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  • br Acknowledgements br Introduction The

    2022-05-18


    Acknowledgements
    Introduction The glutathione S-transferase (GST, EC 2.5.1.18) family of multifunctional enzymes plays a particularly important role in cellular detoxification [1]. Over-expression of the GSTP1 isoform characterizes many human tumor cell lines derived from stomach, brain and colon as well as leukemia [2]. In the clinic, this over-expression is a bad prognostic for patient survival [3]. In addition, GSTP1 is involved in resistance against antineoplastic drugs [4] and against apoptosis by binding to Jun N-terminal kinase (JNK) [5], whereas inhibition of GSTP1 leads to apoptosis in K562 leukemia cells [6]. Transcriptional regulation by SP-1 [7], AP-1 [8] and NF-κB [9] was described to be part of the molecular mechanisms controlling GSTP1 gene expression. GSTP1 down-regulation was also described by methylation of CpG residues within the core promoter in various pathologies, including prostate carcinoma [10] and leukemia [11]. In addition, we recently demonstrated that the erythroid differentiation-specific transcription factor GATA-1 binds to a GATA sequence within the GSTP1 gene promoter with similar variations of GATA-1 binding activity and GSTP1 expression during both induced erythroid- and megakaryocytic K562 cell differentiation [12]. Differentiation therapy aims to induce the reversal of the malignant phenotype by using non toxic doses of differentiating agents and appears to become an alternative strategy to conventional anti-cancer therapies [13], [14]. However, we recently demonstrated that this clinical approach might increase cancer cell resistance by enhanced detoxification potential via over-expressed GSTP1 [12], [15].
    Materials and methods
    Results
    Discussion and conclusion This report compares the effect of different concentrations of butyric Gramine on the K562 cell line which has the capacity to differentiate along the erythroid or megakaryocytic lineage [18]. Compared to other chemical inducers of differentiation exhibiting lineage specificity, butyric acid has the property to induce differentiation differently, depending on the concentration used: low butyric acid concentrations of 0.5–1mM activate the erythroid differentiation process whereas concentrations above 2mM differentiate towards the megakaryocytic lineage. Our results are confirmed by a number of individual reports where butyric acid is used as an erythroid differentiating agent [19], [20] or as a megakaryocytic inducer [21]. In order to elucidate molecular mechanisms related to chemoresistance during chemically induced differentiation of leukemia cells, we previously showed that 12-O-tetradecanoyl phorbol 13-acetate (TPA), a megakaryocytic pathway inducer, leads to decreased GSTP1 expression at both mRNA and protein levels whereas induction of erythroid differentiation by aclarubicin and doxorubicin generates increased GSTP1 gene expression [12] as does the canonical erythroid inducer hemin [15]. Here, our results show a decrease in GSTP1 mRNA expression after butyric acid treatment and this independently of the differentiation lineage. These results were unexpected as earlier reports documented an increase in GSTP1 expression after butyric acid-induced differentiation of a rat liver non parenchymal epithelial cell line [22]. Similarly, in HT-29 colon cancer cells, differentiation by butyric acid activates the extracellular regulated kinase (ERK) 1/2 cell signaling pathway leading to increased GSTP1 expression and enhanced chemoprotection [23]. Caco-2 cells, which are closely related to normal human enterocytes, if differentiated by butyric acid, express higher GSTP1 levels, contributing to enhanced detoxification capacity in the gut [24]. The reduction of GSTP1 expression described in this report can nevertheless be explained by the cell signaling pathways by which butyric acid specifically induces differentiation of K562 cells: ERK and JNK are dephosphorylated and thus inhibited whereas p38 kinases are phosphorylated following butyric acid exposure [20]. According to the literature, neither ERK nor p38 were described to be involved in GSTP1 expression, but we previously published that JNK inhibitors including curcumin down-regulate GSTP1 mRNA expression as well as the essential AP-1 binding [6]. Moreover, chemical inducers of differentiation, like doxorubicin, induce GSTP1 mRNA synthesis [12] while activating JNK and leading to increased binding of AP-1 to a site located at −73 relative to the GSTP1 transcriptional start site [6]. Therefore, inhibition of GSTP1 via JNK by butyric acid is in accordance with previously published data and contributes to explain the decrease of GSTP1 mRNA synthesis. Additionally, butyric acid markedly suppresses tumor necrosis factor (TNF) α-induced activation of NF-κB [25]. We previously published that NF-κB homo- and heterodimers positively regulate GSTP1 expression as well as the TNFα→TNF receptor 1→TNF receptor associated factor (TRAF)→NF-κB inducing kinase (NIK)→IκB kinase (IKK) pathway [9]. Suppression of the TNFα signaling pathway by butyric acid is therefore likely to contribute to the down-regulation of GSTP1 mRNA synthesis in human leukemia cells. Finally, GATA-1 transcription factor was previously described to contribute to GSTP1 up-regulation by chemical inducers including hemin and aclarubicin [12], [15]. A reduced binding onto the GSTP1 GATA-1 site located at −1208 relative to the transcriptional initiation site contributes to a reduction of GSTP1 expression. Published data confirm these results as Busfield et al. [26] previously reported a depletion of GATA-1 transcripts in butyric acid-treated J2E erythroid cells.