Regulation Of Burkitt's Lymphoma By SPK1-S1P Signal And Correlative Studies On Its Mechanism

Although there are considerable progress in cancer therapy, such as surgery, chemotherapy, radio-therapy and bio-targeted therapy in recent years, therapeutic effect of Burkitt's lymphoma (BKL), which is a high malignant lymphoma, has little advancement. BKL has a lot of characteristics, for instance, high malignancy, wide- spread in body, easy recur and secondary drug resistance. Up till now, three years disease free survival (DFS) in general chemotherapy projects for BKL is lower than 50%. Therefore, a safe and specific drug targeted BKL, which aimed to ease the recurrence and resistance of the patient is the goal of this research.At present, studies have shown that sphingolipids is not only an integral part of the cell membrane structure, and is an important signaling molecule. Sphingosine kinase 1 (SPK1) is a key enzyme in modulating sphingolipids balance and catalyzing the formation of sphingosine 1 phosphate (S1P). S1P is believed to serve both as the first or second messenger involved in multiple signal transduction pathways. SPK1- S1P exquisitely regulates genesis development, angiogenesis, allergies reaction, heart development, tumor genesis and progress. These led us to hypothesize that SPK1-S1P signal could offer the regulation of BKL. To this end, we have embarked the studies on the regulation of BKL by SPK1-S1P and correlative molecular mechamisms. The aims of the present study were to identify whether SPK1-S1P signal regulated BKL biological function, and whether SPK1-S1P signal associated with MAPK, Akt, STAT3 pathway. To elucidate these questions may contribute to understand the effect of SPK1-S1P in BKL, and provide experimental basia for BKL potential therapy.First of all, we detected expressions of SPK1-S1P signal associated molecules in BKL cells by RT-PCR. We identified that BKL cell lines like Ramos cells expressed S1P receptors (also named Edgs) including Edg-1/S1P1, Edg-3/S1P3, Edg-5/S1P2, Edg-6/S1P4, Edg-8/S1P5 and SPK1. It indicates that BKL cells may be regulated by SPK1-S1P.To discover the effects of the activation of SPK1-S1P signal to biological func- tions of BKL cells, We examined the impact of exogenous S1P to the proliferatiation of Ramos cells by CCK-8 above all. Results showed after 24h, 48h and 72h culture, the proliferation of 0.01～2.5μmol/L dose groups had no significant statistic differ- ence compared to control group. After that, we investigated that exogenous S1P regu- lated invasion of Ramos cells by Transwells assay. Ramos cells were treated with S1P 0.1～2.5μmol/L for 6.0h. Results showed that S1P promoted cells migration in a dose dependent manner, and up to peak in 1.0μmol/L which chemotaxy was double higher than that of control. In addition, to investigate the effect of S1P protects Ramos cell from apoptosis, Ramos cells apoptosis was applied with Dexamethasone(DEX). Results showed that 10.0μmol/L DEX can significantly induce Ramos cells apoptosis, that was 2.0 fold higher than merely serum-free group. When pretreated with S1P 1.0μmol/L for 2.0h, the apoptotic ratio significantly reduced from 24.5% of DEX group to 10.6%. In short, S1P can promote migration of Ramos cells and protect them from apoptosis induced by DEX.SPK1 is a key rate-limiting enzyme for catalyzing phosphorylating sphingenine to produce S1P. To identify the role of SPK1-S1P signal in regulating Ramos cell functions, we used RNAi technology to induce SPK1 gene silence. Firstly we prepared and purified Ad-H1-SPK1 by cesium chloride gradient centrifugation. The purity of Ad-H1-SPK1 was 1.37, and infection titre (IU/ml) was 2.0×1010. After Ad-H1-SPK1 infecting Ramos cells 48h, Western blot was used to detect the expression of SPK1, and results showed that SPK1 expression was reduced 34%.γ-32P incorporation method was used to determine SPK1 activity. Results showed that the activity of SPK1 reduced 56% compared with control group. It suggested that we successfully interfering SPK1. After Ad-H1-SPK1 or Ad-H1 virus infecting Ramos cells 48h, we seeded Ramos cells in 96 plate and used Transwells assay to determine proliferation and migration ability. Results showed the proliferation of Ad-H1-SPK1 group was slightly lower than Ad-H1 group, but no statistic difference. Nevertheless the migration of Ad-H1-SPK1 group was significant lower than Ad-H1 group. N,N- dimethylsphingosine(DMS), specific inhibitor of SPK, was used to observe the effect of blocking SPK1 on cell apoptosis. These results showed that DMS can induce apop- tosis of Ramos cells. These results suggest that interference or inhibition of SPK1, from the point of negative regulation, can verify that SPK1-S1P promotes Ramos cell migration and protect them from apoptosis.To identify the signal pathways that involved in the S1P regulation of BKL cells, Ramos cells were treated with exogenous S1P, then assayed the phosphorylation level of MAPK, Akt and STAT3. Results showed that S1P could induce rapid phosphory- lation of MAPK, Akt and STAT3 in a certain dose range with peak 0.25μmol/L or 0.5μmol/L, time peak 20 or 30min. In a word, MAPK, Akt and STAT3 participated in the regulation of Ramos cells by S1P.To clarify the mechamism of S1P to regulate the apoptosis, we treated Ramos cells with exogenous S1P, then Western blot was used to detect the expression of anti- apoptosis factor Mcl-1. Results showed that S1P can upregulate Mcl-1 expression of Ramos cells in dose-dependeted and time-dependeted manner, with dose peak 0.5μmol/L, time peak 45min. To elucidate how S1P regulates Mcl-1 expression, we used special blocking agents like PD98059 (MAPK inhibitor), Wortmannin (PI3K inhibitor) and AG490 (JAK inhibitor) to inhibit signal pathways and then investigated the Mcl-1 expression after S1P treatment. Results showed that PD98059, Wortmannin, AG490 could inhibit S1P upregulatd Mcl-1 expression. All above suggested that MAPK, PI3K-Akt and JAK-STAT pathways involved in Mcl-1 regulation by S1P. S1P binds and interacts with five isoforms of S1P receptors expressed in BKL cells, however, it is still unclear which isoform plays an important role in regulating BKL cell function. With regards to this, we used pertussis toxin(PTX), SEW2871(S1P1 receptor agonist), JTE-013(S1P2 receptor antagonist), CAY10444(S1P3 receptor antagonist)to pretreat cells, and then investigated the regulation of S1P to Mcl-1. Results showed that only CAY10444 could significantly inhibit the upregulation of Mcl-1 induced by S1P. It demonstrates that S1P upregulates Mcl-1 expression mainly through Edg-3 by PTX- insensitivity manner.Taken together, our study identified that: 1)BKL cells express 5 isoforms of S1P receptors and SPK1; 2)Exogenous S1P could promote Ramos cells migration and protect DEX-induced apoptosis; 3)After inhibiting SPK1 expression in Ramos cells, cell migration ability declined and cell apoptosis; 4)S1P can upregulate Mcl-1 expression through signal pathways such as MAPK, PI3K-Akt and JAK-STAT; 5)S1P can protect Ramos cell from apoptosis through Edg-3. Therefore, we provide experi- mental proof to targeted therapy of BKL by interfering SPK1-S1P or blocking Edg-3.