Surgical resection remains the best treatment for colorectal metastases isolated to the liver; however, 5-year survival rates following liver resection are only 40% to 50%, with liver recurrence being a significant reason for treatment failure. The ischemia-reperfusion (I/R) injury incurred during liver surgery can lead to cellular dysfunction and elevations in proinflammatory cytokines and matrix metalloproteinases (MMP). In rodents, I/R injury to the liver has been shown to accelerate the outgrowth of implanted tumors. The mechanism for increased tumor growth in the setting of liver I/R injury is unknown. To investigate the effect of I/R on tumor growth, an experimental model was used whereby small hepatic metastases form after 28 days. Mice subjected to 30 min of 70% liver ischemia at the time of tumor inoculation had significantly larger tumor number and volume, and had elevated MMP9 serum and liver tissue MMP9 as evidenced by zymography and quantitative real-time PCR. Mice treated with doxycycline, a broad-spectrum MMP inhibitor, had reduced MMP9 levels and significantly smaller tumor number and volume in the liver. MMP9-null mice were used to determine if the effects of doxycycline were due to the absence of stromalderived MMP9. The MMP9-null mice, with or without doxycycline treatment, had reduced tumor number and volume that was equivalent to wild-type mice treated with doxycycline. These findings indicate that hepatic I/R-induced elevations in MMP9 contribute to the growth of metastatic colorectal carcinoma in the liver and that postresection MMP9 inhibition may be clinically beneficial in preventing recurrence following hepatic surgery. [Cancer Res 2007;67(6):2720-8]
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Ischemia-reperfusion (I/R) injury is a commonly encountered clinical problem in liver surgery and transplantation. The pathogenesis of I/R injury is multifactorial, but mitochondrial Ca(2+) overload plays a central role. We have previously defined a novel pathway for mitochondrial Ca(2+) handling and now further characterize this pathway and investigate a novel Ca(2+)-channel inhibitor, 2-aminoethoxydiphenyl borate (2-APB), for preventing hepatic I/R injury. The effect of 2-APB on cellular and mitochondrial Ca(2+) uptake was evaluated in vitro by using (45)Ca(2+). Subsequently, 2-APB (2 mg/kg) or vehicle was injected into the portal vein of anesthetized rats either before or following 1 h of inflow occlusion to 70% of the liver. After 3 h of reperfusion, liver injury was assessed enzymatically and histologically. Hep G2 cells transfected with green fluorescent protein-tagged cytochrome c were used to evaluate mitochondrial permeability. 2-APB dose-dependently blocked Ca(2+) uptake in isolated liver mitochondria and reduced cellular Ca(2+) accumulation in Hep G2 cells. In vivo I/R increased liver enzymes 10-fold, and 2-APB prevented this when administered pre- or postischemia. 2-APB significantly reduced cellular damage determined by hematoxylin and eosin and terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling staining of liver tissue. In vitro I/R caused a dissociation between cytochrome c and mitochondria in Hep G2 cells that was prevented by administration of 2-APB. These data further establish the role of cellular Ca(2+) uptake and subsequent mitochondrial Ca(2+) overload in I/R injury and identify 2-APB as a novel pharmacological inhibitor of liver I/R injury even when administered following a prolonged ischemic insult.
Background Peripheral blood stem cells (PBSC) have become the preferred stem cell source for autologous hematopoietic transplantation. A critical aspect of this treatment modality is cryopreservation of the stem cell products, which permits temporal separation of the PBSC mobilization/collection phase from the subsequent high-dose therapy. While controlled rate freezing and liquid nitrogen storage have become “routine” practice in many cell processing facilities, there is clearly room for improvement, as current cryopreservation media formulations still result in significant loss and damage to the stem/progenitor cell populations essential for engraftment, and can also expose the patients to relatively undefined serum components and larger volumes of DMSO that can contribute to the morbidity and mortality of the transplant therapy. Methods This study compared cryopreservation of PBSC in a novel intracellular-like, fully defined, serum- and protein-free preservation solution, CryoStor™ (BioLife Solutions, Inc.), with a standard formulation used by the Fred Hutchinson Cancer Research Center (FHCRC). Briefly, human PBSC apheresis specimens were collected and 5 × 107 cells/1 ml sample vial were prepared for cryopreservation in the following solutions: 1) FHCRC standard – Normosol-R, 5% HSA, 10% DMSO, and 2) CryoStor™ CS10 (final diluted conc. of 5% DMSO). A standard controlled-rate freezing program was employed, and frozen vials were stored in the vapor phase of a liquid nitrogen freezer for a minimum of one week. Vials were then thawed and evaluated for TNC, Viability, CD34, and granulocytes by flow cytometry, along with colony-forming activity in methylcellulose. Results The PBSC samples frozen in CryoStor™ CS10 yielded significantly improved post-thaw recoveries for total viable CD34+, CFU, and viable granulocytes. Specifically, relative to the FHCRC standard formulation, cryopreservation with CS10 resulted in an average 1.8 fold increased recovery of viable CD34+ cells (P = 0.005), a 1.5 fold increase in CFU-GM numbers (P = 0.030), and a 2.3 fold increase in granulocyte recovery (P = 0.045). Discussion This study indicates that use of CryoStor™ for cryopreservation can yield significantly improved recovery and in vitro functionality of the stem/progenitor cells in PBSC products. In addition, it is important to note that these improved recoveries were obtained while also not introducing any extra serum or serum-derived proteins, and reducing the final concentration/volume of DMSO by half. Further in vitro and in vivo studies are clearly necessary, however these findings imply use of CryoStor™ for cryopreservation might ultimately result in improved engraftment for those patients with lower content of CD34+ cells in their PBSC collections, along with reducing the requirement for additional apheresis collections, and decreasing the risk of adverse infusion reactions associated with higher exposure to DMSO.
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