Luisa Vicari

Altered Expression of c-IAP1, Survivin, and Smac Contributes to Chemotherapy Resistance in Thyroid Cancer Cells

Resistance to chemotherapy predicts an unfavorable outcome for patients with radioiodine-insensitive thyroid cancer. To investigate the mechanisms underlying this resistance, we evaluated the expression of four different inhibitor of apoptosis proteins, and their antagonist, Smac, in thyroid cancer cells that survived 48 hours of exposure to cisplatin, doxorubicin, or taxol. We found high levels of c-IAP1 after cisplatin treatment and increased expression of survivin following exposure to doxorubicin. Cells that endured treatment with taxol showed reduced expression of Smac and released minimal amounts of this protein from the mitochondria. Down-regulation of c-IAP1 and survivin increased the cytotoxicity of cisplatin and doxorubicin, whereas overexpression of Smac improved the efficacy of taxol. Finally, thyroid cancer cells permanently resistant to doxorubicin or cisplatin showed increased expression of c-IAP1 and survivin, respectively. However, silencing of these proteins by RNA interference restored sensitivity to doxorubicin and cisplatin. Thus, in thyroid cancer cells, early resistance to chemotherapeutic agents requires high levels of c-IAP1 and survivin and low levels of Smac. Furthermore, increased expression of c-IAP1 and survivin contributes to the acquisition of permanent resistance to cytotoxic compounds.

Lipophilic methotrexate conjugates with antitumor activity

Lipophilic methotrexate (MTX)-lipoamino acid conjugates coupled with amide or ester linkages (1a-1r) were synthesised. The inhibitory activity of the conjugates was evaluated on bovine liver DHFR. The in vitro growth inhibitory effect against MTX-sensitive human lymphoblastoid CCRF-CEM cells and an MTX-resistant sub-line (CEM/MTX), which displays defective intracellular transport of MTX, was determined under short-term and continuous (120-h incubation) exposure conditions. The alpha, gamma, or alpha,gamma amide conjugates showed different activity in inhibiting the growth of parent cells. CEM/MTX cells were much less susceptible than CCRF-CEM cells to inhibition by alpha or alpha,gamma-substituted lipoamino acid conjugates, whereas both cell lines were almost equally sensitive to the MTX-gamma conjugates. Although less potent than MTX, they could partially circumvent the impaired transport system. These findings confirm that lipophilic MTX conjugates may be good lead compounds on the drug development for the treatment of some MTX-resistant tumors. Ester-type conjugates displayed an interesting activity against parent CCRF-CEM cells, although they were less potent against the transport-resistant sub-line. Stability studies on these molecules indicated that they are not degraded into MTX in the culture medium, thus suggesting that they are not able to over-cross cell resistance despite of their lipophilicity.

Paclitaxel loading in PLGA nanospheres affected the in vitro drug cell accumulation and antiproliferative activity

BackgroundPTX is one of the most widely used drug in oncology due to its high efficacy against solid tumors and several hematological cancers. PTX is administered in a formulation containing 1:1 Cremophor® EL (polyethoxylated castor oil) and ethanol, often responsible for toxic effects. Its encapsulation in colloidal delivery systems would gain an improved targeting to cancer cells, reducing the dose and frequency of administration.MethodsIn this paper PTX was loaded in PLGA NS. The activity of PTX-NS was assessed in vitro against thyroid, breast and bladder cancer cell lines in cultures. Cell growth was evaluated by MTS assay, intracellular NS uptake was performed using coumarin-6 labelled NS and the amount of intracellular PTX was measured by HPLC.ResultsNS loaded with 3% PTX (w/w) had a mean size < 250 nm and a polydispersity index of 0.4 after freeze-drying with 0.5% HP-Cyd as cryoprotector. PTX encapsulation efficiency was 30% and NS showed a prolonged drug release in vitro. An increase of the cytotoxic effect of PTX-NS was observed with respect to free PTX in all cell lines tested.ConclusionThese findings suggest that the greater biological effect of PTX-NS could be due to higher uptake of the drug inside the cells as shown by intracellular NS uptake and cell accumulation studies.

Antitumor activity of bortezomib alone and in combination with TRAIL in human acute myeloid leukemia.

Acute myeloid leukemia (AML) is a malignant disease characterized by abnormal proliferation of clonal precursor cells. Although different strategies have been adopted to obtain complete remission, the disease actually progresses in about 60–70% of patients. Bortezomib has been used in multiple myeloma and other lymphoid malignancies because of its antitumor activity. Here we examined the sensitivity of bone marrow cells from AML patients (34 patients: 25 newly diagnosed, 4 relapsed, 5 refractory) to bortezomib alone or in combination with TRAIL, a member of the TNF family that induces apoptosis in tumor cells while sparing normal cells. Bortezomib induced cell death in blasts from each patient sample. The cytotoxic effect was dose- and time-dependent (concentration from 0.001 to 10 µM for 24 and 48 h) and was associated with a downregulation of Bcl-xL and Mcl-1, an upregulation of TRAIL-R1, TRAIL-R2, p21, activation of executioner caspases and a loss of the mitochondrial membrane potential. Moreover, low doses of bortezomib primed TRAIL-resistant AML cells for enhanced TRAIL-mediated killing. These results suggest that a combination of proteasome inhibitors and TRAIL could be effective for treating AML patients, even patients who are refractory to conventional chemotherapy.

Aliphatic α,γ-bis(Amides) of Methotrexate. Influence of Chain Length on In-vitro Activity Against Sensitive and Resistant Tumour Cells

The synthesis of short-chain alkyl bis(amides) (heptyl-, isoheptyl-, octyl-) and nonylamide derivatives as lipophilic derivatives of methotrexate is reported. Direct amine substitution on methotrexate diethyl ester and a carbodiimide-assisted coupling method were used. The compounds were screened for in-vitro inhibitory activity against bovine liver dihydrofolate reductase and growth inhibition of human lymphoblastoid methotrexate-sensitive and resistant CCRF-CEM cells and erythroleukaemic K562 cells. The lipophilic methotrexate derivatives, despite showing lower activity against methotrexate-sensitive tumour cell lines, maintained similar activity to methotrexate, even against a methotrexate-transport resistant cell subline, since they can penetrate tumour cells by a passive route, by-passing the deficient cellular carrier system for the folates. Increasing the lipophilicity of methotrexate is a possible strategy to overcome the clinical resistance to the drug by tumour cells.

Effect of Liposomal Delivery on In Vitro Antitumor Activity of Lipophilic Conjugates of Methotrexate with Lipoamino Acids

Some selected lipophilic conjugates of the antifolate drug methotrexate (MTX) with lipoamino acids (LAA), previously described, were incorporated in liposomes with a different composition and charge (neutral, positive, or negative). The properties of the liposomal systems were determined. The inhibitory activity of the conjugates after incorporation in the vesicles was determined in a preliminary assessment against a human erythroleukemic cell line (K562 cells) and compared with the activity of the parent drug and of free conjugates. The influence of liposome surface charge and of the type of conjugate (i.e., in the carboxylic or ester form) on the biological effect is discussed.

Increased phospho-mTOR expression in megakaryocytic cells derived from CD34+ progenitors of essential thrombocythaemia and myelofibrosis patients.

The mammalian target of rapamycin (mTOR) is a serine/ threonine kinase that forms different multiprotein complexes with diverse subunit compositions, downstream substrates and biological effects (Ma & Blenis, 2009). As mTOR is a key regulator of cell growth and metabolism, its improper activation has been previously linked to carcinogenesis (Meric-Bernstam & Gonzalez-Angulo, 2009). Polycythaemia vera (PV), essential thrombocythaemia (ET) and primary myelofibrosis (PMF) are BCR-ABL1-negative myeloproliferative neoplasms (MPNs). ET is characterized by megakaryocytic bone marrow hyperplasia and a sustained elevation of platelet count with a tendency for thrombosis and haemorrhage (Levine & Gilliland, 2008). PMF can arise ‘de novo’ or evolve from PV or ET. In PMF, the abnormal proliferation of an aberrant clone eventually leads to the replacement of the normal bone marrow with connective tissue fibres (Tefferi & Vainchenker, 2011). As the molecular culprit(s) of these disorders still need(s) to be elucidated, current treatment options are not curative and have yet to produce clear benefits in terms of increasing overall survival (Tefferi & Vainchenker, 2011). Mammalian target of rapamycin is a pivotal downstream target of the thrombopoietin receptor (Drayer et al, 2006). Thus, it is not surprising that several studies have previously demonstrated critical contributions of mTOR signalling in the proliferation and differentiation of normal megakaryocyte (MK) cultures (Drayer et al, 2006; Raslova et al, 2006). However, the role of mTOR in the megakaryopoiesis of ET and PMF cells remains to be investigated. As thrombocytosis is a common clinical feature of both ET and PMF, we wanted to establish whether mTOR activation was involved in the increased platelet counts observed in these MPNs. To this end, CD34-positive (CD34+) progenitors were employed to reproduce the different stages of human MK differentiation that generate morphologically and functionally mature platelets (Guerriero et al, 1995; Deutsch & Tomer, 2006). After acquiring informed consent, human CD34+ cells were isolated from healthy donors, ET and PMF patients using the midi-MACS immunomagnetic separation system (Miltenyi Biotec, Auburn, CA, USA) obtaining a >90% pure cell population. Megakaryocytic differentiation was then induced by growing these cells for 14 d in serum-free medium in the presence of bovine serum albumin, insulin, human transferrin, human low-density lipoprotein, and 100 ng/ml thrombopoietin (PeproTech, London, UK). The expression of the CD34 and CD61 surface markers was evaluated by flow cytometry, collecting cells at different time-points (days 0, 3, 12) and using a fluorescein isothiocyanate-labelled anti-CD34 antibody and an anti-CD61 antibody bound to phycoerythrin (Miltenyi Biotec). A gradual decrease in the CD34 signal associated with increased expression of the MK-specific CD61 marker confirmed the purity of the initial population and its progressive megakaryocytic differentiation (Figure S1A). Morphological observations after May–Grunwald Giemsa staining (Sigma, St Louis, MO, USA) correlated with the flow cytometry data, showing large polynucleated MK cells after 12 d of culture (Figure S1B). A unilineage system was subsequently employed to establish if MK cells isolated from patients with ET or PMF expressed higher levels of phosphorylated mTOR (p-mTOR) when compared to healthy individuals. We initially performed an immunofluorescence (IF) analysis in which cells were stained with an anti-p-mTOR (recognizing serine 2448) antibody (Cell Signaling, Danvers, MA, USA) after 3 and 12 d of culture. While we found no differences in mTOR phosphorylation after 3 d (Fig 1A, top panels), p-mTOR was increased in MKs from ET and PMF patients obtained after 12 d of culture (Fig 1A, bottom panels), but not in cells derived from healthy donors. To quantify the qualitative differences detected by IF, we analysed mTOR phosphorylation by flow cytometry, evaluating the xfold increase in median fluorescence intensity over the isotype control (D’Asaro et al, 2010). This analysis confirmed higher p-mTOR expression in ET and PMF-derived MKs generated after 12 d of culture (Fig 1B). To further confirm these data, we performed an anti-pmTOR immunohistochemical analysis on 35 bone marrow specimens derived from patients diagnosed with ET (n = 14) or PMF (n = 21). Three micrometre-thick sections were incubated with the same anti-p-mTOR antibody employed for the IF analyses. We found higher p-mTOR expression in 34 of 35 bone marrow samples as compared to the mostly negative staining observed in healthy individuals (Fig 2). The present study provides the first comparison of p-mTOR expression in MK cells derived from the CD34+ progenitors of healthy donors, ET and PMF patients. Recent evidence suggests that targeting mTOR with the oral inhibitor everolimus may be clinically useful for patients

Pyrrolidinedithiocarbamate Induces Apoptosis in Human Acute Myelogenous Leukemic Cells Affecting NF-κB Activity

Pyrrolidindithiocarbamate (PDTC), is a metal chelator widely used to study the activation of redox sensitive transcription factors. Recently it has been demonstrated that it manifests pro-oxidant properties. The nuclear factor-Kappa B (NF-κB) transcription factor can both promote cell survival and induce apoptosis depending on cell type and context in response to genotoxic stress. In our previous study we reported that in acute myelogenous leukemia CD34+ cells PDTC stimulates apoptosis, whereas in CD34+ cells of healthy volunteers PDTC was ineffective. This cytotoxicity was dependent on the generation of superoxide anion and oxidized glutathione. In this article we have shown that the pro-oxidant effect of PDTC in AML cells induces NF-κB activity. These findings imply a role for NF-κB in the survival of normal cells with respect to leukemic cells, suggesting that NF-κB activity and function differs according to tumor cell phenotype.

Potential Role of Activating Transcription Factor 5 during Osteogenesis.

Human adipose-derived stem cells are an abundant population of stem cells readily isolated from human adipose tissue that can differentiate into connective tissue lineages including bone, cartilage, fat, and muscle. Activating transcription factor 5 is a transcription factor of the ATF/cAMP response element-binding protein (CREB) family. It is transcribed in two types of mRNAs (activating transcription factor 5 isoform 1 and activating transcription factor 5 isoform 2), encoding the same single 30-kDa protein. Although it is well demonstrated that it regulates the proliferation, differentiation, and apoptosis, little is known about its potential role in osteogenic differentiation. The aim of this study was to evaluate the expression levels of the two isoforms and protein during osteogenic differentiation of human adipose-derived stem cells. Our data indicate that activating transcription factor 5 is differentially expressed reaching a peak of expression at the stage of bone mineralization. These findings suggest that activating transcription factor 5 could play an interesting regulatory role during osteogenesis, which would provide a powerful tool to study bone physiology.

The PU.1 transcription factor induces cyclin D2 expression in U937 cells.

The PU.1 transcription factor is expressed in a wide variety of haematopoietic precursors including long-and short-term reconstituting stem cells, the common myeloid and lymphoid progenitors (CMP, CLP), granulocyte–monocyte progenitors (GMP), monocytes, neutrophils and B-lymphocytes. Compelling evidence gathered from both mouse models and human studies has demonstrated that PU.1 is a pivotal component of the intricate network regulating normal and neoplastic haematopoiesis. Physiologically, PU.1 expression contributes to the commitment of the CMPs to granulocytes and monocytes and to B-cell differentiation. These observations derive from studies on PU.1 knockout mice showing that PU.1-null animals present normal erythroid and megakaryocytic cells, but display embryonic/newborn lethality owing to the lack of monocytes, neutrophils and B-lymphocytes. More recently, the role of PU.1 in normal haematopoiesis has been further defined by conditional knockout mice demonstrating that PU.1 expression is critical to perpetuate the haematopoietic stem cell pool and to allow the generation of CMPs and CLPs. PU.1 disruption in CMPs or GMPs blocks their maturation, whereas PU.1 deficiency in CLPs does not affect their ability to differentiate in mature B cells. These results are not surprising considering that PU.1 induces the expression of multiple proteins critically involved in the commitment of the myeloid and lymphoid lineages, including the granulocyte/macrophage colony-stimulating factor receptor (CSF), the macrophage CSF receptor, the granulocyte CSF receptor and the interleukin-7 receptor. PU.1 is also directly involved in the pathogenesis of human leukaemias. Overexpression of PU.1 in mice exposed to the Friend virus results in the development of erythroleukaemia. Likewise, mice harbouring deletions of the upstream regulatory element of the PU.1 promoter that lead to reduced PU.1 expression develop acute myeloid leukaemia (AML) or T-cell lymphoma. Moreover, mutations impairing PU.1 transcriptional activity have been reported in 7% of 126 patients diagnosed with AML. Recent findings have also shown that both AML1/ETO and FLT3-ITD oncoproteins inhibit PU.1 activity, therefore reinforcing the notion that reduced PU.1 function (possibly coupled with mutation-induced haploinsufficiency) may contribute to the development of AML. Although it is well established that PU.1 modulates the proliferation and differentiation of several haematopoietic precursors, the mechanisms responsible for these activities are still partially unclear. As D-type cyclins are critical modulators of cell proliferation favouring cell cycle transition from the G1 to the S phase, we investigated if PU.1 could regulate the levels of D-cyclins. We report that PU.1 induces the expression of cyclin D2 in the U937 promonocytic cell line. A preliminary sequence analysis of the promoter for human cyclin D2 (accession number U47284) revealed two GAGGAA consensus sites for PU.1, one of which is conserved in mice (accession number AFO15788) (Figure 1a, in bold). To determine if PU.1 is able to transactivate this sequence, we transiently expressed the full-length human cyclin D2 promoter (cloned in the PGL2 luciferase vector, gift of Professor M Eilers, Marburg, Germany) in human embryonic kidney (HEK) 293 cells either alone or with PU.1. Luciferase assays performed normalizing transfection efficiency with an expression vector for Renilla demonstrated a reproducible 2.7-fold increase in the activity of the cyclin D2 promoter in the presence of PU.1 (Figure 1b). However, as PU.1 is expressed exclusively in haematopoietic lineages, we wanted to ascertain if these results could be reproduced in a haematopoietic cell line. Previous evidence has shown that treatment of U937 cells with tetradecanoyl-phorbol-13 acetate (TPA) leads to the phosphorylation of PU.1 and induction of its transcriptional activity. Indeed, as had been reported previously, when we carried out an electrophoretic mobility shift assay on U937 cells incubated with 20 ng/ml TPA, we observed an increase in PU.1 transcriptional activity after 9–12 h (not shown). Interestingly, PU.1 phosphorylation by serine–threonine kinases can be monitored by immunoblot because hyperphosphorylated (active) PU.1 exhibits a supershift compared to the unphosphorylated (inactive) protein. We therefore performed an antiPU.1 Western blot on U937 cell lysates derived from cells incubated with TPA and noticed a reproducible increase of the hyperphosphorylated PU.1 protein after 9 h. This increase reached a plateau after a 21-h stimulation (Figure 1c, left panel). When we repeated the immunoblot employing an anticyclin D2 antibody, we found a marked increase in cyclin D2 levels starting from 12 h of TPA incubation, suggesting that in U937 cells activation of PU.1 leads to increased expression of cyclin D2 (Figure 1c, right panel). To exclude that PU.1-independent biological events may be responsible for the observed TPA-mediated induction of cyclin D2 expression, we retrovirally transduced U937 cells with a dominant-negative form of PU.1 (DNPU.1 provided by Professor R Kawara, University of Nebraska Medical Center, USA) that retains the DNA-binding domain of the wild-type protein, but is devoid of both the transactivation and the PEST domains. Control Western blots showed that DNPU.1-infected cells (lanes 5–8) but not mock-infected cells (lanes 1–4) expressed the dominant-negative form of the transcription factor (Figure 1d, left panel). Moreover, this experiment demonstrated that expression of DNPU.1 did not affect TPA-induced phosphorylation of the endogenous PU.1 as incubation with the drug caused PU.1 hyperphosphorylation both in the absence and in the presence of DNPU.1. However, when we analysed cyclin D2 expression levels, we found a marked difference in the two U937 cell populations. In mock-infected U937 exposure to TPA increased cyclin D2 expression as had been observed previously (Figure 1d, right panel, lanes 1–4). On the contrary, U937 cells expressing DNPU.1 did not present increased levels of cyclin D2 upon PU.1 phosphorylation (Figure 1d, right panel, lanes 5–8), indicating that the dominant-negative form of the transcription factor successfully antagonized the effect of the endogenous PU.1 on the cyclin D2 promoter. To further determine the biological effect of PU.1 regulation of cyclin D2 expression in U937 cells, we transduced this cell line with viral particles encoding for DNPU.1 and cyclin D2, alone or in combination. Immunoblotting experiments confirmed proper expression of the expected proteins in each experimental condition (Figure 1e, left panels). Fluorescentactivated cell sorting (FACS) analysis of these different cell populations after an 18-h treatment with TPA revealed that DNPU.1 did not alter the cell cycle distribution of these cells, as Letters to the Editor