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I. Spinello et al.
ers previously identified in LSCs.34 We confirmed that CD147 is expressed at high levels in all AML subtypes (M1, M2, M3, M5 and M5a) analyzed (Table 1). We also report for the first time that CD147 is co-expressed with CD34, and particularly with CD371, in these cells (Table 1 and Figure 6A). It is interesting to note that in 8 non-M3 AMLs analyzed for CD147 and CD371 expression, there was a strong positive correlation between CD147 and CD371 expression (P<0.001) (Table 1). These data suggest
the existence of CD34+/CD147+ and CD371+/CD147+ sub- populations in AMLs that may have functional properties of leukemic stem cells (LSCs).
Then, we investigated the effects of AC-73 used alone or combined with Ara-C and ATO at low concentration in primary AML blasts, and particularly in M2, M3 and M5 AML cells. We found that AC-73 used alone has no signif- icant effect on AML cell apoptosis, unless used at high concentration (10 mM) for AML(7)-M3 and AML(8)-M5
A
Figure 5. AC-73 potentiates the sensitivity of leukemic cells to chemotherapeutic treatment. (A and B) Cell viabil- ity assays on U937 and NB4 leukemic cells, treated 24 hours (h) with AC-73 used alone (2.5 mM), then in combi- nation with Ara-C (AC-73 + Ara- C, left panels) or ATO (AC-73 + ATO, right panels) for 48 h, as compared to treatment with single drug, AC-73 (AC-73 + 0.00 mM Ara-C; AC-73 + 0.00 mM ATO), Ara-C and ATO. (C) Western blot analysis of phos- pho-STAT3(ser727) (pSTAT3(S727), STAT3, phos-
pho-ERK1/2 (pERK) ERK1/2 (ERK) protein expres- sion in U937, NB4 and MV4-11 leukemic cell lines. U937, NB4 and MV4-11 cells were treated for 3 days with AC-73 in combi- nation with ATO (AC-73 + ATO 1 mM), or Ara-C (AC-73 + Ara-C 1 mM), as compared to AC-73 (2.5 mM), ATO (1 mM) or Ara-C (1 mM) used alone or to control (C) leukemic cells. Actin was used as an internal control. Quantification of total and phosphorylated ERK1/2 and STAT3 proteins by densitometry analysis is indicated. (D and E) Analysis of the autophagy flux induces by ATO (1 mM) and AC-73 (5 mM) used alone or in combination in U937 and NB4 leukemic cells compared to control cells (C). (A, B and E) Mean±Standard Error of Mean of three inde- pendent experiments is shown. *P<0.05; **P<0.01; ***P<0.001. ns: not signifi- cant. (C and D) One representa- tive experiment out of three is shown. (C) A.U.: arbitrary units. Ratio pSTAT3/STAT3 and pERK/ERK of three independ- ent experiments is shown. kDa: molecular weights.
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