Sa. Kim et al., INDUCTION OF TOPOISOMERASE II-MEDIATED DNA CLEAVAGE BY A PROTOBERBERINE ALKALOID, BERBERRUBINE, Biochemistry (Easton), 37(46), 1998, pp. 16316-16324
Topoisomerase II is the cytotoxic target for a number of clinically re
levant antitumor drugs. Berberrubine, a protoberberine alkaloid which
exhibits antitumor activity in animal models, has been identified as a
specific poison of topoisomerase II in vitro. Topoisomerase II-mediat
ed DNA cleavage assays showed that berberrubine poisons the enzyme by
stabilizing topoisomerase II-DNA cleavable complexes. Subsequent prote
inase K treatments revealed that berberrubine-induced DNA cleavage was
generated solely by topoisomerase II. Topoisomerase II-mediated DNA r
eligation with elevated temperature ee revealed a substantial reductio
n in DNA cleavage induced by berberrubine, to the extent comparable to
that of other prototypical topoisomerase II poison, etoposide, sugges
ting that DNA cleavage involves stabilization of the reversible enzyme
-DNA cleavable complex. However, the step at which berberrubine induce
s cleavable complex may differ from that of etoposide as revealed by t
he difference in the formation of the intermediate product, nicked DNA
. This suggests that berberrubine's primary mode of linear formation m
ay involve trapping nicked molecules, formed at transition from linear
to covalently closed circular DNA. Unwinding of the duplex DNA by ber
berrubine is consistent with an intercalative binding mode for this co
mpound. In addition to the ability to induce the cleavable complex med
iated with topoisomerase II, berberrubine at high concentrations was s
hown to specifically inhibit topoisomerase II catalytic activity. Berb
errubine, however, did not inhibit topoisomerase I at concentrations u
p to 240 mu M. Cleavage sites induced by topoisomerase II in the prese
nce of berberrubine and etoposide were mapped in DNA. Berberrubine ind
uces DNA cleavage in a site-specific and concentration-dependent manne
r. Comparison of the cleavage pattern of berberrubine with that of eto
poside revealed that they share many common sites of cleavage. Taken t
ogether, these results indicate that berberrubine represents a new cla
ss of antitumor agent which exhibits the topoisomerase II poison activ
ity as well as catalytic inhibition activity and may have a potential
clinical value in cancer treatment.