31preferential mediator for antitumor, and hemoproteins should be ART’s targets in
tumor cells. Validation of such a suggested idea needs to verify the formation of
ART-heme adducts being correlated with the inhibition of a specific hemoprotein
and upregulation of the hemoprotein gene. In the present study, we used ART as a
heme alkylator to investigate an interaction between ART and hemoproteins in the
hepatoma cell line HepG 2. By the parallel detections of ART-heme adducts and
enzymatic activities, we would confirm which hemoproteins are ART’s targets for
exerting antitumor activity.
3.2.2 Results and Analysis
3.2.2.1 Identification of ART-Heme Adducts in Tumor Cells
To testify the interaction of ART with hemoproteins, we monitored the dynamic
fluctuation of a specific absorbance peak of heme (A 415 ) and a unique absorbance
peak of ART-heme adducts (A 476 ) in the tumor cell line HepG 2 after incubation
with 50 μM ART for 24, 48, and 96 h. To prepare a cell lysate for photometric
detection, the in vitro cultured tumor cells were collected and lysed by a simple
freeze-thaw cycle (Zhang and Gerhard 2009 ).
As results, a gradual elevation of A 476 was observed in cells incubated with
ART until 96 h. The readings of A 476 are from 0.042 to 0.110 after 24 h, to 0.123
after 48 h, and to 0.131 after 96 h. A dual-phase change of A 415 was also noticed
with an elevation (0.145) after 24 h and a decline (0.033 and 0.049) after 48 h and
96 h, respectively (Table 3.1). The increase of heme suggests an enhanced biosyn-
thesis of hemoproteins following inhibition by ART.
3.2.2.2 ART’s Threshold Effect and Possible ART-Targeted Enzymes
In the present investigation, we evaluated the effect of different concentrations of
ART on the in vitro propagation of HepG 2 after incubation for 48 h. It was found
Table 3.1 The fluctuation of A 415 and A 476 values of HepG 2 after incubation with 50 μM
artesunate for 24, 48, and 96 h
ART artemisinin. The singular asterisk (*) represents statistically significant difference from the
control (P < 0.05); The double asterisks (**) represent statistically very significant difference
from the control (P < 0.01)
Group A 415 A 476
Control 24 h 0.044 ± 0.001 0.042 ± 0.004
ART 24 h 0.145 ± 0.002* 0.110 ± 0.003**
ART 48 h 0.033 ± 0.001* 0.123 ± 0.002**
ART 96 h 0.049 ± 0.002* 0.131 ± 0.002**3.2 ART Diminishes NO-Conferred Anticytotoxicity ...