Treatment of lung cancer via telomerase inhibition: self-assembled nanoplexes versus polymeric nanoparticles as vectors for 2'-O-Methyl-RNA

Antisense oligonucleotide, 2'-O-Methyl-RNA (OMR), is known as potent telomerase inhibitor for the treatment of lung cancer but limited by poor intracellular uptake. Chitosan-coated polymeric nanoparticles were compared to chitosan solution as non-viral vectors for OMR. The study investigated the role of chitosan properties and concentration in improving the efficiency of the nanocarriers in terms of loading, viability, cellular uptake, and telomerase inhibition in human lung cancer cell lines. Certain concentration of chitosan on nanoparticle surface is necessary to significantly increase the cellular uptake. However, excessive chitosan negatively affected the transfection efficiency. Self-assembled nanoplexes with chitosan polymer are preferentially adsorbed to the cell membrane rather than being internalized. Thus, polymeric nanoparticles proved to be superior to cationic polymers as carrier for antisense oligonucleotides. Charge cannot be considered the principle factor behind improved transfection. Uptake studies carried out on air-interface cell cultures to mimic in vivo conditions supported the results on normal cultures showing enhanced uptake of nanoplexes over naked oligonucleotides. OMR nanoplexes reduced telomerase activity by ～50% in A549 cells concluding the potential of the system as a safe, non-invasive, and efficient treatment for lung carcinoma. These data are prerequisites for the ongoing studies on lung perfusion model and in vivo experiments.     

Tissue slice model of human lung cancer to investigate telomerase inhibition by nanoparticle delivery of antisense 2'-O-methyl-RNA

Nanoparticles delivery of oligonucleotides represents a potential approach for cancer treatment. However, most of the experiments were based on established cancer cell lines and may not reflect the original solid tumor in vivo. Both, tumor microenvironment and tumor cell biological properties in the tumor can influence the delivery efficiency of oligonucleotides. Therefore, it is important to understand the effect of nanoparticles delivery of oligonucleotides on tumor response in intact tissue architecture of individual tumors. We used freshly isolated human tumor tissue slices and primary lung cancer cells from non-small cell lung cancer patients to evaluate this nanocarrier system. Chitosan-coated poly(lactide-co-glycolide) (PLGA) nanoparticles were used to form oligonucleotide-nanoparticle-complexes (nanoplexes) with antisense 2'-O-methyl-RNA (OMR) that can inhibit telomerase activity by binding to the RNA component of telomerase. OMR cellular uptake was strongly enhanced by nanoplexes mediated delivery in both, primary cells and tissue slices. More than 80% of primary cancer cells and 50% of cells in tissue slices showed OMR uptake. Telomerase activity was inhibited by approximately 45% in primary cancer cells and about 40% in tissue slices. Nanoplexes could penetrate into tumor tissue without influencing tissue architecture and the delivered OMR was able to inhibit telomerase activity with relatively low cytotoxicity.     

The influence of chitosan content in cationic chitosan/PLGA nanoparticles on the delivery efficiency of antisense 2′-O-methyl-RNA directed against telomerase in lung cancer cells

Tailorable cationic chitosan/PLGA nanoparticles (CPNP) were used for the delivery of an antisense 2′-O-methyl-RNA (2OMR) directed against RNA template of human telomerase. Here, we describe the influence of the chitosan content on binding efficiency, complex stability, uptake in different human lung cell types and finally demonstrate the efficacy of this nanoplex system.CPNPs were prepared by the emulsion-solvent evaporation method using different amounts of chitosan and purified by preparative size exclusion chromatography. The characterization by photon correlation spectroscopy and zeta potential measurements showed a small increase in size and an increase of zeta potential with increasing amounts of chitosan. Binding efficiency and complex stability with 2OMR was high in water and correlated well with the chitosan content of particles but was weak in physiologically relevant media (PBS and RPMI cell culture medium). However, flow cytometry analysis showed that the uptake of 2OMR into A549 lung cancer cells was considerably higher in combination with nanoparticles and dependent on the amount of chitosan when compared to 2OMR alone. Confocal laser scanning microscopy revealed that the uptake into A549 cells is mediated via complexes of 2OMR and chitosan/PLGA nanoparticles despite the weak binding in cell culture medium. The nanoparticles were well tolerated and efficient in inhibiting telomerase activity.     

Cross-linked polyethylenimine-hexametaphosphate nanoparticles to deliver nucleic acids therapeutics

Branched polyethylenimine (PEI; 25 kDa) as a nonviral vector exhibits high transfection efficiency and is a potential candidate for efficient gene delivery. However, the cytotoxicity of PEI limits its application in vivo. PEI was ionically interacted with hexametaphosphate, a compact molecule with high anionic charge density, to obtain nanoparticles (PEI-HMP). Nanoparticles were assessed for their efficacy in protecting complexed DNA against nucleases. The intracellular trafficking of nanoparticles was monitored by confocal microscopy. The cytotoxicity and transfection efficiency of PEI-HMP nanoparticles were evaluated in vitro. In vitro transfection efficiency of PEI-HMP (7.7%) was ～1.3- to 6.4-folds higher than that of the commercial reagents GenePORTER 2^TM, Fugene^TM, and Superfect^TM. Also, PEI-HMP (7.7%) delivered green fluorescent protein (GFP)-specific small interfering ribonucleic acid (siRNA) in culture cells leading to >80% suppression in GFP gene expression. PEI-HMP nanoparticles protected complexed DNA against DNase for at least 2 hours. A time-course uptake of PEI-HMP (7.7%) nanoparticles showed the internalization of nanoparticles inside the cell nucleus in 2 hours. Thus, PEI-HMP nanoparticles efficiently transfect cells with negligible cytotoxicity and show great promise as nonviral vectors for gene delivery.From the Clinical EditorBranched polyethylenimine (PEI) as a non-viral vector exhibits high transfection efficiency for gene delivery, but its cytotoxicity limits its applications. PEI hexametaphosphate nanoparticles (PEI-HMP) demonstrated a 1.3-6.4 folds higher transfection rate compared to commercial reagents. Overall, PEI-HMP nanoparticles efficiently transfect cells with negligible cytotoxicity and show great promise as non-viral vectors for gene delivery.     

Nuclear translocation of telomerase reverse transcriptase and calcium signaling in repression of telomerase activity in human lung cancer cells by fungal immunomodulatory protein from Ganoderma tsugae

Recombinant fungal immunomodulatory protein, reFIP-gts, was cloned from Ganoderma tsugae and purified. In our previous study, it was shown that reFIP-gts has anti-telomerase effects in A549 cells. Here, we proved that reFIP-gts entry into the cell and localization in endoplasmic reticulum can result in ER stress, thereby increasing ER stress markers (CHOP/GADD153) and intracellular calcium release in A549 cells. The use of calcium chelator restores reFIP-gts-mediated reduction in telomerase activity. These results strongly suggest that ER stress induces intracellular calcium release and results in inhibition of telomerase activity. Although reFIP-gts decreased hTERT mRNA level in both A549 and H1299 cells, only the telomerase activity in A549 cells was inhibited. Surprisingly, we found that reFIP-gts induces translocation of hTERT from the nucleus into the cytosol in A549 cells but not in H1299 cells. Using leptomycin B, nuclear export inhibitor, we showed that hTERT is not transported. Using MG132, a proteasome inhibitor, reFIP-gts also prevents hTERT translocation from proteasome degradation. Taken together, these results indicate that reFIP-gts inhibits telomerase activity in lung cancer cells through nuclear export mechanisms, which might be mediated by ER stress-induced intracellular calcium level.     

α-Conotoxin ImI-modified polymeric micelles as potential nanocarriers for targeted docetaxel delivery to α7-nAChR overexpressed non-small cell lung cancer

A micelle system modified with α-Conotoxin ImI (ImI), a potently antagonist for alpha7 nicotinic acetylcholine receptor (α7-nAChR) previously utilized for targeting breast cancer, was constructed. Its targeting efficiency and cytotoxicity against non-small cell lung cancer (NSCLC) highly expressing α7-nAChR was investigated. A549, a non-small cell lung cancer cell line, was selected as the cell model. The cellular uptake study showed that the optimal modification ratio of ImI on micelle surface was 5% and ImI-modification increased intracellular delivery efficiency to A549 cells via receptor-mediated endocytosis. Intracellular Ca²⁺ transient assay demonstrated that ImI modification led to enhanced molecular interaction between nanocarriers and A549 cells. The in vivo near-infrared fluorescence imaging further revealed that ImI-modified micelles could facilitate the drug accumulation in tumor sites compared with non-modified micelles via α7-nAChR mediation. Moreover, docetaxel (DTX) was loaded in ImI-modified nanomedicines to evaluate its in vitro cytotoxicity. As a result, DTX-loaded ImI-PMs exhibited greater anti-proliferation effect on A549 cells compared with non-modified micelles. Generally, our study proved that ImI-modified micelles had targeting ability to NSCLC in addition to breast cancer and it may provide a promising strategy to deliver drugs to NSCLC overexpressing α7-nAChR.     

多胺类似物CPENSpm通过干扰多胺代谢抑制肺癌细胞的增殖

目的研究多胺类似物CPENSpm对肺癌细胞株A549增殖和细胞凋亡的影响,以探讨CPENSpm抗肿瘤的作用机制。方法MTS法分析细胞的增殖速度,化学分析法测定多胺代谢酶的活性,HPLC法分析细胞内的多胺含量,亚凋亡峰测定法和DNA片段化分析法鉴定细胞程序性凋亡。结果CPENSpm处理A549肺癌细胞可导致:①癌细胞生长抑制并激发细胞凋亡;②抑制多胺合成关键酶ODC的活性,活化多胺降解代谢关键酶SSAT和SMO的活性;③耗竭细胞内多胺含量。SMO抑制剂MDL72527可拮抗CPENSpm对A549细胞的生长抑制作用。结论CPENSpm通过干扰A549细胞的多胺代谢途径,耗竭肿瘤快速生长必需的多胺成分,诱导产生活性氧H2O2从而抑制癌细胞生长并激发细胞程序性凋亡。     

Calcium condensation of DNA complexed with cell-penetrating peptides offers efficient, noncytotoxic gene delivery

Drug delivery strategies using cell-penetrating peptides (CPPs) have been widely explored to improve the intracellular delivery of a large number of cargo molecules. Electrostatic complexation of plasmid DNA using CPPs has been less explored due to the relatively large complexes formed and the low levels of gene expression achieved when using these low-molecular-weight polycations as DNA condensing agents. Here, condensing nascent CPP polyplexes using CaCl(2) produced small and stable nanoparticles leading to gene expression levels higher than observed for control polyethylenimine gene vectors. This simple formulation approach showed negligible cytotoxicity in A549 lung epithelial cells and maintained particle size and transfection efficiency even in the presence of serum.     

An antisense oligonucleotide carrier based on amino silica nanoparticles for antisense inhibition of cancer cells

Antisense oligonucleotides (anti-ODNs), which are able to interfere with gene expression at the mRNA level, have potential activity in the treatment of viral infections or cancer. However, the application of therapies based on anti-ODNs is hampered by their instability to cellular nuclease and their weak intracellular penetration. Among the many efforts to increase their stability and cellular penetration have been modifications of ODNs and introduction of particulate carriers. Here we report an anti-ODNs carrier based on amino silica nanoparticles (NH(2)SiNPs) and its preliminary applications in cancer cells. The positively charged NH(2)SiNPs were synthesized by a water-in-oil microemulsion method. The NH(2)SiNP-ODN complexes were formed by electrostatic interaction, and their cellular uptake was visualized by using fluorescein isothiocyanate (FITC)-labeled ODNs and NH(2)SiNPs doped with rhodamine 6G isothiocyanate (RITC) as fluorescent signal indicators. The antisense inhibition efficiency of anti-ODNs delivered by NH(2)SiNPs was evaluated using MTT (3,4,5-dimethylthiazol-2,5-diphenyl tetrazolium bromide) assay and western blot analysis. Uniform NH(2)SiNPs with an average diameter of 25 nm were obtained and could combine with anti-ODNs to form a bioconjugate favorable for cellular uptake. The NH(2)SiNPs were able to protect anti-ODNs from degradation by DNase I. In vitro experiments showed that the NH(2)SiNPs could greatly improve the inhibition efficiency of anti-ODNs for the proliferation and survivin expression in Hela cells and A549 cells. Compared with liposomes, the NH(2)SiNPs presented a better biocompatibility and had almost no cytotoxicity at the concentrations required for efficient transfection. Our results suggest that the NH(2)SiNPs may be a promising carrier for delivery of anti-ODNs.     

The blockage of survivin and securin expression increases the cytochalasin B-induced cell death and growth inhibition in human cancer cells

Survivin and securin proteins are overexpressed in most cancer cells that have been shown to regulate mitotic progression. In this study, we investigated the roles of survivin and securin on cytochalasin B, a cytokinesis blocker mediating the cytotoxicity and cell growth inhibition in human cancer cells. The human lung carcinoma cell lines A549 and H1299 highly expressed survivin proteins in mitosis and concentrated on the midbodies during cytokinesis. Cytochalasin B significantly decreased cell survival, inhibited cell growth, increased the levels of G(2)/M fractions, and induced binuclei formation in lung carcinoma cells; however, the survivin proteins were concentration-dependently increased by 1 to 5 mug/ml cytochalasin B for 24 h. It is noteworthy that the expression of securin proteins was decreased in cytochalasin B-treated lung carcinoma cells. Transfection of 20 to 40 nM survivin siRNA for 48 h significantly induced the formation of multiple nuclei and apoptosis but decreased the levels of survivin and securin proteins in A549 cells. Cotreatment with survivin small interfering RNA (siRNA) and cytochalasin B increased the cytotoxicity and cell growth inhibition. In addition, the securin-null colorectal carcinoma cells were more susceptible to the cytotoxicity after cytochalasin B and survivin siRNA treatments than the securin-wild-type cells. As a whole, our results indicate that the inhibition of survivin and securin protein expression may increase the cell death and growth inhibition after cytochalasin B treatment in human cancer cells.     

Studies on bioadhesive PLGA nanoparticles: A promising gene delivery system for efficient gene therapy to lung cancer

The study aimed to design novel bioadhesive PLGA nanoparticles for efficient gene delivery to lung cancer cells. The bioadhesive agent and stabilizer, Carbopol 940 was chosen to establish bioadhesive PLGA nanoparticles and Pluronic F68, Pluronic F127 stabilized PLGA nanoparticles were formulated as control. The effects of different surfactants on the physicochemical and biological characterizations of PLGA nanoparticles were compared. All the obtained nanoparticles showed negative surface charge, similar spherical morphology, a relatively narrow particle size distribution, and lower cytotoxicity to A549 cells comparing with Lipofectamine 2000. Carbopol stabilized nanoparticles hold advantages in DNA-binding efficiency (>80%) at an optimal Carbopol concentration, DNA protection from enzymatic degradation in vitro release and better buffering capacity. Most importantly, higher transfection efficiency in A549 cells was observed comparing to Pluronics stabilized nanoparticles or naked DNA, similar to that of Lipofectamine 2000. These results revealed that the bioadhesive PLGA nanoparticles formulated with Carbopol might be a very attractive candidate as a non-viral vector for lung cancer gene therapy and might alleviate the drawbacks of the conventional cationic vectors/DNA complexes for gene delivery in vivo.     

Antioxidant activity of Vaccinium stamineum: exhibition of anticancer capability in human lung and leukemia cells

Fruit of deerberry [Vaccinium stamineum L.] were evaluated for their antioxidant capacity and anticancer properties in JB6 P (+) mouse epidermal cells, human lung and leukemia cells. Deerberries contain potent free radical scavenging activities. Pretreatment of JB6 P (+) mouse epidermal cells with deerberry fruit extracts produced an inhibition on the activation of activator protein-1 (AP-1) and nuclear factor-kappaB (NF-kappaB) induced by either 12- O-tetradecanoylphorbol 13-acetate (TPA) or ultraviolet-B (UVB). Deerberry fruit extracts also blocked TPA- or UVB-induced phosphorylation of ERKs and MEK 1/2, two upstream regulators of AP-1 and inhibited proliferation of human leukemia HL-60 cancer cells and human lung epithelial cancer A549 cells and induced apoptosis of HL-60 cells. These results suggest that the inhibition of TPA- or UVB-induced AP-1 and NF-kappaB activity, inhibition of HL-60 cells and cancer A549 cells proliferation and induction of apoptotic in human leukemia HL-60 cancer cells may be mediated through the ERKs and MEK 1/2 signal pathway.     

Synthesis and characterization of aromatic ring-based cationic lipids for gene delivery in vitro and in vivo

A new series of cationic lipids has been synthesized for gene delivery using 3,5-dihydroxybenzyl alcohol as the backbone and starting material. Using CMV driven expression system and luciferase gene as a reporter, we demonstrated that the transfection activity of these new lipids when formulated with Tween 80 as co-lipid is comparable to that of DOTAP, one of the most commonly used cationic lipids for transfection. Among the four different cell lines tested including murine melanoma BL-6 cells, human embryonic kidney 293 cells, HepG2 and HeLa cells, the highest transgene expression was seen in 293 cells. Results from in vivo experiments using mice as an animal model show that these cationic lipids preferentially transfect the cells in the lung upon tail vein administration. The cationic lipid, N,N,N-trimethyl-N-[3,5-bis(tetradecyloxy)benzyl] ammonium bromide 4c(di-C14:0) with two 14-hydrocarbon chains exhibits the best transfection activity. These results suggest that these new aromatic ring-based cationic lipids are useful transfection reagents for both in vitro and in vivo gene transfer studies.     

整合素配体RGD修饰的阳离子脂质体作为siRNA输送载体的体外研究

本研究构建了能够靶向肿瘤新生血管的RGD修饰阳离子脂质体（RGD-Lipo），作为靶向耐药相关基因MDR1的siRNA输送载体并评价其相关的药剂学性质。该脂质体与siRNA形成的复合物粒径控制在200nm以内，并且对其中所包载的siRNA具有一定的保护作用。体外实验结果表明，经RGD修饰的脂质体（RGD-Lipo）细胞粘附能力显著增强，并可增加细胞内siRNA的转染效果。与利用前插法进行靶向修饰相比，利用后插法进行RGD修饰可有效地改善siRNA的溶酶体释放效率。细胞毒试验结果表明，后插法制备的pRGD-Lipo-siRNA能够有效逆转人卵巢癌SKV03／A细胞的耐药性，并增加阿霉素药物在细胞内的蓄积。体外研究结果证实，使用pRGD-Lipo-siRNA有利于提高耐药细胞对化疗药阿霉素的敏感度，并将有可能应用于临床耐药肿瘤的治疗。     

Cell transfection with polycationic cyclodextrin vectors

Polycationic cyclodextrins (CDs) were complexed with plasmid DNA and their effectiveness as vectors was tested on COS-7 cells. These CDs were modified with pyridylamino, alkylimidazole, methoxyethylamino or primary amine groups at 6-positions of the glucose units. Uncharged CDs, β-CD, hydroxypropyl-β-CD, and dimethyl-β-CD were also tested, but these did not form stable complexes with the DNA and produced only a slight improvement in transfection level over DNA alone. The polycationic CDs neutralised DNA to form stable nanoparticulate complexes. The transfection efficiency of these CDs was dependent on the substituents present, with the most efficient having either an amino, pyridylamino or butylimidazole group at the 6-positions and unmodified 2- and 3-hydroxyls. One of the most effective vectors, heptakispyridylamino CD, produced a 4000-fold increase in transfection level over DNA alone. Levels were improved 10-fold by use of the endosomolytic agent, chloroquine. The transfection efficiency of the best of these systems in serum equals that of DOTAP in serum. Studies with ³²P-labelled plasmid DNA indicate that the polycationic CDs are exceptional promoters of DNA cellular-uptake, the most efficient surpassing DOTAP. Uptake is dependent on proteoglycan-mediated binding to cells. The data imply that intracellular trafficking but not cellular uptake, may be the rate-limiting step in the transfection process. These initial results indicate that CDs are useful templates for further modification to produce molecular constructs capable of enhanced gene delivery.     

顺铂和依托泊苷下调肺癌细胞端粒酶量及端粒酶逆转录酶的表达

目的　观察肺癌化疗的一线药物顺铂 (DDP)和依托泊苷 (VP16 )对肺癌细胞端粒酶量和端粒酶逆转录酶基因表达和蛋白质表达的影响 ,从而进一步探讨这些药物在肺癌治疗中的作用机理。方法用不同浓度的化疗药物处理肺腺癌A5 49细胞 ,用噻唑蓝 (MTT)和流式细胞术观察化疗药物 (DDP和VP16 )对肺癌细胞的生长抑制和细胞凋亡情况 ,分别用端粒重复扩增 (TRAP)法、逆转录 聚合酶链反应 (RT PCR)法和Western印迹杂交检测处理后端粒酶量 ,以及其催化亚基端粒酶逆转录酶 (hTERT)基因和蛋白质的变化。结果　DDP和VP16明显抑制肺癌细胞生长 ,并诱导细胞凋亡 ,有剂量和时间依赖性 .DDP和VP16明显抑制端粒酶量和hTERT基因及蛋白表达 ,尤其以高剂量 (VP16 2 0 0mg·L- 1,DDP5 0mg·L- 1)和低剂量 (VP16 2 0mg·L- 1,DDP 5mg·L- 1)作用的d 5更为明显。结论　VP16和DDP可以抑制肺癌细胞端粒酶量 ,这种作用可能是通过抑制端粒酶的催化亚基hTERT的表达来实现的。端粒酶量测定可能有助于作为判断肺癌化疗时VP16和DDP疗效的观察指标     

两种阳离子脂质体介导基因转染的比较研究

In order to study the important factors involved in cationic liposome-mediated gene transfer,   Lipofectamine 2000 or DOTAP was evaluated using three types of cells (Hep-2, MCF-7 and SW-480) in vitro transfection efficiencies.  Different properties of the two reagents were analyzed and compared by DNA     arrearage assay and MTT assay.  Both Lipofectamine 2000 and DOTAP had strong capability to combine with DNA; Lipofectamine 2000 can get higher transfection efficiency of the three cells by using GFP as report gene, meanwhile, DOTAP can also get higher transfection efficiency against Hep-2 cell.  However, DOTAP showed lower transfection efficiency against MCF-7 and SW-480 cell.  On the other hand, the cytotoxicity assay showed that over 85% cell viability of MCF-7 cell could be achieved both by Lipofectamine 2000 and DOTAP under the optimal transfection condition.  Relatively speaking, Lipofectamine 2000 has very high transfection efficiency in a broad range of cell lines, but because of the special selectivity of cell type on liposome, DOTAP also has a broad application prospect.