ABSTRACT
Many malignant tumors express high levels of the chemokine receptor CXCR4, and the interaction between CXCR4 and its ligand, SDF-1, promotes migration, invasion, and metastasis of breast cancer cells. Therefore, blocking the interaction between CXCR4 and SDF-1 could alter the tumor's metastatic phenotype and control the development and progression of cancers. We used a cellular phenotypic knockout strategy and developed a novel recombinant gene, AdSDF-1α/54/KDEL, which contains an adenovirus vector, a mutant form of SDF-1 that lacks a C-terminal α-helix, and a KDEL tetrapeptide sequence that promotes retention at the endoplasmic reticulum (ER). We hypothesized that SDF-1α/54/KDEL could efficiently block metastasis of breast cancer cells with less inflammatory side effects than SDF-1α/KDEL. Using the MCF-7 cell line, which expresses a stable, high level of CXCR4, we found that SDF-1α/54/KDEL efficiently becomes localized at the ER of tumor cells, where it specifically binds to newly synthesized CXCR4 and prevents it from reaching the cell surface. Chemotaxis and invasion assays revealed that the cells treated with SDF-1α/54/KDEL failed to migrate toward SDF-1. We also found that SDF-1α/54/KDEL impaired lung metastasis of metastatic breast cancer by decreasing CXCR4 on the cell surface. The novel recombinant gene, SDF-1α/54/KDEL, played an instrumental role in blocking SDF-1/CXCR4-mediated cell migration, and we found that this gene-based strategy for targeting the SDF-1/CXCR4 axis offers a very effective alternative method for preventing metastasis of breast cancer and other cancers expressing high levels of CXCR4.
Subject(s)
Breast Neoplasms/genetics , Breast Neoplasms/pathology , Chemokine CXCL12/genetics , Mutation , Receptors, CXCR4/genetics , Receptors, Peptide/genetics , Animals , Breast Neoplasms/metabolism , Cell Movement , Cell Proliferation , Cell Survival/genetics , Disease Models, Animal , Female , Gene Expression , Gene Knockout Techniques , Humans , Mice , Neoplasm Metastasis , Receptors, CXCR4/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolismABSTRACT
Although doxorubicin (Dox) is widely used in clinical treatment for solid tumors, it causes many side-effects such as heart and kidney damage, bone marrow suppression, and drug resistance. Legumain is a lysosomal protease that is elevated and associated with an invasive and metastatic phenotype in a number of solid tumors. In this study, we designed and synthesized a Dox prodrug, N-benzyloxycarbonyl-Ala-Ala-Asn-Doxorubicin (CBZ-AAN-Dox), with 94% purity. Single substrate kinetic assays demonstrated hLegumain-specific enzymatic cleavage and activation of the prodrug in vitro, and this enzymatic cleavage of the prodrug substrate was more sensitive in acidic conditions, releasing more than 70% of Dox after 24 h. Treatment of tumor cells with our prodrug demonstrated a much higher IC50 value, significantly enhanced uptake of the prodrug, and considerably less cellular toxicity compared to Dox treatment alone. Our study presents a novel prodrug, CBZ-AAN-Dox, to potentially increase both the safety and efficacy of clinical treatment of tumors by exploiting the tumor's innate expression of legumain.
Subject(s)
Doxorubicin/analogs & derivatives , Doxorubicin/pharmacology , Oligopeptides/chemical synthesis , Prodrugs/chemical synthesis , Uterine Cervical Neoplasms/drug therapy , Apoptosis/drug effects , Cell Hypoxia/drug effects , Cell Line, Tumor/drug effects , Cell Proliferation/drug effects , Chemistry Techniques, Synthetic , Cysteine Endopeptidases/genetics , Cysteine Endopeptidases/metabolism , Doxorubicin/chemical synthesis , Doxorubicin/chemistry , Drug Screening Assays, Antitumor/methods , Female , Humans , Inhibitory Concentration 50 , Oligopeptides/pharmacology , Prodrugs/pharmacokinetics , Prodrugs/pharmacology , Uterine Cervical Neoplasms/pathologyABSTRACT
BACKGROUND: Strategies that reduce ischemia-reperfusion injury (IRI) have the potential to expand the numbers of available organs for transplantation. Recent reports in rodent models have demonstrated that high-mobility group box 1 (HMGB1) acts as an alarm in initiating the inflammatory response resulting from ischemic injury. The aim of this study was to evaluate the cytoprotective effects of anti-HMGB1 antibodies on renal IRI in preclinical large animals. METHODS: One hundred twenty minutes of warm and 60 min of cold renal ischemia were induced in 8 CLAWN miniature swine. Three of eight animals received intravenous anti-HMGB1 antibody at 1 mg/kg just before the reperfusion of renal blood flow. Renal function was assessed by serum creatinine and renal biopsy. Serum levels of interleukin (IL)-1ß, IL-6, and HMGB1 were measured. RESULTS: The concentration of HMGB1 increased as early as 30 min after reperfusion and before the elevation of IL-1ß and IL-6. Serum creatinine levels were markedly elevated, peaking at a median of 5 days (peak creatinine levels: 11.6 ± 1.6 mg/dL) and recovering by day 14. Anti-HMGB1 antibody injection dramatically decreased renal damage as well as serum levels of HMGB1 associated with IRI. Renal function returned to near normal by day 9, and peak creatinine levels were markedly lower (7.4 ± 0.2 mg/dL), and biopsies possessed fewer pathologic changes when compared to the control group. CONCLUSION: In this study, we demonstrated the beneficial effects of perioperative administration of anti-HMGB1 antibody in reducing renal IRI in a clinically relevant, large animal model.
Subject(s)
Antibodies/immunology , HMGB1 Protein/antagonists & inhibitors , Kidney Diseases/pathology , Kidney/pathology , Reperfusion Injury/pathology , Animals , Apoptosis , Biopsy , Creatinine/blood , Cytoprotection , Disease Models, Animal , Female , HMGB1 Protein/blood , Inflammation , Interleukin-1beta/blood , Interleukin-6/blood , Ischemia , Kidney/immunology , Kidney Diseases/therapy , Male , Renal Circulation , Reperfusion Injury/therapy , Swine , Swine, Miniature , Time FactorsABSTRACT
The highest risk of venous thromboembolism lies with abdominoplasty and liposuction and deaths increase when combined with other procedures. The objective of this study was to evaluate physiologic changes in the deep venous system with compression-garments and asses whether there is a correlation between these procedures, rectus plication, and garments. Part 1 of this article is a retrospective review of all office surgical incidents that resulted in an abdominoplasty and/or liposuction death in the state of Florida over the past 8 years. Part 2 is a clinical study, evaluating the effect compression garments have on the pathophysiology of venous thromboembolism. Duplex ultrasounds were performed with and without garments to evaluate venous changes. Literature was reviewed related to intraabdominal pressure and high-tension abdominal closures and surgeons were polled about their use of rectus plication and garments/binders. In 8 years, 13 patients died related to abdominoplasty, liposuction, or the combination of the 2. In 100% of subjects, ultrasounds showed a decrease in venous flow, proximal vessel dilation, and loss of normal biphasic flow within the popliteal vein. Multiple publications have reported an increased morbidity and mortality related to liposuction and abdominoplasty procedures. The most frequent cause of death with these types of procedures was thromboembolism, and we discuss possible reasons for this increased rate. The exact cause of deep venous thrombosis in these procedures remains unknown, but postoperative garments may affect the physiology of venous flow, and may be involved in the formation of deep venous thrombosis and should be considered along with other patient safety procedures.
