32 3 ART for Antitumor
that the survival rate of tumor cells is highly dependent upon ART’s concentra-
tions, in which 50 μM ART leads to a higher survival rate accounting for nearly
80 %, whereas 100 and 200 μM ART cause the dramatic cell death, with a sur-
vival rate of 40 % for 100 μM ART and of 25 % for 200 μM ART. These results
indicated that there exists a threshold effect of ART on the survival/death fate of
tumor cells.
Considering that NOS is a hemoprotein and ART enables the alkylation of
hemoproteins, we envisaged that ART in an optimal concentration might conjugate
the heme-containing NOS to inhibit its activity, and also induce its expression in
a feedback manner. Therefore, we should verify an elevation of NO levels after
incubation of HepG 2 with a sublethal dose of ART. For this purpose, we incubated
HepG 2 with 50 μM ART for measuring the NO level. As our expectation, a dra-
matic elevation of the NO level was observed up to 35 μM after incubation for
48 h. In contrast, NO was not detected in HepG 2 treated by 100 or 200 μM ART.
These results indicated that 50 μM ART can trigger a protective level of NO from
tumor cells, but 100 and 200 μM represent the lethal doses of ART.
Because CAT that scavenges H 2 O 2 is also a hemoprotein, we measured
the H 2 O 2 level after incubating HepG 2 with 50, 100, or 200 μM ART for 48 h.
However, neither low-dose nor high-dose ART results in a significant change of
H 2 O 2. The level of H 2 O 2 in untreated and 50 μM ART-treated tumor cells is equal
to 53 mM, and that in 100 or 200 μM ART-treated tumor cells is approximately
57 mM. These results suggested that CAT is unlikely implicated in tumor killing
by ART, and H 2 O 2 -mediated apoptosis may not be a major mechanism by which
ART exerts an antitumor effect.
3.2.2.3 Synergistic Effects Through the Combination of ART
with Chemotherapeutics
Although high doses of ART (100–200 μM) are effective on tumor killing, it is
apparently higher than ART’s IC 50 (70 μM), thereby endowing a nonselective
toxicity to normal cells. However, 50 μM ART not only fails to kill tumor cells,
but also promotes tumor growth. To obtain a higher antitumor effect using a lower
dose of ART, we combined 50 μM ART with 10 μM 5-fluorouracil (FLU) for in
vitro antitumor evaluations in HepG 2 cells. Consequently, ART exhibits a signifi-
cant synergistic effect to FLU, in which tumor growth is inhibited by 25–40 %. In
contrary, tumor cells treated by 10 μM FLU alone show an inhibition rate of only
8 %. These results suggested that a sublethal dose of ART could effectively kill
tumor cells as it is combined with FLU.
It was observed that ART + FLU can dramatically decrease the NO level in
HepG 2 , from 16 μM NO in 50 μM ART-treated tumor cells to 11 μM NO in
50 μM ART + 10 μM FLU-treated tumor cells. At the same time, no signifi-
cant change in H 2 O 2 levels was found in tumor cells incubated with ART alone
or ART + FLU. Therefore, ART may not stimulate H 2 O 2 for augmenting FLU’s
antitumor activity.