title: [Ceramide participates in cell programmed death induced by Type II anti-CD20 mAb].
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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).
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Hematology, First Affiliated Hospital of Xinxiang Medical College, Weihui Henan 453100, China huangydoctor@163.com.
authors.forename: Yan
authors.initials: Y
authors.lastname: Huang
authors.affiliation: Department of Hematology, First Affiliated Hospital of Xinxiang Medical College, Weihui Henan 453100, China.
authors.forename: Sun
authors.initials: S
authors.lastname: Wu
authors.affiliation: Department of Hematology, First Affiliated Hospital of Xinxiang Medical College, Weihui Henan 453100, China.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Department of Hematology, First Affiliated Hospital of Xinxiang Medical College, Weihui Henan 453100, China.
authors.forename: Youmei
authors.initials: Y
authors.lastname: Zi
authors.affiliation: Department of Hematology, First Affiliated Hospital of Xinxiang Medical College, Weihui Henan 453100, China.
authors.forename: Man
authors.initials: M
authors.lastname: Yang
authors.affiliation: Department of Hematology, First Affiliated Hospital of Xinxiang Medical College, Weihui Henan 453100, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Guo
authors.affiliation: Department of Hematology, First Affiliated Hospital of Xinxiang Medical College, Weihui Henan 453100, China.
authors.forename: Lingxiu
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Department of Hematology, First Affiliated Hospital of Xinxiang Medical College, Weihui Henan 453100, China.
authors.forename: Lihua
authors.initials: L
authors.lastname: Wang
mesh_terms: D000590:Amino Acid Chloromethyl Ketones; D017209:Apoptosis / Q000187:drug effects*; D045744:Cell Line, Tumor / Q000187:drug effects; D006801:Humans; D008228:Lymphoma, Non-Hodgkin; D000069283:Rituximab / Q000494:pharmacology*; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000494:pharmacology
chemical_list: D000590:Amino Acid Chloromethyl Ketones; C064769:N-acetylsphingosine; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D000069283:Rituximab; D013110:Sphingosine