Liver Metastasis In Nude Mice Research Articles

The regulatory role of cancer stem cell marker gene CXCR4 in the growth and metastasis of gastric cancer

Single-cell RNA sequencing (scRNA-seq) and bulk RNA sequencing (bulk RNA-seq) are increasingly used for screening genes involved in carcinogenesis due to their capacity for dissecting cellular heterogeneity. This study aims to reveal the molecular mechanism of the cancer stem cells (CSCs) marker gene CXCR4 in gastric cancer (GC) growth and metastasis through scRNA-seq combined with bulk RNA-seq. GC-related scRNA-seq data were downloaded from the GEO database, followed by UMAP cluster analysis. Non-malignant cells were excluded by the K-means algorithm. Bulk RNA-seq data and clinical sample information were downloaded from the UCSC Xena database. GO and KEGG pathway analyses validated the correlation between genes and pathways. In vitro and in vivo functional assays were used to examine the effect of perturbed CXCR4 on malignant phenotypes, tumorigenesis, and liver metastasis. A large number of highly variable genes were identified in GC tissue samples. The top 20 principal components were selected, and the cells were clustered into 6 cell types. The C4 cell cluster from malignant epithelial cells might be CSCs. CXCR4 was singled out as a marker gene of CSCs. GC patients with high CXCR4 expression had poor survival. Knockdown of CXCR4 inhibited the malignant phenotypes of CSCs in vitro and curtailed tumorigenesis and liver metastasis in nude mice. CSC marker gene CXCR4 may be a key gene facilitating malignant phenotypes of CSCs, which thus promotes tumor growth and liver metastasis of GC.

Aquaporin 1 is a prognostic marker and inhibits tumour progression through downregulation of Snail expression in intrahepatic cholangiocarcinoma.

Recently, some studies have suggested a link between AQP1 and cancer progression. The aim of the present study was to investigate the influence of AQP1 on the clinicopathology and prognosis of intrahepatic cholangiocarcinoma (ICC) patients. We retrospectively detected the expression of AQP1 protein in 307 patients with ICC who underwent partial hepatectomy. Western blot analysis was used to detect AQP1 protein levels in stable AQP1 overexpression and knockdown cell lines. The influence of AQP1 on the invasion and metastasis ability of ICC cells was assessed by wound-healing and Transwell assays in vitro as well as by a splenic liver metastasis model in vivo. Positive membranous AQP1 expression was identified in 34.2% (105/307) of the ICC specimens. Survival data revealed that positive AQP1 expression was significantly associated with favourable disease-free survival (DFS) and overall survival (OS) (p=0.0290 and p=0003, respectively). Moreover, high AQP1 expression inhibited the invasion and migration of ICC cells in vitro as well as inhibited liver metastasis in nude mice. Mechanistically, high AQP1 expression in ICC cells increased the levels of E-cadherin but decreased the levels of the Snail transcription factor. AQP1 expression is associated with a favourable prognosis in ICC patients. AQP1 inhibits ICC cell invasion, metastasis, and epithelial-mesenchymal transition (EMT) through downregulation of Snail expression.

HDAC8 Promotes Liver Metastasis of Colorectal Cancer via Inhibition of IRF1 and Upregulation of SUCNR1

Histone deacetylases (HDACs) are well-characterized for their involvement in tumor progression. Herein, the current study set out to unravel the association of HDAC8 with colorectal cancer (CRC). Bioinformatics analyses were carried out to retrieve the expression patterns of HDAC8 in CRC and the underlying mechanism. Following expression determination, the specific roles of HDAC8, IRF1, and SUCNR1 in CRC cell functions were analyzed following different interventions. Additionally, tumor formation and liver metastasis in nude mice were operated to verify the fore experiment. Bioinformatics analyses predicted the involvement of the HDAC8/IRF1/SUCNR1 axis in CRC. In vitro cell experiments showed that HDAC8 induced the CRC cell growth by reducing IRF1 expression. Meanwhile, IRF1 limited SUCNR1 expression by binding to its promoter. SUCNR1 triggered the growth and metastasis of CRC by inhibiting cell autophagy. HDAC8 blocked IRF1-mediated SUCNR1 inhibition and thereby inhibited autophagy, accelerating CRC cell growth. Lastly, HDAC8 facilitated the development of CRC and liver metastasis by regulating the IRF1/SUCNR1 axis in vivo. Taken together, our findings highlighted the critical role for the HDAC8/IRF1/SUCNR1 axis in the regulation of autophagy and the resultant liver metastasis in CRC.

Liver Metastasis Formation Is Defined by AMIGO2 Expression via Adhesion to Hepatic Endothelial Cells in Human Gastric and Colorectal Cancer Cells

The adhesion of circulating cancer cells to vascular endothelial cells is an initial and critical step in distant metastases. Amphoterin-induced gene and open reading frame 2 (AMIGO2) was found to regulate tumor cell adhesion to hepatic endothelial cells and act as a driver gene for liver metastasis in mouse cell lines. However, whether the role of AMIGO2 observed in mouse tumor cells can be extrapolated to human cancer cells in vivo has not been verified. In this study, AMIGO2 expression in various human gastric and colorectal cancer cells was found to be closely associated with their adhesion to human hepatic sinusoidal endothelial cells (HHSECs). Constitutive AMIGO2-knockdown clones of human gastric (MKN-45) and colorectal cancer cell lines (DLD-1) were established to examine whether AMIGO2 expression in cancer cells is involved in the adhesion to HHSECs in vitro and the formation of liver metastasis in vivo. All AMIGO2-knockdown cells showed significantly attenuated adhesion to HHSECs. In vivo analysis revealed that intrasplenic inoculation of AMIGO2-knockdown clones could engraft in the spleen but significantly suppressed liver metastasis in nude mice. This study demonstrated that the role of AMIGO2 as a driver gene of liver metastasis in mouse tumor cells can be extrapolated to human cancer cells.

A Regulatory Loop Involving Notch and Wnt Signaling Maintains Leukemia Stem Cells in T-Cell Acute Lymphoblastic Leukemia.

Leukemia-initiating cells play critical role in relapse, resistance to therapies and metastases but the mechanism remains largely elusive. We report that β-catenin is over-expressed in almost all T-ALL patients and flow sorted β-cateninhigh fractions are highly resistant to therapy, leading to liver metastases in nude mice as well as dysregulated lncRNAs. Pharmacological inhibition through XAV-939 as well as si-RNA mediated inhibition of β-catenin is initially effective in re-sensitization to therapy, however, prolonged inhibition shifts dependency from β-catenin to Notch signaling, with particularly high levels of receptors Notch 1 and Notch 2. The results are verifiable in a cohort of T-ALL patients comprising of responders vs. those who have progressed, with β-catenin, Notch 1 and Notch 2 elevated in progressed patients. Further, in patients-derived cells, silencing of Notch 1 or Notch 2 does not counter resistance to β-catenin inhibition, rather pharmacological pan-Notch inhibition is needed to overcome resistance and its effect on in vitro tumor sphere formations as well as in vivo liver metastases. Thus, wnt and Notch signaling are part of a regulatory loop mutually compensating for each other in T-ALL, while ensuring the maintenance of stem cell phenotype.

Metastatic suppression by DOC2B is mediated by inhibition of epithelial-mesenchymal transition and induction of senescence

Senescence induction and epithelial-mesenchymal transition (EMT) events are the opposite sides of the spectrum of cancer phenotypes. The key molecules involved in these processes may get influenced or altered by genetic and epigenetic changes during tumor progression. Double C2-like domain beta (DOC2B), an intracellular vesicle trafficking protein of the double C2 protein family, plays a critical role in exocytosis, neurotransmitter release, and intracellular vesicle trafficking. DOC2B is repressed by DNA promoter hypermethylation and functions as a tumor growth regulator in cervical cancer. To date, the molecular mechanisms of DOC2B in cervical cancer progression and metastasis is elusive. Herein, the biological functions and molecular mechanisms regulated by DOC2B and its impact on senescence and EMT are described. DOC2B inhibition promotes proliferation, growth, and migration by relieving G0/G1-S arrest, actin remodeling, and anoikis resistance in Cal27 cells. It enhanced tumor growth and liver metastasis in nude mice with the concomitant increase in metastasis-associated CD55 and CD61 expression. Inhibition of EMT and promotion of senescence by DOC2B is a calcium-dependent process and accompanied by calcium-mediated interaction between DOC2B and CDH1. In addition, we have identified several EMT and senescence regulators as targets of DOC2B. We show that DOC2B may act as a metastatic suppressor by inhibiting EMT through induction of senescence via DOC2B-calcium-EMT-senescence axis.Graphical abstract

C8orf76 Promotes Gastric Tumorigenicity and Metastasis by Directly Inducing lncRNA DUSP5P1 and Associates with Patient Outcomes.

We identified for the first time that C8orf76 (chromosome 8 open reading frame 76) is preferentially amplified in gastric cancer. We elucidated its role and clinical significance in gastric carcinogenesis. The clinical impact of C8orf76 was assessed in 592 patients with gastric cancer. The biological function of C8orf76 was studied in vitro, in vivo, and in gastric cancer patient-derived organoid models. C8orf76 downstream effector and pathways were identified by RNA sequencing, chromatin immunoprecipitation sequencing, luciferase reporter, and electrophoretic mobility shift assay. C8orf76 was upregulated in 69.74% and 65.71% of two independent cohorts of gastric cancers and was positively associated with C8orf76 amplification. Multivariate analysis showed that gastric cancer patients with C8orf76 amplification (cohort I, n = 129; cohort II, n = 107) or overexpression (n = 356) had a significantly shortened survival. C8orf76 significantly promoted gastric cancer cell proliferation, cell-cycle transformation, and migration/invasion, but suppressed cell apoptosis. Silencing C8orf76 expression exerted opposite effects in vitro and significantly inhibited xenograft tumor growth, lung metastasis, and liver metastasis in nude mice. Silencing C8orf76 also significantly suppressed the growth of patient-derived organoids. Mechanically, C8orf76 activated MAPK/ERK signaling cascade. C8orf76 directly bound to the promoter region of lncRNA dual specificity phosphatase 5 pseudogene 1 (DUSP5P1) with a binding motif of AGGCTG and activated DUSP5P1 transcription. DUSP5P1 induced MAPK/ERK signaling and promoted gastric tumorigenesis. Knockdown DUSP5P1 abrogated the effect of C8orf76 in activating MAPK/ERK cascade and the tumor-promoting function. C8orf76 directly binds to oncogenic lncRNA DUSP5P1 to induce its expression and activates MAPK signaling. C8orf76 plays a pivotal oncogenic role in gastric carcinogenesis and is an independent prognostic factor for gastric cancer patients.

Proteinase-activated receptor 2 promotes tumor cell proliferation and metastasis by inducing epithelial-mesenchymal transition and predicts poor prognosis in hepatocellular carcinoma.

AIMTo clarify the role of proteinase-activated receptor 2 (PAR2) in hepatocellular carcinoma, especially in the process of metastasis.METHODSPAR2 expression levels were assessed by qRT-PCR and immunohistochemistry (IHC) in patient tissues and in hepatocellular carcinoma cell lines SMMC-7721 and HepG2. Cell proliferation and metastasis were assessed both in vitro and in vitro. Immunoblotting was carried out to monitor the levels of mitogen-activated protein kinase (MAPK) and epithelial-mesenchymal transition markers.RESULTSThe prognosis was significantly poorer in patients with high PAR2 levels than in those with low PAR2 levels. Patients with high PAR2 levels had advanced tumor stage (P = 0.001, chi-square test), larger tumor size (P = 0.032, chi-square test), and high microvascular invasion rate (P = 0.037, chi-square test). The proliferation and metastasis ability of SMMC-7721 and HepG2 cells was increased after PAR2 overexpression, while knockdown of PAR2 decreased the proliferation and metastasis ability of SMMC-7721 and HepG2 cells. Knockdown of PAR2 also inhibited hepatocellular carcinoma tumor cell growth and liver metastasis in nude mice. Mechanistically, PAR2 increased the proliferation ability of SMMC-7721 and HepG2 cells via ERK activation. Activated ERK further promoted the epithelial-mesenchymal transition of these cells, which endowed them with enhanced migration and invasion ability.CONCLUSIONThese data suggest that PAR2 plays an important role in the proliferation and metastasis of hepatocellular carcinoma. Therefore, targeting PAR2 may present a favorable target for treatment of this malignancy.

Role of miR-647 in human gastric cancer suppression.

MicroRNAs (miRNAs) regulate various oncogenes concomitantly, resulting in tumor suppression. They regulate proliferation and migration pathways in tumor development, suggesting a potential therapeutic role. In the present study, we found that miR-647 was markedly downregulated in gastric cancer (GC), and was significantly correlated with reduced tumor size and metastasis. In addition, miR-647 was also reduced in GC cell lines. Furthermore, overexpression of miR-647 in the GC cell lines inhibited cell proliferation, promoted cell cycle arrest at the G0/G1 phase and induced cell apoptosis. miR-647 also significantly inhibited tumor growth in vivo. Notably, we found that miR-647 overexpression suppressed the migration and invasion of the cancer cells, particularly liver metastasis in nude mice. miR-647 also reduced the expression levels of genes associated with proliferation and metastasis in tumors, including ANK2, FAK, MMP2, MMP12, CD44 and SNAIL1. Overall, our findings demonstrated that miR-647 exerts powerful antitumorigenic effects in vitro and in vivo, and may represent a promising therapeutic agent against GC.

RAGE-aptamer Attenuates the Growth and Liver Metastasis of Malignant Melanoma in Nude Mice

Epidemiological studies have suggested a link between cumulative diabetic exposure and cancer. The interaction of advanced glycation end products (AGEs) with their receptor (RAGE) may contribute to the phenomenon. We examined the effects of DNA aptamer raised against RAGE (RAGE-aptamer) on growth and liver metastasis of G361 melanoma in nude mice. Malignant melanoma cells were intradermally injected into the upper flank region of nude mice, which received continuous administration of RAGE-aptamer (38.4 pmol/day/g body weight) or vehicle intraperitoneally by an osmotic pump up to 42 d. RAGE-aptamer significantly reduced levels of 8-hydroxy-2′-deoxy-guanosine, AGEs, RAGE, proliferating nuclear antigen, cyclin D1, vascular endothelial growth factor (VEGF), monocyte chemoattractant protein-1 (MCP-1), and CD31 and Mac-3, respective markers of endothelial cells and macrophages in tumors of nude mice, and suppressed proliferation and liver metastasis of malignant melanoma. Furthermore, RAGE-aptamer attenuated AGE-induced oxidative stress generation, proliferation, and VEGF and MCP-1 gene expression in both G361 melanoma cells and endothelial cells. The present findings suggest that RAGE-aptamer could attenuate melanoma growth and liver metastasis in nude mice by suppressing tumor angiogenesis and macrophage infiltration via inhibition of the AGE-RAGE system. RAGE-aptamer may be a novel therapeutic tool for the treatment of malignant melanoma.

Abstract 4111: Imaging of colon cancer-cell mitosis within liver metastasis in nude mice

Abstract Colon cancer frequently results in metastasis to the liver, when it becomes the main cause of death. The tumor cell cycle in primary tumors and metastases are poorly understood. We developed a liver metastasis mouse model using a human colon cancer cell line (HCT-116), which expresses GFP in the nucleus and RFP in the cytoplasm (HCT-116-GFP-RFP). HCT-116 GFP-RFP (2.0 × 106) cells were injected in the spleen of a BALB/c nude mouse. The cells subsequently formed tumor colonies in the liver and retroperitoneum 28 days after cell transplantation. Using an Olympus FV1000 confocal microscope, mitosis of the dual-colored colon cancer cells could be clearly imaged in the liver and other metastases. Multi-nucleated cancer cells, in addition to a mononuclear cancer cells and their mitosis, were observed in the primary tumor and metastasis. Multi-nuclear cells were also observed in cell culture of HCT-116 GFP-RFP cells. Multi-nuclear HCT-116 cells may participate in the tumor growth in vivo. A significant difference in mitotic cells count in the primary tumor and metastasis was not observed. Citation Format: Kosuke Hasegawa, Atsushi Suetsugu, Miki Nakamura, Takuro Matsumoto, Hitomi Aoki, Takahiro Kunisada, Masahito Shimizu, Robert M. Hoffman. Imaging of colon cancer-cell mitosis within liver metastasis in nude mice. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4111.

Abstract 4218: Diffusion-weighted MRI can predict response to aflibercept in in vivo models

Abstract BACKGROUND: The liver is the most frequent metastatic site in colorectal cancer (CRC), and relevant in vivo models are needed to study the efficacy of anticancer drugs in this setting. Aflibercept, targeting vascular endothelial growth factors A and B and placental growth factor, is one of the most recently approved antiangiogenic agents for treatment of metastatic CRC. In the present work, the efficacy of aflibercept was investigated in experimental models of liver metastases in nude mice and growth progression was monitored using non-invasive imaging. METHODS: Liver metastases were established in mice by intrasplenic injection of CRC cell lines HCT116 and HT29 transfected with luciferase. Magnetic resonance imaging (MRI) (7T) and bioluminescent imaging (IVIS spectrum) were used to monitor tumor growth. Aflibercept was delivered intraperitoneally. To further characterize treatment response in metastatic tumors, diffusion-weighted (DW)-MR images were obtained. At termination tumors were sampled for histopathologic analysis. RESULTS: Mice bearing HCT116 xenografts responded well to treatment and a significant increase in survival compared to vehicle treated animals was observed (p< 0.001), in addition to decreased tumor burden. No increase in survival was observed upon aflibercept treatment in HT29 xenografts (p = 0.155), whereas in one of two experiments performed a significant reduction in tumor volume was observed, suggesting that there was a slight response to treatment. T2-weighted MRI was used to quantify tumor volume by manual delineation using the OsiriX software. MRI-assessed tumor volumes correlated well with tumor weight at the time of termination, while for bioluminescence measurements no such correlation was observed. In contrast, bioluminescence was a sensitive detection method early in the experiments when MRI did not show liver tumor. DW-MRI was obtained and apparent diffusion coefficient (ADC) tumor maps were calculated using the nordicIce software. A slight increase in ADC values was observed for HT29 tumors while there was a large increase in ADC values for HCT116 tumors after treatment with aflibercept. This corresponded well with histologic evaluation of tumors, showing increased necrosis in HCT116 tumors, but not in HT29 tumors. CONCLUSION: Our results demonstrate efficacy of aflibercept in an orthotopic model of liver metastases in CRC. MRI could be used to monitor treatment efficacy with high precision, while bioluminescence measurement could detect small-volume disease with high sensitivity, but was less specific in high-volume disease. Interestingly, ADC values obtained from DW-MRI were highly predictive of treatment response by clearly visualizing and quantifying tumor necrosis. Citation Format: Karianne G. Fleten, Kine M. Bakke, Andreas Abildgaard, Gunhild M. Mælandsmo, Katrine Røe Redalen, Kjersti Flatmark. Diffusion-weighted MRI can predict response to aflibercept in in vivo models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4218.

Mesenchymal stem cells promote pancreatic adenocarcinoma cells invasion by transforming growth factor-β1 induced epithelial-mesenchymal transition.

Mesenchymal stem cells (MSCs) could be ideal delivery vehicles for antitumor biological agents in pancreatic adenocarcinoma (PA). While the role of MSCs in tumor growth is elusive. Inflammation is an important feature of PA. In this study, we reported that MSCs pre-stimulated with the combination of TNF-α and IFN-γ promote PA cells invasion. The invasion of PA cell lines were evaluate by wound healing assay and transwell assay in vitro and liver metastasis in nude mice. We observed MSCs pre-stimulated with the combination of TNF-α and IFN-γ promoted PA cells invasion in vitro and in vivo. Consistent with MSCs promoting PA cells invasion, PA cells were found undergo epithelial-mesenchymal transition (EMT). We demonstrated that MSCs pre-stimulated with both of TNF-α and IFN-γ provoked expression transforming growth factor-β1 (TGF-β1). MSCs promoting EMT-mediated PA cells invasion could be reversed by short interfering RNA of TGF-β1. Our results suggest that MSCs could promote PA cells invasion in inflammation microenvironment and should be cautious as delivery vehicles in molecular target therapy.

Adjuvant treatment with tumor-targeting Salmonella typhimurium A1-R reduces recurrence and increases survival after liver metastasis resection in an orthotopic nude mouse model.

Colon cancer liver metastasis is often the lethal aspect of this disease. Well-isolated metastases are candidates for surgical resection, but recurrence is common. Better adjuvant treatment is therefore needed to reduce or prevent recurrence. In the present study, HT-29 human colon cancer cells expressing red fluorescent protein (RFP) were used to establish liver metastases in nude mice. Mice with a single liver metastasis were randomized into bright-light surgery (BLS) or the combination of BLS and adjuvant treatment with tumor-targeting S. typhimurium A1-R. Residual tumor fluorescence after BLS was clearly visualized at high magnification by fluorescence imaging. Adjuvant treatment with S. typhimurium A1-R was highly effective to increase survival and disease-free survival after BLS of liver metastasis. The results suggest the future clinical potential of adjuvant S. typhimurium A1-R treatment after liver metastasis resection.

Expression of the miR-302/367 cluster in glioblastoma cells suppresses tumorigenic gene expression patterns and abolishes transformation related phenotypes.

Cellular transformation is initiated by the activation of oncogenes and a closely associated developmental reprogramming of the epigenetic landscape. Transcription factors, regulators of chromatin states and microRNAs influence cell fates in development and stabilize the phenotypes of normal, differentiated cells and of cancer cells. The miR‐302/367 cluster, predominantly expressed in human embryonic stem cells (hESs), can promote the cellular reprogramming of human and mouse cells and contribute to the generation of iPSC. We have used the epigenetic reprogramming potential of the miR‐302/367 cluster to “de‐program” tumor cells, that is, hift their gene expression pattern towards an alternative program associated with more benign cellular phenotypes. Induction of the miR‐302/367 cluster in extensively mutated U87MG glioblastoma cells drastically suppressed the expression of transformation related proteins, for example, the reprogramming factors OCT3/4, SOX2, KLF4 and c‐MYC, and the transcription factors POU3F2, SALL2 and OLIG2, required for the maintenance of glioblastoma stem‐like tumor propagating cells. It also diminished PI3K/AKT and STAT3 signaling, impeded colony formation in soft agar and cell migration and suppressed pro‐inflammatory cytokine secretion. At the same time, the miR‐302/367 cluster restored the expression of neuronal markers of differentiation. Most notably, miR‐302/367 cluster expressing cells lose their ability to form tumors and to establish liver metastasis in nude mice. The induction of the miR‐302/367 cluster in U87MG glioblastoma cells suppresses the expression of multiple transformation related genes, abolishes the tumor and metastasis formation potential of these cells and can potentially become a new approach for cancer therapy.

Targeting delivery of lipocalin 2-engineered mesenchymal stem cells to colon cancer in order to inhibit liver metastasis in nude mice.

One of the major obstacles in cancer therapy is the lack of anticancer agent specificity to tumor tissues. The strategy of cell-based therapy is a promising therapeutic option for cancer treatment. The specific tumor-oriented migration of mesenchymal stem cells (MSCs) makes them a useful vehicle to deliver anticancer agents. In this study, we genetically manipulated bone marrow-derived mesenchymal stem cells with their lipocalin 2 (Lcn2) in order to inhibit liver metastasis of colon cancer in nude mice. Lcn2 was successfully overexpressed in transfected MSCs. The PCR results of SRY gene confirmed the presence of MSCs in cancer liver tissue. This study showed that Lcn2-engineered MSCs (MSC-Lcn2) not only inhibited liver metastasis of colon cancer but also downregulated the expression of vascular endothelial growth factor (VEGF) in the liver. Overall, MSCs by innate tropism toward cancer cells can deliver the therapeutic agent, Lcn2, and inhibit cancer metastasis. Hence, it could be a new modality for efficient targeted delivery of anticancer agent to liver metastasis.

Purification of a Pd20-TNFα fusion protein that prevents liver metastasis of gastric cancer.

The specific binding peptide pd20 of gastric cancer cells with a high potential for liver metastasis was fused with human tumour necrosis factor (TNF) α, and a prokaryotic expression vector was established to express the pd20-TNFα fusion protein. After purification and identification, the preventive effects of the fusion protein on liver metastasis of gastric cancer were observed in mice. The whole gene synthesis method was used for pd20-TNFα fusion gene preparation, and a pd20-TNFα prokaryotic expression vector was constructed. The vector was induced and expressed in Escherichia coli BL21. The expression products were analysed and verified by SDS-PAGE electrophoresis and Western blot analysis. The Ni-NTA column method was used to purify the fusion protein, and the L929 cytotoxicity method was used to detect biological activity. Flow cytometry apoptosis experiments and invasion assays were performed to observe the effects of the fusion protein on apoptosis and metastasis of gastric cancer cells with high potential for liver metastasis. Thirty nude mice with liver metastasis of gastric cancer were established and then randomly divided into three groups of ten mice each. The Pd20-TNFα recombinant protein (1.2 × 10(6) U/kg day) or standard TNFα (1.2 × 10(6) U/kg day) saline was administered via tail vein injection for 7 consecutive days. The pathological changes in various organs of nude mice were observed 4 weeks later. The size of the gastric cancer, the incidence of liver metastasis and the number of liver metastases were measured and calculated. We successfully constructed a Pd20-TNFα recombinant plasmid and prepared the fusion protein. Detection of the pd20-TNFα protein by immunofluorescence showed a very strong expression in liver tissue, suggesting a targeting of the fusion protein to the liver. The L929 cytotoxicity assays showed that the pd20-TNFα fusion purified protein had a significant lethal effect on L929 cells, with a killing activity of up to 7.6 × 10(6) IU/ml. The apoptosis experiments showed that as the concentration of the fusion protein increased, the early gastric cancer cell apoptosis also increased, with the early apoptosis rate increasing from 5.99 % to 9.04 %. Cell invasion experiments showed that the purified pd20-TNFα fusion protein significantly inhibited the in vitro invasion of XGC9811-L cells, with the penetrating cells being significantly decreased compared with the control group per unit time (P < 0.01). Vector experiments showed that the pd20-TNFα recombinant protein group had significantly reduced cancer lesions and liver metastasis in nude mice compared with the control group. We successfully purified a pd20-TNFα fusion protein and confirmed that it had significant biological activity promoting early gastric cancer cell apoptosis, thereby inhibiting gastric cancer cell invasion.

Effects of γ-synuclein on the tumorigenicity and metastasis of colon cancer SW1116 cells in vitro and in vivo

Recent evidence suggests the involvement of γ-synuclein in tumorigenesis and tumor progression. The present study was designed to further clarify the effects of γ-synuclein on the biological features of colon cancer cells invitro and invivo. We constructed the eukaryotic expression vector and siRNA vector and selected stable transfectants to respectively upregulate and downregulate γ-synuclein expression in SW1116 cells. we found that silencing of γ-synuclein significantly attenuated SW1116 cell growth and colony formation invitro (P<0.05), and overexpression of γ-synuclein moderately enhanced cell growth and colony formation, but not significantly when compared with the parental SW1116 cells and empty vector-transfected cells (P>0.05). Meanwhile, overexpression of γ-synuclein significantly facilitated SW1116 cell migration, invasion and adhesion to human liver sinusoidal endothelial cells (HLSECs) in vitro (P<0.05), and the effects were less attenuated by γ-synuclein knockdown (P>0.05). Furthermore, γ-synuclein promoted these malignant phenotypes in a γ-synuclein expression quantity-dependent manner not only invitro but also in the invivo expression. stable cells were injected subcutaneously into the right flank, and injected intrasplenically in nude mice. γ-synuclein knockdown suppressed the tumorigenicity of SW1116 cells in mice, which presented significantly smaller tumor masses on day6 over a 30-day period, compared with empty vector cells (P<0.05). Meanwhile, overexpression of γ-synuclein led to a profound augmentation of liver metastasis in nude mice, not only in macroscopic appearance but also in the size and weight of livers (P<0.05). These results provide strong evidence that suggests γ-synuclein plays a positive role in the progression of colorectal cancer.

Abstract 3316: The stem cell marker nestin inhibits growth and invasion of malignant melanoma

Abstract BACKGROUND: Nestin, a class VI intermediate filament protein, was first described as a neural stem cell/progenitor cell marker. Nestin was detected in various neoplasms, and its expression level has relevance to the poor prognosis in some tumors, including malignant melanomas. Recently, nestin was reported as one of the cancer stem cell markers in melanomas. Furthermore, nestin is expressed in circulating tumor cells of melanoma patients, and its expression was found to be correlated with the prognosis. These reports indicate that nestin plays important roles in melanomas, and has the possibility of becoming a therapeutic target in melanomas. In the present study, we used a silencing strategy of nestin to clarify the effectiveness of nestin-targeting therapy in malignant melanomas. METHODS: We constructed a plasmid vector of short hairpin RNA (shRNA) tageting nestin and scramble vector as a negative control. Human malignant melanoma cells, A375 cells, were stably transfected with these vectors. Using nestin-shRNA-transfected cells, scramble vector-transfected cells, and wild-type cells, we analyzed cell growth, sphere formation, and invasion in vitro. Activation of the AKT/MAPK pathway was analyzed using a phosphorylated protein array. To determine the effect of nestin-knockdown in vivo, we performed tail vein injection of transfected and wild-type A375 cells into nude mice, and analyzed the metastatic tumor formation in 5 weeks. RESULTS: Nestin shRNA transfection suppressed cell growth, invasion, and sphere formation of A375 cells in vitro. In the protein array, the suppression of nestin decreased the phosphorylation of AKT and p53. The incidence of liver metastasis in nude mice was 100% in wild-type cell-injected mice, 75% in scramble vector-transfected cell-injected mice, and 16.7% in nestin-shRNA-transfected cell-injected mice. CONCLUSION: These findings suggest that nestin regulates cell growth, stemness, invasion, and metastasis of melanomas, in part, by alterling the phosphorylation of the MAPK/AKT pathway. Nestin may be a candidate therapeutic target in malignant melanomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3316. doi:1538-7445.AM2012-3316

Ginsenoside Rg3 inhibits growth of liver metastases in nude mice after surgical removal of primary tumor

Ginsenoside Rg3 inhibits growth of liver metastases in nude mice after surgical removal of primary tumor