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Epithelial to mesenchymal transition (EMT) is the cellular transition process of epithelium-associated phenotypes and behaviors into mesenchymal phenotypes. EMT is linked with cancer, and it is believed to be an important factor facilitating the motility and invasive activity of solid tumor cells. EMT facilitates the capability of cancer cells to metastasize because it promotes cell survival in detached conditions and facilitates the establishment of new tumors. In lung cancer, EMT has garnered considerable attention because of its importance in metastasis and has been recognized as an important target for anticancer drug therapy. Several studies have pointed out the promising activities of natural product-derived compounds and other agents that have EMT-suppressive activities and may facilitate the development of novel strategies for lung cancer management. In this review, we discuss the recent discoveries regarding the fundamental signaling regulating EMT and identify molecular targets for anti-EMT activities of the natural product-derived compounds. We also highlight the anti-EMT effect of natural compounds with their molecular targets and mechanisms of action that may benefit the understanding of and support the development of EMT targeting therapy.

Sericin, a protein found in wastewater from the silk industry, was shown to contain a variety of biological activities, including antioxidant. The enzymatic conditions have been continuously modified to improve antioxidant effect and scavenging capacity against various free radicals of silk sericin protein.Variables in enzymatic reactions, including pH, temperature and enzyme/substrate ratio were analysed to discover the optimum conditions for antioxidant activity of sericin hydrolysates.Hydrolysis reaction catalysed by AlcalaseAmong these three variables, response surface plots demonstrate the major role of temperature on scavenging capacity of sericin hydrolysates. Sericin hydrolysates prepared by using AlcalaseThe acquired RSM information would be of benefit for further developing antioxidant peptide from diverse resources, especially the recycling of waste products from silk industry.

Background Cellular senescence is an aging‐related process found in cancer cells that contributes to irreversible growth arrest and tumor aggressiveness. Recently, calmodulin‐regulated spectrin‐associated protein 3 (CAMSAP3), a minus‐end microtubule‐stabilizing protein, has received increasing attention in cancer cell biology. However, the biological role of CAMSAP3 on senescence in human lung cancer remains incompletely understood. Methods The function of CAMSAP3 on the regulation of cellular senescence‐associated phenotypes in human non‐small cell lung cancer H460 cells were determined in CAMSAP3 deletion (H460/C3ko) cells. The effects of CAMSAP3 on cell proliferation were investigated using MTT and colony formation assays. The cell cycle activity was evaluated by flow cytometry and the senescence‐associated phenotypes were observed by SA‐β‐Gal staining. Quantitative RT‐PCR and westen blot were used to evaluate the expression of cell cycle and senescence markers. Moreover, the interaction of CAMSAP3‐ERK1/2 and possible partner protein was quantified using immunoprecipitation/mass spectrometry and immunofluorescence. Lastly, an xenograft model were performed. Results CAMSAP3 knockout promotes lung cancer cell senescence‐associated phenotypes and induces G1 cell cycle arrest. Mechanistic investigation revealed that phosphorylated ERK (p‐ERK) was markedly downregulated in CAMSAP3‐deleted cells, suppressing cyclin D1 expression levels, and full‐length CAMSAP3 abrogated these phenotypes. Proteomic analysis demonstrated that vimentin, an intermediate filament protein, is required as a scaffold for CAMSAP3‐modulating ERK signaling. Furthermore, an in vivo tumor xenograft experiment showed that tumor initiation is potentially delayed in CAMSAP3 knockout tumors with the downregulation of p‐ERK and cyclin D1, resulting in a senescence‐like phenotype. Conclusion This study is the first to report an intriguing role of CAMSAP3 in lung carcinoma cell senescence‐associated phenotypes via the modulation of p‐ERK/cyclin D1 signaling., Senescence is characterized by an irreversible growth arrest, and the biological role of senescence and its regulation in human lung cancer remains incompletely understood. This study demonstrated that CAMSAP3 knockout promotes lung cancer cell senescence‐associated phenotypes through suppression of ERK/vimentin complex that is required for stabilization of ERK activity.

BACKGROUND/AIM Cancer metastasis is the main cause of mortality in cancer patients. As lung cancer patients are mostly detected at metastatic stages, strategies that inhibit cancer metastasis may offer effective therapies. Activation of FAK and Akt/mTOR pathways promotes the highly metastatic phenotypes of epithelial to mesenchymal transition (EMT). We unraveled EMT inhibitory action of pongamol and the mechanism controlling cell dissemination in lung cancer cells. MATERIALS AND METHODS Cytotoxic and antiproliferative effects of pongamol were determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis and necrosis induction in response to pongamol treatment was observed and visualized by nuclei staining assay. Wound healing migration, invasion, and anchorage-dependent growth assay were conducted to evaluate metastatic behaviors. EMT protein expression and FAK pathway were detected by western blot analysis. RESULTS Pongamol at 0-100 μM exhibited significant inhibition on migration, and invasion of cancer cells. Regarding anoikis resistance potential, the compound significantly inhibited survival and growth of cancer cells in an anchorage-independent manner, as indicated by the depletion of growing colonies in pongamol-pretreated cells. Protein level analysis further showed that pongamol exerted its anti-metastasis effect by inhibiting EMT, as indicated by a decrease of several mesenchymal proteins (N-cadherin, vimentin, Snail, and Slug). Regarding the up-stream mechanisms, we found that pongamol inhibited activation of FAK and Akt/mTOR signaling pathways. CONCLUSION Pongamol exhibits potent anti-metastatic activity through suppressing key potentiating factors of cancer metastasis EMT and FAK.

Cisplatin (CDDP) induces senescence characterized by senescence-associated secretory phenotypes (SASP) and the unfolded protein response (UPR). In this study, we investigated the proteins related to the UPR during the senescence cell fate. Strikingly, we found that one of the critical ER-resident proteins, GRP78/BiP, was significantly altered. Here we show that GRP78 levels differentially expressed depending on non-small lung cancer subtypes. GRP78 indeed regulates the evasion of senescence in adenocarcinoma A549 cells, in which the increased GRP78 levels enable them to re-proliferate after CDDP removal. Conversely, GRP78 is downregulated in the senescence H460 cells, making them lacking senescence evasion capability. We observed that the translational regulation critically contributed to the GRP78 protein levels in CDDP-induces senescence. Furthermore, the increased GRP78 level during senescence confers resistance to senolytic drug, Bortezomib, as observed by a twofold increase in IC50 in A549 senescence cells compared to the wild-type. This observation is also consistent in the cells that have undergone genetic manipulation by transfection with pcDNA3.1(+)-GRP78/BiP plasmids and pSpCas9(BB)-2A-Puro containing guide RNA sequence targeting GRP78 exon 3 to induce the overexpression and downregulation of GRP78 in H460 cells, respectively. Our findings reveal a unique role of GRP78 on the senescence evasion cell fate and senolytic drug resistance after cisplatin-based chemotherapy.

The most prevalent lung cancer is non-small cell lung cancer (NSCLC). This lung cancer type often develops other organ-specific metastases that are critical burdens in the treatment process. Orchid species in the genus

Mantle cell lymphoma (MCL) is an aggressive non-Hodgkin lymphoma with poor prognosis, due to the inevitable development of drug resistance. Despite being the first-in-class proteasome inhibitor for relapsed/refractory MCL, resistance to bortezomib (BTZ) in MCL patients remains a major hurdle of effective therapy, and relapse following BTZ is frequent. Understanding the mechanisms underlying BTZ resistance is, therefore, important for improving the clinical outcome and developing novel therapeutic strategies. Here, we established de novo BTZ-resistant human MCL-derived cells with the highest resistance index of 300-fold compared to parental cells. We provided compelling evidence that both Bcl-xL and Bax are key mediators in determining BTZ sensitivity in MCL cells. Overexpression of antiapoptotic Bcl-xL and depletion of proapoptotic Bax cooperatively protected MCL cells against BTZ-induced apoptosis, causing acquired BTZ resistance, likely by tilting the balance of Bcl-2 family proteins toward antiapoptotic signaling. Bioinformatics analyses suggested that high BCL2L1 (encoded Bcl-xL) and low BAX were, in part, associated with poor prognosis of MCL patients, e.g., when combined with low OGT, which regulates cellular O-GlcNAcylation. Our findings support recent strategies in small molecule drug discovery co-targeting antiapoptotic Bcl-2 family proteins using BH3 mimetics and Bax using Bax activators to overcome cancer drug resistance.

The Akt-mTOR signal is important for the survival and proliferation of cancer cells and has become an interesting drug target. In this study, five resveratrol derivatives were evaluated for anticancer activity and Akt/mTOR targeting activity in non-small lung cancer cell lines. The effects of resveratrol derivatives on cell proliferation were assessed by 2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, nucleus staining, and colony formation assay. Furthermore, the effect of resveratrol derivatives on proliferation-related protein expression was analyzed by immunofluorescence and Western blotting. For the structure–activity relationship (SAR), results reveal that two derivatives of resveratrol which are 4,4′-(ethane-1,2-diyl) bis(2-methoxyphenol) (RD2) and the 4-(3-hydroxy-4-methoxyphenethyl)-2-methoxyphenol (RD3) had very similar structures but exerted different cytotoxicity. The IC50 of RD2 and RD3 were 108.6 ± 10.82 and more than 200 µM in the A549 cell line and 103.5 ± 6.08 and more than 200 µM in H23 cells, respectively. RD2 inhibited cell proliferation and induced apoptosis when compared with the control, while RD3 caused minimal effects. Cells treated with RD2 exhibited apoptotic nuclei in a concomitant with the reduction of cellular p-Akt and p-mTOR. RD3 had minimal effects on such proteins. According to these results, molecular docking analysis revealed a high-affinity interaction between RD2 and an Akt molecule at the ATP-binding and the allosteric sites, indicating this RD2 as a potential Akt inhibitor. This study provides useful information of resveratrol derivatives RD2 for treating lung cancer via Akt/mTOR inhibition.

Background Akt and mTOR are aberrantly activated in cancers and targeting these proteins are interesting for cancer drug discovery. Napabucasin (NB), a phytochemical compound, has been reported as potential anti-cancer agent, however, Akt and mTOR targeting mechanisms remain unclear. Method Apoptosis induction was investigated by Hoechst 33342/PI double staining and annexin V/PI staining with flowcytometry. Autophagy was evaluated by monodansylcadaverine staining and Western blot analysis. Binding affinity of NB and essential signaling proteins (PI3K, Akt, and mTOR) was investigated using molecular docking and confirmed by Western blot analysis. Result A structure modification from changing methyl moiety of acetyl group of NB to hydroxyl moiety of carboxyl group of NB derivative (napabucasin-acid or NB-acid) greatly affected the compound activities. NB showed more potent anti-cancer activity. NB reduced cell viability with an approximately 20 times lower IC50 and inhibited the colony formation capacity much more than NB-acid treated cells. NB induced cell apoptosis, which was accompanied by decrease Bcl‑2 and Mcl-1 and clevage of PARP, while NB-acid show lesser effect on Mcl-1. NB was found to strongly induce autophagy indicated by acidic vesicle staining and the LC3B conversion. Interestingly, computational molecular docking analysis further demonstrated that NB directly bound to Akt and mTOR (complex 1 and 2) proteins at their critical sites indicating that NB targets the upstream regulators of apoptosis and autophagy. The docking results were confirmed by decrease of p-Akt/Akt, p-mTOR/mTOR, and c-Myc a downstream target of Akt protein levels. Conclusion Results show for the first time that NB exerts an anti-cancer activity through the direct interaction to Akt and mTOR proteins. The methyl moiety of acetyl group of NB is required for its potent anti-cancer activities. These data encourage further development of NB compounds for Akt and mTOR driven cancers.

Lung cancer remains a leading cause of death in cancer patients, and deregulation of apoptosis is a serious concern in clinical practice, even though therapeutic intervention has been greatly improved. Plants are a versatile source of biologically active compounds for anticancer drug discovery, and aspiletrein A (AA) is a steroidal saponin isolated from Aspidistra letreae that has a potent cytotoxic effect on various cancer cell lines. In this study, we investigated and determined the underlying molecular mechanism by which AA induces apoptosis. AA strongly induced apoptosis in NSCLC cells by mediating ROS generation and thereby activating AMP-activated protein kinase (AMPK) signaling. Consequently, downstream signaling and levels of phosphorylated mTOR and Bcl-2 were significantly decreased. Pretreatment with either an antioxidant, N-acetylcysteine, or an AMPK inhibitor, compound C, could reverse the apoptosis-inducing effect and counteract the effect of AA on the AMPK signaling pathway. Decreased levels of Bcl-2 were due to AA-mediating Bcl-2 degradation via a ROS/AMPK/mTOR axis-dependent proteasomal mechanism. Consistently, the apoptotic-inducing effect of AA was also observed in patient-derived malignant lung cancer cells, and it suppressed an in vitro 3D-tumorigenesis. This study identified the underlying mechanism of AA on lung cancer apoptosis, thereby facilitating potential research and development of this compound for further clinical implications.

Background/aim Epithelial to mesenchymal transition (EMT), and focal adhesion kinase (FAK) facilitate lung cancer cell motility and survival. We, therefore, investigated the antimigratory effect of 3,4-dihydroxy-5,4'-dimethoxybibenzyl (DS-1) on human lung cancer cells. Materials and methods Cell viability and proliferation were examined by the 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide assay. Filopodia formation, migration, and anchorage-independent growth assays were performed to assess metastatic behaviors while EMT-related proteins, integrins, and FAK-RhoA pathway were evaluated by western blot analysis. Results We found that DS-1 significantly inhibited the proliferation of lung cancer cells compared to the control. The aggressive behavior of cancer cells, including migration and invasion, was significantly reduced by DS-1. Anchorage-independent growth analysis provided evidence that DS-1 suppressed the growth and survival of cancer cells in detached conditions as indicated by the significant reduction in size and number of colonies. With regard to the mechanisms involved, we found that DS-1-suppressed EMT, as indicated by the reduction of EMT markers, namely N-cadherin, SNAIL and SLUG, and increased levels of the epithelial marker, E-cadherin. In addition, DS-1 was shown to reduce the level of integrin β1 protein and FAK activation. Conclusion DS-1 suppressed lung cancer metastasis via suppressing EMT, integrin β1 expression and FAK-related signaling.

Background/aim: Dermal papilla cells (DPCs) regulate hair follicle development. We aimed to investigate the effect of scoparone from Dendrobium densiflorum on DPCs in the induction of stem cell properties and pluripotency-related proteins. Materials and methods: DPC viability was evaluated by the MTT assay. Apoptosis or necrosis of DPCs was determined by Hoecsht33342/PI nuclear staining analysis. Expression of OCT4, NANOG and SOX2 genes was determined using Real-Time Polymerase Chain Reaction (PCR). Immunocytochemistry and western blot analysis were performed to determine pluripotency related proteins. Results: Scoparone increased the expression of pluripotency related transcription factors SOX2 and NANOG, while it had minimal effects on OCT4 levels. Scoparone exerted its stemness-enhancing activity through the up-regulation of Akt-dependent inhibition of GSK3β, resulting in increased cellular levels of β-catenin. Conclusion: Our results show a potential novel activity and mechanism of action of scoparone on human DPCs that could facilitate the development of hair enrichment approaches.

The effects of plant-derived active compounds on cancer cells have been intensively investigated, leading to the possibility of dietary-based cancer prevention regimens and recommendations for patients with cancer. Many studies have revealed that several compounds can attenuate oxidative stress, suppress survival and proliferative signals, and diminish or suppress cancer stem cells (CSCs). These may provide novel lead compounds for drug development and benefit cancer therapy. The important pharmacological shift in anticancer therapy is the transition of drug discovery for cytotoxic drugs toward targeted therapy and more specific therapy like CSC-targeted therapy. Cancer-driven signaling, as well as survival pathways, have become vital targets for targeted therapeutic drug action. Furthermore, in aggressive cancers, such as lung cancer, it was shown that CSCs drive cancer initiation, progression, metastasis, and therapeutic failure. Moreover, plant-derived compounds are found as a component in diet and are considered safe. Here, we review cancer-protective elements found in plants, including phenolic compounds such as curcumin, carotenoids (β-carotene and lycopene), epigallocatechin-3-gallate, ginsenoside Rg3, resveratrol, and sulforaphane, for their possible anticancer, anti-metastasis, and cancer-preventive actions against lung cancer, especially in clinical and molecular pharmacological approaches. This review comprehensively summarizes the anticancer properties, target proteins, and CSC suppression capabilities of these plant-derived compounds that may potentially benefit the development of novel anticancer drugs or dietary recommendations for patients with lung cancer.

Background Calcium is an essential signal transduction element that has been associated with aggressive behaviours in several cancers. Cell motility is a prerequisite for metastasis, the major cause of lung cancer death, yet its association with calcium signalling and underlying regulatory axis remains an unexplored area. Methods Bioinformatics database analyses were employed to assess correlations between calcium influx channels and clinical outcomes in non-small cell lung cancer (NSCLC). Functional and regulatory roles of influx channels in cell migration and invasion were conducted and experimental lung metastasis was examined using in vivo live imaging. Results High expression of TRPM7 channel correlates well with the low survival rate of patients and high metastatic potential. Inhibition of TRPM7 suppresses cell motility in various NSCLC cell lines and patient-derived primary cells and attenuates experimental lung metastases. Mechanistically, TRPM7 acts upstream of O-GlcNAcylation, a post-translational modification and a crucial sensor for metabolic changes. We reveal for the first time that caveolin-1 and c-Myc are favourable molecular targets of TRPM7/O-GlcNAc that regulates NSCLC motility. O-GlcNAcylation of caveolin-1 and c-Myc promotes protein stability by interfering with their ubiquitination and proteasomal degradation. Conclusions TRPM7/O-GlcNAc axis represents a potential novel target for lung cancer therapy that may overcome metastasis.

Background/aim Certain integrins including integrin β3 facilitate movement and survival of metastatic cancer cells. We examined whether benzoxazine dimer analogue N,N-bis(5-ethyl-2-hydroxybenzyl) methylamine (HM) has anti-metastatic effects. Materials and methods Cell viability was examined by the 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay. Wound healing and phalloidin-rhodamine assays were performed to evaluate the migration and filopodia formation, respectively. Anoikis resistance was studied by anchorage-independent growth assay. The expression of proteins regulating migration were examined by western blot. Results HM treatment significantly inhibited growth and survival of detached lung cancer cells as indicated by the reduced colony number and size of anchorage-independent growth analysis. HM inhibited cell migration and suppressed filopodia formation. Protein analysis indicated that the compound dramatically decreased integrin β3 and its related downstream proteins including active focal adhesion kinase (FAK) and active protein kinase B (AKT); however, integrin β1 and α5 were found to be unaltered. Conclusion HM shows a potential in targeting integrin β3 and could be a good candidate for further developed as an anti-metastatic therapy.

Background Gigantol is a pharmacologically active bibenzyl compound exerting potential anticancer activities. At non-toxic concentrations, it reduces cancer stem cell properties and tumorigenicity. The mechanisms of the effects of gigantol on cancer cell growth are largely unknown. This study aimed to unravel the molecular profile and identify the prominent molecular mechanism of the effects of gigantol in controlling lung cancer cell proliferation. Materials and methods Proteomics and bioinformatics analysis were used accompanied by experimental molecular pharmacology approaches. Results Gigantol exhibited antiproliferative effects on human lung cancer cells confirmed by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide proliferation assay and colony growth assay. The protein profile in response to gigantol treatment associated with regulation of cell proliferation was analyzed to determine the prominent protein targets. Among the significant hub proteins, MYC, an important proto-oncogene and proliferation-promoting transcription factor, was down-regulated with the highest number of protein-protein interactions. MYC down-regulation was confirmed by western blot analysis. The up-stream regulator of MYC, Glycogen synthase kinase 3 beta (GSK3β) was found to be responsible for MYC destabilization mediated by gigantol. Gigantol facilitated GSK3β function and resulted in the increase of MYC-ubiquitin complex as evaluated by immunoprecipitation. Conclusion Gigantol was found to inhibit lung cancer proliferation through induction of GSK3β-mediated MYC ubiquitin-proteasome degradation. These data suggest gigantol to be a promising candidate for novel strategy in inhibition of lung cancer.

Background: Neonatal hypoglycemia is found in up to 15% of neonates and 50% of those with risk factors. Hypoglycemia can cause brain damage and increase risk of developmental delay. Nevertheless, the data regarding hypoglycemia prevention by oral sucrose are still limited. The present study aimed to investigate whether oral sucrose solution can prevent hypoglycemia in high-risk infants. Patients and Methods: Four hundred and twenty-five infants with high hypoglycemic risk were randomized into two groups (214 infants in the intervention and 211 infants in the control groups). The intervention group received one dose of 0.8 ml/kg of 24% oral sucrose solution followed by enteral feed and was compared to the control group receiving enteral feed alone. Glucose levels were evaluated by Dextrostrix. Results: There was no significant difference in antenatal and perinatal risk factors of neonatal hypoglycemia between groups. Glucose level on admission was 72.1±20.3 and 72.1±24.1 mg/dl in the intervention and control groups, respectively. Although no significant difference was recognized in terms of capillary blood glucose levels between groups, data analysis revealed that the glucose increase over time was significantly higher in the intervention group at 1 h (mean±SE=3.61±1.27 mg/dl; p

Background/aim Nitric oxide (NO) is recognized as an important biological mediator that exerts several human physiological functions. As its nature is an aqueous soluble gas that can diffuse through cells and tissues, NO can affect cell signaling, the phenotype of cancer and modify surrounding cells. The variety of effects of NO on cancer cell biology has convinced researchers to determine the defined mechanisms of these effects and how to control this mediator for a better understanding as well as for therapeutic gain. Materials and methods We used bioinformatics and pharmacological experiments to elucidate the potential regulation and underlying mechanisms of NO in non-small a lung cancer cell model. Results Using microarrays, we identified a total of 151 NO-regulated genes (80 up-regulated genes, 71 down-regulated genes) with a strong statistically significant difference compared to untreated controls. Among these, the genes activated by a factor of more than five times were: DCBLD2, MGC24975, RAB40AL, PER3, RCN1, MRPL51, PTTG1, KLF5, NFIX. On the other hand, the expression of RBMS2, PDP2, RBAK, ORMDL2, GRPEL2, ZNF514, MTHFD2, POLR2D, RCBTB1, JOSD1, RPS27, GPR4 genes were significantly decreased by a factor of more than five times. Bioinformatics further revealed that NO exposure of lung cancer cells resulted in a change in transcription factors (TFs) and epigenetic modifications (histone modification and miRNA). Interestingly, NO treatment was shown to potentiate cancer stem cell-related genes and transcription factors Oct4, Klf4, and Myc. Conclusion Through this comprehensive approach, the present study illustrated the scheme of how NO affects molecular events in lung cancer cells.

Because available depigmenting agents exhibit short efficacy and serious side effects, sericin, a waste protein from the silk industry, was hydrolyzed using Alcalase

From the aerial parts of

Background Crinum latifolium L. (Amaryllidaceae) has been used in Southeast Asian traditional medicine to alleviate the symptoms of benign prostatic hyperplasia (BPH). The pathological mechanism of BPH is associated with the induction of prostate stromal cell proliferation through transforming growth factor-beta (TGF-β). Standardization as well as investigation of the potential anti-BPH activity of C. latifolium extract could benefit the further development of BPH-related analyses and provide evidence to support the application of this extract for BPH treatment. This study aimed to standardize and investigate the antiproliferative activity of the ethanolic extract of C. latifolium leaves. The major alkaloids isolated from C. latifolium were also explored for their potential use as bioactive markers. Methods Two major alkaloids were isolated from the ethanolic extract of C. latifolium leaves by chromatographic techniques, identified by NMR and MS, and quantified by a validated UHPLC method. Their antiproliferative activity was studied in human prostate stromal cells (WPMY-1) induced by TGF-β. The synergistic effect of combining the two major isolated alkaloids was analyzed by the zero interaction potency (ZIP) model. Results Two alkaloids, lycorine (1) and 6α-hydroxybuphanidrine (2), were isolated from the ethanolic leaf extract of C. latifolium. A UHPLC method for the quantification of (1) and (2) was developed and validated in terms of linearity, precision, and accuracy. The C. latifolium leaf extract contained 0.279 ± 0.003% (1) and 0.232 ± 0.004% (2). The crude extract was more potent than either (1) and (2) alone against TGF-β-treated WPMY-1 cell proliferation. The drug combination study revealed that the greatest synergistic effect of (1) and (2) was achieved at a 1:1 ratio. Conclusions The results of this study support the anti-BPH activity of C. latifolium in traditional medicine and suggest that these the two isolated alkaloids may promote the efficacy of the C. latifolium extract. Additionally, major alkaloids (1) and (2) can be used as bioactive markers for the standardization of C. latifolium extracts.

Triple-negative breast cancer (TNBC) is characterized by excessive accumulation of tumor-infiltrating immune cells, including tumor-associated macrophages (TAMs). TAMs consist of a heterogeneous population with high plasticity and are associated with tumor aggressiveness and poor prognosis. Moreover, breast cancer cells can secrete factors that influence TAM polarization. Therefore, this study aimed to evaluate the crosstalk between cancer cells and macrophages in the context of TNBC. Cytokine-polarized M2 macrophage were used as control. Distinct from the classical M2 macrophage, TAMs generated from TNBC-conditioned media upregulated both M1- and M2-associated genes, and secreted both the anti-inflammatory cytokine interleukin IL-10 and the proinflammatory cytokine IL-6 and tumor necrosis factor- α. Theses TNBC-induced TAMs exert aggressive behavior of TNBC cells. Consistently, TCGA and MTABRIC analyses of human breast cancer revealed upregulation of M1- associated genes in TNBC comparing with non-TNBC. Among these M1-associated genes, CXCL10 and IL1B were revealed to be independent prognostic factors for disease progression. In conclusion, TNBC cells induce macrophage polarization with a mixture of M1 and M2 phenotypes. These cancer-induced TAMs further enhance tumor cell growth and aggressiveness.

Lung cancer is recognized as a major cause of mortality worldwide owing to its metastatic activity. Given the lack of solid information regarding the possible effects of caffeine, one of the most consumed natural psychoactive substances, on molecular signaling pathways implicated in the aggressive behavior of lung cancer, our study aimed to evaluate the effect and mechanism of caffeine on metastasis-related mechanisms. The results revealed that caffeine treatment at concentrations of 0–500 µM caused no direct cytotoxic effects on NCI-H23 cells. Treatment of cells with caffeine showed good potential to inhibit cell proliferation at 48 h and induced significant cell cycle arrest at the G0/G1 phase. Concerning metastasis, caffeine was shown to reduce filopodia formation, inhibit migration and invasion capability, and reduce the ability of cancer cells to survive and grow in an anchorage-independent manner. Moreover, caffeine could attenuate the formation of 3D tumor spheroids in cancer stem cell (CSC)-enriched populations. With regard to mechanisms, we found that caffeine significantly altered the integrin pattern of the treated cells and caused the downregulation of metastasis-associated integrins, namely, integrins αv and β3. Subsequently, the downstream signals, including protein signaling and transcription factors, namely, phosphorylated focal adhesion kinase (p-FAK), phosphorylated protein kinase B (p-Akt), cell division cycle 42 (Cdc42), and c-Myc, were significantly decreased in caffeine-exposed cells. Taken together, our novel data on caffeine-inhibiting mechanism in relation to metastasis in lung cancer could provide insights into the impact of caffeine intake on human diseases and conditions.

In searching for novel targeted therapeutic agents for lung cancer treatment, norcycloartocarpin from Artocarpus gomezianus was reported in this study to promisingly interacted with Akt and exerted the apoptosis induction and epithelial-to-mesenchymal transition suppression. Selective cytotoxic profile of norcycloartocarpin was evidenced with approximately 2-fold higher IC50 in normal dermal papilla cells (DPCs) compared with human lung cancer A549, H460, H23, and H292 cells. We found that norcycloartocarpin suppressed anchorage-independent growth, cell migration, invasion, filopodia formation, and decreased EMT in a dose-dependent manner at 24 h, which were correlated with reduced protein levels of N-cadherin, Vimentin, Slug, p-FAK, p-Akt, as well as Cdc42. In addition, norcycloartocarpin activated apoptosis caspase cascade associating with restoration of p53, down-regulated Bcl-2 and augmented Bax in A549 and H460 cells. Interestingly, norcycloartocarpin showed potential inhibitory role on protein kinase B (Akt) the up-stream dominant molecule controlling EMT and apoptosis. Computational molecular docking analysis further confirmed that norcycloartocarpin has the best binding affinity of -12.52 kcal/mol with Akt protein at its critical active site. As Akt has recently recognized as an attractive molecular target for therapeutic approaches, these findings support its use as a plant-derived anticancer agent in cancer therapy.

Alpha folate receptor (FRα) is currently under investigation as a target for the treatment of patients with non-small-cell lung cancer (NSCLC), since it is highly expressed in tumor cells but is largely absent in normal tissue. In this study, a novel human variable domain of a heavy-chain (VH) antibody fragment specific to FRα was enriched and selected by phage bio-planning. The positive phage clone (3A102 VH) specifically bound to FRα and also cross-reacted with FRβ, as tested by ELISA. Clone 3A102 VH was then successfully expressed as a soluble protein in an E. coli shuffle strain. The obtained soluble 3A102 VH demonstrated a high affinity for FRα with affinity constants (Kaff) values around 7.77 ± 0.25 × 107 M−1, with specific binding against both FRα expressing NSCLC cells and NSCLC patient-derived primary cancer cells, as tested by cell ELISA. In addition, soluble 3A102 VH showed the potential desired property of a targeting molecule by being internalized into FRα-expressing cells, as observed by confocal microscopy. This study inspires the use of phage display to develop human VH antibody (Ab) fragments that might be well suited for drug targeted therapy of NSCLC and other FRα-positive cancer cells.

Background/aim To date, no cell-based assay that focuses on the prime cause of acne initiation through activation of toll-like receptor2 and 4 and interleukin-8 (IL-8) production exists. Herein, we present an assay that evaluates acne by determining TLR2 and 4 expression and activation. Materials and methods Viability of keratinocytes was determined by the MTT assay. IL-8 was evaluated by ELISA. Immunocytochemistry was performed for determining receptor expression. Results Lipoteichoic acid (LTA), peptidoglycan (PGN) and lipopolysaccharide (LPS) induced IL-8 production. Pre-treatment of cells with TLR2 and TLR4 inhibitors, before stimulation, reduced IL-8 production. Zinc gluconate was used for verification. Zinc can significantly suppress IL-8 production in the system. Treatment of cells with LTA+PGN or LPS resulted in increased TLR2 and TLR4 expression on the cell surface. This effect was prevented by zinc treatment. Conclusion The measurement of IL-8 and TLR2 and TLR4 levels can be used for the evaluation of anti-acne treatment.

Autophagy is the multistep mechanism for the elimination of damaged organelles and misfolded proteins. This mechanism is preceded and may induce other program cell deaths such as apoptosis. This study unraveled the potential pharmacological effect of 24MD in inducing the autophagy of lung cancer cells. Results showed that 24MD was concomitant with autophagy induction, indicating by autophagosome staining and the induction of ATG5, ATG7 and ubiquitinated protein, p62 expression after 12-h treatment. LC3-I was strongly conversed to LC3-II, and p62 was downregulated after 24-h treatment. The apoptosis-inducing activity was found after 48-h treatment as indicated by annexin V-FITC/propidium iodide staining and the activation of caspase-3. From a mechanistic perspective, 24-h treatment of 24MD at 60 μM substantially downregulated p-mTOR. Meanwhile, p-PI3K and p-Akt were also suppressed by 24MD at concentrations of 80 and 100 μM, respectively. We further confirmed m-TOR-mediated autophagic activity by comparing the effect of 24MD with rapamycin, a potent standard mTOR1 inhibitor through Western blot and immunofluorescence assays. Although 24MD could not suppress p-mTOR as much as rapamycin, the combination of rapamycin and 24MD could increase the mTOR suppressive activity and LC3 activation. Changing the substituent groups (R groups) from dimethylphenol to ethylphenol in EMD or changing methylazanedyl to cyclohexylazanedyl in 24CD could only induce apoptosis activity but not autophagic inducing activity. We identified 24MD as a novel compound targeting autophagic cell death by affecting mTOR-mediated autophagy.

Integrin-targeting compounds have shown clinically significant benefits in many patients. Here, we examined the activity of millettocalyxin B, extracted from the stem bark of Millettia erythrocalyx, in lung cancer cells.The viability of human lung cancer cells was investigated by the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazoliumbromide (MTT) assay. Migration and invasion assays were performed. Phalloidin-rhodamine staining was used to determine the formation of filopodia. Western blot analysis and immunofluorescence staining were used to identify the signaling proteins involved in migration regulation.Non-toxic concentrations (0-25 μM) of millettocalyxin B reduced migration and invasion of lung cancer A549 cells. Filopodia were significantly reduced in millettocalyxin B-treated cells. The migration regulatory proteins including integrin α5, active FAK, active Akt, and Cdc42 were significantly decreased in Millettocalyxin B-treated cells.Our findings revealed a novel anti-migration and anti-invasion effects and the underlying mechanism of millettocalyxin B, which may be exploited for cancer treatment.

Background Intratumour heterogeneous gene expression among cancer and cancer stem cells (CSCs) can cause failure of current targeted therapies because each drug aims to target the function of a single gene. Long mononucleotide A-T repeats are cis-regulatory transcriptional elements that control many genes, increasing the expression of numerous genes in various cancers, including lung cancer. Therefore, targeting A-T repeats may dysregulate many genes driving cancer development. Here, we tested a peptide nucleic acid (PNA) oligo containing a long A-repeat sequence [A(15)] to disrupt the transcriptional control of the A-T repeat in lung cancer and CSCs. Methods First, we separated CSCs from parental lung cancer cell lines. Then, we evaluated the role of A-T repeat gene regulation by counting the number of repeats in differentially regulated genes between CSCs and the parental cells of the CSCs. After testing the dosage and effect of PNA-A15 on normal and cancer cell toxicity and CSC phenotypes, we analysed genome-wide expression to identify dysregulated genes in CSCs. Results The number of A-T repeats in genes differentially regulated between CSCs and parental cells differed. PNA-A15 was toxic to lung cancer cells and CSCs but not to noncancer cells. Finally, PNA-A15 dysregulated a number of genes in lung CSCs. Conclusion PNA-A15 is a promising novel targeted therapy agent that targets the transcriptional control activity of multiple genes in lung CSCs.

Background/aim c-Met (mesenchymal-epithelial transition factor) facilitates cancer progression and is recognized as a promising drug target. The molecular target of gigantol from Dendrobium draconis in suppressing cancer metastasis is largely unknown. Materials and methods Proteins affected by gigantol treatment were subjected to proteomic and bioinformatic analysis. Protein-Protein interaction (PPI) networks were constructed by the Search Tool for the Retrieval of Interacting Genes (STRING). The Kyoto Encyclopedia of Genes and Genomes (KEGG) database and hub gene were used to enrich the dominant pathways. Western blot analysis and immunofluorescence were used to validate the effect of gigantol on the target protein and signaling. Results Gigantol down-regulates 41 adhesion proteins and 39-migratory proteins, while it up-regulates 30 adhesion-related proteins and 22 proteins controlling cell migration. The key components of our constructed PPI network comprised 41 proteins of cell adhesion enriched in 40 nodes with 25 edges, 39 proteins of cell migration enriched in 39 nodes with 76 edges in down-regulated proteins, 30 proteins of cell adhesion enriched in 30 nodes with 21 edges, and 22 proteins of cell migration enriched in 22 nodes with 22 edges in up-regulated protein. c-Met was identified as a central protein of the PPI network in the largest degree. KEGG mapper further suggested that c-Met, PI3K, and AKT were the regulatory proteins affected by gigantol. To confirm, the effects of gigantol on c-Met, the p-PI3K, PI3K, p-AKT, and AKT proteins were investigated by western blotting and the results showed a consistent effect of gigantol in the suppression of the c-Met/PI3K/AKT signal. Next, immunofluorescence showed a dramatic decrease in c-Met, PI3K and AKT activation in response to gigantol. Conclusion c-Met is an important target of gigantol treatment in lung cancer cells. Gigantol suppresses metastasis-related cell motility through decreasing c-Met resulting in PI3K/AKT signaling disruption.

The incidence of metastasis stage crucially contributes to high recurrence and mortality rate in lung cancer patients. Unfortunately, no available treatment inhibits migration, a key metastasis process in lung cancer. In this study, the effect of 22-O-(N-Boc-L-glycine) ester of renieramycin M (22-Boc-Gly-RM), a semi-synthetic amino ester derivative of bistetrahydroisoquinolinequinone alkaloid isolated from Xestospongia sp., on migratory behavior of human lung cancer cells was investigated. Following 24 h of treatment, 22-Boc-Gly-RM at non-toxic concentrations (0.5-1 μM) effectively restrained motility of human lung cancer H460 cells assessed through wound healing, transwell migration, and multicellular spheroid models. The capability to invade through matrix component was also repressed in H460 cells cultured with 0.1-1 µM 22-Boc-Gly-RM. The dose-dependent reduction of phalloidin-stained actin stress fibers corresponded with the downregulated Rac1-GTP level presented via western blot analysis in 22-Boc-Gly-RM-treated cells. Treatment with 0.1-1 μM of 22-Boc-Gly-RM obviously caused suppression of p-FAK/p-Akt signal and consequent inhibition of epithelial-to-mesenchymal transition (EMT), which was evidenced with augmented level of E-cadherin and reduction of N-cadherin expression. The alteration of invasion-related proteins in 22-Boc-Gly-RM-treated H460 cells was indicated by the diminution of matrix metalloproteinases (MT1-MMP, MMP-2, MMP-7, and MMP-9), as well as the upregulation of tissue inhibitors of metalloproteinases (TIMP), TIMP2, and TIMP3. Thus, 22-Boc-Gly-RM is a promising candidate for anti-metastasis treatment in lung cancer through inhibition of migratory features associated with suppression on EMT.

Parkinson’s disease (PD) is characterized by the progressive degeneration of dopaminergic neurons. The cause of PD is still unclear. Oxidative stress and mitochondrial dysfunction have been linked to the development of PD. Luteolin, a non-toxic flavonoid, has become interested in an alternative medicine, according to its effects on anti-oxidative stress and anti-apoptosis, although the underlying mechanism of luteolin on PD has not been fully elucidated. This study aims to investigate whether luteolin prevents neurotoxicity induction by 1-methyl-4-phenylpyridinium iodide (MPP+), a neurotoxin in neuroblastoma SH-SY5Y cells. The results reveal that luteolin significantly improved cell viability and reduced apoptosis in MPP+-treated cells. Increasing lipid peroxidation and superoxide anion (O2ˉ), including mitochondrial membrane potential (Δψm) disruption, is ameliorated by luteolin treatment. In addition, luteolin attenuated MPP+-induced neurite damage via GAP43 and synapsin-1. Furthermore, Cdk5 is found to be overactivated and correlated with elevation of cleaved caspase-3 activity in MPP+-exposed cells, while phosphorylation of Erk1/2, Drp1, Fak, Akt and GSK3β are inhibited. In contrast, luteolin attenuated Cdk5 overactivation and supported phosphorylated level of Erk1/2, Drp1, Fak, Akt and GSK3β with reducing in cleaved caspase-3 activity. Results indicate that luteolin exerts neuroprotective effects via Cdk5-mediated Erk1/2/Drp1 and Fak/Akt/GSK3β pathways, possibly representing a potential preventive agent for neuronal disorder.

The limitations of cisplatin, a standard chemotherapy for lung cancer, have been documented with serious adverse effects and drug resistance. To address the need for novel therapy, this study firstly reveals the potential of peptide from Lentinus squarrosulus (Mont.) as a chemotherapeutic adjuvant for cisplatin treatment. The purified peptide from L. squarrosulus aqueous extracts was obtained after eluting with 0.4 M NaCl through FPLC equipped with anion exchange column. Preincubation for 24 h with 5 µg/mL of the peptide at prior to treatment with 5 µM cisplatin significantly diminished %cell viability in various human lung cancer cells but not in human dermal papilla and proximal renal cells. Flow cytometry indicated the augmentation of cisplatin-induced apoptosis in lung cancer cells pretreated with peptide from L. squarrosulus. Preculture with the peptide dramatically inhibited colony formation in lung cancer cells derived after cisplatin treatment. Strong suppression on integrin-mediated survival was evidenced with the diminution of integrins (β1, β3, β5, α5, αV) and down-stream signals (p-FAK/FAK, p-Src/Src, p-Akt/Akt) consequence with alteration of p53, Bax, Blc-2 and Mcl-1 in cisplatin-treated lung cancer cells preincubated with peptide from L. squarrosulus. These results support the development of L. squarrosulus peptide as a novel combined chemotherapy with cisplatin for lung cancer treatment.

Background Accumulated evidence demonstrates cisplatin, a recommended chemotherapy, modulating pro-survival autophagic response that contributes to treatment failure in lung cancer patients. However, distinct mechanisms involved in cisplatin-induced autophagy in human lung cancer cells are still unclear. Results Herein, role of autophagy in cisplatin resistance was indicated by a decreased cell viability and increased apoptosis in lung cancer H460 cells pre-incubated with wortmannin, an autophagy inhibitor, prior to treatment with 50 µM cisplatin for 24 h. The elevated level of hydroxyl radicals detected via flow-cytometry corresponded to autophagic response, as evidenced by the formation of autophagosomes and autolysosomes in cisplatin-treated cells. Interestingly, apoptosis resistance, autophagosome formation, and the alteration of the autophagic markers, LC3-II/LC3-I and p62, as well as autophagy-regulating proteins Atg7 and Atg3, induced by cisplatin was abrogated by pretreatment of H460 cells with deferoxamine, a specific hydroxyl radical scavenger. The modulations in autophagic response were also indicated in the cells treated with hydroxyl radicals generated via Fenton reaction, and likewise inhibited by pretreatment with deferoxamine. Conclusions In summary, the possible role of hydroxyl radicals as a key mediator in the autophagic response to cisplatin treatment, which was firstly revealed in this study would benefit for the further development of novel therapies for lung cancer.

The inherent limitations, including serious side-effects and drug resistance, of current chemotherapies necessitate the search for alternative treatments especially for lung cancer. Herein, the anticancer activity of colicin N, bacteria-produced antibiotic peptide, was investigated in various human lung cancer cells. After 24 h of treatment, colicin N at 5&ndash, 15 &micro, M selectively caused cytotoxicity detected by MTT assay in human lung cancer H460, H292 and H23 cells with no noticeable cell death in human dermal papilla DPCs cells. Flow cytometry analysis of annexin V-FITC/propidium iodide indicated that colicin N primarily induced apoptosis in human lung cancer cells. The activation of extrinsic apoptosis evidenced with the reduction of c-FLIP and caspase-8, as well as the modulation of intrinsic apoptosis signaling proteins including Bax and Mcl-1 were observed via Western blot analysis in lung cancer cells cultured with colicin N (10&ndash, M) for 12 h. Moreover, 5&ndash, M of colicin N down-regulated the expression of activated Akt (p-Akt) and its upstream survival molecules, integrin &beta, 1 and &alpha, V in human lung cancer cells. Taken together, colicin N exhibits selective anticancer activity associated with suppression of integrin-modulated survival which potentiate the development of a novel therapy with high safety profile for treatment of human lung cancer.

Background/aim Epithelial to mesenchymal transition (EMT) is a cellular process that facilitates cancer metastasis. Therefore, therapeutic approaches that target EMT have garnered increasing attention. The present study aimed to examine the in vitro effects of ephemeranthol A on cell death, migration, and EMT of lung cancer cells. Materials and methods Ephemeranthol A was isolated from Dendrobium infundibulum. Non-small cell lung cancer cells H460 were treated with ephemeranthol A and apoptosis was evaluated by Hoechst 33342 staining. Anoikis resistance was determined by soft agar assay. Wound healing assay was performed to test the migration. The regulatory proteins of apoptosis and cell motility were determined by western blot. Results Treatment with ephemeranthol A resulted in a concentration-dependent cell apoptosis. At non-toxic concentrations, the compound could inhibit anchorage-independent growth of the cancer cells, as indicated by the decreased colony size and number. Ephemeranthol A also exhibited an inhibitory effect on migration. We further found that ephemeranthol A exerts its antimetastatic effects via inhibition of EMT, as indicated by the markedly decrease of N-cadherin, vimentin, and Slug. Furthermore, the compound suppressed the activation of focal adhesion kinase (FAK) and protein kinase B (Akt) proteins, which are key regulators of cell migration. As for the anticancer activity, ephemeranthol A induced apoptosis by decreasing Bcl-2 followed by the activation of caspase 3 and caspase 9. Conclusion The pro-apoptotic and anti-migratory effects of ephemeranthol A on human lung cancer cells support its use for the development of novel anticancer therapies.

Aberrant cellular Myc (c-Myc) is a common feature in the majority of human cancers and has been linked to oncogenic malignancies. Here, we developed a novel c-Myc-targeting compound

Emerging evidence has provided important information on oncoproteins involved in cancer initiation, progression, metastasis, and resistance to current therapies. C-myc, one of the critical oncoproteins, has been shown to be implicated in enhancing the aggressiveness of many cancers, mainly through its ability to increase cancer cell growth and cellular survival mechanisms. Despite the more precise and earlier detection and the availability of better therapies, lung cancer remains the most dreadful cancer as it causes high mortality rate with relatively poor treatment success. In lung cancer, C-myc is frequently dysregulated and associated with unfavorable patient survival. C-myc plays a role in regulation of lung cancer cell behaviors including growth, resistance, death, and dissemination through the activation of cell cycle driving proteins, an increase in the cellular levels of anti-apoptotic proteins, and the modulation of metabolism. Besides, C-myc has been shown to be important for cancer stem cell (CSC) properties. Taken together, targeting as well as inhibiting C-myc could provide promising means for resolving lung cancer. This review emphasizes on the molecular mechanism by which C-myc influences lung cancer growth, metastasis, drug resistance, and CSC maintenance, and suggests the target proteins that may benefit drug discovery and design.

Hepatocellular carcinoma (HCC) is one of the most common causes for cancer‐related death worldwide with rapidly increasing incidence and mortality rates. As for other types of cancers, also in HCC cancer stem cells (CSCs) are thought to be responsible for tumour initiation, progression and therapy failure. However, as rare subpopulations of tumour tissue, CSCs are difficult to isolate, thus making the development of suitable and reliable model systems necessary. In our study, we generated HepG2 subclones with enriched CSC potential by application of the spheroid formation method and subsequent single‐cell cloning. Analyses in several 2D and 3D cell culture systems as well as a panel of functional assays both in vitro and in vivo revealed that the generated subclones displayed characteristic and sustained features of tumour initiating cells as well as highly aggressive properties related to tumour progression and metastasis. These characteristics could clearly be correlated with the expression of CSC markers that might have prognostic value in the clinical HCC setting. Therefore, we conclude that our CSC enriched HepG2 clones certainly represent suitable model systems to study the role of CSCs during HCC initiation, progression and drug resistance.

Cancer stem cells (CSCs) are unique populations of cells that can self-renew and generate different cancer cell lineages. Although CSCs are believed to be a promising target for novel therapies, the specific mechanisms by which these putative therapeutics could intervene are less clear. Nitric oxide (NO) is a biological mediator frequently up-regulated in tumors and has been linked to cancer aggressiveness. Here, we search for targets of NO that could explain its activity. We find that it directly affects the stability and function of octamer-binding transcription factor 4 (Oct4), known to drive the stemness of lung cancer cells. We demonstrated that NO promotes the CSC-regulatory activity of Oct4 through a mechanism that involves complex formation between Oct4 and the scaffolding protein caveolin-1 (Cav-1). In the absence of NO, Oct4 forms a molecular complex with Cav-1, which promotes the ubiquitin-mediated proteasomal degradation of Oct4. NO promotes Akt-dependent phosphorylation of Cav-1 at tyrosine 14, disrupting the Cav-1:Oct4 complex. Site-directed mutagenesis and computational modeling studies revealed that the hydroxyl moiety at tyrosine 14 of Cav-1 is crucial for its interaction with Oct4. Both removal of the hydroxyl via mutation to phenylalanine and phosphorylation lead to an increase in binding free energy (ΔG(bind)) between Oct4 and Cav-1, destabilizing the complex. Together, these results unveiled a novel mechanism of CSC regulation through NO-mediated stabilization of Oct4, a key stem cell transcription factor, and point to new opportunities to design CSC-related therapeutics.

The novel information regarding molecular and translational research have created a paradigm shift in the understanding of lung cancer biology, revealing the more precise target for anti-cancer drug discovery. Lung cancer is a leading cause of cancer death worldwide accounting for approximately 1 in 5 of all cancer-related deaths. The most important causes of death in such a cancer involves the treatment failure as well as the spreading of cancer cells to distant sites which the cancer stem cell (CSC) within the tumor is accepted as a key driver. CSC is a rare special population of cancer cells exhibiting high tumorigenic properties together with self-renewal and differentiation capability. CSC is not only linked with high tumor-initiating activity, but is also implicated in chemotherapeutic resistance, metastasis, epithelial to mesenchymal transition, and recurrence. Thereafter, novel therapeutic strategies targeting these CSCs are considered in order to improve long-term clinical outcome. Here, we provide sufficient data regarding the biology of CSC in lung cancer, known CSC markers and cellular signals, and promising compounds targeting the stem cell signals in lung cancer that may benefit the development of novel anti-cancer treatment.

Lung cancer appears to have the highest rate of mortality among cancers due to its metastasis capability. To achieve metastasis, cancer cells acquire the ability to undergo a switch from epithelial to mesenchymal behaviour, termed the epithelial-to-mesenchymal transition (EMT), which is associated with poor clinical outcomes. Drug discovery attempts have been made to find potent compounds that will suppress EMT. Cypripedin, a phenanthrenequinone isolated from Thai orchid, Dendrobium densiflorum, exhibits diverse pharmacological activities. In this study, we found that cypripedin attenuated typical mesenchymal phenotypes, including migratory behaviour, of non-small cell lung cancer H460 cells, with a significant reduction of actin stress fibres and focal adhesion and with weakened anchorage-independent growth. Western blot analysis revealed that the negative activity of this compound on EMT was a result of the down-regulation of the EMT markers Slug, N-Cadherin and Vimentin, which was due to ATP-dependent tyrosine kinase (Akt) inactivation. As a consequence, the increase in the Slug degradation rate via a ubiquitin-proteasomal mechanism was encouraged. The observation in another lung cancer H23 cell line also supported this finding, indicating that cypripedin exhibits a promising pharmacological action on lung cancer metastasis that could provide scientific evidence for the further development of this compound.

Background/aim Novel information on the role of endogenous compounds in regulating physiological and pathological process are of interest, as it may lead to the development of better strategies for disease management. The role of angiotensin II and the signaling of type 1 angiotensin II receptor (AGT1R) in T-lymphocyte activation and interleukin-2 (IL-2) production are largely unknown. Materials and methods Jurkat T-cells were treated with AGT1R inhibitor candesartan and stimulated with phorbol myristate acetate (PMA) and ionomycin. T-Cell activation, associated cytokine production and levels of signaling proteins were evaluated by flow cytometry and western blot analysis. Results Candesartan significantly suppressed PMA and ionomycin-induced CD25 expression and IL-2 production. Regarding the molecular mechanism involved, we showed that such suppressive effects of blocking of AGT1R by candesartan resulted in the significant inhibition of ERK activation in PMA-stimulated Jurkat T-cells. The effect of ERK inhibition on T-cell activation was further confirmed. Treatment with FR180204, a specific ERK inhibitor, reduced T-cell activation and IL-2 secretion. Conclusion AGT1R signaling is essential for T-cell activation and IL-2 production, and the inhibition of this pathway suppressed T-cell activation via an ERK-dependent mechanism.

The epithelial-to-mesenchymal transition is proposed to be a key mechanism responsible for metastasis-related deaths. Similarly, cancer stem cells (CSCs) have been proposed to be a key driver of tumor metastasis. However, the link between the two events and their control mechanisms is unclear. We used a three-dimensional (3D) tumor spheroid assay and other CSC-indicating assays to investigate the role of E-cadherin in CSC regulation and its association to epithelial-to-mesenchymal transition in lung cancer cells. Ectopic overexpression and knockdown of E-cadherin were found to promote and retard, respectively, the formation of tumor spheroids in vitro but had opposite effects on tumor formation and metastasis in vivo in a xenograft mouse model. We explored the discrepancy between the in vitro and in vivo results and demonstrated, for the first time, that E-cadherin is required as a component of a major survival pathway under detachment conditions. Downregulation of E-cadherin increased the stemness of lung cancer cells but had an adverse effect on their survival, particularly on non-CSCs. Such downregulation also promoted anoikis resistance and invasiveness of lung cancer cells. These results suggest that anoikis assay could be used as an alternative method for in vitro assessment of CSCs that involves dysregulated adhesion proteins. Our data also suggest that agents that restore E-cadherin expression may be used as therapeutic agents for metastatic cancers.

Aberrant metabolism in hexosamine biosynthetic pathway (HBP) has been observed in several cancers, affecting cellular signaling and tumor progression. However, the role of O-GlcNAcylation, a post-translational modification through HBP flux, in apoptosis remains unclear. Here, we found that hyper-O-GlcNAcylation in lung carcinoma cells by O-GlcNAcase inhibition renders the cells to apoptosis resistance to cisplatin (CDDP). Profiling of various key regulatory proteins revealed an implication of either p53 or c-Myc in the apoptosis regulation by O-GlcNAcylation, independent of p53 status. Using co-immunoprecipitation and correlation analyses, we found that O-GlcNAcylation of p53 under certain cellular contexts, i.e. high p53 activation, promotes its ubiquitin-mediated proteasomal degradation, resulting in a gain of oncogenic and anti-apoptotic functions. By contrast, O-GlcNAcylation of c-Myc inhibits its ubiquitination and subsequent proteasomal degradation. Gene manipulation studies revealed that O-GlcNAcylation of p53/c-Myc is in part a regulator of CDDP-induced apoptosis. Accordingly, we classified CDDP resistance by hyper-O-GlcNAcylation in lung carcinoma cells as either p53 or c-Myc dependence based on their molecular targets. Together, our findings provide novel mechanisms for the regulation of lung cancer cell apoptosis that could be important in understanding clinical drug resistance and suggest O-GlcNAcylation as a potential target for cancer therapy.

Cancer stem cells (CSCs) that possess the ability of self-renewal and multi-potency have been shown to drive tumor progression and metastasis. The majority of recent studies has focused on potential molecules targeting CSCs so as to develop novel strategies for efficient cancer treatment or protection. Here, we show how alpha-lipoic acid (LA), an endogenous mitochondrial anti-oxidant, affects the CSC-like phenotypes of human non-small cell lung cancer-derived H23, H292 and H460 cells. CSC-like phenotypes were verified by anchorage-independent growth, three-dimensional (3D) spheroid formation and the expression of CSC markers. Enriched CSC populations were used to confirm the effects of LA. Protein ubiquitination and degradation were assessed using immunoprecipitation. We found that treatment with LA reduced the CSC-like phenotype, as indicated by a decreased expression of known CSC markers (CD133, CD44, ALDH1A1, Oct-4 and Nanog) in H460 cells. In addition, we found that LA reduced the CSC-related abilities of anchorage-independent growth and 3D spheroid formation, and suppressed factors related to epithelial-mesenchymal transition, such as E-cadherin, Vimentin, Slug and Snail. Mechanistically, we found that LA suppresses CSC through depletion of the cellular stemness proteins β-catenin and Oct-4 via decreasing the level of active (phosphorylated) Akt. This resulted in the induction of GSK3β-dependent β-catenin ubiquitin-proteasomal degradation and a decrease in the stabilized (phosphorylated) form of Oct-4. The effects of LA on the CSC-like phenotypes were confirmed in CSC enriched H460, H292 and H23 non-small cell lung cancer-derived cells. Our data are indicative for a novel regulatory role and underlying mechanism of LA in the negative regulation of a CSC-like phenotype in non-small cell lung cancer-derived cells.

Background/aim Cancer stem cells (CSCs) are a subpopulation of cancer cells that possess self-renewal and differentiation capacities. CSCs contribute to drug-resistance, cancer recurrence and metastasis, thus development of CSC-targeted therapeutic strategies has recently received significant attention in cancer research. In this study, the potential efficacy of renieramycin M (RM) isolated from the sponge Xestospongia species, was examined against lung CSCs. Materials and methods Colony and spheroid formation assays, as well as western blotting analysis of lung CSC protein markers were employed to determine the CSC-like phenotypes of H460 lung cancer cells after treatment with RM at non-toxic concentrations. Results RM treatment reduced significantly colony and spheroid formation of H460 cells. Moreover, the CSC markers CD133, CD44 and ALDH1A1 of CSC-enriched H460 cells were reduced significantly following RM treatment. Conclusion RM could be a potent anti-metastatic agent by suppressing lung CSC-like phenotypes in H460 cells.

Context: Lentinus squarrosulus Mont. (Polyporaceae) is an interesting source of diverse bioactive compounds. Objective: This is the first study of the anticancer activity and underlying mechanism of peptides extracted from Lentinus squarrosuls. Materials and methods: Peptides were isolated from the aqueous extract of L. squarrosulus by employing solid ammonium sulphate precipitation. They were further purified by ion-exchange chromatography on diethylaminoethanol (DEAE)-cellulose and gel filtration chromatography on Sephadex G25. Anticancer activity was investigated in human lung cancer H460, H292 and H23 cells cultured with 0–40 μg/mL of peptide extracts for 24 h. Cell viability and mode of cell death were evaluated by MTT and nuclear staining assay, respectively. Western blotting was used to investigate the alteration of apoptosis-regulating proteins in lung cancer cells treated with peptide extracts (0–20 μg/mL) for 24 h. Results: The cytotoxicity of partially-purified peptide extracts from L. squarrosulus was indicated with IC50 of ∼26.84 ± 2.84, 2.80 ± 2.14 and 18.84 ± 0.30 μg/mL in lung cancer H460, H292 and H23 cells, respectively. The extracts at 20 μg/mL induced apoptosis through the reduction of anti-apoptotic Bcl-2 protein (∼0.5-fold reduction) and up-regulation of BAX (∼4.5-fold induction), a pro-apoptotic protein. Furthermore, L. squarrosulus peptide extracts (20 μg/mL) also decreased the cellular level of death receptor inhibitor c-FLIP (∼0.6-fold reduction). Conclusions and discussion: This study provides the novel anticancer activity and mechanism of L. squarrosulus peptide extracts, which encourage further investigation and development of the extracts for anticancer use.

Individualized proper chemotherapy using in vitro drug sensitivity testing has been proposed as a novel therapeutic modality and shown to have better efficacy than empiric chemotherapy. However, issues around establishing a patient-derived cell culture or xenograft, the timing of the testing obtained, and the validity of testing represent major limitations to translating the use of such a technique to clinical practice.In this study, we assessed the feasibility of an in vitro drug sensitivity technique for testing malignant pleural effusion from advanced-stage non-small cell lung cancer.Our technique was able to produce a turnaround time for in vitro drug sensitivity testing of less than 1 week, with a success rate of more than 90% of cases. Correlated with the individual clinical outcome, using the area under the dose response curve (AUC) could define the level of in vitro drug sensitivity as: responsive (AUC0.25), intermediate response (0.1≤AUC≤0.25), or resistance (AUC0.1).Data obtained from this method of drug testing were correlated with the clinical outcome. The present drug sensitivity evaluation may benefit the development of individual precision chemotherapy.

Background/Aim: Fine airborne particles of Particular Matter of less than 2.5 micrometers (PM(2.5)) have been recognized as a dominant air contamination causing critical health concerns. Herein, we determined whether isovitexin, a natural plant-derived compound could protect PM(2.5)-mediated oxidative stress and induce stemness in epidermal cells. Materials and Methods: Cell viability was detected by the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay. Reactive oxygen species (ROS) were determined by flow cytometry with 2’,7’-dichlorofluorescin diacetate (DCFH-DA). Protein hallmarks of stem cells were examined by western blot analysis. Results: PM(2.5) treatment for 30 min increased the levels of intracellular ROS. Pre-treatment of cells with 10-50 μM of isovitexin dramatically inhibited the ROS induced by PM(2.5). Antioxidant efficacy of isovitexin was also determined by the ROS scavenging activity against 2,2-diphenyl-2-picrylhydrazyl (DPPH), ABTS and superoxide anion radicals. In addition, we found that isovitexin enhanced the stem cell properties of keratinocytes, indicated by the significant increase in the levels of stem cell proteins. Conclusion: Isovitexin can be potentially used as an effective compound for preventing skin damage.