CGP 54626 hydrochloride We recently reported that the human

We recently reported that the human-derived PancCa cell line PANC-1 and the human-derived hepatocellular HepG2 cell line express GPR55 mRNA and protein [21]. In PANC-1 and HepG2 cells, knockdown of GPR55 with specific siRNAs abrogates cellular uptake of Tocrifluor 1117, a selective GPR55 ligand [21]. We have also demonstrated that treatment of these cell lines with (R,R⿿)-4⿲-methoxy-1-naphthylfenoterol (MNF) inhibits cellular uptake of Tocrifluor 1117 in a concentration-dependent manner and has a negative impact on GPR55 signaling, as evidenced by the significant impairment in ligand-inducible changes in p-ERK levels, cell morphology, and migration using scratch wound healing assay [21]. In addition, incubation of HepG2 cells with MNF decreased signaling through the MEK/ERK and PI3K/AKT pathways to produce CGP 54626 hydrochloride arrest and apoptosis [20]. The ability of MNF to block the inducible expression of EGFR protein by a GPR55 ligand and significantly lower GPR55-mediated activation of the EGFR-MEK/ERK cascade in PANC-1 cells [21] led us to an expanded investigation of the effect of MNF on cellular signaling and expression of key tumor biomarkers such as PKM2 and MDR expression and function. We now report that the pharmacological inhibition of GPR55 is a viable approach to the attenuation in PKM2 nuclear accumulation and MDR protein expression in PANC-1 cells. The reduction in both the expression and function of MDR proteins results in increased doxorubicin and gemcitabine cytotoxicity. Incubation with MNF also increased the cytotoxicity of doxorubicin in MDA-MB-231 and U87MG cancer cell lines. The results suggest that in addition to having anti-mitogenic properties, pharmacological inhibitors of GPR55 may be useful in the treatment of MDR disease.
Materials and methods
Results
Discussion In the current study, we demonstrate that GPR55 antagonism decreased the expression of Pgp, BCRP, MRP1 and MRP5 in a human-derived PancCa cell line, PANC-1. Moreover, treatment of PANC-1 cells with the GPR55 agonist AM251 produced a significant increase in BCRP expression, which was blocked by the two GPR55 antagonists tested, MNF and CID (Fig. 4A). Recent reports have indicated a role for basal GPR55 expression and activity on ERK1/2/MAPK signaling and the ability of GPR55 antagonists in blocking agonist-induced increases in the levels of phospho-active ERK [18], [19]. Additionally, we have previously reported that MNF-mediated inhibition of ERK1/2 phosphorylation in PANC-1 cells is specifically modulated by GPR55 antagonism [21]. These findings clearly indicate a link between GPR55 activity and MDR protein expression via the MEK/ERK pathway, which is consistent with the previously identified link between activation of the EGFR/MEK/ERK pathway and increased MDR [7]. Here, PKM2 has been found to be a key factor in linking the GPR55-MEK/ERK nexus with the expression levels of MDR proteins. PKM2 plays a key transcriptional role in metabolic regulation and enhancement in the expression of proteins associated with cellular replication and survival through β-catenin-, HIF-1α- and c-Myc-mediated transcription [13], [14]. The non-receptor tyrosine kinase c-Src mediates β-catenin tyrosine phosphorylation [14] and nuclear PKM2 interacts with tyrosine-phosphorylated β-catenin to enhance its transcriptional activity toward cyclin D1, EGFR, and Pgp [13]. Furthermore, inducible PKM2-HIF-1α interaction in the nucleus leads to higher HIF-1α-dependent expression of Pgp, MRP1 and MRP2 [30], [31], EGFR and PKM2 [13]. PKM2 phosphorylates histone H3, which leads to the dissociation of pre-bound histone deacetylase 3 (HDAC3) accompanied by subsequent monoacetylation of histone H3 at lysine 9 and the transactivation of CCDN1 and MYC. Amplification of c-Myc levels is associated with increased expression of MRP1, MRP2 and BCRP [32]. Our results demonstrate that incubation of PANC-1 cells with MNF and CID led to significant reduction in nuclear PKM2 levels both under basal conditions and upon GPR55 stimulation. This effect is consistent with the GPR55/MEK/ERK pathway resulting in the ERK-mediated phosphorylation of PKM2 at Ser 37, which is necessary for PKM2 nuclear translocation [15]. It follows that the impaired formation of multiprotein complexes involving nuclear PKM2 with tyrosine-phosphorylated β-catenin and/or HIF-1α combined with lower phosphorylation of histone H3 may be responsible for the downregulation in the expression of tumor biomarkers, including MDR proteins, EGFR, and cyclin D1 as well as PKM2. Although cyclin D1 is a target gene of β-catenin (Fig. 1A), the effect of MNF on cyclin D1 was more sensitive than β-catenin. We surmise that small changes either in posttranslational modification of β-catenin, its nuclear translocation and/or association with other transcription factors will have profound effects on its ability to induce target gene expression.