| Objectives:Recently, the target therapies for leukemia include the antibody drugs and inhibitor drugs to specifically expressed molecules of leukemic cells. The complexity and heterogeneity of leukemia pathogenesis have lead to frequent drug resistance.Therefore, development of novel medications which has the potential to become anti-leukemia drugs is needed. ASPNJ(Acidic Serine Protease from Neanthes japonica)was first isolated and purified from Neanthes japonica in our lab. ASPNJ has both strong plasmin and proteolytic enzyme activies. Previous study of ASPNJ on myelogenous leukemia cells(K562 cells, NB4 cells) showed that ASPNJ may inhibit proliferation and induce apoptosis of myelogenous leukemia cells. Further experiments found that the action site of ASPNJ may be on some specific protein of cell membrane, causing cell apoptosis and cell death by degradation of these proteins and alteration of the related cell signal transductons. The effects of ASPNJ on leukemia Jurkat cell were determined and the early mechanism of its inhibitory effects were studied by using membrane and membrance associated proteomics in order to provide an noval research field for the treatment of leukemia, and to further elucidate the pathogenesis of leukemia.Methods:1. MTT was used to measure the effect on the proliferation inhibition of ASPNJ on different cell lines(Raji,U937,Jurkat,4T1,Smmc7721,293T).2. Using fibrin plate method to determine the relationship between ASPNJ enzyme activity and its proliferation inhibitory effects on Jurkat cell.3. Ttrypan blue exclusion method to examine the effect of ASPNJ on growth inhibition of Jurkat cells.4. The morphology of Jurkat cells that had been treated with ASPNJ was observed by Wright-Giemsa staining method.5. Flow cytometry was used to detect the apoptotic effect of ASPNJ on Jurkat cells.6. Vincristine was combined with ASPNJ in treatment of Jurkat cells to examine the synergistic effect.7. Jurkat cells were treated with ASPNJ(4μg/m L, 3h) and then the cell membrane proteins and membrane associated proteins were purified and separated by two-dimensional electrophoresis.8. Obvious differential protein spots were taken and performed the mass spectrometry analysis to identify ASPNJ possible target proteins.9. The 2DE and MS identified proteins were confirmed by Western Blot and RT-PCR.Results:1. ASPNJ has much strong proliferation inhibition effect on leukemic cells(Raji,U937, Jurkat) than other cells(4T1, Smmc7721, 293T), thus leukemic cells are more sensitive to ASPNJ. ASPNJ could obviously inhibite Jutkat cell growth. The inhibitory rates were dosage and time dependent.2. ASPNJ enzyme activity in cell culture supernatant was increased gradually with increasing dosage, but was reduced with time; ASPNJ enzyme activity is the necessary condition for its proliferation inhibition on Jutkat cells. While the proliferation inhibitory effect of ASPNJ on Jutkat cell was increased with increasing time, the fibrinolytic enzyme activity in the cell culture supernatant was decreased with increasing time. These results suggest that effect of inhibition of ASPNJ on Jurkat cell may have some indirect mechanism, in addition to the direct effect that rely on its enzyme activity.3. Morphological changes, such as the cell membrane rupture, the chromatin condensation and nuclear pyknosis, etc were observed. Significant apoptosis induction effect of ASPNJ on Jurkat cell was detected with time and dose dependent manner.4. There had been a significant synergtic tumor suppression effect when vincristine was used in combination with ASPNJ to treat Jutkat cells.5. The 2DE images of Jurkat cell membrane protein detected 44 different protein expression spots(34 higher expressed protein spots in control group,10 higher expressed protein spots in dosing group). The corresponding basic and functional information of the 7 different protein spots were identified through MS analysis and exploring of the database(1-5 higher expressed protein spots in control group, 6-7 higher expressed protein spots in ASPNJ treated group). Their specific functional information are as follows:1) SBP1( Selenium-bingding protein) inhibite tumor cell growth and metastasis by change lipid/glucose metabolic signaling pathways [1].2) SUMF2(Sulfatase-modifying factor 2) interact with IL-13 and IL-13receptor; in the meanwhile restrain IL-13 secretion [2].3) PRDX6(Peroxiredoxin-6) is a bifunctional protein with both glutathione peroxidase(GPx) and-independent Phospholipase A2(i PLA2) activities,may promote tumorigenesis. PRDX6 down regulation can make tumor cells more sensitive to radiation therapy,and PRDX6 result in cell cycle arrest and apoptosis by regulate the activity of i PLA2[3].4) CHP1(Calcium-binding protein) promote tumor angiogenesis, and inhibite the activity of hypoxia-inducible factor(HIF-1α) [4].5) SAR1A(GTP-binding protein SAR1a) participate in protein transport from the endoplasmic reticulum to the Golgi apparatus [5].6) PGRC1(Mebrane-associated progesterone receptor component 1) is a membrane associated progesterone receptor. Protect neurons by blocking the mitochondrial apoptosis pathway [6].7) RSSA(40S ribosomal protein SA) is a kind of protein which participate in 40 s ribosomal assembly and stability. It is also a kind of laminin receptor which mainly exist on the surface of the cell membrane. RSSA participate in adhesion of cells with basement membrane and activate signal transduction pathways [7]. It is over expressed in many cancer cells[8-10].6. PRDX6, CHP1 and RSSA protein were verified by Western Blot and RT-PCR. The three membrane protein expression levels were consistent with the results from 2DE- mass spectrometry. Whereas the m RNA expression level and the total protein expression level were not shown in parallely the same trend.Conclusion:Leukemic cells are more sensitive to ASPNJ. ASPNJ could obviously inhibite Jutkat cells proliferation and growth with dosage and time dependent manner; ASPNJ activity is the necessary condition for ASPNJ to play the inhibitory effects on proliferation and growth of Jutkat cells, which imply the direct mechanism and there may be some other indirect mechanism for the action of ASPNJ.ASPNJ has both effect of necrocytosis and apoptosis on Jurkat cell. The action mechanism of ASPNJ is completely different from vincristine, which disturb protein synthesis and inhibite cell mitosis. Killing tumor cells by different ways will greatly enhance the effects of drugs to kill leukemia cells. Reducing the dose of chemotherapeutic drugs can reduce the toxic and side effects.Seven different proteins were identified by 2DE Mass spectrometry analysis,they are SBP1,SUMF2,PRDX6,CHP1,SARIA,PGRC,RSSA. PRDX6, CHP1 and RSSA protein were verified by Western Blot and RT-PCR. We hypothesized that the indirect mechanism of proliferation and growth inhibition of ASPNJ on Jurkat cell may be associated with these differentially expressed proteins.Therefore, the research on effect and mechanism of ASPNJ to leukemia cells may provide new biomarker proteins for leukemia treatement, and may provide a new approach for the treatment of leukemia, and the antileukemic effect of ASPNJ worth to be studied further. |
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