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Insulin-like growth factor binding protein (IGFBP)-3 protease activity secreted by MCF-7 breast cancer cells:
H Salahifar1, R C Baxter, J L Martin
1Kolling Institute of Medical Research, University of Sydney, Royal North Shore Hospital, St. Leonards, New South Wales, Australia.
Abstract:
The proliferative action of insulin-like growth factors (IGFs) on breast cancer cells is regulated by IGF binding proteins (IGFBPs). This study characterizes the proteolysis of IGFBP-3 by an enzyme secreted by MCF-7 human breast cancer cells. Proteolysis of IGFBP-3 by incubation at 37 C with serum-free medium from MCF-7 cells was maximal at pH 5.0-5.5, with no activity detected below pH 4.5. This enzyme activity resulted in the disappearance of the 40- to 45- and 30-kDa bands of pure plasma-derived IGFBP-3, detectable by immunoblotting after SDS-PAGE, and the appearance of a single 21-kDa immunoreactive species. The 21-kDa protein did not bind IGF-I or IGF-II by ligand blotting. The enzyme activity appeared at 25- to 30-kDa by gel chromatography at pH 6.5 and was inhibited by EDTA and leupeptin, an inhibitor of cysteine and serine proteases, but not by the serine protease inhibitors aprotinin and benzamidine. IGFBP-3 protease activity was inhibited in medium conditioned by cells incubated with 50 ng/ml IGF-I. A similar inhibitory effect was seen under cell-free conditions by adding IGF-I to medium harvested from cells incubated without IGFs. The cell-free inhibition of IGFBP-3 proteolysis by IGFs did not require IGF interaction with the binding protein, because [long Arg3]IGF-I, which binds to IGFBP-3 with less than 0.2% of the potency of IGF-I, inhibited IGFBP-3 proteolysis with 20% of the potency of IGF-I. These results suggest that IGFs may regulate their own activity in breast cancer cells, preventing IGFBP-3 proteolysis by a mechanism that is not receptor mediated and does not require IGF-IGFBP interaction.
Insights
Insulin-like growth factors (IGFs) regulate breast cancer cell proliferation via IGF binding proteins (IGFBPs). This study found that IGFs can prevent the breakdown of IGFBP-3 by an enzyme secreted by breast cancer cells.
Area of Science:
- Endocrinology
- Cancer Biology
- Proteomics
Background:
- Insulin-like growth factors (IGFs) play a role in breast cancer cell proliferation.
- IGF binding proteins (IGFBPs) modulate IGF activity.
- MCF-7 human breast cancer cells secrete enzymes that may affect IGFBPs.
Purpose of the Study:
- To characterize the proteolysis of IGFBP-3 by an enzyme secreted by MCF-7 human breast cancer cells.
- To investigate the regulation of this IGFBP-3 protease activity by IGFs.
Main Methods:
- Incubation of purified IGFBP-3 with serum-free medium from MCF-7 cells at various pH.
- SDS-PAGE and immunoblotting to detect IGFBP-3 fragments.
- Ligand blotting to assess binding of IGF-I and IGF-II to fragments.
- Gel chromatography to determine enzyme size.
- Inhibition studies using EDTA, leupeptin, aprotinin, and benzamidine.
- Experiments with varying concentrations of IGF-I and a modified IGF-I analog ([long Arg3]IGF-I).
Main Results:
- A protease in MCF-7 cell medium maximally degraded IGFBP-3 at pH 5.0-5.5, producing a 21-kDa fragment that did not bind IGFs.
- The enzyme activity was localized to 25- to 30-kDa by gel chromatography and inhibited by EDTA and leupeptin.
- IGF-I inhibited IGFBP-3 proteolysis in a dose-dependent manner, both in conditioned medium and cell-free.
- [long Arg3]IGF-I also inhibited proteolysis, suggesting the mechanism is not solely dependent on IGF-IGFBP interaction or receptor binding.
Conclusions:
- MCF-7 cells secrete an IGFBP-3 protease.
- IGFs can inhibit the proteolysis of IGFBP-3 through a non-receptor-mediated mechanism that does not require direct IGF-IGFBP binding.
- These findings suggest a novel regulatory pathway where IGFs may control their own bioavailability and activity in the breast cancer microenvironment by modulating IGFBP-3 stability.