression-mediated mitochondrial membrane depolarization, caspase-9/-7/-3 activation, and poly(ADP-ribose) polymerase (PARP) cleavage in A549 and H1975 cells. Moreover, treatment of A549 and H1975 cells with DHM induced increase of intracellular peroxide and sustained activation of extracellular signal-regulated kinase (ERK)1/2 and c-Jun N-terminal kinase (JNK)1/2, and the reactive oxygen species scavenger, N-acetylcysteine (NAC), reversed DHM-induced ERK and JNK activation. Furthermore, treatment of cells with specific inhibitors of ERK and JNK or NAC significantly promoted the DHM-induced activation of caspase-9/-7/-3 and PARP cleavage and also sensitized the antitumorigenic effect of DHM on NSCLC cells. These findings define and support a novel function of DHM of inducing mitochondrion-derived apoptosis in human NSCLC cells, and a combination of DHM with ERK and JNK inhibitors should be a good strategy for preventing NSCLC proliferation. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1426-1438, 2017.

MAP3K19 Is a Novel Regulator of TGF-β Signaling That Impacts Bleomycin-Induced Lung Injury and Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive, debilitating disease for which two medications, pirfenidone and nintedanib, have only recently been approved for treatment. The cytokine TGF-β has been shown to be a central mediator in the disease process. We investigated the role of a novel kinase, MAP3K19, upregulated in IPF tissue, in TGF-β-induced signal transduction and in bleomycin-induced pulmonary fibrosis. MAP3K19 has a very limited tissue expression, restricted primarily to the lungs and trachea. In pulmonary tissue, expression was predominantly localized to alveolar and interstitial macrophages, bronchial epithelial cells and type II pneumocytes of the epithelium. MAP3K19 was also found to be overexpressed in bronchoalveolar lavage macrophages from IPF patients compared to normal patients. Treatment of A549 or THP-1 cells with either MAP3K19 siRNA or a highly potent and specific inhibitor reduced phospho-Smad2 & 3 nuclear translocation following TGF-β stimulation. TGF-β-induced gene transcription was also strongly inhibited by both the MAP3K19 inhibitor and nintedanib, whereas pirfenidone had a much less pronounced effect. In combination, the MAP3K19 inhibitor appeared to act synergistically with either pirfenidone or nintedanib, at the level of target gene transcription or protein production. Finally, in an animal model of IPF, inhibition of MAP3K19 strongly attenuated bleomycin-induced pulmonary fibrosis when administered either prophylactically ortherapeutically. In summary, these results strongly suggest that inhibition of MAP3K19 may have a beneficial therapeutic effect in the treatment of IPF and represents a novel strategy to target this disease.

Abstract 3342: Combining Pemetrexed with methoxyamine to enhance the radiosensitization of non-small-cell lung cancer (NSCLC): Preclinical studies in vivo

Abstract Combining Pemetrexed with Methoxyamine to Enhance the Radiosensitization of Non-Small-Cell Lung Cancer (NSCLC): Preclinical Studies in vivo BACKGROUND: Stage III lung cancer is often treated with radiation therapy (RT) and a platin, and recent clinical trials have found benefit for tumors of non-squamous histology of adding Pemetrexed on stage IV disease. Since the cytotoxic effects of Pemetrexed are amplified in the presence of methoxyamine, we tested the ability of methoxyamine to also amplify the radiosensitization provided by Pemetrexed. METHODS: Human lung cancer cells (A549, expressing wild-type p53, or H1299, p53 null) were inoculated into the right flank of female athymic nude mice. When the xenografts reached 100-175 mm3, the mice were randomized (Day 0) into 4 arms, which were given 5 daily doses (Days 1-5) of Pemetrexed (150 mg/kg) and/or methoxyamine (4 mg/kg) or no drugs. On Day 6, half of the tumors of each arm were irradiated (8 Gy single fraction, Cs-137 γ-radiation). Twice weekly the animals were weighed and tumor growth was monitored by calipers. RESULTS: None of the treatments caused animals to lose weight. For A549 xenografts, growth rates for all treatment arms differed significantly from the untreated control growth rate (p&amp;lt;0.0001); tumors treated with Pemetrexed/methoxyamine/RT grew at the slowest rate (8 mm3/day), which was significantly slower than that for RT alone (18 mm3/day), methoxyamine/RT (19.3 mm3/day), or Pemetrexed/RT (15 mm3/day) (p&amp;lt;0.0001). Similar differences in growth rate were found for the H1299 xenografts. The data were also analyzed as the time for the volume of A549 xenografts to reach 400 mm3 or H1299 xenografts to reach 600 mm3. For A549, the median time for the Pemetrexed/methoxyamine/RT arm (38 days) was greater than those produced by no treatment (14 days with 2 cures; p&amp;lt;0.0001), RT alone (21 days with 1 cure; p = 0.05) and Pemetrexed/RT (26 days with no cures; p = 0.055). For H1299, a similar ranking of time-to-endpoint was found. Based on our studies of the two cell lines in vitro, it appears that the radiosensitizing effect of Pemetrexed + methoxyamine results from cell cycle redistribution to the G1/S border and early S-phase along with inhibition of DNA damage repair. CONCLUSIONS: The addition of methoxyamine to the pre-irradiation treatment of A549 and H1299 xenografts significantly reduced the tumor growth rate after RT and extended the tumor growth delay. Since methoxyamine enhanced the radiosensitization by Pemetrexed in both tumor types, p53 is not necessary for this response. Note: This abstract was not presented at the meeting. Citation Format: Ravi Patel, Rutul Patel, Tithi Biswas, Pingfu Fu, Mitchell Machtay, Nancy Oleinick. Combining Pemetrexed with methoxyamine to enhance the radiosensitization of non-small-cell lung cancer (NSCLC): Preclinical studies in vivo . [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3342. doi:10.1158/1538-7445.AM2015-3342

Proliferative and Inhibitory Activity of Siberian ginseng (Eleutherococcus senticosus) Extract on Cancer Cell Lines; A-549, XWLC-05, HCT-116, CNE and Beas-2b.

Siberian ginseng (Eleutherococcus senticosus) is used primarily as an adaptogen herb and also for its immune stimulant properties in Western herbal medicine. Another closely related species used in East Asian medicine systems i.e. Kampo, TCM (Manchuria, Korea, Japan and Ainu of Hokkaido) and also called Siberian ginseng (Acanthopanax senticosus) also displays immune-stimulant and anti-cancer properties. These may affect tumour growth and also provide an anti-fatigue effect for cancer patients, in particular for those suffering from lung cancer. There is some evidence that a carbohydrate in Siberian ginseng may possess not only immune stimulatory but also anti-tumour effects and also display other various anti-cancer properties. Our study aimed to determine the inhibitory and also proliferative effects of a methanol plant extract of Siberan ginseng (E. senticosus) on various cancer and normal cell lines including: A-549 (small cell lung cancer), XWLC-05 (Yunnan lung cancer cell line), CNE (human nasopharyngeal carcinoma cell line), HCT-116 (human colon cancer) and Beas-2b (human lung epithelial). These cell lines were treated with an extract from E. senticosus that was evaporated and re- constituted in DMSO. Treatment of A-549 (small cell lung cancer) cells with E. senticosus methanolic extract showed a concentration-dependent inhibitory trend from 12.5 - 50μg/mL, and then a plateau, whereas at 12.5 and 25 μg/mL, there is a slight growth suppression in QBC-939 cells, but then a steady suppression from 50, 100 and 200μg/mL. Further, in XWLC-05 (Yunnan lung cancer cell line), E. senticosus methanolic extract displayed an inhibitory effect which plateaued with increasing dosage. Next, in CNE (human nasopharyngeal carcinoma cell line) there was a dose dependent proliferative response, whereas in Beas-2 (human lung epithelial cell line), an inhibitory effect. Finally in colon cancer cell line (HCT-116) we observed an initially weak inhibitory effect and then plateau.