Studies indicate secreted cathepsins are involved in metastasis. V-ATPases, which are necessary for activating intracellular cathepsins, also play a role in metastasis and are targeted to the plasma membrane of metastatic breast cancer cells. We are interested in a connection between cell surface V-ATPases, activation of secreted cathepsins and the metastatic phenotype of MDA-MB231 cells. We investigated whether V-ATPase inhibition would reduce the activity of secreted cathepsin B and cathepsin L. Using cell lysates and conditioned media, we measured cathepsin B and L activity within and outside of the cells. We found different forms of cathepsin B and L were secreted representing the pre-pro, pro and active forms of the proteases. Cathepsin B activity was higher than cathepsin L in conditioned media and in cell lysates. V-ATPase inhibition by concanamycin A decreased cathepsin B activity in conditioned media and significantly decreased cathepsin B activity in cell lysates. Cathepsin L activity showed a slight decrease in cell lysates. Changes in the activity of secreted and intracellular cathepsins following V-ATPase inhibition were supported by changes in the amounts of pro and active forms of cathepsin B in conditioned media and cathepsins B and L in cell lysates. Overall, our data shows that inactive forms of cathepsins B and L are secreted from the MB231 cells and V-ATPase activity is important for the activation of secreted cathepsin B. This indicates a connection between cell surface V-ATPases in metastatic breast cancer cells and the function of secreted cathepsin B.
Studies indicate secreted cathepsins are involved in metastasis. V‐ATPases, which are necessary for activating intracellular cathepsins, also play a role in metastasis and are targeted to the plasma membrane of metastatic breast cancer cells. We are interested in a connection between cell surface V‐ATPases, activation of secreted cathepsins and the metastatic phenotype of MDA‐MB231 cells. We investigated whether V‐ATPase inhibition would reduce the activity of secreted cathepsin B and cathepsin L. Using cell lysates and conditioned media, we measured cathepsin B and L activity within and outside of the cells. We found different forms of cathepsin B and L were secreted representing the pre‐pro, pro and active forms of the proteases. Cathepsin B activity was higher than cathepsin L in conditioned media and in cell lysates. V‐ATPase inhibition by concanamycin A decreased cathepsin B activity in conditioned media and significantly decreased cathepsin B activity in cell lysates. Cathepsin L activity showed a slight decrease in cell lysates. Changes in the activity of secreted and intracellular cathepsins following V‐ATPase inhibition were supported by changes in the amounts of pro and active forms of cathepsins B in conditioned media and cathepsin B and L in cell lysates. Overall, our data shows that inactive forms of cathepsins B and L are secreted from the MB231 cells and V‐ATPase activity is important for the activation of secreted cathepsin B. This indicates a connection between cell surface V‐ATPases in metastatic breast cancer cells and the function of secreted cathepsin B.Support or Funding InformationDenison University Anderson Fellowship, Denison University Research FundThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Background The safety and feasibility of sterile, acellular pulley allografts in reconstruction has been previously demonstrated. Comparisons with tendon-based techniques for pulley reconstruction have not been reported. We hypothesized that the use of allograft pulleys would result in reduced procedural time and equivalent clinical outcomes as compared with traditional tendon-based reconstructive techniques. Methods All cases of pulley reconstruction using either allograft pulleys or tendon-based pulley reconstruction between November 2013 and November 2015 were reviewed. Patients who underwent concomitant procedures were excluded. Patient demographics, comorbidities, operative details (tourniquet and total operative times, number of pulleys repaired), postoperative complications (surgical site infection, reoperation, stiffness, and persistent pain), disability of the arm, shoulder and hand scores, and follow-up data were recorded. A P value of <0.05 was considered significant. Results Fifteen pulleys in 10 patients were reconstructed: 5 tendon-based and 5 with allograft. Average length of follow-up was 12.5 ± 2.9 months. There was no difference in patient demographic factors or comorbidities between groups. The most common indication for surgery was trauma. Four of 5 patients in the allograft group had multiple pulleys reconstructed versus 1 in the tendon-based group. One patient in the tendon-based group required reoperation versus 0 in the allograft group. Total operative and tourniquet times were significantly reduced in the allograft group (46 ± 5.5 vs 89 ± 12.9 minutes and 34 ± 6.8 vs 63 ± 5.3 minutes; P = 0.015 and 0.014). Postoperative disability of the arm, shoulder and hand scores were lower in the allograft group (56.8 vs 3.6, P = 0.11). There was no significant difference in postoperative range of motion between groups. Conclusion Pulley reconstruction with allograft is an efficient, technically feasible, reconstructive technique that adheres to the principle of replacing like with like, while eliminating donor site morbidity. Overall operative and tourniquet times were significantly shorter using allograft pulleys for pulley reconstruction.
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