Page 19 - Read Online

Seno et al. Cancer Drug Resist 2019;2:335-50 I http://dx.doi.org/10.20517/cdr.2019.01 Page 339

RESULTS
Identification of drugs effective against miPS-CSCs
The miPS-LLCcm cells are cancer stem-like cells that are spontaneously generated from miPSCs cultured
[8]
in the presence of CM of mouse Lewis lung cancer cells . Given that the parental miPSCs express GFP and
puromycin-resistant gene under the control of the Nanog promoter, GFP-expressing Nanog-positive stem cell
populations of miPS-LLCcm cells could be concentrated when cultured in the presence of puromycin [10,11,18] .
Based on our previous findings, puromycin selection of stem cells was carried out in the presence of CM
prepared from the bulk culture of miPS-LLCcm cells to fully mimic the niche of miPS-LLCcm cells, which
[11]
comprise both differentiated and undifferentiated cell populations . Furthermore, we designed the drug
screening system against puromycin-selected CSCs in medium containing CM from bulk culture of miPS-
CSC [Figure 1A]. The cell viabilities under different conditions were determined as described in “Materials
and Methods”. In the present study, 190 different chemical compounds categorized as anticancer drugs or
cellular signal inhibitors were evaluated against four miPS-CSC cell lines. The analysis identified several
compounds that exhibited significant growth inhibitory or cytotoxic effects on miPS-CSCs [Table 1]. The
results obtained using several anticancer drugs are summarized in Figure 1B. The miPS-CSCs assessed in
the present study were highly sensitive to doxorubicin, daunorubicin, mitomycin C, and actinomycin D,
thereby implying that DNA damage and inhibition of DNA replication and/or transcription are effective
against miPS-CSCs. However, cisplatin, which can also interfere DNA replication, was not effective at the
concentration of 1 μmol/L. The cytotoxic effects of daunorubicin were further analyzed [Figure 1C]. The
IC values of daunorubicin against several different miPS-CSCs in both bulk cell culture and stem cell
50
populations selected by puromycin ranged from 5 to 20 nmol/L. The obtained IC values against L1210 cells
50
was 60 nmol/L, while that against Hela cells was 137 nmol/L; daunorubicin was not effective on BALB/c 3T3
cells, indicating that daunorubicin effectively inhibits the growth of both undifferentiated and differentiated
cells of miPS-CSCs.

Daunorubicin-induced apoptotic cell death of miPS-LLCcm cells
Daunorubicin and doxorubicin are topoisomerase II inhibitors that cause DNA strand breaks and induce
[19]
DNA damage responses and apoptotic cell death . In the present study, we assessed the death of miPS-
LLCcm cells following treatment with 100 nmol/L daunorubicin. Small vesicles that appeared similar to
apoptotic bodies were frequently observed on GFP-positive cells during daunorubicin treatment [Figure 2A].
After 3-h exposure to daunorubicin, significant proportions of annexin V-stained cells were detected for
both GFP-positive and -negative cells [Figure 2B]. DNA fragment isolation could remove intact genomic
DNA by centrifugation immediately after cell lysis, which could significantly increase the sensitivity to allow
the detection of fragmented DNA. Thus, the amounts of intact DNA in healthy cells were very low in the
background such that apoptotic DNA fragmentation was strongly evident [Supplementary Figure 1]. The
typical oligonucleosomal DNA ladder was clearly observed after 12 h of treatment with danuorubicin in both
the bulk cells and stem cells selected by puromycin [Figure 2C]. Taken together, the above results indicated
that miPS-LLCcm cells, including the populations comprising both GFP-negative differentiated cells and
GFP-positive stem cells, underwent apoptotic cell death.

Next, we assessed the involvement of the p53 pathway in the apoptosis of miPS-LLCcm cells. Significant
accumulation of p53 proteins was observed upon treatment with daunorubicin [Figure 2D] and was also
observed in parental miPS and the primary cells established from the tumor formed by transplantation of
[8]
the miPS-LLCcm cells in a nude mouse . The p53 proteins were concentrated in the nuclear fraction after
daunorubicin treatment [Figure 2E]. The p53 transcription factor induces the expression of several genes
involved in DNA damage response, such as apoptosis and cell cycle regulation. In the miPS-LLCcm cells,
the expression of apoptotic factors, such as Noxa, Bax, and Puma, as well as the CDK inhibitor p21, and
Mdm2, a negative regulator of p53, were found to be upregulated upon daunorubicin treatment [Figure 2F].
The induction of Noxa expression was especially prominent, indicating the involvement of the Noxa-