Human Maspin Research Articles

Inhibitory effect of maspinon neovascularization in diabetic retinopathy.

BACKGROUNDDiabetic retinopathy (DR) is a serious and potentially blinding complication of diabetes mellitus. Retinal neovascularization is one of the main pathological features of proliferative DR, and inhibiting retinal neovascularization is a research focus.AIMThe aim was to evaluate the effect of intravitreal injection of recombinant human maspin on neovascularization in DR.METHODSAn oxygen-induced retinopathy (OIR) mouse model was used to simulate neovascularization in DR. New born C57BL/6J mice were randomly divided to a normal control group, a maspin injection OIR group, and an OIR group. The mice in the maspin injection OIR group were injected with recombinant human maspin in the bilateral vitreous cavity on postnatal day P12, and those in the OIR group were injected with sterile phosphate buffered saline. The protein expression of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1-alpha (HIF-1α) in the retina was measured by western blotting, and the mRNA expression of VEGF and HIF-1α was measured by real-time polymerase chain reaction. The vascular cell nuclei that broke through the inner limiting membrane (ILM) were counted in haematoxylin-eosin stained retinal sections.RESULTSIt was found that the number of vascular cell nuclei breaking through the ILM was 31.8 ± 8.75 in the OIR group, which was significantly more than that in the normal control group (P < 0.001). The number of vascular cell nuclei breaking through the ILM was 6.19 ± 2.91 in the maspin injection OIR group, which was significantly less than that in OIR group (P < 0.01). The relative protein and mRNA expression of VEGF and HIF-1α was significantly lower in the retinas in the maspin injection OIR group than in those in the OIR group (P < 0.01).CONCLUSIONMaspin inhibited neovascularization in DR by modulating the HIF-1α/VEGF pathway, which provides a potential and effective strategy for the treatment of DR.

Recombinant human maspin inhibits high glucose-induced oxidative stress and angiogenesis of human retinal microvascular endothelial cells via PI3K/AKT pathway.

Maspin is known as a tumor suppressor and a potent angiogenesis inhibitor, however, its effects on proliferative diabetic retinopathy (PDR) have not been fully elucidated. This study aimed at evaluating the effects of maspin on high glucose-induced oxidative stress and angiogenesis in human retinal microvascular endothelial cells (HRMECs). Herein, HRMECs were treated with 0.25, 0.5, or 1µM recombinant human maspin in the presence of 30mM glucose, and their proliferation, tube formation, and oxidative stress responses were further detected. Our results revealed that maspin inhibited the high glucose-induced proliferation, migration, and tube formation of HRMECs. Maspin also decreased reactive oxygen species, nitric oxide level, and increased glutathione S-transferase activity in HRMECs. Meanwhile, maspin reduced the mRNA and protein levels of hypoxia-inducible factor-1α and vascular endothelial growth factor in high glucose-stimulated cells in a dose-dependent manner. Additionally, the high glucose-induced elevation of phosphorylated phosphoinositide-3-kinase (p-PI3K) and phosphorylated AKT was also suppressed by maspin. In summary, our data suggest that maspin inhibits high glucose-induced proliferation, oxidative stress, and angiogenesis of HRMECs at least by modulating the PI3K/AKT pathway. Maspin may be a potential therapeutic agent for the prevention and treatment of PDR.

Maspin impairs the function of endothelial cells: an implying pathway of preeclampsia

BackgroudWidespread endothelial injury contributes to the occurrence of preeclampsia. Maspin, first identified as a tumor suppressor, plays a critical role in cell invasion and angiogenesis. Our previous studies found that the expression of maspin was increased in preeclampsic placenta. In this research, we studied the function of human umbilical vein endothelial cells (HUVECs) to explore the role and possible mechanism of maspin gene in the pathogenesis of preeclampsia.MethodsHUVECs were treated with different concentration of recombinant human maspin protein (r-maspin) during normoxia and hypoxia, we detected the proliferation, apoptosis, migration and tube formation of HUVECs. We also assessed nitride oxide (NO) synthesis and the expression of matrix metalloproteinase 2 (MMP2) to further explore the underlying molecular mechanism.ResultsThere was only slight maspin expression at mRNA level in HUVECs. Treated HUVECs with r-maspin, the proliferation of HUVECs was significantly promoted both under normoxia and hypoxia. The tubes formed by HUVECs were significantly inhibited and NO synthesis was significantly reduced by r-maspin. Meantime, r-maspin also inhibited MMP2 expression and activity in HUVECs. However, there was no significant change in the migration and apoptosis of HUVECs.ConclusionsMaspin may be an important participant for mediating endothelial function and ultimately leads to the occurence of preeclamsia.

RGD and polyhistidine tumor homing peptides potentiates the action of human Maspin as an antineoplastic candidate.

Maspin, a non-inhibitory member of serine protease family, acts as an effective tumor suppressor by inhibiting cell inhesion and mobility. We found that exogenous wild-type rMaspin had a low effect on tumor growth in vivo. However, when the peptide Arg-Gly-Asp-hexahistidine (RGD-6His) was introduced into rMaspin, the modified rMaspin showed significant inhibitory activity in angiogenic assays and tumor-bearing animal models. Overall, our data suggested that both the RGD and hexahistidine fragments contributed to improve the fusion protein activity and polyhistidine peptide could be considered as flexible linker to separate RGD and Maspin moieties to avoid function interference. Besides, it is an efficient tag to achieve purified recombinant proteins. Furthermore, rMaspin fusing with RGD and hexahistidine could be a viable anticancer candidate.

A Versatile Monoclonal Antibody Specific to Human SERPINB5

Maspin (SERPINB5) is a member of the Clade B subgroup of the large superfamily of serine protease inhibitors. It is proposed that maspin is a tumor suppressor; however, its molecular role remains to be elucidated. Here we report the characterization of a mouse monoclonal antibody directed against human maspin. This antibody, 16F7, recognizes maspin in both its native and denatured form, unlike several other commercial antibodies tested in this study. It will be a useful and versatile tool for future analyses of the biological function of maspin.

G-helix of Maspin Mediates Effects on Cell Migration and Adhesion

Maspin is a member of the serine protease inhibitor (serpin) superfamily that lacks protease inhibitory ability, although displaying tumor metastasis-suppressing activity resulting from its influence on cell migration, invasion, proliferation, apoptosis, and adhesion. The molecular mechanisms of these actions of maspin are as yet undefined. Here, we sought to identify critical functional motifs by the expression of maspin with point mutations at sites potentially involved in protein-protein interactions: the G α-helix (G-helix), an internal salt bridge or the P1 position of the reactive center loop. Our findings indicate that only mutations in the G-helix attenuated inhibition of cell migration by maspin and that this structural element is also involved in the effect of maspin on cell adhesion. The action of maspin on cell migration could be mimicked by a 15-mer G-helix peptide, indicating that the G-helix is both essential and sufficient for this effect. In addition, we provide evidence that the effects of the G-helix of maspin are dependent on β1 integrins. These data reveal that the major extracellular functions associated with the tumor suppressive action of maspin likely involve interactions in which the G-helix plays a key role.

The High Resolution Crystal Structure of the Human Tumor Suppressor Maspin Reveals a Novel Conformational Switch in the G-helix

Maspin is a serpin that acts as a tumor suppressor in a range of human cancers, including tumors of the breast and lung. Maspin is crucial for development, because homozygous loss of the gene is lethal; however, the precise physiological role of the molecule is unclear. To gain insight into the function of human maspin, we have determined its crystal structure in two similar, but non-isomorphous crystal forms, to 2.1- and 2.8-A resolution, respectively. The structure reveals that maspin adopts the native serpin fold in which the reactive center loop is expelled fully from the A beta-sheet, makes minimal contacts with the core of the molecule, and exhibits a high degree of flexibility. A buried salt bridge unique to maspin orthologues causes an unusual bulge in the region around the D and E alpha-helices, an area of the molecule demonstrated in other serpins to be important for cofactor recognition. Strikingly, the structural data reveal that maspin is able to undergo conformational change in and around the G alpha-helix, switching between an open and a closed form. This change dictates the electrostatic character of a putative cofactor binding surface and highlights this region as a likely determinant of maspin function. The high resolution crystal structure of maspin provides a detailed molecular framework to elucidate the mechanism of function of this important tumor suppressor.

The promise and challenge toward the clinical application of maspin in cancer.

Since the identification of the human maspin gene, a decade of extensive research revealed the promise of maspin both as a valuable molecular marker for the diagnosis and prognosis of many types of cancers, and as a tumor suppressor at the level of tumor growth, invasion, angiogenesis, metastasis, and tumor sensitivity to drug-induced apoptosis. This review is intended to summarize the consensus of these findings, and provide an overview of the current challenges toward the clinical application of maspin. Specifically, this review discusses several likely molecular mechanisms underlying (1) the differential regulation of maspin expression, and (2) the biological activities of maspin in tumor progression.

Three-state Unfolding and Self-association of Maspin, a Tumor-suppressing Serpin

Maspin is a tumor suppressor protein expressed by normal human mammary epithelium but not by many breast tumor cell lines. Recombinant human maspin (rMaspin) inhibits tumor cell motility, invasion, and metastasis and thus has potential value as an anti-cancer therapeutic. Maspin is a member of the serpin family and, although the molecular mechanism by which maspin acts is unknown, recent work suggests that tissue plasminogen activator is a potential target. A puzzling observation in previous cell culture studies was loss of rMaspin activity at higher protein concentrations. One hypothesis to explain these results is self-association of rMaspin at the higher concentrations, which would be consistent with the tendency of serpins to form noncovalent polymers. This hypothesis is addressed by examining the relationship between rMaspin stability and self-association. Urea denaturation of rMaspin at pH 7 and 25 degrees C and at protein concentrations ranging from 0.01 to 0.2 mg/ml has been monitored by circular dichroism and intrinsic tryptophan fluorescence. Denaturation profiles show a protein concentration dependence and indicate the presence of at least one unfolding intermediate. The results suggest that destabilization of native monomeric rMaspin leads to partial unfolding and formation of an intermediate which can self-associate.

Maspin polymorphism associated with apoptosis susceptibility and in vivo tumorigenesis

Maspin is a tumor suppressor protein that stimulates apoptosis and inhibits motility, invasion and cancer metastasis. We report on a previously uncharacterized Pro/Ser (C to T) polymorphism at amino acid 176 of the human maspin protein. We analyzed the maspin mutation in 17 cancer cell lines and 36 cancer tissues. Association of polymorphic variants on apoptosis, colony formation and in vivo tumor formation was evaluated. Mutant maspin was found to be frequently expressed in gastric cancer (32/36, 89%). According to predicted maspin tertiary structure, the polymorphic residue is located on the surface of the protein proximal to the reactive site loop domain, and thus may significantly affect the protein interactions of maspin. Stable expression of the Pro and Ser forms of maspin in lung cancer cells revealed that cells expressing Ser176 maspin showed significantly decreased apoptosis and increased colony formation compared with those expressing Pro176 maspin. In a mouse model, cells expressing Ser176 maspin showed a higher rate of tumor formation in vivo than those harboring Pro176 maspin. Therefore, P176S (C526T) substitution of maspin may result in a partial loss of the tumor suppressor function of this protein, contributing to decreased susceptibility to apoptosis and malignant progression.

Rat and human maspins: structures, metastatic suppressor activity and mutation in prostate cancer cells.

The rat homologue of human maspin cDNA was cloned. The deduced amino acid sequence of rat maspin was homologous to human maspin with 88% of the amino acids conserved. Rat maspin mRNA was detected in rat mammary gland, vagina, urinary bladder, thymus, small intestine, skin, ventral prostate, seminal vesicles, and thyroid but not in many other organs, such as heart, lung, liver, brain and kidney. Rat maspin cDNA retrovirally introduced into highly metastatic Dunning AT3.1 rat prostate cancer cells did not suppress metastasis of these tumor cells in Copenhagen rats. Maspin mRNA was detected in 5/10 human prostatic carcinoma tissue samples. Two human prostate cancer cell lines, PC-3 and LNCaP, and two human prostatic carcinoma and two benign prostatic hyperplasia tissue samples contained maspin mRNA having an isoleucine to valine mutation at amino acid 319.

MMaspin: The Mouse Homolog of a Human Tumor Suppressor Gene Inhibits Mammary Tumor Invasion and Motility

The human maspin gene encodes a protein in the serine proteinase inhibitor (serpin) family with tumor-suppressing functions in cell culture and in nude mice. In order to examine the role of maspin in an intact mammal, we cloned and sequenced the cDNA of mouse maspin. The recombinant protein was produced and its activity in cell culture was assessed. Mouse maspin (mMaspin) was cloned by screening a mouse mammary gland cDNA library with the human maspin cDNA probe. Northern blot analysis was used to examine the expression patterns in mouse tissues, mammary epithelial cells, and carcinomas. Recombinant mMaspin protein was produced in E. coli. Invasion and motility assays were used to assess the biological function of mMaspin. mMaspin is 89% homologous with human maspin at the amino acid level. Like its human homolog, mMaspin is expressed in normal mouse mammary epithelial cells and down-regulated in mouse breast tumor cell lines. The expression is altered at different developmental stages in mammary gland. Addition of the recombinant mMaspin protein to mouse tumor cells was shown to inhibit invasion in a dose-dependent manner. As with the human protein, recombinant mMaspin protein also inhibited mouse mammary tumor motility. Deletion in the putative mMaspin reactive site loop (RSL) region resulted in the loss of its inhibitory functions. mMaspin is the mouse homolog of a human tumor suppressor gene. The expression of mMaspin is down-regulated in tumor cells and is altered at different developmental stages of mammary gland. mMaspin has inhibitory properties similar to those of human maspin in cell culture, suggesting that the homologous proteins play similar physiological roles in vivo.