Sprouty 2 protein, but not Sprouty 4, is an independent prognostic biomarker for human epithelial ovarian cancer.

Sprouty proteins are evolutionary-conserved modulators of receptor tyrosine kinase signaling, deregulation of which has been implicated in the pathophysiology of cancer. In the present study, the expression status of Spry2 and Spry4 proteins and its clinical relevance in human epithelial ovarian cancer (EOC) were investigated retrospectively. We examined the immunohistochemical expression of Spry2 and Spry4 in matched tumor and normal tissue samples from 99 patients. The expression of ERK, p-ERK, Ki67, fibroblast growth factor-2, vascular endothelial growth factor and interleukin-6 and their correlation with Sprouty homologs were also evaluated. Moreover, the correlation between Spry2 and Spry4 and the clinicopathological characteristics were analyzed along with their predictive value for overall survival (OS) and disease-free survival (DFS). Our data indicated significant downregulation of Spry2 and Spry4 in tumor tissues (p < 0.0001). A significant inverse correlation was evident between Spry2 and p-ERK/ERK (p = 0.048), Ki67 (p = 0.011), disease stage (p = 0.013), tumor grade (p = 0.003), recurrence (p < 0.001) and post-treatment ascites (p = 0.001), individually. It was found that Spry2 low-expressing patients had significantly poorer OS (p = 0.002) and DFS (p = 0.004) than those with high expression of Spry2. Multivariate analysis showed that high Spry2 (p = 0.018), low stage (p = 0.049) and no residual tumor (p =0.006) were independent prognostic factors for a better OS. With regard to DFS, high Spry2 (p = 0.044) and low stage (p = 0.046) remained as independent predictors. In conclusion, we report for the first time significant downregulation of Spry2 and Spry4 proteins in human EOC. Spry2 expression was revealed to significantly impact tumor behavior with predictive value as an independent prognostic factor for survival and recurrence.

Cytotoxicity and biomechanics of suture anchors used in labral repairs.

BACKGROUND: Biodegradable suture anchors are associated with higher redislocation rates. This study examined whether the biocompatibility and/or biomechanical properties of suture anchors contribute to the increase in complications. METHODS: Human glenohumeral capsule cells were cultured with 4 types of suture anchors, Opus LabraFix (titanium alloy; ArthroCare, Austin, TX, USA), PushLock (poly-ether-ether-ketone; Arthrex, Naples, FL, USA), BioKnotless (poly-l-lactic acid; DePuy Mitek, Warsaw, IN, USA), and Suretac II (polyglycolic acid; Smith & Nephew, London, UK), to measure cell viability and pH. Four groups of 6 ovine shoulders were used to repair the labrum, which was completely detached from the glenoid rim anteroinferiorly and reattached with 2 suture anchors and subject to failure load testing. RESULTS: In cell culture, BioKnotless at 48 and 72 hours (85.2% ± 2.1% and 84.5% ± 3.6%) and Suretac II groups (33.9% ± 3.1% and 42.8% ± 6.4%) had fewer viable cells compared with control (P = .048). The pH of Suretac II was lower than control (7.51 to 7.65) at 24 hours (7.31 ± 0.08, P = .049), 48 hours (7.25 ± 0.02, P = .046), and 72 hours (7.29 ± 0.04, P = .04). During mechanical testing, 83% of repairs failed by the capsule tearing. Among the anchors, the BioKnotless repair group had a significantly lower failure load (37 ± 5 N) compared with the PushLock (61 ± 7 N), Opus (60 ± 6 N), and Suretac II (57 ± 7 N) groups (P = .038). CONCLUSION: BioKnotless and Suretac II anchors are cytotoxic. The BioKnotless biodegradable anchor has significantly lower failure load. Absorbable suture anchors may cause higher redislocation of arthroscopic Bankart repair.

Localization of bone morphogenetic protein 13 in human intervertebral disc and its molecular and functional effects in vitro in 3D culture.

Our laboratory has demonstrated that bone morphogenetic protein 13 prevented the effects of annular injury in an ovine model, maintaining intervertebral disc height, cell numbers and increasing extracellular matrix production compared to degenerated controls. The present study sought to examine the molecular effects of bone morphogenetic protein 13 on human degenerated disc cells and localize its expression in both human degenerate and scoliotic disc tissue. Effect of bone morphogenetic protein 13 on human derived nucleus pulposus, annulus fibrosus and endplate cells cultured in alginate beads was evaluated by changes in proteoglycan and collagen content. Migratory potential of disc cells towards bone morphogenetic protein 13 was also examined. Bone morphogenetic protein 13 induced significant proteoglycan accumulation in nucleus (18%), annulus (21%) and endplate (23%) cells cultured in alginate beads (p<0.05) compared to controls. Further bone morphogenetic protein 13 increased collagen I and II protein expression in nucleus and endplate cells. Nucleus cells displayed a significant chemotactic response towards bone morphogenetic protein 13. The endogenous expression of bone morphogenetic protein 13 in degenerate disc tissue was not different to scoliotic disc. Bone morphogenetic protein 13 has the potential to enhance extracellular matrix accumulation and induce cell migration in certain disc cells.

Sprouty2 protein in prediction of post-treatment ascites in epithelial ovarian cancer treated with adjuvant carbotaxol chemotherapy.

Ascites development and resistance to chemotherapy with carbotaxol are common clinical problems in epithelial ovarian cancer, partly due to the activation of MAPK/ERK signaling. Sprouty proteins are negative modulators of MAPK/ERK pathway, but their role in predicting resistance to carbotaxol chemotherapy and ascites development is unknown. In this study, we evaluated the expression of Sprouty protein isoforms by immunohistochemistry. The associations between the Sprouty expression and the clinicopathological features, including chemoresistance and the presence of ascites, were then explored. We found that the decreased expression of Spry2 was correlated with the post-treatment development of ascites and represented an independent predictor of this condition in carbotaxol-treated patients. However, no association was observed between the Sprouty expression and chemoresistance. In conclusion, our results suggest that Spry2 may be useful for patient follow-up and monitoring as it predicts the development of ascites in epithelial ovarian cancer cases treated with carbotaxol.

Cartilage Derived Morphogenetic Protein-2 Induces Cell Migration and Its Chondrogenic Potential in C28/I2 Cells.

BACKGROUND: Intervertebral disc degeneration is a major cause of low back pain. Previous researches have demonstrated local administration of signalling molecules as potential biological therapies for disc regeneration. Our laboratory has published encouraging results for effectiveness of injection of the cartilage derived morphogenetic protein-2 (CDMP-2) into ovine discs following annular injury. To elucidate the mechanisms underpinning these in vivo effects, this project aimed to investigate the potential of CDMP-2 on cellular migration, proliferation and extracellular matrix production in a human chondrocytic cell line. METHODS: To evaluate cell motility, cells were seeded into Boyden chambers and CDMP-2 as a chemo-attractant or a stimulant was placed into either the bottom or top chambers respectively. Cells that had completed migration through the porous membrane were visualized by immunocytochemical staining and analysed using Image J. The effect of CDMP-2 on cell proliferation, proteoglycan and collagen production, as well as chondrogenic gene expression in human chondrocytic cell line C28/I2 was also examined. RESULTS: The results revealed that cells migrated significantly under the influence of CDMP-2 (200 ng/ml) stimulation compared to control (3-fold increase, p = 0.033) and demonstrated a significant chemotactic movement towards a solution of 200ng/ml CDMP-2 (>2-fold increase, p = 0.027). A 35% increase in C28/I2 proliferation was observed after CDMP-2 stimulation (p < 0.0001) compared to control, and in the presence of 100ng/ml CDMP-2, proteoglycan synthesis had an 8-fold increase (p = 0.048). Similarly, gene expression analysis demonstrated increased expression of aggrecan, collagen types II, X and XXVII, BMPR-1A and BMPR-2 when cells were treated with CDMP-2. CONCLUSION: The study shows that C28/I2 cells can migrate under the influence of CDMP-2 as a chemoattractant or migration stimulator, suggestive of an effect on chondrocytic cells in the intervertebral disc. Further, CDMP-2 can stimulate C28/I2 cells to proliferate and synthesize key extracellular matrix proteins.

Vascular endothelial growth factor expression correlates with serum CA125 and represents a useful tool in prediction of refractoriness to platinum-based chemotherapy and ascites formation in epithelial ovarian cancer.

There is an increasing need for the identification of novel biological markers and potential therapeutic targets in epithelial ovarian cancer (EOC). Given the critical role of growth factors in the biology of EOC, we aimed in the present study to evaluate the intratumoral expressions of vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) proteins and their clinical relevance in a cohort of 100 patients with EOC. All patients received platinum-based chemotherapy after surgery. A comparative immunohistochemical study of normal ovarian and EOC tissues showed that both growth factors were expressed at higher levels in tumor samples. In our statistical analysis, while no association existed between the FGF expression status and the clinicopathological characteristics of patients, intratumoral VEGF was identified as a potential biomarker for the prediction of ascites formation. In addition, the expression status of VEGF appeared to independently predict overall survival and response to chemotherapy. Furthermore, a direct association was demonstrated between the pre-treatment VEGF expression and serum CA125 after three cycles of chemotherapy. In sum, we report for the first time to our knowledge the correlation between intratumoral VEGF and serum CA125 in EOC. Our data also shows the prognostic value of VEGF expression in EOC. These results suggest the potential value of intratumoral VEGF in patient stratification. Dual inhibition of VEGF and CA125 might bring about a better outcome for patients with EOC.

Sprouty 1 predicts prognosis in human epithelial ovarian cancer.

Sprouty proteins are evolutionary-conserved modulators of receptor tyrosine kinase (RTK) signaling. We have previously reported inverse correlation of the Sprouty 1 (Spry1) protein expression with ovarian cancer cell proliferation, migration, invasion and survival. In the present study, the expression status of Spry1 protein and its clinical relevance in patients with epithelial ovarian cancer were explored. Matched tumor and normal tissue samples from 100 patients with epithelial ovarian cancer were immunohistochemically stained for Spry1. Expression of ERK, p-ERK, Ki67, FGF-2, VEGF and IL-6 and their correlation with Spry1 were also evaluated. In addition, correlation between Spry1 and clinicopathological characteristics and predictive significance of Spry1 for overall survival (OS) and disease-free survival (DFS) were analysed. Our data indicated that Spry1 was significantly downregulated in tumor tissues (p=0.004). Spry1 showed significant inverse correlation with p-ERK/ERK (p=0.045), Ki67 (p=0.010), disease stage (p=0.029), tumor grade (p=0.037), recurrence (p=0.001) and lymphovascular invasion (p=0.042). It was revealed that Spry1 low-expressing patients had significantly poorer OS (p=0.010) and DFS (p=0.012) than those with high expression of Spry1. Multivariate analysis showed that high Spry1 (p=0.030), low stage (p=0.048) and no residual tumor (p=0.007) were independent prognostic factors for a better OS, among which high Spry1 (p=0.035) and low stage (p=0.035) remained as independent predictors of DFS, too. We also found that the expression of Spry1 significantly correlates with the expression of Spry2 (p<0.001), but not that of Spry4. In conclusion, we report for the first time to our knowledge that Spry1 protein is downregulated in human epithelial ovarian cancer. Spry1 expression significantly impacts tumor behavior and shows predictive value as an independent prognostic factor for survival and recurrence.

Intratumoral interleukin-6 predicts ascites formation in patients with epithelial ovarian cancer: A potential tool for close monitoring.

BACKGROUND: The implication of IL-6 in the pathogenesis of epithelial ovarian cancer (EOC) is well documented. Accordingly, the clinicopathological significance of this cytokine in patients' ascites fluid or serum has largely been investigated. Since the main source of IL-6 secreted into the biological fluids is the tumor tissue, this study was designed to investigate the status and possible clinical relevance of the IL-6 expression in an array of EOC tissue specimens. METHODS: Tissue samples obtained from ninety-eight consecutive patients with EOC were studied using immunohistochemistry. Clinicopathological characteristics and treatment related factors were collected from patient files. The relationship between the expression of the protein of interest and the study endpoints of disease-free survival (DFS) and overall survival (OS) were analyzed using the Kaplan-Meier method. For evaluating the predictive value of IL-6, logistic regression and cox proportional hazards models were employed. RESULTS: An upregulation of IL-6 expression was observed in EOC tissues as compared with the normal samples (p < 0.0001). As regards the clinical relevance, IL-6 failed to predict OS, DFS and response to the platinum-based chemotherapy in EOC patients. In multivariate analysis, however, IL-6 was identified as an independent predictive factor for the development of post-treatment ascites (p:0.033). CONCLUSIONS: Having the capability to predict the ascites formation, IL-6 might serve as a biomarker and a useful tool in EOC for monitoring purposes. IL-6 targeting for the prevention of the ascites development is a potential avenue for further investigation.

Apoptosis in rotator cuff tendonopathy.

The aim of this study was to investigate the involvement of apoptosis (programmed cell death) in the pathogenesis of rotator cuff disorders. The edges of torn supraspinatus rotator cuff tendons were collected from patients with rotator cuff tear (n = 25). Samples of the intra-articular portion of subscapularis tendons were collected from patients without rotator cuff tear as control (n = 6). To minimize individual variance, we also collected six pairs of supraspinatus tendon and subscapularis tendon from six patients with rotator cuff tears. Apoptosis was detected by in situ DNA end labelling assay and DNA laddering assay. Immunohistochemical staining was performed to identify cells undergoing apoptosis. Control subscapularis tendon had normal morphology. Tendon from torn supraspinatus rotator cuff showed significant mucoid degeneration. Within the areas of degeneration, there were large numbers of apoptotic cells. The percentage of apoptotic cells in the degenerative rotator cuff (34%) was significantly higher than that in controls (13%) (p < 0.001). The excessive apoptosis detected in degenerative rotator cuff tissue was confirmed by DNA laddering assays. This is the first report of excessive apoptosis in degenerating rotator cuff tendon. Cells undergoing apoptosis in rotator cuff were mainly fibroblast-like cells. These finding indicate that apoptosis may play an important role in the pathogenesis of rotator cuff degeneration.

The expression of the Sprouty 1 protein inversely correlates with growth, proliferation, migration and invasion of ovarian cancer cells.

BACKGROUND: Our recent study on a panel of human ovarian cancer cells revealed that SKOV-3 cells barely express the Sprouty isoform 1 (Spry1) while 1A9 cells maintain it at a level similar to normal ovarian cells. Here we investigated the functional outcomes of induced alterations in the expression of Spry1 in the two cell lines in vitro. METHODS: Using the Spry1 specific plasmid and siRNA, the expression of Spry1 was induced and conversely silenced in SKOV-3 and 1A9 cells, respectively. The functional outcome was investigated by means of proliferation, MTT, scratch-wound, migration and invasion assays and selection of the stable clones. Mechanism of the effect was explored by Western blot. RESULTS: In the Spry1-transfected SKOV-3 cells, a significant reduction in growth and proliferation was evident. Stable clones of the Spry1-transfected SKOV-3 were almost undetectable after day 14. The number of migrated and invaded cells and the percentage of the scratch closure were significantly lower in the Spry1-transfected group. Spry1 silencing in 1A9 cells, on the other hand, led to a significant increase in cell growth and proliferation. The number of migrated and invaded cells and the percentage of the scratch closure significantly increased in Spry1-silenced 1A9 group. Mechanistically, overexpression of Bax, activation of caspases 3, 7, 8 and 9, cleavage of PARP and attenuation of Bcl-2 and Bcl-xl were observed along with reduced activation of Erk and Akt and increased amount and activity of PTEN in the Spry1-transfected SKOV-3 cells. CONCLUSIONS: Here, we report the inverse correlation between the expression of Spry1 and growth, proliferation, invasion and migration of ovarian cancer cells.

Initial report on differential expression of sprouty proteins 1 and 2 in human epithelial ovarian cancer cell lines.

Sprouty (Spry) proteins, modulators of receptor tyrosine kinase signaling pathways, have been shown to be deregulated in a variety of pathological conditions including cancer. In the present study we investigated the expression of Spry1 and Spry2 isoforms in a panel of human ovarian cancer cell lines in vitro. Our western blot analysis showed nonuniform patterns of Spry expression in the cancer cells, none of which conformed to the pattern observed in the normal ovarian epithelial cells employed as the control. Among the seven cancer cell lines studied, Spry1 was expressed lower in four cell lines and higher in one as compared with the control. As for Spry2, four cell lines showed lower and two exhibited higher expression. Results from RT-PCR assay raised the possibility that Spry protein levels may not necessarily correspond with its expression at mRNA level. Our immunostaining study revealed that Spry2 was predominantly distributed within the whole cytoplasm in vesicular structures whereas Spry1 was found in both the cytoplasm and nucleus. This might provide clues to further investigation of Spry mode of action and/or function. Collectively, our study unveiled the differential expression of Spry1 and Spry2 proteins in various ovarian cancer cell lines.

The effect of running exercise on intervertebral disc extracellular matrix production in a rat model.

STUDY DESIGN: Using a running rat model, the effects of physical exercise on cellular function and intervertebral disc (IVD) extracellular matrix were studied. OBJECTIVE: To investigate whether 3-weeks treadmill running exercise can stimulate matrix production and cellular proliferation of the IVD. SUMMARY OF BACKGROUND DATA: Appropriate physical exercise plays an important role in the treatment of patients with low back pain-associated IVD disorder. However, it is unknown how regular exercise affects the disc at the cellular level. METHODS: Twelve Sprague-Dawley rats underwent a daily treadmill exercise regime for a total of 3 weeks. Twelve nonexercised rats served as controls. The spinal lumbar IVD were collected and paraffin embedded for histologic analysis. Cell counts were determined on hematoxylin-eosin- and Masson-Trichrome-stained paraffin sections. Protein expression of collagen-I, collagen-II, aggrecan, Sox-9, and Sox-6 was evaluated with immunohistochemical staining. mRNA expression of Sox-9 and collagen-2 were studied by in situ hybridization. Proteoglycans were visualized with Alcian blue. Apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. RESULTS: The cell numbers in the anulus fibrosus (AF) increased by 25% (P < 0.05) after 3 weeks of exercise. Collagen-2 and Sox-9 mRNA were strongly expressed in the nucleus pulposus (NP) samples of the running group, but weakly expressed in the controls. An increase in collagen-II, aggrecan, and Sox-9 protein expression in NP and AF regions of the disc was detected in the exercised rats compared with controls. Quantification of Alcian blue staining demonstrated increased proteoglycan in both NP (8-fold) and AF (7-fold) in the exercised group compared with controls (P < 0.05). In addition, no significant differences were observed between the experimental groups in cellular apoptosis, collagen-I, or Sox-6 expression. CONCLUSION: In this study, increased extracellular matrix production and cell proliferation with no induction of disc cell apoptosis was observed in the lumbar IVD after a 3-week running regimen in rats, suggesting that regular exercise may have an augmentative effect on cells and matrix production.

Posterolateral intertransverse spinal fusion possible in osteoporotic rats with BMP-7 in a higher dose delivered on a composite carrier.

STUDY DESIGN: Posterolateral intertransverse process spinal fusion (PLF) was performed in ovariectomized female rats using recombinant human BMP-7 (OP-1) delivered on a composite carrier. OBJECTIVE: To investigate whether BMP-7 collagen on a composite carrier in a higher dose will enhance posterolateral spinal fusion in an estrogen deficiency rat model. SUMMARY OF BACKGROUND DATA: Osteoporosis is a systemic disease characterized by bone remodeling skewed in favor of excess bone resorption. This makes new bone formation and fixation of metallic implants difficult. Thus, treating osteoporotic patients who require posterior spinal fusion is challenging. Ovariectomized rats have been used as an osteoporotic model for posterolateral intertransverse process fusion. We have demonstrated in the past that endochondral bone formation in osteoporotic rats is delayed when compared with rats without osteoporosis. We have also shown that OP-1 Putty (BMP-7, collagen, and carboxy-methyl-cellulose) can overcome the effects of osteoporosis in a rat fracture model. However, it has not yet been demonstrated whether BMP-7 collagen composite carrier (Calstrux) can achieve a fusion in a process spinal fusion model in osteoporotic bone. METHODS: A total of 42 ovariectomized Sprague-Dawley female rats were randomly assigned to 4 control and 2 experimental groups: (1) no Calstrux, no BMP; (2) 400 mg Calstrux alone; (3) 30 microg lactose + 400 mg Calstrux; (4) 90 microg lactose + 400 mg Calstrux; (5) 30 microg rhBMP-7 + 400 mg Calstrux; and (6) 90 microg rhBMP-7 + 400 mg Calstrux. Spinal fusion was evaluated by manual motion testing, microradiographs, computed tomographic scans, DEXA scans, and histology. RESULTS: Ovariectomized rats receiving Calstrux alone or either dose of lactose and Calstrux did not show spinal fusion. Ovariectomized rats receiving 90 microg BMP-7 + 400 mg Calstrux showed significantly higher fusion rates than these control animals. (P < 0.0001). The rats receiving 30 microg BMP-7 + 400 mg Calstrux exhibited only partial fusion. CONCLUSION: BMP-7, delivered on a composite carrier, is able to overcome the detrimental effects of estrogen deficiency on posterolateral spinal fusion and generate a relatively robust fusion. The effect seems to be dose dependent.

Nucleus pulposus cellular longevity by telomerase gene therapy.

STUDY DESIGN: Nonviral transfection of nucleus pulposus cells with a telomerase expression construct to assess the effects on cellular lifespan, function, karyotypic stability, and transformation properties. OBJECTIVES: To investigate whether telomerase gene therapy can extend the cellular lifespan while retaining functionality of nucleus pulposus cells in a safe manner. SUMMARY OF BACKGROUND DATA: Degeneration of the intervertebral disc is an age-related condition in which cells responsible for the maintenance and health of the disc deteriorate with age. Telomerase can extend the cellular lifespan and function of other musculoskeletal tissues, such as the heart, bones, and connective tissues. Therefore, extension of the cellular lifespan and matrix production of intervertebral disc cells may have the potential to delay the degeneration process. METHODS: Ovine nucleus pulposus cells were lipofectamine transfected in vitro with a human telomerase reverse transcriptase (hTERT) expression construct. Cellular lifespan and matrix transcript levels were determined by cumulative population doublings and real-time RT-PCR, respectively. G1-cell cycle checkpoint, p53 functionality, growth of transfected cells in anchorage-independent or serum starvation conditions, and karyotypic analysis were performed. RESULTS: Transfection was achieved successfully with 340% +/- 7% (mean +/- SD) relative telomerase activity in hTERT-transfected cells. hTERT transfection enabled a 50% extension in mean cellular lifespan and prolonged matrix production of collagen 1 and 2 for more than 282 days. Karyotypic instability was detected but G1-cell cycle checkpoint and p53 was functionally comparable to parental cells with no growth in serum starvation or anchorage-independent conditions. CONCLUSIONS: Telomerase can extend the cellular lifespan of nucleus pulposus cells and prolong the production of extracellular matrix. Safety is still unresolved, as karyotypic instability was detected but no loss of contact inhibition, mitogen dependency, or G1-cell cycle checkpoint control was evident.