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Calreticulin (CALR) is a Ca2+-binding protein that shuttles among cellular compartments with proteins bound to its N/P domains. The knowledge that activation of the human erythropoietin receptor induces Ca2+ fluxes prompted us to investigate the role of CALR in human erythropoiesis. As shown by Western blot analysis, erythroblasts generated in vitro from normal sources and JAK2V617F polycythemia vera (PV) patients expressed robust levels of CALR. However, Ca2+ regulated CALR conformation only in normal cells. Normal erythroblasts expressed mostly the N-terminal domain of CALR (N-CALR) on their cell surface (as shown by flow cytometry) and C-terminal domain (C-CALR) in their cytoplasm (as shown by confocal microscopy) and expression of both epitopes decreased with maturation. In the proerythroblast (proEry) cytoplasm, C-CALR was associated with the glucocorticoid receptor (GR), which initiated the stress response. In these cells, Ca2+ deprivation and inhibition of nuclear export increased GR nuclear localization while decreasing cytoplasmic detection of C-CALR and C-CALR/GR association and proliferation in response to the GR agonist dexamethasone (Dex). C-CALR/GR association and Dex responsiveness were instead increased by Ca2+ and erythropoietin. In contrast, JAK2V617F proErys expressed normal cell-surface levels of N-CALR but barely detectable cytoplasmic levels of C-CALR. These cells contained GR mainly in the nucleus and were Dex unresponsive. Ruxolitinib rescued cytoplasmic detection of C-CALR, C-CALR/GR association, and Dex responsiveness in JAK2V617F proErys and its effects were antagonized by nuclear export and Ca2+ flux inhibitors. These results indicates that Ca2+-induced conformational changes of CALR regulate nuclear export of GR in normal erythroblasts and that JAK2V617F deregulates this function in PV.

Since 2001 the Istituto Superiore di Sanità established a quality assurance programme for molecular genetic testing that covers four pathologies: Cystic Fibrosis (CF), Beta Thalassemia (BT), Fragile X Syndrome (FX), and Familial Adenomatous Polyposis Coli (APC). Since 2009 this activity is an institutional activity and participation is open to both public and private laboratories. Seven rounds have been performed until now and the eighth is in progress. Laboratories receive 4 DNA samples with mock clinical indications. They analyze the samples using their routine procedures. A panel of assessors review the raw data and the reports; all data are managed through a web utility. In 2010 the number of participants was 43, 17, 15, 5 for CF, BT, FX, APC schemes respectively. Genotyping results were correct in 96%, 98.5%, 100%, and 100% of CF, BT, FX, and APC samples, respectively. Interpretation was correct in 74%, 91%, 88%, and 60% of CF, BT, FX, and APC reports, respectively; however in most of them it was not complete but a referral to genetic counseling was given. Reports were satisfactory in more than 60% of samples in all schemes. This work presents the 2010 results in detail comparing our data with those from other European schemes.

In our previous studies, the bisindolic alkaloid voacamine (VOA), isolated from the plant Peschiera fuchsiaefolia, proved to exert a chemosensitizing effect on cultured multidrug resistant (MDR) osteosarcoma cells exposed to doxorubicin (DOX). In particular, VOA was capable of inhibiting P-glycoprotein action in a competitive way, thus explaining the enhancement of the cytotoxic effect induced by DOX on MDR cells. Afterwards, preliminary observations suggested that such an enhancement did not involve the apoptotic process but was due instead to the induction of autophagic cell death. The results of the present investigation demonstrate that the plant alkaloid VOA is an autophagy inducer able to exert apoptosis-independent cytotoxic effect on both wild-type and MDR tumor cells. In fact, under treatment condition causing about 50 percent of cell death, no evidence of apoptosis could be revealed by microscopical observations, Annexin V-FITC labeling and analysis of PARP cleavage, whereas the same cells underwent apoptosis when treated with apoptosis inducers, such as doxorubicin and staurosporine. Conversely, VOA-induced autophagy was clearly evidentiated by electron microscopy observations, monodansylcadaverine staining, LC3 expression, and conversion. These results were confirmed by the analysis of the modulating effects of the pretreatment with autophagy inhibitors prior to VOA administration. In addition, transfection of osteosarcoma cells with siRNA against ATG genes reduced VOA cytotoxicity. In conclusion, considering the very debated dual role of autophagy in cancer cells (protective or lethal, pro- or anti- apoptotic) our findings seem to demonstrate, at least in vitro, that a natural product able to induce autophagy can be effective against drug resistant tumors, either used alone or in association with conventional chemotherapeutics.

Elevated fetal hemoglobin (HbF) ameliorates the clinical severity of hemoglobinopathies such as β-thalassemia and sickle cell anemia. Currently, the only curative approach for individuals under chronic transfusion/chelation support therapy is allogeneic stem cell transplantation. However, recent analyses of heritable variations in HbF levels have provided a new therapeutic target for HbF reactivation: the transcriptional repressor BCL11A. Erythroid-specific BCL11A abrogation is now actively being sought as a therapeutic avenue, but the specific impact of such disruption in humans remains to be determined. Although single nucleotide polymorphisms in BCL11A erythroid regulatory elements have been reported, coding mutations are scarcer. It is thus of great interest that patients have recently been described with microdeletions encompassing BCL11A. These patients display neurodevelopmental abnormalities, but whether they show increased HbF has not been reported. We have examined the hematological phenotype, HbF levels, and erythroid BCL11A expression in 3 such patients. Haploinsufficiency of BCL11A induces only partial developmental g-globin silencing. Of greater interest is that a patient with a downstream deletion exhibits reduced BCL11A expression and increased HbF. Novel erythroid-specific regulatory elements in this region may be required for normal erythroid BCL11A expression, whereas loss of separate elements in the developing brain may explain the neurological phenotype.

Splenomegaly is a major manifestation of primary myelofibrosis (PMF) contrib- uting to clinical symptoms and hematologic abnormalities. The spleen from PMF patients contains increased numbers of hematopoietic stem cells (HSC) and megakar- yocytes. These megakaryocytes express high levels of P-selectin (P-sel) that, by trig- gering neutrophil emperipolesis, may cause TGF-β release and disease progression. This hypothesis was tested by deleting the P-sel gene in the myelofibrosis mouse model carrying the hypomorphic Gata1low mutation that induces megakaryocyte abnormalities that recapitulate those observed in PMF. P-selnullGata1low mice survived splenectomy and lived three months longer than P-selWTGata1low littermates and did not express fibrosis and osteosclerosis in the marrow or splenomegaly. Furthermore, deletion of P-sel disrupted megakaryocyte/neutrophil interactions in spleen, reduced TGF-β content and corrected the HSC distribution that in Gata1low mice, as in PMF patients, is abnormally expanded in spleen. Conversely, pharmacological inhibition of TGF-β reduced P-sel expression in megakaryocytes and corrected HSC distribution. Spleens, but not marrow, of Gata1low mice contained numerous cKITpos activated fibro- cytes, probably of dendritic cell origin, whose membrane protrusions interacted with megakaryocytes establishing niches hosting immature cKITpos hematopoietic cells. These activated fibrocytes were not detected in spleens from P-selnullGata1low or TGF-β-inhibited Gata1low littermates and were observed in spleen, but not in marrow, from PMF patients. Therefore in Gata1low mice, and possibly in PMF, abnormal P-sel expres- sion in megakaryocytes may mediate the pathological cell interactions that increase TGF-β content in MK and favor establishment of a microenvironment that supports myelofibrosis-related HSC in spleen., Italian Journal of Anatomy and Embryology, Vol. 120, No. 1 (Supplement) 2015

Expansion of erythroblasts from human peripheral blood mononuclear cells is 4- to 15-fold more efficient than that of CD34(+) cells purified from peripheral blood mononuclear cells. In addition, purified CD34(+) and CD34(-) populations from blood do not reconstitute this erythroid yield, suggesting a role for feeder cells present in blood mononuclear cells that increase hematopoietic output. Immunodepleting peripheral blood mononuclear cells for CD14(+) cells reduced hematopoietic stem and progenitor cell expansion. Conversely, the yield was increased upon co-culture of CD34(+) cells with CD14(+) cells (full contact or transwell assays) or CD34(+) cells re-constituted in conditioned medium from CD14(+) cells. In particular, CD14(++)CD16(+) intermediate monocytes/macrophages enhanced erythroblast outgrowth from CD34(+) cells. No effect of CD14(+) cells on erythroblasts themselves was observed. However, 2 days of co-culturing CD34(+) and CD14(+) cells increased CD34(+) cell numbers and colony-forming units 5-fold. Proliferation assays suggested that CD14(+) cells sustain CD34(+) cell survival but not proliferation. These data identify previously unrecognized erythroid and non-erythroid CD34(-) and CD34(+) populations in blood that contribute to the erythroid yield. A flow cytometry panel containing CD34/CD36 can be used to follow specific stages during CD34(+) differentiation to erythroblasts. We have shown modulation of hematopoietic stem and progenitor cell survival by CD14(+) cells present in peripheral blood mononuclear cells which can also be found near specific hematopoietic niches in the bone marrow.

Background: Resistance to antitumor agents is a major cause of treatment failure in patients with cancer. Some mechanisms of tumor resistance to cytotoxic drugs may involve increased acidification of extracellular compartments. We investigated whether proton pump inhibitors (PPIs), currently used in the anti-acid treatment of peptic disease, could inhibit the acidification of the tumor microenvironment and increase the sensitivity of tumor cells to cytotoxic agents. Methods: We pretreated cell lines derived from human melanomas, adenocarcinomas, and lymphomas with the PPIs omeprazole, esomeprazole, or pantoprazole and tested their response to cytotoxic drugs in cell death assays. We also evaluated extracellular and intracellular pH and vacuolar-H + -ATPase (V-H + -ATPase) expression, distribution, and activity in PPI-pretreated cells by using western blot analyses, immunocytochemistry, laser scanning confocal analysis, and bioluminescence assays. Finally, we evaluated human melanoma growth and cisplatin sensitivity with or without omeprazole pretreatment in xenografted SCID/SCID mice. Results: PPI pretreatment sensitized tumor cell lines to the effects of cisplatin, 5-fluorouracil, and vinblastine, with an IC 50 value reduction up to 2 logs. PPI pretreatment was associated with the inhibition of V-H + -ATPase activity and increases in both extracellular pH and the pH of lysosomal organelles. PPI pretreatment induced a marked increase in the cytoplasmic retention of the cytotoxic drugs, with clear targeting to the nucleus in the case of doxorubicin. In in vivo experiments, oral pretreatment with omeprazole was able to induce sensitivity of human solid tumors to cisplatin. Conclusion: Our results open new possibilities for the treatment of drug-resistant tumors through combination strategies based on the use of well-tolerated pH modulators such as PPIs.

Particulate matter (PM), a component of urban air pollution that derives primarily from the combustion of fossil fuels, is responsible for a number of health effects in humans. Recent studies have demonstrated that the fine particles (PM(2.5)) present in high numbers in PM samples can be more harmful than larger particles, since they are more efficiently retained in the peripheral lung. In the present study, we have investigated the biological effects of PM(2.5) on human lung epithelial cell line A549. Morphological analysis performed by immunofluorescence and electron microscopy showed that fine particles interact with the cell surface, where they induce evident alterations and, subsequently, are internalized in the cytoplasm. Cytoskeletal components, in particular microfilaments and microtubules, cause modifications upon challenge with PM(2.5). Of interest, an early cell response to the fine particulate is an increase of reactive oxygen species content, which can account for the observed cytoskeletal changes and the production of proinflammatory cytokines in A549 cells. In particular, exposure to PM(2.5) promoted a dose- and time-dependent release of TNF-alpha and IL-6 in the cell medium.

The search for innovative therapeutic approaches based on the use of new substances is gaining more interest in clinical oncology. In this in vitro study the potential anti-tumoral activity of tea tree oil, distilled from Melaleuca alternifolia, was analyzed against human melanoma M14 WT cells and their drug-resistant counterparts, M14 adriamicin-resistant cells. Both sensitive and resistant cells were grown in the presence of tea tree oil at concentrations ranging from 0.005 to 0.03%. Both the complex oil (tea tree oil) and its main active component terpinen-4-ol were able to induce caspase-dependent apoptosis of melanoma cells and this effect was more evident in the resistant variant cell population. Freeze-fracturing and scanning electron microscopy analyses suggested that the effect of the crude oil and of the terpinen-4-ol was mediated by their interaction with plasma membrane and subsequent reorganization of membrane lipids. In conclusion, tea tree oil and terpinen-4-ol are able to impair the growth of human M14 melanoma cells and appear to be more effective on their resistant variants, which express high levels of P-glycoprotein in the plasma membrane, overcoming resistance to caspase-dependent apoptosis exerted by P-glycoprotein-positive tumor cells.

Salivary gland tumours are rare tumours characterized by histopathologic complexity and a wide variety of morphologic features. Studies on genetic changes in different histological subtypes of salivary gland tumours are important to better understand molecular pathogenetic mechanisms and to identify diagnostic and prognostic markers. Data are even more scanty dealing with unusual subtypes of these tumours. The aim of the present study was to analyse two high grade transformation adenoid cystic carcinomas (hgACC) and one hybrid tumour in order to identify, by mutational and microsatellite analysis, genetic alterations in TP53, CDKN2A/ARF, RAS, BRAF, PTEN, MAPK2 and EGFR genes. The two hgACCs showed snps missense in RAS genes and alterations with allelic instability in CDKN2A/ARF; moreover, a double mutation in TP53 was detected in one case. The hybrid tumour showed alterations in CDKN2A/ARF gene and snps missense in NRAS genes. Our data suggest that CDKN2A/ARF pathway might be involved in pathogenesis of the salivary gland tumours analysed. Further molecular analyses of these very rare tumours are necessary to better understand the role of other genetic alterations detected in our study.

Myelofibrosis (MF) is characterized by hyperactivation of thrombopoietin (TPO) signaling, which induces a RPS14 deficiency that de-regulates GATA1 in megakaryocytes by hampering its mRNA translation. As mice carrying the hypomorphic Gata1low mutation, which reduces the levels of Gata1 mRNA in megakaryocytes, develop MF, we investigated whether the TPO axis is hyperactive in this model. Gata1low mice contained two times more Tpo mRNA in liver and TPO in plasma than wild-type littermates. Furthermore, Gata1low LSKs expressed levels of Mpl mRNA (five times greater than normal) and protein (two times lower than normal) similar to those expressed by LSKs from TPO-treated wild-type mice. Gata1low marrow and spleen contained more JAK2/STAT5 than wild-type tissues, an indication that these organs were reach of TPO-responsive cells. Moreover, treatment of Gata1low mice with the JAK inhibitor ruxolitinib reduced their splenomegaly. Also in Gata1low mice activation of the TPO/MPL axis was associated with a RSP14 deficiency and a discordant microarray ribosome signature (reduced RPS24, RPS26 and SBDS expression). Finally, electron microscopy revealed that Gata1low megakaryocytes contained poorly developed endoplasmic reticulum with rare polysomes. In summary, Gata1low mice are a bona fide model of MF, which recapitulates the hyperactivation of the TPO/MPL/JAK2 axis observed in megakaryocytes from myelofibrotic patients.

Our study showed the detection of S. suis in DNA from tissue belonging to 4 patients out of 8 with salivary gland tumors. To our knowledge there are no prevalence data of S. suis in the region where the patients were recruited; however, over the past few years the number of reported S. suis infections in humans has increased significantly (5) and it is worthwhile to report this finding to accumulate significant data about possible risk factors for these rare tumors.

Steady state erythropoiesis is regulated by interaction of EPO with its receptor, EPO-R which activates Ca2+signaling and terminal maturation [Miller, Blood 1989;73:1188]. Under stress, GR switches EPO-R signaling to a proliferation mode allowing erythroblast (Ery) amplification [Zhang, Nature 2013;499:92]. However, for recovery from anemia to occur, a mechanism, still to be identified, must counteract GR and switch EPO-R signaling back to a maturation mode. CALR chaperones other proteins to their active sites[Michalak, Biochem J 1999;344: 281]. By importing GR from the nucleus, CALR resets the stress response of murine fibroblasts. This function is regulated by the exposure of its C-terminal domain (C-CALR) sustained by Ca2+ [Holaska, Mol Cell Biol 2002;22:6286]. Whether CALR regulates nuclear import of GR in human Ery and how this regulation is modulated by Ca2+and EPO is unknown. Studies were performed to fill this gap. By WB, human Ery expanded in vitro from adult blood expressed robust levels of CALR, detectable with N-CALR- and C-CALR antibodies, and of GR. By flow cytometry, N-CALR and C-CALR were detectable on the surface of cells from the proerythroblast (proEry) to polychromatic stage. Expression of N-CALR, the epitope most exposed on the surface, was up-regulated by EPO. By confocal microscopy, both N-CALR and C-CALR were detected in non-permeabilized Ery but only C-CALR was detectable in cells permeabilized to reveal their cytoplasmic content. Intra-cytoplasmic C-CALR staining was robust in proEry, barely detectable in orthochromatic cells and enhanced by adding Ca2+ to the permeabilization buffer. Double staining of C-CALR with GR or LaminB1 (an inner nuclear membrane protein) indicated that C-CALR/GR were associated in the perinuclear area of the cytoplasm but that C-CALR remained distinct from Lamin B1, suggesting that C-CALR/GR co-localize at the cytoplasm/nuclear boundary. These results suggest that C-CALR mediates nuclear export of GR and that this function is regulated by Ca2+. This hypothesis was tested first by determining content and localization of GRα and C-CALR in nuclear and cytoplasmic fractions of Ery treated with Dex, EPO and SCF by WB. Although none of these stimulations altered the total content of these proteins, some of them affected their compartmentalization: Dex rapidly (15') induced GR S211 phosphorylation and increased the levels of GRα in nuclear fractions. These effects were antagonized by addition of the GR inhibitor RU486. The nuclear levels of GRα were modestly increased also by SCF but strongly decreased by EPO. By contrast, regardless of the stimuli, C-CALR was detected mostly in cytoplasmic fractions. The effects of these stimulations were also assessed by confocal microscopy. Exposure to Dex and EPO increased by 15' but decreased by 4h the nuclear localization of GR and significantly increased the levels of C-CALR and of C-CALR/GR co-localization detected in the cytoplasm by 4h, suggesting that C-CALR drives nuclear export of GR. In agreement with this hypothesis, multi-regression analyses comparing single C-CALR and total and cytoplasmic GR signals as independent parameters against merged signals as dependent parameter in 410 individual proEry indicated that the levels of C-CALR/GR co-localization directly correlated with the cytoplasmic levels of both C-CALR and GR but that the greatest p-values were observed for C-CALR (p=0.0000 vs 0.045). Moreover, treatment with the nuclear export inhibitor Leptomycin greatly decreased C-CALR/GR co-localization, increased nuclear localization of GR in proEry, both in untreated and EPO-treated cells, and reduced Ery proliferation in response to Dex down to levels sustained by Dex with RU486. To assess the role of Ca2+ in the nuclear export activity of C-CALR confocal microscopy and proliferation studies on Ery exposed or not to the Ca2+ chelator BAPTA in combination with Ionomycin (Io, a raiser of intracellular Ca2+) or Thapsigargin (TG, a reducer of Ca2+in endoplasmic reticulum) were performed. These treatments greatly decreased C-CALR and C-CALR/GR co-localization in the cytoplasm, increased GR signals in nuclei and reduced the proliferation of Ery in response to Dex. These results suggest that conformation changes of C-CALR induced by Ca2+signaling downstream to EPO-R sustain nuclear export of GR, resetting the stress-response of normal proEry allowing them to undergo terminal maturation. Disclosures No relevant conflicts of interest to declare.

Elevation of cAMP inhibits the proliferation and expression of transformed phenotype in several cell types, including breast cancer cells. Leptin has been shown to act as a mitogen/survival factor in many types of cancer cells. In the present work, we have studied the impact of cAMP elevation on leptin-induced proliferation of breast cancer cells. Here we report that treatment of estrogen receptor negative human breast cancer cell line MDA-MB-231 with leptin or cAMP elevating agents has positive and negative effects on cell proliferation, respectively. Surprisingly, we find that leptin strongly potentiates the anti-proliferative action of cAMP elevating agents, by concurring to cell cycle arrest at G1 phase and inducing apoptosis. Pretreatment with the PKA inhibitor KT-5720 completely prevented the anti-proliferative effects induced by the combination between leptin and cAMP elevating agents. The above anti-proliferative effects were paralleled by the decrease of cyclin D1 and A and by the increase of inhibitor p27kip1 cell cycle regulating protein levels. In these conditions we found also a strong decrease of anti-apopotic Bcl2 protein levels. Altogether, our data extend the evidence of adenylate cyclase/cAMP/PKA as a growth suppressor system and of leptin as a growth promoting factor in breast cancer cells. Remarkably, our results suggest that when cAMP levels are increased, leptin drives cells towards apoptosis, and that targeting both cAMP levels and leptin signalling might represent a simple novel way for therapeutic intervention in breast cancer.

The occurrence of multidrug resistance (MDR) is the major obstacle to successful anthracycline-based cancer chemotherapy. In the present study, we assessed the effects of Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl, TPL), a piperidine nitroxide with growth-inhibitory properties in tumor cell lines, on a number of molecular mechanisms involved in the resistance of human breast adenocarcinoma cell lines to doxorubicin (DOX). Cytotoxicity studies in MCF-7 wildtype and their MDR variant MCF-7 AdrR cells showed a synergistic effect between TPL and DOX when exposure to TPL preceded or was simultaneous with DOX treatment in MCF-7 AdrR cells. This effect of TPL seems to be due in part to its ability to increase peroxide levels and to deplete cellular glutathione pools. In addition, TPL increased DOX accumulation in MCF-7 AdrR cells by interfering with P-glycoprotein-mediated DOX efflux, as evidenced using a specific antibody that recognizes the active form of the protein. TPL was also found to affect the expression levels of proteins involved in response to drug treatment (e.g., p53, bcl2, bax, p21). Taken together, our results indicate that TPL is a potential new agent that may improve the clinical effect of DOX in tumors exhibiting a MDR phenotype.

We have previously reported that interferon-α (IFNα) induces apoptosis and EGF can antagonize this effect in human epidermoid cancer KB cells. Since apoptosis occurs together with cytoskeleton reorganization we have evaluated if IFNα and EGF could modulate cell remodeling in our experimental conditions. We have found that 48 h 1,000 IU/ml IFNα induced structural reorganization of stress fibers and membrane delocalization and partial capping of the actin severing protein gelsolin. The transfection of KB cells with both a wild type (WT) or a C-terminal truncated form of gelsolin caused overexpression of the protein and an increase of both the spontaneous and IFNα-induced apoptosis and cell cytoskeletal modifications. In fact, after 48 h of treatment IFNα induced 45% of apoptotic cell death in parental cells while an approximately 80% of cell population was apoptotic in transfected cells. These effects occurred together with an increase of the expression and consequent degradation of gelsolin. Again the addition of EGF to IFNα-treated transfected cells caused a recovery of the apoptosis. Notably, IFNα and EGF did not modify the expression of other molecules associated to cytoskeleton such as focal adhesion kinase and vinculin. In the same experimental conditions IFNα induced also gelsolin cleavage that occurred together with caspase-3 activation and release of cytochrome c. All these effects were antagonized by the exposure of IFNα-treated KB to 10 nM EGF for the last 12 h. Moreover, the specific inhibition of caspase-3 with 20 μM DEVD completely abrogated apoptosis and gelsolin cleavage induced by IFNα. In conclusion, our data are the first demonstration that IFNα can induce morphological cell changes that are peculiar of apoptosis onset through the caspase-3-mediated cleavage of gelsolin. Furthermore, we have demonstrated that EGF is able to antagonize these effects through the inhibition of caspase-3 activation. J. Cell. Physiol. 201: 71–83, 2004. © 2004 Wiley-Liss, Inc.

Multidrug-resistance (MDR) is largely caused by the efflux of therapeutics from the tumor cell by means of P-glycoprotein (P-gp), resulting in reduced efficacy of the chemotherapy. In order to overcome MDR, substances, such as verapamil and cyclosporin A (CsA), were employed. As these P-gp modulating agents did not seem promising in clinical practice, new compounds with a low degree of undesirable side effects, were introduced. In this study, bisindolic alkaloid voacamine was examined for its possible capability of enhancing the cytotoxic effect of doxorubicin (DOX) on drug resistant cells. Two different pairs of tumor cell lines were analyzed: the parental lymphoblastoid cell line CEM-WT and its MDR derivative CEM-R, the parental osteosarcoma cell line U-2 OS-WT and its resistant counterpart U-2 OS-R. These cell lines were characterized for their morphological features by scanning electron microscopy (SEM) and for the expression of the main drug transporters by flow cytometric analysis. The effects of voacamine on the cell survival and on both accumulation and efflux of DOX were then investigated. The intracellular distribution of DOX, given alone or in association with CsA or voacamine, was observed by laser scanning confocal microscopy. A differential effect of voacamine between sensitive and resistant cells on the intracellular DOX concentration and distribution was shown. In particular, voacamine induced a significant increase of drug retention and intranuclear location in resistant cells. The results of cell survival experiments revealed an enhancement of the cytotoxic effect of DOX induced by voacamine, confirmed by evident morphological changes observed by SEM. These findings suggest promising applications of this natural substance against MDR tumors.

Drug resistance, one of the major obstacle in the successful anticancer therapy, can be observed at the outset of therapy (intrinsic resistance) or after exposure to the antitumor agent (acquired resistance). To gain a better insight into the mechanisms of intrinsic resistance we have analyzed two human cell types derived from untreated tumors: MCF-7 breast cancer and A549 non small cell lung cancer (NSCLC). We have examined: the cytotoxic effect induced by doxorubicin (DOX); the time course of drug accumulation by flow cytometry and intracellular drug distribution by confocal microscopy; the expression and distribution of proteins related to anthracycline resistance, such as P-gp (P-glycoprotein), MRP1 (multidrug resistance-associated protein) and LRP (lung resistance-related protein). The cytotoxicity assays showed that A549 cells were less sensitive than MCF-7 cells to the DOX treatment in agreement with the different DOX uptake. Moreover, while in A549 cells DOX was mostly located in well defined intracytoplasmic vesicles, in MCF-7 cells it was mainly revealed inside the nuclei. The analysis of P-gp and MRP expression did not show significant differences between the two cell lines while a high expression of LRP was detected at the nuclear envelope and cytoplasmic levels in A549 cells. These findings suggest that the lower sensitivity to DOX treatment showed by lung carcinoma cells could be ascribed to drug sequestration by LRP inside the cytoplasmic compartments.

A number of studies have reported that increased P-glycoprotein expression in drug-resistant tumour cells may be associated with decreased expression of a family of surface glycoproteins. However, despite its potential biological and clinical relevance, this phenomenon has not been extensively studied. In this study the phenotypic alterations that are associated with the acquisition of the multidrug-resistant phenotype in tumour cells, together with drug transporter overexpression, were investigated in human melanoma cells. The expression of cell adhesion molecules was analysed in a panel of multidrug-resistant melanoma cell lines (M14Dx) showing different degrees of resistance to doxorubicin and different levels of the expression of the drug transporter P-glycoprotein. In particular, expression of intercellular adhesion molecule-1 (ICAM-1), CD44, very late activation antigen (VLA)-5 and VLA-2 was determined by flow cytometry in the different resistant cell lines. A progressive downregulation of all the adhesion molecules examined was revealed in M14Dx cells, in parallel with an increasing level of expression of the drug transporter P-glycoprotein. The results obtained raise the question of the role of P-glycoprotein in the invasive and metastatic behaviour of tumour cells.

The occurrence of resistance to cytotoxic agents in tumor cells, associated with several phenotypic alterations, is one of the major obstacles to successful anticancer chemotherapy. A new strategy to overcome MDR of human cancer cells was studied, using BSAO, which generates cytotoxic products from spermine, H(2)O(2) and aldehyde(s). The involvement of these products in causing cytotoxicity was investigated in both drug-sensitive (LoVo WT) and drug-resistant (LoVo DX) colon adenocarcinoma cells. Evaluation of clonogenic cell survival showed that LoVo DX cells are more sensitive than LoVo WT cells. Fluorometric assay and treatments performed in the presence of catalase demonstrated that the cytotoxicity was due mainly to the presence of H(2)O(2). Cytotoxicity was eliminated in the presence of both catalase and ALDH. Transmission electron microscopic observations showed more pronounced mitochondrial modifications in drug-resistant than in drug-sensitive cells. Mitochondrial functionality studies performed by flow cytometry after JC-1 labeling revealed basal hyperpolarization of the mitochondrial membrane in LoVo DX cells. After treatment with BSAO and spermine, earlier and higher mitochondrial membrane depolarization was found in LoVo DX cells than in drug-sensitive cells. In addition, higher basal ROS production in LoVo DX cells than in drug-sensitive cells was detected by flow-cytometric analysis, suggesting increased mitochondrial activity in drug-resistant cells. Our results support the hypothesis that mitochondrial functionality affects the sensitivity of cells to the cytotoxic enzymatic oxidation products of spermine, which might be promising anticancer agents, mainly against drug-resistant tumor cells.