Maspin is known to be a tumor suppressive serpin and has been shown to inhibit motility and invasion in prostate and breast cancer by its inhibitory mechanisms that is currently unknown. For cancer cells to properly invade the barrier of the matrix, it requires proteolytic degradation meaning that it needs to break down the matrix. In other words, the more proteolysis meaning the more degradation of the matrix which leads to the invasion of cancer cells. Proteolysis serves as an important factor for degradation that allows cancer cells to move around and invade tissues. One specific proteolytic system is urokinase-plasminogen activator, or uPA. This article addresses the possibility of how maspin targets and may inhibit uPA. This mechanism is how it is possible to block invasion by inhibiting uPA mediated proteolysis and matrix degradation. Mentioned briefly before, maspin is a tumor suppressive serpin known to inhibit the progression of prostate and breast tumors at the steps of invasion and metastasis; however, the mechanism is unknown. It was speculated that maspin may target plasminogen activators that are bound to biological surfaces, for example, the plasma membrane. Other studies have shown that the tumor cell surface-associated uPa is inhibited by recombinant maspin. In addition, the proteolytic inhibitory effect of recombinant maspin was quantitatively consistent with the inhibitory effect of maspin in cell migration. The key question that the article addresses is, does endogenous maspin exert its tumor suppressive function by inhibiting pericellular proteolysis by inhibiting uPA. The data of this article demonstrates that maspin blocks tumor invasion and metastasis by inhibiting pericellular proteolysis.
The parental cell line that was used was the human prostate carcinoma cell DU145 for three following reasons: they do not express detectable endogenous maspin; they have also been shown to respond to the inhibition of recombinant maspin in in vitro invasion and motility assays; LAstly, DU145 cells secrete and rely primarily on uPA to initiate pericellular plasminogen activation. In other words, cell line DU145 was used because it has a low expression of maspin, which serves as an excellent model system to manipulate the concentration of maspin. In addition, other studies have shown that recombinant maspin specifically inhibits the DU145 cell surface-associated uPA. Protein Fractionation and Western Blotting analyses were performed to separate and detect the protein of interest; in this case, maspin. RT-PCR was performed to extract RNA from each cell line. It is an assay that allows one to detect the level of the mRNA using primers specific to the mRNA. Northern Blotting analysis was performed as an additional method of detecting RNA. In Vitro Motility and Invasion Assays were conducted using MICS. This method was used to observe the cells that would migrate between the two chambers and to verify the specificity of the maspin effect in inhibiting tumor cell invasion.
To examine the effects of maspin on cell surface associated uPA, prostate carcinoma cell line DU145 was chosen for the reasons mentioned previously in the materials and methods. Shown in Fig. 1A, maspin was detected in both the total cell lysates as well as the conditioned medium of the maspin transfectant clones. On the other hand, there was no detection of maspin protein in the parental or control cell lines. The RT-PCR performed to detect maspin mRNA in the maspin transfectant clones in Fig. 1B showed a similar expression pattern of maspin displayed in Fig. 1A. To observe the effects of mapsin’s ability to inhibit cell invasion and motility, an in vitro motility and invasion assay was conducted. Shown in Fig. 1C, in comparison to the parental cell line DU145, the maspin transfectant clones displayed a significant inhibition of migrating cells by approximately 60%. In contrast, the mock transfectant clones showed similar results as the parental cell line. The reason for these results is due to the presence of maspin because maspin is a tumor suppressive serpin that inhibits the motility or migration of cells. Those with more maspin have shown significantly reduced levels of migrating cells. Similarly, in Fig. 1D, the maspin transfectant clones exhibited a significant reduction of approximately 40-50% of invading cells. Similar to the migrating cells, the higher the expression of maspin, the lower the levels of invading cells. To confirm the specificity of maspin’s inhibitory effects of the invasive potential, the maspin-neutralizing antibody Abs4A was used in Fig. 1E. The results indicated that there was no significant effect on the mock transfectant clones. On the other hand, Abs4A reversed the invasive potential in the maspin transfectant clones but in a dose-dependent manner. The maspin transfectant clone had very low invasion, but the increase in the dosage of the maspin-neutralizing antibody Abs4A caused the inhibition of invading cells to be lost due to the lost of maspin by Abs4A. As the concentration of the maspin-neutralizing antibody increases, the number of invasive cells increases.
To test whether the biological activity of expressed maspin in inhibiting cell motility and invasion in relation with specific inhibition of uPA on the cell surface, the cell surface-associated plasminogen activation was measured using the coupled colorimetric plasminogen activation assay shown in Fig. 2A. The colorimetric plasminogen activation assay was performed in the presence of uPA-neutralizing antibody or tPA-neutralizing antibody. Untreated cells were used as a positive control. The cell surface-mediated plasminogen activation activity in the three maspin transfectant clones was significantly reduced in comparison to the parental and mock transfectant clones. This information makes sense because the parental and mock transfectant clones lack maspin leading to higher levels of cell surface-mediated plasminogen activation activity. In other words, this means that there is more proteolysis occurring in these cell lines hence more invasion. In the maspin transfectant clones, the data indicates that there are already reduced levels of cell surface-mediated plasminogen activation activity; however, there is further reduction with the addition of the uPA-neutralizing antibody. To confirm that uPA is affected, the cells were pretreated with uPA antibody leading to a reduction of the basal uPA activity in both the parental cell lines and the maspin transfectant clones. Observing the tPA-neutralizing antibody lines, they highly resembled the positive control lines demonstrating that tPA was not a significant factor in reducing the levels of cell surface-mediated plasminogen activation activity. The data supports that the cell surface-mediated plasminogen activation activity was effectively inhibited by the uPA-neutralizing antibody and not by the tPA neutralizing antibody. As a result, these data are consistent with DU145 cell-mediated plasminogen activation is dependent primarily on uPA. In addition, a plasminogen-dependent zymogram was conducted using cell surface eluates to observe the effect of mapsin on uPA present on the cell surface (Fig. 2B). Compared to the uPA standard, the uPA activity in the maspin transfectant clones was significantly diminished compared to the parental and mock transfectant clones. Consequently, the inhibition of the cell surface-mediated plasminogen activation is directly correlated to the reduced levels of cell-associated uPA in the maspin transfectant clones.
Because secreted uPA is in equilibrium with the cell surface and the cell cultured medium, it was necessary to investigate whether maspin further regulates uPA. Using the coupled colorimetric plasminogen activation assay (Fig. 5), it was discovered that the SF-CM of the mock transfectant clones had similar plasminogen activation activity as the parental cell line. In contrast, the SF-CM of the three maspin transfectant clones showed dramatically reduced levels of plasminogen activation activity. As a result, the cultured medium clearly indicates that the presence of activated maspin led to reduced levels of plasminogen activation activity.
The objective of this research was to analyze the effect of endogenous mapsin on pericellular uPA system. Using the stable maspin transfectants from prostate carcinoma cells DU145, a major key finding was that endogenous maspin inhibited the pericellular uPA activity, cell motility and cell invasion. This result serves as a foundation for the rest of the findings. Another major key finding was the direct interaction of mapsin and uPA on the cell surface. The data supports the significant reduction of uPA mediate plasminogen activation activity in both the cell surface and cultured medium. The expression of uPA by malignant cells correlates with an aggressive phenotype that includes increased levels of tumor cell invasion and metastasis through the activation of plasminogen resulting in the degradation of the pericellular matrix. It was also noted that the epithelial cell surface plays a major role in mediating the inhibitory the interaction between endogenous maspin and uPA.