Highly effective inhibition of lung cancer growth and metastasis by systemic delivery of siRNA via multimodal mesoporous silica-based nanocarrier

Lung cancer has been the leading type of cancers with regard to mortality and mobility. New versions of RNAi-based therapy are greatly required to tackle the challenges of lung cancer. In this study, we developed a novel siRNA delivery vector based on our magnetic mesoporous silica nanoparticles (M-MSNs) platform. This nanocarrier was constructed by loading siRNAs into the mesopores of M-MSNs, followed by polyethylenimine (PEI) capping, PEGylation and fusogenic peptide KALA modification. The resultant delivery system exhibited prolonged half-life in bloodstream, enhanced cell membrane translocation and endosomal escapablity, and favorable tissue biocompatibility and biosafety. Systemic application of vascular endothelial growth factor (VEGF) siRNA via this nanocarrier resulted in remarkable tumor suppression, both in subdermal and orthotopic lung cancer models, while tumor metastasis was also significantly reduced, overall leading to improved survival. In addition, the magnetic core of the particles and the functionalized fluorescence markers conveniently enabled in vivo imaging of target tissues. Taken together, this M-MSNs-based siRNA delivery vehicle has shown very favorable applicability for cancer therapy.     

Amygdalin-mediated inhibition of non-small cell lung cancer cell invasion in vitro

Lung cancer is a common malignant tumor claiming the highest fatality worldwide for a long period of time. Unfortunately, most of the current treatment methods are still based on the characteristics of cancer cells in the primary lesion and the prognosis is often much poorer in patients with metastatic cancers. Amygdalin, a natural product of glycosides and lots of evidence shows that amygdalin can inhibit the proliferation of some kinds of cancer cells. In this study, we first obtained the highly metastatic NSCLC cell lines H1299/M and PA/M and further treated these cells with amygdalin. We found that the in vitro proliferability of H1299/M and PA/M was inhibited, but such inhibition required higher concentration of amygdalin. When lower concentration of amygdalin was used for the experiments, we observed that the in vitro invasive and migration capacities of H1299/M and PA/M were significantly inhibited. These results strongly suggested that amygdalin was likely to have anti-metastatic NSCLC effect. This study offers information of the role of amygdalin that may be useful as a therapeutic target in lung tumors.     

微小RNAs对肺癌细胞系侵袭、迁移及凋亡的影响

目的 探讨微小RNAs（miRNAs）对肺癌细胞系侵袭、迁移及凋亡的影响。方法 通过hsa-mir-933、hsa-mir-4700-3p、hsa-mir-3144-3p、hsa-mir-3972和hsa-mir-548a-5p感染目的细胞,流式细胞术检测5种miRNAs对肺癌细胞系的细胞凋亡的影响;Transwell分析5种miRNAs对肺癌细胞系的细胞侵袭效果;划线分析5种miRNAs对肺癌细胞系的细胞迁移的影响;Real-time PCR检测miRNA表达情况。结果 与感染空载体组和无感染的对照组相比,感染了hsa-mir-933、hsa-mir-4700-3p、hsa-mir-3144-3p、hsa-mir-3972和hsa-mir-548a-5p的细胞凋亡明显增加,其中NCI-H460和A549均以转染hsa-mir-4700-3p凋亡率最高;5种miRNAs对肺癌细胞的侵袭数目明显减小;5种miRNAs对肺癌细胞的细胞的迁移效果减弱; Real-time PCR检测hsa-mir-933、hsa-mir-4700-3p、hsa-mir-3144-3p、hsa-mir-3972和hsa-mir-548a-5p处理后相应miRNA的水平表达均相应增加。结论 5种miRNAs能够促进肺癌细胞系的凋亡,降低肺癌细胞系的高侵袭性,抑制肺癌细胞系的迁移,增加对应miRNA的表达。     

Peroxiredoxin 1 promoted tumor metastasis and angiogenesis in colorectal cancer

Peroxiredoxin1 (Prdx1) is a member of the PrdxS family, and it regulates cellular signaling and differentiation. The role of Prdx1in colorectal cancer (CRC) remains unclear. In this study, we investigated the relevance of Prdx1 in the metastasis and angiogenesis of CRC. The expression of Prdx1 in 60 cases human CRC tissues was detected through immunohistochemistry. The tumors that highly expressed Prdx1 (42/60) exhibited higher tumor grade and lymph node metastasis than those with low expression of Prdx1 (18/60) (p?<?0.05). Kaplan–Meier survival analysis showed that the survival time of thePrdx1-positive group was shorter than that of thePrdx1-negative group (p?=?0.046).Moreover, a statistically significant correlation was observed between the Prdx1 expression and microvessel density (p?=?0.004). Transwell migration assay revealed that Prdx1 was down-regulated in the CRC cell line HCT116, thereby suppressing the invasion and migration capacities of tumor cells, whereas Prdx1was up-regulated in HT29 cells, thereby increasing the invasion and migration capacities of tumor cells. The tube formation capacity of human umbilical vein endothelial cells cultured in 3D medium was increased after conditioned medium from overexpressed Prdx1cancer cells was added relative to that when down-regulated Prdx1 cell medium was added (p?<?0.05). In addition, up-regulated Prdx1 increased the protein expression of MMP2, MMP9, and VEGFA. These data suggested that Prdx1 expression predicted poor prognosis by regulating the tumor metastasis and angiogenesis of CRC. Therefore, Prdx1 may serve as a potential therapeutic target.     

Suppression of colorectal cancer subcutaneous xenograft and experimental lung metastasis using nanoparticle-mediated drug delivery to tumor neovasculature

Antiangiogenic therapy is a validated approach for colorectal cancer (CRC) treatment. However, diverse adverse effects inevitably appear due to the off-target effect of the approved antiangiogenic inhibitors on the physiological functions and homeostasis. This study was to investigate a new tumor vessel targeting nanoparticulate drug delivery system, F56 peptide conjugated nanoparticles loading vincristine (F56-VCR-NP), for the effective treatment of CRC subcutaneous xenograft and experimental lung metastasis model. The controlled release behavior and in vivo pharmacokinetic profile of F56-VCR-NP were characterized. The tumor vessel targeting and antiangiogenic activity of F56-VCR-NP was evaluated in human umbilical vein endothelial cells (HUVEC, a classical cell model mimicking tumor vascular EC), subcutaneous human HCT-15 xenograft in immunodeficient nude mice, and experimental CT-26 lung metastasis model in immunocompetent mice. The therapeutic efficacy (animal survival and toxicity) was further investigated in the model of CT-26 lung metastasis in mice. F56-VCR-NP could achieve 30-day controlled drug release in PBS (pH 7.4) and exhibited favorable long-circulating feature in vivo. F56-VCR-NP could accurately target the CRC neovasculature and elicit nanoparticle internalization in the tumor vascular EC, where the antiangiogenic VCR-induced dramatic EC apoptosis and necrosis of CRC tissue. F56-VCR-NP significantly prolonged the mouse survival with no obvious toxicity (weight loss and anepithymia) in the CT-26 lung metastasis mice model, and this pronounced antitumor effect was closely related with the decreased microvessel density in the metastases. The present nanoparticle-based targeted antiangiogenic therapy may provide a new promising approach for the therapy of CRC and lung metastasis, which deserves further translational research.     

Transient RNA silencing of tissue factor pathway inhibitor-2 modulates lung cancer cell invasion

Tissue Factor Pathway Inhibitor-2 (TFPI-2) is a potent inhibitor of plasmin which activates metalloproteinases (MMPs) involved in extracellular matrix (ECM) degradation. Its secretion in ECM makes TFPI-2 a potential inhibitor to regulate tumour invasion and metastasis. Moreover, TFPI-2 is frequently downregulated, particularly in aggressive cancers. In this study, we silenced TFPI-2 in the NCI-H460 non-small cell lung cancer cell line and evaluated the role of TFPI-2 in cell invasion and its impact on MMPs expression. As the effects of siRNA are transient, the consequences of both gene silencing and restoration to normal expression could be studied kinetically in the same cells. We showed that TFPI-2 expression by NCI-H460 cells was effectively downregulated using specific small interfering RNA and this silencing was associated with an increase in the invasive potential of tumour cells while migration was not affected. We also showed that mRNA levels and protein expression of MMP-2, -3, -9, -14 were not influenced by TFPI-2 silencing. Moreover, the gelatinase activity of MMP-2 and MMP-9 was unmodified. In contrast, MMP-1 mRNA levels and protein were significantly and similarly increased in cells transfected with TFPI-2 siRNA. In conclusion, this study confirms that TFPI-2 downregulation can contribute to tumour invasion of lung cancer cells. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Non-small cell lung carcinoma therapy using mTOR-siRNA

Molecular targeting agents play important roles in non-small-cell lung cancer (NSCLC) therapy. Published studies have investigated new drugs categorized as molecular targeting agents that inhibit the mammalian target of rapamycin (mTOR). We focused on a small interfering RNA (siRNA) that specifically inhibits mTOR and has fewer side effects. To evaluate the antitumor effects of the siRNA, cell proliferation, apoptosis, and migration were assessed. In the study group, the siRNA was transfected into NSCLC cells. The number of cells present after 6 days of culture was counted to determine changes in cell proliferation. The level of apoptosis was evaluated by the detection of DNA-histone complexes in the cytoplasmic fraction using an absorption spectrometer. Changes in migration were evaluated by calculating the number of cells that passed through a specific filter using a commercial chemotaxis assay kit. mTOR-siRNA transfection inhibited cell proliferation as indicated by 37.3% (p = 0.034) decrease in the number of cells compared with the control cells. Analysis of the level of apoptosis in NSCLC cells revealed 16.7% (p = 0.016) increase following mTOR-siRNA transfection, and mTOR-siRNA transfection significantly inhibited cell migration by 39.2% (p = 0.0001). We confirmed that mTOR-siRNA induces apoptosis and inhibits the proliferation and migration of NSCLC cells in vitro. Further studies using mTOR-siRNA may aid in the development of an alternative therapy that maximizes the antineoplastic effect of mTOR inhibition.     

全反式维甲酸对克隆化高转移人肺癌细胞浸润和转移的抑制作用

用５μｍｏｌ／Ｌ和１０μｍｏｌ／Ｌ全反式维甲酸（ＲＡ）处理克隆化高转移人肺癌细胞（ＰＧＣＬ３）５天后，细胞的体外生长速度、穿过Ｂｏｙｄｅｎ小室人工基底膜胶的浸润能力都受到一定的抑制，其中尤以１０μｍｏｌ／ＬＲＡ的作用明显。实验性转移显示，ＲＡ体外处理ＰＧＣＬ３细胞可在一定程度上降低其实验性转移的能力。此外通过ＤＮＡ－ＲＮＡ斑点杂交还发现ＰＧＣＬ３细胞经１０μｍｏｌ／ＬＲＡ处理５天后，人组织金属蛋白酶抑制剂基因（Ｔｉｍｐ－１和Ｔｉｍｐ－２）表达有一定水平的增高，这有助于进一步阐明ＲＡ抑制肿瘤细胞浸润和转移的机理。     

miR-let-7d通过调控核受体PPARγ促进肺癌细胞的增殖和侵袭

目的:探讨miR-let-7d对肺癌细胞核受体过氧化物酶体增殖物激活受体γ(PPARγ)的调控及对肺癌细胞增殖和侵袭的影响。方法:用生物信息学分析与PPARγ相关的microRNA,通过质粒报告基因验证miR-let-7d的作用靶点;利用Western blot法筛选出PPARγ表达水平低的肺癌细胞株;通过双萤光素酶标记和Western blot法验证miR-let-7d对肺癌细胞中PPARγ表达的调控作用;通过集落形成实验检测miR-let-7d对肺癌细胞增殖能力的调控作用;通过Transwell侵袭实验检测miR-let-7d对肺癌细胞侵袭能力的作用。结果:生物信息学的分析结果证明miR-let-7d可以调控核受体PPARγ的蛋白表达;在核受体PPARγ的3’UTR包含2个功能性的miR-let-7d结合位点;PPARγ是miR-let-7d的直接靶点,miR-let-7d可在蛋白和mRNA水平直接调控PPARγ的表达;miR-let-7d inhibitor通过升高PPARγ的表达促进肺癌细胞的增殖和侵袭能力。结论:miR-let-7d能够通过靶向增加具有抑癌作用的核受体PPARγ的表达水平,抑制肺癌细胞的增殖和侵袭能力。     

Sirtuin SIRT6 suppresses cell proliferation through inhibition of Twist1 expression in non-small cell lung cancer

SIRT6 is a member of the NAD⁺-dependent class III deacetylase sirtuin family. Current studies have revealed that SIRT6 plays important roles in the epigenetic regulation of genes expression and contribute to the proliferation, differentiation and apoptosis of cancer cells. However, the biological function of SIRT6 in lung cancer has not been elucidated. The present study showed that the mRNA and protein levels of SIRT6 were decreased in human non-small cell lung cancer (NSCLC) tissues and cell lines. MTT assay showed that overexpression of SIRT6 could inhibit the proliferation in NSCLC cells. In contrast, SIRT6 knockdown using small interfering RNA promoted NSCLC cells proliferation. On the molecular level, we found that SIRT6 inhibited the expression of Twist1 both at the mRNA and protein levels in NSCLC cells. Taken together, these results demonstrated for the first time that SIRT6 suppressed NSCLC cells proliferation via down-regulation of Twist1 expression and might provide novel therapeutic targets in the treatment of lung cancer.     

自噬在熊果酸抑制人肺癌PC9细胞增殖中的作用

目的:研究自噬在熊果酸(UA)抑制人肺癌PC9细胞增殖中的作用及机制。方法:应用MTT法和台盼蓝拒染法检测UA对PC9细胞增殖的影响。吖啶橙染色法在荧光显微镜下观察UA对PC9细胞自噬的影响。Western blot检测自噬相关蛋白LC3及ATG5的表达情况。采用自噬抑制剂3-甲基腺嘌呤(3-MA)观察UA对PC9细胞增殖的抑制作用。结果:UA可以显著抑制PC9细胞的活力(P0.05或P0.01),随着给药剂量和时间的增加,UA对PC9细胞的生长抑制率显著上升。UA诱导PC9细胞自噬体表达增加,并诱导自噬相关蛋白LC3-Ⅱ和ATG5表达的增加(P0.01)。自噬抑制剂3-MA提高了UA对PC9细胞的抑制作用(P0.01)。结论:UA抑制PC9细胞的增殖,并诱导细胞发生自噬。UA诱导PC9细胞自噬的机制有可能依赖ATG5细胞自噬途径。自噬抑制剂3-MA能够增强UA对PC9细胞的增殖抑制作用,有望为肺癌的临床治疗提供新的联合治疗方案。     

IL-35通过诱导T细胞分化促进肺癌肿瘤进展

目的 探究肺癌中IL-35对T细胞表型及对癌症进展的影响.方法 建立小鼠肺癌移植瘤模型,施用IL-35中和抗体,监测肿瘤生长情况.并通过检测肿瘤中T细胞的浸润情况及其表型和细胞因子分泌情况,进一步探究对抗肿瘤免疫反应的影响.最后通过对肿瘤浸润T细胞的体外刺激实验,检测浸润T细胞的抗肿瘤活性.结果 瘤体积及瘤重的检测结果...     

趋化因子及其受体CXCL16/CXCR6在人肺癌转移中的作用

目的 探讨CXCL16/CXCR6在肺癌的表达模式及其对肺癌细胞系生物学行为的影响.方法 免疫组织化学技术分析CXCL16/CXCR6蛋白在人肺癌组织的表达;免疫细胞化学分析CXCL16/CXCR6在3种肺癌细胞系A549、95D和H292的表达;MTT试验及迁移、侵袭试验分别分析CXCL16时A549、95D和H292细胞体外增殖活力和侵袭能力的调节作用.结果 人肺癌组织高表达CXCR6和CXCL16蛋白;A549、95D和H292细胞均表达CXCL16和CXCR6蛋白;外源性CXCL16可明显促进A549、95D和H292细胞的体外增殖活力和侵袭能力,且CXCL16中和抗体可以有效阻断CXCL16蛋白对3种肺癌细胞系的刺激作用.结论 CXCL16/CXCR6可能是参与肺癌侵袭转移的分子.     

ICA和PJA对高转移性人肺癌细胞体外侵袭转移能力抑制的研究

目的：将新型抗癌药物淫羊藿甙（ＩＣＡ）和济南假单胞菌制剂（ＰＪＡ）作用于ＰＧ细胞,从肿瘤转移抑制的多个方面和环节探讨ＰＪＡ、ＩＣＡ抗转移作用的机制。方法：采用粘附实验、运动侵袭实验、逆转录ＰＣＲ（ＲＴ－ＰＣＲ）、细胞免疫组化等多种方法进行了检测。结果：ＰＪＡ、ＩＣＡ可降低ＰＧ细胞对胞外基质的粘附性及侵袭、运动能力,减少ＰＧ细胞表面粘附分子ＣＤ４４Ｖ６、ＬＮ－Ｒ及胞浆内ＣＫ１８的表达,同时细胞内ｃ－ｍｙｃ、Ｔｉａｍ－１基因ｍＲＡＮ水平均有不同程度的降低,而Ｎｍ２３－Ｈ１ ｍＲＮＡ水平有不同程度的升高,且两药有明显的协同作用。结论：ＰＪＡ、ＩＣＡ通过对肿瘤转移多个步骤的抑制而发挥抗转移作用。     

Prognostic value of FHIT, CTNNB1, and MUC1 expression in non-small cell lung cancer

Comprehensive expression analysis using microarrays has identified a number of differentially expressed genes in smoke-exposed bronchial epithelium and non-small cell lung cancers (NSCLCs). To evaluate the prognostic relevance of these proteins in NSCLCs, we used immunohistochemistry to investigate the expression of beta-catenin (CTNNB1), dickkopf, Xenopus, homolog of 3 (DKK3 gene), fibroblast growth factor receptor 3 (FGFR3), fragile histidine triad (FHIT), tumor protein p53 (TP53), mucin1 (MUC1), topoisomerase II alpha (TOP2A), and glutathione S-transferase-Pi (GST) in a cohort of patients (n = 125). We correlated the expression data with clinicopathologic features and clinical outcome. In addition, SNaPshot multiplex assays (Applied Biosystems, Darmstadt, Germany) and restriction fragment length polymorphism analysis were used to screen for activating point mutations at the hot spots of FGFR3 in a cohort of 30 samples of NSCLC. Using Kaplan-Meier analysis, we observed significantly better overall survival in adenocarcinomas compared with squamous cell cancers (P = .049). Loss of FHIT expression showed a strong association with shorter overall survival in both histologic types of NSCLC (squamous cell cancers, P < .001; adenocarcinomas, P = .001). In adenocarcinomas, the cytoplasmic expression of beta-catenin was associated with shorter survival (P = .012); MUC1 expression was associated with worse prognosis in patients with squamous cell cancers (P = .002). The nuclear staining of TP53 (P = .008) and TOP2A (P = .059) was associated with cancers without lymphonodal metastases. A correlation with positive staining of TOP2A (P = .03) and FGFR3 positivity (P = .057) was found in adenocarcinomas of male patients. Positive MUC1 stainings were associated with squamous cell cancers of male patients (P = .03). DKK3 expression did not show any significant association with clinical outcome or pathologic features. The screening of the FGFR3 sequence in lung cancers showed only wild-type sequences and did not detect mutations in the known hot spots for FGFR3 mutations. We conclude that the immunohistochemical loss of FHIT expression and the positivity for beta-catenin and MUC1 in NSCLC are useful prognostic markers, whereas the variable expression of TP53, TOP2A, and FGFR3 in relation to the different histologic types of NSCLC and sex of the patients is suggestive for different underlying molecular pathways.     

胃癌干细胞在胃癌侵袭、转移中及血管形成中的作用和机制

BACKGROUND: Although tumor stem cells and their differentiated vascular cells, which are not sensitive to chemotherapy and molecular targeted therapy, reduce overall blood supply of the tumor, these cells facilitate the invasion and metastasis of the tumor, eventually resulting in treatment failure.     

Prognosis of non-small cell lung cancer with synchronous brain metastases treated with gamma knife radiosurgery

The clinical outcome and prognostic factors of patients with synchronous brain metastases from non-small cell lung cancer (NSCLC) who were treated with gamma knife radiosurgery (GKS) were analyzed. A total of 35 patients with NSCLC underwent GKS as an initial treatment for metastatic brain lesions of synchronous onset. The period of survival and various prognostic factors such as age, gender, performance status, multiplicity of the brain lesions, intracranial tumor volume, and extent of the primary tumor were analyzed. The overall median survival time for this series was 12 months (range 0.75 to 43 months) from the diagnosis. Of the 21 patients who were no longer alive at the conclusion of this study, only 7 (33.3%) died of neurological causes. Multivariate analysis of these data revealed that N stage, whole-brain radiotherapy (WBRT), and chemotherapy were significant predictors for survival (p<0.05). Survival of patients with NSCLC and synchronous brain metastases is mainly dependent upon the progression of the systemic disease, provided that the cerebral lesions are treated adequately with local treatment modalities including radiosurgery. Application of radiosurgery as an initial treatment option and aggressive local and systemic modalities to control extracranial disease may improve survival.     

Cyclophilin B promotes cell proliferation,migration, invasion and angiogenesis via regulating the STAT3 pathway in non-small cell lung cancer

Lung cancer is the most common type of cancer and has become the leading cause of cancer-associated mortality worldwide. It has been reported that expression of Cyclophilin B was greatly elevated in the pancreatic cancer patient sera as compared with the healthy volunteer sera. This study aimed to investigate the role and regulatory mechanism of CypB in NSCLC progression. The expression levels of CypB was detected in NSCLC samples and cell lines by ELISA, western blot and immunohistochemistry assay. In addition, CCK8, colony formation, scratch and transwell assays were used to evaluate the proliferation, migration and invasion of A549 cells with CypB silencing. The expression of angiogenesis related proteins and pathway-related factors were detected by western blot. In NSCLC samples, CypB expression was upregulated. The expression of CypB was significantly reduced in the siRNA-cyclophilin B group. In addition, CypB silencing inhibited cell proliferation, migration and invasion. The expression of angiogenesis related proteins and pathway-related factors have also changed significantly. These findings suggested that CypB silencing may suppress the proliferation, invasion, migration and angiogenesis of A549 cells via inhibiting STAT3 pathway.     

健脾化瘀解毒方调控能量代谢抑制肺癌细胞侵袭转移的研究

目的:研究健脾化瘀解毒方的含药血清(JHJRMS)对人肺癌细胞能量代谢的影响.方法:TCGA数据库分析己糖激酶2(HK2)与肺癌患者预后之间的相关性;划痕实验用来检测JHJRMS对肺癌细胞侵袭转移的影响.RT-PCR检测JHJRMS对肺癌细胞中miR-143-3p表达水平的影响.数据库预测miR-143-3p与HK2蛋...