Valproic Acid Increases CD133 Positive Cells that Show Low Sensitivity to Cytostatics in Neuroblastoma

Abstract

Valproic acid (VPA) is a well-known antiepileptic drug that exhibits antitumor activities through its action as a histone deacetylase inhibitor. CD133 is considered to be a cancer stem cell marker in several tumors including neuroblastoma. CD133 transcription is strictly regulated by epigenetic modifications. We evaluated the epigenetic effects of treatment with 1mM VPA and its influence on the expression of CD133 in four human neuroblastoma cell lines. Chemoresistance and cell cycle of CD133+ and CD133− populations were examined by flow cytometry. We performed bisulfite conversion followed by methylation-sensitive high resolution melting analysis to assess the methylation status of CD133 promoters P1 and P3. Our results revealed that VPA induced CD133 expression that was associated with increased acetylation of histones H3 and H4. On treatment with VPA and cytostatics, CD133+ cells were mainly detected in the S and G2/M phases of the cell cycle and they showed less activated caspase-3 compared to CD133− cells. UKF-NB-3 neuroblastoma cells which express CD133 displayed higher colony and neurosphere formation capacities when treated with VPA, unlike IMR-32 which lacks for CD133 protein. Induction of CD133 in UKF-NB-3 was associated with increased expression of phosphorylated Akt and pluripotency transcription factors Nanog, Oct-4 and Sox2. VPA did not induce CD133 expression in cell lines with methylated P1 and P3 promoters, where the CD133 protein was not detected. Applying the demethylating agent 5-aza-2’-deoxycytidine to the cell lines with methylated promoters resulted in CD133 re-expression that was associated with a drop in P1 and P3 methylation level. In conclusion, CD133 expression in neuroblastoma can be regulated by histone acetylation and/or methylation of its CpG promoters. VPA can induce CD133+ cells which display high proliferation potential and low sensitivity to cytostatics in neuroblastoma. These results give new insight into the possible limitations to use VPA in cancer therapy.

Histone Deacetylase Inhibitors as Anticancer Drugs

“…This increase, found in the CD133+ cells was not caused by elimination of CD133− cells. Moreover, VPA treatment enhanced clonogenicity and the ability to generate neurospheres, increased Akt phosphorylation and induced expression of the pluripotency transcription factors (Oct-4 (octamer-binding transcription factor 4), Nanog (Homeobox Transcription Factor Nanog), Sox2 (sex determining region Y)) [ 133 ]. Similar increase of CD 133+ cells was found also in glioblastoma cells treated by VPA [ 134 ].…”

“…Decitabine and VPA both induced apoptosis and the combination increased their effects both in vitro and in vivo on leukemic cells and on medulloblastoma and rhabdomyosarcoma that develop in Ptch1 (Protein patched homolog 1) knockout mice [ 143 , 144 ]. On the contrary, this combination induced the CD133 expression in neuroblastoma cells with the methylated CD133 promotor [ 133 ]. Co-treatment of several cancer cells (prostate, pancreatic, lung and AML) with TSA and decitabine synergistically induced apoptosis [ 52 , 55 , 145 , 146 ].…”

“…The other most important question is whether the pan-HDAC inhibitors or the selective inhibitors will be more efficient in different types of cancers. Furthermore, assumption about the role of some HDAC inhibitors as inducers of cancer stem cells [ 133 , 134 ] or about the phenomenon that HDAC inhibition may enhance the epithelial–mesenchymal transition of cancer cells [ 129 , 130 ] needs further studies. We may conclude that while the preliminary results are promising, large multicentric clinical studies are needed to ascertain whether this treatment offers beneficial clinical outcomes with tolerable side-effect profiles.…”

Cancer stem cells in neuroblastoma therapy resistance

“…Consistently, studies have indicated the criticality of CD133-expressing Side populations in NB progression and therapy response. For instance, studies demonstrated that (1) non-adherent clumps of tumor cells that express CD133, OCT4, pAKT could grow in a serum-free medium with higher colony and neurosphere formation [98] ; (2) CD133 levels were directly associated with NB advanced disease stages and inversely correlated with postoperative survival time [95] ; and (3) CD133 + NB cells were more resistant to cisplatin, carboplatin, DOX, and etoposide (vs. CD133cells) and presented with increased phosphorylation of extracellular signal-regulated kinases (ERK) and P38 mitogen-activated protein kinases (P38) [99] . Consistently, an independent study showed that targeted inhibition of CD133 in NB cells produced increased RET expression and NB differentiation and, this response is mediated through the regulation of the p38 MAPK and phosphoinositide-3-kinase (PI3K)/ AKT pathways [97] .…”

CD133 and MYCN Amplification Induce Chemo-Resistance and Reduce Average Survival Time in Pediatric Neuroblastoma

“…Knocking down of the CD133 in NB cells effectively resulted in cell growth retardation [20]. Up-regulated CD133 decreased sensitivity to cytostatics in clinic [21]. Whether CD133 + cells have effects on responses towards chemotherapy and average survival time need to be demonstrated.…”