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.     

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。     

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.     

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.     

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...     

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.     

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.     

PTTG1通过上皮-间质转化促进非小细胞肺癌的迁移和侵袭

目的 研究PTTG1在非小细胞肺癌侵袭转移中的作用.方法 应用免疫组织化学染色法检测80例NSCLC组织中PTTG1、E-cadherin和Vimentin的表达,并分析其相关性.细胞分组:1)A549/con,转染空载的A549,2)A549/PTTG1,转染目的基因的A549,3)Scr/H1299,转染空载的H1299,4)SiPTTG1/H1299,敲除PTTG1的H1299;用Western blot检测细胞中PTTG1、E-cadherin、Vimentin蛋白的表达情况.结果 NSCLC组织中PTG1蛋白的阳性表达率为85％与E-cadherin的表达呈负相关(P＜0.05)而与Vimentin的表达呈正相关(P＜0.05).Western blot结果显示在SiPTTG1/H1299细胞中PTTG1蛋白表达水平明显降低,Vimentin蛋白表达水平降低而E-cadherin蛋白表达水平升高.同时在A549/PTTG1细胞中Vimentin蛋白表达水平升高而E-cadherin蛋白表达水平降低.结论 PTG1能促进NSCLC EMT的发生,从而促进NSCLC的侵袭和转移.     

microRNA-137 modulates pancreatic cancer cells tumor growth,invasion and sensitivity to chemotherapy

Background: We intended to investigate the role of microRNA 137 (miR-137) in regulating pancreatic cancer cells’ growth in vitro and tumor development in vivo. Methods: QTR-PCR was used to examine the expression of miR-137 in pancreatic cancer cell lines and tumor cells from human patients. Lentivirual vector containing miR-137 mimic was used to overexpress miR-137 in PANC-1 and MIA PaCa-2 cells. The effects of overexpressing miR-137 on pancreatic cancer cell invasion and chemo-sensitivity to 5-fluorouracil (5-FU) were examined by cell migration and survival essays in vitro. The molecular target of miR-137, pleiotropic growth factor (PTN), was down-regulated by siRNA to examine its effects on cancer cell invasion. MIA PaCa-2 cells with endogenously overexpressed miR-137 were transplanted into null mice to examine tumor growth in vivo. Results: We found miR-137 was markedly underexpressed in both pancreatic cancer cell lines and tumor cells from patients. In cancer cells, transfection of lentivirus containing miR-137 mimic was able to markedly upregulate endogenous expression of miR-137, inhibited cancer cell invasion and increased sensitivities to chemotherapy reagent 5-FU. PTN was significantly down-regulated by overexpressing miR-137 in pancreatic cancer cells, and knocking down PTN was effective to rescue the reduced cancer cell invasion ability caused by miR-137 overexpression. More importantly, overexpressing miR-137 led to significant inhibition on tumor formation, including reductions in tumor weight and tumor size in vivo. Conclusion: Our study demonstrated that miR-137 played an important role in pancreatic cancer development. It may become a new therapeutic target for gene therapy in patients suffered from pancreatic cancer.     

Surgical treatment of non-small cell lung cancer with isolated synchronous brain metastases

This study is a retrospective examination of our experiences with patients who underwent treatment of isolated synchronous brain metastases coupled with primary non-small cell lung cancer. From January 1995 to June 2004, 12 patients presented with isolated synchronous brain metastases coupled with primary non-small cell lung cancer. The patient was comprised of 8 men and 4 women. The median age was 52 yr, in a range of 32 to 75 yr. Median follow-up duration was 10.6 months, in a range of 2 to 55.8 months. Recurrence developed in 7 patients, and the median interval from 1st treatment to recurrence was 4.5 months (2.8-6.5 months). The overall 1-yr survival rate was 61.7%. The 1-yr survival rates for pathologic N0 and N1 cases were 75% and 66.7%, respectively. The median survival duration for pathologic N2 was 6.2 months (95% CI, 4.8-7.5 months). The 1-yr survival rate for cases of single brain metastasis was 75%. Based on our current observations, we could speculate that aggressive management of primary non-small cell lung cancer and isolated synchronous brain metastases was beneficial in a selected group of patients, as long as the brain lesions and pulmonary lesions were limited or resectable.     

RhoC is essential for the metastasis of gastric cancer

Rho family members are known to regulate malignant transformation and motility of cancer cells, but the clinicopathological significance of RhoC remains unclear yet in the case of gastric cancer. In this study, we evaluated the protein expression level of RhoC in gastric cancer tissues and cell lines. Results showed that only weak staining of RhoC was detected in 3 of 33 non-tumorous cases by immunohistochemistry. The expression of RhoC was significantly higher in gastric cancer tissues (23/42, 54.8%) than in non-tumorous tissues (p < 0.01). Further analysis demonstrated that RhoC had high specificity (80.0%) in detecting gastric carcinomas with metastatic potential. RhoC was positively expressed in 18 out of 20 metastases (90.0%), even higher than that in primary gastric cancer tissues. Western blot showed that RhoC was up-regulated in five different gastric cancer cell lines but not expressed in SV40-transformed immortal gastric epithelial cell GES-1. Overexpression of RhoC GTPase in GES-1 cells could produce the motile and invasive phenotype but did not alter the monolayer growth rate. To further study the functions of RhoC, we took the powerful siRNA technology to knock down the expression of RhoC in SGC7901 cells. It was shown that down-regulation of RhoC did not affect the proliferation of SGC7901 cells. However, interference of RhoC expression could inhibit migration, invasion, and anchorage-independent growth of SGC7901 cells. In conclusion, RhoC may play a very important role in the metastasis of gastric carcinoma. Therapeutic strategies targeting RhoC and RhoC-mediated pathways may be a novel approach for treating metastasis of gastric cancer.     

Rho GTPases in PC-3 prostate cancer cell morphology,invasion and tumor cell diapedesis

BACKGROUND: The Rho GTPases comprise one of the eight subfamilies of the Ras superfamily of monomeric GTP-binding proteins and are involved in cytoskeletal organization. Previously, using a dominant negative construct, we demonstrated a role for RhoC GTPase in conferring invasive capabilities to PC-3 human prostate cancer cells. Further, we demonstrated that inactivation of RhoC led to morphological changes commensurate with epithelial to mesenchymal transition (EMT) and was accompanied by increased random, linear motility and decreased directed migration and invasion. EMT was related positively to sustained expression and activity of Rac GTPase. In the current study we analyze the individual roles of RhoA, RhoC and Rac1 GTPases in PC-3 cell directed migration, invasion and tumor cell diapedesis across a human bone marrow endothelial cell layer in vitro. RESULTS: Use of specific shRNA directed against RhoA, RhoC or Rac1 GTPases demonstrated a role for each protein in maintaining cell morphology. Furthermore, we demonstrate that RhoC expression and activation is required for directed migration and invasion, while Rac1 expression and activation is required for tumor cell diapedesis. Inhibition of RhoA expression produced a slight increase in invasion and tumor cell diapedesis. CONCLUSIONS: Individual Rho GTPases are required for critical aspects of migration, invasion and tumor cell diapedesis. These data suggest that coordinated activation of individual Rho proteins is required for cells to successfully complete the extravasation process; a key step in distant metastasis.