Cuiping Yang

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.

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.

Effect of down-regulated transcriptional repressor ZEB1 on the epithelial-mesenchymal transition of ovarian cancer cells.

Background Progress has been made against early events of malignant transformation and drug resistance associated with epithelial ovarian cancer; uncontrolled metastases, however, still accounts for most patient deaths. The molecular mechanism that regulates the process of epithelial ovarian cancer metastases is not yet clearly understood. The purpose of this study was to investigate the effect of down-regulating the transcriptional repressor zinc-finger E-box–binding homeobox 1 (ZEB1) on an epithelial-mesenchymal transition (EMT) of human ovarian cancer SKOV3 cell line in vitro and in vivo. Methods The human ovarian cancer cells SKOV3 and HO8910 were transfected with an expression vector-based small hairpin RNA (shRNA) targeting ZEB1 (shZEB1), and the stably transfected cells were selected. Colony-forming, wound-healing, and cellular migration assays were respectively used. The tumorigenicity of shZEB1-SKOV3 was also evaluated in mice. Results The shZEB1-SKOV3 and shZEB1-HO8910 cells showed a lower level of ZEB1 expression and weaker cell migration than the control cells. Moreover, down-regulating ZEB1 expression with shRNA in the cells enhanced the expression of miR-200c that acted as a tumor suppressor to inhibit the epithelial-mesenchymal transition of shZEB1-SKOV3 cells and to block shZEB1-SKOV3 cell metastasis in vivo. The shRNA-mediated down-regulation ZEB1 in SKOV3 cells significantly decreased the tumor growth in the xenograft mice. Conclusion The shZEB1-mediated down-regulation of the ZEB1 expression in the SKOV3 cells may be considered for future clinical trials.

Downregulation of gene MDR1 by shRNA to reverse multidrug-resistance of ovarian cancer A2780 cells

BACKGROUND To explore the effects of downregulated multidrug-resistance P-glycoprotein (MDR1/ABCB1) and reversed multidrug-resistance in human A2780 ovarian cancer cells. MATERIALS AND METHODS Three shRNAs targeting the MDR1 gene were synthesized, and cloned into plasmid pSUPER-enhanced green fluorescent protein 1 (EGFP1). The formed pSUPER-EGFP1-MDR1-shRNAs were transfected into the A2780 cells, respectively, and the quantitative reverse transcription polymerase chain reaction and western blot were used respectively to determine the MDR1 expression. The multidrug-resistance (MDR) of the MDR1-shRNAs transfected A2780 cells to chemotherapy drugs in vitro and in tumor-bearing nude mice were respectively evaluated. RESULTS The MDR1 shRNA expression resulted in decreased P-glycoprotein expression in the transfected A2780 cells. The MDR1-shRNA2 transfected cells showed that the sensitivities to chemotherapy drugs were higher than other shRNAs transfected A2780 cells, and that the formed tumor in mice grew slower than those of other mice after paclitaxel was injected into tumor-bearing nude mice. CONCLUSIONS Our data demonstrates that the RNA interference could knock down gene MDR1 and reduce the P-glycoprotein expression, and partly reverse the MDR of A2780 cells in vitro and in vivo. These results suggest that MDR-1 is an effective therapeutic target for human ovarian caner treatment.

Paclitaxel-Fe3O4 nanoparticles inhibit growth of CD138(-) CD34(-) tumor stem-like cells in multiple myeloma-bearing mice.

Background There is growing evidence that CD138− CD34− cells may actually be tumor stem cells responsible for initiation and relapse of multiple myeloma. However, effective drugs targeted at CD138− CD34− tumor stem cells are yet to be developed. The purpose of this study was to investigate the inhibitory effect of paclitaxel-loaded Fe3O4 nanoparticles (PTX-NPs) on CD138− CD34− tumor stem cells in multiple myeloma-bearing mice. Methods CD138− CD34− cells were isolated from a human U266 multiple myeloma cell line using an immune magnetic bead sorting method and then subcutaneously injected into mice with nonobese diabetic/severe combined immunodeficiency to develop a multiple myeloma-bearing mouse model. The mice were treated with Fe3O4 nanoparticles 2 mg/kg, paclitaxel 4.8 mg/kg, and PTX-NPs 0.64 mg/kg for 2 weeks. Tumor growth, pathological changes, serum and urinary interleukin-6 levels, and molecular expression of caspase-3, caspase-8, and caspase-9 were evaluated. Results CD138− CD34− cells were found to have tumor stem cell characteristics. All the mice developed tumors in 40 days after injection of 1 × 106 CD138− CD34− tumor stem cells. Tumor growth in mice treated with PTX-NPs was significantly inhibited compared with the controls (P < 0.005), and the groups that received nanoparticles alone (P < 0.005) or paclitaxel alone (P < 0.05). In addition, the PTX-NPs markedly inhibited interleukin-6 secretion, increased caspase-8, caspase-9, and caspase-3 expression, and induced apoptosis of tumor cells in the treated mice. Conclusion PTX-NPs proved to be a potent anticancer treatment strategy that may contribute to targeted therapy for multiple myeloma tumor stem cells in future clinical trials.

Target therapy of multiple myeloma by PTX-NPs and ABCG2 antibody in a mouse xenograft model.

Multiple myeloma (MM) remains to be an incurable disease. The purpose of this study was to evaluate the effect of ABCG2 monoclonal antibody (McAb) combined with paclitaxel (PTX) conjugated with Fe3O4 nanoparticles (NPs) on MM progressed from cancer stem cells (CSCs)in non-obese-diabetic/severe-combined-immunodeficiency (NOD/SCID) mouse model. Mice were injected with MM CSCs as marked by CD138−CD34− phenotypes through tail veins. The developed MM mice were examined by micro-computer tomography scanning, ultrasonography and enzyme-linked immunosorbent analysis. These mice were then intravenously treated with different combinations of NPs, PTX, McAb, PTX-NPs and melphalan/prednisone once a week for four weeks. The injected mice developed characteristic MM-associated syndromes, including lytic bone lesions, renal damages and proteinuria. All the treated mice showed decrease in bone lesions, renal damages and anemia but increase in apoptosis compared with the mice treated with NPs only. In particular, the treatment with ABCG2 McAb plus PTX-NPs induced the strongest therapeutic response and had an efficacy even better than that of melphalan/prednisone, a conventional regimen for MM patients. These data suggest that PTX-NPs with ABCG2 McAb can be developed into potential treatment regimens for patients with relapsed/refractory MM.

Targeted therapeutic effect of anti-ABCG2 antibody combined with nano silver and vincristine on mouse myeloma cancer stem cells

Studies from hematopoietic origin malignancies have demonstrated that multiple myeloma contain a rare population of cancer stem cells (CSCs) that are responsible for tumor multiresistance and recurrence. The goal of this study was to investigate targeted therapeutic effect of anti-ABCG2 monoclonal antibody (McAb) combined with silver nanoparticles (AgNPs) and vincristine (VCR) on myeloma CSCs. The characteristics of CD44+ CD24− cells that were isolated from the SP2/0 cells using magnetic activated cell sorting system were first identified. The results showed that the CD44+ CD24− cells exhibited higher proliferation, more colony formation, more side population fraction, and stronger tumorigenicity in BALB/c mice than the control cells. Moreover, CD44+ CD24− cells markedly up-regulated the ABCG2 expression, however, anti-ABCG2 McAb combined with AgNPs and VCR effectively inhibited the CD44+ CD24− cell growth and prolonged the survival of myeloma-bearing mice. We concluded that the CD44+ CD24− cells in mouse myeloma SP2/0 cell line posses CSC properties. Anti-ABCG2 McAb combined with AgNPs and VCR provide an efficient targeted therapeutic method for inhibiting myeloma CD44+ CD24− CSC growth in mice.

ESAT‐6‐gpi DNA Vaccine Augmented the Specific Antitumour Efficacy Induced by the Tumour Vaccine B16F10‐ESAT‐6‐gpi/IL‐21 in a Mouse Model

In this study, we hypothesized that the mice immunized with the glycosylphosphatidylinositol (GPI) anchored 6‐kDa early‐secreted antigenic target (ESAT‐6) DNA vaccine (ESAT‐6‐gpi) and the tumour vaccine B16F10‐ESAT‐6‐gpi/IL‐21 might significantly enhance immune responses and antimelanoma efficacy. Our experimental results indicated that the anti‐ESAT‐6 antibody induced by the DNA vaccine ESAT‐6‐gpi bound ESAT‐6 to the surface of tumour vaccine to activate a complement classical pathway and resulted in the B16F10 tumour cell lysis and apoptosis, which served as a potential trigger for breaking melanomatous immune tolerance to elicit an initiation of natural antimelanoma immunity. Our innovative approach of using the DNA vaccine ESAT‐6‐gpi priming and the tumour vaccine B16F10‐ESAT‐6‐gpi/IL‐21 boosting induced strong antimelanoma immunity that inhibited melanomatous growth. These findings highlighted the DNA vaccine ESAT‐6‐gpi as an immune enhancer to augment the immune efficacy of the tumour vaccine B16F10‐ESAT ‐6‐gpi/IL‐21 against melanoma in a mouse model.