C12 inhibits lung tumor growth and induces
tumor cell apoptosis in vivo
The cytotoxic effects of C12 on tumor cells have
been reported previously [5–8, 26], but whether they
are selective for transformed cells was unknown. To
investigate whether oncogenic transformation influences
the cytotoxicity of C12, we studied normal human
bronchia/tracheal epithelial (NHBE) and corresponding
HBE immortalized and transformed successively by
telomerase, SV40 large T antigen and activated Ras
(H-ras V12) [27–29]. This is a well-established epithelial
cell malignant transformation system related to human
lung cancer. Following C12 treatment, transformed HBE
exhibited higher levels of cell death (Figure 1A and
Supplementary Figure 1A) and caspase-3/7 activation (Figure 1B and
Supplementary Figure 1B) compared with
their untransformed counterparts, indicating that C12
induces apoptosis preferentially in transformed cells.
To investigate the relevance of C12 cytotoxicity
on transformed cells to tumor growth in animals, we
examined the effects of C12 on the growth of established
Lewis Lung Carcinoma (LLC) tumors. As shown in
Figure 1C, transplanted tumors grew much more slowly
in C12-treated mice than in vehicle-treated mice, revealing
a dose-dependent anti-tumor activity of C12 as a single
agent. No significant changes of body weight and organ
weight (spleen, kidney, liver, heart and lung) were
observed for C12-treated C57BL/6 mice (Supplementary
Figure 2A and 2C), showing no evidence of significant
toxicities of C12 administration. To test the involvement
of apoptosis in tumor growth inhibition, established LLC
tumors from mice were analyzed for caspase-3 activation
through immunofluorescence staining (Figure 1D–1E),
western blot (Figure 1F–1G) and TUNEL staining
(Figure 1H– 1I). The percentage of TUNEL-positive cells
and the levels of activated caspase-3 were higher in tumors
from C12– treated mice than in those of the control group,
suggesting that apoptosis is involved in the inhibitory
activity of C12 in vivo.
In order to explore the therapeutic potential of
C12, we studied the combinatorial effects of C12 and
therapeutic drugs. Cell death of human lung tumor cells
A549 and NCI-H1299 treated with various concentrations
of C12 and the inhibitor of Bcl-2 and Bcl-xL ABT-737 at
a constant dose ratio were measured, and the combination
index (CI) values were determined (Supplementary
Figure 3). As shown in Supplementary Figure 3C and 3F,
in the range of tested drug concentrations, CI values
were smaller than 1, indicating that C12 and ABT-737
exhibited synergistic cytotoxic effects on A549 cells and
NCI-H1299 cells.

Explain what the purpose of this set of the experiment is and
summarize the key steps on how they tested it.