Safety profile and long-term engraftment of human CD31+ blood progenitors in bone tissue engineering.

BACKGROUND: Endothelial progenitor cells (EPCs) participate in angiogenesis and induce favorable micro-environments for tissue regeneration. The efficacy of EPCs in regenerative medicine is extensively studied; however, their safety profile remains unknown. Therefore, our aims were to evaluate the safety profile of human peripheral blood-derived EPCs (hEPCs) and to assess the long-term efficacy of hEPCs in bone tissue engineering. METHODS: hEPCs were isolated from peripheral blood, cultured and characterized. ß tricalcium phosphate scaffold (ßTCP, control) or 106 hEPCs loaded onto ßTCP were transplanted in a nude rat calvaria model. New bone formation and blood vessel density were analyzed using histomorphometry and micro-computed tomography (CT). Safety of hEPCs using karyotype analysis, tumorigenecity and biodistribution to target organs was evaluated. RESULTS: On the cellular level, hEPCs retained their karyotype during cell expansion (seven passages). Five months following local hEPC transplantation, on the tissue and organ level, no inflammatory reaction or dysplastic change was evident at the transplanted site or in distant organs. Direct engraftment was evident as CD31 human antigens were detected lining vessel walls in the transplanted site. In distant organs human antigens were absent, negating biodistribution. Bone area fraction and bone height were doubled by hEPC transplantation without affecting mineral density and bone architecture. Additionally, local transplantation of hEPCs increased blood vessel density by nine-fold. CONCLUSIONS: Local transplantation of hEPCs showed a positive safety profile. Furthermore, enhanced angiogenesis and osteogenesis without mineral density change was found. These results bring us one step closer to first-in-human trials using hEPCs for bone regeneration.

Downregulation of PLK1 by RNAi attenuates the tumorigenicity of esophageal squamous cell carcinoma cells via promoting apoptosis and inhibiting angiogenesis.

Polo-like kinase 1(PLK1) is essential for the maintenance of genomic stability during mitosis. PLK1 has been reported to be upregulated in several solid tumors, including esophageal squamous cell carcinoma (ESCC). However, the role of PLK1 in tumorigenesis of ESCC remains undetermined. We used siRNA and lentivirus-mediated PLK1 RNA interference to investigate the tumor suppressor function of PLK1 reduction in ESCC cells. Flow cytometry and Terminal deoxynuleotidyl transferase-mediated nick-end labeling assay in vitro, as well as immunohistochemitry analysis of Caspase-3 and CD31 in s.c. tumor tissue section, were performed. Knock down of PLK1 expression significantly suppressed the ability of ESCC cells to form colonies in plastic and soft agar. PLK1 reduction mediated by lentivirus caused growth suppression of ESCC in nude mice. Caspase-3 upregulation further indicated that dysregulated apoptosis might contribute to reduced tumorigenecity. In particular, downregulation of CD31 suggested that PLK1 reduction-induced angiogenesis inhibition may also contribute, at least in part, to attenuated tumorigenecity. These findings indicate that PLK1 might play roles in tumorigenesis of ESCC and that PLK1 might be a potential gene therapy target in ESCC. Apoptosis induction together with decreased angiogenesis might be involved in the mechanism of tumor suppressor function of RNA interference targeting PLK1.

Downregulation of MED23 promoted the tumorigenecity of esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is one of the most common tumors in the world. It is very urgent to develop new therapeutic strategies. MED23, a component of the mediator complex, is known as a hub to integrate various signaling pathways. However, the function of MED23 in ESCC remains unknown. Here, we found that the expression of MED23 was downregulated in the clinical ESCC samples and the expression of MED23 reversely correlated with tumor size and clinical stage. Moreover, overexpression of MED23 in ESCC cells inhibited cell growth dramatically, while downregulation of MED23 promoted the tumorigenecity of ESCC cells in vitro and in vivo. Mechanistically, knockdown the expression of MED23 inhibited cell apoptosis by downregulation of Bax, activated Caspase 3, activated Caspase 9 and upregulation of cyclinD1 and Bcl2. Taken together, our study revealed the suppressive role of MED23 in ESCC and MED23 might be an important therapeutic target in ESCC.

Lewis Y promotes growth and adhesion of ovarian carcinoma-derived RMG-I cells by upregulating growth factors.

Lewis y (LeY) antigen is a difucosylated oligosaccharide carried by glycoconjugates on the cell surface. Overexpression of LeY is frequently observed in epithelial-derived cancers and has been correlated to the pathological staging and prognosis. However, the effects of LeY on ovarian cancer are not yet clear. Previously, we transfected the ovarian cancer cell line RMG-I with the α1,2-fucosyltransferase gene to obtain stable transfectants, RMG-I-H, that highly express LeY. In the present study, we examined the proliferation, tumorigenesis, adhesion and invasion of the cell lines with treatment of LeY monoclonal antibody (mAb). Additionally, we examined the expression of TGF-ß1, VEGF and b-FGF in xenograft tumors. The results showed that the proliferation and adhesion in vitro were significantly inhibited by treatment of RMG-I-H cells with LeY mAb. When subcutaneously inoculated in nude mice, RMG-I-H cells produced large tumors, while mock-transfected cells RMG-I-C and the parental cells RMG-I produced small tumors. Moreover, the tumor formation by RMG-I-H cells was inhibited by preincubating the cells with LeY mAb. Notably, the expression of TGF-ß1, VEGF and b-FGF all increased in RMG-I-H cells. In conclusion, LeY plays an important role in promoting cell proliferation, tumorigenecity and adhesion, and these effects may be related to increased levels of growth factors. The LeY antibody shows potential application in the treatment of LeY-positive tumors.

Gankyrin drives metabolic reprogramming to promote tumorigenesis, metastasis and drug resistance through activating ß-catenin/c-Myc signaling in human hepatocellular carcinoma.

Gankyrin plays important roles in tumorigenicity and metastasis of hepatocellular carcinoma (HCC). We have for the first time investigated the effects of Gankyrin on glycolysis and glutaminolysis both in vitro and in vivo, including in patient-derived xenografts. We reported Gankyrin increases glucose consumption, lactate production, glutamine consumption and glutamate production in HCC through upregulating the expression of the transporters and enzymes involved in glycolysis and glutaminolysis, including HK2, GLUT1, LDHA, PKM2, ASCT2 and GLS1. We further demonstrated that Gankyrin drives glycolysis and glutaminolysis through upregulating c-Myc via activating ß-catenin signaling. Importantly, we found c-Myc mediated metabolic reprogramming might contribute to the tumorigenicity, metastasis and drug resistance induced by Gankyrin. c-Myc inhibitor synergizes with Sorafenib or Regorafenib to suppress HCC PDX tumors with high Gankyrin levels. We detected a significant correlation between Gankyrin and ß-catenin expression levels in a cohort of HCC biopsies, and combination of these two parameters is a more powerful predictor of poor prognosis. Collectively, our results uncovered that Gankyrin functions as an essential regulator in glycolysis and glutaminolysis via activation of ß-catenin/c-Myc to promotes tumorigenesis, metastasis and drug resistance in human HCC.

TLE1 inhibits anoikis and promotes tumorigenicity in human lung cancer cells through ZEB1-mediated E-cadherin repression.

The Transducin-like enhancer of split 1 (TLE1) corepressor protein is overexpressed in human lung tumors and is a putative lung-specific oncogene. However, the molecular mechanism underlying its oncogenic function remains to be delineated. Here, we report an important role of TLE1 in promoting lung tumorigenesis by a mechanism involving induction of anoikis resistance. Using the human lung adenocarcinoma A549 and immortalized bronchial epithelial BEAS-2B cell lines, we observed that TLE1 inhibits anoikis through transcriptional repression of E-cadherin gene. In support of E-cadherin as a downstream target of TLE1 to block anoikis, forced expression of E-cadherin attenuated TLE1-induced anoikis resistance while E-cadherin downregulation decreased the anoikis sensitivity of TLE1 knockdown cells. Furthermore, we determined that E-cadherin expression is transcriptionally induced upon loss of cell attachment and functions as an effector of anoikis. Loss of E-cadherin via the siRNA strategy or exogenous TLE1 expression was sufficient to attenuate anoikis in A549 and BEAS-2B cells. Importantly, we demonstrated that the ZEB1 transcriptional factor is required for TLE1-mediated E-cadherin repression and anoikis resistance. ZEB1 interacted with and recruited the TLE1 to the E-cadherin promoter to impose histone deacetylation and gene silencing. In vivo, TLE1 strongly promoted tumorigenicity of A549 cells in a ZEB1-dependent manner. Underscoring its role in anoikis insensitivity of lung cancer cells, the TLE1-mediated E-cadherin repression was negatively regulated by the tumor suppressor Bcl-2 inhibitor of transcription 1 (Bit1) to effect anoikis. These findings identify the ZEB1/TLE1/E-cadherin transcriptional mechanism as a novel pathway that promotes anoikis resistance and oncogenicity of lung cancer cells.

Aberrant overexpression of ADAR1 promotes gastric cancer progression by activating mTOR/p70S6K signaling.

ADAR1, one of adenosine deaminases acting on RNA, modulates RNA transcripts through converting adenosine (A) to inosine (I) by deamination. Emerging evidence has implicated that ADAR1 plays an important role in a few of human cancers, however, its expression and physiological significance in gastric cancer remain undefined. In the present study, we demonstrated that ADAR1 was frequently overexpressed in gastric cancer samples by quantitative real-time PCR analysis. In a gastric cancer tissue microarray, ADAR1 staining was closely correlated with tumor stage (P < 0.001) and N classification (P < 0.001). Functional analysis indicated that ADAR1 overexpression promoted cell proliferation and migration in vitro, whereas ADAR1 knockdown resulted in an opposite phenotypes. Furthermore, ADAR1 knockdown also inhibited tumorigenicity and lung metastasis potential of gastric cancer cells in nude mice models. Mechanistically, ADAR1 expression had a significant effect on phosphorylation level of mTOR, p70S kinase, and S6 ribosomal protein, implying its involvement in the regulation of mTOR signaling pathway. We conclude that ADAR1 contributes to gastric cancer development and progression via activating mTOR/p70S6K/S6 ribosomal protein signaling axis. Our findings suggest that ADAR1 may be a valuable biomarker for GC diagnosis and prognosis and may represent a new novel therapeutic opportunities.

p75NTR in isolation and identification of cancer stem cells from human esophageal carcinoma / 第二军医大学学报

Objective: To isolate and identify cancer stem cells from esophageal cancer cells (ECCs) using cell surface marker p75NTR. Methods: ECCs were cultured from surgically resected ECC specimens; ECC cell lines TE-1 and Ecal09 were also cultured. The expression of p75NTR in human ECCs was examined by flow cytometry. p75NTR positive cells were isolated from ECCs using magnetic activated cell sorting (MACS) method. The proliferation, differentiation, and the ability of colony-forming in soft agar of the p75 NTR positive cells were observed. The p75NTR positive cells were injected into BALB/c nude mice subcutaneously to observe their tumorigenesis ability. The survival rates of p75NTR positive and negative cells were assessed after treated with chemotherapy drugs to evaluate the resistance of p75NTR positive cells. Results: Six out of the eight cell lines, including SHEC-4, SHEC-6, SHEC-7, SHEC-8, EcalO9, and TE-1, were positive of p75NTR, with the positive rates being 2.71%, 0.32%, 3.35%, 1.13%, 2.15%, and 0.45%. respectively. It was showed that p75 NTR positive cells possessed higher proliferation ability compared with p75NTR negative cells (P<0.01). The p75NTR positive cells had higher colony-forming ability in soft agar compared with p75NTR negative cells (P<0.01). The p75NTR positive cells demonstrated stronger tumorigenesis ability in nude mice. As few as 2 000 SHEC-7 cells could give rise to new tumors in xenotransplantation, with a tumorigenesis ability 50 times as high as that of the p75NTR negative cells. When treated with chemotherapy drugs for 48 h, p75NTR positive cells had significantly higher survival rate than p75NTR negative cells (P<0.05). Conclusion: The p75NTR positive ECCs possess self-renewal, differentiation, and proliferation abilities; they are strongly resistant to chemotherapy drugs, which gives them strong tumorigenesis ability and the characters of tumor stem cells.

STUDY ON INTERFERON-? GENE-MODIFIED HUMAN TUMOR CELLS / 中国免疫学杂志

The whole length cDNA of human interferon-?(IFN-?) gene including signal peptide was constructed to retroviral vector pLXSN and then packaged with PA317. A Human Hepetoma Cell Line(HHCL) and a human gastric cancer cell line (MKN-45) were infected by using supernatant containing retroviral vector with IFN-?. the gene-modified tumor cell. were isolated by G418 selection. Some changes were found in cell cycle, expression of HLA class Ⅰ and classⅡ, and tumorigenecity. Elevated immunogenecity and abrogated rumerigenecity suggest a means for generating gene-modified tumor cell vaccine. for the treatment of cancer.