Study On The Effect And Mechanism Of Tryptase On The Invasion Of Breast Cancer Cells

Breast cancer threatens the women's health seriously, and its incidence has risen for recent years. The main harm of breast cancer is its metastasis, which refers to that the breast cancer cells invade to the blood vessel, coelom and lymphatic vessel, and then are transported to other tissues or organs through blood or lymph to form a metastatic tumor, which has the same characteristic as the primary tumor. The metastasis is the main death cause of the patients with breast cancer, the most important indication and the basic manifestation of breast cancer.It has been previously reported that the number of mast cells in the breast cancer peritumoral tissue is closely related to the pathological type of breast cancer by our lab. There are much more mast cells in malignant invasive lobular carcinoma or invasive ductal carcinoma than in benign tumors (P<0.01). There are medium numbers of mast cells in less malignant, unremarkable invasive mucinous adenocarcinoma and medullary carcinoma. The relationship between mast cells and the metastasis and invasion of breast cancer has been paid more and more attention in recent years. Tryptase, the main substance degranulated by mast cells, is a serine protease with a trypsin-like enzymatic activity. The effect of tryptase on the invasion of breast caner has been barely reported. In this project, firstly, we extracted tryptase from health human lung; we studied the correlation between tryptase and the pathological grade or lymph node using tissue array and immunohistochemical methods. Lastly, we investigated the effect and mechanism of tryptase on breast cancer cell line MDA-MB-231 in vitro.Our experiment includes two parts.PartⅠ: the extraction and purification of tryptase from human lung. The commercial tryptase is not suitable for our experiment due to its high price and low enzymatic activity. Then we tried to extract tryptase from human lung by antibody affinity chromatography. The human lung was homogenized in cold extraction buffer, mixed with the Agarose linked with tryptase antibody B12 and poured into the affinity chromatography column. The elution product was collected. We detected the molecular weight and activity of tryptase by SDS-PAGE and chromogenic substrate assay respectively. The SDS-PAGE showed a specific 34kD band, which is same with molecular weight of tryptase. The tryptase specific activity is 60 IU/mg, K_m is 0.45mmol/L, V_max is 161.29μmol/min, and all these indices are better than the commercial tryptase.PartⅡ: the study on the effect and mechanism of tryptase on the invasion of breast cancer.①We observed the distribution of tryptase in breast cancer peritumoral tissue by tissue array and immunohistochemical methods. There are more tryptase staining in the breast cancer peritumoral tissue than in the normal breast tissue. Tryptase staining in gradeⅢbreast cancer tissue are much higher than grade I and II with lymph node metastasis, while tryptase staining in grade II breast cancer tissue with lymph node metastasis is 3.6 times of that tissue without lymph node metastasis. Compared with tissue without lymph node metastasis, tryptase staining in breast cancer tissue with lymph node metastasis is doubled. All these results suggested the positive correlation between tryptase and the characteristic, grade and lymph node metastasis of breast cancer.②We investigated the effect of tryptase on the invasion of breast cancer cells in different pH to imitate the surrounding of tumor tissue. The results suggested that the invasion ability of breast cancer line MDA-MB-231 is strongest at pH 7.0 but not other pH. We used this pH as the subsequent experimental condition.③We used transwell invasion model to observe the effect of tryptase on the invasion of breast cancer cell line MDA-MB-231. The result suggested that 50 pmol/L, 500 pmol/L and 1000 pmol/L tryptase promoted the invasion of breast cancer cell dramatically. The promotion was most obvious at 500 pmol/L with time dependent manner.④We studied the effect of tryptase on the proliferation of breast cancer cell line MDA-MB-231. 500pmol/L tryptase can not affect the proliferation of this cancer cell line at pH 7.0.⑤In order to study the mechanism of promoting invasion of breast cancer cell by tryptase, we used transwell mobility model and gelatin zymogram to investigate the effect of tryptase on the cell mobility and extracellular matrix degrading enzyme respectively. The result suggested that tryptase promotes the mobility of breast cancer cell line MDA-MB-231 and activates the MMP-2 of MDA-MB-231 to degrade the extracellular matrix.In conclusion, we extracted tryptase from health human lung successfully. In breast cancer peritumoral tissue, accumulated acid metabolites degranulate the mast cells and up-regulate the activity of tryptase when the cancer cells grow to a certain stage. Tryptase promotes the mobility of cancer cells, activates MMP-2 to degrade the extracellular matrix, and then strengthens the invasion of breast cancer cell. This result is consistent with that more tryptase staining in grade III breast cancer peritumoral tissue than in grade I and II in tissue array. This project studied the effect and mechanism of tryptase on the invasion of breast cancer, which provided a possible target for the future clinical treatment of breast cancer.