Effect of trehalose on cryopreservation of pure peripheral blood stem cells.

Stem cells are an important tool for the study of hematopoiesis. Despite developments in cryopreservation, post-thaw cell death remains a considerable problem. Cryopreservation protocol should limit cell damage due to freezing and ensure the recovery of the functional cell characteristics after thawing. Thus, the use of cryoprotectants is essential. In particular, the efficacy of trehalose has been reported for clinical purposes in blood stem cells. The aim of the current study was to establish an efficient method for biological research based on the use of trehalose, to cryopreserve pure peripheral blood stem cells. The efficacy of trehalose was assessed in vitro and the cell viability was evaluated. The data indicate that trehalose improves cell survival after thawing compared with the standard freezing procedure. These findings could suggest the potential for future trehalose application for research purposes in cell cryopreservation.

Differential expression of two activating transcription factor 5 isoforms in papillary thyroid carcinoma.

BACKGROUND: Activating transcription factor 5 (ATF5) is a member of the activating transcription/cAMP response element-binding protein family of basic leucine zipper proteins that plays an important role in cell survival, differentiation, proliferation, and apoptosis. The ATF5 gene generates two transcripts: ATF5 isoform 1 and ATF5 isoform 2. A number of studies indicate that ATF5 could be an attractive target for therapeutic intervention in several tumor types; however, so far, the role of ATF5 has not been investigated in papillary thyroid carcinoma (PTC). METHODS: Quantitative real-time reverse transcription polymerase chain reaction and immuno-histochemical staining were used to study ATF5 mRNA and protein expression in PTC. RESULTS: We report here that ATF5 is expressed more in PTC tissue than in normal thyroid tissue. Furthermore, this is the first study that describes the presence of both ATF5 isoforms in PTC. CONCLUSION: These findings could provide potential applications in PTC cancer treatment.

Cancer stem cells as a potential therapeutic target in thyroid carcinoma.

A number of studies have indicated that tumor growth and proliferation is dependent on a small subset of cells, defined as cancer stem cells (CSCs). CSCs have the capability to self-renew, and are involved with cancer propagation, relapse and metastatic dissemination. CSCs have been isolated from numerous tissues, including normal and cancerous thyroid tissue. A regulatory network of signaling pathways and microRNAs (miRNAs) control the properties of CSCs. Differentiated thyroid carcinoma is the most common type of endocrine cancer, with an increasing incidence. Anaplastic thyroid carcinoma is the most rare type of endocrine cancer; however, it also exhibits the highest mortality rate among thyroid malignancies, with an extremely short survival time. Thyroid CSCs are invasive and highly resistant to conventional therapies, including radiotherapy and chemotherapy, which results in disease relapse even when the primary lesion has been eradicated. Therefore, targeting thyroid CSCs may represent an effective treatment strategy against aggressive neoplasms, including recurrent and radioresistant tumors. The present review summarizes the current literature regarding thyroid CSCs and discusses therapeutic strategies that target these cells, with a focus on the function of self-renewal pathways and miRNAs. Elucidation of the mechanisms that regulate CSC growth and survival may improve novel therapeutic approaches for treatment-resistant thyroid cancers.

Resistance of papillary thyroid cancer stem cells to chemotherapy.

Thyroid carcinoma is the most common endocrine neoplasm, with the highest mortality rate of all the endocrine cancers. Among the endocrine malignancies, ~80% are papillary thyroid carcinomas (PTCs). In the initiation and progression of this tumor, genetic alterations in the mitogen-associated protein kinase pathway, including RAS point mutations, RET/PTC oncogene rearrangements and BRAF point mutations, play an important role, particularly in deciding targeted therapy. In the present study, a small population of thyroid tumor cells, known as tumor spheres, were isolated and characterized from PTC surgical samples. These spheres can be expanded indefinitely in vitro and give rise to differentiated adherent cells when cultivated in differentiative conditions. The present study showed by reverse transcription-polymerase chain reaction and flow cytometric analysis that the undifferentiated PTC cells exhibited a characteristic antigen expression profile of adult progenitor/stem cells. The cells were more resistant to chemotherapeutics, including bortezomib, taxol, cisplatin, etoposide, doxorubicin and vincristine, than differentiated PTC cells and the majority possessed a quiescent status, as revealed by the various cell cycle characteristics and anti-apoptotic protein expression. Such advances in cancer thyroid stem cell biology may provide relevant information for future targeted therapies.

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.

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 microM 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.

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

BACKGROUND: PTX 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. METHODS: In 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. RESULTS: NS 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. CONCLUSION: These 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.

Proteasome inhibitors synergize with tumor necrosis factor-related apoptosis-induced ligand to induce anaplastic thyroid carcinoma cell death.

CONTEXT: Anaplastic thyroid carcinoma (ATC) is one of the most aggressive types of cancer characterized by complete refractoriness to multimodal treatment approaches. Therapeutic strategies based on the simultaneous use of proteasome inhibitors and death receptor ligands have been shown to induce apoptosis in several tumor types but have not yet been explored in ATC. OBJECTIVE AND METHODS: The aim of this study was to investigate the ability of the proteasome inhibitor Bortezomib to induce apoptosis in ATC cell lines. Bortezomib was used as a single agent or in combination with TNF-related apoptosis-induced ligand (TRAIL), a member of the TNF family that selectively induces tumor cell apoptosis. The molecular effects of Bortezomib were investigated by analyzing the expression of key regulators of cell cycle and apoptosis and the activation of different apoptotic pathways. RESULTS: Bortezomib induced apoptosis in ATC cells at doses achieved in the clinical setting, differently from conventional chemotherapeutic agents. Simultaneous treatment with low doses of Bortezomib and TRAIL had a synergistic effect in inducing massive ATC cell apoptosis. Bortezomib increased the expression of cytotoxic TRAIL receptors, p21 (WAF/CIP1) and proapoptotic second mitochondria-derived activator of caspases/direct inhibitor of apoptosis binding protein with low pI, and reduced the expression of antiapoptotic mediators such as cellular Fas-associated death domain-like IL-1beta converting enzyme inhibitory protein, Bcl-2, Bcl-X(L), and inhibitor of apoptosis-1, thus resulting in cell death induction through the mitochondrial apoptotic pathway. CONCLUSIONS: The combination of proteasome inhibitors and TRAIL synergizes to induce the destruction of chemoresistant neoplastic thyrocytes and could represent a promising therapeutic strategy for the treatment of anaplastic thyroid carcinoma.

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.

Pyrrolidinedithiocarbamate induces apoptosis in human acute myelogenous leukemic cells affecting NF-kappaB 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-kappaB) 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-kappaB activity. These findings imply a role for NF-kappaB in the survival of normal cells with respect to leukemic cells, suggesting that NF-kappaB activity and function differs according to tumor cell phenotype.

Lipophilic conjugates of methotrexate with short-chain alkylamino acids as DHFR inhibitors. Synthesis, biological evaluation, and molecular modeling.

Pursuing previous researches on lipophilic conjugates of methotrexate, aimed at over-crossing a form of transport resistance shown by some tumor cell lines toward the drug, a new series of derivatives is described in which the drug alpha- and gamma-carboxyl groups have been linked through amide bonds to short-chain alpha-alkylamino acids (4-6 carbon atoms). A specific NMR study was performed to delineate the stereochemistry of the conjugates. The inhibitory activity of these compounds against the target enzyme, (bovine liver) dihydrofolate reductase, and a sensitive (CCRF-CEM) and a transport-resistant tumor cell subline (CEM-MTX) were assessed. The conjugates showed the ability of retaining the same inhibitory activity also against the resistant cell subline, against which the parent drug was much less active than against the wild one; the alpha,gamma-bis(hexyl) derivative was the most active term of the series. Docking studies are in agreement with the proposed mode of interaction of these conjugates with the human DHFR.

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.