Proteasome inhibitors abolish cell death downstream of caspase activation during anti-microtubule drug-induced apoptosis in leukemia cells.
OBJECTIVE
Anti-microtubule drugs and proteasome inhibitors are currently among the most intensively studied anti-tumor agents, however little is known about their pharmacological interactions at the cellular level.


METHODS
The human promyelocytic leukemia cell line, HL-60, was exposed to nocodazole or etoposide in combination with proteasome or caspase inhibitors. Apoptotic cell death was detected by flow cytometry as sub-G1 population. Caspase and proteasome activities were monitored by the fluorogenic substrates Ac-DEVD-AMC and Suc-LLVY-AMC, respectively, in cell lysate. Heat shock protein 70 (HSP70) expression was determined by Western blotting.


RESULTS
Nocodazole, a microtubule inhibitor, induced caspase-dependent apoptosis in the HL-60 cell line. At sub-cytotoxic concentrations, proteasome inhibitors, including MG-132 or clasto-beta-lactone, decreased nocodazole-induced apoptotic DNA fragmentation without affecting the induction of caspase-3 activity. In contrast, MG-132 decreased both DNA fragmentation and caspase activation induced by etoposide, a topoisomerase-II inhibitor. HSP70 had previously been found to inhibit apoptosis independently from caspase activation. In this study, MG-132 up-regulated HSP70 protein expression, both in the presence or absence of nocodazole.


CONCLUSIONS
Proteasome inhibitors decreased anti-microtubule agent-induced apoptotic DNA fragmentation downstream of caspase-3 activation, possibly due to increased HSP70 expression. The results indicate that combination treatment with these novel anti-tumor agents in leukemia requires careful evaluation of their molecular interaction at the level of apoptosis induction.
Nagy
Katalin
K
First Department of Pathology and Experimental Cancer Research, Semmelweis University, Budapest, Hungary.
Petak
Istvan
I
Imre
Gergely
G
Barna
Gabor
G
Gezane-Csorba
Maria
M
Sebestyen
Anna
A
Houghton
Janet A
JA
Mihalik
Rudolf
R
Kopper
Laszlo
L
D000590:Amino Acid Chloromethyl Ketones / Q000008:administration & dosage / Q000494:pharmacology; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D002453:Cell Cycle / Q000187:drug effects; D015853:Cysteine Proteinase Inhibitors / Q000008:administration & dosage / Q000494:pharmacology*; D004347:Drug Interactions; D004789:Enzyme Activation; D005047:Etoposide / Q000008:administration & dosage / Q000494:pharmacology; D018922:HL-60 Cells; D018840:HSP70 Heat-Shock Proteins / Q000096:biosynthesis; D006801:Humans; D015473:Leukemia, Promyelocytic, Acute / Q000188:drug therapy* / Q000201:enzymology / Q000473:pathology; D007976:Leupeptins / Q000008:administration & dosage / Q000494:pharmacology; D008870:Microtubules / Q000187:drug effects; D015739:Nocodazole / Q000008:administration & dosage / Q000494:pharmacology*; D061988:Proteasome Inhibitors; D015854:Up-Regulation / Q000187:drug effects
D000590:Amino Acid Chloromethyl Ketones; D061945:Caspase Inhibitors; D015853:Cysteine Proteinase Inhibitors; D018840:HSP70 Heat-Shock Proteins; D007976:Leupeptins; D061988:Proteasome Inhibitors; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D005047:Etoposide; D020169:Caspases; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde; D015739:Nocodazole