Cathepsin K Is Present in Invasive Oral Tongue Squamous Cell Carcinoma In Vivo and In Vitro

Objectives

Cathepsin K, a lysosomal cysteine protease, is expressed in the tumor microenvironment (TME) of skin carcinoma, but nothing is known about cathepsin K in oral tongue squamous cell carcinoma (OTSCC). Our aim was to describe the expression of cathepsin K in invasive OTSCC in vitro and in a series of clinical cancer specimens.

Materials and Methods

OTSCC invasion in vitro was studied using invasive HSC-3 tongue carcinoma cells in 3D organotypic models. In total, 121 mobile tongue OTSCCs and 10 lymph node metastases were analyzed for cathepsin K expression. The association between cathepsin K expression and clinicopathological factors was evaluated.

Results

Cysteine protease inhibitor E64 and cathepsin K silencing significantly (p<0.0001) reduced HSC-3 cell invasion in the 3D models. Cathepsin K was expressed in a majority of carcinoma and metastatic cells, but the expression pattern in carcinoma cells did not correlate with clinical parameters. Instead, the weak expression of cathepsin K in the invasive TME front correlated with increased overall recurrence (p<0.05), and in early-stage tumors this pattern predicted both cancer recurrence and cancer-specific mortality (p<0.05 and p<0.005, respectively).

Conclusions

Cathepsin K is expressed in OTSCC tissue in both carcinoma and TME cells. Although the diminished activity and expression in aggressive tongue HSC-3 cells reduced 3D invasion in vitro, the amount of cathepsin K in carcinoma cells was not associated with the outcome of cancer patients. Instead, cathepsin K in the invasive TME front seems to have a protective role in the complex progression of tongue cancer.     

骨髓间充质干细胞向角膜上皮细胞分化及其免疫调节作用

目的：探讨大鼠骨髓间充质干细胞（rBMMSCs）转分化为角膜上皮的潜能,并在体外共培养体系中研究rBMMSCs对促炎细胞因子干扰素-γ（IFN-γ）和肿瘤坏死因子-α（TNF-α）刺激下的人角膜上皮细胞（hCECs）的免疫调节作用。方法采用聚蔗糖梯密度离心法获得rBMMSCs,并通过上皮细胞培养微环境来诱导rBMMSCs分化为上皮样细胞。通过免疫组织化学方法鉴定CD29、CD34、CK5＆8和ZO-1等标记物在rBMMSCs及诱导的上皮样细胞中的表达。流式细胞术用来分析CD29/CD34的表达及细胞分化过程中表达量的变化。hCECs单独培养或与rBMMSCs共培养,并采用IFN-γ/TNF-α刺激24或48 h。通过流式细胞术来分析细胞间黏附分子-1（ICAM-1）于IFN-γ/TNF-α刺激前后在hCECs上的表达,并通过黏附分析实验验证rBMMSC条件培养基对单核细胞黏附于IFN-γ/TNF-α刺激后的hCECs的作用。多组间比较采用单因素方差分析（ANOVA）,两组间比较采用双侧t检验。结果成功分离rBMMSCs,细胞表达CD29,但不表达CD34。在上皮细胞培养条件中培养5 d,大约4﹪的rBMMSCs可分化为上皮样细胞。此类细胞失去了CD29的标志,转为表达CK5＆8和ZO-1。IFN-γ/TNF-α能显著上调hCECs中ICAM-1的表达,在IFN-γ/TNF-α处理24 h和48 h后,ICAM-1分别呈现10倍和8倍的升高,分别达到4524±554.2和3107±329.6（P=0.0025,0.0014）。但与MSC共同培养时,上调作用被显著抑制,ICAM-1平均值为1356±325.6（24 h）与1323±106.6（48 h）（P=0.0079,0.0024）。MSC条件培养基可显著抑制单核细胞对hCECs的黏附作用,黏附细胞数从（10.01±3.01）×10^3/ml细胞降至（2.21±0.19）×10^3/ml细胞（P=0.0271）。结论rBMMSCs可转分化为角膜上皮样细胞,并抑制由促炎细胞因子诱导的ICAM-1在hCECs上的表达,同时对促炎细胞因子诱导的单核细胞的黏附性具有抑制作用,提示BMMSCs具有在角膜炎症疾病和损伤修复中的治疗潜能。     

Lung Collagens Perpetuate Pulmonary Fibrosis via CD204 and M2 Macrophage Activation

Idiopathic pulmonary fibrosis is characterized by abundant collagen production and accumulation of alternatively activated macrophages (M2) in the lower respiratory tract. Mechanisms as to how alveolar macrophages are activated by collagen breakdown products are unknown. Alveolar macrophages were obtained by bronchoalveolar lavage from 30 patients with idiopathic pulmonary fibrosis (IPF) and 37 healthy donors (HD). Alveolar macrophages were cultured in the presence of collagen type I, III, IV and V monomers w/wo a neutralizing antibody against scavenger receptor I class A (CD204). Culture supernatants were assayed for the M2 markers CCL18, CCL2, and interleukin-1 receptor antagonist (IL-1ra) by ELISA. Furthermore, expression of phospho-Akt was measured using ELISA and expression of CD204 by RT-PCR and flow cytometry. Stimulation with collagen type I and III monomers significantly up-regulated CCL18, IL-1ra production of alveolar macrophages. Furthermore, expression of CCL2 and CD204 were up-regulated by collagen type I exposure. In addition, collagen type I stimulation increased pospho-Akt expression. Collagen type I effects were abrogated by neutralizing antiCD204 and a non-selective Phosphatidylinositide 3-kinase inhibitor (LY294002). Spontaneous CD204 expression of alveolar macrophages was significantly increased in patients with IPF. In conclusion, our findings demonstrate that monomeric collagen type I via CD204 induces phospho-Akt expression shifting alveolar macrophages to the profibrotic M2 type. Innate immune responses induced by collagen monomers might perpetuate pulmonary fibrosis.     

MicroRNA and protein profiles in invasive versus non-invasive oral tongue squamous cell carcinoma cells in vitro

Complex molecular pathways regulate cancer invasion. This study overviewed proteins and microRNAs (miRNAs) involved in oral tongue squamous cell carcinoma (OTSCC) invasion. The human highly aggressive OTSCC cell line HSC-3 was examined in a 3D organotypic human leiomyoma model. Non-invasive and invasive cells were laser-captured and protein expression was analyzed using mass spectrometry-based proteomics and miRNA expression by microarray. In functional studies the 3D invasion assay was replicated after silencing candidate miRNAs, miR-498 and miR-940, in invasive OTSCC cell lines (HSC-3 and SCC-15). Cell migration, proliferation and viability were also studied in the silenced cells. In HSC-3 cells, 67 proteins and 53 miRNAs showed significant fold-changes between non-invasive vs. invasive cells. Pathway enrichment analyses allocated “Focal adhesion” and “ECM-receptor interaction” as most important for invasion. Significantly, in HSC-3 cells, miR-498 silencing decreased the invasion area and miR-940 silencing reduced invasion area and depth. Viability, proliferation and migration weren’t significantly affected. In SCC-15 cells, down-regulation of miR-498 significantly reduced invasion and migration. This study shows HSC-3 specific miRNA and protein expression in invasion, and suggests that miR-498 and miR-940 affect invasion in vitro, the process being more influenced by mir-940 silencing in aggressive HSC-3 cells than in the less invasive SCC-15.     

GDNF secreted by pre-osteoclasts induces migration of bone marrow mesenchymal stem cells and stimulates osteogenesis

Bone resorption is linked to bone formation via temporal and spatial coupling within the remodeling cycle. Several lines of evidence point to the critical role of coupling factors derived from pre-osteoclasts (POCs) during the regulation of bone marrow-derived mesenchymal stem cells (BMMSCs). However, the role of glial cell-derived neurotrophic factor (GDNF) in BMMSCs is not completely understood. Herein, we demonstrate the role of POC-derived GDNF in regulating the migration and osteogenic differentiation of BMMSCs. RNA sequencing revealed GDNF upregulation in POCs compared with monocytes/macrophages. Specifically, BMMSC migration was inhibited by a neutralizing antibody against GDNF in pre-osteoclast-conditioned medium (POC-CM), whereas treatment with a recombinant GDNF enhanced migration and osteogenic differentiation. In addition, POC-CM derived from GDNF knockdowned bone marrow macrophages suppressed BMMSC migration and osteogenic differentiation. SPP86, a small molecule inhibitor, inhibits BMMSC migration and osteogenic differentiation by targeting the receptor tyrosine kinase RET, which is recruited by GDNF into the GFRα1 complex. Overall, this study highlights the role of POC-derived GDNF in BMMSC migration and osteogenic differentiation, suggesting that GDNF regulates bone meta-bolism.     

CD47 Regulates Collagen I-Induced Cyclooxygenase-2 Expression and Intestinal Epithelial Cell Migration

Increased epithelial cell expression of the cyclooxgenase-2 (COX-2) enzyme is a characteristic event of both inflammatory bowel disease and colon cancer. We here report the novel findings that collagen I-induced de novo synthesis of COX-2 in intestinal epithelial cells is inhibited by pertussis toxin (PTX) and by an inhibitory peptide selective for the heterotrimeric Gα_i3-protein. These findings could be explained by a regulatory involvement of the G-protein-dependent integrin-associated protein CD47. In support of this notion, we observed a collagen I-induced association between CD47 and α2 integrins. This association was reduced by a blocking anti-CD47 antibody but not by PTX or a control anti-β2 antibody. Furthermore, a blocking antibody against CD47, dominant negative CD47 or specific siRNA knock down of CD47, significantly reduced collagen I-induced COX-2 expression. COX-2 has previously been shown to regulate intestinal epithelial cell adhesion and migration. Morphological analysis of intestinal cells adhering to collagen I revealed a co-localisation of CD47 and α2 integrins to non-apoptotic membrane blebs enriched in Rho A and F-actin. The blocking CD47 antibody, PTX and a selective COX-2 inhibitor, dramatically inhibited the formation of these blebs. In accordance, migration of these cells on a collagen I-coated surface or through a collagen I gel were significantly reduced by the CD47 blocking antibody, siRNA knock down of CD47 and the COX-2 inhibitor NS-398. In conclusion, we present novel data that identifies the G-protein-dependent CD47 protein as a key regulator of collagen I-induced COX-2 expression and a promoter of intestinal epithelial cell migration.     

Effect of Fatty Acids on Human Bone Marrow Mesenchymal Stem Cell Energy Metabolism and Survival

Successful stem cell therapy requires the optimal proliferation, engraftment, and differentiation of stem cells into the desired cell lineage of tissues. However, stem cell therapy clinical trials to date have had limited success, suggesting that a better understanding of stem cell biology is needed. This includes a better understanding of stem cell energy metabolism because of the importance of energy metabolism in stem cell proliferation and differentiation. We report here the first direct evidence that human bone marrow mesenchymal stem cell (BMMSC) energy metabolism is highly glycolytic with low rates of mitochondrial oxidative metabolism. The contribution of glycolysis to ATP production is greater than 97% in undifferentiated BMMSCs, while glucose and fatty acid oxidation combined only contribute 3% of ATP production. We also assessed the effect of physiological levels of fatty acids on human BMMSC survival and energy metabolism. We found that the saturated fatty acid palmitate induces BMMSC apoptosis and decreases proliferation, an effect prevented by the unsaturated fatty acid oleate. Interestingly, chronic exposure of human BMMSCs to physiological levels of palmitate (for 24 hr) reduces palmitate oxidation rates. This decrease in palmitate oxidation is prevented by chronic exposure of the BMMSCs to oleate. These results suggest that reducing saturated fatty acid oxidation can decrease human BMMSC proliferation and cause cell death. These results also suggest that saturated fatty acids may be involved in the long-term impairment of BMMSC survival in vivo.     

Cellular changes resulting from forced expression of glypican-3 in hepatocellular carcinoma cells

Glypican-3 (GPC3) is a member of the glypican family, which encodes cell-surface heparan-sulfate proteoglycans, and is frequently upregulated in hepatocellular carcinoma (HCC). We have recently reported that blocking endogenous GPC3 expression promotes the growth of HCC cell lines, suggesting that GPC3 plays a negative role in HCC cell proliferation. Here, we report that forced expression of GPC3 reduced the growth of HCC cells. We also found that FGF2-mediated cell proliferation was inhibited by GPC3. In addition, we observed that the adhesion of HCC cells to collagen type I and fibronectin was decreased by GPC3, whereas cellular migration and invasiveness were stimulated. Collectively, these results suggest that progression of hepatocellular carcinoma is associated with upregulation of GPC3.     

白藜芦醇苷通过抑制长链非编码RNA HULC表达抑制肝癌SMMC-7721和HepG2细胞的增殖和侵袭

原发性肝癌是临床上最常见的恶性肿瘤之一,目前仍在寻找有效的治疗手段。我们之前的研究证实白藜芦醇苷能够抑制肝癌细胞的增殖和侵袭,但其具体的分子生物学机制仍不清楚。本文主要探讨白藜芦醇苷调控肝癌细胞系SMMC-7721和HepG2肝癌细胞系增殖能力的分子生物学机制。首先,我们构建大鼠原发性肝癌模型,发现模型组肝组织中长链非编码RNA HULC的表达较正常组明显升高;同时检测白藜芦醇苷预防组（分为低剂量组,中剂量组和高剂量组）肝组织中肝癌细胞中出现异常的高表达(HULC)的表达情况。结果显示,在中、高剂量组中HULC的表达明显降低。在体外实验中,SMMC7721和HepG2中HULC的表达明显较正常肝组织显著增高,然而白藜芦醇苷高剂量组中HULC的表达发生明显降低,同时在SMMC7721和HepG2中加入白藜芦醇苷后,高剂量组中细胞增殖能力明显下降。为了进一步探究HULC在白藜芦醇苷预防肝癌中的功能,我们构建了HULC过表达质粒以及针对HULC的siRNA片段,并验证了过表达和敲低的效率。在使用高剂量白藜芦醇苷处理SMMC7721和HepG2的同时,过表达HULC能够逆转白藜芦醇苷引起的对细胞增殖的抑制,然而敲低HULC则能够更加有效地降低白藜芦醇苷对细胞增殖的抑制效果。这提示我们白藜芦醇苷能够可能通过调控HULC的表达抑制肝癌细胞的增殖和侵袭,二者具有协同作用。本文结果为预防原发性肝癌提供了新的理论依据但其临床疗效还需要进一步验证。     

Type I collagen-induced pro-MMP-2 activation is differentially regulated by H-Ras and N-Ras in human breast epithelial cells

Tumor cell invasion and metastasis are often associated with matrix metalloproteinases (MMPs), among which MMP-2 and MMP-9 are of central importance. We previously showed that H-Ras, but not N-Ras, induced invasion of MCF10A human breast epithelial cells in which the enhanced expression of MMP-2 was involved. MMP-2 is produced as a latent pro-MMP-2 (72 kDa) to be activated resulting the 62 kDa active MMP-2. The present study investigated if H-Ras and/or N-Ras induces pro-MMP-2 activation of MCF10A cells when cultured in two-dimensional gel of type I collagen. Type I collagen induced activation of pro-MMP-2 only in H-Ras MCF10A cells but not in N-Ras MCF10A cells. Induction of active MMP-2 by type I collagen was suppressed by blocking integrin alpha2, indicating the involvement of integrin signaling in pro-MMP-2 activation. Membrane-type (MT)1-MMP and tissue inhibitor of metalloproteinase (TIMP)-2 were up-regulated by H-Ras but not by N-Ras in the type I collagen-coated gel, suggesting that H-Ras-specific up-regulation of MT1-MMP and TIMP-2 may lead to the activation of pro-MMP-2. Since acquisition of pro-MMP-2 activation can be associated with increased malignant progression, these results may help understanding the mechanisms for the cell surface matrix-degrading potential which will be crucial to the prognosis and therapy of breast cancer metastasis.     

Type I collagen regulated dentin matrix protein-1 (Dmp-1) and osteocalcin (OCN) gene expression of rat dental pulp cells

In this study, we investigated the effect of type I collagen on dentin matrix protein-1 (Dmp-1) and osteocalcin (OCN) gene expression of dental pulp cells. The mRNA level of Dmp-1 gene was down-regulated; however, OCN gene expression was up-regulated by the culture of dental pulp cells with type I collagen. These findings imply that type I collagen regulates mRNA level of Dmp-1 and OCN gene that are predominantly expressed in active odontoblasts. The change of gene expression by type I collagen was suppressed by the blocking of collagen-integrin interaction. We could conclude that the effect of type I collagen was mediated via binding of collagen to integrin receptors.     

The combination of angiotensin II and 5-azacytidine promotes cardiomyocyte differentiation of rat bone marrow mesenchymal stem cells

Bone marrow mesenchymal stem cells (BMMSCs) are ideal seed cells for tissue engineering and regenerative medicine. Many studies have shown that 5-azacytidine (5-aza) can induce BMMSCs to differentiate into cardiomyogenic cells, but some issues still remain to be resolved. In this study, we investigated the effects of angiotensin II (Ang II) on the proliferation and differentiation of BMMSCs induced by 5-aza in vitro. BMMSCs were isolated from the bone marrow of Sprague-Dawley rats by density gradient centrifugation. The third-passage cells were divided into four groups: the Ang II group (0. 1 μmol/l) (group A), the 5-aza group (10 μmol/l) (group B), the Ang II combined with 5-aza group (0.1 and 10 μmol/l) (group C), and the untreated group as control. After 24 h of induction, the medium was changed to the complete culture medium without any inductor, and the cells were cultured for 3 weeks. Morphological changes were observed under a phase contrast microscope. The effect of Ang II and 5-aza on BMMSC proliferation was evaluated by the methyl thiazolyl tetrazolium (MTT) assay. Cardiomyogenic cells were identified through immunofluorescence staining, and the induction ratio was examined by flow cytometry. The level of cardiac troponin I (cTnI) was examined by western blotting, and the ultrastructures of the induced cells were viewed with a transmission electron microscope. The MTT assay showed that the cell proliferation in group C outweighed that in either group A or group B, but no significant difference existed between group A and group B. The expression of specific proteins, namely, cTnI and sarcomeric α-actin in induced BMMSCs was verified as positive. Flow cytometry showed that the induction ratio in group C was higher than that in group A or group B. The protein levels of cTnI in groups A, B, and C were significantly higher than those in the control group. Transmission electron microscopy showed that the induced cells had myofilaments, z line-like substances, desmosomes, and gap junctions. Angiotensin II and 5-azacytidine can promote the proliferation and differentiation of BMMSCs into cardiomyocyte-like cells.     

Activated protein C mediates a healing phenotype in cultured tenocytes

Tendon injuries cause considerable morbidity in the general adult population. The tenocytes within the tendon have the full capacity to heal the tendon intrinsically. Activated protein C (APC) plays an important role in coagulation and inflammation and more recently has been shown to promote cutaneous wound healing. In this study we examined whether APC can induce a wound healing phenotype in tenocytes. Sheep tenocytes were treated with APC, endothelial protein C receptor (EPCR) blocking antibody (RCR252) and/or EPCR small interfering (si)RNA. Cell proliferation and migration were measured by crystal violet assay and a scratch wounding assay, respectively. The expression of EPCR, matrix metalloproteinase (MMP)-2, type I collagen and MAP kinase activity were detected by real time PCR, zymography, immunofluorescence, immunohistochemistry and Western blotting. APC stimulated proliferation, MMP-2 activity and type I collagen deposition in a dose-dependent manner and promoted migration of cultured tenocytes. APC dose-dependently stimulated phosphorylated (P)-ERK2 and inhibited P-p38. Interestingly, tenocytes expressed EPCR protein, which was up-regulated by APC. When tenocytes were pre-treated with RCR252 or EPCR siRNA the effect of APC on proliferation, MMP-2 and type 1 collagen synthesis and MAP kinases was blocked. APC promotes the growth, MMP-2 activity, type I collagen deposition and migration of tenocytes. Furthermore, EPCR is expressed by tenocytes and mediates the actions of APC, at least partly by signalling through selective MAP kinases. These data implicate APC as a potential healing agent for injured tendons.     

CCL21/CCR7 Enhances the Proliferation,Migration, and Invasion of Human Bladder Cancer T24 Cells

Objective

To investigate the effects of CCL21/CCR7 on the proliferation, migration, and invasion of T24 cells and the possible associated mechanisms: expression of MMP-2 and MMP-9, and regulation of BCL-2 and BAX proteins.

Methods

T24 cells received corresponding treatments including vehicle control, antibody (20ng/mL CCR7 antibody and 50 ng/ml CCL21), and 50, 100, and 200 ng/ml CCL21. Proliferation was evaluated by MTT assay; cell migration and invasion were assayed using a transwell chamber. Cell apoptosis was induced by Adriamycin (ADM). The rate of cell apoptosis was examined by flow cytometry using annexin V-FITC/PI staining. Western-blot was used to analyze MMP-2 and MMP-9 and BCL-2 and BAX proteins.

Results

CCL21 promoted T24 cell proliferation in concentration-dependent manner with that 200 ng/mL induced the largest amount of proliferation. Significant differences of cell migration were found between CCL21treatment groups and the control group in both the migration and invasion studies (P < 0.001 for all). The expressions of MMP-2 and MMP-9 proteins were significantly increased after CCL21 treatment (p < 0.05 for all). Protein expression of Bcl-21 follows an ascending trend while the expression of Bax follows a descending trend as the concentration of CCL21 increases. No difference was found between the control group and antibody group for all assessments.

Conclusion

CCL21/CCR7 promoted T24 cell proliferation and enhanced its migration and invasion via the increased expression of MMP-2 and MMP-9. CCL21/CCR7 had antiapoptotic activities on T24 cells via regulation of Bcl-2 and Bax proteins. CCL21/CCR7 may promote bladder cancer development and metastasis.     

CD200 positive human mesenchymal stem cells suppress TNF-alpha secretion from CD200 receptor positive macrophage-like cells

Human mesenchymal stem cells (hMSCs) display immunosuppressive properties in vitro and the potential has also been transferred successfully to clinical trials for treatment of autoimmune diseases. OX-2 (CD200), a member of the immunoglobulin superfamily, is widely expressed in several tissues and has recently been found from hMSCs. The CD200 receptor (CD200R) occurs only in myeloid-lineage cells. The CD200-CD200R is involved in down-regulation of several immune cells, especially macrophages. The present study on 20 hMSC lines shows that the CD200 expression pattern varied from high (CD200Hi) to medium (CD200Me) and low (CD200Lo) in bone marrow-derived mesenchymal stem cell (BMMSC) lines, whereas umbilical cord blood derived mesenchymal stem cells (UCBMSCs) were constantly negative for CD200. The role of the CD200-CD200R axis in BMMSCs mediated immunosuppression was studied using THP-1 human macrophages. Interestingly, hMSCs showed greater inhibition of TNF-α secretion in co-cultures with IFN-γ primed THP-1 macrophages when compared to LPS activated cells. The ability of CD200Hi BMMSCs to suppress TNF-α secretion from IFN-γ stimulated THP-1 macrophages was significantly greater when compared to CD200Lo whereas UCBMSCs did not significantly reduce TNF-α secretion. The interference of CD200 binding to the CD200R by anti-CD200 antibody weakened the capability of BMMSCs to inhibit TNF-α secretion from IFN-γ activated THP-1 macrophages. This study clearly demonstrated that the efficiency of BMMSCs to suppress TNF-α secretion of THP-1 macrophages was dependent on the type of stimulus. Moreover, the CD200-CD200r axis could have a previously unidentified role in the BMMSC mediated immunosuppression.     

Degenerate wave and capacitive coupling increase human MSC invasion and proliferation while reducing cytotoxicity in an in vitro wound healing model

Non-unions pose complications in fracture management that can be treated using electrical stimulation (ES). Bone marrow mesenchymal stem cells (BMMSCs) are essential in fracture healing; however, the effect of different clinical ES waveforms on BMMSCs cellular activities remains unknown. We compared the effects of direct current (DC), capacitive coupling (CC), pulsed electromagnetic field (PEMF) and degenerate wave (DW) on cellular activities including cytotoxicity, proliferation, cell-kinetics and apoptosis by stimulating human-BMMSCs 3 hours a day, up to 5 days. In addition, migration and invasion were assessed using fluorescence microscopy and by quantifying gene and protein expression. We found that DW had the greatest proliferative and least apoptotic and cytotoxic effects compared to other waveforms. DC, DW and CC stimulations resulted in a higher number of cells in S phase and G(2)/M phase as shown by cell cycle analysis. CC and DW caused more cells to invade collagen and showed increased MMP-2 and MT1-MMP expression. DC increased cellular migration in a scratch-wound assay and all ES waveforms enhanced expression of migratory genes with DC having the greatest effect. All ES treated cells showed similar progenitor potential as determined by MSC differentiation assay. All above findings were shown to be statistically significant (p<0.05). We conclude that ES can influence BMMSCs activities, especially DW and CC, which show greater invasion and higher cell proliferation compared to other types of ES. Application of DW or CC to the fracture site may help in the recruitment of BMMSCs to the wound that may enhance rate of bone healing at the fracture site.     

Hypoxia promotes HO-8910PM ovarian cancer cell invasion via Snail-mediated MT1-MMP upregulation

The molecular mechanisms of ovarian cancer cell invasion under hypoxia remain unclear. Here we employed a 3D collagen model and chick chorioallantoic membrane (CAM) invasion assay to explore the influence of hypoxia on ovarian cancer cell invasion. Hypoxia (both 1% O₂ and CoCl₂ 150 and 250 µM) induced HO-8910PM ovarian cancer cell invasion in 3D collagen and collagenolysis determined by hydroxyproline. Pretreatment with a hypoxia inducible factor-1α inhibitor, YC-1, or MMP inhibitor, GM6001, significantly inhibited 3D collagen invasion and degradation and cell proliferation. Hypoxia stimulated both mRNA and protein expressions of membrane-type 1 matrix metalloproteinase (MT1-MMP) and promoted MT1-MMP translocation to the cell surface in an YC-1 sensitive manner. MT1-siRNA transfection inhibited hypoxia-induced invasion, proliferation, and collagen degradation of cells in 3D collagen. Hypoxia stimulated Snail mRNA and protein expression as well as translocation to nucleus in an YC-1 sensitive manner. Overexpression of Snail with a recombinant plasmid in HO-8910PM cells resulted in an enhanced invasion in 3D collagen. Transfection with Snail-specific siRNA significantly decreased MT1-MMP expression and 3D collagen invasion. Hypoxia-treated cells significantly broke the upper CAM surface of 11-day-old chick embryos and infiltrated interstitial tissue, completely blocked in the presence of YC-1 or GM6001, or after MT1-MMP siRNA or Snail siRNA transfection. Together, these data suggest that hypoxia promotes HO-8910PM ovarian cancer cell traffic through 3D matrix via Snail-mediated MT1-MMP upregulation, a possible molecular mechanism of ovarian cancer cell invasion under hypoxia.     

Type I collagen-mediated proliferation of PC3 prostate carcinoma cell line: implications for enhanced growth in the bone microenvironment.

Prostate cancer is the second leading cause of male cancer-related deaths in the United States. Interestingly, prostate cancer preferentially metastasizes to bone. Once in the bone microenvironment, advanced prostate cancer becomes highly resistant to therapeutic modalities. Several factors, such as, extracelluar matrix components, have been implicated in the spread and propagation of prostatic carcinoma. The prostate cell line, PC3, adhere and spread on collagen I to a greater degree than on fibronectin (FN) or poly-L-lysine (PLL). Flow cytometry analysis reveals the presence of the alpha(1), alpha(2) and alpha(3) collagen binding integrin subunits. Antibody function blocking studies reveal that PC3 cells can utilize alpha(2)beta(1) and alpha(3)beta(1) integrins to adhere to collagen I. Cells plated on collagen I exhibit increased rates of proliferation over cells plated on FN or tissue culture plastic. Additionally, cells plated on collagen I show increased expression of cyclin D1, a molecule associated with progression through G1 phase of the cell cycle. Inhibitor studies point to a role for phosphatidylinositol 3-kinase (PI3K), map kinase (MAPK) and p70 S6 kinase in collagen I-mediated PC3 cell proliferation and cyclin D1 expression. Type I collagen may facilitate the colonization and growth of metastatic prostate tumor cells in the bone microenvironment.     

Cell proliferation of human bone marrow mesenchymal stem cells on biodegradable microcarriers enhances in vitro differentiation potential

Objectives: For reasons of provision of highly‐specific surface area and three‐dimensional culture, microcarrier culture (MC) has garnered great interest for its potential to expand anchorage‐dependent stem cells. This study utilizes MC for in vitro expansion of human bone marrow mesenchymal stem cells (BMMSCs) and analyses its effects on BMMSC proliferation and differentiation. Materials and methods: Effects of semi‐continuous MC compared to control plate culture (PC) and serial bead‐to‐bead transfer MC (MC bead‐T) on human BMMSCs were investigated. Cell population growth kinetics, cell phenotypes and differentiation potential of cells were assayed. Results: Maximum cell density and overall fold increase in cell population growth were similar between PCs and MCs with similar starting conditions, but lag period of BMMSC growth differed substantially between the two; moreover, MC cells exhibited reduced granularity and higher CXCR4 expression. Differentiation of BMMSCs into osteogenic and adipogenic lineages was enhanced after 3 days in MC. However, MC bead‐T resulted in changes in cell granularity and lower osteogenic and adipogenic differentiation potential. Conclusions: In comparison to PC, MC supported expansion of BMMSCs in an up‐scalable three‐dimensional culture system using a semi‐continuous process, increasing potential for stem cell homing ability and osteogenic and adipogenic differentiation.