Effect of all-trans retinoic acid on the proliferation and differentiation of brain tumor stem cells.
OBJECTIVE
To investigate the effect of all-trans retinoic acid(ATRA) on the proliferation and differentiation of brain tumor stem cells(BTSCs) in vitro.


METHODS
Limiting dilution and clonogenic assay were used to isolate and screen BTSCs from the fresh specimen of human brain glioblastoma. The obtained BTSCs, which were cultured in serum-free medium, were classified into four groups in accordance with the composition of the different treatments. The proliferation of the BTSCs was evaluated by MTT assay. The BTSCs were induced to differentiate in serum-containing medium, and classified into the ATRA group and control group. On the 10th day of induction, the expressions of CD133 and glial fibrillary acidic protein (GFAP) in the differentiated BTSCs were detected by immunofluorescence. The differentiated BTSCs were cultured in serum-free medium, the percentage and the time required for formation of brain tumor spheres (BTS) were observed.


RESULTS
BTSCs obtained by limiting dilution were all identified as CD133-positive by immunofluorescence. In serum-free medium, the proliferation of BTSCs in the ATRA group was observed significantly faster than that in the control group, but slower than that in the growth factor group and ATRA/growth factor group, and the size of the BTS in the ATRA group was smaller than that in the latter two groups(P < 0.01). In serum-containing medium, the expression percentages of CD133 and GFAP in the differentiated BTSCs were (2.29% +/- 0.27%) and (75.60% +/- 4.03%) respectively in the ATRA group, and (7.05% +/- 0.49%) and (12.51% +/- 0.77%) respectively in the control group. The differentiation rate of BTSCs in the ATRA group was significantly higher than that in the control group (P < 0.05), but there was still CD133 expressed in the ATRA group. The differentiated BTSCs could re-form BTSs in serum-free medium. The percentage of BTS formation in the ATRA group was(4.84% +/- 0.32%), significantly lower than that in the control group (17.71% +/- 0.78%) (P < 0.05), and the time required for BTS formation in the ATRA group was (10.07 +/- 1.03)d, significantly longer than that in the control group (4.08 +/- 0.35)d (P < 0.05).


CONCLUSIONS
ATRA can promote the proliferation and induce the differentiation of BTSCs, but the differentiation is incomplete, terminal differentiation cannot be achieved and BTSs can be formed again.
Niu
Chao Shi
CS
Department of Neurosurgery, Anhui Provincial Hospital Affiliated to Anhui Medical University, Hefei, Anhui Province, 230001, China. niuchaoshi@126.com
Li
Ming Wu
MW
Ni
Yong Feng
YF
Chen
Jian Min
JM
Mei
Jia Ming
JM
Li
Jing
J
Fu
Xian Ming
XM
D000071916:AC133 Antigen; D015703:Antigens, CD / Q000032:analysis; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001932:Brain Neoplasms / Q000276:immunology / Q000378:metabolism / Q000473:pathology*; D002454:Cell Differentiation / Q000187:drug effects*; D049109:Cell Proliferation / Q000187:drug effects*; D016895:Culture Media, Serum-Free / Q000378:metabolism; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D005909:Glioblastoma / Q000276:immunology / Q000378:metabolism / Q000473:pathology*; D006023:Glycoproteins / Q000032:analysis; D006801:Humans; D014411:Neoplastic Stem Cells / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D010455:Peptides / Q000032:analysis; D044967:Serum / Q000378:metabolism; D018874:Spheroids, Cellular; D013997:Time Factors; D014212:Tretinoin / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D000071916:AC133 Antigen; D015703:Antigens, CD; D000970:Antineoplastic Agents; D016895:Culture Media, Serum-Free; D005904:Glial Fibrillary Acidic Protein; D006023:Glycoproteins; C000606023:PROM1 protein, human; D010455:Peptides; D014212:Tretinoin