Angiostatin and Endostatin

Angiostatin, a proteolytic fragment of plasminogen composed of kringles 1-4, originally isolated from the urine of C57Bl mice bearing the LLC, was shown to be an inhibitor of angiogenesis in several assays, and to inhibit the growth of tumor metastases, by O' Reilly et al. (109). Angiostatin can be generated in vitro by the limited proteolysis of human plasminogen by elastase (110,111). The enzymatic mechanism by which angiostatin is generated in vivo remains unknown. Angiostatin was shown to inhibit the proliferation of endothelial cells in culture, and to induce tumor dormancy as defined by a balance of apoptosis and proliferation, resulting in no growth in lung metastases (112). O'Reilly et al. (112) went on to show that systemic administration of angiostatin to SCID mice bearing human tumor xenografts could inhibit the growth of those tumors, including PC-3 prostate carcinoma, Clone A colon carcinoma, and MDA-MB breast carcinoma. Administration of human angiostatin also inhibited the growth of the murine LLC, the T214 fibrosarcoma, and the M5076 reticulum cell sarcoma. The angiostatin was administered at a dose of 50 mg/kg every 12 h. Gately et al. (113) reported that human prostate carcinoma cells in culture (PC-3, DU-145, and

Table 20

Number of Intratumoral Blood Vessels as Determined by Immunohistochemical Staining for CD31 of LLC Tumors After Treatment of Animals with Various Potential Antiangiogenic Agents

Treatment group

Number of intratumoral vessels by CD31 positivity

Controls

TNP-470 (30 mg/kg) sc, alternate d 0-8 Angiostatin + TNP-470 TNP-470 + minocycline (10 mg/kg) ip, d 0-8 Angiostatin + TNP-470 + minocycline

LNCaP cells) can cleave human plasminogen to angiostatin through the action of serine protease(s). The Folkman laboratory has investigated the structural requirements for the activity of angiostatin, and found that the activity of this complex protein is centered primarily in kringles 2 and 3 of the protein (114). Kringle 1 has some antiangiogenic activity, but kringle 4 is inactive.

The ability of angiostatin to inhibit blood vessel formation in the LLC was determined by immunohistochemical staining of tumors from animals that had been treated twice per day with recombinant murine angiostatin, and compared with animals treated with TNP-470, TNP-470 along with minocycline, angiostatin along with TNP-470, or with the three-drug combination of angiostastin-TNP-470-minocycline (Table 20). Angiostatin administered at a dose of 10 mg/kg twice per day, beginning the day of tumor implantation, decreased the number of stainable intratumoral vessels to 65% of the number found in the untreated control tumors. TNP-470 deceased the number of intratumoral vessels to 51% of the number found in the controls; angiostatin, along with TNP-470, and TNP-470, along with minocycline, were a bit less effective. The three-drug combination of angiostatin-TNP-470-minocycline decreased the number of stainable intratumoral vessels to 31% of the number found in the untreated control animals. Administration of angiostatin on this same schedule of 10 mg/kg, sc twice per day, beginning the day of tumor cell implantation, resulted in a twofold increase in the killing of EMT-6 murine mammary tumor cells from tumors by cyclophosphamide.

Recently, O'Reilly et al. (115) identified another antiangiogenic protein isolated from a hemangioendothelioma and called endostatin. Endostatin is a smaller protein than angiostatin, and is a 20-kDA C-terminal fragment of collagen XVIII. Endostatin has been shown to inhibit the growth of the LLC when administered at doses of 10 or 20 mg/kg daily by intraperitoneal injection. Similar results were found in animals bearing the T241 fibrosarcoma, the B16 melanoma, and the EOMA hemangioendothelioma.

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