Francesco Fabbri

In vitro and in vivo evaluation of NCX 4040 cytotoxic activity in human colon cancer cell lines

BackgroundNitric oxide-releasing nonsteroidal antiinflammatory drugs (NO-NSAIDs) are reported to be safer than NSAIDs because of their lower gastric toxicity. We compared the effect of a novel NO-releasing derivate, NCX 4040, with that of aspirin and its denitrated analog, NCX 4042, in in vitro and in vivo human colon cancer models and investigated the mechanisms of action underlying its antitumor activity.MethodsIn vitro cytotoxicity was evaluated on a panel of colon cancer lines (LoVo, LoVo Dx, WiDr and LRWZ) by sulforhodamine B assay. Cell cycle perturbations and apoptosis were evaluated by flow cytometry. Protein expression was detected by Western blot. In the in vivo experiments, tumor-bearing mice were treated with NCX 4040, five times a week, for six consecutive weeks.ResultsIn the in vitro studies, aspirin and NCX 4042 did not induce an effect on any of the cell lines, whereas NCX 4040 produced a marked cytostatic dose-related effect, indicating a pivotal role of the -NO2 group. Furthermore, in LoVo and LRWZ cell lines, we observed caspase-9 and -3-mediated apoptosis, whereas no apoptotic effect was observed after drug exposure in WiDr or LoVo Dx cell lines. In in vivo studies, both NCX 4040 and its parental compound were administered per os. NCX 4040 induced a 40% reduction in tumor weight. Conversely, aspirin did not influence tumor growth at all.ConclusionsNCX 4040, but not its parental compound, aspirin, showed an in vitro and in vivo antiproliferative activity, indicating its potential usefulness to treat colon cancer.

Detection and recovery of circulating colon cancer cells using a dielectrophoresis-based device: KRAS mutation status in pure CTCs

The characterization of circulating tumor cells (CTCs) could substantially improve the management of cancer patients. However, their study is still a matter of debate, often due to lymphocyte contamination. In the present paper, an investigation of CTCs was carried out for the first time using DEPArray, a dielectrophoresis-based platform able to detect and sort pure CTCs. Analyses were conducted on peripheral blood (PB) samples from patients with metastatic colon cancer. After 100% pure cell recovery and whole genome amplification, KRAS gene mutation of CTCs was screened and compared to gene status in the primary tumor tissue. CTCs were found in 21 colon cancer patients (52.5%), with more than three tumor cells per 7.5 ml. KRAS gene mutation analysis, showed a mutational concordance between CTCs and primary tumor in 50% of matched cases. The present study demonstrates for the first time the feasibility of analyzing at the molecular level pure CTCs avoiding lymphocyte contamination using an innovative instrumentation, and a KRAS discordance between CTCs and primary tissue. Our results present dielectrophoresis-based procedures as a new standard in single cell analysis and recovery and invite careful reflection on the value of CTCs characterization.

p16INK4A and CDH13 hypermethylation in tumor and serum of non-small cell lung cancer patients

Aberrant promoter hypermethylation of several known or putative tumor suppressor genes occurs frequently during the etiopathogenesis of lung cancer and is a promising tool for cancer detection. In the present study, promoter hypermethylation of p16INK4A and CDH13 genes was investigated in tumor tissue and in matched serum from 61 patients with histologically confirmed non‐small cell lung cancer. Using a fluorescence‐based method of methylation‐specific PCR (F‐MSP), methylation of p16INK4A and CDH13 was detected in 79% and 66% of tumors, respectively, and was not significantly related to conventional clinicopathological characteristics of patients or tumors. Methylation of both genes was observed in 52% of tumors and of at least one gene in 92% of lesions. In matched serum, hypermethylation of p16INK4A and CDH13 was observed in 26% and 23% of patients, respectively, but as they were not associated, the methylation of at least one gene was detected in 39% of patients. In conclusion, the frequency of p16INK4A or CDH13 hypermethylation in patient serum, together with evidence of their early occurrence in lung cancerogenesis and the total lack of methylation in serum from healthy individuals, offer a promising tool for non invasive early detection of lung cancer.

Sequential events of apoptosis involving docetaxel, a microtubule-interfering agent: A cytometric study

BackgroundDespite the great advances in the understanding of programmed cell death, little attention has been paid to the sequence of the events that characterise it. In particular, the course of apoptotic events induced by microtubule-interfering agents such as taxanes is poorly understood. In order to increase such knowledge, we studied a number of independent biochemical and cytological modifications using cytometric methods in a bladder cancer cell line treated with the second generation taxane, docetaxel.ResultsWithin a few hours, drug treatment had induced mitochondrial membrane transition, cell shrinkage and a decrease in granularity. Cell cycle was almost completely blocked in G2/M phase within 24 hours. The hypodiploid peak started to become prominent 48 hours after the treatment. At the same time, the appearance of a DNA ladder demonstrated caspase-dependent chromatin fragmentation. Concurrently, specific cell surface modifications took place, involving at first glycoprotein syalilation and later phospholipid asymmetry. DNA fragmentation was subsequently detected by TUNEL assay. Over time, cell membranes became permeable to propidium iodide. A very similar time-course of apoptotic events was found after treatment of a myelomonocytic cell line with the same drug.ConclusionAfter discussing some characteristics of the methods employed and their limitations, a succession of apoptotic events over time is suggested, in which the collapse of mitochondrial transmembrane potential (Δψm) is the earliest sign of apoptosis.

Role of RAF/MEK/ERK pathway, p-STAT-3 and Mcl-1 in sorafenib activity in human pancreatic cancer cell lines.

Sorafenib is a multikinase inhibitor that has shown promising therapeutic results in different tumor histotypes, both as a single agent or in combination with other treatments. We analyzed the in vitro activity of sorafenib in pancreatic cancer, one of the most lethal and chemo‐radio‐resistant tumors, using four human pancreatic cancer cell lines (t3m4, Capan 1, Capan 2, and MiaPaca 2), characterized by different K‐ras gene status and RAF/MEK/ERK profile. Sorafenib exerted a strong anti‐proliferative effect independently of RAS/RAF/MEK/ERK and induced various degrees of apoptosis in the cell lines. The mechanisms involved were explored in detail in t3m4 and Capan 1, in which sorafenib induced the highest and lowest levels of apoptosis, respectively. In t3m4, the RAF/AKT/STAT‐3 rather than the RAF/MEK/ERK pathway was involved, whereas in Capan 1 cells there was a strong decrease in pMEK and pERK which was not accompanied by an important reduction in RAF, AKT, and STAT‐3 proteins or in their phosphorylation. Moreover, U0126‐induced MEK inhibition did not induce apoptosis in any cell line, reinforcing the hypothesis of a MEK/ERK‐independent mechanism of sorafenib activity. Mcl‐1 appears to play a crucial role in sorafenib‐induced apoptosis. In fact, both protein and mRNA were downregulated in t3m4 and upregulated in Capan 1, in which siRNA‐induced silencing resulted in the same level of apoptosis as observed in t3m4. Our results show that sorafenib exerts anti‐proliferative and pro‐apoptotic activity in pancreatic cancer cells. Used singly or in combination with other drugs, it could therefore represent valid treatment for pancreatic cancer. J. Cell. Physiol. 220: 214–221, 2009.

Addition of 5-fluorouracil to doxorubicin-paclitaxel sequence increases caspase-dependent apoptosis in breast cancer cell lines

IntroductionThe aim of the study was to evaluate the activity of a combination of doxorubicin (Dox), paclitaxel (Pacl) and 5-fluorouracil (5-FU), to define the most effective schedule, and to investigate the mechanisms of action in human breast cancer cells.MethodsThe study was performed on MCF-7 and BRC-230 cell lines. The cytotoxic activity was evaluated by sulphorhodamine B assay and the type of drug interaction was assessed by the median effect principle. Cell cycle perturbation and apoptosis were evaluated by flow cytometry, and apoptosis-related marker (p53, bcl-2, bax, p21), caspase and thymidylate synthase (TS) expression were assessed by western blot.Results5-FU, used as a single agent, exerted a low cytotoxic activity in both cell lines. The Dox→Pacl sequence produced a synergistic cytocidal effect and enhanced the efficacy of subsequent exposure to 5-FU in both cell lines. Specifically, the Dox→Pacl sequence blocked cells in the G2-M phase, and the addition of 5-FU forced the cells to progress through the cell cycle or killed them. Furthermore, Dox→Pacl pretreatment produced a significant reduction in basal TS expression in both cell lines, probably favoring the increase in 5-FU activity. The sequence Dox→Pacl→48-h washout→5-FU produced a synergistic and highly schedule-dependent interaction (combination index < 1), resulting in an induction of apoptosis in both experimental models regardless of hormonal, p53, bcl-2 or bax status. Apoptosis in MCF-7 cells was induced through caspase-9 activation and anti-apoptosis-inducing factor hyperexpression. In the BRC-230 cell line, the apoptotic process was triggered only by a caspase-dependent mechanism. In particular, at the end of the three-drug treatment, caspase-8 activation triggered downstream executioner caspase-3 and, to a lesser degree, caspase-7.ConclusionIn our experimental models, characterized by different biomolecular profiles representing the different biology of human breast cancers, the schedule Dox→Pacl→48-h washout→5-FU was highly active and schedule-dependent and has recently been used to plan a phase I/II clinical protocol.

Epithelial Mesenchymal Transition: a double-edged sword

Epithelial mesenchymal transition (EMT) is a physiological process necessary to normal embryologic development. However in genesis of pathological situations, this transition can be perverted and signaling pathways have different regulations from those of normal physiology. In cancer invasion, such a mechanism leads to generation of circulating tumor cells. Epithelial cancer cells become motile mesenchymal cells able to shed from the primary tumor and enter in the blood circulation. This is the major part of the invasive way of cancer. EMT is also implicated in chronic diseases like fibrosis and particularly renal fibrosis. In adult organisms, healing is based on EMT which is beneficial to repair wounds even if it can sometimes exceed its goal and elicit fibrosis. In this review, we delineate the clinical significance of EMT in both physiological and pathological circumstances.

Mitotic catastrophe and apoptosis induced by docetaxel in hormone-refractory prostate cancer cells.

Studies performed in different experimental and clinical settings have shown that Docetaxel (Doc) is effective in a wide range of tumors and that it exerts its activity through multiple mechanisms of action. However, the sequence of events induced by Doc which leads to cell death is still not fully understood. Moreover, it is not completely clear how Doc induces mitotic catastrophe and whether this process is an end event or followed by apoptosis or necrosis. We investigated the mechanisms by which Doc triggers cell death in hormone‐refractory prostate cancer cells by analyzing cell cycle perturbations, apoptosis‐related marker expression, and morphologic cell alterations. Doc induced a transient increase in G2/M phase followed by the appearance of G0/1 hypo‐ and hyperdiploid cells and increased p21 expression. Time‐ and concentration‐dependent apoptosis was induced in up to 70% of cells, in concomitance with Bcl‐2 phosphorylation, which was followed by caspase‐2 and ‐3 activation. In conclusion, Doc would seem to trigger apoptosis in hormone‐refractory prostate cancer cells via mitotic catastrophe through two forms of mitotic exit, in concomitance with increased p21 expression and caspase‐2 activation. J. Cell. Physiol. 217: 494–501, 2008.

Efficacy of a nitric oxide–releasing nonsteroidal anti-inflammatory drug and cytotoxic drugs in human colon cancer cell lines in vitro and xenografts

We previously showed that NCX 4040 inhibits in vitro and in vivo tumor growth and induces apoptosis in human colon cancer cell lines. On the basis of these results, NCX 4040 antitumor activity in combination with 5-fluorouracil (5-FU) or oxaliplatin was evaluated in vitro and in vivo in human colon cancer models. The cytotoxicity of different NCX 4040 and 5-FU or oxaliplatin combination schemes was evaluated on a panel of colon cancer lines (LoVo, LoVo Dx, WiDr, and LRWZ) by the sulforhodamine B assay, and apoptosis was assessed by flow cytometry. NCX 4040 and 5-FU combination was always additive in vitro regardless of the scheme used. Sequential NCX 4040→oxaliplatin treatment produced a strong synergism in three cell lines, with a ratio index ranging from 3.7 to 4. The synergistic effect was accompanied by apoptosis induction (up to 40%). In the in vivo experiments, xenografted mice were treated with the sequential combination of NCX 4040 and oxaliplatin, and apoptosis was evaluated immunohistochemically in excised tumors. Furthermore, in WiDr xenografts, this sequence caused a significantly higher reduction (∼60%) in tumor growth compared with single-drug treatments and produced extensive apoptotic cell death (15.3%), significantly higher (P < 0.01) than that observed in untreated tumors (2.7%) or in tumors treated with NCX 4040 (5.1%) or oxaliplatin (5.7%) alone. These data show that NCX 4040 sensitizes colon cancer cell lines to the effect of antitumor drugs and suggests that their combination could be useful for the clinical management of colon cancer. [Mol Cancer Ther 2006;5(4):919–26]

Circulating tumor cells and epithelial, mesenchymal and stemness markers: characterization of cell subpopulations

Until now detection and numeration of circulating tumor cells (CTCs) were essentially used as a prognostic factor in cancer progression. To extend the role of these kinds of analysis, it seems necessary to improve analytical methods related to isolation and characterization of CTCs. Discrepancies between published results corroborates this requirement. In this review we suggest a combination of markers able to reach the goal. Moreover to improve the clinical utility of CTC analysis, particularly in the therapeutic follow up of the disease, epithelial mesenchymal transition (EMT) level of a global CTC population should be studied.