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Suppression of tumor growth with recombinant murine angiostatin
Z Wu1, M S O'Reilly, J Folkman
1Department of Surgery, Children's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
Abstract:
Angiostatin, a 38 kDa internal fragment of plasminogen, is an antiangiogenic endothelial cell inhibitor. It regresses several primary and metastatic tumors in mice. To produce recombinant angiostatin for further structural and functional studies, the mouse angiostatin gene preceded by a sequence including a signal peptide of plasminogen was introduced into baculovirus. Recombinant murine angiostatin was purified from the culture medium of angiostatin baculovirus-infected insect cells (yield = 1 mg/liter) with a single-step of lysine-Sepharose chromatography. The angiostatin baculovirus-infected insect cells expressed and secreted a 52 kDa polypeptide that demonstrated all of the biological activities of angiostatin. A partial amino acid sequence of the NH2-terminus of the secreted protein revealed that the signal peptide was recognized and properly cleaved in insect cells. The recombinant murine angiostatin potently inhibited the proliferation of bovine capillary endothelial cells in vitro (half maximal inhibition = 50 ng/ml) and suppressed the growth of primary Lewis lung carcinoma in vivo (6 mg/kg/day, T/C = 0.08).
Insights
Researchers produced recombinant murine angiostatin, an anti-cancer protein, using insect cells. This engineered angiostatin effectively inhibited tumor cell growth in vitro and suppressed primary tumor growth in vivo.
Area of Science:
- Molecular Biology
- Biochemistry
- Cancer Research
Background:
- Angiostatin, a plasminogen fragment, inhibits endothelial cell proliferation and tumor angiogenesis.
- Previous studies demonstrated angiostatin's potential in regressing primary and metastatic tumors in vivo.
- Efficient production of recombinant angiostatin is crucial for detailed structural and functional investigations.
Purpose of the Study:
- To establish a system for producing recombinant murine angiostatin using a baculovirus expression vector.
- To characterize the biological activity of the recombinant protein.
- To evaluate the efficacy of recombinant angiostatin in preclinical cancer models.
Main Methods:
- The mouse angiostatin gene, with a signal peptide sequence, was cloned into a baculovirus vector.
- Recombinant baculovirus was used to infect insect cells for protein expression and secretion.
- Single-step purification using lysine-Sepharose chromatography.
- In vitro proliferation assays using bovine capillary endothelial cells.
- In vivo studies using Lewis lung carcinoma model in mice.
Main Results:
- Recombinant murine angiostatin was successfully expressed and secreted by infected insect cells as a 52 kDa polypeptide.
- Purification yielded 1 mg/liter of recombinant angiostatin with high purity.
- The secreted protein exhibited potent inhibition of endothelial cell proliferation in vitro (IC50 = 50 ng/ml).
- In vivo administration suppressed primary Lewis lung carcinoma growth (T/C = 0.08 at 6 mg/kg/day).
- Signal peptide cleavage was confirmed by N-terminal sequencing, indicating proper processing in insect cells.
Conclusions:
- The baculovirus expression system provides an efficient method for producing biologically active recombinant murine angiostatin.
- Recombinant angiostatin demonstrates potent anti-proliferative effects on endothelial cells and significant anti-tumor activity in vivo.
- This system facilitates further structural and functional studies of angiostatin for potential therapeutic applications.