Melchiori A, Albini A, Ray J M, Stetler-Stevenson W G
Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy.
Cancer Res. 1992 Apr 15;52(8):2353-6.
The metastasis associated 72-kDa type IV collagenase is secreted as a latent proenzyme which is converted to an active 62-kDa form by autoproteolytic removal of an amino terminal profragment. The region immediately upstream from the cleavage site contains a highly conserved peptide sequence, MRKPRCGNPDV, which is present in all known members of the matrix metalloproteinase family. Evidence implicates the cysteine residue of this sequence as critical for maintenance of the latent form through coordination with the catalytic zinc atom of the active site. A synthetic peptide, TMRKPRCGNPDVAN (peptide 74), encompassing this conserved sequence, has been shown to inhibit the activated form of the 72-kDa type IV collagenase in vitro. In the present study we examine the ability of this peptide inhibitor to modulate tumor cell invasiveness. Peptide 74 and the control peptide 78, which contains a single substitution of serine for the "critical" cysteine residue, were added at 30 microM concentrations to the upper compartment of the Boyden chamber in the chemoinvasion assay using HT1080 and A2058 human tumor cells. In this assay a layer of reconstituted basement membrane, Matrigel, is coated onto chemotaxis filters and acts as a barrier to the migration of cells in the Boyden chambers. Only cells with invasive capacity can cross the Matrigel barrier. Peptide 74 containing the cysteine residue inhibited the invasion of both the HT1080 and A2058 cells through the Matrigel barrier; control peptide 78 was not inhibitory. Both peptides were shown to be without cytotoxic action and did not inhibit chemotaxis or affect cell number. This study demonstrates that addition of an excess peptide containing the matrix metalloproteinase prosegment inhibitory sequence can inhibit invasive activity at the cellular level and suggests that this may be a useful strategy to modulate tumor cell invasiveness in vivo.
与转移相关的72 kDa IV型胶原酶以无活性的酶原形式分泌，通过氨基末端前肽片段的自蛋白水解作用转化为有活性的62 kDa形式。切割位点上游紧邻区域包含一个高度保守的肽序列MRKPRCGNPDV，该序列存在于基质金属蛋白酶家族的所有已知成员中。有证据表明该序列中的半胱氨酸残基对于通过与活性位点的催化锌原子配位来维持无活性形式至关重要。一种包含该保守序列的合成肽TMRKPRCGNPDVAN（肽74）已被证明在体外可抑制72 kDa IV型胶原酶的活化形式。在本研究中，我们检测了这种肽抑制剂调节肿瘤细胞侵袭性的能力。在使用HT1080和A2058人肿瘤细胞的化学侵袭试验中，将肽74和对照肽78（其中“关键”半胱氨酸残基被丝氨酸单取代）以30 microM的浓度添加到博伊登小室的上室。在该试验中，一层重组基底膜基质胶被涂覆在趋化性滤膜上，并作为博伊登小室中细胞迁移的屏障。只有具有侵袭能力的细胞才能穿过基质胶屏障。含有半胱氨酸残基的肽74抑制了HT1080和A2058细胞穿过基质胶屏障的侵袭；对照肽78没有抑制作用。两种肽均无细胞毒性作用，不抑制趋化性或影响细胞数量。本研究表明，添加过量的含有基质金属蛋白酶前肽抑制序列的肽可以在细胞水平上抑制侵袭活性，并表明这可能是一种在体内调节肿瘤细胞侵袭性的有用策略。
