Activation of caspase-3 in renal cell carcinoma cells by anthracyclines or 5-fluorouracil.
The caspase family of proteases is speculated to have a crucial role in apoptosis. The effect of treatment with adriamycin (ADR), cisplatin (CDDP), 5-fluorouracil (5-FU), vinblastine (VLB), IFN-alpha, or IFN-gamma on the activation of caspase-3, -6, -8, and -9 in renal cell carcinoma (RCC) cells was investigated, to clarify the mechanisms of chemo- and immunotherapeutic agent-mediated apoptosis. Caspase activity was determined by a quantitative colorimetric assay. Apoptosis was monitored by acridine-orange staining assay. Treatment of ACHN cells with CDDP, VLB, IFN-alpha, or IFN-gamma did not activate caspase-3, but its activity was increased 7.2-fold (p = 0.0001) with ADR and 2.8-fold (p = 0.0385) with 5-FU in comparison with control. Furthermore, when the ADR treatment time was shortened from 24 to 8 or 2 h, the same caspase-3 activation occurred. Activation of caspase-3 was also observed in six freshly isolated human RCC cells after the treatment with ADR. Of the six freshly derived RCC cells treated with 5-FU, caspase-3 activity was increased 3.1-fold (p = 0.0051) and 2.4-fold (p = 0.0346) in two of them, respectively. Epirubicin and pirarubicin, compounds closely related to ADR, also respectively enhanced 4.2-fold (p = 0.0052) and 2.8-fold (p = 0.0147) caspase-3 activity in ACHN cells. The activation of caspase-3 observed with a colorimetric assay was confirmed with immunocytochemical analysis using the anti-active caspase-3 mAb, which specifically recognizes the active form of caspase-3. Furthermore, both active caspase-3 and apoptosis triggered by either ADR or 5-FU were inhibited significantly by the general caspase inhibitor Z-VAD-FMK, or a specific caspase-3 inhibitor DMQD-CHO. These findings provide a mechanistic explanation for anthracyclines and 5-FU induced-apoptosis.
Wu
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Department of Urology, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan.
Kakehi
Y
Y
Mizutani
Y
Y
Lu
J
J
Terachi
T
T
Ogawa
O
O
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D002292:Carcinoma, Renal Cell / Q000201:enzymology* / Q000473:pathology; D053148:Caspase 3; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005472:Fluorouracil / Q000494:pharmacology*; D006801:Humans; D007124:Immunoenzyme Techniques; D016898:Interferon-alpha / Q000494:pharmacology; D007371:Interferon-gamma / Q000494:pharmacology; D007680:Kidney Neoplasms / Q000201:enzymology* / Q000473:pathology; D014407:Tumor Cells, Cultured / Q000201:enzymology / Q000473:pathology
D000590:Amino Acid Chloromethyl Ketones; D000903:Antibiotics, Antineoplastic; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D016898:Interferon-alpha; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D007371:Interferon-gamma; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D005472:Fluorouracil