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Page 344 Seno et al. Cancer Drug Resist 2019;2:335-50 I http://dx.doi.org/10.20517/cdr.2019.01

1. We observed no significant changes in p53 levels in the presence of Z-VAD-FMK, indicating that apoptosis
induction itself was not inhibited [Figure 3D]. Under this condition, the levels of apoptotic fragmented
DNA was significantly reduced [Figure 3E], thereby indicating that the activation of the caspase cascade was
induced but not essential for daunorubicin-induced apoptotic cell death of miPS-LLCcm cells.

We next assessed the degradation of ICAD, another caspase-3 substrate that is required for the release of an
endonuclease CAD, which is responsible for the formation of the apoptotic DNA ladder [23-25] . Whereas the
cleaved PARP-1 levels were readily detectable and significantly increased within the first 3 h of daunorubicin
treatment, the full-length ICAD levels remained stable throughout the treatment period, and no cleaved/
degraded ICAD fragments were observed [Figure 4A]. We observed no reduction in ICAD degradation even
in the concentrated detached cells [Figure 4B].

2+
2+
Ca /Mg -dependent endonucleases have been reported to contribute to apoptotic DNA fragmentation [26-31] ,
[32]
2+
whereas Ca is not required for CAD activity . Next, we assessed the effect of BAPTA-AM, a Ca -
2+
specific cell chelator, on the formation of apoptotic DNA ladder. Apoptosis induced by daunorubicin was
not inhibited in the presence of 10 μmol/L BAPTA-AM based on the cleavage of both caspase-3 and PARP-1
together with positive staining of annexin V-APC [Figure 4C]; however, constitutive levels of full-length of
ICAD were detected [Figure 4D]. Interestingly, DNA fragmentation was suppressed under this condition
2+
2+
[Figure 4E], suggesting the involvement of Ca /Mg -dependent endonucleases during daunorubicin-
induced apoptotic cell death of miPS-LLCcm cells.
Daunorubicin is clinically used for the treatment of acute myeloid leukemia. To evaluate the cytotoxic
effects of daunorubicin, we assessed the apoptotic features in mouse leukemia cell line L1210 cells treated
with daunorubicin. Daunorubicin activated the p53 and caspase cascades in L1210 cells. However, ICAD
degradation was not observed in L1210 cells [Figure 5A]. Interestingly, DNA fragmentation in L1210 cells
was different from that observed in miPS-LLCcm cells. In fact, the oligonucleosomal DNA ladder was
detected even when L1210 cells were cultured for more than 36 h without daunorubicin, thereby indicating
cell death during overgrowth; therefore, this condition should be treated as the background condition. In
the presence of daunorubicin, the smearing DNA fragmentation was prominent after 36 h of treatment,
while the oligonucleosomal DNA ladder was not detected within 24 h [Figure 5B]. For ICAD degradation,
we assessed the apoptosis induced by staurosporine in both miPS-LLCcm and L1210 cells. Although the two
cell lines showed different sensitivities to staurosporine, oligonucleosomal DNA fragmentation was detected
in both cells [Figure 5C]. Under this condition, ICAD levels in miPS-LLCcm cells remained stable, whereas
ICAD levels in L1210 cells were reduced [Figure 5D].

We further tested ICAD degradation and DNA fragmentation in human Hela cells. After treatment of
Hela cells with daunorubicin, we observed time-dependent reduction of full-length ICAD, indicating CAD
activation [Figure 5E]. However, oligonucleosomal DNA fragmentation was not detected [Figure 5F]. Taken
together, our results suggested that daunorubicin affects ICAD degradation depending on the cell type and
that CAD contributes little to the formation of oligonucleosomal DNA ladder during apoptosis of miPS-
LLCcm.

DISCUSSION
The behavior of CSCs is considered to be regulated in the niche by mutual communication between CSCs,
their progenies, and adjacent normal cells. Recent studies have shown that endothelial cells play important
roles in regulating CSC properties as components of the CSC niche [11,13-17] . Various studies, including our
previous studies, indicated that tumor endothelial cells are the progenies of CSCs [9-11,33-35] . Regarding therapy
resistance, studies have shown that both senescent and non-senescent tumor microvascular endothelial
cells that are resistant to radiotherapy and chemotherapy could still contribute to the growth of CSCs in