The intratumor concentration of metformin is difficult to evaluate, but it accumulates in ovarian cancer patient biopsies52

The intratumor concentration of metformin is difficult to evaluate, but it accumulates in ovarian cancer patient biopsies52. monoclonal antibodies that recognize tumor antigens. Our results suggest that metformin could improve cytotoxic lymphocyte-mediated therapy. and genes contain consensus p53 response elements, and p53 probably amplifies transcription of certain human mRNAs16,21. Therefore, p53 is a possible candidate to link metabolism and expression of NKG2DL. Cancer and aging patients have impaired NK function22. This could decrease the clinical activity of antitumor drugs designed to recruit NK cells. In this sense the lack of clinical activity of metformin in certain cancer patients could be related to the advanced patients age at treatment10,23. Therefore, the use of allogeneic NK cells together with metformin could improve clinical treatment. We have developed a protocol to activate and expand umbilical cord blood (UCB)-derived NK cells in vitro24. These expanded NK (eNK) have high cytolytic activity against hematological cancer cells, even those resistant to chemotherapy24C26. We have investigated here if metformin sensitizes hematopoietic cells to CL and the possible mechanism(s) of action. Results Metformin regulates expression of stress ligands and ICAM-1 in leukemic cells We have previously shown in several studies that the presence of wtp53 impacts the effect of several co-treatments involving the so-called metabolic drugs, e.g. metformin20,27,28. Hence, we investigated if p53 status can affect the effect of metformin on ligands that are recognized by cytotoxic lymphocytes. We treated with 2?mM metformin for 3?days 3 acute myeloid leukemia (AML) cell Mevalonic acid lines with different p53 status (OCI-AML3 cells express wt p53, HL60 are p53 null and NB4 express mutant (mut) p5320,29) and analyzed MICA/B and ULBP1 expression on plasma membrane. In addition, we evaluated levels of other ligands such as intercellular adhesion molecule-1 (ICAM-1), a ligand of the lymphocyte function-associated antigen 1 (LFA-1). ICAM-1/LFA-1 interaction is essential SPN for target cell recognition by CL30,31. We studied also MHC-I, called HLA in humans, which is the ligand of the killer-cell immunoglobulin-like receptors (KIRs), the main NK cell inhibitory receptors22 and the myeloid marker CD33. Metformin did not statistically change expression of the last two molecules in any cell line (Fig.?1A and Supplemental Fig.?1). In contrast, it increased expression of ULBP-1 and of the integrin ligand ICAM-1 in all of Mevalonic acid them and of MICA/B in OCI-AML3 cells (Fig.?1A and Supplemental Fig.?1). Of note, metformin was cytostatic, but not cytotoxic, on these leukemic cells at Mevalonic acid the selected dose (Fig.?1B). We next tested lower doses of metformin in the cell line that better respond to treatment, i.e. OCI-AML3 (Fig.?1C). Whereas 1 and 2?mM of metformin gave similar effects, concentrations under 1?mM lacked effect. Therefore, for future experiments we used 2?mM. Open in a separate window Figure 1 Metformin regulated expression of stress ligands and ICAM-I in leukemic cells. (A) Different AML cell lines OCI-AML-3 wtp53, HL-60 nullp53, NB4 mutp53 were treated with 2?mM metformin for 3?days and plasma membrane expression of the stress ligands MICA/B and ULBP1, the integrin ICAM-1, the myeloid marker CD33 and MHC class I HLA were analyzed by FACs analysis. (B) The different cell lines were treated as in (A) and the total number of cells and the percentage of alive cells were measured on the Muse Cell Analyzer. (C) OCI-AML3 cells were treated with different concentrations (0.0316; 0.1; 0.316; 1 and 2?mM) of metformin for 3?days, metformin was added every day and plasma membrane expression of the stress ligands MICA/B and ULBP1, the integrin ICAM-1 and the cell line marker CD33 were analyzed by FACs analysis. (D) Different MM cell lines MM1.S wtp53 and U266 mutp53 and the pro-monocytic myeloid leukemia cell U937 nullp53 were treated with 2?mM metformin for 3?days and the.