Induction of apoptosis by combined treatment with differentiation-inducing agents and interferon-alpha in human lung cancer cells.
BACKGROUND
Differentiation-inducing agents for myeloid leukemic cells, such as dimethyl sulfoxide (DMSO), Na-butyrate and hexamethylene-bis-acetamide (HMBA), barely induce irreversible differentiation or growth inhibition in solid tumors when used alone. However, we previously reported that combined treatment with differentiation-inducing agents and interferon (IFN)-alpha effectively suppressed the growth of human lung cancer cell lines in vitro and in vivo. We show here that combined treatment-induced cell death is caused by apoptosis.


METHODS
Induction of apoptosis was examined by expression of several apoptotic markers.


RESULTS
Combined treatment-induced cell death is caused by apoptosis, which is characterized by the induction of apoptotic morphological changes and the activation of caspase-3-like protease. The expression of Apo2.7, an early apoptotic change, is also induced by this combined treatment, and is suppressed by the addition of Z-Asp-CH2-DCB, a pan caspase inhibitor. The signal transduction pathway of IFN-alpha is rapidly observed in lung cancer cells, although it does not induce significant growth inhibition when used alone. We analyzed the effect of IFN-alpha on the apoptotic pathway and found that IFN-alpha but not DMSO induces the expression of caspase-4. The combination of IFN-alpha and DMSO did not cause further increase in the expression of caspase-4. In many cases of the induction of apoptosis, cytochrome c is released from mitochondria, which induces the formation of large caspase-activating complexes. Caspase-3-like-activities induced by the combination of DMSO or Na-butyrate with IFN-alpha were analyzed by gel filtration and detected equally in fractions with molecular weights of 200-300 kDa, suggesting that caspase-3 is activated in large complexes in lung cancer cells. However, Na-butyrate and HMBA, when used alone, induced the release of cytochrome c and enhanced the loss of mitochondrial membrane potential, whereas DMSO had no effect.


CONCLUSIONS
These results suggest that the combination of differentiation-inducing agents with IFN-alpha effectively induces apoptosis in lung cancer cells whereas the mechanism of such induction varies depending on the differentiation-inducing agent used.
Yamamoto-Yamaguchi
Yuri
Y
Saitama Cancer Center Research Institute, 818 Komuro, Ina, Saitama 362-0806, Japan. yamaguchi@cancer-c.pref.saitama.jp
Okabe-Kado
Junko
J
Kasukabe
Takashi
T
Honma
Yoshio
Y
D000081:Acetamides / Q000494:pharmacology; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D002087:Butyrates / Q000494:pharmacology; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000096:biosynthesis; D053471:Caspases, Initiator; D002454:Cell Differentiation / Q000187:drug effects; D003574:Cytochrome c Group / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000096:biosynthesis; D004121:Dimethyl Sulfoxide / Q000008:administration & dosage; D004790:Enzyme Induction / Q000187:drug effects; D006801:Humans; D050856:Interferon Regulatory Factor-1; D016898:Interferon-alpha / Q000008:administration & dosage; D008175:Lung Neoplasms / Q000188:drug therapy* / Q000201:enzymology / Q000473:pathology*; D010750:Phosphoproteins / Q000096:biosynthesis; D014407:Tumor Cells, Cultured
D000081:Acetamides; D002087:Butyrates; D061945:Caspase Inhibitors; D003574:Cytochrome c Group; D004268:DNA-Binding Proteins; C494257:IRF1 protein, human; D050856:Interferon Regulatory Factor-1; D016898:Interferon-alpha; D010750:Phosphoproteins; C505407:CASP3 protein, human; C093516:CASP4 protein, human; D053148:Caspase 3; D020169:Caspases; D053471:Caspases, Initiator; C014026:hexamethylene bisacetamide; D004121:Dimethyl Sulfoxide