PTTG1 Levels Are Predictive of Saracatinib Sensitivity in Ovarian Cancer Cell Lines.

Src kinase is recognized as a key target for molecular cancer therapy. However, methods to efficiently select patients responsive to Src inhibitors are lacking. We explored the sensitivity of ovarian cancer cell lines to the Src kinase inhibitor saracatinib to identify predictive markers of drug sensitivity using gene microarrays. Pituitary tumor transforming gene 1 (PTTG1) was selected as a potential biomarker as mRNA levels were correlated with saracatinib resistance, as well as higher PTTG1 protein expression. PTTG1 expression was correlated with proliferation, cell division, and mitosis in ovarian cancer tissues data sets. In sensitive cell lines, saracatinib treatment decreased PTTG1 and fibroblast growth factor 2 (FGF2) protein levels. Downregulating PTTG1 by siRNAs increased saracatinib sensitivity in two resistant cell lines. Our results indicate PTTG1 may be a valuable biomarker in ovarian cancer to predict sensitivity to saracatinib, and could form the basis of a targeted prospective saracatinib trial for ovarian cancer.

Neutralizing IL-17 prevents obliterative bronchiolitis in murine orthotopic lung transplantation.

Obliterative bronchiolitis (OB) is the key impediment to the long-term survival of lung transplant recipients and the lack of a robust preclinical model precludes examining OB immunopathogenesis. In the current study, lungs from C57BL/10 H-2(b) mice that are MHC compatible, but minor histocompatability antigen incompatible, were transplanted into C57BL/6 mice. Histological features and cytokine profiles of OB were assessed. Moderate rejection (grade A3) developed by day 14, with evidence of OB at that time point. At 21 days, OB was present in 55% of grafts and moderate to severe rejection (grade A3-A4) was present in all mice. At 28 days, OB was present in 44% of mice and severe rejection (grade A4) was present in all. IL-17A, but not IL-17F, splenic mRNA transcripts and serum protein levels were increased only in mice that developed OB, whereas IL-10 transcripts and protein were increased only in non-OB mice. Neutralizing IL-17 prevented OB, down regulated acute rejection, and upregulated systemic IL-10. Collectively, these data show that transplantation of minor histoincompatible lungs from C57BL/10 mice into C57BL/6 mice results in a highly reproducible preclinical model of OB. In addition, these data indicate that neutralizing IL-17A or augmenting IL-10 could be therapeutic interventions to prevent OB.

Lung transplant metalloproteinase levels are elevated prior to bronchiolitis obliterans syndrome.

Parenchymal disease in the allograft lung is associated with interstitial remodeling believed to be mediated by matrix metalloproteinases (MMPs). Recent studies suggest high levels of MMP-9 are associated with bronchiolitis obliterans syndrome (BOS) in lung transplant recipients. Since BOS occurs late in the posttransplant period and may be preceded by episodes of acute rejection or infection, which are associated with interstitial remodeling, we examined MMP profiles in allograft bronchoalveolar lavage (BAL) fluid in the early posttransplant period (preceding BOS). Gelatin zymography, protein array analysis and specific ELISA on BAL fluids from transplanted lungs indicated that MMP-8, MMP-9 and TIMP-1 were strongly expressed in allograft BAL fluid from stable patients, or those with infection or rejection compared to BAL fluid from normal volunteers. Elevated expression of MMP-8, MMP-9 and TIMP-1 occurred early, and was sustained for the 3.2 years covered in this study. Elevations of MMP-8, MMP-9 and TIMP-1 in the first 2 years posttransplant appear to be associated with lung transplantation itself, and not infection or rejection. These data suggest that ongoing and clinically silent MMP activity could perpetuate progressive disease in the allograft lung.

Severity of medial meniscus damage in the canine knee after anterior cruciate ligament transection.

OBJECTIVE: To examine the relationship between the severity of cartilage damage and the severity of meniscus damage after transection of the anterior cruciate ligament (ACLT) in adult dogs. DESIGN: Data were obtained from 40 dogs which underwent ACLT and from three additional sham-operated dogs that were subjected to arthrotomy but not ligament transection. Joint pathology was analysed 12, 24 or 32 weeks after surgery. The severity of damage to the articular cartilage on the femoral condyle and tibial plateau was graded with a scoring system based on that of the Sociètè Française d'Arthroscopie and meniscus damage was graded on a 0-4 scale. RESULTS: No damage to the meniscus or articular cartilage was observed 12 weeks after surgery in the dogs subjected only to arthrotomy. In contrast, tears of the medial meniscus were observed in two of 10 (20%) dogs examined 12 weeks after ACLT. The incidence of severe tears increased to 86% and 84% after 24 weeks and 32 weeks, respectively. Damage to the lateral meniscus was mild, with only 7.5% of all dogs with a cruciate-deficient knee having a bucket handle or complete tear. Most of the unstable knees exhibited ulceration of the articular cartilage of the femoral condyles and tibial plateaus 12 weeks (mean chondropathy score+/-standard deviation 11.9+/-8.5, N=10), 24 weeks (7.9+/-5.0, N=7), and 32 weeks (7.1+/-5.5, N=23) after ACLT. The mean chondropathy scores for the tibial plateaus were similar to those for the femoral condyles. No correlation was apparent between the severity of cartilage damage and of meniscus damage for either joint surface. CONCLUSION: Damage to the medial meniscus is a consistent feature of the pathology which develops in the canine knee after ACLT, but the severity of cartilage damage is not correlated with the severity of meniscal damage.

Effect of intraarticular hyaluronan injection on synovial fluid hyaluronan in the early stage of canine post-traumatic osteoarthritis.

OBJECTIVE: To determine how the quantity and molecular weight of synovial fluid hyaluronan (HA) within the synovial fluid (SF) of osteoarthritis (OA) joints is affected by intraarticular injection of HA. METHODS: Dogs in which OA was induced by transection of the anterior cruciate ligament received 5 weekly injections of HA (1.5 x 10(6) Da) in saline (10 mg/0.67 ml) or an equal volume of saline into the operated knee, beginning the day after surgery. Immediately before each injection, SF was aspirated and the volume of SF and the concentration of HA was measured (uronic acid), and the molecular weight of the HA in each sample was estimated by electrophoresis in agarose. RESULTS: The volume of SF in the unstable knee increased after surgery, and the molecular weight decreased from approximately 2.5 x 10(6) Da to approximately 2 x 10(6) Da. Injection of HA did not affect the volume of SF or average molecular weight of HA in samples obtained immediately before each injection or at the end of the experiment, 12 weeks after surgery. The SF HA concentration fell from a baseline value of 2.3 +/- 0.1 mg/ml to 1.1 +/- 0.2 mg/ml the day after surgery and remained low throughout the course of injections. The HA concentration 12 weeks after surgery in the HA injected knees was approximately 40% lower than the preoperative value, although it increased slightly relative to saline injected knees (1.4 +/- 0.3 vs 1.1 +/- 0.01 mg/ml, respectively; p = 0.04). CONCLUSION: Intraarticular injection of HA did not alter the volume of SF or molecular weight of HA in SF of OA canine knees, nor did it restore the HA concentration to that of normal canine SF.

Specificity of inhibition of matrix metalloproteinase activity by doxycycline: relationship to structure of the enzyme.

OBJECTIVE: To investigate the inhibition of matrix metalloproteinase 1 (MMP-1), MMP-8, and MMP-13 by doxycycline, and to determine whether the variable hemopexin-like domain of each MMP was responsible for the differences in susceptibility to doxycycline inhibition among these collagenases. METHODS: Recombinant human MMP-1 (collagenase 1), MMP-8 (collagenase 2), and MMP-13 (collagenase 3), truncated forms of MMP-8 and MMP-13 lacking the hemopexin-like domain, and a mutant form of truncated MMP-13 were used in these studies. The activity of the full-length MMP in the presence of doxycycline was tested against type II collagen, a natural substrate for the enzymes. A small peptolide substrate was used to determine which structural features of the MMPs were related to sensitivity to doxycycline inhibition. RESULTS: The activity of MMP-13 and MMP-8 against type II collagen was inhibited by 50-60% by 30 microM doxycycline, while that of MMP-1 was inhibited only 18% by 50 microM doxycycline. In contrast, in experiments with the peptolide substrate, neither full-length nor truncated MMP-13 was inhibited until the concentration of the drug exceeded 90 microM. MMP-8 and truncated MMP-8 were sensitive to inhibition by 30 microM doxycycline, while MMP-1 was slightly inhibited (14%) by 90 microM doxycycline. For MMP-8, inhibition was reversible upon dilution and was independent of the order in which the reagents were added. Kinetic analysis of the inhibition constant (K(i)) of MMP-8 (K(i) = 36 microM) and truncated MMP-8 (K(i) = 77 microM) indicated that inhibition was noncompetitive. CONCLUSION: Significant inhibition of MMP-13 and MMP-8 activity against collagen occurred in vitro at concentrations that were near the concentrations achieved in serum after oral dosing. Studies with truncated enzymes and 2 substrates suggest that doxycycline disrupts the conformation of the hemopexin-like domain of MMP-13 and the catalytic domain of MMP-8.

Diacerhein treatment reduces the severity of osteoarthritis in the canine cruciate-deficiency model of osteoarthritis.

OBJECTIVE: To determine if diacerhein protects against the early stages of joint damage in a canine model of osteoarthritis (OA). METHODS: OA was induced in 20 adult mongrel dogs by transection of the anterior cruciate ligament of the left knee. Beginning the day after surgery, dogs in the active treatment group were dosed twice a day with capsules of diacerhein, providing a total daily dose of 40 mg/kg, for 32 weeks. Dogs in the control group received placebo capsules on the same schedule. Pathology in the unstable knee was assessed arthroscopically 16 weeks after surgery and by direct observation when the dogs were killed 32 weeks after surgery. The severity of gross joint pathology was recorded, and samples of the medial femoral condyle cartilage and the synovial tissue adjacent to the central portion of the medial meniscus were collected for histologic evaluation. Water content and uronic acid concentration of the articular cartilage from the femoral condyle were determined, and collagenolytic activity in extracts of cartilage pooled from the medial and lateral tibial plateaus was assayed against 14C-labeled collagen fibers. RESULTS: Diacerhein treatment slowed the progression of OA, as measured by grading of gross changes in the unstable knee at arthroscopy 16 weeks after cruciate ligament transection (P = 0.04) and at the time the animals were killed, 32 weeks after surgery (P = 0.05). However, 32 weeks after ACL transection, the mean proteoglycan concentration and water content of the OA cartilage and the level of collagenolytic activity in extracts of the cartilage were not significantly different in the diacerhein treatment group than in the placebo treatment group. CONCLUSION: Diacerhein treatment significantly reduced the severity of morphologic changes of OA compared with placebo. These findings support the view that diacerhein may be a disease-modifying drug for OA.

Effect of intraarticular hyaluronan injection in experimental canine osteoarthritis.

OBJECTIVE: To determine if intraarticular injections of hyaluronan (HA) protect against the early stages of joint damage in a canine model of osteoarthritis (OA). METHODS: OA was induced in adult mongrel dogs by transection of the anterior cruciate ligament of the left knee. One group of dogs (n=7) was treated with 5 weekly injections of HA (MW 1,500,000) into the operated knee beginning 1 day after ligament transection. The control group (n=6) was injected with saline on the same schedule. Twelve weeks after surgery, all dogs were killed, the severity of pathologic changes of OA was graded, and composition of the cartilage and extent of aggregation of proteoglycans (PGs) synthesized in vitro by cartilage slices were determined. RESULTS: All dogs showed gross morphologic changes typical of OA in the unstable knee. The severity of joint pathology in HA-treated dogs was comparable with that in the saline-injected controls. In OA cartilage from the saline-treated group, the mean uronic acid concentration was 30-60% greater than that in the contralateral knee. In sharp contrast, the uronic acid concentration in OA cartilage from the HA-treated dogs was 10-30% lower than that in cartilage from the contralateral knee (P=0.02 and P=0.03, respectively, for samples from the medial and lateral femoral condyle). The extent of aggregation of PG synthesized in vitro by cartilage from HA-injected animals was similar to that synthesized by cartilage from the saline-injected dogs. CONCLUSION: In this canine model of OA, the series of intraarticular injections of HA did not alter development of osteophytosis or fibrillation. However, the PG concentration of cartilage in the OA knee was significantly reduced by this treatment, suggesting that HA therapy might adversely affect the biomechanical properties of the cartilage.

Inhibition of recombinant human neutrophil collagenase by doxycycline is pH dependent.

OBJECTIVE: To examine, as part of an evaluation of the role of matrix metalloproteinase (MMP) inhibition in the amelioration of cartilage damage by doxycycline, the effect of pH on the inhibition of activity and reduction in stability of recombinant human neutrophil collagenase (rhMMP-8) by doxycycline in vitro. METHODS: After activation with trypsin, rhMMP-8 was assayed using a peptolide substrate and a colorimetric assay. The rate of hydrolysis in the presence and absence of 30 microM doxycycline was measured over a pH range of 6.5-7.9. The molecular weight changes that accompanied activation of the proenzyme by acetylphenylmercuric acetate (APMA) in the presence and absence of doxycycline at pH 6.9 and 7.5 were studied by Western blotting. RESULTS: At pH values above 7.1, doxycycline inhibited the activity of the enzyme. At pH values below 7.1, no inhibition was observed. When doxycycline was present during activation with APMA at pH 7.5, significant amounts of small (< 30 kDa) fragments were generated. In contrast, when doxycycline was present during activation with APMA at pH 6.9, no small fragments were detected. CONCLUSION: The ability of doxycycline to inhibit matrix rhMMP-8 activity or to promote its degradation is lost at pH values lower than 7. Although relatively high pH values may exist in adult articular in some pathological situations, at lower pH the effect of doxycycline on proenzyme levels in the extracellular matrix may be due to an effect on the regulation of synthesis of the proenzyme, rather than to direct inhibition of the active enzyme or reduction in the level of enzyme by proteolysis.

Synthesis of a phenotypically abnormal type XI: type II collagen ratio by chondrocytes from canine osteoarthritic cartilage.

Collagen synthesis by osteoarthritic cartilage from dogs that had undergone anterior cruciate ligament transection was measured in short-term organ cultures and chondrocyte suspension cultures. Slices of osteoarthritic cartilage from unstable knees 2, 6 or 12 weeks after anterior cruciate ligament transection converted approximately 10% of the total incorporated 14C-proline to 14C-hydroxyproline, while the value for cartilage from the contralateral knee or from knees of normal control dogs was generally less than 1%. The increase in collagen synthesis in the osteoarthritic cartilage was not related to the duration of knee instability, but was greater in grossly fibrillated cartilage than in the total pooled cartilage from the osteoarthritic joint. The collagen that was synthesized was predominantly type II, although some type XI and/or type V collagen was also synthesized. Chondrocytes isolated from osteoarthritic knee cartilage of dogs 12 weeks after anterior cruciate ligament transection showed changes in collagen synthesis similar to those seen in the cartilage organ cultures. As in the organ culture studies, type II collagen was the predominant species synthesized. When cell-associated collagen was considered, type II collagen accounted for 87+/-7% of the total collagen synthesized and retained by the osteoarthritic chondrocytes. The corresponding value for cells from the contralateral knee was 63+/-10%. Since the collagen fiber in articular cartilage is a heteropolymer of type II, type XI and type IX collagen, it is likely that the newly synthesized collagens in this model of osteoarthritis form fibers that are phenotypically different from those in normal articular cartilage.

Cleavage of type XI collagen fibers by gelatinase and by extracts of osteoarthritic canine cartilage.

Gelatinase (matrix metalloproteinase 2) purified from culture medium of MDCK cells by affinity chromatography on gelatin-sepharose was tested against type XI collagen. The purified enzyme-digested native type XI collagen in solution, and as reconstituted fibers, at 30, 34, and 37 degrees C. Both substrates yielded the same digestion products, as characterized by SDS-polyacrylamide gel electrophoresis, but the soluble collagen was cleaved at a higher rate. The first major product seen was an 87-kDa peptide, which was usually associated with one or two peptides migrating between it and alpha 3(XI). With time, a second group of 3 peptides appeared at 78, 75, and 73 kDa. After continued digestion, a third group of peptides was detected with prominent 69- and 67-kDa peptides and minor peptides at 71, 65, and 62 kDa. In overnight (20 hour) digestions, the 60-kDa digestion product accumulated and most of the larger digestion products could no longer be detected. Minor products at 71, 55, and 50 kDa were also noted in these limited digestions. Under the same conditions, denatured type XI was digested to fragments smaller than 13.5 kDa. The enzyme was inhibited by 1,10-phenanthroline or EDTA. Two purified components of cartilage matrix, type II collagen and proteoglycan subunit, as well as crude cartilage homogenates, were not effective inhibitors of the purified enzyme. Similar activity was extracted from canine articular cartilage, and the activity was much stronger in cartilage from osteoarthritic joints than from control joints.