Benzo （a） pyrene induced tumorigenesity of human immortalized oral epithelial cells： transcription profiling

Background The present study was designed to examine and analyze the global gene expression changes during the tumorigenesis of a human immortalized oral epithelial cell line, and search for the possible genes that may play a role in the carcinogenesis of oral cancer associated with benzo （a） pyrene. Methods The human immortalized oral epithelial cells, which have been established through transfection of E6/EZgenes of human papillomavirus type 16 and proved to be non-tumorigenic in nude mice, were treated with benzo （a） pyrene. Tumorigenesity of the treated cells were examined through nude mice subcutaneous injection. The global gene expression profiles of immortalized cells and the tumorigenic cells were acquired through hybridization of a microarray of Affymetrix U133 plus 2.0. The data were analyzed using Spring 7.0 software and treated statistically using one-way analysis of variance （ANOVA）. The differentially expressed genes were classified using a Venn diagram and annotated with gene ontology. Several highlighted genes were validated in cells using a real-time polymerase chain reaction. Results There were 883 differentially expressed genes during the tumorigenesis and most of them changed expression in the early stage of tumorigenesis. These genes mainly involved in macromolecule metabolism and signal transduction, possessed the molecular function of transition metal ion binding, nucleotide binding and kinase activity; their protein products were mainly integral to membranes or localized in the nucleus and cytoskeleton. The expression patterns of IGFBP3, S100A8, MAP2K, KRT6B, GDF15, METwere validated in cells using a real-time polymerase chain reaction; the expression of IGFBP3 was further validated in clinical oral cancer specimens. Conclusions This study provides the global transcription profiling associated with the tumorigenesis of oral epithelial cells exposed to benzo （a） pyrene; IGFBP3 may play a potential role in the initiation of oral cancer related to benzo （a） pyrene exposure.

Establishment and characterization of a canine chondrosarcoma cell line:Mango

In the global progress of bone tumor research,established stable and long-lasting transgenic chondrosarcoma(CSA)cell lines are rare,mainly of murine and human origin,while the establishment of canine CSA cell lines has yet to be reported.This study established a canine CSA cell line to facilitate the basic clinical study of canine CSA.Fifty fve cases of canine osteolytic disease were collected,and more than 10 bone tumor samples from dogs with typical clinical signs were used for primary cell culture.A cell line with stable passaging for more than 100 generations and mouse tumorigenic ability was successfully cultured.According to the clinical characteristics of the dog and the histopathological results of the primary tumor,CSA was diagnosed,and the CSA cell line was designated Mango.Immunohistochemical(IHC)results showed that the immunoreactivity of bone gamma-carboxyglutamate protein(BGLAP),secreted protein acidic and rich in cysteine(SPARC),alkaline phosphatase(ALPL),vimentin(VIM)and S100 were positive.However,the immunoreactivity of pan-cytokeratin(PCK),chromogranin A(CGA),and platelet endothelial cell adhesion molecule-1(CD31)was negative.Immunofuorescence(IF)results showed that the protein expressions in the Mango cell line were consistent with the IHC identifcation of the primary tumor.The Mango cell line’s doubling time was 43.92 h,and the cell formation rate exceeded 20%.There were abnormal chromosome numbers,hetero staining with toluidine blue,and certain calcifcation abilities.It could be passaged stably and continuously without changing the cell morphology and characteristics.In vivo,the cells were successfully injected into the nude mice model with a tumorigenic rate of 100%.The immunophenotype of the xenograft tumor was consistent with that of the primary tumor.Therefore,we efectively established a canine CSA cell line.As a promising cell material,this cell line can be used to construct a tumor-bearing model conducive to the subsequent basic research of canine CSA.Moreover,because of its similarity to human CSA,the animal model of CSA is also indispensable for investigating human CSA.

Cone-rod homeobox transcriptionally activates TCF7 to promote the proliferation of retinal pigment epithelial and retinoblastoma cells in vitro

AIM:To investigate the proliferation regulatory effect of cone-rod homeobox(CRX)in retinal pigment epithelium(RPE)and retinoblastoma(RB)cells to explore the potential application and side effect(oncogenic potential)of CRXbased gene therapy in RPE-based retinopathies.METHODS:Adult human retinal pigment epithelial(ARPE)-19 and human retinal pigment epithelial(RPE)-1 cells and Y79 RB cell were used in the study.Genetic manipulation was performed by lentivirus-based technology.The cell proliferation was determined by a CellTiter-Glo Reagent.The mRNA and protein levels were determined by quantitative real-time polymerase chain reaction(qPCR)and Western blot assay.The transcriptional activity of the promoter was determined by luciferase reporter gene assay.The bindings between CRX and transcription factor 7(TCF7)promoter as well as TCF7 and the promoters of TCF7 target genes were examined by chromatin immunoprecipitation(ChIP)assay.The transcription of the TCF7 was determined by a modified nuclear run-on assay.RESULTS:CRX overexpression and knockdown significantly increased(n=3,P<0.05 in all the cells)and decreased(n=3,P<0.01 in all the cells)the proliferation of RPE and RB cells.CRX overexpression and knockdown significantly increased and deceased the mRNA levels of Wnt signaling target genes[including MYC proto-oncogene(MYC),JUN,FOS like 1(FOSL1),CCND1,cyclin D2(CCND2),cyclin D3(CCND3),cellular communication network factor 4(CCN4),peroxisome proliferator activated receptor delta(PPARD),and matrix metallopeptidase 7(MMP7)]and the luciferase activity driven by the Wnt signaling transcription factor(TCF7).TCF7 overexpression and knockdown significantly increased and decreased the proliferation of RPE and RB cells and depletion of TCF7 significantly abolished the stimulatory effect of CRX on the proliferation of RPE and RB cells.CRX overexpression and knockdown significantly increased and decreased the mRNA level of TCF7 and the promoter of TCF7 was significantly immunoprecipitated by CRX antibody.CONCLUSION:CRX transcriptionally activates TCF7 to promote the proliferation of RPE and RB cells in vitro.CRX is a potential target for RPE-based regenerative medicine.The potential risk of this strategy,tumorigenic potential,should be considered.

Regulation of inflammation and tumorigenesis by the FIPE family of phospholipid transfer proteins

The TIPE （tumor necrosis factor-a-induced protein 8-like） family are newly described regulators of immunity and tumorigenesis consisting of four highly homologous mammalian proteins： TNFAIP8 （tumor necrosis factor-a-induced protein 8）, TIPE1 （TNFAIP8-1ike 1, or TNFAIP8L1）, TIPE2 （TNFAIP8L2） and TIPE3 （TNFAIP8L3）. They are the only known transfer proteins of the lipid secondary messengers PIP2 （phosphatidylinositol 4,5-bisphosphate） and PIP3 （phosphatidylinositol 3,4,5-trisphosphate）. Cell-surface receptors, such as G-protein-coupled receptors and receptor tyrosine kinases, regulate inflammation and cancer via several signaling pathways, including the nuclear factor （NF）-KB and phosphoinositide-3 kinase （PI3K） pathways, the latter of which is upstream of both Akt and STAT3 activation. An expression analysis in humans demonstrated that the TIPE family is dysregulated in cancer and inflammation, and studies both in mice and in vitro have demonstrated that this family of proteins plays a critical role in tumorigenesis and inflammatory responses. In this review, we summarize the current literature for all four family members, with a special focus on the phenotypic manifestations present in the various knockout murine strains, as well as the related cell signalinR that has been elucidated to date.

Annexin A2 silencing inhibits invasion, migration, and tumorigenic potential of hepatoma cells

AIM: To investigate the effects of Annexin A2 (ANXA2) silencing on invasion, migration, and tumorigenic potential of hepatoma cells. METHODS: Human hepatoma cell lines [HepG2, SMMC-7721, SMMC-7402, and MHCC97-H, a novel human hepatocellular carcinoma (HCC) cell line with high metastasis potential] and a normal hepatocyte cell line(LO2) were used in this study. The protein and mRNA expression levels of ANXA2 were analysed by western blotting and real-time polymerase chain reaction, re-spectively. The intracellular distribution profile of ANXA2 expression was determined by immunofluorescence and immunohistochemistry. Short hairpin RNA target-ing ANXA2 was designed and stably transfected into MHCC97-H cells. Cells were cultured for in vitro analy-ses or subcutaneously injected as xenografts in mice for in vivo analyses. Effects of ANXA2 silencing on cell growth were assessed by cell counting kit-8 (CCK-8) as-say (in vitro ) and tumour-growth assay (in vivo ), on cell cycling was assessed by flow cytometry and propidium iodide staining (in vitro ), and on invasion and migration potential were assessed by transwell assay and wound-healing assay, respectively (both in vitro ). RESULTS: The MHCC97-H cells, which are known to have high metastasis potential, showed the highest lev-el of ANXA2 expression among the four HCC cell types examined; compared to the LO2 cells, the MHCC97-H expression level was 8-times higher. The ANXA2 expres-sion was effectively inhibited (about 80%) by ANXA2-specific small hairpin RNA (shRNA). ANXA2 expression in the MHCC97-H cells was mainly localized to the cel-lular membrane and cytoplasm, and some localization was detected in the nucleus. Moreover, the proliferation of MHCC97-H cells was obviously suppressed by shR-NA-mediated ANXA2 silencing in vitro , and the tumour growth inhibition rate was 38.24% in vivo . The per-centage of MHCC97-H cells in S phase dramatically de-creased (to 27.76%) under ANXA2-silenced conditions. Furthermore, ANXA2-silenced MHCC97-H cells showed lower invasiveness (percentage of invading cells de-creased to 52.16%) and suppressed migratory capacity (migration distance decreased to 63.49%). It is also worth noting that shRNA-mediated silencing of ANXA2 in the MHCC97-H cells led to abnormal apoptosis. CONCLUSION: shRNA-mediated silencing of ANXA2suppresses the invasion, migration, and tumorigenic potential of hepatoma cells, and may represent a useful target of future molecular therapies.

CD133^+ gallbladder carcinoma cells exhibit self-renewal ability and tumorigenicity

AIM： To identify cancer stern cells （CSCs） in human gallbladder carcinomas （GBCs）. METHODS： Primary GBC cells were cultured under serum-free conditions to produce floating spheres. The stem-cell properties of the sphere-forming cells, including self-renewal, differentiation potential, chemoresistance and tumorigenicity, were determined in vitro or in vivo. Cell surface expression of CD133 was investigated in primary tumors and in spheroid cells using flow cytometry. The sphere-colony-formation ability and tumorigenicity of CD133＋ cells were assayed.floating spheroids were generated from primary GBC cells, and these sphere-forming cells could generate new progeny spheroids in serum-free media. Spheroid cells were differentiated under serum-containing conditions with downregulation of the stem cell markers Oct-4, Nanog, and nestin （P 〈 0.05）. The differentiated cells showed lower spheroid-colony-formation ability than the original spheroid cells （P 〈 0.05）. Spheroid ceils were more resistant to chemotherapeutic reagents than the congenetic adherent cells （P 〈 0.05）. Flow cytometry showed enriched CD133＋ population in sphereforming cells （P 〈 0.05）. CD133＋ cells possessed high colony-formation ability than the CD133 population （P 〈 0.01）. CD133＋ cells injected into nude mice revealed higher tumorigenicity than their antigen-negative counterparts （P 〈 0.05）. CONCLUSION： CD133 may be a cell surface marker for CSCs in GBC.

Malignant Transformation and Abnormal Expression of Eukaryotic Initiation Factor in Bronchial Epithelial Cells Induced by Cadmium Chloride

Objective To analyze the relationship between malignant transformation and abnormal expression of eukaryotic initiation factor 3 （eIF3 p36） in human bronchial epithelial （16HBE） cells induced by cadmium chloride （CdCl2）. Methods 16HBE cells were treated several times with different concentrations of CdCl2. Tumorigenic potential of transformed cells was identified by assays for anchorage-independent growth in soft agar and for tumorigenicity in nude mice after the 35th passage. Total RNA was isolated from 16HBE cells induced by CdC12, including non-transformed, Cd-transformed, and Cd-tumorigenic cell lines. Special primers for eIF3 p36 were designed and the expression of eIF3 mRNA in different cell lines was detected with fluorescent quantitative-polymerase chain reaction technique （FQ-PCR）. Results The 35th passage of 16HBE cells transformed by CdCl2 exhibited overlapping growth. Compared with the non-transformed cells, colonies of transformed cell lines in soft agar showed statistically significant increases and dose-dependent effects （P〈0.01）. All Cd-induced transformed cell lines formed rumors in nude mice within 2 weeks of inoculation, but none of the mice injected with non-transformed cells showed tumors even after 3 weeks. All tumors were pathologically identified as poorly differentiated squamous cell carcinoma. The eIF3 p36 genes in different stages of 16HBE cells transformed by CdCl2 were elevated as compared with the non-transformed control （P〈0.01）, and the eIF3 expression increased with the degree of cell malignancy. Conclusion CdCl2 is capable of inducing morphological transformation in 16HBE cells and transformed cells are potentially tumorigenic. Over-expression of eIF3 p36 is positively correlated with malignant transformation of 16HBE cells induced by CdCl2 and may be one of the molecular mechanisms potentially responsible for carcinogenesis due to Cd.

Role of liver stem cells in hepatocarcinogenesis

Liver cancer is an aggressive disease with a high mortality rate. Management of liver cancer is strongly dependent on the tumor stage and underlying liver disease. Unfortunately, most cases are discovered when the cancer is already advanced, missing the opportunity for surgical resection. Thus, an improved understanding of the mechanisms responsible for liver cancer initiation and progression will facilitate the detection of more reliable tumor markers and the development of new small molecules for targeted therapy of liver cancer. Recently, there is increasing evidence for the "cancer stem cell hypothesis", which postulates that liver cancer originates from the malignant transformation of liver stem/progenitor cells(liver cancer stem cells). This cancer stem cell model has important significance for understanding the basic biology of liver cancer and has profound importance for the development of new strategies for cancer prevention and treatment. In this review, we highlight recent advances in the role of liver stem cells in hepatocarcinogenesis. Our review of the literature shows that identification of the cellular origin and the signaling pathways involved is challenging issues in liver cancer with pivotal implications in therapeutic perspectives. Although the dedifferentiation of mature hepatocytes/cholangiocytes in hepatocarcinogenesis cannot be excluded, neoplastic transformation of a stem cell subpopulation more easily explains hepatocarcinogenesis. Elimination of liver cancer stem cells in liver cancer could result in the degeneration of downstream cells, which makes them potential targets for liver cancer therapies. Therefore, liver stem cells could represent a new target for therapeutic approaches to liver cancer in the near future.

MUC1 and colorectal cancer pathophysiology considerations

Several lines of evidence point towards a biological role of mucin and particularly MUC1 in colorectal cancer. A positive correlation was described between mucin secretion, proliferation, invasiveness, metastasis and bad prognosis. But, the role of MUC1 in cancer progression is still controversial and somewhat confusing. While Mukherjee and colleagues developed MUC1-specific immune therapy in a CRC model, Lillehoj and coinvestigators showed recently that MUC1 inhibits cell proliferation by a β-catenin-dependent mechanism. In carcinoma cells the polarization of MUC1 is lost and the protein is over expressed at high levels over the entire cell surface. A competitive interaction between MUC1 and E-cadherin, through β-catenin binding, disrupts E-cadherin-mediated cell-cell interactions at sites of MUC1 expression. In addition, the complex of MUC1-β-catenin enters the nucleus and activates T-cell factor/leukocyte enhancing factor 1 transcription factors and activates gene expression. This mechanism may be similar to that just described for DCC and UNC5H, which induced apoptosis when not engaged with their ligand netrin, but mediate signals for proliferation, differentiation or migration when ligand bound.

Overexpression of p42.3 promotes cell growth and tumorigenicity in hepatocellular carcinoma

AIM:To investigate the association of p42.3 expression with clinicopathological characteristics and the biological function of p42.3 in human hepatocellular carcinoma(HCC).METHODS:We used reverse transcription-polymerase chain reaction(RT-PCR),quantitative real-time RT-PCR and western blotting to detect p42.3 mRNA and protein expression in hepatic cell lines.We examined primary HCC samples and matched adjacent normal tissue by immunohistochemistry to investigate the correlation between p42.3 expression and clinicopathological features.HepG2 cells were transfected with a pIRES2EGFP-p42.3 expression vector to examine the function of the p42.3 gene.Transfected cells were analyzed for their viability and malignant transformation abilities by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay,colony formation assay,and tumorigenicity assay in nude mice.RESULTS:p42.3 is differentially expressed in primary HCC tumors and cell lines.Approximately 69.6%(96/138) of cells were p42.3-positive in hepatic tumor tissues,while 30.7%(35/114) were p42.3-positive in tumor-adjacent normal tissues.Clinicopathological characteristics of the HCC specimens revealed a significant correlation between p42.3 expression and tumor differentiation(P = 0.031).However,p42.3 positivity was not related to tumor tumor-node-metastasis classification,hepatitis B virus status,or hepatoma type.Regarding p42.3 overexpression in stably transfected HepG2 cells,we discovered significant enhancement of cancer cell growth and colony formation in vitro,and significantly enhanced tumorigenicity in nude mice.Western blot analysis of cell cycle proteins revealed that enhanced p42.3 levels promote upregulation of proliferating cell nuclear antigen,cyclin B1 and mitotic arrest deficient 2.CONCLUSION:p42.3 promotes tumorigenicity and tumor growth in HCC and may be a potential target for future clinical cancer therapeutics.

What we know about ST13, a co-factor of heat shock protein, or a tumor suppressor?

This article is to summarize the molecular and functional analysis of the gene “suppression of tumorigenicity 13” (ST13). ST13 is in fact the gene encoding Hsp70 interacting protein (Hip), a co-factor (co-chaperone) of the 70-kDa heat shock proteins (Hsc/Hsp70). By collaborating with other positive co-factors such as Hsp40 and the Hsp70-Hsp90 organizing protein (Hop), or competing with negative co-factors such as Bcl2-associated athanogen 1 (Bag1), Hip facilitates may facilitate the chaperone function of Hsc/Hsp70 in protein folding and repair, and in controlling the activity of regulatory proteins such as steroid receptors and regulators of proliferation or apoptosis. Although the nomenclature of ST13 implies a role in the suppression of tumorigenicity (ST), to date available experimental data are not sufficient to support its role in cancer development, except for the possible down-regulation of ST13 in gastric and colorectal cancers. Further investigation of this gene at the physiological level would benefit our understanding of diseases such as endocrinological disorders, cancer, and neurodegeneration commonly associated with protein misfolding.

Serum soluble suppression of tumorigenicity 2 as a novel inflammatory marker predicts the severity of acute pancreatitis

BACKGROUND Acute pancreatitis(AP)is an inflammatory disease in which the regulatory pathway is complex and not well understood.Soluble suppression of tumorigenicity 2(sST2)protein receptor functions as a decoy receptor for interleukin(IL)-33 to prevent IL-33/suppression of tumorigenicity 2L(ST2L)-pathwaymediated T helper(Th)2 immune responses.AIM To investigate the role of sST2 in AP.METHODS We assessed the association between sST2 and severity of AP in 123 patients enrolled in this study.The serum levels of sST2,C-reactive protein(CRP)and Th1-and Th2-related cytokines,including interferon(IFN)-γ,tumor necrosis factor(TNF)-α,IL-2,IL-4,IL-5 and IL-13,were measured by highly sensitive ELISA,and the severity of AP in patients was evaluated by the 2012 Atlanta Classification Criteria.RESULTS Serum sST2 levels were significantly increased in AP patients,and further,these levels were significantly elevated in severe AP(SAP)patients compared to moderately severe AP(MSAP)and mild AP(MAP)patients.Logistic regression showed sST2 was a predictor of SAP[odds ratio(OR):1.003(1.001–1.006),P=0.000].sST2 cutoff point was 1190 pg/mL,and sST2 above this cutoff was associated with SAP.sST2 was also a predictor of any organ failure and mortality during AP[OR:1.006(1.003–1.009),P=0.000,OR:1.002(1.001–1.004),P=0.012,respectively].Additionally,the Th1-related cytokines IFN-γand TNF-αin the SAP group were higher and the Th2-related cytokine IL-4 in the SAP group was significantly lower than those in MSAP and MAP groups.CONCLUSION sST2 may be used as a novel inflammatory marker in predicting AP severity and may regulate the function and differentiation of IL-33/ST2-mediated Th1 and Th2 Lymphocytes in AP homeostasis.

A meta-analysis of soluble suppression of tumorigenicity 2 （sST2） and clinical outcomes in pulmonary hypertension

Suppression of Tumorigenicity 2 （ST2） is a member of the interleukin （IL）-1 receptor family The ST2 receptor exists in two isoforms - ST2 ligand （ST2L） and soluble ST2 （sST2）.ST2L is a membrane receptor and sST2 is a trun- cated receptor which is soluble in the blood, allowing it to be detected in serum. IL-33 is a member of the IL-1 family of ligand and is the fimctional ligand of ST2L receptor. It binds to the ST2L, thereby mediating its immune function.

Tumorigenic bacteria in colorectal cancer:mechanisms and treatments

Colorectal cancer(CRC)is the third most common and the second most fatal cancer.In recent years,more attention has been directed toward the role of gut microbiota in the initiation and development of CRC.Some bacterial species,such as Fusobacterium nucleatum,Escherichia coli,Bacteroides fragilis,Enterococcus faecalis,and Salmonella sp.have been associated with CRC,based upon sequencing studies in CRC patients and functional studies in cell culture and animal models.These bacteria can cause host DNA damage by genotoxic substances,including colibactin secreted by pks+Escherichia coli,B.fragilis toxin(BFT)produced by Bacteroides fragilis,and typhoid toxin(TT)from Salmonella.These bacteria can also indirectly promote CRC by influencing host-signaling pathways,such as E-cadherin/β-catenin,TLR4/MYD88/NF-κB,and SMO/RAS/p38 MAPK.Moreover,some of these bacteria can contribute to CRC progression by helping tumor cells to evade the immune response by suppressing immune cell function,creating a proinflammatory environment,or influencing the autophagy process.Treatments with the classical antibacterial drugs,metronidazole or erythromycin,the antibacterial active ingredients,M13@Ag(electrostatically assembled from inorganic silver nanoparticles and the protein capsid of bacteriophage M13),berberine,and zerumbone,were found to inhibit tumorigenic bacteria to different degrees.In this review,we described progress in elucidating the tumorigenic mechanisms of several CRC-associated bacteria,as well as progress in developing effective antibacterial therapies.Specific bacteria have been shown to be active in the oncogenesis and progression of CRC,and some antibacterial compounds have shown therapeutic potential in bacteria-induced CRC.These bacteria may be useful as biomarkers or therapeutic targets for CRC.

Input of microenvironmental regulation on colorectal cancer: Role of the CCN family

Colorectal cancer(CRC)is a major health problem causing significant morbidity and mortality.Previous results from various studies indicate that CRC tumorigenicity encompasses tumor microenvironment,emphasizing the complex interacting network between cancer cells and nearby host cells,which triggers diverse signaling pathways to promote the growth and spread ofcancer cells.The CCN family proteins share a uniform modular structure,mediating a variety of physiological functions,including proliferation,apoptosis,migration,adhesion,differentiation,and survival.Furthermore,CCN proteins are also involved in CRC initiation and development.Many studies have shown that CCN members,such as CCN1,CCN2,CCN3,Wnt-induced secreted protein(WISP)-1,WISP-2,and WISP-3,are dysregulated in CRC,which implies potential diagnostic markers or therapeutic targets clinically.In this review,we summarize the research findings on the role of CCN family proteins in CRC initiation,development,and progression,highlighting their potential for diagnosis,prognosis,and therapeutic application.

Differentiation and tumorigenicity of neural stem cells from human cord blood mesenchymal stem cells

BACKGROUND： Mesenchymal stem cells （MSCs） are capable of differentiating into a variety of tissues and exhibit low immunogenicity. OBJECTIVE： To investigate isolation and in vitro cultivation methods of human cord blood MSCs, to observe expression of neural stem cell （NSC） marker mRNA under induction, and to detect tumorigenicity in animals. DESIGN, TIME AND SETTING： A cell biological, in vitro trial and a randomized, controlled, in vivo experiment were performed at the Department of Neurology, Daping Hospital at the Third Military Medical University of Chinese PLA from August 2006 to May 2008. MATERIALS： Umbilical cord blood was collected from full-term-delivery fetus at the Department of Gynecology and Obstetrics of Daping Hospital, China. Eighteen BALB/C nu/nu nude mice were randomly assigned to three groups： back subcutaneous, cervical subcutaneous, and control, with 6 mice in each group. METHODS： Monocytes were isolated from heparinized human cord blood samples by density gradient centrifugation and then adherent cultivated in vitro to obtain MSC clones. After the cord blood MSCs were cultured for 7 days with nerve growth factor and retinoic acid to induce differentiation into NSCs, the cells （adjusted density of 1 × 10^7/mL） were prepared into cell suspension. In the back subcutaneous and cervical subcutaneous groups, nude mice were hypodermically injected with a 0.5-mL cell suspension into the back and cervical regions, respectively. In the control group, nude mice received a subcutaneous injection of 0.5 mL physiological saline into the back or cervical regions, respectively. MAIN OUTCOME MEASURES： Cellular morphology was observed by inverted microscopy, cultured cord blood MSCs were examined by flow cytometry, expression of nestin and musashi-1 mRNA was detected by reverse-transcriptase polymerase chain reaction prior to and after induction, and tumorigenicity following cord blood MSC transplantation was assayed by hematoxylin-eosin staining. RESULTS： Following adherent cultivation, the majority of cord blood monocytes became rhombic and strongly expressed CD29, but not CD34, CD1 la, or CD11 b. These results supported previously known characteristics of cord blood MSCs. Following differentiation induction, nestin and musashi-1 were expressed on the surface of NSCs, exhibiting strongest expression at 48 hours, and subsequently reducing expression. Cultured cord blood MSCs were not tumorigenic in the nude mice. Cellular morphology displayed no malignant changes between the control and subcutaneous groups. CONCLUSION： MSCs can be isolated from human cord blood, efficiently expanded under culture conditions, differentiated into NSCs following induction, and display no tumorigenicity in nude mice.

Genotoxic and tumorigenic pyrrolizidine alkaloids in Chinese herbal plants

Pyrrolizidine alkaloids are a class of hepatotoxic and tumorigenic compounds detected in Chinese herbal plants, contaminated foods, and dietary supplements. In this review, the sources, toxicity, genotoxicity, tumorigenicity, and the metabolic pathways, particular the activation pathways leading to hepatotoxicity and tumorigenicity, of pyrrolizidine alkaloids are briefly discussed, with a focus on the most recent important findings concerning the genotoxic mechanism by which riddelliine liver tumors. This mechanism involves the formation of 6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP)-derived DNA adducts and may be general to most carcinogenic pyrrolizidine alkaloids.

c-Met in pancreatic cancer stem cells: Therapeutic implications

Pancreatic cancer is the deadliest solid cancer and currently the fourth most frequent cause of cancer-related deaths. Emerging evidence suggests that cancer stem cells (CSCs) play a crucial role in the development and progression of this disease. The identification of CSC markers could lead to the development of new therapeutic targets. In this study, the authors explore the functional role of c-Met in pancreatic CSCs, by analyzing self-renewal with sphere assays and tumorigenicity capacity in NOD SCID mice. They concluded that c-Met is a novel marker for identifying pancreatic CSCs and c-Met high in a higher tumorigenic cancer cell population. Inhibition of c-Met with XL184 blocks self-renewal capacity in pancreatic CSCs. In pancreatic tumors established in NOD SCID mice, c-Met inhibition slowed tumor growth and reduced the population of CSCs, along with preventing the development of metastases.

Antisense Oligonucleotide Targeting TGF-β1 Abrogates Tumorigenicity of Rhabdomyosarcoma in vivo

OBJECTIVE Over-expression of transforming growth factor β1 （TGF-β1） has been observed in many advanced cancers. The present study was aimed at developing potential antisense oligonucleotides （ASONs） to repress TGF-β1 expression in rhabdomyosarcoma （RMS） RD cells, and to examine their effect on tumorigenicity of RD cells in vivo. METHODS ASONs targeting the region surrounding the start codon of TGF-β1 were synthesized and transferred into cells in the form of complexes with Lipofectamine 2000. The TGF-β1 protein was determined by immunofluorescence and ELISA. The cell viability and cell cycle were examined by MTT and flow cytometry. The RD cells, with or without TGF-β1ASON, in 50 μl of serum-free EMDM medium were injected subcutaneously into the right flank of nude mice. The tumors were then measured and weighed. RESULTS The ASON sequence targeting the first start site at bases 841-855 of the human TGF-β1 gene had the greatest effect on attenuating the expression of TGF-β1 （P 〈 0.05）. The ASONs induced a decrease in OD values after 6 d （P 〈 0.05）. Analysis of the cell cycle revealed that the ASON induced a significant decrease in cells in the S phase and an increase in cells in the G1 phase （P 〈 0,05）. In the nude mice model, the mean tumor volume, after 2 weeks of treatment with Lipofectamine or ASON, decreased to 88.5% or 55% respectively, compared to the control tumor size, resulting in a significant difference （P 〈 0.01）. CONCLUSION The sequence of the ASON, which targeted the start condon at the bases 841-855 of the human TGF-β1 gene, was demonstrated to be a useful agent for studying the regulation of TGF-β1 over-expression in RD cells, and has important therapeutic potential for suppressing the tumorigenicity of human RMS in vivo.

REDUCED TUMORIGENICITY OF METASTATIC HUMAN LUNG CANCER CELL SUBLINE（PGCL3） TRANSFECTED WITH hRARβ GENE

The recombinant PSG5-RARβ plasmid and the G418-resistant PSV2neo Plasmid ( 10 : 1) were cotransfected into PGCL3 cells by copreciprtation with calcium phosphate. The transfectants CR3 and CR4, which expressed the RARβ gene. were identified by Northern blot hybridization. The results showed that the in vitro growth and invasion of CR3 and CR4 were dramatically reduced compared to the control-transfected cell (CSV1). Furthermore, the colony-forming abilities in soft agar and the tumorigenicrty in nude mice of CR3 and CR4 were abrogated. Our results suggests that RARβ functions not only as a receptor mediating the RA action, but also as a suppressor in lung tumorigenesis.