Overexpression of the ABCB1 drug transporter in acute myeloid leukemia cells is associated with downregulation of latrophilin-1

Abstract

Finding new markers with appropriate prognostic levels for the differential diagnosis of neoplastic diseases represents an important issue for biomedical research. Recently, latrophilin-1 (LPHN1) was reported to be expressed in human monocytic leukemia cell lines and in primary human acute myeloid leukemia (AML) cells. However, this expression was found to be absent in healthy leukocytes. LPHN1 was therefore considered a novel biomarker of human AML. In previous papers, we established two P-gp-positive variants (SKM-1/VCR and MOLM-13/VCR) of AML cell lines derived from parental human AML cells SKM-1 and MOLM-13 by selection with VCR. The present paper addresses the measurement of LPHN1 expression in SKM-1 and MOLM-13 cells and their P-gp-positive variants. Both parental AML lines were positive for LPHN1 expression at the mRNA and protein levels. However, the expression of LPHN1 at both the mRNA and protein levels was reduced in both P-gp-positive SKM-1/VCR and MOLM-13/VCR variants of AML cells. Interestingly, we observed an elevation of the latrophilin-3 transcript in P-gp-positive variants of AML cell lines. The combined results suggest that alterations in latrophilin expression occur in AML cells expressing P-gp.

Development of Multidrug Resistance in Acute Myeloid Leukemia Is Associated with Alterations of the LPHN1/GAL-9/TIM-3 Signaling Pathway

“…Consistent with our previous results [ 30 ], massive ABCB1 gene expression was detected only in the resistant sublines (SKM-1/VCR and MOLM-13/VCR) by either RT-PCR or qRT-PCR ( Figure S3 in supplementary files ). ABCB1 protein was also identified by Western blot exclusively in SKM-1/VCR and MOLM-13/VCR cell variants and not in the parental SKM-1 and MOLM-13 cells ( Figure S3 in the supplementary files ).…”

“…The presence of latrophilin-1 has been observed in the plasma membrane of myeloid leukemia blasts from patients with AML but not in healthy mature leukocytes [ 26 ]. We also verified the presence of this protein in the AML lines MOLM-13 and SKM-1 [ 30 ]. However, we found that latrophilin-1 was downregulated in ABCB1-positive MOLM-13/VCR and SKM-1/VCR variants.…”

“…The protein expression of GAL-9, TIM-3, CD44 and ABCB1 was analyzed by Western blotting, and GAPDH was used as an internal loading control, as previously described [ 18 , 30 ]. Cells were lysed in lysis buffer (50 mM Tris-HCl, 5 mM EDTA, 150 mM NaCl, 0.5% Nonidet P-40; pH 8) supplemented with protease inhibitor cocktail (Sigma Aldrich) according to the manufacturer’s protocol.…”

“…Densitometric analysis of the respective bands was performed with ImageJ and normalized to GAPDH levels. All experiments were repeated at least 3 times [ 18 , 30 ].…”

“…Apart from the abovementioned expression of LPHN1 in leukemic myeloid blasts, there are few and often contradictory findings about the expression of this receptor in neoplastic cells [ 29 ]. We previously demonstrated LPHN1 expression in AML SKM-1 and MOLM-13 cells [ 30 ]. However, we showed that LPHN1 expression is downregulated (compared to parental cells) in cell variants of both AML lines (SKM-1/VCR and MOLM-13/VCR) that expressed ABCB1.…”