Anti-CD20 antibody (IDEC-C2B8, rituximab) enhances efficacy of cytotoxic drugs on neoplastic lymphocytes in vitro: role of cytokines, complement, and caspases.
OBJECTIVE
Monoclonal antibody IDEC-C2B8 (rituximab) has been shown to be highly effective in the treatment of non-Hodgkin's lymphomas (NHL). The present study was designed to investigate relationships between the efficacy of IDEC-C2B8 and expression of CD20, presence of complement, and effects of differently acting chemotherapeutic agents used in lymphoma treatment (doxorubicin, mitoxantrone, cladribine, bendamustine).


METHODS
DOHH-2, WSU-NHL and Raji lymphoma cell lines and ex vivo cells from patients with chronic lymphocytic leukemia (CLL) (n=17) and leukemic B-cell lymphomas (n=9) were studied. Additionally, the effect of interleukin (IL)-2, IL-4, IL-6, IL-13, granulocyte/macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor (TNF)alpha on expression of CD20 molecules per cell was determined.


RESULTS
We demonstrate that 10 mg/mL rituximab saturated 80-95% of CD20 molecules per cell in all tested lymphoma samples. Although rituximab induced only a minor increase of apoptosis, combinations of rituximab with different cytotoxic drugs significantly decreased the IC(30)- and IC(50) dosages of the chemotherapeutic agents necessary for induction of apoptosis irrespective of addition of complement, demonstrating a chemosensitizing effect of rituximab in combination with cytotoxic drugs in the neoplastic lymphocytes. This effect seemed to be independent of the percentage of saturated CD20 molecules. After addition of caspase inhibitors to the cell lines incubated with rituximab and cytotoxic agents, caspase-7 and -8 were found, by Western blotting, to be the executioner caspases, possibly explaining the rituximab-sensitized apoptosis. Preincubation of lymphoma cells with cytokines did not alter the expression of CD20; IL-2 and IL-4 even decreased the rate of apoptosis.


CONCLUSIONS
We conclude that rituximab sensitizes lymphoma cells to the effect of differently acting cytotoxic drugs used in lymphoma treatment, that this effect does not require complement, and that caspase-7 and -8 may represent the main executioner caspases in chemosensitization by rituximab.
Chow
Kai U
KU
University Hospital, Department of Internal Medicine III, Hematology and Oncology, Theodor-Stern-Kai 7, 60590 Frankfurt/Main, Germany.
Sommerlad
Wolf Daniel
WD
Boehrer
Simone
S
Schneider
Bernd
B
Seipelt
Gernot
G
Rummel
Mathias Johannes
MJ
Hoelzer
Dieter
D
Mitrou
Paris S
PS
Weidmann
Eckart
E
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology* / Q000627:therapeutic use; D058846:Antibodies, Monoclonal, Murine-Derived; D018951:Antigens, CD20 / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D000951:Antigens, Neoplasm / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D000069461:Bendamustine Hydrochloride; D002051:Burkitt Lymphoma / Q000473:pathology; D053179:Caspase 7; D053181:Caspase 8; D053453:Caspase 9; D061945:Caspase Inhibitors; D020169:Caspases / Q000502:physiology*; D017338:Cladribine / Q000494:pharmacology; D003167:Complement Activation; D003165:Complement System Proteins / Q000494:pharmacology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004317:Doxorubicin / Q000494:pharmacology; D004357:Drug Synergism; D015973:Gene Expression Regulation, Leukemic / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D006801:Humans; D007378:Interleukins / Q000494:pharmacology; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000188:drug therapy / Q000276:immunology / Q000473:pathology*; D016393:Lymphoma, B-Cell / Q000188:drug therapy / Q000276:immunology / Q000473:pathology*; D008224:Lymphoma, Follicular / Q000473:pathology; D008942:Mitoxantrone / Q000494:pharmacology; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors / Q000502:physiology*; D009588:Nitrogen Mustard Compounds / Q000494:pharmacology; D009842:Oligopeptides / Q000494:pharmacology; D011817:Rabbits; D000069283:Rituximab; D014407:Tumor Cells, Cultured / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D000590:Amino Acid Chloromethyl Ketones; D000911:Antibodies, Monoclonal; D058846:Antibodies, Monoclonal, Murine-Derived; D018951:Antigens, CD20; D000951:Antigens, Neoplasm; D000970:Antineoplastic Agents; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D007378:Interleukins; D009363:Neoplasm Proteins; D009588:Nitrogen Mustard Compounds; D009842:Oligopeptides; D014409:Tumor Necrosis Factor-alpha; C099158:acetyl-aspartyl-glutamyl-valyl-aspartal; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D017338:Cladribine; D000069283:Rituximab; D004317:Doxorubicin; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D003165:Complement System Proteins; D000069461:Bendamustine Hydrochloride; D008942:Mitoxantrone; C505520:CASP7 protein, human; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053179:Caspase 7; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases