Destabilizing the interplay between miR-1275 and IGF2BPs by Tamarix articulata and quercetin in hepatocellular carcinoma.

Insulin-like growth factor-2 binding proteins (IGF2BPs) are oncogenic RNA-binding proteins, highly up-regulated in HCC, and were recently validated as direct targets of the tumour suppressor miR-1275. It is worth noting that around 47% of FDA approved anticancer drugs are derived from plants. Modulation by miRNAs and their cellular signalling cascades could constitute new pathways by which these phytochemicals exert their effects. This study aimed to investigate the potential use of Tamarix articulata, quercetin and epigallocatechin gallate (EGCG) in HCC and how these phytochemicals could epigenetically modulate the IGF axis using their impact on miR-1275. T. articulata ethyl acetate fraction significantly reduced the viability of Huh-7 cells compared to control cells. Treatment with T. articulata ethyl acetate fraction, quercetin and EGCG significantly enhanced miR-1275, while suppressed IGF2BP1 and IGF2BP3 mRNA expression levels. In summary, T. articulata, quercetin and EGCG have important implications for HCC molecular-targeted therapy through destabilizing the interplay between miR-1275 and the IGF axis.

Repression of miR-17-5p with elevated expression of E2F-1 and c-MYC in non-metastatic hepatocellular carcinoma and enhancement of cell growth upon reversing this expression pattern.

E2F-1, c-MYC, and miR-17-5p is a triad of two regulatory loops: a negative and a positive loop, where c-MYC induces the expression of E2F-1 that induces the expression of miR-17-5p which in turn reverses the expression of E2F-1 to close the loop. In this study, we investigated this triad for the first time in hepatocellular carcinoma (HCC), where miR-17-5p showed a significant down-regulation in 23 non-metastatic HCC biopsies compared to 10 healthy tissues; however, E2F-1 and c-MYC transcripts were markedly elevated. Forced over-expression of miR-17-5p in HuH-7 cells resulted in enhanced cell proliferation, growth, migration and clonogenicity with concomitant inhibition of E2F-1 and c-MYC transcripts expressions, while antagomirs of miR-17-5p reversed these events. In conclusion, this study revealed a unique pattern of expression for miR-17-5p in non-metastatic HCC patients in contrast to metastatic HCC patients. In addition we show that miR-17-5p is the key player among the triad that tumor growth and spread.

miR-615-5p is restrictedly expressed in cirrhotic and cancerous liver tissues and its overexpression alleviates the tumorigenic effects in hepatocellular carcinoma.

microRNAs aberrant behavior in heptocellular carcinoma (HCC) plays a major role in HCC pathogenesis. miR-615-5p expression has never been evaluated in HCC. We showed that miR-615-5p was preferentially expressed in HCC, cirrhotic liver tissues and HCC cell lines, but undetected in normal livers. Forced miR-615-5p expression in HCC cell lines led to significant decrease in cell growth and migration. In-silico predication revealed insulin-like growth factor-II (IGF-II) as a potential downstream target for miR-615-5p. Forcing the expression of miR-615-5p showed downregulation of IGF-II mRNA, as well as inhibition of the luciferase activity in a luciferase reporter vector harboring the IGF-II-3'UTR target sequence. miR-615-5p acts as tumor-suppressor in HCC through targeting IGF-II.

Expression of insulin-like growth factor-II, matrix metalloproteinases, and their tissue inhibitors as predictive markers in the peripheral blood of HCC patients.

BACKGROUND/AIM: Elevated relative expression of insulin-like growth factor-II (IGF-II) was observed in hepatocellular carcinoma (HCC) liver tissues with a role in neovascularization and associated with poor prognosis. IGF-II is influenced by the proteolytic cleavage of IGF-binding protein 3 and by matrix metalloproteinases (MMP), which are further regulated by their tissue inhibitors tissue inhibitor of metalloprotienase-1 (TIMP-1). Our aim is to study new molecular markers for HCC. PATIENTS/METHODS: RNA was extracted from the peripheral blood for evaluating the relative expression of IGF-II, MMP-9, and TIMP-1 in correlation with clinical staging of 39 HCC patients and 15 healthy controls using TaqMan real-time PCR. RESULTS: The relative expression of IGF-II and MMP-9 mRNA were significantly elevated in HCC patients compared with healthy controls; P-value <0.0001 for both. There was a significant correlation between MMP-9 and different HCC stages. On the other hand, TIMP-1 was significantly down-regulated in HCC patients; P = 0.0003 with the elevation of the IGF-II/TIMP-1 ratio. Significant correlation between TIMP-1 and HCC Stage III and Stage IV was found; P-value = 0.0138. CONCLUSION: These results highlight the importance of profiling the expression of IGF-II, MMP-9, and TIMP-1 in the peripheral blood as prognostic molecular biomarkers in HCC.

Kidney transplantation candidates and cardiovascular risk factors.

OBJECTIVE: We sought to determine the prevalence of cardiovascular disease and risk factors among chronic renal failure (CRF) patients on the transplantation waiting list. METHODS: Fifty CRF patients on chronic hemodialysis who underwent evaluation for transplantation were compared with 60 hypertensive patients matched for age. We used Framingham scoring to calculate the absolute risk; relative risk was calculated based on the low-risk Framingham cohort. RESULTS: According to traditional risk factors, a significant difference was observed in systolic blood pressure and total cholesterol (greater in the hypertensive group), and in the prevalence of the male gender, smoking, and diabetes, which were greater in the CRF group. The latter had a greater degree of left ventricular hypertrophy, lower diastolic blood pressure, and a lower prevalence of familial history of cardiovascular disease and obesity. Patients with CRF had a greater relative risk compared with the Framingham control population, but it did not differ from that observed in the group of hypertensive individuals. CONCLUSION: The prevalence of cardiovascular disease and traditional risk factors is high among renal transplantation candidates. The Framingham equations do not adequately quantify the real cardiovascular risk; other risk factors specific for that population probably contribute to their greater cardiovascular risk.

Enhanced activation of B cells in a granulocyte colony-stimulating factor-mobilized peripheral blood stem cell graft.

In a randomized study that compared human leucocyte antigen-identical allogeneic granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood stem cell (PBSC) versus bone marrow (BM) transplantation, the expression of activation markers, CD23, CD25 and CD45RO by B cells, was compared in blood before and after G-CSF mobilization and in PBSC versus BM grafts. The fractions of CD23+ and CD25+ B cells were higher in PBSC than in BM grafts. Moreover, we observed a G-CSF-induced increase in B-cell fractions in blood as well as in PBSC grafts when compared with BM grafts. Such an enhanced B-cell activation could contribute to the accelerated kinetics of immuno-haematological reconstitution, the occurrence of acute haemolysis in the ABO minor incompatibility setting, as well as the increased incidence of chronic graft-versus-host disease observed after PBSC transplantation.

Allogeneic peripheral blood stem cell transplantation results in less alteration of early T cell compartment homeostasis than bone marrow transplantation.

Since low T cell counts evaluated 1 month after allogeneic bone marrow transplantation (BMT) are associated with an increased risk of leukemia relapse (Powles et al., Blood 1998; 91: 3481-3486), we compared, in a randomized multicentric clinical study, the peripheral blood cells obtained 30 days after allogeneic BMT vs allogeneic G-CSF-mobilized peripheral blood stem cell transplantation (BCT) in an HLA-identical setting. T cell counts were higher 30 days after BCT (718+/-142 cells/microl, n = 20) than after BMT (271+/-53 cells/microl, n = 26, P = 0.006). However, T cells were less activated after BCT than after BMT, as demonstrated by a lower expression level of CD25 and a lower percentage of HLA-DR+ and CD95+ T cells. Furthermore, CD4+, CD8+ and CD45RA+ post-BCT T cell counts correlated with the number of cells infused with the PBSC graft, while such a correlation was not observed between post-BMT counts and BM graft cell numbers, suggesting that the intensity of post-transplant peripheral lymphoid expansion and/or deletion differed between BCT and BMT. A comparison of the input of T cells expressing different CD45 isoforms with the post-transplant cell recovery further confirmed that, within the CD4+ T cell subset, post-transplant expansions occurred at a higher level after BMT than after BCT, affecting mainly the CD4+ CD45RO+ subset. Altogether, our data demonstrate for the first time in a randomized setting that homeostasis of the T cell pool is less altered early after BCT than after BMT. This may have a strong impact on the graft-versus-leukemia (GVL) effect and subsequent relapse rate.

Influence of the hematopoietic stem cell source on early immunohematologic reconstitution after allogeneic transplantation.

Several acute hemolysis episodes, sometimes lethal, have been recently described after transplantation of allogeneic peripheral blood hematopoietic stem cells (PBHSCs). Hemolysis resulted from the production of donor-derived antibodies (Abs) directed at ABO antigens (Ags) present on recipient red blood cells (RBCs). A multicenter randomized phase III clinical study comparing allogeneic PBHSC transplantation (PBHSCT) versus bone marrow hematopoietic stem cell transplantation (BMHSCT) has been conducted in France. In the course of this study, serum anti-A and/or anti-B Ab titers were compared before the conditioning regimen and on day +30 after transplantation in 49 consecutive evaluable PBHSCT (n = 21) or BMHSCT (n = 28) recipients. PBHSCT resulted in a higher frequency of increased anti-A and/or anti-B Ab titers 30 days after transplantation as compared to BMHSCT: 8 (38%) of 21 versus 3 (11%) of 28 (P =.04). In PBHSCT recipients, increased titers were observed mostly after receiving a minor ABO mismatch transplant: 5 of 7 versus 3 of 14 in the absence of any minor ABO mismatch (P =.05), whereas this was not the case after BMHSCT: 1 of 8 versus 2 of 20. Anti-A and/or anti-B serum Abs detectable at day +30 after PBHSCT were always directed against A and/or B Ags absent both on donor and recipient RBCs. Finally, 3 of 21 PBHSCT versus 0 of 28 BMHSCT recipients developed anti-allogeneic RBC Abs other than ABO (P =.07). Overall, the data strongly suggest that immunohematologic reconstitution differs significantly after granulocyte colony-stimulating factor-mobilized PBHSCT when compared to BMHSCT. Such a difference could contribute to the acute hemolysis described after PBHSCT as well as to distinct alloreactivity after PBHSCT.

Effect of granulocyte colony-stimulating factor mobilization on phenotypical and functional properties of immune cells.

Some phenotypic and functional properties of lymphocytes from bone marrow or peripheral blood stem cell donors were compared in a randomized study. Lymphocyte subsets were analyzed by immunocytometry in blood harvested from bone marrow donors (n = 27) and from peripheral blood stem cell donors before and after granulocyte colony-stimulating factor mobilization (n = 23) and in bone marrow and peripheral blood stem cell grafts. Granulocyte colony-stimulating factor mobilization increased the blood T and B, but not NK, lymphocyte counts. All lymphocyte counts were approximately 10-fold higher in peripheral blood stem cell grafts than in bone marrow grafts. Analysis of CD25, CD95, HLA-DR, and CD45RA expression shows that T-cell activation level was lower after granulocyte colony-stimulating factor mobilization. Similarly, granulocyte colony-stimulating factor reduced by twofold to threefold the percentage of interferon-gamma, interleukin-2, and tumor necrosis factor-alpha-secreting cells within the NK, NK-T, and T-cell subsets and severely impaired the potential for interferon-gamma production at the single-cell level. mRNA levels of both type 1 (interferon-gamma, interleukin-2) and type 2 (interleukin-4, interleukin-13) cytokines were approximately 10-fold lower in peripheral blood stem cell grafts than in bone marrow grafts. This reduced potential of cytokine production was not associated with a preferential mobilization of so-called "suppressive" cells (CD3+CD4-CD8-, CD3+CD8+CD56+, or CD3+TCRVA24+CD161+), nor with a modulation of killer cell receptors CD161, NKB1, and CD94 expression by NK, NK-T, or T cells. Our data demonstrate in a randomized setting that quantitative as well as qualitative differences exist between a bone marrow and a peripheral blood stem cell graft, whose ability to produce type 1 and type 2 cytokines is impaired.

Detection of intracellular cytokines in citrated whole blood or marrow samples by flow cytometry.

We have used three-color flow cytometric analysis for the detection of intracellular cytokines (IFN-gamma and IL-4) in CD3(+) cells, after stimulation for 4 h with phorbol 12-myristate 13-acetate (PMA) and ionomycin in the presence of monensin. We report in the present paper a validation study for analysing IFN-gamma and IL-4 production by bone marrow (BM)-derived T cells and peripheral blood T cells after BM transplantation. Using citrate as anticoagulant for blood and marrow sampling interfered with PMA+ionomycin-based cell stimulation. Indeed, removing this anticoagulant by two washes with 10% pooled human AB serum-supplemented RPMI 1640 before cell stimulation improved the percentages of IL-4(+) (0.02+/-0.01% to 0. 47+/-0.17% without and with washes, respectively; p<0.01) and IFN-gamma(+) (6.8+/-2.75% to 39.33+/-4.6%; p<0.01) cells to levels similar to those observed in heparin-based whole blood cultures (0.38+/-0.17% IL-4(+) and 34.27+/-4.96% IFN-gamma(+) cells; p>0.05). Delaying the cell cultures for 24 h did not significantly modify the detection of IFN-gamma in washed whole blood, but significantly altered IFN-gamma secretion in culture supernatants, as assessed by ELISA. Moreover, the percentage of IFN-gamma-producing cells within the CD3(+) lymphocyte population was stable, since similar results were obtained in two or three different independent experiments performed with the same healthy donors. This method was shown to be applicable for different kinds of citrated samples, such as blood or BM-derived cells. Overall, our data suggest that in addition to allowing for the identification of cytokine-producing cell phenotype, intracellular cytokines staining using flow cytometry is more reliable than ELISA for the biological follow-up of clinical samples.