CL1-5 Cells Research Articles

Use microfluidics to study cell migration in response to fluid shear stress gradients

Cells respond not only to biological regulatory factors but also to physical stimuli such as electric fields, light, and shear stress in their environment. These stimuli can lead to cell migration and morphological changes. In the human body, cells encounter fluid shear stress induced by interstitial flow, lymphatic flow, blood flow, or organ-specific conditions within their micro-environments. Therefore, fluid shear stress, a classic mechanical force, has gained significant attention in wound healing and cancer metastasis. In this study, a microfluidic chip was developed to both culture cells and generate a shear stress gradient to direct cell migration. The design of this device’s geometry allows the generation of a shear stress gradient perpendicular to the direction of medium flow. This greatly eliminates the influence of flow-induced cell responses. Using mouse fibroblast cells (NIH3T3) and human lung cancer cells (CL1-5) as models, their migration directionality, migration rates, and alignment in response to the shear stress gradient were investigated. Within the stress range of 0.095–0.155 Pa and a gradient of 0.015 Pa/mm, NIH3T3 cells did not exhibit significant directional migration, differences in migration rates, or specific alignment patterns. In contrast, CL1-5 cells preferred higher shear stress environments and alignment parallel to the medium flow, suggesting that these conditions could induce higher mobility in these cancer cells.

Effects of methylation and transcription factor YY1 on ID2 expression in non-small cell lung carcinoma cells.

The inhibitor of DNA-binding 2 (ID2) plays a major role in tumor dedifferentiation in non-small cell lung cancer (NSCLC). Studies have indicated an inverse correlation between ID2 expression and NSCLC cell invasiveness. However, the mechanisms through which ID2 activation is regulated are currently unclear. We overexpressed ID2 in H1299 cells and extensively characterized their cellular behaviors. By employing a serial deletion approach combined with a reporter assay, we pinpointed the basal promoter region of ID2. We also examined the DNA methylation status of the ID2 promoter to elucidate the epigenetic mechanisms driving ID2 regulation. Our results revealed that ID2 overexpression effectively inhibited the migration, invasion, proliferation, and colony formation abilities of H1299 cells. The region from -243 to +202 played a major role in driving the transcriptional activity of ID2. Sequence analysis results indicated that the transcription factor Yin Yang 1 (YY1) might be crucial in the regulation of ID2 expression. The ectopically expressed YY1 activated both the expression levels of ID2 and the transcriptional activity of the ID2 promoter, potentially contributing to its repressive activity on cancer cell growth. Furthermore, site-directed mutagenesis and chromatin immunoprecipitation assays revealed that YY1 may target the -120 and -76 sites of the ID2 promoter, thereby activating its transcriptional activity. The ID2 promoter regions were also fully methylated in CL1-5 cells, and the methylation level was correlated with the expression levels of the ID2 promoter. Moreover, the YY1-induced suppression of colony formation was counteracted by ID2 knockdown, which suggests that YY1 represses cell colony growth through the regulation of ID2. Our results indicate that YY1 plays a role in transactivating ID2 expression and might also contribute to the repression of colony growth through the regulation of ID2.

Gemcitabine resistance in non-small cell lung cancer is mediated through activation of the PI3K/AKT/NF-κB pathway and suppression of ERK signaling by reactive oxygen species.

Lung cancer is one of the most common cancers in the world. Chemotherapy is a standard clinical treatment. However, tumor cells often develop multidrug resistance after chemotherapy, an inevitable bottleneck in cancer treatment. Therefore, this study used gemcitabine-resistant (GEM-R) CL1-0 lung cancer cells. First, we used flow cytometry and western blot analysis to examine differences in performance between resistant and parental cells. The results showed that compared with parental cells, GEM-R CL1-0 cells significantly enhanced the activation of the AKT pathway, which promoted survival and growth, and decreased the activation of the reactive oxygen species-extracellular signal-regulated kinase(ROS)-ERK pathway. Next, the AKT and ERK pathways'role in tumor growth was further explored in vivo using a xenograft model. The results showed that enhancing AKT and inhibiting ERK activation reduced GEM-induced inhibition of tumor growth. Finally, combining the above results, we found that GEM-R CL1-0 cells showed reduced sensitivity to GEM by activating the phosphatidylinositol 3-kinase/AKT/NF-kB pathway and inhibiting the ROS-ERK pathway leading to resistance against GEM. Therefore, the AKT and ERK pathways are potential targets for improving the sensitivity of cancer cells to anticancer drugs.

Phosphorylation of Bcl-2 by JNK confers gemcitabine resistance in lung cancer cells by reducing autophagy-mediated cell death.

The most common cancer-related death in the world is non-small cell lung cancer (NSCLC). Gemcitabine (GEM) is a common and effective first-line chemotherapeutic drug for the treatment of NSCLC. However, the long-term use of chemotherapeutic drugs in patients usually induces cancer cell drug resistance, leading to poor survival, and prognosis. In this study, to observe and explore the key targets and potential mechanisms of NSCLC resistance to GEM, we first cultured lung cancer CL1-0 cells in a GEM-containing medium to induce CL1-0 cells to develop GEM resistance. Next, we compared protein expression between the parental and GEM-R CL1-0 cell groups. We observed significantly lower expression of autophagy-related proteins in GEM-R CL1-0 cells than in parental CL1-0 cells, indicating that autophagy is associated with GEM resistance in CL1-0 cells. Furthermore, a series of autophagy experiments revealed that GEM-R CL1-0 cells had significantly reduced GEM-induced c-Jun N-terminal kinase phosphorylation, which further affected the phosphorylation of Bcl-2, thereby reducing the dissociation of Bcl-2 and Beclin-1 and ultimately reducing the generation of GEM-induced autophagy-dependent cell death. Our findings suggest that altering the expression of autophagy is a promising therapeutic option for drug-resistant lung cancer.

Coriloxin Exerts Antitumor Effects in Human Lung Adenocarcinoma Cells.

Both in Taiwan and around the world, lung cancer is a primary cause of cancer-related deaths. In Taiwan, the most prevalent form of lung cancer is lung adenocarcinoma, a type of non-small-cell lung carcinoma. Although numerous lung cancer therapies are available, their clinical outcomes are unsatisfactory. Natural products, including fungal metabolites, are excellent sources of pharmaceutical compounds used in cancer treatment. We employed in vitro cell invasion, cell proliferation, cell migration, cell viability, and colony formation assays with the aim of evaluating the effects of coriloxin, isolated from fermented broths of Nectria balsamea YMJ94052402, on human lung adenocarcinoma CL1-5 and/or A549 cells. The potential targets regulated by coriloxin were examined through Western blot analysis. The cytotoxic effect of coriloxin was more efficiently exerted on lung adenocarcinoma cells than on bronchial epithelial cells. Moreover, low-concentration coriloxin significantly suppressed adenocarcinoma cells’ proliferative, migratory, and clonogenic abilities. These inhibitory effects were achieved through ERK/AKT inactivation, epithelial–mesenchymal transition regulation, and HLJ1 expression. Our findings suggest that coriloxin can be used as a multitarget anticancer agent. Further investigations of the application of coriloxin as an adjuvant therapy in lung cancer treatment are warranted.

Microfluidic Determination of Distinct Membrane Transport Properties between Lung Adenocarcinoma Cells CL1-0 and CL1-5.

The cell membrane permeability of a cell type to water (Lp) and cryoprotective agents (Ps), is the key factor that determines the optimal cooling and mass transportation during cryopreservation. The human lung adenocarcinoma cell line, CL1, has been widely used to study the invasive capabilities or drug resistance of lung cancer cells. Therefore, providing accurate databases of the mass transport properties of this specific cell line can be crucial for facilitating either flexible and optimal preservation, or supply. In this study, utilizing our previously proposed noncontact-based micro-vortex system, we focused on comparing the permeability phenomenon between CL1-0 and its more invasive subline, CL1-5, under several different ambient temperatures. Through the assay procedure, the cells of favor were virtually trapped in a hydrodynamic circulation to provide direct inspection using a high-speed camera, and the images were then processed to achieve the observation of a cell’s volume change with respect to time, and in turn, the permeability. Based on the noncontact nature of our system, we were able to manifest more accurate results than their contact-based counterparts, excluding errors involved in estimating the cell geometry. As the results in this experiment showed, the transport phenomena in the CL1-0 and CL1-5 cell lines are mainly composed of simple diffusion through the lipid bilayer, except for the case where CL1-5 were suspended in the cryoprotective agent (CPA) solution, which also demonstrated higher Ps values. The deviated behavior of CL1-5 might be a consequence of the altered expression of aquaporins and the coupling of a cryoprotective agent and water, and has given a vision on possible studies over these properties, and their potential relationship to invasiveness and metastatic stability of the CL1 cell line.

Folding and Unfolding of Exogenous G-Rich Oligonucleotides in Live Cells by Fluorescence Lifetime Imaging Microscopy of o-BMVC Fluorescent Probe.

Guanine-rich oligonucleotides (GROs) can self-associate to form G-quadruplex (G4) structures that have been extensively studied in vitro. To translate the G4 study from in vitro to in live cells, here fluorescence lifetime imaging microscopy (FLIM) of an o-BMVC fluorescent probe is applied to detect G4 structures and to study G4 dynamics in CL1-0 live cells. FLIM images of exogenous GROs show that the exogenous parallel G4 structures that are characterized by the o-BMVC decay times (≥2.4 ns) are detected in the lysosomes of live cells in large quantities, but the exogenous nonparallel G4 structures are hardly detected in the cytoplasm of live cells. In addition, similar results are also observed for the incubation of their single-stranded GROs. In the study of G4 formation by ssHT23 and hairpin WT22, the analyzed binary image can be used to detect very small increases in the number of o-BMVC foci (decay time ≥ 2.4 ns) in the cytoplasm of live cells. However, exogenous ssCMA can form parallel G4 structures that are able to be detected in the lysosomes of live CL1-0 cells in large quantities. Moreover, the photon counts of the o-BMVC signals (decay time ≥ 2.4 ns) that are measured in the FLIM images are used to reveal the transition of the G4 formation of ssCMA and to estimate the unfolding rate of CMA G4s with the addition of anti-CMA into live cells for the first time. Hence, FLIM images of o-BMVC fluorescence hold great promise for the study of G4 dynamics in live cells.

Disruption of Cytosolic Folate Integrity Aggravates Resistance to Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors and Modulates Metastatic Properties in Non-Small-Cell Lung Cancer Cells

Patients with advanced-stage non-small-cell lung cancer (NSCLC) are susceptible to malnutrition and develop folate deficiency (FD). We previously found that folate deprivation induces drug resistance in hepatocellular carcinoma; here, we assessed whether disrupted cytoplasmic folate metabolism could mimic FD-induced metastasis and affect the sensitivity of NSCLC cells to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs). We examined whether cytosolic folate metabolism in NSCLC cells was disrupted by FD or the folate metabolism blocker pemetrexed for 1–4 weeks. Our results revealed an increase in NF-κB overexpression–mediated epithelial-mesenchymal transition biomarkers: N-cadherin, vimentin, matrix metalloproteinases (MMPs), SOX9, and SLUG. This finding suggests that the disruption of folate metabolism can drastically enhance the metastatic properties of NSCLC cells. Cytosolic FD also affected EGFR-TKI cytotoxicity toward NSCLC cells. Because SLUG and N-cadherin are resistance effectors against gefitinib, the effects of SLUG knockdown in folate antagonist–treated CL1-0 cells were evaluated. SLUG knockdown prevented SLUG/NF-κB/SOX9-mediated invasiveness and erlotinib resistance acquisition and significantly reduced pemetrexed-induced gelatinase activity and MMP gene expression. To summarize, our data reveal two unprecedented adverse effects of folate metabolism disruption in NSCLC cells. Thus, the folic acid status of patients with NSCLC under treatment can considerably influence their prognosis.

Novel function of PERP-428 variants impacts lung cancer risk through the differential regulation of PTEN/MDM2/p53-mediated antioxidant activity

Lung cancer is the leading cause of cancer-related deaths worldwide. Identifying genetic risk factors and understanding their mechanisms will help reduce lung cancer incidence. The p53 apoptosis effect is related to PMP-22 (PERP), a tetraspan membrane protein, and an apoptotic effector protein downstream of p53. Although historically considered a tumor suppressor, PERP is highly expressed in lung cancers. Stable knockdown of PERP expression induces CL1-5 and A549 lung cancer cell death, but transient knockdown has no effect. Interestingly, relative to the PERP-428GG genotype, PERP-428CC was associated with the highest lung cancer risk (OR = 5.38; 95% CI = 2.12–13.65, p < 0.001), followed by the PERP-428CG genotype (OR = 2.34; 95% CI = 1.55–3.55, p < 0.001). Ectopic expression of PERP-428G, but not PERP-428C, protects lung cancer cells against ROS-induced DNA damage. Mechanistically, PERP-428 SNPs differentially regulate p53 protein stability. p53 negatively regulates the expression of the antioxidant enzymes catalase (CAT) and glutathione reductase (GR), thereby modulating redox status. p53 protein stability is higher in PERP-428C-expressing cells than in PERP-428G-expressing cells because MDM2 expression is decreased and p53 Ser20 phosphorylation is enhanced in PERP-428C-expressing cells. The MDM2 mRNA level is decreased in PERP-428C-expressing cells via PTEN-mediated downregulation of the MDM2 constitutive p1 promoter. This study reveals that in individuals with PERP-428CC, CAT/GR expression is decreased via the PTEN/MDM2/p53 pathway. These individuals have an increased lung cancer risk. Preventive antioxidants and avoidance of ROS stressors are recommended to prevent lung cancer or other ROS-related chronic diseases.

Extracellular Vesicle miR-200c Enhances Gefitinib Sensitivity in Heterogeneous EGFR-Mutant NSCLC.

Intratumoral heterogeneity in epidermal growth factor receptor (EGFR)-mutant mutant non-small-cell lung cancer (NSCLC) explains the mixed responses to EGFR-tyrosine kinase inhibitors (TKIs). However, some studies showed tumors with low abundances of EGFR mutation still respond to EGFR-TKI, and the mechanism remained undetermined. Extracellular vesicles (EVs) can transmit antiapoptotic signals between drug-resistant and drug-sensitive cells. Herein, we profiled EVs from EGFR-mutant cells to identify a novel mechanism explaining why heterogenous EGFR-mutant NSCLC patients still respond to EGFR-TKIs. We first demonstrated that the EVs from EGFR-mutant changes the wild-type cells’ sensitivity to gefitinib by adding EV directly or coculturing EGFR wild-type (CL1-5) cells and EGFR-mutant (PC9) cells. In animal studies, only the combined treatment of PC9 EV and gefitinib delayed the tumor growth of CL1-5 cells. MicroRNA analysis comparing EV miRNAs from PC9 cells to those from CL1-5 cells showed that mir200 family members are most abundant in PC9 EVs. Furthermore, mir200a and mir200c were found upregulated in plasma EVs from good responders to EGFR-TKIs. Finally, the transfection of CL1-5 cells with miR200c inactivates downstream signaling pathways of EGFR, the EMT pathway, and enhances gefitinib sensitivity. Overall, our results suggest that in heterogeneous EGFR-mutant NSCLC, tumor cells transmit EV miRNAs that may affect sensitivity to EGFR-TKIs and provide potential prognostic biomarkers for EGFR-mutant NSCLC.

KRASG12C Can Either Promote or Impair Cap-Dependent Translation in Two Different Lung Adenocarcinoma Cell Lines.

KRASG12C is among the most common oncogenic mutations in lung adenocarcinoma and a promising target for treatment by small-molecule inhibitors. KRAS oncogenic signaling is responsible for modulation of tumor microenvironment, with translation factors being among the most prominent deregulated targets. In the present study, we used TALENs to edit EGFRWT CL1-5 and A549 cells for integration of a Tet-inducible KRASG12C expression system. Subsequent analysis of both cell lines showed that cap-dependent translation was impaired in CL1-5 cells via involvement of mTORC2 and NF-κB. In contrast, in A549 cells, which additionally harbor the KRASG12S mutation, cap-dependent translation was favored via recruitment of mTORC1, c-MYC and the positive regulation of eIF4F complex. Downregulation of eIF1, eIF5 and eIF5B in the same cell line suggested a stringency loss of start codon selection during scanning of mRNAs. Puromycin staining and polysome profile analysis validated the enhanced translation rates in A549 cells and the impaired cap-dependent translation in CL1-5 cells. Interestingly, elevated translation rates were restored in CL1-5 cells after prolonged induction of KRASG12C through an mTORC1/p70S6K-independent way. Collectively, our results suggest that KRASG12C signaling differentially affects the regulation of the translational machinery. These differences could provide additional insights and facilitate current efforts to effectively target KRAS.

Structural identification of a fucose-containing 1,3-β-mannoglucan from Poria cocos and its anti-lung cancer CL1-5 cells migration via inhibition of TGFβR-mediated signaling

Poria cocos (Polyporacea), is a fungus used in traditional Chinese medicine. A study of the valuable sulfated polysaccharides (SPS) with the structure and pharmaceutical benefits from the mycelial culture conditions of P. cocos was attempted. The SPSs were fractionated by gel filtration chromatography to give a fucose-containing mannoglucan polysaccharide (denoted as FMGP): [Display omitted] The main skeleton was a 1,4-α-Man-interlaced-1,3-β-glucan with interlaced 6-O-α-l-fucosyl 1,4-α-Glc and 1,4-α-Gal branches. FMGP dramatically inhibited cell migration in the highly metastatic human lung cancer cell line CL1-5 cells. Mechanistically, FMGP dramatically downregulated the expression of TGFβRI and inhibited phosphorylation of FAK and AKT. Moreover, FMGP reduced the metastasis-related protein, Slug, expression. This is the first paper reporting a branched 1,3-β-mannoglucan from P. cocos and its anti-lung cancer CL1-5 cells migration activities.

Expression of large tumour suppressor (LATS) kinases modulates chemotherapy response in advanced non-small cell lung cancer.

BackgroundThe Hippo signalling pathway plays an important role in regulating organ size and cell proliferation. Down-regulation of large tumour suppressor (LATS) protein homologs LATS1 or LATS2 has been found in lung cancer. LATS1 and LATS2 are the core components of the Hippo signalling pathway. LATS1 and LATS2 share some conserved structural features and exhibit redundant biological functions. The aim of this study was to dissect the interaction between these two homologs.MethodsIn lung adenocarcinoma (AD) cells, protein expression of LATS1 and LATS2 were determined by western blotting; cell viability and apoptosis were measured by MTT and annexin V staining after treatment with cisplatin; subcellular distributions of LATS proteins were determined by immunofluorescence microscopy; LATS2 expression was modulated by shRNA-mediated knockdown or ectopic expression in cancer cell lines.ResultsManipulation of the expression of these two LATS kinases influenced cisplatin response in advanced lung AD cell lines. High LATS2-to-LATS1 ratio in H2023 cells was associated with cisplatin resistance, while low LATS2-to-LATS1 ratio in CL1-0 and CL83 cells was associated with sensitivity to cisplatin. Manipulating the LATS2-to-LATS1 ratio by LATS2 over-expression in CL1-0 and CL83 rendered them resistant to cisplatin treatment, whereas LATS2 knockdown in H2023 alleviated the LATS2-to-LATS1 ratio and sensitized cancer cells to cisplatin exposure.ConclusionsOur data suggested that the ratio of expression of LATS kinases played a role in the modulation of cisplatin sensitivity in advanced lung AD, and targeting of LATS proteins as a novel therapeutic strategy for lung AD deserves further investigation.

Hypoxia induces tumor cell growth and angiogenesis in non-small cell lung carcinoma via the Akt-PDK1-HIF1α-YKL-40 pathway.

BackgroundAs one of the most common forms of cancer, non-small cell lung carcinoma (NSCLC), is characterized by oxygen deprivation (hypoxia). The transcription factor hypoxia-inducible factor (HIF)-1α is a major mediator which responds hypoxia and regulates many contributing factors. The various modes of hypoxia regulation are frequently the focus of research studies. With reference to previous published research, we hypothesized that hypoxia promotes the growth and angiogenesis of NSCLC via the Akt-PDK1-HIF1α-YKL-40 pathway, and verified it.MethodsWe mainly investigated changes in related factor expression between differently treated CL1-5 cells. We carried out overexpression and underexpression transfection, Western blot, rt-PCR and ELISA, and observed cellular biological behaviors by CCK-8 migration and invasion assay, and tube formation assay.ResultsA hypoxic environment significantly increased the phosphorylation of Akt and PDK1 in mitochondria. The hypoxia-induced accumulation of p-Akt in mitochondria activated PDK1 phosphorylation, promoted the expression of HIF1α, and the expression of YKL-40. The overexpression of YKL-40 promoted the proliferation, migration, invasion and tubule formation of CL1-5 cells.ConclusionsA hypoxic tumor microenvironment can promote the expansion and angiogenesis of NSCLC cells through the Akt-PDK1-HIF1α-YKL-40 pathway. This may provide a new mechanism and potential interventional target for anti-vascular lung cancer therapy.

Cellular processes involved in lung cancer cells exposed to direct current electric field

With the rapid breakthrough of electrochemical treatment of tumors, electric field (EF)-sensitive genes, previously rarely exploited, have become an emerging field recently. Here, we reported our work for the identification of EF-sensitive genes in lung cancer cells. The gene expression profile (GSE33845), in which the human lung cancer CL1-0 cells were treated with a direct current electric field (dcEF) (300 mV/mm) for 2 h, was retrieved from GEO database. Differentially expressed genes (DEGs) were acquired, followed by Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes pathway (KEGG) and protein-protein interaction (PPI) analysis. Hub genes were acquired and analyzed by various tools including the Human Protein Atlas, Kaplan-Meier analysis, Cytoscape, FunRich, Oncomine and cBioPortal. Subsequently, three-dimensional protein models of hub genes were modeled by Modeller 9.20 and Rosetta 3.9. Finally, a 100 ns molecular dynamics simulation for each hub protein was performed with GROMACS 2018.2. A total of 257 DEGs were acquired and analyzed by GO, KEGG and PPI. Then, 10 hub genes were obtained, and the signal pathway analysis showed that two inflammatory pathways were activated: the FoxO signaling pathway and the AGE-RAGE signaling pathway. The molecular dynamic analysis including RMSD and the radius of gyration hinted that the 3D structures of hub proteins were built. Overall, our work identified EF-sensitive genes in lung cancer cells and identified that the inflammatory state of tumor cells may be involved in the feedback mechanism of lung cancer cells in response to electric field stimulation. In addition, qualified three-dimensional protein models of hub genes were also constructed, which will be helpful in understanding the complex effects of dcEF on human lung cancer CL1-0 cells.

Overexpression of PSAT1 promotes metastasis of lung adenocarcinoma by suppressing the IRF1-IFNγ axis.

An increasing number of enzymes involved in serine biosynthesis have been identified and correlated with malignant evolution in various types of cancer. Here we showed that the overexpression of phosphoserine aminotransferase 1 (PSAT1) is widely found in lung cancer tissues compared with nontumor tissues and predicts a poorer prognosis in patients with lung adenocarcinoma. PSAT1 expression was examined in a tissue microarray by immunohistochemistry. The data show that the knockdown of PSAT1 dramatically inhibits the in vitro and in vivo metastatic potential of highly metastatic lung cancer cells; conversely, the enforced expression of exogenous PSAT1 predominantly enhances the metastatic potential of lung cancer cells. Importantly, manipulating PSAT1 expression regulates the in vivo tumor metastatic abilities in lung cancer cells. Adjusting the glucose and glutamine concentrations did not alter the PSAT1-driven cell invasion properties, indicating that this process might not rely on the activation of its enzymatic function. RNA microarray analysis of transcriptional profiling from PSAT1 alternation in CL1-5 and CL1-0 cells demonstrated that interferon regulatory factor 1 (IRF1) acts as a crucial regulator of PSAT1-induced gene expression upon metastatic progression. Decreasing the IRF1-IFIH1 axis compromised the PSAT1-prompted transcriptional reprogramming in cancer cells. Our results identify PSAT1 as a key regulator by a novel PSAT1/IRF1 axis in lung cancer progression, which may serve as a potential biomarker and therapeutic target for the treatment of lung cancer patients.

Nano-localized single-cell nano-electroporation.

The ability to deliver foreign cargos into single living cells is of great interest in cell biology and therapeutic research. Here, we have reported a single or multiple position based nano-localized single-cell nano-electroporation platform. The device consists of an array of triangular shape ITO nano-electrodes with a 70 nm gap between two nano-electrodes, each having a 40 nm tip diameter. The voltage is applied between nano-electrodes to generate an intense electric field, which electroporates multiple nano-localized regions of the targeted single-cell membrane, and biomolecules are gently delivered into cells by pressurizing pump flow, without affecting cell viability. The platform successfully delivers dyes, QDs, and plasmids into different cell types with the variation of field strength, pulse duration, and the number of pulses. This new approach allows us to analyze delivery of different biomolecules into single living cells with high transfection efficiency (>96%, for CL1-0 cells) and high cell viability (∼98%), which are potentially beneficial for cellular therapy and diagnostic purposes.

Emetine Synergizes with Cisplatin to Enhance Anti-Cancer Efficacy against Lung Cancer Cells.

Cisplatin is still the primary therapeutic choice for advanced lung cancers without driver mutations. The occurrence of cisplatin resistance is a major clinical problem in lung cancer treatment. The natural extracted agent emetine reportedly has anticancer effects. This study aimed to explore the possible role of emetine in cisplatin resistance. We used cell viability, Western blot, and Wnt reporter assays to show that emetine suppresses proliferation, β-catenin expression, and Wnt/β-catenin signaling in non-small cell lung cancer (NSCLC). The synergism of emetine and cisplatin was assessed by constructing isobolograms and calculating combination index (CI) values using the Chou-Talalay method. Emetine effectively synergized with cisplatin to suppress the proliferation of cancer cells. Furthermore, nuclear β-catenin and cancer stem cell-related markers were upregulated in the cisplatin-resistant subpopulation of CL1-0 cells. Emetine enhanced the anticancer efficacy of cisplatin and synergized with cisplatin in the cisplatin-resistant subpopulation of CL1-0 cells. Taken together, these data suggest that emetine could suppress the growth of NSCLC cells through the Wnt/β-catenin pathway and contribute to a synergistic effect in combination with cisplatin.

Antitumor agent yatein from Calocedrus formosana Florin leaf induces apoptosis in non-small-cell lung cancer cells

Calocedrus formosana Florin is a softwood tree species with high economic value in Taiwan. Several bioactivities of the extracts of C. formosana have been reported; however, only one study focused on the anti-non-small-cell lung cancer cells’ (anti-NSCLC) effect of C. formosana extract and its active phytocompound. In the present study, the anti-lung cancer effects of C. formosana leaf extract and its active derivative yatein were evaluated. The results revealed that the n-hexane fraction of the crude extract exhibited the highest cytotoxicity potential against two non-small-cell lung cancer (NSCLC) cell lines, namely A549 and CL1-5. Yatein, isolated from the n-hexane fraction, exhibited the highest cytotoxicity in the A549 and CL1-5 cells. In addition, the CL1-5 cells were more sensitive than the A549 cells after yatein treatment. Flow cytometry results revealed that yatein induced apoptosis in the two cell lines. Furthermore, expression of regulatory proteins related to apoptosis, such as caspase 3, caspase 8, caspase 9, and poly (ADP-ribose) polymerase (PARP), increased in the A549 and CL1-5 cells after yatein treatment. These findings provide insight into the in vitro anti-lung tumor efficacy of yatein, thus rendering this phytocompound a potential anticancer lead compound for NSCLC treatment.

Molecular Mechanisms Underlying Yatein-Induced Cell-Cycle Arrest and Microtubule Destabilization in Human Lung Adenocarcinoma Cells.

Yatein is an antitumor agent isolated from Calocedrus formosana Florin leaves extract. In our previous study, we found that yatein inhibited the growth of human lung adenocarcinoma A549 and CL1-5 cells by inducing intrinsic and extrinsic apoptotic pathways. To further uncover the effects and mechanisms of yatein-induced inhibition on A549 and CL1-5 cell growth, we evaluated yatein-mediated antitumor activity in vivo and the regulatory effects of yatein on cell-cycle progression and microtubule dynamics. Flow cytometry and western blotting revealed that yatein induces G2/M arrest in A549 and CL1-5 cells. Yatein also destabilized microtubules and interfered with microtubule dynamics in the two cell lines. Furthermore, we evaluated the antitumor activity of yatein in vivo using a xenograft mouse model and found that yatein treatment altered cyclin B/Cdc2 complex expression and significantly inhibited tumor growth. Taken together, our results suggested that yatein effectively inhibited the growth of A549 and CL1-5 cells possibly by disrupting cell-cycle progression and microtubule dynamics.