Human amniotic membrane conditioned medium inhibits proliferation and modulates related microRNAs expression in hepatocarcinoma cells

The placental stem cells have called the focus of attention for their therapeutic potential to treat different diseases, including cancer. There is plenty evidence about the antiproliferative, antiangiogenic and proapoptotic properties of the amniotic membrane. Liver cancer is the fifth cause of cancer in the world, with a poor prognosis and survival. Alternative treatments to radio- or chemotherapy have been searched. In this work we aimed to study the antiproliferative properties of the human amniotic membrane conditioned medium (AM-CM) in hepatocarcinoma cells. In addition, we have analyzed the regulation of pro and antiOncomiRs expression involved in hepatocarcinoma physiology. We have determined by 3H-thymidine incorporation assay that AM-CM inhibits DNA synthesis in HepG2 cells after 72 h of treatment. AM-CM pure or diluted at 50% and 25% also diminished HepG2 and HuH-7 cells viability and cell number. Furthermore, AM-CM induced cell cycle arrest in G2/M. When proliferation mechanisms were analyzed we found that AM-CM reduced the expression of both Cyclin D1 mRNA and protein. Nuclear expression of Ki-67 was also reduced. We observed that this CM was able to promote the expression of p53 and p21 mRNA and proteins, leading to cell growth arrest. Moreover, AM-CM induced an increase in nuclear p21 localization, observed by immunofluorescence. As p53 levels were increased, Mdm-2 expression was downregulated. Interestingly, HepG2 and HuH-7 cells treatment with AM-CM during 24 and 72 h produced an upregulation of antiOncomiRs 15a and 210, and a downregulation of proOncomiRs 206 and 145. We provide new evidence about the promising novel applications of human amniotic membrane in liver cancer.

Determination of the Activity Profile of Bosutinib, Dasatinib and Nilotinib against 18 Imatinib Resistant Bcr/Abl Mutants

The treatment of Chronic Myeloid Leukemia (CML) has been radically modified by the discovery of imatinib (IM), a selective inhibitor of the fusion protein Bcr-Abl, the cause of the disease. A variable portion of CML patients experience resistance to IM therapy. Resistance can arise from different mechanisms but in the vast majority of cases is due to point mutations into the protein sequence that alter directly or indirectly the drug-protein binding. Mutation sites can be schematically clustered in four region: the P loop, the IM binding site, the catalytic domain and the activation loop (A loop). At present more than 70 mutations conferring different levels of resistance have been found in CML patients. Recently, several new inhibitors have been developed in order to obtain an increased potency and a broad range of activity against IM resistant mutants. Nilotinib (NIL) is an IM derivative about 30-fold more potent than IM. Dasatinib (DAS) is a dual-specific Src/Abl inhibitor, structurally unrelated to IM and characterized by an activity 100 to 300-fold higher than IM. Bosutinib (BOS) is a dual Src/Abl inhibitor that shows an activity 10 to 30-fold higher than IM. It is known that resistance to second generation TKIs can also arise and the analysis of mutation profiles reveals substantial differences among different TKIs. Presently the choice of a TKI to treat a patient resistant to IM is mostly based on an empirical basis, e.g. the fact that a certain patient has not been previously exposed to that particular TKI. The possibility to directly compare the different activities of TKIs against a given mutation is of remarkable importance in clinical practice. Such a tool could be used similarly to an antibiogram for bacterial diseases, guiding the choice of the most appropriate inhibitor for each patient. In our study, we investigated the activity of BOS, DAS, IM and NIL against a panel of 18 mutated forms of BCR/ABL chosen to cover the most common mutations found in patients. Stable Ba/F3 transfectant cell lines were generated and the TKIs antiproliferative activity was determined by tritiated thymidine incorporation assay. The relative IC50 increase over wild type BCR/ABL (Relative Resistance RR) was calculated. We classified the RR values in three categories: sensitive (RR≤2), resistant (between 2.01 and 10) or highly resistant (>10) as presented in the table. IC50-fold increase (WT=1) Imatinib Bosutinib Dasatinib Nilotinib Parental 10.78 38.31 >50 38.45 WT 1 1 1 1 P-LOOP L248V 3.54 2.97 5.11 2.80 G250E 6.86 4.31 4.45 4.56 Q252H 1.39 0.81 3.05 2.64 Y253F 3.58 0.96 1.58 3.23 E255K 6.02 9.47 5.61 6.69 E255V 16.99 5.53 3.44 10.31 D276G 2.18 0.60 1.44 2.00 C-Helix E279K 3.55 0.95 1.64 2.05 V299L 1.54 26.10 8.65 1.34 Active site T3151 17.50 45.42 75.03 39.41 F317L 2.60 2.42 4.46 2.22 SH2-contact M351T 1.76 0.70 0.88 0.44 Active site F359V 2.86 0.93 1.49 5.16 A-LOOP L384M 1.28 0.47 2.21 2.33 H396P 2.43 0.43 1.07 2.41 H396R 3.91 0.81 1.63 3.10 G398R 0.35 1.16 0.69 0.49 C terminal lobe F486S 8.10 2.31 3.04 1.85 Sensitive ≤2 Resistant 2.01–10 Highly resistant >10 (Updated table available online at http://www.dimep.medicina.unimib.it/en/staff_174.php?docente_id=32) Our study points out at the differences in the activity spectrum of the 4 TKIs against the 18 Bcr/Abl mutations considered. The activity pattern presented in this work will help to reach a rational and tailored therapy offering to physicians a tool to use the new TKIs in the most efficient way for their patients.

Zidovudine Impairs Adipogenic Differentiation through Inhibition of Clonal Expansion

ABSTRACT Lipoatrophy is a prevalent side effect of treatment with thymidine analogues. We wished to confine the time point of the antiadipogenic effect of zidovudine (AZT) during adipogenesis and to evaluate the antiproliferative effect of AZT on adipocyte homeostasis. We investigated the effects of AZT on adipogenesis in 3T3-F442A cells and studied their proliferation, differentiation, viability, and adiponectin expression. Cells were exposed to AZT (1 μM, 3 μM, 6 μM, and 180 μM), stavudine (d4T; 3 μM), or dideoxycytosine (ddC; 0.1 μM) for up to 15 days. Differentiation was assessed by real-time PCR and quantification of triglyceride accumulation. Proliferation and clonal expansion were determined by a [3H]thymidine incorporation assay. When they were induced to differentiate in the presence of AZT at the maximum concentration in plasma (C max) and lower concentrations, 3T3-F442A preadipocytes failed to accumulate cytoplasmic triacylglycerol and failed to express normal levels of the later adipogenic transcription factors, CCAAT/enhancer-binding protein α and peroxisome proliferator-activated receptor γ. AZT exerted an inhibitory effect on the completion of the mitotic clonal expansion, which resulted in incomplete 3T3-F442A differentiation and, finally, a reduction in the level of adiponectin expression. In addition, AZT impaired the constitutive proliferation in murine and primary human subcutaneous preadipocytes. In contrast, incubation with d4T and ddC at the C max did not affect either preadipocyte proliferation or clonal expansion and differentiation. We conclude that the antiproliferative and antiadipogenetic effects of AZT on murine and primary human preadipocytes reveal the impact of the drug on fat tissue regeneration. These effects of the drug are expected to contribute to disturbed adipose tissue homeostasis and to be influenced by differential drug concentration and penetration in individual patients.

Characterization of CD4+ T Cell Response Induced by p210-Derived Peptide Vaccinations in CML Patients.

We have previously shown that vaccination of chronic myelogenous leukaemia (CML) patients with CMLVAX100 (a mixture of 5 p210-b3a2 breakpoint derived peptides) plus GM-CSF was able to induce an evident and durable peptide-specific CD4+ T cell response in the majority of patients. Peptide-specific CD4+ T cell proliferation was measured by standard [3H] thymidine incorporation assay and this response was mainly mediated by a 25 mer long breakpoint peptide (b3a2-25) included in the vaccine. In this pilot clinical trial we showed that about 60% of 28 CML patients vaccinated while on imatinib, showed a reduction of their long lasting molecular residual disease after immunization (first 6 vaccinations) and about 25% of them achieved in addition a complete molecular response. To investigate the contribute of this peptide specific CD4+ T cell response in antitumor activity, we further characterize peptide-induced proliferating CD4+ cells. Briefly, in 10 CMLVAX100 vaccinated patients in which a strong b3a2-25 specific CD4+ proliferation was previously observed, we freshly isolated peripheral blood CD4+ T cells and we cultured them 4 days only in the presence or not of b3a2-25 vaccine peptide or control PR-25 peptide. Afterwards, each culture condition was analyzed both for the co-expression of CD25, perforin and FOXP3 molecules and for standard [3H] thymidine incorporation assay. As expected all patients showed a strong b3a2-25 specific CD4+ proliferation (mean stimulation index 43 -range 19–81-). When flow cytometric analysis was performed, we observed a consistent increase of two main CD4+ T cells subsets only in the b3a2-25 stimulated CD4+ T cells, not in “no peptide” or “control peptide” stimulated CD4+ T cells. a “small-size” CD4+/perforin+ T cell population (raising from a median of 1.06% (range 0.21–5.2) to 2.32% (range 0.49–6.2) with very likely cytotoxic activity a “large-size” CD4+/CD25+/FOXP3+ T cell population (raising from 0.29% (range 0.03–0.66) to 5.65% (range 0.71–9.5) with potential regulatory function. Since the large size of these cells and their high proliferative activity would argue against a regulatory features and considering that the immunophenotypic profile of Tregs in human is not yet fully defined we performed a functional test to measure a potential regulatory activity of these vaccine-induced T cell population. Thus, we evaluated the ability of b3a2-25 specific CD4+ T cells to inhibit the growth of CFSE labelled normal subjects naïve CD4+ T cells stimulated with autologous CD3-depleted APCs and antiCD3 MoAb. In our experimental conditions, naïve CFSE CD4+ T cells equally proliferated in the presence of b3a2-25 specific CD4+ cells, “no peptide” CD4+ cells or PR-25 control peptide CD4+ cells from vaccinated patients and the rate of proliferation was similar to the one observed in co-culture. experiments with allogeneic normal CD4+ cells. Our data suggest that the immune response induced in CML patients by CMLVAX100 vaccinations, consists of an increase of peptide-specific cytotoxic CD4+ T cells and a much more evident augment of CD4+/CD25+/FOXP3+ T cells that despite resembling a Treg phenotype display apparently no suppressive activity. The exact role of this peptide-specific CD4+ T cells subset in the context of CMLVAX100 mediated immune response needs to be further elucidated.

Anti-metastatic Effects of Fuzhengfangaitang on Human Fibrosarcoma Cells HT1080

Fuzhengfangaitang (FZFAT) is used to inhibit recurrence and metastasis of cancer in the clinic. By applying an in vitro invasion assay model, we examined the antimetastatic effect of FZFAT. In the 3H-thymidine incorporation assay, FZFAT-treated groups showed a decreased DNA synthesis rate compared with the control group (F-value 87.42981, P-value 2.02E-08, F0.05(3,12) 3.4903). Gelatin zymogram assay showed that FZFAT decreased the gelatinolytic activity of matrix metalloproteinases-9 (MMP-9) from human fibrosarcoma cell line (HT-1080), at concentrations of 200 and 400 μg/ml. In the MMPs dot blotting assay, FZFAT inhibited the expression of MMP-1 at concentration of 100 μg/ml, and MMP-9 at concentrations of 200 and 400 μg/ml. Western blots for AP-1 and its signal mediators Erk and JNK showed that expression of Fos and JNK were decreased by the addition of FZFAT at 300 μg/ml, whereas Erk was not. Therefore it was evident that FZFAT regulated the expression of MMP-9 through its transcription factor AP-1 and the signal mediator JNK. We examined whether FZFAT inhibited the invasion of HT-1080 cells through matrigel precoated transwell chambers. The results showed that FZFAT effectively inhibited the invasion of HT-1080 cells as compared with the control phorbol 12-myristate-13-acetate (+PMA) groups (t-value 5.871584, P-value 0.013901, t0.05(2) 2.919987). From our research, part of the mechanism underlying the antimetastatic effect of FZFAT has been elucidated in vitro.

Streptozotocin and Alloxan-based Selection Improves Toxin Resistance of Insulin-Producing RINm Cells

The aim of our study was to develop a method for selection of subpopulations of insulin producing RINm cells with higher resistance to beta cell toxins. Cells, resistant to streptozotocin (RINmS) and alloxan (RINmA), were obtained by repeated exposure of parental RINm cells to these two toxins, while the defense capacity, was estimated by the MTT colorimetric method, and[H3]-thymidine incorporation assay. We found that RINmS and RINmA displayed higher resistance to both streptozotocin (STZ) and alloxan (AL) when compared to the parental RINm cells. In contrast, no differences in sensitivity to hydrogen peroxide were found between toxin selected and parental cells. Partial protection from the toxic effect of STZ and AL was obtained only in the parental RINm cells after preincubation of cells with the unmetabolizable 3- O-methyl-glucose. The possibility that GLUT-2 is involved in cell sensitivity to toxins was confirmed by Western blot analysis, which showed higher expression of GLUT-2 in parental RINm compared to RINmS and RINmA cells. In addition to the higher cell defense property evidenced in the selected cells, we also found higher insulin content and insulin secretion in both RINmS and RINmA cells when compared to the parental RINm cells. In conclusion, STZ and AL treatment can be used for selection of cell sub-populations with higher cell defense properties and hormone production. The different GLUT-2 expression in parental and re sistant cells suggest involvement of GLUT-2 in mechanisms of cell response to different toxins.