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independent of changes in the related mRNA level
(Figures 3 and 5). The increased activity of
caspase-3 was negated via overexpression of DFF-
45. DFF-45 is the natural inhibitor of DFF-40
(CAD) (Liu et al., 1997). In addition, the increased
expression of DFF-45, along with a modest
increase (for sulfabenzamide) and a significant
decrease (for doxorubicin) in DFF-40 expression
(Figure 5), indicated that the increased activity of
caspase-3 could be blocked by the increased
expression ratio of DFF-45/DFF-40. This r<strong>edu</strong>ces
the level of active DFF-40 to trigger DNA
fragmentation and appearance of apoptotic
symptoms. Furthermore, it has been reported that
caspases are activated during autophagy in dying
cells and are suppressed for apoptosis induction
(Martin et al., 2004; Yu et al., 2004). Therefore, it
can be d<strong>edu</strong>ced that during autophagy, the effects
of activated caspase-3 on their downstream
substrates (like DFF-40) should be suppressed by
special factors (e.g. DFF-45 in T-47D cells) only in
those cellular routes which are involved in the
appearance of apoptosis symptoms (e.g. DNA
fragmentation).
Cell cycle arrest, an important cellular target
affected by sulfabenzamide and doxorubicin, was
analyzed using flow cytometry. Incubation of T-
47D cells with 0.33 µM doxorubicin resulted in a
significant accumulation of cells in S phase, and to
a lesser extent in G2/M phase of the cell cycle
(Figure 4B and Table 2). Thus, doxorubicin exerts
its antiproliferative action mainly through cell cycle
arrest. Induced mitotic catastrophe following
increased activation of cyclinB1/Cdc2 may occur
while cells are delayed, particularly in G2 phase of
the cell cycle (Lindqvist et al., 2007). The induced
G2/M arrest along with down regulation of
cyclinB1 expression confirmed that anticancer
activity of doxorubicin is not via mitotic
catastrophe (Figure 5 and Table 2). In contrast to
doxorubicin, minimal cell cycle arrest in S and
G2/M phases (totally 18%) was observed in T-47D
cells incubated with <strong>10</strong>.8 mM sulfabenzamide
(Figure 4B and Table2). Thus, cell cycle arrest
could not be mainly responsible for a 50%
r<strong>edu</strong>ction in cell viability in the presence of
sulfabenzamide.
As we know, when apoptosis is blocked or
delayed autophagy triggered and vice versa. These
possibilities are consistent with our findings
regarding lack of apoptosis in drug treated cells and
induction of autophagy. The induced
overexpression of ATG5 supported that autophagy
Sulfabenzamide promotes autophagic cell death 51
triggered in the presence of sulfabenzamide (Figure
5). This could be probably occurred through the
increase in Bax activity working on mitochondrial
membrane to result in activation of caspase-3 for
PARP1 and DNA-PK deactivation and autophagy
induction. It has been reported that induction of
autophagy by PUMA (the p53-inducible BH3-only
protein) depends on Bax/Bak and can be
reproduced by overexpression of Bax (Yee et al.,
2009). Here, in doxorubicin treatment, increase in
Bax activity could be occurred in parallel with the
increment of Bax transcripts affecting on the cells
for caspase-3 activation and changing the cell's
destiny toward autophagy (Figure 5). This notion
could be also supplied in sulfabenzamide treatment
aside from the mild decrease in Bax expression
because activation of the existed Bax molecules in
the cells could be happened for caspase-3 activation
and autophagy induction (Figure 5). It has been
also reported that proteolytic cleavage of PARP1,
performed by caspase-3, produces specific
proteolytic cleavage fragments which are involved
in the cell’s decision to change its fate from
apoptosis toward autophagy (Munoz-Gamez et al.,
2009; Chaitanya et al., 20<strong>10</strong>). Induction of
autophagic cell death is dependent on DNA-PK
inhibition (Daido et al., 2005). Thus, the increased
activity of caspase-3 could finally deactivate
PARP1 (has a decreased and constant expression
level in sulfabenzamide and doxorubicin
treatments, respectively) and DNA-PK (has an
increased and invariable expression level in
sulfabenzamide and doxorubicin treatments,
respectively) until apoptosis was blocked and
autophagy induced (Figures 3 and 5).
Despite the existence of some controversies
regarding the possible role of autophagy in tumor
progression by promoting cell survival, autophagy
can exist as a backup mechanism promoting
cellular death when other mortality mechanisms are
not functional. Hyperactivation of autophagy above
the threshold point leads to unlimited self-eating of
the cells causing autophagy or type II programmed
cell death (Hoyer-Hansen et al., 2005; Maiuri et al.,
20<strong>10</strong>). Based on our data, downregulation of AKT1
and AKT2 as well as upregulation of PTEN in
sulfabenzamide treated cells indicated that cell
survival pathways were slowed down (Figure 5). In
addition, down regulation of bcl-2 was happened
along with the induction of autophagy (Figure 5). It
has been reported that targeted silencing of bcl-2
expression (an anti-autophagic gene) in human
breast cancer cells with RNA-interference has