benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and Lymphoma--Follicular

Motexafin gadolinium (MGd, Xcytrin) is a tumor-localizing redox mediator that catalyzes the oxidation of intracellular reducing molecules including NADPH, ascorbate, protein and non-protein thiols, generating reactive oxygen species (ROS). MGd localizes to tumors and cooperates with radiation and chemotherapy to kill tumor cells in tissue culture and animal models. In this report, we demonstrate that MGd triggers the mitochondrial apoptotic pathway in the HF-1 lymphoma cell line as determined by loss of mitochondrial membrane potential, release of cytochrome c from mitochondria, activation of caspase-9 prior to caspase-8, cleavage of PARP and annexin V binding. There was minimal effect on MGd-induced apoptosis by the caspase inhibitor z-VAD-fmk, even though caspase-3 activity (as measured by DEVD-cleavage) was completely inhibited. However, MGd-induced apoptosis was reduced to baseline levels by the more potent caspase inhibitor Q-VD-OPh, demonstrating that MGd-induced apoptosis is indeed caspase-dependent. Apoptosis induced by dexamethasone, doxorubicin and etoposide (mediated through the mitochondrial pathway) was also more sensitive to inhibition by Q-VD-OPh than z-VAD-fmk. Our results demonstrating differential sensitivity of drug-induced apoptosis to caspase inhibitors suggest that the term "caspase-independent apoptosis" cannot be solely defined as apoptosis that is not inhibited by z-VAD-fmk as has been utilized in some published studies.

Topics: Amino Acid Chloromethyl Ketones; Annexin A5; Antineoplastic Agents; Apoptosis; Caspase Inhibitors; Caspases; Cell Line, Tumor; Drug Resistance, Neoplasm; Humans; Lymphoma, Follicular; Lymphoma, Large B-Cell, Diffuse; Metalloporphyrins; Mitochondria; Poly(ADP-ribose) Polymerases; Quinolines

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).. 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.. 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.. 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.

Topics: Amino Acid Chloromethyl Ketones; Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Murine-Derived; Antigens, CD20; Antigens, Neoplasm; Antineoplastic Agents; Apoptosis; Bendamustine Hydrochloride; Burkitt Lymphoma; Caspase 7; Caspase 8; Caspase 9; Caspase Inhibitors; Caspases; Cladribine; Complement Activation; Complement System Proteins; Cysteine Proteinase Inhibitors; Doxorubicin; Drug Synergism; Gene Expression Regulation, Leukemic; Gene Expression Regulation, Neoplastic; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Interleukins; Leukemia, Lymphocytic, Chronic, B-Cell; Lymphoma, B-Cell; Lymphoma, Follicular; Mitoxantrone; Neoplasm Proteins; Nitrogen Mustard Compounds; Oligopeptides; Rabbits; Rituximab; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha