Jun Dou

MiR‐7, Inhibited Indirectly by LincRNA HOTAIR, Directly Inhibits SETDB1 and Reverses the EMT of Breast Cancer Stem Cells by Downregulating the STAT3 Pathway

Epithelial–mesenchymal transition (EMT) contributes to tumor invasion and metastasis in many cancers and correlates highly with the acquisition of cancer stem cell (CSC) characteristics. EMT also correlates with changes in specific microRNAs (miRNAs) that have already been integrated into tumorigenic programs as either oncogenes or tumor suppressor genes. Here, we show that miR‐7, which was downregulated in breast CSCs (BCSCs) isolated from the human MCF‐7 and MDA‐MB‐231 cell lines, inhibited cell invasion and metastasis, decreased the BCSC population and partially reversed EMT in MDA‐MB‐231 cells by directly targeting the oncogene, SETDB1. The conspicuous epigenetic transition induced by miR‐7 overexpression was found not only in MDA‐MB‐231 cells but also in BCSC xenograft tumors. MiR‐7 inhibited the metastasis of BCSCs in lungs, kidneys, and adrenal glands of NOD/SCID mice. ChIP‐polymerase chain reaction result suggested that the SETDB1 induced STAT3 expression by binding to the promoter of STAT3. MiR‐7‐mediated downregulation of SETDB1 resulted in the suppression of STAT3, which led to the downregulation of c‐myc, twist, and mir‐9. In addition, the downregulation of miR‐7 in BCSCs may be indirectly attributed to lincRNA HOTAIR by modulating the expression of HoxD10 that promotes the expression of miR‐7. These findings demonstrate that miR‐7 was a tumor suppressor and that the overexpression of miR‐7 might serve as a good strategy for treating highly invasive breast cancer. Stem Cells 2014;32:2858–2868

MicroRNA-200c overexpression inhibits tumorigenicity and metastasis of CD117 + CD44 + ovarian cancer stem cells by regulating epithelial-mesenchymal transition

BackgroundCancer stem cells (CSCs) are believed to be ‘seed cell’ in cancer recurrence and metastasis. MicroRNAs (miRNAs) can play an important role in the progression of primary tumor towards metastasis by regulating the epithelial-mesenchymal transition (EMT). The goal of this study was to investigate the effect of miRNA-200c overexpression on the EMT, tumorigenicity and metastasis of epithelial ovarian cancer (EOC) CSCs.MethodsThe EOC CD117+CD44+CSCs were isolated from the human ovarian cancer cell line SKOV3 by using a magnetic-activated cell sorting system, and the lentivirus miR-200c transduced CSCs were then selected for the study. The assays of colony forming, wound healing, cellular migration in vitro and tumor progression in vivo were performed.ResultsThe miR-200c expression was reduced in the CD117+CD44+CSCs compared with the non-CD117+CD44+CSCs. However, the stable overexpression of the miR-200c in the CD117+CD44+CSCs resulted in a significant down-regulation of ZEB-1 and the Vimentin expression, an upregulation of the E-cadherin expression as well as a decrease of colony forming, migratory and invasion in vitro. Importantly, the miR-200c overexpression significantly inhibited the CD117+CD44+CSCs xenograft growth and lung metastasis in vivo in nude mice by inhibition of the EMT. In addition, the down-regulation of ZEB-1 showed the same efficacy as the miR-200c overexpression in the CD117+CD44+CSCs.ConclusionThese findings from this study suggest that the miR-200c overexpression may be considered a critical approach for the EOC CD117+CD44+CSCs in clinical trials.

Evaluation of characteristics of CD44+CD117+ ovarian cancer stem cells in three dimensional basement membrane extract scaffold versus two dimensional monocultures

BackgroundCancer stem cells (CSCs) are thought to be capable of surviving conventional chemotherapeutic treatments because the cells have more resistant to anticancer drugs than common cancer cells. Most in vitro studies in experimental cancer cells have been done in a two-dimensional (2D) monocultures, while accumulating evidence suggests that cancer cells behave differently when they are grown within a three-dimensional (3D) culture system.ResultsThe CD44+CD117+cells isolated from human epithelial ovarian cancer SKOV-3 cell line using magnetic-activated cell sorting were found to grow faster than the SKOV-3 cells in the 3D culture and in the nude mice. Anticancer drugs 5FU, docetaxel, cisplatin, and carboplatin were seen to inhibit growth of the CD44+CD117+ cells by 50% in the 2D culture with IC50 concentration, whereas, in the 3D culture, the four drugs inhibited the cell growth by only 34.4%, 40.8%, 34.8% and 21.9% at 3D one, respectively. Effect of paclitaxel on the CD44+CD117+cell viability indicated that fewer cells underwent apoptosis in 3D culture than that in 2D one. In addition, anticancer drugs markedly increased the expression of ABCG2 and ABCB1 of CD44+CD117+cells in 3D culture.ConclusionOur assay demonstrated that human epithelial ovarian cancer CD44+CD117+cells possessed the properties of CSCs that exhibited more chemoresistance in the 3D culture than that of in 2D one. The 3D culture provides a realistic model for study of the CSC response to anticancer drugs.

An ocular mucosal administration of nanoparticles containing DNA vaccine pRSC-gD-IL-21 confers protection against mucosal challenge with herpes simplex virus type 1 in mice

Herpes stromal keratitis (HSK) is a chronic inflammatory process caused by the infection of herpes simplex virus type 1 (HSV-1). Development of a HSV-1 vaccine is a priority because these infections are common and cannot be well prevented. It appears that the potential of nanocarriers in DNA vaccination will be required to augment the immune response to DNA vaccines. Therefore, in the study, nanoparticles Fe(3)O(4) coated with glutamic acid, DNA vaccine pRSC-gD-IL-21 and polyethylenimine were prepared and immunized in the mice by ocular mucosal administration. The immune responses and protection efficiency against HSV-1 challenge were also tested. The results showed that the nanoparticles containing DNA vaccine pRSC-gD-IL-21 induced mice to generate higher levels of specific neutralizing antibody, sIgA in tears, and IFN-γ, IL-4 in serum, and to enhance the cytotoxicities of NK cells and splenocytes as well as splenocyte proliferative response to glycoprotein D compared with those of the control mice. More importantly, the mice immunized with the experimental vaccine showed less HSK degree than that of the control mice after HSV-1 challenge of the murine ocular mucosa. In conclusion, an ocular mucosal administration of nanoparticles containing DNA vaccine confers strong specific immune responses and effective inhibition of HSK in a HSV-1 infected murine model.

Using ABCG2-molecule-expressing side population cells to identify cancer stem-like cells in a human ovarian cell line

CSCs (cancer stem cells) are a small subset of cells within a tumour that possesses the characteristics of stem cells and are considered to be responsible for resistance to chemoradiation. Identification of CSCs through stem cell characteristics might have relevant clinical implications. In this study, SP (side population) cells were sorted from a human ovarian cancer cell line by FACS to determine whether cancer stem cell‐like SP cells were present. A very small fraction of SP cells (2.6%) was detected in A2780 cells. SP cells possessed the following characteristics: highly proliferative activity, marked ability for self‐renewal in soft agar and culture medium, high expression of ABCG2, drug resistance to vinblastine in vitro, and strong tumourigenic potential in Balb/c nude mice. It is concluded that there exists in the A2780 cell line a small number of SP cells with high expression of ABCG2. The cells have the characteristics of cancer stem‐like cells, and identification and cloning of such human SP cells can help in improving therapeutic approaches to ovarian cancer in patients.

Enhancing therapy of B16F10 melanoma efficacy through tumor vaccine expressing GPI-anchored IL-21 and secreting GM-CSF in mouse model

In the present study, we developed the tumor vaccine expressing IL-21 in the GPI-anchored form together with secreting GM-CSFs and investigated its antitumor efficacy in C57BL/6 mouse model. The fusion genes containing IL-21 and the GPI anchor signal sequence were acquired by overlaping PCR, inserted into the downstream of two multi-clone sites in recombinant plasmid pRSC/GM-CSFs to form pRSC/IL-21-gpi-GM-CSFs that was transfected into the B16F10 cells. The tumor cell vaccine B16F10/IL-21-gpi-GM-CSFs was identified by reverse transcription PCR, IFA and FCM, respectively. The results showed that the pRSC/IL-21-gpi-GM-CSFs had no cell cycle and proliferative state impact on the B16F10 cells after transfected, and that the tumor vaccine B16F10/IL-21-gpi-GM-CSFs increased the cytotoxicities of NK cells and CD8(+)CTL, enhanced the level of serum IFN-gamma, augmented therapy of tumor effect and prolonged survival time in the tumor-bearing mice immunized with the tumor vaccine B16F10/IL-21-gpi-GM-CSFs. The data that we presented here provided a rationale and practical platform for clinical testing of enhancing cell therapy of B16F10 melanoma efficacy by modified tumor vaccine expressing GPI-anchored IL-21 and secreting GM-CSF.

Identifying tumor stem-like cells in mouse melanoma cell lines by analyzing the characteristics of side population cells

Increasingly more evidence shows that TSCs possess the characteristics of stem‐like cells. However, a link between stem cells and TSCs remains to be shown. We have sorted SP cells and non‐SP cells from the B16F10 cell lines by FACS, and then studied their cellular biological characteristics by using a SFCM culture method, proliferative assay in vitro, clone formation assays in soft agar and normal media, tumorigenic assays in C57BL/6 mice, and resistance to chemotherapy assay in vitro, the quantitative detecting expression of ABCG2 and their CD phenotype analysis by a FCM. We detected 0.96% SP cells in the B16F10 cells and found that they had obvious differences in characteristics from non‐SP cells. They possessed a marked capacity for self‐renewal in soft agar and culture medium, strong tumorigenic potential in C57BL/6 mice, apparent resistance to vinblastin in vitro, upregulated ABCG2 expression, and a high expression of CD44+CD133+CD24+ phenotypes. We conclude that there were a few of SP cells that had the characteristics of tumor stem‐like cells which may provide a useful tool and a readily accessible source for further study when specific TSCs markers are unknown.

Anti-ABCG2 monoclonal antibody in combination with paclitaxel nanoparticles against cancer stem-like cell activity in multiple myeloma

AIM To investigate the effects of anti-ABCG2 monoclonal antibodies (mAbs) in combination with paclitaxel iron oxide nanoparticles (PTX-NPs) on CD138(-)CD34(-) multiple myeloma (MM) cancer stem cells (CSCs) in JJN3 cells. MATERIALS & METHODS PTX-NPs were prepared using the hydrophobic interaction of the polyoxypropylene chain and oleic acid on the surface of iron oxide NPs and were targeted to the ABCG2 transporter overexpressing MM CSCs with mAbs. RESULTS The data showed that MM CSCs have strong drug resistance and tumorigenicity compared with non-MM CSCs. PTX-NPs combined with mAbs led to a significant reduction in the tumor volume, a visible alleviation of lytic bone lesions and a markedly increased survival rate in contrast to using a single agent in MM CSCs when it was transplanted to nonobese diabetic/severe combined immunodeficiency mice. CONCLUSION This study is the first to report on the anti-MM CSC activity by PTX-NPs as a single agent or used together with anti-ABCG2 mAbs to treat MM. These findings provide a rationale for future clinical trials.

Observation of ovarian cancer stem cell behavior and investigation of potential mechanisms of drug resistance in three-dimensional cell culture.

Cancer cells behave differently in a three-dimensional (3D) cell culture compared with in the conventional two-dimensional (2D) one. Accumulated evidences indicate that the characteristics of cancer stem cells (CSCs) are different from common cancer cells due to their ability to produce tumors and resist chemoradiation. The objective of this work was to observe CSC behavior and investigate the potential mechanisms of CSC drug resistance in 3D versus 2D in vitro environment. We first demonstrated that the CD44(+)CD117(+)cells isolated from the human epithelial ovarian cancer HO8910 cell line have the properties of CSCs that revealed faster growth, larger tumorsphere and stronger survival potential in the hypoxic environment in 3D cell culture as well as more powerful tumorigenicity in a xenograft mice than the HO8910 cells. The CD44(+)CD117(+)CSCs also exhibited high chemoresistance to anticancer drugs when the cells were incubated with 5-fluorouracil, cisplatin and carboplatin, respectively in 3D versus 2D environment. This might be associated with the high expression of ABCG2, ABCB1 and the high expression of MMP-2 and MMP-9 in CD44(+)CD117(+)CSCs. Overall, these results suggest the advantages of using 3D culture model to accurately display CSC behavior in vitro. 3D model may improve the efficacy of screening anticancer drugs for treatment of ovarian CSCs.

Antitumor efficacy induced by human ovarian cancer cells secreting IL-21 alone or combination with GM-CSF cytokines in nude mice model.

The ovarian cancer cells (SKOV3) secreting IL-21 alone or combination with GM-CSF cytokines was developed and its antitumor effect was evaluated in the nude mice. The gene of IL-21 was amplified from plasmid pRSC-IL-21 by PCR, cloned into the plasmid pRSC-GM-CSF, and the plasmid pRSC-GM-CSF-IL21 was constructed. The plasmids of pRSC-GM-CSF, pRSC-IL21, pRSC-GM-CSF-IL21 and pRSC were respectively transfected into the SKOV3 cells and antitumor efficacy induced by the SKOV3 cells secreting IL-21 or combination with GM-CSF was evaluated by surveying the tumor growth and the nude mices survival. The results indicated that the secreted IL-21 and GM-CSF were functional because the culture supernatant of SKOV3 cells transfected with the plasmid pRSC-GM-CSF-IL21 enhanced NK cytotoxicity in vitro. The expressions of MIC A/B, NKG2D and ICAM-1 molecules on the SKOV3 cells were up-regulated. The level of IFN-gamma and TNF-alpha, the NK cytotoxicity and the antitumor efficacy were significantly increased in the null mice inoculated with the SKOV3 cells secreting both IL-21 and GM-CSF in comparison with the nude mice inoculated with the other different SKOV3 cells. We concluded that the SKOV3 cells genetically engineered to secrete biologically active IL-21 and GM-CSF elicited antitumor immunity effectively through enhancing NK cytotoxicity, promoting the expressions of MIC A/B , ICAM-1 and NKG2D molecules as well as elevating level of IFN-gamma and TNF-alpha in the nude mice model.