Gefitinib loaded PLGA and chitosan coated PLGA nanoparticles with magnified cytotoxicity against A549 lung cancer cell lines

In the current study, gefitinib loaded PLGA nanoparticles (GFT-PLGA-NPs) and chitosan coated PLGA nanoparticles (GFT-CS-PLGA-NPs) were synthesized to investigate the role of surface charge of NPs for developing drug delivery system for non-small-cell lung cancer (NSCLC). The developed NPs were evaluated for their size, PDI, zeta potential (ZP), drug entrapment, drug loading, DSC, FTIR, XRD, in vitro release profile, and morphology. The anti-cancer activity of GFT loaded PLGA NPs and GFT loaded CS-PLGA-NPs were examined in human A549 lung cancer cell lines. In vitro release studies of GFT-CS-PLGA-NPs showed more sustained release in comparison to GFT-PLGA-NPs due surface charge attraction of chitosan. In addition, viability of A549 cells decreases significantly with the increasing concentration of GFT-PLGA NPs and GFT-CS-PLGA-NPs when compared to that of pure GFT and blank PLGA NPs. In addition, the microscopic analysis and counting of viable cells also validate the cytotoxicity of the developed NPs. This investigation proved that the developed NPs would be efficient carriers to deliver GFT with improved efficacy against NSCLC.     

RING finger protein 38 induces the drug resistance of cisplatin in non-small-cell lung cancer

Cisplatin resistance of non-small-cell lung cancer (NSCLC) needs to be well elucidated. RING finger protein (RNF38) has been proposed as a biomarker of NSCLC poor prognosis. However, its role in drug resistance in NSCLC is poorly understood. RNF38 expression was detected in normal lung epithelial cell and four NSCLC cell lines. RNF38 was stably overexpressed in A549 and H460 cells or silenced in H1975 and cisplatin-resistant A549 cells (A549-CDDP resistant) using lentiviral vectors. RNF38 expression levels were determined using quantitative real-time polymerase chain reaction and western blotting analysis. Cell viability in response to different concentrations of cisplatin was evaluated by Cell Counting Kit-8 assay. RNF38 expression levels were markedly elevated in NSCLC cells and cells harboring high RNF38 were less sensitive to cisplatin. Overexpression of RNF38 reduced, while RNF38 silencing increased the drug sensitivity of cisplatin in NSCLC cells. Cisplatin-resistant cells expressed high RNF38 level. RNF38 silencing promoted cell apoptosis and enhanced the drug sensitivity of cisplatin in cisplatin-resistant NSCLC cells. These findings indicate that RNF38 might induce cisplatin resistance of NSCLC cells via promoting cell apoptosis and RNF38 could be a novel target for rectify cisplatin resistance in NSCLC cases.     

Aptamer-Dendrimer Bioconjugates for Targeted Delivery of miR-34a Expressing Plasmid and Antitumor Effects in Non-Small Cell Lung Cancer Cells

Metastasis and drug resistance are major barriers for the treatment of non-small cell lung cancer (NSCLC). To explore new therapeutic options, we successfully encapsulated MicroRNA-34a (miR-34a), a potent endogenous tumor suppressor in NSCLC into S6 aptamer-conjugated dendrimer to form lung cancer-targeted gene delivery nanoparticles (PAM-Ap/pMiR-34a NPs). PAM-Ap/pMiR-34a NPs had a diameter of 100–200 nm and Zeta potential of ~30 mV at applied N/P ratio. The aptamer conjugation significantly improved cellular uptake as well as gene transfection efficiency of PAM-Ap/pMiR-34a NPs in cultured NSCLC cells. We showed that PAM-Ap/pMiR-34a NPs enhanced the regulation of targeted genes, BCL-2 and p53 in vitro. In addition, we revealed PAM-Ap/pMiR-34a NPs significantly inhibited cell growth, migration, invasion and induced apoptosis of lung cancer cells compared with non-targeted NPs. The method provided a novel therapeutic strategy for the experimental treatment of NSCLC.     

mTOR inhibitors radiosensitize PTEN‐deficient non‐small‐cell lung cancer cells harboring an EGFR activating mutation by inducing autophagy

Clinical resistance to gefitinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), in patients with lung cancer has been linked to acquisition of the T790M resistance mutation in activated EGFR or amplification of MET. Phosphatase and tensin homolog (PTEN) loss has been recently reported as a gefitinib resistance mechanism in lung cancer. The aim of this study was to evaluate the efficacy of radiotherapy in non‐small‐cell lung cancer (NSCLC) with acquired gefitinib resistance caused by PTEN deficiency to suggest radiotherapy as an alternative to EGFR TKIs. PTEN deficient‐mediated gefitinib resistance was generated in HCC827 cells, an EGFR TKI sensitive NSCLC cell line, by PTEN knockdown with a lentiviral vector expressing short hairpin RNA‐targeting PTEN. The impact of PTEN knockdown on sensitivity to radiation in the presence or absence of PTEN downstream signaling inhibitors was investigated. PTEN knockdown conferred acquired resistance not only to gefitinib but also to radiation on HCC827 cells. mTOR inhibitors alone failed to reduce HCC827 cell viability, regardless of PTEN expression, but ameliorated PTEN knockdown‐induced radioresistance. PTEN knockdown‐mediated radioresistance was accompanied by repression of radiation‐induced cytotoxic autophagy, and treatment with mTOR inhibitors released the repression of cytotoxic autophagy to overcome PTEN knockdown‐induced radioresistance in HCC827 cells. These results suggest that inhibiting mTOR signaling could be an effective strategy to radiosensitize NSCLC harboring the EGFR activating mutation that acquires resistance to both TKIs and radiotherapy due to PTEN loss or inactivation mutations. J. Cell. Biochem. 114: 1248–1256, 2013. © 2012 Wiley Periodicals, Inc.     

The naturally occurring xanthone α-mangostin induces ROS-mediated cytotoxicity in non-small scale lung cancer cells

Small cell lung cancer (NSCLC) accounts for 85% of total deaths globally, and recent studies indicate the increasing risks of NSCLC in China and South Asian countries. Hence, development of new therapeutics against NSCLC has been a major concern. α-Mangostin, a naturally occurring xanthone, found abundantly in pericarps of mangosteen fruit is well known for its medicinal importance. The anticancer properties of α-mangostin against several types of cancer are also well documented. But the mechanism of action of α-mangostin against lung cancer is not well understood and requires further investigation. Therefore in the present study, we explored the therapeutic potential of α-mangostin against A549 cells. Treatment of A549 cells with α-mangostin resulted in a dose-dependent loss of cell viability, while the non-malignant cells such as hPBMC and WI-38 remained unaffected. Further we observed that the ROS plays an important role in α-mangostin -induced apoptosis in A549 cells, and administration of N-acetyl cysteine significantly abrogates α-mangostin -mediated cytotoxicity in lung cancer cells. Overall, α-mangostin induces ROS-mediated cytotoxicity in NSCLC cells.     

Lentiviral delivery of a shRNA sequence analogous to miR-4319/miR-125-5p induces apoptosis in NSCLC cells by arresting G2/M phase

In this study, we explored the therapeutic potential of microRNA (miR) analogs against non–small-cell lung cancer (NSCLC) using lentiviral delivery of short hairpin RNA (shRNA). By using A549 as a model cell line, we used analogs and mimics of miR-4319/miR-125-5p to target the tumorigenic RAF1 gene. Lentiviral vectors carrying shRNA of a highly efficient miRNA analog of miR-4319/miR-125-5p, Analog2, were constructed to infect A549 cells. Our results showed that, compared with the noncancerous bronchial epithelial cell line 16HBE, lentivirus delivering Analog2 shRNA induced significant G2/M arrest and subsequent apoptosis in A549 cells, but not in 16HBE cells. Western blot analysis revealed that key factors regulating cell cycle were downregulated following RAF1 inhibition. In vivo xenograft experiments showed that lentivirus carrying Analog2 shRNA markedly decreased tumor size. Therefore, lentiviral delivery of Analog2 shRNA is a valid RNA interference-based treatment against NSCLC with high potency and specificity.     

C1632 suppresses the migration and proliferation of non‐small‐cell lung cancer cells involving LIN28 and FGFR1 pathway

Chemoresistance and migration represent major obstacles in the therapy of non‐small‐cell lung cancer (NSCLC), which accounts for approximately 85% of lung cancer patients in clinic. In the present study, we report that the compound C1632 is preferentially distributed in the lung after oral administration in vivo with high bioavailability and limited inhibitory effects on CYP450 isoenzymes. We found that C1632 could simultaneously inhibit the expression of LIN28 and block FGFR1 signalling transduction in NSCLC A549 and A549R cells, resulting in significant decreases in the phosphorylation of focal adhesion kinase and the expression of matrix metalloproteinase‐9. Consequently, C1632 effectively inhibited the migration and invasion of A549 and A549R cells. Meanwhile, C1632 significantly suppressed the cell viability and the colony formation of A549 and A549R cells by inhibiting DNA replication and inducing G0/G1 cell cycle arrest. Interestingly, compared with A549 cells, C1632 possesses the same or even better anti‐migration and anti‐proliferation effects on A549R cells, regardless of drug resistance. In addition, C1632 also displayed the capacity to inhibit the growth of A549R xenograft tumours in mice. Altogether, these findings reveal the potential of C1632 as a promising anti‐NSCLC agent, especially for chemotherapy‐resistant NSCLC treatment.     

Induction of cell size vesicles from human lymphoma cell lines and their application to drug carriers

Sodium butyrate (NaB) induced the membrane enclosed cell size vesicles from several IgM producing cell lines. We considered the application of the cell-derived vesicles (CDVs) to drug delivery system (DDS) using the lung cancer specific IgM producing AE6 cell line. Microscopic observation showed that the DiI fluorescence labeled AE6 vesicles were incorporated into the lung cancer cell line A549. The anticancer drug, actinomycin D (actD), contained in AE6 and Ramos vesicles decreased the A549 cell viability to 46 and 62% of control without actD, respectively. The cytotoxic effect in AE6 vesicles was superior to that in the Ramos vesicles that have the lung cancer non-specific IgM on their surfaces. However, the result of the Ramos vesicles suggests that the surface molecules other than IgM may interact with the A549 cells. In our method for vesicle production, more specific and abundant antibodies mounted vesicles can be generated by transfection of their genes into cells followed by NaB treatment. These suggest that the CDVs may be useful for the development of a drug carrier for DDS.     

Action of solamargine on TNFs and cisplatin-resistant human lung cancer cells

A loss of TNF receptors expression has been found in advanced lung cancers, and human A549 lung adenocarcinoma cells are resistant to the cytotoxic effects of TNF-alpha and cisplatin. Here, the mechanisms of the drug resistance of A549 were extensively studied by gene modulation of the cells by solamargine (SM) which was isolated from Solanum incanum herb. SM induced morphological changes of chromatin condensation, DNA fragmentation, and sub-G(1) peak in a DNA histogram of A549 cells, indicating cell death by apoptosis. SM elevated the expressions of TNF-R1 and -R2 and overcame the resistance of A549 cells to TNF-alpha and -beta. The recruitment of TRADD, FADD, and activation of caspase-8 and -3 in SM-treated A549 cells evidenced the activation of TNFRs signal transduction. In addition, release of cytochrome c from mitochondria, down-expression of Bcl-2 and Bcl-x(L), up-regulation of Bax, and caspase-9 activities were observed in SM-treated A549 cells. Combinational treatment of SM and cisplatin synergistically enhanced caspase-8, -9, and -3 activities in A549 cells. Thus, SM sensitizes A549 cells through TNFRs and mitochondria-mediated pathways and may have anticancer potential against TNFs- and cisplatin-resistance lung cancer cells.     

Cytotoxic activity of gypenosides and gynogenin against non-small cell lung carcinoma A549 cells

The activity of gypenosides and gynogenin of Gynostemma pentaphyllum against non-small cell lung carcinoma (NSCLC) A549 cells was investigated to identify the structural characteristics of gypenosides and gynogenin to have anti-NSCLC activity. Of the tested dammarane-type compounds, 20S-dammar-24-en-2α,3β,12β,20-tetrol showed the strongest activity against A549 cells. Based on the structure and cytotoxic activity relationships of gypenosides and gynogenin, the OH group in C-2, the connected sugar number and the configuration in C-20 were important for cytotoxic activity against A549 cells.     

Dihydroartemisinin Inhibits Glucose Uptake and Cooperates with Glycolysis Inhibitor to Induce Apoptosis in Non-Small Cell Lung Carcinoma Cells

Despite recent advances in the therapy of non-small cell lung cancer (NSCLC), the chemotherapy efficacy against NSCLC is still unsatisfactory. Previous studies show the herbal antimalarial drug dihydroartemisinin (DHA) displays cytotoxic to multiple human tumors. Here, we showed that DHA decreased cell viability and colony formation, induced apoptosis in A549 and PC-9 cells. Additionally, we first revealed DHA inhibited glucose uptake in NSCLC cells. Moreover, glycolytic metabolism was attenuated by DHA, including inhibition of ATP and lactate production. Consequently, we demonstrated that the phosphorylated forms of both S6 ribosomal protein and mechanistic target of rapamycin (mTOR), and GLUT1 levels were abrogated by DHA treatment in NSCLC cells. Furthermore, the upregulation of mTOR activation by high expressed Rheb increased the level of glycolytic metabolism and cell viability inhibited by DHA. These results suggested that DHA-suppressed glycolytic metabolism might be associated with mTOR activation and GLUT1 expression. Besides, we showed GLUT1 overexpression significantly attenuated DHA-triggered NSCLC cells apoptosis. Notably, DHA synergized with 2-Deoxy-D-glucose (2DG, a glycolysis inhibitor) to reduce cell viability and increase cell apoptosis in A549 and PC-9 cells. However, the combination of the two compounds displayed minimal toxicity to WI-38 cells, a normal lung fibroblast cell line. More importantly, 2DG synergistically potentiated DHA-induced activation of caspase-9, -8 and -3, as well as the levels of both cytochrome c and AIF of cytoplasm. However, 2DG failed to increase the reactive oxygen species (ROS) levels elicited by DHA. Overall, the data shown above indicated DHA plus 2DG induced apoptosis was involved in both extrinsic and intrinsic apoptosis pathways in NSCLC cells.     

Viridiflorol induces anti-neoplastic effects on breast,lung, and brain cancer cells through apoptosis

All active natural molecules are not fully exploited as therapeutic agents, causing delays in the advancement of anticancer drug discovery. Viridiflorol is a natural volatile element that may work as anti-cancer compound. We tested the anticancer properties of viridiflorol at different concentrations ranging from 0.03 to 300 μM in vitro on three cancer cells including breast (MCF-7), lung (A549) and brain (Daoy). The cancer cells responses were documented after treatment using MTT and Annexin V assays. Viridiflorol showed cytotoxic effects against all tested cell lines, reducing cell viability in a concentration-dependent manner with variable IC₅₀ values. Daoy and A549 cell lines were more sensitive to viridiflorol when compared with temozolomide and doxorubicin, respectively. Viridiflorol demonstrated the highest anticancer activity against the Daoy cells with an estimated IC₅₀ of 0.1 µM followed by MCF-7 at 10 µM, and A549 at 30 µM. In addition, upon exposure to concentrations ranging from 30 µM to 300 µM of viridiflorol, early and late apoptotic cell death was induced in a concentration dependent manner in Daoy (55.8%-72.1%), MCF-7 (36.2%-72.7%) and A459 (35%-98.9%) cell lines, respectively. In conclusion, viridiflorol demonstrates cytotoxic and apoptotic ability in three different cancer cell lines (brain, breast and lung).     

α7-Nicotinic acetylcholine receptor antagonist QND7 suppresses non-small cell lung cancer cell proliferation and migration via inhibition of Akt/mTOR signaling

Lung cancer, one of the most commonly found carcinoma type, has the highest mortality rate in cancer patients worldwide. Therapeutic interventions targeting to lung cancer become remaining the world significant challenge. Recently, the α7-nicotinic acetylcholine receptor (α7-nAChR) was reported to play an important role in the mechanism underlying lung cancer progression, being intriguing drug target for lung cancer therapy. Hence, the top four α7-nAChR antagonists (QND7, PPRD10, PPRD11 and PPRD12) among our previously developed ligands were proceeded to the in vitro anti-cancer evaluations in non-small cell lung cancer (NSCLC) cell lines (H460 and A549). In this study, we found that QND7 exhibited the highest cytotoxic effect and induced cell apoptosis in both cell lines at a level comparable to cisplatin, whereas the PPRD compounds showed much lower cytotoxicity. Low doses of QND7 and PPRD11 were able to suppress H460 and A549 cell proliferation, whereas PPRD10 and PPRD12 were considered ineffective. In an in vitro wound healing assay, QND7-treatment showed the greatest suppression of H460 and A549 cell migration. The variations in the anti-cancer activities of PPRD compounds might be, at least in part of, their non-selective antagonisms to serotonin receptor (5-HT3) and α4β2-nAChR. Further investigation revealed that QND7 was able to minimize protein kinase B/mammalian target of rapamycin (Akt/mTOR) activity, in correlating to its anti-cancer effects. These findings warrant QND7 for further preclinical evaluation and demonstrate the potential of α7-nAChR as cancer drug target.     

SPARCL1通过MEK/ERK信号通路调节非小细胞肺癌细胞的增殖、凋亡和侵袭

摘要 目的：分析富含半胱氨酸的酸性分泌蛋白类似蛋白1（SPARCL1）对非小细胞肺癌（NSCLC）细胞增殖、凋亡、侵袭的影响,并探讨分裂原活化抑制剂（MEK）/细胞外调节蛋白激酶（ERK）通路在其中发挥的作用。方法：收集2019年9月~2021年6月期间本院接受手术治疗的84例NSCLC患者癌组织与相应癌旁组织,实时定量逆转录聚合酶链反应（qRT-PCR）法测定并比较各组织以及正常肺上皮细胞HBEpiC、NSCLC细胞A549、HCC827、H1299、H292中SPARCL1 信使RNA（mRNA）表达水平,选取A549、HCC827培养并分组,分为对照组、NC siRNA组、SPARCL1 siRNA组、U0126组（MEK/ERK特异性抑制剂）、SPARCL1 siRNA加U0126组,细胞计数法（CCK8）以及平板克隆法测定A549、HCC827细胞增殖,流式细胞仪测定A549、HCC827细胞凋亡,Transwell小室法测定A549、HCC827细胞侵袭能力,蛋白质印迹法（western blot）检测SPARCL1、p-MEK、MEK、p-ERK1/2、ERK1/2蛋白表达。结果：SPARCL1在NSCLC组织中mRNA表达水平低于癌旁组织（P＜0.05）;与HBEpiC细胞相比,NSCLC细胞A549、HCC827、H1299、H292细胞中SPARCL1 mRNA表达水平降低（P＜0.05）;与对照组相比,SPARCL1 siRNA组A549、HCC827细胞SPARCL1 mRNA表达水平与蛋白表达、凋亡率降低（P＜0.05）,OD₄₅₀、克隆形成数、侵袭细胞数、p-MEK/MEK、p-ERK1/2/ERK1/2蛋白表达升高（P＜0.05）,U0126组A549、HCC827细胞SPARCL1 mRNA表达水平与蛋白表达、凋亡率升高（P＜0.05）,OD₄₅₀、克隆形成数、侵袭细胞数、p-MEK/MEK、p-ERK1/2/ERK1/2蛋白表达降低（P＜0.05）;与SPARCL1 siRNA组相比,SPARCL1 siRNA加U0126组A549、HCC827细胞SPARCL1 mRNA表达水平与蛋白表达、凋亡率升高（P＜0.05）,OD₄₅₀、克隆形成数、侵袭细胞数、p-MEK/MEK、p-ERK1/2/ERK1/2蛋白表达降低（P＜0.05）。结论：SPARCL1可能通过调控MEK/ERK通路影响NSCLC A549、HCC827细胞增殖、侵袭与凋亡。     

Biosynthesized silver nanoparticles using Caulerpa taxifolia against A549 lung cancer cell line through cytotoxicity effect/morphological damage

The Caulerpa taxifolia is excellent marine green algae, which produced enormous bioactive compounds with more biological activities. Also, it is an excellent source for synthesis of Ag NPs with increased bioactivity against various infections. In our study, the marine algae marine algae Caulerpa taxifolia mediated Ag NPs was synthesized effectively. The synthesized Ag NPs was characterized well using UV-spectrometer and X-ray powder diffraction (XRD) and confirmed as synthesized particle was Ag NPs. The available structure of the Ag NPs was morphologically identified by scanning electron microscope (SEM), and exact minimum size, polydispersive spherical shape of the entire Ag NPs structure was confirmed by Transmission electron microscope (TEM). Further, the anti-cancer efficiency of biosynthesized Ag NPs against A549 lung cancer cells was found at 40 µg/mL concentration by cytotoxicity experiment. In addition, the phase contrast images of the result were supported the Ag NPs, which damaged the A549 morphologically clearly. Finally, florescence microscopic images were effectively proved the anti-cancerous effect against A549 lung cancer cells due to the condensed morphology of increased death cells. All the confirmed in-vitro results were clearly stated that the Caulerpa taxifolia mediated Ag NPs has superior anti-cancer agent against A549 lung cancer cells.     

Dendritic cells efficiently acquire and present antigen derived from lung cancer cells and induce antigen-specific T-cell responses

Active immunotherapy of cancer requires the availability of a source of tumor antigens. To date, no such antigen associated with lung cancer has been identified. We have therefore investigated the ability of dendritic cells (DC) to capture whole irradiated human lung tumor cells and to present a defined surrogate antigen derived from the ingested tumor cells. We also describe an in vitro system using a modified human adenocarcinoma cell line (A549-M1) that expresses the well-characterized, immunogenic influenza M1 matrix protein as a surrogate tumor antigen. Peripheral blood monocyte-derived DC, when co-cultured with sub-lethally irradiated A549 cells or primary lung tumor cells derived from surgical resection of non-small cell carcinoma (NSCLC), efficiently ingested the tumor cells as determined by flow cytometry analysis and confocal microscopic examination. More importantly, DC loaded with irradiated A549-M1 cells efficiently processed and presented tumor cell-derived M1 antigen to T cells and elicited antigen-specific immune responses that included IFNgamma release from an M1-specific T-cell line, expansion of M1 peptide-specific Vbeta17+ and CD8+ peripheral T cells and generation of M1-specific cytotoxic T lymphocytes (CTL). We also compared DC loaded with irradiated tumor cells to those loaded with tumor cell lysate or killed tumor cells and found that irradiated lung tumor cells as a source of tumor antigen for DC loading is superior to tumor cell lysate or killed tumor cells in efficient induction of antigen-specific T-cell responses. Our results demonstrate the feasibility of using lung tumor cell-loaded DC to induce immune responses against lung cancer-associated antigens and support ongoing efforts to develop a DC-based lung cancer vaccine.     

Effects of transport inhibitors on the cellular uptake of carboxylated polystyrene nanoparticles in different cell lines

Nanotechnology is expected to play a vital role in the rapidly developing field of nanomedicine, creating innovative solutions and therapies for currently untreatable diseases, and providing new tools for various biomedical applications, such as drug delivery and gene therapy. In order to optimize the efficacy of nanoparticle (NP) delivery to cells, it is necessary to understand the mechanisms by which NPs are internalized by cells, as this will likely determine their ultimate sub-cellular fate and localisation. Here we have used pharmacological inhibitors of some of the major endocytic pathways to investigate nanoparticle uptake mechanisms in a range of representative human cell lines, including HeLa (cervical cancer), A549 (lung carcinoma) and 1321N1 (brain astrocytoma). Chlorpromazine and genistein were used to inhibit clathrin and caveolin mediated endocytosis, respectively. Cytochalasin A and nocodazole were used to inhibit, respectively, the polymerisation of actin and microtubule cytoskeleton. Uptake experiments were performed systematically across the different cell lines, using carboxylated polystyrene NPs of 40 nm and 200 nm diameters, as model NPs of sizes comparable to typical endocytic cargoes. The results clearly indicated that, in all cases and cell types, NPs entered cells via active energy dependent processes. NP uptake in HeLa and 1321N1 cells was strongly affected by actin depolymerisation, while A549 cells showed a stronger inhibition of NP uptake (in comparison to the other cell types) after microtubule disruption and treatment with genistein. A strong reduction of NP uptake was observed after chlorpromazine treatment only in the case of 1321N1 cells. These outcomes suggested that the same NP might exploit different uptake mechanisms to enter different cell types.