| Send E-mail to: markland@usc.edu | |
| Telephone: 323-442-1607 | Fax: 323-442-1610 |
| Office: CRL 106 | Mail Code: 9075 HSC |
Education:
BS 1957 Agricultural & Biological Chemistry - Pennsylvania State University, State College, Pennsylvania
PhD 1964 Physiological Chemistry - Johns Hopkins University, Baltimore, Maryland
Postdoctoral Research Fellowship:
1964-1966 University of California, Los Angeles
Started at USC: 1973
Research Topics: Gene Therapy, Protein Chemistry/Enzymology, Cancer Treatment, Signal Transduction, Cardiovascular & Skeletal Muscle Diseases
Research Description
DIRECT ACTING BLOOD CLOT DISSOLVING ENZYME:
Pharmacologic dissolution of an established thrombus has become an accepted therapeutic approach for many patients who develop arterial or venous thrombotic occlusive disease. The major hypothesis of our work is that direct-acting fibrinolytic enzymes offer a unique alternative approach for thrombolytic therapy with theoretical, as well as practical, advantages over presently available plasminogen activator (PA)-based thrombolytic agents, such as tissue plasminogen activator. Fibrolase is a 23 kDa, direct-acting, fibrinolytic metalloproteinase, found in southern copperhead snake venom, that cleaves fibrin independently of plasmin(ogen). It is not inactivated by serine proteinase inhibitors in the blood, but is inhibited by α2-macroglobulin. Fibrolase lyses fibrin by selective cleavage of peptide bonds in the alpha and beta chains. The thrombolytic actions of fibrolase have been demonstrated in animal models of arterial and venous thrombosis (in the canine and the rabbit). Fibrolase does not activate platelets or coagulation factors, nor does it lyse red blood cells. The recombinant version of fibrolase, named alfimeprase, has been produced by a large Biotech Company using a yeast expression system and the recombinant version which is identical to fibrolase except for the truncation of two amino-terminal amino acids and an aditional change in the new amino-terminus, is being tested in patients for peripheral arterial occlusive disease (PAO) and stroke in two Phase III trials. The clinical trials are ongoing.
We are presently involved in studies with a colleague on the University Park Campus to determine the x-ray structure of the enzyme and have grown crystals that diffract to 1.7Ao. We are also collaborating with an investigator in the Viterbi Engineering School to synthesize a chemically modified form of fibrolase, which can be site-specifically photoactivated. That is we should be able to deliver the modified, inactive form of the enzyme and specifically photoactivate it at the site of a coronary thrombus (or heart attack). Our hope is that this would lead to opening of the obstructed coronary artery and serve as an effective form of therapy.
DISINTEGRINS - EFFECTIVE INHIBITORS OF CANCER GROWTH AND ANGIOGENESIS:
Spread of cancer to remote sites, e.g. bone, lungs, liver, brain, is a characteristic of malignancy and often leads to inoperable disease. Control of metastasis offers an important avenue for cancer treatment. The first step in metastasis involves adhesion of the cancer cells to tissue around the primary tumor site. The cancer cells secrete digestive enzymes that degrade the surrounding tissues allowing the tumor cells to invade into these tissues. Eventually, the tumor cells enter the blood or lymphatic system where they repeat the adhesion and invasion steps at a distant (metastatic) site. Agents that block any of the above steps should act to inhibit metastasis. In order for a tumor mass to grow beyond a size of 1-2 mm3 the development of a vascular network is required. This process is called angiogenesis or neovascularization. Tumor cells at either a primary site or at metastatic sites induce the formation of new blood vessels, and these vessels supply nutrients and growth factors to the tumor and, importantly, serve as a route for tumor dissemination (metastasis). Since cancer-induced angiogenesis is essential for progressive growth of cancer, therapies that block new blood vessel growth into the tumor will also inhibit tumor growth and are of considerable interest to the clinical community since they may provide a unique and practical way for long term control of cancer. Anti-angiogenic therapy promotes long-term dormancy of the tumor and is non-cytotoxic thereby avoiding side effects, such as gastrointestinal problems, loss of hair and bone marrow suppression, that accompany chemotherapy. Further, successfully blocking development of the vascular network may provide a useful alternative to chemotherapy that acts directly on tumor cells; attacking vascular cells would avoid the problem of acquired resistance to chemotherapy that results from genetic instability of tumor cells. We have been studying a protein from southern copperhead snake venom possessing potent anti-tumor activity that we call contortrostatin. Contortrostatin (CN) is a member of a family of peptides called disintegrins that are found in snake venoms. Members of this family are distinguished by the presence of an amino acid sequence, arginine-glycine-aspartic acid (RGD, that enables them to bind to cell surface receptors called integrins found on cancer cells and newly growing blood vessel (angiogenic) cells. Integrins mediate interactions between cells and their surroundings, and on cancer cells they play important roles in tumor invasion and spread. We postulated that since contortrostatin disrupts integrin interactions, it should block cancer or newly growing blood vessel cell integrins and may, therefore, have significant anti-angiogenic and anti-metastatic activity. In fact, we have shown that CN acts as an effective inhibitor of breast cancer progression. Importantly, CN displays impressive inhibitory activity on the growth of new blood vessels into the breast cancer. The RGD sequence in contortrostatin plays a critical role in binding to and inhibiting growth and spread of breast cancer cells. CN is a homodimer with two identical chains held together by two inter-chain disulfide bonds. We recently succeeded in producing a recombinant version of CN using an E. coli expression system. The recombinant protein is a monomer, called vicrostatin (VN), designed with a slightly different amino acid sequence. Using a delivery system in which VN is encapsulated in unilamellar lipid particles (liposomes), we have shown (see figure below) that intravenous delivery of the liposomal formulation (LVN) twice weekly in an animal model of human, metastatic breast cancer leads to close to 80% inhibition of tumor growth and over 90% inhibition of angiogenesis. Recently we have been examining the effect of VN on gene expression, using DNA array technology, and have some interesting preliminary findings. Presently we are using human breast, prostate and ovarian cancer and glioma (a devastating brain tumor) animal models to demonstrate the anti-tumor and anti-angiogenic activities of VN. We are also examining the mechanism of VN induction of integrin-mediated signal transduction pathways in both tumor and endothelial cells. Finally, in collaboration with an investigator on the Medical School Campus we are determining the three-dimensional structure of VN, which will ultimately enable us to examine the role of specific amino acids in biological activity by use of site-directed mutagenesis.
Figure legend: In Panel A we show the effect of liposomal VN (LVN) on breast cancer growth using MDA-MB-435 human, metastatic breast cancer cells implanted in the mammary fat pads of mice. LVN was delivered intravenously twice per week starting the 3rd week using 100ug per dose over a period of 7 weeks. For comparison the effect of phosphate buffered saline (PBS, control) and liposomal contortrostatin are shown (LCN). In Panel B, the effect of LVN on angiogenesis in the breast tumor in the nude mice is assessed by immunohistochemistry using CD31, a protein found on the surface of endothelial cells. Tumor sections were removed at the conclusion of the experiment and immunostained for CD31 as an indication of neovascularization. As can be seen, VN inhibited tumor growth by ~80% and angiogenesis by ~~90%.
Selected Publications
Moiseeva N, Bau R, Swenson SD, Markland FS Jr, Choe JY, Liu ZJ, Allaire M. - Structure of acostatin, a dimeric disintegrin from Southern copperhead (Agkistrodon contortrix contortrix), at 1.7 A resolution. - Acta Crystallogr D Biol Crystallogr [ 2008 ] Apr;64(Pt 4):466-70 . PubMed
Swenson S, Ramu S, Markland FS. - Anti-angiogenesis and RGD-containing snake venom disintegrins. - Curr Pharm Des [ 2007 ] 13(28):2860-71 . PubMed
Minea R, Swenson S, Costa F, Chen TC, Markland FS. - Development of a novel recombinant disintegrin, contortrostatin, as an effective anti-tumor and anti-angiogenic agent. - Pathophysiol Haemost Thromb [ 2005 ] 34(4-5):177-83 . PubMed
Swenson S, Costa F, Ernst W, Fujii G, Markland FS. - Contortrostatin, a snake venom disintegrin with anti-angiogenic and anti-tumor activity. - Pathophysiol Haemost Thromb [ 2005 ] 34(4-5):169-76 . PubMed
Pyrko P, Wang W, Markland FS, Swenson SD, Schmitmeier S, Schönthal AH, Chen TC. - The role of contortrostatin, a snake venom disintegrin, in the inhibition of tumor progression and prolongation of survival in a rodent glioma model. - J Neurosurg [ 2005 ] Sep;103(3):526-37 . PubMed
Schmitmeier S, Markland FS, Schönthal AH, Chen TC. - Potent mimicry of fibronectin-induced intracellular signaling in glioma cells by the homodimeric snake venom disintegrin contortrostatin. - Neurosurgery [ 2005 ] Jul;57(1):141-53; discussion 141-53 . PubMed
Swenson S, Markland FS Jr. - Snake venom fibrin(ogen)olytic enzymes. - Toxicon [ 2005 ] Jun 15;45(8):1021-39 . PubMed
Golubkov V, Garcia A, Markland FS. - Action of fenretinide (4-HPR) on ovarian cancer and endothelial cells. - Anticancer Res [ 2005 ] Jan-Feb;25(1A):249-53 . PubMed
Swenson S, Toombs CF, Pena L, Johansson J, Markland FS Jr. - Alpha-fibrinogenases. - Curr Drug Targets Cardiovasc Haematol Disord [ 2004 ] Dec;4(4):417-35 . PubMed
Swenson S, Costa F, Minea R, Sherwin RP, Ernst W, Fujii G, Yang D, Markland FS Jr. - Intravenous liposomal delivery of the snake venom disintegrin contortrostatin limits breast cancer progression. - Mol Cancer Ther [ 2004 ] Apr;3(4):499-511 . PubMed
