| 1. |
- Denmeade, Samuel R., et al.
(författare)
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Enzymatic activation of a doxorubicin-peptide prodrug by prostate- specific antigen
- 1998
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Ingår i: Cancer Research. - 0008-5472. ; 58:12, s. 2537-2540
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Tidskriftsartikel (refereegranskat)abstract
- New approaches to target cytotoxic therapy specifically to metastatic prostate cancer sites are urgently needed. As such an approach, an inactive prodrug was synthesized by coupling the primary amine of doxorubicin to the COOH-terminal carboxyl of a seven-amino acid peptide carrier (i.e., Mu-His- Ser-Ser-Lys-Leu-Gln-Leu). The seven-amino acid peptide was documented to be hydrolyzable specifically by the serine protease prostate-specific antigen (PSA) to liberate the active cytotoxin L-leucyl-doxorubicin. Primary cultures of PC-82 human prostate cancer cells secreted high levels of enzymatically active PSA (i.e., 70 ± 5 ng of enzymatically active PSA/106 cells/24 h), whereas LNCaP human prostate cancer ells produced lower levels of enzymatically active PSA (i.e., 2.3 ± 1 ng/106 cells/24 h). LNCaP cells, however, secreted sufficient amounts of enzymatically active PSA to activate the doxorubicin prodrug to a cytotoxic form in vitro. The specificity of the cytotoxic response to the prodrug was demonstrated by the fact that 70 nM of the prodrug killed 50% of the PSA-producing LNCaP cells, whereas doses as high as 1 ♂ had no cytotoxic effect on PSA-nonproducing TSU human prostate cancer cells in vitro.
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| 2. |
- Denmeade, Samuel R., et al.
(författare)
-
Specific and efficient peptide substrates for assaying the proteolytic activity of prostate-specific antigen
- 1997
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Ingår i: Cancer Research. - 0008-5472. ; 57:21, s. 4924-4930
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Tidskriftsartikel (refereegranskat)abstract
- Prostate-specific antigen (PSA) is a serine protease secreted by hath normal prostate glandular celts and prostate cancer cells. The major proteolytic substrates for PSA are the gel-forming proteins in semen, semenogelin (Sg) I and II. On the basis of the PSA cleavage map for Sg I and II, a series of small peptides (ie., ≤ 7 amino acids) was synthesized and coupled at the COOH terminus to 7-amino-4-methyl coumarin. Using these fluorescently tagged substrates, K(m)s and k(cm)s were determined for PSA hydrolysis, and the substrates were also tested for activity against a panel of purified proteases. Previously, a variety of chymotrypsin substrates have been used to assay the enzymatic activity of PSA. The present studies have identified a peptide sequence with a high degree of specificity for PSA (i.e., no detectable hydrolysis by chymotrypsin) and improved K(m)s and k(cat)s over previously used substrates. On the basis of these parameters, the best peptide substrate for PSA has the amino acid sequence HSSKLQ. Using PC-82 human prostate cancer xenografts and human prostate tissues, this PSA substrate was used to document that prostate cancer cells secrete enzymatically active PSA into the extracellular fluid but that once in the blood, PSA is not enzymatically active. On the basis of this information, it should be possible to use the HSSKLQ peptide as a carrier to target peptide- coupled prodrugs for selective activation within sites of PSA-secreting, metastatic prostate cancer cells and not within the blood or other nonprostatic normal tissues.
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