The Research On Functions Of Cathepsin B And Its Binding Proteins In Apoptosis Of Ovarian Cancer Cells.

Apoptosis is an active form of cell death that plays an essential role in development and survival by eliminating damaged or otherwise unwanted cells. Although caspases are well established as the main players in apoptosis, other proteases such as calpains, cathepsins may account for alternative types of apoptosis. Cathepsins, in particular cathepsin B (CTSB), activate caspase-dependent or caspase-independent pathways of cell death. Until now, it is not very clear about the mechanisms of cathepsins-mediated apoptosis.PP203 and TSRC1 are recently cloned novel human genes, and their Genbank accession numbers are AF258553 and BC027478 respectively. PP203 encodes 707 amino acids and has 1 BTB domain and 3 WD40 domains; while TSRC1 encodes 424 amono acids and has 5 repeated TSP1 domains. The transcription levels of PP203 and TSRC1 genes were examined by MTC panels analysis (Clontech). There are no transcriptions of PP203 detected in ovary and colon, and PP203 is expressed at low level in prostate, testis and skeleton muscle, and at high level in the left 11 types of tissues examined. Whereas, the transcriptions of TSRC1 are abundant in all examined tissues except for brain. PP203-interacting protein CTSB and TSRC1-interacting protein bikunin were identified by yeast two-hybrid method through screening human fetal liver cDNA library. Furthermore, it was found that CTSB could also interact with TSRC1 and bikunin in yeast respectively. The interactions of CTSB with PP203, TSRC1 or bikunin were confirmed by GST pull-down assay in vitro and coimmunoprecipitation in vivo. And the facts that CTSB were colocalized with PP203, TSRC1 or bikunin in cellular lysosomes gave more supports for the interactions. Tumor necrosis factor (TNF) is a pleiotropic cytokine capable of eliciting complex and diverse cellular events, including apoptosis, cell growth and proinflammatorygenes expression. Recently increasing evidence suggests that lysosomal cysteine proteases cathepsins were released in cytosol when cells were treated with TNF , activating the apoptotic pathway and leading to cell death. MTT methods were used to screen ovarian cancer cells OV-90, T0V-112D, prostatic cancer cells PC-3, hepatoma cells Hep3B and SMMC-7721 for their susceptivity to TNF cytotoxicity. It was found that all examined cancer cells were resistant to treatment of TNF alone. However, when the transcription inhibitor actinomycin D was supplemented to the medium, OV-90, PC-3 and SMMC-7721 cells were susceptible to TNF-induced death. Either specific cathepsin B inhibitor CA074Me (25 μM) or pan-caspases inhibitor zVAD-fmk (1 μM) could reduce the death of these three TNF-sensitive cell lines. Furthermore, the results of apoptosis detected by PI-FACS also showed that 25 μM CA074Me and 1 μM zVAD-fmk suppressed TNF-mediated OV-90 cells apoptosis. The highly sensitive AO (acridine orange)-relocation method and AO-uptake method were used to detect the integrity of lysosome in OV-90 cells during apoptosis. And the results indicate that lysosomes undergo a permeabilization process in TNF-treated OV-90 cells, and inhibitors against caspases or CTSB protect lysosomes from rupture. The destability of lysosomes in apoptosis is also demonstrated by the fact that TSRC1-GFP protein , which is colocalized with CTSB in lysosomes displaying punctate fluorescent appearance, redistribute to the cytosol of OV-90 cells after TNF treatment.CTSB has been testified to play a role in TNF-induced apoptosis of OV-90 cells. In order to understand factors involved in the cathepsin B-mediated death pathway, we investigated the functions of CTSB-binding partners playing in OV-90 cells apoptosis. As results, overexpression of bikunin or PP203 suppresses TNF-triggered apoptosis in OV-90 cells, to the contrary, overexpression of TSRC1 enhances cells apoptosis. Meanwhile, effects of overexpression of PP203, bikunin and TSRC1 on cellular CTSB activity were investigated respectively. The results show that overexpression of PP203 or TSRC1 has no influence on cellular CTSB enzyme activity, whereas in OV-90 cells with bikunin overexpression CTSB activity is reduced efficiently. Accordingly, bikunin might suppress TNF-mediated apoptosis of OV-90 cells byinhibiting enzyme activity of CTSB.CTSB has two opposing roles in malignancy, one role is that its proapoptotic features can reduce malignancy; while the other is that expression level of CTSB is upregulated in many human cancers, and secreted CTSB protein enhance tumor invasion and metastasis. Our initial results demonstrate that CTSB and its candidate regulators, PP203, bikunin and TSRC1, participate in apoptosis pathway of OV-90 cells. The understanding about apoptotic mechanism mediated by CTSB will be very important for successful treatment of cancers.