Steroidal saponins from Trillium tschonoskii rhizome repress cancer stemness and proliferation of intrahepatic cholangiocarcinoma.

A phytochemical study was carried out on the extract of Trillium tschonoskii rhizomes, resulting in the isolation of thirty-six steroidal glycosides (1-36). Their structures were established mainly by spectroscopic analyses as well as necessary chemical evidence, of which 1-25 were identified as new analogues. Herein, all the isolated analogues were screened for the cytotoxicity against intrahepatic cholangiocarcinoma (ICC) cell lines of HuCCT1 and RBE through tumor colony formation and CCK-8 survival analysis, and the results demonstrated that three compounds 9, 12, and 26 significantly repressed tumor colony and sphere formation in both cell lines, respectively. Furthermore, the three analogues possessed a remarkable inhibitory role of organoid formation established from hydrodynamic induced mouse primary intrahepatic cholangiocarcinoma. Moreover, the functional assays of flow cytometry analysis, cancer stemness related gene expression, and western blotting assays all indicated that compound 26 could significantly repress cancer stem markers. Taken together, these results demonstrate that steroidal glycosides derived from T. tschonoskii rhizomes could be potentially implicated in human ICC therapy.

Molecular hydrogen suppresses glioblastoma growth via inducing the glioma stem-like cell differentiation.

BACKGROUND: Glioblastoma (GBM) is the most common type of primary malignant brain tumor. Molecular hydrogen has been considered a preventive and therapeutic medical gas in many diseases including cancer. In our study, we sought to assess the potential role of molecular hydrogen on GBM. METHODS: The in vivo studies were performed using a rat orthotopic glioma model and a mouse subcutaneous xenograft model. Animals inhaled hydrogen gas (67%) 1 h two times per day. MR imaging studies were performed to determine the tumor volume. Immunohistochemistry (IHC), immunofluorescence staining, and flow cytometry analysis were conducted to determine the expression of surface markers. Sphere formation assay was performed to assess the cancer stem cell self-renewal capacity. Assays for cell migration, invasion, and colony formation were conducted. RESULTS: The in vivo study showed that hydrogen inhalation could effectively suppress GBM tumor growth and prolong the survival of mice with GBM. IHC and immunofluorescence staining demonstrated that hydrogen treatment markedly downregulated the expression of markers involved in stemness (CD133, Nestin), proliferation (ki67), and angiogenesis (CD34) and also upregulated GFAP expression, a marker of differentiation. Similar results were obtained in the in vitro studies. The sphere-forming ability of glioma cells was also suppressed by hydrogen treatment. Moreover, hydrogen treatment also suppressed the migration, invasion, and colony-forming ability of glioma cells. CONCLUSIONS: Together, these results indicated that molecular hydrogen may serve as a potential anti-tumor agent in the treatment of GBM.

Loss of G3BP1 suppresses proliferation, migration, and invasion of esophageal cancer cells via Wnt/ß-catenin and PI3K/AKT signaling pathways.

Accumulating evidence suggests that Ras GTPase-activating protein SH3 domain-binding protein 1 (G3BP1) is very crucial to regulate tumorigenesis and metastasis. Recently, many research works have suggested that G3BP1 is overexpressed in many human cancers including esophageal cancer. Nevertheless, the functional roles of G3BP1 in esophageal cancer are still unknown. Here, the results suggested that silencing of G3BP1 inhibited proliferation, migration, and invasion of esophageal cancer cells, whereas overexpression of G3BP1 led to opposite effects on the growth and metastasis. Surprisingly, G3BP1-depletion had no effect on cell death but caused the arrest of cell cycle in the G0 /G1 phase and increased the levels of p53 and p21. In addition, loss of G3BP1 led to a significant elevation of E-cadherin and decrease of N-cadherin, Vimentin, Snail, MMP-9, and MMP-2. Mechanistically, loss of G3BP1 dramatically suppressed Wnt-stimulated T-cell factor/lymphoid enhancer factor (TCF/LEF) transcription factor activity and downregulated its target genes including c-Myc, Axin2, and cyclin D1. Moreover, knockdown of G3BP1 downregulated the expression levels of p-PI3K, p-AKT, and p-GSK-3ß, but the total PI3K, AKT, and GSK-3ß were not changed. Furthermore, our data proved that the promoting effects of G3BP1-overexpression on cell proliferation, migration, and invasion could be rescued by PI3K inhibitor LY294002 treatment. Collectively, our results here elucidate that G3BP1-depletion suppresses proliferation, migration, and invasion capabilities of esophageal cancer cells via the inactivation of Wnt/ß-catenin and PI3K/AKT signaling pathways. Furthermore, our findings imply that G3BP1 can participate in the regulation of esophageal cancer progression, and will be taken as a promising target to treat esophageal cancer.

Adipose mesenchymal stem cell-derived exosomes stimulated by hydrogen peroxide enhanced skin flap recovery in ischemia-reperfusion injury.

BACKGROUND: Mesenchymal stem cell (MSC)-derived exosomes have been recognized as new candidates for the treatment of ischemic disease or injury and may be an alternative treatment for cell therapy. This aim of the study was to evaluate whether exosomes derived from adipose mesenchymal stem cell (ADSC) can protect the skin flap during ischemia-reperfusion (I/R) injury and induce neovascularization. METHODS: To investigate the effects of exosomes in the I/R injury of flap transplantation in vivo, flaps were subjected to 6 h of ischemia by ligating the left superficial inferior epigastric vessels (SIEA) followed by blood perfusion. Exosomes derived from normal ADSC (ADSC-exos) and exosomes derived from ADSC preconditioned with H2O2 (H2O2-ADSC-exos) were injected into the flaps. Then, the blood perfusion unit (BPU) of the flaps was measured by Laser Doppler Perfusion Imaging (LDPI) and microvessel density was determined by the endothelial with cell marker CD31 with Immunohistochemistry (IHC) staining. Inflammatory cell infiltration of the skin flap and apoptosis were detected by hematoxylin & eosin staining (H&E) and the TdT-mediated biotinylated dUTP nick end-labeling (TUNEL) technique. RESULTS: In vivo, exosomes significantly increased flap survival and capillary density compared to I/R on postoperative day 5, and decreased the inflammatory reaction and apoptosis in the skin flap (P < 0.05). Furthermore, H2O2-ADSC-exos had better outcomes compared to normal exosomes (P < 0.05). ADSC-exos could significantly increase human umbilical vein endothelial cell (HUVEC) proliferation (P < 0.05), but no statistic difference was found in exosomes derived from different microenvironments (P > 0.05). HUVEC co-cultured with H2O2-ADSC-exos increased the migration ratio and generated more cord-like structures compared to ADSC-exos and the control group (P < 0.05). CONCLUSION: ADSC-exos can enhance skin flap survival, promote neovascularization and alleviate the inflammation reaction and apoptosis in the skin flap after I/R injury. The use of a specific microenvironment for in vitro stem cell culture, such as one containing a low concentration of H2O2, will facilitate the development of customized exosomes for cell-free therapeutic applications in skin flap transplantation.

Co-transplantation of exosomes derived from hypoxia-preconditioned adipose mesenchymal stem cells promotes neovascularization and graft survival in fat grafting.

BACKGROUND: Adipose-derived stromal cells (ADSCs)-derived exosomes (ADSC-Exos) account for the proangiogenic potential of stem cell. This study aimed to investigate the effect of ADSC-derived exosomes (ADSC-Exos) on the survival in fat grafting. METHODS: A nude mouse model of subcutaneous fat grafting was adopted. Hypoxic preconditioned ADSC-Exos and ADSC-Exos were injected around the grafted tissue. The fat graft sample was weighed and examined by hematoxylin and eosin (H&E) staining and immunohistochemistry. Laser Doppler flowmetry and CD31 immunofluorescence staining were used to analyze neovascularization. RESULTS: ADSC-Exo and hypoxic ADSC-Exo groups had a significantly higher weight of fat graft and more perilipin-positive adipocytes than the control groups from 2 to 8 weeks after grafting, and the hypoxic ADSC-Exo group had better outcomes (all P < 0.05). H&E staining showed that ADSC-Exos attenuated infiltration of inflammatory cells around the fat grafts. Laser Doppler flowmetry showed that the two ADSC-Exo groups had better blood perfusion in the graft tissue than the control groups (all P < 0.05). Immunofluorescence demonstrated that the hypoxic ADSC-Exo group had significantly more CD31-positive cells than the ADSC-Exo group. In vitro study showed that hypoxic ADSC-Exos treatment significantly increased the migration (at 12 and 24 h) and in vitro capillary network formation (at 12 h) in the human umbilical vein endothelial cells (HUVECs) as compared with the ADSC-Exo group and control group (all P < 0.05). CONCLUSIONS: Co-transplantation of ADSC-Exos can effectively promote the survival of graft, neovascularization and attenuated inflammation in the fat grafts. Hypoxia treatment can further enhance the beneficial effect of ADSC-Exos.

MicroRNA-125b attenuates epithelial-mesenchymal transitions and targets stem-like liver cancer cells through small mothers against decapentaplegic 2 and 4.

UNLABELLED: Emerging evidence suggests that epithelial-mesenchymal transitions (EMTs) play important roles in tumor metastasis and recurrence. Understanding molecular mechanisms that regulate the EMT process is crucial for improving treatment of hepatocellular carcinoma (HCC). MicroRNAs (miRNAs) play important roles in HCC; however, the mechanisms by which miRNAs target the EMT and their therapeutic potential remains largely unknown. To better explore the roles of miRNAs in the EMT process, we established an EMT model in HCC cells by transforming growth factor beta 1 treatment and found that several tumor-related miRNAs were significantly decreased. Among these miRNAs, miR-125b expression was most strongly suppressed. We also found down-regulation of miR-125b in most HCC cells and clinical specimens, which correlated with cellular differentiation in HCC patients. We then demonstrated that miR-125b overexpression attenuated EMT phenotype in HCC cancer cells, whereas knockdown of miR-125b promoted the EMT phenotype in vitro and in vivo. Moreover, we found that miR-125b attenuated EMT-associated traits, including chemoresistance, migration, and stemness in HCC cells, and negatively correlated with EMT and cancer stem cell (CSC) marker expressions in HCC specimens. miR-125b overexpression could inhibit CSC generation and decrease tumor incidence in the mouse xenograft model. Mechanistically, our data revealed that miR-125b suppressed EMT and EMT-associated traits of HCC cells by targeting small mothers against decapentaplegic (SMAD)2 and 4. Most important, the therapeutic delivery of synthetic miR-125b mimics decreased the target molecule of CSC and inhibited metastasis in the mice model. These findings suggest a potential therapeutic treatment of miR-125b for liver cancer. CONCLUSION: miR-125b exerts inhibitory effects on EMT and EMT-associated traits in HCC by SMAD2 and 4. Ectopic expression of miR-125b provides a promising strategy to treat HCC.

Hepatocellular carcinoma-associated mesenchymal stem cells promote hepatocarcinoma progression: role of the S100A4-miR155-SOCS1-MMP9 axis.

UNLABELLED: Cancer-associated mesenchymal stem cells (MSCs) play a pivotal role in modulating tumor progression. However, the interactions between liver cancer-associated MSCs (LC-MSCs) and hepatocellular carcinoma (HCC) remain unreported. Here, we identified the presence of MSCs in HCC tissues. We also showed that LC-MSCs significantly enhanced tumor growth in vivo and promoted tumor sphere formation in vitro. LC-MSCs also promoted HCC metastasis in an orthotopic liver transplantation model. Complementary DNA (cDNA) microarray analysis showed that S100A4 expression was significantly higher in LC-MSCs compared with liver normal MSCs (LN-MSCs) from adjacent cancer-free tissues. Importantly, the inhibition of S100A4 led to a reduction of proliferation and invasion of HCC cells, while exogenous S100A4 expression in HCC cells resulted in heavier tumors and more metastasis sites. Our results indicate that S100A4 secreted from LC-MSCs can promote HCC cell proliferation and invasion. We then found the expression of oncogenic microRNA (miR)-155 in HCC cells was significantly up-regulated by coculture with LC-MSCs and by S100A4 ectopic overexpression. The invasion-promoting effects of S100A4 were significantly attenuated by a miR-155 inhibitor. These results suggest that S100A4 exerts its effects through the regulation of miR-155 expression in HCC cells. We demonstrate that S100A4 secreted from LC-MSCs promotes the expression of miR-155, which mediates the down-regulation of suppressor of cytokine signaling 1, leading to the subsequent activation of STAT3 signaling. This promotes the expression of matrix metalloproteinases 9, which results in increased tumor invasiveness. CONCLUSION: S100A4 secreted from LC-MSCs is involved in the modulation of HCC progression, and may be a potential therapeutic target. (HEPATOLOGY 2013).

SPINDLIN1 promotes cancer cell proliferation through activation of WNT/TCF-4 signaling.

SPINDLIN1, a new member of the SPIN/SSTY gene family, was first identified as a gene highly expressed in ovarian cancer cells. We have previously shown that it is involved in the process of spindle organization and chromosomal stability and plays a role in the development of cancer. Nevertheless, the mechanisms underlying its oncogenic role are still largely unknown. Here, we first showed that expression of SPINDLIN1 is upregulated in clinical tumors. Ectopic expression of SPINDLIN1 promoted cancer cell proliferation and activated WNT/T-cell factor (TCF)-4 signaling. The Ser84 and Ser99 amino acids within SPINDLIN1 were further identified as the key functional sites in WNT/TCF-4 signaling activation. Mutation of these two sites of SPINDLIN1 abolished its effects on promoting WNT/TCF-4 signaling and cancer cell proliferation. We further found that Aurora-A could interact with and phosphorylate SPINDLIN1 at its key functional sites, Ser84 and Ser99, suggesting that phosphorylation of SPINDLIN1 is involved in its oncogenic function. Collectively, these results suggest that SPINDLIN1, which may be a novel substrate of the Aurora-A kinase, promotes cancer cell growth through WNT/TCF-4 signaling activation.

Mesenchymal stem cells from primary breast cancer tissue promote cancer proliferation and enhance mammosphere formation partially via EGF/EGFR/Akt pathway.

Mesenchymal stem cells (MSCs) play a critical role in promoting cancer progression. However, it is not clear whether MSCs are located in breast cancer tissues and correlated with tumor proliferation. The aim of this study was to investigate the presence of MSCs in breast cancer tissues and evaluate their interactions with cancer cells. We successfully isolated and identified MSCs from primary breast cancer tissues. Breast cancer-associated MSCs (BC-MSCs) showed homogenous immunophenotype, and possessed tri-lineage differentiation potential (osteoblast, adipocyte, and chondrocyte). When co-transplanted with cancer cells in a xenograft model in vivo, BC-MSCs significantly increased the volume and weight of tumors. We observed that BC-MSCs stimulated mammosphere formation in the transwell co-culture system in vitro. This effect was significantly suppressed by the EGF receptor inhibitor. We verified that BC-MSCs could secrete EGF and activate cancer cell's EGF receptors. Furthermore, our data showed that EGF derived from BC-MSCs could promote mammosphere formation via the PI3K/Akt signaling pathway. Our results confirmed the presence of MSC in primary breast cancer tissues, and they could provide a favorable microenvironment for tumor cell growth in vivo, partially enhance mammosphere formation via the EGF/EGFR/Akt pathway.

SIRT1 is required for long-term growth of human mesenchymal stem cells.

Human mesenchymal stem cells (MSCs) have therapeutic potential because of their ability to self-renew and differentiate into multiple tissues. However, senescence often occurs in MSCs when they are cultured in vitro and the molecular mechanisms underlying this effect remain unclear. In this study, we found that NAD-dependent protein deacetylase SIRT1 is differentially expressed in both human bone marrow-derived MSCs (B-MSCs) and adipose tissue-derived MSCs after increasing passages of cell culture. Using lentiviral shRNA we demonstrated that selective knockdown of SIRT1 in human MSCs at early passage slows down cell growth and accelerates cellular senescence. Conversely, overexpression of SIRT1 delays senescence in B-MSCs that have undergone prolonged in vitro culturing and the cells do not lose adipogenic and osteogenic potential. In addition, we found that the delayed accumulation of the protein p16 is involved in the effect of SIRT1. However, resveratrol, which has been used as an activator of SIRT1 deacetylase activity, only transiently promotes proliferation of B-MSCs. Our findings will help us understand the role of SIRT1 in the aging of normal diploid cells and may contribute to the prevention of human MSCs senescence thus benefiting MSCs-based tissue engineering and therapies.

Epimorphin promotes human hepatocellular carcinoma invasion and metastasis through activation of focal adhesion kinase/extracellular signal-regulated kinase/matrix metalloproteinase-9 axis.

UNLABELLED: The high incidence rate of hepatocellular carcinoma (HCC) is mainly the result of frequent metastasis and tumor recurrence. Unfortunately, the underlying molecular mechanisms driving HCC metastasis are still not fully understood. It has been demonstrated that tumor stroma cells contribute to primary tumor growth and metastasis. Within the HCC environment, activated hepatic stellate cells (HSCs) can release a number of molecules and enhance cancer cell proliferation and invasiveness in a paracrine manner. Here, for the first time, we demonstrate that epimorphin (EPM; also called syntaxin-2), an extracellular protein, is strongly elevated in activated HSCs within tumor stroma. We show that knockdown of EPM expression in HSCs substantially abolishes their effects on cancer cell invasion and metastasis. Ectopic expression of EPM in HCC cancer cells enhances their invasiveness; we demonstrate that the cells expressing EPM have markedly increased metastasis potential. Furthermore, EPM-mediated invasion and metastasis of cancer cells is found to require up-regulation of matrix metalloproteinase-9 (MMP-9) through the activation of focal adhesion kinase (FAK)/extracellular signal-regulated kinase (ERK) axis. CONCLUSION: Our results show that EPM, secreted by activated HSCs within HCC stroma, promotes invasion and metastasis of cancer cells by activating MMP-9 expression through the FAK-ERK pathway.

Migration of dorsal aorta mesenchymal stem cells induced by mouse embryonic circulation.

Mesenchymal stem cells (MSCs) represent powerful tools for regenerative medicine for their differentiation and migration capacity. However, ontogeny and migration of MSCs in mammalian mid-gestation conceptus is poorly understood. We identified canonical MSCs in the mouse embryonic day (E) 11.5 dorsal aorta (DA). They possessed homogenous immunophenotype (CD45(-)CD31(-)Flk-1(-)CD44(+)CD29(+)), expressed perivascular markers (α-SMA(+)NG2(+)PDGFRß(+)PDGFRα(+)), and had tri-lineage differentiation potential (osteoblasts, adipocytes, and chondrocytes). Of interest, MSCs were also detected in E12.5-E13.5 embryonic circulation, 24 hr later than in DA, suggesting migration like hematopoietic stem cells. Functionally, E12.5 embryonic blood could trigger efficient migration of DA-MSCs through platelet-derived growth factor (PDGF) receptor-, transforming growth factor-beta receptor-, but not basic fibroblast growth factor receptor-mediated signaling. Moreover, downstream JNK and AKT signaling pathway played important roles in embryonic blood- or PDGF-mediated migration of DA-derived MSCs. Taken together, these results revealed that clonal MSCs developed in the mouse DA. More importantly, the embryonic circulation, in addition to its conventional transporting roles, could modulate migration of MSC during early embryogenesis.

Characterization of OP9 as authentic mesenchymal stem cell line.

Mesenchymal stem cells (MSCs) are multipotent stem cells capable of differentiating into various cell types, including osteocytes, chondrocytes, adipocytes, myocytes, and tenocytes. However, the difficulty or failure in expanding the mouse MSCs in vitro greatly hampered important research in animal models. The OP9, a stromal cell line from mouse bone marrow, has hematopoietic supportive capacity. Here, we report that the OP9 has the immunophenotype (CD45(-), CD11b(-), FLK-1(-), CD31(-), CD34(-), CD44(+), CD29(+), Sca-1(+), CD86(-), and MHCII(-)) identical to canonical mouse MSCs. The expression of CD140a(+), CD140b(+), alpha-SMA(+) and Calponin(+) suggested the perivascular origin of OP9. Functionally, the OP9 had strong clonogenic ability and could be induced into osteocytes, chondrocytes and adipocytes. The lymphocyte transformation test (LTT) and mixed leukocyte reaction (MLR) showed that the OP9 could suppress T lymphocyte proliferation stimulated by nonspecific mitogens (PHA) or allogeneic lymphocytes (BALB/c T cells). Finally, the migration of OP9 could be efficiently induced by bFGF, IGF-1, IL-3, PDGF-BB, TGF-beta1 and TGF-beta3. In conclusion, the OP9 were bona fide MSCs, and such homogenous cell line will be helpful to delineate biological features of MSCs at the stem cell level.

[Study on migration property of mesenchymal stem cells-review].

Mesenchymal stem cells (MSCs) are multipotent stem cells which can support hematopoiesis, have immunomodulatory property, may differentiate into osteocytes, chondrocytes and adipocytes, and specifically migrate to damage sites and tumor site, but the mechanism involved in the regulation of migration of MSCs still remains unelucidated. Understanding the fundamental mechanisms underlying MSCs migration holds the promise of developing novel clinical strategies which can deliver antitumor proteins to suppress tumor growth. In this review, the MSC migration in vitro mediated by growth factors, chemokines, adhesion molecules and toll-like receptors are summarized.