Download Possible involvement of stem-like population with elevated ALDH1

Possible involvement of stem-like population with elevated ALDH1 and DNA repair enzymes for
drug resistance in human sarcoma
+1Kanya Honoki; 1Hiromasa Fujii; 1Akira Kido;
Shinji Tsukamoto; 1Yasuhito Tanaka; 2Toshifumi Tsujiuchi
+1Nara Medical University, Kashihara, Japan, 2Kinki University, Higashi-Osaka, Japan
[email protected]
1
The idea of stem cells being the cell of origin for cancers has been
proposed decades ago (1), and this idea has been supported by the
cancer stem model which depicts the presence of rare side-population
within the tumor can initiate and generate the cancer tissues (2). The
presence of cancer stem cell population has been described in several
malignancies like acute myeloid leukemia, breast cancer, brain tumors,
etc. Recently, high ALDH1 activity has been found in stem cell
populations in acute myeloid leukemia and breast cancers as well as
their counterparts of mammary and hematopoietic stem cells in
relation to drug resistance and poor outcome of the patients (3). Thus,
ALDH1 has been proposed as a common marker for both normal and
malignant stem cells. In the current study, we investigated whether the
populations with high ALDH1 activity are present in human sarcoma
cell lines using Aldefluor assay system and if they possess the stem
cell properties through characterization of sphere cells formed in an
anchorage-independent, serum-starved condition, and also tested their
capacity of drug-resistance to hypothesize possible involvement of
stem-like cells with effective detoxification and DNA repair for drug
resistance in sarcomas.
Formation and Stem-like characterization of sarcospheres. The
spherical colonies were formed in several days after culturing in
anchorage-dependent, serum starved condition at the frequencies of
~0.25% for MG63 and ~0.78% for HT1080, respectively. Dissociated
sphere cells were able to grow monolayer condition and then formed
spheres repeatedly by re-culturing in an anchorage-dependent, serum
starved condition, showing their self-renewal ability. RT-PCR
revealed that sphere forming cells showed increased expression of
stem cell-related genes; Nanog, Oct3/4, STAT3 and Sox2 compared to
monolayer cells, suggesting their stem-like properties. In addition,
those sphere cells showed increased expression of ALDH1 and DNA
repair enzyme genes MLH1 and MSH2 compared to monolayer cells
Drug resistance of spheres from human osteosaroma cell line. Both
CDDP and DXR inhibited the growth of MG63 up to 68% and 30%,
respectively at a concentration of 10µM for 48h treatment. In contrast,
sphere cells from MG63 showed strong resistance to both agents,
suggesting stem-like sphere forming cells with high ALDH activity
showed resistance to CDDP and DXR, both are key drugs for sarcoma
treatment.
MATERIALS and METHODS
Cell Culture. Human osteosarcoma cell line MG63 and
fibrosarcoma cell line HT1080 from ATCC were used in this study.
Aldefluor assay; Separation of the ALDH- positive and negative
populations by FACS. The Aldefluor (StemCell Technologies,
Durham, NC) was used to isolate the population with a high ALDH
enzymatic activity. The sorting gates were established using the
Aldefluor-stained cells treated with DEAB as negative controls.
Labeled cells were sorted with FACSaria to be fractioned into
ALDH-positive and negative cell populations. Both populations were
then re-cultured into monolayer conditions, and again harvested and
underwent to Aldefluor assay to determine whether both population of
ALDH positive and negative could differentiate into original cell
populations.
Sphere formation assay. The cells were inoculated into N2 /1%
methylcellulose medium without serum at a density of 1 x 105
cells/well in ultra low attachment 6-well plates (Corning Inc., Corning,
NY). Self-renewal ability of sphere cells were determined by
secondary sphere formation ability through the repeating dissociation
and re-culturing in anchorage-independent, serum-starved condition
on ultra low attachment plates.
Reverse transcription-polymerase chain reaction (RT-PCR).
Semi-quantitative RT-PCR was performed for following genes
expression, Nanog; Oct3/4; STAT3; Sox2; ALDH1, respectively. The
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used
as an internal control to adjust the amounts of template.
Efficacy of drugs on monolayer cells and spheres. Cells were
exposed to 10µl of cisplatin (CDDP) or doxorubicin (DXR) at a final
concentration of 10µM for 48 h. Both drug-treated and non-treated
cell viabilities were measured by MTS colorimetric assay using
CellTiter 96 Aqueous One Solution Cell Proliferation Assay reagent
(Promega, Madison, WI).
DISCUSSION
Cancer stem cell hypothesis depicts that tumors originate in either
tissue stem cells or progenitor cells through the deregulation of their
properties of stemness. Inversely, it is likely that cancer stem cells
have a phenotype defined by the cell of origin (stem cells or early
progenitors) and retain the stem cell properties of self-renew, capacity
to differentiate, drug resistance, and initiate the tumor growth giving
rise to a heterogeneous population of cancer cells. The presence of
stem-like cells in human bone sarcomas has been described
previously (4). The current study demonstrated that both human
osteosarcoma MG63 and human fibrosarcoma HT1080 contained the
subpopulations with high ALDH activity at the rates of around 10%
by Aldefluor assay. Sphere forming populations showed increased
expression of stem cell-related genes and capacity of self renewal,
thus they are considered to possess stem-like properties. These
stem-like cells showed drug-resistance with increased expression of
ALDH1 mRNA and DNA repair enzymes, MLH1 and MSH2
compared to monolayer cells. The frequencies of these populations
were much smaller compared to the ratio of the populations with high
ALDH1 activity, suggesting that the population with high ALDH
activity contains stem-like populations, but not entirely represents
them. The possible mechanism of drug resistance in stem cells has
been proposed: (i) activation and/or over-expression of cell membrane
drug efflux transporters, (ii) altered expression of detoxifying
enzymes, (iii) efficient DNA repair and defective apoptosis regulatory
proteins, and (vi) ‘niche’ protection of target cells. Current study
demonstrates the increased expression of detoxifying enzyme of
ALDH1 and DNA repair enzymes MLH1 and MSH2 in stem-like
populations, suggesting that the presence of stem-like cells in human
sarcomas with efficient detoxification of the drug and DNA repair
could mainly contribute to the drug resistance in human sarcomas.
The results from this study may provide future directions of further
investigation targeting sarcoma stem cells.
RESULTS
Separation and Differentiation of the ALDH-positive and negative
populations from human sarcoma cell lines. The results of Aldefluor
assay showed that both cell lines contained the high ALDH activity
populations at the frequency of 11% in MG63 and 9% in HT1080,
respectively. Then, we separated the high and low ALDH activity
populations in MG63 osteosarcoma cell line through cell sorting by
FACSaria, and confirmed that ALDH1 mRNA expression levels were
correlated with cell sorting by ALDH activity. After sorting of the
cells, both high and low ALDH activity cells were cultured in
monolayer condition, then harvested and analyzed by Aldefluor assay
at about 70% confluent. Both cell populations showed the same
pattern of ALDH positive and negative population as parental MG63
cells, suggesting both populations possess the differentiation capacity.
ACKNOWLEDGEMENTS
All authors have no declaration of conflicts of interest. This study
was supported by a Grant-in-Aid for Scientific Research (No.
20591765 to KH) from Ministry of Education, Culture, Sports,
Science and Technology, Japan.
REFERENCES
1. Sell S. Crit Rev Oncol Hematol. 51: 1, 2004
2. Reya T, Morison SJ, Clarke MF, et al. Nature 414: 105, 2001
3. Ginestier C, Hur MH, et al. Cell Stem Cell 1: 555, 2007
4. Gibbs, CP, et al. Neoplasia 7:967, 2005
Poster No. 1665 • 56th Annual Meeting of the Orthopaedic Research Society