benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and Lymphoma--Non-Hodgkin

To explore the exact mechanisms of programmed cell death (PCD) induced by Type II anti-CD20 mAb in CD20+ non-Hodgkin lymphoma (NHL) cells, and to provide theoretical basis for anti-tumor ability of new CD20 mAb.
. After incubation with Rituximab (a Type I anti-CD20 mAb) and Tositumomab (a Type II anti-CD20 mAb), Raji cells were stained by annexin V & propidium iodide (PI). The ratio of programmed death cells were measured by two channel flow cytometry (FCM). Before the treatment of anti-CD20 mAbs, Raji cells was incubated with a caspase inhibitor carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]- fluoromethylketone (Z-VAD-FMK) and a dihydroceramide synthase inhibitor fumonisin B1 (FB1) for 30 minutes to assess their inhibitory effect on PCD. High performance liquid chromatography (HPLC) was utilized to compare the ratio of programmed death cells between the pretreatment group (treated by Rituximab and Tositumomab) and the non-pretreatment group. The anti-CD20 mAbs-treated Raji cells were collected, and the ceramide levels in the Raji cells in the different pretreatment groups were also examined by HPLC, and the inhibitory effect of FB1 on the changes of ceramide levels in the Raji cells was measured. The Raji cells were incubated with different concentration C2-ceramide, C2-Ceramide-induced PCD was also evaluated by annexin V & PI staining after 16 hours. 
. Tositumomab (10 µg/mL) but not Rituximab (10 µg/mL) can induce significant PCD (28.6±4.2)% in Raji cells, with significant difference (t=26.48, P<0.01), which cannot be blocked by Z-VAD-FMK with a concentration range from 10 to 30 µmol/L (F=3.01, P>0.05). The cellular ceramide levels in Raji cells were significantly elevated after the treatment of Tositumomab (t=28.48, P<0.01). C2-ceramide can significantly induce PCD in Raji cells in a dose-dependent manner with a concentration range from 5 to 40 µmol/L (F=2.71, P>0.05). The dihydroceramide synthase inhibitor FB1 can significantly inhibit the elevated cellular ceramide levels (F=20.18, P<0.01) and cell programmed death induced by Tositumomab (F=17.02, P<0.01).
. Type II but not Type I anti-CD20 mAbs can induce caspase independent PCD in CD20+ NHL cells through the elevation of cellular ceramide levels. The PCD is not associated with classic caspase pathway.. 目的：探讨II型CD20抗体诱导靶细胞程序性死亡的机制，为制备具有较强肿瘤杀伤能力的新型CD20抗体提供理论依据。方法：将淋巴瘤细胞Raji分别与 I型CD20抗体利妥昔单抗(Rituximab)和II型CD20抗体托西莫单抗(Tositumomab)孵育后，采用磷脂酰丝氨酸V(annexin V)和碘化丙啶(propidium iodide，PI)双染，流式细胞仪比较两种抗体处理后程序性死亡细胞所占比例；在CD20抗体处理前，用工作浓度的caspase抑制剂Z-VAD-FMK (carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone)或伏马毒素(fumonisin B1，FB1)预处理Raji细胞，并采用流式细胞仪比较预处理组与未预处理组淋巴瘤细胞程序性死亡所占的比例。收集CD20抗体处理过的Raji细胞，应用高效液相色谱法检测不同的CD20抗体处理组间淋巴瘤细胞内神经酰胺(ceramide)的水平，并用相同方法检测FB1对Tositumomab引起淋巴瘤细胞内ceramide水平变化的抑制作用。用不同浓度的C2-ceramide与淋巴瘤细胞进行孵育，16 h后用annexin V & PI双染检测其诱导靶细胞程序性死亡的能力。结果：10 µg/mL的I型抗体Rituximab几乎不能诱导Raji细胞的程序性死亡，而相同浓度的II型CD20抗体Tositumomab能够诱导(28.6±4.2)%的Raji细胞产生程序性死亡，两者差异有统计学意义(t=26.48，P<0.01)，这种程序性死亡不能被caspase抑制剂Z-VAD-FMK所抑制(F=3.01，P>0.05)。Tositumomab处理后能够引起Raji细胞内ceramide水平的升高(t=28.48，P<0.01)，且5~40 µmol/L的C2-ceramide能够通过剂量依赖性的关系诱导Raji细胞的程序性死亡(F=2.71，P>0.05)。Ceramide合成的FB1可以显著抑制Tositumomab引起的Raji细胞内ceramide水平的升高(F=20.18，P<0.01)，进而显著抑制Tositumomab介导的程序性死亡(F=17.02，P<0.01)。结论：II型CD20抗体Tositumomab而非I型CD20抗体Rituximab可以通过介导靶细胞内ceramide水平的升高，诱导靶细胞的程序性死亡，这种程序性死亡与经典的caspase通路无关。.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Cell Line, Tumor; Humans; Lymphoma, Non-Hodgkin; Rituximab; Sphingosine

Sphingolipid metabolites are important regulators of cell growth and apoptosis. To clarify the biological roles of cell surface sialylation in the effects of sphingomyelinase (SM) treatment on cell viability, the human diffuse large B cell lymphoma cell line, HBL-2 with or without treatment with Vibrio cholerae neuraminidase, was incubated with exogenous bacterial SM which is a key enzyme of ceramide production from sphingolipids in cell membranes. SM treatment enhanced viability of HBL-2 cells compared to non-treatment after 6 h of incubation. On the other hand, viability of HBL-2 cells was decreased by SM treatment with neuraminidase pre-treatment after 6 and 24 h of incubation, and ceramide production on cell surfaces of SM treated cells was enhanced by neuraminidase treatment as shown by flow cytometric analysis. Furthermore, treatment with D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, an inhibitor which specifically reduces the activity of UDP-glucose:ceramide glucosyltransferase in combination with SM treatment, causes the viability of HBL-2 cells to be decreased more with neuraminidase pre-treatment than without it. Exogenous C6-ceramide induced HBL-2 cell death, and there was no difference in the effects of C6-ceramide after 6 h of incubation between treatment and non-treatment with neuraminidase. Together these data suggest that alteration in susceptibility of HBL-2 cells to SM by neuraminidase treatment may precede the process of ceramide production, and that cell death through the activation of SM, which induces ceramide production, is regulated by cell surface sialylation in DLBCL.

Topics: Amino Acid Chloromethyl Ketones; Biotinylation; Cell Death; Cell Line, Tumor; Cell Membrane; Cell Survival; Ceramides; Enzyme Inhibitors; Flow Cytometry; Glucose; Glucosyltransferases; Humans; Lectins; Lymphoma; Lymphoma, B-Cell; Lymphoma, Non-Hodgkin; Models, Biological; Neuraminidase; Sialic Acids; Sphingomyelin Phosphodiesterase; Temperature; Time Factors; Uridine Diphosphate