Amalia Azzariti

Cytochrome c Is Released from Mitochondria in a Reactive Oxygen Species (ROS)-dependent Fashion and Can Operate as a ROS Scavenger and as a Respiratory Substrate in Cerebellar Neurons Undergoing Excitotoxic Death

In rat cerebellar granule cells both reactive oxygen species production and release of cytochrome c take place during glutamate toxicity. This investigation was aimed (i) to ascertain whether and how these two processes are related and (ii) to gain insight into the role played by the released cytochrome c in the onset of neurotoxicity. Cytochrome c release takes place owing to the generation of reactive oxygen species both in glutamate-treated cerebellar granule cells and in sister control cultures incubated in the presence of the reactive oxygen species-generating system consisting of xanthine plus xanthine oxidase. In the early phase of neurotoxicity (30-min glutamate exposure) about 40% of the maximum (as measured at 3 h of glutamate exposure) cytochrome c release was found to occur in cerebellar granule cells from mitochondria that were essentially coupled and intact and that had a negligible production of oxygen free radicals. Contrarily, mitochondria from cells treated with glutamate for 3 h were mostly uncoupled and produced reactive oxygen species at a high rate. The cytosolic fraction containing the released cytochrome c was able to transfer electrons from superoxide anion to molecular oxygen via the respiratory chain and was found to partially prevent glutamate toxicity when added externally to cerebellar neurons undergoing necrosis. In the light of these findings, we propose that in the early phase of neurotoxicity, cytochromec release can be part of a cellular and mitochondrial defense mechanism against oxidative stress.

Cyclohexylpiperazine derivative PB28, a σ2 agonist and σ1 antagonist receptor, inhibits cell growth, modulates P-glycoprotein, and synergizes with anthracyclines in breast cancer

σ Ligands have recently been shown to have cytotoxic activity, to induce ceramide-dependent/caspase-independent apoptosis, and to down-regulate P-glycoprotein (P-gp) mRNA levels in some mouse and human models. In this study, we verified whether a mixed σ2 agonist/σ1 antagonist, PB28, was able to have antitumor activity and to enhance anthracycline efficacy in two human breast cancer cell lines, MCF7 and MCF7 ADR, both characterized by significant σ2 receptor expression, by high and low σ1 receptor expression, and low and high P-gp expression, respectively. In both cell lines, PB28 showed high σ2 receptor affinity and low σ1 receptor affinity; furthermore, it inhibited cell growth with a clear effect at 48 hours (IC50 in nanomolar range), a consistent time exposure-independent increase of G0-G1-phase fraction (of ∼20% of both cell lines) and caspase-independent apoptosis (15% increased after 1-day drug exposure). PB28 also reduced P-gp expression in a concentration- and time-dependent manner (∼60% in MCF7 and 90% in MCF7 ADR). We showed also a strong synergism between PB28 and doxorubicin by adopting either simultaneous or sequential schedules of the two drugs. We suggest that this synergism could depend on PB28-induced increase of intracellular accumulation of doxorubicin (∼50% in MCF7 and 75% in MCF7 ADR by flow cytometry analysis). In conclusion, we suggest that the σ2 agonist PB28 could be an interesting antitumor agent either in monotherapy or in combination with conventional drugs. [Mol Cancer Ther 2006;5(7):1807–16]

4-Biphenyl and 2-naphthyl substituted 6,7-dimethoxytetrahydroisoquinoline derivatives as potent P-gp modulators

Starting from lead compound 1 (EC(50)=1.64 microM), its non-basic nucleus has been conformationally restricted by 4-biphenyl and 2-naphthyl moieties. In each series we investigated if the presence of H-bond donor or acceptor substituents, the basicity and the lipophilicity (clogP) were correlated with the P-gp inhibiting activity of tested compounds. In the biphenyl series, derivative 4d displayed the best results (EC(50)=0.05 microM). The corresponding amide 3d was found less active (EC(50)=3.5 microM) ascertaining the importance of basicity in this series whilst the presence of hydroxy or methoxy substituents seems to be negligible. In the naphthyl series, both the basicity and the presence of H-bond donor or acceptor groups seem to be negligible. Moreover, the lipophilicity did not influence the P-gp inhibition activity of each series. Specific biological assays have been carried out to establish the P-gp interacting mechanism of tested compounds discriminating between substrates and inhibitors. Moreover, compound 4d displayed a potent P-gp inhibition activity with good selectivity towards BCRP pump.

Synergic antiproliferative and antiangiogenic effects of EGFR and mTor inhibitors on pancreatic cancer cells

The in vitro efficacy of both EGFR inhibitor gefitinib and mTor inhibitor rapamycin, either administrated alone or in different combination schedules, was analysed in four pancreas cancer cell lines. Both drugs were found to induce cell growth inhibition, apoptosis as well as a slight but stable accumulation of cells in the G0/G1 phase. In all cell lines, neither gefitinib nor rapamycin affected EGFR and the expression of its downstream effectors. By contrast, gefitinib inhibited in a fast and completely way p-EGFR and partially p-Akt while a 3 days-rapamycin exposure resulted in the inhibition of the expression of both mTor and p70S6K. Moreover, after early stimulation, the mTor inhibitor produced a progressive, and almost complete inhibition of p-Akt. The analysis of combined gefitinib and rapamycin administration showed a clear schedule-dependent activity which turned out to be synergic only when gefitinib was given before rapamycin. This synergism seemed to depend on increase of both p-Akt and p70S6K inhibition, the greater the induction of apoptosis, the higher the decrease in cell cycle rate. Moreover, the antiangiogenic activity of the two drugs given in combination was demonstrated by a strong reduction of VEGF release which turned out to be more pronounced in the synergic schedule, and HIF-1alpha inhibition-independent. Our results suggest that the schedule of gefitinib followed by rapamycin, acting at different levels of the EGFR cellular pathway, could induce antitumor and antiangiogenic effects of clinical interest in the pancreas cancer model.

Laminin‐5 stimulates hepatocellular carcinoma growth through a different function of α6β4 and α3β1 integrins

Hepatocellular carcinoma (HCC) growth severely affects prognosis. Ki‐67, a known marker of cell proliferation, is a negative prognostic factor in HCC. Growth factors such as the epidermal growth factor (EGF) induce HCC cell proliferation but do not explain the great heterogeneity of HCC growth. Laminin‐5 (Ln‐5) is an extracellular matrix protein (ECM) present in the tissue microenvironment of HCC. The two main receptors for Ln‐5, integrins α3β1 and α6β4, are expressed on the cell surface of HCC cells. The aim of this study is to investigate an alternative mechanism of HCC growth whereby Ln‐5 promotes HCC cell proliferation through α3β1 and α6β4. HCC tissues containing Ln‐5 display a larger diameter and higher number of positive cells for Ki‐67, a well known proliferative index, as determined by double immunofluorescence staining and real‐time PCR on microdissected tissues. In vitro, Ln‐5, but not collagen I, collagen IV or fibronectin, induces proliferation as much as EGF does, via Erk phosphorylation as a consequence of β4 integrin phosphorylation. However, the two HCC cell lines do not proliferate in presence of Ln‐5 despite β4 integrin and Erk1/2 activation. After transfection with α3 integrin, in the presence of Ln‐5 one of these HCC cell lines acquires a proliferative activity whereas one of the proliferative HCC cell lines, knocked‐down for α3 integrin, loses its proliferative activity. Conclusions: Our study suggests a new mechanism of HCC growth whereby Ln‐5 stimulates proliferation via a different function of α6β4 and α3β1. (HEPATOLOGY 2007.)

Co-expression of CD133+/CD44+ in human colon cancer and liver metastasis

Although relatively good therapeutic results are achieved in non‐advanced cancer, the prognosis of the advanced colon cancer still remains poor, dependent on local or distant recurrence of the disease. One of the factors responsible for recurrence is supposed to be cancer stem cells (CSCs) or tumor‐initiating cells, which are a population of cancer cells with ability to perpetuate themselves through self‐renewal and to generate differentiated cells, thought to be responsible for tumor recurrence. This study globally approach the possible role of tissue‐derived stem cells in the initiation of colon cancer and its metastatic process in the liver. Fresh surgical specimens from colon cancer, non‐tumor tissue and liver metastasis were obtained directly from the operating room, examined, and immediately processed. CSCs were selected under serum‐free conditions and characterized by CD44 and CD133 expression levels. CD133+/CD44+ cell populations were then investigated in paraffin‐embedded tissues and circulating tumor cells isolated from peripheral blood of the same group of colon cancer patients. Our data demonstrate that metastatic properties of cell populations from blood and liver metastasis, differently from primitive tumors, seem to be strictly related to the phenotype CD133 positive and CD44 positive. J. Cell. Physiol. 228: 408–415, 2013.

Target Therapies in Pancreatic Carcinoma

Pancreatic ductal adenocarcinoma (PDAC) occurs in the majority of cases with early locoregional spread and distant metastases at diagnosis, leading to dismal prognosis and limited treatment options. Traditional cytotoxic chemotherapy provides only modest benefit to patients with PDAC. Identification of different molecular pathways, overexpressed in pancreatic cancer cells, has provided the opportunity to develop targeted therapies (monoclonal antibodies and small-molecule inhibitors) and peculiar new class of taxanes with a crucial therapeutic role in this cancer setting. A phase III trial has shown that erlotinib in combination with gemcitabine was clinically irrelevant and skin toxicity can be a positive prognostic factor. Moreover, the combination of cetuximab or erlotinib with radiotherapy in advanced pancreatic cancer has shown to be synergistic and a reversal of radio-resistance has been suggested by inhibition of VEGF/EGFR pathway. To overcome EGFR-inhibition therapy resistance several alternative pathways targets are under investigation (IGF- 1R, MMPs, Hedgehog proteins, m-TOR, MEK, COX-2) and provide the rationale for clinical use in phase II/III studies. Also nab-paclitaxel, a new taxanes class, uses high pancreatic albumin-binding protein SPARC levels to act in cancer cells with a less toxic and more effective dose with respect to classic taxanes. Understanding of molecular pathogenesis of pancreatic adenocarcinoma continues to expand. However, many promising data in preclinic and phase I/II trials did not yield promise in phase III trials, suggesting that identification of predictive biomarkers for these new agents is mandatory. The knowledge of biologic and molecular aspects of pancreatic cancer can be the basis for future therapeutic developments.

Optimize radiochemotherapy in pancreatic cancer: PARP inhibitors a new therapeutic opportunity

Cancer cells may use PARP enzymes and Homologous Recombination to repair single and double strand breaks caused by genotoxic insults. In this study, the PARP‐1 inhibitor Rucaparib was utilized to increase the sensitivity to chemoradiotherapy treatment in BRCA‐2‐deficient and ‐proficient pancreatic cancer cells. We used the pancreatic cancer cell lines, Capan‐1 with mutated BRCA‐2 and Panc‐1, AsPC‐1 and MiaPaCa‐2 with BRCA‐1/2 wild type. Cells were treated with Rucaparib and/or radiotherapy (4–10 Gy) plus Gemcitabine then the capability to proliferate was evaluated by colony formation, cell counting and MTT assays. Flow cytometry, immunocytochemistry and western blotting were utilized to assess cell response to Rucaparib plus irradiation. The antitumour effectiveness of combining the PARP‐1 inhibitor before, together and after radiotherapy evidenced the first as the optimal schedule in blocking cell growth. Pre‐exposure to Rucaparib increased the cytotoxicity of Gemcitabine plus radiotherapy by heavily inducing the accumulation of cells in G2/M phase, impairing mitosis and finally inducing apoptosis and authophagy. The upregulation of p‐Akt and downregulation of p53 were evidenced in MiaPaCa‐2 which displayed replication stress features. For the first time, the rationale of using a PARP inhibitor as chemoradiosensitizer in pancreatic cancer models has been hypothesized and demonstrated.

EGFR and VEGFR as potential target for biological therapies in HCC cells

Hepatocellular carcinoma (HCC) is a highly malignant cancer with poor prognosis. Inhibitors of EGFR and VEGFR for HCC treatment are currently under investigation. Gefitinib and vandetanib inhibit migration of HCC cells on Laminin-5 and Fibronectin, and invasion through matrigel. Both drugs inhibit p-EGFR after short time, while their efficacy on p-Erk1/2 and p-Akt is progressive and stable over time. PI3K/Akt and MEK/Erk1/2 inhibitors, inhibit migration and invasion as well as inducing de-phosphorylation of downstream effectors. Finally, both inhibitors, vandetanib and gefitinib down-regulated the secretion of matrix metalloproteases MMP-2 and MMP-9. All these biological effects seem to depend on the activity of gefitinib and vandetanib blocking activity towards p-EGFR mediated pathways.

Sigma-2 Receptor Agonists as Possible Antitumor Agents in Resistant Tumors: Hints for Collateral Sensitivity

With the aim of contributing to the development of novel antitumor agents, high‐affinity σ2 receptor agonists were developed, with 6,7‐dimethoxy‐2‐[4‐[1‐(4‐fluorophenyl)‐1H‐indol‐3‐yl]butyl]‐1,2,3,4‐tetrahydroisoquinoline (15) and 9‐[4‐(6,7‐dimethoxy‐1,2,3,4‐tetrahydroisoquinolin‐2‐yl)butyl]‐9H‐carbazole (25) showing exceptional selectivity for the σ2 subtype. Most of the compounds displayed notable antiproliferative activity in human MCF7 breast adenocarcinoma cells, with similar activity in the corresponding doxorubicin‐resistant MCF7adr cell line. Surprisingly, a few compounds, including 25, displayed enhanced activity in MCF7adr cells over parent cells, recalling the phenomenon of collateral sensitivity, which is under study for the treatment of drug‐resistant tumors. All of the compounds showed interaction with P‐glycoprotein (P‐gp), and 15 and 25, with the greatest activity, were able to revert P‐gp‐mediated resistance and reestablish the antitumor effect of doxorubicin in MCF7adr cells. We therefore identified a series of σ2 receptor agonists endowed with intriguing antitumor properties; these compounds deserve further investigation for the development of alternate strategies against multidrug‐ resistant cancers.